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

Abstract

Background: Both 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.

Aim: The aim of this study was to investigate and quantify the analytical variation in OSA and CSA.

Methods: Factors 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.

Results: On 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.

Conclusions: In 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.

Keywords: FVIII measurements; factor VIII; haemophilia A.

Figure 1

The coefficient of variation (CV) is higher when FVIII activity levels are lower. A, The CVs were calculated for both one‐stage assay (OSA) and chromogenic stage assay (CSA). The circles indicate the CVs calculated from measurements with the OSA. The squares reflect the CVs calculated from measurements with the chromogenic substrate assay (CSA). B, The CV of the OSA was also calculated when FVIII activity levels were measured with products from Siemens (circles), Stago (squares) and Werfen (triangles)

Figure 2

The distribution of the FVIII activity levels measured by one‐stage assay (OSA). FVIII levels are shown when measured with company set‐ups from Siemens, Stago or Werfen

Figure 3

Combination of deficient plasma, equipment, calibrator and activator from Werfen causes lower factor VIII (FVIII) activity levels when FVIII >0.40 IU/mL compared to Stago and Siemens. The red dots are the results from each laboratory. The black line represents the median. The error bars represent the interquartile range. Statistical significance is indicated as *P < 0.05, **P < 0.01, *** P < 0.001

Figure 4

No consistent differences in factor VIII (FVIII) activity levels between mostly wide used chromogenic assays. The red dots are the results from each laboratory. The black line represents the median. The error bars represent the interquartile range. Statistical significance is indicated as *P < 0.05, **P < 0.01, *** P < 0.001

Figure 5

Exchange of deficient plasma into a system set‐up with equipment of another company does not change the factor VIII (FVIII) activity levels. Deficient plasma was exchanged and used in the one‐stage assay (OSA) set‐up of another company. Samples measured with Werfen equipment had lower FVIII activity levels compared to samples measured with Siemens or Stago. Triangles represent FVIII activity levels measured with a deficient plasma from Werfen. Squares represent FVIII activity levels measured with a deficient plasma from Stago. Circles represent FVIII activity levels measured with a deficient plasma from Siemens