Sensitivity of a New Global Assay for Protein C (PC) Pathway Abnormalities and Potential Significance of an Abnormal Test Result as a Risk Factor/Marker for Thrombosis Independently of Its Sensitivity for PC Pathway Abnormalities.
Authors: Toulon Pierre A. , Rosencher Nadia , Guigon Aurélie , Simon Arnaud 
Published: April 2020

Abstract
Abnormalities of the protein C (PC) anticoagulant pathway i.e. PC deficiency, PS deficiency and APC-resistant factor Va (FV Leiden) are among the most frequent biological risk factors for venous thromboembolism (VTE) in Caucasian patients but normal results are found in more more than 70% of the cases. This highlights the potential usefulness of a simple assay to screen globally for these abnormalities as this would rationalize the use of expensive individual assays. The HemosIL Thrombopath (Instrumentation Laboratory) is a new chromogenic global assay based on the ability of endogenous APC generated after activation of PC by a snake venom (Protac) to reduce the thrombin generation induced by a reagent containing tissue factor. 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 (PCI%) which corresponds to the ratio [OD B - OD A]/OD B x 100. The assay was performed on the ACL9000 analyzer (IL) using 85% as the cut-off level (PCI%) as we previously determined. Using various lyophilized plasma samples (n=8) both in the normal and abnormal ranges, the within-run precision (evaluated as the CV%, n=17) was found in the range from 1.2% to 2.8% and the between-run precision (CV%, n=48) in the range from 1.6% to 6.3%. The assay sensitivity for PC pathway abnormalities was studied by evaluating a selection of frozen plasma samples from 144 patients with a history of VTE (to be expended). All patients with FV Leiden, PC deficiency (1 with a borderline value), combined defects and lupus anticoagulant (LA) had a decreased response to the assay (PCI% &lt;85%), and the same applied to 28 of the 36 patients with PS deficiency. However, 6 of the 40 patients without any PC pathway abnormality had a decreased response to the assay. So the test sensitivity for PC pathway abnormalities was 91.4% (95%CI=84.2–96), its specificity 85.0% (95%CI=70.2–94.3), its negative predictive value 79.1% (95%CI=63.9–90) and its positive predictive value 94.1% (95%CI=87.5–97.8). Moreover, most (69%) of 13 additional patients carrying the G20210A prothrombin mutation had a decreased test result.
PC Def. (n=20) PS Def. (n=36) PC+PS Def. (n=8) FV Leiden (n=27) LA (n=13) No Abnormality (n=40) PCI% (Med/Range) 71.8 (53.8 – 85.3) 74.3 (48.5 – 95.3) 43.5 (19.9 – 70.7) 69.9 (43.8 – 80.9) 69.2 (53.6 – 82.9) 90.5 (73.5 – 98.4) PCI%&lt;85 (n, %) 19/20 (95%) 28/36 (78%) 8/8 (100%) 27/27 (100%) 13/13 (100%) 6/40 (15%)
In an attempt to evaluate the clinical relevance of a decreased PCI% in patients without any PC pathway abnormality, the HemosIL Thrombopath assay was performed pre-operatively in patients who underwent major orthopedic surgery. From the preliminary results available, test result was normal in all of the first 34 enrolled patients except in one woman (PCI%=54%) who developped post-operative deep vein thrombosis after total hip replacement and who had no isolated risk factor for thrombosis. This was the only thrombotic episode recorded in the series with a formal 3-month follow-up. These preliminary results suggest that the HemosIL Thrombopath assay is highly sensitive for PC pathway abnormalities and that an abnormal test result could be in itself a risk factor/marker for VTE (p&lt;0.05, Fischer’s exact test).