Patients with Mild Hemophilia a with Discrepant FVIII Assays: Thrombin Generation and Bleeding Phenotype

Introduction: Patients with mild hemophilia A (MHA) have factor VIII (FVIII) levels between 6 and 40%. FVIII clotting activity can be measured by one-stage (OS), two-stage (TS) and chromogenic substrate (CSA) assays. Discrepancy between assays was reported, raising the issue of which is the most adequate to make diagnosis and predict bleeding tendency. Thrombin generation assay (TGA) was investigated as a tool to discriminate bleeding phenotype in patients with hemophilia.

Aim: This single-center study was aimed at ascertaining the prevalence of discrepant cases among patients with MHA and at correlating FVIII and TGA results with the bleeding phenotype.

Methods and Results: 134 consecutive MHA patients, aged 1-81 years (median 40) and diagnosed on the basis of FVIII:OS >5% were recruited between March 2012 and April 2013 during outpatient follow-up visits. Patients who had developed anti-FVIII inhibitors were excluded. Data on bleeding history were collected from medical files. At recruitment, the bleeding score was assessed and blood drawn in a non-bleeding state to measure FVIII:OS, FVIII:CSA and TGA. Genetic analysis was performed to ascertain FVIII gene defects. Thirty-six patients (26%) aged 2-76 years (median 40) had assay discrepancy being the FVIII:OS/CSA ratio <0.5 in 2 and >2.0 in 34. The former were brothers aged 27 and 34 years, with Arg1689His mutation and similar FVIII and TGA values (FVIII:OS 31 and 34%; FVIII:CSA 70 and 85%; ETP 1629 and 1475 nMxmin; peak 163 and 173 nM). The youngest bled after tooth extraction and had 2 post-traumatic haemorrhages, while the oldest never bled despite surgery/dental procedures. Among the 34 patients with FVIII:OS/CSA >2.0, 6 (18%) had FVIII:OS >40% despite a bleeding history and 2 (6%) never bled; the latter belong to a triplet of brothers, all with similar FVIII and TGA values (FVIII:OS 24-28%; FVIII:CSA 9% in all; ETP 1447-1496 nMxmin; peak 106-116 nM). Among 98 non-discrepant patients aged 1-81 years (median 40), the median FVIII:OS/CSA ratio was 1,45 (IQR: 1,26-1,65) and only 4 (4%) never bled. Major features of patients with FVIII:OS/CSA ratio >2.0 (from 26 families) and non-discrepant patients (from 90 families) are shown in the table. Three families contributed to both groups.

Conclusions: In this series of MHA patients, one third were discrepant and 20% of them would not have been diagnosed by FVIII:OS. TGA values were consistent with FVIII:CSA and concordant with the bleeding score. However these potentially misdiagnosed patients represented a minority of the whole study population.