The development of a screening test for haemostasis using physiological levels of tissue factor

Recent work has highlighted the central role of tissue factor (TF) in the physiological activation of haemostasis. This study was designed to investigate the use of physiological levels of TF as the triggering factor in the laboratory assessment of haemostasis. Endpoints based upon fibrin polymerisation and thrombin generation were selected as these have been shown to reflect the combined effect of the components of the haemostatic process. Sample quality was found to be of the utmost importance when picomolar concentrations of TF were used to trigger an assay. Sample mediated contact activation was found to be a major problem. The use of corn trypsin inhibitor (CTI) at a final concentration of 18. 3ýtg/ml of whole blood was found to be optimum for the elimination of the effect of factor XII activation which occurred during sample collection and subsequent manipulation. In the absence of CTI significant thrombin generation was evident (1425nM. min, SID 582) resulting from intrinsic sample activation. TF concentration was optimised for the detection of both hypercoagulable and hypocoagulable samples using the calibrated automated thrombin generation method (CAT), rotational thromboelastometry (ROTEM) and a clot kinetic assay (CKA) as global tests of haemostasis. The incorporation of thrombomodulin (TM) into the reagent was found to improve the assay sensitivity to hypercoagulable samples. Final concentrations of TF / TM were found to differ between the CAT assay of thrombin generation and the ROTEM and CKA assays of fibrin polymerisation. The optimum reagent for the CAT assay was found to be 15pM TF in combination with 7nM TM. Although this combination was able to discriminate hypocoagulable, hypercoagulable and normal individuals, the high assay coefficient of variance of around 50% was unacceptable when the hypocoagulable samples were assessed. This variance could be halved by the use of 4pM TF in the absence of TM for this patient group. The ROTEM and CKA were optimised at much lower reagent concentrations (2pm TF / 0.5nM TM and IpM TF / 0.5pM Tm respectively). However, neither of these assays of fibrin polymerisation was able to fully discriminate between mild haemophiliacs and normal individuals. These optimised reagents were used to assess 54 patients previously found to have suffered a venous thromboembolic event (VTE). It was found that each assay identified >20% of the VTE patients with values above the upper limit of normality. The ability of the assays to detect an identifiable thrombophilic defect, within the VTE patient samples, varied from 0-100% depending upon the defect / assay combination. The assays were clearly identifying different patient populations. The question as to which, if any, are most at risk remains to be answered and a follow-up prospective study has been initiated in conjunction with Haemostasis Unit at the University of Leiden to attempt to answer this question. Both the CAT and CKA were also used to assess haemostasis in 13 severe haemophiliacs undergoing routine assessment of post infusion recovery or full product half life studies. The CKA was found to be uninformative in these patients. However, the CAT using both 15pM TF / 7nM TM and 4pM TF reagents could be used to monitor factor concentrate infusion. The longer half-life calculated using the CAT rather than conventional single factor assays suggested that haemophiliacs retain an enhanced haemostatic function beyond the time calculated from traditional half-life calculations. A follow-up study has been initiated to ftuther investigate this finding. This study supports the view that assays triggered using low TF concentrations may produce more clinically relevant findings that the current "routine" screening tests.