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. 2025 Mar;31(2):319-327.
doi: 10.1111/hae.70007. Epub 2025 Feb 14.

Factor VIII Activity and Factor VIII Inhibitors Can Be Measured Accurately in Plasma Containing Mim8 by Using Specific Chromogenic Assays

William Pickering  1 , Mary Robinson  1 , Caroline Cogswell  1 , Wan Hui Ong Clausen  2 , Karin Nana Weldingh  3 , Mirella Ezban  3
Affiliations

Factor VIII Activity and Factor VIII Inhibitors Can Be Measured Accurately in Plasma Containing Mim8 by Using Specific Chromogenic Assays

William Pickering et al. Haemophilia. 2025 Mar.

Abstract

Introduction: Patients with Haemophilia A treated with Mim8 may require concomitant Factor VIII (FVIII) replacement; hence, accurate assessment of FVIII activity (FVIII:C) and FVIII inhibitors in the presence of Mim8 is important.

Aim: Assess which chromogenic substrate assays (CSAs) accurately monitor FVIII:C and FVIII inhibitor levels in the presence of Mim8.

Methods: FVIII and Mim8 were spiked into congenital FVIII-deficient plasma to obtain mixtures containing various FVIII:C levels (0-100 IU/dL) and Mim8 concentrations (0, 3, 6 and 12 µg/mL). Five CSAs using different activated Factor IX and X sources (bovine, bovine-human or human) were used to measure FVIII:C. Bovine and bovine-human CSAs were then used to measure FVIII:C of standard half-life (SHL) and extended half-life (EHL) FVIII products. FVIII inhibitor levels were assessed using two different bovine CSAs in congenital FVIII-deficient plasma spiked with various Mim8 concentrations (5, 10, 20 and 40 µg/mL) and FVIII inhibitor levels (0.2, 1.0 and 4.8 BU).

Results: High levels of interference were observed using human CSAs. Bovine CSAs accurately measured FVIII:C of SHL and EHL FVIII products in the presence of Mim8 without interference. In bovine-human CSAs, interference was observed at 5 IU/dL FVIII, increasing up to four-fold with increasing Mim8 levels. FVIII inhibitor levels were accurately measured using bovine CSAs without Mim8 interference.

Conclusion: FVIII:C of SHL and EHL products and FVIII inhibitor levels can be accurately monitored in the presence of Mim8 using bovine CSAs at all FVIII levels, and bovine-human CSAs at FVIII concentrations >20 IU/dL.

Keywords: FVIII inhibitor; Factor VIII; Factor VIII assays; Haemophilia A; bispecific antibodies; chromogenic assays; procoagulant activity.

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Conflict of interest statement

William Pickering, Mary Robinson and Caroline Cogswell are employees of Labcorp Colorado Coagulation. Wan Hui Ong Clausen, Karin Nana Weldingh and Mirella Ezban are employees of Novo Nordisk A/S.

Figures

FIGURE 1
FIGURE 1
Measurement of FVIII activity (A) and FVIII inhibitors (B) by CSAs in the presence of Mim8. CSA, chromogenic substrate assay; FVIII, Factor VIII; FIXa, activated Factor IX; FX, Factor X; FXa, activated Factor X; PwHA, patients with Haemophilia A.
FIGURE 2
FIGURE 2
Effect of Mim8 on SHL FVIII (turoctocog alfa) recovery in human‐based FVIII CSAs. Results depicted are means of triplicate measurements over one analytical run. CSA, chromogenic substrate assay; FVIII, Factor VIII; SHL, standard half‐life.
FIGURE 3
FIGURE 3
Effect of Mim8 on SHL FVIII (turoctocog alfa) recovery using bovine–human CSAs (A) and effect of Mim8 on FVIII at concentrations where FVIII molecules outcompete Mim8 using bovine–human CSAs (CRYOcheck) (B). FVIII recovery above the red line is deemed to show interference. The grey horizontal dotted line shows a reference point for an FVIII activity ratio of 1.0 (FVIII activity in the presence of Mim8:FVIII activity in the absence of Mim8). Results depicted are means (±standard deviation) of triplicate measurements over one analytical run. CSA, chromogenic substrate assay; FVIII, Factor VIII; SHL, standard half‐life.
FIGURE 4
FIGURE 4
Effect of Mim8 on SHL FVIII (turoctocog alfa) recovery using bovine‐based reagents. FVIII recovery above the red line is deemed to show interference. The grey horizontal dotted line shows a reference point for an FVIII activity ratio of 1.0 (FVIII activity in the presence of Mim8:FVIII activity in the absence of Mim8). Results depicted are means of triplicate measurements over one analytical run. FVIII, Factor VIII; SHL, standard half‐life.
FIGURE 5
FIGURE 5
For bovine–human chromogenic FVIII assays, FVIII concentrations ≤20 IU/dL showed a Mim8 dose‐dependent increase in interference across all SHL (turoctocog alfa and octocog alfa) and EHL (turoctocog alfa pegol and efmoroctocog alfa) products (A); and the bovine chromogenic FVIII assays showed no notable interference at all FVIII/Mim8 concentrations for all SHL (turoctocog alfa and octocog alfa) and EHL (turoctocog alfa pegol and efmoroctocog alfa) products (B). FVIII recovery above the red line is deemed to show interference. The grey horizontal dotted line shows a reference point for an FVIII activity ratio of 1.0 (FVIII activity in the presence of Mim8:FVIII activity in the absence of Mim8). Results depicted are means of triplicate measurements over one analytical run. EHL, extended half‐life; FVIII, Factor VIII; SHL, standard half‐life.
FIGURE 6
FIGURE 6
No Mim8 interference was shown across concentrations with varying FVIII inhibitor levels. Data presented from the FVIII Chromogenic Assay (Siemens Healthineers, Germany). The grey dotted lines represent Mim8 unspiked sample values. The red dotted lines represent the acceptable error margin from unspiked Mim8 sample values. Results depicted are means of triplicate measurements over three analytical runs. FVIII, Factor VIII; NA, not applicable; RE, relative error; SD, standard deviation.
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