A New Chromogenic Assay (HemosIL ThromboPath) Is Sensitive to Major Prothrombotic Risk Factors Affecting the Protein C Pathway. Results of a Multicenter Study.

Currently, screening for abnormalities of the protein C (PC) pathway is based on determination of PC, PS and factor V (FV) Leiden-related activated (A) PC resistance. However, more than 70% of the Caucasian patients with thrombophilia yield normal results. This highlights the potential usefulness of a simple global screening assay for these abnormalities rationalizing the use of expensive individual assays. The HemosIL ThromboPath assay (Instrumentation Laboratory) is a new chromogenic assay designed to globally evaluate the functionality of the PC pathway. It is based on the ability of endogenous APC generated after activation of PC by a snake venom extract (Protac) to reduce the thrombin generation induced by a reagent containing tissue factor. Briefly, optical density is measured after addition of a thrombin-specific chromogenic substrate in the presence (OD A) or absence (OD B) of Protac. It is recommended by the test manufacturer to express results as the Protac- Induced Coagulation Inhibition percentage (PiCI%) which corresponds to the ratio [OD B − OD A]/OD B × 100. The within-run and the between run precision is below 6.3% using commercially-available plasma samples both in the normal and pathological ranges. This multicenter trial involving three laboratories was designed in order to determine the ability of this assay to distinguish between subjects with and without PC pathway abnormalities. For that purpose, we retrospectively tested frozen plasma samples obtained from patients with a history of venous thromboembolism and patients family members who were referred to the different participating laboratories in order to screen for biological risk factors for thrombosis, as well as from healthy controls. The ThromboPath assay was locally performed, blinded of the specific test results. In all of three centers, test results were significantly lower (p<0.0001) in subjects who presented with any confirmed PC pathway abnormality than in those without. The cut-off value for the PiCI% which provided the best sensitivity-to-specificity ratio was defined in each participating center as the mean value minus 1 SD using a panel of 30 normal samples. It was found to provide a sensitivity-to-specificity ratio similar to that obtained using ROC-analysis. The assay performed well in carriers of the FV Leiden mutation (n=80), patients with PC deficiency (n=40), combined defects (n=55) and lupus anticoagulant (n=44), with test results below the locally defined cut-off values in 98.8%, 95.0%, 100% and 100% of the subjects, respectively. The assay sensitivity for PS deficiency (n=62) was lower (88.1%). Interestingly, 13.6% of the 272 patients without any PC pathway abnormality had a test result below the locally defined cut-off level. So, using the locally defined cut-off values for the PiCI%, the test sensitivity for all PC pathway abnormalities was 96.1% (95%CI=93.1–98.0), its specificity 86.4% (95%CI=81.8–90.2), its negative predictive value 95.5% (95%CI=92.2–97.7) and its positive predictive value 88.0% (95%CI=83.8–91.4). These results suggest that the HemosIL Thrombopath assay could be useful as part of a prothrombotic screening algorithm. However, further larger-scale prospective studies would be required before drawing definite recommendations concerning the economic impact of the implementation of the HemosIL ThromboPath assay as part of the screening strategy of thrombophilia.