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Thromb Haemost 2019; 119(05): 779-785
DOI: 10.1055/s-0039-1681101
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

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

Laura Hille
1   Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
,
Marco Cederqvist
1   Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
,
Julia Hromek
1   Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
,
Christian Stratz   ∗
1   Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
,
Dietmar Trenk
1   Department of Cardiology and Angiology II, Clinical Pharmacology, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
,
Thomas G. Nührenberg
2   Department of Cardiology and Angiology II, University Heart Centre Freiburg • Bad Krozingen, Bad Krozingen, Germany
› Author Affiliations Funding This study was supported by the PharmCompNet Baden-Württemberg: Kompetenznetzwerk Pharmakologie Baden-Württemberg - Wirkstoffnetzwerke als Grundlagen der individualisierten Arzneistofftherapie and the University Heart Center Freiburg - Bad Krozingen.
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Publication History

01 November 2018

19 January 2019

Publication Date:
01 April 2019 (online)

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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

Note

Part of the data was presented as a poster at the Congress of the European Society of Cardiology 2017 in Barcelona (Spain).


∗ Christian Stratz current address is Novartis AG, Fabrikstrasse, Basel, Switzerland.


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