Optimization of pre-analytical heat treatment for inhibitor detection in haemophilia A

P Batty; D P Hart; S Platton

doi:10.1111/ijlh.12862

Optimization of pre-analytical heat treatment for inhibitor detection in haemophilia A

Int J Lab Hematol. 2018 Oct;40(5):561-568. doi: 10.1111/ijlh.12862. Epub 2018 May 18.

Authors

P Batty^{1

2}, D P Hart^{1

2}, S Platton²

Affiliations

¹ The Royal London Hospital Haemophilia Centre, Barts and The London School of Medicine and Dentistry, QMUL, London, UK.
² The Royal London Hospital Haemophilia Centre, Barts Health, The Royal London Hospital, London, UK.

PMID: 29777571
DOI: 10.1111/ijlh.12862

Abstract

Introduction: Factor VIII (FVIII) antibody formation is the greatest clinical and laboratory challenge within the haemophilia centre. The Nijmegen-Bethesda assay (NBA) is the gold standard for inhibitor quantification, but affected by pre-analytical variables including a patient's FVIII activity (FVIII:C). Pre-analytical heat treatment (PHT) provides a methodology for inhibitor testing when measurable FVIII:C is present.

Methods: We evaluated the effect of different PHT conditions (time/temperature) on FVIII:C as well as on potency of inhibitory activity in samples containing FVIII:C (endogenous pooled plasma and exogenous recombinant FVIII (rFL-FVIII) concentrate) or FVIII inhibitor.

Results: PHT of endogenous FVIII at 37°C, 47°C and 52°C resulted in declining measurable FVIII:C at 120 minutes (69%, 57% and 13% of the original FVIII:C, respectively). Incubation at 56°C resulted in FVIII:C ≤ 1IU/dL after 60 minutes for endogenous FVIII and 120 minutes for rFL-FVIII. Incubation at 58°C resulted in FVIII:C < 1IU/dL at 15-30 minutes for endogenous FVIII and at 30-60 minutes for rFL-FVIII. No difference was seen for inhibitor detection following PHT (56°C or 58°C) by NBA or anti-FVIII IgG ELISA.

Conclusion: PHT at 58°C for 30 minutes demonstrated consistent reduction in FVIII:C < 1IU/dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.

Keywords: Bethesda; haemophilia; heat treatment; inhibitors; pre-analytical.