Explain why there is discordance in FVIII activity after AAV gene therapy between one-stage (OS) and chromogenic substrate (CS) assays.

Answer: After valoctocogene roxaparvovec (AAV5-FVIII-SQ), transgene-produced FVIII measures ~1.6-fold higher by OS than CS.

• Recombinant BDD-FVIII and native FVIII measure comparably by both assays.
• Transgene FVIII accelerates early FXa and thrombin generation, so clot forms sooner. OS reads at 1–2 minutes and captures this; CS reads color at 5 minutes and misses it, reporting lower activity.
• Both assays are clinically valid — joint bleed frequency correlates with each. CS is the trial standard (conservative, works across non-native FVIIIs), but many labs don't run it.

Related: 0001 — FIX-Padua assay discrepancy — also chromogenic-reads-lower after gene therapy, but from the variant's hyperfunctionality rather than assay read timing.

Source: Gene-produced FVIII: measure for measure. Blood. 2020;136(22):2483 — commentary on Rosen S et al. Activity of transgene-produced B-domain-deleted factor VIII in human plasma following AAV5 gene therapy. Blood. 2020;136(22):2524 — review