PB0393 - Use of spectral flow cytometry to immunophenotype platelets

Background: Platelet function is mediated through expression of specialised surface markers and dedicated signalling pathways. Changes in expression levels of these surface markers following platelet activation have been shown to underlie haemostatic response and thrombus formation.

Aims: To develop an assay to immunophenotype platelets using spectral flow cytometry.

Methods: Blood was taken via venepuncture from healthy volunteers (n=23) and centrifuged to produce platelet-rich plasma. Spectral flow cytometry (Cytek Aurora) was used to simultaneously determine the expression levels of 14 markers in resting platelets and platelets activated with ADP (adenosine diphosphate), TRAP-6 amide (thrombin receptor-activating peptide ligand), U46619 (thromboxane A2 receptor ligand), PAR-4 (thrombin receptor PAR-4 ligand), and CRP-XL (collagen-related peptide). Median fluorescence intensity (MFI) was recorded, analysed with NovoExpress software and GraphPad Prism software using an ANOVA or mixed-effects analysis, and presented as mean±s.e.m.

Results: Following platelet activation, changes in surface marker expression varied according to agonist stimulation. In general terms, expression of 9 markers (CD9, CD36, CD63, CD107a, CD62P, CD29, CD41, CD61, PAC-1) increased following activation, whilst the expression of 4 markers (CD31, CD42a, CD42b, GPVI) decreased, and that of CLEC-2 remained unchanged. The largest increase in expression of CD62P was in response to TRAP-6 (9818±2140 to 53186±2165, p< 0.0001), and that of PAC-1 to PAR-4 (633±90 to 4866±494, p< 0.0001). The largest decrease in CD42b was in response to PAR-4 (14014±1171 to 6447±530, p< 0.0001).

Conclusion(s): Using spectral flow cytometry, we have developed an assay that can concurrently characterise expression of 14 platelet surface markers in a low-volume sample. It reliably detects changes in response to activation that are consistent with existing literature. Such an assay will be a powerful tool not only in investigating basal and activation-dependent platelet heterogeneity, but also in detecting abnormalities in surface marker expression associated with conditions such as inherited bleeding disorders or immune thrombocytopaenia.