pt aptt.pptx New coagulation tests 22222

Coagulation tests

1. PT,APTT,CLOT RETRACTION TEST,CLOT LYSIS,PRINCIPLES OF FACTOR ASSAY

2. Coagulation cascade

3. Clotting Screen Prothrombin time Assess extrinsic(factor vii) and common(factor x,v,prothrombin,fibrinogen ) pathway of coagulation PT is the time taken by citrated plasma to clot after addition of tissue thromboplastin and calcium

4. Principle Test determines the overall efficiency of extrinsic and common pathway

5. Equipment Water bath at 37°C Test tubes (75 × 12 mm) Stopwatch

6. Reagents Thromboplastin reagent: This contains tissue factor and phospholipids and is available commercially Calcium chloride 0.025 mol/liter

7. Specimen Platelet-poor citrated plasma

8. Method Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 37°C Add 0.1 ml of thromboplastin reagent and mix After 1 minute, add 0.1 ml of calcium chloride solution. Immediately start the stopwatch and record the time required for clot formation Normal range: 11-16 seconds

9. Causes of prolongation of PT Treatment with oral anticoagulants Liver disease Vitamin K deficiency Disseminated intravascular coagulation Inherited deficiency of factors in extrinsic and common pathways

10. Uses of PT Screenimg test for coagulation disorders To monitor patients who are on oral anticoagulant therapy Oral anticoagulants inhibit carboxylation of vitamin K-dependent factors (Factors II, VII, IX, and X) and make these factors inactive To assess liver function: site of synthesis of various coagulation factors, including vitamin K dependent proteins

11. Detection of vitamin K deficiency: PT measures three of the four vitamin K-dependent factors (i.e. II, VII, and X) To screen for hereditary deficiency of coagulation factors VII, X, V, prothrombin, and fibrinogen

12. Activated Partial Thromboplastin Time (APTT) APTT is a measure of coagulation factors in intrinsic pathway (F XII, F XI, high molecular weight kininogen, prekallikrein , F IX, and F VIII) and common pathway (F X, F V, prothrombin, and fibrinogen)

13. Principle Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured

14. Equipment : Same as for PT Reagents Kaolin 5 gm/ liter : This is a contact activator Phospholipid: Various APTT reagents are available commercially, which contain phospholipids Calcium chloride 0.025 mol/ liter Specimen : Citrated platelet poor plasma

15. Method Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 37°C Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution. Incubate at 37°C in the waterbath for 10 minutes After exactly 10 minutes, add 0.2 ml of phospholipid calcium chloride mixture, start the stopwatch, and note the clotting time Normal range: 30-40 seconds

16. Causes of prolongation of APTT Hemophilia A or B Deficiencies of other coagulation factors in intrinsic and common pathways Presence of coagulation inhibitors Heparin therapy Disseminated intravascular coagulation Liver disease

17. Uses of APTT Screening for hereditary disorders of coagulation:haemophilia A and B To monitor heparin therapy : Heparin potentiates the action of natural anticoagulant antithrombin III which is an inhibitor of thrombin and activated factors IX, X, and XI. Full dose heparin therapy needs monitoring by APTT to maintain the dose in the therapeutic range (1.5 to 2.5 times the upper reference limit of APTT) Screening for circulating inhibitors of coagulation: APTT prolonged

18. Factor Assays Deficiency of particular factor is confirmed by 1 stage factor assays 2 stage factor assays Chromogenic factor assays

19. Principles of factor assay If screening test indicate coagulation defect,plasma concentration of coagulation factors should be assayed The assay of a clotting factor relies upon measuring the degree of correction of the PT or APTT when plasma is added to a plasma sample specifically deficient in the factor to be measured

20. Assays based on PT Isolated prolonged PT-one stage factor vii assay If reduced factor vii-immune assays of factor vii,family study

21. One stage assay of factor vii Principle: Assay of factor vii is based on PT Assay compares the ability of dilutions of the patient’s plasma and of a standard plasma to correct the PT of a substrate plasma

22. Reagents: PPP from patient Standard plasma Factor vii deficient plasma Barbitone buffered saline Thromboplastin Cacl2

23. Method Prepare 1 in 5,1 in 10,1 in 20 and 1 in 40 dilutions of standard and test plasma in buffered saline Transfer 0.1ml of each dilution to a glass tube and add to it 0.1ml of deficient (substrate) plasma Mix and allow to warm to 37°C Add 0.1ml of dilute thromboplastin and start the stopwatch

24. Record the clotting time If the thromboplastin does not contain calcium, start the stopwatch after adding 0.1ml of CaCl2 A blank must be included with every assay and all tests must be carried out in duplicate and in balanced order

25. Result calculation Plot the clotting times of the test and standard against the concentration of factor VII on log/log graph paper Factor vii level should be compared to normal

26. Assays based on APTT An APTT-based assay (e.g. for FVIII) may be indicated after obtaining correction of a prolonged APTT by mixing with another plasma An assay for FVIII is described, easily adapted to FIX, FXI or contact factor assays by substituting the relevant factor deficient plasma

27. One stage assay for factor viii Principle Based on APTT Same as earlier

28. Reagents PPP from patient Standard/reference plasma FVIII-deficient plasma (substrate plasma) Barbitone buffered saline. See page Reagents for APTT Plastic tubes-To avoid contact activation while preparing samples Ice bath.

29. Method Place the APTT reagent and CaCl2 at 37°C and the patient’s, standard and substrate plasma in the ice bath until used Make 1 in 10 dilutions of the test and standard plasma in buffered saline in plastic tubes in the ice bath Using 0.2ml volumes, make doubling dilutions in buffered saline to obtain 1 in 20 and 1 in 40 dilutions

30. Place 0.1ml of the three dilutions (1 in 10, 1 in 20 and 1 in 40) in glass tubes If the test plasma is suspected of having a very low FVIII content, make 1 in 5, 10 and 20 dilutions of the test instead Add to each dilution 0.1ml of freshly reconstituted or thawed substrate plasma and warm up at 37°C Perform APTTs according to the laboratory protocol following a balanced order of duplicates

31. Result calculation Plot the clotting times of the test and standard against the concentration of FVIII on semi-log paper

32. Two stage and chromogenic assays

33. CLOT RETRACTION TEST Measure of platelet function Test measures the amount of time it takes for a blood clot to pull away from the walls of a test tube(shrinking) Completed in 18-24 hours

34. Principle After coagulation of blood, clot under the action of thrombasthenin undergoes contraction,starts retracting within 1 hr 50% retraction in 2 to 4 hrs Completed in 18 to 24 hrs with separation of serum Subsequently fibrin clot dissolves due to fibrinolysis, RBCs sink to bottom

35. Procedure Place a tube containing blood in a water bath at 37 degree celcius Examine clot after 1 hr and after 24 hrs Note the size,consistency of clot,nature of retraction Continue observation of clot for 72 hrs to assess clot lysis

36. Normal clot retraction shows more than 50% of serum separated at the end of 24 hrs

37. Fibrinolysis-clot lysis Lysis and removal of blood clot after stoppage of bleeding and healing of vascular wall By plasmin- present as inactive plasminogen in plasma Plasminogen gets activated by tPA Plasmin breaks down fibrin and interferes with its polymerisation

38. Tests for fibrinolysis Euglobulin clot lysis time Plasma euglobulin contains plasminogen activators and fibrinogen Rate of lysis of a fibrin clot prepared from euglobulin fraction is a measure of plasminogen activators EACA can inhibit plasminogen activators but not free plasmin Shortened euglobulin lysis time in presence of EACA indicate presence of free plasmin

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