A Standardized Kit for a Chromogenic Modified Nijmegen-Bethesda Assay: Repeatability, Reproducibility, and Analytical Sensitivity

Antibody-based inhibitor response to Factor VIII replacement therapy is one of the most common complications in the treatment of persons with hemophilia A (PwHA). Accordingly, accurate quantification of FVIII inhibitor antibody titer is essential in guiding patient treatment. Testing for FVIII inhibitors is not standardized; typically, FVIII inhibitor titers are measured using non-standardized LDTs (Laboratory Developed Tests). These LDTs vary in the processing of patient plasma, the reagents used to perform the assay, and the method of FVIII activity measurement.

Here we report the development of a standardized set of reagents to perform a chromogenic modified Nijmegen-Bethesda assay (MNBA). The kit consists of imidazole-buffered pooled normal plasma (IB-PNP) with a consistent normal level of FVIII activity, and an imidazole-buffered bovine serum albumin solution (IB-BSA) as a diluent. Also included in the kit are a positive FVIII inhibitor plasma control (1.2 - 1.8 BU/mL) and an inhibitor-free negative plasma control.

To perform the assay, test plasma was heat inactivated at 56 °C for 30 minutes to minimize residual FVIII activity. The inactivated plasma was then centrifuged (2700 x g,5 min.) and the supernatant used to make a series of two-fold serial dilutions with IB-BSA. The dilutions were each mixed with an equal volume of IB-PNP to form test mixes and incubated in a 37 °C water bath to allow time for the inhibitor antibodies from the patient plasma to inactivate FVIII in the IB-PNP. After two hours, the reaction was halted by cooling the test mixes in an ice bath. The FVIII activity of the test mixes was then measured using a Siemens FVIII Chromogenic Assay on a Siemens BCS XP analyzer. The residual FVIII activity of each test mix was calculated by comparing its FVIII activity to the activity of a control mix containing no test plasma (1:1 mixture of IB-BSA and IB-PNP).

Here we report the use of this kit to perform a comprehensive study examining the repeatability, reproducibility, and analytical sensitivity of a chromogenic MNBA performed according to CLSI guidelines (CLSI EP05 and EP17). Inhibitor-negative and inhibitor-positive plasma from PwHA were combined to yield a panel of test plasmas at four different levels of inhibitor: below cutoff level (~0.3 BU/mL), low positive (~1.2 BU/mL), mid positive (~5 BU/mL) and a high positive (~8 BU/mL). To assess the repeatability of the assay, we measured each of these test plasmas using three lots of MNBA kit and one lot of FVIII chromogenic kit for a total of 240 titer determinations (3 lots × 20 days × 2 runs × 2 replicates). Inhibitor measurements of the panel of test plasmas showed within-lot precision of less than 10% (CV), and the kit controls were identified correctly each time. The reproducibility of the assay was determined in a study conducted across three different laboratories using different BCS XP analyzers and operators but the same panel of test plasmas. The limit of blank (LoB) and the limit of detection (LoD) of the assay were also determined using multiple low titer plasmas from unique donors. The assay system can reliably distinguish inhibitor titers as low as 0.2 BU/mL (LoD) in plasma from PwHA with no history of inhibitors.

The chromogenic MNBA kit shows potential for labs seeking repeatable and reproducible FVIII inhibitor measurement that can otherwise vary significantly within or between labs. Standardization of reagents and protocol yields consistent results and is suitable for multi-center inhibitor studies in PwHA.

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