2. Part 1-
1) Constituents of hemostasis ~ Role players
2) Normal coagulation homeostasis
3) Bleeding disorders- introduction & classification
4) Clinical evaluation of bleeding disorders
Part 2-
1) Lab investigations:-
I) Screening
A. Screening tests for primary hemostasis
B. Screening tests for secondary hemostasis
II) Specific tests
A. Tests for platelet functions
B. For coagulation factors
C. For fibrinolysis.
2) Different scenarios, Summary and conclusion
6. Screening tests for primary hemostasis are -
I. Bleeding time
II. Peripheral blood smear examination
III. Platelet count
IV. Mean Platelet volume
V. Platelet distribution width
VI. Reticulated platelets
VII. Platelet function analysis
VIII. Tests for Vessel wall disorder
7. Bleeding time
ï The bleeding time assesses primary hemostasis ï Assesses adequate functioning of platelets and
blood vessels
ï 3 methods available ï Dukeâs, Ivyâs, and template.
ï Dukeâs methodï Measures bleeding time following ear lobe puncture ~ is not advocated since it
cannot be standardized and can cause a large local hematoma.
ï Ivyâs methodï 3 punctures are made on the volar surface of the forearm with a lancet (cutting
depth 2-2.5 mm) under standardized venous pressure (40 mm Hg) and the average time required
for bleeding to cease from the puncture sites is measured.
ï Template methodï uses a special surgical blade, which makes a larger cut (5 mm long and 1 mm
deep) ~ Although template method is better, it can produce a large scar and even a keloid in
predisposed individuals.
ï Reference range: 2-7 minutes (Ivyâs) ~ in most BT <4 mins. It should be reported in minutes or
nearest half minute. If bleeding continues beyond 20 minutes, BT should be reported as >20
minutes and the test is discontinued.
8. Bleeding time.. contâd..
ï BT provides a measure of the efficiency of the vascular and platelet phases.
ï So BT is prolonged in vascular and platelet disorders.
ï However, it canât discriminate between vascular defects, thrombocytopenia, and
platelet dysfunction.
ï The bleeding time leaves much to be desired in terms of reproducibility, because no
two skin areas are exactly the same and it is impossible to produce a truly standard
wound.
9. Peripheral blood smear examination
Cytopenia Anemia, Leukopenia,Thrombocytopenia
RBCs Fragmented RBCs ï DIC with thrombocytopenia.
Schistocytes ï also found in thrombotic
thrombocytopenic purpura.
WBCs Abnormal cells with thrombocytosis/thrombocytopenia
(leukemias)
Platelets Normal count ï 1 platelet per 500-1000 RBCs
Giant platelets ï Myeloproliferative neoplasms
Bernard soulier syndrome
ITP
Discrete isolated
platelets without
clumping in finger prick
smear
Glanzmannâs thrombasthenia
Uremia
Microthrombocytopenia Wiskott-Aldrich Syndrome
Schistocytes inTTP
Giant platelet
10. Platelet count
ï2 methods available â 1) Manual & 2) Automated
Manual method
ï¶ Principle:-
Whole blood(venous) sample is mixed with a diluent.
Diluent ï 1% ammonium oxalate (in which RBCs are lysed).
0.38 ml of 1% ammonium oxalate taken in a test tube ~ added 20 ÎŒl of
well-mixed anticoagulated(EDTA) venous blood(NOT capillary) and
mix thoroughly.
Dilution of blood is 1:20.
An improved Neubauer counting chamber is filled with the mixture, and
platelets are counted under the microscope ~ Result is expressed as
platelets/ÎŒl (or /liter).
Automated method
ï¶ By ï Automated hematology analyzers
ï¶ Requiredï Small aperture tube. A threshold needs to be set to exclude
larger red cells and smaller debris.
(Platelet counting is usually based on the principle of aperture impedance)
11. What is thrombocytopenia?
ï¶ A count <100,000 /ÎŒL is generally considered to constitute thrombocytopenia.
ï¶Platelet counts in the range of 20,000 to 50,000/ÎŒL can aggravate posttraumatic
bleeding.
ï¶Platelet counts <20,000 /ÎŒL may be associated with spontaneous (nontraumatic) bleeding.
What is pseudothrombocytopenia?
Pseudothrombocytopenia or spurious thrombocytopenia is characterized by clumping of
platelets which mimics a low platelet count.
Causes?
1) PLATELET CLUMPING ~ use of automated counters.
2) Platelet satellitism
3) Use of EDTA as an anticoagulant
Also a/w platelet cold agglutinins, and with multiple myeloma.
12. Platelet satellitism ï a rare condition that occurs when an
IgG antibody forms in the presence of EDTA. IgG antibody is
directed against the gp IIb/IIIa ï As the antibody coats the
platelets ï the platelets rosette around segmented
neutrophils, bands, and sometimes around monocytes.
Antibody-coated platelets that are huddled around white blood
cells (WBCs) will not be counted as platelets by automated
equipment and the platelet count will be falsely decreased. If a
peripheral blood smear is reviewed, platelets will be observed
attached toWBCs..
What to do then?
If either platelet satellitism or platelet clumping is observed on
the peripheral smear, the sample could be recollected using
sodium citrate as the anticoagulant. Platelets can then be
counted using the automated method.
The platelet count from a tube that contains liquid sodium citrate
will need to be corrected for the dilutional effect of the citrate. This
can be accomplished by multiplying the platelet count that is
obtained from the automated analyzer by 1.1.
13.
14.
15. MPV (mean platelet volume)
Normal rangeï 7.5-11.5 fL
MPV increased MPV normal/decreased
1. myeloproliferative disorders
2. peripheral destruction of platelets
3. Bernard-Soulier syndrome
1. Thrombocytopenia due to impaired
platelet production
2. Sepsis
Platelet distribution width(PDW)
Measure of degree of variation in platelet size.
PDW increased PDW normal
Myeloproliferative disorder Secondary or reactive thrombocytosis
Reticulated plateletsï Reticulated platelets are immature platelets circulating in blood; they reflect
the activity of megakaryopoiesis in the bone marrow.
Increased Normal
Peripheral destruction Aplastic anemia
17. PLATELET FUNCTION ANALYZER (PFA)100
âą In vitro system for measuring plt-vwf function.
âą Assesses both platelet adhesion & aggregation
âą More sensitive than BT to asses Primary hemostasis.
âą The membrane is coated with collagen & epinephrine or collagen & ADP.
âą It reproduces platelet vwf function under high shear rate.
âą Time req. for closure of full aperture is closure time
âą Normal closure time: 1 to 3 mins.
19. Tests for Vessel wall disorder
LABORATORY TESTS:
ïTPC, PT, APTT,TT are usually normal.
ïThe only test of any use is BT.
ïBT may be normal or increased.
HESS` CAPILLARY FRAGILITY TEST:
ïCuff is wrapped in upper arm and pressure is maintained midway b/w systolic and
diastolic BP for 15 minutes, 4 cm below the elbow joint, a circle of 2.5 cm diameter is
drawn on the anterior aspect of forearm.
ïUpto 10 new hemorrhagic spots are normal.
But >20 new spots are always pathological.
ïThis is positive in increased capillary fragility, ITP.
21. Screening tests for secondary hemostasis are -
I. Clotting time
II. Prothrombin time (PT) and Activated partial thromboplastin time (aPTT)
III. Thrombin Time (TT)
22. ï In general, meticulous performance of coagulation tests is more important than the exact
technique chosen.
ï A poorly collected blood sample is a far more common cause of inaccurate results than is
technical error.
ï With the exception of one assay for fibrin degradation products (FDPs), all coagulation tests
are performed on citrated plasma, most commonly obtained using blue-top vacuum blood
collection tubes that pull in nine parts of blood to one part citrate (9:1).
ï The International Society for Thrombosis and Haemostasis recommends the routine use of
3.2% sodium citrate.
ï A pool of freshly frozen citrated plasma from several normal donors is a suitable control for
screening procedures in most laboratories. Lyophilized control plasma and borderline
abnormal control plasmas are available commercially
23. Collection of blood for coagulation studies
ï Venous blood is collected from antecubital fossa with a plastic or polypropylene syringe and a large bore
needle (20 or 21 G in adults, 22 or 23 G in infants).
ï Blood should never be collected from indwelling intravenous lines, as these often contain heparin.
ï Glass syringe should not be used for collection since it activates coagulation.
ï The blood is drawn gently but quickly after a single, smooth venepuncture.
ï The needle is detached from the syringe, and the sample is passed gently into the plastic container.
ï After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5
times.
ï The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood
proportion being 1:9.
ï Most coagulation studies require platelet poor plasma (PPP).
ï To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes.
ï Coagulation studies are carried out within 2 hours of collection of sample.
24. Clotting Time
ï This is a crude test and is now replaced by activated partial thromboplastin time.
ï Clotting time measures the time required for the blood to clot in a glass test tube kept at
37°C.
ï The expected range for clotting time is 4-10 mins.
ï Prolongation of clotting time only occurs in severe deficiency of a clotting factor and is
normal in mild or moderate deficiency.
25. PROTHROMBIN TIME(PT)
ï PT assesses coagulation factors in extrinsic pathway
(F VII) and common pathway.
ï Principle:- Tissue thromboplastin and calcium are
added to platelet poor plasma and clotting time is
determined.
ï Method:-
1. Deliver 0.1 ml of plasma in a glass test tube kept
in water bath at 37°C.
2. Add 0.1 ml of thromboplastin reagent and mix.
3. After 1 minute, add 0.1 ml of calcium chloride
solution.
4. Immediately start the stopwatch and record the
time required for clot formation.
26. CONCEPT OF INR
1. The international normalized ratio (INR) was introduced in an attempt to standardize the PT.
2. In its original manifestation, the PT was very variable because different thromboplastins were non-standardized
and derived from many varied sources. PTs performed on the identical specimen by different laboratories were
inconsistent.
3. Calculation ~ INR = [ PT (patient) / PT (Control) ]ISI
The INR has no units (it is a ratio) and is determined to one decimal place.
**ISI, or international sensitivity index is a function of the thromboplastin reagent.
** NORMAL RANGEï PT 11-16 seconds INR 0.9 â 1.1
INR should be maintained in the therapeutic range.. Providing adequate anticoagulation.
27. Causes of prolongation of PT
1. Treatment with oral anticoagulants
2. Liver disease
3. Vitamin K deficiency
4. Disseminated intravascular coagulation
5. Inherited deficiency of factors in extrinsic and common pathways.
Uses of PT
1. To monitor patients who are on oral anticoagulant therapy
2. To assess liver function
3. Detection of vitamin K deficiency
4. To screen for hereditary deficiency of coagulation factors
28. Significance
ïReflects efficiency of Intrinsic and Common pathway.
ïSensitive to changes in Factor VIII,IX,XI,XII.
ïAlso sensitive to heparin & circulating anticoagulants.
Principle
The test measures the clotting time of plasma after the activation of contact factors
(Kaolin/Silica/Ellagic acid) and the addition of phospholipid and CaCl2, but without
added tissue thromboplastin.
So it indicates the overall efficiency of the Intrinsic pathway.
Normal range
26 to 40 seconds.
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)
29. Uses of APTT:-
1. Screening for hereditary disorders of coagulation
2. To monitor heparin therapy
3. Screening for circulating inhibitors of coagulation
30. aPTT is prolonged in:-
1. Inherited deficiencies of factor VIII (Hemophilia A) and Factor IX (Hemophilia B)
2. Non specific inhibitor antibodies against F VIII e.g. Lupus inhibitor
(Donât act directly but block interaction of FVIII with other clotting factors)
3. DIC
4. Heparin
(ï Inhibits factor XII, XI and X through antithrombin III & Heparin therapy is monitored
through aPTT)
5. Vit K deficiency
6. Massive transfusion of plasma depleted stored blood.
31. Significance:-
ï Asses the final step of coagulation i.e. conversion of fibrinogen to fibrin in presence of
thrombin.
ïBypasses Extrinsic & Intrinsic pathway.
Principle:-
ïThrombin is added to plasma and the clotting time is measured.
ïTT is affected by the concentration and reaction of fibrinogen and by the presence of
inhibitory substances including fibrinogen/fibrin degradation products(FDPs) and heparin.
Normal range:-
A patientâs TT should be within ± 3 seconds of control.
Times of 20 s and longer are definitely abnormal.
THROMBIN TIME(TT)
33. ï It is a prothrombin test similar to the (one-stage) prothrombin time
ï But performed with Russell's viper venom (Stypven) as the thromboplastic agent
ï Useful in defining deficiencies of blood coagulation factor X.
What is Stypven time test?
34. Usually done by CLAUSS TECHNIQUE.
Principle
Diluted plasma is clotted with a strong thrombin solution.
The plasma must be diluted to give a low level of any inhibitors(e.g. FDPs and heparin).
A strong thrombin solution must be used so that the clotting time over a wide range is
independent of the thrombin concentration.
Normal range
1.8 to 3.6 g/l
Interpretation
ï¶Sensitive to inherited Dysfibrinogenaemia.
ï¶Insensitive to Heparin unless the level is very high(>0.8”/”l).
ï¶High level of FDP(>190”g/ml) may also interfare with the result.
Fibrinogen Assay
37. Specific tests for platelet functions are -
I. Test for platelet adhesion (Glass bead column test)
II. Test for platelet aggregation ~ Platelet Aggregometry
III. Tests for platelet release reaction
IV. Tests for clot retraction
V. Tests for platelet procoagulant activity
VI. Flow Cytometry ~ GP receptor status
VII. Tests for defects in Arachidonic acid metabolism
38. I. Test for platelet adhesion (Glass bead column test)
Normals:
>25 % retention of platelets Normal
Abnormals:
<25% retention is observed with vWD
39. II.Test for platelet aggregation ~ Platelet Aggregometry
ï Gold standard of platelet function testing and
is still the most used test for the identification
and diagnosis of platelet function defects.
ï Platelet rich plasma (PRP) is stirred within a
cuvette located between a light source and a
detector.
ï After addition of a various panel of agonists,
such as collagen, ADP, thrombin, ristocetin,
epinephrine, and arachidonic acid, the
platelets aggregate and light transmission
increases.
A light beam, passing through the PRP
sample, is measured.
As platelets aggregate, more light passes.
This determines the percentage of
aggregation.
40. ï The platelet aggregation pattern is thought as a primary response to an exogenous
agonist, followed by a secondary response to the release of dense granule contents.
ï This biphasic response can be masked if high concentrations of agonists are
added.
ï Parameters measured include the rate or slope of aggregation (%/min) and the
maximal amplitude (%) or percentage of aggregation after a fixed period of time,
usually 6â10min.
Test for platelet aggregation ~ .. Contâd..
42. Test for platelet aggregation ~ .. Contâd..
Various normal curves
Aggregating agents
used
Curve type
ADP / Epinephrine Biphasic
Collagen / ristocetin /
Arachidonic acid
Monophasic curve
43. Abnormal platelet aggregation tests ~
Scenario 1. ~ Glanzman Thrombasthenia
1. In Glanzmannâs thrombasthenia, Platelet GPIIb/IIIa (important for platelet aggregation) is defective.
3. All agents induce aggregation through this receptor except ristocetin.
4. Hence in Glanzmannâs thrombasthenia, aggregation is defective for all agents except ristocetin.
44. Abnormal platelet aggregation tests ~
Scenario 2. ~ BERNARD SOULIER SYNDROME AND VON WILLEBRAND DISEASE
1. In Bernard soulier syndrome there is deficiency of GpIb/IX, whereas in von willebrand disease, there
is deficiency of von willebrand factor.
2. Aggregation is there in response to all agents except ristocetin
BS and vWD can be
differentiated by addition of
normal plasma.
If the aggregation is seen now,
it means the disease is vWD
because normal plasma is a
source of vWF.
Then how can we
differentiate between these 2
conditions?
45. Abnormal platelet aggregation tests ~
Scenario 3. ~ STORAGE POOL DEFECTS
1. In this disorder ï defective granule release from platelets
2. Due to defective release reaction, secondary aggregation is defective
3. Hence there is no secondary wave in response to ADP/epinephrine/collagen and only partial
aggregation in response to ristocetin.
47. III. Tests for platelet release reaction
Indirect Tests
The secondary wave of aggregation in response to ADP/epinephrine/collagen is a
measure of release reaction.
Direct Tests
1. estimation of secretion of dense core granules
2. estimation of secretion of alpha granules
Abnormals:
1. Storage pool deficiency
2. Aspirin like defect (COX deficiency)
48. IV. Tests for clot retraction
ï Clot retraction is the "shrinking" of a blood clot over a number of days. In so doing, the
edges of the blood vessel wall at the point of injury are slowly brought together again to
repair the damage.
ï Clot retraction is dependent on ï release of multiple coagulation factors from
platelets trapped in the fibrin mesh of the clot. Thus, failure to retract can be a sign
of thrombocytopenia or Glanzmannâs thrombasthenia.
49. V. Tests for platelet procoagulant activity
This test measures amount of residual prothrombin activity after whole blood is allowed to clot completely.
Whole blood In plain tube ï allowed to clot ï serum with residual prothrombin ï Perform PT (PT1)
Citrated plasma (same pt) ï Perform PT (PT2)
PT1
= PLATELET PROCOAGULANT ACTIVITY
PT2
Interpretation:-
Presence of residual prothrombin (PCA >1) may be due to deficiency of coagulation factors or of platelet
phospholipids.
50. âą Advantages:
-Small volume of whole blood and
-Independence of platelet count.
âą Most commonly used routine
flow cytometry tests are the
quantification of the basal
platelet GP receptor status and
the determination of the platelet
granule composition.
VI. Flow Cytometry ~ GP receptor status
51. The most common platelet activation markers assessed by flow cytometry
are -
âą the conformational change of GP IIb/IIIa into its active state (measured with monoclonal antibody
PAC-1),
âą P-selectin expression on the platelet surface (as a marker of đŒ-granule secretion),
âą platelet-leukocyte conjugates,
âą microparticle examination,
âą exposure of anionic, negatively charged phospholipids on the platelet surface (procoagulant
activity),
âą phosphorylation of vasodilator stimulated phosphoprotein-phosphorylation (VASP-P) (Bio- Cytex,
Marseille, France), as a marker of P2Y12 receptor activation-dependent signaling
Flow Cytometry.. Contâd..
52. VII. Tests for defects in Arachidonic acid metabolism
1. Arachidonic acid is necessary for TXA2 generation which activates platelets.
2. Normal aggregation in response to Arachidonic acid shows defects in arachidonic acid
metabolism pathway
54. 1) Coagulation factor assays
2) Quantitative estimation of fibrinogen
3) Thromboplastin generation test (TGT)
4) Mixing test based on PT/aPTT
5) FXIII Qualitative assay (Urea clot lysis test)
6) Paracoagulation tests (protamine sulphate test & ethanol gelation test)
Specific tests for ~ Coagulation factors
55. Coagulation factor assays
Principle:-
Patientâs plasma + standard plasma deficient factor to be tested in various dilutions
Perform PT and aPTT
NORMAL PROLONGED
Patientâs plasma contains that factor Patientâs plasma doesnât contain that factor
56. Quantitative estimation of fibrinogen
Methods:-
1. Coagulable protein method (based on thrombin time)
2. Immunological method (RIA based)
3. Heat precipitation test
4. Chemical precipitation test
5. Weighing of clot
Normals:-
Fibrinogen levels 200-400 mg/dl
Abnormals:-
Decreased fibrinogen Afibrinogenemia, dysfibrinogenemia, DIC
Increased fibrinogen MI, trauma, neoplasia, inflammatory conditions
58. If the stage I is
defective then PT
and aPTT are done
and substitutions
studies are carried
out to determine
deficient factors in
plasma and serum.
TGT.. Contâd..
ï¶ prepared serum contains FV andVIII
ï¶ adsorbed plasma â FIX and X
ï¶ Both contain â FXI and XII
60. Mixing Study based on PT/aPTT
Done to differentiate clotting
factor deficiency from
prolonged PT/aPTT due to
inhibitors
61. FXIII Qualitative assay (Urea clot lysis test)
ï Done when all other tests for hemostasis are normal.
ï FXIII provides stability to clot formed.
Method:-
71. 3PTâN
APTTâ PROLONGED
TTâN
FIBRINOGENâN
PLT COUNTâN
Interpretation
1. Congenital deficiency of F VIII, F IX, prekallikrein or HMWK.
2. von Willebrand disease.
3. Heparin-either pt. is on t/t or contaminated sample.
4. Circulating anticoagulants-
Specific (Anti factor VIII).
Non-specific(Antiphospholipid Ab).
78. ï¶ Reptilase time (RT) is a blood test used to detect deficiency or abnormalities in fibrinogen,
especially in cases of heparin contamination.
ï¶ Reptilase, an enzyme found in the venom of Bothrops snakes, has activity similar to thrombin.
ï¶ Unlike thrombin, reptilase is resistant to inhibition by antithrombin III.
ï¶ Thus, the reptilase time is not prolonged in blood samples containing heparin, hirudin, or
direct thrombin inhibitors, whereas the thrombin time will be prolonged in these samples.
ï¶ Reptilase also differs from thrombin by releasing fibrinopeptide A, but not fibrinopeptide B, in
its cleavage of fibrinogen.
ï¶ Other causes of prolonged reptilase time include the presence of fibrin degradation products,
which interfere with fibrin polymerization.
REPTILASE OR ANCORDTIME
84. FDP ASSAY
Plasminogen
Plasmin
Fibrinogen/ FDP X,Y,D,E
Fibrin
Principle
A Suspension of latex particles sensitized with specific Ab to FDP
fragments D & E.
Suspension mixed on a glass slide with a dilution of test serum.
Agglutination indicates presence of FDPs.
85. Interpretation of FDPAssessment
Agglutination with 1 in 5 dilution:-FDP >10”g/ml
Agglutination with 1 in 20 dilution:-FDP>40”g/ml
ïNormal range- <10 ”g/ml
ï10-40 ”g/ml- Acute myocardial Infarction.
Acute venous thromboembolism.
Acute pneumonia.
ï>40 ”g/ml- DIC.
Thrombolytic therapy with Streptokinase .
86. D-DIMER ASSAY
Identical to FDP except latex particles are coated with a Monoclonal Antibody
specifically directed against Fibrin D-dimer in human.
Normal range < 200mg/l.