No 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.

URL: https//www.clinicaltrials.gov. Unique Identifier: NCT: 01959451.

Background This study evaluated the impact of a dedicated outpatient service on vaccination uptake after splenectomy and on the incidence of postsplenectomy sepsis. Methods From 2009 to 2016 at the University Hospital Freiburg (Germany), asplenic patients were referred to a dedicated outpatient service, provided with comprehensive preventive care including vaccinations, and enrolled in a prospective cohort study. The impact of the service on vaccination uptake and the occurrence of severe sepsis/septic shock was compared between patients who had splenectomy (or were asplenic) within 3 months of study entry (“early study entry”) and those who had splenectomy (or were asplenic) &gt;3 months before study entry (“delayed study entry”). Results A total of 459 asplenic patients were enrolled, and 426 patients were followed prospectively over a median period of 2.9 years. Pneumococcal vaccine uptake within 3 months of splenectomy or first diagnosis of asplenia was 27% vs 71% among delayed study entry and early study entry patients, respectively (P &lt; .001). Forty-four episodes of severe sepsis or septic shock occurred in study patients: 22 after study entry and 22 before study entry. Streptococcus pneumoniae was more frequent among sepsis episodes that occurred before study entry (8/22) than after study entry (1/22 episodes). For episodes occurring after study entry, only a higher Charlson comorbidity index score was significantly associated with severe sepsis/septic shock postsplenectomy. Conclusions With dedicated outpatient care, high uptake of pneumococcal vaccination postsplenectomy was achieved. Sepsis episodes were largely of nonpneumococcal etiology in patients who had received dedicated postsplenectomy care.