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.
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
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. 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.
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.
In frail elderly patients who are stable on warfarin for stroke prevention from atrial fibrillation, can you switch them to a DOAC? And does that lead to harm?¶
Answer: Switching is probably reasonable for most, but monitor closely in the first ~60 days and prefer apixaban.
- Background: DOACs are standard of care for stroke prevention in AF requiring anticoagulation (multiple RCTs).
- FRAIL-AF (large open-label RCT): in frail older adults already stable on a VKA, switching to a DOAC may lead to more bleeding without a reduction in thromboembolic events.
- Caveats of FRAIL-AF — early termination (can overestimate treatment effect), and early overlap of DOAC and VKA exposure (investigators addressed this after the first 102 patients were randomized). Two further points from the critique in Can J Cardiol:
Although subgroup analysis provided no statistical evidence for heterogeneity of treatment effect, the hazard ratio for bleeding with individual DOACs ranged from 1.10 to 2.17 and the confidence levels were wide, so a type 2 error cannot be excluded.
The population of FRAIL-AF was stable while receiving a VKA and monitored in a specialized anticoagulant clinic. Therefore, the results might not apply to recently diagnosed AF in patients with frailty, nor even to stable patients receiving a VKA who are managed in a community setting
- Conflicting later evidence:
- COMBINED-AF — individual-patient-data systematic review and meta-analysis of the landmark AF trials plus COMBINE-AF (~6000 frail patients): less stroke/systemic embolism, fatal bleeding, intracranial hemorrhage, and death with DOACs.
- Retrospective database study (~165,000 patients): switching to apixaban performed very well, while switching to rivaroxaban led to more bleeding; most of the excess risk occurred within the first 60 days.
- Bottom line: switching is probably OK, but monitor closely within the first 60 days. Apixaban preferred.
Source: FRAIL-AF randomized controlled trial (Circulation)
What tests for qualitative platelet function defects do we have available, and how can they influence management in clinic?¶
Answer: A tiered set of assays — from a whole-blood screen to disorder-specific confirmatory tests.
- PFA-100/200: originally developed to replace the bleeding time, now used to screen for platelet function defects. Whole blood is aspirated at high shear through cartridges with a membrane coated with either collagen/epinephrine or collagen/ADP; shear stress drives adhesion, activation, and aggregation. Pros: small volume, automated, good screening, relatively insensitive to clotting factor deficiency, high NPV — with exceptions (storage pool disease, secretion defect, type 1 VWD).
- Flow cytometry: detects surface membrane glycoproteins; most commonly to identify lack of CD41/CD61 (Glanzmann thrombasthenia) or CD42b (Bernard-Soulier).
- Light transmission aggregometry (LTA).
- VerifyNow (intra-operative).
- Other tests: electron microscopy / nucleotide assays (not very helpful clinically), and the platelet procoagulant activity assay (e.g. Scott syndrome).
- Genetic testing: an evolving topic.
Source: Platelet function testing — introduction (Practical-Haemostasis)
Is IVIG use in ITP associated with increased risk of thrombosis?¶
Answer: IVIG has generally been associated with thrombosis (venous and arterial), but ITP itself carries a baseline thrombotic risk, and treating the ITP — including with IVIG — likely lowers overall risk. Bottom line: treat the ITP.
- ITP itself raises thrombotic risk vs. the general population:
The annualized incidence was 0.41-0.67 for venous thromboembolism (VTE) and 0.96-1.15 for arterial thrombosis (AT), whereas the control populations had 0.28-0.42 and 0.67-0.91, respectively, showing a slightly but statistically significantly higher risk of VTE and possibly AT in ITP patients
— Ghanima et al., Thrombopoietin receptor agonists: ten years later (Haematologica 2019)
The incidence rate of thrombosis was 2.71 (95% CI, 1.97-3.72) (0.66 [95% CI, 0.33-1.26] for arterial thromboembolism and 2.05 [95% CI, 1.42-2.95] for VTE) per 100 person-years.
— Thrombosis in patients with immune thrombocytopenia: incidence, risk, and clinical outcomes
- TPO-receptor agonists add further risk:
The incidence per 100 patient-years (censoring after first TEE) ranged from 3.1 to 4.2 with romiplostim and was 2.9 in the single eltrombopag study. Without censoring after first event, the incidence ranged from 4.1 to 7.5 with romiplostim and 3.4 with eltrombopag. In a pooled analysis of romiplostim studies, an incidence rate per 100 patient-years of 5.5 was reported for both patients exposed to romiplostim or to placebo/SoC.
The TEE events were neither associated with thrombocytosis nor with a higher dose of TPO-RA. At least 30-50% of cases occurred in patients with lower than normal platelet counts. In general, TEE events tended to happen in the first year of treatment, creating a trend towards lower incidence figures with more prolonged exposure time.
— both from Ghanima et al. (Haematologica 2019)
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In summary, although they have not been substantiated in properly designed trials, the annualized thrombosis rates in adults appear to be 2-3 times higher (annualized incidence rate of TEE of 4-7%) with TPO-RA treatment than in an ITP population not treated with TPO-RA, and even higher if compared to non-ITP control populations.
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IVIG: IVIG use has generally been associated with an increased risk of thrombosis, both venous and arterial. The risk is probably not additive — TPO agonists raise risk but likely don't compound the inherent risk of ITP itself (clinical judgment).
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Treating ITP likely lowers thrombotic risk: a large retrospective review (~56,000 ITP hospitalizations) found IVIG lowered the risk of thrombosis — Sanjeevi et al., Impact of IVIG on VTE/PE in ITP — an NIS 2020 Database Analysis (Blood 2024).
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Bottom line: treat the ITP.