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

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

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

5. 4 Approach to Hemostasis Disorders Clinical: Clinical: – History taking – Physical Examination Laboratory Laboratory

6. 5 History Taking Family history Family history Drugs used Drugs used Previous diseases Previous diseases Symptoms Symptoms

7. 6 Physical Examination Petechaie Petechaie Ecchimosis Ecchimosis Hematom Hematom Epistaxis Epistaxis Gingival bleeding Gingival bleeding Metro-menorrhagia Metro-menorrhagia

8. 7 Hemostasis Test Screening Screening Confirmatory Confirmatory

9. Hemostasis Tests Screening assays in hemostasis: 1.Patients without any signs/symptoms  preoperative. 2.Patient with bleeding 3.Monitoring of anticoagulant therapy 4.Disseminated Intravascular Coagulation 5.Thrombophilia 6.Inhibitor (Lupus Anticoagulant, Anti Phospholipid Antibody)

10. 9 Hemostasis Screening Test 1.Tourniquet Test 2.Bleeding Time 3.Clotting Time 4.Clot Retraction 5.Platelet Count* 6.PT* 7.APTT* 8.TT* 9.Fibrinogen* 10.Euglobulin Clot Lysis Test 11.D-Dimer*Thrombelastography

11. 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.

12. Purpose To evaluate capillary resistance Equipments Sphygmomanometer Stethoscope Stopwatch Marker

13. 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

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

15. TOURNIQUET TEST 5 cm SYSTOLICDIASTOLIC 5 min 100 mmHg Leave for 5 min petechiae

16. Bleeding Time Duke Method Ivy Method

17. 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.

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

19. 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.

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

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

23. 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.

24. 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.

25. 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

26. 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.

27. 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.

28. 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

29. 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.

30. - 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 BP : 40 mmHg BLOOD LANCET 2. IVY METHOD :

31. 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.

32. Sample : 5 ml blood obtained from vein Instruments : 1.A set of devices for blood sampling from vein. 2.Centrifuge glass test tube. 3.Stick (lidi) 4.Centrifuge

33. Procedure 1.Obtain 5 mL of venous blood. 2.Take off the needle and transfer blood into the test tube. Put the tube on the rack. 3.Leave at room temperature for about 1,5-2 hours. 4.After 2 hours, separate the blood clot from the test tube’s wall carefully, placed the blood clot into another test tube. 5.Centrifuge the serum for about 5 min/1000 rpm. 6.Measure how much (mL) serum was performed and calculate the percentage.

35. Hemocytometer Platelet Count (manual)

36. MANUAL REAGENT TROMBOCYTE

37. Clotting Time Bedside Clotting Time Clotting Time Lee & White Method

38. 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

39. Equipments 1.Clean, oil free and smooth object glass. 2.Stopwatch. 3.Needle. 4.Sterile blood lancet. 5.Cotton ball soaked in alcohol 70 %.

40. PROCEDURE 1.Clean the finger tip with70% of alcohol, let it dry. 2.Puncture with blood lancet, 3 mm in depth. 3.Wipe the first blood drop with dry cotton. 4.Drop the next blood drop onto the object glass (4-5 mm in diameter) and start stopwatch immediately as the blood touch glass surface. 5.Put the second drop beside the first one with same size diameter. 6.Watch the fibrin thread formed in the second drop of blood by tilt it with a needle every 30 sec. 7.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. 8.Record the time.

41. Clotting Time : Lee & White blood 3 ml 37 o C 1 cc 1 cc 1cc N : 5 – 11 min

42. Coagulation Tests PT APTT TT Manual Automatic (Coagulometer)

43. Coagulometer

44. 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

45. 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 = { ----------- } PT pat PT n ISI

46. 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

47. 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

48. 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).

49. SUBSTITUTION TEST FACTOR DEFICIENT XII PTT A P T N TT N NP C AP C AS C XIANNCCC IXANNCUCC VIIIANNCCUC XAANCC VAANCC VIINA---- IIAANCUC IAAACC HEPARIN OR INHIBITOR RESEMBLE HEPARIN AAAUC-- PTT - PARTIAL THROMBOPLASTIN TIME NP - NORMAL PLASMA PT - PROTHROMBIN TIME AP - ADSORBED PLASMA TT - THROMBIN TIME AS - AGED SERUM A = ABNORMAL N = NORMAL C = CONTROL UC= UNCONTROL

50. 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

51. D-dimer

52. Thromboelastography Screening & control therapy Easy to perform, no reagent needed, fast Record clot formation and converse to graph TEG ruler Conversion table

53. PISTON CUVET

55. 1. TEG NORMAL PATTERN r = reaction time (start to amplitudo 1 mm) k = coagulation time (end of r to amplitudo 20 mm) m.a = maximum amplitudo (mm) m. e= maximum elasticity 100 x a m. e = ------------- 100 - a r k 20 mm m. a

56. r k m. a 2. THROMBOCYTOPENIA r = normal k = normal/prolonged m.a. = shortened 3. HYPERFIBRINOLYSIS m.a r k r = normal k = normal m.a. = previously normal, but suddenly become shortened

57. r k 4. HEMOPHILIA r = prolonged k = prolonged m.a. = normal/shortened m. a 5. HYPERCOAGULATION r k m.a r = shortened k = shortened m.a = prolonged

58. Normal Hemophilia Thrombocytopenia Hyperfibrinolysis Hypercoagulation TEG pattern

59. Confirmatory Tests Vascular Platelet : – Platelet adhesion – Platelet aggregation Coagulation : – Substitution test – Factor Assay Fibrinolysis

60. TestResultDifferential diagnosis PTN - Normal- Von Willebrand disease (mild) APTTN - Mild trombocyte disfunction - Fibrinolysis abnormality TTN Fibr.N - Vaskuler abnormality Tromb.N 1

61. TestResultDifferential diagnosis PTL - F VII deficiency APTTN - Initial phase of oral anticoagulant therapy TTN - Lupus anticoagulant Fibr.N - Faktor II, V or X deficiency (mild) Tromb.N 2

62. TestResultDifferential diagnosis PTN - Faktor VIII, IX, XI, XII, Prekalikrein, HMWK deficiency APTTL - Circulating anticoagulant - Von Willebrand disease TTN - Faktor II, V atau X deficiency (mild) Fibr.N Tromb.3N 3

63. TestResultDifferential diagnosis PTL - Vit.K deficiency- Combined F V and F VIII deficiency APTTL - Oral antikoagulant TTN - F V, II, X deficiency Fibr.N - Liver disfunction  coagulation factor deficiency Tromb.N 4

64. TestResultDifferential diagnosis PTL - Heparin APTTL - Liver disease TTL - Fibrinogen deficiency Fibr.N/A - Polimerization fibrin inhibition Tromb.N - Hyperfibrinolisis 5

65. TestResultDifferential diagnosis PTN - Trombocytopenia APTTN TTN Fibr.N Tromb.Low 6

66. TestResult Differential diagnosis PTL - Massive transfusion APTTL - Liver disease TTN Fibr.N/A Tromb.Low 7

67. TestResultDifferential diagnosis PTL - DIC APTTL - Acute liver disease TTL Fibr.Low Tromb.Low 8

68. Thank You