Platelet activation and blood extracellular vesicles: The influence of venepuncture and short blood storage.

Extracellular vesicles (EVs) as membrane-bound particles released by various cells are potential tools for diagnosis and treatment. Blood cells, particularly platelets, are the source of circulating EVs. MATERIAL: EVs were enriched with gradient ultracentrifugation and measured by nanoparticle tracking assay. A flow cytometric multiplex assay was used for cellular source determination. Activation of platelets was measured as a percentage of CD62p+/CD61+ platelets and correlated with the concentration and size of released EVs. RESULTS: In general there was no statistically significant correlation between EVs` concentration and degree of platelet activation. EVs from different cellular sources were detected. Comparing different needle thicknesses, there was a decrease in the EVs concentration for the 16G needle versus the 21G needle, but no difference was observed for EVs` size and phenotype or platelets activation. During blood storage, platelet activation increased, but there was no effect on the EVs` concentration, size, or phenotype. CONCLUSIONS: Preanalytical factors like needle thickness and storage time can affect the MVs' properties. Activation of platelets during blood collection or blood storage occurs; however, it is difficult to determine its effect on the physiological properties of EVs since the mechanisms of EVs` biogenesis and especially clearness are not precisely known.

Markers of Mitochondrial Injury and Neurological Outcomes of Comatose Patients after Cardiac Arrest.

Background and Objectives: Most patients who are successfully resuscitated from cardiac arrest remain comatose, and only half regain consciousness 72 h after the arrest. Neuroprognostication methods can be complex and even inconclusive. As mitochondrial components have been identified as markers of post-cardiac-arrest injury and associated with survival, we aimed to investigate cytochrome c and mtDNA in comatose patients after cardiac arrest to compare neurological outcomes and to evaluate the markers' neuroprognostic value. Materials and Methods: This prospective observational study included 86 comatose post-cardiac-arrest patients and 10 healthy controls. Cytochrome c and mtDNA were determined at admission. Neuron-specific enolase (NSE) was measured after 72 h. Additional neuroprognostication methods were performed when patients remained unconscious. Cerebral performance category (CPC) was determined. Results: Cytochrome c was elevated in patients compared to healthy controls (2.029 [0.85-4.97] ng/mL vs. 0 [0.0-0.16], p < 0.001) but not mtDNA (95,228 [52,566-194,060] vs. 41,466 [28,199-104,708] copies/µL, p = 0.074). Compared to patients with CPC 1-2, patients with CPC 3-5 had higher cytochrome c (1.735 [0.717-3.40] vs. 4.109 [1.149-8.457] ng/mL, p = 0.011), with no differences in mtDNA (87,855 [47,598-172,464] vs. 126,452 [69,447-260,334] copies/µL, p = 0.208). Patients with CPC 1-2 and CPC 3-5 differed in all neuroprognostication methods. In patients with good vs. poor neurological outcome, ROC AUC was 0.664 (p = 0.011) for cytochrome c, 0.582 (p = 0.208) for mtDNA, and 0.860 (p < 0.001) for NSE. The correlation between NSE and cytochrome c was moderate, with a coefficient of 0.576 (p < 0.001). Conclusions: Cytochrome c was higher in comatose patients after cardiac arrest compared to healthy controls and higher in post-cardiac-arrest patients with poor neurological outcomes. Although cytochrome c correlated with NSE, its neuroprognostic value was poor. We found no differences in mtDNA.

Heparin-induced thrombocytopenia: effect of heat inactivation of patient's serum on functional flow cytometric assay results.

BACKGROUND: Heat inactivation of a patient's sample is not systematically performed in the diagnostics of heparin-induced thrombocytopenia (HIT). Some authors recommend that the patient's sample is heat-inactivated to avoid the effect of thrombin on platelet activation in a functional assay. Others do not find this additional step essential or even advise against it. MATERIAL AND METHODS: An enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based functional assay with CD62P as a marker of platelet activation were performed. Forty-seven patients with suspected HIT and three healthy controls were included in the study. Each serum sample was divided into two aliquots: one was heat-inactivated and the other was not. Both aliquots were tested in parallel using the same donor platelets from four randomly selected individuals. We designed an index of platelet activation for both protocols to assess platelet activation in the assay and to compare the results. RESULTS: We observed a higher percentage of platelet activation in heat-inactivated compared to non-heat-inactivated sera. This phenomenon was seen in low and high heparin steps, although it did not occur for all samples. There were discrepant results in seven samples, which tested negative in the non-heat-inactivated protocol and positive in the heat-inactivated protocol. There was no case in which the result of a non-heat-inactivated aliquot was positive and the corresponding heat-inactivated aliquot was negative. DISCUSSION: Due to the higher percentages of donor platelet activation, all seven non-compliant cases met our current criteria for a positive result. However, those results were probably false-positive based on ELISA optical density value and 4T score. Therefore, in the current settings, heat inactivation of serum is not suitable for our flow cytometric functional assay since it can cause an elevated risk of creating false-positive results.

Haemato-biochemical profile and acid-base status of Croatian spotted goats of different ages.

The aim of the present research was to determine the haemato-biochemical profile and blood acid-base status of Croatian spotted goats in a traditional Mediterranean production system. The 60 non-gravid female Croatian spotted goats of different ages were included in the research. They were divided into four groups of 15 goats according to age: group I - ≤ 1  year old; group II - 2-3 years; group III - 3-6 years; and group IV - 7-10 years. Haematological parameters were determined in whole blood, biochemical parameters in serum and acid-base status in plasma by automatic analyser. Total leukocyte number (WBC), haemoglobin (HGB) and mean corpuscular volume (MCV) in the blood were the highest, while mean haemoglobin concentration in erythrocytes (MCHCs) was the lowest in yearlings compared to other groups. Concentrations of urea, Mg, Cl, non-esterified fatty acids (NEFAs) and lactate were the highest in yearlings. Concentrations of Ca, Na, total cholesterol, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and beta hydroxybutyrate (BHB) as well as the activity of alanine aminotransferase (ALT) were higher in older goats compared to yearlings, while the opposite was determined for the activities of creatine kinase (CK) and alkaline phosphatase (ALP). Values of pH, the strong ion difference (SID), anion gap (AG) and z  values as well as the content of HCO 3 and total pressure of carbon dioxide ( ctCO 2 ) were higher in older goats compared to yearlings. The results obtained may help in monitoring the health and nutritional status and improve the management of Croatian spotted goats. Based on the results of the present study, the effect of age needs to be included in the model when preparing the reference values for the haemato-biochemical profile and acid-base status of goats.