Development of a whole platelet ELISA to detect circulating activated platelets

Area of Science:

• Biochemistry
• Immunology
• Hematology

Background:

• P-selectin is a marker of activated endothelial cells and platelets.
• Flow cytometry is a common method for detecting activated platelets using P-selectin antibodies.
• A need exists for alternative methods to quantify platelet activation.

Purpose of the Study:

• To develop and validate a whole platelet enzyme-linked immunosorbent assay (ELISA) for measuring P-selectin on activated platelets.
• To assess the stability and accuracy of the developed ELISA method.
• To evaluate the utility of the ELISA in detecting platelet activation in patients with unstable angina.

Main Methods:

• Whole platelet ELISA was developed using platelet-rich plasma samples.
• Samples were fixed and analyzed within 3 hours or after storage at -70°C for up to 10 months.
• ELISA accuracy and sensitivity were validated against fluorescence-activated flow cytometry.

Main Results:

• The developed whole platelet ELISA demonstrated high interassay correlation (r = 0.98 +/- 0.03).
• The ELISA is highly sensitive (< or = 0.5%) and comparable to flow cytometry for P-selectin detection.
• Patients with unstable angina showed significantly higher levels of circulating activated platelets compared to controls.

Conclusions:

• A whole platelet ELISA is a reliable and sensitive method for quantifying P-selectin on activated platelets.
• The ELISA method is stable after prolonged storage and comparable to flow cytometry.
• This assay can effectively detect elevated platelet activation in clinical conditions like unstable angina.