Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels

Area of Science:

• Biochemistry
• Hematology
• Clinical Diagnostics

Background:

• Traditional one-point clot time assays have limitations in detecting thrombotic disorders.
• Microplate-based plasma clotting (MTP) assays offer a more comprehensive analysis of fibrin formation.
• Current MTP parameter derivation relies on subjective graphical analysis.

Purpose of the Study:

• To investigate the sensitivity and reproducibility of MTP clotting assays.
• To evaluate MTP assays for measuring tissue factor (TF) levels in biological samples.
• To develop a quantitative method for analyzing MTP clotting progress curves.

Main Methods:

• MTP plasma clotting assays were performed using absorbance measurements.
• Progress curves were analyzed by fitting a logistic equation derived from an autocatalytic model.
• Parameters including maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm), and maximum rate (vm) were calculated.
• TF levels were assessed in cell culture medium and patient urine.

Main Results:

• The logistic equation provided a robust analysis of MTP clotting progress curves.
• Derived parameters (Fm, k, tm, vm) exhibited power-law relationships with TF concentration.
• Increasing TF concentration showed limiting effects on clotting parameters.
• Log/log plots of tm and k against TF concentration yielded standard curves for TF quantification.

Conclusions:

• Fitting a logistic equation to MTP clotting assays enables quantitative and reproducible analysis.
• This method is suitable for measuring TF levels in various biological matrices.
• The developed approach offers an improvement over subjective graphical analysis for clotting assays.