P6042Establishing Syto 13 as an alternative dye for the cytometric evaluation of RNA-rich immature platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

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

Thrombosis and Haemostasis ◽

10.1055/s-0039-1681101 ◽

2019 ◽

Vol 119 (05) ◽

pp. 779-785 ◽

Cited By ~ 6

Author(s):

Laura Hille ◽

Marco Cederqvist ◽

Julia Hromek ◽

Christian Stratz ◽

Dietmar Trenk ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Reactivity ◽

Dependent Manner ◽

Reticulated Platelets ◽

Automated Assay ◽

Rna Quantification ◽

Time Period ◽

Immature Platelet Fraction ◽

Syto 13

AbstractReticulated 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.

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Effect of nutrient and stress factors on polysaccharides synthesis in Proteus mirabilis biofilm.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1935 ◽

2014 ◽

Vol 61 (1) ◽

Cited By ~ 11

Author(s):

Magdalena Moryl ◽

Aleksandra Kaleta ◽

Kacper Strzelecki ◽

Sylwia Różalska ◽

Antoni Różalski

Keyword(s):

Proteus Mirabilis ◽

Stress Factors ◽

Nutrient Depletion ◽

Optimal Conditions ◽

Fluorescent Staining ◽

Tryptone Soya Broth ◽

Nutritional Conditions ◽

Additional Amount ◽

Syto 13 ◽

Modified Media

The extracellular matrix in biofilm consists of water, proteins, polysaccharides, nucleic acids and phospholipids. Synthesis of these components is influenced by many factors, e.g. environment conditions or carbon source. The aim of the study was to analyse polysaccharides levels in Proteus mirabilis biofilms after exposure to stress and nutritional conditions. Biofilms of 22 P. mirabilis strains were cultivated for 24, 48, 72 hours, 1 and 2 weeks in tryptone soya broth or in modified media containing an additional amount of nutrients (glucose, albumin) or stress factors (cefotaxime, pH 4, nutrient depletion). Proteins and total polysaccharides levels were studied by Lowry and the phenol-sulphuric acid methods, respectively. Glycoproteins levels were calculated by ELLA with the use of selected lectins (WGA and HPA). For CLSM analysis dual fluorescent staining was applied with SYTO 13 and WGA-TRITC. In optimal conditions the levels of polysaccharides were from 0 to 442 μg/mg of protein and differed depending on the strains and cultivation time. The agents used in this study had a significant impact on the polysaccharides synthesis in the P. mirabilis biofilm. Among all studied components (depending on tested methods), glucose and cefotaxime stimulated the greatest production of polysaccharides by P. mirabilis strains (more than a twofold increase). For most tested strains the highest amounts of sugars were detected after one week of incubation. CLSM analysis confirmed the overproduction of N-acetyloglucosamine in biofilms after cultivation in nutrient and stress conditions, with the level 111-1134%, which varied depending on the P. mirabilis strain and the test factor.

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Immature Platelets and Risk of Cardiovascular Events among Patients with Ischemic Heart Disease: A Systematic Review

Thrombosis and Haemostasis ◽

10.1055/s-0040-1721386 ◽

2020 ◽

Author(s):

Julie Faber ◽

Anne-Mette Hvas ◽

Steen Dalby Kristensen ◽

Erik Lerkevang Grove ◽

Kasper Adelborg

Keyword(s):

Systematic Review ◽

Heart Disease ◽

Ischemic Heart Disease ◽

Cardiovascular Events ◽

Ischemic Heart ◽

Cochrane Library ◽

Coronary Syndrome ◽

Immature Platelet Fraction ◽

Immature Platelets ◽

Stable Cad

Abstract Background Immature platelets are larger and may be more thrombogenic than mature platelets. This systematic review included studies on the association between mean platelet volume (MPV), immature platelet count (IPC), and immature platelet fraction (IPF) and the risk of major cardiovascular events (MACEs) in patients with acute coronary syndrome (ACS) or stable coronary artery disease (CAD). Methods The literature search included studies in PubMed, Embase, Web of Science, and Cochrane Library. The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Effect estimates that included multivariate adjusted odds ratios, relative risks, or hazard ratios were extracted. Results Forty-two studies were identified. High MPV was positively associated with MACE in 20 of 26 studies of patients with ACS, four of five studies in patients with stable CAD, and in all six studies comprising a combined population with ACS and stable CAD. Using continuous models of MPV in patients with ACS, effect estimates varied from 0.90 (95% confidence interval [CI]: 0.95–1.03) to 1.66 (95% CI: 1.32–2.09). The strength of these associations was broadly similar among patients with stable CAD and in combined populations. Five studies investigated IPC or IPF as exposures and all reported positive associations with MACE among patients with ACS, stable CAD, or in combined populations. Conclusion This review demonstrated clear evidence for positive associations between measures of immature platelets and subsequent risk of MACE in acute and stable ischemic heart disease patients.

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Immature platelets: a review of the available evidence

Thrombosis Research ◽

10.1016/j.thromres.2020.06.048 ◽

2020 ◽

Vol 195 ◽

pp. 43-50

Author(s):

Naoufal Benlachgar ◽

Kamal Doghmi ◽

Azlarab Masrar ◽

El Mehdi Mahtat ◽

Hicham Harmouche ◽

...

Keyword(s):

Immature Platelets

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Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry.

Applied and Environmental Microbiology ◽

10.1128/aem.63.11.4608-4611.1997 ◽

1997 ◽

Vol 63 (11) ◽

pp. 4608-4611 ◽

Cited By ~ 49

Author(s):

T Guindulain ◽

J Comas ◽

J Vives-Rego

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Nucleic Acid ◽

Acid Dyes ◽

Syto 13

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Mature and immature platelets during the first week after birth and incidence of patent ductus arteriosus

Cardiology in the Young ◽

10.1017/s1047951120000943 ◽

2020 ◽

Vol 30 (6) ◽

pp. 769-773 ◽

Cited By ~ 1

Author(s):

Hannes Sallmon ◽

Boris Metze ◽

Petra Koehne ◽

Bernd Opgen-Rhein ◽

Katja Weiss ◽

...

Keyword(s):

Platelet Count ◽

Birth Weight ◽

Patent Ductus Arteriosus ◽

Very Low Birth Weight ◽

Ductus Arteriosus ◽

Postnatal Life ◽

Platelet Counts ◽

Immature Platelet Fraction ◽

Immature Platelets ◽

Patent Ductus

AbstractBackground:Thrombocytopenia is a risk factor for patent ductus arteriosus. Immature and mature platelets exhibit distinct haemostatic properties; however, whether platelet maturity plays a role in postnatal, ductus arteriosus closure is unknown.Methods:In this observational study, counts of immature and mature platelets (=total platelet count − immature platelet count) were assessed on days 1, 3, and 7 of life in very low birth weight infants (<1500 g birth weight). We performed echocardiographic screening for haemodynamically significant patent ductus arteriosus on day 7.Results:Counts of mature platelets did not differ on day 1 in infants with (n = 24) and without (n = 45) haemodynamically significant patent ductus arteriosus, while infants with significant patent ductus arteriosus exhibited lower counts of mature platelet on postnatal days 3 and 7. Relative counts of immature platelets (fraction, in %) were higher in infants with patent ductus arteriosus on day 7 but not on days 1 and 3. Receiver operating characteristic curve analysis unraveled associations between both lower mature platelet counts and higher immature platelet fraction (percentage) values on days 3 and 7, with haemodynamically significant ductus arteriosus. Logistic regression analysis revealed that mature platelet counts, but not immature platelet fraction values, were independent predictors of haemodynamically significant patent ductus arteriosus.Conclusion:During the first week of postnatal life, lower counts of mature platelets and higher immature platelet fraction values are associated with haemodynamically significant patent ductus arteriosus. Lower counts of mature platelet were found to be independent predictors of haemodynamically significant patent ductus arteriosus.

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