Aim: A pilot project in our laboratory showed that flow cytometry could be utilized to perform platelet aggregation studies in thrombocytopenic samples. The aim of this study is to establish reference ranges and test thrombocytopenic patients with bleeding phenotype.

Method: Normal donor samples (n=10) and four thrombocytopenic patients had blood collected into 3.2% sodium citrate tubes. Whole blood aggregometry was performed with agonists and control (saline). After addition of the agonists, aliquots were taken at various time points and fixed with a buffered glutaraldehyde solution. Platelet marker CD42a-PE was added and analysed by flow cytometry for loss of platelet events, which correlated with aggregation. Patients were tested concurrently with a normal donor.

Results: After addition of agonists Collagen 1.0 ug/mL, ADP 5.0 uM, TRAP 10 uM and Arachidonic acid 1.6 uM, testing of normal donors at 10 seconds, 30 seconds, 1 minute and 5 minutes established the reference ranges. Complete aggregation (90 – 100%) was achieved within 30 seconds after addition of any of the tested agonists. In some donors this was achieved at 10 seconds.

Of the thrombocytopenic patients (platelet count range 49 - 119 x 10⁹/L), two patients showed normal aggregation by flow cytometry. Another two patients demonstrated reduced aggregation with Collagen (1.0 and 0.5 ug/mL), and Collagen (1.0 ug/mL) and ADP (5.0 uM) respectively. This reflected results observed where traditional light transmission aggregation could be performed.

Conclusion: Platelet function testing by flow cytometry is suitable for thrombocytopenic patients who otherwise could not be tested with traditional aggregometry methods. Ongoing validation is needed with a larger patient cohort.