Abstract Number: PB0471
Meeting: ISTH 2021 Congress
Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia - Basic
Background: In recent years, the treatment of hemophilia A has trended toward the use of truncated forms of FVIII where most or all of the B-domain has been deleted (BDD-FVIII), while FVIII coagulant activity is retained. The use of BDD-FVIII constructs present challenges for existing commercial FVIII antigen kits, where their recognition can be reduced. A new 2nd generation ELISA kit (F8PLUS-AG) was developed with the intent to provide a more uniform recognition of native, full-length, and BDD-FVIII preparations, while using a WHO-traceable plasma-based calibrator. The method and assay performance of the 1st (FVIII-AG) and 2nd generation (F8PLUS-AG) kits are similar with regard to sensitivity (0.008 IU/mL) and non-interference by von Willebrand factor. The distinguishing feature of the new kit is the improved recognition of BDD-FVIII.
Aims: To assess the 2nd generation antigen kit (F8PLUS-AG) in the quantification of various therapeutic FVIII products currently available.
Methods: In this study, plasma-derived (pdFVIII), recombinant full-length (rflFVIII) and BDD-FVIII replacement products were reconstituted and spiked into congenital FVIII-deficient plasma to achieve activity levels of 1.0, 0.3, and 0.05 IU/mL based on the product assigned activity potency. FVIII antigen was measured in both 1st and 2nd generation kits and expressed as a ratio of antigen to the expected activity.
Results: Both antigen kits gave similar recoveries for pdFVIII and rflFVIII, which were consistent with the expected activity. The exception was porcine rFVIII, which was not recognized with either of these kits (Table 1).
| FVIII Sample | Description | FVIII Ag:Ac Ratio by 1st Gen (FVIII-AG) Kit | FVIII Ag:Ac Ratio by New 2nd Gen (F8PLUS-AG) Kit | ||||
|---|---|---|---|---|---|---|---|
| NPP | Normal Pooled Plasma | 1.50 | 1.33 | ||||
| Expected FVIII Activity Values | 1.00 IU/mL | 0.30 IU/mL | 0.05 IU/mL | 1.00 IU/mL | 0.30 IU/mL | 0.05 IU/mL | |
| pdFVIII-1 | Plasma-Derived FVIII/VWF Complex | 1.20 | 1.07 | 1.04 | 1.32 | 1.13 | 1.16 |
| pdFVIII-2 | Plasma-Derived FVIII/VWF Complex | 0.72 | 0.63 | 0.62 | 1.04 | 0.83 | 0.84 |
| rflFVIII-1 | Recombinant Full-Length FVIII | 1.43 | 1.40 | 1.34 | 1.16 | 1.00 | 1.02 |
| rflFVIII-2 | Recombinant Full-Length FVIII | 1.57 | 1.50 | 1.26 | 1.24 | 1.00 | 0.86 |
| rflFVIII-PEG | PEGylated Recombinant Full-Length FVIII | 1.37 | 1.26 | 1.06 | 1.22 | 0.97 | 0.90 |
| rFVIII-Porcine | Recombinant Porcine FVIII Sequence | not detectable | not detectable | not detectable | not detectable | not detectable | not detectable |
Ratios (FVIII Antigen : Expected FVIII Activity) of pdFVIII, rflFVIII, PEGylated FVIII, and porcine rFVIII as determined using 1st generation (FVIII-AG) and 2nd generation (F8PLUS-AG) antigen kits.
The BDD-FVIII products tested in the 1st generation kit were significantly under-recovered, confirming earlier observations. In contrast, these samples tested in the F8PLUS-AG kit demonstrated considerably higher recoveries, with antigen values more in line with the expected activity values (Table 2).
| FVIII Sample | Description | FVIII Ag:Ac Ratio by 1st Gen (FVIII-AG) Kit | FVIII Ag:Ac Ratio by New 2nd Gen (F8PLUS-AG) Kit | ||||
|---|---|---|---|---|---|---|---|
| NPP | Normal Pooled Plasma | 1.50 | 1.33 | ||||
| Expected FVIII Activity Values | 1.00 IU/mL | 0.30 IU/mL | 0.05 IU/mL | 1.00 IU/mL | 0.30 IU/mL | 0.05 IU/mL | |
| BDDrFVIII-1 | Recombinant B-Domain Deleted FVIII | 0.30 | 0.28 | 0.26 | 0.79 | 0.70 | 0.74 |
| BDDrFVIII-2 | Recombinant B-Domain Deleted FVIII | 0.26 | 0.21 | 0.20 | 0.81 | 0.67 | 0.66 |
| BDDrFVIII-3 | Recombinant B-Domain Deleted FVIII | 0.26 | 0.20 | 0.20 | 0.78 | 0.60 | 0.52 |
| BDDrFVIII-4 | Recombinant B-Domain Deleted FVIII | 0.32 | 0.28 | 0.24 | 0.87 | 0.70 | 0.66 |
| BDDrFVIII-5 | Recombinant B-Domain Deleted FVIII | 0.27 | 0.22 | 0.20 | 0.71 | 0.57 | 0.56 |
Ratios (FVIII Antigen : Expected FVIII Activity) of five different BDD-FVIII products determined using 1st generation (FVIII-AG) and 2nd generation (F8PLUS-AG) antigen kits.
Conclusions: We verified significantly improved recognition of various BDD-FVIII constructs using the 2nd generation antigen kit, demonstrating its versatility in modern hemophilia research.
To cite this abstract in AMA style:
Ni R, Erb P, Della Maestra J, Atkinson H, Foulon D, M Black K, Hoogendoorn H, Matino D. Comparative Recovery of FVIII Antigen with Various FVIII Therapeutic Concentrates Using an Improved 2nd Generation ELISA Kit [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/comparative-recovery-of-fviii-antigen-with-various-fviii-therapeutic-concentrates-using-an-improved-2nd-generation-elisa-kit/. Accessed November 29, 2023.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/comparative-recovery-of-fviii-antigen-with-various-fviii-therapeutic-concentrates-using-an-improved-2nd-generation-elisa-kit/