Durability of humoral and cellular immunity six months after the BNT162b2 bivalent booster.

Studies about the duration of the humoral and cellular response following the bivalent booster administration are still scarce. We aimed at assessing the humoral and cellular response in a cohort of healthcare workers that received this booster. Blood samples were collected before the administration of the bivalent booster from Pfizer-BioNTech and after 14, 28, 90, and 180 days. Neutralizing antibodies against either the D614G strain, the delta variant, the BA.5 variant, or the XBB.1.5 subvariant were measured. The cellular response was assessed by measurement of the release of interferon gamma from T cells in response to an in vitro SARS-CoV-2 stimulation. A substantial waning of neutralizing antibodies was observed after 6 months (23.1-fold decrease), especially considering the XBB.1.5 subvariant. The estimated T1/2 of neutralizing antibodies was 16.1 days (95% CI = 10.2-38.4 days). Although most participants still present a robust cellular response after 6 months (i.e., 95%), a significant decrease was also observed compared to the peak response (0.95 vs. 0.41 UI/L, p = 0.0083). A significant waning of the humoral and cellular response was observed after 6 months. These data can also help competent national authorities in their recommendation regarding the administration of an additional booster.

Vaccine-induced binding and neutralizing antibodies against Omicron 6 months after a homologous BNT162b2 booster.

Evidence about the long-term persistence of the booster-mediated immunity against Omicron is mandatory for pandemic management and deployment of vaccination strategies. A total of 155 healthcare professionals (104 COVID-19 naive and 51 with a history of SARS-CoV-2 infection) received a homologous BNT162b2 booster. Binding antibodies against the spike protein and neutralizing antibodies against Omicron were measured at several time points before and up to 6 months after the booster. Geometric mean titers of measured antibodies were correlated to vaccine efficacy (VE) against symptomatic disease. Compared to the highest response, a significant 10.2- and 11.5-fold decrease in neutralizing titers was observed after 6 months in participants with and without history of SARS-CoV-2 infection. A corresponding 2.5- and 2.9-fold decrease in binding antibodies was observed. The estimated T1/2 of neutralizing antibodies in participants with and without history of SARS-CoV-2 infection was 42 (95% confidence interval [CI]: 25-137) and 36 days (95% CI: 25-65). Estimated T1/2 were longer for binding antibodies: 168 (95% CI: 116-303) and 139 days (95% CI: 113-180), respectively. Both binding and neutralizing antibodies were strongly correlated to VE (r = 0.83 and 0.89). However, binding and neutralizing antibodies were modestly correlated, and a high proportion of subjects (36.7%) with high binding antibody titers (i.e., >8434 BAU/ml) did not have neutralizing activity. A considerable decay of the humoral response was observed 6 months after the booster, and was strongly correlated with VE. Our study also shows that commercial assays available in clinical laboratories might require adaptation to better predict neutralization in the Omicron era.

Clinical Chemistry and Laboratory Medicine celebrates 60 years - narrative review devoted to the contribution of the journal to the diagnosis of SARS-CoV-2.

This review is an integral part of the special issue for the 60 years of the journal Clinical Chemistry and Laboratory Medicine (CCLM). The aim of the review is to highlight the role of the clinical laboratory since the emergence of the "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), which causes Coronavirus disease 2019 (COVID-19), with special focus on the contribution of the journal in generating knowledge in SARS-CoV-2 diagnosis. As of October 30, 2022, a total of 186 CCLM publications were dedicated to COVID-19. Of importance, major International Federation of Clinical Chemistry (IFCC) guidelines related to the diagnosis of COVID-19 were published in CCLM. Between early-2020 and late October 2022, COVID-19 publications represented around 27% of all articles in CCLM, highlighting the willingness of the editorial board to help the field in order to better describe and diagnose this new emerging disease. First launched in 1963 under the name "Zeitschrift für Klinische Chemie", the Journal was entirely devoted to clinical chemistry in the strict sense. The various topics published in relation to COVID-19 including its diagnosis, its impact on biochemical or hematological measures, as well as biosafety measures, is the perfect example that shows that the journal has greatly diversified over time.

Peri-infection titers of neutralizing and binding antibodies as a predictor of COVID-19 breakthrough infections in vaccinated healthcare professionals: importance of the timing.

OBJECTIVES: The BNT162b2 messenger RNA vaccine is highly effective in reducing COVID-19 infection, hospitalization and death. However, many subjects developed a breakthrough infection despite a full vaccination scheme. Since the waned efficacy of mRNA vaccines is correlated with the decrease of antibodies occurring over time, we aimed at evaluating whether lower levels of antibodies were associated with an increased risk of breakthrough infection in a cohort of breakthrough subjects that received three vaccine doses. METHODS: Total binding antibodies against the RBD of the S1 subunit (Roche Diagnostics, Machelen, Belgium) and neutralizing antibodies using the Omicron B.1.1.529 variant pseudovirus were measured. Based on individual kinetic curves, the antibody titer of each subject was interpolated just before the breakthrough infection and compared to a matched-control group that did not develop a breakthrough infection. RESULTS: Lower levels of total binding and neutralizing antibodies were observed compared to the control group (6.900 [95% CI; 5.101-9.470] vs. 11.395 BAU/mL [8.627-15.050] [p=0.0301] and 26.6 [18.0-39.3] vs. 59.5 dilution titer-1 [32.3-110] [p=0.0042], respectively). The difference between breakthrough and control subjects was mostly observed for neutralizing antibodies before three months after the homologous booster administration (46.5 [18.2-119] vs. 381 [285-509] [p=0.0156]). Considering the measurement of total binding antibodies before 3 months, there was no significant difference (p=0.4375). CONCLUSIONS: In conclusion, our results showed that subjects that developed a breakthrough infection had lower levels of neutralizing and total binding antibodies compared to controls. The difference was mostly noticeable considering neutralizing antibodies, especially for infections occurring before 3 months after the booster administration.

Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration.

OBJECTIVES: To assess the long-term humoral immunity induced by booster administration, as well as the ability of binding antibody and surrogate virus neutralization tests (sVNT) to predict neutralizing antibodies (NAbs) against the SARS-CoV-2 Omicron variant. METHODS: A total of 269 sera samples were analyzed from 64 healthcare workers who had received a homologous booster dose of BNT162b2. Neutralizing antibodies assessed by sVNT and anti-RBD IgG measured with the sCOVG assay (Siemens Healthineers®) were analyzed at five timepoints; before and up to 6 months following the booster. Antibody titers were correlated with neutralizing antibodies against the Omicron BA.1 variant obtained by pseudovirus neutralization test (pVNT) as a reference method. RESULTS: While Wild-type sVNT percentage of inhibition (POI) remained above 98.6% throughout the follow-up period after booster administration, anti-RBD IgG and NAbs assessed by Omicron BA.1 pVNT showed respectively a 3.4-fold and 13.3-fold decrease after 6 months compared to the peak reached at day 14. NAbs assessed by Omicron sVNT followed a steady decline until reaching a POI of 53.4%. Anti-RBD IgG and Omicron sVNT assays were strongly correlated (r=0.90) and performed similarly to predict the presence of neutralizing antibodies with Omicron pVNT (area under the ROC: 0.82 for both assays). In addition, new adapted cut-off values of anti-RBD IgG (>1,276 BAU/mL) and Omicron sVNT (POI>46.6%) were found to be better predictors of neutralizing activity. CONCLUSIONS: This study showed a significant drop in humoral immunity 6 months after booster administration. Anti-RBD IgG and Omicron sVNT assays were highly correlated and could predict neutralizing activity with moderate performance.

Dexamethasone Modulates the Cytokine Response but Not COVID-19-Induced Coagulopathy in Critically Ill.

Severe forms of coronavirus 2019 (COVID-19) disease are caused by an exaggerated systemic inflammatory response and subsequent inflammation-related coagulopathy. Anti-inflammatory treatment with low dose dexamethasone has been shown to reduce mortality in COVID-19 patients requiring oxygen therapy. However, the mechanisms of action of corticosteroids have not been extensively studied in critically ill patients in the context of COVID-19. Plasma biomarkers of inflammatory and immune responses, endothelial and platelet activation, neutrophil extracellular trap formation, and coagulopathy were compared between patients treated or not by systemic dexamethasone for severe forms of COVID-19. Dexamethasone treatment significantly reduced the inflammatory and lymphoid immune response in critical COVID-19 patients but had little effect on the myeloid immune response and no effect on endothelial activation, platelet activation, neutrophil extracellular trap formation, and coagulopathy. The benefits of low dose dexamethasone on outcome in critical COVID-19 can be partially explained by a modulation of the inflammatory response but not by reduction of coagulopathy. Future studies should explore the impact of combining dexamethasone with other immunomodulatory or anticoagulant drugs in severe COVID-19.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

A novel estetrol-containing combined oral contraceptive: European expert panel review.

PURPOSE: Despite considerable advances in recently developed combined oral contraceptives (COCs), resulting in lower rates of adverse events while maintaining contraceptive efficacy, there is interest in further innovation. MATERIALS AND METHODS: Estetrol (E4), a native oestrogen, and progestin drospirenone (DRSP) were combined in a new COC. A European expert panel reviewed the pharmacology, efficacy, and safety and tolerability of this combination. Their findings are presented as a narrative review. RESULTS: E4 15 mg/DRSP 3 mg in a 24/4 regimen provided effective contraception with good cycle control, characterised by a predictable regular bleeding pattern and minimal unscheduled bleeding, together with a good safety profile. The combination was associated with high user satisfaction, well-being, and minimal changes in body weight. The effects on endocrine and metabolic parameters were limited, and the combination was found to have a limited impact on liver function and lipid and carbohydrate metabolism. Moreover, its effect on several haemostatic parameters was lower than that of comparators containing ethinyl oestradiol (EE) 20 µg/DRSP 3 mg and EE 30 µg/levonorgestrel 150 µg. CONCLUSION: E4 15 mg/DRSP 3 mg provides safe and effective contraception, with high user satisfaction and predictable bleeding. Further research will evaluate the long-term safety of the COC.

A novel estetrol-containing combined oral contraceptive: European expert panel review.

PURPOSE: Despite considerable advances in recently developed combined oral contraceptives (COCs), resulting in lower rates of adverse events while maintaining contraceptive efficacy, there is interest in further innovation. MATERIALS AND METHODS: Estetrol (E4), a native oestrogen, and progestin drospirenone (DRSP) were combined in a new COC. A European expert panel reviewed the pharmacology, efficacy, and safety and tolerability of this combination. Their findings are presented as a narrative review. RESULTS: E4 15mg/DRSP 3 mg in a 24/4 regimen provided effective contraception with good cycle control, characterised by a predictable regular bleeding pattern and minimal unscheduled bleeding, together with a good safety profile. The combination was associated with high user satisfaction, wellbeing, and minimal changes in body weight. The effects on endocrine and metabolic parameters were limited, and the combination was found to have a limited impact on liver function and lipid and carbohydrate metabolism. Moreover, its effect on several haemostatic parameters was lower than that of comparators containing ethinyl oestradiol (EE) 20mg/DRSP 3 mg and EE 30mg/levonorgestrel 150mg. CONCLUSION: E4 15 mg/DRSP 3 mg provides safe and effective contraception, with high user satisfaction and predictable bleeding. Further research will evaluate the long-term safety of the COC.

Analytical and clinical validation of an ELISA for specific SARS-CoV-2 IgG, IgA, and IgM antibodies.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests is massive. The external validation of their performance is needed before use in clinical routine practice. Our study aims at assessing the analytical and clinical performance of two enzyme-linked immunosorbent assay tests detecting antibodies directed against the virus nucleocapsid protein: The NovaLisa SARS-CoV-2 immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) test (NovaTec) allowing a separate detection of each antibody and the Platelia SARS-CoV-2 Total Ab test (Bio-Rad) detecting total antibodies (IgM, IgA, and IgG). Two-hundred and eight coronavirus disease 2019 samples from 48 quantitative reverse transcription-polymerase chain reaction (RT-qPCR) confirmed patients were used to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 79) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. In addition, using receiver operator characteristic curves, adapted cut-off for improvement of the performances were proposed. The kinetics of these antibodies was also assessed over 8 weeks. Two weeks after the RT-qPCR positive detection, the NovaLisa test shows a sensitivity and specificity of 94.9% (95% confidence interval [CI]: 83.1%-98.6%) and 96.2% (95% CI: 89.4%-98.7%) for IgG, of 89.7% (95% CI: 76.4%-95.9%) and 98.7% (95% CI: 93.2%-98.8%) for IgA, and of 48.7% (95% CI: 33.9%-63.8%) and 98.7% (95% CI: 93.2%-99.8%) for IgM. With the Platelia system, the specificity and sensitivity were 97.4% (95% CI: 92.1%-99.7%) and 94.9% (95% CI: 87.7%-98.0%) for total antibodies using the adapted cut-offs. The NovaLisa and the Platelia tests have satisfactory analytical performances. The clinical performances are excellent for IgG, IgA, and total antibodies especially if the cut-off is optimized.

Clinical performance of three fully automated anti-SARS-CoV-2 immunoassays targeting the nucleocapsid or spike proteins.

This study assesses the clinical performance of three anti-SARS-CoV-2 assays, namely EUROIMMUN anti-SARS-CoV-2 nucleocapsid (IgG) ELISA, Elecsys anti-SARS-CoV-2 nucleocapsid (total antibodies) assay, and LIAISON anti-SARS-CoV-2 spike proteins S1 and S2 (IgG) assay. One hundred and thirty-seven coronavirus disease 2019 (COVID-19) samples from 96 reverse-transcription polymerase chain reaction confirmed patients were chosen to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 141) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. None of these tests demonstrated a sufficiently high clinical sensitivity to diagnose acute infection. Fourteen days since symptom onset, we did not find any significant difference between the three techniques in terms of sensitivities. However, Elecsys performed better in terms of specificity. All three anti-SARS-CoV-2 assays had equivalent sensitivities 14 days from symptom onset to diagnose past-COVID-19 infection. We also confirmed that anti-SARS-CoV-2 determination before Day 14 is of less clinical interest.

Clinical performance of the Panbio assay for the detection of SARS-CoV-2 IgM and IgG in COVID-19 patients.

Following the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, numerous serological tests have been developed, including rapid diagnostic tests. This study aims at assessing the clinical performance of the Panbio immunoglobulin G (IgG)/IgM coronavirus disease 2019 (COVID-19) test (Abbott), a rapid lateral flow assay for the qualitative detection of IgG and IgM against SARS-CoV-2. One hundred and thirty-eight samples from 95 COVID-19 patients with a positive SARS-CoV-2 reverse-transcriptase polymerase chain reaction were analyzed to assess the clinical sensitivity. Seventy-six pre-COVID-19 samples were used to evaluate the clinical specificity. Two independent and blinded raters determined visually the presence or absence of the IgG, IgM, and control lines for each test after 10 and 20 min. The sensitivity obtained from collected samples more than 14 days after the onset of symptoms was 95.2% for IgG. IgM was less frequently detected (highest sensitivity of 20.5%). The specificities obtained were 98.7% and 100% for IgG and IgM, respectively. In addition, the sensitivity of the assay was better when the reading was performed at 20 min than at 10 min, whereas the specificity was unchanged. The Panbio COVID-19 IgG/IgM rapid test detects IgG with high sensitivity 14 days since symptom onset but presents a low sensitivity for IgM. The specificity was excellent for both IgG and IgM.

An original multiplex method to assess five different SARS-CoV-2 antibodies.

OBJECTIVES: Accurate SARS-CoV-2 serological assays are urgently needed to help diagnose infection, determine past exposure of populations and assess the response to future vaccines. The study aims at assessing the performance of the multiplex D-tek COVIDOT 5 IgG assay for the detection of SARS-CoV-2 IgG antibodies (N, S1+S2, S1, S2 and RBD). METHODS: Sensitivity and dynamic trend to seropositivity were evaluated in 218 samples obtained from 46 rRT-PCR confirmed COVID-19 patients. Non-SARS-CoV-2 sera (n=118) collected before the COVID-19 pandemic with a potential cross-reaction to the SARS-CoV-2 immunoassay were included in the specificity analysis. RESULTS: A gradual dynamic trend since symptom onset was observed for all IgG antibodies. Sensitivities before day 14 were suboptimal. At ≥21 days, sensitivities reached 100% (93.4-100%) for N, S1+S2, S2 and RBD-directed IgG and 96.3% (87.3-99.6%) for S1-directed IgG. In 42 out of 46 patients (91.3%), all five antibodies were detected at ≥14 days. The four remaining patients had between 2 and 4 positive antibodies at their respective maximal follow-up period. The specificity was 100 % for S1+S2, S2 and RBD, 98.3% for N and 92.4% (86.0-96.5%) for S1-directed IgG. The combined use of antigens increases the early sensitivity whilst enforcing high specificity. CONCLUSIONS: Sensitivities at ≥21 days and specificities were excellent, especially for N, S1+S2, S2 and RBD-directed IgG. Caution is however required when interpreting single S1-directed reactivities. Using a multiplex assay complies with the orthogonal testing algorithm of the CDC and allows a better and critical interpretation of the serological status of a patient.

Oral Contraceptives and Venous Thromboembolism: Focus on Testing that May Enable Prediction and Assessment of the Risk.

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia.

Ecarin based coagulation testing.

Ecarin is derived from venom of Echis carinatus, and will activate prothrombin into meizothrombin which will then cleave fibrinogen to result in clot formation. Ecarin based testing has been described for decades, but these assays were typically restricted to reference or speciality coagulation laboratories. This test was initially described for the assessment of direct thrombin inhibitors (eg, bivalirudin lepirudin, or argatroban) and was not affected by heparins or heparinoids. Ecarin based assays were rarely used for anticoagulation monitoring until the emergence of the direct oral thrombin inhibitor dabigatran etexilate in 2010. As this test was mentioned in the prescribing information for dabigatran etexilate, there was increased interest for use by clinical laboratories as the preferred method for assessing the anticoagulant effect of this drug. The purpose of this document is to review the current status of ecarin based assays for assessing dabigatran. This is with the understanding that these methods can also be exploited for determining the anticoagulation effect of parenteral direct thrombin inhibitors, such as argatroban and bivalirudin.

Validation of a chemiluminescent assay for specific SARS-CoV-2 antibody.

Objectives Faced with the COVID-19 pandemic and its impact on the availability and quality of both therapeutic and diagnostic methods, the Belgian authorities have decided to launch a procedure for additional evaluation of the performance of serological tests offered for sale on the national territory. This has been proposed with a double aim: (1) an in-depth verification of the analytical and clinical performances presented by the manufacturer and (2) an economy of scale in terms of centralized validation for all the laboratories using the tests subject to evaluation. Methods A retrospective validation study was conducted including the serum of 125 patients in order to determine the analytical and clinical performances of the LIAISON®SARS-CoV-2 from DiaSorin® detecting anti-SARS-CoV-2 IgG and to compare its clinical performance with the enzyme-linked immunosorbent assay (ELISA) test from Euroimmun®, one of the first commercially available tests allowing the detection of anti-SARS-CoV-2 IgA and IgG. Results The performances of the LIAISON®SARS-CoV-2 satisfied all the acceptance criteria and provided "real world" analytical and clinical performances very close to the ones reported by the manufacturer in its insert kit. Comparison between the LIAISON®SARS-CoV-2 and the ELISA method did not reveal any difference between the two techniques in terms of sensitivities and specificities regarding the determination of the IgG. Conclusions This study reports the validation of the LIAISON®SARS-CoV-2 allowing to detect IgG antibodies specifically directed against SARS-CoV-2. The analytical and clinical performances are excellent, and the automation of the test offers important rates, ideal for absorbing an extension of testing.

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.

Background Regulatory 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. Methods The 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. Results Repeatability 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. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Importance of measuring pharmacologically active metabolites of edoxaban: development and validation of an ultra-high-performance liquid chromatography coupled with a tandem mass spectrometry method.

Although DOACs do not require regular measurements of their blood concentrations, clinical situations may require an assessment of their concentration. Among the factor Xa inhibitors, edoxaban is the only compound for which some metabolites (e.g. edoxaban-M4) are reported to be pharmacologically active. Therefore, their contribution could interfere with assays used for the estimation of edoxaban concentration. In addition, drug interactions may alter the metabolite/parent compound ratio making the sole estimation of edoxaban concentration, a poor assessment of the overall anticoagulation. To develop a validated UHPLC-MS/MS method to quantify simultaneously edoxaban and its more relevant M4-metabolite in human plasma. Electrospray ionization and chromatographic separation were optimized for the simultaneous dosage of edoxaban and edoxaban-M4. The method was validated according to regulatory guidelines for bioanalytical method validation. The total run time was 6 min. The method was validated for calibration curves, precision, accuracy, carry-over, selectivity, matrix effect and short-time stability. This method permits quantification of edoxaban and edoxaban-M4 providing complementary information about the inhibitory effect of this active metabolite in chronometric or chromogenic assays. Although patients treated with edoxaban exhibits usually low concentrations of active metabolites, the measurement of edoxaban-M4 is interesting; especially in case of drug interactions. Indeed, concomitant prescriptions of edoxaban and carbamazepine or rifampicin is frequent and may lead to disturbance of the estimations of edoxaban concentration by chromogenic anti-Xa assays. Therefore, patients are at risk of having inadequate control of anticoagulation supporting the need of measuring the most representative edoxaban metabolite concomitantly to the parent compound.