Nijmegen-Bethesda Assay

Beginning with a 2016 Quick Question and continuing to our most recent post in March, 2018, we’ve devoted altogether six entries to the 2012 CDC refinements of the Nijmegen-Bethesda assay. These have culminated in the following material, which appears in Fritsma GA, Chapter 41, Laboratory Evaluation of Hemostasis; in Keohane EM, Otto C, Walenga JM. Rodak’s Hematology: Clinical Principles and Applications, Sixth Edition, Elsevier, 2019. This latest edition makes its debut at the 2019 American Society for Clinical Laboratory Science Clinical Laboratory Educators’ Conference, February 21–23 in Baltimore, commorating the life of its original editor, Bernadette Rodak, who passed away in March, 2016.

Here is the new NBA material, paraphrased from Chapter 41:

“A median 40% of severe hemophilia patients are tested for inhibitors at US hemophilia centers using the Bethesda titer. Regrettably, the traditional method is characterized by a 32% false-positive rate, a 5% false-negative rate, and an inter-laboratory coefficient of variation that exceeds 50%. The Bethesda titer is affected by residual patient plasma coagulation factor, dabigatran, UFH, coexistence of lupus anticoagulant, differences in inhibitor epitope specificity, and neutralizing versus non-neutralizing inhibitor kinetics.

Refinements to the Bethesda titer were offered in 1995, whereupon the procedure was renamed the Nijmegen-Bethesda assay (NBA). In 2012 the US Centers for Disease Control and Prevention expanded on these modifications to reduce NBA variability and improve specificity. The 1995 and 2012 refinements combined are the following:

• Include an inhibitor positive control in all NBA runs.
• Heat patient plasma at 56°C for 30 minutes and centrifuge to eliminate residual factor VIII.
• Serially dilute heated patient plasma in FVIII-depleted (-deficient) plasma, not imidazole buffer, or alternatively dilute in bovine serum albumin (BSA).
• Serially dilute unheated commercial 100 Nijmegen-Bethesda unit (NBU)/dL–positive PNP in FVIII-deficient plasma or BSA.
• Incubate at 37°C for 2 hours, then perform the PTT-based FVIII assay.
• Convert residual FVIII activity to NBU per milliliter.

Few laboratories have made this full conversion; however, 70% of US laboratories use a “hybrid” NBA that substitutes imidazole buffered saline diluent, pH 7.4, for factor VIII–deficient plasma to reduce expense. For these laboratory managers, BSA may become an acceptable alternative.

Promising additional approaches are the fluorogenic and chromogenic NBA. Instead of employing the PTT, the operator adds FXa fluorogenic or chromogenic substrate with thrombin inhibitor and stopping buffer to measure FXa production as a surrogate to FVIII activity. One fluorogenic or chromogenic Bethesda unit (CBU) equals the level of inhibitor per milliliter of patient PPP that inactivates 50% of FVIII in 1 mL of PNP.”

We thank our Fritsma Factor participants for your anecdotal contributions to this material and we hope it contributes to the continual improvement of inhibitor analysis.