Correction of microplate location effects improves performance of the thrombin generation test
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ORIGINAL BASIC RESEARCH
Open Access
Correction of microplate location effects improves performance of the thrombin generation test Yideng Liang, Samuel A Woodle, Alexey M Shibeko, Timothy K Lee and Mikhail V Ovanesov*
Abstract Background: Microplate-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. Methods: In 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. Results: Location 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. Conclusions: Use 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. Keywords: Thrombin generation test, Location effects, Immunoglobulin, Thrombogenicity
Background Thrombin generation test (TGT) measures kinetics of thrombin activity during coagulation of a blood plasma sample mixed with activators of blood coagulation [1]. The TGT is widely used in clinical research to measure global hemostatic potential in blood coagulation disorders either for diagnostic purposes [2,3] or as means of treatment monitoring [4,5]. More recently, TGT became a useful tool in drug development and control of drug potency and quality in drug manufacture [6-9]. Although proposed in the 1950s [10], the test gained popularity
* Correspondence: [email protected] Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 29 Lincoln Drive, N29/306, Bethesda, MD 20892, USA
only a decade ago after the technique was revolutionized with the introduction of fluorogenic thrombin substrates and microtiter plate reader format [2]. However, the convenience of the microtiter plate technology can be deceiving: microtiter plate assays are prone to a special kind of variability caused by the uneven microenvironments in different wells of the plate [11]. In order to describe the location-based effects f
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