Development of a microplate coagulation assay for Factor V in human plasma
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ORIGINAL BASIC RESEARCH
Open Access
Development of a microplate coagulation assay for Factor V in human plasma Derek Tilley1, Irina Levit2 and John A Samis1,2*
Abstract Background: Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. Methods: The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. Results: The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Conclusions: The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement of the functional coagulation activity of FV in human plasma.
Background The use of microplate-based assays for quantification of enzyme activity is now common practice in many laboratories for enzyme linked immunosorbant assays [1] and colorimetric enzyme assays using chromogenic substrates [2]. The two main advantages for microplate-based assays are use of small sample volumes and that multiple samples may be analyzed simultaneously. Several microplate assays have been previously reported for measurement of clot lysis [3], platelet aggregation [4], the activity of Factor (F) VII, VIII, IX and IXa, X, XI, XII, and activated * Correspondence: [email protected] 1 Applied Bioscience, Faculty of Science, University of Ontario Institute of Technology, Oshawa, ON. L1H7K4. Canada Full list of author information is available at the end of the article
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