Interlaboratory variability of activated protein C resistance using the ETP-based APC resistance assay.

BACKGROUND AND OBJECTIVE: Although the endogenous thrombin potential (ETP)-based activated protein C (APC) resistance is recommended for the development of steroid contraceptive agents, one of the main limitations of this technique was its lack of standardization, which hampered study-to-study comparison. A validated methodology that meets all the regulatory requirements in terms of analytical performances has been developed recently. To ensure a wide implementation of this test, the assessment of the interlaboratory variability was needed. METHOD: The assay was implemented in three testing laboratories. First, dose-response curves were performed to locally define APC concentration leading to 90% of ETP inhibition on healthy donors. Intra- and inter-run repeatability were assessed on a reference plasma and three quality controls. To investigate the variability in results among the different testing units, 60 donor samples were analyzed at each site. RESULTS: The APC concentration leading to 90% of ETP inhibition was defined at 1.21 µg/ml and 1.14 µg/ml in the two receiving units. Intra- and inter-run repeatability showed standard deviation below 3%. Analyses of the 60 donor samples showed no statistically significant difference. The sensitivity of the test in the different laboratories was maintained and subgroup analyses still reported significant differences depending on hormonal status of donors. CONCLUSION: This study is the first reporting the interlaboratory variability of the ETP-based APC resistance assay. Data revealed excellent intra- and interlaboratory reproducibility. These results support the concept that this blood coagulation test provides an appropriate sensitivity irrespective of the laboratory in which analyses are performed.

[Study of thrombin generation in healthy children]. / Étude de la génération de thrombine dans une population pédiatrique.

Exploring coagulation in newborns and children is a challenge due to the low levels of both procoagulant factors and inhibitors. Conventional coagulation tests might be inadequate to explore all of these changes. The aim of the study is to evaluate the thrombin generation assay, a global test to explore coagulation, in a pediatric population (n=586) compared to an adult population (n=166). The thrombin generation assays were performed using Calibrated Automated Thrombography with two different tissue factor concentrations (1 and 5 pM), with and without thrombomodulin (TM). In the absence of TM, the endogenous thrombin potential (ETP) is significantly lower in the pediatric population, reflecting the decrease in procoagulant factors. In the presence of TM, ETP values in pediatric subjects are within the reference range of adult values. The thrombin generation assay demonstrates that coagulation balance is maintained in the pediatric population.

Measurements of eftrenonacog alfa by 19 different combinations reagents/instrument: A single-centre study.

INTRODUCTION: Recombinant factor IX Fc fusion protein (rFIXFc) is an extended half-life concentrate for the treatment of haemophilia B (HB). rFIXFc activity monitoring is crucial in several clinical situations. However, differences were observed between one-stage clotting (OSC) and chromogenic assays, but not for all factor IX (FIX) concentrations. AIMS: To compare rFIXFc measurements obtained using different instruments and common OSC and chromogenic asssays. METHODS: FIX:C measurements were performed in rFIXFc-spiked plasma aliquots (targeted FIX levels of 1.5, 1, 0.5, 0.2, 0.05, 0.02 and 0.01 IU/mL) and plasma samples collected from two patients with HB at various time points after rFIXFc infusion, using three instruments (STA-R MAX, ACLTOP700 and CS2100i) and common clotting and chromogenic FIX:C assays. RESULTS: The same reagent could give different FIX:C measurements when adapted to different instruments. Moreover, the same reagent/instrument combination could give different results depending of the FIX concentration. For OSC assays, only STA-Cephascreen on STA-R MAX and CS2100i, SynthAFax on ACLTOP 700 and Actin on CS2100i provided acceptable recoveries for all rFIXFc concentrations. The chromogenic assays ROX-FIX and Biophen FIX:C underestimated rFIXFc for concentrations lower than 0.05 and 0.2 IU/mL, respectively. CONCLUSIONS: Our study demonstrates that the same reagent adapted to different instruments could lead to different rFIXFc values. As rFIXFc under/overestimation could be associated with inappropriate treatment or biased calculation of pharmacokinetic parameters, the reagent/instrument combination used by haemostasis laboratories should be considered and regularly evaluated by external quality assessment programmes.

Hydrogenotrophic methanogens of the mammalian gut: Functionally similar, thermodynamically different-A modelling approach.

Methanogenic archaea occupy a functionally important niche in the gut microbial ecosystem of mammals. Our purpose was to quantitatively characterize the dynamics of methanogenesis by integrating microbiology, thermodynamics and mathematical modelling. For that, in vitro growth experiments were performed with pure cultures of key methanogens from the human and ruminant gut, namely Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanobacterium formicium. Microcalorimetric experiments were performed to quantify the methanogenesis heat flux. We constructed an energetic-based mathematical model of methanogenesis. Our model captured efficiently the dynamics of methanogenesis with average concordance correlation coefficients of 0.95 for CO2, 0.98 for H2 and 0.97 for CH4. Together, experimental data and model enabled us to quantify metabolism kinetics and energetic patterns that were specific and distinct for each species despite their use of analogous methane-producing pathways. Then, we tested in silico the interactions between these methanogens under an in vivo simulation scenario using a theoretical modelling exercise. In silico simulations suggest that the classical competitive exclusion principle is inapplicable to gut ecosystems and that kinetic information alone cannot explain gut ecological aspects such as microbial coexistence. We suggest that ecological models of gut ecosystems require the integration of microbial kinetics with nonlinear behaviours related to spatial and temporal variations taking place in mammalian guts. Our work provides novel information on the thermodynamics and dynamics of methanogens. This understanding will be useful to construct new gut models with enhanced prediction capabilities and could have practical applications for promoting gut health in mammals and mitigating ruminant methane emissions.