Agreement between one stage and chromogenic assays in samples from patients receiving recombinant porcine FVIII (Obizur, Susoctocog-alfa).

INTRODUCTION: Recombinant porcine FVIII (rpFVIII) (Obizur, Susoctcog-alfa, Takeda, Japan) is licensed for the treatment of bleeding in acquired Haemophilia A (AHA). The summary of product characteristics state that monitoring should be by one stage assay (OSA) rather than chromogenic assay (CSA). CSA have been shown to underestimate activity when rpFVIII is added to plasma in vitro. METHODS: Samples from three AHA patients (n = 21) (pre- and post rpFVIII) were assessed using FVIII:C assays; OSA methods: Actin, Actin FS, Actin FSL and Pathromtin SL performed on CS5100i (Sysmex, Kobe, Japan); APTT-SP, SynthASil and SynthAFax performed on ACL TOP (Werfen, Barcelona, Spain). CSA methods on CS5100i: Siemens Chromogenic Assay, Biophen FVIII:C, Technochrom FVIII:C; on ACL TOP: Rox Factor VIII, Coamatic Factor VIII and CRYOcheck Factor VIII. RESULTS: OSA and CSA varied according to reagent used median OSA 61 IU/dL (range 41.5-81 IU/dL (ANOVA p < 0.0001)) median CSA 46.5 IU/dL (range of method specific medians 36.5-84 IU/dL (ANOVA p < 0.0001)). Amongst OSA, Actin FS was associated with the highest FVIII:C, APTT-SP was associated with the lowest. Variation in CSA results by different methods was also seen with highest FVIII:C levels obtained using the Technochrom FVIII:C and the lowest levels obtained with Siemens Assay. CONCLUSION: The relationship between OSA and CSA was not consistent between method or patient. Previously there has been reports of underestimation by CSA in in vitro spiked samples. Investigation into concentration of phospholipids in the APTT reagents may explain some of these variations.

How to assess parallelism in factor assays: coefficient of variation of results with different dilutions or slope ratio?

INTRODUCTION: Non-parallelism in factor assays can lead to incorrect factor activities. Parallelism can be assessed by calculating the coefficient of variation (CV) of results obtained on 3 dilutions of the same sample. Some authors have proposed that if there is <15% then the average activity is reportable. Some analysers use a slope ratio (SR) to calculate parallelism, with an acceptance range of approximately 0.9-1.1. METHODS: We evaluated CV and SR in one stage FII-FXII assays on Sysmex CS5100i using Innovin or Actin FS. Frozen normal and pathological plasmas, plasmas containing Direct Oral Anticoagulants, Direct Thrombin Inhibitors or Lupus Anticoagulant were analysed to assess possible non-parallelism. RESULTS: In plasmas with factor levels >25 IU/dl (plus no interfering substances) all CVs were < 15%. One sample (low factor activities 10-15 IU/dl), had CVs > 15% in FII, FVII and FXII assays only. SR outside of 0.9-1.1 were seen in FII and FXII assays at different levels of clotting factor including some within the normal range. Non-parallelism was detected more frequently with SR than CV for those with interfering substances. CONCLUSIONS: SR outside of 0.9-1.1 were seen in different levels of clotting factors, including samples which did not contain interfering substances. The target of 15% CV was a better discriminator than a SR for acceptance. When factor levels were reduced to around 10-15 IU/dl, a target 20 %CV was more appropriate than 15%. It might be appropriate for laboratories to assess locally whether their acceptance criteria need to be wider at low levels of clotting factors.