Development of a rapid and fully automated factor VIII inhibitor assay, insensitive to emicizumab, and a lowest level of quantification of 0.2 BU/mL.

BACKGROUND: Factor (F)VIII inhibitors are measured using labor- and resource-expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titer inhibitors and show high variations in quality surveys, mainly because of manual assay procedures. OBJECTIVES: The goal of this study was the development of a fast and fully automated FVIII inhibitor assay by using recombinant (r)FVIII as substrate and dedicated equipment for execution of the test. METHODS: A new rapid, fully automated, FVIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII; Kovaltry, Bayer) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of von Willebrand factor. Dramatic shortening of incubation time facilitated full automation on an analyzer capable of 3 subsequent sample dilution steps and 3 reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated for 10 minutes at 37 °C, whereafter remaining FVIII activity was analyzed with a kinetic chromogenic assay, allowing inhibitor activity calculation without preceding FVIII activity calibration, using a Ceveron s100 analyzer (Technoclone). RESULTS: Mean titer in 60 nonhemophiliacs was 0.0 BU/mL (SD, 0.1), yielding a limit of blank of 0.1 BU/mL and lower limit of quantification of 0.2 BU/mL. Analyses were performed with the new method and a Nijmegen assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type I inhibitor samples was 1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive samples. Reproducibility was tested by analyzing 7 samples in 3 laboratories for 5 days, twice daily; coefficients of variation of all samples were <15%. CONCLUSION: We present development data of a sensitive and specific rapid, automated FVIII inhibitor assay generating results within 20 minutes that is less resource-intensive than standard assays with potential to improve assay variability.

The Nijmegen ultra-sensitive Bethesda Assay detects very low-titer factor VIII inhibitors in patients with congenital and acquired hemophilia A.

BACKGROUND: An inhibitor can develop in congenital hemophilia A (HA) patients against exogenous infused factor (F)VIII, whereas in acquired HA (AHA) inhibitors initially develop against endogenous FVIII. Inhibitors can be detected with the Nijmegen Bethesda Assay (NBA), which has an international cut-off level of 0.60 Nijmegen Bethesda Units/mL (NBU/mL). Thereby, very low-titer inhibitors may remain undetected. AIM: To describe the design and validation of the Nijmegen ultra-sensitive Bethesda Assay (NusBA) for the detection of very low-titer inhibitors. METHODS: The NusBA is a modification of the NBA in which the ratio of patient plasma to normal pooled plasma is changed from 1:1 to 9:1. Analytical validation was performed according to the CLSI EP10 guideline in order to determine trueness and reproducibility. Clinical validation was performed in two cohorts of congenital HA patients (82 adults) with pharmacokinetic data and four AHA patients. The limit of quantitation (LOQ) was determined by measuring plasma samples spiked with inhibitor levels in the low range (0.05-0.80 NBU/mL). RESULTS: The LOQ for the NusBA was 0.10 NusBU/mL, with a coefficient of variation of 24.2 %. Seven (8.5 %) congenital HA patients had a positive NusBA result, of which only one was detected with the NBA. There was no correlation between NusBA and FVIII half-life. In three of the AHA patients the NusBA remained positive, when the NBA became negative. DISCUSSION: The NusBA is able to detect very low-titer FVIII inhibitors of ≥0.10 NBU/mL. Thereby, it may have added value in early inhibitor detection and therapy adjustments in patients with congenital HA and AHA.

Use of the platelet function analyzer to minimize bleeding complications after renal biopsy.

BACKGROUND: The bleeding time is frequently used to screen primary haemostasis before surgical procedures, although it poorly predicts the risk of hemorrhage. The platelet function analyzer (PFA), which is also used to screen primary haemostasis, has a higher sensitivity and other advantages, like patient friendliness, higher degree of objectivity and analytical reliability, but needs more extensive clinical validation. METHODS: We compared the predictive values of the PFA-CTs (closure times) and bleeding time for bleeding events after renal biopsy. We prospectively evaluated the complications in patients that underwent a renal biopsy and were screened with PFA in advance (n=170). For comparison we used a historical cohort of patients screened with the bleeding time (n=132). RESULTS: When the PFA-CTs were normal, 26.0% of the patients had a mild bleeding event after the biopsy, which did not differ from the event rate with a normal bleeding time (29.4%). When one or both PFA-CTs were prolonged, 51.3% of the patients had post-biopsy bleeding events independently of the measures to correct the closure time(s), significantly more than with either a prolonged bleeding time (26.7%) or normal PFA-CTs (26.0%). CONCLUSION: For bleeding events, the PFA has a higher positive and similar negative predictive value compared to the bleeding time. Taken into account the additional advantages of the PFA like patient friendliness and better analytical qualities, we prefer the PFA over the bleeding time as a screening tool for primary haemostasis before performing a renal biopsy.