Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets

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.

Ultrastructural, transcriptional, and functional differences between human reticulated and non‐reticulated platelets

“…Blood samples were processed within 10 minutes from sampling. Washed platelets were obtained and diluted to 5 × 10 4 platelets/μL as described before 29 …”

“…Platelets were stained and sorted into a RP-and non-RP-fraction as described before. 29 In brief, unfixed washed platelets as well as platelet rich plasma were stained with the nucleic acid binding fluorescent dye SYTO 13 (12.5 nmol/L final concentration; Thermo Fisher). After incubation for 90 minutes at room temperature in the dark, fluorescence was quantified in the FL-1 fluorescein isothiocyanate channel…”

Evaluation of Chemo‐ and Photo‐toxicity of a Live Fluorescent Dye for Cell Analysis

“…Since primary cells retain their endothelial characteristics for a limited number of passages in vitro, the natural choice for live cell studies is the use of chemical dyes that, once added to the cell culture, render the cell membrane, organelles or nucleus intensely fluorescent. A widely used compound for live cell staining is SYTO 13 (9,15,16). However, its viability in combination with endothelial cells has not yet been reported.…”

Reticulated Platelets—Which Functions Have Been Established by In Vivo and In Vitro Data?

“…The nucleic acid dye SYTO 13 is an alternative for staining RP which proves to have several advantages over TO [ 35 ]. SYTO13 shows high stability over time, facilitating extended experimental analysis (for instance making it more suitable for sorting) and higher quantum yield.…”