Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet...

Area of Science:

• Hematology
• Immunology
• Biotechnology

Background:

• Flow cytometry offers advanced platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine, and predicting thrombosis.
• A significant challenge is establishing flow cytometry as a routine diagnostic test.

Purpose of the Study:

• To standardize the preparation, optimization, and validation of a flow cytometry-based platelet function test kit.
• To determine reference values for this test in a healthy population.

Main Methods:

• Developed and stored test kits with agonists and antibodies against platelet activation markers (P-selectin, activated αIIbβ3, GPIb).
• Quantified platelet activation using flow cytometry on diluted whole blood.
• Included anti-mouse Ig κ particles for test stability validation and result standardization.
• Determined reference intervals in 129 healthy individuals.

Main Results:

• Stable and reproducible platelet function test results were achieved when blood was stored at room temperature for up to 4 hours and tested at 37°C (intra-assay %CV <5%, inter-assay %CV <10%).
• Test kits demonstrated stable fluorescence after antibody storage for up to 12 months.
• Inter-individual variation in measured platelet activation markers ranged from 23% to 37%.

Conclusions:

• This study advances flow cytometry platelet function testing from a research tool to a diagnostic test.
• Reproducible measurements are achievable with prepared and stored kits when performed at 37°C.
• Established reference ranges can be validated in clinical settings, with ongoing research exploring detection of reduced platelet reactivity in bleeding disorders.