A blinded in vitro study with Refacto® mock plasma samples: similar FVIII results between the chromogenic assay and a one-stage assay when using a higher cephalin dilution

Summary.  Assay of factor VIII (FVIII) in patient samples is routinely carried out using the one-stage assay rather than the chromogenic substrate assay. The introduction of new FVIII preparations for the treatment of haemophilia A, including immunopurified FVIII and particularly, recombinant FVIII (rFVIII) concentrates, has led to discrepancies between the results obtained with the two assays. In patients treated with rFVIII concentrates, FVIII levels measured with the one-stage assay can be 20–50% lower than those measured with the chromogenic assay. In this study, the one-stage assay was performed with cephalin dilutions higher than those recommended by the manufacturer. B-domain-deleted recombinant FVIII, Refacto®, was diluted to eight different concentrations, ranging from 1–100 IU dL−1, in FVIII-deficient plasma and the FVIII activity of the eight solutions was determined by the chromogenic method in a central laboratory. Aliquots were then assayed by the one-stage method in the four participating laboratories, using different dilutions of CK-Prest®. When CK-Prest® was reconstituted according to the manufacturer's recommendations (dilution 1 : 1), the difference between the one-stage and chromogenic methods was close to 30%. CK-Prest® cephalin dilutions of 1 : 5 and 1 : 8 gave very similar results with the two methods, without increasing the interlaboratory coefficient of variation. These findings confirm the influence of phospholipids on the one-stage assay, particularly the importance of using a phospholipid concentration close to the physiological value in platelets. Thismodified one-stage method may therefore offer an alternative to the use of a concentrate-specific standard.