Laboratory Investigations – all that a blood banker needs to Know

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

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

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

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 * 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 - 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- 1st for blood counts and 2nd 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 370C water bath. A circulating water bath is ideal. • Keep test plasma or control plasma and reagents at 370C 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

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 = PRISI

36. aPTT • Time taken by a recalcified citrated plasma to clot in presence of negatively charged - contactactivator (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 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 - Lupus anticoagulant (antiphospholipid antibody) - Factor Inhibitor ( antibodies to Clotting factors) - 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 plasmasupplied 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 Mixing = 40 sec (Keep the “Mix” and individual plasmas 2 hours in water bath) 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 - Fasterturn around time (TAT) 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)--------- Stationary cup with blood - oscillated ( 4o 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 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)

49. Normal TEG

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