In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one‐stage and two‐stage factor VIII assay results

Abstract

Summary In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one‐stage assay are more than double than those by two‐stage assay. This may be due to the longer incubation times (10–12 min) in the two‐stage assay. This study aimed to determine the time course of the activation phase of the two‐stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one‐stage and two‐stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short‐ or long‐incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23–56%, mean 41%) than after 10 min (19–41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21–64%, mean 37%) than with the longer incubation times usually used (13–29%, mean 23%). These time‐course experiments have verified that the longer incubation time used in the two‐stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

The “normal” factor VIII concentration in plasma

“…Nontheless, the maximum activity of the intrinsic FXase does not last for more than 1.5-2.0 min and is observed after a 0.5-1.0 min lag phase (S. Butenas, unpublished observation). Thus, it is not surprising that the results of this assay are influenced by the incubation time of FX with the intrinsic FXase [3] and that a longer incubation time gives lower results for FVIII activity in hemophilia A patient plasma [9]. …”

“…These discrepancies are dependent upon the concentration of FVIII in test plasmas [6], standards used [7, 8] and incubation times in the chromogenic assay [3, 9]. The nature, purity and structure of natural and recombinant FVIII proteins are also confounders for these two assays as well [8, 10, 11].…”

Diagnosis of factor VIII deficiency

“…No significant differences in the results between the two methods were detected in the blood of donors with normal or high FVIII levels, in patients with a chronic liver disease and in normal persons with high FVIII levels after treatment with desmopressin. However, in about 1/3 of the patients with mild haemophilia caused by a single-point mutation, a discrepancy is described between the one-stage and two-stage assay (both the two-stage clotting assay and chromogenic assay) [29]. Usually, the activity with the chromogenic assay is lower compared with the results of the one-stage assay and at least 17 different mutations have been identified so far with discrepant assay data.…”

“…Usually, the activity with the chromogenic assay is lower compared with the results of the one-stage assay and at least 17 different mutations have been identified so far with discrepant assay data. Most mutations appear in the interface between the subunits of the FVIII molecule [29]. The discrepancy can, at least partly, be explained by increased instability of the mutated molecule.…”

Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A

“…A missed diagnosis may occur when one‐stage FVIII results fall within the normal reference range (Keeling et al. , 1999; Goodeve & Peake, 2003; Rodgers et al. , 2007), or are only slightly reduced.…”