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

Thrombosis Journal

Mikhail V Ovanesov¹  Timothy K Lee¹  Alexey M Shibeko¹  Samuel A Woodle¹  Yideng Liang¹ 

[1] 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

关键词: Thrombogenicity;    Immunoglobulin;    Location effects;    Thrombin generation test;   

Others  :  838563
DOI  :  10.1186/1477-9560-11-12

 received in 2013-02-08, accepted in 2013-05-11,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Liang et al.; licensee BioMed Central Ltd.

【 图 表 】

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