Hematology

Presented by
Alyazeed hussein, BSc
 Tubes and anticoagulants
➢ EDTA (Ethylene Diamine Tetra-Acetate)
➢ Mechanism: forming Ca salts to remove Ca.
➢ Uses: CBC, PCR, blood films and HbA1c.
➢ Sodium citrate:
➢ Anticoagulant: 3.2%
➢ Mechanism: Calcium chelation.
➢ Use: Coagulation studies and platelet function.
➢Black:
➢ 3.8% of sodium citrate
➢Action: Remove calcium.
➢Uses: Erythrocyte Sedimentation Rate (ESR).

➢Heparin:
➢ Sodium Heparin or Lithium Heparin
➢Mechanism: inactivation of thrombin and
thromboplastin.
➢Uses: Use for osmatic fragility test and blood gases
Hemolyzed, clotted and lipemic sample!!!!
 Red blood cells and RBCs indices:
• Morphology characteristics:
• Cell size: 7-8 µm.
• No nucleus.
• Cytoplasm: pink to salmon to
light red.
• Zone of central pallor is one-
third of cell diameter.
• Reference values:
• Adult male: 4.7-6.1 million/µL.
• Adult female: 4.2-5.4
million/µL.
• Newborn: 4.4-5.8 million/µL.
• Hemoglobin, M: 14-17 g/dL, F: 12.5-15g/dL.
• Hematocrit (HCT) is the volume of whole blood occupied by packed RBCs.
RBC Indices
• MCV (mean corpuscular volume): Reference range is 80-100 femtoliters (fL), and it is an
indicator of the average cell (RBCs).
a. Increased in megaloblastic anemia, hemolytic anemia with reticulocytosis, liver
disease, and normal newborn.
b. Decreased in iron deficiency anemia, thalassemia, sideroblastic anemia, and lead
poisoning.
• MCH (mean corpuscular hemoglobin): Reference range is 26-34 picograms (pg), and it
is an indicator of the average weight of hemoglobin in individual RBCs.
a. Increased in macrocytic anemia.
b. Decreased in microcytic, hypochromic anemia.
• MCHC (mean corpuscular hemoglobin concentration): Reference range is 32-37 g/dL,
and it is a measure of the average concentration of hemoglobin in grams per deciliter.
a. 32-37 g/dL MCHC indicates normochromic RBCs.
b. Lesser than ( <) 32 g/dL MCHC indicates hypochromic RBCs, which is seen in iron
deficiency and thalassemia.
c. Greater than(>) 37 g/dL MCHC indicates a possible error in RBC or hemoglobin
measurement, or the presence of spherocytes.
• RDW (RBC distribution width): Reference range is 11.5-14.5 %,
a. Increased proportional to the degree of anisocytosis (variation in size).
b. High RDW: Post-treatment (e.g., Iron, B 12, or folic acid therapy), in the presence of two
concurrent deficiencies (iron and folic acid deficiencies).
Normal MCV Low MCV shift left High MCV shift right
White blood cells:

• WBCs count: 4,800-10,800 Corrected WBC count:

cells/µL. It is required if the NRBCs greater than 5/100 WBCs
• Differential count: count, the automated counter count the NRBCs as a
• Neutrophil: 50-70%, WBCs gives a false high count.
Corrected WBCs = WBCs count / (100 + NRBCs per
• Lymphocytes: 20-40%, 100 WBCs) x 100.
• Monocytes: 2-9% Example: WBC count is 15,000. the number of NRBCs
• Bands: 2-6% per 100 WBCs was 20,15,000 / (100 + 20) x 100 =
• Eosinophils: 1-4% 12,500.
• Basophils: 0-1%
• Platelets:
• PLT count: 150,000-450,000/µL.
• Normal platelets histogram displays cells from (2-20 fL), over
20 fL indicates platelets clumping and giant platelets.
Reticulocytes:
Non nucleated polychromatic cell.
Reticulocyte count: count reticulocytes in 1000 RBCs, then divide by 10, e.g. 25 retics/1000RBCS:
25/10= 2.5%. Normal range: adult: 0.5-2.5%, infants: 2-6%.
➢ Clinical value: To exclude the hemolysis in normocytic/normochromic anemia.
o Reticulocytosis (increased RBC Production):
1. Acute blood loss or hemorrhage
2. Acute Hemolytic Anemia (Microangiopathic Anemia)
3. Hemoglobinopathy:
o Sickle Cell Anemia
o Thalassemia major
4. Post-Anemia Treatment:
o Folate Supplementation
o Iron Supplementation
o Vitamin B12 Supplementation
5. Membrane defect: hereditary spherocytosis(increase osmatic fragility) test.
 RETICULOCYTE
• Reticulocytes are visualized by supra-vital staining (such as new methylene blue, Brilliant
Cresyl Blue) that precipitate the RNA and forming a filamentous network of reticulum.
• On Wright stain. The Reticulocyte appears polychromatophilic or as Macrocytic blue red
cell.

Supravital stain
ESR: Erythrocyte Sedimentation Rate
 False increased ESR False decreased ESR
Tube tilted (not vertical position) Air bubbles in the tube
Time Vibration of tube during test
RT >25 C RT <20 C
Clotted sample
Hemoglobinopathies
 Structure of Hemoglobin
• Hemoglobin molecule is tetramer
consisting of two pairs of similar
polypeptide chains called globin chains. Hb type Adult reference Newborn reference
• To each of the four chains is attached
Hb A >95% 10-40%
heme which is a complex of iron in
ferrous from and protoporphyrin.
• The major (96%) type of hemoglobin Hb F 0-2% 60-90%
present in adults is called HbA it has
✓ 2 alpha globin chains and 2 beta globin Hb A2 0-3.5%
chains (α2β2).
❖ In 7 months the HbF switch to HbA.
 Sickle cell anemia
Hb S disease: formed when a valine is substituted for a glutamic acid at the 6th
position in the beta chain,(low O2 > insolubility of Hb S > polymerization)
Sickle anemia: 25%
 Lab diagnosis:
• Normocytic/normochromic RBCs, sickle
cells.
• Sickle solubility test: positive (depend on
the Hb S solubility and concentration).
Hb-electrophoresis:Hb S: 90-100%.
• Sickle solubility test and sickling test:
blood mixed with a reducing solution
(sodium metabisulfute), if there is HbS it
will precipitate and form a turbidity.
• Hb-electrophoresis: Hb S 90-100%.
• Hb-F vs Hb-S!!
 Favism

G6PD - Pathophysiology
• G-6-PD enzyme reduces NADP to NADPH producing
sufficient reduced Glutathione (GSSH). Reduced
glutathione protects the RBC from oxidative injury.
• Heinz bodies – denatured hemoglobin that precipitates
out.
• Bite cells-RBC’s with a piece cut out (where Heinz
bodies were located) abnormal cells, gets trapped in
the spleen and destroyed.
• Normocytic, normochromic anemia.
 Beta & Alpha thalassemia
• Beta thalassemia:
The gene of beta globin protein is missing, resulting excess alpha globin in RBCs.
• Alpha thalassemia:
Alpha globin protein is missing > excess beta globin.

β thalassemia (Hb electrophoresis)

HEMOGLOBI MAJOR MINOR NORMAL

N
Hb F 10-98% Variable <1%

Hb A Absent 80-90% 97%

Hb A2 Variable 5-10% 1-3%

(increased)
 The α thalassemia:Genetic, clinical,
and laboratory findings:
Disorder Genotype MCV Anemia Hb electrophoresis

Silent carrier α α/α - NL None Normal <3%Hb Barts at

birth
Minor αα / - - or Low Mild Normal
α-/α- 3 to 8% Hb Barts at birth

Hb H disease α-/-- Low Moderate 5 to 30% HbH present in

(deletional) adults
20 to 40% Hb Barts at birth

Major (fetal --/-- Low Fatal Hb Barts, and HbH present

hydrops) HbA, HbF, and HbA2 are
absent
 Life span of RBCs: 90-120 days
Classification
according to ANEMIAS: Classifications
etiological
ground: Anemia: classification according to MCV
• Nutritional
• Aplastic
• Hemorrhagic Macrocytic anemia Normocytic anemia Microcytic anemia
• Hemolytic (MCV>100) (80<MCV<100) (MCV<80)

Deficiency of vit Acute blood Heme synthesis

B12, folic acid, loss, chronic defect (i.e., iron
or IF. diseases, bone deficiency, chronic
Hypothyroidism. Marrow failure, diseases)
Alcoholism. Liver hemolysis Globin synthesis
diseases. Drugs defect (i.e.,
that inhibit DNA thalassemia)
replication (i.e., Sideroblastic
methotrexate, defect
zidovudine)
Red blood cell morphology
 Anisochromia

• Normochromic: normal Hb, normal central pallor, seen in: hemolytic

anemia, hemoglobinopathies, MCHC: 32-36 g/dL.
• Hypochromic: low Hb, increase central pallor, seen in: IDA, lead
poisoning, alpha thalassemia.
• Hyperchromic: full hemoglbinized RBC, spherocytes.
• Polychromasia: (reticulocytes) slight large, blue to gray color, residual
RNA, seen in: increased erythropoiesis, hemolysis, acute and chronic
hemorrhage.

▪ Poikilocytosis: Variation in RBC shape, in severe anemia, hemolytic

state.
• Acanthocytes: 3-5 irregular spikes soicules, hemolytic anemia, alcoholic cirrhosis, vitamin E deficiency, sever
liver disease, pyruvate kinase deficiency.
• Blister cell: containing a vacuole, on one side (removing of inclusion within the RBC, seen in: G6PD deficiency,
hemoglobinopathies.
• Target cell: like archery target, central area of Hb surrounded by a peripheral ring of Hb, seen in: thalassemia,
IDA, liver disease, hemoglobinopathies.
• Teardrop cell: with a single elongated point or tail, seen in: primary myelofibrosis, megaloblastic anemia,
thalassemia.
• Sickle cell: crescent or banana shape, no central pallor, seen in: sickle cell anemia.
• Burr (echinocytes): regular 10-30 short projections evenly distributed, seen in: uremia, cancer of the stomach,
sever burns, hepatitis, renal failure, hypothyroidism.
• Elliptocytes: rod or cigar shape, seen in: hereditary elliptocytosis, IDA, thalassemia, primary myelofibrosis.
• Ovalocytes: egg or oval shaped wider than elliptocyte, seen in: megaloblastic anemia, myelodysplastic
syndrome, thalassemia.
• Schistocytes: fragments of RBCs, seen in: DIC, TTP, HUS, MAHA, burns, heart valves.
• Spherocytes: small with no central pallor, condensed Hb (hyperchromic), seen in: hereditary spherocytosis,
immune hemolytic anemia, autoimmune hemolytic anemia, sever burns.
• Stomatocytes: slit like area of central pallor, seen in: hereditary stomatocytosis, acute alcoholism, Rh null
disease, liver disease, cirrhosis.
 Acanthocytes Blister cell Target cell

Tear drop cell Sickle cell Burr cell

(echinocytes)
Elliptocytes and schistocyte
ovalocytes

stomatocyte spherocytes
 Rouleaux formation in
MM

Cold agglutination disease

Red cell inclusions and parasites:
• Basophilic stippling: dark purple color, (RNA), seen in
thalassemia, lead poisoning.
• Cabot ring: oval or eight ring, nuclear membrane remnants,
seen in: lead poisoning, megaloblastic anemia.
• Heinze bodies: denatured Hb, seen in: G6PD deficiency.
• Hemoglobin C: rod shape crystal, seen in: HbC disease, HbSC
disease.
• HbH: golf ball like, seen in: HbH disease, alpha thalassemia.
• Howell-jolly bodies: nuclear remnants of DNA, hemplytic
anemia, megaloblastic anemia, thalassemia, sickle cell anemia.
• Pappenheimer bodies: iron after ineffective erythropoiesis.
Basophilic stippling Cabot ring Heinz bodies

Hb-C Hb-SC Hb-H

Parasites:

Microfilaria
Trypomastigotes (trypanosomes) Leishmaniasis (Amastigotes)
inside macrophage
Babesia
Ring stages in thick film Ring stages in thin film
White blood cells
(Morphology)
 Leukopoiesis

1. Granulopoiesis-myelopoiesis: (myeloblast, promyelocyte,

myelocyte, metamyelocyte, band cells, mature WBCs: neutrophil,
eosinophil and basophil).
2. Monopoiesis: (monoblast, promonocyte, and monocyte).
3. Lymphopoiesis: (lymphoblast, prolymphocyte, small and
large lymphocytes).
myeloblasts
promyelocytes
 myelocyte

metamyelocytes
Band cells
Mature monocytes
 Lymphoblasts

prolymphocytes

Plasma cell

Reactive lymphocyte
Blood smear under microscope
Allergy and
leukemia
Chronic
infection: TB

Bacterial
infection viral

Allergy and parasitic

infections(helminths)
 Qualitative WBCs disorders and
WBCs inclusions.

1. May-Hegglin anomaly.
2. Chediak-Higashi syndrome.
3. Alder-Reilly anomaly.
4. Pelger-Huet anomaly.
May-Hegglin anomaly, congenital defect:
• Autosomal dominant Mutation in MYH9 gene in chromosome 22,
a cytoskeletal protein in platelets that may be responsible for the
platelets abnormal diameter.
• Thrombocytopenia.
• Purpura and bleeding.
• Giant platelets.
• Large basophilic cytoplasmic (granulocyte) inclusion body called
Dohle body.
Chediak-Higashi syndrome:
• Rare autosomal recessive.
• Recurrent bacterial infection (defective chemotaxis) impaired
motility, defect in granulocyte degranulation.
• Oculocutaneous albinism.
• Giant lysosomal granules in granulocyte, monocyte,
lymphocyte, melanocyte, tissue macrophage and platelets.
• Moderate Neutropenia and thrombocytopenia.
Alder-Reilly anomaly:
• Autosomal recessive disorder of mucopolysaccharidosis.
• Dark staining coarse cytoplasmic granules in neutrophils,
eosinophils, basophils, monocytes and lymphocytes.
• Precipitation of mucopolysaccharides (Hunter and Hurler
syndromes).
Pelger-Huet anomaly:
• Autosomal dominant disorder of neutrophil morphology,
bilobed nucleus or a nucleus with no lobulation.
• 70-90% of the neutrophils are bilobed (sun glasses) or
have no lobulation of the nucleus.
Hypersegmented neutrophil:
• May hereditary autosomal dominant or acquired which
seen in anemia such as megaloblastic anemia.
• Neutrophil with six or more nuclear lobes.
Platelets and
morphology
• 8-20 PLT/OIF: adequate.
• 150,000-450,000: adequate,
• > 450,000: increased,
• >150,000: decreased.
 Pseudo-Thrombocytopenia
Platelet clumps seen in EDTA anti-
coagulated blood sample in a patient
with EDTA Dependent platelet
agglutinins.
No platelet clumps seen and platelet count
normal in the blood sample from the same
patient anti-coagulated with sodium citrate
 Giant platelet

Bizarre platelet
Disorder Pathophysiology Platelet count Platelet Platelet aggregation
morphology
Bernard-Soulier Lack of functional glycoprotein Normal to Giant platelets Abnormal with
(GP) Ib/IX/V on plt surface decrease ristocetin
prevents interaction with VWF.
Abnormal plt adhesion to
collagen.
Glanzmann Deficiency or abnormality of plt Normal Normal Abnormal with ADP,
thromboasthemia membrane GP IIb/IIIa. Fibrinogen collagen, and
can’t attach to plt surface & epinephrine
initiate plt aggregation.
Leukemia
 Acute myelogenous leukemia(AML) 55 – 60 years

Acute lymphoid lukemia(ALL) 2 – 10 years

Chronic myelogenous leukemia(CML) > 40 years

Chronic lymphoid leukemia(CLL) > 55 years(65 – 70)

✓ FAB classification: ≥ 30% blasts,

✓ WHO classification: ≥ 20% blasts.
Presence of Auer rods in myeloblast
differentiate AML from ALL
CLL: PBS show smudge cells or basket cells > prevented
by addition of Albumin.
Epstein Barr Virus(EBV), Infectious mononucleosis,
PBS: Atypical lymphocytes.
Diagnosed by heterophile Abs(monospot test).
Hodgkin lymphoma and Reed–Sternberg cell.
Owl eye.

“ Reed-Sternberg cell’’
Leukoerythroblastic reaction
Immature WBCs + immature RBCs
• Chronic myeloid leukemia (CML) is an acquired clonal
myeloproliferative neoplasm of the pluripotent hematopoietic stem
cell.
• It is characterized by anemia, extreme leukocytosis, granulocytic
immaturity, basophilia, thrombocytosis and splenomegaly.
• LAP (NAP) score is markedly reduced vs leukemoid reaction.
• It is distinguished from other chronic myeloproliferative neoplasm
by the presence of Philadelphia chromosome in more than 90% of
cases.
• Philadelphia chromosome result from a translocation between long
arm of chromosome 9 and chromosome 22 t (9; 22). The resulting
BCR-ABL1 fusion gene is responsible for autophosphorylation and
constitutive tyrosine kinase activity (p210BCR/ABL). The net
outcome is neoplastic cell proliferation of mainly myeloid
precursors and inhibition of apoptosis.
 JAK2 (Janus kinase) oncogene

• JAK2(V617F) mutation.
• Polycythemia vera (PV).
• High RBCs (major).
• Hb > 20 g/dL.
• High WBCs and PLTs.
• chronic idiopathic myelofibrosis.
• Essential thrombocythemia.
 Flow cytometry

The flow cytometer combines the principles of cell counting

and sorting based on both physical and immunologic
features. Using laminar flow technology, it can routinely
measure 500 to 10,000 cell/second and thus evaluate several
hundred thousand cells in a typical acquisition.
Flow cytometry can evaluate the size and volume as well as
their internal structures. Size and volume are measured from
the forward scatter of the laser beam, while structures in the
cell, such as mitochondria, endoplasmic reticulum and
granules, all deflect the incident light beam and contribute to
the measurement of cell complexity known as side scatter.
How dose a flow cytometer work (principle)?
The flow cytometer is composed of three main systems:
1. Fluidics.
2. Optics.
3. Electronics.
• Fluidics: cells are withdrawn from sample tube and entre the flow
cell where they form a column surrounded by sheath fluid usually
saline, the cell pass through a laser beam, deflecting the light,
cells pass one by one (hydrodynamic focusing).
Signals measurements:
• cell size, forward scatter (FSC), the cells interrupt the laser light
path, which is directly proportional to the size and volume of
cells.
• Cell complexity/granularity: side scatter (SSC), light encounters
the internal structures such as nucleus, specific granules. The light
is scattered in all directions. The more complex of the cell, the
greater the degree of side scatter.
+ CD38 (myeloblasts)

(TdT)

Hairy cell leukemia: CD123, CD24,

CD11c, CD103, CD19.
 Hemostasis
Everybody talks about it, nobody understand it.
HEMOSTASIS
Note that!
• V & VIII are labile factors.
• Factor VIII only factor not
synthesized in the liver.
• Factor X: the convergence
point for both pathways.
• Vitamin K dependent factors:
(X, IX, VII, II, protein C and
S)
 EVALUATION TESTS FOR SECONDARY HEMOSTASIS:
A/ Prothrombin Time (PT): PT measures the clotting time from the
activation of factor VII, through the formation of fibrin clot, is a useful
screening test to know the efficiency of the extrinsic and common
pathways (I, XI, VII, X, but mainly VII). Also used to follow up the
course of oral anticoagulant therapy by (vitamin K antagonists
warfarin/coumadin).
• Principle: VII react with the tissue factor in the presence of the
phospholipids and Cacl2 to activate X and the coagulation mechanism
continue from here to form the clot.
• Reagents: platelets poor plasma (PPP) from citrated blood,
thromboplastin: phospholipids with tissue factor and calcium chloride
“0.025 molar” (keep refrigerated when not used).
• Reference range: 12-14 sec. note that the PT is up to 16 sec at
birth, gradually shortens into the adult normal range by the age
of 6 months.
• Interpretation: common cause of prolonged PT:
1. Oral anticoagulant therapy (warfarin), by blocking the ability
of vitamin K to carboxylate the vitamin K dependent clotting
factors.
2. Vitamin K deficiency.
3. Liver diseases (obstructive).
4. Disseminated intravascular coagulopathy (DIC).
5. Lupus anticoagulant.
6. Deficiency in factors I, II, V, VII (X PT and APTT be
prolonged).
• Normal range:
0.8-1.2
• Therapeutic:
2-3
B/ Activated Partial Thromboplastin Time (APTT):
• Screening test for intrinsic/common pathways (factors XII, XI,
prekallikrein, HMWK, IX, VIII, X, V, II, and I. Monitors heparin
therapy.
• Screen for lupus anticoagulant.
• Principle: measures the clotting time of PPP after activation of
the contact factor with partial thromboplastin (containing
phospholipid “cevalin” and activator “koalin” which assistment
the intrinsic/common pathways.
• Reagents:
a. Cephalin as phospholipids.
b. Partial thromboplastin containing activator (kaolin).
c. Calcium chloride (0.025M).
• Reference range: 25-40 sec.
• Prolonged aPTT can indicate:
1. Anticoagulant therapy (Heparin) or contamination of heparin.
2. Vitamin K deficiency.
3. Liver disease.
4. DIC.
5. Circulating anticoagulant (anti-factor VIII) “lupus”.
6. Deficiency of coagulation factors other than VII.
C/ Thrombin time (TT) or Thrombin clotting time (TCT):
The time require for the thrombin to cleave the fibrinogen into
fibrinopeptide A & B to form the clot. Measure the value of
fibrinogen, monitor fibrinogen abnormalities: hereditary, in the
structure or acquired, liver disease or hepatic cell carcinoma.
• Principle: the test measure the clotting time of plasma after
adding thrombin.
• Reagents: Bovine thrombin solution.
• Reference range: 12-16 sec.
• Prolongation:
1. Hypofibrinogenemia (DIC).
2. High FDPs (DIC).
3. Presence of heparin.
4. Abnormal fibrinogen.
 Precautions
• The ratio in blood collection tubes must be maintained at a 9:1 ratio of
blood to 3.2% sodium citrate anticoagulant or excess citrate will bind
calcium chloride in the reagents for PT and aPTT, causing falsely long
coagulation times.
• Specimens must be processed as soon as possible following blood
collection. Recommendations include processing within 2 hours or 2-5
days at -20° C. for aPTT and 24 hours for PT. Centrifuge to obtain
platelet-poor plasma, and remove plasma from cells; can freeze plasma
at -20° C.
• Testing must be performed at 37° C. Enzyme reactions work best at
37 °C. Labile factors V and VIII will break down at temperatures
above 37 ° C or more than 5 mins of incubation. Factors VII and XI
will be activated at cold temperatures.
Bleeding disorders:
1/ von Willebrand disease:
• Decreased vWF.
• Variable or prolonged aPTT.
• Prolonged bleeding time (BT).
2/ hemophilia :A, classic hemophilia: (80% of the hemophilias):
• Prolonged aPTT.
• Decreased VIII (factor assay).
3/ hemophilia B, Christmas disease: (20% of the hemophilias):
• Prolonged aPTT.
• Decreased IX.
4/ hemophilia C:
• Prolonged aPTT.
• Decreased XI.
X-linked recessive, females are
carriers
5/ Deficiencies of factors XII, prekallikrein, and HMWK:
• Prolonged aPTT
6/ Factor X (Stuart-Prower) deficiency:
• Prolonged PT and aPTT.
7/ Factor V: Prolonged PT and aPTT.
8/ Factor II (prothrombin) deficiency: Prolonged PT and aPTT.
9/ Afibrinogenemia:
• prolonged PT, aPTT and TT,
• no fibrinogen.
10/ Hypofibrinogenemia:
• normal PT and aPTT,
• prolonged TT,
• normal fibrinogen.
11/ Dysfibrinogenemia:
• normal PT and aPTT,
• prolonged TT,
• fibrinogen variable.
12/ Liver disease:
• Prolonged PT, aPTT, bleeding time.
13/ Vitamin K deficiency or warfarin:
• Prolonged PT (VII, X, II).
• prolonged aPTT (IX, X, II).
14/ Disseminated intravascular coagulation (sepsis):
• Prolonged PT, aPTT, and TT.
• Decreased fibrinogen. Fibrin and fibrinogen degradation products
are present (abnormal high D-dimer and FDP tests). Schistocytes
form when RB Cs are fragmented by intravascular clots.
15/ Primary fibrinogenolysis:
• Plasminogen is inappropriately activated to plasmin in the
absence of clot formation. Plasmin circulates free in
plasma and destroys factors I, V, and VIII.
• PT, aPTT, and thrombin time are prolonged, and fibrinogen.
• Fibrinogen degradation products are present (abnormal FDP
test), but fibrin degradation products are absent (normal D-
dimer because there is no clot formation).
• Mixing study: is performed when the PT or aPTT is prolonged
to differentiate a factor deficiency from a circulating inhibitor.
• Principle: Patient plasma is mixed with normal pooled plasma
(FNP) in ratio of 50:50 and test is repeated.
1. Shortening of the time into the reference range (correction)
indicates a factor deficiency (hereditary, or acquired causes
such as warfarin therapy or liver disease).
2. Partial or no correction indicates a circulating inhibitor
(heparin, lupus inhibitor, VIII inhibitor, IX inhibitor).
• Fibrinogen level is a quantitative test for fibrinogen.
Fibrinogen can be quantitatively measured by a modification of
the thrombin time by diluting the plasma, because the thrombin
clotting time of diluted plasma is inversely proportional to the
concentration of fibrinogen (principle of Clauss method). 200-
400 mg/dL.
• Factor assays are used to confirm a suspected factor
deficiency, as suggested by a mixing study that shows
correction.
• Lupus anticoagulant: Lupus anticoagulant is directed
against the phospholipid-dependent coagulation factors.
• APTT is sensitive for LA, anti-phospholipid:
prolonged without correction.
• Dilute Russell viper venom test (dRVVT): is a
sensitive test that uses snake venom as the reagent to
activate factor X in the cascade. If the lupus inhibitor
is present, the venom is neutralized, and the test is
prolonged.
Protein C and S + thrombomodulin (thrombophilic), they
want you to bleed.
• The protein C and S are vitamin K dependent factors and
they are important in stopping coagulation by inactivating
factors V and VIII.
• Deficiency of these factors lead to hypercoagulable state
(deep vein thrombosis).
 EVALUATION TESTS FOR THE FIBRINOLYTIC
SYSTEM
Fibrin Degradation Products (FDPs):
• D-Dimer Assay:
• Presence of D-dimer requires recent intravascular
coagulation.
• < 0.5 mg/dL or < 500 µg/dL.
• High D-dimer: DIC, severe sepsis, malignancies, liver
disease and normally in pregnancy.
Anticoagulant PT aPTT
Heparin Normal Prolonged

Warfarin Prolonged Normal

(Coumadin)
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