1. HAEMOPHILIA TREATMENT CENTRE
LAB FACILITIES
DR. MEENA BEEBI
PATHOLOGIST
HEMOPHILIA TREATMENT CENTRE, D.H. ALUVA

2. Government Hospital, Aluva

3. FACTOR ASSAY
PRINCIPLE Based on a comparison of the ability of dilutions of standard and test plasma to correct the APTT of a plasma known to be totally deficient in FVIII but containing all other factors

4. REAGENTS
Standard plasma(PNP)
Factor VIII Def. plasma
APTT reagent and Cacl2
Imidazole Buffer
Patient plasma

5. PLASTIC TUBES
1/10 1/20 1/ µI 500µl 500µl 100µl 500µl 500µl

6. GLASS TUBES
1/ / /40
100µL of dilution +100µL of FVIII plasma
Do APTT

8. Straight lines, parallel to other ,should be obtained Non parallel lines: Converging type-technical error inhibitor F.VIII <1% Diverging type- activated sample

9. Read off the conc of the test sample from the graph. The conc
Read off the conc of the test sample from the graph.The conc.of the factor in standard plasma is already known.
So Factor level in test sample/factor level in standard sample x100 gives you the actual factor level in patient
Eg : /85 x100=6 IU/dl

10. Fully automated coagulometer
STAGO –DESTINY PLUS

11. Refrigerated centrifuge
Water Bath

12. Factor Levels
Factor Level <1IU/dl _severe Hemophilia
Factor Level 1_5IU/dl _Moderate Hemophilia
Factor Level 5_50IU/dl_Mild Hemophilia

13. Factor Assay in Cryoprecipitate
Cryoprecipitate usually show a Factor VIII level in the range IU/dl
In this case, a predilution of the sample to1in5 or1in 10 will reduce the conc., to a level such that the factor assay is possible.The prediluted sample is further diluted 1/10,1/20,1/40 in assay buffer and the assay done

14. Predilution can be done with either Assay
buffer or Factor VIII Def Plasma(preffered)

15. Inhibitor Screening
Inhibitors are Neutralizing Antibodies in patients who have been transfused with exogenous replacement factors
Incidence of inhibitors in hemophilia A-20-35%
Hemophilia B-1-6%

16. IN INHIBITORS
Inhibitors are IgG antibodies reacting with the active sites of FVIII molecule primarily with the epitopes in the A2,A3,C1 and C2 domains
Factor VIII combines with its inhibitors in a time dependant fashion
If the patient´s inhibitor level is low,a higher dose of FVIII may be able to neutralize the inhibitors and allow haemostasis.Higher inhibitor levels require the use of bypassing agents

17. An inhibitor is suspected when it is difficult to control bleeding with the usual dose of clotting factor concentrate in a patient or if there are increased incidence of bleeding
Affected by age,duration of treatment,genetic factors,concentrate type,environmental etc.

18. l Laboratory Diagnosis
Screening
Principle:
APTT is determined on 50:50 incubated mix at 37º C as well as freshly prepared mix, after incubating at 37ºC.The degree of correction of each mix is compared
Reagents
PNP
Test Plasma
Reagents for APTT

19. Plastic Tubes
A B C
0.5 ml plasma ml
test plasma :50 MIX

20. Incubate for 2 hours at 37 deg
Make a50:50 mix from tubes A and B at the end of second hour and mark it as D
Perform an APTT in duplicate on A,B,Dand C(in that order)

21. SAMPLES 1 2 3
TEST PLASMA 90
FRESH MIX 45 48 70
INCUBATED MIX
Normal Plasma APTT Sec.

22. A difference of more than 5 seconds is considered positive.
Classical Bethesda assay
Modified Nijmigen Assay

23. Mixing studies showing presence of an inhibitor
Incubation Mixture
APTT at the start of incubation (in seconds)
APTT after 2 hours(in seconds)
Normal plasma 32 40
Factor VIII Def. plasma 90 95
50:50 Mix of Normal plasma and Def plasma
No Inhibitor 37 45
1BU 53
5BU 43 64
20BU 54 92

24. Fi Fibrinogen Assay PRINCIPLE(Modified Clauss Assay)
Dilutions of standard normal plasma with known Fibrinogen content are prepared and clotting time measured
REAGENTS
Reference Plasma
Thrombin Solution of conc.30IU/ml- 100IU/ml
Imidazole Buffer

25. METHOD
Prepare 1/5,1/10.1/15,1/20 dilutions of reference plasma in Imidazole buffer
Do Thrombin time at37ºC
Plot on a log log graph paper with time in sec on X axis and fibrinogen conc. On Y axis.1/10 dilution represents the standard value(1.5g/l-3.5g/l)
Dilute the test plasma (usually1/10 dilution) and do TT .Plot on the graph paper and find the value in seconds

26. Contd….
If you expect low value of fibrinogen then dilution of 1/5 or1/2 are taken accordingly and the conc obtained from the graph is multiplied by 5/10 and 2/10 respectively

27. THANK YOU