Methodological shortcomings in assessment of factor VIII concentrate inhibition

Dear Editor, With interest we read the recent article by Salvagno et al. that compared the in vitro procoagulant potency of four different factor VIII (FVIII) products in the presence of FVIII inhibitor-containing plasma of various titres [1]. The authors suggest that FVIII products high in von Willebrand Factor (VWF) content are less sensitive to alloantibody inhibition compared with products with little or no VWF on basis of lower inhibitor titres with the Malmö assay and more inhibitor resistance in the thrombin generation assay (TGA). Based on these findings, the authors conclude that VWF-containing FVIII concentrates may have a treatment advantage for patients with inhibitors directed to the C2 domain of FVIII. Unfortunately however, the methods described show some striking methodological limitations and therefore the results cannot, to our opinion, be converted into putative clinical effects. Of foremost concern is that the experiments described in this study were performed in mixtures with varying levels of VWF because of the different dilution rates of the patient plasma samples in combination with the use of VWF-free diluent, resulting in very low native VWF content in the highly diluted samples 2, 3, 4, 6, 9 and 11, and the variable VWF content of the FVIII concentrates. Especially in the mixtures of VWF-free FVIII products (Hemofil-M and Kogenate ) and the samples with high inhibitor titres the total absence of VWF may lead to increased neutralization of FVIII both by temperature-dependent inactivation because of the non-physiological saline medium and the increased binding of the inhibitors to the unprotected light chain of the FVIII molecule. These two phenomena may explain the high Malmö inhibitor titres in these assays compared with the titres in assays with the VWF-containing FVIII products Haemate-P and Fanhdi . Our hypothesis is supported by the results of the five remaining low-level inhibitor samples with titres that were more or less similar for all FVIII products and the fact that patient samples with inhibitors directed against other domains do not show these large discrepancies. This effect has already been described by our group for the Nijmegen assay resulting in lower FVIII inhibitor titres when using VWF-free FVIII-deficient plasma as control sample [2] and one can imagine that this, mutatis mutandis, also holds true for the Malmö assay. In the TGA the samples were diluted to 1 MIU ml measured against Hemofil-M and the resistance of different products against the inhibitors were tested measuring the thrombin generation. However, as we think that the measured inhibitor activities of the samples are not comparable, because of the shortcomings in the inhibitor assay, no conclusion can be drawn from these experiments. The differences seen with the TGA probably reflect the differences in real inhibitor titre more than the inhibitor resistance of the different products. A more reliable approach in our opinion would be standardization of the inhibitor titre with a product independent assay like the Nijmegen assay or an Elisa assay. Altogether, our major concern is that for the inhibitor assay the test conditions do not mimic the likely natural situation as in the circulation VWF is present in great excess compared with FVIII. VWF will bind and stabilize rapidly FVIII after infusion as was demonstrated clearly in pharmacokinetic studies describing a correlation between patients preinfusion VWF levels and the observed plasma half-life of infused recombinant FVIII [3,4]. We suggest that the effect of VWF levels in inhibitor assays will be further investigated in a larger cohort in order to Correspondence: Dr. Bert Verbruggen, Radbound University Nijmegen, Medical Centre, Dpt. Laboratory for Haematology, Geert Grooteplein 8, 6525 GA Nijmegen. Tel. : +31 24 361 4796; fax: +31 24 356 8408; e-mail: H.Verbruggen@chl.umcn. nl