Background: One-stage clotting FIX assays (OSA) are used for diagnosis of hemophilia B and monitoring clotting factor concentrate (CFC). Selected extended half-life CFC require chromogenic assay (CSA), which are not US FDA approved.

Aims: We present our in-house validation of a CSA.

Methods: The ROX FIX, (Rossix AB, Sweden) was optimized on the IL-ACL TOP 700. Following CLSI guidelines (CLSI EP28, EP05, EP15, EP09C, EP06). Assays were performed on CFC (ALPROLIX^® and IDELVION^®) spiked in FIX deficient plasma (n=32), proficiency testing samples (n=3) and waste de-identified patient plasma samples (n=33). Linearity was performed on one commercial normal pool sample and 3 de-identified individual patient samples with elevated OSA (in triplicate at a minimum of 10 different dilutions with diluent buffer). Reference range was established following CLSI guidelines (n=120). Analytical sensitivity was carried out on diluent buffer, FIX deficient plasma, and control material diluted to approximately 1%; each material had multiple replicates tested on multiple lots of reagent.

Results: Reference range was established at 65–140% and analytical sensitivity was achievable down to 1% (CV:4.9%). Reportable range was established at 1–200%. Intra/inter CV’s ≤6% were obtained for results ranging 9.8 – 107.2% (Table 1). Accuracy results compared with the FIX OSA (Figure 1): All Data (r²=0.874, slope=0.984, avg% diff=3.5%); Known Assigned and Patient Samples (r²=0.958, slope=0.893, avg% diff=-8.1%); ALPROLIX^® (r²=0.990, slope=1.205, avg% diff=-6.6%); IDELVION^® (r²=0.995, slope=2.035, avg% diff=46.3%). Linearity average r²=0.998 and average slope=1.02 for all samples with results ranging from 0.7–174.8%.

Conclusions: Our CSA kit validation efforts met acceptance criteria for non-spiked patient samples. Data demonstrates the CSA assay can be optimized to provide reliable FIX activity estimates for baseline and ALPROLIX^® with an acceptable CV and lower limit of detection of 1% activity. Additional studies are needed to further understand differences between the OSA and CSA when IDELVION^® is present.

Intra and Inter Precision

Regression Analysis of Accuracy Studies

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/laboratory-verification-of-a-chromogenic-factor-ix-assay-kit/