Performance of a New Global Chromogenic Assay for Protein C Pathway (HemosIL ThromboPath) in the Diagnosis of Lupus Anticoagulant and Antiphospholipid Antibodies.
Authors: Toulon Pierre A. , Martin-Toutain Isabelle , Piette Jean-Charles , Diemert Marie-Claude , Ankri Annick 
Published: April 2020

Abstract
Background: 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 x 100. Previous studies demonstrated a high sensitivity (over 96%) for PC pathway abnormalities i.e. PC/PS deficiency, and FV Leiden-related APC Resistance, but also for lupus anticoagulant (LA), known to be associated with acquired APC Resistance.
The aim of the present study was to evaluate the sensitivity of that assay for antiphospholipid antibodies (APL), especially in connection with the occurrence of thrombotic complications. Actually, APL is a heterogeneous family of antibodies which have a significant impact on coagulation tests (LA) or could be detected using ELISA anticardiolipin assays (ACL).
Methods: We retrospectively evaluated the frozen plasma samples from 178 patients previously diagnosed as positive for LA or as having high ACL levels. There were 48 M and 130 F with a mean age=43 years (range 14 – 87). None was on vitamin K-antagonist. Screening for LA, performed according to the ISTH criteria (Thromb Haemost1995;74:1185), was positive in 126 patients (LA+ patients) and negative in 52 patients, all of the latter having high ACL levels (LA- patients). Among LA+ patients, 41 had antiphospholipid syndrome (APS) i.e. 18 with a history of venous thrombosis (VT), 10 with arterial thrombosis (AT), 7 with obstetrical complication (OB) and 6 with combined vascular complications (CVC). The same applied to 39 LA- patients with high APL levels i.e. VT (n=13), AT (n=8), OB (n=12) and CVC (n=6). The control group consisted of 29 age- and sex-matched healthy subjects without a history of vascular or auto-immune disease. The HemosIL ThromboPath assay was performed by an operator blinded of the specific biological test results and of the clinical status of the patients.
Results: Test result was significantly lower in LA+ patients than in LA- patients or in healthy controls (Table). Similarly, the percentage of abnormal test results i.e. PICI% below 85.0% as the cut-off value, was significantly higher in LA+ patients than in the two other groups (p&lt;0.0001 in both cases). That cut-off value for the PICI% was determined, according to the recommendations of the reagent manufacturer, as the mean minus 1 SD of the values measured in the plasma of 29 healthy control subjects. Despite significantly lower PICI% in LA- patients than in controls (p&lt;0.05), the proportion of abnormal test results was not significantly different (p&lt;0.10) in these two groups.
LA+ Patients (n=126) LA-Patients (n=52) Controls (n=29) Test Result (PICI%) 74.5 ± 13.1 86.7 ± 9.4 90.3 ± 5.3 PICI%&lt;85% (n, %) 99 (78.6%) 15 (28.8%) 3 (10.3%)
Moreover, test result was not significantly different in LA+ patients with and without a history of vascular thrombosis (PICI%=74.4 ± 12.9, n=51 vs. 74.5 ± 13.5 n=75, p=0.91) and the same applied for the percentage of abnormal test results (n=41/51 (80.4%) vs. n=58/75 (77.3%); p=0.83). There was no significant difference between LA+ patients with venous, arterial thrombosis and obstetrical complications.
Conclusion: The HemosIL ThromboPath assay was highly sensitive for LA (78.6%) but it did not allow to distinguish between LA+ patients those with APS. It was found to be weakly sensitive (28.8%) to high ACL levels without LA activity, despite a higher percentage of abnormal test results. The potential interest of the HemosIL ThromboPath assay as part of the screening strategy of LA deserves to be further investigated prospectively.