Microkinetic coagulation assays for human and zebrafish plasma

Area of Science:

• Biochemistry
• Hematology
• Zebrafish models

Background:

• Traditional coagulation assays like prothrombin time (PT) and partial thromboplastin time (PTT) require 100μl of human plasma.
• Existing methods for zebrafish plasma coagulation often involve dilution and exogenous fibrinogen, altering physiological conditions.

Purpose of the Study:

• To develop a microkinetic coagulation assay using only 1μl of plasma.
• To enable accurate measurement of coagulation in both human and zebrafish plasma under conditions mimicking PT and PTT.
• To validate the assay's performance and its ability to derive clotting times comparable to established methods.

Main Methods:

• Utilized a Take3 plate with micro-volume wells (≤6μl) compatible with microplate readers.
• Performed kinetic measurements of fibrin formation using 1μl of citrated human or zebrafish plasma.
• Activated plasma with thromboplastin, Dade ACTIN, or Russell's viper venom activator and CaCl2, optimizing reaction volumes to 3-4μl.

Main Results:

• Developed kinetic curves for PT, PTT, and Russell's viper venom time in both human and zebrafish plasma.
• Demonstrated that the microkinetic assay results were comparable to established assays using dilute plasma.
• Confirmed assay sensitivity to heparin inhibition and reduced activity in factor-deficient plasmas, validating its accuracy.

Conclusions:

• Established a novel microkinetic coagulation assay requiring minimal plasma volume (1μl).
• The assay accurately measures blood coagulation activity in human and zebrafish models.
• This method is suitable for analyzing small blood samples, such as those obtained from finger or heel pricks.