Evaluation of Pre-analytical Variables in a Commercial Thrombin Generation Assay

Abstract Background There is minimal data on the influence of pre-analytical variables on the use of calibrated automated thrombography (CAT), to measure thrombin generation. Objectives To evaluate the impact of centrifugation methods, time after collection, and contact activation inhibition on the CAT assay performed using two commercial reagents. Methods and Results Six different methods of plasma separation were examined. Thrombin generation triggered by a 5 pM tissue factor reagent was not greatly affected by plasma separation method, with similar results obtained with all methods apart from single centrifugation and membrane filtration. Membrane filtration increased APTT and is not recommended. Extended double centrifugation at higher speed was required to minimise the impact of residual phospholipid with 1 pM tissue factor trigger, particularly with inhibition of contact activation. The effect of a delay of up to 24 hours in preparing plasma was assessed. No significant difference in results was observed among samples processed between 0.5 and 6 hours after blood collection into plastic Vacuette® tubes. The presence or absence of corn trypsin inhibitor had a significant impact on all parameters with 1 pM tissue factor trigger, with minor differences seen on Peak and ttPeak results using 5 pM tissue factor. Conclusions The impact of pre-analytical variables on thrombin generation results is dependent on the concentration of tissue factor in the trigger reagent used. Results with 1 pM tissue factor are particularly sensitive to centrifugation method and contact activation, and standardisation is required to allow large collaborative studies to be performed.