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Optimization of pre‐analytical heat treatment for inhibitor detection in haemophilia A
Author(s) -
Batty P.,
Hart D. P.,
Platton S.
Publication year - 2018
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/ijlh.12862
Subject(s) - haemophilia a , endogeny , haemophilia , recombinant dna , potency , incubation , antibody , chemistry , medicine , pharmacology , immunology , in vitro , biochemistry , surgery , gene
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 ≤ 1 IU / dL after 60 minutes for endogenous FVIII and 120 minutes for rFL ‐ FVIII . Incubation at 58°C resulted in FVIII :C < 1 IU / 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 < 1 IU / dL without appearing to affect inhibitor detection. Laboratory awareness of differences in thermostability of different sources of FVIII is important when choosing PHT conditions.