MTAP 2: BLOOD BANKING

Speaker: Michael John Aguilar

into bicarbonate. Bicarbonate excess is called as

HISTORY metabolic alkalosis.
FIRST BLOOD TRANSFUSION • Lewisohn determined the amount of citrate needed
GOAL • Safe blood transfusion 1915 for AC and demonstrated non-toxicity in small
• First documented blood transfusion (BT) between amounts
human recipient and human donor • Rous and Turner introduced a citrate dextrose
1492
• Happened in ANCIENT ROME solution for blood preservation.
o Something to do with POPE INNOCENT VII 1916 o During this time, citrate dextrose has no known use
• Happened in 1942 o Dextrose (carbohydrate) will serve as addition
• First documented recipient of human blood from 3 source of ATP
young male donors 1930 • Dextrose function was fully understood.
POPE
o The 3 human donors dies from severe blood loss • According to scientists, dextrose maintains RBC
INNOCENT VII
and all of them (including Pope Innocent VII) died viability by maintaining cell membrane integrity by
oAHFPope Innocent VII dies of massive/severe IN VIVO providing additional source of ATP.
coagulation and severe transfusion reaction o Red cells rely on metabolic pathway to maintain
1667 TO 1829 • TRANSFUSION WAS FORBIDDEN their structure, so they won’t collapse. They need
• James Blundell of England transfused blood to a source of fuel coming from dextrose.
human (successfully) 1943 • Loutit and Mollison of England introduced the
1829 formula ACD (Acid-Citrate-Dextrose)
• The patient is a female suffering from postpartum
bleeding with the donor being her husband o ACD is like a first-generation anticoagulant, hindi
pa gano’n kaganda although nowadays there are
ANTICOAGULANT (AC) countries using ACD especially those that depend
• Chemical agent that prevents blood clotting and at the same time always in need of blood units;
• Extends life of blood IN VITRO these are countries subjected to war.
• Braxton Hicks recommended sodium phosphate as • Characteristics of ACD:
1869 AC o pH 5.0 (acidic)
o First example of blood preservation research o Poor 2,3-DPG preservative (one of the primary
factors considered for blood preservation)
• Karl Landsteiner discovered the ABO BGS
1901 o 2,3-DPG depletes in 2-3 weeks of storage (results
• Win a nobel award in a shift to the LEFT = ↑ O2 affinity of Hb, ↓ O2
• Hustin reported the use of sodium citrate as an AC in tissues)
solution o Poor O2 transfer (disadvantage)
o Sodium citrate is considered as a major AC 1957 • Gibson introduced CPD (Citrate-Phosphate-
nowadays Dextrose)
o Citrate, on itself, is toxic to humans o Phosphate in the lab, is always a buffer, as well
o Clinical chemistry: massive blood transfusion in as dihydrogen phosphate in the body.
1914
a narrow period can lead to acid base disturbance o The addition of phosphate makes the
which is a case of metabolic alkalosis anticoagulant less acidic.
o Bicarbonate is the basic component of your
• Characteristics of CPD:
plasma
o Less acidic = more superior preservative of 2-3
o Citrate present in the blood bag will circulate
months
towards on your liver. This will then be converted

MTAP2: BLOOD BANKING 1

  Shows more advantages especially for Platelets, mabilis palitan. RBCs, medyo matagal palitan.
platelets since platelets undergo spontaneous Sa processing ng whole blood you will always end up with pRBC, platelets,
activation when lost in certain pH range hence, and plasma.
the acidic of ACD should be buffered or
phosphate.
Note:
o 2 weeks of storage
What are the 4 basic chemicals/components in your blood bags?
DEVICES FOR BLOOD TRANSFUSION • Citrate / Sodium citrate / Citric acid
• Designed to aid the doctor in blood transfusion. • Monobasic sodium phosphate
• Dextrose
• Carried out vein-to-vein transfusion using multiple • Adenine
syringes and a special cannula for puncturing the vein
Edward E.
through the skin. Major anticoagulants that are FDA-approved
Lindemann
• He needed many skilled assistants and is time • ACD-A
consuming
• CPD
• Designed a syringe-valve apparatus so that
• CP2D
Unger transfusion from donor to patient is possible without
the assistance of a physician. • CPDA-I
Note: o The only anticoa gulant with adenine.
• Dr. Charles Drew was appointed director of the first American Red
Cross Blood Bank at Presbyterian Hospital (NYC) in February 1941 The name will tell the chemical present in your anticoagulant. CPDA-I lang
(Board exam recall) ang may adenine.

Note: Anticoagulant Storage Time

• Following blood donation, you will get 450 mL ± 10% of whole blood ACD-A 21 days
approximately 1 pint. Nowadays we also use, 500 mL ± 10%. CPD
• Primary consideration, since we’re talking about blood volume, is the
CP2D
hematocrit of your patient.
• The logic is, you have to collect a certain amount of blood that won’t be CPDA-I 35 days
harmful to your patient. Because if you collect this amount of blood from Note:
your patient, they can just replenish it in 2 days, then (Nawala audio nya) Pag nagdagdag ka ng adenine, yung 21 days magiging 35 days. Naka-base
• According to Harmening, the minimum hematocrit of your patient should yon sa function ng individual chemicals present sa blood bag. May
be approximately 38%. The logic here is, what if you’re going to use the nacocover na function yung adenine kaya mas tumatagal.
500 mL? You’re going to adjust the anticoagulant concentration:
o 450 mL ± 10%  63 mL (anticoagulant) Chemicals in Anticoagulant Solutions
o 500 mL ± 10%  70 mL (anticoagulant) Chemical Function Present in
Note: ACD-A CPD CP2D CPDA-1
Now, kung kaya i-replenish in within 2 days (initial replacement, lalo na ang Citrate • Chelates calcium.
plts), ano yung cut-off para sa safe blood donation? Alam natin that donor (Sodium • Prevents clotting X X X X
red cells are replaced within 1-2 months after donation. Hence, a volunteer citrate/Citric
donor can donate whole blood every 8 weeks. acid)
• Replenished RBCs: within 2 days (initial replenishment) Monobasic • Maintains pH during
sodium storage. X X X X
• Replaced RBCs: 1-2 months after donation
phosphate
MTAP2: IMMUNOLOGY SEROLOGY 2
 • Necessary for three most
maintenance of important RBC
adequate levels of 2,3- properties:
DPG.  RBC
o During storage, we deformability –
avoid the depletion of requires energy.
2,3-DPG because  RBC
that will decrese the permeability –
O2 delivery to the requires energy.
tissues from the  Maintenance of
blood bag. stable
o RBC’s primary hemoglobin
function concerning oxygen
blood transfusion is dissociation
to restore the curve – does not
oxygen carrying require energy.
capacity of the • Optimal production of
patient. ATP (extend shelf-life
o If there is no 2,3- from 21 to 35 days).
DPG, the • ADP + P = ATP
administration of o Extra phosphate will
packed RBCs would bind with the help of
be rendered Adenine energy. X
insignificant. o Adenine is important
o Right after blood in order to have
transfusion, 2,3-DPG adenosine that will
restore its levels. bind to phosphate to
o 2,3-DPG is being have adenosine
maintained because triphosphate.
during storage, the ACD-A = Acid Citrate-Dextrose (Formula A); CPD = Citrate-phosphate dextrose; CP2D = Citrate
RBCs are in vitro Phosphate Double Dextrose; CPDA-1 = Citrate-Phosphate-Dextrose-Adenine
(Harmening, Chapter 1, page 9)
environment.
• Substrate for ATP ANTIHUMAN GLOBULIN TEST (AHG)
production for cellular
respiration (cellular Additional Notes
energy). • With the identification of Weak D Phenotypes, you need indirect
o This is needed antiglobulin test (IAT).
because RBCs
• In ABO antibodies, we know that IgM is the naturally occurring antibody
Dextrose during storage relies X X X X
on the metabolic
Factors Affecting Agglutination of RBCs
pathways in order to
survive and this 1. Antibody • IgM: average length of 1000 Angstrom
metabolic pathway is Length • IgG: average length of 250 Angstrom
essential for the • Low Zeta Potential = more optimum reaction
2. Zeta Potential
maintenance of would be

MTAP2: IMMUNOLOGY SEROLOGY 3

 • A1 = 0.81-1.7x106 • These can serve as an antigen as well and can also
• A2 = 0.24-0.29x106 induce immune response once transferred to another
3. Position & organism.
• B = 0.75 x 106
Number of
• D = 9.9-33.3x103 Ex. 1 – Rabbit/ • If a human serum is transfused in this another
Antigen Sites
• Kell = 3.5-6x103 Murine organism, an “alloimmunization” may be induced
o 2nd most common BGS outside ABO into it—leading to the production of antibodies
• Most RBC antigen have HIGH-binding constant against the antibodies that is present in the human
4. Length of • Therefore, the optimum incubation time is up to serum that was transfuse.
Incubation 30-120 mins (when only saline is used) o What if ang nature nung Ab na nanggaling sa
Time • Or 10-15 mins (when enhancement media are human serum ay IgG? Ano yung ipo-produce
used) nung another organism? It would be Anti-IgG.
• IgM: reacts optimally @ cold temp • AHG content = Anti-IgG
5. Reaction Temp o An antibody against another antibody produced
• IgG: reacts @ warm temp
by other organisms against human antibodies.
• Anti-M: 6.0-6.5
Ex. 2 – HDFN • The fetal cells contain IgG attached to it. How will
6. pH o pH & glucose resistant
detection HDFN be detected if IgG couldn’t produce an
• Anti-D: 6.5-7.0
agglutination by itself? The anti-IgG would bind to
• Point of equivalence reach when: it in order to cross-link the other IgG—therefore
7. Concentrations
o Antibody: 2 drops of serum + 5% w/v RCS observing an “agglutination”.
of Antigen &
o Low tittered: 133:1 (2 drops of serum + 3%
Antibody 1) Direct AHG • In vivo sensitization
RCS) Testing (DAT) • Clinical applications (for detection):
o Hemolytic Disease of the Fetus and Newborn
ANTIHUMAN GLOBULIN (AHG)
o Hemolytic Transfusion Reaction (recent)
• Aka Coomb’s test
o Autoimmune Hemolytic Anemia
Personalities Involved  Common ground of these three: Coating of
1945 • Coombs, Mourant, & Race RBCs with IgG occurred within the patient/
o Described the use of AHG reagent to detect weak/ patient’s blood stream.
non-agglutinating antibodies/ alloantibodies (IgG) 2) Indirect AHG • In vitro sensitization
o IgG – non-agglutinating antibodies or coat Testing (IAT) o Sensitization/binding of IgG with RBCs
incomplete antibodies. occurred prior to the addition of AHG reagent.
o Phases of AHG assay: Saline  Immediate Spin
• Diagnostic applications (as a part of testing):
 37C Phase  AHG
o Antibody screening and identification
 React agad sa Saline and IS, IgM siya
o Antibody titration
 Non-agglutinating antibodies/ incomplete
o Red blood cell phenotyping
antibody in relation to IgM = IgG
o Compatibility testing
alloantibodies
 Alloantibodies are only considered as
GOAL OF BLOOD BANKING
clinically significant if it belongs to the
• Safety practices are employed to prevent alloimmunization from
secondary immune response (IgG)
occurring during and/or after transfusion which may lead to hemolytic
 IgG4: clinically insignificant in BB; only
transfusion reactions or HDFN
interested in IgG1& IgG3 (only IgGs that can
cross the placenta)
Anti-Human Globulin Testing
Human Serum • Source of antibodies; are protein in nature

MTAP2: IMMUNOLOGY SEROLOGY 4

 AHG REAGENT is one clone of plasma cell, the antibody produced against
POLYSPECIFIC AHG RABBIT POLYCLONAL the stimulating antigen has only one specificity (even if that
- Anti-IgG and anti-C3d/C3b antigen has many different epitopes, the antibody will only
- AHG is against 2 components correspond/bind/interact to one epitope)

Note:
RABBIT POLYCLONAL + MURINE MONOCLONAL BLEND • Polyclonal (many clones) – the variety of cells (a lot of plasma cells) that produces the
same AHG (antibodies) that can recognize different antigenic determinants or
recognize the same portion of the same antigen but with different affinities
• Monoclonal (single clone) – only has a single plasma cell that can only produce a
single monoclonal antibody that recognizes a single epitope
MONOSPECIFIC ANTI-IgG (rabbit polyclonal source) o Attached to a cancer cell and becomes a hybridoma

Extra Information for AHG reagents

MONOSPECIFIC AHG

ANTI-IgG (gamma clone source) • Monoclonal because you only have a single plasma cell
o A single plasma cell can only produce a single type of monoclonal
antibody that can only recognize a single epitope from the original
antigen
o This single plasma cell will be attached to a cancer cell and will become
ANTI-COMPLEMENT (anti-C3d) hybridoma
• Polyclonal means different plasma cells produce the same antibody
(which in this case is anti-IgG)
o Various plasma cells encounter the same antigen (IgG from human) and
they all produce anti-IgG
o The ff are the properties of the antibodies produced by the plasma cells:
TRANSER’S NOTE: (Marami kinatak si sir pero basically ganto lang siya  Recognize different antigenic determinants (epitope) within the
kasimple) same antigen
• The terms polyspecific and monospecific refers to the specificity of  Recognize the same portion of antigen but with different affinities or
the antibodies present within the reagent. strengths
o Polyspecific means the antibody can attach to antigens
with different epitopes (EX: attach to both IgG and C3d). • Multiple rabbits were exposed to serum (source of
o Monospecific means that the antibody can only attach to antigen) and various plasma cells produced anti-IgG
one distinct epitope on an antigen (EX: attach to IgG or C3d and anti-C3d upon harvesting.
Rabbit
only) o The property of the anti-IgG produced is that it can
polyclonal
• The terms polyclonal and monoclonal refers to the way that the detect any place in the original antigen where the
antibodies were produced. rabbits were exposed to.
o Polyclonal means that many different clones of plasma • In a nutshell: Clonality relates to the production
cells with different specificities have produced antibodies Rabbit • Source of anti-IgG is polyclonal (various rabbits were
against one antigen (one antigen stimulates production but polyclonal + produce it) but the source of anti-C3d is from the
that antigen has different epitopes therefore creating murine murine (hybridoma)
antibodies that correspond/bind/interact to each epitope) monoclonal o Polyspecific since may dalawang bagay na kayang
o Monoclonal means that it is made from one clone of blend dumikit sa ibang bagay
plasma cell fused with a myeloma cell (hybridoma). Since it

MTAP2: IMMUNOLOGY SEROLOGY 5

 o Hence, 2 specificity (polyspecific) but different Lambda light chains) since all antibody classes
sources or manner of production have light chains
Monospecific • A lot of plasma cells of rabbits produce anti-IgG but no ● This is the minor advantage of
anti-IgG compliment activity (anti-complement) Polyspecific AHG
rabbit o Specificity is monospecific
polyclonal MONOSPECIFIC AHG
• Production of anti-IgG that is monoclonal (murine ● It contains EITHER anti-IgG or anti-C3d.
Anti-IgG
monoclonal) ○ How does it detect anti-IgG? Its specificity is directed to or contains
Gamma
clone • It only has anti-IgG and no anti-complement hence still reactivity against the Fc fragment of the gamma heavy chain.
monospecific ■ There are monospecific anti-IgG AHG reagents with a label
• Clonality means kung paano lang naproduce, wala siyang kinalaman sa of gamma heavy chain specific.
specificity. ● It pertains to the reactivity
● Has no anti-complement activity
• Polyclonal – produced by rabbit
• Monoclonal – produced by murine
● Anti-complement reagents
○ Specificity for C3d or C3b
Note: ■ Anti-C3b
……cont: Ang magdidictate ng specificity, kung ano yung laman. Kapag ■ Anti-C3d
IgG lang yung laman, Monospecific, regardless of the production. ○ Has no Anti-IgG, hence, monospecific
Kaya nagend up sa dalawa, merong anti-IgG na rapid polyclonal, ○ If the reagent is monospecific, it is a blend of monoclonal Anti-C3d
meron anti-IgG na gamma clone / Murine Monoclonal and Anti-C3b
Anti-complement: only has anti-complement, and it is monospecific ○ If the nature of your alloantibody is IgM, they can bind thus
Kapag pinagsama ang dalawang monospecific (anti-IgG and anti- activating complement pathway
complement), polyspecific na ba yon? ○ If nature of alloantibody is IgG, they cant bind and activate
YES, it is already polyspecific, because it is isolated in complement in BB/IH except the Kidd BGS alloantibodies
specificity ■ Madedetect mo siya dahil sa anticomplement ng
NO RELATIONSHIP between polyspecific and clonality AHG mo
clonality - paano naproduce
kung didikit sa dalawa = polyspecific; kung didikit sa isa= PREPARATION OF AHG
monospecific
If nag inject ka ng human globulin sa isang foreign organism, yung human Ab
will behave as a foreign Ag.
POLYSPECIFIC AHG
• The foreign organism will induce a secondary immune response for
● It contains antibodies to human IgG (Anti - IgG) and to the C3d the antibodoy, making anti-human globulin (AHG)
component (Anti-C3d) of the human complement • Two options for AHG production:
○ Since anti-IgG lang, it con tains any reactivity against IgM and 1) Pool of human IgG + rabbits (has plasma cells that produces antibodies
IgG. Commercially prepared polyspecific AHG contains little, if = polyclonal antibodies
any reactivity against IgA and IgM 2) Post-immunization: Single plasma cell (already produces antibody such
■ Kung meron man ma detect na IgA and IgM, as IgG) + spleen or liver cell of murine = hybridoma technology = hybridoma
mahirap na. produces monoclonal antibodies
○ Polyspecific mixture may contain antibody activity / reactivity to DEFINITION OF TERMS
Kappa and Lambda light chains • Polyclonal antibodies are mixture of antibodies from different plasma
■ If you observed reactivity with IgA and IgM clones
molecules, it came from there (Kappa and o An antibody that can recognize different antigenic determinants

MTAP2: IMMUNOLOGY SEROLOGY 6

• Monoclonal antibodies are antibodies that can recognize only a single • However, its disadvantages are:
epitope o You are running the risk sa presence ng heterospecific antibody.
If you run the process of polyspecific, polyclonal blend, you are
Polyspecific, polyclonal IgG, such as polyspecific antihuman globulin running the risk of producing heterospecific antibodies (specificity for
(AHG) something that you don’t need).
• The manner in which one produces polyspecific AHG, if the nature is o HOW DO YOU RESOLVE FOR THE PRESENCE OF
polyclonal method, involves the following: HETEROSPECIFIC ANTIBODIES FOR THE PRODUCTION OF
o Take 2 colonies of rabbit: Rabbit’s colony 1 and 2 POLYSPECIFIC POLYCLONAL ANTIHUMAN GLOBULIN
 Rabbit colony 1: injects purified human IgG REAGENT?
 Rabbit colony 2: injects purified human C3  Perform absorption panel with A1, B, and O cells to remove the
heterospecific antibodies.
o Rabbit colony 1 produces monospecific, polyclonal anti-IgG,
because the source of stimulation is IgG only o Another disadvantage is that it is hard to determine the optimal
 Many plasma cells in rabbit 1 are triggered, the nature of antibody reactivity of the final product.
produced is monospecific  To determine the optimal reactivity, perform antibody dilution
 But because many clones yung nagproduce, its nature is polyclonal  Perform block titration
o Rabbit colony 2: produces monospecific, polyclonal anti-C3
 Lymphoproliferative organs of rabbits have multiple, there are many • HOW DO YOU DETERMINE THE POTENCY OF ANTI-C3 FROM THE
clones that produces anti-C3 because only C3 yung nagtrigger POOL OF POLYCLONAL POLYSPECIFIC?
o Perform another dilution procedure. However, it is not accurate.
o Mixing them together, produces POLYSPECIFIC, POLYCLONAL AHG
BLEND • One of the most observable disadvantage of the POLYSPECIFIC anti-
C3d and anti-C3b is that it is very hard for you to determine the potency
Continuation, anti IgG polyclonal polyspecific blend if they are polyclonal in nature
• Since IgG molecules have a distinct property of being heterogenous
o Heterogenous because there is IgG1 and IgG4 • Napansin niyo kanina meron tayong second reagent (polyspecific IgG
o Kaya Maganda ung polyspecific tsaka polyclonal kasi ung + rabbit/murine monoclonal blend) that is what resolved the problem
pinangstimulate mo ng anti IgG production nanggaling sa many • Dito yung anti-C3d niya is nanggaling sa murine monoclonal and that is
donors. Ang mangyayari, ung result ng preparation of polyclonal the reason on why you can now determine the potency. So pinaghalo
polyspecific is antihuman globulin mo naging polyspecific.
o Ang property ng antihuman globulin is kaya nyang makarecognize ng • Very important personality is Kohler and Milstein (board exam recall
IgG tapos kaya nyang makapag recognize ng maraming types ng question), they were the ones who develop the monoclonal antibody
IgG kasi ang IgG nyo by nature maraming subclass tapos nanggaling technique production which is the HYBRIDOMA TECHNOLOGY
sa maraming donors
• Diba tinatry natin mag identify ng maraming alloantibodies, kung • Kohler and Milstein - Hybridoma technology
maraming plasma cell ung nag produce, ibig sabihin, ung immunized • It is called hybrid due to the fusion of cell types
rabbits nyo ngayon, ang naproduce nya is an anti-IgG that can recognize • And “oma” as it uses cancer cells
a lot of antigenic determinants from a lot of IgGs.
• Given na heterogenous ung IgG kasi maraming subclass, ung
purified IgG mga IgG pero maraming pinanggalingan.
• Rabbits are exposed to many IgG and many variants of IgG pero
monospecific pa rin.

MTAP2: IMMUNOLOGY SEROLOGY 7

HOW DOES IT WORK?
-Ngayon gagamit tayo ng murine, we will utilize 2 murines which is murine
1 and murine 2. in murine 1 we put COMPLEMENT(C3) and in murine 2 we
put IgG
-they will be subjected to immunization following alloimmunization

- dito kukuha tayo ng isang pirasong cell that can produce 1 type of antibody.

-Then the single antibody producing cell (from spleen or liver) will be
placed now in a MYELOMA cell line. Then we would culture it and end
up with a cell that MULTIPLY INDEFINITELY and it will produce an
antibody. And now when we join the two together what will be produced is
the ANTI-HUMAN GLOBULIN that is POLYSPECIFIC and
MONOCLONAL.

Murine 1 Murine 2
C3 IgG
Monoclonal/ Monoclonal/
monospecific Anti-C3 monospecific Anti-IgG

MTAP2: IMMUNOLOGY SEROLOGY 8

MTAP 2: BLOOD BANK (IMMUNOHEMATOLOGY)
Speaker: Michael John Aguilar, RMT

BLOOD GROUP SYSTEMS (BGS) • Besides the ABO BGS, all of the clinically significant
BGS are considered significant if they are present in
HISTORY (TIMELINE) RBCs. Why? Because in blood banking, most of time
we transfuse RBCs. So kapag sumablay ka ng
• Karl Landsteiner discovered A and B antigens; identification ng BG na nasa RBC, kasalanan mo yon
1901
classified the blood groups. as MedTech.

Additional Note: Must to Know about the ABO BGS

• IMMUNOHEMATOLOGY • Antibody production occurs without stimulation
o Branch of science dealing with the study of RBC antigens and its • May lead to severe transfusion reaction
corresponding antibodies • Unique; only BGS in which individual produced antibodies against an
o The reason why it’s called Immunohematology is because it hinges antigen that is absent on the RBC surface
from the principles of Immuno-Sero, Hematology, Molecular
1. Antibody production does not require prior stimulation.
diagnostics/biology, and Cytogenetics.
• ABO antibodies are always present following the Landsteiner law.
• Besides the ABO BGS, almost all clinically significant alloantibodies
• Von Descatello and Sturle
require prior exposure or stimulation in order for their production. In
o Discovered red cell antigens which was
1902 short, ang ABO hindi mo kailangan ma-expose sa foreign antigen, meron
designated as blood group AB.
kang antibody. Meanwhile, for the rest of the BGS that are clinically
• AB – rarest phenotype
significant, kailangan mo laging ma-expose.
• Landsteiner and Wiener • ABO antibodies are always present: “naturally-occurring”
AROUND 1940 o Discovered another blood antigen, Rh system
• (1) You will always have an antibody against an
(named after the RHESUS monkey)
antigen that you do not have.
• Dr. Charles Drew Landsteiner • (2) You will never have an antibody against the antigen
o Was appointed director of the first American Red Law/Rule you possess.
FEBRUARY Cross Blood Bank at Presbyterian Hospital (NYC)
o Logic: Kapag blood type A ka, hindi ka
1941 • Dr. Drew’s programs became the model for the magkakaroon ng anti-A sa serum mo.
national volunteer blood donor program of the
• What are the 2 possible reasons of being exposed to
American Red Cross Exposure to
foreign antigen?
Foreign
(1) Pregnancy
Additional Note: Antigen
(2) Blood transfusion
• Blood-Borne Infection
2. May lead to severe transfusion reaction.
o Sexually acquired and blood transfusion transmitted (e.g. Syphilis,
Hepatitis B, HIV) • The nature of your ABO naturally-occurring antibodies is IgM.
o IgM can always bind complement. They are efficient in complement
activation.
ABO BLOOD GROUP SYSTEM
• Besides the ABO BGS, almost all clinically significant blood groups
• Most important system in transfusion and transplantation therapy
cannot bind complement.
o ABO lang ang clinically significant na kayang mag-bind ng
• Kapag magt-transfuse ka ng blood, you have to test
complement.
for ABO. Another thing, if magt-transfuse ka ng tissue,
o For the rest of the clinically significant blood groups, they can’t bind
Purpose they must be ABO-compatible. Because your ABH
complement, except for Kidd.
substances are also produced by other tissue type.
They are still immunogenic.
MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 1
 • Besides the ABO BGS, almost all clinically significant blood groups, ang • In paternity testing there is two criteria:
type ng hemolysis nila is extravascular. Inclusion • Not used as direct inclusion tool of paternal chain.
o ABO: Intravascular hemolysis • ABO blood group used in direct exclusion of
o Other clin. Sig. BGS: Extravascular hemolysis paternal chain.
• There is the involvement of myelolymphoproliferative organs (spleen o You can use this to say if the individual is your child.
or liver). • Blood type should not manifest to the offspring.
What Type of • Intravascular Hemolysis • Two possible reasons why exclusion happens:
ASHTR? o This is due to complement activation. o Technical error
• What type of hemolytic transfusion reaction can you o Man is not the biological father.
Exclusion
observe following • Examples:
o Acute Severe Hemolytic Transfusion Reaction 1. Parent blood type – O x A
• Ex. Yung blood type A nilagyan mo ng blood type B. o Possible blood type of children – A and O
What do you think will happen? Magwawala yung o Exclusion – AB and B
ABO -
naturally-occurring IgM, mag-i-induce ng complement 2. Parent blood type – B x AB
Incompatible
activation, then magkakaroon ng hemolytic reactions. o Possible blood type of children – A, B, AB
Transfusion
Eh palaging nandoon, hinihintay lang yung o Exclusion – O
incompatible blood type. Anong mangyayari? Acute.
Now yung severity, it correlates well sa kung gaano B. Three (3) Systems of Nomenclature
karaming blood ang na-transfuse. Severe Hemolytic • Landsteiner – developed O, A, B, AB
Transfusion Reaction. • Jansky – developed roman numeral I (corresponds to O), II, III, IV
• Moss – developed roman numeral IV (corresponds to O), III, II, I.
Board Recall Question/s: Landsteiner O A B AB
Q: What is the most important BGS in transfusion and transplantation Jansky I II III IV
therapy? Moss IV III II I
A: ABO Blood Group System (BGS)
Board Recall Question/s:
ANTIGENS Q: Manner of inheritance?
A: Codominance / Codominant manner
ANTIGEN INHERITANCE o All clinically significant BGS (including ABO), their manner of
• ABH genes are located on chromosome 9 (long arm) inheritance is codominant expression.
• ABH genes are inherited in a codominant manner following simple Q: Who described the manner of inheritance concerning ABO blood
Mendelian genetics laws group system?
A: Bernstein (1924)
Additional Note:
• There are currently 36 BGS.
• Sa boards, remeber ISBT number, BGS, and location of chromosome.

A. Codominant (Bernstein, 1924)

• Almost all clinically significant blood group systems, their manner of
inheritance is always codominant expression.
• ABH codominant
o 50% haploid females + 50% haploid males
• Has three (3) systems of nomenclature.

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 2

 A. ABO GROUPS OF OFFSPRING FROM VARIOUS POSSIBLE ABO • Phenotype – serologically detected/tested antigen in the RBC surface.
MATINGS • Genotype – overall genetic composition of an individual
o Homozygous – combination of similar allele (double dose = strong
reaction)
o Heterozygous – combination of two different alleles (single dose =
weaker reaction)
o Allele – one of two or more different genes occupying the same
locus in the chromosome.

Parent Alleles A B O
AA AB AO
A
(A) (AB) (A)
AB BB BO
B
(AB) (B) (B)
AO BO OO
O
(A) (B) (O)

B. FORMATION OF ABH ANTIGENS

• ABO genes code NOT for the antigen themselves but

Glycosyl- for the production of glycosyltransferases that
transferase add/transfer immunodominant sugars to a basic
precursor substance.
• Precursor substance – required in order for the
proper manifestation of the A and/or B phenotype.
• Identity – Lacto-N-neotetraosylceramide (Type 2
chain paragloboside, beta 1-4 linkage)
• Also called “acceptors”
• Purpose: Site of attachment of the immunodominant
sugar
• Oligosaccharide structure composed of 4 (four)
interlink sugars.
Paragloboside • In order for A&/B phenotypes to manifest, we need the
or Glycans corresponding genes
o To determine the phenotype of the patient, we
(Harmening, Chapter 6, Page 123) utilize serologic reactions (Ag-Ab reaction)
o Since our concern are red cells (forward typing):
Blood Type/Phenotype Genotype ▪ Red cells (unknown Ag) + Reagent (known
O OO antisera/ source of Ab) = Ag-Ab binding →
A AA, AO serologic reaction
B BB, BO ▪ Serologic reaction → determines the
AB AB phenotype
• Blood type – actual physical manifestation of an inherited gene/sets of o Now, in order for the antigen to be present on the
genes. red cell, we need genes

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 3

 ▪ Genes produce particular antigen on the RBC C. CHARACTERISTICS OF ABH ANTIGENS
surface (specifically the genes produce the 1. Can be demonstrated as early as 2nd month of fetal life
glycosyltransferases that would determine the 2. Persist throughout life unaltered. However, antibodies and antigens may
immunodominant sugar → immunodominant be found acquired characteristics to leukemia and cancer (CLL – leukemia
sugar would determine the antigen present on that would commonly alter ABO type or ABH antigens)
the RBC) o In leukemia, red cells and leukemia cells both originate from the bone
marrow. It could be due to the leukemic process (cancer/hematologic
Gene Glycotransferase Immunodominant Sugar neoplasm), it can induce this erythropoietic process.
L-fucose o If there is a co-existing myeloid/myelocytic neoplasm (blood cancer),
H gene L-fucosyltransferase it could have an effect on the proper production of red cells—one of
(H antigen specificity)
N-acetylgalactosaminyl N-acteylgalactosamine the many reasons for the alteration of ABH antigens.
A gene 3. May be found at secretions of people who are secretors (SE gene –
transferase (A antigen specificity)
D-galactose secretor status)
B gene D-galactosyltransferase 4. May be found on bacteria and other species (seen at the bacterial capsule
(B antigen specificity)
and cell wall).
Additional Notes:
• Glycosyltransferase: able to transfer/ add sugars from the plasma to Antigens
the RBC surface to the basic precursor substance • Weakly expressed during fetal development, unstable,
A Antigen
• 99.9% of all individuals possess H gene and development will be stabilized 1 year after birth.
• 0.01% of the population that doesn’t possess the H gene = H null/ B Antigen • Strongly expressed and stable along with H antigen
BOMBAY • Required for the attachment of A and B antigens to the
• H gene is needed as it coats for L-fucosyltransferase paragloboside
(glycosyltransferase) that will transfer a sugar w/ the ceramide o In order to identify these antigens, the
• 1st you need a ceramide → next the H gene is needed → next the A corresponding anti-seras must be used: Anti-A,
and/or B gene Anti-B, and the Anti-H.
• H gene → codes for L-fucosyltransferase → L-fucose placed on the H antigen • If your blood type is AB; (+) rare phenotype—chances
ceramide → doon pa lang pwede mag-bind yung immunodominant sugar are that the glycosyltransferases corresponding the A
ng A or B & B gene will have a negative effect with each other;
• Therefore, for individual’s w/o H GENE, even if A or B gene is present, competing for the H substance which leads to the
the phenotype would not be manifested differences in between the antigenic sites of A, B, and
AB.
A gene, RBC surface: Secretors
H gene • RBC surface → L-fucose → N-acetylgalactosamine • Possess sese genes
B gene, RBC surface: o If you possess a secretor status, you can have A,
B, H soluble substances in your bodily secretions
H gene • RBC surface →L-fucose → D-galactose
Se Genes (saliva, tears, urine, digestive juices, bile, milk,
amniotic fluid, and pathologic fluids like pleural
• On the 87th day of fetal life, attachment of immunodominant sugars
fluid, peritoneal/ascitic fluid, pericardial fluid,
occurs on the RBC membrane & it is dependent of the ABH genes
ovarian cyst, etc.).
inherited.
Glycoproteins
o On the early days of fetal, A & B gene already work/manifest;
although they are not that optimal which is why for the forward • Possess ABH soluble substances
ABH Soluble
grouping of ABO for neonates, the strongest reaction would only be o If the antigens are bound to the RBCs, its nature
Substances
2+. would be glycolipids (ceramide + sugar).

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 4

 ANTIBODIES NATURALLY-OCCURRING VERSUS IMMUNE-TYPE

A. ABO ANTIBODIES NATURALLY-OCCURRING IMMUNE-TYPE

• Are mostly naturally occurring antibodies that are detectable 3 to 6 • Usually IgG in nature
months after birth following exposure to ABO-like antigens in the • Usually IgM in nature • Requires prior exposure for
environment. • Present without any prior production
• Undetectable fetal antibodies at birth, but maternally-derived antibodies exposure • EX: Group O developing anti-A
may be detected (i.e. IgG1 and IgG3, not IgG4 because it cannot cross • EX: Anti-A in Group B, Anti-B in post-transfusion with Group A
the placenta) Group A unit, “O” mother developing anti-B
o Reverse grouping should not be performed for neonates. while pregnant with “B” baby
• Mostly IgM in nature and react best at RT or below (cold temp)
• IgM = 4 deg C or lower B. GENERAL CHARACTERISTICS OF ABO ANTIBODIES
o Anti-A: IgM in nature (naturally-occurring in “B”)
o Anti-B: IgM in nature (naturally-occurring in “A”) 1 • Not normally present at birth (If present, it is maternally derived)
o Anti-A,B: IgG in nature (but naturally-occurring in “O”) 2 • They develop 3-6 months after BIRTH
3 • React better at RT; mostly IgM
GROUP ANTIGEN ANTIBODY
• They occur in two forms: naturally occurring antibodies, and
O H Anti-A, Anti-B, Anti-A,B immune antibodies
A A, H Anti-B • Immune antibodies occur due to (IgG):
B B, H Anti-A o Transfusion
AB A, B, H None 4 o Previous pregnancies
• Immune antibodies – Alloimmunization occurred = mild
1. The RBC antigen determines the blood type of the incompatibility with ABO
individual (i.e., presence of A antigen is seen in • Mild Incompatibility: presence of IgG from immunization of
Group A individuals, and so on). incompatible AB phenotypes
2. There will never be an antibody present against the 5 • They are present in some plants and animals
Landsteiner RBC antigen present in an individual (i.e., Group A
• They are present in LOW TITER or even absent in some conditions
Law individuals will never have anti-A).
(e.g. hematologic dyscracias, Burton’s agammaglobulinemia or
3. There will always be an antibody present against
congenital agammaglobulinemia)
the missing RBC antigen (i.e., Group A individuals 6
will always have anti-B). • Talking about discrepancies - missing ABO antibodies
Take Note: Number 2 and 3 has the antithesis concept. • The patients have an abnormality concerning immunodeficiency or
• Arranged according to clinical significance of production of antibodies
immunogenicity (descending): 1st = ABO, 2nd = Rh,
3rd. Kell Must to Know
1. ABO IgM • Seen in A, B, O
2. Rh • Type of ABO antibody seen in patients with history of
Immunogenicity IgG
3. Kell, Duffy, Kidd transfusion, and patients with previous pregnancies
4. S,s Mild
• Presence of IgG in the form of antibodies
5. U Incompatibility
6. Lub Lectins • Plants
7. I, P1P, M, N, Le, Lua Prolectins • Animals (mostly seen in nails)

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 5

 BLOOD TYPING 2. Indirect/ Reverse/ Serum Grouping
• Routine blood banking procedure in which antigens or antibodies are • Screening procedure in blood typing
detected using commercial/lab prepared reagents. • To detect unknown antibodies using lab prepared known RCS
Specimen • Patient’s serum
TWO (2) PROCEDURES
Reagents • Lab prepared RCS (aka Known Red Cells)
1. Direct/ Forward/ Cell Grouping
Additional Note:
• Screening procedure in blood typing
• Rough rules concerning Forward & Reverse Typing
• To detect unknown RBC antigen using commercially prepared reagents
• You should always add the clear solution first before the red cells
o In order to make sure that the source of antibody then the antigen
Specimen • Patient’s red cells (usually in suspension/RCS)
were added
Reagents • Anti-A, Anti-B, Anti-A,B (typing sera) o In order to prevent false-negative reaction
Reagents Used ▪ For ex. The reagent is colored clear such as Anti-D in which the
• Used to detect A antigens negative reaction is only “no agglutination”. If the Anti-D was not
• Blue in color due to the dyes that can be used: added, it might be interpreted as D-negative instead of D-positive
o Methylene blue • Weak D Phenotyping
Anti-A
o Bromophenol blue o If the donor is typed as D “negative”, you have to confirm if it really is
o Typan blue negative by performing a workup because what if they are weak D
o Thymol blue ▪ No workup: IF the donor is Du but classified as D “negative”, they
• Detects B antigen really are D “positive” which can cause immunization in a D
Anti-B
• Yellow in color due to acriflavine dye “negative” patient
• Detects A and B antigens o If the recipient/patient is classified as D “negative”, no further workup
Anti-A,B
• Usually colorless or clear in color • There is no risk since the patient is still going to receive a D “negative:
blood bag = no immunization
Additional Note:
Q: What is the preservative used in the Anti-A or Anti-B sera? FORWARD AND REVERSE GROUPINGS
A: Sodium azide FORWARD /CELL INDIRECT/ REVERSE/
o Preservative – prevents the growth of molds in the reagent BLOOD TYPING SERUM TYPING
• Proper mindset for ABO phenotyping (forward): TYPE ANTI- A B O
ANTI-A ANTI-B
o You are using a monoclonal antisera = potent = optimal reaction A,B CELLS CELLS CELLS
capabilities = highly specific O 0 0 0 + + 0
▪ Hence, the expected reaction should always be 3+ or 4+ A + 0 + 0 + 0
• If the serological reaction in forward grouping is in the range of 1+ or 2+, B 0 + + + 0 0
it warrants a subgroup investigation AB + + + 0 0 0
• Anti-A,B
o Mainstream reagent in AB phenotyping POLYSPECIFIC AND MONOSPECIFIC AHG REAGENT
o A single cross-reacting antibody in which is IgG in nature, capable of
agglutinating A and/or B positive phenotypes • If you want to get the specificity for IgG opt to
o Not a combination of Anti-A and Anti-B choose the polyclonal to capture
o Used in subgroups investigations in the confirmation of weak A IgG
• IgG is heterogenous; to capture different variant of
phenotypes IgG
• Last rule concerning the subgroups: The serologic reaction of weak C3 • Use monoclonal
A subgroup parallels the serologic reaction of weak B subgroups

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 6

 Detection of • Combination of the two (blend) 2. Zeta Potential
both IgG and C3 • Polyclonal anti-IgG with murine monoclonal anti-C3 • The more optimum reaction would be
Low Zeta
o Inversely proportional to the strength of the
Potential
Additional Note: serologic reaction
• Monospecific and polyspecific have the same production process they
only differ in their specificity. 3. Position and Number of Antigenic Site
A1 • 0.81-1.7 x 106
FACTORS THAT AFFECT THE STRENGTH THE AB-AG • 0.24–0.29 x 106
A2
• Whether you use AHG or not you are still looking for agglutination. o Subgroups of A contains of small antigenic site
B • 0.75 x 106
REACTIONS IN BLOOD TYPING D • 9.9–33.3 x 103
• In blood banking we are always looking for two types of reaction: • 3.5 – 6 x 103 (2nd most common BGS outside
• Most common antigen-antibody reaction that is ABO)
detected in blood typing o The number of antigenic site is directly
• Appears macroscopically as CLUMPS proportional to the strength of serologic reaction
• Appears due to lattice formation (cross linking of o Logic for positioned: The more exposed the
Agglutination antibodies due to IgM) antigenic site = The optimal reaction
Kell
o Not using antihuman globulin (AHG) reagent, o Memorize the antigenic site of ABO, Rh, kell
but if you are going to use AHG, lattice o ABO is the most significant followed by RH and
formation will only happen after the addition of then KELL
AHG. o ABO ultra-significant followed by RH
• Observed in blood typing when the supernatant • KELL, DY, KIDD is the most significant and must
appears clear red or pink (background) treat them as a group
• Rare antigen-antibody reaction
• Takes place when there is a strong agglutination 4. Length of Incubation Time
graded 4+ an when there is complement activation • Most RBC antigen have HIGH-binding constant
Hemolysis o The reason why we incorporate C3d or C3b o The binding constant is a pulse-repulsion that antigen and
o C3 = amplification stage alloantibody have to overcome in order to induce antibody antigen
▪ This makes complete sense since the end binding = agglutination
of the complement activation there would o Optimal incubation time must be observed to induce agglutination
be a cell membrane lysis (final product) reaction
▪ C3b has opsonic property Optimal • 30-120 mins – when only saline is used
Incubation Time • 10-15 mins – when enhancement media are used
FACTORS AFFECTING AGGLUTINATION OF RBCS • Helps in shortening the incubation time by
Potentiators overcoming the high binding constant of your red
1. Antibody Length (BAS, PEG, blood cell antigen
• Average length of 1000 Angstrom LISS) • E.g. 10-15 minutes can be achieved if you use
IgM
o Malaki - largest LISS (Low Ionic Saline Solution)
• Average length of 250 Angstrom
IgG • The larger the antibody the efficient it is in inducing
agglutination patterns.

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 7

 5. Reaction Temperature • AHG goal is to detect alloantibodies in the patient’s plasma/serum.
• Reacts optimally at cold temp or below (25°C o So, if you are trying to increase the ratio of your serum to cell, the
IgM
below) sensitivity of AHG increases but you are trying to increase the risk of
• Reacts at warm temp (37°C) prozone (Ab excess) → ↑ ratio of serum:cell = ↑ Sn, ↑ risk of
o If an alloantibody is clinically significant it will prozone
always react at body temperature • Use of red cell suspended in saline:
o With an exception of ABO blood group because o Without use of potentiator; the saline is the only source of antigen
this will serve as your bondage point o Achieve 133:1 via 4 drops serum + 1 drop of 3% w/v RCS)
o Natural occurring antibodies on ABO is IgM but o ↑ Sensitivity and without running the risk of prozone
they can induce acute hemolytic transfusion
reaction (AHTR) regardless of the temperature 8. Reaction Medium/Potentiators
o Other blood group with the nature of IgM will not • They can be used to enhance the reaction between antigen and antibody
work unless the body temperature is at if AHG procedure is used.
IgG
refrigeration temp hence it is not insignificant • These are large molecules dissolved in plasma that
o Even if they have an alloantibodies unless the are responsible regulation of colloid osmotic
body reaches a refrigeration temp it will not pressure.
react o Colloid – viscous; as if you put your Ag-Ab in a
o IgG will always respond because your body will viscous solution.
always maintain a minimum of 37°C; Albumin o Principle: Antigen-antibody will come in close
• If you have an alloantibodies that reacts at 37°C contact so you can induce an agglutination
and you received an incompatible blood, chances reaction.
are it will induce a hemolytic transfusion • Concentration of albumin to achieve the said
reaction. phenomena: 2 gtts serum + 2 gtts 22% w/v BSA
(Bovine Serum Albumin)
6. pH • 3 primary advantages of LISS:
• Effect of pH in agglutination procedures: Alloantibodies will always react 1. Highly sensitive
at physiologic pH 7.2 to 7.4 in concern with the acid-base homeostasis. 2. Decreased the incubation time in as much as
• The ultra normal pH of the plasma is pH 7.4, we are trying to mimic 10-15 mins (from 30-60 mins. to 10-15 mins.)
something that happens inside the body. making it convenient
Anti-M • 6.0 to 6.5 (pH and glucose resistant) 3. Economical
Anti-D • 6.5 to 7.0 • Most common potentiator nowadays.
Most • Principle: It can reduce the zeta potential of RBC.
antibodies • 7.2 to 7.4 (physiologic pH) Low Ionic o ↓ zeta potential = ↑ agglutination reaction
react best at: Strength o By reducing the zeta potential, you are
Solution (LISS) increasing the antibody uptake.
7. Concentrations of Antigen and Antibody • If it is used, consider the optimal antigen to
• Prevention of prozone (antibody excess) and postzone (antigen antibody ratio. LISS and the concentration of
excess) hence we are trying to pay attention to our red cells if we are antibody comes hand in hand to get the desired
looking for agglutination reaction. reaction.
• Point of equivalence reach when: • ↑ ratio of serum to red cells = ↓ sensitivity of LISS
• 2 drops of serum + 5% w/v RCS (3-5% in other o The ratio becomes sensitive when using LISS
Antibody because when you increase the ratio of the
references)
• 133:1 (achieved by 4 drops serum + 1 drop of 3% serum to red cells, the sensitivity decreases
Low Tittered
w/v RCS)

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 8

 • The optimal reaction conditions for LISS can be Because instead of your AHG binding to sensitized red
achieved if you are going to use: 2 gtts serum + 2 cells, it will bind to the free-floating antibodies that is in
gtts 3% v/v RCS (red cells suspension) your residual serum or plasma.
• Polyethylene glycol (PEG) is a water-soluble • What is the purpose why the inadequate washing may
False
linear polymer. result into a false negative reaction?
Negative (-)
• Drawback: PEG is the most common cause of o It could neutralize the AHG reagent, because
d/t
FALSE POSITIVE reaction concerning the Indirect instead of your AHG reacting to your sensitized red
Inadequate
Antiglobulin Test. cells, it will react with your residual or unbound
Washing
o It removes the water; hence, it is a strong serum globulins.
reagent.
• Since PEG is a highly potent potentiator, you will Board Recall Question/s:
remove a phase in the Indirect antiglobulin test if Q: What type of reagent serves as a check in order to confirm adequate
you are using PEG. If you’re going to use PEG in washing?
Indirect antiglobulin test, you are going to omit the A: Coomb’s reagent Check cells / Control check cell / Check cells
37˚C incubation step. Incubation time will depend Q: What is the nature of your Coomb’s check cells?
Polyethylene
upon the potentiator. A: Blood group O sensitized red cells. Meaning, there are red cells that
glycol (PEG)
o If you use saline, the incubation time will range are type O and there are antibodies that are already coating it.
from 30-120 minutes, since there is no
potentiator involved. 10. Properties of your saline for washing
o If you’re going to use LISS and PEG, you can • You can use distilled water as buffer for 24 hours once you opened it.
reduce the incubation time to 10-15 minutes. You can use it to wash something else.
• If you’re going to use potentiators, except for PEG, • Saline for washing in blood bank, it goes like this, when you open saline
the reaction time temperature is 37˚C (Albumin for 24 hours, the open expiration of it would only be 30 days.
and LISS). That is why it is except, because you will • In blood bank, you must use fresh saline. If your saline becomes old, the
remove the incubation time. pH goes lower, then that means you can’t get the best reaction pH for
• Extended incubation time (>40 minutes) for LISS, the most clinically significant antibodies.
can induce antibody elution. If you have this, then • One of the main causes of bacterial contamination in AHG is saline for
what you’re doing is nonsense. washing (because it’s just water + salt).
pH Property of
Board Recall Question/s: • 7.2-7.4
Saline
Q: What is the principle behind Polyethylene glycol? False Positive
A: It removes the water surrounding your red blood cells. If you remove • Bacterially contaminated NSS for AHG
(+) AHG
the water surrounding your RBCs, they will be concentrated more. When
RBCs have greater concentration, they will have closer contact with each 11. Centrifugation for Reading
other, hence it will induce a greater agglutination reaction Immediate • Since you perform IAT, you spin it (Immediate spin
Under the context of potentiators, if the boards are going to ask from you: Spin Phase phase)
Q: What is the most common cause of false-positive reaction?
A: The answer is Polyethylene glycol.
Board Recall Question/s:
Q: What is the proper relative centrifugal force for how many seconds
9. RBC Washing in AHG?
• To remove free unbound globulins. A: 1,000 RCF for 20 seconds
Purpose of
RBC washing • Why do you have to wash your RBCs? o Although in Harmening, 500 RCF for 15-20 seconds. If you don’t
If you don’t remove the water, chances are inadequate want to get a mistake search the choices for an answer that is in
in AHG
washing may result to FALSE NEGATIVE reaction. between 500-1,000 RCF for 15-20 seconds.

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 9

 12. Addition of AHG DISTINCTION OF ABH ANTIGENS AND ABH SOLUBLE SUBSTANCES
Purpose • In order to prevent antibody elution
• Add AHG immediately after cell washing, because if ABH SOLUBLE
ABH ANTIGENS
you take too long the one you’re detecting might elute. SUBSTANCES
So that you may capture the bound antibodies. Found In RBCs, epithelium, In all body secretions,
• In technical manual, they say its 2 drops of AHG, the where? tissues, BM, other cells mostly plasma
Procedure
one you should use for IAT, for that matter, because it Secreted
Glycolipids Glycoproteins
is due to the fact that you wash. Since you washed substances
and you removed a lot of bounded antibodies. In order 1st sugar in
to capture it you must use 2 volumes (drops) of AHG the precursor Glucose N-acetylgalactosamine
substance
% GRADE SCORE DESCRIPTION OF APPEARANCE Precursor 1st carbon of Type 2
Type 1 and Type 2
One solid clump, no free cells, clear chain carbon-carbon linkage
100 4+ 12
supernatant 1st carbon-galactose
Several large clumps, clear attached to the 3rd carbon of
75 3+ 10
supernatant Linkage Beta 1->4 linkage galactosamine
Many medium-sized clumps, clear
50 2+ 8
supernatant 1-3 linkage
Numerous small clumps, cloudy red Regulating
25 1+ 5 Zz gene Se gene
supernatant gene
Numerous very small clumps easily
<25 +/- or w+ 3
dispersed, cloudy red supernatant RED CELL PRECURSOR SUBSTANCE (PARAGLOBOSIDE)
Appears negative macroscopically, but
1
agglutination visible microscopically TYPE 2 PRECURSOR CHAIN
0 0
Negative, no agglutination seen • Beta 1-4 linkage: #1 C of D-galactose
macroscopically or microscopically • Connects to #4 C of N-acetylglucosamine
Complete hemolysis, no intact RBCs • Structure:
H
remaining Fucose –– D-galactose
Partial hemolysis, some RBCs still N-acetylglucosamine –– D-glucose
PH intact; but hemolysis visible in D-galactose
supernatant Glucose
Mixed field–mixtures of agglutinated Red cell membrane (protein backbone)
mf
and unagglutinated cells TYPE 1 PRECURSOR CHAIN
Sometimes the notations “S” for strong or “W” for weak are used to denote • Beta 1-3 linkage: #1 C of D-galactose
slight variations between reaction grades. For example, a reaction between 1+ • Connects to #3 C of N-acetylglucosamine
and 2+ might be noted as 1+s or 2+w • Structure:
Galactose
INTERACTIONS OF THE SESE, ZZ, AND ABH GENES N-acetylglucosamine
Galactose
• Regulated the formation of H antigen and N-acetylgalactosamine
Sese System
subsequently, of A and B antigens in secretory cells Red cell membrane (protein backbone)
Zz System • Regulated the production of H antigens on RBCs

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 10

 ABO SUBGROUPS BOMBAY (OH) PHENOTYPE
• Antigens under the ABO system which reacts less strongly to commercially • The allele h is very rare and does not produce the L-fucose necessary for
typing sera the formation of H structure
o Blood type O = strongest reaction to anti-H • The genotype hh or Hnull is also known as the Bombay phenotype or Oh
o Highly recessive – Mumbai, India
REACTIVITY OF ANTI-H ANTISERA OR ANTI-H LECTIN
WITH ABO BLOOD GROUPS GENERAL CHARACTERISTICS OF OH PHENOTYPES

Greatest amount of H Ag Least amount of H Ag 1 • Absence of H, A, and B antigens

O > A2 > B > A2B > A1 > A1B 2 • Presence of Anti-H, Anti-A, and Anti-B in serum
3 • Strong reactivity with Anti-I reagents
Additional Note: o When you are Bombay phenotype, you have no A, B, or H kaya
• O contains the greatest amount of H Ag because yun lang yung kaya may magwawalang antigen which is I kaya it will manifest strong
niyang ioffer. reactivity with Anti-I.
o If you’re blood Group O, you only have H gene and no A & B gene. 4 • A recessive mode of inheritance
You will only have ceramide and L-fucose. 5 • No reaction with Anti-H lectin
• Antisera is used to identify phenotype. When you use anti-H, nagwawala o The panel to use to differentiate between a normal group O and
yung blood group O since the H antigen is exposed. A Bombay phenotype is anti-H lectin
• However, if your phenotype is A1B, you have galactosamine and o May “h” yung normal group O, however, walang “h” yung
galactose. Bombay phenotype kaya walang didikitan yung Ulex. Hence,
Natatakpan yung H antigenic sites and yung naooffer niya lang ay A and B. when you use Ulex panel, it will only react with normal blood
Walang madikitan yung anti-H lectin that is specific for A1B. group O.

Dolichus biflorus • Anti-A1 Additional Note:

Bandeiraea simplicifolia • Anti-B • Anti-H = naturally occurring
Vicia graminea • Anti-N • Bombay phenotype RBCs possess I antigen-like specificity (hence,
Ulex europaeus • Anti-H the strong reactivity with Anti-I reagents)

Additional Note: PARA BOMBAY PHENOTYPE

• What is the primary clinical application for the utility of anti-A1 lectin?
o Used for the initial differentiation of A1 and A2 Classic
H-Partially Para Bombay
• There’s a reason why Dolichus biflorus only reacts to A1. Anti-A1 lectin Bombay
is used to differentiate A1 and A2. Genes hh/sese hh/sese (weak var.) hh/Se
o Anti-A1 reacts to A1 but not with A2. Glycosylated Weak A and/or B Weak A and/or
0
• When you performed forward grouping and the result is not 3+ to 4+ but ferases transferase B transferase
1+ to 2+, you have to do anti-A1 lectin panel. • Classic Bombay is H-deficient, non-secretor
o When lectin reacts with A1, it is definitely A1. When A1 lectin did not • Para Bombay is H-deficient, secretor
react, it may be A2 or the rest of the weak subgroups. o Although H deficient, you were able to retained the synthesis of Type
This is where you’ll performed subgroup investigation and you have to be 1 H antigen on the mucosa of your secretions
mindful of the general properties.

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 11

 ABO DISCREPANCIES
• ABO discrepancies are ambiguous which means there is no clear line that Conditions leading to • Antibodies are missing
separates these discrepancies. immunodeficiency • Group 1 most common
o Huwag niyo siya itake for instance ito yung nasa condition sa Group 1 Conditions leading to
hindi na siya mag eexist sa another group. • Most common: Group 3
hyperactive immune system
o The goal is to identify which is the most common.
• Any deviation from the expected pattern of antigen on the cell and the o Check if patient is suffering from hematologic neoplasm (blood
opposite antibody in the serum cancer)
o You always expect a serologic reaction. In forward, 3+ or 4+ • Can lead to group 1 up until group 4 discrepancies.
• Check transfusion history
Additional Note: 7
o If patient has transfusion history, it is suspected of alloantibody
• When performing Reverse grouping, wala naman nakukuha ng 3+ or 4+,
thus, this is where confusion arises – Group 1 discrepancy Additional Note:
• If the antigen is weak or absent, the FORWARD typing result is
GENERAL RULES TO RESOLVE ABO DISCREPANCY discrepant while the REVERSE typing is correct
• Always re-test first • If the antibody is weak or absent, the REVERSE typing is discrepant
1
o To avoid procedural error while the FORWARD typing is correct
• Check for clerical / technical errors
2
o Always recheck the label STEPS IN CHECKING OF DISCREPANCY RESULT
• Weakest reaction is usually the one in doubt • Check the autocontrol
o Doubtful reaction = 2+ or +/- 1
o To rule out autoantibodies
o True reaction = 4+ 2 • Check the control panels (anti-A,B and O cells)
• REMEMBER: 3 • Determine the FORWARD typing reaction
o If the antigen is weak or absent, the forward typing is
4 • Determine the REVERSE typing reaction
3 discrepant, while the reverse typing is correct.
o If the antibody is weak or absent, the reverse typing is
5 • Decide whether there is discrepancy
discrepant, while the forward typing is correct.
CLASSIFICATION OF ABO DISCREPANCIES
• If the reactions are far from each other, either there’s something
wrong with what you are doing or there’s something wrong inherent
with your specimen. GROUP 1 DISCREPANCY
• Check the results of the screening cells and autocontrol panel • Due to weakly reacting or missing antibodies
4 o Autocontrol Panel: (Px Serum + Px RBCs) = detects • Reverse typing is discrepant
autoantibodies • Most commonly encountered
• Check the patient’s age • If the blood group of the patient is group O, there is a chance that strong
o If a healthy human being is <50 years old: serologic reactions are acquired (4+ in both A and B). If the patient’s
▪ The lower the chance for alloantibody detection phenotype is blood group A, the expected maximum reaction is 2+, due
5 to reacting to B cells
▪ The lesser the chance for an immune type of disorder; it
could be that he has no immune deficiency or he has no ○ in between A cells and B cells, B cells contain lower antigenic sites
hypersensitivity type of condition. compared to A1 cells
• Check the patient’s diagnosis • If the patient’s phenotype is O, you can still expect 4+ (blood type is O)
o Check if autoimmune disease, leukemia, lymphoma, B-cell • In reverse typing, if the patient’s blood type is A
6 abnormality, sepsis ○ it will not react with A1 cells; it will react with the B cells ( 2+ only)
o If you are looking at the patient’s diagnosis, you are looking for ○ A1 serum contains anti-B
two types of conditions:

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 12

 ○ B cells (fewer antigenic sites compared to A). therefore, you can GROUP 2 DISCREPANCY
only get a maximum of 2+ • due to missing antigen (forward typing) and is the
• In reverse typing, if the patient’s blood group is B • forward typing is discrepant
○ Reaction with A1 is 3+ • It is found in the following:
○ Serum in B contains anti-A (millions of antigenic sites) 1 • Subgroups
• Average antigenic sites in A1 is 1,170,000 Sa • Leukemias
• It is found in the following: o Nagwawala yung myeloid or lymphoid cell line sa loob ng bone
1 • Newborn 2 marrow
2 • Elderly patients (patients older than 65 yrs of age) o Developing erythroblastic islands would get disrupted hence
3 • Leukemic patients demonstrating hypogammaglobulinemia sooner or later baby red blood cells could lose their antigens
4 • Patients with lymphoma 3 • Hodgekin’s Disease (not defenitive), Reedstern Berg Cells
5 • Patients using immunosuppressive drugs • Acquired B phenomena (in GIT obstruction, colon, or rectum CA)
4
6 • Patients with congenital agammaglobulinemia – applicable to affected RBC
7 • Patients with immunodeficiency (in general) • Antibodies to low incidence antigen (may be present in reagent
5
• Patients who undergone BM transplant who develop antisera)
hypogammaglobulinemia from therapy • Excess Blood Group Specific Soluble Substances (BGSS) – in
o Following bone marrow transplantation, they would take cases of stomach or pancreas CA
8 6
immunosuppressive drugs in order to prevent rejection o Yung secretions nagkakaroon ng BGSS hence neutraalizing
reactions hence becoming immunodeficient following the the reagent (Anti-A)
theraphy.
• CHIMERISM Board Recall Question:
o Also categorized under Group 2 Q: Least frequently encountered?
9 o A rare group 1 discrepancy A: Group 2
o Presence of two cell populations in single individual like in
cases of fraternal twins (true chimerism) ACQUIRED B PHENOMENON
a) When bacterial enzymes (of Proteus vulgaris) modify – most
Additional Note: common (Positive for D-acetylose)
• ARTIFICIAL OR FALSE CHIMERISM b) Increased permeability of the intestinal wall causes adsorption of B-like
o Pregnant women bacterial polysaccharide onto RBCs of A or B patients (P. vulgaris or E.
o Blood transfusion (e.g. O to A or B) coli O86)
o BM transplant - What If nag BM transplant tas magkaiba ng PRINCIPLE: Bacterial enzyme modification
blood type pero inaccept ng donor, hence producing blood cells CAUSES OF ACQUIRED B (EPIC)
with different population (immune tolerance) • E. coli infection (O86)
o Exchange transfusion • Proteus vulgaris infection (most common cause)
o Fetal-maternal bleeding (most common, when the placenta o Both bacterias contain modification enzymes
ruptures) o Example: the original blood type of the patient is A because the
▪ Immunotolerance – allows the presence of foreign antigens in terminal sugar is N-acetyl galactosamine. Yung bacterial
the individual circulation modification enzyme na D-acetylase???
▪ The two cell populations are both recognized as SELF, and the o If may D-acetylase yung bacteria, yung N-acetyl galactosamine
individuals do not make Anti-A or Anti-B mawawalan nang N-acetyl, matitira nalang is galactose
o Ano yung D specificity? = galactose

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 13

 o From blood type A, inalis ng modifying enzyme yung acetyl part, SALINE REPLACEMENT TEST
yung naging specificity tuloy is D-galactose. So saan ngayon • Used to check rouleaux formation or pseudoagglutination
nagrereact yung cells? Sa Anti-B = “Acquired B” • 2 to 3 drops
• Intestinal infection (severe) • WASHING OF CORD BLOOD with saline (saline repeats)
• Carcinoma of the colon o Harmening = 6 to 8 times
• Acquired A is caused by Proteus mirabilis • AABB = 8 to 10 times
o Check patient’s history • True rouleaux will disperse
Example: Result
• No dispersion = agglutination
Anti-A Anti-B Anti-A,B Washing of • Saline repeats
4+ +/- + Cord Blood • Harmening = 6 to 8 times
with saline • AABB = 8 to 10 times
GROUP 3 DISCREPANCY
• Anything that has something to do with increased protein concentration
in the patient’s plasma GROUP 4 DISCREPANCY
o Too much protein in the plasma is called • Discrepancy is mostly seen in forward typing
Hyperproteinemia (rare) • Due to miscellaneous problems seen in:
o If protein is deficient it is known as Hypoproteinemia • Polyagglutination (Hubener-Thomsen Friedenreich
(common) phenomenon)
o In terms of being rare mas madalas kang mawalan ng o spontaneous RBC agglutination by most normal human
proteins compare sa sumobra serum
o Ang hyperproteinemia is a rare condition relative to o occurs owing to exposure of a hidden RBC antigen in patients
hypoproteinemia with bacterial and viral infections
▪ Example: fractitious hyperproteinemia = baka
dehydration lang • Exposure/presence of cryptantigen
o First look at the patient’s condition and think for any (hidden antigen)
possible condition that could lead to increase production of • (+) bacterial infection = (+) enzymes
Polyagglutination
proteins that ends up in the plasma that can modify the RBC antigen,
• Caused by protein or abnormalities resulting to rouleaux formation exposing others (i.e. cryptantigen)
• Reverse typing is discrepant
• It is found in: 1 Polyagglutinable • RBCs with exposed cryptantigens
• Increased globulin (e.g. multiple myeloma, Waldenstrom’s Cells
1 THREE TYPES OF POLYAGGLUTINABLE RBCS:
macroglobulinemia)
2 • Increased fibrinogen / Hyperfibrinogenemia • Three mechanisms wherein you will have polyagglutination
• Presence of plasma expanders (e.g. dextran, PVP) – synthetic • Bacterial = Clostridium spp., Vibrio
volume expanders Microbially- spp., Pneumococci spp.
o Used in cases of massive bleeding; for volume replacement associated • Viral = Influenza
3
o Pag-nasaksak ka, the body first tries to prevent hypovolemia • Produce neuraminidase that
(i.e. decreased plasma volume) promotes exposure of cryptantigens
o It is harder to have hypovolemic shock than other imbalances Non-microbially • Impaired hematopoiesis
• Wharton’s jelly mucopolysaccharide substance present on cord associated
4 blood Genetic • Ab necessary gene is inherited
o To resolve: Saline Replacement test
2 • Cold reacting antibodies (allo and auto)
3 • Unexpected ABO isoantigen
MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 14
 • Warm autoantibodies • Functions to serve as a co-stimulator and co-expressor
• RBCs with cis-AB phenotype in order for the proper manifestation of the Rh D and CE
4 o In AB cis-phenotype = 1 parent with AB genes + 1 parent with phenotypes
O genes • Pag walang RHAG gene, it manifests as Rh Null
o Due to codominant expression of these ABO genes Syndrome
• Gene product: glycosylated protein
Board Recall Question/s: • Classified as co-expressor and co-stimulator
Q: Which group of ABO discrepancy is cis-AB phenotype classified? o Works just like the H gene
A: Group 4
Q: Second most important blood group following ABO If not italicized NOMENCLATURE
= phenotype
A: Rh Blood Group System 1. Fisher-Race/DCE Terminology (England)
Q: Most common cause of HDFN • Where DCE terminology started
A: Rh Blood Group System • Rh antigens are produced by three closely linked sets of alleles
• The five major antigens defined are D, C, E, c, and e (all are
Board Recall Question/s: distinct/unique to each other)
• The most common cause of hemolytic disease of the fetus and
newborn? Board Recall Question/s:
o Rh Blood Group Q: Which of the following naming classification system is based on
o If there is 100% HDFN, if ABO incompatibility its frequency is 0.008%. the genetic background of your Rh blood group?
o Meanwhile, for Rh blood group, 97% can be attributed to it • Fisher-Race
• Non-genetically associated Rosenfield and ISBT
RH BLOOD GROUP SYSTEM Q: Which of the following naming classification system offers a
• Represents a critical component of pre-transfusion testing distinct advantage by giving numerical data that can be incorporated
• Is the most complex and most extensive in HIS/Inventory?
• In 2014, there was 50 antigens, now, there is 61. • Rosenfield
• It is inherited in codominant manner and is the most clinically significant • Utilizes integers and the presence of absence of a particular phenotype
blood group Q: What is the ISBT number for Rh blood group system?
• 004 (Old system)
RH BGS IS REGULATED BY 3 GENES • RH (New system)
• Function of Rh proteins is to maintain the structural integrity of RBCs
• If it is not available, the RBCs become vulnerable and easily lysed *For Boards, either itatanong ung nomenclature based on genetic
• Gene responsible for the presence or absence of D background or genetic basis
• 85% of individuals have the D gene, corresponding to Q: What is the genetic background for your Fisher-Race terminology?
Rh positive (mostly seen in Asians) • The Rh antigens are produced by three closely linked sets of alleles
RHD • 15% of individuals lack the D gene, corresponding to Rh • Each gene is responsible for the production of a single antigen that will
negative (mostly seen in Europeans) on the red blood cell membrane
• Gene product: non-glycosylated proteins (protein o Antithetical relationships of antigens
without sugar) o Occurs if a protein can only present a single antigenic determinant
• Responsible for the inheritance of Cc Ee antigens
RHCE
• Gene product: non-glycosylated proteins
RHAG • RHAG (Rh Associated Glycoprotein)

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 15

 ANTITHETICAL RELATIONSHIP • “E” is indicated by the presence of (rh”) double prime
• If a protein can only present a single antigenic determinant • If there is both “C and E” it is represented by “z” or “y”
• Example if you only have the “D gene” which has the “C, c” and you have • If there is small letter “c” denoted as (hr’)
the “E, e” gene then you have 3 rh proteins • If there is small ltter “e” denoted as (hr’’)
• So pag na-inherit mo yung D gene sa red cells mo magkakaroon ka ng “D • THE INTEGERS ALWAYS COME WITH BIG LETTERS AND THE
antigen”: so magiging phenotype mo is “D” so ibig sabihin kapag PRIME (‘ AND ‘’) WILL ALWAYS GO WITH THE SMALL LETTERS
nainherit mo ang “C gene” so sa red cells mo dapat meron lang “C” then • Zero (0) and “r” indicates small letters (c and e)
ang magiging phenotype mo nyan ay only “D and C” • Example : Capital “R” and 0 (Ro): pag merong R it denotes D and yung
“0” we have small “c” and “e”
• Small letter is only there to denote the ABSENCE of
Small Letter
“D” WIENER FISHER-RACE ROSENFIELD
Rh0 D Rh1
RULES IN NAMING CLASSIFICATION SYSTEM rh’ C Rh2
• The patients phenotype will always correlate with the serologic reaction rh’’ E Rh3
• It is virtually impossible to determine the patient’s genotype using hr' c Rh4
serology (only patient’s phenotype) hr" e Rh5

Additional Note: SHORTHAND WIENER BLOOD FACTORS FISHER-RACE

• Basically yung antithetical relationship ay dapat sa isang protein may R0 Rh0 Rhohr’hr’’ Dce
both big and small letter “c”(dapat isa lang ang antigen) R1 Rh1 Rhorh’hr’’ DCe
R2 Rh2 Rhohr’rh’’ DcE
2. Wiener/Rh-Hr Terminology (US) Rz Rhz Rhorh’rh’’ DCE
• Nowadays: Modified wiener (wala na yung Hr r rh hr’hr’’ dce
terminology/nomenclature)
Modern
• We are only relying on the MODIFIED RH terminology: RHNULL SYNDROME
Wiener
so dahil naiwan ka with the RH, ang ibang tawag ngayon • It expresses no Rh antigen on RBC and the phenotype is expressed as
sa wiener nomenclature “---/---"
Genetic • The RH antigen has series of blood factors in which each • Has many problems; clinical manifestations
Background factor is an antigen recognized by an antibody Clinical Manifestations:
• For example, naka-inherit ka na ng RH gene so alam na • The poikilocyte involved in this syndrome is stomatocytosis
natin na magcocode sya for an agglutinogen (traditional • Mild reticulocytosis as part of the compensatory mechanism of the body
term for an antigen) due to mildly compensated hemolytic anemia
• The logic behind this if na-inherit mo yung “RH 0” gene, • Slight to moderate decrease in hemoglobin and hematocrit
How to
ano daw yung mapupunta sa red cell surface mo? So
visualize? • As part of the compensatory mechanism, Hemoglobin F is increased
ang mangyayari according to wiener sa isang antigen
maraming coat factors • Those suffering from hemolytic anemia have a decreased serum
haptoglobin
• You have produced 1 gene -> produces 1 antigen ->
that antigen has 3 factors (Rh 0, hr”1 and hr”2) pero • Elevation of total bilirubin levels
ngayon wala na kayong makikitang hr puro “RH” na.
Board Recall Question/s:
RULES IN CONVERTING FISHER TO WIENER Q: Who were the first individuals who first described hemolytic
• “R” indicates that the red blood cell contains the “D” antigen transfusion reactions concerning the Rh Incompatibility?
A: Levine and Stetson
• “C” is indicated by the presence of (rh’) single prime

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 16

Q: These individuals described an antibody made by guinea pigs
made in when they transfused Rhesus Macaque monkeys blood
cells
A: Landsteiner and Weiner

MTAP2: BLOOD BANK (IMMUNOHEMATOLOGY) 17