Assay discrepancy in mild haemophilia A

Abstract

There are three main methods used to assay factor VIII (FVIII) activity: the one-stage and two-stage clotting assays and the two-stage chromogenic method. The most commonly used assay for the diagnosis of haemophilia A is the automated one-stage FVIII assay. The classical two-stage FVIII assays are less frequently used. The chromogenic FVIII:C assay is a variant of the two-stage assay. It is easier to use and therefore used more commonly. Recently significant assay discrepancy has been recognised in the FVIII:C measurements in approximately one-third of mild haemophilia A patients. This so-called discrepant mild haemophilia A is characterised by a high ratio of one-stage/two-stage assay with one-stage FVIII levels that are typically more than double those of the two-stage coagulation assay. There are several mutations that destabilise the FVIIIa structure that can explain this result of a more pronounced decrease of the chromogenic FVIII:C activity compared with the one-stage activity. These mutations are clustered at the interfaces of the A1, A2 and A3 domains of the FVIII protein. The inverse discrepancy, where the one-stage assay gives lower FVIII:C results than the chromogenic assay, seems to be associated with mutations found close to important sites for thrombin cleavage or FIX binding. We are carrying out a study of mild haemophilia A samples from the Malmö Haemophilia Centre of families with a unique F8 genotype. The activity of FVIII will be measured using a chromogenic assay and two different one-stage assays. We hope to estimate the true size of assay discrepancy.

Aim: This project will review assay discrepancy in mild/moderate haemophilia A and the risk of misdiagnosis. The overall aim is to estimate the size of the problem and to learn from the literature and experiences from our centre as well as to suggest recommendations on how to avoid misdiagnosis.

Keywords: assays; discrepancy; haemophilia A; misdiagnosis.