PB1099 - The validation of linear One-Stage Clotting Assays (OSCA) using fully automated Multi-Dilution Analysis (MDA) in the quantification of factor activity

Background: When determining factor coagulant activities using One-Stage Clotting Assays (OSCA), at least three dilutions of test plasma should be prepared to determine linearity and assess parallelism to the reference. The linear region of the reference curves and testing of plasma using Multi-Dilution Analysis (MDA) results are essential in the assessment of parallelism using the slope ratio (SR) and linearity using the R-value of the reference curve.

Aims: The validation of linear OSCA using fully automated MDA in the quantification of factor activity.

Methods: Linear MDA OSCA on a CN-6000 were evaluated against point-to-point reference plot, single dilution analysis on a CS-5100. The eight extrinsic and intrinsic pathway OSCAs were tested using Siemens Dade® Actin® FS and Innovin® reagents, respectively. Comparability of at least 156 samples for each assay was performed for all factors (1,460 samples in total). On the CN-6000, Lower limits of quantification (LLoQ) and linearity evaluations were performed using MDA and sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned.

Results: Excellent correlation and LLoQ for all MDA OSCA were achieved; r2 >0.995 and < 0.9IU/dL, respectively. Acceptable between-run imprecision was obtained using commercial controls with normal and abnormal activity for each analyte (%CV < 3%) for all eight assays using MDA, and no carryover was observed.

Conclusion(s): Linear OSCA using MDA allows for the detection of sample activation and presence of inhibition (Figure 1), presence of which can be easily missed using point-to-point interpolation and single dilutions, leading to misinterpretation of results. Inhibitory activity is commonly observed with interfering anticoagulants, antibodies to coagulation factors and lupus anticoagulants. Using point-to-point interpolation, loss of linearity below 10IU/dL compromises differentiation between severe, moderate, and mild forms of factor deficiencies (Figure 2). This deterioration of precision justifies the need for linear OSCA using MDA as clinically imperative.