Correction of microplate location effects improves performance of the thrombin generation test

Abstract

BackgroundMicroplate-based thrombin generation test (TGT) is widely used as clinical measure of global hemostatic potential and it becomes a useful tool for control of drug potency and quality by drug manufactures. However, the convenience of the microtiter plate technology can be deceiving: microplate assays are prone to location-based variability in different parts of the microtiter plate.MethodsIn this report, we evaluated the well-to-well consistency of the TGT variant specifically applied to the quantitative detection of the thrombogenic substances in the immune globulin product. We also studied the utility of previously described microplate layout designs in the TGT experiment.ResultsLocation of the sample on the microplate (location effect) contributes to the variability of TGT measurements. Use of manual pipetting techniques and applications of the TGT to the evaluation of procoagulant enzymatic substances are especially sensitive. The effects were not sensitive to temperature or choice of microplate reader. Smallest location effects were observed with automated dispenser-based calibrated thrombogram instrument. Even for an automated instrument, the use of calibration curve resulted in up to 30% bias in thrombogenic potency assignment.ConclusionsUse of symmetrical version of the strip-plot layout was demonstrated to help to minimize location artifacts even under the worst-case conditions. Strip-plot layouts are required for quantitative thrombin-generation based bioassays used in the biotechnological field.

High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure

“…3). This was consistent with the well-known row and/or column effects within microplates as well as systematic differences between them (25)(26)(27). Surface plasmon resonance analysis showed that mAb 2D7 was specific to human NT-proUcn2 and did not cross-react with the bovine, mouse, or rat counterparts (see online Supplemental Fig.…”

Optimization of the thrombin generation test components to measure potency of factor VIII concentrates

“…To further improve the robustness of the TGT-based potency assay, we used automated dispenser to replace the manual step of row-by-row plasma recalcification which we previously found responsible for TGT variability [27]. In the automated version, a 96-channel robotic pipettor allowed rapid and simultaneous mixing of FVIII samples with recalcified plasma in all wells of a microplate [18,19].…”

Effect of the injectable contraceptive depot‐medroxyprogesterone acetate on coagulation parameters in new users

“…The thrombin generation test (TGT) is a new technique that measures the kinetics of thrombin activity during coagulation in a plasma sample mixed with blood coagulation activators. It has been widely used in clinical trials to measure the overall hemostatic potential in blood coagulation disorders, both for diagnostic purposes and as a means of monitoring treatment …”

“…It has been widely used in clinical trials to measure the overall hemostatic potential in blood coagulation disorders, both for diagnostic purposes and as a means of monitoring treatment. 20 In view of the controversial clinical findings and the difficulty in conducting large epidemiological studies that would provide strong evidence on the relation between the use of DMPA and VTE and arterial thrombosis, the objective of this study was to assess the effects of DMPA on selected blood coagulation parameters, including evaluation of TGT, in young and healthy new users and to compare them with a group of non-users.…”