1 INTRO TO IMMUNOHEMATOLOGY • Performs the first successful whole blood transfusion to treat

hemophilia.
IMMUHOME LEC//MAAM SALTIN 1867
•
•
Joseph Lister
Uses antiseptics to control infection during transfusions.
OUTLINE 1873- US physicians transfuse milk (from cows, goats, and humans)
A. Historical Background and the current trends in Transfusion Medicine 1880
B. Blood Preservation Saline infusion replaces milk as a “blood substitute” due to the
1884
C. Review of Basic Genetics increased frequency of adverse reactions to milk.
D. Review of Fundamentals of Immunology
E. Review of Molecular Biology BLOOD GROUPS
• Karl Landsteiner
HISTORICAL BACKGROUND AND THE CURRENT TRENDS IN TRANSFUSION 1900 • Discovered the first three human blood groups, A, B, and C.
MEDICINE (Blood type C was later changed to O.)
• Blood • Edward Lindeman
→ Ancient Egyptians bathed in it • The first to succeed in designing a device for transfusion.
→ Aristocrats drank it 1901 • Performed a vein-to-vein blood transfusion by using multiple
→ Authors and playwrights used it as themes syringes & a special canula for transfusion from donor to
patient.
→ Modern humanity transfuses it
• Alfred Decastello and Adriano Sturli
• Human blood transfusion is the process of transferring blood or blood-based 1902
• Added AB as the fourth human blood group.
products from an individual into the circulatory system of another.
• Hektoen
• Over the past 100 years, blood transfusion has grown from the transfusion of → Suggested that the safety of transfusion might be
small amounts of fresh whole blood, to one of the most common therapeutic improved by cross matching blood between donors and
medical practices. patients to exclude incompatible mixtures.
• Reuben Ottenberg
EARLY TRANSFUSION 1907
→ performed the first blood transfusion using blood typing
• Pope Innocent VII and crossmatching in New York.
→ First blood transfusion recorded → Ottenberg also observed the mendelian inheritance of
blood groups and recognized the “universal” utility of
→ Blood was taken from 3 young men; unfortunately, all
group O donors.
four (including the Pope) died.
• Moreschi
• Clotting was the principal obstacle to overcome. 1908
1492 • Described the antiglobulin reaction.
• Attempts to find non-toxic anticoagulants began in 1868, when
Braxton Hicks recommended sodium phosphate. • Dr. Karl Landsteiner
• This was perhaps the first example of blood preservation 1990 • published the first of a series of papers demonstrating
research presence of the ABO blood group system.
• Landsteiner, Weiner, Levine and Stetson
1939-1940
• William Harvey • Discovered the Rh blood group system.
1628 1927-1947 The MNSs and P systems were discovered
• Discovered the circulation of blood.
The first recorded successful blood transfusion occurred in • Coombs, Mourant, and Race-
1665 1945 • Described the use of antihuman globulin sera to detect IgG
England.
• Jean Denis antibodies in compatibility testing.
1667 • Published in the Philosophical Transactions his experience in
Paris with transfusing lamb blood. BLOOD STORAGE
• Dr. James Blundell • Alexis Carrel
1908
1818- • The first to successfully transfuse human blood into a patient • Performed direct transfusion.
with postpartum hemorrhage. 1914 • Hustin
1840 • Samuel Armstrong Lane

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 • Long-term anticoagulants, among them sodium citrate, were 1967 Concentrated Rh immune globulin was introduced commercially.
developed. Dry-heated, lyophilized factor VIII and IX concentrates became
1985
• Lewisohn available.
• Determined the minimum amount of citrate needed for Genetically engineered (recombinant) factor VIII became
1915 1993
anticoagulation and demonstrated its nontoxicity in small available.
amouts 1998 Factor IX became available
• Rous and Turner
• Introduced citrate-dextrose solution for the preservation of BLOOD COMPONENT THERAPY
blood • Roger Lee and Paul Dudley White
1916 1912
• However, the function of glucose in RBC metabolism was • Developed the Lee-White clotting time.
not understood until 1930s. therefore, the common practice • Walter and Murphy
of using glucose in the preservative solution was delayed 1950 • Introduced the use of plastic bags as a replacement for
1932 The first blood bank was established in a Leningrad hospital glass bottles.
• Bernard Fantus Concentrated blood platelets were recognized as useful for the
1961 treatment of thrombocytopenia.
1937 • Established the first hospital blood bank in the United
States. The first antihemophilic factor (AHF) concentrate to treat
• Dr. Charles Drew 1962 coagulation disorders in hemophilia patients was developed
• Developed techniques on blood transfusion and blood through fractionation.
preservation during WWII Plasmapheresis was introduced as a means of collecting plasma
1964
1941 • Led to the establishment of a widespread system of blood for fractionation.
banks. Rh immune globulin was commercially introduced to prevent Rh
1967
• Director of the first American Red Cross blood bank at disease in the newborns of Rh-negative women.
Presbyterian Hospital Hepatitis B surface antigen (HBsAg) testing of donated blood
1971
• Loutit and Mollison begins.
1943
• Developed acid citrate dextrose (ACD) solution. 1972 Platelets for transfusion were collected by apheresis.
• Audrey Smith
1950 • Reported the use of glycerol cryoprotectant for freezing red ADVERSE EFFECT OF TRANSFUSION
blood cells. • Greenwalt et al.
• Gibson et al 1962 • Demonstrated leukocyte reduction filters prevented febrile
• developed citrate-phosphate-dextrose (CPD) reactions.
1957 • CPD eventually replaced ACD and became commonly used • Graw et al.
1970
preservative for storage of blood/red cells in liquid form. • Used irradiation to prevent TA-GVHD.
• Shelf-life of blood stored in CPD at 2-4 °C was 21 day. 1971 Commercial testing for hepatitis B surface antigen began.
• A new anticoagulant preservative, CPDA-1, extends the First Acquired Immune Deficiency Syndrome (AIDS) case was
1981
shelf life of whole blood and red blood cells to 35 days, reported.
increasing the blood supply and facilitating resource sharing 1983- Transfusion-transmitted HIV was described.
1979 among blood banks. Human Immunodeficiency Virus (HIV) was identified as the
1984
• The addition of adenine improved the synthesis of ATP in cause of AIDS.
the stored blood, which prolonged the storage of blood/red A test for the HIV antibody was introduced; FDA approves
cells at 2-4 °C to 35 days. 1985 enzyme-linked immunosorbent assay (ELISA), first blood-
screening test to detect HIV antibodies.
BLOOD DERIVATIVES Two tests that screen for indirect evidence of hepatitis were
• Edwin Cohn 1987 developed and implemented, hepatitis B core antibody (anti-
1940 • Developed the cold ethanol fractionation process, called HBc) and the alanine aminotransferase test (ALT).
Cohn fractionation. Testing of donated blood for human-Tlymphotropic-virus-I-
1989
antibody (anti-HTLV-I) begins.
• Pool and Shannon
1961 1990 Testing for hepatitis C became routine.
• Revolutionized the treatment of hemophilia A.
2002 West Nile virus identified as transfusion transmissible.
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What are the different obstacles that they need to overcome in order to have a DONATION PROCESS
successful blood transfusion? STEP 1: Educational Materials
1. Problem with the clotting • Educational (ex. AABB pamphlet “An Important Message to Blood Donors”)
2. Use of Anticoagulant that contains information on the risks of infectious diseases transmitted by
3. Compatibility Testing blood transfusion, including the symptoms and signs of AIDs, is given to
4. Antiseptic Techniques each prospective donor to read.
5. Blood Preservation
6. Component Therapy- in cases of Transfusion Overload
STEP 2: The Donor Health History Questionnaire
ADVANCES IN BLOOD TRANSFUSION • A uniform donor history questionnaire, designed to as questions that protect
● Today, the blood transfusion community continues to advance its transfusion the health of both the donor and the recipients, is given to every donor.
systems, guided by the: • The health history questionnaire is used to identify donors who have been
→ ISBT (International Society of Blood Transfusion), exposed to diseases that can be transmitted in blood
→ AABB (American Association of Blood Banks), ● Example of blood-transmitted diseases;
→ FDA (Food and Drug Administration), and 1. Variant Creutzfeldt-Jakob
→ Other federal and professional organizations. 2. West Nile Virus
● Researchers are establishing new surveillance systems that record data and 3. Malaria
transfusion outcomes (haemovigilance system). 4. Babesiosis
5. Chagas Disease
→ They are offering more personalized treatment, limiting transfusions
based on careful assessment of need, and ultimately improving
patient care.
STEP 3: The Abbreviated Physical Examination
● In addition to infectious disease risks, treating physicians must also manage
other risks, such as post-transfusion reactions. o The abbreviated physical examination for donors includes;
→ These include: 1. Blood pressure, Pulse, and Temperature readings;
▪ Transfusion-related lung injury (TRALI); 2. Hemoglobin or Hematocrit level;
▪ Transfusion associated cardiac overload (TACO); and 3. Inspection of the arms for skin lesions
▪ Post-transfusion iron overload. ● Currently 10 screening tests for infectious disease are performed on each
● To minimize these risks, researchers studying the body’s immune response to unit of donated blood (See Table 1-1; Harmening, 6th Edition)
transfusions have found that modifying the blood prior to transfusion can
reduce reactions.
BLOOD PRESERVATION
→ In particular, removing white blood cells or radiating blood.
→ Recently, studies found that using male plasma and platelets may RBC BIOLOGY
eliminate the transmission of certain antibodies that can cause
reactions and are found only in previously pregnant women and Three areas of RBC biology are crucial for normal erythrocyte survival and function:
transfused males. (1) Normal chemical composition and structure of the RBC membrane
● Over the last 15 years, we’ve seen a shift from whole blood–derived platelets (2) Hemoglobin structure and function
obtained through whole-blood collection to single-donor platelets obtained (3) RBC metabolism Defects in any or all of these areas will result in RBCs
through apheresis. surviving less than the normal 120 days in circulation.
→ Blood components collected by apheresis offer several advantages
over manually collected whole blood.
→ Because apheresis separates the components, the blood doesn’t Normal chemical composition and structure of the RBC membrane
need further processing. RBC MEMBRANES
● Many apheresis devices allow collection of plasma and RBCs simultaneously • Represents a semipermeable lipid bilayer supported by a protein meshlike
with platelets. Although components still need to be tested, labeled, and stored cytoskeleton structure
appropriately, apheresis eliminates one processing step. Some apheresis
• Phospholipids and their orientation
devices also leukoreduce RBCs and platelets, eliminating the need to filter
• Integral and peripheral proteins
them in the laboratory.
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 • Membrane deformability
• 52% protein, 40% lipid content and 8% carbohydrate

CHONs and lipids Asymmetrically organized

LIPIDS Unequally distributed
EXTERNAL Glycolipids and choline phospholipid
LAYER
INTERNAL Amino phospholipid
CYTOPLASMIC
MEMBRANE
BIOCHEMICAL CHONs, lipids and CHO
COMPOSITION
Schematic illustration of red blood cell membrane depicting the composition and
Phospholipids arrangement of RBC membrane proteins.
o The main lipid components of the membrane, are arranged in a bilayer
structure comprising the framework in which globular proteins traverse RBC CHARACTERISTICS
and move. • The normal chemical composition and the structural arrangement and molecular
Integral Membrane Proteins interactions of the erythrocyte membrane are crucial to the normal length of RBC
o Proteins that extend from the outer surface and span the entire survival of 120 days in circulation.
membrane to the inner cytoplasmic side of the RBC • In addition, they maintain a critical role in two important RBC characteristics:
Peripheral Proteins deformability and permeability.
o Second class of membrane proteins, found beneath the lipid bilayer
o located and limited to the cytoplasmic surface of the membrane forming
the RBC cytoskeleton • To remain viable, normal RBCs must also remain
DEFORMABILITY flexible, deformable, and permeable.
• The loss of adenosine triphosphate (ATP) (energy)
Integral Proteins Peripheral Proteins levels leads to a decrease in the phosphorylation of
Glycophorin A Spectrin spectrin and, in turn, a loss of membrane deformability.
Glycophorin B Actin (band 5) • An accumulation or increase in deposition of membrane
Glycophorin C Ankyrin (band 2.1) calcium also results, causing an increase in membrane
Anion-exchange-channel protein (band Band 4.1 and 4.2 rigidity and loss of pliability.
3) Band 6 → These cells are at a marked disadvantage
Adducin when they pass through the small (3 to 5 μm in
diameter) sinusoidal orifices of the spleen, an
organ that functions in extravascular
NOTE (from the book): sequestration and removal of aged, damaged,
• Red blood cells are a product of a differentiation process that starts in the bone or less deformable RBCs or fragments of their
marrow where hematopoietic stem cells differentiate to nucleate RBCs. After membrane.
extrusion of nuclei and degradation of endoplasmic reticulum, reticulocytes • The loss of RBC membrane is exemplified by the
emerge in the circulation; here they rapidly develop into mature RBCs. formation of “spherocytes” (cells with a reduced surface-
• Despite these features, the protein and lipid composition of the RBCs is to-volume ratio) and “bite cells,” in which the removal of
subject to change during its life-time. This can be particularly observed at the a portion of membrane has left a permanent indentation
level of its plasma membrane. in the remaining cell membrane.
• Changes in red blood cell morphology occurred as quickly as 22 days. → The survival of these forms is also shortened.
→ This alteration can be harmful because red blood cells are similar in size to PERMEABILITY • The permeability properties of the RBC membrane and
the diameter of small capillaries; therefore, red blood cells have to change the active RBC cation transport prevent colloid
shape to get through the capillaries. hemolysis and control the volume of the RBC.

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 → Any abnormality that increases permeability or • Each iron can bind one molecule of oxygen
alters cationic transport may decrease RBC • Since it has 4 iron, 1 hemoglobin can be bind 4 molecules of iron
survival.
• The RBC membrane is freely permeable to water and HEMOGLOBIN FUNCTION
anions. Hemoglobin’s primary function is gas transport:
• Chloride (Cl–) and bicarbonate (HCO3–) can traverse → Oxygen (O2) delivery from the lungs to the tissues
the membrane in less than a second. → Transport of Carbon dioxide (CO2) from tissues to the lungs
• The RBC membrane is relatively impermeable to
cations such as sodium (Na+) and potassium (K+). 2,3-DPG
• RBC volume and water homeostasis are maintained by • One of the most important controls of hemoglobin affinity for oxygen is
controlling the intracellular concentrations of sodium the RBC organic phosphate 2,3-DPG.
and potassium. • A carbon molecule important in erythrocyte metabolism.
→ The erythrocyte intracellular-to-extracellular o It binds to deoxygenated hemoglobin and increases oxygen off-loading
ratios for Na+ and K+ are 1:12 and 25:1, from hemoglobin into the tissues.
respectively. o As erythrocyte storage time increases, the levels of 2,3-DPG decrease.
• The 300 cationic pumps, which actively transport Na+ → Transfusion of 2,3-DPG-depleted blood may shift the oxygen–
out of the cell and K+ into the cell, require energy in the hemoglobin dissociation curve to the left.
form of ATP. → As a result, red blood cells will have difficulty in unloading oxygen
• Calcium (Ca2+) is also actively pumped from the interior from hemoglobin into the tissues.
of the RBC through energy-dependent calcium-ATPase → The unloading of oxygen by hemoglobin is accompanied by
pumps. Calmodulin, a cytoplasmic calcium-binding widening of a space between chains and the binding of 2,3- DPG
protein, is speculated to control these pumps and to on a mole-for-mole basis, with the formation of anionic salt
prevent excessive intracellular Ca2+ buildup, which bridges between the chains.
changes the shape and makes it more rigid.
• When RBCs are ATP depleted, Ca2+ and Na+ are FORMS
allowed to accumulate intracellularly, and K+ and water Tense” Form The resulting conformation of the deoxyhemoglobin
are lost, resulting in a dehydrated rigid cell subsequently molecule is known as the tense (T) form, which has a lower
sequestered by the spleen, resulting in a decrease in affinity for oxygen.
RBC survival.
“Relaxed” Form • When hemoglobin loads oxygen and becomes
oxyhemoglobin
• established salt bridges are broken
HEMOGLOBIN STRUCTURE AND FUNCTION • chains are pulled together, expelling 2-3-DPG.
HEMOGLOBIN • This is the relaxed (R) form of the hemoglobin
Hemoglobin is the major protein in RBCs, and it gives to red cells the ability to molecule, which has a higher affinity for oxygen.
transport oxygen and carbon dioxide to and from tissues.

HEMOGLOBIN-OXYGEN DISSOCIATION CURVE

• These allosteric changes that occur as the hemoglobin loads and unloads
oxygen are referred to as the respiratory movement.
• Are not directly proportional to the partial pressure of oxygen (pO2) in its
environment but instead exhibit a sigmoid-curve relationship, known as the
hemoglobin-oxygen dissociation curve.
• The shape of this curve is very important physiologically because it permits a
considerable amount of oxygen to be delivered to the tissues with a small drop
in oxygen tension.

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 → For example, in the environment of the lungs, where the oxygen (pO2) Hemoglobin-dissociation curve
tension, measured in millimeters of mercury (mmHg), is nearly 100 mm
Hg, the hemoglobin molecule is almost 100% saturated with oxygen. o The effect of hydrogen ions, 2, 3-bisphosphoglycerate, and carbon dioxide
(H++BPG +CO2) is to promote a right shift.
• As the RBCs travel to the tissues, where the (pO2) drops to an average of 40
o If man had the hemoglobin of the lug worm (left shift), he would die of
mm Hg (mean venous oxygen tension), the hemoglobin saturation drops to anoxia.
approximately 75% saturation, releasing about 25% of the oxygen to the
tissues. RBC METABOLIC PATHWAY
• This is the normal situation of oxygen delivery at a basal metabolic rate. • RBCs produce ATP anaerobically by the breakdown of glucose, thus not
using any of the oxygen for its own metabolism.
LIGANDS → Erythrocytes do not have mitochondria, which is the site of aerobic
• The normal position of the oxygen dissociation curve depends on three respiration for oxidative metabolism
different ligands normally found within the RBC: H+ ions, CO2, and organic • Anaerobic metabolism allows red blood cells to deliver 100% of the oxygen
phosphates. to the organ sites. RBC metabolism may be divided into the anaerobic
• Of these three ligands, 2,3-DPG plays the most important physiological role. glycolytic pathway and three ancillary pathways that serve to maintain the
Normal hemoglobin function depends on adequate 2, 3-DPG levels in the structure and function of hemoglobin:
RBC. 1. The Pentose Phosphate pathway
2. The Methemoglobin Reductase pathway
SHIFT TO THE RIGHT 3. The Luebering-Rapoport shunt.
• All of these processes are essential if the erythrocyte is to transport oxygen and
• In situations such as hypoxia, a compensatory “shift to the right” of the
to maintain critical physical characteristics for its survival.
hemoglobin-oxygen dissociation curve alleviates the tissue oxygen deficit.
→ Glycolysis generates about 90% of the ATP needed by the RBC.
This rightward shift of the curve, mediated by:
→ Approximately 10% is provided by the pentose phosphate pathway.
→ Increased levels of 2,3-DPG; → The methemoglobin reductase pathway is another important
→ Decreases hemoglobin’s affinity for the oxygen molecule and; pathway of RBC metabolism, and a defect can affect RBC post-
→ Increases oxygen delivery to the tissues. transfusion survival and function.
→ Leubering-Rapoport shunt, allows accumulation of 2,3-DPG
SHIFT TO THE LEFT • RBC metabolism includes the glycolytic pathways producing both energy (as
• A “shift to the left” of the hemoglobin-oxygen dissociation curve results, adenosine 5′- triphosphate, or ATP) and oxidation-reduction intermediates that
conversely; support oxygen transport and membrane flexibility. RBCs interact with their
→ Increase in hemoglobin oxygen affinity and a decrease in oxygen environment by changing shape in response to pH and by secreting ATP in
delivery to the tissues. response to sheer forces and nitric oxide in response to hypoxia.
→ With such a dissociation curve, RBCs are much less efficient because • At the end of their life span, RBCs undergo programmed cell death in two
ways:
only 12% of the oxygen can be released to the tissues.
→ By racemization of negatively charged membrane phospholipids in
→ Multiple transfusions of 2,3-DPG–depleted stored blood can shift the
response to calcium or low concentrations of ATP, and
oxygen dissociation curve to the left.
→ By the active loss of membrane through microvesiculation.

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 LUEBERING-RAPAPORT PATHWAY
• An offshoot of the Embden-Meyerhof pathway is the Luebering-Rapaport
bypass.
• This pathway permits the accumulation of an important RBC organic
phosphate, 2,3-diphosphoglycerate (2,3- DPG).
• Has a significant effect on the affinity of hemoglobin for oxygen and
therefore affects how well RBCs function post-transfusion.
• The 2,3- DPG alternate route does not yield ATP, but does modulate levels
of 2,3-DPG.
• Erythrocyte 2,3-DPG concentration is especially sensitive to pH because
ANAEROBIC GLYCOLYTIC PATHWAY the enzymes catalyzing its production are inhibited or stimulated by
• In RBCs, which lack mitochondria and oxidative metabolism, pyruvate is hydrogen ions.
reduced to lactic acid, a three-carbon hydroxyacid, the product of anaerobic • Defects in the Luebering-Rapaport bypass can affect the levels of 2,3-DPG
glycolysis. available to erythrocytes. Red blood cell 2,3-DPG regulates oxygen release
• Each mole of glucose yields 2 moles of lactate, which are then excreted into depending on the needs of tissues.
blood. • Whenever the peripheral tissues have an increased amount of
→ Two molecules of lactic acid contain exactly the same number of deoxygenated blood (deoxyhemoglobin), glycolysis is stimulated and 2,3-
carbons, hydrogens, and oxygens as one molecule of glucose; DPG levels rise.
→ there is sufficient free energy available from the cleavage and → Again, pH changes within the cell probably contribute to this
rearrangement of the glucose molecule to produce 2 moles of ATP process.
per mole of glucose converted into lactate. → The result is that 2,3- DPG attaches to deoxyhemoglobin and
• The RBC uses most of this ATP to maintain electrochemical and ion gradients causes hemoglobin to resist binding to oxygen. This decrease in
across its plasma membrane. oxygen affinity by hemoglobin increases oxygen release to tissues.
• One mole of glucose is converted to 2 moles of lactate during anaerobic
glycolysis.
• No oxygen is consumed, nor is CO2 produced in this pathway. There is a net
yield of 2 mole of ATP per mole of glucose converted to lactate.

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 METHEMOGLOBIN REDUCTASE PATHWAY
• An important auxiliary process of erythrocyte metabolism is the
methemoglobin reductase pathway.
• This pathway maintains heme iron in the reduced, or active state (ferrous).
• It requires reduced pyridine nucleotides generated from the Embden-Meyerhof
pathway.
• Unlike the hexose monophosphate shunt, which provides a mechanism for
preventing the denaturation of the globin component of hemoglobin, the
methemoglobin reductase pathway ensures that the iron (heme) in the
hemoglobin molecule does not become oxidized. Methemoglobin with iron in
the ferric state is useless as an oxygen carrier. This pathway uses the enzyme
methemoglobin reductase and NAD to maintain hemoglobin in a reduced
2,3-BPG state.
The normal position of the oxygen dissociation curve depends on three different
ligands normally found within the RBC:
a. H ions
b. CO2
c. Organic phosphates
Of these three ligands, 2,3-DPG plays the most important physiological role. Normal
hemoglobin function depends on adequate 2,3-DPG levels in the RBC
2,3-DPG helps release oxygen from hemoglobin

HEXOSE MONOPHOSPHATE SHUNT/ PATHWAY

• Red blood cells are capable of limited aerobic glycolysis
• Also called the phosphogluconate pathway or the pentose phosphate shunt.
• The major role of the hexose monophosphate shunt, is the generation of
reduced nicotinamide adenine dinucleotide phosphate (NADPH). THE IMPORTANCE OF 2-3 DPG LEVELS IN TRANSFUSED BLOOD
→ Erythrocyte NADPH converts oxidized glutathione to reduced • The two main regulators of oxygen uptake and delivery are the pH of tissues
glutathione, the major red blood cell antioxidant. and the content of 2,3-diphosphoglycerate (2,3-DPG) in red cells.
→ Red blood cell enzymes, and especially hemoglobin, are protected • The pH of blood is kept relatively constant at the slightly alkaline level of about
from oxidant damage through the action of glutathione, which 7.4 (pH less than 7 indicates acidity, more than 7 alkalinity). The effect of pH
maintains hemoglobin in a reduced, active form. on the ability of hemoglobin to bind oxygen is called the Bohr effect:
• Although oxidants are damaging to cells, cells commonly produce them. → When pH is low, hemoglobin binds oxygen less strongly, and when pH
→ Macrophages, for example, produce them in response to infection. is high (as in the lungs), hemoglobin binds more tightly to oxygen.
→ Erythrocytes even produce them when certain drugs are present. → The Bohr effect is due to changes in the shape of the hemoglobin
• If the level of reduced glutathione is not sufficient to neutralize intracellular molecule as the pH of its environment changes. The oxygen affinity of
red blood cell oxidants, globin will denature and precipitate as Heinz bodies, hemoglobin is also regulated by 2,3-DPG, a simple molecule produced
ultimately producing membrane damage. The levels of nicotinamide adenine by the red cell when it metabolizes glucose.
dinucleotide phosphate (NADP)/NADPH regulate the amount of glucose • Effect of 2,3-DPG is to reduce the oxygen affinity of hemoglobin. When the
metabolized by the hexose monophosphate shunt. availability of oxygen to tissues is reduced, the red cell responds by
• As NADPH generates reduced glutathione, NADP is produced, which synthesizing more 2,3-DPG, a process that occurs over a period of hours to
stimulates glucose metabolism in the hexose monophosphate shunt. This days. 2,3-DPG is an intermediary metabolite in the Embden–Meyerhof
mechanism arms the red blood cell with more reducing capability during an glycolytic pathway in the red cells, which affects haemoglobin affinity for
oxidative challenge. oxygen.

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 ↑ Concentration of 2,3-DPG ↓ Concentration of 2,3-DPG STORAGE LESION
o decreases the affinity and thus o Has the opposite effect. A blanket term used to encompass all of the “bad” things that happen to RBCs during
increases the fraction of o Caused by acidosis. storage. These “bad” things include:
haemoglobin-bound oxygen (1) Decreased concentrations of ATP and 2,3- diphosphoglycerate (2,3-DPG)
available to the tissues (2) Increased concentrations of extracellular potassium, changes in cell
o found in response to hypoxia or shape
anemia (3) Loss of RBC viability
(4) Hemolysis
Effects of 2,3-DPG

BLOOD PRESERVATION CLINICAL SIGNIFICANCE

The goal of blood preservation is to provide viable and functional blood components
Elevated • Not usually of clinical significance
extracellular • However, can be fatal if RBC concentrates are
for patients requiring blood transfusion.
potassium infused in large volumes through central vascular
concentrations in catheters
RBC VIABILITY stored RBC units • As in neonatal exchange transfusions or when
• A measure of in vivo RBC survival following transfusion.
priming cardiopulmonary bypass machines
• Because blood must be stored from the time of donation until the time of Hemolysis • Reduction of the relative number of stored RBCs that
transfusion, the viability of RBCs must be maintained during the storage time survive when returned to circulation
as well. • Release of harmful breakdown products
→ To maintain optimum viability, blood is stored in the liquid state between Free hemoglobin • Vasoconstriction
1°C and 6°C for a specific number of days, as determined by the • Endothelial cell activation
preservative solution(s) used. • Renal tubular damage
→ 2,3-DPG is re-formed in stored RBCs after in vivo circulation Free membrane • Are procoagulant
phospholipids • Clinical consequences of the early forms of RBC
FACTORS AFFECTING THE RATE OF 2,3-DPG RESTORATION shape change or reduced 2,3-DPG concentrations
(1) Recipient’s Acid-Base status are much less clear.
(2) Phosphorus Metabolism Loss of viability Reduces the effective transfused RBC dose, but the
(3) Degree of Anemia during storage consequences of administering intact but nonviable cells
(4) Overall severity of the disorder are not known

• RBC clearance occurs within the first hour after transfusion

• Approximately 220 to 250 mg of iron in one RBC unit
• Rapid RBC clearance of a single unit of blood delivers a massive load of iron to
the monocyte and macrophage system

Food and Drug Administration requirements

1. Average 24-hour post transfusion RBC survival of more than 75%
2. Free Hemoglobin less then 1% of total hemoglobin

Determination of Post-Transfusion RBC survival

1. Taking RBCs from healthy subjects
2. Storage of RBCs labeled with radioisotopes
3. Reinfusion back to the original donor
4. Measurement 24 hours after transfusion

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 ANTICOAGULANT PRESERVATIVE SOLUTIONS IN BLOOD BAGS NAME ABBREVIATION STORAGE TIME
ANTICOAGULANTS (DAYS)
1. Prevent blood from clotting 1. Acid -citrate-dextrose ACD 21
2. Also used to preserve the life and survival of RBCs so as to have the 2. Citrate-Phosphate-Dextrose CPD 21
maximum post transfusion survival. 3. Citrate-Phosphate-Dextrose- CPDA-1 35
Adenine
CITRATE-BASED ANTICOAGULANTS 4. Citrate-Phosphate-Double- CP2D 21
• are used for the collection of blood for transfusion Dextrose
• Citrate binds calcium, preventing the activation of plasma coagulation factors. ANTICOAGULANT PRESERVATIVE SOLUTION
• Most common anticoagulant used in apheresis blood collection
o Sodium citrate (4%) is used for plasma apheresis • 2,3-DPG levels necessary for optimal hemoglobin oxygen delivery are not
o ACD-A is used for platelet apheresis. reached immediately
• EDTA, which binds calcium more strongly compared to citrate, is not used for • Approximately 24H to restore normal. 2,3-DPG levels after transfusion
the collection of blood transfusion products. • 2,3-DPG concentrations after transfusion reach normal levels as early as
• Heparin is rarely used for the collection of blood transfusion components as its 6H post-transfusion
effective anticoagulant half-life (approximately an hour) is limited.
ACID CITRATE DEXTROSE (ACD):
• Whole blood is collected into a primary blood bag containing a citrate based • The original ACD solution had a pH of 5;
anticoagulant (CPD, CPDA-1 or CP2D) and adequate mixing is ensured stored • Was made with citric acid, dextrose, and sodium citrate; and stably survived
at 1 to 6 degree Celsius. The anticoagulant solution may contain nutrients such being autoclaved.
as glucose and adenine. • It was used primarily for glass bottle storage of whole blood and allowed for
72 % RBC survival after 21 days with little hemolysis.
• Additional cell specific nutrients are supplied to the stored red cells and platelets
by the additive solutions in the respective red cell and platelet satellite bags.
• Incorporation of Adenine & its effects on glycolysis and ATP levels CITRATE PHOSPHATE DEXTROSE (CPD)
→ Adenine incorporated into the CPD solution (CPDA-1) increases ADP • (CPD) Citrate–phosphate–dextrose (CPD), is the mainstay of blood
Levels, thereby driving glycolysis toward the synthesis of ATP. preservation.
• Pathophysiologic effects of the transfusion of RBCs with low 2,3-DPG levels & • Citrate works as an anticoagulant by binding to and inhibiting the function of
increased affinity for Oxygen: calcium (factor IV).
→ ↑ Cardiac Output • Phosphate stabilizes pH which maintains proper levels of 2,3-DPG.
→ ↓Mixed venous (pO2) tension • The dextrose component is necessary for red blood cell ATP production.
→ ↑Cardiac output & ↓pO2 tension • If adenine, a purine nucleotide, is added to CPD (CPD-A), storage time
jumps from 21 days to 35 days.
• Plastic containers used for storage of blood affects its viability
→ Adenine assists in the production of ATP.
• di (ethylhexyl)-phthalate (DEHP) in PVC bags
→ Plasticizer
→ Leach from the plastic into the lipids of the plasma medium and
RBC membranes of the blood during storage
→ Break at low temperature
• Added to the RBCs after removal of the plasma
• Without ADSOL:
→ Decreased nutrients necessary to maintain RBCs during storage
→ Decreased in viability, particularly in the last 2 weeks of storage.

CITRATE PHOSPHATE DEXTROSE ADENINE (CPDA-1)

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 • CPD to which adenine (A) is added, becomes CPDA-1 (the‘1’ signifies the • Two of the solutions (Adsol, Optisol) contain adenine, glucose, saline, and
formula used) and improves the synthesis of ATP. CPDA-1 is usually used mannitol.
when the collected donation is to be stored as whole blood. → Reason for their development: removal of the plasma component
during the preparation of RBC concentrates removed much of the
nutrients needed to maintain RBCs during storage. Also overcome
NOTE: the problem of high viscosity of RBC concentrates.
• The volume of anticoagulant required to prevent clotting and preserve With CPD anticoagulant in the primary bag, the additive solution used is SAGM
red cells is dependent on the volume of blood taken from the donor. (saline, adenine, glucose, mannitol).
• Some collection bags are designed for the collection of 500 mL blood
and contain 70 mL anticoagulant; others are designed for 450 mL
collections and contain 63 mL anticoagulant. ADVANTAGES
• If smaller quantities of blood are to be drawn, then the volume of • Extends the storage of RBCs and lowers the viscosity of packed
anticoagulant is reduced proportionately. 15 ml of ACD, 14ml of CPD or red cells for ease of transfusion. Maximum amount of fresh plasma is
CPDA is used for preserving 100ml of blood. harvested – platelets and cryoprecipitate. Red cell concentrates that are
prepared from whole blood donations collected into CPD are suspended
in additive solution for improved storage and shelf life.

ACTION OF INGREDIENTS OF ANTICOAGULANT SOLUTION • When plasma is removed after the centrifugation of whole blood
CITRATE Acts by chelating Calcium. donations, most of the anticoagulant and nutrients in CPD are removed
DEXTROSE Necessary for the metabolism of stored RBCs. It passes from along with it. At this stage, blood has been effectively anticoagulated so
plasma into the red cells and is utilized for energy production. the presence of CPD is no longer required by the red cell concentrate
The principal pathway being Anaerobic glycolysis. remaining in the primary collection bag. However, the red cells need
CITRIC Prevents carmalization of glucose in citrate dextrose solution nutrients to survive, and should also be suspended in sufficient fluid to
ACID during autoclaving. allow for normal flow characteristics. This is achieved by the use of
ADENINE Improves the viability of red cells. additive solutions.
CPDA – 2 Here, the amount of Adenine is increased to 0.55g and that of
dextrose to 44.6g.
→ This is a better anticoagulant preservative solution than
CPDA–1. • Additive solutions (AS) vary in composition depending on the supplier. They
are sometimes referred to by their brand names or simply as SAGM (saline,
adenine, glucose and mannitol) or AS-1, AS-3, AS-5 and so on. The typical
composition of an additive solution is as follows:
STORAGE TEMPERATURES
• Collection of blood into anticoagulant-preservatives maintains component
function and viability only if storage is within the correct temperature range. SALINE the fluid in which the red cells are suspended
• Low temperature storage slows glycolytic activity and allows the shelf life of to provide the desired flow rate conditions.
the component to be extended. GLUCOSE (OR DEXTROSE) Provides the basic nutrients for glycolysis.
• Low temperatures also retard bacterial proliferation, should bacteria have ADENINE AND MANNITOL assist in the process of ATP generation.
gained access during the time of donation; either from the venipuncture site,
the donor’s circulation or other source.
• When an additive system is used then the blood donation is collected in
CPD, which has no adenine.
Additive Solution → The unit is processed within 24 hours of collection and the adenine is
• Additive solutions are preserving solutions that are added to the RBCs added with the red cell additive solution.
after removal of the plasma with/without platelets. Additive solutions replace → The volume of additive solution required to preserve red cells during
nutrients lost when the plasma is removed from red blood cells. When storage varies according to the volume of the whole blood donation.
additive solutions are added, red blood cell’s storage time can be increased → Red cells from a donation of 500 mL require about 111 mL of
from 35 days to 42 days. additive solution, whereas 450 mL donations need 100 mL.

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 • Red cell concentrates and platelet concentrates are stored as fresh For Platelet Concentrates
products ready for transfusion, although in special circumstances can be • Shelf life at +22°C ± 2°C is determined by its efficacy when transfused.
frozen using specific cryopreservation procedures. • This may be related to platelet viability and function during storage in the
→ Red cell concentrates are stored refrigerated (2– 6 °C) for up to 42 correct conditions of temperature and motion, and is considered to be up to 7
days (maximum duration may vary depending on the type of additive days.
solution used and/or local regulatory criteria). • Most blood services allocate a 5-day shelf life to limit the increased risk of
Platelet concentrates are stored at 20–24 °C for up to 5 days, with continuous bacterial growth resulting from the room temperature storage requirement.
gentle agitation to maintain maximum biological function. For Plasma
• The levels of stable clotting factors (FII, FVII, FIX, FX and fibrinogen) are
During storage, red cells and platelets continue to metabolize and undergo a range quite well maintained at +4°C ± 2°C.
of physiochemical changes, collectively referred to as the 'storage lesion’. • Labile clotting factors (FV, FVIII) deteriorate to levels that are not useful if
→ The storage lesion ultimately affects the in vivo function and survival of not frozen within 24 hours of collection.
transfused red cells and platelets and thus limits their shelf life.
• Shelf life of labile coagulation factors is up to 3 years if frozen (colder than –
25°C).
• However, a storage temperature that does not reach –25°C but is colder
CURRENTLY APPROVED ADSOL:
than –18°C reduces shelf life to 3 months.
• Adsol (AS-1) – Baxter Healthcare
• Nutricel (AS-3) – Pall Corporation
• Optisol (AS- 5) – Teruo Corporation RBC FREEZING
• For Autologous Units & storage of rare blood types
AS-1 AS-3 AS-5 • Involves the addition of a cryoprotective agent to RBCs that are less than 6
Storage period (days) 42 42 42 days old
pH (measured at 37°C) 6.6 6.5 6.5 • 20% or 40% Glycerol
24-hour survival (%) 83 85.1 80 → Cryoprotectant
ATP (% initial) 68 67 68.5 • Rapid freezing and stored in a freezer
2,3-DPG (% initial) 6 6 5 • Transfusion of frozen cells must be followed by deglycerolization
Hemolysis 0.5 0.7 0.6 • Cryoprotectant us systematically replaced with decreasing concentrations
Red Cell Additives: Biochemical Characteristics of saline
• Current Trend: FDA licenses frozen RBCs for a period of 10 years from the
SHELF LIFE OF WHOLE BLOOD COMPONENTS date of freezing
• SHELF LIFE is the maximum allowable storage time that a blood product • Upon addition of glycerol or saline solutions, the outdating period of thawed
may be stored, provided that the requirements of temperature, preservative RBCs has been 24 hours
solutions and physical environment are met. • RBCs in CPD or CPDA-1 are glycerolized and frozen within 6 days of whole
• Because of the great diversity in collection containers and anticoagulant blood collection
preservatives, as well as storage capabilities within different blood services,
the shelf life of different components varies considerably. Shelf-life RBC REJUVENATION
specifications must comply with local standards. • Process of enhancing and restoring ATP & 2,3-DPG levels
• RBCs (liquid state) can be rejuvenated at outdate or up to 3 days after
For Red Cells outdate
• Shelf life varies at +4°C ± 2°C according to anticoagulant/preservative and → Ex. Rejuvesol
additive solution used. • Incubation of RBC unit with 50 mL of the rejuvenating solution for 1 hour at
• The requirement that determines shelf life is that at least 75% of red cells 37°C
transfused at the end of the proposed storage period must still be in • Following Rejuvenation, the RBCs can be washed with Saline & Transfused
circulation 24 hours after transfusion. within 24 hours
• This interprets as a shelf life of 21 days for ACD, 28 days for CPD, 35 days
for CPDA-1 and 42 days for CPD replaced with a suitable additive solution.

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 PLATELET STORAGE → Sepsis due to contaminated platelets: most common infectious
MAJOR CHALLENGE IN PLATELET STORAGE complication of transfusion
• 5-day shelf life (in the US) → 10-40% of patients transfused with a bacterially contaminated platelet unit
• Bacterial contamination at incubation of 22° develop life-threatening sepsis
• A varying degree of platelet activation/ aggregation
• Release of intracellular granules
INDICATIONS FOR BLOOD COMPONENTS
• Suppression in ATP &ADP levels
RED BLOOD CELL TRANSFUSION
• Are used to treat hemorrhage and to improve oxygen delivery to tissues.
CLINICAL USE OF PLATELET • It should be based on the patient's clinical condition.
• Treatment of bleeding associated with thrombocytopenia • Indications for transfusion include:
• Transfused to hematology-oncology thrombocytopenic patients to prevent → Symptomatic Anemia (causing shortness of breath, dizziness,
bleeding congestive heart failure, and decreased exercise tolerance)
• Current Trend: Platelets are prepared as concentrates from whole blood → Acute Sickle Cell Crisis
& increasingly by apheresis → Acute Blood Loss of more than 30 percent of blood volume. Fresh
frozen plasma infusion can be used for reversal of anticoagulant
effects.
CURRENT CONDITION OF PLATELET PRESERVATION
PLATELET CONCENTRATES FRESH FROZEN PLASMA
• Prepared from whole blood and apheresis components For reversal of anticoagulant effects
• Storage: 20-24°C with continuous agitation for up to 5 days
• Expiration time (FDA): midnight of Day 5
• Should contain a minimum of 5.5 x 10^10 platelets in a volume of between PLATELET TRANSFUSION
45 & 65 mL • Indicated to prevent hemorrhage in patients with thrombocytopenia or
platelet function defects.
• Cryoprecipitate is used in cases of hypofibrinogenemia, which most often
RATIONALE AND ADVANE CONCEPT FOR PRESERVATION occurs in the setting of massive hemorrhage or consumptive coagulopathy.
• Maintenance of pH • Transfusion-related infections are less common than non-infectious
→ Key parameter for retaining platelet viability in vivo when platelets complications.
were stored at 20-24°C → All noninfectious complications of transfusion are classified as
• pH 6.2: current standard for maintaining satisfactory platelet viability noninfectious serious hazards of transfusion.
• Second generation storage containers → Acute complications occur within minutes to 24 hours of the
→ Increased gas transport properties transfusion
→ Delayed complications may develop days, months, or even years
MEASUREMENT OF VIABILITY AND FUNCTIONAL PROPERTIES OF STORED later.
PLATELETS • Platelet transfusion may be indicated to prevent hemorrhage in patients with
• To assess platelet viability thrombocytopenia or platelet function defects.
→ Pretransfusion and posttransfusion platelet counts (1 hr and/or • Contraindications to platelet transfusion include thrombotic
thrombocytopenic purpura and heparin-induced thrombocytopenia.
24hrs)
→ Should not be transfused in patients unless a life-threatening
→ Expressing the difference based on the # of platelets transfused
hemorrhage has occurred.
CCI → Transfusion of platelets in these conditions can result in further
• Room temperature-stored vs. cold-stored platelets thrombosis.
→ One unit of apheresis platelets should increase the platelet count in
PLATELET STORAGE AND BACTERIAL CONTAMINATION
adults by 30 to 60 × 103 per μL (30 to 60 × 109 per L).
Storage of platelets at 20-24°C may cause bacterial growth
→ In neonates, transfusing 5 to 10 mL per kg of platelets should
→ Room temperature storage and the presence of Oxygen can encourage
increase the platelet count by 50 to 100 × 103 per μL (50 to 100 ×
bacterial proliferation
109 per L).

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 → One apheresis platelet collection is equivalent to six pooled random FORMULA:
donor platelet concentrates.

DISEASES IMPORTANT IN BLOOD BANKING

o Immunodeficiency
o Hypersensitivity Units (m2/μL) are usually omitted when reporting the result.
o Monoclonal and polyclonal gammopathies
o Autoimmune disease
Example:
o Hemolytic Disease of Newborn (HDN) A patient with 10,000/uL platelet count has a body surface are (BSA) of 1.3 meters
Further discuss in Module 1- Lesson 4
squared. Six units of platelets are given. The 1-hr port transfusion platelet count is
50,000. What is the patient’s corrected count? Is platelet transfusion effective?
IMPORTANCE OF PLATELET CORRECTED COUNT INCREMENT (CCI)
Answer: 50,00/Ul-10,000uL x1.32 m2 / 6 units x 0.55 unit (0.55 is constant)
• Refractory is defined as failure to achieve acceptable increase in platelet
= 15, 578/uL
count following platelet transfusion on at least two occasions and no alternate
cause for refractoriness such as fever, sepsis, DIC, bleeding, splenomegaly,
or drug interaction (e.g. amphotericin B).
BASIC GENETICS MOLECULAR GENETICS: BASIC GENETIC COMPONENTS
• Platelet count must be measured within one hour after transfusion. Response
to platelet transfusion is typically measured within one hour. However, a DNA
sample collected 10 minutes after transfusion yields similar information and • Deoxyribonucleic acid, the main building block of genetic material
may be easier to obtain routinely. A post-transfusion platelet count can • Composed of four building blocks: adenine, guanine, cytosine, and
alternatively be taken at 20 hours. thymine. These building blocks are bases.
→ Each base attaches to a sugar molecule and a phosphate
Rule of thumb: molecule. These building blocks form strings. Following the “stringing”
• A unit apheresis platelet (or a pool of 6 platelet concentrates) should of a single strand, there is a “pairing” process where the partner for
achieve an increment of 30,000 to 50,000/μL in an average adult. each building block attaches and forms a double strand.
Alternative way to determine whether a patient is refractory is by → The pairing that occurs is specific: adenine is partnered with
calculating the corrected count increment or CCI. guanine and thymine is partnered with cytosine. Through the
process of binding, the strands twist and form a double helix.
• Corrected count increment (CCI) and percent platelet recovery (PPR) are • The double helix DNA forms an actual unit of inheritance, a gene.
measures of response to platelet transfusion that "correct" the count
increment for blood volume and number of platelets transfused.
• Corrected count increment (CCI) is a measure of the expected increase in
platelets following a platelet transfusion.
→ The “count increment” refers to the increase in platelets following a
transfusion.
→ The “correction” is based on the patient’s size and the number of
platelets transfused.
→ It is used because CCI is a more accurate measure of
refractoriness, as it adjusts for the number of platelets transfused and
the patient's blood volume.
→ It can guide the decision to pursue platelets with improved
compatibility (i.e., HLA-matched platelets).

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 GENES • The location of a specific gene on the chromosome is known as its genetic
• Genetics is the study of inheritance (transmission of characteristics) from locus.
parent to offspring • The alternate gene forms for a specific locus are known as alleles.
→ It is based on the biochemical structure of chromatin → for example, eye color might be determined by allele: blue, brown,
→ Chromatin is composed of nucleic acids, structural proteins and hazel, or green.
enzymes. • A single allele for eye color would be inherited from each parent. This
• The gene is an area of DNA that controls a trait or characteristic. simple pattern of inheritance of a single allele from each parent will be
• The product of the gene is usually a protein or RNA. applicable for blood group antigen inheritance.
• Most of antigens in the various blood group systems generally follow • When multiple alleles exist at a single locus, this is known as
straightforward inheritance patterns, usually of a codominant nature polymorphism.
• The understanding of inheritance of blood group antigens and the testing of → Some systems are more polymorphic than others.
disease markers at the molecular level are based on the science of genetics → A trait that has ten possible alleles at a single locus will be more
• Levels of genetics polymorphic than a trait with four possible alleles.
o Population → Example of a polymorphic gene is the ABO blood group
o Cellular • Antithetical genes are a pair (or more than 2) of genes that are loated in
o Molecular different alleles
→ Opposite form of gene
Major areas of population genetics of concern to blood banking:
1. Pioneering work of Linnaeus and Darwin
2. Mendel’s Laws of inheritance POLYMORPHISM
3. Hardy-Weinberg principle • of a locus determines the likelihood that two individuals will be found with an
4. Inheritance patters identical allelic composition.
• Hundreds of genes comprise each chromosome.
CENTRAL DOGMA OF MOLECULAR BIOLOGY • Humans have 23 pairs of chromosomes.
• First described by James Watson and Francis Crick → Each individual inherits one half of his or her chromosomes from each
• Framework for understanding the transfer of sequence information parent.
between Information-carrying Biopolymers in living organisms → The pairs consist of 22 pairs of autosomal chromosomes and one
pair of sex chromosomes.
DNA → With rare exception, genes that code for blood group antigens and the
• Dictate the synthesis of RNA. production of blood group antigens are found on the autosomal
• Produce new DNA by replication chromosomes.
• Produce RNA by transcription

RNA
• Dictate the synthesis of Proteins
• Produces proteins by translation

PROTEINS
• Dictate the function of the cell

GENE EXPRESSION
• With inherited physical traits, there are certain patterns of expression.
Gene expression of simple physical traits typically follows one of three
patterns.
• A gene is dominant if it is always expressed even when found in
combination with a second gene. In this case, the second gene is not
expressed.

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 • The “silent” recessive gene can only be expressed if two identical genes • The concept of zygosity can be related to antigen strength.
are present. → There are some gene products that exhibit dosage.
• A third level of gene expression is codominant. → When genes are inherited as homozygous (i.e. two identical alleles
coding for the same product), the individual is said to have a “double
dose” of this product. The expression of the trait or product is stronger
CODOMINANCE when present in the homozygous state.
→ is the expression of two different genes that are inherited at the same loci → In comparison, an individual that inherits a heterozygous set of genes
on a pair of chromosomes. (i.e. two different alleles coding for two different products) is said to
→ One gene is not dominant over its allele, and the products of both genes have a “single dose” of each gene since only one gene for each
are seen at the phenotypic level product has been inherited. In this case, the expression of each trait
or product is weaker than when two identical genes are inherited.
With rare exceptions, blood group systems are expressed as codominant → Dosage is exhibited with some blood group systems. Red cells that
characteristics. For example, when an individual inherits an A gene from one are heterozygous for a specific antigen will demonstrate a weaker
parent and a B gene from the other parent, the genotype is AB. The expressed reaction than the homozygous cells when they react with the specific
blood type, or phenotype, will be AB. In this case, neither the A nor B gene is antisera.
expressed in a dominant manner. • This is an important concept in blood group testing.
→ Interactions may occur between two genes.
→ Interactions are dependent on location of the inherited genes. The
AMORPH are genes that do not code for the production of any detectable likelihood of an interaction occurring is determined by the proximity
product. These genes appear to be recessive. of the genes on the autosomal chromosomes.
When the amorphic gene is inherited in conjunction with an allele that does
produce a detectable product, the detectable product of that allele is Cis-
expressed. This allele is not dominant over the amorph nor is the amorph → Relationship of two genes that are inherited on the same chromosomes
recessive to the expressed allele. A common example of an amorph is the gene → Gene expression is stronger compared to Trans genes
that codes for the O blood group. When inherited in a homozygous state, two O
→ C in cis position to D: DCe/dce
genes, there is no detectable product.
Trans-
→ Genes that are inherited on different/ seperate chromosomes.
→ C in trans position to D: Dce/dCe
HARDY-WEINBERG PRINCIPLE
• Mathematical equation to predict allele distribution and frequencies in
a population
• The steric arrangement of two genes may create a weakened expression of
• Allows for the study of Mendellian inheritance in great detail
one of the gene products.
→ Predictions are not always accurate
o This is a position effect or steric hindrance of that particular gene
• Formula: p+q=1 product.
→ p = percentage/ gene frequency for the dominant allele • An example of gene interaction exists within the Rh blood group system.
→ q = percentage/ gene frequency for the recessive allele o D and C are specific genes in this system.
• Also expressed by: p2+2pq+q2=1 o All genes in the Rh system are inherited on the same
→ p2 = Homozygous dominant allele (AA) chromosome.
→ q2 = Homozygous recessive allele (aa) o When C and D genes are inherited trans to each other (C on one
→ 2pq = Heterozygous allele (Aa) chromosome and D on the opposite homologous chromosome), the
steric effect will weaken the expression of the D antigen.
ZYGOSITY o When the genetic relationship is cis (D and C on the same
• describes the similarity or dissimilarity of genes at an allelic position on two homologous chromosome), there is no effect on the antigen
homologous chromosomes. expression. For each genetic characteristic, an individual receives a
→ When the genes are identical, they are said to be homozygous. gene from each of his or her parents. The sum total of both genes is
Conversely, when the genes are different, they are said to be known as the individual’s genotype.
heterozygous.

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• For example, the inheritance of a “blue” gene from the mother and a “blue” MENDEL’S 1ST LAW: INDEPENDENT SEGREGATION
gene from the father produces a genotype of “blue/blue.” This individual is • The first generation in the study, called the parental, pure, or P1 generation,
homozygous for the “blue” allele. consisted of all red or all white flowers that bred true for many generations.
• An individual that inherits a P gene from his or her mother and a Q gene from • The plants were either homozygous for red flowers (RR, a dominant trait;
his or her father has a genotype of P/Q and is heterozygous for the P/Q dominant traits are usually written with uppercase letters). or homozygous for
alleles. white flowers (rr, a recessive trait; recessive traits are usually written with
lowercase letters).
o When these plants were crossbred, the second generation, called
NOTE: first-filial, or F1, had flowers that were all red.
• The frequency of each genotype is reflective of the degree of o Thus the dominant trait was the only trait observed.
polymorphism within that system. • When plants from the F1 generation were crossbred to each other, the
• Systems that display a greater degree of polymorphism (i.e. more alleles second-filial, or F2, generation, of plants had flowers that were red and white
at each locus) will have a lower frequency for each allele than those that in the ratio of 3:1.
are less polymorphic. o All the plants from the F1 generation are heterozygous (or hybrid) for
flower color (Rr).
o The F2 generation has a ratio of three red-flowered plants to one
white flowered plant.
PHENOTYPE o This is because the plants that have the R gene, either RR
• The phenotype is a function of gene expression. homozygous or Rr heterozygous, will have red flowers because the
o The product of a recessive gene will not be expressed in a red gene is dominant.
phenotype. o Only when the red gene is absent and the white gene occurs in
• The dominant gene will produce a detectable product, whether in the duplicate, as in the rr homozygous white-flowered plant, will the
homozygous or heterozygous state. recessive white gene expression be visible as a phenotype.
• If the alleles are codominant, both will be expressed in the phenotype. • This illustrates Mendel’s first law, the law of independent segregation.
o In a system where the alleles are codominant, an individual with a o Therefore, each gene is passed on to the next generation on its own.
genotype of Z/Y will have a phenotype of ZY while an individual o Specifically, Mendel’s first law shows that alleles of genes have no
with the genotype Z/Z will have a phenotype of Z. permanent effect on one another when present in the same plant but
segregate unchanged by passing into different gametes.
REVIEW OF GENETICS: POPULATION GENETICS: MENDEL’S LAWS • An intermediate situation can also occur when alleles exhibit partial
dominance.
• Gregor Mendel is recognized as the father of genetics. o This is observed when the phenotype of a heterozygous organism is
o The results of his genetic research can be directly applied to the a mixture of both homozygous phenotypes seen in the P1
inheritance of blood group antigens. generation.
o Mendel was an Austrian monk and mathematician who used sweet o An example of this is plants with red and white flowers that have
pea plants growing in a monastery garden to study physical offspring with pink flowers or flowers that have red and white
traits in organisms and how they are inherited. sections.
• He first described the law known as independent segregation which refers to o It is important to remember that although the phenotype does not
the transmission of a trait between generations in a predictable fashion. show dominance or recessive traits, the F1 generation has the
• He determined the physical traits to be due to factors he called elementen heterozygous genotype of Rr. It is essential to understand how a
(eventually known as genes) within the cell. genotype can influence a phenotype, and using flower color is a good
• He studied the inheritance of several readily observable pea plant (a good basic model system to study this.
model organism) characteristics
o notably flower color, seed color, and seed shape—and based his
first law of inheritance, the law of independent or random
segregation, on these results.

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• Unlike the flower color of many types of plants, most blood group genes are → Specifically, if a homozygote that is dominant for two different
inherited in a codominant manner. characteristics is crossed with a homozygote that is recessive for
o In codominance, both alleles are expressed, and their gene both characteristics, the F1 generation consists of plants whose
products are seen at the phenotypic level. phenotype is the same as that of the dominant parent.
o In this case, one gene is not dominant over its allele, and the protein → However, when the F1 generation is crossed in the F2 generation,
products of both genes are seen at the phenotypic level. two general classes of offspring are found.
o An example of this is the MNSs blood group system, in which a • One is the parental type; the other is a new phenotype called a reciprocal
heterozygous MN individual would type as both M and N antigen type and represents plants with the dominant feature of one plant and the
positive. recessive feature of another plant.
o Blood group antigens are inherited in this fashion with only rare → Recombinant types occur in both possible combinations.
variations. The outcome of this type of inheritance can be predicted • Mendel formulated this law by doing studies with different types of seeds
by a Punnett Square. produced by peas and noted that they can be colored green or yellow and
textured smooth or wrinkled in any combination.
• Genes located on different chromosomes are inherited separately and
PUNNETT SQUARES
expressed discreetly from one another.
• Prediction of possible genotypes and phenotypes in an offspring can be
→ In most cases, this applies to the blood group antigens.
performed using a Punnett Square.
• Even blood groups that are inherited on the same chromosome, such as Rh
• In order to use the Punnett Square as a predictive tool, the user must know
and Duffy (Chromosome #1), are inherited as separate entities.
the exact genotype or inferred genotype of both parents.
→ One is not dependent on the other for inheritance or expression.
• Using this information, a Punnett Square can be set up to predict the
→ Separate genes account for the inheritance of blood group
likelihood of the genotype and phenotype of the offspring.
system antigens.

PEDIGREE CHARTS
• A method for tracking family history and inheritance patterns is a
pedigree chart.
• It is a visual representation of the parents and the possible genotypes and LINKED GENES
phenotypes for the offspring. • Genes are in very close proximity, that are inherited as a unit rather than
• This chart illustrates the inheritance patterns of all the family members as separate entities.
and can be used for visualization of inherited traits, including blood group • Independent assortment does not occur with antigens that are linked.
systems. • For example, the two genes that code for M/N and S/s antigen pairs are
→ The pedigree chart is useful since it is more detailed than the very close to one another. They are inherited as a “package” from each
Punnett Square. parent.
• Two genes are linked if the products appear with greater frequency
MENDEL’S 2ND LAW: INDEPENDENT ASSORTMENT than expected if inherited independently
• The second concept of Mendelian genetics/ Mendel’s second law → This deviance from anticipated frequencies is termed linkage
• States that genes for different traits are inherited separately from each disequilibrium.
other. HAPLOTYPE
→ This allows for all possible combinations of genes to occur in the → Set of genes inherited via one of the two parental gamates
offspring.

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• Linkage can be determined by examining the frequency of the antigen or MULTIPLE ALLELES
product in the general population. • Although individual humans (and all diploid organisms) can only have two
• A more complex example of linkage disequilibrium occurs within the HLA alleles for a given gene, multiple alleles may exist at the population level,
antigen system. such that many combinations of two alleles are observed.
→ The HLA-A and HLA-B antigens are more closely linked than the M/N o Mendel implied that only two alleles, one dominant and one recessive,
and S/s genes. could exist for a given gene, which is an oversimplification.
→ An individual’s haplotype is the set of HLA antigens inherited from one • Note that when many alleles exist for the same gene, the convention is to
parent. denote the most common phenotype or genotype in the natural population as
→ For example, the mother of an offspring may be typed as HLA-A3, A69; the wild type (often abbreviated “+”).
B7, B45. This mother may pass along to herprogeny the haplotype A3, • All other phenotypes or genotypes are considered variants (mutants) of this
B7, or A69, B45 but neverA3, B45, or A69, B7. typical form, meaning they deviate from the wild type.
o The variant may be recessive or dominant to the wild-type allele.
• An example of multiple alleles is the ABO blood-type system in humans.
o In this case, there are three alleles circulating in the population. The IA
allele codes for A molecules on the red blood cells, the IB allele codes
for B molecules on the surface of red blood cells, and the i allele codes
for no molecules on the red blood cells.
o In this case, the IA and IB alleles are codominant with each other and
are both dominant over the i allele.
o Although there are three alleles present in a population, each individual
only gets two of the alleles from their parents.
o This produces the genotypes and phenotypes shown in the table below.

DOSAGE EFFECT
• Excess unbound immunoglobulin that leads to Prozone effect
• An antibody gives a stronger reaction with RBC double-dosed for the
target antigen
• A homozygous recessive trait may be expressed more strongly than
a heterozygous trait
• Affected by inheritance of genotype
CONCEPTS OF DOMINANCE AND RECESSIVE TRAITS
• A variation on incomplete dominance is wherein both alleles for the same
characteristic are simultaneously expressed in the heterozygote.
• An example of codominance occurs in the ABO blood groups of humans.
The A and B alleles are expressed in the form of A or B molecules present on
the surface of red blood cells.
• Homozygotes (IAIA and IBIB) express either the A or the B phenotype, and
heterozygotes (IAIB) express both phenotypes equally.
→ The IAIB individual has blood type AB.
• In a self-cross between heterozygotes expressing a codominant trait, the three
possible offspring genotypes are phenotypically distinct.
→ However, the [Link] genotypic ratio characteristic of a Mendelian
monohybrid cross still applies.

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PROZONE EFFECT
• effectiveness of antibodies to form immune complexes is sometimes
Note:
impaired when concentrations of an antibody or an antigen are very
high • that more than one gene can affect a particular trait (part of a phenotype),
such as the height of an individual; all relevant genes can be considered as
part of the genotype for that trait.
• Notice that instead of three genotypes, there are six different genotypes • Depending on the alleles inherited, an organism can be either homozygous or
when there are three alleles. heterozygous for a specific trait.
• The number of possible phenotypes depends on the dominance • The presence of two identical alleles results in a homozygous genotype (i.e.,
relationships between the three alleles. AA), and the phenotype is group A blood. On the other hand, the inheritance
GENOME of different alleles from each parent gives a heterozygous genotype.
• It takes two gametes to make a fertilized egg with the correct (2N) number
of chromosomes in the nucleus of a cell.
o Therefore, each parent contributes only half (1N) of the inherited • Another important concept is that of the “silent” gene, or amorph, and the term
genetic information, or genes, to each child. hemizygous.
• In order to be completely healthy, each child must have the correct • An AMORPH is a gene that does not produce any obvious, easily detectable
number of genes and chromosomes (2N), without major mutations traits and is seen only at the phenotypic level when the individual is
affecting necessary biochemical systems. homozygous for the trait.
o At the smallest level, genes are composed of discrete units of • HEMIZYGOUS refers to the condition when one chromosome has a copy of the
DNA arranged in a linear fashion, similar to a strand of pearls, gene and the other chromosome has that gene deleted or absent.
with structural proteins wrapped around the DNA at specific
intervals to pack it into tightly wound bundles.

CHROMOSOMES • are DNA that is organized at a higher level

• Each chromosome being one incredibly long strand of
duplex (double-stranded) DNA.

GENE • is a section, often very large, of DNA along the REVIEW OF GENETICS: CELLULAR GENETICS: CELL DIVISION
chromosome. It controls a trait or characteristic
• The specific sequence of nucleotides and the location In eukaryotic cells such as human cells, the cell cycle is divided into four distinct
on the chromosome determines a gene. stages and is represented by a clock or circular scheme indicating that it can repeat
• In addition, each gene has specific and general itself or can be stopped at any one point in the cycle.
sequences that occur upstream (before the start site)
and downstream (after the termination signals) that G0 / resting stage The first step, the state of cells not actively dividing.
contribute to how the gene functions G1 pre-replication stage
S Phase The step at which DNA is synthesized
RNA is the product of a gene, that codes for proteins G2 Postreplication stage
LOCUS (plural = is the specific location of a gene on a chromosome M phase mitosis occurs
loci)
ALLELES are specific locus formed by an alternate gene. They one or
several different forms of the same gene at each locus • Chromosomes are in the interphase stage of mitosis in the span from G0 to
the end of the G2 phase.
GENOTYPE is the sequence of DNA that is inherited. • Cells that are completely mature and no longer need to divide to increase their
PHENOTYPE is anything that is physically manifested; the ratio of muscle numbers, such as nerve cells, can remain in the G0 stage for a very long
fibers; the level of hormones produced; and such obvious time.
traits as eye, skin, and hair color. • It is a hallmark of cancer cells, such as the transformed cells seen in the
various leukemias and solid tumors, that they can go through the stages of

20 | P a g e
 cell division much faster than non-transformed, normal cells and therefore • If cells with 2N chromosomes were paired, the resulting daughter cells would
outgrow them. have 4N chromosomes, which would not be viable. Therefore, gametes carry
• In this way, they take up the bulk of nutrients needed by the nontransformed a haploid number of chromosomes, 1N, so that when they combine, the
cells and crowd them out of existence and potentially overgrow the adult resulting cell has a 2N configuration.
organism in which they occur. • Meiosis only occurs in the germinal tissues and is important for reproduction.
• Without the complicated process of meiosis, there would be no change from
MITOSIS generation to generation, and evolution would not occur or happen too slowly
• The process by which cells divide to create identical daughter cells is called for organisms to adapt to environmental changes.
mitosis
• During cell division, the chromosomes are reproduced in such a way that all MEIOSIS I • The first stages of meiosis are nearly identical to
daughter cells are genetically identical to the parent cell. those in mitosis, in which chromatin is condensed,
o Without maintaining the same number and type of chromosomes, homologous chromosomes are paired in prophase,
the daughter cells would not be viable. and chromosomes are aligned along the center of the
• The chromosomes are duplicated, and one of each pair is passed to the cell
daughter cells. • However, there is no centromere division, and at
• During the process of mitosis, quantitatively and qualitatively identical anaphase and telophase, the cell divides and enters
DNA is delivered to daughter cells formed by cell division. interphase once again, in which there is no
• The complex process of mitosis is usually divided into a series of stages, replication of DNA.
characterized by the appearance and movement of the chromosomes.
PROPHASE II Chromosomes are condensed
• DNA is in the form of chromatin and is dispersed METAPHASE II centromeres divide and chromosomes line up along the
throughout the nucleus. center again
INTERPHASE • This is the stage of the DNA when cells are not ANAPHASE II Chromosomes are pulled to opposite ends of the cell
actively dividing. New DNA is synthesized by a TELOPHASE II The two cells divide, giving rise to four 1N daughter cells.
process called replication. In addition, during meiosis, crossing over and recombination can happen between
• The chromatin condenses to form chromosomes. In maternal- and paternal-derived chromosomes.
PROPHASE
prophase, the nuclear envelope starts to break down. o This allows for the creation of new DNA sequences that are different from
• The chromosomes are lined up along the middle of the the parent strains. Combined with random segregation, it is possible to
nucleus and paired with the corresponding have very large numbers of new DNA sequences.
METAPHASE
chromosome. In this stage, chromosome preparations o In humans with 23 pairs of chromosomes, the total possible number is
are made for chromosome analysis in cytogenetics. several million.
• The cellular spindle apparatus is formed and the PATTERNS OF INHERITANCE
chromosomes are pulled to opposite ends of the cell. AUTOSOMAL • Genes expressed with equal frequency in
ANAPHASE
The cell becomes pinched in the middle, and cell DOMINANT males and females
division starts to take place
• Non-sex chromosome
• the cell is pulled apart, division is complete, and the
• One parent can transmit a trait to a son or
chromosomes and cytoplasm are separated into two
TELOPHASE daughter
new daughter cells.
SEX-LINKED DOMINANT • Trait carried on the X chromosome
• Father to daughter transmission only
MEIOSIS • No father to son transmission
• A different process is used to produce the gametes or sex cells. SEX-LINKED • Trait carried on the X chromosome
• The process results in four unique, rather than two identical, daughter RECESSIVE • Carrier mother to son transmission
cells. • Traits are exhibited most commonly in males
o The uniqueness of the daughter cells generated with meiosis • Example: Hemophilia A
allows for great genetic diversity in organisms and controls the
number of chromosomes within dividing cells.

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 CELLS AND ORGANS OF THE IMMUNE SYSTEM
FUNDAMENTALS OF IMMUNOLOGY Different types of immune system cells can be distinguished by the membrane
markers they possess known as clusters of differentiation (CD) markers and are
IMMUNITY detected by immunotyping methods.
• Refers to the process by which a host organism protects itself from attacks
by external and internal agents.
• Confers protection from non-self and abnormal self-elements which are Types of Cells:
controlled at different levels. 1. Lymphocytes (B cell/T cell)
• The response and elimination of organisms and unwanted cells is 2. Plasma cell
accomplished through cellular and/or humoral mechanisms. 3. Natural Killer cells
4. Antigen-presenting cells (APCs)
CELLULAR IMMUNITY 5. Immune system organs
• Mediated by various IS cells, such as macrophage, T cells, and dendritic → Primary lymphoid organs
cells. → Secondary lymphoid organs
• Lymphokines- are other effector molecules that play critical roles in the 6. MHC class I and class II antigens
cellular system by activating and deactivating different cells, which allows
cells to communicate throughout the host body. It includes cytokines and
B CELLS
chemokines.
• Antibodies are secreted by mature B cells called plasma cells and bind to
antigens in specific manner.
HUMORAL IMMUNITY
• When the receptor on the B cell reacts with a specific antigen and
• Consists of the fluid parts of the IS, such as antibodies and complement
recognizes it, the B cell is activated to divide. The cells produced from this
components found in plasma, saliva, and other secretions.
rapid division mature into plasma cells and memory B cells.
• Antibody- also known as immunoglobulins (immune because of their
• Memory B cells have antibody on their surfaces that is of the same
function and globulin because they are a type of globular soluble protein).
conformation as that of the B cell from which they were derived.
Its function is to bind to foreign molecules called antigens.
→ Antigen-antibody reactions are specific. Only one antibody reacts with
one antigen or one part (an epitope/antigenic determinant) of a T CELLS
complex antigen. • They require help in the form of cell membrane proteins known as major
histocompability complex (MHC) molecules
INNATE IMMUNITY • MHC 2 classes
• It is the immediate line of immune defense. o Class 1: found in all nucleated cells except trophoblast and sperm
• First line of defense, not specific. and play in the role of cytotoxic T-cell function
• It doesn’t need modification to function to the same antigen o Class 2: found in APC (Antigen Presenting Cell), essential in
presenting processed antigens to CD4 cells and are necessary for
PHYSICAL BARRIERS BIOCHEMICAL BARRIERS both the B and T-cells function
(1) skin bactericidal enzyme (lysozymes, • The MHC genes (region of genome that encodes HLA) determine the human
(2) mucous membrane RNase, fatty acid, sweat, digestive leukocyte antigens (HLA) present on leukocytes and other cells; they have
(3) cilia lining enzyme, stomach acid, and vaginal been known for many years to cause rejection of tissue grafts.
(4) cough mechanism low ph) • T cell mediated immunity involve response against fungal and viral infection,
intracellular parasite, tissue grafts, and tumor.

ANTIGEN PRESENTING CELLS (APCs)

ACQUIRED/ ADAPTIVE IMMUNITY • Several types of leukocytes that function as APCs including macrophage,
• Acquired refers to the fact that the immunity is acquired via specific contact neutrophil and some of the B-cells
with a pathogen or aberrant cell. • They are also specialized cells capable of antigen presentation like dendritic
• Adaptive refers to the ability to adapt to and destroy new complex cell (skin), Langerhans cell, glial cell, Kupffer cell and osteoclast
pathogens, although it must first react to them through complex recognitions • They would first phagocytize the foreign material and process it internally
processes. and with the help of MHC present short peptides sequence of the antigen to
their cell membrane.
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 • The T-helper cell can now recognize the antigen in the context of MHC IMMUNOGLOBULIN STRUCTURE
presentation and they would respond to it by appropriating immune reaction • Basic Ig structural unit is composed of four polypeptide chains; two
identical light chains (MW=approximately 22,500 daltons) and two
LYMPHOID ORGANS identical heavy chains (MW= app. 50,000 to 75,000 daltons).
PRIMARY SECONDARY • Covalent disulfide bonding holds the light and heavy chains together. The
Thymus Spleen covalent disulfide linkages in Ig molecules provide greater structural strength
Bone marrow Lymph nodes than hydrogen bonding and van der Waals forces. However, they limit the
Mucosa associated tissue flexibility of the Ig molecule.
• The heavy chains are also interconnected by disulfide linkages in the hinge
CYTOKINES region of the molecule.
• Soluble proteins, peptides that function as powerful mediators of immune • Two types of light chains: kappa and lambda (both are present in all
system classes of immunoglobulins, regardless of the heavy chain classification).
o Regulation of growth, mobility and differentiation of leukocytes • Ig molecules are protein and therefore have two terminal regions: the amino
• If produced by lymphocytes, it is known lymphokines (-NH2) terminal and the carboxyl (-COOH) terminal.
• If produced by monocytes and macrophage, it is known as monokines • Enzyme papain splits the antibody molecule at the hinge to give three
• Examples: interlukins, interferon, CSF (colony-stimulating factor) fragments: one crystallizable Fc fragment and two antigen binding fragments,
Fab.
CHARACTERISTICS OF IMMUNOGLOBULINS • Domains of the immunoglobulins are the regions of the light and heavy
• A complex protein produced by plasma cells, with specificity to antigens that chains that are folded into compact globular loop structures.
stimulate their production
• 2 functions:
(1) To combine with antigen
(2) Mediate various biochemical effects
• 20% Immunoglobulins present to an individual
• Antibodies bind antigen, fix complement, facilitate phagocytosis and
neutralize toxic substances in the circulation.
• They are classified according to the molecular structure of their heavy chains.
Five classifications:

IgG Most concentrated in serum (80%) of total serum Ig.

(gamma heavy • secreted by the plasma in the blood
chain) • acts in long term immune system
• ability to cross the placenta
IgA Is next with 13% (although majority it is major found in
(alpha heavy chain) body secretions)
• found in mucous, saliva, tears
• present in secretions
IgM 6%
(mu heavy chain) • responsible for the early stages of immunity
1gD 1%
(delta heavy chain) • receptor in B cells
• triggers or activates basophils or mast cells
• found in the membrane of immature B cell
• helps in the activation of plasma cell
IgE Least common, <1%
(epsilon heavy • present against allergic reaction
chain) • protect against parasitic worms

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 IMMUNOGLOBULINS SIGNIFICANT FOR BLOOD BANK • Functional differences between the subclasses include the ability to fix
• All immunoglobulins can be significant for transfusion medicine; however, complement and cross the placenta.
IgG, IgM and IgA have the most significance.
• Most clinically significant antibodies that react at body temperature (37⁰C)
are IgG isotype and are capable of destroying transfused antigen-positive IgA
RBCs, causing anemia and transfusion reactions of various severities. • Like IgM, IgA exists in two main forms, a monomer and polymer form, as
IgM dimers or trimers composed of two or three identical monomers, respectively,
• IgM antibodies are most commonly encountered as naturally occurring joined by a J chain.
antibodies in the ABO system and are believed to be produced in response • IgA is located in different parts of the IS depending on subclass.
to commonly occurring antigens like intestinal flora and pollen grains. o Serum IgA is found in both monomeric and polymeric forms;
o Other blood groups such as Lewis, Ii, P and MNS may also produce → Serum IgA can cause problems when transfused in plasma
IgM antibodies, which usually react best at ambient temperature product to patient having deficient IgA can cause fatal
(22⁰C to 24⁰C) anaphylaxis and can induce IgG rbc hemolysis
• The primary testing problem encountered with IgM antibodies is that they can o Secretory IgA is usually found in the mucosal tissues of the body.
interfere with the detection of clinically significant IgG antibodies by masking → Its polymer form acquires a glycoprotein secretory component
their reactivity. Unlike IgG, IgM exist both monomeric and polymeric forms as it passes through epithelial cell walls of mucosal tissues and
(as pentamers) containing a J (joining) chain. appears in nearly all body fluids, including saliva, tears,
• The pentameric form can be dissociated through cleavage of covalent bonds bronchial secretions, prostatic fluid, vaginal secretions, and the
interconnecting the monomeric subunits and the J chain by chemical mucous secretions of the small intestine.
treatment with sulfhydryl reducing reagents such as ß-2-mercaptoethanol (2- • Also, anti-IgA antibodies can cause severe problems if transfused in
ME) or dithiothreitol (DTT). These reagents can distinguish a mixture of IgM plasma products to patients who are deficient in IgA, as potentially fatal
and IgG antibodies because only IgM is removed by the use of such anaphylaxis can result. Another reason for the importance of IgA is that IgA
compounds; therefore, the removal allows unexpected IgG to be detected. can increase the effect of IgG-induced RBC hemolysis.

IgG IgE
• IgG antibodies are significant in transfusion medicine because they are the • IgE is normally found only in monomeric form in trace concentrations in
class of immunoglobulins that are made in response to transfusion with non- serum, about 0.004% of total immunoglobulins, and is important in allergic
self and therefore are incompatible RBCs and other blood products. reactions.
• IgG antibodies are important in hemolytic disease of the newborn (HDN) • The Fc portion of the IgE molecule attaches to basophils and mast cells and
because antibodies can be formed in response against alloantigens on fetal facilitates histamine release when an allergen binds to the Fab portion of the
RBCs that enter the mother’s circulation, usually during delivery. molecule and cross- links with a second molecule on the cell surface.
• IgG has the greatest number of subclasses: IgG1, IgG2 , IgG3 , and IgG4 , • Histamine is critical for bringing about an allergic reaction.
and all four are easily separated by electrophoresis. → Although hemolytic transfusion reactions are not caused by IgE,
o The small differences in the chemical structure within the constant urticaria may occur because of the presence of IgE antibodies.
regions of the gamma heavy chains designate the various → Because IgE causes transfusion reactions by release of histamines,
subclasses, and the number of disulfide bonds between the two patients who have several allergic reactions to blood products can
heavy chains in the hinge region of the molecule constitutes one of be pretreated with antihistamines to counteract the response when
the main differences between subclasses. receiving blood products.

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 IgD IMMUNOGLOBULIN Fc Receptors
• IgD, present as less than 1 percent of serum immunoglobulins, appears to • Macrophages and monocytes have receptors for the attachment and can
have functions that deal primarily with maturation of B cells into plasma cells. bind CH3 domain of the Fc portion.
IgD is usually found bound to the membrane of immature B cells. Therefore, • Only the IgG1 and IgG3 subclasses are capable of attachment to
IgD may be necessary for regulatory roles during B-cell differentiation and phagocytic receptors. This is one way that incompatible RBCs coated with
antibody production but is probably the least significant for blood banking. IgG antibody are removed by phagocytosis.
• The other phagocytic cells with Fc receptors include neutrophils, NK cells
CHARACTERISTICS IgG1 IgG2 IgG3 IgG4 and mature B cells.

Proportion of 65-70 23-28 4-7 3-4 COMPLEMENT SYSTEM

total serum IgG • Is a complex group of over 20 circulating and cell membrane proteins that
(%) have a multitude of functions within the immune response
• Primary roles include direct lysis of cells, bacteria, and enveloped viruses as
Complement ++ + +++ 0 well as assisting with opsonization to facilitate phagocytosis.
fixation (classic • Another role is production of peptide fragment split products, which play roles
pathway) in inflammatory responses such as increased vascular permeability, smooth
muscle contraction, chemotaxis, migration and adherence.
Binding to +++ ++ +++ + • The complement proteins are activated in a cascade of events through three
macrophage FC main activation pathways pathways: the classical, alternative and lectin
receptors pathways.
• The three pathways converge at the activation of the complement C3:
Ability to cross + + + +
placenta
ACTIVATION PATHWAYS
Dominant antibody activities: Classical pathway The classical pathway is activated by binding of an
Anti-Rh ++ 0 + + antigen with an IgM, IgG1 or IgG3 antibody.
Anti-factor VII 0 0 0 +
Alternative pathways Alternative pathway is activated by high molecular
Anti-dextran 0 + 0 0 weight molecules with repeating units found on the
Anti-Kell + 0 0 0 surfaces of target cells.
Anti-Duffy + 0 0 0 Lectin pathway The lectin pathway is activated by attachment of
plasma mannose-binding lectin(MBL) to microbes.
Anti-platelet 0 0 + 0 MBL in turn activates proteins of the classical
Biological half-life 21 21 7-8 21 pathway.
(days)
IMMUNOGLOBULIN VARIATIONS

Isotype (class → Refers to variants present in all members of a

variation) species, including the different heavy and light
chains and the different subclasses.

Allotypic → Present primarily in the constant region not all

variants occur in all members of a species.

Idiopathic → which determines the antigen-binding specificity

regions and is specific for each antibody molecule

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 Binding of complement by RBC antibodies • The supernatant from the cell culture contains antibody from a single type of
B cell, clonally expanded, and therefore with the same variable region and
• Activation by igG having a single epitope specificity.
• Activation by IgM • This results in a monoclonal antibody suspension. Monoclonal antibodies are
• IgG Rh antibodies preferred in testing because they are highly specific, well characterized, and
• IgM lewis antibodies uniformly reactive. Most reagents used today are monoclonal in nature.
• ABO antibodies

CHARACTERISTICS OF ANTIGENS Naturally Occurring and Immune Antibodies

• The immune response is initiated by the presentation of an antigen (can • RBC antibodies are considered naturally occurring when they are found in
initiate formation of and react with an antibody) or immunogen (can initiate the serum of individuals who have never been previously exposed to RBC
an immune response) to the IS and the IS determining that the antigen is antigens by transfusion, injection, or pregnancy. These antibodies are
non-self. probably produced in response to substances in the environment that are
• The immune reaction to any immunogen, including antigens, is determined highly similar to RBC antigens such as pollen grains and parts of bacteria
by the host response as well as by several biochemical and physical membranes.
characteristics of the immunogen. • Most naturally occurring antibodies are IgM cold agglutinins, which react
• Properties that influence the amount and type of immune response such as: best at room temperature or lower; activate complement, and when active at
→ size, complexity 37⁰C may be hemolytic. In blood banking, the common naturally occurring
→ conformation antibodies react with antigens of the ABH, Hh, Ii, Lewis, MN, and P blood
→ charge group systems.
• RBC antibodies are considered immune when found in the serum of
→ accessibility
individuals who have been transfused or pregnant. These antigens are not
→ solubility
generally found in nature, and their molecular makeup is unique to human
→ digestibility RBCs.
→ biochemical composition • Most immune RBC antibodies are IgG antibodies that react best at 37⁰C and
• Molecules that are too small cannot stimulate antibody production. require the use of antihuman globulin sera (Coombs sera) for detection. The
• Immunogens having a molecular weight (MW) less than 10,000 daltons (D), most common immune antibodies encountered in testing include those that
for example, are called haptens and usually do not elicit an immune react with the Rh, Kell, Duffy, Kidd, and Ss blood group systems.
response on their own. A hapten coupled with a carrier protein having a MW
greater than 10,000 D, however, can produce a reaction.
• The biochemical composition of the stimulus plays a role in immune Unexpected Antibodies
stimulation. • All other antibodies directed against RBC antigens are considered
• Remember that RBC antigens are very diverse in structure and composition unexpected and must be detected and identified before blood can be safely
and may be proteins (such as the Rh, M, and N blood group substances) transfused, even if antibodies react at room temperature or only with Coombs
or glycolipids (such as the ABH, Lewis, Ii, and P blood group sera.
substances). • Also, autoreactive antibodies must be investigated. The reactivity of
• Human leukocyte antigens (HLAs) are glycoproteins. Because of these unexpected antibodies is highly varied and unpredictable as they may be
differences in structure, conformation, and molecular nature, not all blood either isotype IgM or IgG; rarely, both may be present in the same sample.
group substances are equally immunogenic in-vivo. These antibodies may be able to hemolyze, agglutinate, or sensitize RBCs.
• Some antibodies require special reagents to enhance their reactivity and
detection. Due to the enormous polymorphism of the human population, a
CHARACTERISTICS of Blood Group diversity of RBC antigens exists, requiring a variety of standardized
Polyclonal and Monoclonal Antibodies immunologic techniques and reagents for their detection and identification.
• Polyclonal or serum antibodies and are produced in response to a single
antigen with more than one epitope.
• Monoclonal antibodies- are produced by isolating individual B cells from a
polyclonal population and propagating them in cell culture with hybridoma
technology

26 | P a g e
 ALLOANTIBODIES AND AUTOANTIBODIES HEMAGGLUTINATION
ALLOANTIBODIES • Are methods for the analysis of blood group antigen-antibody responses and
typing for ABO, Rh, and other blood group antigens is accomplished by red
• Alloantibodies are produced after exposure to genetically different, or non- cell agglutination reactions.
self, antigens of the same species, such as a different RBC antigen after → Agglutination is a straightforward process and can be shown to
transfusion. develop in two stages. In the first stage, called sensitization, antigen
• Transfused components may elicit the formation of alloantibodies against binding to the antibody occurs.
antigens (red cell, white cell and platelets) not present in the recipient. → Epitopes on the surfaces of RBC membranes combine with the
antigen combining sites (Fab region) on the variable regions of the
immunoglobulin heavy and light.
AUTOANTIBODIES
→ Antigen and antibody are held together by various non-covalent
• Autoantibodies are produced in response to self-antigens. They can cause bonds, and no visible agglutination is seen at this stage. In the
reactions in the recipient if they have a specificity that is common to the second stage, a lattice-type structure composed of multiple
transfused blood. antigen-antibody bridges between RBC antigens and antibodies is
• Some autoantibodies do not have a detectable specificity and are referred to formed. A network of these bridges forms, and visible agglutination is
as pan- or polyagglutinins. present during this stage.
• Autoantibodies can react at different temperatures, and cold or warm
autoantibodies may both be present.
PRECIPITATION REACTION
The development of an insoluble antigen-antibody complex, resulting from the mixing
CHARACTERISTICS OF ANTIGEN-ANTIBODY REACTIONS of equivalent amounts of soluble antigen and antibody.
• The antigen-binding site of the antibody molecule is uniquely designed to
recognize a corresponding antigen; this antibody amino acid sequence
AGGLUTINATION INHIBITION
cannot be changed without altering its specificity. The extent of the reciprocal
relationship, also called the fit between the antigen and its binding site on the • A method in which a positive reaction is the opposite of what is normally
antibody, is often referred to as a lock and key mechanism. observed in agglutination. Agglutination is inhibited when an antigen-antibody
reaction has previously occurred in a test system and prevents agglutination.
→ Affinity, strength of a single antigen-antibody bond produced by the
summation of attractive and repulsive forces • The antigen and antibody cannot bind because another substrate has been
added to the reaction mixture and blocks the formation of antigen-antibody
→ Avidity, express the binding strength of the multivalent antigen with
agglutinates
anti-sera

ANTIBODY SPECIFICITY RIA, ELISA AND IF TECHNIQUES

radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or
enzyme immunoassay (EIA), and immunofluorescence (IF) techniques are
DETECTION OF RBC ANTIGEN-ANTIBODY REACTIONS immunologic methods based on quantization of antigen or antibody by the use of a
• Various factors influence detection of RBC antigen-antibody reactions. radioisotope, enzyme, or fluorescent label, respectively.
These include having a correct sample (a sample that is stored under the
right conditions) and the proper reagents performing the correct test and
understanding how the test should be done. FACTORS THAT INFLUENCE AGGLUTINATION REACTIONS
Typical of most biochemical reaction systems, agglutination reactions are influenced
by the concentration of the reactants (antigen and antibody) as well as by
TRADITIONAL LABORATORY METHODS factors such as pH, temperature, and ionic strength. The surface charge,
• Commonly used techniques including hemagglutination (a special type of antibody isotype, RBC antigen dosage, and the use of various enhancement media,
agglutination), precipitation, agglutination inhibition, and hemolysis. antihuman globulin reagents, and enzymes are all important in antigen-antibody
Other techniques such as radioimmunoassay (RIA), enzyme-linked reactions.
immunosorbent assay (ELISA) or enzyme immunoassay (EIA), and
immunofluorescence (IF), which quantifies antigen or antibody with the use
of a radioisotope, enzyme, or fluorescent label, respectively, may be used in
automated or semiautomated blood banking instrumentation.

27 | P a g e
 Centrifugation • Many of the commercially available enhancement media accomplish this by
• It decreases reaction time by increasing the gravitational forces on the reducing the zeta potential of RBC membranes. The net negative charge
reactants as well as bringing reactants closer together. Under the right surrounding RBCs (and most other human cells) in a cationic media is part
centrifugation conditions, sensitized RBCs overcome their natural repulsive of the force that repels RBCs from each other and is due to sialic acid
effect (zeta potential) for each other and agglutinate more efficiently molecules on the surface of RBCs.

FACTORS THAT INFLUENCE AGGLUTINATION REACTIONS: ENHANCEMENT

Antigen-Antibody Ratio MEDIA
• Antigen and antibody have optimal concentrations; in ideal reactive Protein Media
conditions, an equivalent amount of antigen and antibody binds. Any • Colloidal substances, or colloids are a type of clear solution that contains
deviation from this decreases the efficiency of the reaction and a loss of the particles permanently suspended in solution. Colloidal particles are usually
zone of equivalence between antigen and antibody ratio that is necessary large moieties like proteins as compared with the more familiar crystalloids,
for agglutination reactions to occur. which usually have small, highly soluble molecules, such as glucose, that are
→ An excess of unbound immunoglobulin leads to a prozone effect, easily dialyzed.
and a surplus of antigen (antigen-binding sites) leads to a postzone • The colloidal solutes can be charged or neutral and go into solution because
effect. of their microscopic size. Colloids include albumin, polyethylene glycol
→ Another reason antigen amount may be altered is due to weak (PEG), polybrene, polyvinylpyrrolidone (PVP), and protamine. These
expression of antigen on RBCs (dosage effect). Weak expression substances work by increasing the dielectric constant (a measure of electrical
occurs as a result of the inheritance of genotypes that give rise to conductivity), which then reduces the zeta potential of the RBC.
heterozygous expression of RBC antigens and resultant weaker
phenotypes.
LISS Media
• Low Ionic Strength Solution (LISS) Media (LISS), or low salt media,
Effect of pH generally contain 0.2 percent sodium chloride.
The ideal pH of a test system for antigen-antibody reactions is a range between 6.5 • They decrease the ionic strength of a reaction medium, which reduces the
and 7.5, which is similar to the pH of normal plasma or serum. Exceptions include zeta potential and therefore allows antibodies to react more efficiently with
some anti-M and some Pr(Sp1) group antibodies that show stronger reactivity below RBC membrane antigens.
pH 6.5.
• LISS media are often used because they result in an increased rate of
antibody uptake during sensitization and a decreased reaction incubation
Temperature time (from 30 to 60 minutes to 5 to 15 minutes as compared with protein
Different isotypes of antibodies may exhibit optimal reactivity at different potentiators such as albumin).
temperatures. IgM antibodies usually react optimally at ambient temperatures or • However, they can result in false-positive reactions and may cause wasted
below 22⁰C, whereas IgG antibodies usually require 37⁰C. time if reactions have to be repeated with albumin.

Ig type Polyethylene Glycol (PEG)

• Examples of IgM antibodies that have importance in blood banking include • Polyethylene Glycol (PEG) and Polybrene are macromolecule additives
those against the ABH, Ii, MN, Lewis (Lea , Leb ), Lutheran (Lua ), and P used with LISS to bring sensitized RBCs closer to each other to facilitate
blood group antigens. antibody cross-linking and agglutination reactions. T
• Important IgG antibodies are those directed against Ss, Kell (Kk, Jsa , Jsb • they are often used in place of albumin and have some advantages and
, Kpa , Kpb ), Rh (DCEce), Lutheran (Lub ), Duffy (Fya , Fyb ), and Kidd possible drawbacks. Polybrene can detect ABO incompatibility as well as
(Jka , Jkb ) antigen. clinically significant IgG alloantibodies, whereas PEG produces very specific
reactions with reduction in false positive or nonspecific reactions.
• PEG is considered to be more effective than albumin, LISS, or polybrene for
Enhancement media detection of weak antibodies.
• Agglutination reactions for IgM antibodies and their corresponding RBC
antigens are easily accomplished in saline medium as these antibodies
usually do not need enhancement or modifications to react strongly with
antigens.

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 Proteolytic Enzymes DISEASES IMPORTANT IN BLOOD BANK SEROLOGIC TESTING
• Are protein molecules that function by altering reaction conditions and bring Immunodeficiency diseases
about changes in other molecules without being changed themselves. Can result from various defects in the IS at many different levels of immune function
• Enzymes used in the detection and identification of blood group antibodies and may be congenital or acquired. They can result from defects in either innate or
include ficin (isolated from fig plants), papain (from papaya), trypsin adaptive immunity or both; the cause is often not known.
(from pig stomach), and bromelin (from pineapple).
• The use of enzymes provides enhanced antibody reactivity to Rh, Kidd, P1,
Lewis, and I antigens and destroys or decreases reactivity to Fya , Fyb, M, Hypersensitivity (or allergy)
N, and S antigens An inflammatory response to a foreign antigen and can be cell- or antibody-
mediated or both. There are four different types of hypersensitive reactions, and the
symptoms and treatment required for each are different. All four types can be
Antihuman Globulin (AHG) Reagents caused by blood product transfusions and may be the first sign of a transfusion
• When RBCs become coated with antibody or complement or both but do reaction.
not agglutinate in regular testing, special reagents are needed to produce
agglutination. Type I Reactions • Also called anaphylaxis or immediate hypersensitivity,
• The direct antihuman globulin (AHG) test is designed to determine if RBCs involves histamine release by mast cells or basophils
are coated with antibody or complement or both. with surface IgE antibody.
→ Polyspecific AHG can determine if RBCs have been sensitized • It can occur in IgA-deficient individuals who receive
with IgG antibody or complement (components C3b or C3d) or both. plasma products containing IgA. Urticarial reactions
→ Monospecific AHG reagents react only with RBCs sensitized with (skin rashes) may also result from transfusion of
IgG or complement. certain food allergens or drugs in plasma products
Type II Reactions • Reaction can involve IgG or IgM antibody with
complement, phagocytes, and proteolytic enzymes.
Chemical Reduction of IgG and IgM Molecules • HDN or transfusion reactions caused by blood group
• The reagents generally act on covalent sulfhydryl bonds and facilitate antibodies as well as autoimmune hemolytic reactions
antibody identification by removal of either IgG or IgM antibodies.\ are all type II reactions.
• Dithiothreitol (DTT) and ß-2-mercaptoethanol (2-ME) are thiol reducing
agents that break the disulfide bonds of the J (joining) chain of the IgM Type III • Involve phagocytes and IgG and IgM and complement.
molecule but leave the IgG molecule intact Reactions Type III reactions result in tissue damage from the
• Another reagent, ZZAP, which consists of a thiol reagent plus a formation of immune complexes of antigen-antibody
proteolytic enzyme, causes the dissociation of IgG molecules from the aggregates, complement, and phagocytes and are
surface of sensitized RBCs and alters the surface antigens of the RBC. therefore very serious.
• Chemical reduction of the disulfide bond of the IgG molecule is also used to • Penicillin and other drug induced antibodies can lead
produce chemically modified reagents that react with RBCs in saline. to hemolytic reactions through type III hypersensitivity.
Sulfhydryl compounds reduce the strong but less flexible covalent disulfide
bonds in the hinge region of the IgG molecule, allowing the Fab portions Type IV • The type IV reaction involves only T cell–mediated
more flexibility in facilitating agglutination reactions. Reactions responses and their cytokines and can be fatal if
untreated. The most important type IV reaction is graft
versus-host, of which there is also more than one type
NEW AND NONTRADITIONAL LABORATORY METHODS
(1) Flow Cytometry
(2) Solid-Phase Adherence Monoclonal and Polyclonal Gammopathies
(3) Gel Test • Plasma cell neoplasms result in proliferation of abnormal immunoglobulin
(4) RBC Affinity Column Test (or gamma globulin) from either a single B cell clone (monoclonal
gammopathies) or multiple clones (in polyclonal gammopathies) and may be
a specific isotype or only light or heavy chain molecules.
• Increased serum viscosity is a result of these diseases and can interfere
with testing. The increased concentrations of serum proteins can cause
nonspecific aggregation (as opposed to agglutination) of erythrocytes called
29 | P a g e
 rouleaux, which is seen as a stacking of RBCs, like a stacking of coins. It Max Delbrück of • Confirmation that DNA was the genetic material
often occurs in multiple myeloma patients. Vanderbilt University, came from the “phage group”.
• If rouleaux is suspected, the saline replacement technique may be needed Salvador Luria of • Coincided in their interest to research
to distinguish true cell agglutination from nonspecific aggregation. Indiana University, bacteriophages, viruses that infect bacteria.
and Alfred Hershey of • Bacteriophages are very small viruses that
Carnegie’s consist only of a DNA core enclosed in a protein
Autoimmune Disease Department of capsule. It was known that, during bacterial
• Autoantibodies are produced against the host’s own cells and tissues. Genetics at Cold infection, the phage particles reproduce inside
• It is unknown why this loss of tolerance to self-antigens occurs, but there are Spring Harbor the cell and lyse the bacteria to release a new
many possible explanations such as aberrant antigen presentation, failure generation of viruses.
to obtain clonal deletion, anti-idiotypic network breakdown, and cross
reactivity between self and non self -antigens. Alfred Hershey and • 1952
• Autoimmune hemolytic anemias are an important problem in testing and Martha Chase • they reported an experiment that convinced the
transfusion scientific community that DNA was indeed the
genetic material.
Hemolytic Disease of the Newborn
• (HDN) can result when the maternal IS produces an antibody directed at an
antigen present on fetal cells but absent from maternal cells.
• The mother is exposed to fetal RBCs as a result of feto-maternal transfer of
cells during pregnancy or childbirth. DNA STRUCTURE
o Maternal memory cells can cause a stronger response during a 1950 • the chemical composition of nucleic acids was known.
second pregnancy if the fetus is positive for the sensitizing antigens. • Nucleic acids were known to be long molecules composed
IgG1, IgG3, and IgG4 are capable of crossing the placenta and of three distinct chemical subunits:
attaching to fetal RBCs whereas IgG2 and IgM are not. • Five-carbon sugar
o Severe HDN is most often associated with IgG1 antibodies and may • Acidic phosphate
require exchange transfusion • Four types of nitrogen-rich bases.
• Two forms of nucleic acids were differentiated by their
sugar composition: RNA contained ribose and DNA 2-
MOLECULAR BIOLOGY deoxyribose.
• Both possessed adenine, guanine, and cytosine. RNA
DNA: THE GENETIC MATERIAL contained uracil, and DNA contained thymine.

Fred Griffith • 1928 • Erwin Chargaff of Columbia University

(1) A British microbiologist that presented a model
1950
• He reported a consistent one-to-one ratio of adenine to
system that was key to demonstrating that DNA thymine and guanine to cytosine in DNA samples from
is the genetic material. different organisms
(2) He used two naturally occurring strains of • Maurice Wilkins and Rosalind Franklin
pneumococcus bacterium that differed in their
1951
• He produced x-ray diffraction photographs of DNA, which
infectivity of mice. The virulent smooth (S) suggested a helical molecule with repeats of 34 angstroms
strain, which has a smooth polysaccharide (Å) and a width of 20 Å.
capsule, kills mice through pneumonia.
(3) The nonvirulent rough (R) strain lacks the outer • , James Watson (trained in the phage group) and Francis
capsule and is nonlethal to mice. 1953
Crick (a physicist trained in x-ray crystallography, published
a paper in the journal Nature in which they assembled the
Oswald T. Avery • 1944 puzzle pieces of DNA structure.)
and his group • Rockefeller Institute were able to reproduce this • They proposed that the DNA molecule was a helix. DNA
transformation in vitro. was a helical ladder, the rails of which were built from
alternating units of deoxyribose and phosphate.

30 | P a g e
 • Each rung of the ladder was composed of a pair of
nucleotides (a base pair) held together by hydrogen bonds.
The double helix consisted of two strands of nucleotides
that ran in opposite directions (antiparallel).
• Consistent with the 34-Å repeat from x-ray diffraction, 10
base pairs were stacked on top of each other at each turn
of a helix.
• In agreement with Chargaff’s observation, adenine always Scientists have added further qualifications to the central dogma:
paired with thymine, and guanine always paired with 1. Genes are not “fixed.”
cytosine. Thus the nucleotide alphabet of one half of the 2. DNA sequences and protein amino acid sequences are not entirely
DNA helix determined the alphabet of the other half. collinear.
3. Some RNA molecules display catalytic activity; by “RNA interference,”
small RNA molecules help to regulate gene expression.

DNA FUNCTION
• Genes must be replicated and passed down to each new cell and each new
generation.
• Genes must also provide the information for protein synthesis.
DNA REPLICATION
Expression of Genetic Information • The Watson-Crick structure revealed how DNA could replicate.
• Genes act by determining the sequence of amino acids and therefore the • The DNA molecule is made of two antiparallel complementary strands.
structure of proteins. Cytosine always pairs with guanine, and thymine always pairs with adenine.
• Knowledge of the structure of DNA revealed that the genetic information • The information in one DNA strand predicts the information in the other strand.
must be specified by the order of the four bases (A, C, G, and T). During replication, the hydrogen bonds break, the strands separate, and each
• Proteins are polymers of 20 amino acids, the sequence of which determines one functions as template for the synthesis of another complementary half
their structure and function. molecule.
• Because DNA was in the nucleus, separated from the cytoplasm by the • Two identical DNA molecules are generated, each containing an original
nuclear membrane, an intermediary molecule had to be responsible for strand and a new complementary strand, each to be passed to a daughter cell
conveying the genetic information from the nucleus to the cytoplasm. • Because each daughter double helix contains an “old” strand and a newly
• RNA was a good candidate for this role. Its structure suggested that RNA
synthesized strand, this model of replication is called semiconservative.
could be produced from a DNA template and that RNA was located mainly
in the cytoplasm, where protein synthesis occurred. DNA TRANSCRIPTION
• Transcription is the process by which DNA is copied (transcribed) to mRNA,
which carries the information needed for protein synthesis. Transcription takes
The Central Dogma of Molecular Biology place in two broad steps.
• The Central Dogma of Molecular Biology states that: DNA makes RNA → First, pre-messenger RNA is formed, with the involvement of RNA
makes proteins. polymerase enzymes. The process relies on Watson-Crick base
• Deciphering the genetic code confirmed the central dogma of molecular pairing, and the resultant single strand of RNA is the reverse-
biology first formulated by Francis Crick. complement of the original DNA sequence.
• The genetic material is DNA. → The pre-messenger RNA is then "edited" to produce the desired mRNA
• DNA is self-replicating and is transcribed into mRNA, which in turn serves molecule in a process called RNA splicing.
as a template for the synthesis of proteins. → Reverse transcription
• Although Crick’s central dogma remains true, the knowledge acquired in the • RNA is "reverse transcribed" into DNA
following → catalyzed by reverse transcriptase enzymes, allows retroviruses,
• years has refined and enlarged it, a process that continues now and into the including the human immunodeficiency virus (HIV), to use RNA as their
future. genetic material

31 | P a g e
 → Reverse transcriptase enzymes have also found applications in endonucleases and the fragments separated by agarose
biotechnology, allowing scientists to convert RNA to DNA for gel electrophoresis.
techniques such as PCR. • The gel is then placed over a nitrocellulose or nylon
membrane and overlaid with transfer buffer. The DNA
fragments are transferred or “blotted” when vacuum is
applied by the flow of transfer buffer.
DNA TRANSLATION • The membrane-bound fragments have the same relative
• The mRNA formed in transcription is transported out of the nucleus, into positions as the fragments separated by size on the gel.
the cytoplasm, to the ribosome (the cell's protein synthesis factory). Here, •
it directs protein synthesis. Northern • This technique is a variation of the Southern blotting and
• Messenger RNA is not directly involved in protein synthesis − transfer RNA Blotting is used for detection of RNA instead of DNA.
(tRNA) is required for this. • Total cellular RNAs are extracted and fractioned by size
• The process by which mRNA directs protein synthesis with the assistance through gel electrophoresis.
of tRNA is called translation. • The RNAs are then blotted onto a filter and detected by
hybridization with a labeled probe. This technique is used
for studying gene expression.
RECOMBINANT DNA DNA • also called gene chips, allow tens of thousands of genes
The big breakthrough came with the development of recombinant DNA microarrays to be analyzed simultaneously.
technology.
• A gene chip consists of a glass slide or a membrane filter
• DNA from one organism, humans for example, can be “cut and pasted” into
onto which oligonucleotides or fragments of cDNA are
a carrier DNA molecule or vector. printed by a robot in small spots at high density.
• The new DNA molecule, which is a “recombinant” of the original DNA with • Because a chip can contain more than 10,000 unique
the vector DNA, can be introduced into another, usually simpler, host DNA sequences, scientists can produce DNA
organism. Because the genetic code is almost universal, the host organism microarrays containing sequences representing all the
treats the gene as its own. This technique is called molecular cloning. genes in a cellular genome.
• Cloning is the reproduction of daughter cells from one single cell by fission • A common application of this technique is the study of
or mitotic division, giving rise to a population of genetically identical clones. differential gene expression; for example, the comparison
• In DNA cloning, the DNA fragment of interest carried by the vector is of the genes expressed by a normal cell as opposed to
introduced into a host cell. Successive divisions of the host cell create a those of a tumor cell.
population of clones containing the DNA fragment of interest.
Fluorescent in • In this technique, scientists use fluorescent probes to
DETECTION OF NUCLEIC ACIDS AND PROTEINS situ detect homologous DNA or RNA sequences in
Hybridization chromosomes or intact cells.
Nucleic Acid Hybridization (FISH) • In this case, the hybridization of the probe to specific
• Base pairing between complementary strands of DNA or RNA allows the
cells or sub-cellular structures is determined by direct
specific detection of nucleic acid sequences. microscopic examination.
• Double-stranded nucleic acids denature at high temperature (90⁰ to 100⁰C)
and renature when cooled to form double-stranded molecules as dictated
by complementary base pairing.
• Nucleic acid hybrids can be formed between two strands of DNA, two PCR-Based Techniques
strands of RNA, or one strand of RNA and one of DNA. DNA or RNA Amplification of DNA by PCR allows the detection of even single copies of
sequences complementary to any purified DNA fragment can be detected DNA molecules.
by nucleic acid hybridization. The specificity of the PCR amplification depends on the primers that
hybridize to complementary sequences of the template molecule spanning
the target DNA fragment.
Southern • In this technique developed by E. M. Southern, the DNA With carefully chosen primers, PCR can be used to selectively amplify
Blotting to be analyzed is digested with one or more restriction DNA molecules from complex mixtures, such as genomic DNA from cell
extracts.
32 | P a g e
 Red Cell Genotyping
Reverse • By adding a step of cDNA synthesis by reverse In cases where serology is not possible or not sensitive enough or where
Transcription transcription (RT) prior to the PCR amplification, discrepancies occur, genotyping provides results that can be used to obtain a blood
single copies of RNA can be detected. By RT- type on a donor or patient.
PCR,
• RNA molecules can be specifically amplified from
total RNA obtained from cell extracts or tissue Applications for genotyping:
sections.
Real-Time PCR in • the product formed in each cycle of amplification is 1. Fetal DNA typing
real-time PCR (also detected by fluorescence at the same time that it 2. Blood group typing of donors for alloimmunized patients
called RT-PCR) is produced. 3. Screening blood donors to locate rare blood group phenotypes
• Combined with reverse transcription, this method 4. Screening blood inventory for antigen negative units
allows the quantification of specific mRNA in 5. Determining the frequency of blood group polymorphism
complex mixtures and is extremely useful in the 6. Determining zygosity for the fathers of fetuses at risk for HDFN
study of gene expression. 7. Blood group typing of patients with autoimmune hemolytic anemia and
other disease.
• the high sensitivity of PCR-based DNA and RNA
detection techniques makes them valuable for
early detection of transfusion-transmitted viruses, REVIEW QUIZ
such as HIV and hepatitis B and C.
• Nucleic acid testing (NAT) is rapidly becoming a What class of immunoglobulin is capable of crossing the igG
standard method in blood banks. It allows the placenta?
detection of pathogens before the appearance of a What is responsible for recognition of antibody-binding Variable region
testable immune response, such as screening of site to homologous antigen?
antibodies. At what temperature do igM antibodies react? 22 degrees C
• Reducing the “window” period (during which What immunoglobulin react at body temperature? IgG
donors can be infected but do not yet test positive) A Person with genotype MM shows a 3+ reaction when Dosage effect
helps to enhance the safety of blood products. RBC are mixed with the M antibody whereas a person
with the genotype MN shows only 1+ reaction. What
phenomenon is occurring?
What is the purpose of the antihuman globulin AHG test -
in blood banking?
Antibodies as Probes for Proteins Which immunoglobulin is a pentameric configuration igM
Gene expression at the protein level can be studied by using labeled antibodies as What cells are responsible for mounting secondary Memory B cells
probes. In particular, monoclonal antibodies are widely used in the technique called response when expose to the same antigen?
immunoblotting. The portion of the immunoglobulin molecule that Heavy chain
determine class is the?

Recombinant DNA Libraries

• A way of isolating single genes is by the production of recombinant DNA
libraries. Instead of trying to fish one gene out of a mass of genomic DNA or
cDNA, each gene is physically separated and introduced into a vector.
• The target gene is selected by screening each of the individual pieces with
a specific probe. Recombinant DNA libraries are collections of clones that
contain all genomic or mRNA sequences of a particular cell type.
• Clones containing a specific gene are identified by hybridization with a
labeled probe, such as a cDNA or genomic clone or a PCR product.

33 | P a g e
 ● The first of the Kell blood group system antibodies and the
associated antigen were reported only weeks after Coombs had
described the test
OUTLINE 1908 Moreschi
A. ANTIGLOBULIN TEST ● Described the Principle of Coombs test.
B. ABO BLOOD GROUP SYSTEM ● Moreschi’s studies involved the use of rabbit antigoat serum to
C. RH-HR BLOOD GROUP SYSTEM agglutinate rabbit RBCs that were sensitized with low
non-agglutinating doses of goat anti-rabbit RBC serum. The
antiglobulin test can be used to detect RBCs sensitized with IgG
alloantibodies, IgG autoantibodies, and complement components.
ANTI-GLOBULIN TEST
● Antiglobulin test (also called Coombs’ test)
● Based on the principle that: anti-human globulins (AHGs) obtained from
HOW DOES THE AHG REAGENT WAS PRODUCED?
immunized nonhuman species bind to human globulins such as IgG or
● It involves the injection of Human Serum into the Rabbit to produce an
complement, either free in serum or attached to antigens on red blood cells Anti-human Serum.
(RBCs).
● After the adsorption, to remove the heterospecific antibodies and the dilution
● There are two major types of blood group antibodies, IgM and IgG.
to avoid the Prozone Effect
o Because of their large pentamer structure, IgM antibodies bind to o Prozone Effect- excessive Antibodies
corresponding antigen and directly agglutinate RBCs suspended in
● AHG Serum still remains sufficient activity to permit cross linking of the
saline.
adjacent RBCs synthesize with IgG Antibodies.
o IgG antibodies are termed non-agglutinating because their monomer
o This can later on produce the heme agglutination.
structure is too small to agglutinate sensitized RBCs directly.
● The addition of AHG containing anti-IgG to RBCs sensitized with IgG AHG able to detect in vivo and in vitro sensitization of RBCs
antibodies allows for hemagglutination of these sensitized cells. Some blood
group antibodies have the ability to bind complement to the RBC membrane. IN VIVO: DIRECT ANTIGLOBULIN IN VITRO: INDIRECT ANTIGLOBULIN
Antiglobulin tests detect IgG and/or complement-sensitized RBCs. TEST (DAT) TEST (IAT)
One-stage procedure Two-stage procedure
HISTORY
● Before the discovery of the antiglobulin test, only IgM antibodies had been
detected.
AHG REAGENTS
● The introduction of the antiglobulin test permitted the detection of
non-agglutinating IgG antibodies and led to the discovery and POLYSPECIFIC ● Contains antibody to human IgG and to the C3d
characterization of many new blood group systems. AHG component of human complement.
● Other anticomplement antibodies, such as anti-C3b,
1945 Coombs and associates
anti-C4b, and anti-C4d, may also be present.
● Described the use of the antiglobulin test for the detection of weak
● Commercially prepared polyspecific AHG contains little,
and nonagglutinating Rh antibodies in serum
if any, activity against IgA and IgM heavy chains.
1946 Coombs and coworkers ● However, the polyspecific mixture may contain antibody
● Described the use of AHG to detect in-vivo sensitization of the activity to kappa and lambda light chains common to all
RBCs of babies suffering from hemolytic disease of the newborn immunoglobulin classes, thus reacting with IgA or IgM
(HDN). molecules.

1 | Page
MONOSPECIFIC ● Contains only one antibody specificity: either anti-IgG or POLYCLONAL ● Polyclonal AHG is usually prepared in rabbits, although
AHG antibodies to specific complement components such as AHG when large volumes of antibody are required, sheep or
C3b or C3d. PRODUCTION goats may be used.
● Licensed monospecific AHG reagents in common use ● In contrast with the early production methods, in which a
are anti-IgG and anti-C3bC3d crude globulin fraction of serum was used as the
immunogen, modern production commences with the
purification of the immunogen from a large pool of normal
Anti-IgG Reagents labeled anti-IgG contain no
sera.
Reagent anticomplement activity.
Anti-IgG reagents contain antibodies
specific for the Fc fragment of the MONOCLONAL ● The monoclonal antibody technique devised by Kohler
gamma heavy chain of the IgG molecule. AHG and Milstein has been used to produce AHG and has
If not labeled “gamma heavy PRODUCTION proved particularly useful in producing high-titer
chain–specific,” anti-IgG may contain antibodies with well-defined specificities to IgG and to the
anti–light chain specificity and therefore fragments of C3.
react with cells sensitized with IgM and ● Monoclonal antibody production begins with the
IgA as well as with IgG. immunization of laboratory animals, usually mice, with
purified human globulin.
● After a suitable immune response, mouse spleen cells
Anti-Comple anti-C3b-C3d reagents, are reactive
containing antibody-secreting lymphocytes are fused with
ment against the designated complement
components only and contain no activity myeloma cells.
against human immunoglobulins. ● The resulting “hybridomas” are screened for antibodies
with the required specificity and affinity. The
antibody-secreting clones may then be propagated in
tissue culture or by inoculation into mice, in which case
PREPARATION OF AHG the antibody is collected as ascites.
● are a mixture of antibodies from different plasma cell
clones
POLYCLONAL ● The resulting polyclonal antibodies recognize different
ANTIBODIES
antigenic determinants (epitopes), or the same portion
of the antigen but with different affinities.

MONOCLONAL ● are derived from one clone of plasma cells and

ANTIBODIES recognize a single epitope.
● Hybridoma technology can be used to produce
monoclonal antiglobulin serum

PREPARATION OF POLYSPECIFIC AHG

PREPARATION OF MONOSPECIFIC AHG

2 | Page
 ● It is prepared by a production process similar to that described for polyspecific ● Also, five antibodies of antiKell, anti-Jka, and Fya
AHG; however, it contains only one antibody specificity. specificities were detected when using LISS, but not
● Monospecific anti-IgG is usually of polyclonal origin; however, monoclonal albumin, with both polyspecific AHG and antiIgG.
anti-IgG has been prepared effectively by hybridoma technology. → Their results concluded that some clinically
● Monospecific anticomplement reagents are often a blend of monoclonal significant antibodies are detected with the
anti-C3b and monoclonal anti-C3d. anticomplement component of AHG but not with
anti-IgG.
ANTIBODIES REQUIRED IN AHG
● Also determined the number of false-positive reactions
Anti-IgG ● HG must contain antibody activity to non-agglutinating obtained when using polyspecific AHG versus anti-IgG with
blood group antibodies. The majority of these LISS and albumin.
antibodies are a mixture of IgG1 and IgG3 subclass. ● False-positive reactions were defined as those caused by
● Rarely, non-agglutinating IgM antibodies may be antibodies with no definable specificity or by antibodies
found; however, they have always been shown to fix considered to be
complement and may be detected by anticomplement. ● clinically insignificant because of optimum reactivity at cold
● IgA antibodies with Rh specificity have been reported; temperatures (anti-I, anti-H, anti-P1, anti-M).
however, IgG antibody activity has always been → Of the unwanted positive reactions, 93% were
present as well. shown to be caused by C3 on the cells.
● The only RBC alloantibodies that have been reported
Engelfriet and ● have also shown that degradation of C3b to C3d can occur
as being solely IgA have been examples of anti-Pr,12 others in vitro, providing that the incubation period is greater than
and those antibodies were agglutinating.
1 hour.
● IgA autoantibodies have been reported, although very ● In 1976, Garratty and Petz confirmed the need for anti-C3d
rarely.
activity in AHG for use in the DAT. They also confirmed
→ Therefore, anti-IgG activity must be present in
Engelfriet’s observation that, given sufficient time,
the AHG reagent.
cell-bound C3b could be degraded to C3d in vitro
Anti-Complement ● Some antibodies “fix” complement components to the
RBC membrane after complexing of the antibody with
its corresponding antigen. NOTE:
● These membrane-bound complement components can ● The detection of C3d on the RBC membrane is important in the investigation of
be detected by the anticomplement activity in AHG. both warm and cold autoimmune hemolytic anemia (AIHA). Many cases of
warm AIHA are associated with both IgG and C3d coating the RBCs. In cold
AIHA, C3d may be the only globulin detectable on the RBC.
USE OF POLYSPECIFIC VS. MONOSPECIFIC AHG IN THE IAT ● In the investigation of AIHA, a DAT is performed initially with polyspecific AHG.
If globulins are detected on the RBC membrane, follow-up testing with
Petz and ● Compared monospecific anti-IgG with polyspecific AHG. monospecific AHG (antiIgG, anti-C3d) is performed to identify the coating
coworkers ● They also compared the albumin technique with low ionic proteins. Although the RBCs of most patients with AIHA are coated with IgG,
strength solutions (LISS)-suspended RBCs. the cells of some patients will exhibit both IgG and complement coating or
● Four Jka antibodies were detected with polyspecific but not complement alone. The presence of complement alone may support the
with monospecific anti-IgG using albumin or diagnosis of AIHA, rendering the finding significant
LISS-suspended RBCs.
● An additional anti-Jka was detected only with polyspecific PRINCIPLES OF THE ANTIGLOBULIN TEST
AHG when using LISS but not with albumin. 1. Antibody molecules and complement components are globulins.

3 | Page
 2. Injecting an animal with human globulin stimulates the animal to produce 3. Recent transfusion history
antibody to the foreign protein (i.e., AHG). Serologic tests employ a variety of
AHG reagents reactive with various human globulins, including anti-IgG,
antibody to the C3d component of human complement, and polyspecific
reagents that contain both anti-IgG and anti-C3d activity.
3. AHG reacts with human globulin molecules, either bound to RBCs or free in
serum.
4. Washed RBCs coated with human globulin are agglutinated by AHG

DIRECT ANTIGLOBULIN TEST (DAT)

● Detects in-vivo sensitization of RBCs with IgG and/or complement
components.
o Detects antibodies bound to RBC surface
● Not required tests in routine in pretransfusion
● Clinical conditions that can result in in-vivo coating of RBCs with antibody
and/or complement are:
(1) Hemolytic disease of the newborn (HDN)
(2) Hemolytic transfusion reaction (HTR)
(3) Autoimmune and drug-induced AIHA.
● Initial DATs include testing one drop of a 3 to 5 percent suspension of washed
RBCs with polyspecific (anti-IgG, anti-C3d) reagent.
● Positive results are monitored by a DAT panel using monospecific anti-IgG and
anti-C3d to determine the specific type of protein sensitizing the cell.
● The saline control serves to detect spontaneous agglutination of cells or
reactions occurring without the addition of AHG reagents.
● In warm AIHA, including drug-induced hemolytic anemia, the RBCs may be
coated with IgG or C3d, or both.
● For the case of HDN:
o The RBC of the Fetus contains antigens to its surface. This will bind
to the Antibodies formed by the mother
o After the Antigen-Antibody binding, the Anti-IgG found in Coomb’s
Reagent will bind itself to the Antibody of the mother.
o This will lead to the formation of agglutination

EVALUATION OF POSITIVE DAT

The American Association of Blood Banks Technical Manual states that: “The results
of serological tests are not diagnostic; their significance can only be assessed in INDIRECT ANTIGLOBULIN TEST (IAT)
● The IAT is performed to determine in-vitro sensitization of RBCs
relationship to the patient’s clinical condition:
1. Knowledge of your patient’s diagnosis ● Used in the following situations:
2. Drug therapy

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 (1) Detection of incomplete (non-agglutinating) antibodies to potential Ratio of serum to ● increasing the ratio of serum to cells increases the
donor RBCs (compatibility testing) or to screening cells (antibody cells sensitivity of the test system.
screen) in serum ● 2 drops of serum and 1 5% RBC suspension
(2) Determination of RBC phenotype using known antisera (e.g., Kell ● When using cells suspended in saline, it is often
typing, weak D testing) advantageous to increase the ratio of serum to cells in
(3) Titration of incomplete antibodies an effort to detect weak antibodies (e.g., 4 drops of
serum with 1 drop of a 3% [v/v] cell suspension will give
2 STAGES OF IAT
a ratio of 133:1).
Stage 1 ● We have the serum of the patient, it contains free Antibody,
Reaction medium 1. Albumin
this will be collected and allowed to be mixed with the ● allowing antibody coated to come on close
blood of the donor.
contact
● The Antigen present at the surface of the donor’s blood will
● stroup and macIlroy
bind to the Antibody of the patient.
● decreasing incubation time but may miss
several clinically significant antibodies
STAGE 2 After the Antibody-Antigen binding, the Coomb’s Reagent containing 2. Low Ionic strength solutions (LISS)
Anti-IgG will now bind to the Antibody present in the patient. ● enhance the antibody uptake and allow the
incubation time to be decreased from 30-60
mins – 10-15 mins by reducing zeta potentials
● low and messeter
● Has critical requirement with serum-to-cell
ratio
● Optimum reactions: 2 drops of serum and 2
drops of 3% cell suspension
● Increasing the serum-to-cell ratio increased
the ionic strength of the reaction mixture,
leading to a decrease in sensitivity and
counteracting the shortened incubation time of
the test.
● Common practice: LISS as additive agent
3. Polyethylene glycol (PEG)
● water soluble linear polymer
● used as additive to increased antibody uptake
by removing the water molecules that surround
the red cell
● Anti-IgG is the AHG reagent of choice with
PEG testing to avoid false-positive reactions
● PEG may cause aggregation of RBCs, reading
for agglutination following 37°C incubation in
the IAT is omitted

FACTORS AFFECTING THE ANTIGLOBULIN TEST

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 ● PEG increases the detection of clinically The test can be carried through to an AHG technique if
significant antibodies while decreasing required.
detection of clinically insignificant antibodies. ● If this is performed, a monospecific anti-IgG reagent must be
used because the low ionic conditions cause considerable
amounts of C4 and C3 to coat the cells and would give
Temperature IgG optimal temperature at 37 degrees Celsius also for false-positive reactions if a polyspecific reagent were used.
complement activation
Incubation time ● For cells suspended in saline, incubation times may
Enzyme-Linke ● An RBC suspension is added to a microtiter well and
vary between 30 and 120 minutes d Antiglobulin washed with saline.
● The majority of clinically significant antibodies can be Test (ELAT) ● AHG, which has been labeled with an enzyme, is added.
detected after 30 minutes
● The enzyme labeled AHG will bind to IgG-sensitized RBCs.
● Extended incubation in the LISS technique shown to
● Excess antibody is removed, and enzyme substrate is
cause antibody to elute from the RBCs, decreasing the
added.
sensitivity of the test
● The amount of color produced is measured
Washing of RBC ● For both IAT and DAT spectrophotometrically and is proportional to the amount of
● removes free unbound serum globulins. antibody present.
● Inadequate washing may result in a false-negative ● The optical density is usually measured at 405 nm. The
reaction because of neutralization of the AHG reagent number of IgG molecules per RBC can also be determined
by residual unbound serum globulins from this procedure.
● one of the most important steps in testing
● Should be performed immediately after being removed
Solid Phase ● Solid-phase technology may be used for the performance of
from the incubator and in as short a time as possible to
antiglobulin tests. Several different techniques have been
minimize the elution of low-affinity antibodies.
reported using either test tubes or microplates.
Addition of AHG immediately after washing to minimize the chance of ● With the availability of microplate readers, this modification
antibody eluting from the cell and subsequently neutralizing lends itself to the introduction of semi-automation.
the AHG reagent ● Direct and indirect tests can be performed using solid-phase
Centrifugation for crucial step in the technique methodology.
reading ● In the former, antibody is attached to a microplate well, and
RBCs are added. If antibody is specific for antigen on RBCs,
MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES the bottom of the well will be covered with suspension; if no
such specificity occurs, RBCs will settle to the bottom of the
Low Ionic ● In 1980, Lalezari and Jiang reported on the adaptation of the well.
Polybrene automated low ionic polybrene (LIP) technique for use as a ● In the latter, known RBCs are bound to a well that has been
Technique manual procedure. treated with glutaraldehyde or poly L-lysine. Test serum is
● The technique relies on low ionic conditions to rapidly added to RBC-coated wells, and if antibody in serum is
sensitize cells with antibody. specific for antigen on fixed RBCs, a positive reaction
● Polybrene, a potent rouleaux-forming reagent, is added to occurs.
allow the sensitized cells to approach each other to permit ● Positive: well will be covered with the suspension
cross-linking by the attached antibody. ● Negative: red cell at the bottom of the well
● A high ionic strength solution is then added to reverse the
rouleaux; however, if agglutination is present, it will remain.

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Anti-Complem ● The gel test is a process to detect RBC antigen-antibody
ent reactions by means of using a chamber filled with
polyacrylamide gel.
● The gel acts as a trap; free unagglutinated RBCs form
pellets in the bottom of the tube, whereas agglutinated
RBCs are trapped in the tube for hours.
● Therefore, negative reactions appear as pellets in the
bottom of the microtube, and positive reactions are fixed in
the gel.
● There are three different types of gel tests: neutral, specific,
and antiglobulin.
● A neutral gel does not contain any specific reagent and acts
only by its property of trapping agglutinates.
● The main applications of neutral gel tests are antibody
screening and identification with enzyme-treated or
untreated RBCs and reverse ABO typing.
● Specific gel tests use a specific reagent incorporated into
the gel and are useful for antigen determination.
● Gel low ionic antiglobulin test (GLIAT) may be used for IAT
and DAT, AHG reagent is incorporated into the gel

SUMMARY:

● The antiglobulin test is used to detect RBCs sensitized by IgG

alloantibodies, IgG autoantibodies, and complement components.
● AHG reagents containing anti-IgG are needed for the detection of IgG
antibodies because the IgG monomeric structure is too small to directly
agglutinate sensitized RBCs.
● Polyspecific AHG sera contain antibodies to human IgG and the C3d
component of human complement.
● Monospecific AHG sera contain only one antibody specificity: either anti-IgG
or antibody to anti–C3b–C3d.
● Classic AHG sera (polyclonal) are prepared by injecting human globulins
into rabbits, and an immune stimulus triggers production of antibody to
human serum.
● hybridoma technology is used to produce monoclonal antiglobulin serum. α

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 ● The DAT detects in vivo sensitization of RBCs with IgG or complement o He recognized three groups—A, B, and O—based on their reactions
components. Clinical conditions that can result in a positive DAT include to each other. He was inadvertently the first individual to perform
HDFN, HTR, and AIHA. forward and reverse grouping.
● The IAT detects in vitro sensitization of RBCs and can be applied to ● ABO forward and reverse grouping tests must be performed on all donors and
compatibility testing, antibody screen, antibody identification, RBC patients. ABO grouping is the most frequently performed test in the blood
phenotyping, and titration studies. bank.
● A positive DAT is followed by a DAT panel using monospecific anti-IgG and ● There is always an inverse reciprocal relationship between the forward and
anti-C3d to determine the specific type of protein sensitizing the RBC. reverse type; thus, one serves as a check on the other.
● EDTA should be used to collect blood samples for the DAT to avoid in vitro ● Landsteiner’s rule: if an individual has the antigen, the individual will not
complement attachment assocated with refrigerated, clotted specimens. have the antibody
● There are multiple sources of error that can be introduced into the AHG
procedure. NOTE:
Forward grouping (front type)
● LISS, PEG, polybrene, and albumin can all be used as enhancement media
using known sources of commercial antisera (anti-A, anti-B) to detect antigens on
for AHG testing, with each having its own advantages and disadvantages. an individual’s RBCs.
● Conventional tube testing, gel technology, enzyme-Linked technology, and
solid-phase testing are available methods to use in AHG testing. Reverse grouping (back type)
● Method-dependent antibodies do exist and should be evaluated on a detecting ABO antibodies in the patient’s serum by using known reagent RBCs,
case-by-case basis. namely A1 and B cells.

AB Group
● Red cells of the A group clump with donor blood of the B group; those of the
B group clump with blood of the A group; those of the AB group clump with
those of the A or the B group because AB cells contain both A and B
antigens
● Those of the O group do not generally clump with any group, because they
do not contain either A or B antigens.

IMPORTANCE OF ANTIGENS AND ANTIBODIES

ABO BLOOD GROUP SYSTEM
● The red cells of an individual contain antigens on their surfaces that
● Most important in transfusion and transplantation
correspond to their blood group and antibodies in the serum that identify and
● Antibodies in serum to antigens that are absent from their RBCs combine with the antigen sites on the surfaces of red cells of another type
ABO BLOOD GROUP
● Antibodies are part of the circulating plasma proteins known as
● ISBT #001
immunoglobulins, which are classified by molecular size and weight and by
● Classification of blood based on inherited differences (polymorphisms) in
several other biochemical properties.
antigens on the surfaces of the red blood cells ● Most blood group antibodies are found either on immunoglobulin G (IgG) or
● Inherited differences of white blood cells (leukocytes), platelets
immunoglobulin M (IgM) molecules,
(thrombocytes), and plasma proteins also constitute blood groups. o Occasionally the immunoglobulin A (IgA) class may exhibit blood
● The human ABO blood groups were discovered by Austrian-born American group specificity.
biologist Karl Landsteiner in 1901.
● Naturally occurring antibodies are the result of immunization by substances in
o Landsteiner found that there are substances in the blood, antigens nature that have structures similar to human blood groups.
and antibodies that induce clumping of red cells when red cells of o Present in an individual despite the fact that there has been no
one type are added to those of a second type. previous exposure to the corresponding red cell antigens

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 o Example: anti-A in the plasma of people of blood group B and anti-B o Full expression between 2-4 years of age
in the plasma of people of blood group A. ● Newborns demonstrate weaker antigens, but ABO antigens are fully developed
● Immune antibodies are evoked by exposure to the corresponding red cell by two to four years of age
antigen. ● One factor contributing to the difference in ABO antigen strength between
● Immunization (i.e., the production of antibodies in response to antigen) against newborns and adults is the number of branched oligosaccharides.
blood group antigens in humans can occur as a result of pregnancy, blood o Adults demonstrate greater numbers of branched chains compared to
transfusion, or deliberate immunization. newborns, who have more linear chains.
o The combination of pregnancy and transfusion is a particularly potent o Adults have more branched chains and, hence, the ability to add on
stimulus. more terminal sugars and produce more antigens.
● Individual blood group antigens vary in their antigenic potential o Newborns and infants have fewer antigen sites on their red cells.
o For example, some of the antigens belonging to the Rh and ABO ● The branched chains permit attachment of more molecules to determine H
systems are strongly immunogenic (i.e., capable of inducing antibody antigen specificity.
formation), whereas the antigens of the Kidd and Duffy blood group ● Following H antigen development, the A and/or B specific molecule may be
systems are much weaker immunogens. attached.
o The blood group antigens are not restricted solely to red cells or even
INHERITANCE OF ABH ANTIGENS
to hematopoietic tissues.
● As Bernstein (1924) discovered, ABO antigens are inherited in a simple
● The antigens of the ABO system are widely distributed throughout the tissues
and have been unequivocally identified on platelets and white cells (both Mendelian fashion from an individual’s parents.
o Each individual possesses a pair of genes.
lymphocytes and polymorphonuclear leukocytes) and in skin, the epithelial
o Each gene occupies an identical locus on chromosome 9.
(lining) cells of the gastrointestinal tract, the kidney, the urinary tract, and the
o There are three possible genes that can be inherited: A, B, and O.
lining of the blood vessels.
● Evidence for the presence of the antigens of other blood group systems on ● A and B genes produce a detectable product while the O gene is an amorph
that does not produce a detectable product or antigen
cells other than red cells is less well substantiated.
o The expression of the A and B genes is codominant.
● Among the red cell antigens, only those of the ABO system are regarded as
● The H antigen is required to produce A and/or B antigens .
tissue antigens
o Therefore, need to be considered in organ transplantation. o The H and SE gene is also inherited in Mendelian fashion and
occupies a locus on chromosome 19.
NOTE: o H and Se not part of ABO system
Agglutination o Each parent contributes one gene, either H or h.
The reaction between red cells and corresponding antibodies that cause the o The possible genetic combinations are HH, Hh, or hh.
clumping of RBC ● Individuals who are genetically either HH or Hh will produce the H antigen, and
it can be detected on their red cells.
Agglutinogens
Antigens on the surfaces of red cells responsible for agglutination ● The frequency of occurrence of the H antigen in the Caucasian population is
greater than 99.99%.
● Individuals inheriting an hh genotype do not produce the H antigen and have
ABO ANTIGENS the Bombay Phenotype, Oh.
● Located on the surface of the red blood cell. ● Anti-H – Frequently demonstrated on the plasma of an individual with a
● Also present on lymphocytes, thrombocytes, organs, endothelial cells, and Bombay phenotype
epithelial cells.
● First detected by Landsteiner as he performed his mixing tests BIOCHEMICAL AND STRUCTURAL DEVELOPMENT OF ABH ANTIGENS
● The biochemistry and structure of ABO antigens are well-established. ● Expression of A, B, and H genes does not result in the direct production of
● Antigens of the ABO system are well-developed in adults. antigens.
o They are detectable at 5 to 6 weeks of gestation.

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 o Rather, each gene codes for the production of an enzyme known as ● These ABO antibodies were originally thought to be natural antibodies formed
a transferase (Glycoseryltransferase) with no apparent antigenic stimulus.
● Each transferase catalyzes the transfer of a carbohydrate molecule to an ● Since the antibodies are not stimulated by exposure to red cells, they may also
oligosaccharide chain. The attached carbohydrate provides antigenic be considered non-red cell stimulated antibodies.
specificity. o However, some form of an antigenic stimulus must exist.
● The O gene codes for an enzymatically inactive protein and, hence, no o The proposed mechanism is environmental.
antigen is produced. ● These “naturally occurring” substances resemble A and B antigens and
stimulate the production of complementary antibodies to the antigens that are
DEVELOPMENT OF H ANTIGEN
not present on the red cell surface.
● The H allele codes for the transferase, L-fucosyltransferase.
● IgM (most); some IgG
o This enzyme catalyzes the formation of the H antigen by transfer of o Activates complement
L-fucose to either type one or type two oligosaccharide chains.
o Capable of hemolysis
o The L-fucose is the immunodominant sugar for the H antigen. It is
o React at room temperature
the sugar that confers antigenic specificity to the H antigen.
o Too low for detection until 3-6 months of age
● 2 alleles ▪ Peaks at 5-10 years of age
o The H antigen serves as a precursor for A and B antigens .
▪ Decline later in life
o The h allele is an amorph and does not produce a detectable
product. NOTE:
Newborns have no ABO antibodies. When newborns are tested, only a forward
DEVELOPMENT OF A AND B ANTIGENS
group is performed. Newborns may exhibit passive ABO antibodies that have crossed
● The H antigen oligosaccharide chain serves as a precursor for both the A
the placental barrier. Reverse grouping of a newborn or umbilical cord serum
and B antigens.
indicates the blood group of the mother. The child will begin antibody production, and
● The A and B alleles each code for a transferase that attaches a sugar
have a detectable titer, at three to six months of age. ABO antibody production peaks
molecule to the terminal end of the H antigen oligosaccharide chain, which
at age five to ten years of age and continues in immunocompetent individuals
forms either the A or B antigen.
throughout life. Titers begin to wane in the elderly.
o The A allele codes for N-acetylgalactosamine transferase.
o This transferase attaches N-acetyl-D-galactosamine to the H
antigen forming the A antigen.
THE IMMUNOGLOBULIN CLASSES OF ABO ANTIBODIES IN GROUP ABO
o The B allele codes for D-galactosyltransferase.
o This transferase attaches D-galactose to the H antigen forming the IMMUNOGLOBULIN CLASS
B antigen. ● ABO antibodies are typically isoagglutinins.
● The product of the O allele is an enzymatically inactive protein. o They are saline agglutinins with optimal reactivity at 4°C.
o Hence, this allele produces no detectable antigen (amorph) ● These naturally occurring antibodies are mostly IgM isotype
● Conversely, group O cells contain the most H antigen. This results from no o IgG and IgA classes of ABO antibodies have been detected.
conversion of H antigen to A and/or B antigens. ● The development of IgG antibodies occurs without apparent antigen
o In comparison, group A1 B cells have the least amount of H antigen exposure via transfusion of incompatible red cells or fetal maternal
since quantitatively the most H is converted to A1 and B antigens. incompatibility.
ABO ANTIBODIES IMMUNIGLOBULIN CLASSESS
● Antibodies directed against ABO antigens are the most important antibodies in Anti-A,B ● Group O individuals do not have A or B antigens on their cells.
transfusion medicine. o Consequently, they produce anti-A, anti-B, and anti-A,B.
● The ABO blood group presents a unique situation in Immunohematology. ● Anti-A,B is an antibody that has cross- reactivity with A and B
● It is the only example of a blood group where each individual produces cells.
antibodies to antigens not present on the red cells.

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 ● This cross-reactive antibody detects a common molecular ● The most common scenario is a group O mother and a group A baby.
structure in both antigens. o ABO hemolytic disease may affect a woman’s first pregnancy. This is
o Although the antibody reacts with both antigens, it cannot in contrast to Rh HDFN where the antigenic stimulation usually occurs
be divided into individual components (i.e. anti-A plus in the first pregnancy and subsequent antigen-positive newborns are
anti-B). affected.
o Anti-A,B is used as a third antisera in forward grouping. ● Hemolytic transfusion reaction occurs when a recipient is transfused with red
o Anti-A,B is not required in forward grouping. cells that are an ABO group incompatible with the antibodies in his or her
● Since it is a valuable reagent for determining subgroups of A and serum. Because of the complement-binding ability of the ABO antibodies, this
B, anti-A,B is often included as a routine part of forward grouping. is always a life-threatening situation.
● Monoclonal anti-A,B have replaced the use of human anti-A, B in ● As the recipient antibodies react with the incompatible red cells, complement
forward grouping. is activated and in vivo hemolysis, agglutination, and red blood cell
destruction occurs.
● ABO compatibility is also significant in solid organ transplantation.
● As per Landsteiner’s Law, group B and O individuals produce
o For most organs, an ideal scenario for transplant is an ABO compatible
Anti-A1 anti-A.
solid organ.
● This anti-A can be separated by absorption procedures.
o Post-transfusion antibody titer, and pheresis to reduce the titer of the
o These absorption procedures can produce two
incompatible antibody, will assist in achieving a positive outcome when
components of the antibody found in group B and O
an ABO incompatible organ is transplanted.
individuals.
o These components are anti-A and anti-A1. EFFECTS OF AGE ON THE PRODUCTION OF ISOAGGLUTININS
● The anti-A1 antibody reacts specifically with A1 cells and not with
A2 cells or cells from other subgroups of A. ISOAGGLUTININS ● Isoagglutinins are antibodies produced by an individual
● Like other ABO antibodies, this antibody reacts optimally at room that cause agglutination of RBCs in other individuals.
temperature or colder. ● People possess isoagglutinins directed toward the A or
● Anti-A1 is not considered clinically significant as it relates to B antigen absent from their own RBCs.
transfusion. ● For example, type B or O individuals will usually
o It is, however, significant when it causes incompatible possess anti-A. The anti-A is formed in response to
crossmatches at the immediate spin phase. exposure to A-like antigenic structures found in
● Antibodies to other A subgroups, such as A2, are not produced. ubiquitous non-red cell biologic entities (eg, bacteria).
These subgroups have the A antigen but in reduced amounts. ● Isoagglutinins present in the newborn are passively
o Therefore, transfusion of A1 individuals with A2 cells will not acquired from maternal circulation.
stimulate the production of anti-A2 since both A1 and A2 o Such passively acquired isoagglutinins will
individuals have the A antigen in common. gradually disappear, and the infant will begin to
produce isoagglutinins at 3 to 6 months of age.
● Isoagglutinins production may vary in patients with
certain pathologic conditions.
o Decreased levels of isoagglutinins may be seen
CLINICAL SIGNIFICANCE OF ABO ANTIBODIES in patients with acquired and congenital
● ABO antibodies are capable of causing both Hemolytic Disease of the Fetus hypogammaglobulinemia and
and Newborn (HDFN) and Hemolytic Transfusion Reactions (HTR). agammaglobulinemia.
● These issues explain the clinical significance of “naturally occurring”
o Some individuals with roundworm infections will
antibodies. have elevated levels of anti-A.
● HDFN usually presents itself with a maternal antibody of an IgG isotype that
corresponds to an antigen on the surface of the baby’s red cells.

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 ● In contrast, anti-H in non-Bombay
ANTI-A AND ● Anti-A and anti-B are found in the sera of individuals individuals is usually IgM and
ANTI-B who lack the corresponding antigens. clinically insignificant.
● They are produced in response to environmental ● Anti-IH is not uncommonly found in
stimulants, such as bacteria, and have therefore been patient sera and is usually IgM;
termed natural antibodies. compatible blood is easily found
● Antibody production begins after birth, reaching a peak among donors of identical ABO type
at age 5 to 10 and declining with increasing age.
● The antibodies formed to carbohydrate antigens are
mostly immunoglobulin M (IgM).
o IgM antibodies activate complement, which, in
INHERITANCE OF THE ABO BLOOD GROUPS
conjunction with the high density of ABO antigen
● The theory for the inheritance of the ABO blood groups was first described by
sites on RBCs,
Bernstein in 1924.
o is responsible for the severe, life-threatening
transfusion reactions that may be caused by o He demonstrated that an individual inherits one ABO gene from each
parent and that these two genes determine which ABO antigens are
ABO-incompatible transfusions.
present on the RBC membrane.
● The inheritance of ABO genes, therefore, follows simple Mendelian genetics.
Hemolytic ● Hemolytic disease of the newborn ● ABO, like most other blood group systems, is codominant in expression.
disease of caused by ABO antibodies is ● One position, or locus, on each chromosome 9 is occupied by an A, B, or O
the Newborn usually mild, for the following gene.
reasons: ● The O gene is considered an amorph, as no detectable antigen is produced in
o placental transfer is limited to response to the inheritance of this gene.
the fraction of IgG anti-A and o Therefore, the group O phenotype is an autosomal recessive trait with
anti-B found in maternal serum the inheritance of two O genes that are nonfunctional.
o fetal ABO antigens are not fully o The designations group A and B refer to phenotypes, whereas AA,
developed BO, and OO denote genotypes.
● ABO tissue antigens provide o In the case of an O individual, both phenotype and genotype are the
additional targets for the antibodies. same, because that individual would have to be homozygous for the
● ABO-HDN is most often seen in O gene.
non-group O infants of group O ● An individual who has the phenotype A (or B) can have the genotype AA or AO
mothers, because anti-A, anti-B, (or BB or BO).
and anti-A,B of group O mothers
often has a significant IgG ABO Blood Antigen A Antigen B Antibody Antibody
Type anti-A anti-B
component A YES NO NO YES
B NO YES YES NO
Bombay ● Potent anti-H (along with anti-A and
O NO NO YES YES
Phenotype anti-B) found in Oh (Bombay) or AB YES YES NO NO
para-Bombay nonsecretors destroys For example;
transfused RBCs of any ABO group, ● People with type A blood will have the A antigen on the surface of their red
so these individuals must be cells (as shown in the table below).
transfused only with blood of the o As a result, anti-A antibodies will not be produced by them because
Bombay phenotype. they would cause the destruction of their own blood.

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 ● However, if B type blood is injected into their systems, anti-B antibodies in o These genes do not actually code for the production of antigens but
their plasma will recognize it as alien and burst or agglutinate the introduced rather produce specific glycosyltransferases that add sugars to a
red cells in order to cleanse the blood of alien protein. basic precursor substance.
● A, B, and H antigens are formed from the same basic precursor material
ABO Blood Antigen A Antigen B Antibody Antibody (called a paragloboside or glycan) to which sugars are attached in response to
Type anti-A anti-B
specific enzyme transferases elicited by an inherited gene
A YES NO NO YES
B NO YES YES NO H AND SE ● The H antigen is actually the precursor structure on which
O NO NO YES YES ANTIGEN A and B antigens are made.
AB YES YES NO NO
o Inheritance of the H gene results in the formation of
● Individuals with type O blood do not produce ABO antigens.
the H antigen.
● Therefore, their blood normally will not be rejected when it is given to others
o The H and Se genes are closely linked and located
with different ABO types.
on chromosome 19, in contrast to ABO genes,
o As a result, type O people are universal donors for transfusions, but
which are located on chromosome 9.
they can receive only type O blood themselves.
o The H and Se genes are not part of the ABO
● Those who have type AB blood do not make any ABO antibodies. Their blood
system; however, their inheritance does influence A
does not discriminate against any other ABO type.
and B antigen expression.
o Consequently, they are universal receivers for transfusions, but
their blood will be agglutinated when given to people with every PRECURSOR ● The precursor substance on erythrocytes is referred to as
other type because they produce both kinds of antigens. SUBSTANCE type 2.
o This means that the terminal galactose on the
ABO Blood Antigen A Antigen B Antibody Antibody precursor substance is attached to the
Type anti-A anti-B N-acetylglucosamine in a beta 1 → 4 linkage .
A YES NO NO YES
o Found in body fluids and secretions
B NO YES YES NO
● A type 1 precursor substance refers to a beta 1 → 3
O NO NO YES YES
linkage between galactose and N-acetylglucosamine.
AB YES YES NO NO
● It is easy and inexpensive to determine an individual's ABO type from a few o RBCs. Body fluids and secretions
● ABH antigens on the RBC are constructed on
drops of blood.
oligosaccharide chains of a type 2 precursor substance.
o A serum containing anti-A antibodies is mixed with some of the
blood.
o Another serum with anti-B antibodies is mixed with the remaining
sample. ABH ANTIGEN ● Found on RBCs, lymphocytes, platelets, tissue cell and
● Whether or not agglutination occurs in either sample indicates the ABO type. DEVELOPMENT bone
● It is a simple process of elimination of the possibilities. ● Glycolipid/glycoprotein
● For instance, if an individual's blood sample is agglutinated by the anti-A ● The ABH antigens develop early in fetal life but do not
antibody, but not the anti-B antibody, it means that the A antigen is present increase much in strength during the gestational period.
but not the B antigen. Therefore, the blood type is A o Developed in utero at 5-6 weeks of gestation
● The RBCs of the newborn have been estimated to carry
FORMATION OF ABH ANTIGENS ON THE RBCS FROM PRECURSOR anywhere from 25% to 50% of the number of antigenic
SUBSTANCE TO IMMUNODOMINANT SUGARS sites found on the adult RBC.
● The formation of ABH antigens results from the interaction of genes at three ● As a result, reactions of newborn RBCs with ABO reagent
separate loci (ABO, Hh, and Se).
antisera are frequently weaker than reactions with adult
cells.

13 | Page
 ● The expression of A and B antigens on the RBCs is fully ● The O gene at the ABO locus, which is sometimes referred
developed by 2 to 4 years of age and remains constant to as an amorph, does not elicit the production of a
throughout life. catalytically active polypeptide transferase; therefore, the H
● In addition to age, the phenotypic expression of ABH substance remains unmodified.
antigens may vary with race, genetic interaction, and ● Consequently, the O blood group has the highest
disease states concentration of H antigen.
o The H substance (L-fucose) must be formed for the
other sugars to be attached in response to an inherited
ABH-SOLUBLE ANTIGENS
● Can be found in all body secretions A and/or B gene.
● Dependent on ABO gene inherited and Se gene ● The H gene is present in more than 99.99% of the random
(secretor gene) population. Its allele, “h,” is quite rare, and the genotype hh
● It is in the presence of the Se gene specified is extremely rare.
a-2-L-fucosyltransferase that determines whether ● The term Bombay has been used to refer to the phenotype
ABH-soluble substances will be secreted that lacks normal expression of the ABH antigens because
● Sese genotype are termed non-secretors of the inheritance of the hh genotype.
o The hh genotype does not elicit the production of
α-2-L-fucosyltransferase.
o Therefore, L-fucose is not added to the type 2 chain,
and H substance is not expressed on the RBC.
SUMMARY OF GLYCOSYLTRANSFERASES AND IMMUNODOMINANT SUGARS o Even though Bombay (hh) individuals may inherit
RESPONSIBLE FOR H, A AND B ANTIGEN SPECIFICITIES ABO genes, normal expression, as reflected in the
formation of A, B, or H antigens, does not occur.
GEN GLYCOSYLTRANSFERASE IMMUNODOMINANT ANTIGEN
E SUGAR
H a-2-L-fucosyltransferase L-fucose H
A a-3-N acetylgalactosaminylferase N-acetyl-D-galactosa A BLOOD ● In the formation of blood group A, the A gene (AA or AO)
mine GROUP A codes for the production of α-3-N-acetylgalac -
B a-3-D-galactosyltransferase D-galactose B
tosaminyltransferase, which transfers an N-acetyl-D-galac -
tosamine(GalNAc) sugar to the H substance.
o This sugar is responsible for A specificity (blood
INTERACTION OF Hh AND ABO GENES
group A).
Blood Group ● Individuals who are blood group O inherit at least one H o The A-specific immunodominant sugar is linked to
O gene (genotype HH or Hh) and two O genes. a type 2 precursor substance that now contains H
● The H gene elicits the production of an enzyme called substance through the action of the H gene.
α-2-L-fucosyltransferase, which transfers the sugar L-fucose ● The A gene tends to elicit higher concentrations of
to an oligosaccharide chain on the terminal galactose of transferase than the B gene. This leads to the conversion of
type 2 chains. practically all of the H antigen on the RBC to A antigen sites.
o The sugars that occupy the terminal positions of this ● As many as 810,000 to 1,170,000 antigen sites exist on an
precursor chain and confer blood group specificity are A1 adult RBC in response to inherited genes.
called the immunodominant sugars.
o Therefore, L-fucose is the sugar responsible for H BLOOD ● Individuals who are blood group B inherit a B gene (BB or
specificity (blood group O). GROUP B BO) that codes for the production of
α-3-D-galactosyltransferase, which attaches D-galactose

14 | Page
 (Gal) sugar to the H substance previously placed on the PRINCIPLE ● Human red blood cells possessing the H antigen will
type 2 precursor substance through the action of the H agglutinate in the presence of seed extract (lectins)
gene. containing phytohaemagglutinin specifically directed
o This sugar is responsible for B specificity (blood towards it.
group B). ● Water soluble H substance present in saliva neutralises
o Anywhere from 610,000 to 830,000 B antigen sites Anti-H lectin.
exist on a B adult RBC in response to the conversion ● Agglutination of red blood cells / Neutralization of Anti-H
of the H antigen by the α-3-Dgalactosyltransferase lectin by saliva is a positive test result and indicates the
produced by the B gene. presence of
● When both A and B genes are inherited, the B enzyme ● H substance on/in the red cell / saliva respectively.
(α-3-D-galactosyltransferase) seems to compete more ● No agglutination / Neutralization of Anti-H lectin is a
efficiently for the H substance than the A enzyme negative test result and indicates the absence of H
(α-3-Nacetylgalactosaminyltransferase). substance on / in the red cell / saliva respectively.
o Therefore, the average number of A antigens on an
AB adult cell is approximately 600,000 sites, compared
with an average of 720,000 B antigen sites.
NOTE:
● In vitro diagnostic reagent for laboratory and professional use only.
o Not for medicinal use.
REACTIVITY OF ULEX EUROPAEUS WITH THE VARIOUS ABO GROUPS ● The reagent contains sodium azide 0.1% as preservative. Avoid contact with
● The H antigen is a basic blood group antigen present in human beings. There skin and mucosa.
is considerable variation in the H antigen content in different individuals of the o On disposal flush with large quantities of water.
same ABO group but the general pattern indicates their strength as ● Extreme turbidity may indicate microbial contamination / reagent deterioration.
O>A2>A2B>B>A1>A1B. Such reagents should be discarded
● Water soluble H substance can also be demonstrated in saliva or body fluids
of individuals who are secretors.
● Human red blood cells that do not agglutinate with Anti-H lectin are classified ABO SUBGROUPS
as Bombay Phenotype (Oh). ● Differ in the amount of Ag expressed on the RBC
o The Bombay Phenotype is more common in India than other parts ● Shows weaker variable serologic reactivity with the commonly used human
of the world and the estimated gene frequency of Oh phenotype in polyclonal anti-A, anti-B and anti-A, B reagents
Bombay is 0.0066%. A SUBGROUPS

REAGENT ● Anti-H lectin is a ready to use purified extract of Ulex A1 AND A2 SUBGROUPS
europaeus seeds. It contains a phytohaemagglutinin, which ● Group A antigens can be differentiated into multiple subgroups. The two
is virtually specific for the H antigen on human red blood
major subgroups are
cells.
o A1, 80% of Group A individuals, and
● Anti-H lectin is used for recognition of the H antigen on o A2, 20% of group A individuals
human red blood cells. ● Persons typing as AB can be divided into the same percentages of A antigen
● It is useful, especially for assessing the H secretor status of
presentation.
group 'O' individuals and also in differential grouping of Aint o A1B make up approximately 80% and
subgroup along with Anti-A1 lectin. o A2B are 20% of all AB individuals.
● The remaining group A individuals fall into one of many minor subgroups.

15 | Page
 o A1 and A2 antigens have qualitative and quantitative differences. ● The lectin, Dolichos biflorus, is used to obtain an extract
with anti-A1 specificity.
DIFFERENCES OF A1 AND A2 ANTIGENS
● Dolichos biflorus will react specifically with A1 cells and
Qualitative ● a subgroup phenotype is distinguished from A1 phenotype
will be negative with A2 cells.
differences of by a glycolipid antigen expression because of the
A1 and A2 structural differences in the branching of the
antigens
oligosaccharide chain
● The structural difference explain why the A subgroup ● A2 individuals can develop antibodies to the A1 antigens.
individuals often make the anti-A1 ● The typical reaction pattern of reverse grouping in a group A individual is no
● The reagent dolichos biflorus lectin will distinguish A1 agglutination with the A cells (no anti-A) and agglutination with B cells (anti-B
from A2 present).
● When red cells are qualitatively tested for antigens, A1 o In A2 persons with an anti-A1, the A cells will also be agglutinated in the
and A2 red cells have differing amounts of antigens on the reverse grouping.
cell surface. o This discrepancy should be confirmed by testing the red cells with the
o The A1 gene produces a transferase that has a Dolichos biflorus lectin.
greater ability to convert H antigen to A antigen
than the A2 gene.
o This quantitative difference results from the kinetics
QUANTITATIVE QUALITATIVE
of the reaction catalyzed by each of the
↓ Number of Antigen Sites Differences in the precursor
transferases. oligosaccharide chains
● There are also differences in the quantity of transferase ↓ Amount of Transferase Enzyme Subtle differences in transferase
produced. enzymes
● Individuals exhibiting the A1 phenotype have five to ten ↓ Amount of Branching Formation of anti-A1, in a percentage of
times more transferase than those with an A2 phenotype. some subgroups
● Two mutations have been detected that produce this
A2 phenotype:
REACTIONS
o a Pro156Leu substitution
o a single nucleotide deletion (nucleotide 1060). BLOOD Anti-A reagent Anti-A lectin reagent Dolichus Biflorus
● These substitutions are responsible for the decreased GROUP (anti-A plus Anti-A1) lectin
enzyme activity that differentiates A2 from A1 cells. A1 Positive Positive Agglutinate
A2 Positive Negative No agglutinate
Quantitative ● For quantitative test, A2 produces so little antigen
differences of because the number of A antigen is reduced in A2.
A1 and A2 ● There is a little amount of antigen in A2 than in A1 LECTINS IN BLOOD BANKING
antigens
● A2 antigens are composed mainly of linear ● Dolichus biflorus – agglutinated A1 and A1B
oligosaccharide chains ● Bandeiraea simplicifolia – agglutinated B cells
● A1 cells have a greater number of branched chains ● Ulex europaeus – agglutinateds O cells and other ABO blood groups
o In routine testing, this qualitative difference is not depending on the amount of H ag available
detectable but can be determined biochemically.
● Typing of A1 and A2 cells is unremarkable with routine AMOUNT OF H ANTIGEN
antisera. ● O>A2>B>A2B>A1>A1B
● Both A1 and A2 cells will react equally with anti-A and ● Greatest amount of H to least amount of H
anti-A,B.
ADDITIONAL A SUBGROUPS

16 | Page
● Occurring subgroups of A exist less frequently. WEAK B SUBGROUPS
o These subgroups are also genetically controlled. ● With the addition of the anti-A and anti-B reagent to the cell, there can be
● Subgroups of A include Aintr, A3, Ax , Am, Aend, Ael,and Abantu. two reactions: weak agglutination or no agglutination at all.
o These subgroups follow the patterns of A1 and A2 with regard to
quantitative and qualitative antigenic differences. Weak ● B(3) – mix field reaction with anti-A and anti-B
o The cells of these subgroups exhibit fewer antigen sites on their ● B(x)– weak agglutination with anti-A and anti-AB
surface while many demonstrate an anti-A1 in the plasma. only
● Adsorption and elution techniques may be necessary for detection of antigens
on the surface of red cells. No agglutination ● B(m) – demonstrates the presence of A-substance
● The classification of subgroups is based on reactions of the patient’s red cells ● B(el) – shows the presence of H-substance
with anti-A, anti-B, anti-A,B, and anti-A1 antisera as well as A1, A2, and B
reverse grouping cells. -Refer to reference book for further information
o While testing for subgroups of A, a mixed field agglutination reaction
may be noted.
o A3 cells will demonstrate this pattern of agglutination with anti-A and
THE BOMBAY PHENOTYPES (OH)
anti-A,B.
● The Bombay phenotype was first reported by Bhende in 1952 in Bombay, India.
● SUBGROUPS OF B
● It represents the inheritance of a double dose of the h gene, producing the very
o Very rare and encountered less frequently than subgroups of A.
rare genotype hh.
o Methods of detection and classification are similar to those used for the
o As a result, the ABO genes cannot be expressed, and ABH antigens
subgroups of A.
cannot be formed, since there is no H antigen made in the Bombay
WEAK SUBGROUPS OF A phenotype.
● The prevalence of A subgroups of A weaker than A1 and A2 is less than 1%. ● More than 130 Bombay phenotypes have been reported in various parts of the
● Fewer antigen sites on the RBC means weaker reactions with antisera. world. These RBCs are devoid of normal ABH antigens and, therefore, fail to
● It is possible for an Ax donor to be mistyped as O. react with anti-A, anti-B, and anti-H.
● This unit could then be transfused into an O recipient, who has anti-A,B. ● In RBC testing using anti-A and anti-B, the Bombay would phenotype as an O
● The anti-A,B antibody in the recipient could agglutinate and lyse the donor blood group.
Ax RBCs and cause intravascular hemolysis o Using conventional or non-potent anti-A and anti-B sera would result in
the Bombay phenotype to be mistaken as O-blood group.
Subgroup Laboratory Results Number of A o To differentiate group O from Bombay, use anti-A, anti-B, and anti-H
Antigens sites
since group O (with H-antigens) would always have an agglutination
A3 Mixed field reaction with anti-A and most 35,000 per RBC
with anti-H
anti-A,B reagents
o However, the RBCs of the Bombay phenotype (Oh) do not react with the
Ax Characteristically not agglutinated with anti-A but 4000
do agglutinate with most examples of anti-A,B anti-H lectin (Ulex europaeus), unlike those of the normal group O
Aend Mixed field reaction with anti-A and anti-A,B. A 3500 individual, which react strongly with anti-H lectin.
end is inherited as an allele at the ABO locus. ● Bombay serum contains antiA, anti-B, anti-A,B, and anti-H.
Anti-A1 is found in some sera. Only H is found in ● Unlike the anti-H found occasionally in the serum of A1 and A1B individuals, the
secretions. Bombay anti-H can often be potent and reacts strongly at 37°C.
Am Characteristically not agglutinated, or very 200-1900 ● It is an IgM antibody that can bind complement and cause RBC lysis.
weakly agglutinated by all anti-A and anti-A,B ● Transfusing normal group O blood (with the highest concentration of H antigen)
reagents. Usually do NOT produce anti-A1 in
sera. to a Bombay recipient (anti-H in the serum) would cause immediate cell lysis.
o Therefore, only blood from another Bombay individual will be compatible
and can be transfused to a Bombay recipient.

17 | Page
● ABH substance is also absent in saliva. o The weakening of the antigen tends to follow the course of the
● The (Oh) Bombay phenotype is inherited as an autosomal recessive trait. disease.
● The underlying molecular defect is most commonly a mutation in the gene FUT1 o The antigen strength will increase again as the patient enters
(H gene), which produces a silenced gene that is incapable of coding for the remission.
enzyme, α(1,2)fucosyltransferase (H transferase). ● The isoagglutinins (anti-A, anti-B, or anti-A,B) also may be weak or absent in
● This enzyme catalyzes the transfer of fucose in an α-1,2 linkage to the terminal those leukemias demonstrating hypogammaglobulinemia, such as chronic
galactose of the precursor molecule on RBCs forming the H antigen. lymphocytic leukemia (CLL).
● This mutation underlying the Bombay phenotype is also associated with a ● Various lymphomas, such as the malignant (non-Hodgkin’s) variety, may
silenced FUT2 gene (Se gene), which normally encodes a very similar yield weak isoagglutinins, owing to moderate decreases in the gamma
α(1,2)fucosyltransferase and normally transfers a fucose to form H antigens in globulin fraction.
secretions when active. ● Also, immunodeficiency diseases, such as congenital agammaglobulinemia,
● When family studies demonstrate which ABO genes are inherited in the Bombay will also yield weak or absent isoagglutinins.
phenotype, the genes are written as superscripts (OhA, OhB, OhAB). o If this problem is suspected, a simple serum protein electrophoresis
will confirm or rule out this condition.
● Individuals with intestinal obstruction, carcinoma of the colon or rectum, or
EFFECTS OF DISEASE ON THE EXPRESSION OF ABH ANTIGENS AND other disorders of the lower intestinal tract may have increased permeability
ANTIBODIES of the intestinal wall, which allows passage of the bacterial polysaccharides
from Escherichia coli serotype O86 into the patient’s circulation.
ABH ANTIGENS AND ANTIBODIES IN DISEASE ● This results in the acquired B phenomenon in group A1 individuals.
● Associations between ABH antigens and practically any disorder known to o The patient’s group A red cells absorb the B-like polysaccharide,
man can be found throughout medical literature. which reacts with human-source anti-B.
● Even more profound are the associations of blood group specificity with such ● A lack of detectable ABO antigens can occur in patients with carcinoma of
things as a more pronounced: the stomach or pancreas.
o “hangover” in A blood groups o The patient’s red cell antigens have not been changed, but the
o “criminality” in group B blood groups serum contains excessive amounts of blood group–specific soluble
o “good teeth” in group O individuals. substances (BGSS) that may neutralize the antisera utilized in the
● There are also several papers correlating blood groups with personality forward grouping.
traits. It is no surprise that many scientists refer to these associations as a ● All these disease states previously mentioned may result in discrepancies
part of blood group mythology. between the forward and reverse groupings, indicating that the patient’s red
● However, more relevant associations between blood groups and disease are cell group is not what it seems.
important to the blood banker in terms of blood group serology. ● All ABO discrepancies must be resolved before blood for transfusion is
● Various disease states seem to alter red cell antigens and result in released for that patient.
progressively weaker reactions or additional acquired pseudoantigens, o In some cases, secretor or molecular studies may help confirm the
which can be seen during forward grouping. patient’s true ABO group.
● Leukemia, chromosome 9 translocations, and any hemolytic disease that
induces stress hematopoiesis (e.g., thalassemia) have been shown to LABORATORY TESTS TO DETECT AND IDENTIFY ABO ANTIGENS AND
depress antigen strength. ANTIBODIES
o Often the cells will appear to show a mixed-field agglutination (tiny
ENZYME-CONVERTED O CELLS (ECO)
agglutinates in a sea of unagglutinated cells). ● Blood group O is considered the universal donor because it can be
● Hodgkin’s disease also has been reported to weaken or depress ABH red transfused to patients of all ABO types. Therefore, enzymes that remove
cell antigens, resulting in variable reactions during forward grouping similar terminal carbohydrates from the nonreducing end of carbohydrate chains
to those found in leukemia.

18 | Page
 could be used to remove terminal A and B sugars to convert the blood ● When utilizing gel technology, the cell suspension consists
supply to all universal group O units. of a 0.8% suspension of washed patient cells in the
● An enzyme from coffee beans, α-galactosidase, has been the most manufacturer’s recommended diluent.
successful at removing galactose to convert blood group B to group O. ● These cells are applied to the anti-A and anti-B tubes of the
● RBCs treated in this manner have normal survival when transfused to group gel card. In both methods, centrifugation is applied and
B, A, or O recipients. results interpreted.
● Removal of N-acetylgalactosamine to convert group A to group O has been
much more problematic, owing to the inaccessibility of the carbohydrates on
ANTISERA ● When performing tube typing, three antiseras are
internal branching chains, especially those found on A1 cells.
available for ABO forward grouping.
● The procedures required to convert B to O, which include exposure to low
pH followed by numerous washings, make them impractical for general use. ● Forward grouping may be performed using all three, or
in the case of patients or transfusion recipients anti-A
This is an active area of research and alternative enzymes and improved
and anti-B are used
methodologies are under investigation.
● Antisera are combined in a 1:1 ratio with the patient’s
BLOOD TYPING cell suspension.
● Blood typing is a method to tell what type of blood you have. ● When evaluating reaction patterns, the antigens on the
● Is done so you can safely donate your blood or receive a blood transfusion. cells are reacting with the specific antibodies in the
● It is also done to see if you have a substance called Rh factor on the surface antisera.
of your red blood cells. o Group A individual has the A antigen and
● Your blood type is based on whether or not certain proteins are on your red reacts with both anti-A and anti-A,B.
blood cells. o A group O will react with no antisera since
o These proteins are called antigens. Your blood type (or blood these cells have neither A nor B antigens.
group) depends on what types your parents passed down to you. ● The inclusion of anti-A,B antisera in forward grouping
is significant.
FORWARD AND REVERSE GROUPING o It is not a mixture of anti-A and anti-B, but
ABO ● As previously described, ABO antigens are present on the rather a separate antibody that will react with
FORWARD surface of red cells, while the antibodies are found in both the A and B antigens.
GROUPING plasma or serum. ● It is included in forward grouping and serves two
● Routine testing for antigens and antibodies is performed as purposes:
a forward and reverse grouping, respectively. (1) To confirm the results of the anti-A and anti-B.
● The forward grouping is a test performed for antigens using (2) To detect weak subgroups of A and B.
known antisera with patient’s cells that may contain ● These subgroups may demonstrate a positive reaction
unknown antigens. with anti-A,B but not with anti-A and anti-B.
● Test methods for forward grouping include:
(1) Tube typing
(2) Gel Technology
(3) Automation
(4) Solid Phase Technology
● ABO forward grouping with tube typing uses a saline REACTION PATTERNS FOR ABO GROUP
suspension of 3 to 5% washed patient red cells. These BLOOD GROUP ANTI-A ANTI-B ANTI-A,B
cells are combined in a 1:1 ratio with commercial antisera. A POSITIVE NEGATIVE POSITIVE
B NEGATIVE POSITIVE POSITIVE

19 | Page
 AB POSITIVE POSITIVE POSITIVE The result is evaluated by examining the tubes for hemolysis and agglutination.
O NEGATIVE NEGATIVE NEGATIVE NOTE:
● Recall that antibodies present in the test plasma correspond to antigens
In Forwarding Typing the Antigens are used as Antisera. missing on the red cell surface.
(1) Blood Type O has no agglutination ● For example, group A has the A antigen and the B antigen is not present.
(2) Blood Type A only has the reaction seen in Anti-A since Blood type A ● The corresponding B antibody is demonstrated in the group A individual’s
contains Antigen A plasma.
(3) Blood Type B only has the reaction seen in Anti-B since Blood type B ● When the plasma reacts with the reagent red blood cells, the B antibody
contains Antigen B reacts with specific antigens on the B cells, but not antigens on the A cells.
(4) Blood Type AB has both the Antigen AB therefore it is present in both ● Therefore, a positive reaction will be seen in the B tube, but not in the A tube.
reactions. ● For the reverse typing, this is the detection of antibodies in our serum
● Both positive reaction for Blood type O since it contains both Anti A,B
MOLECULAR TESTING
● In Blood type A, only B cells has the reaction since Blood type A individual has
● As molecular diagnostic testing continues to develop, applications to ABO the Anti B
forward grouping may become commonplace in the laboratory.
● In Blood type B, only A cells has the reaction since Blood type B has the Anti-
● Molecular testing has the potential to solve typing discrepancy in recently or
A
chronically transfused patients.
● In Blood type AB, there is no reaction for the reverse typing since Blood type
● These patients present unique challenges to the blood bank, since typing AB contains no any antibodies in their serum
through traditional methods frequently produces discrepant or erroneous
results.

POLYMERASE CHAIN REACTION (PCR) METHODS

● Have been proven to be more reliable than traditional serological methods For the reverse typing, this is the detection of antibodies in our serum
in resolution of typing discrepancies in recipients that have been transfused ● Both positive reaction for Blood type O since it contains both Anti A,B
within the last 30 days. ● In Blood type A, only B cells has the reaction since Blood type A individual
● As PCR test results are available within several hours, this testing method has the Anti B
presents a promising future for resolution of discrepancies that were ● In Blood type B, only A cells has the reaction since Blood type B has the
previously major compatibility challenges Anti- A
● In Blood type AB, there is no reaction for the reverse typing since Blood type
AB contains no any antibodies in their serum
REVERSE GROUPING
● ABO reverse grouping uses patient plasma combined in a 2:1 ratio with ABO DISCREPANCIES
● ABO discrepancies occur when unexpected reactions occur in the forward
commercially prepared cells.
and reverse grouping.
● The cells are packaged in sets of two (A1 and B) or three (A1, A2, and B).
● These can be due to:
The cells are used to detect unknown antibodies in the plasma
. o Problems with the patient’s serum (reverse grouping)
o Problems with the patient’s red cells (forward grouping)
o Problems with both the serum and cells.
INTERPRETATION OF REVERSE GROUPING TEST RESULTS ● The unexpected reaction can be due to an extra positive reaction or a weak
BLOOD GROUP A1 CELLS B CELLS or missing reaction in the forward and reverse grouping.
A NEGATIVE POSITIVE ● All ABO discrepancies must be resolved prior to reporting a patient or donor
B POSITIVE NEGATIVE ABO group.
O POSITIVE POSITIVE
AB NEGATIVE NEGATIVE TECHNICAL ERRORS

20 | Page
 ● Technical errors can also cause ABO discrepancies. When a reaction in the serum grouping is weak or missing, a
● This includes errors in labeling the blood sample at the patient’s bedside or group I discrepancy should be suspected, because, normally,
in the laboratory; therefore, patient and sample identification are essential! RBC and serum grouping reactions are very strong (4+).
● Other errors include the failure to add reagents or the addition of incorrect One of the reasons for the missing or weak isoagglutinin is that
reagents or sample. Therefore, it is recommended that serum and antiserum the patient has depressed antibody production or cannot
be added first, then the patient or reagent red cells. produce the ABO antibodies.
● It is also recommended that results be recorded immediately to avoid Common populations with discrepancies in this group are:
transcription errors. (1) Newborns (the production of ABO antibodies is not
● In addition, contaminated reagents can cause errors in testing. Therefore, detectable until 4 to 6 months of age)
looking at all the reagent vials when performing ABO testing and during (2) Elderly patients (the production of ABO antibodies is
quality control testing is extremely important. depressed)
(3) Patients with a leukemia (e.g., chronic lymphocytic
leukemia) or lymphoma (e.g., malignant lymphoma)
demonstrating hypogammaglobulinemia
RESOLUTION
(4) Patients using immunosuppressive drugs that yield
● If the initial test was performed using RBCs suspended in serum or plasma, hypogammaglobulinemia
repeat testing the same sample using a saline suspension of RBCs can (5) Patients with congenital or acquired
usually resolve the ABO discrepancy. agammaglobulinemia or immunodeficiency diseases
● It is important to make sure that any and all technical factors that may have (6) Patients with bone marrow or stem cell transplantations
given rise to the ABO discrepancy are reviewed and corrected. (patients develop hypogammaglobulinemia from
● It is also essential to acquire information regarding the patient’s age, therapy and start producing a different RBC population
diagnosis, transfusion history, medications, and history of pregnancy. from that of the transplanted bone marrow)
● If the discrepancy persists and appears to be due to an error in specimen (7) Patients whose existing ABO antibodies may have been
collection or identification, a new sample should be drawn from the patient diluted by plasma transfusion or exchange transfusion
and the RBC and serum testing repeated.
● When a discrepancy is encountered, results must be recorded, but ABO subgroups Resolution of Common Group I
interpretation of the ABO type must be delayed until the discrepancy is Discrepancies
resolved. ● Obtaining the patient’s history may resolve this type
● If blood is from a potential transfusion recipient, it may be necessary to of discrepancy, such as a newborn sample that
administer group O–compatible RBCs before the discrepancy is resolved. would not have ABO antibodies in the serum until the
● In general, when investigating ABO discrepancies, it should be noted that child was 4 to 6 months of age.
RBC and serum grouping reactions are very strong (3+ to 4+); therefore, the ● If the history indicates an elderly individual, or the
weaker reactions usually represent the discrepancy. diagnosis indicates hypogammaglobulinemia, then
the best way to resolve this discrepancy is to
Categories of ABO Discrepancies
enhance the weak or missing reaction in the serum.
ABO discrepancies may be arbitrarily divided into four major categories: group I, o This is usually performed by incubating the
group II, group III, and group IV discrepancies patient serum with reagent A1 and B cells at
room temperature for approximately 15 to
GROUP I associated with unexpected reactions in the reverse grouping 30 minutes or by adding one or two drops
due to weakly reacting or missing antibodies more plasma or serum to the test.
more common than those in the other groups listed.

21 | Page
 o If there is still no reaction after o Include group O and autologous cells as
centrifugation, the serum-cell mixtures can controls. RBCs can also be pretreated with
be incubated at 4°C for 15 minutes. enzymes and retested with reagent antisera.
● An auto control and O cell control must always be ● The acquired B antigen arises when bacterial
tested concurrently with the reverse typing when enzymes modify the immunodominant blood group A
trying to solve the discrepancy, since the lower sugar (N-acetyl-D-galactosamine) into
temperature of testing will most likely enhance the D-galactosamine, which is sufficiently similar to the
reactivity of other commonly occurring cold group B sugar (D-galactose) and cross-reacts with
agglutinins (such as anti-I) that react with all adult anti-B antisera.
RBCs. ● This pseudo-B antigen is formed at the expense of
● The red cell results present a group O individual, and the A1 antigen and disappears after recovery.
the serum results present an AB individual. Since ● The reaction of the appropriate antiserum with these
serum problems are more common, it is more likely acquired antigens demonstrates a weak reaction,
that the serum immunoglobulins are decreased. often yielding a mixed-field appearance. Blood group
reagents of a monoclonal anti-B clone (ES4) strongly
agglutinate cells with the acquired B antigen.
● The pH of reagents containing ES4 has been
GROUP II ● unexpected reactions in the forward grouping due to weakly
lowered; consequently, only those cells with the
reacting or missing antigens. This group of discrepancies is
strongest examples of acquired B antigen react with
probably the least frequently encountered. Some of the
the antisera.
causes of discrepancies in this group include:
● Testing the patient’s serum or plasma against
● Subgroups of A (or B) may be present
autologous RBCs gives a negative reaction, because
• Leukemias may yield weakened A or B antigens,
the anti-B in the serum does not agglutinate the
and Hodgkin’s disease has been reported in some
patient’s RBCs with the acquired B antigen.
cases to mimic the depression of antigens found in
● The acquired B antigen is also not agglutinated when
leukemia.
reacted with anti-B that has a pH greater than 8.5 or
• The “acquired B” phenomenon will show weak
less than 6.40.
reactions with anti-B antisera and is most often
● Secretor studies can be performed when trying to
associated with diseases of the digestive tract
characterize the acquired B phenomenon.
(e.g., cancer of the colon).
● If the patient is in fact a secretor, only the A
substance is secreted in the acquired B
ABO subgroups Resolution of Common Group II phenomenon.
● The agglutination of weakly reactive antigens with ● Treating RBCs with acetic anhydride reacetylates the
the reagent antisera can be enhanced by incubating surface molecules, then markedly decreases the
the reactivity of the cells tested with anti-B.
test mixture at room temperature for up to 30 ● The reactivity of normal B cells is not affected by
minutes, which will increase the association of the treatment with acetic anhydride.
antibody
with the RBC antigen. Rare Group II Discrepancies
● Weakly reactive or missing reactions in RBC
● If it is still negative, incubate the text mixture at 4°C
grouping may be due to excess amounts of blood
for 15 to 30 minutes.
group–specific soluble (BGSS) substances present

22 | Page
 in the plasma, which sometimes occurs with certain O cells instead of the expected group of either B or
diseases, such as O.
carcinoma of the stomach and pancreas. ● Detecting a separate cell population may be easy or
● Excess amounts of BGSS substances will neutralize difficult, depending on what percentage of cells of the
the reagent anti-A or anti-B, leaving no unbound minor population are present.
antibody to react with the patient cells. ● Reactions from chimerism are typically mixed field.
● This yields a false-negative or weak reaction in the ● True chimerism, which occurs in twins, is rarely
forward grouping. found, and the two cell populations will exist
● Washing the patient cells free of the BGSS throughout the lives of the individuals. In utero
substances with saline should alleviate the problem, exchange of blood occurs because of vascular
resulting in correlating forward and reverse anastomosis.
groupings. ● As a result, two cell
● Antibodies to low-incidence antigens in reagent populations emerge, both of which are recognized as
anti-A or anti-B may also result in weakly reactive or self, and the individuals do not make anti-A or anti-B.
missing reactions in RBC grouping. ● Therefore, expected isoagglutinin are not present in
● It is impossible for manufacturers to screen reagent the reverse grouping, depending on the percentage
antisera against all known RBC antigens. of the population of red cells that exist in each twin. If
● It has been reported (although rarely) that this the patient or donor has no history of a twin, then the
additional antibody in the reagent antisera has chimera may be due to dispermy (two sperm
reacted with the corresponding low-incidence antigen fertilizing one egg) and indicates mosaicism. More
present on the patient’s RBCs. commonly, artificial chimeras occur, which yield
● This gives an unexpected reaction of the patient’s mixed cell populations as a result of:
cells with anti-A or anti-B, or both, mimicking the a. Blood transfusions (e.g., group O cells
presence of a weak antigen. given to an A or B patient)
● The best way to resolve this discrepancy is by b. Transplanted bone marrows or peripheral
repeating the forward type, using antisera with a blood stem cells of a different ABO type
different lot number. c. Exchange transfusions
● If the cause of the discrepancy is a low-incidence d. Fetal-maternal bleeding
antibody in the reagent antisera reacting with a
low-incidence antigen on the patient’s cells, the
GROUP III ● These discrepancies between forward and reverse
antibody probably will not be present in a different lot
number of reagent. groupings are caused by protein or plasma abnormalities
and result in rouleaux formation or pseudo agglutination,
● This is only seen when human source antiserum is
attributable to:
used.
● Most ABO reagents in use today are monoclonal (1) Elevated levels of globulin from certain disease states,
such as multiple myeloma, Waldenström’s
antibodies, and these reagents are free of
macroglobulinemia, other plasma cell dyscrasias, and
contaminating antibodies to low-incidence antigens.
certain moderately advanced cases of Hodgkin’s
lymphomas
Chimerism is defined as the presence of two cell populations (2) Elevated levels of fibrinogen
in a single individual. (3) Plasma expanders, such as dextran and
● It was discovered in twins (born to a group O mother polyvinylpyrrolidone
and group B father) who had a mixture of both B and (4) Wharton’s jelly in cord blood samples

23 | Page
 transplant
(3) Unexpected ABO isoagglutinin
RESOLUTION (4) Unexpected non-ABO alloantibodies
Rouleaux is a stacking of erythrocytes that adhere in a
coinlike fashion, giving the appearance of agglutination.
● It can be observed on microscopic examination. Cell RESOLUTION
grouping can usually be accomplished by washing ● Potent cold autoantibodies can cause spontaneous
the patient’s RBCs several times with saline. agglutination of the patient’s cells.
● Performing a saline replacement technique will free ● These cells often yield a positive direct Coombs’s or
the cells in the case of rouleaux formation in the antiglobulin test. If the antibody in the serum reacts with
reverse type. In this procedure, serum is removed all adult cells—for example, anti-I—the reagent A1 and B
and replaced by an equal volume of saline. In true cells used in the reverse grouping also agglutinate.
agglutination, RBC clumping will remain after the ● To resolve this discrepancy, the patient’s RBCs could be
addition of saline. incubated at 37°C for a short period, then washed with
● Rouleaux can be a nuisance in the laboratory, since saline at 37°C three times and retyped. If this is not
it is an in vitro problem observed during laboratory successful in resolving the forward type, the patient’s
testing. RBCs can be treated with 0.01 M dithiothreitol (DTT) to
● It is not an in vivo problem for the patient. disperse IgM-related agglutination.
● As for the serum, the reagent RBCs and serum can be
Cord blood samples received in the laboratory can also warmed to 37°C, then mixed, tested, and read at 37°C.
pose a problem in ABO testing, since cord cells may be The test can be converted to the antihuman globulin
contaminated with a substance called Wharton’s jelly, which phase if necessary. Weakly reactive anti-A or anti-B may
may cause the red cells to aggregate. not react at 37°C, which is outside their optimum thermal
● Washing cord cells six to eight times with saline should range.
alleviate spontaneous rouleaux due to Wharton’s jelly. ● If the reverse typing is still negative (and a positive result
● This substance is a viscous mucopolysaccharide material was expected), a cold auto absorption (patient cells with
present on cord blood cells, and thorough washing should patient serum) could be performed to remove the cold
result in an accurate ABO grouping. autoantibody from the serum.
● However, because testing is usually not performed on ● The absorbed serum can then be used to repeat the
cord serum (because the antibodies detected are usually serum typing at room temperature.
of maternal origin), reverse grouping may still not ● Unexpected ABO isoagglutinin in the patient’s serum
correlate with the RBC forward grouping. react at room temperature with the corresponding antigen
present on the reagent cells.
● Examples of this type of ABO discrepancy include A2 and
These discrepancies between forward and reverse groupings are A2B individuals, who can produce naturally occurring
GROUP IV
due to miscellaneous problems and have the anti-A1, or A1 and A1B, individuals who may produce
following causes: naturally occurring anti-H. Serum grouping can be
(1) Cold reactive autoantibodies in which RBCs are so repeated using at least three examples of A1, A2, B cells;
heavily coated with antibody that they spontaneously O cells; and an autologous control (patient’s serum mixed
agglutinate, independent of the specificity of the reagent with patient’s RBCs).
antibody. ● The specificity of the antibody can be determined by
(2) Patient has circulating RBCs of more than one ABO examining the pattern of reactivity (e.g., if the antibody
group due to RBC transfusion or marrow/stem cell

24 | Page
 agglutinates only A1 cells, it can most likely be identified (analogous to A2 cells) and a weak B antigen. The B antigen
as anti-A1). usually yields a weaker reaction with the anti-B
● The patient’s RBCs can be tested with Dolichos biflorus from random donors, with mixed-field agglutination typical of
subgroup B3reported in several cases. Weak
to confirm the presence of the ABO subgroup.
anti-B (present in the serum of most cis-AB individuals) leads to
● Dolichos biflorus will agglutinate cells of the A1 but not an ABO discrepancy in the reverse grouping.
the A2 phenotype. The serum of most cis-AB individuals contains a weak anti-B,
● Unexpected alloantibodies in the patient’s serum other which reacts with all ordinary B RBCs, yet not
than ABO isoagglutinin (e.g., anti-M) may cause a with cis-AB RBCs. A and B transferase levels are lower than
● discrepancy in the reverse grouping. those found in ordinary group AB sera. Various
● Reverse grouping cells possess other antigens in addition hypotheses have been offered to explain the cis-AB phenotype.
Many favors an unequal crossing over between
to A1 and B, and it is possible that other unexpected
the A and B gene with gene fusion and the formation of a new
antibodies present in the patient’s serum will react with gene. However, the banding pattern of the
these cells. distal end of the long arm of chromosome 9 representing the
● In this situation, an antibody identification panel should be ABO locus is normal. There have been other
performed with the patient’s serum. examples of cis-ABs that do not fit the above scenario. In these
● Once the unexpected alloantibodies are identified, examples, there was a point mutation at the
reagent A1 and B cells negative for the corresponding ABO locus, and an enzyme was produced that could transfer
both A-specific and B-specific sugars to the
antigen can be used in the reverse grouping.
precursor molecule. Many families have been reported in other
RARE GROUP IV DISCREPANCIES parts of the world, with a high incidence of cis-
AB being found in Japan.
Antibodies other than anti-A and anti-B may react to form
antigen-antibody complexes that may then adsorb
onto patient’s RBCs. One example is an individual who has an
RH-HR BLOOD GROUP SYSTEM
antibody against acriflavine, the yellow dye used
in some commercial anti-B reagents. The acriflavine-ant HISTORY OF THE RH SYSTEM
acriflavine complex attaches to the patient’s RBCs, causing
● Before 1939, the only significant blood group antigens recognized were
agglutination in the forward type. Washing the patient’s cells
three times with saline and then retyping those of the ABO system.
them should resolve this discrepancy. Cis-AB refers to the ● Transfusion medicine was thus based on matching ABO groups.
inheritance of both AB genes from one parent
carried on one chromosome and an O gene inherited from the LEVINE AND STETSON
other parent. This results in the offspring ● Described a hemolytic transfusion reaction in an obstetrical patient.
inheriting three ABO genes instead of two. The designation o After delivery of a stillborn infant, a woman required transfusions.
cis-AB is used to distinguish this mode of Her husband, who had the same ABO type, was selected as her
inheritance from the more usual AB phenotype in which the donor.
alleles are located on different chromosomes. The o After transfusion, the recipient demonstrated the classic symptoms
cisAB phenotype was first discovered in 1964, when a Polish
of an acute hemolytic transfusion reaction.
family was described in which the father was
group O, and the mother was group AB and gave birth to ● Subsequently, an antibody was isolated from the mother’s serum that
children who were all group AB. It was resolved by reacted both at 37C and at 20C with the father’s RBCs.
the fact that the A and B genes were inherited together and were ● It was postulated that the fetus and the father possessed a common factor
both on the same, or cis, chromosome; thus that the mother lacked.
the term cis-AB. RBCs with the cis-AB phenotype (a rare o While the mother carried the fetus, she was exposed to this factor
occurrence) express a weakly reactive A antigen and subsequently built an antibody that reacted against the

25 | Page
 transfused RBCs from the father, which resulted in the hemolytic o Now it is known that “d” represents the absence of D antigen.
transfusion reaction. o The phenotype (blood type observed during testing) of a given RBC
is defined by the presence of D, C, c, E, and e expression.
LANDSTEINER AND WIENER
● According to the Fisher-Race proposal, each person inherits a set of Rh
● Reported on an antibody made by guinea pigs and rabbits when they were
genes from each parent (i.e., one D or d, one C or c, and one E or e).
transfused with rhesus monkey RBCs a year after Levine and Stetson’s o Because Rh genes are codominant, each inherited gene expresses
discovery.
its corresponding antigen on the RBC.
● This antibody, which agglutinated 85 percent of human RBCs, was named
o The combination of maternal and paternal haplotypes determines
Rh. one’s genotype (the Rh genes inherited from each parent) and
● Demonstrated that the agglutinin that had caused the hemolytic transfusion
dictates one’s phenotype (the antigens expressed on the RBC that
reaction and the antibody described by Landsteiner and Wiener appeared to
can be detected serologically).
define the same blood group.
● An individual’s Rh phenotype is reported as DCE rather than CDE because
o Many years later it was recognized that the two antibodies were Fisher postulated that the C/c locus lies between D/d and E/e loci. This
different.
information is based on frequencies of the various gene combinations.
o However, the name Rh was retained for the human-produced
● Rh-positive person exhibiting a deletion phenotype such as these is written
antibody, and the anti-rhesus antibody formed by the animals was
-De or -DE, CD- or cD-, or -D-, respectively.
renamed anti-LW in honor of those first reporting it (Landsteiner and o The last is sometimes referred to as a double deletion.
Wiener).
o The person expressing no Rh antigens on the RBC is said to be
● Further research resulted in defining Rh as a primary cause of hemolytic
Rhnull, and the phenotype may be written as –––/–––.
disease of the newborn (HDN, also called erythroblastosis fetalis) and a ● Weakened expression of all Rh antigens of an individual has also been
significant cause of hemolytic transfusion reactions.
reported.
● By the mid-1940s, five antigens made up the Rh system. Today the Rh blood
o These individuals are said to have the Rhmod phenotype, and
group system is made up of nearly 50 different specificities
there is no unique way of indicating this using the Fisher-Race
NOMENCLATURES OF THE RH SYSTEM terminology
The terminologies used to describe the Rh system are derived from four sets of
investigators:
● Two of the terminologies are based on the postulated genetic mechanisms WIENER: THE RH-HR TERMINOLOGY
of the Rh system. ● Wiener believed that the gene responsible for defining Rh actually produced
● The third terminology describes only the presence or absence of a given an agglutinogen that contained a series of blood factors.
antigen. ● According to Rh-Hr terminology, this Rh gene produces at least three factors
● The fourth is the result of the combined efforts of the International Society of within an agglutinogen.
Blood Transfusion (ISBT) Working Party on Terminology for Red Cell ● The agglutinogen may be considered the phenotypic expression of the
Surface Antigens. haplotype. Each factor is an antigen recognized by an antibody. Antibodies
can recognize single or multiple factors (antigens).
Fisher-Race: The DCE Terminology
● In the early 1940s, Fisher and Race were investigating the antigens found
on human RBCs, including the newly defined Rh antigen. ● When describing an agglutinogen, the uppercase R denotes the presence
o They postulated that the antigens of the system were produced by of the original factor, the D antigen.
three closely linked sets of alleles. ● The lowercase r indicates the absence of the D antigen.
● Each gene was responsible for producing a product (or antigen) on the RBC ● The presence of uppercase C is indicated by a one (1) or a single prime
surface. Each antigen and corresponding gene were given the same letter (′).
designation (when referring to the gene, the letter is italicized). ● Lowercase c is implied when there is no 1 or ′ indicated
● Fisher and Race named the antigens of the system D, d, C, c, and E and e.

26 | Page
 ● A minus sign preceding the number indicates that the antigen was tested for
but was not present.
● When referring to the Rh antigens (or factors) in Wiener nomenclature,
o The phenotype D CE c e or DccEe or R2 r would be written
o the single prime (′) refers to either C or c,
RH:1,-2,3,4,5.
o The double prime (′′) to either E or e.
● When referring to a gene, an allele, or a haplotype, the symbols are
o If the r precedes the h (i.e., rh′ or rh′′), this refers to the C or E italicized, followed by a space or asterisk, and then the numbers of the
antigens, respectively.
specificities are separated by commas.
o When the h precedes the r, this refers to either the c (hr′) or e (hr′′)
● R1 or DCe would be RH 1,2,5.
antigen.
● Rh0 is equivalent to D. In the Wiener nomenclature, there is no designation PROPOSED GENETIC PATHWAYS
for the absence of D antigen.
MECHANISMS OF ANTIGEN PRODUCTION
ROSENFIELD AND COWORKERS: ALPHA/ NUMERIC TERMINOLOGY ● Two theories of Rh genetic control were initially postulated.
● As the Rh blood group system expanded, it became more difficult to assign o Wiener postulated that a single gene produces a single product that
names to new antigens using existing terminologies. contains separately recognizable factors.
● In the early 1960s Rosenfield and associates proposed a system that o In contrast, Fisher and Race proposed that the Rh locus contains
assigns a number to each antigen of the Rh system in order of its discovery three distinct genes that control the production of their respective
or recognized relationship to the Rh system. antigens.
o This system has no genetic basis, but simply demonstrates the ● It is currently accepted that only two closely linked genes located on
presence or absence of the antigen on the RBC. chromosome 1 control the expression of Rh; one gene (RHD) codes for the
o A minus sign preceding a number designates absence of the presence or absence of the D polypeptides and the second gene (RHCE) for
antigen. either RHCe, RHcE, RHce, or RHCE polypeptides.
● For the five major antigens, D is assigned Rh1, C is Rh2, E is Rh3, c is Rh4, ● Another gene (RHAG), which resides on chromosome 6, produces a
and e is Rh5. Rh-associated glycoprotein that is very similar in structure to the Rh proteins
● For RBCs that type D C E c negative, e negative, the Rosenfield designation and within the RBC membrane forms complexes with the Rh polypeptides.
is Rh: 1, 2, 3, 4, 5. If the sample was not tested for e, the designation would RHAG is called a coexpressor and must be present for the
be Rh: 1, 2, 3, 4. All Rh system antigens have been assigned a number. ● successful expression of the Rh antigens but by itself does not express any
of the Rh antigens.
INTERNATIONAL SOCIETY OF BLOOD TRANSFUSION: NUMERIC
● The Rh genes are inherited as codominant alleles. Offspring inherit one Rh
TERMINOLOGY
haplotype from each parent. In rare instances, individuals express no Rh
● The ISBT adopted a six-digit number for each authenticated blood group antigens on their RBCs. These individuals are said to have the Rhnull
specificity. phenotype.
o The first three numbers represent the system and the remaining
three the antigenic specificity.
o The number 004 was assigned to the Rh blood group system, and
then each antigen assigned to the Rh system was given a unique
BIOCHEMISTRY OF THE RH ANTIGENS
number to complete the six-digit computer number.
o 001 was assigned to ------- ● Rh antigens are nonglycosylated protein.
o Therefore, D is RH1, C is RH2, and so forth. ● This means that there are no carbohydrates attached to the protein.
● The phenotype designation includes the alphabetical symbol that denotes ● The Rh antigens are transmembrane polypeptides and are an integral part of
the RBC membrane.
the blood group, followed by a colon and then the specificity numbers of the
antigens defined.

27 | Page
 ● The gene products of RHD and RHCE are remarkably similar in that both ● Is required when testing donor bloods
encode for proteins composed of 417 amino acids that traverse the cell ● Blood for transfusion is considered Rhpositive if either the D or the weak-D
membrane 12 times and that their sequence differs by only 44 base pairs. test is positive.
● Any donor blood sample that is typed Rh0(D)-negative by the slide or rapid
WEAK D: VARIATIONS OF THE RH0 (D) ANTIGEN EXPRESSION
tube method must be tested further by an indirect antihuman globulin
RBCs carrying the weaker D antigen have historically been referred to as having the
technique.
Du type.
o Now they are referred to as expressing weak D and are considered NOTE:
Rh-positive. ● If both tests are negative, the donor sample is considered Rh-negative.
o Three different mechanisms have been described that can explain the ● If the donor sample tests positive in any phase of Rh0(D) testing, the
weakened expression of the D antigen: sample is considered Rh-positive.

1. GENETIC WEAK D
● Inheritance of D genes that code for a weakened expression of the D
● Determining the Rh0(D) status (including weak-D status) of obstetric patients
antigen.
is critical.
● The D antigens expressed appear to be complete but few in number. On a
● All Rh-negative, weak D–negative obstetric patients are candidates for Rh
molecular level, these quantitative differences in D expression are attributed
immune globulin (RhIg) (a drug injected to prevent Rh-negative individuals
to mutations of the Rh polypeptide.
who are exposed to Rh-positive RBCs from developing antiD).
● Inheritance of these genes can be tracked vertically from one generation to
o Likewise, when the mother is Rh-negative and the newborn is typed
the next and are seen most frequently in blacks. The genetic weak D is rare
Rh-negative, the weak-D status of the newborn must be determined
and seldom found in whites.
to assess the likelihood of maternal sensitization and the need for
2. C TRANS Rh immune globulin prophylaxis for the mother.
o If a newborn’s cells are coated with maternal IgG anti-D in utero,
● Position effect or gene interaction effect
very few D antigen sites are available to react with reagent anti-D
● In individuals showing the gene interaction weak D, the allele carrying D is
(termed “blocking phenomena”).
trans (or in the opposite haplotype) to the allele carrying C
● Elution of the sensitizing antibody (removing the antibody) and identifying it
● for example, Dce/dCe.
as anti-D will verify that the infant’s RBCs are D-positive.
● The Rh antigen on the RBC is normal, but the steric arrangement of the C
antigen in relationship to the D antigen appears to interfere with the
expression of the D antigen.
DETECTION OF RH ANTIBODIES AND ANTIGENS
3. PARTIAL D (D MOSAIC)
Rh Antibodies
● One or more of the D epitopes within the entire D protein is either missing
● Although the Rh system was first recognized by saline tests used to detect
and/or is altered.
IgM antibodies, most Rh antibodies are IgG immunoglobulins and react
● Cells with a partial-D antigen usually type weaker than expected or may not
optimally at 37 degrees C or after antiglobulin testing.
react at all when routine procedures are used with most commercial anti-D
● Rh antibodies are usually produced following exposure of the individual’s
reagents.
immune system to foreign RBCs, through either transfusion or pregnancy.
Rh antigens are highly immunogenic; the D antigen is the most potent.
● Exposure to less than 0.1 mL of Rh-positive RBCs can stimulate antibody
production in an Rh-negative person.
● IgG1, IgG2, IgG3, and IgG4 subclasses of Rh antibodies have been
reported. IgG1 and IgG3 are of the greatest clinical significance because the
DETERMINATION OF D STATUS

28 | Page
 reticuloendothelial system rapidly clears RBCs coated with IgG1 and IgG3 control reacted, the test result was invalid and had to be repeated using a
from the circulation. different technique or reagent anti-D. The major advantages of high-protein
● As with most blood group antigen sensitization, IgM Rh antibodies are anti-D reagents are reduced incubation time and the ability to perform
formed initially, followed by a transition to IgG. weak-D testing and slide typing with the same reagent.
● Rh antibodies do not bind complement. For complement to be fixed (or the ● In the late 1970s, scientists chemically modified the IgG anti-D molecule by
complement cascade activated), two IgG immunoglobulins must attach to an breaking the disulfide bonds that maintain the antibody’s rigid shape.
RBC in close proximity. Rh antigens (to which the antibody would attach) are ● Monoclonal antibody reagents have become available more recently. These
not situated on the RBC surface this closely reagents are derived from single clones of antibody-producing cells.
● Therefore, when an Rh antibody coats the RBCs, intravascular,
complement-mediated hemolysis does not occur. CLINICAL CONSIDERATIONS
● RBC destruction resulting from Rh antibodies is primarily extravascular. TRANSFUSION REACTIONS
● Because Rh antibodies are primarily IgG and can traverse the placenta and ● Rh antigens are highly immunogenic. The D antigen is the most
because Rh antigens are well developed early in fetal life, Rh antibodies immunogenic antigen outside the ABO system.
formed by Rh-negative pregnant women do cross the placenta and may coat ● Circulating antibody appears within 120 days of a primary exposure and
fetal RBCs that carry the corresponding antigen. within 2 to 7 days after a secondary exposure.
● This results in the fetal cells testing positive by the direct antiglobulin test ● Rh-mediated hemolytic transfusion reactions, whether caused by primary
and in HDN, the coated fetal cells are removed prematurely from the fetal. sensitization or secondary immunization, usually result in extravascular
Until the discovery of Rh immune globulin, anti-D was the most frequent destruction of immunoglobulin-coated RBCs.
cause of HDN. ● The transfusion recipient may have an unexplained fever, a mild bilirubin
elevation, and a decrease in hemoglobin and haptoglobin.
RH ANTIGEN TYPING REAGENTS
● The direct antihuman globulin test is usually positive, and the antibody
● The reagents used to type for D and for the other Rh antigens may be
screen may or may not demonstrate circulating antibody.
derived from a variety of sources.
● The reagents may be high-protein–based or low-protein–based, HEMOLYTIC DISEASE OF THE NEWBORN (HDN)
saline-based, chemically modified, monoclonal, or blends of monoclonals. ● HDN caused by Rh antibodies is often severe because the Rh antigens are
● Saline reactive reagents, which contain IgM immunoglobulin, were the first well developed on fetal cells, and Rh antibodies are primarily IgG, which
typing reagents available to test for the D antigen. readily cross the placenta.
o Saline anti-D has the advantage of being lowprotein–based and ● After years of research, a method was developed to prevent susceptible
can be used to test cells that are coated with IgG antibody. The (Rh0 D-negative) mothers from forming antiD, thus preventing Rh0(D) HDN.
primary disadvantages of saline typing reagents are their limited ● Rh-immune globulin, a purified preparation of IgG anti-D, is given to a
availability, cost of production, and lengthy incubation time. D-negative woman during pregnancy and following delivery of a D positive
Because saline anti-D is an IgM immunoglobulin, it cannot be used fetus. Rh-immune globulin is effective only in preventing anti-D HDN
for weak-D typing.
● In the 1940s, high-protein anti-D reagents were developed. RH DEFICIENCY SYNDROME: RHNULL AND RHMOD
o Human plasma containing high-titer D-specific antibody is used as ● It is the rare individual who fails to express any Rh antigens on the RBC
the raw material. surface or exhibits a severely reduced expression of all Rh antigens. The
o Potentiators of bovine albumin and macromolecular additives such individuals who lack all Rh antigens on their RBCs are said to have the
as dextran or polyvinylpyrrolidone are added to the source material Rhnull syndrome, which can be produced by two different genetic
to optimize reactivity in the standard slide and rapid tube tests. mechanisms:
o The presence of potentiators and the higher protein concentration, ● In the regulator-type Rhnull syndrome, there is a mutation in the RHAG
however, increase the likelihood of false positive reactions. gene. This results in no Rh polypeptides or RHAG antigen expression on the
● To assess the validity of the high-protein Rh typing results, a control reagent RBCs, even though these individuals usually have a normal complement of
was manufactured and had to be tested in parallel with each Rh test. If the

29 | Page
 RHD and RHCE genes. These individuals can pass the normal RHD and ● The f antigen is expressed on the RBC when both c and e are present on
RHCE genes to their children. the same haplotype (i.e., cis position); it has been called a compound
● In the second type of Rhnull syndrome (the amorphic type), there is a antigen.
mutation in each of the RHCE genes and a deletion of the RHD gene. The ● Phenotypically, the following samples appear the same when tested with the
RHAG gene is normal. five major Rh antisera: Dce/DCE and DcE/DCe. However, when tested with
● Individuals with the Rhnull syndrome demonstrate a mild compensated anti-f, only the former reacts.
haemolytic anemia, reticulocytosis, stomatocytosis, a slight-to-moderate ● Anti-f has been reported to cause HDN and transfusion reactions.
decrease in haemoglobin and hematocrit levels, an increase in hemoglobin
rhi (Ce)
F, a decrease in serum haptoglobin, and possibly an elevated bilirubin level.
● When transfusion of individuals with Rhnull syndrome is necessary, only ● Similar to f, rhi is present when C and e are in the cis configuration, has
Rhnull blood can be given. been called a compound antigen, and is a single entity.
● A sample with the phenotype D C E c e can be either DcE/DCe or Dce/DCE.
Anti-rhi reacts only with Dce/DCe RBCs.
NOTE:
● Individuals of the Rhmod phenotype have a partial suppression of Rh
gene expression and exhibit features similar to those with the Rhnull G
syndrome ● G is an antigen that is present on most D-positive and all Cpositive RBCs. In
● However, the clinical symptoms are usually less severe and rarely the test tube, anti-G reacts as though it were a combination of anti-C plus
clinically remarkable. anti-D.
● 33 RBCs classified as Rhmod do not completely lack Rh or LW antigens. ● Was originally described in an rr person who received Dccee RBCs.
● Rhnull and Rhmod RBCs exhibit other blood group antigens; however, S, Subsequently, the recipient produced an antibody that appeared to be
s, and U antigen expression may be depressed. ● anti-D plus anti-C, which should be impossible because the C antigen was
● 34 Rhnull RBCs are negative for FY5. not on the transfused RBCs.

Rh:13, Rh:14, Rh:15, Rh:16 Rh:13, Rh:14, Rh:15, and Rh:16

UNUSUAL PHENOTYPES AND RARE ALLELES
● Define four different parts of the D mosaic, as it was originally described.
Cw o Although these parts are included in the partial-D categories II to
● Originally considered an allele at the C/c locus.
VII as defined by Tippett and Sanger, 25, 27 they are not directly
● Later studies showed that it can be expressed in combination with both C
comparable.
and c and in the absence of either allele.
● Now known that the relationship between C/c and Cw is only phenotypic and Hr0 Hr0
that
● Antithetical to the high-incidence antigen MAR. antigen present on all RBCs with the “common” Rh phenotypes (e.g., R1R1 , R2R2 ,
● Found in about 2 percent of whites and is very rare in blacks. rr).40 When RBCs phenotype as D--, the most potent antibody they make is often one
● Anti-Cw has been identified in individuals without known exposure to foreign directed against Hr0.
RBCs as well as after transfusion or pregnancy.
● Anti-Cw may show dosage (i.e., reacting more strongly with cells from Rh:23, Rh:30, Rh:40
individuals who are homozygous for Cw). Because of the low incidence of all low-frequency antigens associated with a specific category of partial-D. Rh:23
Cw, Cw antigen–negative blood is readily available. (also known as Wiel and Dw) is an antigenic marker for category Va partialD.41 Rh:30
f (ce)

30 | Page
(also known as Goa or Dcor) is a marker for category Iva partial-D.42 Rh:40 (also ● Many examples lacking all Cc or Ee often have an unusually strong D
known as Tar or Targett) is a marker for category VII. antigen expression, frequently called exalted D.
● The deletion phenotype is indicated by the use of a dash (–), as in the
Rh:33 following examples:
● The low-incidence antigen Rh:33 is associated with a rare variant of the R0
● DC–, Dc–, D–E, D--. The antibody made by D-- people is called anti–Rh 17
(Dce) gene called R0 Har. 44 R0 Har gene codes for normal amounts of c,
or anti-Hr0.
reduced amounts of e, reduced f, reduced Hr0, and reduced amounts of D
● A variation has been recognized within the deletion D--, called D••.
antigen. ● The D antigen in the D•• is stronger than that in DC–, D–E, Dc–, or D–e
● The D reactions are frequently so weak that the cells are frequently
samples but weaker than that of D-- samples.
mistakenly typed as Rh negative.
● A low-incidence antigen called Evans (Rh:37) accompanies the Rh structure
● To denote the weakened expression of an antigen in Fisher-Race of D•• cells.
nomenclature the letter is placed in parentheses. The R0 Har gene
● Transfusion of individuals with a Rh deletion or D•• phenotype is difficult if
expresses (D)c(e) and has been found in whites.
multiple antibodies are present; blood of a similar phenotype would be
Rh:32 required.
● Rh:32 is a low-frequency antigen associated with a variant of the R1
THE LW ANTIGEN
[D(C)(e)] gene that is called RN.
● LW systems.
● The C antigen and e antigen are expressed weakly. ● Anti-LW reacts strongly with most D-positive RBCs, weakly (sometimes not
● The D antigen expression is exaggerated or exalted. This gene has been
at all) with Rh-negative RBCs, and never with Rhnull cells.
found primarily in blacks.
● The independent segregation of LW from the Rh blood group genes was
e Variants established by a family study on a D-positive LW-negative woman; other
● It appears, especially in the black population, that the e antigen may exhibit family studies support this point.
the same mosaic quality described for D. Because of these variations, e ● There are three alleles at the LW locus: LWa , LWb , and LW (a silent allele).
typings can be unreliable. Persons lacking LW antigen altogether are LW/LW and express no LW on
● Among the variants at the e locus are hrs, hrB, and VS(es ), with a variant the RBCs.
R0 or r gene making e plus one or the other of these pieces. ● LWa is very common, and LWb is rare. When the Rhnull is present due to
● Such variants are usually recognized when they make antibodies that the suppression mechanism, the Rh and LW genes are not expressed on the
behave as anti-e, even though their RBCs type as e-positive with routine Rh RBC; however, the Rh and LW genes are normal and, when passed to
typing reagents. offspring, can be expressed normally.
● The amorphic Rhnull individual inherits the rr genes, which do not express
V, VS Rh antigens; therefore, LW genes cannot be expressed.
● The V(ceS) antigen is found in about 30 percent of randomly selected ● Anti-LW usually reacts more strongly with D-positive RBCs than with
American blacks. D-negative adult RBCs.
● In selected individuals, it appears to be the serologic counterpart of f ● A weak anti-LW may react only with D-positive RBCs, and enhancement
because it is present when c is cis with eS. techniques may be required to demonstrate its reactivity with Dnegative
● (The VS(eS) antigen is also relatively common in blacks with the VS cells.
antibody, reacting with all V-positive RBCs and additionally with r′s RBCs.48) ● Anti-LW reacts equally well with cord cells regardless of their D type.
Although the relationship of V to VS remains somewhat less than clear, both ● This is an important characteristic to remember when trying to differentiate
are markers associated with the black population. anti-LW from anti-D.
● Also, anti-LW more frequently appears as an autoantibody, which does not
Deletions
present clinical problems.
● There are very uncommon phenotypes that demonstrate no Cc and/or Ee
reactivity.

31 | Page
 APPENDICES
R2r DcE/dce Rh: 1, -2, 3, 4, 5, 9
Wiener Fisher-Race Wiener Fisher-Race R2R2 DcE/DcE Rh: 1, -2, 3, 4, -5, 2
Ro Dce r dce RARER GENOTYPES
R1 DCe r’ dCe r’r dCe/dce Rh: -1, 2, -3, 4, 5, 1
R2 DcE r” dcE r’r’ dCe/dCe Rh: -1, 2, -3, -4, 5, 0.01
Rz DCE R^y dCE r”r dcE/dCe Rh: -1, -2, 3, 4, 5, 1

r”r” dcE/dcE Rh: -1, -2, 3, 4, -5, 0.03

ISBT FISHER-RACE WIENER ROSENFIELD R0r Dce/dce Rh: 1, -2, -3, 4, 5, 2
004001 D Rho Rh1 R0R0 Dce/Dce Rh: 1, -2, -3, 4, 5, 0.1
004002 C rh’ Rh2 ryr dCE/dce Rh: -1, 2, 3, 4, 5, rare
004003 E rh” Rh3

004004 c hr’ Rh4

004005 e hr” Rh5

COMMON Rh TYPES BY THREE NOMENCLATURES

COMMON GENOTYPE

WIENER FISHER-RACE ROSENFIELD FREQUENCY (%)

(approx., White)

R1r DCe/dce Rh: 1,2, -3, 4, 5 33

R1R1 DCe/DCe Rh: 1, 2, -3, -4, 5, 18

rr dce/dce Rh: -1, -2, -3, 4, 5, 15

R1R2 DCe/DcE Rh: 1, 2, 3, 4, 5, 11

32 | Page
 MODULE 4: PRE-TRANSFUSION TESTING
MLS 18, March 17, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
The crossmatch is an actual mixing of the recipient and donor’s blood to
Legend: insure in vitro compatibility.
Transcription Bullet Only a part of compatibility testing
Notes/Module Packet Bullet Check for any untoward reaction between the donor and recipient’s
PPT blood
MODULE OUTLINE
I. Compatibility Testing SEROLOGIC CROSSMATCH
II. Antibody Screening Two main functions of the serologic crossmatch test:
III. Selection of Appropriate Donor Units o It is a final check of ABO compatibility between donor and patient.
IV. Routine Blood Bank Laboratory o It may detect the presence of an antibody in the patient’s serum
that will react with antigens on the donor RBCs but that was not
LEARNING OUTCOMES detected in antibody screening because the corresponding antigen
1. Define compatibility test. was lacking from the screening cells.
2. Identify tests included in compatibility test. Consists of mixing the patient’s serum with the donor’s RBC
3. Describe appropriate methods for proper patient identification in
sample collection.
4. Outline the procedure for testing donor and patient specimens.
Major Crossmatch
An in-vitro determination that combines recipient’s plasma with a
5. Compare and contrast major and minor crossmatch procedures.
5% cell suspension of the donor’s cells.
6. Resolve incompatibilities in the crossmatch.
This test is performed at different phases in the same manner as the
7. State the limitations of compatibility testing procedures.
antibody screen and antibody identification.
8. Discuss future issues of compatibility testing.
The immediate spin crossmatch is the first phase of the major
crossmatch procedure.
o This procedure provides a determination of compatibility
COMPATIBILITY TESTING
that encompasses ABO incompatibility.
Series of procedures designed to ensure the safety of blood for
The final phase is the anti-human globulin (AHG) test.
transfusion.
o Antibodies present in the recipient that are reactive at the
Vital part of the blood bank laboratory. AHG phase are most likely to cause an in vivo reaction
o The determination of compatibility of a unit of blood for transfusion
is performed each time a unit of red blood cells is to be transfused.
Minor Crossmatch
Included in the compatibility test is a crossmatch.
Performed by mixing donor plasma with cells from the recipient.
The minor crossmatch is no longer performed.
CROSSMATCHING/CROSSMATCH TESTING The majority of cellular products transfused are red blood cells
The crossmatch test is the testing of the patient’s serum with the donor rather than whole blood.
RBCs, including an antiglobulin phase or simply an immediate spin phase
to confirm ABO compatibility.
 The volume of donor plasma transfused with red cell products is Adding ethylenediaminetetraacetic to the test system reportedly
very small and not significant enough to induce a reaction with eliminates some of the false-positive reactions, thus improving the
recipient cells sensitivity of the immediate spin crossmatch.
2-3 drops of serum sample with a drop of a wash of 2-5% RBC
Compatibility test results: suspension; observe presence of hemolysis in the centrifuge
Compatibility test procedures are performed and the results accomplished by mixing the recipient’s serum with the donor’s RBC
assessed. and centrifuging immediately (no incubation)
Compatible units of red cells will have no agglutination or hemolysis
at any phase of testing. Type-And-Screen Procedure
Tests that have either hemagglutination or hemolysis noted are Involves testing the patient’s blood sample for ABO, Rh, and
incompatible and should not be transfused without further clinically significant unexpected antibodies.
evaluation. The patient sample is then stored in the blood bank refrigerator for
The cause of the incompatibility should be determined and future crossmatch if blood is needed for transfusion.
documented. The type-and-screen procedure has application for patients
undergoing many elective procedures who may need blood;
because the process may not always require transfusion, the
Objective of testing: to select donor units that can provide maximal crossmatch is not performed until necessary.
benefit to the patient, which should be kept in mind when developing
the test protocol.
37°C Incubation with the use of enhancement media:
Enhancement Media – there is the option to use 22% albumin, low
Conventional Crossmatching ionic saline solution, or polyethylene glycol
Three Phases: Immediate Spin Crossmatch, 37°C Incubation with
the use of enhancement media, Antiglobulin Crossmatch Antiglobulin Crossmatch
The antiglobulin crossmatch procedure begins in the same manner
Immediate Spin Crossmatch as the immediate spin crossmatch, continues to a 37°C incubation,
This is accomplished by mixing the recipient’s serum with donor and finishes with an antiglobulin test.
RBCs and centrifuging immediately. Several enhancement media may be applied to boost antigen-
Absence of hemolysis or agglutination indicates ABO compatibility. antibody reactions
The immediate spin does not detect all ABO incompatibilities o These may include albumin, low ionic strength solution
False reactions may be seen in: (LISS), polyethylene glycol, and polybrene
o The presence of other immediate spin-reactive antibodies For greatest sensitivity, an antihuman globulin (AHG) reagent
(e.g., autoanti-I) containing both anti-IgG and anticomplement may be selected for
o Patients with hyperimmune ABO antibodies the final phase of this crossmatch method.
o May be seen when the procedure is not performed correctly An autocontrol, consisting of the patient’s own cells and serum, may
(e.g., delay in centrifugation or reading) be tested in parallel with the crossmatch test.
o When rouleaux is observed Used to detect (unagglutinated IgG) antibodies that were not
o When infants’ specimens are tested. detected with 37°C Incubation and Immediate spin crossmatch

#MLSWATER2022 Page 2 of 16
 Computer Crossmatch Blood Sample Collection and Labelling
Uses computer technology that is developed and increases
sensitivity of tests Acceptable collection tubes for pre-transfusion testing are:
As computer technology has developed and the sensitivity of Plain Red Tube (No Anticoagulant; No Serum Separator)
antibody detection has increased, a process for computer or Yellow Top Tube (Acid Citrate Dextrose or ACD, Formula B)
electronic crossmatch has been developed. Pink or Purple Top Tube (EDTA)
The electronic crossmatch is a viable option when there is no Blue Top Tube (Sodium Citrate)
current or previous history of clinically significant antibodies.
One of the primary advantages is maximum utilization of blood Historically, the use of anticoagulant for crossmatching was
supply. discouraged, since the binding of calcium inhibited the activation of
When the electronic crossmatch is used with appropriate patients, complement in the compatibility test.
wastage of units by holding units for specific patients is significantly o The clinical significance of these complement-binding
reduced. antibodies has been determined insignificant. Hence, the
Many believe that the computer crossmatch is safer than the use of anticoagulated samples has become acceptable.
immediate spin because of the integrity of the computer software Anticoagulated samples are readily available for crossmatching in an
to detect ABO incompatibility between the sample submitted for emergency situation since the time required for a sample to clot
pretransfusion testing and the donor unit. prior to processing may delay testing and treatment.
The computer crossmatch compares recent ABO serologic results o In addition, the formation of fibrin that may interfere with
and interpretations on file for both the donor and the recipient testing is less likely when using an anticoagulated specimen.
being matched and determines compatibility based on this The sample should not be contaminated with IV solutions
comparison. o A sample may be collected from an infusion line only if the
Advantages of the Electronic Crossmatch: line is flushed with saline and a volume of blood equal to
a. More efficient management of blood inventory twice the test volume is withdrawn and discarded prior to
b. Less wastage of blood components due to outdate obtaining the sample.
c. More efficient use of technician time The collection process should employ a needle of sufficient size to
d. Increase in staffing flexibility avoid hemolyzing the red cells in the sample.
e. Smaller recipient sample required o The presence of hemolysis in plasma of the original sample
f. Potential for a centralized transfusion service data bank may mask hemolysis resulting from an antigen-antibody
accessible by all interested parties interaction in the testing process.
g. annual savings Lipemic serum may also provide an environment that is difficult for
h. Reduced sample requirements evaluation of serological reactions.
Reduced expenses o At times it may be necessary to accept test samples with
i. Reduced handling of biological materials some amount of hemolysis or lipemia.
j. Elimination of false reactions associated with the immediate o Each institution should establish criteria and procedures for
spin crossmatch. acceptable samples
Individual institutions should establish the maximum age for pre-
transfusion testing samples.

#MLSWATER2022 Page 3 of 16
 Patient specimen free transfusion testing begins with properly These two independent identifiers should be confirmed at the
labelled, properly collected and identified patient specimen; patient time of collection and the blood sample labeled with this unique
identification is first in compatibility testing information:
Proper identification of the patient is imperative; patient must have the date of collection
a wristband with the identification information, tube should be The phlebotomist’s signature.
labelled on the bedside immediately after the sample was drawn This labeling must take place at the bedside.
Label should include the date, time of collection, name of the
person collected, and sample should not be collected into a Most institutions use hospital identification bands to identify
previously labeled tube. patients
During collection serum is a preferred specimen for compatibility o Past records should be compared to current test results.
testing, hemolysis should be avoided, and blood should not be The requirement further states that the current ABO and Rh
drawn from intravenous site. type must be compared to results obtained within the past
Stop the infusion for 5-10 minutes before collecting blood and then 12 months.
the lye should be flush with normal saline and first 5-10ml blood lye o Discrepancies are investigated and resolved before any
should be discarded. units are issued for transfusion.
o Red top for serum sample The AABB Standards require that all donor blood have the ABO and
o Yellow, pink and purple top used for collecting from donor Rh confirmed from an attached segment prior to transfusion of any
Fresh sample (not older than 72 hours) should be the best option whole blood or red blood cell component.
used for compatibility testing if patient has previously undergone o The standards do not require that the institution issuing the
transfusion or transfusion history is unknown. blood for transfusion perform a test for the confirmation of
o Also, the same for pregnant women the weak D antigen
For sample storage, EED standards states that patient sample According to the AABB, patient samples must be tested for ABO, Rh,
should be stored between 1° C and 6° Celsius for at least 7 days and unexpected antibodies to red cell antigens prior to transfusion
following transfusion. of red cells.
Lipemic samples or lipemia can interfere with the testing o The weak D test is not required, but the antibody screen
Hemolyzed sample should also be avoided must include “incubation at 37°C preceding an antiglobulin
test using reagent red cells that are not pooled.”
AABB Standards for Blood Bank and Transfusion Services, 25th Edition, The compatibility test procedure is only applicable for red cell
states that a recipient sample must be: products that are to be transfused
a. Collected within three (3) days of the scheduled transfusion if o Plasma based products such as cryoprecipitate, platelet
the patient has been transfused or pregnant in the preceding concentrates, and frozen plasma contain virtually no red
three (3) months or if the history is uncertain or unavailable. cells and require no compatibility testing.
The day of collection is labeled as day zero (0). o These components have been screened for antibodies and
b. These patient samples, as well as a segment from any red cell infectious diseases. Hence, they are transfused as ABO-
containing component, are all refrigerated and retained for at compatible.
least seven (7) days after transfusion. o Platelet-pheresis products and granulocyte concentrates
may contain some red cells, but require crossmatching only
The AABB designates that the request for blood and blood products if more than 2 ml of red cells are present.
contain sufficient information to uniquely identify the patient. This
includes a minimum of two independent identifiers:
#MLSWATER2022 Page 4 of 16
 PROBLEM SOLVING INCOMPATIBLE CROSSMATCHES o The procedure must include a system to ascertain that the
autologous units are administered before any allogeneic units
PROBLEM are issued.
POSSIBLE CAUSE RESOLUTION o The administration of allogeneic units before the stored
ENCOUNTERED
ABO typing Patient identification Repeat the ABO autologous units presents a liability risk for the administering
error group of the institution.
Sample identification recipient
error Recheck the label COMPATIBILITY TESTING OF NEONATES UNDER 4 MONTHS OF AGE
Choice of unit of of the selected unit Neonates require special consideration
incompatible ABO Recollect a sample Antibodies present have originated from the mother since the
group from the recipient neonate is unable to produce their own antibodies.
Unexpected Cold alloantibody Test with panel Neonates actually have the antibody of their mother
antibodies when Cold autoantibody cells and Initial pretransfusion testing uses the neonate’s red cells for ABO
antibody screen is anti-A1 in an A2 determine clinical and Rh typing.
negative at AHG individual significance of Reverse grouping is not performed on neonates due to the lack of
antibody antibody production.
Prewarm Antibody screen may be performed using either the infant or
compatibility maternal plasma.
testing If the initial antibody screen is negative, there is no need to perform
Test plasma of additional antibody screens or crossmatches on the neonate during
recipient with A2 this admission, so long as ABO-compatible and Rh identical red cell
cells products are administered.
Positive AHG phase If the antibody screen is positive, the antibody must be identified
Positive DAT in donor Perform DAT test
of compatibility and antigen negative units administered for assured compatibility.
Antibody to low on donor
test Additionally, an antiglobulin crossmatch should be performed and
incidence antigen in Choose alternate
only compatible units released.
recipient with antigen unit for
Crossmatching of all units needs to be continued until the maternal
positive cells in donor compatibility test
antibody is no longer detectable in the infant’s plasma.
Units of blood are crossmatched and divided since only small
COMPATIBILITY TESTING FOR AUTOLOGOUS TRANSFUSION quantities are administered to neonates.
Autologous donation is the process of collecting units of blood and Blood bags with multiple satellite bags or “pedi-packs” are
storing them for transfusion to the original donor commercially available for this purpose.
o This procedure is most often used for patients anticipating o This further reduces the possibility of incompatibility or
elective surgery. disease transmission
o Procedures to ensure that the collected units are transfused CMV negative units for use specifically in neonates may be obtained
to the intended recipient are established by the transfusing from the component source
institution.
o This procedure may be computerized with all records retained
electronically.

#MLSWATER2022 Page 5 of 16
 CAUSES OF POSITIVE RESULTS IN THE SEROLOGIC CROSSMATCH Goal of Antibody Screening
Incorrect ABO grouping of the patient or donor. o To detect as many clinically significant antibodies as
An alloantibody in the patient’s serum reacting with the possible
corresponding antigen on donor RBCs. o To detect as few insignificant
An autoantibody in the patient’s serum reacting with the o To detect the red cell antibody other than anti a and anti b
corresponding antigen on donor RBCs.
Prior coating of the donor RBCs with protein, resulting in a positive SELECTION OF APPROPRIATE DONOR UNITS
antihuman globulin test. The first choice for transfusion is blood and blood components of
Abnormalities in the patient’s serum. the patient’s own ABO and Rh group. This is defined as ABO group–
Contaminants in the test system specific.
When blood and blood components of the patient’s ABO blood
ANTIBODY SCREEN group are not available or some other reason precludes their use,
The recipient’s serum or plasma must be tested for clinically units selected must lack any antigen against which the recipient has
significant unexpected antibodies. a clinically significant antibody.
Usually done or is part of the antibody testing When a recipient must be given blood of a different ABO group,
The object of the antibody screening test is to detect as many only packed RBCs can be given.
clinically significant unexpected antibodies as possible. Whole blood cannot be administered in these situations because
In general, “clinically significant unexpected antibody” refers to incompatible, preformed ABO antibodies are present in the whole-
antibodies that are reactive at 37°C or in the antihuman globulin blood plasma.
test and are known to have caused a transfusion reaction or o Ex. Group A whole blood cannot be transfused into a group
unacceptably short survival of transfused RBCs AB recipient, because the plasma of the group A whole
Detection of unexpected antibodies is important for the selection of blood has anti-B antibodies present.
donor RBCs that will have the best survival rate in the patient’s Group O packed RBCs can be safely used for all patients; however,
circulation and reduce the risk of hemolytic transfusion reaction. conservation of a limited supply of group O blood should dictate its
Antibody screening or antibody panels are used use for recipients of other ABO types only in special circumstances.
If ABO group–specific blood is not available or is in low supply,
Antibody screening tests should demonstrate the presence of all
alternative blood groups are chosen
potentially clinically significant alloantibodies in the recipient’s
Rh-negative blood can be given to Rh-positive patients; however,
serum or plasma and indicate the need for further studies.
good inventory management should conserve this limited resource
All antibodies encountered in the screening test must be identified
for use in Rh-negative recipients.
to determine potential clinical significance and to decide whether
o But if the Rh-negative unit is near expiration, the unit
there is a need to select antigen-negative units for transfusion.
should be given rather than wasted.
Multiple antibodies are more commonly found in patients older
Rh-positive blood should not be given to Rh-negative female
than 60 years old or have undergone transfusion multiple times
patients of childbearing age.
are usually the ones detected with unexpected reactions
Transfusion of Rh-negative male patients and female patients
In antibody identification we make use of screening cells (cells that
beyond menopause with Rh-positive blood is acceptable as long as
have no antigen present)
no preformed anti-D is demonstrable in their sera.
3 Phases Involved (Immediate screen, 37°C, Antihuman globulin
phase)

#MLSWATER2022 Page 6 of 16
 BLOOD BANK LABORATORY Donor Processing
A facility involved in the collection, storage, processing, and Before unit blood can be placed in general inventory, testing must
distribution of human blood and blood products for transfusion be performed
Blood Processing centers utilize the testing methods that will
BLOOD BANK AREA AND FUNCTION provide the safest blood products for patients
AREA FUNCTIONS Product Labelling
Component Preparation Separation of whole blood into packed Appropriate ABO and Rh type
And Storage RBCs, plasma, platelets and Expiration date
cryoprecipitate Stored at proper temperature
Storage of blood products at
appropriate temperatures Issue of Blood Products
Apheresis procedure The individual in the blood bank who will issue the blood product
Donor Processing Donor units tested for: inspects the unit for any abnormal appearance and verifies that all
ABO and Rh required transfusion forms and labels are complete and that they
Antibody Screen adequately identify the transfusion recipient.
Serologic Test for Syphilis
Transfusion-Transmitted viruses Personnel Requirements
Product Labelling RBCs and any other component are Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88)
labeled establishes personnel qualifications for laboratories performing
Products stored at their proper certain types of testing:
temperatures o include gel technologies
Main Laboratory Patient Samples tested for: o Solid phase RBC adherence.
o ABO and Rh
o Antibody Screen Standard Operating Procedures
o Crossmatch These manuals, usually located at the workbench and accessible to
o DAT all personnel, contain information outlining the operations of the
o Prenatal Evaluation laboratory; details on how, when and why particular activities are
o Postpartum Evaluation done; and procedures for all tests performed.
o Cord blood studies SOP manuals are integral components of any blood bank
o Issue blood products laboratory’s quality assurance program.
Reference Laboratory Resolution of: They are reviewed at least annually and updated on a regular basis
o ABO and Rh discrepancy to reflect changes in operations and implementation of new
o Antibody identification regulations.
o Positive DAT
o Warm autoantibodies
o Cold autoantibodies
o Transfusion reaction

#MLSWATER2022 Page 7 of 16
 Equipment o In addition to patient information, the label should also
Blood Storage Refrigerator include the date and time of collection and the name of the
Donor Couches person who collected the sample
Dielectric Tube Sealer 2. Collection
Blood Mixer & Collector o Serum is the preferred specimen for compatibility testing.
Platelet Incubator o Hemolysis should be avoided.
Platelet Agitator o Blood should not be drawn from an intravenous site unless
Plasma Expressor absolutely necessary. In such a case, the infusion should be
ELISA Reader with washer stopped, the line should be flushed with normal saline, and
Refrigerated Centrifuge the first 5 to 10 mL of blood should be discarded before the
Binocular Microscope CH 21 specimen is collected.
Universal Hot air oven 3. Age of specimen
Bacteriological Incubation o The freshest sample possible should be used for compatibility
Centrifuge Machine testing.
Rh view box o If the patient has previously undergone transfusion or if the
transfusion history is unknown, the sample should be no older
than 72 hours.
SUPPLEMENTARY NOTES: PRE TRANSFUSION TEST ING o Pregnant patients should also be tested with samples not
CROSSMATCHING more than 72 hours old
4. Sample storage
Only a part of compatibility testing.
o The AABB requires that patient samples must be stored
Type and Screen procedure has emerged as an acceptable
between 1◦C and 6◦C for at least 7 days following
alternative to crossmatching blood
transfusion.
Major Crossmatch
COMPATIBILITY TESTING:
o DONOR RBCS + PATIENT SERUM
Minor Crossmatch for homologous transfusion (ALLOGENIC):
o DONOR SERUM + PATIENTS RBCS ABO and Rh on donor units
Autocontrol ABO and Rh on recipient
o PATIENT RBCS + PATIENT SERUM Antibody screening of recipient
Antibody identification
Autocontrol
SPECIMEN COLLECTION Crossmatch
Pretransfusion testing begins with a properly collected and
identified patient specimen for autologous transfusion:
ABO and Rh on autologous units
1. Patient identification ABO and Rh on recipient
o The tube should be labelled at bedside immediately after the Antibody screening and major crossmatch
sample is drawn o not required, although an immediate spin major crossmatch
is often performed
#MLSWATER2022 Page 8 of 16
for neonatal transfusion: FDA Licensed Screening Cells
ABO and Rh on the infant unspooled reagent red cells
Antibody screen on the infant or the mother 2-3 vials (R1R1, R2R2,rr)
o If the antibody screen is negative, a crossmatch is not detects clinically significant Ab
necessary reagents red cell express the ff: D,C,E, c,e
o If the donor cells are not group O, the infant must be tested M,N,S,s,P1,Lea,Leb,K,k,Fya,Fyb,Jka,Jkb
for anti-A and anti-B antibodies. If either is present, ABO- Autocontol not required
compatible RBCs should be used. A crossmatch is not DAT-not required
necessary
NOTE: Group O cells are used so that anti-A and anti-B will not interfere
ANTIBODY DETECTION in the detection of antibodies to other blood group systems.
Antibody detection/ antibody screening is testing the patient’s
serum against 2 or 3 reagent group O screening cells. ANTIGRAM/ WORKSHEET
Screening cells are commercially prepared cells suspension from It is a listing of the antigen make up of each screening cells that is
individual donor that is phenotyped for most common antigens. provided with every lot of screening cells.
In test tube testing: 3 phases Can be used as a worksheet during antibody detection process.
o Immediate spin o Caution: Lot number must match against the screen cells in
o 37C incubation use
o AHG Sample (link to worksheet):
Testing can also be: gel technique or solid phase method [Link]
Performed as part of: Hi?usp=sharing
o testing to provide compatible red cell transfusion
o prenatal evaluation
o evaluation of HDFN ANTIBODY IDENTIFICAT ION
o resolution of transfusion reactions ANTIBODY PANEL
o processing of donor units
Just an expanded antibody screen
Uses group O reagent RBCs
Uses Antibody Panels - used to identify an unexpected antibody
RBCs from 8-20 donors
detected by the antibody screening
Patient serum or plasma
Expanded version of antibody screening
Autocontrol
antibody screening uses screening cells 2-3 reagents of group O
IS / 37 C / AHG if tubes
screening cells
AHG only if gel or solid phase
Antibody panel uses 8-20 screening cells, these panels usually
Reactions documented on a sheet that outlines every RBC’s
contain 10-15 or 8-20 vials of group O cells each of which yield a
phenotype
different antigen reaction pattern

Each of the panel cells has been antigen typed (shown on antigram)
o + refers to the presence of the antigen
o 0 refers to the absence of the antigen
#MLSWATER2022 Page 9 of 16
 INTERPRETING ANTIBODY PANELS
1. Check History
Will give you clue what possible Ab is involved.
Check racial profile, recent transfusion, pregnancy and recent
bacterial or viral infection.
Notes the previous condition of the patient.
o Ex. pregnant: Rh blood group system is involved
Important also to note the racial profile of the patient since African-
American lacks Duffy antigen and Whites has high frequency
antigen

All cells are negative at AHG, so add “Check” Cells 2. Check Autocontrol
Check cells are added to all negative tubes. Negative - alloantibody
Check cells are cells coated with IgG and should react positively with Positive – autoantibody or DTR (i.e., alloantibodies)
the AHG in the tube.
o If check cells are negative, the procedure was not Check if the reaction if zero or plus or positive
performed correctly and should be repeated. If the reaction is positive that would indicate that there is presence
of autoantibody
If the reaction is negative there might be the presence of
alloantibody
o Another thing for negative control, it would show a uniform
reaction that means all cells are reactive. It would suggest there
might be presence of multiple alloantibodies and single
alloantibodies related with high frequency antigen
Antibodies will only react with cells that have the corresponding
antigen; antibodies will not react with cells that do not have the
antigen

3. Check the Reaction Phase

IS – cold reacting (IgM); clinically insignificant
37° - cold (some have higher thermal range) or warm reacting (IgG)
AHG – warm (IgG) and significant Ab. It detects Ab that have been
coated/sensitized RBCs but were not visible as agglutinates

4. Look at the general pattern (Reaction strength)

1 consistent strength (Uniform)– one antibody
Different strengths(Mixed reaction)s – multiple antibodies or
dosage

#MLSWATER2022 Page 10 of 16
 NOTE:
5. Ruling Out Strength of reaction may be due to “dosage”
Look at the first completely negative cells ( panel cells that has 0 or If panel cells are homozygous, a strong reaction may be seen
no reaction) If panel cells are heterozygous, reaction may be weak or even
Go blood group by blood group or paired antigen non-reactive
Panel cells that are heterozygous should not be crossed out
Example case: because antibody may be too weak to react

6. Look what IS there (Look at the usual Ab reactivity)

Cell # 3 and 4 shows double dosage (homozygous Lea) Lea with

(+) reaction should be eliminated since the IS results should be
negative. Leb cannot be rule out since it has the same reaction
with IS results.
Cell # 5 and 7 shows single dose (heterozygous) cannot be ruled
out.
Cell #9 and 10 shows double dosage (homozygous) Leb with (+)
reaction can be ruled out.

7. Look for Matching Pattern

Single antibodies usually shows a pattern that matches one of the
antigens.
Multiple antibodies are more difficult to match because they often
show mixed reaction strengths.

#MLSWATER2022 Page 11 of 16
 RULE OF THREE: FOR EXAMPLE:
Patient Serum must be: Let’s say you ran a panel and identified 3 different antibodies:
o Positive with 3 cells with the antigen anti-S, anti-Jka, and anti-P1
o Negative with 3 cells without the antigen o Selected cells could help…
o If there are not enough cells in the panel to fulfill the rule,
then additional cells from another panel could be used. Selected cells S Jka P1 IS LISS AHG
37
8. Use Special Techniques if Necessary #1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
9. Ensure statistical significance #8 0 0 + 0 0 0 (check cell +)

SPECIAL TECHNIQUES These results show that instead of 3 antibodies, there are actually
2: anti-S and anti-Jka
PHENOTYPING
In addition to the rule of three, antigen typing the patient red cells
can also confirm an antibody
NEUTRALIZATION/ INHIBITION
Only perform this if the patient has NOT been recently transfused
Other substances in the body and in nature have antigenic
(donor cells could react)
structures similar to RBC Antigens which can neutralize antibodies
If reagent antisera (of the suspected antibody) is added to the
present in serum, allowing separation
patient RBCs, a negative reaction should result.

NOTE: Individuals DO NOT make allo-antibodies against antigens SUBSTANCE ANTIBODY NEUTRALIZED
they have – Landsteiner Rule Anti P1
Hydatid cyst fluid, pigeon droppings,
turtledove’s egg whites
MULTIPLE ANTIBODIES
Selected cells are chosen from other panel or screening cells to Plasma/serum;secretor saliva Anti-Le
confirm or eliminate the antibody
Plasma or serum from Ch/Rg(+) individuals Anti-Chido; Anti-Rodgers
The cells are “selected” from other panels because of their
characteristics Anti-Sda
Urine, guinea pig urine
The number of selected cells needed depends on how may
antibodies are identified Human breast milk Anti0I

Secretor saliva ABH antibodies

#MLSWATER2022 Page 12 of 16
ENZYMES (PROTEOLYTIC) AUTOANTIBODIES
Can be used to enhance or destroy certain blood group antigens; Autoantibodies can be cold or warm reacting
Enzymes remove the sialic acid from the RBC membrane, thus A positive autocontrol or DAT may indicate that an auto-antibody is
“destroying” it and allowing other antigens to be “enhanced” present
Several enzymes exist: Sometimes the autocontrol may be positive, but the antibody
o Ficin (figs) screening may be negative, meaning something is coating the RBC
o Bromelin (pineapple)
o Papain (papaya) WHAT CAN THE DAT TELL US?
Although not always performed in routine pretransfusion testing,
DESTROYED ENHANCED NO EFFECT a positive DAT can offer valuable information
If the patient has been transfused, the patient may have an
MN, Duffy, Xg, JMH, alloantibody coating the transfused cells
ABO, Rh, Lewis, P, Ii,
Ss, Chido/Rodgers, Pr, Kell, Diego, Colton ,U If the patient has NOT been transfused, the patient may have an
Kidd
Lutheran autoantibody coating their own cells

One-stage: Enzyme is added directly to the serum/cell mixture Note: Auto-antibodies can sometimes “mask” clinically significant allo-
Two-stage: Panel cells are pre-treated with enzyme, incubated and antibodies, so it’s important to differentiate between auto- and allo-
washed. Patient serum is added to panel cells and tested antibodies

SULFHYDRYL REAGENTS
Cleave the disulfide bonds of IgM molecules and help differentiate COLD AUTOANTIBODIES
between IgM and IgG antibodies React at room temperature with most (if not all) of the panel cells
Good to use when you have both IgG and IgM antibodies and give a positive autocontrol
(warm/cold) The DAT is usually positive with anti-C3 AHG (detects complement)
Dithiothreitol (DTT) is a thiol and will denature Kell antigens Could be due to Mycoplasma pneumoniae, infectious mono, or cold
2-mercaptoethanol (2-ME) agglutinin disease
A combination of proteolytic enzymes and DTT
Denatures Kell, M, N, S, Duffy and other less frequent blood group
antigens
Does not denature the Kx antigen
Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that autoantibody can
then be adsorbed onto another RBC

#MLSWATER2022 Page 13 of 16
AVOIDING REACTIVITY ELUTION (WHENEVER DAT IS POSITIVE)
Cold autoantibodies can be a nuisance at times. Here are a few ways to Elution techniques “free” antibodies from the sensitized red cells so
avoid a reaction: that the antibodies can be identified.
Use anti-IgG AHG instead of polyspecific. Most cold antibodies Testing the eluate is useful in investigations of positive DATs
react with polyspecific AHG and anti-C AHG because they fix o HDN
complement o Transfusion reactions
Skipping the IS phase avoids the attachment of cold o Autoimmune disease
autoantibodies to the red cells The red cells can also be used after elution for RBC phenotyping if
Use 22% BSA instead of LISS needed
If the antibodies remain, then prewarmed techniques can be When tested with panel cells, the eluate usually remains reactive
performed: with all cells if a warm autoantibody is present
Red cells, serum, and saline are incubated at 37° before being Types of Elution:
combined Acid elutions (glycine acid)
Autoadsorption is another technique in which the autoantibody is o Most common
removed from the patients serum using their own red cells o Lowers pH, causing antibody to dissociate
The serum can be used to identify any underlying alloantibodies Organic solvents (ether, chloroform)
o Dissolve bilipid layer of RBC
Heat (conformational change)
Freeze-Thaw (lyses cells)
WARM AUTOANTIBODIES
More common that cold autoantibodies
Positive DAT due to IgG antibodies coating the red cell
Again, the majority of panel or screening cells will be positive ADSORPTION
The Rh system (e antigen) seems to be the main target although Adsorption procedures can be used to investigate underlying
others occur alloantibodies
Cause warm autoimmune hemolytic anemia (WAIHA) ZZAP or chloroquine diphosphate can be used to dissociate IgG
Sources of Warm autoantibodies: antibodies from the RBC (may take several repeats)
o Idiopathic After the patient RBCs are incubated, the adsorbed serum is tested
o Known disorder (SLE, RA, leukemias, UC, pregnancy, with panel cells to ID the alloantibody (if present)
infectious diseases, etc) Two Types:
o Medications Autoadsorption
o No recent transfusion
o Autoantibodies are removed using patient RBCs, so
alloantibodies can be identified
Allogenic (Differential) adsorption
o If recently transfused
o Uses other cells with the patients serum

#MLSWATER2022 Page 14 of 16
 ANTIBODY PANEL VS. ANTIBODY SCREEN
CHLOROQUINE DIPHOSPHATE o Group O reagents but with antibody panel it uses 10-15 or 8-20 vials
Quinilone derivative often used as an antimalarial or donors blood compared into 2-3 vials in antibody screening.
May not remove autoantibody completely from DAT positive cells o Patient serum is also used just like in antibody screening.
Partial removal may be enough to antigen type the cells or to be o It also involves 3 phases: immediate spin, 37° Celsius, and
used for autoadsorption of warm autoantibodies antihuman globulin
o We use 10 panel cells, the number 11 is usually the auto control
o Group O cells are used so that anti-A and anti-B will not interfere in
the detection of antibody to the other blood group system
ADD-ONS FROM MA’AM JOANA’S DISCUSSION o Each of the panel cells have been antigen type
When do we need to identify the antibody?
ANTIGRAM/WORKSHEET
COMMON REASONS TO IDENTIFY AN ANTIBODY PANEL TESTING o The table is called the antigram
o Uses Antibody Panels - are used to identify an unexpected antibody o antigram or worksheet is a listing of the antigen make up of each of
detected by the antibody screening the screening cells or panel cells that is provided with a lot of
o This is usually done following a positive antibody screening when screening cells. This can be used as a worksheet during antibody
testing would suggest a presence of new antibody and confirm a detection process
previously identified antibody o (+) positive or plus sign symbol: refers to presence of antigen
o (0) zero: refers to the absence of antigen
o Auto controls are located below, it is used to determine the
Why do we need to identify the antibody present in a reaction?
antibody present is an autoantibody or alloantibody
o it is needed for transfusion processes
o it is essential component for the compatibility testing There is the presence of autoantibody if the auto control with
o identification of any unexpected antibodies in the patient’s serum the patient’s RBC will result a (+) positive reaction
If (0) zero or no agglutination that would occur during the auto
In antibody panel testing, it is basic to know what is alloantibody and control testing would mean that the antibody present is
autoantibody alloantibody
o Alloantibodies – are antibodies against RBC agent not present on o It is also needed to check in the antigram the reaction in different
patient’s own RBC phases: immediate spin, 37° Celsius, and antihuman globulin
o Autoantibodies- are antibodies against the RBC antigen present on o 10 panel: a drop in each panel cells and 2 drops of patient’s serum
patient’s own RBC
IMMEDIATE SPIN PHASE
RBC screening o Detect clinically insignificant cold antibodies usually IgM in nature
o uses 2-3 screening cells to detect if any antibodies are present in the and it involves immediate centrifugation of the mixture at room
serum temperature for 20 seconds using the centrifuge
o it follows the concept about when detecting/identifying the There are some laboratories that will not require immediate
antibodies, test the patient’s serum (contains the unknown), weigh spin phase they directly undergo LISS phase and anti-human
the reagents RBC (contains the known) globulin

#MLSWATER2022 Page 15 of 16
37° CELSIUS WITH ENHANCEMENT MEDIUM DIFFERENT GRADING FOR AGGLUTINATION
o it detects the warm reactive IgG antibodies 1 CLUMP 4+
Enhancement Media are potentiators that added to the cells IF DISTANCED 3+
serum mixture before the 37° Celsius or incubation to increase SCATTERED 2+
the sensitivity of the test system HAS AGGLUTINATION BUT VERY 1+
MINIMAL
DIFFERENT ENHANCEMENT MEDIA NO AGGLUTINATION REPORTED AS NEGATIVE
1. 20% Albumin
DO NOT FORGET TO ADD THE CHECK CELLS TO ANY NEGATIVE AHG
o Incubation period 30-60 minutes, works in decreasing zeta
o Example: Encounter a reaction that can be only seen in Immediate
potential Spin Phase
o Low Ionic Strength Solution/ Saline Solution: Incubation time 10- o No reaction was recorded for the LISS and no reaction was also seen in
15 minutes and it contains .2% of Sodium Chloride. Meaning it anti-human globulin.
has low ionic content compared to the normal saline solution o To confirm that the procedure is performed correctly, it doesn’t need
that contains glycine, dextrose or glucose in addition to saline to repeat and check cells must be performed. Since check cells are added
reduce the zeta potential in all negative tubes
o increases the amount of the antibody taken up by the red cell Check cells – are cells coated with IgG and should react
during the sensitization process positively with anti-human globulin reagent within the tube.
o Sensitization – is the first page in the agglutination process If the check cells are negative, procedure was not
performed correctly and should be repeated
2. Polyethylene Glycol
-END OF TRANSCRIPTION -
o Works in removing water from the test system thereby
ඞ
concentrating any antibodies that are present in the reaction.
More sensitive compared to LISS and 22% albumin
Disadvantage: don’t usually used in high motile patients
having high protein levels just like in the cases of multiple
myeloma since it can interfere in the testing procedure

3. Antiglobulin phase
o Detects non-agglutinating warm reactive antibodies that are
possibly not detected with 37° Celsius incubation period.
o It would also detect antibodies that has been coated or
sensitized, RBC that did not show any visible agglutination.

#MLSWATER2022 Page 16 of 16
 MODULE 4: PRE-TRANSFUSION TESTING
MLS 18, April 25, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend: Three (3) Definitions of Blood Bank

Transcription Bullet 1. It refers to a section of hospital laboratory responsible for the storage
Notes/Module Packet Bullet and issue of blood products
PPT
LESSON OUTLINE May perform many other tasks which includes pre-transfusion
I. Routine Blood Bank Laboratory analysis of potential recipients to screen for blood type (i.e. Rh
A. Blood Bank Are and Function type). And also, the presence of antibodies against red cell antigens.
B. Equipment There are many blood banks which are involved in preparation for
advanced components for infusion such as stem cells, harvested
BLOOD BANK LABORATORY from peripheral blood or bone marrow.
A facility involved in the collection, storage, processing, and o Not Available in Iloilo
distribution of human blood and blood products for transfusion
2. Refers to blood collection organizations that operate more on the
Blood banking is a process that takes place in the laboratory supply side of the transfusion process
that ensures donated blood or blood products are safe before Red Cross
being used for transfusion or other medical procedures. o Good example/ representation of the second definition
Blood bank and transfusion services are regulated by a of blood bank
number of federal agencies
3. Reference for the medical practice surrounding blood collection and
Food and Drug Administration (FDA) transfusion
Which has regulatory responsibility for blood collectors and Sometimes called “Transfusion Medicine”
manufacturers of blood components.
Its inspectors ensure compliance with regulations. What is Blood Banking?
Is a facility wherein donor recruitment is made
American Association of Blood Bank (AABB) Where donors properly bleed
Is a key international association of blood banks including hospitals Pre-transfusion testing is performed
and community blood centers Facility where product making is performed
Transfusion and transplantation services and also individuals Blood products are stored properly
involved in the said services Documentation is made
Establishes standards of care for patients and donors in all aspects o Only section of laboratory wherein there is an interview.
of blood banking (i.e. transfusion medicine, hematopoietic, cellular o Donors need to sign for consent
and gene therapy and lastly, tissue transplantation) Facility wherein the blood and its products are issued
Take note: a blood bank may be a separate free-standing facility or
part of a larger laboratory in a hospital
 o Free standing facility (e.g. Red cross) Product Labelling RBCs and any other component are
o Part of a larger laboratory in a hospital (e.g. Western Visayas labeled
Medical Central Blood bank). Products stored at their proper
o Commercial blood banks- these are blood banks that exist for temperatures
“profit” or selling blood but this type of blood bank facility is Main Laboratory Patient Samples tested for:
not available in our country. o ABO and Rh
o Hospital Based Blood bank- it is a blood bank which is located o Antibody Screen
in the premises of a hospital which can perform compatibility o Crossmatch
testing of blood. o DAT
o Prenatal Evaluation
o Blood collection unit- is an institution or facility duly
o Postpartum Evaluation
authorized by the Department of Health to recruit and screen
o Cord blood studies
donors and collect blood. (e.g. Red Cross qualified as a blood
o Issue blood products
collection unit
Reference Laboratory Resolution of:
o ABO and Rh discrepancy
What are the responsibilities of a hospital blood bank? o Antibody identification
1. Rapid response to urgent request for blood components o Positive DAT
2. Checking for pre- transfusion samples and requests. o Warm autoantibodies
3. Assessing of immunological compatibility between donor and o Cold autoantibodies
patient o Transfusion reaction
4. Selecting suitable blood components for each clinical condition.
o Example: If packed RBC is only need then only packed RBC Donor Processing
must require; no need of whole blood or plasma Before unit blood can be placed in general inventory, testing must
5. Safe delivery and handling of blood component be performed
Blood Processing centers utilize the testing methods that will
provide the safest blood products for patients
BLOOD BANK AREA AND FUNCTION
Product Labelling
AREA FUNCTIONS
Appropriate ABO and Rh type
Component Preparation Separation of whole blood into packed Expiration date
And Storage RBCs, plasma, platelets and Stored at proper temperature
cryoprecipitate
Storage of blood products at
appropriate temperatures
Issue of Blood Products
The individual in the blood bank who will issue the blood product
Apheresis procedure
inspects the unit for any abnormal appearance and verifies that all
Donor Processing Donor units tested for:
required transfusion forms and labels are complete and that they
ABO and Rh
adequately identify the transfusion recipient.
Antibody Screen
Serologic Test for Syphilis
Transfusion-Transmitted viruses

#MLSWATER2022 Page 2 of 4
Personnel Requirements Equipment Description
Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) A designated temperature-controlled refrigerator
Blood Storage
establishes personnel qualifications for laboratories performing equipment specifically designed to store blood
Refrigerator
certain types of testing: banks. The blood refrigerator operates at a
o include gel technologies temperature between 2 and 6°C.
o Solid phase RBC adherence. Specially designed to make blood withdrawal stir,
Donor Couches safe and provides a comfortable position for the
Standard Operating Procedures donor.
These manuals, usually located at the workbench and accessible to Dielectric Tube Seals the tube of the blood bag without causing
all personnel, contain information outlining the operations of the Sealer hemolysis and leakage of the blood.
laboratory; details on how, when and why particular activities are Composed of a support structure, supporting the
done; and procedures for all tests performed. components of the mixer, a tubing fastening and
Blood Mixer &
SOP manuals are integral components of any blood bank guiding arrangement also configured to secure a
Collector
laboratory’s quality assurance program. blood collecting tubing in the mixer.
They are reviewed at least annually and updated on a regular basis It also comprises a tray adapted to house a blood
to reflect changes in operations and implementation of new collecting bag.
regulations. Platelet Incubator Provides accurate and stable storage conditions
for platelets.
Equipment Designed for storage of platelets concentrates
Platelet Agitator
Blood Storage Refrigerator and they are designed to provide continuous,
Donor Couches gentle, horizontal motion of your platelets.
Dielectric Tube Sealer Plasma Expressor Mechanical accessories for transferring of plasma
Blood Mixer & Collector to the satellite bags.
Platelet Incubator An ELISA reader measures and quantitates the
Platelet Agitator color differences in the 12 wells of the plate.
Plasma Expressor ELISA Reader with They emit light at 1 wavelength and measure the
ELISA Reader with washer washer amount of light absorbed and reflected by an
Refrigerated Centrifuge object such as a protein.
Binocular Microscope CH 21 So, a spectrophotometer measures the ultraviolet
Universal Hot air oven and visible light.
Bacteriological Incubation Refrigerated This refrigeration constitutes an important added
Centrifuge Machine Centrifuge feature as any laboratory centrifuge.
Rh view box An optical microscope with two eyepieces,
Binocular
significantly improving viewing and cuts down on
Microscope CH 21
eye strain.
Universal Hot air Used to sterilize equipment and materials used in
oven the medical field, especially for the cleaning of
test tubes (washed the sterilized).

#MLSWATER2022 Page 3 of 4
 Hot air oven is a type of dry heat sterilization.
Dry heat sterilization is used on equipment that
can’t be wet and on materials that will not melt,
catch fire or change form when exposed to high
temperature.

Items that are sterilized in the hot air oven

include glassware, petri dishes, pipettes, test
tubes, and powders like zinc, zinc oxides, starch.
Also includes materials that contain oil or metal
equipment like scissors, blade, scalpels, glass
and test tubes.

Bacteriological Basically used for the incubation of the biological

Incubation products under controlled conditions.

Centrifuge Machine Laboratory device that is used for separation of

fluids, gas, or liquid based on density.
Comes with a soft fluorescent glare-free bulb that
provides excellent uniforms like illumination and
a temperature indicator that easily and
Rh view box accurately monitors the viewing area. Also
regulates the temperature to compensate for
ambient temperature changes
(Ranges from 45°C to 50°C).

-END OF TRANSCRIPTION-
ඞ

#MLSWATER2022 Page 4 of 4
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 1)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
Has regulatory authority over drugs, biologics and devices as
Legend: mandated by the Federal Food, Drug and Cosmetic (FDC) Act of
Transcription Bullet 1938 and the Public Health Services (PHS) Act of 1944.
Notes/Module Packet Bullet Blood establishments in the US are therefore:
PPT • Regulated by the FDA
• Required to follow federal regulations provided by the FDA in
MODULE OUTLINE
the composite form of the CFR (Code of Federal Regulations)
I. Organizations that Regulate or Accredit the Immunohematology
• Inspected at regular intervals by agents of the FDA
Laboratory
Blood establishments that ship products across state lines must be
II. Blood Donor Selection
licensed by the FDA, while blood banks and hospital transfusion
III. Types of Donation
services that operate within state boundaries must be registered
IV. Blood Component
with the FDA
V. Safe Storage of Blood
Hospital transfusion services that do not perform any
VI. Packing Blood Components for Transportation
manufacturing processes as defined by the FDA are not required to
be licensed or registered, though they must be in compliance with
LEARNING OUTCOMES applicable portions of the CFR and state regulations.
At the end of this module, the student shall be able to:
1. Identify the organizations that regulate or accredit the A government agency that regulates and establishes blood products
immunohematology laboratory. and its guidelines, transportation temperature, storage temperature
2. State the minimum acceptable levels for the following tests in and the products used in blood bank laboratory
allogenic & autologous donation Established CBER (Center for Biologics Evaluation and Research) is
3. Identify the medical history information that would cause responsible for the regulation of collection of the blood and blood
permanent deferral or temporary deferral and state the length of components that is being used in the transfusion and for the
deferral period. manufacture of the pharmaceutical derived from the blood and
4. Explain the reasons for permanent or temporary deferral given the blood components. It developed and enforces quality standard and
various medical conditions. inspection in blood establishments and monitor reports about
errors, accidents and adverse clinical events that occurs in the blood
transfusion practice. In hospital transfusion services it will not
ORGANIZATIONS THAT REGULATE OR ACCREDIT THE
perform any manufacturing processes defined by the FDA are not
IMMUNOHEMATOLOGY LABORATORY
required to licensed or registered.
FDA
Food and Drug Administration AABB (American Association of Blood Banks)
An organizational arm of the Department of Health and Human A professional organization which serves a number of key functions
Services (HHS) often referred to as the Agency. for the blood industry, including education, supporting science and
research and its dissemination, and public advocacy.

#MLSWATER2022 Page 1 of 16
 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
• The National Blood Foundation (NBF), a subsidiary of AABB,
was created with the sole mission of providing seed funding Donor Requirements
and financial support for scientific inquiry related to 1. Identification card
transfusion medicine Includes photographic identification of the donor

College of American Pathologists (CAP) 2. Donor registration information

Participates in laboratory accreditation and proficiency testing. Includes full name, date and time of donation, address,
The CAP program uses a system of peer review, and participating telephone/cell phone number, sex, age and date of birth
institutions must provide inspectors for future accreditation visits.
3. Consent Form
BLOOD DONOR SELECTIO N
Two questions that must be answered before proceeding with the 4. Additional Information
blood donation: The name of the patient whom the blood is intended
1. Will a donation of approximately 450 mL of whole blood at this time Race of the donor for the rare phenotype
be harmful to the donor? CMV status
2. Could blood drawn from this time potentially transmit a disease to
the recipient Calculations for the Blood Volume:
Calculate adjusted blood volume and anticoagulant:
If “yes”: Defer donor: temporarily or permanently In athletes, pulse rate of <50 bpm is considered normal and not a factor
for deferral
If “No”: Proceed to Blood Donation
Volume to Collect:
General Requirements for Donation
𝑑𝑜𝑛𝑜𝑟 ′ 𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑘𝑔
= 𝑥 450 𝑚𝐿
General Appearance: Observe the prospective donor for the 50 𝑘𝑔
presence of excessive anxiety, drug or alcohol
influence, or nervousness.
Age At least 17-18 years old Reduced Volume of Anticoagulant:
Oral Temperature < 37.5 °C or 99.5 °F
Blood Pressure Less than or equal 180/100 mmHg 𝑉𝑜𝑙𝑢𝑚𝑒 𝑡𝑜 𝐶𝑜𝑙𝑙𝑒𝑐𝑡
= 𝑥 63 𝑚𝐿
450 𝑘𝑔
Hemoglobin ≥12.5%
Hematocrit ≥ 38%
Pulse 50-100beats/minute Volume of Solution to be removed:
Weight ≥ 110 lbs or ≥ 50 kg
= 63 𝑚𝐿 − 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡
Volume Maximum of 525 mL can be collected
WB Donation Maximum of 10.5 mL of blood /kg of donor See Appendices for Sample Problem
weight

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Blood Collection Process Position needle at 10-20° angle
1. Donor Registration 16-gauge needle usually used for blood donation; instruct
donor to open-and-close their fists every 10-20secs
2. Educational material is distributed to the donor.
Mix the unit periodically 1-2 times per minute.
If the prospective donor shows symptoms of an infectious disease,
o Blood Mixer – automatically mixes the blood with
the donor is excluded from the donation.
anticoagulant
Explain to the donor what might happen to his blood since it will
o If there is no blood mixer, the medical technologist will do
undergo different blood test
the mixing manually.
They must also sign the consent form allowing for the blood
Amount of blood collected: 450mL ± 10%
collection as well as other blood tests
Duration of collection: 7-10 minutes
Medical technologists must further explain to the donor the
o If the correct duration time is not observed, the blood
other blood tests to be conducted to his blood
collected would not be suitable for platelet
concentration/concentrate preparation, FFP, or
3. Actual Donor selection/ Identification cryoprecipitate (>15mins of blood collection would make
No fasting is required for blood donation, but donor must eat the blood not viable, especially in cryoprecipitate).
Donors who have not eaten food for 4hrs or more are required
to eat before blood collection Color Coding for Blood Bags

4. Blood Collection FDA (1985) *RA 1517

Use Aseptic Technique: Blood Type Color Label Blood Type Color Label
o PVP Iodine compound or chlorhexidine gluconate & A Yellow A Blue
Isopropyl alcohol B Pink B Yellow
o Scrub site at least 4 cm in all directions for 20 seconds AB White AB Pink
o Apply tourniquet or blood pressure cuffs: 40-60 mmHg O Blue O White
o Position the needle at 10-20 ° angle *Blood Bank Law; the standard/ what used
Mix the unit periodically 1-2 times per minute
Amount of blood collected: 450 mL ± 10% Donor Deferral
Duration of collection: 7-10 mins
o Component must be prepared after collection TEMPORARY DEFERRAL
EXAMPLE: Donor has received a blood transfusion; defer for 12
Blood collection facilities confirms the identity of the donor months from date of transfusion
through the photographic identification such as the use of Tooth Extraction
showing their driver’s license for confirmation Persons who had experienced convulsions
The antecubital fossa should be free from lesions, scars and
track marks INDEFINITE DEFERRAL
During blood collection 2 blood bank personnel, 1 Medical These donors may be eligible to donate autologous blood
Technologist and 1 Head of Blood Bank is required to be The donor is unable to donate their blood for some reason and
present. They do interview and look for multiple needle marks, there is no specified period of time due to regulatory requirements;
unexplained weight loss or if the patient is immunosuppressed, but donors are still eligible to donate autologous blood
oral thrush or Kaposi sarcoma
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
PERMANENT DEFERRAL
TEMPORARY DEFERRAL
EXAMPLE: Donor states that he or she has hepatitis C; defer Deferral if the donor received the following vaccine:
permanently 2-week deferral Smallpox, Polio, Measles, Mumps, Influenza
The donor will never be eligible to donate blood for someone else; 4-week deferral German Measles, Chicken-pox
donor is only eligible to donate autologous blood
12-month deferral Rabies vaccine if given after the bite of a
o Definite disease or habits strongly associated with bloodborne
rabid animal
pathogens
May donate if afebrile Diphtheria, Pertussis, Typhoid, Tetanus,
o Narcotic addiction
Cholera, Influenza
o Alcohol addiction
1 Year Skin penetration w/ instruments
o Received pituitary growth hormones of human origin
(Possible exposure to contaminated w/blood (surgery)
o History of chaga’s disease, malaria, or babesiosis; cancer,
Hepatitis, HIV or Malaria) Closed contact with persons with viral
leukemia, lymphoma
hepatitis
o Persons w severe thrombocytopenia
(+) STS- 12 months from completion of
therapy
PERMANENT DEFERRAL Traveled to endemic area for malaria w/
Definite disease or Viral Disease or w/o antimalarial drugs
habits strongly Hepatitis after the age of 11
≥72 hours in correctional institution
associated with (+) confirmatory test for HbsAg
bloodborne pathogens Repeatedly (+) test for anti-Hbc Female donor who had sex with bisexual
Gave only unit to recipient who men
developed post transfusion hepatitis and Anyone who had given someone money
HIV or drugs in payment for sexual contact
Present and Past infection with Hepa C 3 Years Visit/ immigrant from area endemic for
and HIV (Possible exposure to malaria
Narcotic addiction malaria) Had malaria, but presently asymptomatic
Alcohol addiction
Other diseases: **plasma preparation w/o red cells are
Received pituitary growth hormone of exempted from these restriction
human origin No deferral if afebrile Diphtheria, pertussis, typhoid, tetanus,
History of Chaga’s disease, babesiosis or cholera, influenza, previous history of tb
malaria that has been successfully treated and is
History of cancer, leukemia, or lymphoma no longer active
Person’s with severe thrombocytopenia

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Intake of Medication injections given over
a 6-month period
Medication Indication Deferral Period Unlicensed Vaccine Research Proposal 12 months unless
Proscar (Finasteride) Prostate Gland 2 months otherwise
Avodart (Dutasteride) enlargement 6 months indicated by
Propecia Baldness 2 months medical director
Accutane (Amnesteem, Severe Acne 2 months
Claravis, Sotret, How it affects donation?
Isotretinoin) HBIG does not prevent Hepatitis B in every case
Soriatane (Acitretin) Severe Psoriasis 3 years
Tegison (Etretinate) Permanent Contraceptive Pills
May donate anytime
How it affects donation?
These medications can cause birth defects. Donated blood with high Highly Allergenic Drugs like Penicillin, Aspirin, and others
enough levels to damage the unborn baby if transfused to a May donate anytime provided the blood collected will not be used
pregnant woman. to prepare platelets
Once medications are cleared from the body, one can donate again Donor may donate only after 24 hours of taking the medications.

Medication Indication Deferral Period Piroxicam

Growth Hormone from Delayed impaired Permanent Deferred until TB is completely cured
Human Pituitary Glands growth (used only until
1985) TYPES OF DONATION
Insulin from cows Indefinite
ALLOGENIC DONATION
(Bovine or beef insulin)
Genetically different individual but same specie
Blood is taken from an individual of the same specie as the recipient
How it affects donation?
Can lead to development of rare nervous system disorder called CJD
(Creutzfeldt-Jakob Disease) REQUIREMENTS FOR ALLOGENIC DONATION
If Insulin was imported from countries infected in which Mad Cow
Disease had been found, it could contain material from infected Philippine Standard AABB Standards
cattle Age 16-65 years old At least 17 years old
MCD can be transmitted through blood transfusion Temperature ≤37.5 °C or ≤99.5 °F ≤37.5 °C or ≤99.5 °F
Pulse Rate 60-100 bpm 50-100 bpm
Medication Indication Deferral Period Hemoglobin ≥ 12.5 g/dL ≥ 12.5 g/dL
HBIg Following exposure 12 months after
Blood Pressure
to hepatitis B receipt of vaccine
Systolic 90- 160 mmHg ≤ 180mm Hg
Different for
Diastolic 60-100 mmHg ≤ 100 mm Hg
Hepatitis B vaccine,
which is a series of 3

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
AUTOLOGOUS DONATION 3. INTRAOPERATIVE COLLECTION
Autologous means “self” Involved collecting shed blood from the surgical site, a device that
Blood is given to the recipient came from the recipient himself utilizes vacuum is used to collect shed blood.
REQUIREMENTS FOR AUTOLOGOUS DONATION Blood is the washed with saline and concentrated to reach
hematocrit of 50-60%, then reinfusing those cells immediately.
Collection and Reinfusion of lost blood during a surgery
Age No age requirement
Hemoglobin 11 g/dL
Hematocrit 33% 4. POSTOPERATIVE COLLECTION
General Condition: Patient should have no condition predisposing to Collected from a drainage tube placed at the surgical site.
bacteremia or any form of severe cardiovascular/ pulmonary It is reinfused with or without processing, via a microaggregate filter
condition. to screen out any debris
Single unit is removed at a time, with at least 3 days intervals
Final phlebotomy must be at least 72hrs before surgery DIRECT DONATION
A direct donation is a unit collected under the same requirements as
those for allogenic donors, except that the unit collected is directed
TYPES OF AUTOLOGOUS DONATION towards a specific patient.
The patient selects his/her own donor for an anticipated non-
1. PREOPERATIVE COLLECTION emergency transfusion
Removal of blood 5-6 weeks immediately preceding a scheduled, Donor is typically a friend or relative of the patient
elective surgical procedure
The storage of blood and its components before an elective surgery; APHERESIS DONATION
blood is used during or after surgery Aphaeresis= “taking away”
o Possible Problems: presurgical anemia or hypovolemia can An effective mechanism for collecting a specific blood component
occur, clinical identification errors, outdating of liquid-stored while returning the remaining whole blood components back to the
blood, homologous blood transfusion instead of autologous patient
Amount of time for a particular procedure can range from 45-120
2. ACUTE NORMOVOLEMIC HEMODILUTION minutes
Collection of whole blood with the concurrent infusion of crystalloid ACD: Most common and Primary Anticoagulant in Apheresis
or colloid solutions procedure
The idea is that the patient bleeds more dilute blood during the It is the withdrawal of blood from a donor removing selected
procedure, and the patient’s heart may pump more effectively due components and reinfusion of the remaining components back to
to decreased blood viscosity the donor
Isovolemic Hemodilution
o Multiple units of blood are collected; the blood is reinfused Methods of Centrifugation
near-end the surgery
1. INTERMITTENT FLOW CENTRIFUGATION
The last unit collected must be the first unit to go back
Blood is drawn and reinfused through the same needle
Once the desired component is separated, the remaining
components are reinfused to the donor

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The blood is processed in batches or cycle and requires only 1
venipuncture. Donor’s RBCs should be ABO- and Rh-compatible, relatively fresh
Spins, process in the machine and gives back the unused part to the (leukoreduced, negative for hemoglobin S and partially phenotype-
donor. The blood is return to the same site where it was extracted. matched for the Rh (C, c, E, e) and K1 antigens
To stop the blood from coagulating, anticoagulant is automatically
mixed with the blood as it pumps the blood into the apheresis WBC PHERESIS (LEUKAPHERESIS)
machine. The granulocyte concentrate from leukapheresis are needed for
patients who have conditions such as severe neutropenia or
2. CONTINUOUS FLOW CENTRIFUGATION (CFC) conditions that are unresponsive to antibiotic.
Involve withdrawal and processing and reinfusing of blood to the Storage time is 24 hours at 20-24°Celsius.
individual simultaneously Compatibility testing is typically performed for WBC pheresis.
PLASMAPHERESIS (PLASMA EXCHANGE)
Equivalent of at least two whole-blood derived plasma units
o Infrequent plasmapheresis
o Frequent or Serial
FDA recommends 12 L (14.4 L for donors weighing more than 175
pounds) as the maximum allowable plasma volume donated per
year
Maximum of 2 donation in a 7 day period with 2 days gap period is
allowed.

THERAPEUTIC PROCEDURES
The rationale of Therapeutic Apheresis (TA) is based on the
following:
o A pathologic substance exists in the blood that contributes
Types of Apheresis to a disease process or its symptoms
PLATELETPHERESIS (THROMBOCYTOAPHERESIS) o The substance can be more effectively removed by
Donor’s platelet count: at least 150,000/ µL apheresis than by the body’s own homeostatic mechanisms
Donor should have not taken aspirin 3 days before donation
Interval of at least 2 days 1. Therapeutic Plasma Exchange (TPE)
pH ≥ 6.2 Removal and retention of the plasma, with return of all cellular
components to the patient.
To remove the agent in the plasma, such as an antibody, toxin, or
RBC PHERESIS (ERYTHROCYTAPHERESIS) abnormal protein, that is causing the clinical symptoms.

REQUIREMENTS FOR ERYTHROCYTAPHERESIS Factors Removed by Therapeutic Plasmapheresis

Height Weight Hct Immune Complexes SLE
Male 5’11” 130 lbs 40% Alloantibodies Antibody
Female 5’5” 150 lbs 40% Autoantibody Gullain-Barré syndrome
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Immunoglobulin causing Waldenstrōm’s macroglobulinemia NAUSEA OR VOMITING
hypersensitivity Management:
Protein-bound toxins or drugs Amanita mushroom poisoning, 1. Instruct the donor to breathe slowly
barbiturate poisoning 2. Apply cold compresses to the forehead
LPPs Familial hypercholesterolemia, 3. Turn the donor’s head to one side and provide an appropriate
hypertriglyceridemia receptacle
Phytanic Acid Refsum’s disease 4. The donor may be given water after vomiting has ceased

LOSS OF CONSCIOUSNESS
2. Therapeutic Platelet Pheresis Management:
Thrombocytosis (at least 500,000/ µL) can occur in myeloproliferative 1. Check vital signs frequently
disorders (ET, PV, CML) or as a reactive process in response to 2. Administer 95% O2 and 5% CO2
splenectomy, infection, chronic inflammation, or malignancy
SEVERE REACTIONS
3. Therapeutic Leukapheresis
CONVULSIONS
Elevated level of WBCs placed the patient at risk for complication
Management:
associated with leukostasis, including organ dysfunction due to the
1. Call for help immediately; notify blood bank physician
formation of microthrombi in the pulmonary and cerebral
2. Try and restrain the donor to prevent injury to self or others
microvasculature
3. Ensure an adequate airway

STEM CELL PHERESIS

CARDIAC OR RESPIRATORY
Management:
DONOR REACTIONS
1. Perform CPR until medical help arrives
MILD REACTION
HEMATOMA
SYNCOPE Management:
Management:
1. Remove the tourniquet and withdraw needle
2. Place cold compression on the donor’s forehead POST DONOR CARE
3. Raise the donor’s legs above the level of the head Raise arm and apply pressure on the puncture site after collection.
4. Loosen tight clothing and secure airway Rest after blood collection, reclining for few minutes, then sit upright
5. Monitor vital signs and allow to talk.
Instruct the donor to drink a lot of water, refrain from smoking and
TWITCHING OR MUSCLE SPASMS avoid strenuous work or driving.
Management:
1. Disengage the hyperventilation sequence by conversing with
the donor and having the donor breathe into a paper bag.

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NOTES FROM MODULE PACKET:
Blood Donor Selection Key principles of blood donor selection:
The primary responsibility of a blood transfusion service is to The health and safety of the donor as well as the recipient must
provide a safe, sufficient and timely supply of blood and blood be safeguarded
products Only individuals in good health should be accepted as donors of
The purpose of blood donor selection is to: whole blood and blood components
o Protect donor health and safety by collecting blood only The selection of blood donors should be based on regularly
from healthy individuals reviewed selection criteria, without discrimination of any kind
o Ensure patient safety by collecting blood only from donors including gender, race, nationality or religion
whose donations, when transfused, will be safe for the A prospective donor’s health status and medical history should be
recipients evaluated for each donation, on the day of donation prior to
o Identify any factors that might make an individual blood collection
unsuitable as a donor, either temporarily or permanently The blood donor selection (BTS) should provide appropriate
o Reduce the unnecessary deferral of safe and healthy donors donor information and a simple donor questionnaire for health
o Ensure the quality of blood products derived from whole and risk assessment and obtain the donor’s informed consent to
blood and apheresis donations blood donation
o Minimize the wastage of resources resulting from the Staff should be suitably qualified and trained in the donor
collection of unsuitable donations. selection process
Blood donors have a responsibility to self-defer if they are aware of Good communication should be established between the BTS
having been exposed to any risk of an infection or a known health staff and the donor, and donor confidentiality should be assured
condition or treatment that could influence their suitability to The BTS has a duty of care to provide counselling to all deferred
donate blood. donors and referral for their further management.
Blood donors also have the right to withdraw at any stage of the
donation process. Steps Involved in The Donor Selection Process
A donor questionnaire is the key tool in donor selection for Compliance with all donor selection criteria is crucial to ensure a
assessing donor health and safety and for reducing the risk of safe blood donation process and outcomes.
transmission of infection, in particular for infections for which no
Donor registration
suitable screening tests are available.
All prospective donors who meet the general criteria for blood
o The use of a donor questionnaire prompts donor selection
donation such as age and good health should be registered when
staff to ask important questions and carefully assess the
they attend a blood donation session, even if they are subsequently
donor’s health.
not accepted for donation.
o By presenting all relevant information in a standard format,
Essential donor registration information includes the individual’s full
a donor questionnaire facilitates decisions on the
name, date of birth, gender and contact details.
acceptance or deferral of the donor.
During donor registration, prospective donors should be provided
Effective public information and donor education are the first steps
with donor information and education materials and the donor
in the process of donor selection.
questionnaire, which should be completed on each occasion of
donation.

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Pre-donation information donor selection process; and donor’s duties, responsibilities and
The process of donor selection begins even before donors come to rights
give blood through public awareness campaigns and donor Options for the donor to decide about blood donation prior to
education. proceeding further, to withdraw or self-defer at any time during
At the donation session, pre-donation information should be or after the donation process, without any undue embarrassment
provided either orally or through printed, graphic, audio-visual or or questioning
online materials, presented in a simple and clear format and in Transfusion-transmissible infections, including HIV, HBV, HCV and
appropriate languages. syphilis, routes of their transmission, natural history and
Pre-donation information provides an opportunity for the prevention; types of screening tests performed; and window
prospective donors to know about health conditions or high-risk period of infection and alternative testing sites for individuals
behaviour that would make them unsuitable to donate blood. seeking to ascertain their infection status
This information assists the donors in deciding whether to self- Possible consequences for donors and the donated blood in the
defer; case of abnormal TTI test results; the mechanism for notification
• It may also assist in donor return if they understand the about abnormal test results and post-donation counselling,
reason why they should not donate blood on this occasion assurance of confidentiality and if necessary, referral for further
testing, treatment and care
Objectives of Pre-donation Information: The possibility of adverse donor reactions.
Increase donor awareness of the donor selection criteria, the
process of blood donation and the tests that will be performed on Completion of donor questionnaire
donors’ blood Each prospective blood donor should complete a donor
Encourage prospective donors to inform the BTS of any medical questionnaire to provide information in relation to the donor
conditions or TTI-related risks that may affect their suitability to selection criteria defined in the national guidelines.
donate blood In most situations, the donor questionnaire is given to donors at the
Encourage individuals to self-defer from blood donation if they time of registration for completion before the donor interview and
recognize that they are not suitable to donate blood due to assessment.
general health or medical conditions or risk for TTI. Alternatively, the donor questionnaire may be sent to the donor’s
residence to be completed before donation.
Pre-donation Information Coverage: • This has the advantage of allowing donors time to think about
Nature and use of blood and its components; the need for the answers and saves time at a blood donation session.
voluntary nonremunerated blood donors; and the importance of • However, donors may misunderstand some of the questions
maintaining healthy lifestyles and self-defer for the wrong reasons.
The blood donation process, including the donor questionnaire, The donor questionnaire may also be administered electronically as
donor medical history, health and risk assessment, venipuncture, a computer-based questionnaire.
blood collection as whole blood or apheresis procedure, post- It is essential that donors are aware of the importance of the
donation care and the screening tests performed on donated questionnaire, the significance of the questions and the need for
blood providing accurate information
Rationale for the donor questionnaire and pre-donation health The information provided by the donor can then be further
assessment and the importance of donor compliance in the elaborated on during the interview.

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Donor interview and pre-donation counselling The basic health check also enables an assessment to be made of
The completed donor questionnaire should be reviewed prior to any physical disabilities that may impede the donation process, such
donation in a one-to-one confidential interview between the donor as:
and a donor selection staff member so that an assessment can be • Mobility: the donor should be able to easily access the donor
made of the donor’s general health, medical history and any TTI bed or couch
risks. • Sight or hearing impairment: assistance should be provided
It also provides an opportunity to check whether the donor has by a staff member.
understood the questions and has answered them correctly. Issues that require special attention during donor health and risk
Assurance about the confidentiality of the donor’s medical history is assessment include:
essential. • The prevalent culture and context of the environment for
Pre-donation counselling is an integral part of the donor interview. donation; in some situations, a donor may simply be
It enables donor selection staff to: overawed by the medical setting and procedures
• Check that the donor has understood all questions and • The provision of sufficient privacy and assurance of
responded accurately to the questionnaire confidentiality to make the donor comfortable when
• Answer the donor’s questions and provide reassurance in case answering probing and sensitive questions
of anxiety • Identifying and overcoming language barriers or lack of
• Explain reasons for any deferral and give advice about further understanding of questions in the donor questionnaire
medical care, if needed • Ensuring good communication by using simple jargon-free
• Ensure that the donor is able to give informed consent to language and explaining any medical terms.
donate and recognizes that his/her signature is an affirmation
that responses provided to the questionnaire are accurate. Informed consent
Informed consent is a voluntary agreement given by the prospective
Donor health and risk assessment donor to the donation of blood, to the testing of a blood sample for
The assessment of donor suitability and deferral, where TTI, for the transfusion of the donated blood to patients and if
appropriate, aims to exclude donations from individuals at risk of required, for the use of the blood for additional tests, quality
TTI, particularly from those with recently acquired infection that assurance or research purposes.
cannot or may not be detected by routine screening tests or with To obtain informed consent, the BTS should provide the following
infections for which no effective blood screening tests are available. minimum information to the potential donor:
The donor assessment not only enables the review of the donor’s • The donation process and potential adverse donor reactions
medical history and medications, but also provides an opportunity • The tests that will be performed (TTI and others) on the
for a basic health check to assess whether the donor is in general samples taken from the donated blood and the reasons for
good health. these tests
Any signs of debility, under-nutrition, pallor, jaundice, cyanosis, • Confidentiality of all personal information, including test
dyspnea or intoxication from alcohol or drugs should also be noted results.
Physical examination, weighing and/or measurement of vital signs The donor should sign and provide informed consent to the
(pulse, blood pressure) are part of the basic health check and are donation of blood or blood components on a voluntary basis.
carried out at this stage. Informed consent signifies that the donor has understood the
The venipuncture site should be examined to check that the donor’s questionnaire, has provided accurate answers and is willing to
veins are accessible and suitable to enable easy venipuncture. donate blood.

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It also indicates that the donor understands the blood donation Criteria for Blood Donor Selection
process, the possibility of adverse reactions to blood donation, the
Age
risks of the transmission of infections through donated blood and
The lower age limit for blood donation in most countries is 18 years,
the implications of any abnormalities that may be detected during
although in some countries national legislation permits 16–17 year-
the donation process and blood screening, and is providing consent
olds to donate provided that they fulfil the physical and
for post-donation notification and counselling, if detected to have a
haematological criteria required and that appropriate consent is
positive viral infection marker or any other abnormality.
obtained.
• Adolescents of either gender are at risk of iron deficiency
Donor Deferral:
during the pubertal growth spurt when the average daily total
Donors who do not to meet the selection criteria should be
requirement of absorbed elemental iron is 1.50 mg/day for
deferred on a temporary or permanent basis.
males aged 15–17 years and 1.62 mg/day for females
All deferred donors should be treated with respect and care in a
Upper age limits for blood donation of between 60 and 70 years
confidential manner and should be given a clear explanation of
have been implemented in the past because of concerns regarding
the reason for deferral and an opportunity to ask questions.
the increasing incidence of cardiovascular disease with age and the
They should be informed whether the deferral is to safeguard
potential risk of adverse reactions, which are more likely in first-
their own health and/or that of the recipient.
time donors.
It is the responsibility of the BTS to ensure that donors who are
• The usual upper age limit for blood donation is 65 years
deferred due to medical conditions are referred for further
• First-time donors older than 60 years and regular donors over
investigations and management, as appropriate.
the age of 65 may be accepted at the discretion of the
Temporarily deferred donors should be advised on when they
responsible physician
could donate and encouraged to return
• First-time donors over 60 years should make their first
A donor deferral registry (DDR) is a confidential list of donors
donation at a donation site where a physician is available
who are positive for a transfusion-transmissible infection and
who have been permanently deferred.
A DDR is used to monitor the incidence and prevalence of such DONOR APPEARANCE AND INSPECTION
infections in the donor population and may also assist in Prospective donors should be accepted only if they appear to be in
identifying areas that require strengthening in the donor good health and comply with donor selection criteria
selection process. The prospective donor should appear generally well and should not
be febrile, breathless or suffering from a persistent cough.
The colour of exposed skin and mucous membranes should be
normal, with no jaundice, cyanosis, flushing or pallor, and no signs
of skin infection, rash or obviously enlarged lymph nodes.
• If body piercings or tattoos are present, the risk of
transfusion-transmissible infections (TTI) should be assessed

MINOR ILLNESSES
Individuals with a history of recent infection: defer for 14 days
following full recovery and cessation of any therapy, including
antibiotics

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• Minor non-specific symptoms (e.g. general malaise, pain, DONOR IRON STATUS
fever, headache, cough, diarrhoea) may indicate the presence Haemoglobin screening safeguards anaemic individuals from
of an acute infection that may be transmissible by transfusion. donating blood and also protects returning donors from donation-
• Donors should be asked to confirm that they are free from induced iron deficiency (DIID), the depletion of iron stores by
such symptoms on the day of donation and that they have repeated donations.
fully recovered from any recent infection(s). Collecting a unit of blood from a donor with a normal haemoglobin
• Individuals suffering from minor illnesses and not feeling well level also provides good quality blood components, with adequate
should not donate blood. and consistent haemoglobin content in the collected blood.
In determining the lower limits of haemoglobin for whole blood
WEIGHT donation and implementing haemoglobin screening, the BTS should
Prospective donors of whole blood donations should weigh at least consider:
45 kg to donate 350 ml ± 10% and 50 kg to donate 450 ml ± 10% • A haemoglobin level of not less than 12.0 g/dl for females and
Prospective donors of apheresis platelet or plasma donations should not less than 13.0 g/dl for males as the threshold
weigh at least 50 kg Only sterile disposable lancets should be used for blood sampling
Prospective donors of double red cell apheresis donations should
have an estimated blood volume of more than 5 litres; this FLUID INTAKE AND FOOD
requirement is generally met by non-obese individuals weighing Most BTS guidelines recommend that donors should maintain their
more than 70 kg. usual food and fluid intake before donation but should avoid heavy
or fatty meals which may result in a lipaemic donation that may
VITAL SIGNS need to be discarded
PULSE The BTS should consider providing 500 ml drinking water to donors
• A normal pulse rate of 50–100 per minute and a regular before donation to minimize the risk of vasovagal reactions
rhythm are indicators of good health; many BTS recommend
that these are examined prior to donation. PREGNANCY, LACTATION AND MENSTRUATION
• The ability to detect significant abnormalities of pulse rate or The average woman needs about 350–500 mg additional iron to
rhythm depends on the skill and experience of staff. maintain iron balance during pregnancy.
BODY TEMPERATURE Female donors should be deferred during pregnancy and for a
• A prospective donor who is febrile – defined as a core oral sufficient time after delivery (or following abortion or miscarriage)
temperature more than 37.5°C– is by definition unwell and and during lactation to allow for the recovery of iron stores.
should be deferred. Menstruation is not a reason for deferral. However, women who
• Fever can indicate any number of medical conditions and report regular excessive menstrual bleeding and are found to have
infections, but is usually associated with other symptoms low haemoglobin levels should not donate blood and should be
BLOOD PRESSURE (BP) referred for medical assessment.
• A normal blood pressure (systolic 180 mmHg, diastolic 100 Contracting and relaxing the muscles in the legs, arms and abdomen
mmHg) is generally regarded as an indicator of good health. during donation may reduce the risk of vasovagal reactions,
particularly among female donors
The BTS should encourage donors to practise applied muscle
tension during blood donation

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Accept female donors during menstruation, provided that they feel NOTE:
well and meet the minimum haemoglobin level for blood donation One unit of whole blood can be broken down into one unit of
Defer female donors during pregnancy and up to 6 months after packed red cells, one unit of platelets, and one unit of fresh
delivery or termination of pregnancy frozen plasma/cryoprecipitate.
Defer female donors during lactation

Reducing the risk of transfusion-associated acute lung injury (TRALI) Methods for collection of blood for preparation of blood components:
The gender of the donor may influence the type of blood Single whole blood donation
component prepared from the donation.
After collection of a unit of whole blood in the primary bag, blood
Plasma-rich blood components from multiparous women are
components can be separated from one another by differential
more likely to cause TRALI and related disorders than those from
centrifugation due to differences in their specific gravities.
males, because plasma from such women is likely to contain
After their separation, various components can be transferred from
alloimmune-reactive antibodies; these include antibodies to
one bag to another in a closed circuit thus avoiding exposure to the
human leucocyte antigens (HLA) or to human neutrophil antigens
external environment and maintaining the sterility.
(HNA), which are transferred passively during transfusion, to the
Blood should be processed for component separation within 6 hours of
possible detriment of a recipient who possesses the
collection
corresponding antigen.

Apheresis
Apheresis donation uses a specialized programmable machine that
Frequency of Donation processes over 3.5 L of the donors' blood and separates whole blood
The minimum interval between donations of whole blood should be into the blood component that is required (either up to 750 mL plasma
12 weeks for males and 16 weeks for females or 100–400 mL platelets), returning the remainder to the donor
The minimum interval between donations of platelets should be 4 (sometimes with saline fluid replacement).
weeks Depending on the component that is separated and removed, the
The minimum interval between donations of plasma should be 2 procedure is called plateletpheresis, leukapheresis, or plasmapheresis.
weeks Blood is withdrawn and returned via the 16-gauge apheresis needle
The minimum interval before an apheresis platelet or plasma (single arm/needle procedure).
donation should be 4 weeks following a whole blood donation, an Whole blood is mixed with anticoagulant and the required blood
apheresis red cell donation or a failed return of red cells during component (plasma or platelets) is collected into the attached plastic
apheresis blood bag.
In determining the frequency of donation and whether iron Red cell products can also be collected via apheresis, although this is
supplementation is given, the BTS should consider: less common than plasma and platelet apheresis.
• The need for longer donation intervals for young donors and Apheresis donation takes an average of 1 hour.
female donors of childbearing age Upon completion of the apheresis collection, the blood component
• Assessing the feasibility and affordability of providing iron requires no additional processing and has been depleted of the
supplementation to donors susceptible to donation-induced majority of white blood cells during the collection via filtration and/or
iron deficiency, especially women, adolescents, and repeat centrifugal conditions built in to the apheresis procedure.
and regular donors

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
Double red blood cell donation Directed or designated donation
A person donates twice as many red blood cells as with a single Family members or friends can donate blood specifically for one
donation of whole blood. another if the recipient's and donor's blood types and Rh factors are
This double donation is possible because the person gives only red compatible.
blood cells rather than whole blood. For some recipients, knowing who donated the blood is comforting,
Whole blood is drawn from the donor, and a machine that separates the although a donation from a family member or friend is not necessarily
blood into its components selectively removes the red blood cells and safer than one from an unrelated person.
returns the rest of the blood components (platelets and plasma) to the Blood from a family member is tested, as are all blood samples, and
donor. then treated with radiation to prevent graft-versus-host disease, which,
Some fluid is also given to the donor intravenously because otherwise, although rare, occurs more often when the recipient and donor are
the donor's blood pressure could become low enough to cause related.
symptoms, such as light-headedness or loss of consciousness.
After double red blood cell donation, people may be less able to Whole Blood
exercise vigorously for a few days. One unit of donor blood collected in a suitable anticoagulant-
Double red blood cell donation can be done as often as once every 112 preservative solution (citrate phosphate dextrose adenine or CPDA-1).
days (every 16 weeks). Whole blood donation involves collection of 450 mL (±10%) blood from
Some experts recommend that people take iron supplements after the antecubital vein via a 16- gauge needle into the attached specialized
double red cell donation so that their body can replace the donated red PVC plastic blood collection bag typically containing 63 mL
blood cells more rapidly. anticoagulant.
Whole blood donation usually takes 5–10 min.
Autologous transfusion The donated whole blood is then processed into components including
Donors are recipients of their own blood. red cell concentrate, platelet concentrate and plasma, via centrifugation
The person takes iron pills after donating the blood to help the body Processing of whole blood must commence within 24 hours of blood
replenish the lost blood cells before surgery. donation in order to maintain maximum quality of the components.
Also, during some types of surgery and in certain kinds of injuries, blood Whole blood is stored in an approved blood bank refrigerator at 1°-6°C.
that is lost can be collected, washed, and immediately given back to the If collected in ACD or CPD, shelf-life is 21 days and CPDA-1 is 35 days
person (intraoperative blood salvage). It does not contain functionally effective platelets and labile coagulation
An autologous transfusion eliminates the risks of incompatibility and factors (Factor V and Factor VIII).
blood-borne disease (unless the wrong blood is given by mistake). Transfusion of whole blood should commence within 30 minutes of
However, doctors do not use this technique as often as standard removal from the refrigerator and should be complete within 4 hours of
transfusion because the general blood supply is very safe due to starting.
rigorous donor screening and testing. Transfusion of one unit raises hemoglobin by 1 gm/dl or hematocrit by
In addition, older people may not tolerate donating blood befor surgery 3%.
because they are more likely to have side effects during donation such Indications
as low blood pressure and fainting. • Acute blood loss with hypovolemia
Older people are also more likely to have fewer blood cells than normal • Exchange transfusion in neonates
(a low blood count) to begin with. • Non-availability of red cell concentrate or suspension
Also, autologous transfusion is more expensive than standard
transfusion.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
APPENDICES Sample Problem # 2:
Mathew, weighing 95 lbs, wants to donate blood for a friend. How
Sample Problem # 1: much must be removed from the blood bag?
Given: 40kg individual that to donate a blood
a. Volume to Collect: 50 kg = 110 lbs
40 𝑘𝑔 a. Volume to Collect:
= 𝑥 450 𝑚𝐿
50 𝑘𝑔 95 𝑙𝑏𝑠
= 𝑥 450 𝑚𝐿
110 𝑙𝑏𝑠
360 mL volume of blood to be collected
388.64 mL volume of blood to be collected
b. Reduced Volume of Anticoagulant:
360 𝑚𝐿 d. Reduced Volume of Anticoagulant:
= 𝑥 63 𝑚𝐿
450 𝑘𝑔 388.64 𝑚𝐿
= 𝑥 63 𝑚𝐿
50.4 mL 450 𝑚𝐿
54.4 mL
c. Volume of Solution to be removed: e. Volume of Solution to be removed:
= 63 𝑚𝐿 − 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 = 63 𝑚𝐿 − 54.4 𝑚𝐿

Answer: 12.6 mL Answer: 8.6 mL

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
BLOOD COMPONENTS
Legend: Cellular Components
Transcription Bullet
Notes/Module Packet Bullet Legend
PPT Red cells
o Packed red cells
MODULE OUTLINE
o Red cells in additive solution (Red cell suspension)
I. Organizations that Regulate or Accredit the Immunohematology
o Leukocyte-poor red cells
Laboratory
o Washed red cells
II. Blood Donor Selection
o Frozen red cells
III. Types of Donation
o Irradiated red cells
IV. Blood Component
Platelets
V. Safe Storage of Blood
o Platelet concentrate (Random donor platelets prepared
VI. Packing Blood Components for Transportation
from whole blood unit)
o Apheresis platelets (Single donor platelets) (SDP)
LEARNING OUTCOMES
Granulocytes
At the end of this module, the student shall be able to:
o granulocyte concentrate
1. Identify the organizations that regulate or accredit the
immunohematology laboratory.
2. State the minimum acceptable levels for the following tests in Cellular Components: Red Cells
allogenic & autologous donation Packed red cells
3. Identify the medical history information that would cause INDICATIONS:
permanent deferral or temporary deferral and state the length of • Anemia: Chronic severe anemia, severe anemia with
deferral period. congestive cardiac failure, anemia in elderly
4. Explain the reasons for permanent or temporary deferral given the • Acute blood loss (transfused along with a crystalloid or a
various medical conditions. colloid solution)

Packed red cells are prepared by removing most of the plasma from
one unit of whole blood
Whole blood is either allowed to sediment overnight in a
refrigerator at 1-6°C or is spun in a refrigerated centrifuge.
Supernatant plasma is then separated from red cells in a closed
system by transferring it to the attached empty satellite bag.
Red cells and a small amount of plasma are left behind in the
primary blood bag.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Packed red cells have a high viscosity and therefore the rate of Such red cells are used for IgA-deficient individuals who have
infusion is slow. developed anti-IgA antibodies, as exposure will lead to anaphylaxis.
Transfusion of one unit of red cells increases hemoglobin by 1 gm%
(or increases hematocrit by 3%). Frozen red cells
If a cryoprotective agent such as glycerol is added, red cells can be
Red cells in additive solution (Red cell suspension) stored frozen for up to 10 years.
INDICATIONS: This method can be used for storage for donor red cells with rare
• Anemia: Chronic severe anemia, severe anemia with blood groups, for future autologous transfusion, and for individuals
congestive cardiac failure, anemia in elderly who have repeated febrile nonhemolytic transfusion reactions.
• Acute blood loss (transfused along with a crystalloid or a
colloid solution) Irradiated red cells
Gamma-irradiation of red cells inactivates lymphocytes and
These are red cells with minimal residual plasma and an additive prevents graft vs. host disease.
solution (SAG-M which contains saline, adenine, glucose, and Irradiated red cells are indicated for intrauterine or premature
mannitol). neonate transfusions, and in individuals with immunodeficiency,
This increases shelf life from 35 days to 42 days. and in those receiving blood from first-degree relative donors.
After collection of whole blood in the primary collection bag
(containing CPDA-1), maximum amount of plasma is removed (after
Cellular Components: Platelets
centrifugation) and transferred to one satellite bag.
The additive solution from the second satellite bag is transferred INDICATIONS:
into the primary collection bag (containing packed red cells) in a • Bleeding due to decreased platelet production
closed system. • Bleeding in hereditary disorders of platelet function
• Massive blood transfusion
Leukocyte-poor red cells
INDICATIONS: Platelet concentrate (Random donor platelets prepared from whole
• Prevention of HLA immunization in patients who are likely to blood unit)
receive allogeneic bone marrow transplantation Platelet concentrates can be obtained from single donor units or by
• Prevention of febrile nonhemolytic transfusion reactions in plateletpheresis.
persons receiving multiple transfusions One unit of whole blood is centrifuged (light spin) to obtain platelet-
• Prevention of transmission of cytomegalovirus. rich plasma (PRP).
PRP is then transferred to the attached satellite bag and spun (high
Leukocyte-poor red cells contain < 5 × 106 white cells per bag. spin) to get platelets at the bottom and supernatant plasma.
Methods for leukocyte depletion are: Most of the supernatant is returned back to the primary collection
• Leukocyte-reduction filters bag or to another satellite bag, leaving behind 50-60 ml of plasma
• Removal of buffy coat with the platelets.
Platelets are stored at 20°-24°C with continuous agitation (in a
storage device called platelet agitator).
Washed red cells
Maximum period of storage is 5 days.
Red cells can be washed with normal saline to remove plasma
Transfusion of one unit will raise the platelet count in the recipient
proteins, white cells, and platelets.
by about 5000/μl.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
The usual adult dose is 4-6 units of platelet concentrate (or 1 FFP is prepared from whole blood within 6 hours of collection
unit/10 kg of body weight). because after this time labile coagulation factors are lost.
These units (which are from different donors) are pooled into one Plasma is separated from whole blood by centrifugation, expressed
bag before transfusion. into the attached satellite bag, and rapidly frozen at –20°C or at
This dose will raise the platelet count by 20,000 to 40,000/μl. lower temperature.
FFP contains all the coagulation factors.
Apheresis platelets (Single donor platelets) FFP can be stored for 1 year if temperature is maintained below –
In platelet pheresis, a donor is connected to a blood cell separator 25°C.
machine in which whole blood is collected in an anticoagulant When required for transfusion, FFP is thawed between 30- 37°C and
solution, platelets are separated and retained, and remaining then stored in the refrigerator at 2-6°C.
components are returned back to the donor. Since labile coagulation factors rapidly deteriorate, FFP should be
With this method, a large number of platelets can be obtained from transfused within 2 hours of thawing.
a single donor (equivalent to 6 units of platelet concentrate).
This method is especially suitable if HLA-matched platelets are Cryoprecipitate
required (i.e. if patient has developed refractoriness to platelet INDICATIONS:
transfusion due to the formation of alloantibodies against HLA • F VIII deficiency (if F VIII concentrate is not available)
antigens). • Von Willebrand disease
• Deficiency of fibrinogen.

Cellular Components: Granulocytes Cryoprecipitate is prepared from plasma that has been freshly
separated (within 6 hours of collection) by rapidly freezing it at -
Granulocyte Concentrate
20°C or lower and thawing it slowly at 4-6°C.
A white flocculent precipitate and plasma are obtained.
The mixture is centrifuged and supernatant plasma is removed
Plasma Components leaving behind sediment of cryoprecipitate suspended in 10-20 ml
Legend of plasma.
Fresh frozen plasma (FFP) The unit is then refrozen (-20°C or colder) and can be stored at this
Cryoprecipitate temperature for 1 year.
When needed, cryoprecipitate is thawed at 30-37°C, required
Fresh frozen plasma (FFP) donations are pooled and transfused to the patient.
INDICATIONS: Cryoprecipitate contains
• Multiple coagulation factor deficiencies: liver disease, • F VIII
warfarin overdose, massive blood transfusion • von Willebrand factor
• Disseminated intravascular coagulation • fibrinogen
• Inherited deficiency of a coagulation factor for which no • F XIII
specific replacement therapy is available • fibronectin.
• Thrombotic thrombocytopenic purpura

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Plasma Derivatives A serious risk of PCC is thrombotic complications due to the
Legend presence of small amounts of activated coagulation factors.
Human albumin solutions
F VIII concentrate Immunoglobulins
F IX concentrate Immunoglobulins are obtained by cold ethanol fractionation of large
Prothrombin complex concentrate (PCC) pools of human plasma.
Immunoglobulins They are of two types: specific and nonspecific.
o Non-specific immunoglobulins
o Specific immunoglobulins Non-specific immunoglobulins:
Plasma derivatives are manufactured by fractionation of large INDICATIONS:
volumes of pooled human plasma. • passive prophylaxis of viral infections like hepatitis, rubella,
and measles
Human albumin solutions • treatment of hypogammaglobulinaemia,
Albumin is prepared by cold ethanol fractionation of pooled plasma • autoimmune thrombocytopaenic purpura to induce a rise
and is sterilized during manufacture to destroy viruses and bacteria. in platelet count
Albumin is used as a replacement fluid in therapeutic plasma • neonatal sepsis
exchange, and for treatment of diuretic-resistant edema of
hypoproteinemia. These are derived from the pooled plasma of non-selected
donors.
F VIII concentrate
Freeze-dried F VIII concentrate is prepared by fractionation from Specific immunoglobulins:
large pools of fresh frozen plasma. They are obtained from donors who have selected high titer IgG
To reduce the risk of transmission of viral infections, it is treated antibodies.
with heat or chemicals during manufacturing process. Anti-RhD immunoglobulin is prepared from plasma of Rh-
F VIII concentrate is the treatment of choice for treatment of negative donors who have produced anti-D following
hemophilia A and severe von Willebrand disease. immunization; it is used for prevention of sensitization to RhD
antigen in Rhnegative women giving birth to a Rh-positive baby.
Other specific immunoglobulins include hepatitis B immune
F IX concentrate
globulin, varicella-zoster immune globulin, and tetanus immune
globulin that are used for passive prophylaxis of infections.
Prothrombin complex concentrate (PCC)
MAIN USES/ INDICATIONS:
• Deficiency of F IX
• Deficiency of F VIII with development of inhibitors against F
VIII
• Inherited deficiency of factors II, VII, and X.

PCC contains factors II, VII, IX, and X, and also protein C and S.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
SAFE STORAGE OF BLOO D Factor V and Factor VIII, however, which are essential in the clotting
Whole blood mechanism, will deteriorate and diminish in quantity if they are not
Whole blood and red cells must always be stored at a temperature stored at –20 °C or lower and greatly reduce the clotting activity of
between +1°C and +6 °C. the plasma.
The main reasons for giving a blood transfusion are to restore or FFP may be given to a patient to restore or help to maintain
help to maintain the body’s oxygen-carrying capacity and the coagulation factors such as Factor V or Factor VIII.
volume of blood circulating around the body. Plasma should not be used as a volume expander unless crystalloids
• If blood is not stored at between +1 °C and +6 °C, its oxygen- and colloids are unavailable
carrying ability is greatly reduced.
• The anticoagulant/preservative solution in the blood bag Cryoprecipitate
contains nutrients for the blood during storage and stops the Cryoprecipitate is the cold insoluble portion of plasma remaining
blood from clotting. after FFP has been thawed between +1 °C and +6 °C and is useful for
• The red cells can only carry and deliver oxygen if they remain correcting certain coagulation defects.
viable: that is, if they retain the same properties as they have It contains approximately 50% of Factor VIII and von Willebrand
during their normal circulation in the body. Factor, 20–40% of fibrinogen and some of the Factor XIII originally
Another important reason for storing blood between +1 °C and present in the fresh plasma.
+6 °C is to keep the growth of any bacterial contamination in the Plasma is separated from red cells within 6 to 8 hours of collecting
unit of blood to a minimum. blood.
• If blood is stored above +6 °C, bacteria that may have The plasma is frozen solid rapidly, certainly within 30 minutes of
inadvertently entered the unit during collection may grow to separation from the cells.
such an extent that transfusion of the contaminated blood The plasma is then thawed slowly at below +4 °C.
could be fatal. In order to get the maximum yield of Factor VIII in the
The lower limit of +1 °C is also very important. cryoprecipitate from a blood unit it is important to adhere strictly to
• This is because red cells are very sensitive to freezing. the standard procedures for the collection, storage and processing
• If they are allowed to freeze, the red cell membranes rupture of the component.
and the haemoglobin is released; that is, the cells are The stability on storage is dependent on the storage temperature
haemolysed. available.
• The transfusion of haemolysed blood can also be fatal. • The optimal storage temperature is below –30 °C.

Fresh frozen plasma Platelet concentrates

Fresh frozen plasma (FFP) is plasma that has been separated from a Platelet transfusions are used to prevent spontaneous bleeding or
unit of whole blood within 6 to 8 hours of collection, and has been to stop bleeding in patients with established thrombocytopenia or
rapidly frozen and maintained at all times at a temperature of – platelet dysfunction
20 °C or lower. • Ex., hypoplastic anaemia or bone marrow failure – due to
There is no lower temperature limit for the storage of FFP, although replacement with malignant cells or to the effects of
the optimal temperature is –30 °C or lower chemotherapy.
Plasma contains water, electrolytes, clotting factors and other Both manual and automated methods can be used in the
proteins (mostly albumin), most of which are stable at refrigerator preparation of platelet concentrates.
temperature, i.e. +1 °C to +6°C. Lower temperatures adversely affect platelet function and viability.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
• For this reason, whole blood should be kept at between Storage conditions and expiry dates should also be strictly adhered
+20 °C and +24 °C until it is processed into platelet to in order to prevent septic shock for the recipient.
concentrates and other blood components. After the hermetic seal is broken, platelet concentrates should be
Platelet-rich plasma must be separated from whole blood by transfused as soon as possible, but definitely within a maximum of 4
centrifugation within 8 hours of phlebotomy. hours of storage at between +20 °C and +24 °C.
Additional centrifugation and removal of most of the supernatant
plasma may then concentrate the platelets. Plasma derivatives
Platelet concentrates should be stored at a temperature of between Unlike blood components, plasma derivatives such as albumin or
+20 °C and +24 °C with continuous agitation. immunoglobulin are concentrated, sterile specific proteins,
• This is essential to prevent platelet aggregation which results obtained from large pools of donor plasma through a complex
in loss of viability. pharmaceutical process called plasma fractionation.
The shelf life and transport conditions differ according to the type of They are used to treat patients with specific protein deficiencies or
plastic bag used to store the component. requirements for passive immunity.
Platelet concentrates stored at between +20 °C and +24 °C maintain
their function and viability better than refrigerated platelet
concentrates. PACKING BLOOD COMPONENTS FOR TRANSPORTATION
Current plasticizers used in the manufacture of plastic bags allow for The following general observations must be kept in mind:
storage of up to five days, because gaseous exchange takes place
between the container and the environment and this results in the Label the container THIS WAY UP with an arrow.
maintenance of pH in the component, which is critical for platelet Ice should be placed above the blood because cool air moves
storage. downwards.
o Cubed wet ice may be better than chipped or broken ice for
Note:
long distance shipments of blood because it melts more
If no platelet agitator or rotator is available, it is not possible to store
slowly.
platelets.
o Ice packs can be used at –5 °C or below.
Once prepared, they must be transfused immediately unless the
The recommended storage conditions must be maintained when
blood bank is equipped with:
blood is moved from one location to another, including:
an air-conditioned facility with a temperature monitoring system
o from a mobile or satellite collection site to the laboratory
that will maintain an ambient temperature of between +20 °C
o from the blood bank to a different facility (to a hospital or
and +24 °C or
clinic or another blood bank)
- a platelet incubator that will keep the platelet concentrates at a
o from the blood bank to hospital wards or operating rooms
temperature of between +20°C and +24 °C.

Since platelet concentrates are stored at room temperature, they

pose a greater risk for bacterial proliferation.
SOPs on the cleaning of the venipuncture site prior to donation
must be strictly followed, and the disinfectant in use must undergo
regular quality control checks.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 2)
Red cell components: at no point should ice be allowed to come
into direct contact with the blood as the red cells nearest to the ice
may freeze and haemolyse.
o In boxes shipped long distances or at high environmental
temperatures, the volume of ice should at least equal that
of the blood.
o In an insulated container, the temperature can be
considered to be in the +2 °C to +10 °C range as long as
unmelted ice is still present on arrival at destination.

Plasma: there should be at least as much wet ice in the cold box as
there is plasma.
o It is important to protect the frozen plasma units during
transportation.
o If possible, they should have been placed in cardboard
boxes before freezing to protect the bags from developing
small cracks.
o A simple method to determine if plasma units have thawed
and refrozen is to place a rubber band around the unit at
the time of preparation.
o Once the unit freezes it leaves an indentation at the sides.
o If the unit has thawed, or thawed and refrozen, the
indentation will not be there.

Platelets: containers for transporting platelets should be

equilibrated at a temperature of +20 °C to +24 °C before use.
o If outdoor temperatures are extremely high, special
chemical, coolant pouches are available that may be
shipped with platelets and will maintain temperatures of
approximately +20 °C to +24 °C for up to 12 hours.
o Also available are containers with a power source that
maintains temperatures between +20 °C and +24 °C.
o Platelets should reach their destination within 24 hours,
which is the maximum time allowed without agitation.

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 3)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend:
Transcription Bullet 14. State the expected incremental increase of a
Notes/Module Packet Bullet a) patient’s hematocrit level following transfusion of each unit of RBCs
PPT and
MODULE OUTLINE b) platelet count following transfusion of each unit of platelet.
15. Identify the group of recipients who are at high risk of infection from
I. Test required for donations
transfusion of cytomegalovirus-positive RBcs or platelets.
II. Therapeutic Apheresis
16. Explain the role of irradiation in the prevention of tansfusion-
III. Transfusion Therapy
associated graft- versus-host disease
17. State the respective blood products of choice for treating von
MODULE OUTLINE Willebrand’s disease, hemophilia A, and hemophilia B
At the end of this modules, the student shall be able to: 18. List the main advantages and disadvantages of autologous
1. Differentiate among the four different methods of autologous blood transfusion.
donation. 19. Identify the most important factors to consider when emergency
2. Describe the procedure for a whole blood donation and post transfusion is indicated.
phlebotomy care instructions for the donor. 20. Define massive transfusion.
3. Differentiate mild, moderate, and severe donor reactions and list
recommended treatments for each.
4. List the tests required for allogenic, autologous, and apheresis
TESTS REQUIRED FOR DONATIONS
donortions.
5. State the acceptable interval of donation for allogenic and apheresis ABO/Rh
donors. Testing for the donor’s ABO group must include both forward and
6. List the labeling criteria for a unit allogenic and autologous blood. reverse grouping.
7. Define apheresis; leukapheresis, platelepheresis, plamapheresis, & The ABO group must be determined by testing the donor RBCs with
erythcypheresis. anti-A and anti-B reagents and by testing the donor serum or
8. Compare and contrast the procedures of continuous flow plasma with reagent A1 cells and B cells
centrifugation and intermittent flow centrifugation. The donor’s Rh type should be determined by testing with anti-D
9. List the components that can be collected using apheresis technology. reagent at the immediate spin phase.
10. Explain the rationale for the basic types of therapeutic apharesis.
11. Explain the adverse effects of apharesis. Antibody Screen
12. Describe the blood products that are currently available for Donors with a history of pregnancy or transfusion must be tested
therapeutic us - the composition and approximate volume of each. for unexpected antibodies to RBC antigens
13. Select the appropriate blood product for patient with specific Uses a pooled cell reagent:
disorders. o This reagent contains two or three individual cells pooled
into a single reagent.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o The pooled screening cell reagent contains all of the Deferral
required antigens and is capable of detecting the presence A positive result for anti-HBc along with a positive HBsAg would place
of the clinically significant alloantibodies. the donor in a permanently deferred category.
The antibody screen performed on a recipient prior to transfusion A positive test for anti-HBc without an accompanying positive test for
uses two or three individual reagent cells HBsAg or HBC is not cause for donor deferral unless it occurs on more
than one occasion or on two consecutive tests.
HBsAg o The products will not be used for transfusion even if the donor is not
Methods currently available include: deferred for a positive result
o Enzyme immunoassay (EIA)
o Chemiluminescence (ChLIA)
o Nucleic acid amplification (NAT) Anti-HCV
Is able to detect small amounts of viral nucleic acid in blood before
Deferral antibodies or viral proteins such as HCV core antigen are detectable
If the EIA or ChLIA tests are initially positive, they must be repeated by current methods.
in duplicate and a confirmatory (neutralization) test must be Screening tests for anti-HCV involve:
performed. o EIA
o If the confirmatory test is positive, the donor is considered to be o ChLIA
infected (acute or chronic) with the hepatitis B virus (HBV) and must o NAT
be permanently deferred Confirmatory methods include:
NAT is not mandated for donor screening o RIBA (recombinant immunoblot assay)
o If the initial HBsAg testing is reactive but the confirmatory testing is o HCV RNA
not (unconfirmed positive), all the current products are discarded
but the donor need not be permanently deferred, provided the Deferral
antibody to hepatitis B core (anti-HBc) testing is also nonreactive. An individual who is positive by RIBA is considered to have the
o The donor is deferred for 8 weeks and may be reinstated if the next HCV antibody and would be indefinitely deferred as a blood
HBsAg testing is nonreactive. donor
Donors who test repeat reactive for HCV screening tests must be
deferred, and the components or products prepared are
Anti-HBc discarded.
The methods employed are similar to those for HBsAg. If the confirmatory RIBA or NAT testing are nonreactive, the
Presence of HBc antibody in the donor serum suggests the donor may be considered for reentry.
possibility of HBV infection, either acute or chronic.

Anti-HIV-1/2
All donor units must be screened for the presence of the human
immunodeficiency virus (HIV-1/2) antibody using an FDA-approved
method.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o If the initial screening test is negative, the unit is suitable for Any individual units that test reactive should be discarded, and the
transfusion; donor should be deferred from further donations for 120 days
o If it is positive, the test must be repeated in duplicate.
Screening tests include: Syphilis
o EIA A serologic test for syphilis is done because the disease is
o ChLIA characterized as being sexually transmitted and places the donor at
o NAT (run using 16 to 24 donation samples per pool) higher risk for possible exposure to hepatitis and HIV
Confirmation tests for HIV include: Screening tests include:
o Western blot (Wb) o Rapid plasma regain (RPR)
o Immunofluorescence assay (IFA) o Venereal Disease Research Laboratory (VDRL).
o Results are expressed as positive, negative, or indeterminate o Both tests are based on Reagin, or antibody directed toward
cardiolipin particles.
Anti-HTLV-I/II o Cardiolipin-like antibodies have been documented in
HTLV-I virus or human T-cell lymphotrophic virus type I is the persons with untreated syphilis infections.
causative agent of adult T-cell leukemia o Antibody will agglutinate cardiolipin carbon particles in the
Associated with a neurological disorder called HTLV-associated form of visible flocculation.
myelopathy. The confirmatory test for syphilis is:
HTLV-II has been shown to have about 60% homology with type I o FTA-ABS or fluorescent treponemal antibody absorption
and is prevalent among intravenous drug users in the United States. test
Persons can contract both viruses from transfusion via infected o Indirect immunofluorescence is used to detect antibodies to
lymphocytes the spirochete T. pallidum, the agent that causes syphilis
Screening test methodologies include: o If the STS or VDRL screening test is reactive or
o EIA indeterminate, the components from that donation must be
o ChLIA discarded and the donor deferred unless a confirmatory
Confirmatory tests include: FTA-ABS test is nonreactive.
o Western blot o If the FTA-ABS test is nonreactive, the donor may be
o RIPA reentered and the components labeled as reactive with the
o NAT testing screening test.
o The donor of a confirmed reactive test may be reentered
WNV RNA into the donor pool after 12 months and documentation of
Test include: completion of treatment
Mini-pool (MP-NAT)
o Uses pools of 6 to 16 donor samples T. cruzi (Chaga’s Disease)
o Is routinely used when risk of WNV infection in the Chagas’ disease is a parasitic infection that is endemic to Mexico
geographical area is low and Central and South America
The infection is generally mild but persists, without symptoms,
Individual donor (ID-NAT) method throughout the infected individual’s life and can be transmitted
o Recommended when the risk is high through blood transfusions
All donors (allogeneic and autologous) are to be tested once.

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE
o If the results are nonreactive, the donor need not be to remove a particular blood component for therapeutic purposes
retested with each donation. (therapeutic apheresis).
o This is because most donors who are infected in the United The process of removing plasma is termed plasmapheresis. In a
States have a chronic infection that was acquired when similar manner, platelets (plateletpheresis), red blood cells
residing in a country where the disease is endemic. (erythrocytapheresis), or leukocytes (leukapheresis) can be
o Repeat reactive donors must be deferred indefinitely and removed (or collected) using apheresis technology.
the products quarantined and destroyed Citrate is used as the primary anticoagulant in apheresis procedures.
o The binding of calcium ions by citrate inhibits the calcium-
Platelet Bacterial Detection dependent coagulation cascade
Platelet components are particularly at risk because they are stored o Citrate is mixed with the blood immediately as it is removed
at room temperature from the donor’s (or patient’s) vein, and effectively
For apheresis-derived platelets, it is recommended that a culture anticoagulates the blood before it enters the apheresis
method be used that would usually be performed by the collecting machine
facility. The variables that are considered during an apheresis procedure
For whole blood–derived platelets, there are two choices. include:
For platelets that are pooled within 4 hours of transfusion, a new o Centrifuge speed and diameter
PGD test (Verax) can be used. o Duration of dwell time of the blood in the centrifuge
o It is a relatively easy test to perform, requires about 45 o Type of solutions added, such as anticoagulants or
minutes, and can be done by a transfusion service at the sedimenting agents
time of issue. o Cellular content or plasma volume of the patient or donor
For platelets that are pooled and stored as a pre-pooled product, a
culture-based test is recommended. Methods of Centrifugation
o Typically performed by the collection facility
INTERMITTENT FLOW CENTRIFUGATION (IFC)
Blood is processed in batches or cycles
PROCEDURES USED TO SEPARATE BLOOD COMPONENTS
Whole blood is drawn from an individual with the assistance of a
Apheresis pump
A procedure in which whole blood is removed from the body and To keep the blood from clotting, an anticoagulant is mixed with the
passed through an apparatus that separates out one (or more) blood as it is pumped into a centrifuge bowl through the inlet port
particular blood constituent. Can be performed as a single-needle procedure with only one
Returns the remainder of the constituents to the individual’s venipuncture
circulation. This is advantageous when collecting apheresis products from blood
It allows a larger volume of specific components to be collected donors.
It permits the removal of disease-causing or unwanted cellular or If both arms are used (double-needle procedure: one for
plasma constituents from a patient. phlebotomy and one for reinfusion), the amount of time for the
It is used to harvest stem cells from the peripheral blood of donors apheresis can be reduced
and patients, avoiding the need for extraction from the bone Procedure involved complete a cycle before beginning the next one
marrow Usually smaller and more mobile
Apheresis can be performed on a donor to collect a specific blood A single venipuncture may be used
component (donor apheresis), or it can be performed on a patient

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CONTINUOUS FLOW CENTRIFUGATION (CFC) RED BLOOD CELLS
The processes of blood withdrawal, processing, and reinfusion are RBCs collected by apheresis are typically collected as a double unit
performed simultaneously in an ongoing manner. (termed a 2RBC or double RBC procedure)
Because blood is drawn and returned continuously during a A clinical advantage to the collection of apheresis RBCs is reduced
procedure, two venipuncture sites are necessary donor exposure for the recipient since the patient can potentially
Separation of the components is performed by centrifugation, and receive two units from the same individual
the specific component is diverted and retained in a collection bag. Since the volume of RBCs being collected during a 2RBC procedure
The remainder of the blood is reinfused to the individual via the is greater than it would be for a whole blood donation, the
second venipuncture site. requirements for donor hematocrit are more stringent.
Two venipunctures are usually required o The hematocrit must be at least 40% regardless of gender,
and the level (hemoglobin or hematocrit) must be
Membrane filtration technology can also be used to separate determined by a quantitative method;
blood components o The use of copper sulfate is not acceptable
Membrane separators are typically composed of bundles of hollow If two units of RBCs are collected by apheresis, the donor must wait
fibers or flat plate membranes with specific pore sizes 16 weeks before providing another donation that includes RBCs.
This technology lends itself well to the collection of plasma, since If one RBC and one plasma and/or platelet unit are collected, the
pores can be sized to prevent the passage of even small cellular donor must wait 56 days before donating another red cell product
elements.
Filtration has several advantages: PLASMA
o The collection of a cell-free product In a plasmapheresis procedure, whole blood from the donor is
o The ability to selectively remove specific plasma proteins centrifuged, the plasma is diverted into a collection bag, and the
by varying the pore size cellular components (RBCs, platelets, WBCs) are returned to the
donor.
COMPONENT COLLECTION o This allows a larger volume of plasma to be collected from a
A qualified, licensed physician must be responsible for all aspects of donor, such that each apheresis unit (“jumbo” plasma) is
the apheresis program the volume equivalent of at least two whole-blood-derived
Operators of apheresis instruments may include: plasma units.
o Medical technologists (clinical laboratory scientists) Purposes:
o Nurses o It can be used to augment the inventory of fresh frozen
o Technicians trained on the job plasma (FFP) of a particular ABO group, especially group AB
Written, informed consent must be obtained from the donor o To prepare immune globulin to provide prophylaxis against
The apheresis procedure is more comfortable for the donor: infectious organisms in exposed individuals
o Since the needle used for venous access is typically of a o Is used commercially to collect plasma for further
larger gauge (i.e., smaller size) than for whole blood manufacturing into such products as intravenous immune
collection globulin (IVIG), hepatitis immune globulin, and Rh-immune
o The infusion of saline during the procedure helps reduce globulin.
donor reactions due to hypovolemia With infrequent plasmapheresis, donation occurs no more than
once every 4 weeks, and the donor requirements are the same as
for whole blood

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With frequent, or serial, plasmapheresis, donation occurs more o To protect the donor, if a double or triple apheresis platelet
frequently than once every 4 weeks is collected, 7 (rather than 2) days must elapse before the
o There must be at least 2 days between procedures and no donor is again eligible to provide apheresis platelets
more than two procedures in a 7-day period o A donor may undergo no more than 24 plateletpheresis
procedures in a rolling 12-month period
PLATELETS The total volume of plasma collected during any one procedure
Platelets obtained by an apheresis procedure provide the equivalent cannot exceed 500 mL (or 600 mL if the donor weighs 175 lb or
of six to eight whole-blood-derived platelets (random-donor more) or the volume of plasma cleared by the FDA for the
platelets) instrument
o This significantly decreases the donor exposure for a patient
In a plateletpheresis procedure, platelets along with a portion of GRANULOCYTES
plasma are removed, and the remaining RBCs, WBCs, and majority Granulocyte transfusions have been shown to be beneficial in some
of the plasma are returned to the donor severely neutropenic patients (neutrophil count less than 500/µL)
o The platelets are suspended in donor plasma in a collection who meet the following criteria:
bag specifically designed for platelet storage o Documented (clinically or by culture) infection for 24 to 48
A routine plateletpheresis procedure typically takes 45 to 90 hours that is unresponsive to standard antibiotic or
minutes antifungal therapy
Donor selection criteria for the plateletpheresis donor are the same o Bone marrow demonstrates myeloid hypoplasia
as for whole blood donation, with two additional requirements o There is a reasonable chance of bone marrow recovery (i.e.,
o Prior to each plateletpheresis procedure, a sample must be the neutropenia is reversible)
collected to determine the donor’s platelet count. Granulocyte transfusions have also shown favorable results in the
o The platelet count must be at least 150,000/µL in order to treatment of neutropenic neonates with sepsis
provide an adequate platelet collection and for the donor to Collection of granulocytes by apheresis provides a higher yield
safely undergo the collection procedure. product
o If the donor’s platelet count is less than 150,000/µL, he or Apheresis collection requires close communication between the
she is deferred from platelet donation until a subsequent blood center or apheresis center, the blood bank physician, and the
count is at least 150,000/µL patient’s physician
Plateletpheresis collections should not be performed on potential A minimum therapeutic dose is 1 × 10¹⁰ granulocytes per day
donors taking medications that interfere with platelet function, as The granulocyte yield is influenced by the donor’s neutrophil count
this would result in production of a suboptimal and therapeutically and the collection process
ineffective patient product During centrifugation of whole blood, granulocytes are found in the
Antiplatelet medications have differing deferral time periods: buffy coat between the RBC and plasma layers (see Fig. 14–2).
o 48 hours for aspirin, aspirin-containing medications, and the Adding the red cell sedimenting agent, hydroxyethyl starch (HES),
anti-inflammatory drug Feldene allows better separation of layers, resulting in an improved yield
o 14 days for clopidogrel (Plavix), ticlopidine (Ticlid), ticagrelor with reduced RBC contamination.31,32 Since HES is added directly
(Brilinta), and prasugrel to the apheresis circuit, some amount will enter the donor’s
The interval between plateletpheresis procedures must be at least 2 circulation during the collection procedure and ultimately be
days with no more than two procedures in a 7-day period removed by the reticuloendothelial system. Immediate side effects

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of HES are due to its colloid properties, including circulatory volume
expansion, with headaches and peripheral edema Adequate vascular access is mandatory during TA, as larger volumes
of blood are processed and the duration of the procedure is longer
THERAPEUTIC APHERESIS (TA) than for donor apheresis
Like donor apheresis, this involves the removal of a specific blood
component, with return of the remaining blood constituents to the Vascular access can be obtained via:
patient Peripheral veins
o However, with TA, since the component being removed is o If only one to two TA procedures are to be
considered pathological (or contributing to the patient’s performed, peripheral access can be used
underlying disease state), significantly larger volumes of o In such instances, two venous sites are necessary—
blood must be processed in order to remove as much of the one for removal and one for return
offending agent as possible Central veins
The TA procedure is classified according to the blood component A combination of both
removed:
o A cytapheresis procedure may be used to selectively Therapeutic plasma exchange (TPE) is the removal and retention of
remove RBCs, WBCs, or platelets the plasma, with return of all cellular components to the patient
o A plasmapheresis procedure is used to remove plasma o This is the most common TA procedure performed
when the pathological substance is found in the circulation o The purpose is to remove the agent in the plasma, such as an
o The indication categories for therapeutic apheresis are as antibody, toxin, or abnormal protein, that is causing the
follows: clinical symptoms
o TPE is also used to replace a normal factor or substance that
Category I Apheresis is standard and acceptable, either as may be missing or deficient in the patient’s plasma
primary therapy or as a first-line adjunct to other o During the TPE procedure, replacement fluids are being
initial therapies administered to maintain the patient’s intravascular volume,
Efficacy is based on well-designed randomized resulting in dilution of plasma proteins
controlled clinical trials or a broad base of published Therapeutic plateletpheresis can be used to treat patients who
experience have abnormally elevated platelet counts with related symptoms
Category II Apheresis is generally accepted in a supportive role or o During a plateletpheresis procedure, the platelet count will
as second-line therapy, rather than first-line therapy be decreased by 30% to 60%
Category III Apheresis is not clearly indicated based on insufficient Therapeutic leukapheresis has been used to treat patients with
evidence, conflicting results, or inability to document a hyperleukocytosis, defined as a WBC or circulating blast count of over
favorable risk-to-benefit ratio 100,000/µL
Decision-making should be individualized Erythrocytapheresis, or red cell exchange, removes a large number
Category IV Apheresis has been demonstrated to lack efficacy or of RBCs from the patient and returns the patient’s plasma and
be harmful, and should be discouraged in these platelets along with compatible allogeneic donor RBCs
disorders o The procedure is most commonly performed in patients
Clinical applications should be undertaken only under with sickle cell disease in order to decrease the number of
an approved research protocol hemoglobin S–containing RBCs, thereby treating or
preventing the complications (acute chest syndrome,

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impending stroke, unrelenting painful crisis) associated with Transfusion therapy is used primarily to treat two conditions:
the disease o Inadequate oxygen-carrying capacity because of anemia or
blood loss
The therapeutic goal is to decrease the level of hemoglobin S to less o Insufficient coagulation proteins or platelets to provide
than 30%: adequate hemostasis
This is usually accomplished with a single red cell exchange Blood and blood products are considered to be drugs because of
procedure, requiring from six to ten RBC units, depending on the their use in the treatment of disease
patient’s age and red cell volume How do we prepare blood components product in transfusion
The donor RBCs selected for transfusion should be ABO- and Rh- therapy?
compatible, relatively fresh (less than 10 days is preferable to
allow maximum in vivo survival), leukocyte-reduced, negative for Whole Blood
hemoglobin S (donor does not have sickle cell trait), and partially Whole blood should be used to replace the loss of both RBC mass
phenotype-matched for the Rh (C, c, E, e) and K1 antigens to and plasma volume
avoid future alloimmunization A definite contraindication to the use of whole blood is severe
The procedure can be considered for treatment of overwhelming chronic anemia
malaria or Babesia infections For a 70-kg (155-lb) adult, each unit of whole blood should increase
o Both of these protozoa infect red blood cells, and a the hematocrit level 3% or hemoglobin 1 g/dL.
pronounced parasitemia can occur o After transfusion, the increase may not be apparent until 48
o Red cell exchange has been shown to be beneficial for to 72 hours when the patient’s blood volume adjusts to
treating these patients when the parasite load is greater normal
than 10%
o A 1.5- to 2-volume red cell exchange should significantly If given that the RBC mass is only affected, plasma volume is
decrease the parasite load increased already. Transfusing and inducing can’t be done to the
Red cell exchange can be used to remove incompatible RBCs from whole blood of the patient
a patient’s circulation Patients suffering from severe chronic anemia has a reduce RBC
count/mass but even though the RBC mass is reduced but the plasma
volume is increased due to the compensation of the body
The patient will suffer from circulatory volume overload once
TRANSFUSION THERAPY transfused with plasma, causing for the patients to develop adverse
reaction such as pulmonary edema and heart failure
TRANSFUSION MEDICINE
It happens mostly in patients with pre-existing heart or kidney failure
Was created because there are people who are in need of blood and
other blood components in the treatment of disease
Red Blood Cells
A multi-disciplinary branch of medicine that is concern with the
RBCs are indicated for increasing the RBC mass in patients who
transfusion of blood and the blood components or products. Including require increased oxygen-carrying capacity
the proper selection and utilization of blood components as well as the The decreased RBC mass may be caused by:
removal of blood or blood components in the treatment of the o Decreased bone marrow production (leukemia or aplastic
diseases anemia)
o Decreased RBC survival (hemolytic anemia)
o Surgical or traumatic bleeding
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Transfusion of RBCs is contraindicated in patients who are well Freezing RBCs allows the long-term storage of rare blood donor
compensated for the anemia units, autologous units, and units for special purposes, such as
RBCs should not be used to treat nutritional anemia, such as iron intrauterine transfusion
deficiency or pernicious anemia, unless the patient shows sign of
decompensation (need for increased oxygen-carrying capacity) Platelets and Plateletpheresis
RBC transfusion is not to be used to enhance general well-being, Platelets are essential for the formation of the primary hemostatic
promote wound healing, prevent infection, expand blood volume plug and maintenance of normal hemostasis
when oxygen-carrying capacity is adequate, or prevent future Patients with severe thrombocytopenia (low platelet count) or
anemia. abnormal platelet function may have petechiae, ecchymoses, and
Each unit of transfused RBCs is expected to increase the hemoglobin mucosal or spontaneous hemorrhage
level 1 g/dL and the hematocrit level 3% in the typical 70-kg (154-lb) The thrombocytopenia may be caused by decreased platelet
human, the same as whole blood production (e.g., after chemotherapy for malignancy) or increased
In pediatric patients, a dose of 10 to 15 mL/kg will increase the destruction (e.g., disseminated intravascular coagulation [DIC])
hemoglobin about 2 to 3 g/dL or the hematocrit 6% to 9% o Massive transfusion may also cause thrombocytopenia
because of the rapid consumption of platelets for hemostasis
Leukocyte-Reduced RBCs and the dilution of the platelets by resuscitation fluids and
Donor leukocytes may cause: RBC transfusion
o Febrile nonhemolytic transfusion reactions Platelet transfusions are indicated for patients who are bleeding
o Transfusion-associated graft-versus-host disease (TA-GVHD) because of thrombocytopenia or abnormally functioning platelets
o Transfusion-related immune suppression One plateletpheresis should increase the adult patient’s platelet
In addition, human leukocyte antigens (HLA) are responsible for HLA count to 20,000 to 60,000/µL
alloimmunization o Each unit of platelets from whole blood must contain at least
Leukocytes may harbor cytomegalovirus (CMV) 5.5 × 10¹⁰ platelets and should increase the platelet count by
o To reduce HLA alloimmunization and CMV transmission, the 5,000 to 10,000/µL in a 70-kg human
leukocyte content must be reduced to less than 5 × 10⁶,
which can be achieved by using one of several leukocyte Granulocyte Pheresis
reduction filters Newborn infants may develop overwhelming infection with
o With these filters, most RBC units are less than 1 × 10⁶; neutropenia because of their limited bone marrow reserve for
some are 1 × 10⁴ leukocytes neutrophil production
o In addition, neonatal neutrophils have impaired function
Washed RBCs and Frozen/Deglycerolized RBCs o Studies show granulocyte transfusions to be beneficial for
Patients who have severe allergic (anaphylactic) transfusion these patients
For an adult or a child, the usual dose is one granulocyte pheresis
reactions to ordinary units of RBCs may benefit from receiving
product daily for 4 or more days
washed RBCs
o For neonates, a portion of a granulocyte pheresis unit is usually
The washing process removes plasma proteins, the cause of most
given once or twice
allergic reactions
Granulocyte components should be administered as soon as
Washed RBCs are used for the rare patient who has had moderate
possible and within 24 hours of collection
to severe allergic transfusion reactions and has anti-IgA antibodies
The granulocyte pheresis needs to be crossmatched because of the
because of IgA deficiency
significant content of RBCs
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The neutrophil count will increase to 1,000/µL or more in response Fibrinogen replacement may be required in patients with liver
to infusion of granulocyte colony-stimulating factor (GCSF)– failure, DIC, or massive transfusion and in rare patients with
mobilized granulocyte pheresis congenital fibrinogen deficiency
Cryoprecipitate was used as a source for fibrin sealant, which uses
Plasma cryoprecipitate as the source of fibrinogen
Plasma includes fresh frozen plasma, plasma 24 (frozen within 24 Cryoprecipitate was originally prepared as a source of factor VIII
hours), and thawed plasma Each unit of cryoprecipitate must contain at least 80 units of factor
o Plasma and plasma 24 contain all coagulation factors VIII
o After fresh frozen plasma and plasma 24 are thawed, they can
become thawed plasma and stored for 5 days at 4°C Factor VIII
o The 5-day storage reduces outdating and allows rapid response Patients with hemophilia A or factor VIII deficiency have
to urgent orders for bleeding patients spontaneous hemorrhages that are treated with recombinant or
o Thawed plasma after 5-day storage has less factor V and VIII human plasma–derived factor VIII replacement
but is still therapeutic Factor VIII is treated by different methods, such as pasteurization,
Plasma can be used to treat multiple coagulation deficiencies nanofiltration, and solvent detergent, to ensure sterility for HIV,
occurring in patients with liver failure, DIC, vitamin K deficiency, hepatitis B virus, and hepatitis C virus (HCV)
warfarin overdose, or massive transfusion Both the plasma derived and recombinant factor VIII are stored at
Plasma is the product of choice for patients with multiple-factor refrigerator temperatures and are reconstituted with saline at the
deficiencies and hemorrhage or impending surgery time of infusion
Usually 4 to 6 units of plasma will effectively control hemostasis
Congenital coagulation factor deficiencies are rarely treated with Factor IX
plasma, because the dose requirement for surgical procedures and Factor IX complex (prothrombin complex) is prepared from pooled
serious bleeding is so great as to cause pulmonary edema as a result plasma using various methods of separation and viral inactivation
of volume overload, even in a young individual with a healthy The prothrombin complex contains factors II, VII, IX, and X;
cardiovascular system however, the product is recommended for factor IX–deficient
Plasma is sometimes used as a replacement fluid during plasma patients (hemophilia B), patients with factor VII or X deficiency
exchange (rare), and selected patients with factor VIII inhibitors or reversal of
Plasma should not be used for blood volume expansion or protein warfarin overdose
replacement because safer products are available for these
purposes—serum albumin, synthetic colloids, and balanced salt Antithrombin and Other Concentrates
solutions—none of which transmit disease or cause severe allergic Antithrombin is a protease inhibitor with activity toward thrombin
reactions or transfusion-associated acute lung injury Heparin accelerates the binding and inactivation of thrombin by
Plasma should be ABO-compatible with the recipient’s RBCs, but the antithrombin.
Rh type can be disregarded The hereditary deficiency of antithrombin is associated with venous
thromboses, whereas the acquired deficiency is seen most
Cryoprecipitate frequently with DIC
Cryoprecipitate is used primarily for fibrinogen replacement Thawed plasma is an alternative source of antithrombin
The AABB requires that at least 150 mg of fibrinogen be in each unit Protein C and protein S are vitamin K–dependent proteins
of cryoprecipitate synthesized in the liver

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Protein S functions as a cofactor for activated protein C, which, in
turn, inactivates factors V and VIII, thus preventing thrombus
formation

Albumin
Albumin is prepared by chemical and physical fractionation of pooled
plasma
Albumin is available as a 5% or a 25% solution, of which 96% of the
protein content is albumin
Albumin may be used to treat patients requiring volume replacement
Albumin can also be used in the treatment of burn patients to replace
colloid pressure

Immune Globulin
Immune globulin prepared from pooled plasma is primarily IgG
Products are available for intramuscular or intravenous
administration
o The intramuscular product must not be given intravenously
because severe anaphylactic reactions may occur
o The intravenous product must be given slowly to lessen the risk
of reaction
Immune globulin is used for patients with congenital
hypogammaglobulinemia and for patients exposed to diseases such
as hepatitis A or measles
For hypogammaglobulinemia, monthly injections are usually given
because of the 22-day half-life of IgG
The recommended dose is 0.7 mL/kg intramuscularly or 100 mg/kg
intravenously
The intravenous preparation of immune globulin is used increasingly
in the therapy of autoimmune diseases, such as immune
thrombocytopenia and myasthenia gravis
Rh immune globulin (RhIg) was developed to protect the Rh-
negative female who is pregnant or who delivers a Rh-positive
infant
Rh immune globulin products, which can be administered
intravenously or intramuscularly, are approved for use in idiopathic
thrombocytopenic purpura patients who are Rh (+)

-END OF TRANSCRIPTION-

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
MLS 18, April 6, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend:
Transcription Bullet During Transfusion
Notes/Module Packet Bullet Positive identification
PPT Identification of the patient and also the patient’s blood specimen
prior to transfusion as well as the blood unit that is used in transfusion
MODULE OUTLINE
I. Adverse Effects (page 4)
Clerical errors represent the main cause of transfusion related death
A. Immediate HTR
and acute hemolytic reaction. Final clerical check is performed at
B. Delayed HTR
II. Other Related Disease (page 5) the bedside patient done by nurse and it also double checks the
A. Transfusion-Transmitted Disease blood bag tag attached to the component to be transfused against
B. Hemolytic Disease of the Newborn the patient’s arm band
C. Autoimmune Hemolytic Anemia The medtech’s job is only in the issuance of the blood, to check if
the blood is viable (check for presence of any contaminants,
LEARNING OUTCOMES bacterial contamination and clots that might cause problem during
At the end of this module, the student shall be able to: transfusion).
1. Define transfusion reaction. The specimen must be collected within 3 days of the scheduled
2. Explain the risks of transfusions. transfusion and the blood can’t be used when it exceeds 3 days. The
3. Compare and contrast immediate and delayed HTR; HTR and non- donor’s blood must be kept stoppered or sealed and stored in 1-6°C
HTR
for at least 7 days post transfusion
4. Differentiate the clinical signs & symptoms of the various types of
Even though the transfusion is done, pre-transfusion blood
transfusion reactions.
5. List laboratory findings associated with acute transfusion reactions specimen is prohibited to put into waste
All specimen that is used in pre-transfusion must be kept for 7 days
because if ever there are adverse reactions during transfusion, there
WHAT TO DO DURING TRANSFUSION? would be specimen that can be used as the basis to compare the
after checking if the donated blood can be transfused to the recipient, result in the post transfusion specimens
proper procedure in the transfusing of the blood must also be done
aside from screening or testing the blood being qualified in transfusion, Venous access
proper procedure in transfusing the blood must be observed Should be established before a blood component is issued
take into consideration the precautionary measures transfusion, as well The selection of the location and the type of access are dependent
as cases where transfusion reaction might occur on the volume, timing expected duration of the transfusion therapy
Peripheral access with an 18 gauge needle or catheter is typically
sufficient

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It is desirable for high volume administration or for long term Automatic temperature control is set when the blood is warmed at
therapy 42°C
Never use water bath as blood warmer or microwave
Intravenous transfusion device
Should be set before the blood is issued
Rate and length of transfusion
Isotonic saline or 5% albumin should be used as an intravenous
Blood components are infused slowly for the first 10-15 minutes
solution to dilute the blood components. Because it is not
Approximately 2mL for the first 15mins.
preferable to transfuse blood that is too viscous since it is not
Slow transfusion is needed for the nurses and physician to observe
advisable
any sign of transfusion reaction
Do not use 5% dextrose solution in water also known as D5W (a
Subsequently the administration rate maybe increased and the
hypotonic solution)
desirable time to complete the red cell transfusion should be
o that can cause hemolysis of the RBC
finished within 2 hours
Do not use ringer solution, since it contains calcium which initiates
For platelets and plasma, since it is less in volume compared to red
coagulation
cell transfusion, the transfusion of platelets and plasma should fall
No medication must be added or administered in the same line with
within 30-60 minutes
the blood component
NOTE: Any transfusion should be completed within 4 hours of
initiation
Clot-screen filters o Exceeding to 4 hours would put the patients at risk for
All blood components must be filtered to remove the clots by using bacteremia or septicemia
the clot screen filters. At least 75% of the transfused red cells remains in the recipient’s
70 -170 microns of filters are used to remove gross clots and cellular circulation 24 hours after the transfusion
debris
SPECIAL CONSIDERATION IN TRANSFUSION
Vital signs Emergency transfusion
Pulse rate, respiratory rate, blood pressure, temperature are If patients Abo and Rh group is unknown, during emergency
checked by the nurse to note if there would be any signs of transfusion patients are given group O Rh negative red cells since
transfusion reaction group O/ blood type O individuals are considered to be as universal
donor (they do not contain any antigen)
Blood warmers Emergency situations or transfusion require group O Rh negative
Cold blood can cause hypothermia in the patient red cell if the patient’s Abo or Rh is unknown
Hypothermia can lead to cardiac arrhythmia and haemorrhage Massive transfusion
Blood should be set at 37°C defined as the administration of enough blood or components
Blood must not be allowed to warm above 38°C because it can within less than 24 hours to reconstitute a complete volume
induce physical or chemical damage in our cells, that can make the replacement
blood unit not viable to transfusion anymore in adults, this requires 8-10 units of blood

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
Adverse Effects of Massive Transfusion Intrauterine Transfusion
1. Citrate toxicity and Hypocalcemia done when the baby inside the womb is suffering severe anemia
o Citrate toxicity associated with hemolytic disease of the fetus or the newborn
caused by the presence of citrate in the anticoagulant Is done by:
preservative solution o intraperitoneally inject RBCs into the fetal peritoneal cavity
patients experiencing hypothermia, 2,3-DPG depletion, where RBC can be absorbed to the circulation or perform
coagulation factor depletion, as well as depletion of platelets cordocentesis
due to delusional effects because of extremely massive Process where donor RBC is directly injected into
transfusion the fetal umbilical vein
there could also be accumulation of biochemical and
microaggregates Purpose of the Intrauterine Transfusion
to maintain fetal hemoglobin above 10g/dL
Neonatal RBC transfusion once initiated, the procedure must be repeated every 2-4 weeks
Done in neonates less than seven days old to reduce the risk of until the 34-36 gestation week is reached or until the fetal lung has
hyperkalemia and to maximize 2,3-DPG matured
For O-negative or blood types compatible with the mother, the
infant is given CMV-negative or leukocyte-reduced blood WHAT TO DO WHEN THIN GS GO WRONG?
o should be hemoglobin S negative for hypoxic and acidotic
newborns Bedside Procedures Transfusion Reactions
Prepare aliquots since neonates require small volume of RBCs Investigation Work up
o dosage: 10-15 mL / kg over 2-3 hours 1. Stop transfusion 1. Clerical checks
2. Keep intravenous line open 2. Visual inspection
Exchange Transfusion
with physiologic saline 3. DAT
used in the treatment for both anemia and hyperbilirubinemia that
4. Repeat ABO /Rh
characterizes hemolytic disease of the fetus and the newborn 3. Notify patient’s physician and
5. Repeat compatibility testing
small amounts of blood are removed from the baby and then blood bank 6. Repeat crossmatch
exchanged with the donor’s blood 4. Take care of the patient per 7. Bilirubin test in blood
Main purpose or the indication: physician’s order 8. Urine test
o to replace the antibody-coated red cells with the compatible 5. Perform bedside clerical checks 9. Hemoglobin and hematocrit
donor cells 6. Specimen to be submitted Post
o to remove bilirubin in order to prevent Kernicterus transfusion venous blood. First
o to remove the circulating maternal antibody in the baby’s
voided urine and blood bags
plasma
7. Document reaction
o to suppress the erythropoiesis or the production of
incompatible red blood cells
Greater than 0.5 mg/dL per hour rise in bilirubin or 10 mg/dL in the
first 24hrs amount of bilirubin would be indicative that the baby is
in need of exchange transfusion

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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
ADVERSE EFFECTS Transfusion-Related Acute Lung Injury (TRALI)
Transfusion Reaction attributed to the administration of donor plasma containing high
Defined as any transfusion-related adverse event that occurs during concentrations of leukoagglutinins
or after the transfusion of whole blood, blood components or Cause: anti-leukocyte antibodies act on endothelial cells of lungs;
human-derived plasma products. emboli form
They are classified according to the time interval between Remedy: Leukopoor RBCs
transfusion and the presentation of adverse effects
Non-Immunologic Reaction
IMMEDIATE (Acute) – DELAYED Bacterial Contamination
signs and symptoms presenting after 24 hours of transfusion Contamination of blood products from donors with transient
within 24 hours of transfusion. bacteremia during phlebotomy, preparation and processing,
Immunologic Non- Immunologic Non-Immunologic thawing.
Immunologic Cause: endotoxins of psychrophilic organisms; Yersinia
Hemolytic- Bacterial Hemolytic TA- Hemosiderosis enterocolitica, E. coli, Pseudomonas spp.
Febrile contamination TA-GVHD Disease Transmission Prevention: Aseptic techniques, visual inspection and infusion
within 4 hours
Allergic Circulatory Post-
Overload transfusion Transfusion Associated Circulatory Overload (TACO)
TRALI Purpura Associated with rapid infusion of large volumes of blood products.
PCITR Children, elderly patients and cardiac disease patients
Treatment can include intravenous diuretics and therapeutic
Immediate (Acute) Transfusion Reaction phlebotomy
Immunologic Reaction
Febrile Non-Hemolytic Physical or Chemical Induced Transfusion Reaction (PCITR)
Increase in temperature of 1 degree Celsius associated with Causes: mechanical damage (infusion through small bore), osmotic
transfusion that cannot be explained by other condition. or chemical damage, thermal trauma, citrate toxicity
Most common adverse reaction seen
Cause: Anti-leukocyte antibodies Includes physical damage to the RBC due to intravascular lysis
Remedy: Leukopoor RBCs Cause: proliferation of T-cells reacts against foreign tissue of the
host recipient
ALLERGIC VS. ANAPHYLACTIC REACTIONS Prevention: Irradiated blood components
ALLERGIC ALLERGIC
Reaction between recipient Afebrile reaction that occurs only Delayed Immunologic Reaction
antibody and transfused donor after infusion of only few mL of Immunologic Reaction
plasma proteins blood Transfusion-Associated Graft vs. Host Disease (TA-GVHD)
Cause: Donor plasma with foreign Cause: IgA deficient patient with Certain susceptible recipients with compromised immune systems
proteins anti-IgA are transfused with blood or blood components containing
Remedy: Washed RBCs Remedy: Washed RBCs
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immunocompetent lymphocytes which engraft in recipient’s tissue Hepatitis
and multiply. Inflammation of the liver
Cause: Proliferation of T cells that reacts against foreign tissue of Symptoms: jaundice, dark urine, hepatomegaly, anorexia, malaise,
the host recipient fever, etc.
Prevention: Irradiated blood components
Hepatitis A
Picornaviridae
Post- Transfusion Purpura Small, non-enveloped, single stranded enterovirus RNA
Previously immunized patients to platelet antigens through Most common
pregnancy or transfusion produce antibodies once stimulated, Not screened for blood units
destroying patient’s own platelets.
Cause: Platelet antibodies
Hepatitis B
Therapy: Exchange transfusion, corticosteroids, intravenous
A partially double stranded circular DNA virus of the Hepadnaviridae
immunoglobulin

Hepatitis C
Non-Immunologic Reaction
Flaviviridae virus family. RNA virus.
TA- Hemosiderosis Diagnosis depends on biochemical changes suggestive of HCV,
Iron overload accumulating in the mitochondria of cells in organs detection of HCV RNA or anti-HCV in serum.
like the liver, heart and endocrine gland. EIA, ChLIA and RIBA (Recombinant immunoblot assays)
Individuals that are chronically transfused are at risk example
thalassemia major, sickle-cell anemia and hemoglobinopathies. Hepatitis D, E, G
Iron chelating agent like Desferrioxamine can be used for therapy. HDV is a defective, single-stranded RNA virus that is found only in
the patients with HBV infection.
OTHER RELATED DISEASE o It requires HBsAg in order to synthesize an envelope
Transfusion-Transmitted Disease protein. It was previously called the delta antigen. If HBV
Hepatitis B,C,D and HDV are contracted concurrently, this co-infection.
CMV HEV is a member of the Calciviridae family of nonenveloped RNA
EBV viruses.
HTLV 1 and 2 GB virus C (GBV-C) and hepatitis G virus (HGV) are two genotypes of
HIV the same enveloped RNA virus that belongs to the Flaviviridae
T. pallidum family.
Plasmodium spp.
B. microti
T. cruzi
T. gondii

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o Primary infection with HIV may be asymptomatic or result in a
Hepatitis Route of Transmission mild, chronic lymphadenopathy with symptoms similar to those
seen in infectious mononucleosis.
A fecal/oral Symptoms may occur within 6 to 12 weeks of infection
and persist for a few days to 2 weeks. HIV enters the cell
B parenterally
by the binding of the virus glycoprotein 120 with cell
surface receptors. Cells possessing these receptors
C parenterally
include CD4+ lymphocytes, macrophages, and other
D parenterally antigen-presenting cells.
Expansion of donor screening protocols in 1996 to include
E fecal/oral p24 core antigen testing reduced the window period from
an estimated 22 days to 16 days
G parenterally
Refer to Appendices for Decision Tree for Anti-HIV-1/HIV-2 testing of donor
blood.
Diagnosis
HBV DNA first marker to appear- PCR
HBsAg- 2 to 12 weeks post-exposure during acute stage; HTLV I and II (Human T-Cell Lymphotropic Viruses Types I/II)
undetectable in 12 -20 weeks after the development of anti-HBsAg HTLV-I and HTLV-II are RNA retroviruses.
HbeAg appears after the HBsAg in recovering patients, disappears o HTLV-I causes a T-cell proliferation with persistent infection.
before HbsAg. Once the RNA has been transcribed into DNA, it is integrated
HbcAg is present in the serum but is undetectable. randomly into the host cell’s genome.
Once integrated into the DNA, the provirus can either complete its
HIV Type 1 and 2 replication cycle or remain latent for many years.
HTLV-I was the first retrovirus to be associated with a human
HIV is a retrovirus that is spherical in shape, with an approximate
disease.
diameter of 100 nm. That association was with adult T-cell lymphoma/ leukemia (ATL), a
It consists of an envelope of glycoproteins, core proteins, and an highly aggressive, mature T-cell non-Hodgkin’s lymphoma with a
inner core of viral RNA and reverse transcriptase. leukemic phase.
o Infection with the virus causes a slowly progressing immune HTLV-II is similar to type-I and is prevalent in intravenous drug
disorder. users.
o The causative viruses, HIV-1 and HIV-2, are similar in structure,
Refer to Appendices for Flowchart of HTLV Testing
varying primarily in the envelope proteins
o The use of very sensitive serologic testing in screening the blood
supply has resulted in an extremely low risk of HIV transmission
o Positive screening tests are repeated in duplicate, and if at least
one of the duplicates also tests positive, WB or
immunofluorescent antibody assay is used for confirmation.
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West Nile Virus EBV
WNV is a member of the Flavivirus family and is a human, avian, and EBV has been called the “kissing disease” because the virus usually
equine neuropathogen. replicates in the cells of the oropharynx, possibly in infected B cells.
It is a single-stranded RNA lipid-enveloped virion that is common in The virus is shed in the saliva and is most frequently associated with
Africa, West Asia, and the Middle East. infectious mononucleosis.
WNV is usually subclinical but may cause a mild flu-like disease.
Mosquitoes have been found to carry WNV, the genus Culex is the Human Herpesvirus 6 (HHV-6) and 8 (HHV-8) Human herpesvirus 6
chief vector. (HHV-6)
The infection in humans has an incubation period of approximately
Is a very common virus that causes a lifelong infection.
3 to 14 days following the mosquito bite, with symptoms lasting 3 to
After infection, the virus replicates in the salivary gland and then
6 days.
remains latent in lymphocytes, monocytes, and perhaps other
o Other animals can become infected, including horses, cats,
tissues, without obvious pathology.
dogs, bats, chipmunks, skunks, squirrels, and domestic birds
HHV-6 causes roseola infantum, also known as exanthem subitum
and rabbits.
or sixth disease.
CMV
Herpesvirus group Syphilis
CMV can remain latent in the tissues and leukocytes for years, with
Treponema pallidum, the causative agent of syphilis, is a spirochete.
reactivation occurring due to severe immune system impairment.
It is usually spread through sexual contact but can be transmitted
Those at the highest risk of a CMV infection are individuals receiving
through blood transfusions.
allogeneic marrow transplants and the fetus.
Spirochete cannot live in blood stored for 72 -96 hours at 1-6⁰C-
o CMV-seronegative recipients transplanted with
which would make platelets the only component capable of
CMVseronegative allogeneic marrow are at risk if they
transmitting the infection.
receive untested and non-WBC-reduced blood components.
Still part of the test since donor positive for syphilis are considered
high risk individuals.
Parvovirus B19
Human B19 parvovirus (B19) is a small, single-stranded DNA non- Tick-Borne Bacterial Agents
enveloped virus and is the only known pathogenic human
Lyme disease, Rocky Mountain spotted fever (RMSF), and
parvovirus.
ehrlichioses are all bacterial diseases spread by a tick bite.
It causes a common childhood illness called “fifth disease” and is
Lyme disease is caused by the spirochete Borrelia burgdorferi and
usually transmitted through respiratory secretions.
RMSF (Rickettsia rickettsii) and ehrlichioses (Ehrlichia species) are
o Fifth disease presents with a mild rash described as “slapped
caused by bacteria that are obligate intracellular pathogens.
cheek” when occurring on the face and a lacy red rash when
occurring on the trunk and limbs.
o The virus must enter the cell through a specific cell receptor. B19 Transfusion-Associated Parasites
parvovirus enters the red blood cell (RBC) via the P antigen and At least three parasites have been associated with transfusionassociated
replicates in the erythroid progenitor cells. infections:
1. Babesia microti
Babesiosis, a zoonotic disease, is usually transmitted by the bite of
an infected deer tick.
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 MODULE 5: BLOOD DONATION & TRANSFUSION PRACTICE (part 4)
Infection is caused by the protozoan parasite, Babesia, which infects During gestation, and particularly at delivery when the placenta
the RBCs. separates from the uterus, variable numbers of fetal RBCs enter the
Babesia infection may also be acquired by blood transfusion and maternal circulation
solid organ transplant. These fetal cells, carrying D antigen inherited from the father,
immunize the mother and stimulate the production of anti-D.
2. Trypanosoma cruzi Once the mother is immunized to D antigen, all subsequent
Trypanosoma cruzi is a flagellate protozoan that is the etiologic offspring inheriting the D antigen will be affected.
agent of Chagas disease (American trypanosomiasis). The maternal anti-D crosses the placenta and binds to the fetal Rh-
The reduviid bug bite produces a localized nodule, referred to as a positive cells.
chagoma. The sensitized RBCs are destroyed by the fetal reticuloendothelial
system, resulting in anemia
3. Malaria (Plasmodium species)
Malaria, another intraerythrocytic protozoan infection, may be
caused by several species of the genus Plasmodium (P. malaria, P.
falciparum, P. vivax, and P. ovale).
Natural transmission occurs through the bite of a female Anopheles
mosquito, but infection may also occur following transfusion of
infected blood.

Prion Disease
Creutzfeldt-Jakob Disease (CJD)
Creutzfeldt-Jakob Disease (CJD) is one of the transmissible
spongiform encephalopathies (TSE). These are rare diseases
characterized by fatal neuro-degeneration resulting in spongelike
lesions in the brain.

Hemolytic Disease of the Newborn and Fetus (HDFN)

destruction of the RBCS of the fetus and neonate by the antibodies
produce by the mother
Only the antibodies if the immunoglobulin G (IgG) class are actively
transported across the placenta

Usually in Rh(D) HDN, the Rh-positive firstborn infant of a Rh-

negative mother is unaffected because the mother has not yet been
immunized.

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Characteristics of HDN INDICATION
Anemia Antenatal RhIg should be given early in the third trimester, or at
• compensated anemia (increased immature RBCs leads to about 28 weeks’ gestation. The dose does not pose a risk
erythroblastosis fetalis) to the fetus, inasmuch as this amount will cause a titer of
Generalized edema and cardiac feature (hydrops fetalis) only 1 or 2 in the mother.
Increased urinary bilirubin Post-partum The Rh-negative nonimmunized mother should receive
Deposition in brain tissue ( kernicterus) RhIg soon after delivery of an Rh-positive infant. The
Hepatosplenomegaly (extramedullary hematopoiesis) recommended interval is within 72 hours after delivery.

Serological Testing Dose and Administration

Mother Newborn Infant 15 mL of packed RBCs or 30 mL of whole blood
ABO, Rh, Antibody Screen ABO This is equal to 300 g of the World Health Organization (WHO)
Antibody Identification Rh reference material.
Paternal Phenotype and DAT
Genotype The total fetal blood volume is estimated to be less than 5 mL at 12
Elution weeks.
Fetal DNA Testing
Antibody Titers o The microdose can be used for abortions and ectopic
pregnancies before the 12th week of gestation.
RhIg An IV preparation of RhIg is approved for use in the United States.
Active immunization induced by RBC antigen can be prevented by This product also contains 300ug in each vial and can be
the concurrent administration of the corresponding RBC antibody. administered either intramuscularly or intravenously.
This principle has been used to prevent immunization to D antigen Massive fetomaternal hemorrhages of more than 30 mL of whole
by the use of high-titered RhIg. blood occur in less than 1 percent of deliveries.
These massive hemorrhages can lead to immunization if adequate
Mechanism RhIg is not administered.
o A maternal sample should be obtained within 1 hour of
o The administered RhIg attaches to the fetal Rh-positive RBCs in delivery to test for massive fetomaternal hemorrhage by a
the maternal circulation. The antibody coated RBCs are removed
screening test, such as the rosette technique. If positive,
by the macrophages in the maternal spleen. The RBC antigens
quantitation of the hemorrhage must be done by Kleihauer-
are thus unavailable for dendritic cells to present antigen to T
helper cells. Betke or similar test or by flow cytometry

Kleihauer-Betke test
Kleihauer-Betke test, a maternal blood smear is treated with acid and then
stained with counterstain.
Fetal cells contain fetal hemoglobin (Hgb F) that is resistant to acid
and will remain pink.

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The maternal cells will appear as ghosts. Bilirubin at
Normal Elevated
birth
Anemia at
No Yes
birth
DAT Weakly positive/ negative Positive
Spherocytosis Yes Rare
Phototherapy Phototherapy,
Therapy Exchange/ intrauterine
transfusion

Immune Hemolytic Anemia

A simpler way to calculate the dose is to multiply the fetal cell
Shortened RBC survival mediated through immune response.
percentage by 50, which gives the volume of fetomaternal
specifically by humoral antibody
hemorrhage in milliliters.
If needed, additional vials of RhIg should be administered within 72 3 TYPES
hours of delivery or as soon as possible. Alloimmune Patient produces alloantibodies to foreign or non-self
The RhIg must be injected according to the product label. The IV RBC antigens introduced into the circulation, most often
product can also be given intramuscularly. through transfusion or pregnancy
The intramuscular form must be given intramuscularly only.
IV injections of intramuscular preparations can cause severe
anaphylactic reactions because of the anticomplementary activity of Autoimmune Patient produces antibodies against his or her own RBC
these products. antigens
RhIg also contains IgA and may be contraindicated in patients with
anti-IgA and IgA deficiency who have had anaphylactic reactions to Drug-induced production of antibodies to a particular drug or drug
blood products. complex with ensuing damage to the individual’s RBCs

Comparison of ABO vs. Rh HDN Refer to Appendices for Diagram on Drug-Induced Hemolytic Anemia

Characteristics ABO Rh Drug-Dependent or Immune Complex (“Innocent Bystander”)

Non immune/immune IgG Immune IgG anti-D Mechanism
Antibody
anti-A, B The antibody (IgG or IgM) produced recognizes determinants on the
Mother= O Mother= Rh negative drug. If the patient ingests the same drug (or a drug bearing the same
Blood group haptenic group) after immunization, the formation of a drug-antidrug
Baby= A/B/AB Baby= Rh positive
First pregnancy and First pregnancy not complex may occur.
Obstetric The complement cascade may be activated because of this antigen-
subsequent pregnancies affected; rare
history antibody interaction. RBCs are thought to be involved in this process
may be affected
Disease only as “innocent bystanders.”
predicted by No Yes
titers

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The soluble drug-antidrug complex nonspecifically adsorbs loosely to
the RBC surface. Complement, when activated, sensitizes the cell and
may cause lysis.

Membrane Modification (Nonimmunologic Protein Adsorption)

It is hypothesized that the cephalosporins, especially cephalothin
(Keflin), both operate through the drug-adsorption mechanism and are
able to modify RBCs so that plasma proteins (e.g., IgG, IgM, IgA, and
complement) can bind to the membrane.

Autoimmune Hemolytic Anemia (AIHA)

Warm Autoimmune Hemolytic Anemia (WAIHA)
DAT: Positive
Donath- Landsteiner test: Negative
IgG antibody
Optimal temperature: 37 degrees Celsius
Rh, Kell
extravascular hemolysis; splenic

Cold Hemaglutinin Disease

DAT: Positive
Donath- Landsteiner test: Negative
IgM antibody
Optimal Temperature: 0-4 degrees Celsius
I blood group
Extravascular hemolysis; hepatic

Paroxysmal Cold Hemoglobinuria

DAT: Positive
Donath- Landsteiner test: Positive
Involves biphasic IgG
May occur on both 0-4 degrees Celsius; 37 degrees Celsius
P blood group
Intravascular

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APPENDICES

Softcopy version:
[Link]
QBcUZC-oe?usp=sharing

-END OF TRANSCRIPTION-

#MLSWATER2022 Page 12 of 12
1
PREPARATION OF WASHED RED BLOOD CELL (8) nescofilm/parafilm
SUSPENSION (9) 5-ml EDTA tube
(10) serological pipettes (1ml, 2ml, 5 ml)
IMMUNOHEMA LAB// MAAM PALOMAR SPECIMEN Whole blood
RED CELL SUSPENSION
• A common reagent used for many serological procedures. WORKING 0.85%NaCl solution (NaCl- Raw Chemicals, Distilled Water)
• Provides the appropriate serum to cell ratio to allow for grading and
REAGENT → general cell suspending medium
→ should be freshly prepared each day and should not
interpretation of tests results.
be stored
o The ratio of serum to red cells may affect the sensitivity of
To prepare 1000 mL of NaCl/NSS
agglutination tests.
• This procedure applies to all serological testing that requires red cells • 8.5 g NaCl crystals which must be dissolved in 1000mL
suspension preparation (i.e. ABO blood grouping, crossmatching). of distilled water (no other than distilled water)
• take note if the NSS is pharmaceutically purchased and
• It is the responsibility of the Medical Technologist, in the laboratory performing
traditionally made and stored until consumed that may
a given test, to prepare the appropriate red cell suspension.
have pH variations and can’t be controlled because it is
• For best results, red cell suspensions should be used for testing on the day of unbuffered
preparation • Washing RBCs with stock saline may yield a pH below
o The suspension need not be exactly 5%; an approximation achieves or above the normal range required and may cause in
the appropriate serum-to-cell ratio for most test procedures. accurate result
• Reversely used as an indicator to demonstrate antigen-antibody reactions in
vitro 2 COMMON FORMULATIONS OF SALINE SOLUTIONS
• Saline solutions that is used is preferred to be fresh to avoid changes in pH 1. NSS (Normal Saline Solution)
that may affect the result → Physiologic saline or isotonic solution
• It is a source of antigen to which the corresponding antibody can react to → chemically 0.85% or 0.90% weight per volume NaCl
produce agglutination or hemolysis in vitro → A normal saline solution is 0.85% NaCl solution, that
• This suspension can exist in different concentrations; there is 2%, 3%, 4% and means a 0.85 g of NaCl IN 100mL distilled water
5% and can reach until 10%. → Is more commonly used because it is inexpensive
and easy to make
o Can be prepared using an anticoagulated blood
2. PBS (Phosphate Buffered Saline)
• 2-5% RBC suspension provides optimal antigen concentration for various
→ Water-based salt solution
blood bank laboratory applications and includes ABO typing and cross
→ Contains NaCl and Sodium phosphate
matching
→ In another formulation it contains Potassium Chloride
• Cell suspension should be between 2 and 5% per standard tube agglutination and Potassium Phosphate
test with an ideal strength of about 3% → Buffer Phosphate group helps maintain the pH
between 7.2 and 7.4
PROCEDURE
→ Phosphate Buffered Saline (PBS) is an ideal diluent
PURPOSE OF • Remove plasma, microaggregates, cytokines, and that is used in the preparation of washed red cell
WASHING unwanted antibodies that may interfere with antigen- suspension because its pH is maintained at a certain
RBCs antibody reaction period
• The solution should be freshly prepared each day and → The Osmolality and Iron concentration of the solution
should not be stored. usually match those of the human body which is
MATERIALS (1) 10-ml test tubes isotonic.
NEEDED: (2) venipuncture kit
(3) test tube rack CAUSES OF • Bacterial contamination
(4) Markers FALSE → Positive result
(5) centrifuge plastic POSITIVE OR → Causing agglutination
(6) droppers NEGATIVE
(7) Forceps RESULT

1|Page
 → Discoloration of blood samples, foul smell and • Resuspend the cell button thoroughly between washes
cloudiness of serum = indications that the sample is before adding more saline to ensure complete washing
bacterially contaminated • do not attempt to mix a tube full of saline
• Improper centrifugation • do not mix cells using the fingers as a stopper
→ Over centrifugation - difficult in resuspending the → use nescofilm instead
cell • Prevent cells from spraying out during centrifugation:
→ Under centrifugation – agglutination → fill the tubes no more than 3⁄4 full
• Improper storage of material • Ensure good resuspension of cells:
• Blood groups substances in plasma neutralize the anti- → add the saline in a more forceful manner or forceful
serum leading to false negative result stream
• Hemolysis of red cells during washing may lead to an
inaccurate result. PROCEDURE 1. Collect 5ml of blood and place in an EDTA tube. Label the
tube with patient’s name.
INTERFERING SUBSTANCES 2. Spin for 5 minutes at 5000rpm and separate plasma from
Wharton’s jelly the red cells. Make sure that there is absence of hemolysis
→ An interfering protein on the plasma.
→ Cause red cells to non-specifically aggregate, 3. Fill the test tube with 3⁄4 full of saline solution with the use
leading to error of a dropper to re-suspend the cells. Mix well by inversion.
Rouleaux-promoting substances Avoid contamination of tubes when dispensing saline into
→ Globulins several tubes.
→ Fibrinogen 4. Spin for 5 minutes. Note the color of saline wash on top. It
→ when elevated, identification of reaction is difficult/ must not be hemolyzed.
misleading 5. Remove the saline supernatant on top. Following the
TUBE • Between 2-5% cell suspension provides optimum antigen removal of saline, gently re-suspend the red cell button.
EXAMINATION 6. Fill the tube with additional 3⁄4 full of saline solution. Make
concentration for the tube method for red blood cells typing.
PROCEDURES sure that all cells are uniformly re-suspended by inverting
• Preparation of a 2%-5% cell suspension provides cells in
USING 3-5% the tube for at least three times.
an optimum concentration to detect weak antibodies
RBC 7. Repeat procedure 4 to 6 to complete at least three washes
• May be from clotted whole blood, anticoagulated patient
SUSPENSION or until the saline supernatant is clear. After the last wash,
samples or from sealed segment on the donor unit
remove the supernatant on top.
(1) ABO and Rh typing
8. Label another clean and dry 10 ml test tube with “2%”
(2) Direct antiglobulin test
including patient’s name. With the use of a serological
(3) Donor unit compatibility (crossmatch)
pipet, prepare a 2% red cell suspension by aspirating 4.9
(4) Red cell phenotyping
ml of NSS added with 0.1 ml of packed RBC to make a final
volume of 5 ml.
FOR 0.8% RED (1) Direct antiglobulin test
9. Cover the tube with nescofilm or parafilm and invert for at
CELL (2) Donor unit compatibility test (crossmatch)
least 5 times.
SUSPENSION (3) Indirect antiglobulin test
10. Repeat procedure 8 and 9 in the preparation of 5%, and
(4) Identification panel test cells
10% washed red cell suspension with a final volume of 5
ml.
QUALITY • Visually compare the 3-5% red cell suspension to
CONTROL commercially prepared red cell suspension to ensure the IMPORTANT • Mix the red cell suspension immediately before performing
appropriate strength has been achieved REMINDERS the serological procedure.
• The 3% red cell suspension must be well mixed prior to
• Compare the red cell suspension to a commercial 3%
using for Testing
suspension if necessary, to ensure the appropriate strength
has been achieved.
If using washed 3% RCS:
• Adjust to a 3-5% suspension by adding saline or
• To prevent contamination: do not touch tubes with the tip of
centrifuging to remove saline.
the saline bottle

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 • Avoid contamination of tubes when dispensing saline into
several tubes
• Variation in red cell concentrations can markedly affect the
sensitivity of test results
→ red cell suspension is too high conc. = mask with
agglutination
→ red cell suspension is too low conc. = becomes difficult
to visualize and in extreme cases, a weak positive can
fail to be detected
• Red blood cell suspension prepared from cord blood
specimen: Requires a minimum of three washes

TEMPERATURE STORAGE
• Reagent red cells should not be left at ambient
temperature before an extended period of time
• Commercial reagents should be stored according to
the manufacturers’ instructions and should be
refrigerated after use
→ To avoid deterioration
→ The higher the ambient temperature, the
occurred reagents will deteriorate
→ The samples should not be left at ambient
temperature or incubated with tests at 37 C
unless appropriate

-end of transcription-

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2
•
DEMONSTRATION OF HEMATOLOGIC
REACTIONS
IMMUNOHEMA 2 LAB//MAAM PALOMAR
INTRODUCTION
The two common immunologic reactions observed in performing laboratory
tests involving blood products are:
1. Agglutination
2. hemolysis
• In some tests, agglutination and hemolysis are considered positive reactions
and therefore should always be given emphasis HEMOLYSIS
• Hemolysis is the breakdown or destruction of red blood cells so that the contained
2 COMMON REACTIONS oxygen carrying pigment which is the hemoglobin freed into the surrounding
AGGLUTINATION Refers to a reaction wherein particles form a clump due to medium
antigen-antibody reaction o In Erythroblastosis fetalis or HDN, a mismatched in antibody compatibility
test between the fetal and maternal blood result in the destruction of the
HEMOLYSIS Pertains to the destruction of red blood cells resulting to fetal red blood cells by maternal antibodies that cross the placenta.
the liberation of hemoglobin in the surrounding fluid. o In In vitro Hemolysis, this can result from the lysis of Red Blood Cells
It may occur in vitro or in vivo during collection and handling of blood sample
o In In vivo Hemolysis, occurs at the rate of erythrocyte destruction is
increased thereby increasing the erythrocyte life span
AGGLUTINATION • not all hemolysis is mediated by the Immune response some can be mediated
• In blood banking, since the reaction involves the clumping of red blood cells, with erythrocyte fragility or the shearing effect.
the term applied to is hemagglutination
• Agglutination reactions produce visible aggregates of antibody-antigen 3 CATEGORIES OF IMMUNE RESPONSE HEMOLYSIS
complexes when the antibodies or the antigens are conjugated to the carrier When the Immune system mediates hemolysis, it will generally fall into 3 Categories.
(artificial or biological).
o Artificial carriers – examples are latex and charcoals. Autoimmune • When the immune system produces an antibody which
o Biological carriers – example erythrocytes Hemolysis binds to the patient’s endogenous (own) red blood cells
• When these antibodies bind they can cause either:
STEPS OF AGGLUTINATION o Intravascular Hemolysis - immediate RBC
1. SENSITIZATION • Initial Binding of antibody to their corresponding destruction within the circulatory system itself.
antigenic site o Extravascular Hemolysis - Shortens the life span
• Epitope on the RBC membrane of RBCs usually due to the increase clearance of
• No visible clumping the spleen

2. LATTICE • Antibody molecules crosslinked with RBCs

Alloimmune • When the immune system produces an antibody as a
FORMATION (large Hemolysis result to the exposure of non-self (exogenous) RBC
forming a lattice
aggregates antigen
• Visible clumping or agglutination
o A very good example is during blood transfusion
or pregnancy
FACTORS AFFECTING AGGLUTINATION
o Hemolytic Transfusion Reaction- If the patient is
1. Temperature of the reaction (denatured at 50 degrees Celsius)
re-exposed to the non-self-antigen, the
2. Incubation time
antibodies can mediate the destruction of red
3. pH (range: 6-8)
blood cells
4. ionic strength
o HDFN- can result to fetal hydrops
5. distance between red cells
6. optimal concentration of antigen and antibody
7. effect of centrifugation
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Drug-Induced Occurs when the immune systems produced the RBC PROCEDURES
Hemolysis antibodies after exposure to pharmaceutical agent such as AGGUTINATIONS REACTIONS
antibiotics
A. SLIDE • The slide test is relatively the least sensitive method
METHOD among others for BG determination, but due to its prompt
MATERIALS NEEDED results, it is very much valuable in emergency cases.
WORKING REAGENT • 0.85%-0.9% NaCl solution • In this method, a glass slide or white porcelain support is
• Distilled water divided into three parts, as for each part, a drop of donor
• Anti-D typing sera: diluted to 1:10 and 1:50 or recipient blood is mixed with anti-A, anti-B and anti-D
separately.
• The agglutination or blood clumping pattern can be
visually observed from which the ABO and rhesus D
(RhD) type of blood can be determined.
• The test completes in 5–10 min and is inexpensive, which
requires only a small volume of blood typing reagents.
• However, it is an insensitive method and only useful in
preliminary BG matching for getting an early result.
• The test cannot be conducted for weakly or rarely reactive
antigens from which the results are difficult to interpret
SPECIMEN • Whole blood (EDTA TUBE) o Additionally, a low titer of anti-A or anti-B could
• 5% Red blood cell suspension from an Rh (+) lead to false positive or false negative results.
individual • Although the slide test is useful for outdoor blood typing, it
is not reliable enough for completely safe transfusion
STANDARD • Reading of agglutination and hemolytic reactions
PROCEDURE should be done properly as this can lead to an STEPS
erroneous result. 1. Divide one glass slide into two equal parts.
• Reactions should not be graded until the entire cell 2. Label the first part as “U” (unknown) and the other
button is dislodged from the bottom of the tube. part as “NC” (negative control)
o This is done by gently shaking, rolling or 3. Place one drop of Anti-D on the part labeled “U”
tilting the tubes containing the serum and red and one drop of NSS on the part labeled “NC”
blood cells. 4. Place one of undiluted blood sample on each part
• Vigorously shaking must be avoided as this can of the glass slide.
cause dispersion of immune complexes. 5. Mix each part with the use of an applicator stick.
• Reactions must be read and recorded immediately 6. Observe the mixture macroscopically and
microscopically using LPO.
7. Read and record results within two minutes as
positive or negative. No need of grading the slide
agglutination reaction

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B. TUBE • In comparison to the slide test, the tube test is more HEMOLYTIC REACTIONS
METHO sensitive and reliable; STEPS 1. Prepare two 5ml test tubes and label as “P” (positive)
o Therefore, it can be used conveniently for blood and “N” (negative”
transfusion. 2. Add 5 drops of 5% red cell suspension of an Rh (+)
• In this method, both forward (cell), as well as reverse blood sample on each tube.
(serum) grouping is carried out. 3. Add 1 ml of distilled water to the tube labeled “P”.
• The forward grouping suggests the presence or absence 4. Place 1 ml of NSS to the tube labeled “N”
of A and B antigens in RBCs, whereas reverse grouping 5. Cover each tube with nescofilm and mix by tapping on
indicates the presence or absences of anti-A and anti-B in the palm
serum. 6. Centrifuge for 15-30 seconds
• In Forward Grouping, Blood cells are placed in two test 7. After centrifugation, carefully remove the tubes from the
tubes along with saline as a diluent media, and then one centrifuge.
drop of each anti-A and anti-B is added separately in 8. DO NOT dislodge the cell button.
these samples. 9. Examine for the characteristics of the supernatant.
o These tubes are subjected to centrifugation for few 10. Interpret and record results
minutes, and then, the resultant matrix is gently INTERPRETATION
shaken for observing agglutination. OF RESULTS INTRPTTION RBC SUPERNATANT
o The purpose of centrifugation is to ensure Negative Present and colorless
enhanced chemical interactions, particularly for for Hemolysis intact
weaker antibodies to react, thus leading to Positive Few to absent Reddish or
agglutination. for Hemolysis pinkish
• In a similar fashion, Reverse Grouping can be performed.
Here, the blood serum is treated against RBC reagent
groups of A1 and B, and the subsequent agglutination
pattern is monitored.
• In general, the tube method is much more sensitive than
the slide test and requires a low volume of reagents, and
some unexpected antigens can also be detected;
therefore, it is a better option for safer transfusions.
• However, in infants, reverse grouping is somewhat
difficult to perform and generally not recommended, since
they produce insufficient amounts of antibodies to be
determined.
STEPS
1. Prepare two 5ml test tubes. Label as “1:10” and “1:50”
2. Add one drop of 1:10 dilution of anti-D to the tube
labeled “1:10”
3. Add one drop of 1:50 of anti-D to the tube labeled
“1:50”
4. Place one drop of 5% red blood cell suspension from
an Rh (+) individual to each test tube.
5. Cover each tube with nescofilm and mix the tube by
tapping on the palm.
6. Centrifuge at 3,400 rpm for 15-30 seconds.
7. Gently dislodged the cell button.
8. Read the results by grading the agglutination reaction.
Refer to table #1 below for the grading of results.
9. Record results.

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 ACTIVITY
-SALINE AGGLUTINATION TEST-
1. Draw small amount of anticoagulated blood and put a drop of it in a clean
glass slide
2. Draw a small amount of saline solution and put a drop of it directly on to the
glass slide
3. Mix the slide by slowly rotating it back and forth
4. Check under the microscope

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3 ABO GROUPING-FORWARD TYPING o Lacks a nucleus (anucleated)
o Contains hemoglobin, a red iron-rich protein that binds oxygen

IMMUNOHEMA LAB//MAAM PALOMAR ABO BLOOD GROUP

BLOOD TYPING • “A”, “B” and “O” were discovered by Karl Landsteiner in 1900
• A test that determines the person’s blood type • “AB” was discovered by Descastello and Sturli in 1902
o Also called as blood grouping • Most important blood group system in blood transfusion
• Classification of blood based on the presence or absence of: • Associated antibodies are usually IgM
(1) Antibodies • Landsteiner’s Law
(2) Inherited antigenic substances on the surface of the RBC’s o antibodies are present in the plasma only when the corresponding
• it is essential in need of blood transfusion or if planning to donate blood antigens are not present in the erythrocytes
o since not all blood types are compatible, it is important to know your • ABO blood group remain of prime importance in transfusion medicine
blood group o It is the Most immunogenic among the blood group antigens
• also done to determine if the patient has Rh factor on the surface of the RBC o There are 4 blood groups defined by the ABO system:
• recorded as the classification of blood in terms of distinctive inherited
characteristic that are associated with the antigens located in the surface of CAN
ANTIBODIES
BLOOD ANTIGEN RECEIVE CAN GIVE
the RBC (In the
GROUP (On the RBC surface) BLOOD BLOOD TO
plasma)
FROM
WHAT IS BLOOD? type O and
• Blood is a red substance that circulates in the human body and it composed GROUP Anti-B / A and AB
A-ag (A-antigen) A
of cells that helps us survive. A B-ab blood
• 4.5 L – 5 L in the body (tip: blood has 5 letters so 5 L) B-ag (B-antigen) on the Anti-A / type O and B and AB
Group B
• fluid that transports oxygen, nutrients to the cells and carries away carbon red blood cells A-ab B individuals
dioxide and other waste products no O, A, B and
Group B and AB
• pumped by the heart to all parts of the body of which it is returned to the heart has A&B antigen antibodies AB
AB individuals
to repeat the process
• Both a tissue and a fluid has both
Group O O only O, A, B, AB
o It is a tissue because it is a collection of similar specialized cells that no antigens A&B
serves particular function and these cells are suspended in a liquid antibodies
matrix which is called the plasma which makes the blood fluid
o The fluid portion plasma is a clear, slightly sticky, yellowish liquid
• Red cells constitute about 45% volume of the blood and the remaining white Blood Group
blood cells (leukocytes) and the platelets (thrombocytes) constitutes the Can give blood to: Can receive blood from:
remaining 1% A+ A+, AB+ A+, A-, O+, O-
• Composed of: B+ B+, AB+ B+, B-, O+, O-
(1) mostly of plasma AB+ AB+ Everyone
(2) Platelets (helps the blood to clot) O+ A+, B+, AB+, O+ O+, O-
(3) White Blood Cells (fight against infection) A- A+, A-, AB+, AB- A-, O-
(4) Red Blood Cells (Transport Oxygen throughout our body) B- B+, B-, AB+, AB- B-, O
AB- AB+, AB- A-, B-, AB-, O-
WHAT ARE RED BLOOD CELLS? O- Everyone O
• The most common cells in the human blood
• Carries oxygen throughout the body
• differentiated from each other on the basis of their surface antigen structure ADDITIONAL INFO
• A mature human red blood cells is small, round, biconcave and appears • AB blood type
dumbbell shape in profile o UNIVERSAL RECIPIENT
o The biconcave shape allows oxygen exchange at a constant rate o Rarest of the ABO blood group system
o Covered in membrane composed of lipids and proteins o Least requested blood type in hospitals

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 • O Blood type • In forward typing, the individual’s cells are set up against serum containing
o Most common blood group in the ABO blood group system known antibody (e.g. antisera-A and anti-sera B).
o O blood group red blood cells are compatible and versatile
LANDSTEINER’S LAW
• states that antibodies are present in the plasma only when the corresponding
2 TYPES OF BLOOD TYPING antigens are not present in the erythrocytes.
• To determine the individual’s blood group, some type of substance must be
FORWARD TYPING REVERSE TYPING available to show what antigens are present on the RBC.
• Uses anti-sera • Uses known cells o These substances used as indicator are called antiserum (plural
• Detects for the presence of antigen • Detects the presence of Antibody antisera).
• Anti-Sera (made from Dolichos • A cross check for forward typing o An antiserum is a prepared and highly purified solution of antibody
biflorus extract) • Not recommended when typing and is named based on the antibody it contains.
• Anti-sera B (made from Ulex newborns and infants under the
europaeus extract) age of 4 months because they
have not yet developed the proper PREPARATION OF ANTISERUM
antibodies • Monoclonal antisera are produced by hybridization, a fusion of a single clone
of human neoplastic antibody-producing cells with sensitized splenic
lymphocytes obtained from a rodent species.
RULES BEFORE PERFORMING BLOOD TYPING • Serum is collected from humans who have been sensitized to an antigen
(1) Always run control through transfusion, pregnancy, or intramuscular injection.
(2) Check for the expiration date of anti-sera • Animals are deliberately inoculated with antigen, and the resulting serum,
(3) Always add serum or antisera before adding cells which contains antibody, is purified and standardized for use as an antiserum.
(4) Always examine tube after serum has been added to ensure that none has
been missed ANTISERA REQUIREMENTS
(5) When dropping the anti-sera/ red cells to the tubes, be sure that the tubes The following are the characteristics of an ideal antisera:
are distant with each other 1. It must be specific for the antigen to be detected;
2. It must have a sufficient concentration, titer, to detect an antigen;
ABO GROUPING – FORWARD TYPING 3. It must have a certain avidity for, or strength of reaction with, corresponding
• Blood groups and the inherent differences in human blood from one individual RBCs;
to another were first discovered by a German scientist, Karl Landsteiner, in 4. It must also be sterile, clear, provided in a good container with dropper, and
1900. stable;
o He recognized three different “groups”, named according to the 5. It should be marked with expiration date and must not be used after this
antigen present on the red cells. Individuals who possessed the “A” date;
antigen (i.e., their cells showed agglutination with anti-A) were 6. It must be stored at 4°C when not in use
classified as belonging to group “A”.
• Individuals who possessed the “B” antigen (i.e., their cells showed
agglutination with anti-B) were classified as belonging to group “B”. PRINCIPLE
• Certain individuals showed no agglutination with either anti-A or anti-B, and • The individual’s red cells are combined with serum containing known
they were classified as belonging to group “O”— the symbol “O” denoting antibody.
zero—or the lack of A and B antigens on the red cells. o Forward Typing is done using the known anti-sera.
• A fourth group was discovered by Landsteiner’s pupils, von Decastello and o Presence of agglutination and/or hemolysis on the red cells with a
Sturli in 1902. particular antiserum indicates the presence of corresponding
o Individuals in this group showed agglutination with both anti-A and antigen.
anti-B, and the group was called group “AB”. • Uses the patient’s red blood cells
• It was discovered that individuals who possessed A antigen on their red cells o All red blood cells contain antigens that are specific to the patient’s
also possessed anti-B in their serum; individuals who possessed B antigen blood type
had anti-A in their serum; individuals with both A and B antigens showed
neither anti-A nor anti-B (see Appendices section).

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 o When antibody A & antibody B reagents is added to the patients procedure for at least three times or until a clear
red blood cells, the antigens on the cells will cause to react with the supernatant is acquired.
antibodies (8) After the last wash, gently re-suspend the red cells with
• The procedure is based on the principle of agglutination. ¾ full of saline.
• This is to detect ABO antigens present in the patient’s red blood cells. (9) Prepare another 2 clean test tubes and label with anti-A
• Testing with both Anti-A and Anti-B is necessary. and anti-B including patient’s name.
o To determine if the RBC possess or lack the A or B blood group (10) Add one drop of anti-A grouping serum to the tube
antigens labeled “Anti-A” and one drop of Anti-B grouping serum
• Absence of agglutination = negative test result to the tube labeled “Anti-B”.
o Indicates that the corresponding antigen is not demonstrable NOTE: To avoid contamination, make sure that the
• Agglutination of the Red Blood Cells w/ a given reagent = positive result tubes are well-away from each other.
o indicates the presence of the corresponding antigen on the RBC (11) Add one drop of a washed red cell suspension from the
individual under test to each test tube.
MATERIALS NOTE: Allow sufficient distance between the tube
containing the antisera and the dropper used for the
Working reagent: (1) 0.85-0.9% NaCl solution suspension to avoid contamination
(2) Antisera A and antisera B (must be stored at 4°C) (12) Mix each tube thoroughly by tapping the tube on the
palm.
Specimen Used: 5% RBC suspension (freshly prepared) (13) Centrifuge for 15-30 seconds at 3,500 rpm.
(14) After centrifugation, gently re-suspend the tube by
Materials and (1) 5-mL test tubes shaking.
Equipment: (2) Serological pipettes (15) Observe and grade the agglutination present
(3) Venipuncture kit test macroscopically. (see Table 1 from activity #2 for the
(4) Tube rack grading)
(5) Plastic droppers (16) Interpret (see table below) and record results
(6) EDTA tubes
(7) Centrifuge INTERPRETATION Anti-A Anti-B Blood group
(8) Nescofilm/parafilm OF RESULTS - - O
(9) Forceps + - A
(10) Red top tubes - + B
+ + AB
• Positive (+) = presence of agglutination and/or
hemolysis
PROCEDURE • Negative (-) = absence of agglutination and/or
hemolysis
A. TUBE (1) Collect at least 2-mL of blood and place in an EDTA
METHOD tube.
(2) Spin for 5 minutes. After centrifugation, remove the
B. SLIDE (1) A clean and dry glass slide is divided into two sections
plasma on top.
METHOD with a glass marking pencil. The sections are labeled as
(3) Prepare another clean and dry 5-mL tube. Label with
anti-A and anti-B to identify the antisera.
the patient’s name.
(2) Place one drop of anti-A serum and one drop of anti-B
(4) Add 2 drops packed red cells into the labeled test tube.
serum in the center of the corresponding section of the
(5) Fill test tube with ¾ full of saline to re-suspend the cells.
slide. Antiserum must be taken first to ensure that no
Avoid contamination of tubes when dispensing saline
reagents are missed.
into several tubes.
(3) Add one drop of blood sample to be tested to each drop
(6) Centrifuge the tube for 5 minutes to obtain clear
of antiserum.
supernatant and a defined red cell button. Decant the
(4) Mix antiserum and blood by using a separate stick or a
supernatant.
separate corner of a slide for each section over an area
(7) Following the removal of supernatant, gently re-
about 1 inch in diameter.
suspend the red cell button. Repeat the RBC washing
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 (5) By tilting the slide backwards and forwards, examine for
agglutination after exactly two minutes.

INTERPRETATION Anti-A Anti-B Blood Group

OF RESULTS - - O
+ - A
- + B
+ + AB

• Positive (+): Little clumps of red cells are seen floating

in a clear liquid.
• Negative (–): Red cells are floating homogeneously in
a uniform suspension.

ADDITIVES USED IN BLOOD TYPING:

Freshly collected RBCs Preferred for testing and may be collected as

clotted samples or in anticoagulant
Clotted samples or samples May be used 5 days after collection
collected in ACD
EDTA or Sodium Citrate Should be tested within 3 days
Heparin or Oxalate May be used within 2 days
Donor red blood cells May be tested up to the expiration date of the
collected in CPDA-1 or CP2D unit
or CPD

---end of transcription-

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4 ABO BLOOD GROUPING-REVERSE TYPING Materials and (1) 5-mL test tubes
Equipment: (2) Serological pipettes (1 mL, 5 mL)
IMMUNOHEMA LAB//MAAM PALOMAR (3) Venipuncture kit
(4) Test tube rack
INTRODUCTION: ABO GROUPING – REVERSE TYPING (5) Plastic droppers
• The presence of these environmentally stimulated anti-A and anti-B (6) EDTA tubes
antibodies in the serum/plasma of immunocompetent individuals makes the (7) Centrifuge
ABO system unique among RBC groups. (8) Forceps
• Anti-A and anti-B are very potent, and transfusing RBCs with the antigen to a (9) Marker
person with antibody (ABO incompatible) would result to immediate and (10) Applicator sticks
severe hemolytic transfusion reactions that could be fatal to the patient. (11) Red top tubes
• An individual of unknown ABO blood group is usually tested by forward typing
and by reverse grouping. Preparation of (1) Collect at least 2 mL of blood from group A
• In reverse grouping, the individual’s serum is tested against cells with known known “A” and individual.
antigen. known “B” cells: (2) In a clean and dry 5-mL tube, prepare a washed
• Red blood cells from group A and group B individuals are being used as an RBC suspension from the collected blood sample.
indicator. This will serve as the known A cells.
o If there is a positive reaction with known group A cells, the serum (3) Follow the same procedure in the preparation of
contains anti-A anti bodies. known B cells. Take note that the blood sample must
o If there is a positive reaction with known group B cells, the serum come from a group B individual.
contains anti-B antibodies.
o If serum reacts with both known group A and group B cells, both anti- Tube Method (1) Collect at least 3-mL of blood and place in a red top
A and anti-B antibodies are present. tube.
o Absence of reaction with both known group A and group B cells (2) Let it stand for 20-30 minutes. Spin to separate
indicates that the serum does not possess both the antibodies. serum.
• This method is also known as antibody, serum, indirect, or back typing (3) Prepare two clean test tubes and label with “known
reactions. A” and “known B” including patient’s name.
• Patient’s serum is used (4) Add two drops of serum from the individual under
o serum contains the antibodies that react w the reagent RBC which test to each test tube.
are coated with antigen A or B (5) Add two drops of 5% known A cells to the tube
o aka “A” cells or “B” cells labeled “known A” and two drops of 5% known B
• Type of Antibodies present in the Patient’s serum will determine which reagent cells to the tube labeled “known B”.
RBC will cause agglutination and will therefore confirm the blood type. Note: Ensure that the tubes are well away from each
• Results from Reverse typing are opposite from the results of Forward typing other upon the addition of known cells. Allow sufficient
o Not considered to be the blood type but the confirmation for the distance between the tube containing the serum and the
results of forward typing droppers used for known cells to avoid contamination.
(6) Mix each tube thoroughly by shaking or by tapping
PRINCIPLE on the palm.
(7) Centrifuge for 15-30 seconds at 3,500 rpm.
• The individual’s serum is combined with red cells possessing known antigen.
(8) Carefully remove the tubes from the centrifuge.
• Presence of agglutination is associated with the presence of the
Observe for the presence of hemolysis.
corresponding antibody.
(9) After checking for hemolysis, gently resuspend the
• This test is used to enhance agglutination in weakly reacting serum specimen tube.
(10) Observe and grade the agglutination reaction
TUBE METHOD macroscopically (see Table 1). 17. Interpret (see
Working reagent: (1) 5% known A and known B cells Table below) and record results.
(2) 0.85% NaCl solution
Specimen Used: Serum

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Tube Method: Known A Cells Known B Cells Blood Group • Exists when the result of an ABO RBC Typing or Forward Typing does not
Interpretation of + + O agree with the result of the plasma typing or reverse typing
Results - + A • Can be encountered when ABO typing is performed on samples from blood
+ - B or tissue donors
- - AB • When a discrepancy is observed, it must be determined if the problem is
• Positive (+) = presence of agglutination and/or associated with the forward type, reverse type or both
hemolysis
• Negative (-) = absence of agglutination and/or APPROACHES THAT CAN BE FOLLOWED WHEN AN ABO DISCREPANCY IS
hemolysis ENCOUNTERED:
(1) Repeat the test with the same sample
QUALITY CONTROL - from the beginning since the plasma or RBC suspensions can be
• to recognize reagent deterioration, the reactivity of all blood grouping mixed up within the laboratory
reagents should be confirmed on each day of use by testing known positive (2) Repeat the test with a new sample or review the patient’s medical record
and negative controls (3) Review prior in-house laboratory testing record
• Problems regarding Rouleaux Formation: (4) May contact other healthcare facilities
o add 1-3 drops of saline to the tubes that have rouleaux formation (5) Verify the reagent and the equipment performance
o then mix and centrifuge
o Rouleaux Formation tends to disperse after this NOTE:
▪ however, Antibody-mediated agglutination will remain • When repeating the test, prepare a fresh RBC suspension
• When doing Blood Typing: o because a mix up in the sample identification may have occurred
o If anti-A is positive, “A1” cells are positive = presence of A2 (?) during initial testing
o Crossmatching a group A person with A units and the units are • In most laboratories, they perform initial forward typing using normal saline
incompatible = A2 is or may be present washed RBC
o when initial typing was performed using plasma suspended RBC
ADDITIONAL INFORMATION o if the result is the same as the first, request for a new sample for
• ABO and Rh testing require both the serum and the RBC testing
o Specimens should be collected in a vacuum tube with no
preservative COMMON SOURCES OF TECHNICAL ERRORS
o preferably on a red top tube 1. Misidentification of patient
o EDTA (lavender top) can be used as well 2. Incorrect identification of blood specimens, tubes or slides
• Upon using red top tube: 3. Cell suspension either too heavy or too light
o Do not use gel separator tubes 4. Mix up samples
▪ will put a layer of gel between the RBC and the serum 5. Missed observation of hemolysis
when centrifuged 6. Failure to add reagents
▪ may make RBC difficult to obtain 7. Failure to add sample
▪ the gel may mix in the RBC and cause negative reading to 8. Failure to follow manufacturers’ instructions
look positive 9. Uncalibrated centrifuge
▪ will cause false negative result 10. Over-centrifugation or Under-centrifugation
• Test the specimen ASAP; if there is delay in testing, properly store the 11. Contaminated reagents
specimen in a refrigerator 12. Warming during centrifugation
13. Technical problems should be ruled out with a check of the reagents,
ABO DISCREPANCIES equipment and repeating the testing
• Occurs when the unexpected reaction occurs in the forward and reverse
FOUR MAJOR CATEGORIES OF ABO DISCREPANCY
grouping
• Can be resolve by repeating the test using saline suspension
Group 1 • When the reaction in the serum grouping is weak or
missing, group I discrepancy should be suspected
• Essential to acquire patient’s information
• RBC and serum grouping are very strong (4+)
• If the error persists, extract another sample from the patient and repeat the
test • most common
• Most common cause are technical errors.
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 • mainly seen in reverse grouping due to weak or missing (2) transplanted BM
antibodies (3) fetal-maternal bleeding
(4) exchange transfusion
Cause • Weak reacting or Missing antibodies
• Low antibodies or error to produce antibodies (1.) CHIMERA → Is an organism that arises from
the fusion of more than one
Commonly (1) newborns blastocyst or zygotes following
Found In (2) elderly patients sexual reproduction
(3) px’s with leukemia or lymphoma, → Or also called single organism
(4) patient with bone marrow or stem cell transplantations, etc. containing two population of
genetically distinct cells
Resolution (1) Obtain patient’s history diagnosis originating from two different
(2) Incubate patient’s serum at room temp to enhance reaction zygotes
(3) Add more plasma or serum to the test
(4) If no reaction: incubate at 4°C (2.) MICRO → These cells may travel from the
• For newborns, only forward grouping is done until 4 CHIMERISM mother’s or fetus’ bloodstream
months of age and migrate to different organs.
• Can be solved by enhancing the serum grouping → They may remain in a mother’s
reaction. This can be achieved by incubating the cells body or a child’s body a
serum mixture at low temp. decade or more following child
• Another procedure: prolonging the incubation at room birth.
temp. 30 mins to 1 hr. 22°C
Note: incubation should be done for 15-30 mins (3.) ARTIFICIAL → Occurs when a person
CHIMERISM receives a blood transfusion,
Example Reaction of patient’s cell Reaction of patients’ stem cell transplant, or a bone
(forward typing) serum marrow transplant from another
(reverse typing) person and absorbs some of
Anti-A Anti-B A1 cells B cells the person’s cells
0 0 0 0
Probable group: O (elderly or newborn)
(4.) TWIN → It can occur when a pair of
Group II • Associated with unexpected reaction on forward grouping CHIMERISM twins is conceived and one
• Weak reacting or missing antigens embryo dies in the womb.
• Antibodies to low incidence antigens → The surviving cell may absorb
• seen least common some of the cells of its
• the variation in agglutination reactivity occurs due to deceased twin and this
differences in amounts of antigen present on RBCs or in givesthe surviving twin fetus
secretion two sets of cells.
→ Its own cell and some of its
CHIMERISM twin.
• occurs when cells of different donor origin consist in the
same organism
• most commonly occurs in pregnant women
• Presence of two cell populations in a single individual Causes (1) subgroups of A and B
• Cause of a weak or missing ABO isoagglutinin • Subgroups of A are more common than B
• True chimerism rarely found / occurs in twins • two most important subgroups clinically are A1 and
• Artificial chimerism occurs: A2
(1) BT (Type “O” given to “A” and “B” individuals) • A1 is distinguish by its reactivity with the lectin
Dolichos biflorus
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 • All other A subgroups non-reactive with Dolichos are → The saline disperses the cells. The tube is mixed,
referred to as A2 centrifuge and re-examined for agglutination
• A2 shows increased reactivity with lectin Ulex
europaeus Example Reaction of patient’s cell Reaction of patients’ serum
(2) leukemia’s may yield weakened A and B antigens (forward typing) (Reverse typing)
(3) acquired B phenomenon (cancer of the colon) Anti-A Anti-B A1 cells B cells
(4) Excess amount of BGSS in the plasma (carcinoma of 4+ 4+ 2+ 2+
stomach and pancreas) Probable group: AB

Resolution (1) Incubate the test mixture at room temp for up to 30 mins
Group IV (1) Cold reactive autoantibodies
(2) No reaction: incubate at 4°C for 15-30 mins
miscellaneou (2) Unexpected ABO Isoagglutinins
(3) Wash red cell to remove excess BGSS
s (3) Unexpected non-ABO alloantibodies
(4) Repeat the test using antisera with different lot number
(4) Poly agglutination (bacterial infection)
(5) For subgroups A or B, this can dissolve by repeating blood
grouping by using washed cells, or anti AB, antisera and • Poly Agglutination is due to exposure of hidden
anti-A1 lectin or by absorption-elution. erythrocytes antigens like the T antigens in bacterial
(6) Wash patient’s RBCs several times with saline are viral infections
(7) Will free the cells from rouleaux formation • If poly agglutination is present, its symptoms is
(8) In true agglutination, cells will remain clumped suggestive of infection
(9) Wash cord cells with saline 6-8 times to remove the (5) Ab’s other than anti-A and anti-B may react to form Ag-Ab
rouleaux formation due to Wharton’s jelly complexes
• E.g some individuals have Ab’s against acriflavine,
Example Reaction of patient’s cell Reaction of patients’ anti-acriflavin complex attaches to the Px’s RBC and
(forward typing) serum causes agglutination
(reverse typing) (6) Also caused by A2 or A3 individual with anti-A antibodies,
Anti-A Anti-B A1 cells B cells naturally occurring or irregular antibodies reacting at room
0 0 0 3+ temperature
Probable group: O
Note: The Auto control and DAT are negative
Group III Results in rouleaux formation or pseudoagglutination
How to resolve poly agglutination:
Caused by (1) Elevated levels of globulin from certain diseases (multiple
Use various lectins like glycine soja or arachis hypogaea
myeloma, Waldenstrom’s macroglobulinemia, and other
plasma dyscarias, advance cases of Hodgkin’s lymphomas)
(2) Elevated levels of fibrinogen
Resolution • pre-warming at 37 degree Celsius will result in elimination of
(3) Plasma expanders alloantibodies
(4) Wharton’s jelly in cord blood samples • for polyagglutination and rouleaux formation: perform 3x
saline technique
Resolution (1) Wash patient’s RBCs several times with saline
• Will free the cells from rouleaux formation Example Reaction of patient’s cell Reaction of patients’ serum
• In true agglutination, cells will remain clumped (forward typing) (Reverse typing)
(2) Wash cord cells with saline 6-8 times to remove the Anti-A Anti-B A1 cells B cells
rouleaux formation due to Wharton’s jelly 2+ 4+ 4+ 2+
• If the serum or reverse grouping is affected you Probable blood group: B
perform saline replacement technique
How to perform saline replacement technique: -end of transcription-
→ The reagent cells and the patient’s serum are
centrifuged to allow antigen and antibody to react.
→ Then the serum is removed and replaced by an equal
volume of saline.

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 Module Notes: PPT Presentation:
Gloves Capillary tube
Eye Goggles Blood lancets
CuSO4 Crystals Plastic droppers
COPPER SULFATE(CUSO4) METHOD Hot Water 100 ml beaker
● It is best to use freshly prepared CuSO4 Measuring spoons/cups
● Based on specific gravity of blood Beaker
● It is the simplest of methods that ensures that the donor meets the minimum Glass Container
hemoglobin level of 12.5g/dl with specific gravity of 1.053 at temperatures
25° Celsius
● Prepare by allowing ambient temperature especially when it is exposed
temperature in transport WORKING REAGENT
● Mix well before using, then transfer to Coplin jar and keep the jar covered
when not in use Copper sulphate standards ([Link]. of 1.053 and 1.055)
● If the solution is used in a higher temperature the specific gravity will be
lower, will result to collection of some units from donors whose hemoglobin SPECIMEN
level maybe inadequate for donation
Capillary blood
● Ideal long storage temperature is 15-25° Celsius
PROCEDURE
PRINCIPLE
According to Module Notes According to the Module Notes
● A drop of whole blood when dropped into a solution of CuSO4 which has a
given specific gravity, will maintain its own density for approximately 15 1. Get 1 part of CuSO4 crystal then pour it to 6 parts of hot water in a beaker
seconds. 2. Mix it until all crystals are dissolved
● The density of the drop is directly proportional to the amount of hemoglobin 3. Let it cool
in that drop. If the drop is denser than the specific gravity of the solution, the 4. Put it in a container with cover (ready to use)
drop sinks to the bottom, if not, it will float on top. Note: It is best to use freshly prepared CuSO4.
● Plasma or Whole Blood dropped into a solution of Copper Sulfate of known
gravity is encased in a sack of copper proteinate and the gravity of the According to PPT Presentation
discrete drop is not changed for about 15 seconds
● The rise or fall of the drop during this interval (within 15 seconds) shows 1. Perform capillary puncture and fill 1-2 capillary tubes with 2/3 of whole blood
whether it is lighter or heavier than the solution 2. Allow a drop of blood to fall approximately 1 cm above the standard copper
sulphate solution.
According to PPT Presentation 3. Use copper sulphate solution with a specific gravity of 1.053 for female and
male 1.055
● When a drop of protein solution is immersed in a solution of copper sulphate,
4. Observe the behavior of the drop in the copper sulphate solution. It will sink
a sac of copper proteinate forms on the surface of the drop and prevents a
beneath the surface from the momentum of its fall.
change in its contents for twenty to thirty seconds
5. Within 5 seconds, it will rise slightly if it is lighter than the solution, fall slowly
● If the specific gravity of the protein exceeds that of the copper sulfate
if it's heavier, or remain suspended if its density is equal to that of the copper
solution the drops sinks; when the protein is lighter than the copper sulphate
sulphate solution.
solution, the drop tends to rise in the solution
o A drop of blood with hemoglobin of 12.5 g/dL or higher should sink
it 10-15 seconds INTERPRETATION OF RESULTS
o A drop of blood with hemoglobin <12.5 g/dL should float
● For male donors:
o Whole blood sinks- at least 13.5 g/dL of hemoglobin
o Whole blood floats- less than 13.5 g/dL of hemoglobin

● For Female donors

MATERIALS AND EQUIPMENT
o Whole blood sinks- at least 12.5 g/dL of hemoglobin
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 o Whole blood floats- less than 12.5 g/dL

NOTE:
● if the drop of blood sinks, accept the donor;
● if it floats, reject the donor
Advantages of Copper Sulphate Method:
1. Measurements are sufficiently accurate for any clinical application
2. The manipulation and interpretations are extremely simple
3. A stable base is not required, e.g., the test may be performed on
shipboard
4. The apparatus and materials are readily obtainable and economical
5. Not temperature correction is needed
6. Calibrated pipettes are not necessary

-End of transcription-

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 ● Major crossmatch includes mixing of donor’s RBCs with the patient’s serum
● You need RBC from donors in EDTA and citrate
● Is to find out any incompatibility of the RBCs with the patient’s serum to
avoid blood transfusion reactions
INTRODUCTION
● The term compatibility test and crossmatch are sometimes interchangeably MINOR CROSSMATCH
used; they should be clearly differentiated. ● Consisting of mixing the donor’s plasma with the patient’s red cells, detects
● A crossmatch procedure is only part of the compatibility test. antibodies in the donor which may be capable of affecting the recipient’s red
● Compatibility test consists of the following: cells.
o review of patient’s blood bank history and records; ● Compares donor’s serum to recipient’s erythrocytes and this check for
o ABO and Rh grouping of the recipient and donor; preformed antibodies in donor’s serum that could hemolyzed recipients red
o antibody screening of the recipient’s and donor’s serum cells
o the crossmatch procedure itself ● It is rarely requested, when compatibility of the recipients red cells is tested
against the donor’s serum
COMPATIBILITY TESTING
● it refers to the serologic aspect of pretransfusion testing Note: In major crossmatch, donor erythrocytes are washed and incubated with
● Is performed to determine if a particular unit of blood can be transferred recipient’s serum. In minor crossmatch the donor’s serum is incubated with washed
safely to a patient recipients erythrocytes.
● Includes ABO Rh typing,
● Antibody screening - used for unexpected red blood cell antibodies that PRINCIPLE OF COMPATIBILITY TESTING
would cause problem to the recipient ● Serum of the recipients is tested against the red cells of the donor under
● Crossmatching - performed to determine if the patient has antibodies that different condition to establish crossmatching and non-agglutination
react with the donor’s cells ● If there is agglutination in any of the conditions this would indicate the
presence of incompatible antibody in patient whether natural or immune
CROSSMATCHING
● The crossmatch is the most important and most frequently performed 3 PHASES OF COMPATIBILITY TESTING
procedure in the routine blood bank laboratory.
● Two main functions of the crossmatch test can be cited: Saline phase it is where the immunologic reaction between the red cells
(1) It is a final check of ABO compatibility between donor and patient suspended in saline medium and the antibody occurs at
(2) It may detect the presence of an antibody in the patient’s serum room temperature
that will react with antigens on the donor RBCs but that was not Thermal phase it is where the red cells are suspended in the antibody with
detected in antibody screening because the corresponding antigen with protein 22% albumin and incubated for 30 minutes at 37° Celsius
was lacking from screening cells AHG phase where incubated cells are washed to remove the free
● The objective of crossmatch is to select donor units that are able to provide globulin and reacted with AHG serum or Coomb’s reagent.
maximal benefit to the patient. Many procedures are available which can, in
combination, constitute a satisfactory crossmatch.
● Transfusion reactions may occur if antibodies in the recipient’s serum are not SALINE TECHNIQUE
recognized or detected in the crossmatch. ● It is designed to detect IgM antibodies which react at room temperature or
o This could result in a reduced survival or rapid destruction lower.
transfused cells, and even in death of the patient. ● Agglutination or hemolysis at 22° Celsius will detect ABO incompatibility and
o It is therefore essential that the crossmatch provide conditions is usually due to incorrect typing.
suitable for the optimum reactivity of all clinically significant ● Would also detect cold agglutinin and saline actin antibodies such as anti-M,
antibodies anti-N, anti-S, anti-LU, anti-F, and anti-LE
● Saline crossmatch at 37°Celsius would detect the saline acting anti Rh
TWO TYPES OF CROSSMATCH antibodies, some anti-M, anti-N and anti-S, anti-K, and anti-LE antibodies
MAJOR CROSSMATCH
● The Major Crossmatch is the most important.
● The purpose of “major” crossmatch is to prevent a transfusion reaction by
detecting antibodies in the patient’s serum which would reduce the survival
of the donor’s red cells, and to ensure maximum benefit to the patient. ALBUMIN TECHNIQUE

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 ● Bovine albumin acts to increase the dielectric constant of the medium and so 13. Rh viewbox
therefore it would allow the Igg antibodies to be demonstrated and most IgG 14. Marker
antibodies are detected using the albumin technique.
● Albumin crossmatch at 37 ° Celsius detects most anti Rh antibodies, some SALINE TECHNIQUE PROCEDURES
anti M, anti N, anti S, and Anti LE antibodies A. Technique No. 1: Saline (Immediate Spin)
1. Prepare a clean test tube and label with patient’s name.
ANTI-GLOBULIN TECHNIQUE 2. Add two drops of serum from the individual under test to the tube.
● Would detect the IgG antibodies of donors who do not react in either the 3. Add one drop of a washed 5% suspension of the donor’s red cells.
saline of high protein media or which react weakly or variably in these 4. Mix the tube thoroughly by shaking or by tapping on the palm.
medias and it would detect antibodies which bind compliment but which may 5. Centrifuge for 15-30 seconds at 3,500 rpm.
not be capable of binding to the cells insufficient amount to give a positive 6. Observe for the presence of hemolysis and/or agglutination macroscopically
test with the use of optical aid. (Examine negative reactions microscopically).
● It could detect anti FY, anti JK, and most anti K antibodies 7. Interpret and record results

ENZYME TECHNIQUE
● Serve as an important back up test for the indirect antiglobulin test and
detects many clinically significant IgG and saline inactive IgM antibodies
● Enzyme used: Trypsin, Papain, Ficin, and Guamulene

CROSSMATCHING PROCEDURE
PRINCIPLE

Major Crossmatch Detection of antibodies in the PATIENT’S serum which

would reduce the survival of the donor’s red cell, and to
ensure maximum benefit to the patient
Minor Crossmatch Detection of antibodies in the DONOR’S serum which
may be capable of affecting the recipient’s red cells.

WORKING REAGENT:
● 0.85% NaCl solution
● Donor’s 5% red cell suspension
● Distilled water
Technique 1: Interpretation of Results
SPECIMEN USED: ● Presence of hemolysis and/or agglutination = INCOMPATIBLE
Serum ● Absence of hemolysis and/or agglutination = COMPATIBLE
MATERIALS AND EQUIPMENT: B. Technique No. 2: Saline (22°C Incubation)
1. 5-mL test tubes 1. Place two drops of the patient’s serum in a clean test tube.
2. serological pipettes 2. Add one drop of a washed 5% suspension of donor’s red cells.
3. venipuncture kit 3. Mix the tube thoroughly by shaking or by tapping on the palm.
4. test tube rack 4. Incubate at room temperature for 15 to 30 minutes.
5. thermometer 5. Centrifuge at 3,400 rpm for 15 seconds.
6. plastic droppers 6. Examine macroscopically for hemolysis and/or agglutination using optical aid
7. EDTA or viewbox. (Examine negative reactions microscopically)
8. centrifuge 7. Interpret and record results
9. waterbath 8. The only difference between the 1st Technique (Saline immediate spin) and
10. nescofilm/parafilm this technique is that you need to incubate at 15-30 minutes at room
11. forceps temperature and centrifuge at 3400rpm for 15 seconds.
12. applicator sticks

2 | Page
 ● Need 2 drops of anti-human globulin(green). This anti-human globulin(green)
act as enhancement media especially if ever the patient has a past transfusion
or patients having beta-thalassemia or dialysis patients.
● Patients having these conditions have medicines mixed in their blood as a result
their blood will not interact immediately with saline so that’s why anti-human
globulin(green) is added to enhance the reaction.

Technique 3: Interpretation of Results

● Presence of hemolysis and/or agglutination = INCOMPATIBLE
● Absence of hemolysis and/or agglutination = COMPATIBLE

SALINE TECHNIQUE: APPLICATION AND LIMITATIONS

● Certain blood group antibodies react at body temperature in-vivo, however
they may give optimal reactions in vitro or temperature below that at the
● Here we detect only IgM Antibodies reactive at 22 Degrees celsius and body.
clinically significant IgG Antibodies reactive at 37° Celsius is NOT detected. ● Designed to detect IgM antibodies that react optically at room temperature or
● We also check here the ABO Incompatibility lower.
● That is why we performed immediate spinning of 22 degrees to 37 degrees
Technique 2: Interpretation of Results incubation because they react with temperature.
● Presence of hemolysis and/or agglutination = INCOMPATIBLE ● Serves to detect major ABO grouping error since incompatibility in this
● Absence of hemolysis and/or agglutination = COMPATIBLE phase will result in potentially serious situations if red cells at an incorrect
ABO group have been mistakenly selected for crossmatching.
C. Technique No.3: Saline (37°C Incubation) ● Saline techniques are inadequate since clinically significant IgG antibodies
1. Place two drops of the patient’s serum in a clean test tube. are not detected in this phase
2. Add one drop of a washed 5% suspension of donor’s red cells.
3. Mix the tube thoroughly by shaking or by tapping on the palm. -end of transcription-
4. Incubate at 37°C using water bath for 15 to 30 minutes.
5. Centrifuge at 3400 rpm for 15 seconds.
6. Examine macroscopically for hemolysis and/or agglutination using optical aid
or viewbox. (Examine negative reactions microscopically)
7. Interpret and record results.
In this technique, we utilize our water bath and incubate it at 37° Celsius for 15-30
minutes

3 | Page
 MODULE 9: THE ANTIGLOBULIN TEST
MLS 18, June 8, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`
Involves red cells coated with incomplete antibodies- those that fail
Legend: to agglutinate red cells suspended in saline.
Transcription Bullet • These antibodies are usually IgG and are agglutinated by the
Notes/Module Packet Bullet anti-IgG-I antiglobulin serum through the linking of the IgG
PPT molecules on neighboring red cells
Antiglobulin reagents is a pool of serum made from two different
MODULE OUTLINE colonies of rabbits.
I. Antiglobulin Test • One group has been immunized with human beta globulin
II. Anti-Human Globulin • The other with human gamma globulin.
III. 2 Types of Antiglobulin Test • The animals build antibodies against the human globulin thus
IV. Sources of Error in the Antiglobulin Technique the reagent is called “antihuman globulin”
Also known as Coombs’ Test
LEARNING OUTCOMES Reagent used is Anti Human Globulin (AHG)
At the end of this module, the student shall be able to: Made by injecting human globulin into animals that will produce
1. Detect and identify the unexpected antibodies. Polyclonal Antibodies Specific for Human Immunoglobulins and
2. Perform the two kinds of Antiglobulin Technique. Human Complement System Factors
3. Trace the sources of error in the Antiglobulin Technique.
4. Interpret results based on the procedure they perform. Principle

ANTIGLOBULIN TEST
The most important and most widely used serological procedure in
modern bloodbanking.
o Most essential part of any crossmatch procedure.
Discovered in 1908 by Moreschi and was rediscovered and
introduced into clinical medicine by Coombs’, Mourant and Race in
1945
Used for the detection and identification of unexpected antibodies,
and for detecting red cell antigens not demonstrated by other
techniques, and for special studies such as the antiglobulin
consumption test, mixed agglutination reactions and platelet
antibody tests.
Red cells coated with complement or IgG antibodies do not
It detects the red cells sensitized by IgG alloantibodies, IgG
agglutinate directly when centrifuge and these cells are sensitize
autoantibodies and/or complement components which do or do not
with IgG or complement
react either in saline or high protein media, or which react weakly or
variably in these media.
#MLSWATER2022 Page 1 of 8
 MODULE 9: THE ANTIGLOBULIN TEST
In order for agglutination to occur, an additional antibody which Working Reagent: Albumin Phase
reacts with the Fc portion of the IgG antibody or with the C3b/C3b Meant to detect IgG antibodies that are too small to cause direct
component complement must be added to the system and will form agglutination of red cell suspended in saline
a bridge between the antibodies or the complement coating the red The source of albumin used in the lab test are cows/bovine
cells that will cause agglutination Mechanism of action of albumin is it increases the di- electric
constant and decreases the zeta potential to allow the red cells to
Materials and Equipment Needed come closer.
Materials and Equipment: Being a dipolar molecule, albumin can dissipate energy as it rotates
o 5-ml test tubes and reduces the thickness of the ionic clouds surrounding each cell
o serological pipettes (1ml, 2ml, 5ml) Once the red cell is close enough, the small IgG molecule can span
o red top tubes the distance between the cells and causes agglutination
o Test tube rack Antibodies detected including IgG or H antibodies or any IgM
o thermometer antibody that react at 37°C
o Plastic droppers Albumin is the poorest in detecting antibodies because both the
o Centrifuge IgM and IgG antibodies react best in other phases
o Water bath although it is meant to detect IgG antibodies, the only IgG antibody
o EDTA tube that can react in albumin are the Rh antibodies
o Forceps
o Applicator Sticks ANTI-HUMAN GLOBULIN
o nescofilm/parafilm 1. Anti-IgG
o Rh viewbox Contains antibody reactivity against like IgG chains and thus may
o Marker also agglutinate IgA or IgM-coated red blood cells
2. Anti C3b
Working Reagent: 3. Anti-complement component
o 0.85%-0.9% NaCl solution Consist of Murine Monoclonal IgM anti-C3b antibody reactive with
o Distilled water C3b and C3d coated red blood cells
o Bovine albumin Preservative used: 0.1% sodium acetate
o Antihuman globulin
ANTIGLOBULIN REAGENTS
Take note: POLYSPECIFIC ANTI-GLOBULIN REAGENTS
This reagent is for in vitro diagnostic use DAT determine that either IgG or complement molecules attached
Store it at 2-8°C to the red cells in vivo
Do not use the reagents beyond the expiration date Contains both Anti-IgG and Anti-C3d antibodies
Do not use if it is already turbid Detects IgG and C3d antibodies
Do not dilute the reagents Conventionally, AHG reagents are manufactured by injecting rabbits
Do not use specimens coated with gel separators with human globulin that will stimulate the production of antibodies
Do not pipette by mouth When using these reagents, the antigen binding sites or the Fab
(Further discussion below) sites of the anti-globulin molecule attached to either the Fc portion

#MLSWATER2022 Page 2 of 8
 MODULE 9: THE ANTIGLOBULIN TEST
of the bound antibody or to the complement on 2 adjacent RBCs Anti-complement reagent:
and it will form a lattice that allows for the visible agglutination o eg. Anti-C3d and Anti C3b – reactive against only the
Polyspecific AHG reagents are blends of anti-imunoglobulin and designated complement components and contains no
anti-C3. And are used to detect IgG antibodies activity against the human immunoglobulin
if the DAT is positive, IT SHOULD BE REPEATED using monospecific
reagents to determine the specific protein causing the positive IgG SENSITIZED RED CELLS
reaction Control system for Anti-globulin test interpreted as negative
Anti IgG reagents detect IgG antibody sensitization only Additive system for negative anti-globulin test to control the
Anti-complement reagents detect complement sensitization only possibility of false negative-result
IgM and IgA bounded to the RBCs are not detected with these 3 POTENTIAL REASONS FOR FALSE NEGATIVE
methods 1. Failure to add anti-globulin reagent
Monoclonal anti-C3 detects activated complement component 2. Failure of the added anti-globulin reagent to react
which may or may not indicate IgM immunoglobulin in complement 3. Failure to wash red cells adequately
When we say Polyspecific, it contains both the anti-IgG and the anti- Are used for the control of the indirect and direct antiglobulin test
C3d These cells are used to confirm the validity of negative antiglobulin
Monospecific, it contains 1 specificity either the anti IgG or anti C3d test by demonstrating the anti-IgG activity of the anti-human
Both the Polyspecific and Monospecific AHG are available reagent used in the test
commercially known IgG sensitized cells are added in all AHG test that are
Positive direct anti-globulin test with polyspecific AHG should be negative both macroscopically and microscopically, the test is
further tested with monospecific reagents since additional testing centrifuged and read macroscopically in which agglutination must
provides further information in determining the type of immune occur
haemolytic anemia involved These cells control two as is of the test
These reagents are mainly used in anti-globulin test in saline, 1. Control the AHG serum was added to the test
albumin or LISS 2. Controls the washing is adequate and no unbound protein
was left behind which would neutralize the AHG serum
MONOSPECIFIC ANTI-GLOBULIN REAGENTS if no agglutination is the result, AHG test is invalid and it must be
Prepared by separating the specificities of the Polyspecific Anti- totally repeated from the start
human globulin Reagents When IgG sensitized RBCs are added to the negative anti globulin
Anti-IgG monospecific – contains antibodies to human gamma test, the resulting agglutination would indicate both the presence
chains and the absence of the anti-human globulin
Anti-C3b and Anti-C3d – contains no reactivity to human
immunoglobulin molecules ANTIBODY POTENTIATORS
Detect complement proteins attach to red cells surface What are Potentiators?
Contains no complement activity Are reagents used to adjust the in vitro test environment to
Contains antibody specific for the Fc fragment of the gamma heavy promote agglutination
chain of the IgG molecule
Zeta potential
Term used to describe the degree of electrostatic repulsion
between adjacent similarly charged particles.
#MLSWATER2022 Page 3 of 8
 MODULE 9: THE ANTIGLOBULIN TEST
In terms of RBCs, negatively charged RBCs and clouds of positive ion Take note:
surrounding them keep adjacent RBC a certain distance away from If using LISS, to avoid problems with cold agglutinins, the reagents
each other and this distance is significant because in a typical blood must first be brought to room temperature before use.
sample RBCs are not close enough for the IgG antibodies to cross o must be thawed first.
link them When using LISS, before positive reactions are investigated, the test
Crosslinking is needed for RBC agglutination to detect potentially should be repeated using pre-warm technique.
dangerous antibodies o To prevent cold clinically insignificant antibodies from
reacting.
Potentiators Enhancement Reagent
Decrease the charges surrounding RBC and allows them to get 2. PEG – Polyethylene Glycol
closer to adjacent RBCs Increases sensitivity of antibody detection and identification
Potentiators are used in immunohematology laboratory in cross Enhances warm autoantibodies
match test, antibody detection, antibody detection and in titration Water soluble polymer that eliminates water, allowing RBCs to get
procedures. closer together
1. LISS – Low Ionic Strength Saline PEG is more effective than LISS or albumin in detecting weak
Commonly used potentiators with the antibody screen because it antibodies.
speaks the agglutination, economical and provides good sensitivity PEG is added after the immediate spin phase and then incubated.
Enhance cold autoantibodies
Take note:
Consists of glycine in an albumin solution There is no 37 °C interpretation when using PEG.
Decreases the zeta potential but also shortens the incubation time o because centrifuging RBCs with added PEG could cause
required to detect most antibodies False-Positive reactions
Increases the speed of antibody sensitization which can also o instead of interpreting, RBCs are washed to remove PEG
increase the possibility of agglutination and excess antibodies before proceeding to AAHG phase
By using a low ionic strength saline, that is iso-osmotic, the rate PEG has been reported to increase the sensitivity of IAT
and extent of the first stage of antigen-antibody reaction which is
sensitization is enhanced 3. ENZYMES – Proteolytic
LISS has pure ions which reduces the shielding effect that ions have Commonly used in the immunohematology laboratory
and thus, will prevent the oppositely charged antigen-antibody from Can be used as additional tools for investigating complex antibody
coming together problems
Enhances the cold and warm autoantibodies
ADVANTAGES FOUR IMPORTANT ENZYMES
it decreases incubation time of 10-15 minutes as compared to 30-45 Enzymes Sources Order of Effectiveness in
minutes if doing the saline base test Detecting IgG Antibodies
it increases in sensitivity for most clinically significant antibodies Bromelin Pineapples 3
Ficin Figs (Ficus insipida) 1 (most effective)
DISADVANTAGES Papain Papaya 2
there is increase sensitivity for cold clinically insignificant antibodies Trypsin Lining of Hog’s Stomach 4 (least)
there is non-specific binding of compliment if ion strength is too low
#MLSWATER2022 Page 4 of 8
 MODULE 9: THE ANTIGLOBULIN TEST
These enzymes, when added to a blood sample will remove sialic 2 TYPES OF ANTIGLOBULIN TEST
acid. Direct Antiglobulin Test (Direct Coombs’ Test)
o The removal of sialic acid will decrease the negative surface Used to demonstrate whether the red cells have been coated or
charge making cell agglutination easier. sensitized with antibody or complement in vivo.
o The enzymes will enhance the reactivity of Rh, I, P, Lewis and The test is useful in:
Kidd antibodies. o The diagnosis of hemolytic disease of the newborn (HDFN), to
o These enzymes will also destroy the M, N, Ss and the FY slow whether fetal cells are sensitized with maternal antibody
antigens. o In autoimmune hemolytic anemia
Proteolytic enzymes enhance the breast or inhibit the antigen o In the investigation of red cell sensitization caused by drugs
antibody complex formation by removing the negatively-charged and immediate and delayed transfusion reactions; DAT does
molecules from red cell membranes; not require in vitro incubation
o Also by the denaturing antigenic determinance from red Used to detect antibodies that are stopped to the surface of the red
blood cell surface; blood cells
o Ficin and papain are the most commonly used enzymes in These antibodies destroy the red blood cells and cause anemia
blood bank laboratories Test that is done in newborn blood sample, especially when the
newborn is experiencing jaundice
4. BSA – Bovine Serum Albumin Direct antiglobulin test (DAT) is used to demonstrate in vivo coating
22% or 30% concentration of red blood cells w IgG antibodies and complement
Use as potentiators is less common than the other enhancement This assay uses the Coombs’ reagent incubated in the patient’s
media washed RBCs; other reagents used are monospecific reagents (to
Enhances the sensitivity of the IAT for a wide range of antibody detect bound IgG or bound complement), polyspecific reagent that
specificities
can simultaneously detect IgG and C3
normal findings are negative and no agglutination
Albumin can be added before the 37°C incubation
o to decrease the zeta potential and enhance reactivity. Principle:
BSA is prepared from bovine serum or plasma The direct antiglobulin test (DAT) is used to detect in vivo
Promotes agglutination by dispersing some of the positively- sensitization of the red blood cells with antibody, usually IgG, or
charged ions surrounding each negatively-charged cells which may complement degradation products such as C3d or C3dg. Presence of
allow the small IgG molecules to bridge the gap between the red complement pathway and produced split products.
cells (lattice formation/agglutination) DAT testing aids to diagnose autoimmune hemolytic anemia, HDN,
Was primarily used to enhance the binding of RH system antibodies transfusion reactions, or other type II hypersensitivity reactions

ADVANTAGE Specimen Used:

Does not enhance warm auto-antibodies o Patient- 5% red cell suspension
o Donor – Serum
DISADVANTAGE • Anticoagulated with EDTA
time-consuming because incubation time is 30-60 minutes

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 MODULE 9: THE ANTIGLOBULIN TEST
Procedure Indirect Antiglobulin Test (Indirect Coombs’ Test)
1. Prepare a clean test tube and label with patient’s name. Used for the detection of free antibodies in the serum that may
2. Add one drop of a washed 5% red cell suspension from the patient cause red cell sensitization in vitro
to be tested to the tube. Used to detect antibodies which are freely circulating in the plasma
3. Add one drop of an antiglobulin reagent to the tube. Note: Ensure or antisera and reacts with specific antigen RBCs in vitro.
the reagent’s dropper has a sufficient distance from the tube It is utilized to:
containing red cells suspension. o Test for the presence of antibody in the patient donor/serum
4. Mix well by shaking the test tube. (antibody screen)
5. Centrifuge at 3400rpm for 15-30 seconds. o Identifying the presence of antibody in the patient’s serum
6. Loosen the button by gently shaking the test tube or by tapping on against the donor unit cells (crossmatching)
the palm until no cells remain at the bottom of the tube. o Identifying antigens present on the patient’s RBCs, such as
7. Examine for the presence of agglutination and/or hemolysis. weak D and Kell (phenotyping)
8. Interpret and record results. IAT requires in vitro incubation
o Note: The manner in which the red cells are dislodged from the
bottom of the tube is of great importance. The tube should be This test is performed to detect presence of Rh-antibodies or other
held at an angle and gently shaken back and forth until an even antibodies in patient’s serum in case of the following:
suspension of cells or agglutinates is observed. 1. To check whether a Rh-negative woman (married to a Rh-
Graded from 0 to +4 (presence of agglutination is positive; positive husband) has developed Anti-Rh antibodies
absence is negative). 2. Anti-D may be produced in the blood of any Rh-negative
o EDTA is necessary to chelate calcium so that in vitro C3 person by exposure to D antigen by:
fixation will not occur a. Transfusion of Rh-positive blood
o Classic tube testing – RBCs to be tested should be washed b. Pregnancy, in infant is Rh-positive (if father is Rh-positive)
with saline (to remove excess unbound IgG or complement c. Abortion of Rh-positive fetus
present in the serum that could bind and inhibit the
reactivity of the added reagent) Principle:
o After washing, proceed ASAP before any bound antibody The indirect coombs’ test (also known as the indirect test or IAT) is
has a chance to dissociate off the patient’s RBCs used to detect in-vitro antibody-antigen reactions. It is used to
o Inadequate RBC washing and a delay in test performance detect very low concentration of antibodies present in a patient’s
could lead to a false-negative result plasma/serum prior to a blood transfusion. In antenatal care, the
9. Add IgG sensitized red cells as a control; centrifuge and read. If IAT is used to screen pregnant women for antibodies that may
negative result is obtained, the test result is invalid. cause hemolytic disease of the newborn.
o Note: If monospecific anti-complement reagents are used, The IAT can also be used for compatibility testing, antibody
complement-sensitized red cells should be substituted for identification, RBC phenotyping, and titration studies.
immunoglobulin-sensitized red cells.
Specimen Used:
Interpretation of Results • Patient- Serum
Positive (+) = presence of agglutination and/or hemolysis • Donor – 5% RBC suspension
Negative (-) = absence of agglutination and/or hemolysis

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 MODULE 9: THE ANTIGLOBULIN TEST
AAHG used in IAT is anti-IgG o Meaning there are antibodies in the red blood cells and may
o Anti-IgG will bind to the patient’s IgG antibody if present and have a condition that causes the destruction of the red blood
will facilitate macroscopic agglutination cells by the immune system therefore there is hemolysis.
o IgG antibodies present in the patient’s serum or plasma are
considered clinically significant SOURCES OF ERROR IN THE ANTIGLOBULIN TEC HNIQUE
False –Positive Reactions:
Procedure 1. Traces of species-specific antibodies (hetero-agglutinins) due to
1. Place 2 to 6 drops of patient’s serum in a clean test tube. improper preparation of the antiglobulin serum.
2. Add one drop of a washed 5% suspension of donor’s red cells. 2. Enzyme-treated red cells (because of greater sensitivity) reacting
3. Mix the tube thoroughly. with residual antispecies antibodies.
4. Incubate at 37 degrees Celsius for 15 to 60 minutes. 3. Test cells that have a positive direct antiglobulin test.
5. Centrifuge immediately upon removal from the incubator or 4. Extreme reticulocytosis bound to reticulocytes reading with anti-
waterbath at 3400 rpm for 15 seconds. transferrin in the antiglobulin serum.
6. Examine macroscopically for hemolysis and/or agglutination using 5. Colloidal silica in saline stored in glass bottles that is leached from
optical aid or viewbox. (Examine negative reactions microscopically) the container
7. Record results. 6. Metallic ions in saline stored in metal containers or used in
8. If agglutination is observed, wash the mixture for three or four equipment with metal parts. This may bring about nonspecific
times with saline. Decant each wash as completely as possible. protein sensitization of red cells.
9. Add one drop of antiglobulin reagent. 7. Improperly cleaned glass-wares or other forms of contamination of
10. Mix well.
cells, serum, or reagents.
11. Centrifuge at 3400 rpm for 15-30 seconds.
8. Over centrifugation.
12. Examine for hemolysis and/or agglutination using an optical aid.
9. Auto-agglutination before washing that may persist through
13. Interpret and record results.
14. Add IgG sensitized red cells as a control; centrifuge and read. If washing phase, clotted blood samples may react with anti-
negative result is obtained, the test result is invalid. complement in the anti-globulin serum.
• Note: If monospecific anti-complement reagents are used, o A small percentage of samples from segments of plastic
complement-sensitized red cells should be substituted for tubing
immunoglobulin-sensitized red cells. o i.e. containing blood stored at 40 degrees with
anticoagulant will react with ant C4d.
Interpretation of Results DAT can be positive in patients without (AIHA) Autoimmune
Positive (+) = presence of agglutination and/or hemolysis Hemolytic Anemia
Negative (-) = absence of agglutination and/or hemolysis Improper sample: Clotted cells, that can cause positive reactions
Spontaneous RBC agglutination
Positive results – clumping or agglutination during a direct Coombs’ Elevation of serum immunoglobulin
test Administration of intravenous immune globulin
Negative results – there is no clumping of cells, no agglutination Elevated serum globulin and BUN levels
o Meaning no antibodies in the red blood cells Contaminated reagents
Insufficient washing of the patient’s RBC

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 MODULE 9: THE ANTIGLOBULIN TEST
If the tubes were left to stand following centrifugation or if the RBCs Video of Activity
were left in suspension in an extended period before testing [Link]
If the test is delayed, separate the plasma so that the RBC will not Instructions
stay in the suspension • Discuss the principle based on the video.
• What are the materials needed to perform this procedure?
False –Negative Reactions: • Outline the procedure completely.
1. Incorrect technique for the particular antibody involved. • Why are you performing this test?
2. Inadequate washing of the red cells causing neutralization of the -END OF TRANSCRIPTION-
antiglobulin serum by trace amounts of residual globulin.
3. Insufficient active complement when the particular antibody is
detectable only in the presence of active complement or insufficient
anticomplement in the antiglobulin reagent.
4. Improper storage of test cells, test serum, or antiglobulin reagent,
or all of these resulting in loss reactivity.
5. Delays or interruptions in the test procedure, particularly during
the washing phase which may result in the elusion in the antibody
from the red cells.
6. Failure to add antiglobulin reagent.
7. Red cell suspensions are too heavy (which may not permit
optimum coating with antibody) or too weak (which may make
reading difficult)
8. Under-centrifugation or over-centrifugation.
o The latter is due to the excessive force required to
resuspend the red cells.
9. Contamination of the antiglobulin reagent with human serum.
10. Incorrect incubation temperature (not allowing for maximum
coating of the red cells) or fluctuating incubation temperatures.
11. Insufficient incubation time.
12. Prozone Effect
o Note: This should not be a problem with licensed products
provided that the manufacturer’s directions are followed
13. Failure to check negative reactions microscopically.

Delay of adding antiglobulin reagent after the washing step

Improper specimen agitation at the time of result interpretation

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 MODULE 10: GEL COLUMN AGGLUTINATION TEST (DG Therm and Spin)
MLS 18, June 8, 2021
BSMLS 3B | BY MLSWATER
University of San Agustin-Iloilo`

Legend: Application of Gel Technology

Transcription Bullet ABO forward and reverse typing
Notes/Module Packet Bullet Rh typing
PPT Direct antiglobulin testing
Antibody screening
MODULE OUTLINE
Antibody Identification
I. History
Compatibility Testing
II. Principle
III. Do’s and Don’ts
Used for any immunohematological test that has hemagglutination
IV. Procedure
as its end point this includes ABO and RH typing and also typing for
V. Interpretation of Results
other blood group systems, antibody screening and identification,
and also compatibility testing including crossmatching.
LEARNING OUTCOMES
At the end of this module, the student shall be able to:
1. Accurately perform gel column agglutination test. LECTINS
2. Detect recipient antibodies directed against antigens on the Useful alternative to anti-sera for blood typing
donor’s red cells using gel column agglutination test. First discovered in plant extract
3. Identify a compatible and incompatible blood transfusion Identifies antigens present on patient or donor’s red cells
reactions. Advantages:
4. Correctly interpret the results for the compatibility test Standardization, stability, smaller sample volume, ease of
performance and analysis, and rapidity

GEL COLUMN AGGLUTINATION TEST Disadvantages:

Detects the RBC antigen-antibody reaction using a chamber filled It does require specialized equipment
with polyacrylamide gel
It gives more reproducible and standardized test results
What is a Gel Card?
Utilizes the differential migration of RBC agglutinates thru a small
A gel held in a microtubule contain in a plastic card, each
microtube containing dextran acrylamide gel
microtubules contains sephadex gel or dextran acrylamide gel
The gel acts as a trap where in free non-agglutinated RBC form
prepared in a buffer solution such as LISS or saline.
pellets at the bottom of the tube which is a negative reaction;
Preservatives used sodium azide
o If the agglutinated RBCs remained at the top of the tube or
Specific reagents are specific antisera
in trap in the gel it is a positive reaction
Sedimenting agent is Bovine Serum Albumin

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 MODULE 10: GEL COLUMN AGGLUTINATION TEST (DG Therm and Spin)
History Is a controlled centrifugation of predetermined reagents and RBCs
1985- Dr Yves Lapierre developed gel test through a Dextran Acrylamide Gel
o Investigated ways to trap RBC agglutinates during The gel uses a process known as molecular sieving to separate the
standardized sedimentation or centrifugation RBCs based on agglutinated particle size with larger agglutinated
o Gelatin, acrylamide gel and glass beads RBCs located at the top of the gel and a non-agglutinated cells
o Gel particles were found to be the ideal material for forming a pellet at the bottom.
trapping agglutinates The antibody screening and panel cells made specifically for gel
1988 – Dr. Lapierre worked with DiaMed AG to develop and testing contain an antigen type cell diluted into a LISS solution
produce the gel test in Europe Also, the pre-made gel card contains polyacrylamide gel covered in
September 1994 – the FDA granted Micro Typing Systems (MTS) a
an AHG gel solution.
license to manufacture and distribute Antiglobulin anti-igG gel cards
In January 1995, the distribution rights were sold to Ortho
DO’S AND DON’TS
Diagnostic Systems (company that distributes test kits for HIV,
The Do’s
Hepatitis, and BDRL)
Always place the gel card in upright position.
Spin the gel card before use if you see bubbles in the column.
Principle
The principle of the test is based on the gel technique described by Store the red cells at 2-8 degrees Celsius after use.
Yves Lapierre in 1985 for detecting red blood cell agglutination Ensure that the cards and reagents are stored according to
reactions. conditions:
The DG Gel 8 plastic cards are composed of eight microtubes. o DG Gel Coombs, CT, T/S Poly, Diana Fluid: 20-25 degrees
o Each microtube is made of a chamber, also known as Celsius
incubator chamber, at the top of a long and narrow o Other cards, reagents, red cells: 2-8 deg. Celsius
microtube, referred to as the column. Use the column as soon as you can when you remove the foil.
o Buffered gel solutions containing polyclonal anti-human Cards must be at room temperature before use.
globulin have been prefilled into the micro tubes of the plastic Centrifuge the cards within 30 minutes after adding reagents, cells
card. or plasma into the column.
The agglutination occurs when the red blood cells sensitized in vivo Check expiry of cards, diluent and reagents before use.
or in vitro by human IgG antibodies react with the anti-human Labelling of sample must be correct.
globulin present in the gel solution. Ensure there are no clots in cells from donor bags.
The gel column acts as a filter that traps agglutinated red blood Ensure there is no contamination in diluent.
cells as they pass through the gel column during the centrifugation
The matrix ABO RHD group card it contains 6 or 8 (depends on the
of the card.
manufacturer) micro tubes pre filled with gel in a suitable buffer
The gel column separates agglutinated red blood cells from non-
containing specific monoclonal anti A, anti B, and Anti D antibodies.
agglutinated red blood cells based on size.
Any agglutinated red blood cells are captured at the top of or along Sample Collection
the gel column, and non-agglutinated red blood reach the bottom of
There is no special preparation of the patient that is required prior
the micro tube forming a pellet
to sample collection and samples should preferably be collected as
soon as possible

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 MODULE 10: GEL COLUMN AGGLUTINATION TEST (DG Therm and Spin)
If there is delay the sample should be stored at 2-8° C or it may be
used within 7 days of collection

Preferably the blood sample should be drawn into Citrate, EDTA, or

CPDA anticoagulant.

Do not use hemolyzed or contaminated samples

Sample Preparation
If the serum is used instead of plasma
o the serum must be cleared by centrifuging for 10 minutes NOTE: Pipette the test sample, plasma or serum using the pipette held
to avoid presence of fibrin residues which might interfere vertically, directly above the cells and slightly to one side of the incubation
with the test results chamber. Do not pipette directly onto the gel surface or into the cell
suspension as this may lead to unexpected and/or false positive results.
The Don’ts:
Do not freeze or store cards near any heat source. Pipetting
o Freezing or evaporation of the Gel card due to the exposure Variations in pipetting decreases the sensitivity of the gel antibodies
of heat may impede the passage of un-agglutinated RBCs screen
through the Gel cards. Therefore, it will increase the probability of failure to detect an
Do not use cards if: antibody particularly if the antibody is weakly reactive
o The cards appear damaged. Reagent screening cells are pipetted vertically into the microwell,
o There is evidence of drying of the column. this would allow the RBCs suspension to fall into the column of the
if used can also lead to erroneous results microwell and rest directly on top of the dextran acrylamide gel
This technique represents the elimination of the air gap between the
o The cards appear discolored.
cells and gel column
o The seal on the foil has been broken
Plasma containing antibody was pipetted 45°angle of the bowl wall
Gel cards showing damaged aluminum foils should
of the microwell
be discarded also bubbles in gel card may interfere Care must be taken in pipetting to ensure that the tip of the pipette
the passage of non-agglutinated RBCs doesn’t touch the microwell
Bacterial or other contamination of reagents during use it may
cause a false positive or a false negative result.
PROCEDURE
Fibrin residues in the serum or red cell aggregation in the red cell
suspension can trap non-agglutinated cells presenting a pink line on Antiglobulin Major Crossmatch Tests
top of the gel 1. Allow DG Gel 8 Anti-IgG (Rabbit) cards, additional reagents and the
Lipemic or Icteric samples samples to reach room temperature (18-25 ⁰C).
2. Identify the cards to be used and the samples to be tested.
3. Prepare a 1% donor’s red blood cell suspension in Grifol’s Diluent
(10 µL of packed red blood cells in 1 mL of Grifols Diluent).
4. Remove the foil seal from the complete DG Gel 8 card or from the
individual microtubes to be used for testing. Carefully peel off the

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 MODULE 10: GEL COLUMN AGGLUTINATION TEST (DG Therm and Spin)
aluminum film to prevent cross- contamination of the microtube A well-defined band of agglutinated red blood cells in
contents among them. 4+ the top part of the gel column. A few agglutinated
5. Ensure the re-suspension of the red blood cells before use. cells may be visible below the band
6. Dispense 50 μL of the donor's 1% red blood cell suspension into the
corresponding microtube. Mixed-field. A band of red blood cells at the top part
7. Add 25 μL of the recipient’s serum or plasma into the microtubes. Mf of the gel or dispersed throughout the gel column, and
8. Carefully dispense the red blood cell suspension and the serum or a pellet in the bottom as a negative result
plasma, avoiding contact of the pipette tip with the wall or the
contents of the microtubes to prevent carryover. Hemolysis in the microtube with very few or no red
9. Incubate 15 minutes at 37 ºC using DG THERM incubator. H blood cells in the gel column. Report if hemolysis is
10. Centrifuge the gel card in the DG SPIN centrifuge for 9 minutes. present in the microtube but not in the original sample
11. After centrifugation, remove the gel card from the centrifuge.
12. Read, interpret and record the results. Positive Reaction:
Agglutinated RBCs form a clear line on the surface of the gel or get
Interpretation of Results/ Reaction Grades this burst in the gel

Well-defined pellet of non-agglutinated red blood

Negative Reaction:
cells at the bottom of the gel column and no visible
Agglutinated RBCs settle at the bottom of the microtube, take note:
Negative 0 agglutinated cells in the rest of the gel column. (All the
the reading and interpretation of results must be done after
RBCs passed through and form a compact button at
centrifugation process only.
the bottom the column)
Barely visible small-sized clumps of agglutinated cells Note:
in the lower part of the gel column and a pellet of There is no need for multiple washing of red cells before adding
w+ unagglutinated cells at the bottom. (most of into the AHG serum and there is no need to add sensitized
agglutinated RBCs are in the lower third part of the controlled cell to all negative AHG test.
column) When using gel technology: less specimen volume is required and
also there is greater uniformity between repeated test. There are
Some small-sized clumps of agglutinated cells most also no variations among technologies in reading and grading the
frequently in the lower half of the gel column. A small agglutination
1+
Positive pellet may also be observed at the bottom of the gel Disadvantage: a special centrifuge is needed to accommodate the
column micro tube card. Also a special incubator is needed to incubate the
micro tube cards and the pipettes to dispose serum and red cell
Small or medium-sized clumps of agglutinated cells
suspension is also needed. It is also expensive
2+ throughout the gel column. A few unagglutinated
cells may be visible at the bottom of the gel column

Medium-sized clumps of agglutinated cells in the

3+
upper half of the gel column

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 MODULE 10: GEL COLUMN AGGLUTINATION TEST (DG Therm and Spin)

Video of Activity
[Link]
Instructions
• What is the principle behind gel column technology?
• Why is washing not necessary in the procedure?
• Why is it necessary to incubate the test cards before
centrifugation?
• Why are red cells added first in the test cards before plasma?
• What is the purpose of spinning the gel cards at a 90°angle?

-END OF TRANSCRIPTION-

#MLSWATER2022 Page 5 of 5
 ● They are immunoglobulins which are formed in response to a viral, bacterial
or spirochete infection and are capable of inducing PCH which is an
THE DONATH-LANDSTEINER autoimmune hemolytic anemia.

1 TECHNIQUE (QUALITATIVE)
IH LAB// MAAM PALOMAR
●

●
●
This auto-antibody binds to red blood cells during exposure to cold
temperatures.
Antibody produced in PCH
Biphasic activity
● Has a specificity of Autoanti-P

3 ●

●
Binds to RBC antigens at temperatures less than 37 °Celsius but does not
cause hemolysis until the coated RBCs are heated to 37°Celsius.
The IgG antibody binds to the P antigen fixing the complement at cold
temperatures and when heated at 37°Celsius, it will activate the membrane
attack complex and lyses the RBCs.
INTRODUCTION ● Hemolysis due to DL antibodies are stronger than that caused by anti-I
● Paroxysmal cold hemoglobinuria (Donath-Landsteiner syndrome) is a type of antibodies. This is because the DL antibodies are capable of detaching from
autoimmune hemolytic anemia characterized by the passage of urine lysed RBCs and then re-attaching to fresh red cells which will cause
containing hemoglobin or methemoglobin in solution (Hemoglobinuria) as a additional hemolysis.
result of exposure to cold. ● Serology testing for your DL antibodies is characterized by a positive DAT
● The more familiar type of PCH occurs secondary to syphilis. The less which is due to complement.
familiar type occurs in association with chronic hemolytic anemia of the ● During routine testing, this auto antibody is not easily identified using your
“cold” antibody type, the cold-hemagglutinin syndrome. This type is usually plasma or eluted plasma.
idiopathic.
● In 1904, Donath and Landsteiner reported that hemolysin in PCH was PAROXYSMAL COLD HEMOGLOBINURIA
probably due to an autolysin which unites with the patient’s red cells at low ● (Donath-Landsteiner Syndrome)
temperatures. This was confirmed by many workers. The test for the ● Described in 1904
detection of the hemolysin is called Donath-Landsteiner test and the ● Type of autoimmune hemolytic anemia
antibody is called the Donath-Landsteiner (DL) antibody. ● Extremely rare cold-reactive AIHA
● Characterized by acute episodes of massive hemolysis that occur after
DL Test exposure to cold
● Donath Landsteiner Test o Caused by the presence of cold reacting auto antibodies in the
● Is a serology test which is used to detect the presence of a biphasic blood and it is characterized by the sudden presence of
hemolysin hemoglobinuria which typically occur after exposure to cold
● This auto antibody is seen in patients with PCH or Paroxysmal Cold temperatures.
Hemoglobinuria ● It is IgG antibody that attached to RBCs only at temperatures of 15 ° Celsius
● The test relies on the characteristic code binding of an IgG auto antibody ● The antibody is directed against the P antigen
with specificity to the P blood group antigen ● Complement is always fixed and binds to RBCs at low temperatures
● This autoantibody causes complement mutated red blood cell lysis when o Acute cases of PCH are characterized by an abrupt onset with
warmed to body temperature. features of severe intravascular hemolysis and it also includes high
fever, chills, back or leg pain. Hemoglobinuria occurs and it will
DONATH-LANDSTEINER ANTIBODY reduce dark red to black urine.
● Seen in Paroxysmal Cold Hemoglobinuria (PCH) ● Occurs secondary to Syphilis
● The term PCH was named for a recurrent complication in late-stage or ● Cold Antibody-type (DL Antibody)
congenital syphilis: sudden attacks of constitutional symptoms and ● Cold-hemagglutinin Syndrome
hemoglobinuria precipitated by exposure to cold temperatures ● Idiopathic type
● With the advent of antibiotics and the almost complete eradication of late o Hemoglobinuria, Hemoglobinemia, Jaundice and Pallor are
syphilis, this chronic relapsing from of PCH is now extremely rare common clinical findings in acute PCH.
● PCH now tends to be an acute non-recurring illness affecting the pediatric o A particular significance is that Hemoglobinuria is found in almost
population all cases in childhood.
● Additional symptoms that may occur: tingling in the hands and feet, condition
marked by feeling of coldness or numbness of the hands, nose and ears.

1 | Page
 This is in response to cold temperatures and we referred to this as the 12. nescofilm/parafilm
Raynaud’s phenomenon. 13. Rh viewbox
● Another symptom is called Cold urticaria, it is a skin condition which is 14. marker
marked by reddening and the itching of the skin in response to cold 15. crushed ice
temperatures.
PROCEDURE:
PCH CLINICAL SIGNS BLOOD SMEARS DL test was developed on the characteristic by phasing hemolysis that was seen in
Hemoglobinuria Polychromasia vivo when PCA shows highly associated mid-syphylis and hemoglobinuria was
Bilirubinemia Presence of NRBC directly related to exposure to cold
Anemia: Hb level 4-5 g/dL poikilocytosis
Splenomegaly
BASIC STEPS OF THE TEST
● The DL Test is positive 1. Incubate the patient’s sample at a cold temperature to allow the antibody to
● Lactate dehydrogenase is high attach
● Hepatosplenomegaly can be attributed to an underlying lymphoproliferative or 2. To incubate the sample at a body temperature to activate compliment and
other neoplastic process detect the presence of RBC lysis
● Examination of your blood of the peripheral blood smear may reveal the - The patient’s sample has to be kept at 37 degrees Celsius after
presence of poikilocytosis, spherocytes of polychromasia, and nucleated RBCs collection

TWO DL TEST METHODS

● Your DL Test should be considered when a patient presents with symptoms
including recurrent fever, chills, abdominal pain, and laboratory findings of an Direct DL test Direct DL test
intravascular hemolytic anemia with both hemoglobinemia and ● primarily used as an initial ● Was developed to increase the
hemoglobinuria. screening test. sensitivity and specificity of the test
● The patient’s RBC samples should have a positive DAT due to C3. ● When this test is negative DL test system
● No demonstrable auto antibody activity by routine methods. is performed. ● There are modifications of indirect
● The positive DL test occurs when DL test, this includes:
PRINCIPLE only the sample in incubated at 0 ° o Using the enzyme treated
● Freshly collected serum contains hemolysin which in the presence of Celsius and then at 37 °Celsius reagent RBCs,
complement is capable of destroying red cells when high titer naturally occurring shows hemolysis o Performing a two-stage
anti-A and anti-B antibodies are present. test,
● The IgG auto antibodies that cause PCH act as biphasic hemolysins in vitro. It o Testing the DL antibody by
binds to the red cells at cold temperatures and as the test is warmed, 37°Celsius using IAT or indirect anti
complement is activated and lysis of the red cells occur. globulin test.

Specimen Used:
● Whole blood (red top tube)
Enzyme treated indirect the use of enzyme treated reagent O RBCs is one
Materials and Equipment: DL test means of increasing the sensitivity of indirect DL test
1. 5ml test tubes Two stage indirect DL not often performed in blood bank laboratories
2. serological pipettes test
3. venipuncture kit IAT can be used for the detection of the allo-antibody
4. Test tube rack because the antibody is IgG.
5. thermometer IAT should be performed with anti IgG reagent after
6. plastic droppers the low temperature incubation
7. Centrifuge
8. waterbath DIRECT DL TEST
9. Red top tubes 1. Label two clean and dry tubes as Tube 1 and Tube 2 including patient’s
10. Forceps name.
11. applicator sticks

2 | Page
 2. Before extracting the blood, pre-warm both tubes into a waterbath with 37 ● Blocking of C3BG will prevent the activation of the complement complex and
degrees Celsius temperature for 5 minutes. lysis of RBCs
3. Extract 5 ml venous blood from the individual under test and divide the blood
immediately between the two tubes warmed at 37 degrees Celsius. INDIRECT DL TEST
4. Leave Tube 1 at 37 degrees Celsius for 1 ½ hour (it removed together with ● Can be falsely negative when the antibody titer is low.
Tube 2 later). ● False negative results can also occur because of auto absorption of
5. Place Tube 2 at 0 degrees Celsius in crushed ice for 30 minutes. antibody. This is when serum separation is not carried out strictly at 37
6. After 30 minutes, without disturbing the clot, transfer Tube 2 to the water degrees Celsius.
bath at 37 degrees Celsius for one hour. ● Neutralization of Anti-P -False negative results can also occur when there is
7. After incubation, remove both tubes from the waterbath. neutralization of anti-P by globoside in fresh serum
8. Centrifuge both tubes for 5 minutes. o can be avoided by performing the two stage DL test.
9. Examine for the presence of hemolysis. ● Use of IAT can lead to false positive result due to carry over of direct
agglutination by code IgM antibody
Interpretation of Results
● Both direct and indirect DL test can be falsely positive when lysis occurs this
Tube 1 (control) Tube 2 (Test) is because of the presence of a code reacting IgM autoantibody and this is
Positive No hemolysis With hemolysis usually seen with patients with Cold Agglutinin Disease.
Negative No hemolysis No hemolysis
TREATMENT
Inconclusive With hemolysis With hemolysis ● Requires supportive treatment
● Disorders terminates when underlying illness is resolves
● Tube which was exposed only to 37°Celsius must not have hemolysis
because this will serve as the control (tube 1) CHRONIC PCH ACUTE PCH
● Tube 2 is exposed at 0 degrees then up to 37 degrees
Avoid exposure to cold temperature Steroids and blood transfusion
● If both tube has hemolysis result will be inconclusive because tube 1 must
not have hemolysis. Tube 1 will serve as control
Treatment for Donath Landsteiner PCH is supportive and this will include
SOURCES OF ERROR environmental warning and RBC transfusion as needed

FALSE POSITIVE: FALSE NEGATIVES PNH (PAROXYSMAL NOCTURNAL HEMOGLOBINURIA) FROM PCH
If patient has cold with broad thermal ● Low antibody titer ● Autoantibody is not implicated with PNH
amplitude ● Inhibition of the antibody by ● Involves membrane defect
neutralizing carbohydrate antigens ● RBC destruction is complement-mediated
present in fresh donor serum ● Absence or reduced amount of complement regulatory protein
● Transience of autoantibody (may ● Has similar presentation of hemoglobinuria, hemoglobinemia, and Anemia
be only present in plasma for a few secondary to intravascular hemolysis
days) ● But PNH pathogenesis is dependent on PIGA (phosphatidylinositol glycan
class A)
● Manifest with persistent cytopenia and also absent bone marrow iron
NOTE: Direct DL test ● DAT results are negative as well as the DL antibody test is also negative
Is more prone to false negative results than the indirect DL test. This finding could be
due to low antibody titer, also low compliment level or C3BG presence on the patients -end of transcription-
RBC.

C3BG
● Is protective by preventing complement mediated lysis.
● There is resistance of the RBC lysis due to C3BG coating with RBC
membrane. So therefore, this will mean it will block C3BG from binding on
RBCs.

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