Week of 13–19 July 2026¶
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
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
Can we use Altuviiio (efanesoctocog alfa) for patients who have a history of inhibitors and underwent ITI?¶
Answer: There is no prospective trial evidence — but the limited retrospective data are encouraging.
- XTEND-1 excluded patients with a detected inhibitor or any prior history of inhibitors, and also excluded patients on emicizumab. Enrolled patients had ≥150 exposure days.
- One retrospective study suggests it is effective — 7 patients who switched from emicizumab and 5 who previously underwent ITI:
Five patients had a history of FVIII inhibitors, and all achieved excellent hemostasis without inhibitor recurrence. One of the five initially had rapid clearance of Efa, necessitating more frequent dosing at 50 IU/kg every five days to achieve clinical goals. Longitudinal monitoring of FVIII levels revealed reduced clearance after 1 year of prophylaxis, and he returned to standard weekly dosing. We hypothesise that the structure of Efa may have shielded immunogenic epitopes, providing additional effective immune tolerance of trace persistent neutralising or clearance FVIII antibodies, leading to normalisation of half-life over time.
— Haemophilia, doi:10.1111/hae.70175
Ongoing trials to watch: XTEND-ed extension · joint health · physical activity · and, most relevant here, efanesoctocog alfa for ITI.
Source: Retrospective cohort, Haemophilia, doi:10.1111/hae.70175 — primary-literature. Pivotal trial: XTEND-1, NEJM, doi:10.1056/NEJMoa2209226 (excluded this population).
What is the F8 gene duplication (FVIII Padua)?¶
Answer: A 23.4-kb tandem duplication of the proximal F8 gene reported in 2 Italian families. It is the first thrombophilic defect described in F8, designated FVIII Padua.
- Phenotype: severe thrombophilia with extreme, persistently elevated FVIII (antigen and activity >400%) as the only thrombophilic defect. The proband had recurrent VTE before age 50.
- Genetics: the duplication cosegregated with high FVIII levels and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII ≥250% found a second family carrying the same rearrangement on the same genetic background suggesting a founder effect.
- Mechanism: carriers show ≥2-fold upregulation of F8 mRNA, consistent with open chromatin signatures and enhancer elements inside the duplicated region.
Related — do not confuse with FIX-Padua. Same eponym, unrelated entity: FVIII Padua is an F8 duplication causing thrombophilia via overexpression; FIX-Padua is a hyperfunctional F9 point variant deliberately used in hemophilia B gene therapy.
Source: Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia. Blood. 2021;137(17):2383 — primary-literature
How can someone who has a low factor VIII level of around 40% be considered to possibly have type 2N VWD?¶
Answer: Since type 2N VWD follows a recessive inheritance pattern, a clinical diagnosis requires a homozygous or compound heterozygous state. Therefore someone who is a carrier may seem to have mild hemophilia when it is type 2N.
Type 2N arises from D'-D3 domain variants that impair VWF binding to FVIII. In the German type 2 VWD cohort, genotype sorted the phenotype into tiers. FVIII:C/VWF:Ag ratio is what separates them:
- Homozygous / compound heterozygous for two 2N variants (e.g. p.Arg854Gln, p.Arg816Trp): FVIII:C 2–25%, VWF:Ag 50–166%, ratio 0.01–0.4. The classic severe picture.
- 2N variant compound heterozygous with a quantitative VWF variant (nonsense, small deletion, splice site, or the type 1 clearance variant p.Tyr1584Cys): FVIII:C 10–50%, VWF:Ag 29–74%, ratio 0.2–1.3.
- Heterozygous carriers (8 with p.Arg854Gln, 2 with p.Cys1225Gly): a carrier state, not a 2N diagnosis. Notably they had a normal average FVIII:C/VWF:Ag ratio. Diagnosis of suspected 2N should be confirmed by DNA testing, per the ISTH-SSC VWF guidelines.
Source: Yadegari H et al. Landscape and spectrum of VWF variants in type 2 von Willebrand disease: insights from a German patient cohort. Thromb Haemost. 2025;126(2):156–178. doi:10.1055/a-2616-5161 — primary-literature