Analytical variation in factor VIII one‐stage and chromogenic assays: Experiences from the ECAT external quality assessment programme

Abstract

BackgroundBoth one‐stage (OSA) and chromogenic substrate assays (CSA) are used to measure factor VIII (FVIII) activity. Factors explaining analytical variation in FVIII activity levels are still to be completely elucidated.AimThe aim of this study was to investigate and quantify the analytical variation in OSA and CSA.MethodsFactors determining analytical variation were studied in sixteen lyophilized plasma samples (FVIII activity <0.01‐1.94 IU/mL) and distributed by the ECAT surveys. To elucidate the causes of OSA variation, we exchanged deficient plasma between three company set‐ups.ResultsOn average, 206 (range 164‐230) laboratories used the OSA to measure FVIII activity and 30 (range 12‐51) used CSA. The coefficient of variation of OSA and CSA increased with lower FVIII levels (FVIII <0.05 IU/mL). This resulted in misclassification of a severe haemophilia A sample into a moderate or mild haemophilia A sample in 4/30 (13.3%) of CSA measurements, while this was 37/139 (26.6%) for OSA. OSA measurements performed with reagents and equipment from Werfen showed slightly lower FVIII activity (0.93, IQR 0.88‐0.98 IU/mL) compared to measurements with Stago (1.07, IQR 1.02‐1.14 IU/mL) and Siemens (1.03, IQR 0.97‐1.07 IU/mL). Part of this difference is explained by the value of the calibrator. For CSA, the measured FVIII levels were similar using the different kits.ConclusionsIn the lower range (<0.05 IU/mL), analytical variation of FVIII measurements is high in both OSA and CSA measurements. The variation in FVIII activity levels was partly explained by specific manufacturers. Further standardization of FVIII measurements and understanding of analytical variation is required.

Estimation of Nuwiq®(simoctocog alfa) activity using one‐stage and chromogenic assays—Results from an international comparative field study

“…The CSA is generally assumed to be more sensitive at low concentrations . However, results from previous field studies are inconsistent, and recent data from proficiency testing indicate that both OSAs and CSAs show high variability at low concentrations . It has been speculated that high variability at low target concentrations in the CSA may be due to the absence of very low concentration samples in calibration curves, and in fact, according to the questionnaire data, the lowest concentration used for calibration was ≥2.5 IU/dL for almost one‐third of CSAs in this study (data not shown).…”

“…OSAs and CSAs show high variability at low concentrations. 28 It has been speculated that high variability at low target concentrations in the CSA may be due to the absence of very low concentration samples in calibration curves, 25 and in fact, according to the questionnaire data, the lowest concentration used for calibration was ≥2.5 IU/dL for almost one-third of CSAs in this study (data not shown). Independently of this, it is important to note that for samples c Some values were excluded from the analysis because exact FVIII:C values were not provided; therefore, n is less than the n listed in the column title (OSA: n = 48 for Nuwiq ® 1 IU/dL and n = 47 for Advate ® 1 IU/dL; CSA: n = 37 for both 1 IU/dL samples for interlaboratory CVs, n = 36 for both 1 IU/ dL samples for intralaboratory CVs, and n = 40 for both 5 IU/dL samples).…”

“…Published results for interlaboratory CVs range from 10% to 34% for the OSA and from 5% to 51% for the CSA, with the highest values consistently observed for the lowest concentrations. High interlaboratory CVs at low sample concentrations have also been reported in proficiency testing, with values of up to approximately 120% for samples with concentrations below 20 IU/dL . For intralaboratory precision, previously reported values for Advate ® range from 5% to 13% for the OSA and from 2% to 15% for the CSA.…”

The traceability of commercial plasma calibrators to the plasma International Standards for factor VIII and factor IX

“…Previous scheme reports have suggested that the calibrator used within each assay may be one of the biggest sources of variation. 6,9 The present, single centre, study was set up to investigate the traceability of the IU between the International Standards for FVIII and FIX and commonly used commercial plasma calibrators.…”

“…Of the various coagulometer instruments available, machines from three manufacturers are most commonly used: Siemens, Stago and Werfen. 6 Often, laboratories choose reagents from the same manufacturer as their assay instrument. 4 Finally, the assays may be affected by the choice of calibrator used.…”