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

Government Hospital, Aluva

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

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

PLASTIC TUBES 1/10 1/20 1/40 500 500 900 900µI 500µl 500µl 100µl 500µl 500µl

GLASS TUBES 1/10 1/20 1/40 100µL of dilution +100µL of FVIII plasma Do APTT

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

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 : 7/85 x100=6 IU/dl

Fully automated coagulometer STAGO –DESTINY PLUS

Refrigerated centrifuge Water Bath

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

Factor Assay in Cryoprecipitate Cryoprecipitate usually show a Factor VIII level in the range 200-1000IU/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

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

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%

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

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.

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

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

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)

SAMPLES 1 2 3 TEST PLASMA 90 FRESH MIX 45 48 70 INCUBATED MIX Normal Plasma APTT 35 Sec.

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

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

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

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

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

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