The current international guidelines do not define the type of assay to be used in the diagnosis of mild Hemophilia A and can misclassify some individuals as normal.
Factor VIII (FVIII) is an essential blood-clotting protein and can be measured using the one-stage clotting assay (FVIII:C1), two-stage clotting assay (FVIII:C2) and the chromogenic (amidolytic) assay (FVIII:CR).
Hematologists at the University of Sheffield (UK) investigated the incidence of assay discrepancy, assessed the impact of alternative reagents on FVIII:C activity assays and determined the usefulness of global assays of hemostasis in mild hemophilia A. They measured FVIII:C in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was determined by two-fold or greater differences between one-stage and two-stage FVIII:C.
Activated partial thromboplastin times (APTT) and FVIII:C1 assays were performed with Actin FS APTT reagents and FVIII-deficient plasma (both from Siemens; Marburg, Germany). The FVIII:C2 assays were performed by an in-house method using bovine factor V and platelet substitute (Diagnostic Reagents; Thame, UK). The FVIII:CR assays were performed with three commercial kits Siemens, Coamatic (Chromogenix; Bedford, MA, USA) and Technochrom (Pathway Diagnostics; Dorking, UK).
Genetic analysis of Factor 8 (F8) mutation was undertaken using either confirmation sensitive gel electrophoresis (CSGE) followed by DNA sequencing of amplicons displaying a migration shift or by direct sequencing of the entire coding region and intron-exon boundaries of F8. Rotational thromboelastometry and calibrated automated thrombography were also performed.
There were 84 individuals whose FVIII:C was between 5 and 50 IU/dL by at least one method, and were included in this study; 59 had less than a two-fold difference between FVIII:C1 and FVIII:C2 assays and were classified as congruent. Assay discrepancy was observed in 31% of individuals; 12% with lower two-stage FVIII:C and 19% with lower one-stage. However, an individual''s genotype did not always predict their phenotype. Chromogenic assays were shown to be a suitable alternative to the two-stage test. Poor sensitivity to hemophilia was demonstrated by thromboelastometry. Thirty percent of patients exhibited significant two-fold assay discrepancy.
The authors concluded that 4% of patients would not be diagnosed by one-stage FVIII:C assays. They recommend that laboratories should utilize both one stage and chromogenic or two-stage assays in the diagnosis of patients with mild bleeding disorder and possibly hemophilia A. The study was published on June 28, 2013, in the journal Haematologica.