An Update on Laboratory Diagnostics in Haemophilia A and B

Abstract

Haemophilia A (HA) and B (HB) are X-linked hereditary bleeding disorders caused by lack of activity of coagulation factors VIII (FVIII) or IX (FIX), respectively. Besides conventional products, modern replacement therapies include FVIII or FIX concentrates with an extended half-life (EHL-FVIII/FIX). Two main strategies for measuring plasma FVIII or FIX activity are applied: the one-stage clotting assay (OSCA) and the chromogenic substrate assay (CSA), both calibrated against plasma (FVIII/FIX) standards. Due to the structural modifications of EHL-FVIII/FIX, reagent-dependent assay discrepancies have been described when measuring the activity of these molecules. Assay discrepancies have also been observed in FVIII/FIX gene therapy approaches. On the other hand, nonfactor replacement by the bispecific antibody emicizumab, a FVIIIa-mimicking molecule, artificially shortens activated partial thromboplastin time-based clotting times, making standard OSCAs inapplicable for analysis of samples from patients treated with this drug. In this review, we aim to give an overview on both, the currently applied and future therapies in HA and HB with or without inhibitors and corresponding test systems suitable for accompanying diagnostics.

Fig. 1

Schematic overview on the principles of FVIII/FIX one-stage clotting (OSC) and (two-stage) chromogenic substrate (CS) assays . ( A ) OSC assays. Due to dilution of the patient sample in FVIII- or FIX-deficient plasma (containing defined amounts of the remaining coagulation factors [grey characters]), either FVIII or FIX stemming from the patient sample (black characters) becomes the only variable that influences the activated partial thromboplastin time (aPTT) of the mixture: in brief, addition of the aPTT reagent activates the intrinsic, non-Ca ²⁺ -dependent pathway of the mixture (down to FXI[a]) during initial incubation. After recalcification, the clotting time, that, inter alia, depends on the thrombin-based feedback activation (dotted lines) of cofactors FV and FVIII (italic characters), is measured and FVIII or FIX activities calculated from corresponding plasma-based standard curves. Although the aPTT is a two-step process, aPTT-based determination of factor activity is designated as one-stage clotting assay since the activity of the forming tenase-complex (FIXa + FVIIIa) is reflected in one go by the clotting time measured after recalcification. ( B and C ) (Two-stage) CS assays for the determination of FVIII or FIX activity, respectively. During the first stage, plasma samples are diluted and reagents (factors) that allow for activation of plasma FVIII or FIX (bold characters), assembly of the tenase complex as well as activation of FX are added (characters with grey background). After incubation, the amount of generated FXa is quantified during the second stage of the procedure by addition of a chromogenic peptide substrate (CS). Underlying original plasma FVIII or FIX activities are again calculated from corresponding plasma calibrators.

Fig. 2

Principle of the (Nijmegen)-Bethesda Assay for quantitative determination of inhibitory antibodies against FVIII or FIX. (A) In an exemplary procedure, a heat-inactivated patient plasma sample with an inhibitor titre of 3 Bethesda units (BU)/mL is serially diluted in the respective factor-deficient plasma (or buffer) and the single dilutions are mixed with equal amounts (1 + 1) of (buffered) pooled plasma ([B]PP). As a control, the (B)PP is mixed 1 + 1 with factor-deficient plasma. After incubation (e.g., 2 hours at 37 °C for FVIII inhibitor testing, no such long incubation required for FIX inhibitor testing), (residual) factor activities are measured and calculated relative (%) to that of the control reaction (relative activities [RA], panel C ). ( B ) Inhibitor titres are usually given as BU/mL according to the definition that one BU/mL inhibits the FVIII/FIX activity by 50% (50% RA) and two BE/mL by further 50% (25% RA). The corresponding ideal interpolation function (including 0 BU/mL = 100% RA) that allows the calculation of BU/mL from determined RA values is shown in the graph (RA [ y -axis, log ₁₀ ] versus BU/mL [ x -axis]). The defined range of valid RAs is limited from 75% down to 25%, necessitating the testing of sample dilutions in case of inhibitor titres of > 2 BU/mL. ( C ) Measured factor activities and calculated RA values for the exemplary inhibitor-positive plasma sample (dilutions). Only the 1:4 dilution shows a RA value (60%) within the defined acceptable range of 25 to 75%. Based on the formula shown in panel B , a BU/mL titre of 0.74 is calculated, corresponding to 3 BU/mL in the original sample after correction for the initial dilution.