1. Laboratory Investigations – all that a blood banker needs to Know Department of Clinical Pathology & Blood Bank C M C Vellore India

2. Tests for Appropriate use of :- Red Cells PlateletsPlasmaCryo Factor Concentrate By pass agents – rFVIIa/ FEIBA Others – Tranexamic acid, DDAVP

3. Red Cells and Platelets Hb/ PCV/ RCC Platelet count

4. The Coulter Principle

5. Sensing Zone Red Blood Cell A red cell passes through RBC aperture Oscilloscope Ohm’s law: Voltage = Current X resistance

6. Oscilloscope Sensing Zone Neutrophil A White cell passes through WBC aperture Oscilloscope Thresholds for sensing the voltage can be adjusted

7. Applying Thresholds to separate Plt from RBC 20 fl 2 fl Base line RBC of various sizes Platelets

11. Platelet count and Platelet morphology and numbers on smear Manual or Machine

12. Platelet Histogram

13. Laboratory Evaluation of Haemostasis

14. Screening Tests for Hemostasis Platelet count Bleeding time (BT) - Plt adh-vWF,Gp1b/ Plt aggr- GpIIb-IIIa/ Plt secretion Prothrombin time (PT) - Extrinsic pathway factors-VII,V,X,II and Fibrinogen Activated Partial thromboplastin time (aPTT) – Intrinsic pathway factors XII,XI,IX,VIII,V,X,II,Fibrinogen Most important is - History

15. Value of history in Diagnosis An abnormal coagulation parameter has no major diagnostic value if the patient is not bleeding has not been a bleeder ( no past history of bleeding) not likely to be a bleeder –( no family or medication history)

16. History Key to understanding and diagnosing hemorrhagic disorders * Without any symptoms an abnormal coagulation test result has no value * Without any symptoms an abnormal coagulation test result has no value * With a good history you may just need basic tests of haemostasis in the laboratory for diagnosis * With a good history you may just need basic tests of haemostasis in the laboratory for diagnosis

17. Bleeding Time Time taken by a standardized skin wound to stop bleeding Standard Skin wound – 2.5 to 3 mm deep and 1.0 to 1.5 mm wide (Ivy’s Method) Lancet tip is 3 mm long and 1.5 mm wide at the base

18. Bleeding Time (Modified Ivy’s Method) BP cuff to 40 mm Hg Select an area avoiding any vein or angiomas Clean the Volar aspect Stab confidently three times and start Stop watch at the end of the third wound. At least one wound is STANDARD

19. Results of Bleeding Time Stop the Stopwatch when all the wounds stops bleeding Normal – 2 to 6 minutes Ideal to establish your own range Prolongation – a) Vascular defect b) Adhesion Defect – VWF (Von Willebrand disease) or Platelet adhesion molecule Gp1b (Bernard Soulier Syndrome) c) Aggregation defect – Platelet aggregation receptors GpIIbIIIa (Glanzmann Thrombasthenia) d) Platelet Secretion Defect – Platelet Granule deficiency (Gray platelet) or Aspirin

20. Interpretation and further Specific Tests - Von Willebrand Factor assays - Platelet Aggregometry studies - Platelet Aggregometry studies - Clot Retraction – absent in aggregation defects

21. Tests for Secondary haemostatic Factor defects - All these Factors are present in the plasma Tests for Clotting factors - plasma based tests Important – care in preparing plasma Starts at Blood sampling Avoid contamination by Tissue and avoid activation

22. Preparing plasma Anticoagulate blood in Trisodium Citrate to prevent clotting of blood to be tested by chelating Calcium. One part Citrate and nine part Blood from veins Carefully collected to prevent activation of factors Two syringe method- 1 st for blood counts and 2 nd for Coagulation studies

23. Preparing Plasma Centrifuge at high speed (1500 – 2000g for 10 min) - To separate Plasma as Platelet Poor Plasma Test immediately before less stable factors are lost. If not keep in ice bath

24. Plasma Based Tests All clotting tests to be done in a 37 0 C water bath. A circulating water bath is ideal. Keep test plasma or control plasma and reagents at 37 0 C at least 5 min before doing the tests

25. Activators

26. Plasma Based Tests – Adding the activator

27. Timing

28. Automated Coagulometers

29. CMC-EQAS Total No. of Participants 400 Total No. of Participants in 2010 231 Information available 158

31. Prothrombin Time Time taken by a recalcified citrated plasma to clot in presence of Tissue Factor. Tests the Extrinsic Pathway – Tissue Factor will activate the extrinsic pathway by activating FVII onwards Tissue Factor - Thromboplastin with its bound negatively charged phospholipid

32. Results Normal Range – 9 to 12 seconds or 12-15 seconds (Depending on the Reagent) RESULTING Raw Data- Patient time and Reference range. Prothrombin ratio (PR) = Patient time Control time >1.2 (3 Seconds more than Control) Prolonged PT indicates deficiency of FVII, FX, FV, FII and Fibrinogen

33. Factors affecting - PT Result Variety of reagents (containing different thromboplastin) are available in the market some are good and some are bad giving different PR (Prothrombin Ratio) on the same sample. Ideally a good screening test reagent should be very sensitive so that all levels of low factors can be picked up when a PT is done.

34. Sensitivity of a PT reagent Most sensitive PT reagent – picks up the mildest of deficiency and exaggerates the difference between ranges of deficiency.( PT is abnormal even if FVII is 40% and there is a significant difference in PT time when levels are 13% and 17%) Least sensitive PT reagent- misses deficiency and will not show the difference between ranges of deficiency.( PT is normal even if FVII is 23% and there is no significant difference in PT time when levels are 11% and 17%)- BAD Reagent

35. PT Result - ISI and INR International sensitivity index (ISI)- value that is assigned indicating the sensitivity of the reagent. Most sensitive reagent has ISI close to a value of 1 Less sensitive reagents will be more than 1.4 onwards International normalized ratio (INR) is normalizing a PT expressed as PR by incorporating the allocated sensitivity (ISI) of the PT reagent used = PR ISI

36. aPTT Time taken by a recalcified citrated plasma to clot in presence of negatively charged - contact activator (Silica, Kaolin, Ellagic acid) - phospholipid (Partial Thromboplastin) Tests the Intrinsic Pathway - Contact activator will activate the intrinsic pathway by activating FXII onwards with the help of phospholipids

37. Results Normal range – 27 – 37 Seconds (Different for different Reagents) Resulting Raw Data- Patient time and Reference range Ratio ->1.2 (more than 6 secs difference from the Control time) Prolonged aPTT indicates deficiency of FXII, FXI, FIX, FVIII, FX, FV, FII and Fibrinogen

38. Results What next If results are abnormal (Prolonged time) Prolonged aPTT indicates deficiency of FXII, FXI, FIX, FVIII, FX, FV, FII and Fibrinogen Prolonged PT indicates deficiency of FVII, FX, FV, FII and Fibrinogen A Thrombin time is done since it picks Fibrinogen deficiency. Time taken by plasma to clot when thrombin is added – this is the time taken by thrombin to convert F’gen to Fibrin.

39. Mixing/Correction Studies Done when PT/APTT are prolonged Mixing studies :- Mix equal volumes of test (abnormal) plasma and control pool plasma (where all factors are present in normal quantity) and repeat the test. Rationale: - if the prolongation in time is due to deficiency of factor (s) in the test plasma, normal plasma will provide the deficient factor when they are mixed correcting the prolonged time - if the prolongation in time is due to deficiency of factor (s) in the test plasma, normal plasma will provide the deficient factor when they are mixed correcting the prolonged time - if the prolongation in time is due to an inhibitor (antibody to factor or heparin) the normal plasma will also be inhibited and the prolonged time will remain prolonged - if the prolongation in time is due to an inhibitor (antibody to factor or heparin) the normal plasma will also be inhibited and the prolonged time will remain prolonged Correction of time: Deficiency of factor No correction: Inhibitor

40. No correction Whatever caused the abnormal (prolonged) timing is also causing an abnormality to normal plasma after the patients plasma was mixed in it. - Heparin - Heparin - Lupus anticoagulant (antiphospholipid antibody) - Lupus anticoagulant (antiphospholipid antibody) - Factor Inhibitor ( antibodies to Clotting factors) - Factor Inhibitor ( antibodies to Clotting factors) - FDP/D-Dimer - FDP/D-Dimer

41. Correction Studies Direct interpretation to therapy Patient who is bleeding with a prolongation in plasma clotting tests Correction of the time by mixing studies in which the normal plasma supplied the deficient factor will directly mean that if you give (transfuse) the patient - normal plasma (FFP) his clotting defect, that lead to the prolongation of the time, will get corrected and the bleeding will stop. Non Correction of time – No benefit of transfusing FFP. Use safer products –Heparin – use Protamin –Inhibitor – other agents Standard Mixing studies – will miss a FVIII inhibitor

42. FVIII inhibitor Late acting Picked only on incubation, for e.g, Patient – aPTT = 120 sec Control= 30 sec Patient – aPTT = 120 sec Control= 30 sec Mixing= 40 sec Mixing= 40 sec (Keep the “Mix” and individual plasmas 2 hours in water bath) (Keep the “Mix” and individual plasmas 2 hours in water bath) aPTT of Incubated Mix= 102 sec aPTT of Incubated Mix= 102 sec

43. Definitive (Specific) Tests Specific Factor assays Inhibitor assay

44. Quantify Inhibitors – Bethesda Assay Quantify the amount of inhibitors Principle of the assay - Ability of a patients plasma to neutralize FVIII in normal plasma (Plasma ≈ 100% FVIII) –on mixing and incubating for 2 hrs Bethesda Units <5 BU low responder Treatment

45. Urea clot solubility (Qualitative assay for FXIII) Fibrin is held by weak forces till FXIII introduces bonds between them. Weak forces will dissolve in low ionic strength solution but not the strong bonds. Weak ionic strength solution is – 5 Molar Urea Incubate clot in 5M Urea overnight. FXIII deficiency- no clot seen the next day (the clot dissolves) but the clot in a normal plasma appears the same.

46. Are these tests Useful ? Issues:- - Availability - Reliability - Faster turn around time (TAT) Limitation exists for FFP/Cryo Limitation exists for FFP/Cryo ( These tests are necessary in blood banks with components for use in ensuring quality control of these products – Coag Factor assays etc )

47. Any Alternative At least start with Red cells and Platelets - Hb/Hct - Platelet count - Reliability - Faster TAT - Quality Assurance as per ISO-15189 NABL Look at alternative POC/ NPT (Point of Care/ Near Patient Testing) devices – ROTEM/TEG - Thromboelastography

48. Thromboelastography (ROTEM/TEG)--------- Clot activation by TF (at Physiological level) CT/R- Clot time Rate of Clotting – CFT/k and α angle – rate of thrombin generation MA/MCF – Clot strength (Plt/Fib) Stationary cup with blood - oscillated ( 4 o to45’ -10 secs) Pin is suspended - torsion wire - monitored for motion. Measure the torque of rotation - transmitted from the immersed pin – after fibrin platelet bonding has linked the cup and pin together

49. Normal TEG

50. Thromboelastogram TEG (Whole Blood) 30% 20% 10% 5% 2% 1% 0.5% 0.2% FVIII deficient blood spiked with Recombinate

51. Hypocoagulable

52. Hypofibrinogenemia

53. GlanzmannThrombasthenia

54. FXIII deficiency

55. Primary Fibrinolysis

56. Mr.S – with DU perforation Post –op in SICU shifted to MICU due to frank sepsis Coagulogram only mildly abnormal. Over days he uses either red cell or FFP if to undergo a procedure. 14 days later - significant ooze from all puncture sites. Coagulogram still mildly abnormal – given 4 FFP and 8 cryo. No change – in bleeding but improved coagulogram 12.2/34/275/7.7/45,000 -? microvascular bleeding TEG – since they still wanted to give FFP Informed that nothing in the repertoire of the blood bank to turn the situation around Advised rFVIIa

57. Mr.HKP – post Subtotal colectomy in SICU Persistant collection in the drain. Dark coloured. Monitoring coagulation and Hb and using Blood and Blood products. Persistantly coagulopathic with fibrinogen at 75 – risk of Dilutional Coagulopathy But best was later in the evening when 16/40/242/7.5/48,000 but collections were same TEG – still significantly hypocoagulable rFVIIa Re-explored the next day and a open vessel was ligated.

58. PRE & POST FEIBA/rFVIIa TEG – 798938B Pre – 4.9.10 Pre – 1.9.10 P0st – 4.9.10 Pre – 7.9.10 Pre–2.9.10 Post –1.9.10 Post – 2.9.10 Post – 7.9.10

59. Ms.M refered with atonic PPH and 11 Red cell unit transfused Coags – 32/93/43/3.1/120,000 Cryoprecipatate – 10 rushed Continued to ooze in ICU after hysterectomy and another 5 units of Red cells but still mildly coagulopathic 17/42/124/10.5/35,000 Did not give rFVIIa Given 3 PRC and 4 cryo