HEMATOPOIESIS COMMITTED GF/ MATURE

 Building of blood components PROGENITOR CELLS INTERLEUKINS CELL

 Intrauterine hematopoiesis – begins after conception CFU-Meg
Thrombopoietin,
Thrombocytes
GM-CSF
 19th day of gestation (2nd week of fetal GM-CSF,
life) formation of blood islands in yolk CFU-GM CFU-M M-CSF Monocytes
sac (mesodermal extraembryonic IL-3
layer), aggregation of primitive GM-CSF,
nucleated erythroblast (first cell) CFU-GM CFU-G G-CSF Neutrophils
Mesoblastic
 Yolk sac - Erythroblasts (immature red IL-3
(yolk sac)
blood cells) come from mesodermal Erythropoietin
phase
cells lining the yolk sac. Chief site BFU-E CFU-E GM-CSF Erythrocytes
 Embryonic hemoglobin: IL-3
o Gower I GM-CSF
o Gower 2 CFU-Eo IL-3 Eosinophils
o Portland hemoglobin IL-5
 Begins at 6 weeks of gestation CFU-Bs
IL-3
Basophils
 Production of red blood cells, IL-4
Hepatic granulocytes, monocytes, and
phase megakaryocytes
 Alpha and gamma globin chain  Erythrocyte - Composed of beta polypeptide chain, alpha
production (Hemoglobin F ) polypeptide chain, oxygen, cell membrane and iron
 Begins around 5th month of gestation (where 4 oxygen binds)
 Bone marrow producing mainly
Myeloid/
granulocytes
Medullary ANEMIAS
phase  M:E ratio - 3:1 / 4:1
 6th months of age - gamma-beta globin  Anemias is defined as a decrease in erythrocytes and
chain switch hemoglobin, resulting in decreased oxygen delivery to the
tissues
 Impoverished condition of the blood caused by reduction
PEDIATRIC AND ADULT HEMATOPOIESIS in RBC, hemoglobin or both. It is considered present if the
BONE MARROW Hgb content of the Hct is below the lower limit of the 95%
reference interval for the individual’s age, sex and
 Newborn: 80 – 90% of bone marrow is active bone geographic location.
marrow  Total erythropoiesis – total production of the RBCs;
 Young adult (age 20): 60% of bone marrow is active measured through M:E, fecal urobilinogen and plasma
o hematopoiesis: proximal ends of large flat bones, iron
pelvis, sternum) o Urobilinogen came from degraded RBCs
 Older adult (age 55): 40% bone marrow is active and  Effective erythropoiesis – production of RBCs that
60% is fat reach the circulation of the peripheral blood as normal
 CELLULARITY: ratio of marrow cells to fat (red o Measured through RBC turnover utilization of iron,
marrow/yellow marrow) reticulocyte count and RBC lifespan
o Normocellular: marrow has 30 – 70% hematopoietic  Can be classified morphologically using RBC indices
cells (MCH, MCV and MCHC) or etiology/cause
o Hypercellular/hyperplastic: marrow has >70% o Anemia can be caused by:
hematopoietic cells a. Decreased RBC production; problem in
o Hypocellular/hypoplastic: marrow has <30% production site
hematopoietic cells b. Increased RBC destruction; healthy cells but
o Aplastic: marrow has few or no hematopoietic cells can’t finish the lifespan
 M:E (Myeloid:Erythroid) ratio: ratio of granulocytes and  Anemia is suspected when the hemoglobin level is <12
their precursors to nucleated erythroid precursors g/dL in men or <11 g/dL in women
o Normal: 3:1 or 4:1  CLINICAL SIGNS & SYMPTOMS GENERAL:
o Lymphocytes and monocytes are excluded from a. Low Hgb conc, & blood volume
the M:E ratio b. Fatigue
 Hematopoiesis: c. palpitation
1) Pluripotential stem cells d. Dyspnea on exertion
2) Committed progenitor cells (Lymphoid or Myeloid) e. Faintness
3) Mature blood cells f. Vertigo
 Reviewed through PBS – identify the g. Headache
morphology of blast cells (especially RBC)  COMMON:
 If there’s discrepancy check the hemoglobin, a. Pallor
indices b. Rapid bounding pulse
 Differential count – count the number WBC c. Low blood pressure (Normal: 120/80)
types; should also observe the morphology of d. Slight fever
RBCs and platelets e. Some dependent edema
f. Systolic murmurs
 MORPHOLOGIC CLASSIFICATION OF ANEMIA c. Hemolytic diseases - increased retic production
MACROCYTIC NORMOCHROMIC ANEMIA can’t keep pace with loss of RBCs peripherally
d. Hypoplastic bone marrow (aplastic anemia)
 MCV is >96 fL, MCHC is normal (more accurate) e. Infiltrated bone marrow (leukemia)
 the size of RBC is large with normal hemoglobin content f. Endocrine abnormality
g. Chronic disorders
h. Renal disorders
1. Megaloblastic anemia – erythroblast shows abnormality i. Liver diseases
and maturation is delayed; lifestyle or inherited  Laboratory findings:
o Causes: o Plasma volume and red cell volume
a. Vitamin B12 deficiency – pernicious anemia o Hematocrit is normal.
b. Folic acid deficiency – nutritional megaloblastic o  Platelet count
anemia o  Plasma fibrinogen level.
c. Abnormalities of Vit B12 or folate metabolism o Neutrophilic leukocytosis is present.
d. Inherited disorders of DNA synthesis o Normocytes and normochromic cells are present
e. Drug-induced disorders of DNA synthesis
o Laboratory Findings %
SERUM FERR BM
 Red cells are macrocytic – MCV >95 fl. and DISORDER
IRON
TIBC SATURA-
ITIN IRON
often as high as 120 - 140 fl. TION
 Macrocytes are typically oval shape. Iron
 Reticulocyte count is low in relation to the Deficiency  
degree of anemia. Anemia
 Total white cell count and platelet counts may 
Anemia of
be moderately reduced, especially in severely Normal Normal to
Chronic Normal to 
anemic patients. to ↓ 
Disorder
o Peripheral Smears Sideroblastic
 Show normal RBC in morphology despite the   
Anemia
drop of Hemoglobin, Hematocrit and RBC count. Normal Normal
  bone marrow activity ( of Reticulocyte Thalassemia Normal to 
to  to 
count), without increased red cell or hemoglobin
breakdown
2. Non-megaloblastic anemia - membrane changes due to ANEMIA ACCORDING TO CAUSE
disruption of the cholesterol-to-phospholipid ratio
o Rare for MCV to be >115 fL I. IMPAIRED RBC PRODUCTION ANEMIA
o Shows macrocytosis and normoblastic rather than 1. IRON DEFICIENCY ANEMIA (IDA)
megaloblastic
o Causes:  Most common form of anemia
a. Accelerated erythropoiesis – after accident  Microcytic/hypochromic anemia
b. Increased membrane surface area
c. Obscure causes (hypoplastic & aplastic
DECREASED INCREASED
anemias)
Ferritin
d. Alcohol
Hemoglobin/Hematocrit
e. Liver disease RDW
RBC Indices
f. Cytotoxic drugs Reticulocyte Count
TIBC

MICROCYTIC HYPOCHROMIC ANEMIA Serum Iron

 MCV is <80 fl
2. ANEMIA OF CHRONIC DISEASE (ACD)
 Decreased size and hemoglobin content
 Causes:  Inability to use available normal RBC
a. Iron deficiency
 Impaired release of storage iron associated with 
b. Disorder of globin synthesis as in thalassemia
c. Disorders or porphyrin & heme synthesis as in HEPCIDIN levels
sideroblastic anemia  Normocytic/normochromic anemia, or slightly
d. Other disorders of iron metabolism microcytic/hypochromic anemia
 Peripheral smears show:  Associated with persistent infections, chronic
o anisocytosis and poikilocytosis inflammatory disorders
o the red blood cells are microcytic since many are
smaller than the nucleus of the lymphocyte DECREASED INCREASED
o the erythrocytes are hypochromic with an increased Serum iron ESR
central pallor TIBC Ferritin
o elliptocytic and pencil shaped forms are present

NORMOCYTIC NORMOCHROMIC ANEMIA 3. SIDEROBLASTIC ANEMIA

 MCV is 80 - 90 fl.  Caused by blocks on the protoporphyrin pathway
  reticulocyte count resulting in defective hemoglobin synthesis and iron
 Causes: overload
a. Recent blood loss – very acutely with hypovolemia,
 Excess iron accumulates in the mitochondrial region of
may have normal blood count, will become anemic
with volume replenishment immature erythrocytes (ringed sideroblasts)
b. Overexpansion of plasma volume as in pregnancy  2 RBC populations (dimorphic) are seen
 Microcytic/hypochromic anemia
 POST HEMORRHAGIC ANEMIA
DECREASED INCREASED
Ferritin ACUTE POSTHEMORRHAGIC ANEMIA
TIBC
Serum Iron  if blood is lost over a short period of time in amount
sufficient to cause anemia.

4. THALASSEMIA LABORATORY FINDINGS

 Abrupt  of Hgb, Hct and RBC count
 is an inherited blood disorder that causes your body to   bone marrow activity, without increase red cell or
have less hemoglobin than normal. hemoglobin breakdown.
5. LEAD POISONING PERIPHERAL BLOOD SMEAR:
 Shows normal red cells
 Multiple blocks in the protoporphyrin pathway   reticulocyte count
 Normocytic/normochromic anemia with characteristic CHRONIC POSTHEMORRHAGIC ANEMIA
course basophilic stippling  if blood is lost in small amount over an extended period
of time, both the clinical and hematologic features that
6. PORPHYRIAS characterize acute posthemorrhagic anemia are lacking
 about to menopause or problem in reproductive (tumor)
 Group of inherited disorders characterized by a block in
the protoporphyrin pathway LABORATORY FINDINGS:
 Heme precursors before the block accumulate in the  WBC count is normal or slightly 
tissues, and large amounts are excreted in urine/feces   Platelet count
 Photosensitivity, abdominal pain, CNS disorders
 Hematologic findings are insignificant PERIPHERAL BLOOD SMEAR:
 reticulocyte count may be normal or slightly increased
7. MEGALOBLASTIC ANEMIAS  red blood cells at first normochromic and normocytic and
gradually the newly formed red cells become microcytic
 Defective DNA synthesis causes abnormal nuclear and hypochromic
maturation
 RNA synthesis is normal, so the cytoplasm is not affected
 Nucleus matures slower than the cytoplasm III. ACCELERATED DESTRUCTION OF RBC
(asynchronism)  Could be inherited, infection or toxic problem
 Caused by either Vitamin B12 deficiency or folic acid  Cannot complete 120 days lifespan
deficiency  Hemolytic anemia - may be due to:
 Macrocytic/normochromic anemia I. Intrinsic hemolytic anemias - defect of red cell
itself, usually hereditary & grouped as membrane,
8. APLASTIC ANEMIA metabolic or hemoglobin defects.
II. Extrinsic hemolytic anemias - a factor outside the
 Bone marrow failure red cell & acting upon it. Almost always acquired.
  in hemoglobin/hematocrit and reticulocytes
 Normocytic/Normochromic anemia
I. INTRINSIC HEMOLYTIC ANEMIA

9. MYELOPHTHISIC (BM REPLACEMENT) ANEMIA A. Due to Membrane Disorders

 Hypoproliferative anemia caused by replacement of bone

1. HEREDITARY SPHEROCYTOSIS
marrow hematopoietic cells by malignant cells or  Aka: Congenital Hemolytic Jaundice or Anemia
 inherited as a non-sex-linked dominant trait
fibrotic tissue
 most common in North Europeans
 Normocytic/normochromic anemia
 spherocytes die prematurely
 Splenomegaly - Happens in liver
II. BLOOD LOSS ANEMIAS
LABORATORY FINDINGS:
  Osmotic Fragility Test
ACUTE BLOOD LOSS CHRONIC BLOOD LOSS
 (-) Direct Coomb’s (antiglobulin test is negative)
Gradual loss of blood
 MCV is normal;  MCHC.
Sudden loss of blood Normocytic/normochromi
resulting from trauma or c anemia (initially)  WBC, platelet counts are normal except during periods
other severe forms of injury Microcytic/hypochromic of hemolysis.
Normocytic/Normochromi (caused by gradual loss of   Total bilirubin
c anemia iron)  Reticulocytes are usually 5 - 20%

PERIPHERAL SMEARS: Blood film shows

microspherocytes.
2. PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
 AKA: Machiafava-Micheli syndrome
 chronic intravascular hemolysis
 nocturnal hemoglobinuria occurs during sleep or after
awakening
 Low pH of plasma due to red cells → Depressed  membrane abnormalities due to absence of all Rh-Hr
respiration → Retention of carbon dioxide → Acidosis antigens on the red cells.
LABORATORY FINDINGS
 (+) sucrose hemolysis test or Ham’s acidified serum test LABORATORY FINDINGS:
or sugar water test.   Reticulocyte count
  WBC count and platelet count   autohemolysis & Osmotic Fragility
 Hemosiderinuria is a feature.
PERIPHERAL SMEARS:
PERIPHERAL SMEARS:  mild, chronic normocytic normochromic hemolytic
  Reticulocyte count anemia
 Normocytic–normochromic anemia is present.  smear shows stomatocytes & spherocytes
3. HEREDITARY 8. HIGH PHOSPHATIDYLCHOLINE HEMOLYTIC
ELLIPTOCYTOSIS/OVALOCYTOSIS ANEMIA
 Dominant - associated w/ severe hemolytic anemia in  inherited
infants  represents an imbalance in the membrane
 defect involves the impaired association of spectrin phospholipids in the red cells
dimers resulting in free, unconnected dimers.  anemia may increase due to infection or under condition
of stress.
LABORATORY FINDINGS:
  Osmotic fragility test PERIPHERAL SMEARS: Causes mild anemia w/
 Autohemolysis of red cells is present. morphologically normal cells

PERIPHERAL SMEARS:
 non-hypochromic elliptocytes are abundant on blood B. Due to Metabolic Disorders
films.
 Reticulocytes and NRBC are normal in shape. 1. G6PD DEFICIENCY
4. HEREDITARY PYROPOIKILOCYTOSIS  inherited sex-linked
 complex heterogeneous disorder which is ubiquitous
 Rare, moderately severe congenital hemolytic anemia  most commonly seen in enzyme deficient hemolytic
 Inherited as recessive autosomal traits anemia
 Occurs in blacks  Type A (black, not severe), Type B (Mediterranean, risk
for severe oxidant hemolysis) & Favism (life
threatening after eating fava beans)

PERIPHERAL SMEARS:
 microcytosis, striking micropoikilocytosis &
fragmentation LABORATORY TEST:
5. HEREDITARY STOMATOCYTOSIS  Methyl violet or crystal violet stains
(HYDROCYTOSIS)  Dye Reduction test
 Ascorbate Cyanide test
 Rare congenital anemia  Fluorescent Spot test
 Inherited as recessive autosomal trait caused by   Quantitative Assay of G6PD
sodium &  potassium due to increased permeability of 2. PYRUVATE KINASE DEFICIENCY
membrane  the most common red cell enzyme deficiency involving
 10 - 30% red cells appear as mouth like the Embden-Meyerhof glycolytic pathway
 inherited disorder
LABORATORY FINDINGS:  PK converted to phosphophenolpyruvate to pyruvate
  Osmotic Fragility Test in the EMP w/ the production of ATP
 Reticulocyte count may be normal or    ATP → no ATP for Na/K pump → RBC loses its
flexibility → Spiculated
PERIPHERAL SMEARS: 10 - 30% red cells appear as  mild to moderately hemolytic anemia w/ splenomegaly
mouth like linear pallor (stomatocytes) instead of the
normal central round pale area.

6. HEREDITARY ACANTHOCYTOSIS
PERIPHERAL SMEARS:
(ABETALIPOPROTEINEMIA)
 No notable red cell abnormalities until after
 caused by absence of beta-lipoprotein splenomegaly
 associated w/ plasma lipid abnormalities  Ecchinocytes - Irregularly contracted red cells
  total lipid, cholesterol &  Crenated red cells may be prominent
phospholipids
 Autohemolysis occurs LABORATORY FINDINGS AND TESTS:
 Reticulocyte count ranges from   Reticulocyte count
normal to   Quantitative assay of PK
 Presence of mild anemia
 (+) Fluorescent test
 EDTA is used
7. RHNULL DISEASE
 inherited due to gene suppression or presence of silent
Rh gene (Xo)
 3. PYRIMIDINE-5-NUCLEOTIDASE (PN)  Lab evaluation: CBC, retic count, peripheral blood film
DEFICIENCY review, chemistry panel, iron studies, vit b12 and
 inherited; caused by an abnormality in nucleotide folate levels, free erythrocyte porphyrin
metabolism o Hypoproliferative anemia
 acquired occurs in lead poisoning & responsible for the o Most common form of anemia in the hospitalized
basophilic stippling geriatric population
o Impaired erythropoietin-dependent erythrocytosis
LABORATORY FINDINGS: – involved in the pathogenesis of this disease
 Reticulocytosis is observed
 Positive in the demonstration of decreased 1. IRON DEFICIENCY ANEMIA
Nucleosidase activities
 Affects both erythrocytes and metabolic pathways of iron-
PERIPHERAL SMEARS: There are marked dependent tissue enzymes
basophilic stipplings in red cells  Results from conditions leading to chronic gastrointestinal
4. GLUCOSE PHOSPHATE ISOMERASE blood loss, including long term use of nonsteroidal
DEFICIENCY inflammatory medications, gastritis, peptic ulcer disease,
 this causes an abnormality in anaerobic glycolysis gastropharyngeal reflux disease and angiodysplasia
causes a moderately severe anemia
2. INEFFECTIVE ERYTHROPOIESIS
LABORATORY FINDINGS:  Reticulocyte count
PERIPHERAL SMEAR: Red cells show anisocytosis and  Sideroblastic anemia – impaired heme synthesis and
poikilocytosis abnormal globin synthesis
5. TRIOSEPHOSPHATE ISOMERASE,  Megaloblastic anemia – defective DNA synthesis;
HEXOKINASE AND DIPHOSPHOGLYCERATE results in ineffective erythropoiesis (deficiency of vitamin
MUTASE DEFICIENCY B12 and folate)
 other enzyme deficiencies that involve anaerobic
glycolysis 3. VITAMIN B12 DEFICIENCY
6. GLUTATHIONE SYNTHASE, GLUTATHIONE
PEROXIDASE AND GLUTATHIONE REDUCTASE  Megaloblastic anemia in 5 -10% of elderly
DEFICIENCIES  Attributed to inadequate intestinal absorption of bound
 show hemolytic anemia vitamin B12 and pernicious anemia
 these enzymes are required in the HMP like G-6-PD,  Loss of gastric acid
hemolysis increase due to oxidant drug exposure or  bacterial growth: Helicobacter pylori
infection.
4. FOLATE DEFICIENCY

II. EXTRINSIC HEMOLYTIC ANEMIA  Develops from inadequate dietary intake

 Alcoholic elderly are more prone because alcohol
 Causes: interferes with folate absorption
a. Chemical agents – drugs and chemicals
b. Physical agents – heat, trauma 5. HEMOLYTIC ANEMIA
c. Vegetable and animal poisons
d. Infectious agents – malarial parasite, bacteria  Shortened RBC survival time
e. Presence of autoantibodies, isoantibodies or drug-  Drug induced hemolytic anemia – high doses of
related antibodies antibiotics, non steroidal inflammatory drugs, quinidine,
phenacetin, etc.
ANEMIA AND THE ELDERLY  Also results from collagen vascular disease, infections,
 Hemoglobin < 13 g/dL in males and <12g/dL in females chronic lymphocytic leukemia
 Factors contributing:  Major types:
a.  bone marrow function o Caused by immunologic mechanisms
b.  in physical activity o Due to intrinsic effects
c. Nutritional deficiencies o Resulting from extrinsic factors
d. Cardiovascular disease
e. Chronic inflammatory disorders
 Most common among elderly
a. Anemia of chronic inflammation
b. Iron deficiency anemia
c. Unexplained anemia
 Ineffective erythropoiesis
a. Vitamin b12 deficiency
b. Sideroblastic anemia
c. Thalassemia
 Hypoproliferation
a. Secondary to iron deficiency
b. Vitamin b12 or folate deficiency
c. Renal failure
d. Hypothyroidism
e. Chronic inflammation
f. Endocrine disease
 HEMOSTASIS SPECIMEN COLLECTION AND  QUALITATIVE ASSESSMENT OF PLATELET
MANAGEMENT ERRORS THAT REQUIRE SPECIMEN o Platelet count (N) - Only if patient have normal
REJECTION platelet count but have bleeding risk or history
ERROR COMMENTS  RV: 150 – 400,000/uL
 Whole-blood volume <90% of required volume o Suggestive of bleeding history → assessment of
or less than manufacturer specified minimum. platelet function test is conducted
PT and PTT are falsely prolonged.
Short draw
 Ratio of anticoagulant to blood: PLATELET FUNCTION TESTS
o 3.2% Sodium citrate = 1:9  Inaccurate especially when using medication, low
o 3.8% citrated = 1:4 fibrinogen and non-functional platelets
 Each specimen is inspected visually before
Specimen centrifugation or during analysis; even a small OBSOLETE BLEEDING TIME TEST
clot clot interferes with hemostasis  Reflects the platelet function
 Fibrinogen is already used  An infrequently performed in vivo measurement of platelet
 Pink or red plasma indicates in vitro activation adhesion and aggregation on locally injured vascular
of platelets and coagulation; unpredictable subendothelium
Visible
hemostasis test interference. Further, hemolysis
hemolysis  Provides an estimate of the integrity of the platelet plug
interferes with optical endpoint coagulometer
results.  Measures the interaction between the capillaries and
 Optical instruments may fail to measure clots in platelets
cloudy or highly colored specimens.  Performed bedside as requirement for pre-surgical
 Interfere in turbidimetry or impendence procedure
Lipemia or
 Interferes with chromogenic substrate
icterus
methods. DUKE’S METHOD
 The practitioner must employ an electro-
mechanical detection method instrument.  PROCEDURE:
 Blood stasis activates endothelial cells and  1) Phlebotomist’s uses a lancet to make a SMALL,
Tourniquet CONTROLLED puncture wound (earlobe or
the concentration of von Willebrand factor and
application fingertip).
fibrinogen, falsely shortening clot-based test
>1 minute  Side of ring finger (walang kalyo and not often
results.
 Storage at refrigerator temperatures causes used)
Specimen precipitation of large von Willebrand factor  Do not wipe the 1st drop
storage at multimers, activation of coagulation factor VII, 2) Puncture site is blotted with filter paper every 30
1–6° C activation of platelets, and destruction of seconds until the bleeding stopped.
platelet integrity.  Wag pigain
Specimen  Storage at above standard room temperature 3) The time where the bleeding stops is the one that will
storage at causes coagulation factors V and VIII to be recorded
>25° C deteriorate.  No standardization of puncture site leading to inaccuracy.
If shallow = shortened, if deep = prolonged
HEMOSTASIS SPECIMEN STORAGE TIMES AND  Reference Interval: 2 – 9 minutes (universally accepted)
TEMPERATURES
SPECIMEN
APPLICATION TEMP
STABILITY
Test within 24 hour,
maintain upright and
sealed
PT with no UFH
Plasma should be
separated
PTT with no UFH Test within 4 hour,
Factor assays maintain upright and STANDARDIZED/MODIFIED IVY METHOD
15C – 25C
sealed
Whole-blood (can be  A blood pressure cuff is inflated to 40 mmHg
aggregometry room
no need to centrifuge  A calibrated spring-loaded lancet was triggered on the
temperature)
PTT for monitoring volar surface of the forearm a few inches distal to the
Centrifuge to separate
UFH antecubital crease
plasma within 1 hour,
PT when UFH is o 3 mm deep; use 2 puncture site
test within 4 hour
present  A puncture site is blotted with filter paper every 30
Wait 30 mins after seconds until the bleeding stopped
Optical platelet
centrifugation, test
aggregometry using  Reference interval: 2 – 9 minutes
within 4 hour of
PRP
collection  Prone to hematoma
Storage in household
freezer
2 weeks (separated) -20C CLOT RETRACTION
Storage for 6 months 6 months or indefinite -70C  Degree of clot retraction is directly proportional to the
PRP, Platelet-rich plasma; PT, prothrombin time; PTT, partial number of platelets and inversely proportional to the
thromboplastin time, UFH, hematocrit and the level of fibrinogen.
unfractionated heparin.
 Use 3 mL and red top
*perform coagulation studies before CBC
 Obsolete Test
 Evaluates how well platelets keep the clot adhered to the PLATELET AGGREGOMETRY USING PRP
sides of specimen tube
 Examine clot at 1, 2, 4 and 24 hours for clot retraction  Using a specialized photometer: Light-Transmittance
o If there’s problem – no clotting after 24 hrs Aggregometer
 By 30 minutes: clot starts to shrink  Sodium citrate-anticoagulated blood – Centrifuged to
 After clot forms, remaining 40 - 60% consists of serum get the PRP
and RBC "fall - out" from clot o Requires 9 – 12 mL of WHOLE BLOOD: to produce a
 MUST HAVE normal fibrinogen and hematocrit for test sufficient PRP
be accurate o PRP: plasma with platelet count of 200,000-
300,000/μL
CAPILLARY FRAGILITY TEST  Specimen must be test within 4 HOURS of collection to
avoid IN VITRO platelet activation & loss of normal
 a.k.a Rumpel–Leede Capillary Fragility Test Or activity.
Tourniquet Test  If  aggregation =  transmittance (platelet quality is
 The test is defined by the WHO as one of the necessary directly proportional to the transmittance)
requisites for diagnosis of dengue fever as screening test
 A blood pressure cuff is applied and inflated to a point WHOLE-BLOOD PLATELET AGGREGOMETRY
between the systolic and diastolic blood pressures for 5
minutes  Measured by ELECTRICAL IMPEDANCE
 Petechiae: <3 mm diameter   impedance is DIRECTLY PROPORTIONAL to platelet
 Purpura: 3 mm – 1 cm aggregation.
 Ecchymosis: >1 cm o Uses a rag. When aggregating agents are added, the
 POSITIVE: 10 or more petechiae per square inch platelets will aggregate on the sides of the rag →
 DENGUE HEMORRHAGIC FEVER: 20 or more impeding the light
petechiae per 1 square inch  Specimen is collected in 3.2% sodium citrate & held at
 Does not have high specificity – you can have petechiae 15C to 25C until testing.
from tourniquet alone  Aggregometry should be started immediately and must be
completed within 4 hours of specimen collection (for
 Tourniquet Test (CDC Procedure)
undiminished ex vivo platelet activity).
1) Take the patient's blood pressure and record it, for
example, 100/70.  Most specimens for whole blood aggregometry are mixed
2) Inflate the cuff to a point midway between SBP and 1:1 with normal saline before testing
DBP and maintain for 5 minutes. (100 + 70) ÷ 2 = 85 o platelet count is < 100,000/mL the specimen is tested
mm Hg undiluted
3) Reduce and wait 2 minutes.
PLATELET LUMIAGGREGOMETRY
4) Count petechiae below antecubital fossa.
 Do not count near the edges of tourniquet  Diagnosis of platelet dysfunction
5) A positive test is 10 or more petechiae per 1 square
 Simultaneous measurement of platelet aggregation &
inch.
secretion of ATP from activated platelet dense
EVALUATION OF PRIMARY HEMOSTASIS granules.
PLATELET ADHESION  Same lang, transmittance of light
 Adherence of platelets to glass surfaces
PLATELET AGONISTS IN AGGREGPMETRY
 Counting the number of platelets BEFORE and AFTER
exposure to glass beads.  Agonists used to activate platelets:
 Not a reliable method. a. Thrombin or Synthetic TRAP (Thrombin-Receptor-
Activating Peptide)
1. SALZMANN METHOD b. ADP
o Platelet adhesiveness to glass beads (diagnosis of c. Epinephrine
von Willebrand’s Disease) – but NOT RELIABLE d. Collagen
o Platelet count before and after exposure to beads e. Arachidonic Acid
o Retention of functional platelet: >25% platelet  used to check for deficiencies in the eicosanoid
adhere to the glass beads synthesis pathway.
f. Ristocetin
PLATELET AGGREGATION
 used to check for abnormalities of plasma VWF
 Based on some variations of Born Method
in VWD
 Principle: Platelet rich plasma is treated with known
 These platelet agonists are used to test for abnormalities
aggregating agents.
in specific membrane binding sites.
 Aggregated: Cloudiness or Turbidity can be measured by
spectrophotometer. (aggregation  =  light passes VON WILLEBRAND FACTOR ACTIVITY ASSA Y
through the sample) VWF ANTIGEN (VWF:Ag) IMMUNOASSAY
 Aggregating Agents: (Aggregates platelets)
a. ADP  Collagen-Binding Enzyme-Linked Immunosorbent
b. Collagen Assay (ELISA) - Introduced as an alternative procedure
c. Epinephrine for RCo Assay
d. Snake Venom  vWF-Antigen: marker of generalized endothelial
e. Thrombin dysfunction.
f. Ristocetin
 CLOT-BASED COAGULATION FACTOR VIII ASSAY 2. Capillary Tube/ Dale & Laidaws Method
o PROCEDURE:
 Measure the activity of Factor VIII 1) Make a skin puncture. Wipe off the 1st drop of
blood.
FUNCTIONAL VWF RISTOCETIN COFACTOR 2) Fill a non-heparinized capillary tube ¾ with
ASSAY (VWF:RCo) blood.
3) Start timing as soon as blood enters the tube.
 Measures vWF 4) Set the capillary tube aside in a horizontal
 Mediated agglutination of platelets in the presence of an position for 2 minutes.
antibiotic (ristocetin). 5) Break off about 1 cm of the capillary tube at 30-
 Most commonly used assay for the measurement of the second interval until fibrin thread bridges the
functional activity of vWF. broken ends of the capillary tube.
6) Record the coagulation time.
DILUTE RISTOCETIN-INDUCED PLATELET o NV: 2 - 4 minutes
AGGREGATION ASSAY (RIPA)
VENOM ACTIVATED ASSAYS
 Also called: RISTOCETIN RESPONSE CURVE REPTILASE TIME (PEFAKIT REPTILASE
 Employed to diagnose VWD subtype 2B TIME/PENTAPHARM)

 Reptilase is a thrombin-like enzyme, batroxobin, isolated

CLOT-BASED SCREENING TESTS FOR from the venom of that Bothrops atrox catalyzes the
COAGULATION DISORDERS conversion of fibrinogen to fibrin
 For secondary hemostasis
RUSSEL VIPER VENOM TEST
MACRO METHOD
 Russell viper venom (RVV) from the Daboia russelli
1. Lee-White Whole Blood for Coagulation Time Test viper, which triggers coagulation at the level of factor X,
o Used as clotting test before but obsolete now – still was once used as an alternative to the PT.
used by some doctors for pre-surgical (PT is the  The assay was named the STYPVEN TIME, but is now
proper) obsolete.
A. Single Tube Method
 PROCEDURE: PROTHROMBIN TIME (PT) TEST
1) 3 mL of whole blood in a test tube  Early coagulation test developed by Dr. A.J. Quick
2) Tilt every 30 seconds.  Evaluates the function of Extrinsic & Common Pathway
3) Observed for clotting.  Major Use: Monitor Warfarin (Coumadin) Anticoagulant
4) Record time of clotting. Therapy
o Three Tube Method - modified o Vitamin K antagonist, it blocks the action of vitamin K
 PROCEDURE:  Pre-surgery coagulation screening test
1) Label 3 tubes (1, 2, 3). Place the clean dry  PRINCIPLE OF PT
tubes in a 37C water bath. o The hemostasis pathway is activated when damage
- If bedside, no need for water bath occurs to blood vessel endothelium or to tissue.
- Should be vertical upright and in a flat o The extrinsic pathway is activated by tissue
surface thromboplastin (factor III) in the presence of
2) Obtain a 3 mL venous blood (syringe). calcium ions.
3) Place 1 mL of blood into each tube starting o Factor X, a proenzyme, is converted to the enzyme
with TUBE 1. Xa, which in turn converts prothrombin to the enzyme
4) Start TIMING as the blood is delivered into thrombin.
TUBE 1.  Prothrombin (II) is produced in liver
5) Gently tilt TUBE 3 every 30 seconds until o Thrombin then acts on fibrinogen to form fibrin
blood solidifies. Handle TUBE 2 in the same monomers that make up the initial clot.
way.  PT Reagent
6) Finally, TILT TUBE 2 until blood forms a
o Contains a commercial preparation of tissue
SOLID CLOT.
thromboplastin and calcium chloride, substitutes
7) STOP timing and record the coagulation
for the tissue thromboplastin and calcium ions (Ca++)
time.
that are coagulation activators of the extrinsic
 NV: 7 - 15 minutes
pathway.
MICRO METHOD Anticoagulated (N) Plasma + PT Reagent → Fibrin Clot
1. DROPP OR SLIDE METHOD  PROCEDURE: Using pre-warmed citrated plasma, add
o PROCEDURE: PT reagent. Observe for the formation of clot (gel-like).
1) Perform a skin puncture. Discard the 1st drop of  Deficiency in Extrinsic or Common Pathway =  PT
blood.  Reference Value:
2) Place a drop of blood on a clean glass slide. o 3.2% Citrated Plasma: 10 - 13 seconds
Start timing.
3) Draw the tip of the lancet across the drop of
blood at 30-second interval until fibrin threads
cling to the tip.
4) Stop timing and record the coagulation time.
 International Normalized Ratio
o NV: 2 - 4 minutes
 o Calculated value that allows standardization of  The assay is preceded by a thrombin time to detect
prothrombin time reporting among different therapeutic heparin or a direct thrombin inhibitor.
laboratories.  PROCEDURE
1) Measure APTT or PT
2) Mix an equal volume of the patient’s plasma and
normal pooled plasma (NPP)
3) Measure APTT
4) Incubate at 37C for 2 hours. Then measure APTT
o International Sensitivity Index (ISI) - Assigned by again
the manufacturer to each reagent lot is used with the  If the APTT after mixing and incubation is corrected =
prothrombin time to calculate the INR the 1st APTT is prolonged (there’s factor deficiency =
o Reference Value: INR: 1.0 – 1.4 substitution studies)

ACTIVATED PARTIAL THROMBOPLASTIN TIME

(APTT) TEST
 Is a coagulation test used to monitor low-dose heparin
therapy and to screen for function of the intrinsic and
common pathways of hemostasis
 Partial thromboplastin - Lipid portion of tissue
thromboplastin, is the reagent used in performing the
INHIBITOR OR PTT / APTT PTT / APTT
APTT.
DEFICIENCY AFTER MIXING AFTER INCU.
 Partial Thromboplastin Reagent Factor Deficiency C
o Manufactured from rabbit or bovine brain tissue as C
FVIII Inhibitor
well as from vegetable sources such as soybeans. FV (Leiden Inhibitor) NC
o Performs the function of platelet factor 3 (PF3) in NC
Lupus Anticoagulant
the APTT test *FVIII deficiency is the most common cause of prolonged APTT
o Contains activators such as: kaolin or silica to
activate the contact factors in the intrinsic pathway SUBSTITUTION STUDIES
o Calcium chloride (CaCl2): 2nd reagent used in the  Substitution studies and factor assays can be used to
test and is added to supply the ionized calcium identify specific coagulation factor deficiencies.
required to activate prothrombin in the common  PRINCIPLE OF SUBSTITUTION STUDY
pathway. o Substitution studies may be performed using
 Performed by combining citrated patient plasma with 2 adsorbed plasma and aged serum with the AP to
reagents: partial thromboplastin and calcium chloride identify deficiencies of blood coagulation.
(CaCl2) o Substitution studies may also be performed using
o Add reagent 1 in citrated plasma. Incubate. Add adsorbed plasma with the PT to identify a factor
reagent 2 then start timing until gel-like clot. VII deficiency.
o Pre-warmed patient plasma is combined with partial
thromboplastin and allowed to react → activation of WHEN DOING MIXING/SUBSTITUTION TEST
factors in the intrinsic pathway begins (activation that ABSENT PRESENT
would be initiated by PF3 in vivo) Fresh Plasma All Coagulation Factors
o CaCl2 → added to plasma-partial thromboplastin Aged Plasma V, VIII I, II, VII, IX, X, XI, XII
mixture to allow the reaction necessary for fibrin clot Factor V & VIII – Labile;  upon storage
formation. Adsorbed
 Reference Value: 20 - 35 Seconds II, VII, IX, X I, V, VIII, XI, XII, XIII
Plasma
Fresh Serum I, V, VIII, XIII II, VII, IX, X, XI, XII

THROMBIN TIME (TT) TEST Factor II - <20% present in fresh serum / 80% are consumed
during coagulation
 PRINCIPLE OF TT: The thrombin time test determines
Other Coagulation Factors – entirely consumed during
the rate of thrombin-induce cleavage of fibrinogen to fibrin
coagulation
monomers and the subsequent polymerization of
hydrogen-bonded fibrin polymers. Aged Serum I, II, V, VIII, XIII VII, IX, X, XI, XII
 Used to measure the availability of functional fibrinogen Factor II – entirely consumed during the coagulation process
 PPP + Thrombin reagent = clot *LV = factor V is the Labile factor
 Prolonged in cases of:
o Low levels of fibrinogen
o Non-functional fibrinogen
o Presence of heparin, fibrin/fibrinogen products,
thrombolytic agents (streptokinase)

MIXING STUDIES
 AKA: Circulating Anticoagulant Screen, Screening for
Circulating Inhibitor
 PRINCIPLE OF MIXING STUDY
o PTT mixing study is indicated when the PTT is
prolonged in the absence of heparin therapy.
 Fresh /
Aged Aged Adsorbed
Factor PT APTT TT Normal
Plasma Serum Plasma
Plasma
I A N C NC C
II A A C NC
V A A C NC C
VII A N A C NC
VIII N A A C NC C
IX N A A C NC
X A A C NC
XI N A A C
XII N A A C
*to memorize easily: yung fresh plasma, aged plasma, etc. yung reagent mo. Memorize mo yung absent na factors per reagent sa 1st table. Ex. FII
is not present sa aged serum + adsorbed plasma = Not Corrected.

 Plasma level of plasminogen are

quantified using a chromogenic
Corrected: Present in the reagent substrate method
Not Corrected: Not present in the reagent  Plasminogen is activated to Plasmin
APTT: Measure intrinsic and common pathway by: reagent Streptokinase or UPA
PT: measure extrinsic and common pathway (in vivo)
APTT – prolonged  Activated Plasmin is measure by
PT – prolonged substrate para-nitroaniline (R-pNA)
Problem: Common pathway (X, V, II, I, XIII) → producing a YELLOW COLOR.
Find the Common Factor:  Chromogenic Plasminogen Assay:
o Fresh Plasma (C) – (X, V, II, I) semi-functional assessment of
o Adsorbed Plasma (NC) – (II, VII, IX, X) plasminogen activity.
o Aged Serum (C) – (VII, IX, X, XI, XII)  R-pNA designates a chromogenic
APTT – prolonged PLASMINOGEN
ASSAY substrate
PT – normal o R indicates a peptide sequence
Problem: Intrinsic pathway (XII, XI, IX, VIII) (Val Leu-Lys) specific to the
Find the Common Factor: enzyme being measured
o Fresh Plasma (C) – XII, XI, IX, VIII  Plasmin recognizes the Val-
o Adsorbed Plasma (NC) – IX, X Leu-Lys amide sequence as
o Aged Serum (C) – IX, XI, XII its enzymatic cleavage site,
APTT – normal releasing the pNA, which
PT – prolonged generates a yellow color.
Problem: Extrinsic pathway (VII) o pNA is the chromophore
*wag na isama yung factors na naman involve sa pathway like if
intrinsic, wag mo na ilist yung FVII pababa Plasminogen + Streptokinase (U-PA) →
*if NC = i-list na agad yung absent factors, if C = lagay yung present
Plasmin + (R-pNA) substrate →
YELLOW COLOR
LABORATORY DIAGNOSIS OF FIBRINOLYSIS  Plasma level of TPA are quantified
Fibrinolysis generates: FDPs and D- using a chromogenic substrate
D-DIMER dimer at concentrations >200 ng/mL. method
IMMUNOASSAY  To assay, plasma that contains TPA
 FDP & D-Dimer:
– product of a. Acute/Chronic DIC
is added to plasminogen to produce
stabilized fibrin plasmin
b. Systemic Fibrinolysis
degradation
c. Deep Vein Thrombosis (DVT)  Plasmin activity is measured using
d. Pulmonary Embolism the same chromogenic substrate in
TISSUE the plasminogen assay
Principle of Quantitative D-Dimer Assay: PLASMINOGEN  Plasminogen is activated to Plasmin
ACTIVATOR by: TPA
Microlatex particles in buffered saline are coated with ASSAY  Activated Plasmin is measure by
monoclonal anti-D-dimer antibodies. The coated substrate para-nitroaniline (R-pNA)
particles are agglutinated by patient plasma D-dimer; the → producing a YELLOW COLOR.
resultant turbidity is measured by turbidimetric or
 Chromogenic Plasminogen Assay:
nephelometric technology.
semi-functional assessment of
FDPs may be detected using a plasminogen activity.
FDP qualitative visible agglutination
IMMUNOASSAY immunoassay that detects FDPs in Plasminogen + TPA → Plasmin + (R-
serum and urine. pNA) substrate → YELLOW COLOR
*UPA = Urokinase Plasminogen Activator)
Thrombo-Wellcotest:
* TPA = Tissue Plasminogen Activator
A slide agglutination method in which the practitioner mixes
1 drop of sample with 1 drop of latex suspension. The
polystyrene latex particles in buffered saline are coated
with polyclonal antibodies specific for D and E fragments
calibrated to detect FDPs at a concentration of 2 mg/mL or
greater by the appearance of visible agglutination.
 ANEMIA INEFFECTIVE AND INSUFFICIENT ERY.
 anemia - from anaimia, meaning “without blood.”  Erythropoiesis - marrow erythroid proliferative activity.
 manifestation of an underlying condition or deficiency. o occurs in bone marrow under the control of erythropoietin
 Anemia - reduction in the hemoglobin content of blood caused by and other growth factors and cytokines
decrease in the RBC count, Hgb concentration, and hematocrit  Erythropoiesis is effective - bone marrow is able to produce
functional RBCs that replace the daily loss of RBCs.
HISTORY AND CLINICAL FINDINGS  Ineffective erythropoiesis - production of erythroid precursor
 History and physical examination - important components for cells that are defective.
clinical diagnosis of anemia. o often undergo apoptosis in the bone marrow before they
o decrease in oxygen = decreases the energy available to have a chance to mature to the reticulocyte stage and be
individuals to perform day-to-day activities. released into the peripheral circulation.
 iron deficiency can lead to pica - cravings for unusual o Ex: Megaloblastic anemia, thalassemia (deficient globin
substances such as ice (pagophagia), cornstarch, or clay. chain synthesis), and sideroblastic anemia (deficient
 may be asymptomatic seen in mild or slowly progressive protoporphyrin synthesis) –
anemias  blood Hgb concentration is low → triggers an increase in
 evaluated closely during the physical examination - skin (for erythropoietin production → erythropoietic activity
petechiae), eyes (for pallor, jaundice, and hemorrhage), and  Insufficient erythropoiesis - decrease in the number of
mouth (for mucosal bleeding) erythroid precursors in the bone marrow, resulting in decreased
o search for sternal tenderness, lymphadenopathy, cardiac RBC production and anemia.
murmurs or arrhythmias, splenomegaly, and hepatomegaly. o deficiency of iron (inadequate intake, malabsorption,
o Jaundice - important for the assessment of anemia, may be chronic bleeding); deficiency of erythropoietin (renal
due to increased RBC destruction = hemolytic disease); or loss of the erythroid precursors due to
 Measuring vital signs is also a crucial component autoimmune reaction (aplastic anemia, acquired pure red
o rapid fall in hemoglobin concentration have tachycardia cell aplasia) or infection (parvovirus B19).
(fast heart rate)  can also be suppressed by infiltration of leukemia cells or with
o anemia is long-standing - heart rate may be normal because nonhematopoietic cells (metastatic tumors, granulomas, or
of the body’s ability to compensate for the anemia fibrosis) = myelophthisic anemia with characteristic teardrop
 Moderate anemias (Hgb 7 - 10 g/dL) - cause pallor of RBCs.
conjunctivae and nail beds but may not produce clinical BLOOD LOSS AND HEMOLYSIS
symptoms
 Increased hemolysis → shortened RBC life span → increasing
 Severe anemias (Hgb <7 g/dL) produce tachycardia,
the risk for anemia.
hypotension, and other symptoms of volume loss
 Chronic blood loss induces iron deficiency as a cause of
anemia.
PHYSIOLOGIC ADAPTATIONS  bone marrow may be inadequate to compensate for a sudden
 trauma, blood volume decreases and hypotension develops excessive RBC loss
= decreased blood supply to the brain and heart.  intrinsic defects in the RBC membrane, enzyme systems, or
 immediate adaptation: sympathetic overdrive → increasing heart hemoglobin, or extrinsic causes such as antibody-mediated
rate, respiratory rate, and cardiac output. processes, mechanical fragmentation, or infection-related
 Severe anemia - blood is shunted to vital organ for survival destruction.
(brain, muscle, and heart)
 tissue hypoxia → increase in RBC 2,3-bisphosphoglycerate LABORATORY DIAGNOSIS OF ANEMIA
that shifts the oxygen dissociation curve to the right (decreased
oxygen affinity of hemoglobin) and results in increased oxygen CBC WITH RED BLOOD CELL INDICES
delivery to tissues  MCV - most important of these indices, a measure of the average
 significant mechanism in chronic anemias → relatively RBC volume in femtoliters (fL).
asymptomatic  Reticulocyte count - performed for every patient with anemia.
 persistent and severe anemia - strain on the heart can  RBC histogram – if healthy, distribution is Gaussian.
ultimately lead to cardiac failure. o Abnormalities include a shift in the curve to the left (smaller
 Reduced oxygen delivery to tissues caused by reduced cell population or microcytosis) or to the right (larger cell
hemoglobin concentration → increase in erythropoietin secretion population or macrocytosis).
o Erythropoietin - stimulates the erythroid precursors in the o widening of the curve - population of RBCs have different
bone marrow, which leads to the release of more RBCs into volumes causing a greater variation of RBC volume about
the circulation the mean.
 Rapid blood loss - hemoglobin and hematocrit initially o histogram complements the PBS examination
unchanged  RDW - coefficient of variation of RBC volume expressed as a
ADAPTATIONS TO ANEMIA percentage.
Response to Acute (Sudden) Loss of Blood o increased RDW correlates with anisocytosis (variation in
The following adaptations occur in minutes to hours: RBC diameter) on the peripheral blood film.
o  in heart rate, respiratory rate, and cardiac output, which
increases the flow of oxygenated blood RETICULOCYTE COUNT
o Redistribution of blood flow from skin to essential organs
(brain, heart, muscles)  assess the bone marrow’s ability to increase RBC production in
response to an anemia.
Response to Slowly Developing Anemia  still contain residual RNA to complete the production of
The following adaptations occur over days to weeks: hemoglobin.
o  in erythrocyte 2,3-bisphosphoglycerate which decreases  Normally circulate peripherally for only 1 day while completing
hemoglobin’s affinity for oxygen and allows more oxygen their development.
release to tissues  adult reference interval: 0.5% to 2.5%
o  in erythropoietin production by kidneys, which increases  newborn reference interval: 1.5% to 6.0%
erythropoiesis and promotes release of more red blood cells  absolute reticulocyte count: 20 - 115 x 109/L
into circulation  Severe anemia - producing  numbers of reticulocytes
 2 successive corrections to obtain a better representation of RBC
MECHANISMS OF ANEMIA production - obtain a corrected reticulocyte count
 RBC production requires several nutritional factors such as iron, o corrects for the degree of anemia (reticulocyte percentage
vitamin B12, and folate. x hematocrit/45).
 Globin (polypeptide chain) synthesis must also function normally. o released prematurely and remain in the circulation 2 to 3
days - corrected reticulocyte count/maturation time to
 excessive bleeding or hemolysis - bone marrow must increase
determine the reticulocyte production index (RPI)
RBC production to compensate
o RPI - better indication of the rate of RBC production than is
the corrected reticulocyte count.
 immature reticulocyte fraction (IRF) – fraction of immature Sideroblastic anemia
reticulocytes among the total circulating reticulocytes in Thalassemia/Hb E
automated analyzer disease and trait
o helpful in assessing early bone marrow response after Abnormal variation in RBC
Poikilocytosis Severe anemia
treatment for anemia shape
 Determine whether an anemia is due to an RBC production Extensive burns (along
defect or to premature hemolysis and shortened survival defect. with schistocytes)
o shortened RBC survival (hemolytic anemias) - increasing Small, round, dense RBC Hereditary
RBC production →  reticulocytes Spherocyte
with no central pallor spherocytosis
  reticulocyte count - hallmark of the hemolytic Immune hemolytic
anemias, anemia
o Chronic blood loss - leads to iron deficiency and a Hereditary
subsequent  reticulocyte count elliptocytosis or
o Low retic count - decreased production of normal RBCs, Elliptocyte, Elliptical (cigar-shaped) ovalocytosis
either insufficient or ineffective erythropoiesis. ovalocyte oval (egg-shaped) Iron deficiency anemia
Myelophthisic anemias
Absolute Thalassemia major
= [reticulocytes (%)/100] x RBC 20–115 x
reticulocyte Hereditary
count (x 1012/L) 109/L
count stomatocytosis
Corrected Rh-deficiency
= Reticulocytes (%) x patient’s
reticulocyte RBC with slit-like area of syndrome
HCT(%)/45 Stomatocyte
count (%) central pallor Acquired
In anemic stomatocytosis (liver
Reticulocyte
= corrected reticulocyte patients, disease, alcoholism)
production
count/maturation time RPI should Artifact
index (RPI)
be >3 Thin, dense, elongated Sickle cell anemia
Mean cell Sickle cell RBC pointed at each end; Sickle cell–b-
HCT (%) x 10/RBC count (x
volume 80–100 fL may be curved thalassemia
1012/L)
(MCV) (fL) Hexagonal crystal of
Mean cell Hb C crystal dense hemoglobin formed Hb C disease
= HGB (g/dL) x 10/RBC count (x
hemoglobin 26–32 pg within the RBC membrane
1012/L)
(MCH) (pg) Fingerlike or quartz-like
Mean cell crystal of dense
Hb SC crystal Hb SC diseas
hemoglobin 32–36 hemoglobin protruding
= HGB (g/dL) x 100/HCT (%)
concentration g/dL from the RBC membrane
(MCHC) (g/dL) RBC with hemoglobin
Hemoglobinopathies
Target cell concentrated in the center
Liver disease
PERIPHERAL BLOOD FILM EXAMINATION (codocyte) and around the periphery
Thalassemia
resembling a target
 RBC diameter, shape, color, and inclusions. Fragmented RBC due to Microangiopathic
Schistocyte
rupture in the peripheral hemolytic anemia*
 verify the results produced by automated analyzers. (schizocyte)
circulation (along with
 Normal RBCs on Wright-stained blood film - nearly uniform,
microspherocytes)
ranging from 7 to 8 mm in diameter.
Helmet cell RBC fragment in shape of Macroangiopathic
 Small or microcytic cells: <6 mm in diameter, (keratocyte) a helmet hemolytic anemia**
 large or macrocytic RBCs: >8 mm in diameter. Extensive burns
 shape abnormalities (sickle cells, spherocytes, schistocytes, RBC with membrane Hb C disease
and oval macrocytes) and RBC inclusions (malarial parasites, Folded cell
folded over Hb SC disease
basophilic stippling, and Howell-Jolly bodies) can be detected Severe liver disease
 hypersegmented neutrophils - seen in vitamin B12 or folate Small, dense RBC with
(spur cell anemia)
deficiency Acanthocyte few irregularly spaced
Neuroacanthocytosis
 blast cells and  platelets - indication of acute leukemia. (spur cell) projections of varying
(abetalipoproteinemia,
length
McLeod syndrome)
Hemolytic RBC with blunt or pointed,
Abnormal variation in RBC
Anisocytosis IDA short projections that are
volume or diameter
Megaloblastic usually evenly spaced Uremia
Bone marrow failure Burr cell
over the surface of cell; Pyruvate kinase
Chronic liver disease (echinocyte)
Large RBC (>8 mm in present in all fields of deficiency
Megaloblastic anemia blood film but in variable
Macrocyte diameter)
Myelodysplastic numbers per field
MCV >100 fL
syndrome Primary myelofibrosis
Reticulocytosis RBC with a single pointed
Teardrop cell Myelophthisic anemia
Oval extension resembling a
Large oval RBC Megaloblastic anemia (dacryocyte) Thalassemia
macrocyte teardrop or pear
Megaloblastic anemia
Small RBC (<6 mm in Anemia of chronic
Microcyte diameter), inflammation
MCV <80 fL IDA
SUPRAVITAL STAIN WRIGHT STAIN COMPOSITION
Bluish tinge throughout
Dark blue granules and Hemolytic anemia
Diffuse cytoplasm; also called
filaments in cytoplasm (seen in RNA After treatment for iron, vitamin
basophilia polychromasia (seen in
reticulocytes) B12, or folate deficiency
polychromatic erythrocytes)
Lead poisoning
Dark blue-purple, fine or
Dark blue-purple, fine or coarse Thalassemia
Basophilic coarse punctate granules Precipitated
punctate granules distributed Hemoglobinopathies
stippling distributed throughout RNA
throughout cytoplasm Megaloblastic anemia
cytoplasm
Myelodysplastic syndrome
Hyposplenism Postsplenectomy
Howell-Jolly Dark blue-purple dense, round granule; usually one per cell; DNA (nuclear Megaloblastic anemia Hemolytic
body occasionally multiple fragment) anemia Thalassemia
Myelodysplastic syndrome
 Glucose-6-phosphate
Round, dark blue-purple
Denatured dehydrogenase deficiency
Heinz body granule attached to inner RBC Not visible
hemoglobin Unstable hemoglobins Oxidant
membrane
drugs/chemicals
Sideroblastic anemia
Hemoglobinopathies
Pappenheimer Irregular clusters of small, light to dark blue granules, often near
Iron Thalassemias Megaloblastic
bodies periphery of cell
anemia Myelodysplastic syndrome
Hyposplenism Post-splenectomy
Remnant of Megaloblastic anemia
Cabot ring Rings or figure-eights
mitotic spindle Myelodysplastic syndromes

Fine, evenly dispersed, dark Precipitate of b-

Hb H blue granules; imparts “golf Not visible globin chains Hb H disease
ball” appearance to RBCs of hemoglobin

protoporphyrin synthesis (sideroblastic anemia, lead

BONE MARROW EXAMINATION poisoning).
o Globin chain synthesis - insufficient or defective in
thalassemia and in Hb E disease.
 patient with an unexplained anemia associated with or without
o Iron deficiency - most common cause of microcytic anemia
other cytopenias, fever of unknown origin, or suspected
o manifested only by reduced iron stores.
hematologic neoplasm.
2. Macrocytic anemias
 evaluates hematopoiesis and can determine whether there is an
o MCV >100 fL with large RBCs (>8 mm in diameter).
infiltration of abnormal cells into the bone marrow
o result in megaloblastic or nonmegaloblastic red cell
 include
development in bone marrow.
o abnormal cellularity (e.g., hypocellularity in aplastic
o Megaloblastic anemias - impair synthesis of DNA, such as
anemia) vitamin B12 and folate deficiency or myelodysplasia.
o evidence of ineffective erythropoiesis and megaloblastic
 All cells of the body are affected
changes (folate/vitamin B12 deficiency or myelodysplastic
 Pernicious anemia - one cause of vitamin B12
syndromes)
deficiency
o lack of iron on iron stains of bone marrow (the gold
 pregnancy - leading cause of folate deficiency.
standard for diagnosis of iron deficiency)
 oval macrocytes and hypersegmented neutrophils
o presence of granulomata, fibrosis, infectious agents,
and megaloblasts in the bone marrow.
and tumor cells
 MCV up to 150 fL
 Other tests - immunophenotyping of membrane antigens by flow o Nonmegaloblastic forms - typically related to membrane
cytometry, cytogenetic studies, and molecular analysis to changes caused by disruption of the cholesterol-to-
detect specific genetic mutations and chromosome abnormalities phospholipid ratio.
in leukemia cells  mostly round, and the erythroid precursors do not
OTHER LABORATORY TESTS display megaloblastic changes.
 seen in patients with chronic liver disease, alcohol
 Routine urinalysis (detect hemoglobinuria or an increase in abuse, and bone marrow failure.
urobilinogen) with a microscopic examination (to detect  rare for the MCV to be >115 fL in nonmegaloblastic
hematuria or hemosiderin) and analysis of stool (to detect anemias.
occult blood or intestinal parasites). 3. Normocytic anemias
 Chemistry studies - serum haptoglobin, lactate dehydrogenase, o MCV of 80 - 100 fL.
and unconjugated bilirubin (to detect excessive hemolysis) and o result of the premature destruction and shortened survival of
renal and hepatic function tests. RBCs (hemolytic anemias), and an  reticulocyte count.
 Patients having undergone gastric bypass surgery for obesity o hemolytic anemias divided into
- insufficient copper is more common that can cause anemia. a. intrinsic causes (membrane defects,
 Iron studies (including serum iron, total iron-binding capacity, hemoglobinopathies, and enzyme deficiencies)
transferrin saturation, and serum ferritin) - low reticulocyte count b. extrinsic causes (immune and nonimmune RBC
and a microcytic anemia. injury).
 Serum vitamin B12 and serum folate assays – for macrocytic o Direct antiglobulin test - differentiate immune-mediated
anemia with a  reticulocyte count RBC destruction from other causes of hemolysis.
 direct antiglobulin test - differentiate autoimmune hemolytic o Other normocytic anemias result of a decreased production
anemias from other hemolytic anemias. of RBCs →  reticulocyte count

APPROACH TO EVALUATING ANEMIAS CLASSIFICATION OF ANEMIAS AND THE RETIC.

 begins with taking a complete history and performing a physical
examination  Absolute reticulocyte - classifying anemias into decreased or
 strict vegetarian - not be getting enough vitamin B12, with ineffective RBC production ( reticulocyte count) and excessive
alcoholism not be getting enough folate RBC loss ( reticulocyte count).
 large spleen - indication of hereditary spherocytosis  Reticulocyte count is decreased → MCV can further classify
 stool positive for occult blood - iron deficiency. the anemia into three subgroups: normocytic anemias, microcytic
 STEPS anemias, and macrocytic anemias.
1) accurate measurement of the hemoglobin concentration,  Excessive RBC loss category includes acute hemorrhage and
hematocrit, MCV, and RBC count - reduction of 10% or the hemolytic anemias with shortened RBC survival.
more may be the first clue
2) Reticulocyte count and a peripheral blood film examination CLASSIFICATION RBC DISTRIBUTION WIDTH

MORPHOLOGIC CLASSIFICATION (MVC)  help determine the cause of an anemia in conjunction with the
MCV.
1. Microcytic anemias  subclassified by the RDW as homogeneous (normal RDW) or
o MCV of <80 fL with small RBCs (<6 mm in diameter) heterogeneous (increased or high RDW ).
o Microcytosis is often associated with hypochromia and an  Decreased MCV with an increased RDW = iron deficiency
MCHC of <32 g/dL. PATHOPHYSIOLOGIC CLASSIFICATION
o reduced hemoglobin synthesis  grouped by the mechanism causing the anemia.
o Heme synthesis - diminished in iron deficiency, iron  Decreased RBC production → inappropriately  reticulocyte
sequestration (chronic inflammatory states), and defective counts (disorders of DNA synthesis and aplastic anemia) and
 are distinguished from other anemias caused by increased RBC o localized bleeding seldom implies a blood vessel defect.
destruction (intrinsic and extrinsic abnormalities of RBCs) or  Qualitative platelet defect -  platelet count
acute blood loss, which have  reticulocyte counts. (thrombocytopenia), or a coagulation factor deficiency cause
systemic and not localized bleeding.
 Generalized bleeding - Bleeding from multiple sites,
spontaneous and recurring bleeds, or a hemorrhage that requires
physical intervention
o disorder of primary hemostasis such as a blood vessel or
platelet defect or thrombocytopenia; or secondary
hemostasis characterized by single or multiple coagulation
factor deficiencies or uncontrolled fibrinolysis.
MUCOCUTANEOUS VS. ANATOMIC HEMORRHAGE
 Generalized bleeding may exhibit either a mucocutaneous
(skin or at body orifices) or anatomic (in soft tissue, muscles,
joints, deep tissue) pattern.
 Mucocutaneous bleeding into the skin
o petechiae, red pinpoint spots
o purpura, purple skin lesions >3 mm diameter
o ecchymoses (bruises) >1 cm, typically seen after trauma
o unprovoked presence of more than one such lesion may
indicate a disorder of primary hemostasis.
 Other symptoms of a primary hemostasis defect -
bleeding from the gums, epistaxis (uncontrolled
nosebleed), hematemesis (vomiting of blood), blood in
the urine or stool, and menorrhagia (profuse
menstrual flow).
 nosebleeds are common - primary hemostatic defect
when occur repeatedly, last longer than 10 minutes,
suggest a involve both nostrils
o Mucocutaneous hemorrhage - most likely to be
associated with thrombocytopenia (platelet count
<50,000/mL), qualitative platelet disorders, VWD, or
vascular disorders such as scurvy or telangiectasia.
 patient history and physical examination may distinguish between
mucocutaneous and anatomic bleeding
 Anatomic (soft tissue) hemorrhage - seen in acquired or
congenital defects in secondary hemostasis such as plasma
coagulation factor deficiencies (coagulopathies).
o recurrent or excessive bleeding after minor trauma
MCV o bleeds into joints, body cavities, muscles, or the central
DECREASED NORMAL INCREASED nervous system. Joint bleeds (hemarthroses) cause
Anemia of swelling and acute pain.
a- or b-
thalassemia
chronic o Recurrent hemarthroses - inflammation → permanent
trait
inflammation Aplastic anemia cartilage damage that immobilizes the joint → Bleeds into
RDW Anemia of
Anemia of renal Chronic liver soft tissues such as muscle or fat → nerve compression and
disease disease subsequent temporary or permanent loss of function.
Normal chronic
inflammation
Acute Alcoholism o Bleeding into the central nervous system → headaches,
hemorrhage Chemotherapy confusion, seizures, and coma
Hb E
disease/trait
Hereditary o Bleeds into the kidney → hematuria and may be
spherocytosis associated with acute renal failure.

 CBC + platelet count, PT, PTT, TT, and fibrinogen assay

Early iron, folate,  Some add thromboelastography - performed using the
or vitamin B12 Folate or vitamin thromboelastograph (TEG) or thromboelastometry (TEM)
deficiency B12 deficiency using the rotational thromboelastometer (ROTEM)
Mixed deficiency Myelodysplastic o TEG and TEM are coagulometers that measure whole blood
Iron deficiency of iron 1 vitamin syndrome clotting, a process called global hemostasis
RDW
Sickle cell–b- B12 or folate Cold agglutinin o Both report clot onset dynamics, clot strength, and
Increased
thalassemia Sickle cell disease fibrinolysis in 15 to 30 minutes.
anemia Chronic liver o require interpretation by experienced laboratory
Hb SC disease disease practitioners.
Myelodysplastic Chemotherapy
syndrome GENERALIZED BLEEDING SIGNS
Purpura—recurrent, chronic bruising in multiple locations; called
petechiae when ,3 mm, purpura when 3 mm to 1 cm, and
ecchymoses when .1 cm in diameter
HEMORRHAGIC DISORDERS Epistaxis—nosebleeds that are recurrent, bleed from both nostrils,
last longer than 10 minutes, or require physical intervention
BLEEDING SYMPTOMS Recurrent or excessive bleeding from trauma, surgery, or dental
 Hemorrhage - excessive bleeding that requires medical or extraction
physical intervention. Bleeding into multiple body cavities, joints, or soft tissue
o may be local or general, mucocutaneous or anatomic, Simultaneous bleeding from several sites
acquired or congenital. Menorrhagia (menstrual hemorrhage)
 Congenital - bleeding result from primary (platelet- Bleeding that is delayed or recurrent
related) or secondary (coagulation factor-related) or Bleeding that is inappropriately brisk
from unregulated fibrinolysis Bleeding for no apparent reason
 Congenital hemorrhage - provoked or spontaneous. Hematemesis (vomiting of blood)
LOCALIZED VS. GENERALIZED HEMORRHAGE
 Localized bleeding/hemorrhage - Bleeding from a single
location usually indicates injury, infection, tumor, or an isolated
blood vessel defect
o Ex: inadequately cauterized or ineffectively sutured
surgical site or an arteriovenous malformation .
 ASSAY ASSESSES TIC: MASSIVE TRANSFUSION
Hemoglobin, Anemia associated with chronic
hematocrit; bleeding or a hemolytic anemia; bone  Trauma centers publish and maintain massive transfusion
reticulocyte count marrow response protocols (MTPs) for TIC management
Platelet count Thrombocytopenia  Massive hemorrhage definition
o blood loss exceeding total blood volume within 24 hours
Prothrombin time Clotting time prolonged in deficiencies
o loss of 50% of blood volume within a 3-hour
(PT) of factors II (prothrombin), V, VII, or X o blood loss >150 mL/min, or blood loss that necessitates
Partial plasma and platelet transfusion.
Clotting time prolonged in deficiencies
thromboplastin  MTP → otherwise healthy trauma victim whose
of all factors except VII and XIII
time (PTT) o systolic blood pressure is <90 mmHg
prolonged by unfractionated heparin o pulse is >120 beats/min
therapy, dysfibrinogenemia, o pH is <7.25
Thrombin time (TT) o hematocrit is <32%
hypofibrinogenemia, and
o hemoglobin is <10 g/dL
afibrinogenemia; qualitative o urine output is diminished
Reduced in dysfibrinogenemia, o PT is prolonged to >1.5 times or generates an INR of 1.5 or
Fibrinogen assay
hypofibrinogenemia, and greater.
(FG)
afibrinogenemia; quantitative result  assessment of blood consumption (ABC) score assesses 1
point each for up to four nonlaboratory parameters - Any 2 of the
ACQUIRED VS CONGENITAL BLEEDING DIS. 4 parameters activates the MTP.
 begin after childhood - some disease or physical trauma, and o penetrating mechanism
are not duplicated in relatives, they are probably acquired o positive focused assessment sonography for trauma (FAST)
o adult patient seeks treatment of generalized o arrival systolic blood pressure of 90 mm Hg or less,
hemorrhage - first looks for an underlying condition, o arrival heart rate of ≥120 beats/min.
disease, drug effect, or event and records a personal and  unmatched thawed group AB or group A plasma - warmed
family history and administered to the victim en route or immediately on
 patient history - age; sex; current or past pregnancy; a hospital arrival.
systemic disorder such as diabetes or cancer; trauma;  continue administering equal amounts (1:1:1) of warmed RBCs,
and exposure to drugs, including prescription drugs, plasma, and random donor platelet concentrate
over-the-counter nutritional supplements, alcohol o RBCs - not exceed the volumes of the other components.
abuse, and drugs of abuse. o most instances, clinicians administer pheresis platelet
o physician determines the trigger, location, and volume of concentrate - equivalent of 4 to 6 random platelet
bleeding and then orders initial hemostasis laboratory concentrate preparations practical component ratio is
assays actually 6:6:1, with 1 representing pheresis platelet
 Congenital hemorrhagic disorders - uncommon, usually concentrate.
diagnosed in infancy or during the first years of life.
o may be first-degree relatives with similar symptoms TIC: PLASMA
o lead to recurrent hemorrhages may be spontaneous or
may occur after minor injury or in unexpected locations,  Plasma is a key TIC management component.
o with mild congenital hemorrhagic disorders - no  separate and freeze plasma within 24 hours of collection aka FP-
symptoms until they reach adulthood or experience some 24/FFP
physical challenge, such as trauma, dental extraction, or a  FP-24 - thawed and stored at 1°C to 6°C for up to 5 days aka
surgical procedure. thawed plasma.
o most common congenital deficiencies - VWD, factor VIII  VWF and coagulation factor V and VIII - activities decline to
and factor IX deficiencies and platelet function disorders 60% after 5 days of refrigerator storage
o Inherited deficiencies of fibrinogen, prothrombin, and factors o thawed plasma require supplementation with factor
V, VII, X, XI, and XIII are rare. concentrates, especially in patients with VWD or
hemophilia.
TIC: PLATELET CONCENTRATE

 administered in equal proportion with red cells and plasma

 Some stipulates that platelet concentrates only be administered
when the platelet count is <50,000/µL
 limited and costly to manage
 platelets halt microvascular bleeding
 Platelet concentrate therapy - generally ineffective when the
patient has immune thrombocytopenia, TTP, or heparin-
induced thrombocytopenia
ACQUIRED COAGULOPATHIES o therapeutic platelets are rapidly consumed, and their
 Chronic disorders commonly associated with bleeding - liver administration is contraindicated
disease, vitamin K deficiency, and renal failure. TIC: CONCENTRATES
TRAUMA-INDUCED COAGULOPATHY
 unintentional injury - leading cause of death among those aged  to reduce the risk of transfusion-associated circulatory
1 to 45 years. overload (TACO) and transfusion-related acute lung injury
 accounts for most instances of fatal hemorrhage (TRALI), improve patient outcomes, and conserve resources -
 Coagulopathy - any single or multiple coagulation factor or employ concentrates to augment or even replace component
platelet deficiency administration in TIC.
 triggered by the combination of injury-related acute 1. ADAMTS13 concentrate - early TIC intervention and may
inflammation, hypothermia, acidosis, and hypoperfusion reduce the need for MTP
(poor distribution of blood to tissues) → systemic shock → 2. Activated prothrombin complex concentrate (APCC)
acute reduction of ADAMTS13 (aka VWF cleaving protease) o used at a dosage of 50 units/kg every 12 hours, not to
with rise in ultra-large VWF multimers and VWF-triggered platelet exceed 200 units/kg in 24 hours.
activation. o Overdose → risk disseminated intravascular coagulation
o Shock → tissue factor release, coagulation factor activation, (DIC)
loss of coagulation control proteins, and hyperfibrinolysis. 3. Nonactivated prothrombin complex concentrates (PCCs)
o resembles the pathophysiology of thrombotic such as four-factor concentrate Kcentra
thrombocytopenic purpura (TTP), as well as conditions o safer
generated by major surgery, ruptured aortic aneurysm, o to treat hemorrhage in Coumadin overdose, but its use in
gastrointestinal bleeding, esophageal varices, and TIC is off-label
postpartum hemorrhage → massive transfusion required.
 o ADAMTS13 concentrate and PCCs - may be used in
conjunction with the antifibrinolytic lysine analog  Fibrinogen - APR that becomes elevated in early or mild liver
tranexamic acid (TXA). disease.
4. TXA - effective and commonly employed for TIC, though an off- o Moderately and severely diseased liver produces fibrinogen
label application. that is coated with excessive sialic acid, a condition called
o Postpartum hemorrhage - managed with TXA. dysfibrinogenemia
5. Cryoprecipitate  soft tissue bleeding
o when there is microvascular bleeding and the fibrinogen  prolonged TT
concentration is <100 mg/dL.  prolonged reptilase clotting time
o Postpartum hemorrhage - plunging fibrinogen levels signal o End-stage liver disease - fibrinogen level may fall to 100
the risk of major blood loss. mg/dL, a mark of liver failure
o 15 to 20 mL cryoprecipitate unit = 150 to 250 mg of  VWF and factors VIII and XIII
fibrinogen o APR, unaffected or elevated in mild to moderate liver
o risk of TACO is lower disease.
o target fibrinogen level of 100 mg/dL should be maintained o VWF - from endothelial cells and megakaryocytes and is
o some recommend 200 mg/dL in postpartum hemorrhage stored in endothelial cells and platelets.

6. Willebrand factor and FVIII concentrates - when the patient PLATELET ABNORMALITIES IN LIVER DISEASE
has a preexisting deficiency
a. Recombinant activated coagulation factor VII (rFVIIa)
 Thrombocytopenia
 For treating hemophilia A or B when anti-FVIII or FIX o Platelet counts of <150,000/mL - due to shortened platelet
inhibitors are present
survival and sequestration associated with portal
 application in the treatment of TIC is off-label
hypertension and resultant hepatosplenomegaly
 30 mg/kg - rapidly effective in halting microvascular o Alcohol toxicity suppresses platelet production
hemorrhage in nonhemophilic trauma victims
 Platelet aggregation and secretion properties are often
 does not cause DIC.
suppressed → reduced platelet aggregometry and
 possible link between off-label NovoSeven use and
lumiaggregometry results
arterial and venous thrombosis in patients with existing
o Aggregometry may be used to predict bleeding and
thrombotic risk factors.
thrombosis risk (platelets are hyperactive)
TIC: MONITORING THERAPY DISSEMINATED INTRAVASCULAR COAGULATION
1. TEG or TEM technology - monitor the effects of plasma, platelet
 Chronic or compensated DIC - significant complication of liver
concentrate, PCC, activated PCC, four-factor PCC, TXA, and
disease
rFVIIa.
o caused by decreased liver production of regulatory
2. Fibrinogen assay - Cryoprecipitate efficacy
antithrombin, protein C, or protein S
3. platelet count, PT, and PTT - monitor the effectiveness of all
o release of activated procoagulants from degenerating liver
TIC therapy indirectly
cells.
4. Platelet aggregometry
o failing liver cannot clear activated coagulation factors
o used to measure post-therapy platelet function
o  D-dimers - hallmark of unregulated coagulation and
o coagulation factor assays are valuable as follow-ups to PT
fibrinolysis.
and PTT to determine whether the target activity of 30
units/dL has been met for each.  primary or metastatic liver cancer
o TEG and TEM provide immediate feedback and may be o hepatocytes produce nonspecific procoagulant substances
more sensitive to small physiologic improvements. → trigger chronic DIC → ischemic complications.
 ADAMTS13 assays - necessary in monitoring ADAMTS13  Acute, uncompensated DIC
concentrate therapy. o PT, PTT, and TT are prolonged
o fibrinogen level is reduced to <100 mg/dL

o  D-dimers.
LIVER DISEASE COAGULOPATHY  hemostatic deficiencies - corrected temporarily by administering
 bleeding associated with liver disease may be localized or RBCs, plasma, PCC, TXA, platelet concentrates, or
generalized, mucocutaneous or anatomic. antithrombin concentrates
 Esophageal varices - Enlarged and collateral esophageal o include synthetic ATryn and plasma-derived Thrombate III
vessels; complication of chronic alcoholic cirrhosis HEMOSTASIS LABORATORY TESTS
o hemorrhaging from varices is localized bleeding, not a
coagulopathy
1. PT
 Mucocutaneous bleeding occurs in liver disease-associated
2. PTT
thrombocytopenia, accompanied by  platelet function.
3. TT
 Anatomic bleeding - consequence of procoagulant dysfunction 4. fibrinogen concentration
and deficiency. 5. platelet count
PROCOAGULANT DEFICIENCY IN LIVER DISEASE 6. D-dimer concentration
7. Factor V and VII assays
 Hepatitis, cirrhosis, obstructive jaundice, cancer, poisoning, o differentiate liver disease from vitamin K deficiency
and congenital disorders of bilirubin metabolism - suppress o decreased in liver disease
the biosynthetic function of hepatocytes, reducing either the 8. Plasminogen deficiency and  D-dimer - confirm systemic
concentrations or activities of the plasma coagulation factors to fibrinolysis
less than hemostatic levels (40 units/dL) 9. reptilase time test - may be useful to confirm dysfibrinogenemia
 Liver disease - alters the production of the vitamin K-dependent o duplicates the thrombin time
factors II, VII, IX, and X and control proteins C, S, and Z. o Bothrops venom triggers fibrin polymerization by cleaving
o produced in their des-g-carboxyl forms, which cannot fibrinopeptide A but not fibrinopeptide B from the
participate in coagulation fibrinogen molecule
 At the onset of liver disease o unaffected by standard unfractionated heparin therapy
o Factor VII (shortest plasma half-life at 6 hours) - first and can be used to assess fibrinogen function
coagulation factor to exhibit decreased activity. >400 mg/dL in early, mild liver disease
o PT - particularly sensitive to factor VII activity, prolonged in <200 mg/dL in moderate to severe liver
mild liver disease; sensitive early marker. Fibrinogen
disease, causing hypofibrinogenemia or
o Vitamin K may become deficient when the diet is limited.
o Vitamin K deficiency independent of liver disease produces
dysfibrinogenemia
a similar effect on the PT. Prolonged in dysfibrinogenemia,
 Declining coagulation factor V activity - more specific marker hypofibrinogenemia, elevation of fibrin
Thrombin time
of liver disease because factor V is non-vitamin K dependent degradation products, and therapy with
and is not affected by dietary vitamin K deficiency. unfractionated heparin
o factor V activity assay with the factor VII assay - used to
distinguish liver disease from vitamin K deficiency
 Prolonged in hypofibrinogenemia, o Desmopressin acetate - administered intravenously
significantly prolonged in (DDAVP) or intranasally →  the plasma concentration of
Reptilase time VWF high-molecular-weight multimers.
dysfibrinogenemia; not affected by
o VWF - aids platelet adhesion and aggregation.
heparin; assay rarely used o Patients with renal failure should not take aspirin,
Prolonged even in mild liver disease due clopidogrel, prasugrel, ticagrelor, or other platelet
to des-g-carboxyl factors replacing inhibitors → increase the risk of hemorrhage.
Prothrombin normal factors II (prothrombin), VII, IX, NEPHROTIC SYNDROME AND HEMORRHAGE
time (PT) and X. Report PT in seconds, not
international normalized ratio (INR), when  Nephrotic syndrome - increased glomerular permeability
testing for liver disease associated with a variety of conditions
Mildly prolonged in severe liver disease o chronic glomerulonephritis
Partial o diabetic glomerulosclerosis
due to disseminated intravascular
thromboplastin o systemic lupus erythematosus
coagulation (DIC) or des-g-carboxyl
time (PTT) o amyloidosis
factors II (prothrombin), IX, and X o renal vein thrombosis
Factor V level becomes reduced in liver  low-molecular-weight proteins → glomerulus → glomerular filtrate
disease, but is unaffected by vitamin K and the urine.
Factor V assay
deficiency so the factor V level helps  Coagulation factors II, VII, IX, X, and XII detected in the urine &
distinguish the conditions coagulation regulatory proteins antithrombin and protein C.
Mild thrombocytopenia, platelet count  loss of regulatory proteins leads to venous thrombosis
Platelet count
<150,000/mL VITAMIN K DEFICIENCY AND HEMORRHAGE
Mild suppression of platelet aggregation  Vitamin K - required for normal function of the vitamin K-
Platelet dependent coagulation factors
and secretion in response to most
aggregometry  Body stores are limited, and become exhausted when diet is
agonists
interrupted
D-dimer >240 ng/mL by quantitative assay
o Ex: fed only with parenteral (intravenous) nutrition for period
of time or when people embark upon fad diets.
HEMOSTATIC TREATMENT TO RESOLVE LIVER  fat soluble and requires bile salts for absorption
DISEASE-RELATED HEMORRHAGE  biliary duct obstruction (atresia), fat malabsorption, and
chronic diarrhea → cause vitamin K deficiency
1. Oral or intravenous vitamin K therapy  Broad-spectrum antibiotics - disrupt normal gut flora; destroy
o correct the bleeding associated with nonfunctional des-g- bacteria that produce vitamin K.
carboxyl factors II, VII, IX, and X HDN CAUSED BY VITAMIN K DEFICIENCY
o therapeutic effect of vitamin K is short lived
o severe liver disease
2. plasma transfusion - provides all of the coagulation factors in  newborns are constitutionally vitamin K deficient.
hemostatic concentrations  activity levels of factors II, VII, IX, and X are lower in normal
o VWF and factors V and VIII may be reduced newborns
o plasma is unable to return the PT to normal level (short half-  Breastfeeding prolongs the deficiency because maternal
life of factor VII) antibodies delay the establishment of gut flora.
o 1 unit of plasma = volume of 200 to 280 mL VITAMIN K ANTAGONISTS: COUMADIN
o typical adult plasma dose = 2 units
o depending on the indication and the ability of the patient’s  g-carboxylation cycle of coagulation factors - interrupted by
cardiac and renal system to rapidly excrete excess fluid. coumarin-type oral anticoagulants such as warfarin (Coumadin)
o TACO o disrupt the vitamin K epoxide reductase and vitamin K
 occur when 30 mL/kg has been administered quinone reductase reactions
 occur with smaller volumes in patients with  liver releases dysfunctional des-g-carboxyl factors II, VII, IX, and
compromised cardiac or kidney function. X and proteins C, S, and Z (proteins induced by vitamin K
3. cryoprecipitate or fibrinogen concentrate: fibrinogen level is antagonists /PIVKA factors)
<50 mg/dL  Therapeutic overdose of warfarin-containing rat poisons
o Plasma and cryoprecipitate - risk of virus transmission o moderate to severe hemorrhage because of the lack of
o allergic transfusion reactions are more common with functional K-dependent factors.
plasma-containing products. o brodifacoum or “superwarfarin,” - often used as a
4. platelet concentrates, PCC, antithrombin concentrate, rFVIIa, rodenticide, lasts for weeks to months
and TXA. o treatment - repeated administration of vitamin K with follow-
CHRONIC RENAL FAILURE AND HEMORRHAGE up PT monitoring.
 Chronic Renal failure  Inadvertent Coumadin overdose - the single most common
o associated with platelet dysfunction and mild to moderate reason for hemorrhage-associated emergency.
mucocutaneous bleeding DETECTION OF VIT-K DEFICIENCY OR PIVKA FACTORS
o Platelet adhesion and aggregation are suppressed -
guanidinosuccinic acid or phenolic compounds coat the  PT: prolonged with or without a prolonged PTT
platelets.  platelet-free normal plasma is combined with patient plasma
o RBC mass (anemia) and thrombocytopenia → “corrected” PT and PTT results
o Dialysis, RBC transfusions, or erythropoietin therapy -  Single-factor assays - detect low factor VII, then decreases in
may correct factors IX, X, and II
 Hemostasis activation syndromes  oral or intravenous vitamin K (emergency) - standard therapy
o deposit fibrin in the renal microvasculature reduce for vitamin K deficiency
glomerular function. o synthesis of coagulation factors requires at least 3 hours
o Examples: DIC, hemolytic uremic syndrome, and TTP → o severe bleeding - plasma or four-factor PCC may be
cause thrombocytopenia → mucocutaneous bleeding administered
o Fibrin is also deposited in renal transplant rejection and in o TEG or TEM - primary assays for plasma or four-factor PCC
the glomerulonephritis syndrome of systemic lupus efficacy
erythematosus. o PT/INR - patient’s recovery may be monitored indirectly
 Laboratory tests - little predictive or management value
o bleeding time test – prolonged AUTOANTI-FACTOR VIII INHIBITOR AND ACQUIRED
o Platelet aggregometry – nonpredictive
o PT and PTT – normal HEMOPHILIA
 Management of renal failure-related bleeding  Acquired autoantibodies that inhibit factors II, V, VIII, IX, and XIII
o Renal dialysis - temporarily activates platelets and improve and VWF
platelet function, when anemia is well controlled.  Autoanti-factor VIII - most common
o diagnostic of acquired hemophilia
 o older than 60 and have no apparent underlying disease.  inhibitor titers <5 Nijmegen-Bethesda units (NBU) - respond
 Acquired hemophilia - associated with RA, inflammatory bowel to administration of DDAVP or factor VIII concentrates
disease, SLE, or lymphoproliferative disease. o close monitoring of response to therapy with serial
o Pregnancy trigger acquired hemophilia 2 to 5 months after coagulation factor VIII activity assays is warranted.
delivery.  Immune tolerance therapy - reduce the inhibitor titer.
o Patients with inhibitor autoantibodies - prescribed  Plasma exchange - used in severe cases, less reliable than the
immunosuppressive therapy response to immune tolerance therapy
o Autoantibodies develop after pregnancy disappear
spontaneously. ACQUIRED VON WILLEBRAND DISEASE
o sudden and severe bleeding in soft tissues
 Acquired VWF deficiency - similar to those of congenital VWD
o bleeding in the gastrointestinal or genitourinary tract. o Autoimmune
o even when treated, remains fatal o Benign monoclonal gammopathies
o Congenital heart disease
CLOT-BASED ASSAYS TO DETECT ACQUIRED o Hypothyroidism
HEMOPHILIA o Intestinal angiodysplasia
o Lupus erythematosus
 PT, PTT, and TT - Recommended testing for onset of anatomic o Lymphoproliferative
hemorrhage that resembles acquired hemophilia. o Myeloproliferative disorders
 factor VIII inhibitor → PTT is prolonged, the PT and TT are o Pesticide exposure
likely normal. o Uremia
 Factor assay - reveal factor VIII activity to be <40 units/dL o Wilms tumor
 decreased VWF production; adsorption of VWF to abnormal cell
1. Clot-based mixing studies surfaces (lymphoproliferative disorders) and Wilms tumor; or,
o confirm the presence of the inhibitor. in <2% of specific VWF autoantibody
o PTT prolongation - corrected by the addition of NP to  moderate to severe mucocutaneous bleeding
specimen in a 1:1 ratio → but PTT becomes prolonged  recent onset of bleeding who has no hemorrhage-related medical
after incubation at 37°C for 2 hours. history.
o occurs because factor VIII autoantibodies are IgG4 isotype  PTT: prolonged → factor VIII: <40 units/dL
- time and temperature dependent. o VWF serves as the factor VIII carrier molecule.
o high-avidity inhibitors - may immediately prolong the  diagnosis is based on a finding of diminished VWF activity and
mixture’s PTT; incubated mixing study is unnecessary. diminished VWF antigen by immunoassay
2. Factor VIII neutralization by an autoantibody is nonlinear.  treatment
o early rapid loss of FVIII activity → residual activity remains = 1. DDAVP or plasma-derived factor VIII/VWF concentrate
reaction has reached equilibrium (type II kinetics) such as Humate-P, Wilate, or Alphanate is effective
o alloantibodies to factor VIII develop in 30% of patients with 2. Cryoprecipitate - no longer recommended for treatment of
severe hemophilia VWD because it does not undergo viral inactivation.
o type I kinetics - response to factor VIII concentrate therapy;
linear in vitro neutralization of factor VIII activity over 2 hours DISSEMINATED INTRAVASCULAR COAGULATION
→ complete inactivation.
 classified as a thrombotic disorder
o type I kinetics - in vitro measurement is relatively reliable
o type II kinetics - titration of inhibitor activity is
semiquantitative
3. Quantification of autoanti-VIII inhibitors CONGENITAL COAGULOPATHIES
o using the Nijmegen-Bethesda assay - measure inhibitors VON WILLEBRAND DISEASE
in hemophilic patients with alloantibodies to factor VIII  most prevalent inherited mucocutaneous bleeding disorder.
(hemophilia A with factor VIII inhibitors)  Types: quantitative (type 1) and qualitative (functional, type
2) VWF abnormalities.
INHIBITORS OTHER THAN AUTOANTI–FACTOR VIII   platelet adhesion to injured vessel walls, impairing primary
hemostasis.
1. Antiprothrombin (II) antibodies  prevalence in women who report menorrhagia is 24%
o PT: prolong  VWD inheritance is autosomal dominant and affects both sexes
o develop as lupus anticoagulant variants with prothrombin MOLECULAR BIOLOGY AND FUNCTIONS OF VWF
specificity
o associated with thrombosis  multimeric glycoprotein
o few experience bleeding  molecular mass: 500k to 20m Da
o reduced prothrombin activity by clot-based assay  largest molecule in human plasma
o enzyme immunoassay – detect antiprothrombin antibodies  plasma concentration: 0.5 to 1.0 mg/dL
o Positive lupus anticoagulant test - confirm the diagnosis.
 synthesized in the endoplasmic reticulum of endothelial cells
o rare to detect antiprothrombin antibodies that are not
and stored in cytoplasmic Weibel-Palade bodies.
associated with lupus anticoagulant.
 synthesized in megakaryocytes and stored in the a-granules of
2. Factor XIII inhibitors
platelets
o extremely rare but cause lifethreatening bleeding
o arise spontaneously or in association with autoimmune or  Weibel-Palade bodies and a-granules release VWF in response
lymphoproliferative disorders. to a variety of hemostatic and inflammatory stimuli.
o Autoanti-factor XIII - Patients receiving isoniazid treatment  VWF gene - 52 exons (178 kilobase pairs) on chromosome 12.
for tuberculosis. o translated protein is a monomer of 2813 amino acids
3. Autoantibodies to factor V composed of four structural domains, A through D.
o arise spontaneously in autoimmune disorders and after o become glycosylated → dimers and oligomers → storage
exposure to bovine thrombin in fibrin glue. organelles, → ultralarge VWF multimers.
o Fibrin glue-generated autoantibodies - disappeared once o VWF antigen II - cleaved from the end of domain D
manufacturer began to use plasma-derived human o VWD mutations may occur anywhere on the VWF gene.
thrombin o Blood flow shear unfold UL-VWF → ADAMTS13 cleaves
4. Autoanti-factor X antibodies the linear UL-VWF multimers
o factor X deficiency in amyloidosis - absorptive mechanism.  ADAMTS13 deficiency → retention of circulating UL-
 acquired inhibitors - mixing studies show uncorrected VWF multimers → thrombotic thrombocytopenic
prolongation without incubation, and inhibitor titers may be purpura
determined by the Nijmegen-Bethesda assay  Also modulate acute inflammation, stroke, and
myocardial infarction.
ACQUIRED HEMOPHILIA MANAGEMENT
 primary function - mediate platelet adhesion to
subendothelial collagen in areas of high flow rate and
 Activated PCC or rFVIIa - bypass the coagulation factor VIII high shear force
inhibitor in acquired hemophilia and → control acute bleeding. o Domain A - receptor site for collagen and a binding site
(ligand) for platelet receptor GPIb/IX/V and heparin
 o Domain C - site that binds platelet receptor GPIIb/IIIa; o moderate to markedly reduced VWF activity
provides the carrier site for factor VIII
 protects from proteolysis → half-life 8 to 12 hours II. Subtype 2B
when bound. o mutations within the A1 domain raise the
 released → unfolds and binds fibrillar intimal collagen → affinity of VWF for platelet GPIb/IX/V →
platelets adhere through their GPIb/IX/V site to the VWF “carpet “gain-of-function” mutations.
→ platelets become activated → express second VWF binding o HMW-VWF multimers spontaneously bind
site (GPIIb/IIIa) → binds arginine-glycine-aspartic acid resting platelets.
sequences in VWF and fibrinogen → irreversible platelet- to- o VWF unavailable for normal platelet adhesion
platelet aggregation. o Electrophoretic multimer pattern - lack of
HMW-VWF multimers, but intermediate-
Customary designation for the combination of factor VIII molecular-weight multimers still present
FVIII/VWF o Moderate thrombocytopenia = chronic
and VWF
Procoagulant factor VIII, transported on VWF. Factor VIII platelet activation → bind the endothelium
binds activated factor IX to form the complex of VIIIa- and become cleared.
FVIII o Ristocetin-induced (RIPA) platelet
IXa, which digests and activates factor X. Factor VIII
deficiency is called hemophilia A. agglutination assay – confirmatory testing
Epitope that is the antigenic target for the VWF o platelet-type VWD (PT-VWD) or pseudo-
VWF:Ag VWD - raises GPIb affinity for normal HMW -
immunoassay
Factor VIII coagulant activity as measured in a clot- VWF multimers creates a clinically similar
FVIII:C disorder
based factor assay
The following assays measure VWF activity, which is compared to
VWF:Ag to distinguish qualitative and quantitative VWF deficiency. Most III. Subtype 2M
VWF activity assays measure the presence of high molecular weight o qualitative VWF - poor platelet receptor
VWF multimers, which are the most active multimers in platelet adhesion binding despite generating a normal
Quantitative ristocetin cofactor activity, also called multimeric distribution pattern in
VWF activity. VWF activity is measured by the ability of electrophoresis
VWF:RCo o feature that separates it from type 1 -
ristocetin to cause agglutination of reagent platelets by
the patient’s VWF. discrepancy between the concentration of
Collagen binding assay, a second VWF activity assay. VWF:Ag and its activity as measured using
VWF:CB Large VWF multimers bind immobilized target collagen, the VWF ristocetin cofactor assay
predominantly collagen III.
IV. Subtype 2N (Normandy variant; autosomal
Automated nephelometric activity assay that employs
hemophilia).
VWF:Immuno latex microparticles and monoclonal anti-
o autosomal VWF gene missense mutation in
activity glycoprotein I–VWF receptor, a third method for
the D9 domain → impairs the protein’s factor
assaying VWF activity
VIII binding site function.
Activity assay that employs ristocetin-triggered binding
VWF:GPIbR o factor VIII deficiency despite a normal
of rGPIb, detected by LIA or CLIA.
VWF:Ag concentration, normal VWF activity,
Activity assay that employs recombinant gain-of-function
and a normal multimeric pattern.
VWF:GPIbM GPIb that binds the VWF A1 domain without the need
o aka autosomal hemophilia -
for ristocetin. Reaction is detected using LIA
indistinguishable from the symptoms of
Ristocetin-induced platelet aggregometry, uses ristocetin hemophilia except that it affects both men
and patient’s own platelets, in contrast to the VWF:RCo, and women.
which uses reagent platelets. This assay is modified by o Subtype 2N is suspected when: girl or woman
RIPA using low ristocetin concentrations to identify VWD is diagnosed with hemophilia subsequent to
subtype 2B in which VWF multimers exhibit increased anatomic bleeding symptoms
avidity for the platelet receptor site. This method is also o boys or men - misdiagnosed as a hemophilia
called the ristocetin response curve A sufferer fails to respond to factor VIII
concentrate therapy.
PATHOPHYSIOLOGY OF VWF o confirmed using a molecular assay that
detects the specific mutation responsible for
 mucocutaneous hemorrhage: epistaxis, ecchymosis, the abnormal FVIII binding function.
menorrhagia, hematemesis, gastrointestinal, and surgical Type 3  “Null allele” VWF gene translation or deletion
bleeding. mutations
 creates factor VIII deficiency - inability to protect unbound factor  severe mucocutaneous and anatomic
VIII from proteolysis. hemorrhage in compound heterozygotes or
 <30 units/dL → anatomic bleeding into joints and body cavities homozygotes
 most rare form of VWD
VW DISEASE TYPES AND SUBTYPES  VWF concentration measured by immunoassay
or by activity assay is <10%
Type 1  quantitative VWF deficiency  Factor VIII is diminished or absent, and primary
 several autosomal dominant frameshifts, and secondary hemostasis is impaired.
nonsense mutations, or deletions
 comprises 40% to 70% of VWD cases LABORATORY DETECTION AND CLASSIFICATION
  all VWF multimers and factor VIII
 mild to moderate systemic bleeding after dental  Definitive diagnosis - depends on personal and family history of
extraction or surgery. mucocutaneous bleeding and  VWF concentration or activity
 Women menorrhagia → postpartum (function).
hemorrhage, leads to the diagnosis of VWD. 1. complete blood count - to rule out thrombocytopenia as the
Type 2  4 qualitative VWF abnormalities. cause of mucocutaneous bleeding
 VWF levels is normal or moderately decreased, 2. PT and PTT - to rule out a coagulation factor deficiency other
but VWF function is consistently reduced. than VWF.
I. Subtype 2A 3. Bleeding time test and the PFA-100 or other functional platelet
o autosomal dominant point mutations in the A2 assays – no longer recommended
and D1 structural domains
o susceptible to increased proteolysis by  standard VWD test panel must incorporate at least 3 primary
ADAMTS13 → predominance of small- assays:
molecular-weight multimers → less platelet o VWF:Ag
adhesion o VWF activity by ristocetin cofactor assay (VWF:RCo)
o normal or slightly reduced VWF antigen levels o coagulation factor VIII activity
(immunoassay)
 4. Quantitative VWF:Ag assay - most prominent primary VWD ooffers smaller VWF detection limits and less variability.
laboratory profile. oAll VWF activity assays rely on GPIb binding avidity, a
o Use enzyme immunoassay (EIA) methodology - surrogate for VWF activity.
traditional reference method, batched and performed 8. When the ratio of the VWF:RCo, VWF:GPIbR, or VWF:GPIbM
manually assay value to the VWF:Ag concentration (ex:
o automated LIA VWF:GPIbR/VWF:Ag) is <0.5, 0.6, or 0.7 → infers qualitative or
o chemiluminescence immunoassay - possesses the best type 2 VWD.
sensitivity to VWF concentrations <10% and the best 9. Low-dose RIPA/ristocetin response curve - identifies subtype
precision. 2B.
5. factor VIII assay o performed PRP
o parallel VWF:Ag and VWF:RCo results in VWD types 1 and o subtype 2B - agglutinate in response to <0.5 mg/mL
3 ristocetin or 0.1 mg/mL ristocetin.
o parallel VWF:Ag in subtypes 2A, 2B, and 2M o normal platelets agglutinate only at ristocetin
o  in VWD subtype 2N concentrations >0.5 mg/mL
o subtype 2A - not agglutinate to ristocetin at all.
6. VWF:RCo assay - employs ristocetin 10. sodium dodecyl sulfate– polyacrylamide gel electrophoresis
o Ristocetin - unfolds the VWF molecule → HMW-VWF - VWF multimer analysis
multimers bind reagent platelet membrane GPIb/IX/V o secondary confirmatory procedure that helps establish VWD
o performed using a platelet aggregometer → measures type 2
platelet agglutination → quantitative measure of VWF o differentiates between VWD subtypes 2A, 2B, and perhaps
function 2M.
o uses preserved platelet suspension o intermediate multimers are present in the electrophoretic
o partially replaced by automated ristocetin-triggered pattern of subtype 2B VWD samples but are absent from
nonplatelet rGPIb-based LIA and CLIA methods subtype 2A VWD.
7. VWF:GPIbM/gain-of-function high-affinity rGPIb - alternative o type 2M - pattern is normal despite the reduced function to
o GPIb binds the A1 domain of native VWF without the need concentration ratio.
for ristocetin.
LABORATORY TEST TYPE 1 SUBTYPE 2A SUBTYPE 2B SUBTYPE 2M SUBTYPE 2N TYPE 3
VWF:RCo, VWF:CB,
VWF:Immunoactivity, Very low or
Low Low Low Normal
VWF:GPIbR, or absent
Low
VWF:GPIbM
Normal to slightly Normal to slightly Very low or
VWF:Ag Normal Normal
decreased decreased absent
VWF activity to VWF:Ag
>0.5 <0.5 <0.5 <0.5 >0.5 N/A
ratio
Platelet count Normal   Normal Normal
Normal to
Partial thromboplastin Normal to slightly Normal Normal to slightly
Normal slightly Prolonged
time prolonged prolonged
prolonged
RIPA     Normal Absent
Factor VIII activity Slightly low Normal Normal Normal  <10 units/dL
Large and
VWF multimers Normal pattern intermediate Large forms absent Normal pattern Normal pattern All forms absent
forms absent

PITFALLS IN VON WILLEBRAND DISEASE DIAGNOSIS O 36% – 157%

A 48% – 234%
 genetic penetrance, ABO blood group, inflammation, B 57% – 241%
hormones, age, and physical stress influence VWF activity. AB 64% – 238%
 Raised estrogen levels during the 2nd and 3rd trimesters of
Population based: “Low VWF” <50%
pregnancy nearly normalize plasma VWF activity even in women
with moderate VWF deficiency. Population based: VWD <30%
 VWF concentration and function decrease rapidly after delivery
→ acute postpartum hemorrhage VON WILLEBRAND DISEASE TREATMENT
 Oral contraceptives and hormone replacement therapy →
raise VWF activity  Mild bleeding - limb elevation, pressure, and application of ice
  packs (PRICE - protection, rest, ice, compression, and elevation).
o acute inflammation - postoperatively, trauma, or during  Moderate bleeding
infection. o estrogen and desmopressin acetate → release of VWF
o Physical stress - cold, exertion, or a child’s crying or from storage organelles.
struggling during venipuncture  control diabetes mellitus and bedwetting → release of
o allows the tourniquet to remain tied for >1 minute VWF from storage organelles is a side effect.
  - stored in the refrigerator before testing.  DDAVP (oral) or Stimate (oral spray) - effective for
 variability in the results of the VWF:RCo assay type 1 and subtype 2M and 2A VWD
o poor reproducibility of results of the VWF:RCo assay →  contraindicated for subtype 2B – releases
develop VWF collagen-binding (VWF:CB) assay. abnormal VWF with increased affinity for platelet
 Use type III collagen as its solid-phase target antigen GPIb/IX/V receptors → intensify
- binds predominantly HMW-VWF multimers thrombocytopenia → increased platelet activation
 provides better precision  repeated doses may lead to hyponatremia (low serum
 detects abnormalities of VWF collagen binding sodium) - monitor and regulate electrolytes
 requires standardization o Therapeutic dosages are monitored using serial VWF:Ag
 low VWF – to prevent misdiagnosis of Type 1 VWD assays.
o VWF activity and antigen concentrations - 30%-50% of  lysine analogs e-aminocaproic acid and tranexamic acid
normal o inhibit fibrinolysis and may help control bleeding when used
o ratio of VWF:RCo to VWF:Ag: >0.5 alone or in conjunction with desmopressin acetate
o factor VIII activity: >50 units/dL o using nonbiologic preparations is preferred over human
o definite type 1 VWD diagnosis - <30% of normal plasma-derived biologic therapy
 Rheumatoid factor and heterophile antibodies - false o nonbiologics eliminate the risk of viral disease transmission
positives. and circumvent religious objections
 Type 3 VWD – misdiagnosed as Hemophilia A  mixture of VWF and FVIII: Humate-P, Alphanate, and Wilate
 VWF activity varies by ABO blood group o severe VWD (type 3) and subtype 2B
 o Laboratory monitoring by the VWF:Ag assay  Severe hemophilia usually is diagnosed in the first year of life
 Vonvendi  Sample requirement - unhemolyzed specimen of at least 2 mL
o recombinant VWF that provides no accompanying factor VIII from tiny veins and by the predictably low newborn levels of
o initial Vonvendi dose - 40 to 80 IU/kg body weight every 8 some coagulation factors.
to 24 hours,  PT and PTT prolonged
o If the factor VIII concentration is <40 IU/dL – give  severity of hemophilia A symptoms is inversely proportional to
recombinant VWF-free factor VIII within 10 minutes of FVIII activity.
completing Vonvendi infusion at a ratio of 1.3:1 o activity level of <1 unit/dL as severe, associated with
 Recombinant and affinity-purified factor VIII concentrates - spontaneous or exaggerated bleeding in the neonatal
contain no VWF and cannot be used to treat VWD period.
 Cryoprecipitate and plasma - risk of virus transmission, and o Activity levels of 1 unit/dL - 5 units/dL are seen in
may cause TACO. moderate hemophilia; early childhood.
o activity levels of 5 to 40 units/dL - mild hemophilia;
TYPE PRIMARY APPROACH OTHER hemorrhage follows significant trauma
Estrogen, DDAVP, EACA, or Factor VIII/VWF HEMOPHILIA A COMPLICATIONS
1
TXA concentrate
Estrogen, DDAVP, EACA, or Factor VIII/VWF  debilitating and progressive musculoskeletal lesions and
2A deformities and neurologic deficiencies
TXA concentrate
 chronic hepatitis often resulted from repeated exposure to blood
2B Factor VIII/VWF concentrate EACA
Estrogen, DDAVP, EACA, or Factor VIII/VWF HEMOPHILIA A LABORATORY DIAGNOSIS
2M
TXA concentrate
2N Factor VIII concentrate EACA  PT, PTT, the fibrinogen assay, and TT
Platelet  hemophilia A and B
3 Factor VIII/VWF concentrate o
transfusions PT, fibrinogen, and TT - normal
o PTT prolonged; reagent is sensitive to factor VIII and factor
HEMOPHILIA A (FACTOR VIII DEFICIENCY) IX deficiencies at or <40 units/dL plasma activity level.
 Clot- based factor VIII and factor IX assays replaced with
 Hemophilia - congenital single-factor deficiencies marked by
chromogenic factor VIII and factor IX assays; greater
anatomic soft tissue bleeding
o mostly males accuracy and precision.
o 85% are deficient in factor VIII
o 14% are deficient in factor IX
o 1% are deficient in factor XI DEFICIENT
PT PTT TT REFLEX TEST
 classic hemophilia or hemophilia A - Congenital deficiency of
factor VIII Fibrinogen Prolonged Fibrinogen assay
FACTOR VIII STRUCTURE AND FUNCTION Prothrombin, V,
Prothrombin Prolonged Normal
VII, and X assays
 two-chain, 285,000-Dalton from the X chromosome. Prothrombin, V,
V Prolonged Normal
 coagulation cascade is activated → thrombin cleaves plasma VII, X assays
FVIII → releases B domain → leaves behind a calcium- VII Prolonged Normal VII assay
dependent heterodimer → detaches from VWF carrier → bind VIII, IX, and XI
VIII Normal Prolonged Normal
phosphatidyl serine and factor IXa. assay
o VIIIa/IXa complex/tenase complex - cleaves and activates VIII, IX, and XI
IX Normal Prolonged Normal
coagulation factor X assays
 FVIII deficiency → slows coagulation pathway’s production of X Prolonged Normal
Prothrombin, V,
thrombin → hemorrhage. VII, and X assays
 blood collected in standard citrate-dextrose-phosphate VIII, IX, and XI
XI Normal Prolonged Normal
preservative provides 30 units/dL FVIII activity after 28 days’ assays
storage = approximately 50 units/dL in leukodepleted donor XIII quantitative
XIII Normal
blood. assay
HEMOPHILIA A GENETICS
HEMOPHILIA A CARRIER DETECTION
 186 kilobases of the X chromosome
 mutations result in quantitative disorders in which the FVIII  ratio of FVIII activity to VWF:Ag concentration (FVIII to
coagulant activity and antigen concentration match, but in rare VWF:Ag)
cases. o VWF production is unaffected by FVIII deficiency.
 cross-reacting material positive disorders - qualitative or o ratio is below the lower limit - likely to be a carrier.
structural FVIII abnormalities; low activity despite normal antigen o May be influenced by excessive lyonization, variation in
concentration VWF production, and analytical variables
 All sons of men with hemophilia A and non-carrier women are o if carrier status is suspected and the FVIII to VWF:Ag ratio is
normal, whereas all daughters are obligate carriers of the more than lower limit → genetic testing to detect
disease. polymorphisms associated
 VWD of the Normandy (2N) subtype may mimic hemophilia A HEMOPHILIA A THERAPY
in males and females.
HEMOPHILIA A CLINICAL MANIFESTATIONS  non-biologic oral desmopressin acetate (DDAVP) or Stimate
- mild to moderate hemophilia patients’ FVIII activity rises
 anatomic bleeds with deep muscle and joint hemorrhages o may be administered in combination with an antifibrinolytic
(eaminocaproic acid or tranexamic acid)
 hematomas
 coagulation factor concentrates - in severe hemophilia
 wound oozing after trauma or surgery
 Human plasma-derived FVIII (pdFVIII) concentrates -
 bleeding into the central nervous system, peritoneum,
Alphanate, Hemofil-M, Kogenate FS, Humate-P, and Wilate.
gastrointestinal tract, and kidneys
o Alphanate, Humate-P, and Wilate contain VWF, fibrinogen,
 Acute joint bleeds (hemarthroses) are exquisitely painful and
and noncoagulant proteins in addition to FVIII and are used
cause temporary immobilization.
to treat VWD.
o Chronic joint bleeds cause inflammation and eventual
o from healthy plasma donors using chemical fractionation.
permanent loss of mobility
o processed using immunoaffinity columns, pasteurization,
 bleeding into muscles may cause nerve compression injury,
and the solvent-detergent.
with first temporary and then lasting disability.
o All pdFVIII concentrates undergo viral inactivation
 Cranial bleeds lead to severe, debilitating, and durable o may transmit non-lipid viruses such as parvovirus B19 and
neurologic symptoms - loss of memory, paralysis, seizures, and hepatitis A virus.
coma, and may be rapidly fatal. o On demand therapy
 o target activity level: >75 units/dL  activated PCC dosage should not exceed 200 units/kg
o FVIII has a half-life of 8 to 12 hours, twice-a-day infusions per day; distributed in 2 to 4 injections, because it may
may be required. trigger DIC.
 recombinant FVIII (rFVIII) concentrates  NovoSeven - promotes thrombin formation through the
o first rFVIII products employed human or animal serum and tissue factor pathway.
albumin in their manufacturing process - carry the risk of o Emicizumab - “factor VIII mimetic” bispecific antibody that
viral disease transmission. bypasses factor VIII by binding factor X with IXa.
 Helixate FS, Kogenate FS, and Recombinate and  Hemophilic boys older than 12 with inhibitors - used
Bioclate. subcutaneous prophylactic HemLibra once a week
o rFVIII products free of all human protein - Advate.
o B-domain-deleted (BDD- rFVIII) preparations - less HEMOPHILIA B (FACTOR IX DEFICIENCY)
immunogenic.  also called Christmas disease,
 ReFacto - produced using Chinese hamster ovary  deficiency of factor IX (FIX), one of the vitamin K-dependent
(CHO) cells grown in human albumin serine proteases.
 Xyntha - produced using CHO cells grown in synthetic,
 Factor IX is a substrate for both factors XIa and VIIa
albumin-free culture medium.
 reduces thrombin production and causes soft tissue anatomic
 prophylactic regimens, administering 3 infusions per week to bleeding that is indistinguishable from that in hemophilia A.
maintain their FVIII activity at hemostatic levels.
 a sex-linked
 Eloctate - extended half-life rFVIII
 Determination of female carrier status is less successful in
o BDD-rFVIII fused with the Fc region of human IgG1 (BDD-
hemophilia B than in hemophilia A
rFVIIIFc). Fc fragment binds the neonatal Fc receptor
 DNA analysis - used to establish carrier status
(FcRn) extending the plasma half-life to 20 hours and
reducing the infusion frequency to once every 4 to 5 days.  PTT: prolonged
 Adynovate  PT, fibrinogen assay, and TT are normal
o rFVIII conjugated with polyethylene glycol (PEGylation)  Factor IX assay should be performed even if PTT is within the
o extends the rFVIII half-life to 20 hours and reduces the reference range, because the PTT reagent may be insensitive to
infusion rate to approximately twice weekly. factor IX deficiencies at the level of 30 units/dL.
o triggered only transient inhibitors  Immunine and Mononine - plasma-derived, immunopurified FIX
 Afstyla concentrates.
o single-chain rFVIII o Repeat doses of FIX are given every 24 hours
o binds VWF with greater avidity → extended half-life and  BeneFix - recombinant FIX concentrate (rFIX) grown in CHO
induction of fewer inhibitors, closely resemble native FVIII cells in the absence of human or animal protein.
 half-life rFVIII formulations owe their pharmacokinetics to VWF, o supports prophylactic administration at a rate of 2 to 3
whose half-life of 12 to 15 hours infusions per week.
 When hematologists treat hemophilia, they base FVIII  Two extended half-life preparations - Fc-fusion Alprolix and
concentrate dosage calculations on the international unit of FVIII albumin-fusion Idelvion
activity = 100 units/dL. o administration once every 2 weeks.
 FIX therapy - measured using the one-stage clot assay
o chromogenic assays with improved accuracy, low-level
sensitivity, and reproducibility are in development.
 Nijmegen- Bethesda assay – detects FIX inhibitors
o Treatment: PCC or rFVIIa and immunomodulation
therapy

HEMOPHILIA A AND FVIII INHIBITORS HEMOPHILIA C (ROSENTHAL SYNDROME, FACTOR

XI DEFICIENCY)
 Alloantibody inhibitors of FVIII arise in response to treatment in
 autosomal dominant hemophilia
30% of patients with severe hemophilia and 3% of those with
 mild to moderate bleeding symptoms.
moderate hemophilia.
 described in Ashkenazi Jews
 presence of an inhibitor - when bleeding persists or when the
plasma FVIII activity fails to rise to the target level  treat with frequent plasma infusions during bleeds and times of
hemostatic challenge
 Most FVIII inhibitors are IgG4, non-complement-fixing, warm-
reacting antibodies.  PTT is prolonged
 PT is normal
 inhibitor detection
1) one-stage clot-based FVIII assay OTHER CONGENITAL SINGLE-FACTOR DEF.
o FVIII activity >30 units/dL = no inhibitor is present.  autosomal recessive mutations, and are often associated with
o <30 units/dL → perform mixing studies. consanguinity.
o prolonged PTT – mixed 1:1 with NP, incubated 2 hours at  immunoassays - to distinguish among the more prevalent
37°C, and the PTT is measured. quantitative and the less prevalent qualitative abnormalities.
 no inhibitor is present - within 10% of the incubated  dysproteinemias - qualitative disorders, the ratio of factor
NP PTT activity to antigen is <0.7.
o inhibitor is present - FVIII from the NP is partially  Fibrinogen - measured using the Clauss clot-based assay, a
neutralized and the mixture’s PTT remains prolonged modification of the thrombin time, also may be measured by
2) Nijmegen-Bethesda assay - used to quantitate the inhibitor. turbidimetry or immunoassay.
o NP providing 100 units/dL factor activity is mixed at 
increasing dilutions in a series of tubes with the full-strength 1. factor V deficiency
patient plasma. o platelet function diminished
o expresses the titer as Nijmegen-Bethesda units (NBUs). o prolonged bleeding time test but normal platelet
o adequately monitors therapy. aggregation.
o Low responders - inhibitor titers of 5 NBUs or less and o PT and PTT are prolonged.
inhibitor titers do not increase significantly after FVIII o platelet concentrate - effective form of therapy
administration. 2. combined factor V and VIII deficiency
o High responders - inhibitor titers that exceed 5 NBUs and o genetic defect traced to chromosome 18 that affects
their antibody titers further rise in response to therapy. transport of both factors by a common protein in the Golgi
HEMOPHILIA A TREATMENT IF WITH INHIBITORS apparatus
3. Factor VII deficiency - moderate to severe anatomic
 Low responders - administration of large doses of FVIII hemorrhage.
concentrate o half-life of factor VII is 6 hours
 High responders - gain no benefit from FVIII concentrates and o NovoSeven at 30 mg/mL and non-activated four-factor
instead are treated with activated PCC, or rFVIIa, all generate PCC preparations – effective, provide target factor VII level
thrombin despite the presence of FVIII inhibitors. of 10 units/dL to 30 units/dL.
o Many factor VII deficiencies are dysproteinemias.
o PT is prolonged in factor VII deficiency.
 o Factor XIII deficiency occurs in three forms
4. Factor X deficiency o normal PT, PTT, and TT
o treated with plasma or non-activated PCC → therapeutic o form weak (non-cross-linked) clots that dissolve within 2
levels of 10 units/dL to 40 units/dL. hours when suspended in a 5M urea solution - traditional
o half-life of factor X is 24 to 40 hours. factor XIII screening assay
o Acquired factor X deficiency - described in amyloidosis, in o confirm factor XIII deficiency - factor activity may be
paraproteinemia, and antifungal drug therapy. measured using a chromogenic substrate assay
o PT and PTT are both prolonged (Behrichrom FXIII assay)
o Russell viper venom time test - clotting time is prolonged 6. autosomally inherited deficiencies of the fibrinolytic regulatory
in deficiencies of factors X and V, prothrombin, and proteins a2-antiplasmin and plasminogen activator inhibitor-1
fibrinogen. (PAI-1) have been reported to cause moderate to severe
 Useful for factor VII deficiency - does not prolong the bleeding. Both are rare and may be diagnosed using
Russell viper venom clotting time chromogenic substrate assays.
5. Plasma factor XIII - tetramer of paired a and b monomers.
o a-chain - contains the active enzyme site
o b-chain - binding and stabilizing portion.
FACTOR
DEFICIENCY SYMPTOMS THERAPY
LEVELS
No measurable Severe anatomic
Afibrinogenemia CRYO or FG
fibrinogen bleeding
concentrate to
Moderate systemic
Hypofibrinogenemia raise to 100 mg/dL
bleeding
CRYO or FG
FG activity
concentrate to
assay ,100
Mild systemic control bleeding.
Dysfibrinogenemia mg/dL
bleeding Treat underlying
cause such as
liver disease
PCC or plasma to
Factor II , 30 Mild systemic
Prothrombin deficiency raise factor II to 75
units/dL bleeding
units/dL
Platelet
concentrate or
Factor V , 30 Mild systemic
Factor V deficiency plasma to raise
units/dL bleeding
factor V to 75
units/dL
NovoSeven, PCC,
Moderate to four-factor PCC,
Factor VII , 30
Factor VII deficiency severe anatomic plasma to raise
units/dL
bleeding factor VII to 75
units/dL
PCC or plasma to
Factor X , 30 Severe anatomic
Factor X deficiency raise factor X to 75
units/dL bleeding
units/dL
Plasma to raise
Factor XI , 50 Variable anatomic
Factor XI deficiency factor XI to 75
units/dL bleeding
units/dL
Moderate to
Factor XIII , 1 severe systemic Plasma or CRYO
Factor XIII deficiency
units/dL bleeding, poor every 3 weeks
wound healing

FACTOR XIII DEFICIENCY

Type of Factor XIII
Incidence b-Protein a-Protein
Deficiency Activity
Type I Rare Absent Absent Absent
Type II Infrequent Absent Normal Low
Type III Rare Low Absent Low