Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

Abstract

The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.

Development and validation of a new assay for assessing clot integrity

“…Nevertheless, in some circumstances, such as haemophilia and in point of care testing, other methods may be better [19,20]. Indeed, as shown in this paper, the large CVs in the TEG data have a significant impact on the reproducibility of the results.…”

Role of chromogenic assays in haemophilia A and B diagnosis

“…However, there has been a gradual accumulation of data to demonstrate that in approximately 20%‐50% of cases with mild haemophilia A there are significant discrepancies in FVIII:C results dependent on the method principle used. A number of reports have described cases with the OSA being two‐ to fivefold higher than the results achieved with the TSA . Family members generally all have the same classification as either equivalent or discrepant.…”

“…There are a number of commercially available chromogenic assays which differ in the source of purified proteins, the process to activate FVIII, sample diluent, plasma dilution, calcium concentration and incubation time. Limited studies have directly compared the sensitivity of different chromogenic kits in the measurement of FVIII:C in subgroups of haemophilia A . To the author's knowledge, there have been no comparisons of the precision or sensitivity of different chromogenic kits in the measurement of recombinant factor replacement therapy; however, some data are available for extended half‐life products .…”

Assaying FVIII activity: one method is not enough, and never was

“…Centres that have always only had the one-stage clotting assay available are often not convinced about the utility of the chromogenic assay because they see it as an expensive, complicated luxury, primarily developed for research purposes. Several haemophilia centres, however, have now shown significant FVIII:C discrepancies in up to a third of their mild haemophilia A patients [3][4][5]. Two types of discrepancy exist: the classical type where the chromogenic assay is lower than the one-stage clotting assay and the reverse pattern which is rarer [5].…”

“…The FVIII:C discrepancy is sometimes of clinical significance such as when the one-stage clotting assay is normal and the chromogenic assay reduced [4,5] or when the chromogenic is so low that desmopressin response is unlikely to be effective while the one-stage clotting assay shows good response. In the reverse situation, the one-stage assay may be reduced but the chromogenic assay is normal and often these individuals do not bleed excessively despite the diagnosis of haemophilia A based on a mutation in the FVIII gene and a reduced one-stage FVIII:C [6,7].…”