[WFH LAB Manual] Two stage clotting Assay for Factor VIII:C

#PRINCIPLE

This assay was fi rst described in 1955 as a modifi cation of the thromboplastin dilution test. The two-stage FVIII:C assay is based on the principle that the amount of FVIII present in the system is rate-limiting during clotting of a test mixture containing FX, activated FIX, phospholipid, calcium, and FV in excess. Adsorption of plasma by aluminium hydroxide removes activated factors and vitamin K–dependant factors. This is necessary to remove prothrombin so that none is present in the initial incubation mixture. Without this step, the mixture would contain all the components required for fi brin to form, and the mixture would clot.

The dilutions of adsorbed standard and test plasma are incubated with the combined reagent in the 1st stage. This generates FXa. A source of prothrombin and fi brinogen from pooled normal plasma is added in the 2nd stage, which allows a clot to form and for which the resulting clotting time is

dependent on the initial amount of factor VIII:C.

#REAGENTS

 Combined reagent (see “Production of Combined Reagent”on page 73)

 Owren’s Veronal buffer

 Pooled normal plasma (substrate plasma): store deep frozen

 Standard or reference plasma

 Internal quality control (IQC)

 0.0125M Cacl2 (for example, 1 in 2 dilution of 0.025M CaCl2 as used in APTT testing)

 Alumina hydroxide suspension (e.g. SIGMA catalogue code A8222): store at room temperature)

#SAMPLE REQUIREMENTS

Citrated plasma, which can be stored frozen prior to testing, if required. 

DIAGNOSIS OF HEMOPHILIA AND OTHER 72 BLEEDING DISORDERS

#METHOD

1. Reconstitute standard plasma with distilled water 10 minutes before use, and combined reagent at least 15 minutes before use with 3 ml of 0.0125M CaCl2. 

2. Prepare IQC plasma and substrate plasma. If any are frozen, thaw at 37°C for 5 minutes before use.

3. Label suffi cient disposable plastic tubes for each of the samples, IQC, and standard.

4. Place 0.45 ml of standard, IQC, and patient plasma into each plastic tube.

5. Add 50 μl of well-shaken alumina to each tube and mix well.

6. For small volumes of plasma, use 0.225 ml plasma and 25 μl alumina.

7. Incubate at 37°C for 3 minutes, then spin at 5000 g for 2 minutes in a suitable centrifuge.

8. Immediately transfer the supernatant plasma to plastic cups or tubes compatible with the analyser being used. Take care not to disturb the sedimented alumina.

Note: The following steps are based on use of a Sysmex CA series analyser. The test can be run on instrumentation from a number of other manufacturers. One co-author of this manual has successfully run assays on analysers from Instrumentation Laboratory, and most likely the method would be compatible with other types of analyser. Therefore, this specifi c method is included as an illustrative example.

9. Load combined reagent, assay buffer, and pooled normal plasma/substrate onto auto analyser.

10. Adsorbed plasmas are presented for analysis in the following order: standard, IQC, patient plasmas, and lastly, a second standard. Samples with normal concentrations of FVIII are normally assayed using three different dilutions in the range of 1/50–1/400. For low levels of FVIII (< 0.05 IU/dl) dilutions of 1/10, 1/20, and 1/50 might be needed. For raised levels (>1.5 IU/dl), dilutions of 1/800–1/3200 might be needed. Otherwise, patient dose response lines may not be parallel to the

standard line.

#RESULTS

Plotting of results and calculations of FVIII activity are as described in the one-stage assay (Section 23).