The document discusses tests used to evaluate bleeding disorders. It begins by describing normal hemostasis and the role of platelets and coagulation factors. It then discusses specific bleeding disorders that can result from platelet defects, coagulation factor deficiencies, or fragile blood vessels. The document outlines several laboratory tests used to evaluate hemostasis, including bleeding time, clotting time, platelet count, prothrombin time, activated partial thromboplastin time, thrombin time, and capillary fragility test. Each test is described in terms of its methodology, what coagulation or platelet pathways it evaluates, and how results are interpreted to identify potential causes of bleeding disorders.

1. Tests of
bleeding
disorders
Dr. Chintan Parmar
-1st year Resident,
-Dept. of Physiology.
-Dt. :10/02/2011

2. OUTLINE
• Normal Hemostasis
• Coagulation Factors
• Coagulation Pathway
• Bleeding Disorders
• Laboratory Tests

3. Normal Hemostasis
• Trauma, disease or surgery – Disruption of
vascular endothelial lining
• Primary – Platelet Plug Formation – within
seconds – capillaries, small arterioles &
venules
- Platelet adhesion, granule release &
aggregation
- Role of vWF ( Von Willebrand Factor )
- Role of fibrinogen

4. Normal Hemostasis
• Secondary – plasma coagulation
system – fibrin formation – several
minutes – larger vessels
• Interlinking – Activated platelets
accelerate plasma coagulation &
products of plasma coagulation
reaction such as thrombin induce
platelet activation.

5. 5
Normal Hemostasis
BV Injury
Platelet
Activation
Plt-Fusion
Blood Vessel
Constriction
Coagulation
Activation
Stable Hemostatic Plug
Thromibn,
Fibrin
Reduced
Blood flow
Tissue
Factor,
blood
Primary hemostatic plug
Neural

6. Coagulation Factors
• Factor I : Fibrinogen
• Factor II : Prothrombin
• Factor III : Thromboplastin ( Tissue Factor )
• Factor IV : Calcium
• Factor V : Labile factor or proaccelerin
• Factor VII : Stable factor or proconvertin
• Factor VIII : Antihemophilic globulin (AHA)
• Factor IX : Christmas factor (AHB)
• Factor X : Stuart-Prower factor
• Factor XI : Plasma thromboplastin antecedent (AHC)
• Factor XII : Hageman factor
• Factor XIII : Fibrin-stabilizing factor

8. Bleeding Disorders
• Increased fragility of vessels
– Aging
– Medications (steroids)
• Platelet defect (deficiency)
– Low platelet count
– Medication (aspirin, clopidogrel)
– Congenital platelet defect
• Deranged clotting
– Factor deficiency
– Medication (coumarin)
– Coagulation inhibitor

9. Coagulation Factor
Disorders
• Acquired
– Vitamin K deficiency
– Parenchymal disease of liver
• Hereditary deficiencies
– Factor VIII : hemophilia A
– Factor IX : hemophilia B
– Factor XI : hemophilia C
–Von Willebrand’s Disease

10. Laboratory tests
1. Tests of the Vascular Platelet Phase of
Hemostasis:
• BBlleeeeddiinngg TTiimmee ((BBTT)
2. Tests of the Coagulation Cascade:
• CClloottttiinngg TTiimmee (( CCTT) oorr CCooaagguullaattiioonn ttiimmee
• AAccttiivvaatteedd PPaarrttiiaall TThhrroommbbooppllaassttiinn TTiimmee
((AAPPTTTT)..
• PPrrootthhrroommbbiinn TTiimmee ((PPTT)
• TThhrroommbbiinn TTiimmee ((TTTT)

11. Laboratory tests
3. OTHERS:
- Platelet Count
- Capillary fragility test
- Clot Solubility Test
- Clot Retraction Test
- Thromboplastin Generation Test
- Factors, fibrinogen, anticoagulants assay
- Platelet function tests (aggregation)
- Tests for Liver function

12. 1. Bleeding Time
• It is the time taken from the puncture of the
blood vessel to the stoppage of bleeding.
• The bleeding time test is a useful tool to test
for platelet plug formation and capillary
integrity.
• BT is more imp. than CT.
• CT concerns the blood only i.e. how firm
the the clot is formed, whereas BT involves
the interaction of blood with the injured
tissues.

13. Duke Method
• With the Duke method, the patient is
pricked with a special needle or lancet,
preferably on the earlobe or fingertip,
after having been swabbed with alcohol.
• The prick is about 3-4 mm deep. The
patient then wipes the blood every 30
seconds with a filter paper.
• The test ends when bleeding stops. The
usual time is about 2-6 minutes.

14. Ivy method
٠ Clean the anterior surface of the forearm with spirit.
• The blood pressure cuff is placed on the upper arm
and inflated to 40 mmHg.
• A lancet or scalpel blade is used to make a shallow
incision that is 1 millimeter deep on the anterior of
the forearm.
• The time from when the incision is made until all
bleeding has stopped is measured and is called the
bleeding time. Every 30 seconds, filter paper or a
paper towel is used to draw off the blood.
• Normal BT by this method is 3-6 minutes.

15. Bleeding Time

16. Bleeding Time
• A prolonged bleeding time may be a result from
decreased number of thrombocytes or impaired
blood vessels.
• Bleeding time is affected by platelet function,
certain vascular disorders and von Willebrand
Disease, not by other coagulation factors.
• Diseases that cause prolonged bleeding time include
thrombocytopenia, disseminated intravascular
coagulation (DIC).
• Aspirin and other cyclooxygenase inhibitors can
prolong bleeding time significantly.

17. Bleeding Time
• People with von Willebrand disease usually
experience increased bleeding time.
• Von Willebrand factor is a platelet agglutination
protein, but this is not considered an effective
diagnostic test for this condition.
• It is also prolonged in hypofibrinogenemia.
• Many experts regard the bleeding time as useless, in
that it does not predict surgical bleeding.
• Role in post Myocardial Infraction patients.

18. 2. Clotting Time
• It is the time taken from the puncture of the blood
vessel to the formation of a fibrin thread.
• A. Capillary Glass Tube Method : Here the blood is
collected in capillary tube & total time is noted to
form FIBRIN THREADS on breaking tube every 30
seconds. N : 3-8 minutes
• B. Lee & White method : Here venous blood is
collected in 8 mm diameter glass tube, rocked in a
water bath at 37°C & time is noted from the time of
vene puncture till the blood stops flowing. N : 6-12
minutes.

19. Clotting Time
• Mechanism Involved is INTRINSIC Pathway.
• CT depends on presence of all clotting factors.
• It gets prolonged in :
- 1. Deficiency of clotting factors – Hemophilia.
- 2. Vitamin K Deficiency – Factor II, VII, IX & X.
- 3. Anticoagulant overdose.
• BT & CT is measured before surgery & liver or
bone marrow biopsy.
• PURPURA : BT increased, CT normal.
• HEMOPHILIA : BT normal, CT increased.

20. Clotting Time

21. 3. Capillary Fragility Test
• A tourniquet test (also known as a
Rumpel-Leede Capillary-Fragility Test or
Hess capillary fragility test) determines
capillary fragility.
• It is a clinical diagnostic method to
determine a patient's haemorrhagic
tendency.
• It assesses fragility of capillary walls and
is used to identify thrombocytopenia.

22. Capillary Fragility Test
• The test is defined by the WHO as
one of the necessary requisites for
diagnosis of Dengue fever.
• A blood pressure cuff is applied and
inflated to a point between the
systolic and diastolic blood pressures
for five minutes.
• The test is positive if there are 10 or
more petechiae per square inch.
• In DHF the test usually gives a
definite positive result with 20
petechiae or more.

23. 4. Prothombin Time
• The prothrombin time was discovered by Dr. Armand Quick
and colleagues in 1935 and a second method was published
by Dr.Paul Owren also called the "p and p" or "prothrombin
and proconvertin" method.
• It aided in the identification of the anticoagulants dicumarol
and warfarin and was used subsequently as a measure of
activity for warfarin when used therapeutically.
• The INR was introduced in the early 1980s when it turned
out that there was a large degree of variation between the
various prothrombin time assays, a discrepancy mainly due
to problems with the purity of the thromboplastin (tissue
factor) concentrate.
• The INR became widely accepted worldwide, especially
after endorsement by the World Health Organisation.

24. Prothombin Time
• The prothrombin time (PT) and its derived
measures of prothrombin ratio (PR) and
international normalised ratio (INR) are measures
of the extrinsic pathway of coagulation.
• They are used to determine the clotting tendency of
blood, in the measure of warfarin dosage, liver
damage and vitamin K status.
• PT measures factors I, II, V, VII and X.
• The reference range for prothrombin time is usually
around 11-16 seconds; the normal range for the INR
is 0.8–1.2.

25. Prothombin Time
• Method
• The prothrombin time is most commonly measured using
blood plasma. Blood is drawn into a test tube containing
liquid citrate, which acts as an anticoagulant by binding the
calcium in a sample.
• The blood is mixed, then centrifuged to separate blood cells
from plasma.
• The plasma is analyzed by an automated instrument at
37°C. An excess of calcium is added to reverse the effects
of citrate , which enables the blood to clot again.
• Tissue factor (also known as factor III) is added, and the
time the sample takes to clot is measured.
• The prothrombin ratio is the prothrombin time for a patient,
divided by the result for control plasma.

26. Prothombin Time

27. Prothombin Time
• International normalised ratio
• The result (in seconds) for a prothrombin time performed on
a normal individual will vary depending on what type of
analytical system it is performed.
• This is due to the differences between different batches of
manufacturer's tissue factor used in the reagent to perform
the test.
• The INR was devised to standardize the results. Each
manufacturer assigns an ISI value (International Sensitivity
Index) for any tissue factor they manufacture. The ISI value
indicates how a particular batch of tissue factor compares to
an internationally standardized sample. The ISI is usually
between 1.0 and 2.0.

28. Prothombin Time

29. Prothombin Time
• Interpretation
• The prothrombin time is the time it takes plasma to clot
after addition of tissue factor obtained from animals. This
measures the quality of the extrinsic pathway as well as the
common pathway of coagulation.
• The speed of the extrinsic pathway is greatly affected by
levels of factor VII in the body. Factor VII has a short half-life
and its synthesis requires vitamin K.
• The prothrombin time can be prolonged as a result of
deficiencies in vitamin K, which can be caused by warfarin,
malabsorption, newborns.
• In addition, poor factor VII synthesis due to liver disease or
increased consumption in disseminated intravascular
coagulation may prolong the PT.

30. Prothombin Time

31. 5. APTT
• The partial thromboplastin time (PTT) or activated
partial thromboplastin time (aPTT or APTT) is a
performance indicator measuring the efficacy of both
the intrinsic and the common coagulation pathways.
• Apart from detecting abnormalities in blood clotting, it
is also used to monitor the treatment effects with
heparin, a major anticoagulant.
• Kaolin cephalin clotting time (KccT) is a historic
name for the activated partial thromboplastin time.
• The aPTT was first described in 1953 by researchers at
the University of North Carolina at Chapel Hill.

32. APTT
• Method
• Collection of blood samples is done in vacu-tubes with
oxalate or citrate to arrest coagulation by binding
calcium.
• In order to activate the intrinsic pathway, phospholipid,
an activator such as silica, celite, kaolin, ellagic acid
and calcium to reverse the anticoagulant effect of the
oxalate are mixed into the plasma sample.
• The time is measured until a thrombus (clot) forms.
• The test is termed "partial" due to the absence of tissue
factor from the reaction mixture.

33. APTT
• After
centrifugation, the
Plasma contains all
the intrinsic
coagulation factors
except calcium
(removed during
anticoagulation)
and the platelets
(removed during
centrifugation).

34. APTT
• Interpretation
• Normal value : 25 – 39 seconds.
• Normal PTT times require the presence of the following
coagulation factors: I, II, V, VIII, IX, X, XI & XII.
• Deficiencies in factors VII or XIII will not be detected with
the PTT test.
• Prolonged APTT may indicate:
- use of heparin
- coagulation factor deficiency (e.g. hemophilia A , B )
- antiphospholipid antibody especially lupus anticoagulant,
which increases propensity to thrombosis.

35. APTT
• To distinguish the causes, mixing tests are
performed, in which the patient's plasma is mixed
initially at a 50 : 50 dilution with normal plasma.
• If the abnormality does not disappear, the sample is
said to contain an "inhibitor" , either heparin,
antiphospholipid antibodies or coagulation factor
specific inhibitors.
• If it does correct, a factor deficiency is more likely.
• Deficiencies of factors VIII, IX, XI and XII and
rarely von Willebrand factor, if causing a low factor
VIII level, may lead to a prolonged aPTT, that
corrects on mixing studies.

36. APTT

37. APTT

38. APTT

39. 6. Thrombin Time
• The Thrombin Time (TT) , is a blood test which measures
the time it takes for a clot to form in the plasma from a
blood sample in anticoagulant which had added an excess of
thrombin.
• This test is repeated with pooled plasma from normal
patients.
• The difference in time between the test and the 'normal'
indicates an abnormality in the conversion of fibrinogen (a
soluble protein) to fibrin, an insoluble protein.
• This test is also known as the Thrombin Clotting Time
(TCT).

40. Thrombin Time
• Thrombin time compares a patient's rate of clot
formation to that of a sample of normal pooled
plasma.
• Thrombin is added to the samples of plasma. If the
plasma does not clot immediately, a quantitative
(fibrinogen deficiency) or qualitative (dysfunctional
fibrinogen) defect is present.
• If a patient is receiving heparin, a substance derived
from snake venom called reptilase is used instead of
thrombin.
• Reptilase has a similar action to thrombin but unlike
thrombin, it is not inhibited by heparin.

41. Thrombin Time
• The thrombin time is used to diagnose bleeding
disorders and to assess the effectiveness of fibrinolytic
therapy.
• The time between the addition of the thrombin and the
clot formation is recorded as the thrombin clotting time
• Normal values for thrombin time are 10 to 15 seconds.
• If reptilase is used, the reptilase time should be between
15 and 20 seconds.
• Thrombin time can be prolonged by : heparin, FDPs,
factor XIII deficiency and fibrinogen deficiency /
abnormality.

42. Thrombin Time

43. 7. Platelet Count
• Platelet Count can be determined by improved
Neubauer’s counting chamber with RBC
pipette & 1% ammonium oxalate.
• They can be seen as tiny diameter, well
seprated, highly refractile rounded bodies with
silvery appearance.
• N : 1.5 – 4 lacs / cumm.
• Leishman stain : in clumps, blue cytoplasm,
reddish purple granules, no nucleus.

44. Platelet Count

45. Platelet Count
• Surgical bleeding usually does
not occur until the platelet count
is less than 50,000.
• Spontaneous bleeding does not
occur until the platelet count is
less than 10,000-20,000.

46. Platelet Count
• THOMBOCYTOPENIA :
• Bone marrow depression – Aplastic anemia
• Hypersplenism
• Viral infections
• Drugs : Aspirin, Heparin, Chemotherapy
• ITP
• TTP
• HUS
• DIC

47. 8. Thromboplastin Generation Test
• This test measures generation of
thromboplastin – intrinsic mechanism
• N : 12 sec or less
• Prolonged TGT : deficiency of factor V, VIII,
IX & X.
• Hemophilia : PT normal, TGT prolonged
• Pure factor VII def. : PT prolonged, TGT
normal
• Factor X def. : Both PT & TGT prolonged

48. 9. Clot Solubility Test
• This test detects def. of factor XIII.
• The test is based on the solubility of non
crosslinked fibrin.
• Test plasma is clotted in either calcium chloride
or thrombin, then suspended at 37°C in either
urea, 1% monochloroacetic acid or 2% acetic
acid.
• If the clot dissolves in one of the solvents, this
indicates either low levels or total absence of
factor XIII.
• Confirmed by different Assays.

49. Clot Solubility Test
• Low Factor XIII Levels are seen in :
- Individuals with inherited F XIII deficiency - umbilical
stump bleeding
- F XIII inhibitors – Isoniazid , sodium valproate ,
phenytoin , penicillin or procainamide.
- Henoch-Schoenlein purpura ( HSP )
- In patients on and following cardiopulmonary bypass
- Chronic inflammatory bowel disease
- Severe GvHD of the gut
- Levels fall in pregnancy. Severe inherited F XIII
deficiency is associated with recurrent miscarriage
- Excessive activation, as seen in DIC.
• LATE BLEEDERS

50. 10. Clot Retraction Test
• This test measures time needed for contraction
of an undisturbed clot.
• It indicates function & number of platelets.
• N : begins within 2 hrs, completed within 24
hrs.
• Clot Retraction retarded in thrombocytopenia.
• Clot is soft & small in thromboasthenia –
functional disturbance of platelets.

51. Summary
• CT, APTT increased – Hemophilia A or B,
factor deficiency or inhibitors – factor assays
• BT, APTT increased – vWD – VIII, vW factor
assay ; platelet aggregation test
• PT, APTT increased – def. factor II, V, X ; def
vit. K ; liver disease ; warfarin – factor assays
• PT increased – def. factor VII – factor VII
assay
• BT, CT, PT, TT, APTT increased –
fibrinogenemia – Physio-chemical methods

52. Summary
• Clot solubility test positive – def. factor XIII –
factor XIII assay
• BT increased, tourniquet / clot retraction test
positive – thromboasthenia – platelet function tests
• BT increased, tourniquet / clot retraction test
positive, platelet count reduced – thrombocytopenia,
platelet function defects - platelet function tests,
platelet morphology
• BT, CT, PT, TT, APTT increased ; platelet count
reduced – liver disease, DIC - LFTs

53. • Take home message - All Bleeding
stops…. Eventually