A Standardized Kit for a Chromogenic Modified Nijmegen-Bethesda Assay: Repeatability, Reproducibility, and Analytical Sensitivity
Bibliographic record
Abstract
Abstract 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. Table Table. Disclosures No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.015 | 0.017 |
| Meta-epidemiology (narrow) | 0.003 | 0.002 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.007 | 0.004 |
| Science and technology studies | 0.001 | 0.002 |
| Scholarly communication | 0.002 | 0.001 |
| Open science | 0.003 | 0.002 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.007 | 0.006 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".