Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need

Abstract

BackgroundRegulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay.MethodsThe validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool.ResultsRepeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R2 > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay.ConclusionsThis validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Proof of concept of a new scale for the harmonization and the standardization of the ETP‐based APC resistance

“…Our previous study showed that a validated methodology of ETP‐based APC resistance assay enables batch‐to‐batch management while maintaining the sensitivity, specificity, and robustness over a batch change . Based on 86 measurements (38 for batch 202685 and 48 for batch 202947), the ETP ratio did not show a significant difference between the 2 batches used.…”

“…27,28 Hence, our group recently proposed a validated methodology that meets all the regulatory requirements in terms of analytical performances. 29 However, different units have been reported in the literature for the expression of the ETP-based APC resistance results. Among these different units, the so-called "normalized APC sensitivity ratio" (nAPCsr) is certainly the most popular.…”

“…Indeed, we demonstrated that the choice of the reference plasma highly affects the nAPCsr of the sample. 29 Our approach introduces a new scale called the nAPCsr 10 which avoids the additional variability introduced by the use of a reference plasma. The aim of this study is to investigate a series of individual plasma samples and to compare the results obtained by the traditional nAPCsr and the newly proposed nAPCsr 10 .…”

“…Indeed, we demonstrated that the choice of the reference plasma highly affects the nAPCsr of the sample . Our approach introduces a new scale called the nAPCsr 10 which avoids the additional variability introduced by the use of a reference plasma.…”

“…One of the main limitations of this technique was the lack of standardization and harmonization, which hampered study‐to‐study comparison . Hence, our group recently proposed a validated methodology that meets all the regulatory requirements in terms of analytical performances …”