Prothrombin time and aptt

Prothrombin time and aptt

1. PT & APTT P. SUNIL KUMAR Haematology & Transfusion medicine St.John’s Medical College Bangalore 12/6/2017 1 SUNIL KUMAR P

2. Importance of PT/ Clinical Significance Prothrombin time (PT) is a blood test that measures how long it takes blood to clot. A Prothrombin time test can be used to check for bleeding problems. PT is also used to check whether medicine to prevent blood clots is working. A PT test may also be called an INR test. 12/6/2017 2 SUNIL KUMAR P

3. PROTHROMBIN TIME- ( Extrinsic) QUICK (one stage ) method. Introduced by Dr. Armand Quick in 1935. Time required for clotting of citrated plasma after addition of calcium and tissue thromboplastin. 12/6/2017 SUNIL KUMAR P 3

4. Automated method Electromechanical Impedance steel ball a steel ball rotates in magnetic field until formation of fibrin clot. Optical methods- Turbidimetric method Nephelometric/ light scattering 12/6/2017 4 SUNIL KUMAR P

5. PT by Quick Onestage Method 12/6/2017 5 SUNIL KUMAR P

6. Reagents& Requirements Thromboplastin -CaCl 2mixture reagent ) Control plasma Test tubes, 12mm x 75mm Stopwatch Centrifuge Micropipette Micropipette tips 3.2% sodium citrate anticoagulated blood sample Water bath 37 o C 12/6/2017 6 SUNIL KUMAR P

7. Principle : The calcium in whole blood is bound by sodium citrate, thus preventing coagulation. Tissue Thromboplastin, to which calcium has been added, is mixed with the plasma, and the clotting time is noted. 12/6/2017 7 SUNIL KUMAR P

8. 12/6/2017 8 SUNIL KUMAR P

9. 12/6/2017 9 SUNIL KUMAR P

10. 12/6/2017 10 SUNIL KUMAR P

11. 12/6/2017 11 SUNIL KUMAR P

12. Specimen collection and storage : Specimen collected by veinpuncture using vacutainer and mix the sample .Centrifuge at 2500 rpm -3000 rpm for 15 minutes. 12/6/2017 12 SUNIL KUMAR P

13. Specimen required Blood anticoagulation with 3.2% sodium citrate in the ratio of 1 part of citrate to 9 parts of blood is used . It is important that this ratio be maintained accurately. An anticoagulant correction is required when PCV is high or low. Formula for PCV Correction -100 -PCV for 1 ml blood 595-PCV 12/6/2017 13 SUNIL KUMAR P

14. 12/6/2017 14 SUNIL KUMAR P

15. Rejection criteria Haemolysed sample. Clotted sample. Inadequate sample. 12/6/2017 15 SUNIL KUMAR P

16. Quick’ Method (Manual ) Centrifuge the blood sample at 2500 -3000 RPM for 15 minutes. Separate the plasma from the cells as soon as possible. Label 2 test tubes as test tube No.1 & 2 . Add 0.1 ml of patient plasma to each Label another test tube as control. Add control plasma 0.1 ml . Incubate at 37 o C for 1 min. Add 0.2 ml of prewarmed thromboplastin reagent into the tube. Start the stop watch. 12/6/2017 16 SUNIL KUMAR P

17. Mix the tube and shake it in water bath for 5-6 sec, take out the test tube & observe for clot formation against light. Run the duplicate test & control in the same way. Take the average of the two test readings. 12/6/2017 17 SUNIL KUMAR P

18. PROCEDURE SL NO REAGENT TEST – I TEST - II CONTROL 1 PATEINT PLASMA 0.1 ML 0.1 ML -- 2 CONTROL PLASMA --- ---- 0.1 ML INCUBATE AT 37 o C FOR 60 SEC OR 1 MIN 3 CaCl 2 0.025M 0.1 ML 0.1 ML 0.1 ML START STOP WATCH 12/6/2017 18 SUNIL KUMAR P

19. Biological reference Interval: or Normal Range 11-14 Sec . Critical values for urgent clinical notification : > 60sec. 12/6/2017 19 SUNIL KUMAR P

20. Inr - international normalized ratio Introduced in1983 by WHO PT results of diff labs using diff thromboplastin reagent may lead to diff result even when plasma warfarin conc. is same, so leads to missinterpretation Therefore all thromboplastin reagents distributed are caliberated against WHO reference preparation The caliberation no: is called international sensitivity index (ISI) 12/6/2017 20 SUNIL KUMAR P

21. INR= PT patient ISI geometric mean of normal PT ISI value is provided by manufacturer 12/6/2017 21 SUNIL KUMAR P

22. Uses of inr Allows PT results to be compared among labs accros world Used to monitor patients taking oral anticoagulant, it is not used for initial evaluation of hemostatic system. DISADVANTAGE- Incorrectly calculated INR values Incorrect ISI value assinged to thromboplastin reagent 12/6/2017 22 SUNIL KUMAR P

23. Clinical significance/ Increased (PT) in DIC Liver diseases Vitamin K deficiency Oral anticoagulant therapy FV, FVII, F X deficiency 12/6/2017 23 SUNIL KUMAR P

24. ACTIVATED PARTIAL THROBOPLASTIN TIME (APTT). Intrinsic 12/6/2017 24 SUNIL KUMAR P

25. Principle : The calcium in a whole blood sample is bound by sodium citrate, thus preventing coagulation. The plasma, after centrifugation, contains all intrinsic coagulation factors except calcium and platelets. In the APTT test, partial thromboplastin (a phospholipid substitute) and an activator (to ensure maximum activation) are added to the plasma allowing the coagulation cascade to begin. During incubation, Factors XII, PK and XI are activated, building up the levels of XIa in the reaction tube. Once CaCl2 is added, the rest of the coagulation cascade is allowed to continue and timing of the event is obtained. The time required for the plasma to clot is the activated partial thromboplastin time. 12/6/2017 25 SUNIL KUMAR P

26. 12/6/2017 26 SUNIL KUMAR P

27. Type of specimen : 3.2 % tri sodium citrate anti-coagulated sample, random sample. 12/6/2017 27 SUNIL KUMAR P

28. Specimen collection and storage : Sample collected by vein puncture in the citrate vacutainer and mixed gently by inverting the tube several times. Separate the plasma by centrifuging at 2500-3000 RPM for 15 minutes. Stable for 4 hours at 4 o C and 1 month at -30 o C. 12/6/2017 28 SUNIL KUMAR P

29. Rejection criteria : Hemolysed sample. Clotted sample. Inadequate sample. 12/6/2017 29 SUNIL KUMAR P

30. Reagents required. Phospholipid or Partial thromboplastin containing an activator (commercially available) Platelet poor plasma from the patient & control plasma. CaCl 2 0.025M solution freshly prepared. Control samples (normal and abnormal) Test tubes, 12mm x 75mm Stopwatch 12/6/2017 30 SUNIL KUMAR P

31. PROCEDURE 1. 0.1 ml test plasma in 2 tubes test 1 & 2 , add 0.1 ml of control plasma in 2 tubes labeled control 1 & 2. 2. 0.1 ml of APTT reagent is added to the 1 first. Incubate at 37 o C , stop watch is started as soon as the reagent is added. Incubate the tube labeled test “2” one minute after test 1. 3. At the end of 3 minutes add 0.1 ml of prewarmed CaCl 2 & start the stop watch. 12/6/2017 31 SUNIL KUMAR P

32. 4. Mix the tube gently in the water bath for 20 seconds. 5. Take out & observe for clot. 6. Take the average of the 2 readings. 12/6/2017 32 SUNIL KUMAR P

33. PROCEDURE SL NO REAGENT TEST - 1 TEST- II CONTROL - I CONTROL -II 1 PATIENT PLASMA 0.1 ML 0.1 ML - - 2 CONTROL PLASMA - -- 0.1 ML 0.1 ML 3. APTT REAGENT 0.1 ML 0.1 ML 0.1 ML 0.1 ML INCUBATE AT 37 o C FOR 180 SE OR 3 MIN 4 CaCl 2 0.025M 0.1 ML 0.1 ML 0.1 ML 0.1 ML START STOP WATCH 12/6/2017 33 SUNIL KUMAR P

34. Normal range Normal range : 25 – 35 seconds 12/6/2017 34 SUNIL KUMAR P

35. Clinical significance DIC Haemophilia A & B VWD Liver diseases Massive transfusion of whole blood Administration of Heparin F IX, F XI, F XII 12/6/2017 35 SUNIL KUMAR P

36. 12/6/2017 36 SUNIL KUMAR P

37. THANKS 12/6/2017 37 SUNIL KUMAR P