Why is there a discrepancy between clot-based one-stage assays and chromogenic assays in patients who have undergone gene therapy for hemophilia B?

Answer: In the reported study, the chromogenic-based assay yielded lower FIX activity levels than the one-stage clotting assay. Proposed explanations include:

• The enhanced activity of recombinant and transgene-expressed FIX-Padua depends on the interaction between FXIa and FVIIIa. Differing efficiencies of generating FIXa or FVIIIa by different activator reagents may therefore produce larger discrepancies when measuring FIX-Padua.
• The hyperactivity of FIX-Padua likely facilitates the detection of small differences between one-stage clotting assays.

The impact of specific differences in assay methodologies, instruments, and reagents on the measurement of FIX-Padua activity is not known at this time and could not be determined from the analyses of limited subject samples. Clinical correlation of subject outcomes with determined FIX:C will be important.

Related — do not confuse with FVIII Padua. Same eponym, unrelated entity: FIX-Padua is a hyperfunctional F9 point variant used in hemophilia B gene therapy; FVIII Padua is an F8 duplication causing thrombophilia.

Source: Robinson MM et al. Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua. J Thromb Haemost. 2021. doi:10.1111/jth.15281 — primary-literature