Effects of pre‐analytical heat treatment in factor VIII (FVIII) inhibitor assays on FVIII antibody levels

The use of pre‐analytical heat treatment ( PHT ) with the Nijmegen‐Bethesda assay ( NBA ) for inhibitors to factor VIII ( FVIII ) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56°C for 30 minutes and heating to 58°C for 90 minutes. Data examining the effects of PHT on anti‐ FVIII IgG 4 , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited. Aim To assess the effect of PHT on the levels of detectable anti‐ FVIII IgG 4 . Methods Nijmegen‐Bethesda assay‐positive specimens were incubated at 56, 58 or 60°C for 90 minutes, and anti‐ FVIII IgG 4 was measured by fluorescence immunoassay ( FLI ) at 30‐minute intervals. The effects of PHT on the ability of recombinant FVIII ( rFVIII ) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested. Results Levels of anti‐ FVIII IgG 4 showed little change following incubations at 56°C (mean 101% of original value at 30 minutes and 100% at 60 minutes) but decreased upon exposure to 58°C (mean 85% at 30 minutes and 66% at 60 minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII . Conclusion The optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.