1. Basic coagulation techniques and Quality control issues
Dr. Shrimati Shetty
Deputy Director
National Institute of Immunohematology ( ICMR)
KEM Hospital, Mumbai

2. Final Diagnosis of a bleeding disorder

3. Anticoagulant
0.129 M tri sodium Citrate at a ratio of 9 parts blood to 1 part anticoagulant is used for all coagulation tests.
Why not EDTA or Heparin?
EDTA irreversibly chelates Ca ions
Heparin activates antithrombin which is an inhibitor of coagulation

4. Anticoagulant… If the HCT is above 55%
Anticoagulant vol. [x] = 100 – hematocrit x total vol. of anticoagulated blood required Example: Patient hematocrit = 60%
x 5.0 = 0.37 mL

5. Preanalytical variables
Sample hemolysis/ lipaemic sample
Improper proportion of Anticoagulant to blood
Prolonged time interval before testing
Difficult punctures
Freeze thawing the samples

6. Coagulation Tests
Screening coagulation tests
Confirmatory Tests

7. When a bleeding patient walks in, what should be the initial tests to be performed?
Screening tests
Peripheral smear ( Bernards Soulier syndrome, macrothrombocytopenia, leukemia , thrombocytopenia )
Complete Blood Count ( BSS, MTCP, Leukemias, thrombocytopenia) PT
APTT TT
FXIII screening

9. Only PT is abnormal Congenital Causes Factor VII deficiency
Acquired Causes
Liver disease
Warfarin
DIC
Inhibitors LA
Malignancy

10. PT - INR
PT measures the deficiency of all VK dependent coagulation factors and also the integrity of extrinsic pathway
INR = [Patient PT]ISI
[Control PT]

11. Only APTT is abnormal Congenital Causes
Factor VIII/ IX/XI/XII contact factors
Acquired Causes
Liver disease
Warfarin
DIC
Heparin
Inhibitors to factors, LA

12. PT & APTT prolonged Congenital causes
i.Combined deficiency of V & VIII
ii. Factor X deficiency
iii. Factor V deficiency
iv. Multiple VK dependent clotting factor deficiency
Acquired causes
Liver disease
Warfarin
DIC
Heparin
Inhibitors to factors
vitamin K deficiency

13. PT, APTT, TT prolonged Congenital causes
i. Afibrinogenemia/ dysfibrinogenemia
ii. Factor II deficiency
Acquired causes
DIC
Liver disease

14. Screening for F XIII deficiency
Clot solubility test
Clot formation with either thrombin or CaCl2
Solubility of the clot using 2% acetic acid , 1% mono chloroacetic acid or urea
After 24 hours , the clot is observed for solubility
Different sensitivities with different clotting reagents & solvents
Severe factor deficiency sometimes gets misdiagnosed as F XIII deficiency
ELISA test is the sensitive assay for detecting F XIII deficiency

15. Confirmatory tests-Factor Assays
Reagents required
Normal pooled plasma or unicalibrator
Factor Deficient plasma
APTT reagent
CaCl2

16. Factor assays…. Factor VIII/IX/XI/XII – APTT based
Factor II/VII – PT mode
Factor V/X – can be both PT/APTT mode

17. Factor VIII Graph

18. Interpretation of factor results
At birth, activities of the vitamin K dependent factors II, VII, IX, and X and the concentrations of the contact factors XI and XII are reduced to about 50% of normal adult values. The levels of the factors V, VIII, XIII, and fibrinogen are similar to adult values
Plasma concentrations of the naturally occurring anticoagulant proteins (antithrombin, protein C, and protein S) are significantly lower at birth than during the adult years
Most blood coagulation factors and fibrinogen increase during pregnancy. Factor (F) XI is the only blood coagulation factor that decreases.
Malignancies

19. Interpretation
Should we go ahead with factor assays even when screening tests are normal?
yes, in case of any clinical indication we should do the specific factors even if PT/APTT is normal

20. NPP 20-25 healthy donors , blood group matched
Compare it with unicalibrator with known factor values
Never use a single individual sample as control

21. Deficient Plasma& APTT reagents
Should have 0% FVIII
Negative for TTD/inhibitors
Different APTT reagents have different sensitivities

22. Factor Assays Severe <1% factor Moderate 1-5% Mild 6-40%
About 5% of the patients are clinically mild despite having <1% factor

23. Can we diagnose a patient as HA or HB without doing factor assays?
Mixing Studies
BaSO4 adsorbed normal plasma, deficient in factors VII, IX, X and prothrombin
Aged normal serum, deficient in factors V and VIII, prothrombin, and fibrinogen

24. Mixing Studies…. Result Interpretation NPP+Adsorbed Plasma Correction
F VIII deficiency
NPP+ Aged Serum
No Correction
No Correction
F IX deficiency

25. Mixing Studies….. Mixture Result Interpretation
NPP + F VIII Def Plasma
Correction
F IX deficiency
NPP + F IX Def Plasma
F VIII deficiency

26. Platelet Aggregation tests
Highly variable results
Diet, Medication, physical activity
Platelet receptor studies to confirm diagnosis
Always confirm diagnosis by other tests
Never give a diagnosis based on platelet aggregation alone!

27. Disorders diagnosed by platelet aggregation and receptor studies
Von Willebrand disease
Glanzmanns thrombasthenia
Bernard Soulier syndrome
Storage pool defect
Cyclooxygenase deficiency
others

29. What are the other supporting tests?
Platelet receptor studies using antibodies specific for platelet receptors
GP 1b/IX for diagnosis of BSS (CD 42)
GP IIb/IIIa for diagnosis of GT( CD41, CD61)
collagen receptors ( CD36)

30. Diagnosis of VWD
Platelet aggregation with Ristocetin ( 1.25 mg/ml) absent or reduced
Type IIb shows increased aggregation with 0.5 mg/ml whereas in normal cases there is no aggregation
VWF by Electrophoresis sensitive only for severe def
ELISA is the test of conirmation
RCof , collagen binding ELISA and Multimer analysis to subtype

31. Diagnosis of BSS Giant platelets in peripheral smear
Normal or reduced platelet count
Absence of aggregation with 1.25 mg/ml risticetin
Absence of GP 1b/IX receptors by flow cytometry

32. Diagnosis of GT
Absence of aggregation with 6uM ADP, 4ug/mL collagen and 0.75mM arachdonic acid
Absence of GP IIb/IIIa receptors by flow cytometry

33. Storage pool defect
Primary phase aggregation with all agonists

34. Screening for inhibitors ( Mixing studies)
NPP and patient plasma mixed and APTT performed at 0 hour, 1 hour and 2 hour
Should exclude Lupus anticoagulants
FVIII inhibitors are generally progressive , FIX inhibitors/LA immediate acting

35. Screening for inhibitors
0 hr
1 hr
2 hrs
NPP
Patient
Separately incubated
Incubated Mix

36. The Bethesda Assay 36

37. Specialized Investigations
Thromboelastography

38. Thromboelastography

39. Thrombinoscope

40. Thrombinoscope

41. PFA 100

42. Quality control exercises
IQC
EQC

43. Some examples Case 1 Peripheral smear- giant platelets seen
Platelet count 130X103/uL
PT – C14 Secs/ P 15 secs ;
APTT – C29 secs/P 32 secs;
TT – C 16 secs/P 15 secs
F XIII - N
RIPA – Ristocetin 5%
ADP, collagen, AA %
GP1b/IX receptors – highly reduced
Diagnosis : BSS

44. Some examples ….. Case 2 PS – normal Platelets 260X 103/ul
PT C14 secs/ P 32 secs
APTT C 30 secs P 58 secs
TT C 15 secs/ P 16 secs
F XIII - N
FX 96%; F V 15%
F VIII 8%
Diagnosis: Combined deficiency of F V and VIII

45. Thank you