Abstract
Reticulated platelets reflect the rate of platelet turnover and represent the youngest
circulating platelets in peripheral blood. Reticulated platelets contain residual
ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner
and can be transcribed into proteins even in the absence of a nucleus. An increased
proportion of reticulated platelets is associated with higher platelet reactivity,
cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX
haematology analyser) is available for analysis. This method, however, is not suitable
for extended laboratory investigations like subsequent cell sorting. Flow cytometry
analysis after staining with thiazole orange (TO) is frequently used in such settings
despite several limitations. Here, we describe a new assay for determination of reticulated
platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare
it with SYSMEX and TO staining as current standards. A significant correlation between
immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results
obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation
between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled
sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification
of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations
was stable under various experimental settings. In summary, determination of reticulated
platelets with the new SYTO 13 assay offers distinct technical advantages enabling
further laboratory processing.
Keywords
reticulated platelets - immature platelet fraction - staining
