Laboratory Approach to Patient with Bleeding (Hemostasis) Disorder

1. Laboratory Approach to Patient with Bleeding (Hemostasis) Disorder Dr.Nadjwa ZD, SpPK-K

2. Hemostasis is achieved by highly integrated and regulated interaction of • Blood vessels • Platelets • Coagulation proteins • Fibrinolysis • Natural anticoagulant

3. Hemostasis Disorders • Bleeding and/or thrombosis • Bleding • Vascular • Platelet*(ITP) • Coagulation factors*(vWD,Hemophilia) • Thrombosis* • Fibrinolysis • Natural anticoagulant • Both DIC

4. Approach to Hemostasis Disorders • Clinical: • History taking • Physical Examination • Laboratory

5. History Taking • Family history • Drugs used • Previous diseases • Symptoms

6. Physical Examination • Petechaie • Ecchimosis • Hematom • Epistaxis • Gingival bleeding • Metro-menorrhagia

7. Hemostasis Test • Screening • Confirmatory

8. Hemostasis Tests Screening assays in hemostasis: • Patients without any signs/symptoms  preoperative. • Patient with bleeding • Monitoring of anticoagulant therapy • Disseminated Intravascular Coagulation • Thrombophilia • Inhibitor (Lupus Anticoagulant, Anti Phospholipid Antibody)

9. Tourniquet Test Bleeding Time Clotting Time Clot Retraction Platelet Count* PT* APTT* TT* Fibrinogen* Euglobulin Clot Lysis Test D-Dimer* Thrombelastography Hemostasis Screening Test

10. Tourniquete Test = Capillary Resistance Test. = Rumpel Leede Test = Hess’s Test Principle : This test measures the ability of the capillaries to resist pressure. In healthy individu, the capillaries in the arm will resist a pressure of 100 mmHg. If the capillaries can not resist, they will break or rupture, tiny spot will then appear. These spots are hemorrhages or petechiae.

11. Purpose • To evaluate capillary resistance Equipments • Sphygmomanometer • Stethoscope • Stopwatch • Marker

12. Procedure : • Draw with marker a circle (5 cm in diameter) in the volar surface of the forearm, about 5 cm below the elbow. • Apply a blood pressure cuff on the upper arm, determine a systolic and diastolic pressures, and then deflate to a pressure midway between the two. • Leave the inflated cuff in place for 5 minutes • Remove the cuff, wait for 5 minute

13. Interpretation • Inspect the circle for petechiae • Normal : < 10 petechiae • > 10 petechiae  abnormal, due to : • Increased capillary resistance • Decreased platelet number

14. 5 cm 5 cm TOURNIQUET TEST SYSTOLIC DIASTOLIC 100 mmHg 5 min Leave for 5 min petechiae

15. Bleeding Time • Duke Method • Ivy Method

16. BLEEDING TIME (Duke’s Method) • Principle : The skin is incised, blood flowing out is aspirated with a filter paper, and then the time until hemostasis is measured. • Purpose : To evaluate platelet and vascular ability in performing platelet plug.

17. Equipments • Blood lancet. • Cotton ball soaked in alcohol 70%. • Circular filter paper. • Stop watch

18. Procedure • The lobe of the ear is cleaned with 70% of alcohol and let dried • Use a blood lancet to puncture earlobe to a depth of 3 mm. Start timing (stop watch) • Every half minute, gently apply the edge of a small disc of filter paper to the drops of blood from the earlobe – do not touch the skin. Use a fresh edge of filter paper disc for each half-minute blotting.

19. BLEEDING TIME  VASCULAR  PLATELET 1. DUKE METHOD (ear lobe) Every 30 sec FILTER PAPER 5 mm

21. Interpretation • Time in minutes equals number of blots divided by 2 • Normal : 1-3 minutes

22. When the blood spot becomes 1 mm or smaller, stop the stop watch. • If the bleeding doesn’t stop in 10 min., discontinue testing. Indicate the result as 10 min or longer. • Cover the wound with a sterile gauze for a while, hemostasis should be confirmed, after which the patient may leave.

23. Note : • The size of the blood spot about 1 cm in diameter is desirable, but becomes larger in some cases. However bleeding usually stops for several minutes regardless of the size. • Don’t wipe off the blood. Gently touch. Note so as not to touch the wound.

24. Bleeding time (Ivy Method) • Ivy Method is more reliable than Duke Method. • Equipment : • Sphygmomanometer • Sterile disposable lancet (capable of making an incision of 1 mm wide and 3 mm deep) • Circular filter paper • Alcohol 70% • Cotton wool or surgical gauze

25. Procedure : • Place the sphygmomanometer cuff on the patient’s arm, above the elbow. Inflate the cuff to 40 mm of mercury and hold this exact pressure for the entire period. • Choose an area approximately 3 fingerwidths below the bend in the elbow on the volar surface. • Clean the area with the alcohol sponge.

26. Hold the skin tightly by grasping the under- side of the arm firmly and make 2 separate punctures 5 to 10 cm apart, in quick succession, using the disposable lancet. Start the stopwatch. • Note : it is essential for you to be purposeful and precise as tentative jabs may not bleed satisfactorily. Avoid any subcutaneous veins. The disposable lancet available for this purpose can be inserted freely to its maximum depth without fear of penetrating too deeply.

27. Blot the blood from each incision site on a separate piece of circular filter paper every 30 sec. The filter paper should not touch the incision point at any time • When bleeding ceases, stop the watch and release the blood pressure cuff. • Record the bleeding times of the 2 punctures

28. Interpretation • Normal : up to 11 min. • If bleeding continuous for > 15 min, apply pressure to the wound site, and report the result as greater than 15 min.

29. 2. IVY METHOD : BLOOD LANCET BP : 40 mmHg - 3 Cm • Drops of blood that come from the incision point is blotted with the circular filter paper every 30seconds • NORMAL:1-7 Minute • The depth of the puncture must be deep enough, so that it leaves a blot with a diameter of 5 mm

30. CLOT RETRACTION Principle • When whole blood is allowed to clot spontaneously, the initial coagulum is composed of all elements of the blood. • With time the coagulum reduces in mass, and fluid serum is expressed from the clot, and its volume stated in %. • This is due to an action of platelets on the fibrin network.

31. Sample :5 ml blood obtained from vein Instruments : • A set of devices for blood sampling from vein. • Centrifuge glass test tube. • Stick (lidi) • Centrifuge

32. Procedure • Obtain 5 mL of venous blood. • Take off the needle and transfer blood into the test tube. Put the tube on the rack. • Leave at room temperature for about 1,5-2 hours. • After 2 hours, separate the blood clot from the test tube’s wall carefully, placed the blood clot into another test tube. • Centrifuge the serum for about 5 min/1000 rpm. • Measure how much (mL) serum was performed and calculate the percentage.

34. Platelet Count (manual) Hemocytometer

35. MANUAL REAGENT HAYEM TURK AM OKSALAT TROMBOCYTE

36. Clotting Time

37. BEDSIDE CLOTTING TIME Principle : • Record the time interval from the blood contact with glass surface, until fibrin network is performed at the room temperature. Sample : • Capillary blood

38. Equipments • Clean, oil free and smooth object glass. • Stopwatch. • Needle. • Sterile blood lancet. • Cotton ball soaked in alcohol 70 %.

39. PROCEDURE • Clean the finger tip with70% of alcohol, let it dry. • Puncture with blood lancet, 3 mm in depth. • Wipe the first blood drop with dry cotton. • Drop the next blood drop onto the object glass (4-5 mm in diameter) and start stopwatch immediately as the blood touch glass surface. • Put the second drop beside the first one with same size diameter. • Watch the fibrin thread formed in the second drop of blood by tilt it with a needle every 30 sec. • When the fibrin was performed, do the same step to the first blood drop and stop the stopwatch if the fibrin was seen in the first drop. • Record the time.

40. !....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!....!.... Clotting Time : Lee & White blood 3 ml 1 cc 1 cc 1cc 37oC N : 5 – 11 min

41. Coagulation Tests PT APTT TT • Manual • Automatic (Coagulometer)

42. Coagulometer

43. Prothrombin Time (PT) • Screening test for the extrinsic and common pathways of coagulation (factors II, VII, V, X). • Limited sensitivity to fibrinogen. • Normal range : 11-13 sec

44. INR (International Normalized Ratio) To overcome some of the difficulties with the variability of thromboplastin  normalizing the responses of thromboplastin reagents against an international standard. • INR = { -----------} PTpat ISI PTn

45. ISI(International Sensitivity Index) • Needs to be developed for each thromboplastin reagent and instrument combination used in performing PT and calculation of INR. • Ideal reagent  ISI < 1.7

46. Activated Partial Thromboplastin Time (aPTT) • Screening test for the intrinsic and common pathways of coagulation (factors XII, XI, IX, VIII, X, V and II). • Limited sensitivity to fibrinogen. • Maybe normal in some cases of vWD • Normal range : < 35 sec

47. Substitution Test(Mixing Study) • APTT • To evaluate factors deficiency • Additional reagents : • Adsorbed Plasma (consist of factors : I, V, VIII, XI, XII). • Aged Serum (consist of factors : VII, IX, X, XI, XII).

48. SUBSTITUTION TEST FACTOR DEFICIENT PTT P T TT NP AP AS XII A N N C C C XI A N N C C C IX A N N C UC C VIII A N N C C UC X A A N C UC C V A A N C C UC VII N A - - - - II A A N C UC UC I A A A C C UC A = ABNORMAL N = NORMAL C = CONTROL UC= UNCONTROL HEPARIN OR INHIBITOR RESEMBLE HEPARIN A A A UC - - PTT - PARTIAL THROMBOPLASTIN TIME NP - NORMAL PLASMA PT - PROTHROMBIN TIME AP - ADSORBED PLASMA TT - THROMBIN TIME AS - AGED SERUM

49. Thrombin Time (TT) • Identified stage 3 defects in the coagulation mechanism • Clinical significant  Prolonged TT : • Decreased fibrinogen concentration • Presence of dysfunctional fibrinogen • Presence of heparin • Presence of FDP

50. D-dimer