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

Abstract

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. Nonparallelism 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.

Interferences by factor VIII and lupus anticoagulant in the modified one‐stage assay for emicizumab

“…According to the guidelines, the three results can only be averaged reliably if the CV is below 15%. 9 In fact, the CVs were below 15% for all LA-negative samples but over 15% for all LA-positive samples (Figure 2B3); this confirmed that LA interfered with the emicizumab m-OSA. Additional dilutions were performed until two consecutive results fell within 15% of each other.…”

“…The parallelism between the test line and the standard material line in the plot of clotting time (on the y axis) versus dilution (on the x axis) must then be assessed. 9 Increasing dilutions of all the tested LA-positive plasmas led to increasing emicizumab concentrations (Figure B1 and 2B2). Parallelism was assessed by calculating the coefficient of variation (CV) for the results of the first three dilutions (1/10, 1/20, and 1/40).…”

In vitro differential inhibition of the factor XI activity assay in the setting of a lupus anticoagulant

“…Per Clinical and Laboratory Standards Institute (CLSI) guidelines, a minimum of three sample dilutions is required to demonstrate parallelism on factor assays [6]. Parallelism is defined by a coefficient of variation (CV) of <15-20% of the average factor activity between consecutive sample dilutions [7]. Because of their interference with aPTT, the presence of LAC may interfere with aPTT clot-based factor assays by nonspecific inhibition [2], demonstrating nonparallelism.…”