Platelets isolation and ectonucleotidase assay: Revealing functional aspects of the communication between the vasculature and the immune system.

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5'-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.

Zebrafish Thrombocyte Transcriptome Analysis and Functional Genomics.

Our laboratory is interested in investigating the maturation process of zebrafish thrombocytes, which are functional equivalents to human platelets. We have adopted the zebrafish model to gain insights into mammalian platelet production, or thrombopoiesis. Notably, zebrafish exhibit two distinct populations of thrombocytes in their circulating blood: young and mature thrombocytes. This observation is intriguing because maturation appears to occur in circulation, yet the precise mechanisms governing this maturation remain elusive. Our goal is to understand the mechanisms underlying thrombocyte maturation by conducting single-cell RNA sequencing (scRNA-Seq) on young and mature thrombocytes, analyzing these transcriptomes to identify genes specific to each thrombocyte population, and elucidating the role of these genes in the maturation process, by quantifying thrombocyte numbers after the piggyback knockdown of each of these genes. In this chapter, we present a comprehensive, step-by-step protocol detailing the multifaceted methodology involved in understanding thrombocyte maturation, which encompasses the collection of zebrafish blood, the separation of young and mature thrombocytes using flow cytometry, scRNA-Seq analysis of these distinct thrombocyte populations, identification of genes specific to young and mature thrombocytes, and subsequent validation through gene knockdown techniques.

Pediatric tuberculosis in a high burden setting: Socio-spatial distribution, blood elements features, and outcomes.

Background: The childhood tuberculosis (TB) epidemic has been long neglected. Data on pediatric tuberculosis is needed to develop effective strategies against TB. Methods: We retrospectively reviewed 200 medical records from children aged 0-15 years who suffered from tuberculosis between 2011 and 2021 in Libreville, Gabon. We collected and analyzed socio-demographic data and clinical data. Results: 141 children files were selected (43 % girls and 57 % boys). The mean age of the patients was 9.2 years (CI: 8.5-10). Sixty per cent (60 %) of cases were from precarious housing areas, 35.34 % from mixed housing areas, and 4.51 % from residential. The cure rate was 75.24 %, 9.52 % relapsed, and 15.24 % died. Deaths were significantly higher in older children (Dunn's post-test p < 0.01). Children who recovered had higher haemoglobin and platelet counts than those who died (Dunn's test: haemoglobin p < 0.0001; thrombocytes p < 0.05). The haemoglobin threshold value of 5.5 g/dL identified children death with up to 80 % sensitivity and 86 % specificity. Thrombocytes count identified children's death with a sensitivity of 80 % and a specificity of 51 %. Conclusion: Precariousness is associated with childhood tuberculosis. The directly observed therapy (DOTS) in older children should be reinforced to limit tuberculosis-associated deaths. Haemoglobin concentration and platelet are vital prognosis markers in pediatric tuberculosis.

Delayed cold-stored vs. room temperature stored platelet transfusions in bleeding adult cardiac surgery patients-a randomized multicentre pilot study (PLTS-1).

BACKGROUND: Platelets stored at 1-6 °C are hypothesized to be more hemostatically active than standard room temperature platelets (RTP) stored at 20-24 °C. Recent studies suggest converting RTP to cold-stored platelets (Delayed Cold-Stored Platelets, DCSP) may be an important way of extending platelet lifespan and increasing platelet supply while also activating and priming platelets for the treatment of acute bleeding. However, there is little clinical trial data supporting the efficacy and safety of DCSP compared to standard RTP. METHODS: This protocol details the design of a multicentre, two-arm, parallel-group, randomized, active-control, blinded, internal pilot trial to be conducted at two cardiac surgery centers in Canada. The study will randomize 50 adult (≥ 18 years old) patients undergoing at least moderately complex cardiac surgery with cardiopulmonary bypass and requiring platelet transfusion to receive either RTP as per standard of care (control group) or DCSP (intervention group). Patients randomized to the intervention group will receive ABO-identical, buffy-coat, pathogen-reduced, platelets in platelet additive solution maintained at 22 °C for up to 4 days then placed at 4 °C for a minimum of 24 h, with expiration at 14 days after collection. The duration of the intervention is from the termination of cardiopulmonary bypass to 24 h after, with a maximum of two doses of DCSP. Thereafter, all patients will receive RTP. The aim of this pilot is to assess the feasibility of a future RCT comparing the hemostatic effectiveness of DCSP to RTP (defined as the total number of allogeneic blood products transfused within 24 h after CPB) as well as safety. Specifically, the feasibility objectives of this pilot study are to determine (1) recruitment of ≥ 15% eligible patients per center per month); (2) appropriate platelet product available for ≥ 90% of patients randomized to the cold-stored platelet group; (3) Adherence to randomization assignment (> 90% of patients administered assigned product). DISCUSSION: DCSP represents a promising logistical solution to address platelet supply shortages and a potentially more efficacious option for the management of active bleeding. No prospective clinical studies on this topic have been conducted. This proposed internal pilot study will assess the feasibility of a larger definitive study. TRIAL REGISTRATION: NCT06147531 (clinicaltrials.gov).

The Prognostic Value of Perioperative Platelet and Leukocyte Values in Patients Undergoing Radical Cystectomy: A Prospective Long-Term Cohort Study.

INTRODUCTION: Studies assessing the impact of preoperative and first-day postoperative values of leukocytes, thrombocytes, and platelet/leukocyte ratio (PLR) after radical cystectomy (RC) are sparse. We aimed to assess the impact of these factors on long-term survival after RC. METHODS: An analysis of patients undergoing open RC from 2004 to 2023 at our center was performed. Leukocytosis was defined as ≥8,000 leukocytes/µL and thrombocytosis as ≥400,000 thrombocytes/µL. Similarly, the cutoff for PLR was set at 28. A multivariable Cox regression analysis was performed to assess the role of leukocytosis, thrombocytosis, and PLR on long-term survival after RC. For all analyses, hazard ratios (HRs) with the corresponding 95% confidence intervals (CIs) were estimated. RESULTS: A total of 1,817 patients with a median age of 70 years (interquartile range [IQR]: 62-77) were included. Overall, 804 (44%), 175 (10%), and 1,296 (71%) patients presented with leukocytosis, thrombocytosis, and PLR ≥28 preoperatively. Accordingly, 1,414 (78%), 37 (2%), and 249 (14%) patients presented with leukocytosis, thrombocytosis, and PLR ≥28 on the first day after RC. At a median follow-up of 26 months (IQR: 8-68) after RC, 896 (49%) patients died. In the multivariate Cox regression analysis after adjusting for major perioperative risk factors, only preoperative leukocytosis (HR: 1.3, 95% CI: 1.1-1.6, p = 0.01), as well as both preoperative and first-day thrombocytosis (HR: 2.1, 95% CI: 1.5-2.9, and HR: 2.8, 95% CI: 1.6-5.1, p < 0.001, accordingly) were associated with worse overall survival. CONCLUSION: PLR should not be used as a prognostic marker for survival after RC. On the contrary, preoperative leukocytosis, as well as preoperative and first-day thrombocytosis should raise awareness among clinicians performing RC since they were independently associated with worse survival after RC.

Platelet count and platelet-to-lymphocyte ratio at the onset of a severe COPD exacerbation are unrelated to the time till the next moderate or severe relapse.

PURPOSE: Acute exacerbations (AE) are severe complications of chronic obstructive pulmonary disease (COPD); however, the need for biomarkers which predict them is still unmet. High platelet count (PLC) and platelet-to-lymphocyte ratio (PLR) are associated with higher mortality in patients with COPD. We investigated if PLC and PLR at the onset of a severe AE could predict the time of the next relapse. METHODS: In a prospective observational cohort study, data of 152 patients hospitalized with AECOPD were collected, and patients were divided into PLC-low (<239 â€‹× â€‹109/L, n â€‹= â€‹51), PLC-medium (239-297 â€‹× â€‹109/L, n â€‹= â€‹51) and PLC-high (>297 â€‹× â€‹109/L, n â€‹= â€‹50) or PLR-low (<147, N â€‹= â€‹51), PLR-medium (147-295, n â€‹= â€‹51) and PLR high (>295, n â€‹= â€‹50) groups based on PLC and PLR tertiles using admission laboratory results. Clinical characteristics and the time to the next severe or moderate AE within 52 weeks were compared among subgroups using log-rank test. RESULTS: PLC and PLR tertiles did not differ in clinical characteristics or the time till the next AE (p â€‹> â€‹0.05). PLC and PLR showed a direct weak correlation to neutrophil count (Pearson r â€‹= â€‹0.26, p â€‹< â€‹0.01 and r â€‹= â€‹0.20, p â€‹= â€‹0.01) and PLC also demonstrated a weak relationship to white blood cell counts (Pearson r â€‹= â€‹0.29, p â€‹< â€‹0.001). However, PLR presented an inverse relationship to monocyte and eosinophil counts (r â€‹= â€‹-0.32, p â€‹< â€‹0.001 and r â€‹= â€‹-0.17, p â€‹= â€‹0.03). CONCLUSION: PLC and PLR do not predict the time till the next relapse; however, they may reflect on neutrophilic inflammatory response during an exacerbation of COPD.

Biology and functions of fish thrombocytes: A review.

This comprehensive review examines the role of fish thrombocytes, cells considered functionally analogous to platelets in terms of coagulation, but which differ in their origin and morphology. Despite the evolutionary distance between teleosts and mammals, genomic studies reveal conserved patterns in blood coagulation, although there are exceptions such as the absence of factors belonging to the contact system. Beyond coagulation, fish thrombocytes have important immunological functions. These cells express both proinflammatory genes and genes involved in antigen presentation, suggesting a role in both innate and adaptive immune responses. Moreover, having demonstrated their phagocytic abilities, crucial in the fight against pathogenic microorganisms, underscores their multifaceted involvement in immunity. Finally, the need for further research on the functions of these cells is highlighted, in order to better understand their involvement in maintaining the health of aquaculture fish. The use of standardized and automated methods for the analysis of these activities is advocated, emphaiszing their potential to facilitate the early detection of stress or infection, thus minimizing the economic losses that these adverse situations can generate in the field of aquaculture.

Injections of Platelet-Rich Plasma: An Emerging Novel Biological Cure for Low Back Pain?

Autologous platelet-rich plasma (PRP) injections have emerged as a new biological intervention for many musculoskeletal conditions, such as low back pain (LBP), and have garnered significant attention in recent research endeavors. The recognition of PRP's use is progressively growing; nonetheless, comprehensive clinical validation is required to establish its uses and efficiency. This article offers a thorough evaluation regarding the assurance as well as the efficacy of PRP therapy in the management of low back pain. It specifically focuses on the analysis of clinical trials undertaken in this field.

The effect of normobaric hypoxia on acute exercise-induced changes in blood sphingoid base-1-phosphates metabolism in cyclists.

Extracellular sphingosine-1-phosphate (S1P) emerged as an important regulator of muscle function. We previously found that plasma S1P concentration is elevated in response to acute exercise and training. Interestingly, hypoxia, which is commonly utilized in training programs, induces a similar effect. Therefore, the aim of the current study was to determine the effect of normobaric hypoxia on exercise-induced changes in blood sphingolipid metabolism. Fifteen male competitive cyclists performed a graded cycling exercise until exhaustion (GE) and a simulated 30 km individual time trial (TT) in either normoxic or hypoxic (FiO2 = 16.5%) conditions. Blood samples were taken before the exercise, following its cessation, and after 30 min of recovery. We found that TT increased dihydrosphingosine-1-phosphate (dhS1P) concentration in plasma (both HDL- and albumin-bound) and blood cells, as well as the rate of dhS1P release from erythrocytes, regardless of oxygen availability. Plasma concentration of S1P was, however, reduced during the recovery phase, and this trend was augmented by hypoxia. On the other hand, GE in normoxia induced a selective increase in HDL-bound S1P. This effect disappeared when the exercise was performed in hypoxia, and it was associated with reduced S1P level in platelets and erythrocytes. We conclude that submaximal exercise elevates total plasma dhS1P concentration via increased availability of dihydrosphingosine resulting in enhanced dhS1P synthesis and release by blood cells. Maximal exercise, on the other hand, induces a selective increase in HDL-bound S1P, which is a consequence of mechanisms not related to blood cells. We also conclude that hypoxia reduces post-exercise plasma S1P concentration.

Feasibility and comparability of different platelet function tests in acute stroke with or without prior antiplatelet therapy.

Background: The clinical course of ischemic and hemorrhagic strokes can be influenced by the coagulation status of individual patients. The prior use of antiplatelet therapy (APT) such as acetylsalicylic acid (ASA) or P2Y12-antagonists has been inconsistently described as possibly increasing the risk of hemorrhagic transformation or expansion. Since clinical studies describing prior use of antiplatelet medication are overwhelmingly lacking specific functional tests, we aimed to implement testing in routine stroke care. Methods: We used fluorescence-activated cell sorting (FACS) with antibodies against CD61 for thrombocyte identification and CD62p or platelet activation complex-1 (PAC-1) to determine platelet activation. Aggregometry and automated platelet functioning analyzer (PFA-200) were employed to test thrombocyte reactivity. FACS and aggregometry samples were stimulated in vitro with arachidonic acid (AA) and adenosine diphosphate (ADP) to measure increase in CD62p-/PAC-1-expression or aggregation, respectively. Results: Between February and July 2023, 20 blood samples (n = 11 ischemic strokes; n = 7 hemorrhagic strokes; n = 2 controls) were acquired and analyzed within 24 h of symptom onset. N = 11 patients had taken ASA, n = 8 patients no APT and n = 1 ASA+clopidogrel. ASA intake compared to no APT was associated with lower CD62p expression after stimulation with AA on FACS analysis (median 15.8% [interquartile range {IQR} 12.6-37.2%] vs. 40.1% [IQR 20.3-56.3%]; p = 0.020), lower platelet aggregation (9.0% [IQR 7.0-12.0%] vs. 88.5% [IQR 11.8-92.0%]; p = 0.015) and longer time to plug formation with PFA-200 (248.0 s [IQR 157.0-297] vs. 121.5 s [IQR 99.8-174.3]; p = 0.027). Significant correlations were noted between AA-induced CD62p expression and aggregometry analysis (n = 18; ρ = 0.714; p < 0.001) as well as a negative correlation between CD62p increase and PFA clot formation time (n = 18; ρ = -0.613; p = 0.007). Sensitivity for ASA intake was highest for PFA (81.8% for values ≥155.5 s). The combination of ASA + clopidogrel also affected ADP-induced CD62p and PAC-1 expression. Conclusion: In the clinical setting it is feasible to use differentiated platelet analytics to determine alterations caused by antiplatelet therapy. Among the tests under investigation, PFA-200 showed the highest sensitivity for the intake of ASA in stroke patients. FACS analysis on the other hand might be able to provide a more nuanced approach to altered platelet reactivity.

Reproducibility and stability of the immature platelet fraction using Sysmex XN-10.

BACKGROUND: The Immature Platelet Fraction (IPF) is an indicator of thrombopoiesis which is a useful parameter in thrombocytopenia. It demonstrates compensatory mechanisms in production of platelets, but currently not implemented in routine clinical practice. The aim of this study was to establish the reproducibility and stability of IPF, for both percentage (%-IPF) and absolute (A-IPF) measurements.Material/methods: A total of 71 samples, of which 45 for reproducibility and 26 for stability analysis, were assayed for full blood count using the Sysmex XN-10 analyser at room temperature (RT:19-25 °C). For reproducibility analysis, IPF measurements were analysed 11 times by different appraisers using the same sample, while for stability analysis, IPF was measured over fourteen hourly-intervals up to 24 h (n = 21) and then separately extended beyond the point of stability to 72 h (n = 5). RESULTS: Reproducibility analysis of %-IPF and A-IPF (n = 45) showed very reliable results, with the range of mean CV% values between 1.25-8.90% and 1.70-9.96%, respectively. On the other hand, overall, stability analysis of %-IPF and A-IPF (n = 21) at RT over 24 h showed reliable results, with pooled mean CV% values of 1.32% and 1.43%, respectively, with no significant difference between %-IPF and A-IPF (p = 0.767 and p = 0.821). All %-IPF and A-IPF values had exceeded the set acceptance criterion of stability (CV% ≥ 10.0%) before 72 h. CONCLUSIONS: Overall, %-IPF and A-IPF reproducibility and storage at RT for 24 h predominantly demonstrates the suitability of their usage for testing on the Sysmex XN-series analysers.

Convolutional neuronal network for identifying single-cell-platelet-platelet-aggregates in human whole blood using imaging flow cytometry.

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups.

Composition of thrombi in zebrafish: similarities and distinctions with mammals.

BACKGROUND: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known. OBJECTIVES: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo. METHODS: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo. RESULTS: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbß3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen. CONCLUSION: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo.

Discovery of seven hox genes in zebrafish thrombopoiesis.

Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.

Thrombocytes and Platelet-Rich Plasma as Modulators of Reproduction and Fertility.

Thrombocytes play an essential role in hemostasis and thrombosis. Moreover, the controlled activation of thrombocytes is required in reproduction and fertility. The platelet-activating factor and the controlled activation of platelets have important roles in folliculogenesis, ovulation, placental development, implantation and embryo development. Activated platelets accumulate in the follicular vessels surrounding the follicle and, due to its released soluble molecules (factors, mediators, chemokines, cytokines, neurotransmitters), locally increase oocyte maturation and hormone secretion. Furthermore, activated platelets are involved in the pathogenesis of ovarian hyperstimulation syndrome (OHSS) and preeclampsia. Low-dose aspirin can prevent OHSS during ovulation induction, while intrauterine or intraovarian administration of platelet-rich plasma (PRP) increases the endometrium thickness and receptivity as well as oocyte maturation. Activated thrombocytes rapidly release the contents of intracellular granules and have multiple adhesion molecules and receptors on their surface. Considering the numerous homeostatic endocrine functions of thrombocytes, it is reasonable to suppose a platelet-associated regulatory system (PARS) in reproduction. Although we are far from a complete understanding of the regulatory processes, the results of PARS research and the therapeutic application of aspirin and PRP during in vitro fertilization are promising.

CD41+ extracellular vesicles produced by avian thrombocytes contain microRNAs.

Avians have thrombocytes in their blood circulation rather than mammalian platelets. However, many details of thrombocyte characteristics have not been determined. Here, chicken thrombocytes were isolated, and extracellular vesicle (EV) production was investigated. The thrombocyte-specific markers cd41 and cd61 were expressed in the yolk sac at 24 h. According to the embryonic developmental stage, the cd41-expressing tissues changed from the yolk sac to the bone marrow and spleen. Accordingly, the bone marrow and spleen were the main tissues producing thrombocytes in adult chickens. Avian thrombocytes were separated from adult spleen cells through a combination of discontinuous density gradient centrifugation, phagocytic cell removal, and fluorescence-activated cell sorting. Isolated thrombocytes produced CD41+ EVs (CD41+ EVs), and the CD41+ EVs also expressed CD9. Microarray analysis revealed that CD41+ EVs contain many microRNAs. Macrophage lines (RAW264.7) phagocytosed CD41+ EVs, and their phagocytosis and migration activity were suppressed. Microarray analysis also revealed that EVs altered gene expression in macrophages. These data indicated that the CD41+ EV was a carrier of microRNAs produced from thrombocytes and affected the cell characteristics of the received cells. Therefore, the CD41+ EVs of avians worked as a communication tool.

[Platelet-rich plasma (PRP) : Compositional analysis with different dietary habits and timing of blood sampling]. / "Platelet-rich plasma" (PRP) : Analyse der Zusammensetzung bei unterschiedlichem Ernährungsverhalten und Blutentnahmezeitpunkt.

The variability of PRP is a major contributor to the lack of evidence regarding the therapeutic effect of PRP in musculoskeletal diseases. In a large study, we are currently investigating factors that may influence PRP variability. Interim results showed that concentrations of IL­6, but not IGF­1 or cellular constituents, were significantly decreased in PRP samples from vegans compared with omnivores and tended to be decreased compared to samples from vegetarians. This suggests that diet may have a significant influence on therapeutically active PRP constituents. However, the constituents studied here did not appear to be significantly affected by the timing of the sampling. Identification of significant variables affecting PRP composition will be critical to provide sufficient medical evidence for the therapeutic effects of PRP in orthopedic conditions.

A retrospective evaluation of phenobarbital-induced hematologic changes in 69 cats.

BACKGROUND: Phenobarbital (PB) is used as a first-line treatment for recurrent epileptic seizures in cats. While hematologic abnormalities are well-known side effects of antiepileptic therapy with PB in humans and dogs, little is known about such alterations in cats. OBJECTIVES: The aim of this retrospective study was to investigate the prevalence and clinical relevance of cytopenia during PB treatment in cats. METHODS: In this single-center, retrospective clinical study, 69 cats-with suspected idiopathic epilepsy admitted to the Small Animal Clinic of the University of Veterinary Medicine in Vienna (VMU)-were included. A complete blood count for each patient was performed, and changes in hematocrit, leukocytes, neutrophils, and thrombocytes were documented and graded. RESULTS: Fifty-three out of 69 cats (76.8%) showed cytopenias with a reduction of at least one cell fraction during PB treatment. The most frequent change was neutropenia (60%), followed by leukopenia (49.3%), thrombocytopenia (24.1%), and anemia (20.3%). Most of the changes were mild or moderate; only one patient (1.5%) showed severe leukopenia and neutropenia, and one was a life-threatening neutropenia (1.5%) with a serum PB concentration within or even below the therapeutic range. These patients did not present with clinical symptoms other than those related to epileptic episodes. Cats who received combination therapy showed lower hematocrits than those who received monotherapy. A tendency for leukocytes and neutrophils to decrease during PB treatment was also seen. CONCLUSIONS: Blood cytopenias may frequently occur in cats on chronic PB therapy, even when serum drug levels are within the therapeutic range. However, clinical signs are typically mild to moderate and rarely severe.

Thrombocyte decrease and hemoglobin level as simple non-inflammatory predictors of anastomotic leakage in rectal-cancer surger.

PURPOSE: The study aimed to determine a simple diagnostic test that could predict the risk of anastomotic leakage in early postoperative period. METHODS: A single-center, retrospective study was conducted. The electronic medical records of patients who underwent resection for rectal tumor between January 1, 2016, and December 31, 2021, in University Hospital Olomouc, were reviewed. The data included risk factors for leakage and laboratory parameters commonly obtained. RESULTS: The decrease in platelets was significant as for the possibility of being a marker of anastomotic leakage; OR = 0.980 (p = 0.036). A decrease of 34 or higher predicts leakage with a sensitivity of 45 % (95 % CI: 23.1-68.5 %) and specificity of 81.1 % (95 % CI: 75.2-86.1 %). Postoperative leukocyte blood level (OR = 1.134; p = 0.019) and leukocyte level on postoperative day 1 (OR = 1.184; p = 0.023) were significant predictors for leakage. WBC values ≥ 8.8 predict leakage with a sensitivity of 70.0 % (95 % CI: 45.7-88.1 %) and specificity of 55.3 % (95 % CI: 48.4-62.0 %).  Hemoglobin blood level ≤ 79.5 predicts leakage with a sensitivity of 70.0 % (95 % CI: 45.7-88.1 %) and specificity of 62.2 % (95 % CI: 55.5-68.7 %). CONCLUSION: Despite the fact that the specificity and sensitivity of the followed parameters are low, they could serve as markers useful for early diagnosis or suspicion for leakage (Tab. 5, Fig. 3, Ref. 14).

Gas plasma-induced platelet activation corresponds to reactive species profiles and lipid oxidation.

Surgical-induced hemostasis is a critical step in the closure of incisions, which is frequently achieved via electrocauterization and subsequent tissue necrotization. The latter is associated with postoperative complications. Recent in vivo work suggested reactive species-producing gas plasma technology as a pro-homeostatic agent acting via platelet activation. However, it remained elusive how platelet activation is linked to lipid and protein oxidation and the reactive species compositions. A direct relation between the reactive species composition and platelet activation was revealed by assessing the production of several reactive species and by using antioxidants. In addition, platelet lipidome and proteome analysis identified significantly regulated key lipids in the platelet activation pathway, such as diacylglycerols and phosphatidylinositol as well as oxylipins like thromboxanes. Lipid oxidation products mainly derived from phosphatidylethanolamine and phosphatidylserine species were observed at modest levels. In addition, oxidative post-translational modifications were identified on key proteins of the hemostasis machinery. This study provides new insights into oxidation-induced platelet activation in general and suggests a potential role of those processes in gas plasma-mediated hemostasis in particular.