Tests of bleeding disorders

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

OUTLINE

• Normal Hemostasis

• Coagulation Factors

• Coagulation Pathway

• Bleeding Disorders

• Laboratory Tests

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

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

Normal HemostasisBV Injury

PlateletActivation

Plt-Fusion

Blood Vessel Constriction

CoagulationActivation

Stable Hemostatic Plug

Thromibn,

Fibrin

Reduced

Blood flow

Tissue Factor, blood

Primary hemostatic plug

Neural

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

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

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

Laboratory tests

1. Tests of the Vascular Platelet Phase of Hemostasis:

• Bleeding Time (BT)Bleeding Time (BT)2. Tests of the Coagulation Cascade:• Clotting Time ( CT) or Coagulation timeClotting Time ( CT) or Coagulation time• Activated Partial Thromboplastin Time Activated Partial Thromboplastin Time

(APTT).(APTT).• Prothrombin Time (PT)Prothrombin Time (PT)• Thrombin Time (TT)Thrombin Time (TT)

Laboratory tests3. 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

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.

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.

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.

Bleeding Time

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.

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.

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.

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.

Clotting Time

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.

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.

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.

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.

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.

Prothombin Time

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.

Prothombin Time

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.

Prothombin Time

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.

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.

APTT

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

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.

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.

APTT

APTT

APTT

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).

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.

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.

Thrombin Time

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.

Platelet Count

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.

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

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

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.

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

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.

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

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

• Take home message - All Bleeding stops…. Eventually