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.

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.

• References

• 1 Hoffmann JJ. Reticulated platelets: analytical aspects and clinical utility. Clin Chem Lab Med 2014; 52 (08) 1107-1117
• 2 D'Souza C, Briggs C, Machin SJ. Platelets: the few, the young, and the active. Clin Lab Med 2015; 35 (01) 123-131
• 3 Perl L, Lerman-Shivek H, Rechavia E. , et al. Response to prasugrel and levels of circulating reticulated platelets in patients with ST-segment elevation myocardial infarction. J Am Coll Cardiol 2014; 63 (06) 513-517
• 4 Angénieux C, Maître B, Eckly A, Lanza F, Gachet C, de la Salle H. Time-dependent decay of mRNA and ribosomal RNA during platelet aging and its correlation with translation activity. PLoS One 2016; 11 (01) e0148064
• 5 Stratz C, Bömicke T, Younas I. , et al. Comparison of immature platelet count to established predictors of platelet reactivity during thienopyridine therapy. J Am Coll Cardiol 2016; 68 (03) 286-293
• 6 Armstrong PC, Hoefer T, Knowles RB. , et al. Newly formed reticulated platelets undermine pharmacokinetically short-lived antiplatelet therapies. Arterioscler Thromb Vasc Biol 2017; 37 (05) 949-956
• 7 Lakkis N, Dokainish H, Abuzahra M. , et al. Reticulated platelets in acute coronary syndrome: a marker of platelet activity. J Am Coll Cardiol 2004; 44 (10) 2091-2093
• 8 Grove EL, Hvas AM, Kristensen SD. Immature platelets in patients with acute coronary syndromes. Thromb Haemost 2009; 101 (01) 151-156
• 9 Cesari F, Marcucci R, Gori AM. , et al. Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 Study. Thromb Haemost 2013; 109 (05) 846-853
• 10 Ibrahim H, Schutt RC, Hannawi B, DeLao T, Barker CM, Kleiman NS. Association of immature platelets with adverse cardiovascular outcomes. J Am Coll Cardiol 2014; 64 (20) 2122-2129
• 11 Ingram M, Coopersmith A. Reticulated platelets following acute blood loss. Br J Haematol 1969; 17 (03) 225-229
• 12 Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990; 75 (01) 116-121
• 13 Robinson MS, Mackie IJ, Khair K. , et al. Flow cytometric analysis of reticulated platelets: evidence for a large proportion of non-specific labelling of dense granules by fluorescent dyes. Br J Haematol 1998; 100 (02) 351-357
• 14 Ault KA, Rinder HM, Mitchell J, Carmody MB, Vary CP, Hillman RS. The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis. Am J Clin Pathol 1992; 98 (06) 637-646
• 15 Dale GL, Friese P, Hynes LA, Burstein SA. Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old. Blood 1995; 85 (07) 1822-1825
• 16 Guthikonda S, Alviar CL, Vaduganathan M. , et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. J Am Coll Cardiol 2008; 52 (09) 743-749
• 17 Bernlochner I, Goedel A, Plischke C. , et al. Impact of immature platelets on platelet response to ticagrelor and prasugrel in patients with acute coronary syndrome. Eur Heart J 2015; 36 (45) 3202-3210