Assessment of Red Blood Cell Aggregation in Preeclampsia by Microfluidic Image Flow Analysis-Impact of Oxidative Stress on Disease Severity.

Preeclampsia (PE) is a hypertensive disease characterized by proteinuria, endothelial dysfunction, and placental hypoxia. Reduced placental blood flow causes changes in red blood cell (RBC) rheological characteristics. Herein, we used microfluidics techniques and new image flow analysis to evaluate RBC aggregation in preeclamptic and normotensive pregnant women. The results demonstrate that RBC aggregation depends on the disease severity and was higher in patients with preterm birth and low birth weight. The RBC aggregation indices (EAI) at low shear rates were higher for non-severe (0.107 ± 0.01) and severe PE (0.149 ± 0.05) versus controls (0.085 ± 0.01; p < 0.05). The significantly more undispersed RBC aggregates were found at high shear rates for non-severe (18.1 ± 5.5) and severe PE (25.7 ± 5.8) versus controls (14.4 ± 4.1; p < 0.05). The model experiment with in-vitro-induced oxidative stress in RBCs demonstrated that the elevated aggregation in PE RBCs can be partially due to the effect of oxidation. The results revealed that RBCs from PE patients become significantly more adhesive, forming large, branched aggregates at a low shear rate. Significantly more undispersed RBC aggregates at high shear rates indicate the formation of stable RBC clusters, drastically more pronounced in patients with severe PE. Our findings demonstrate that altered RBC aggregation contributes to preeclampsia severity.

Development of a Risk Assessment Model for Predicting Red Blood Cell Transfusion in Neonatal Patients.

BACKGROUND: The goal was to develop a risk assessment model for predicting red blood cell (RBC) transfusion in neonatal patients to assist hospital blood supply departments in providing small portions of RBCs to those requiring RBC transfusion on time. METHODS: Clinical information was collected from 1,201 children admitted to the neonatal unit. Clinical factors associated with predicting RBC transfusion were screened, and prediction models were developed using stepwise and multifactorial logistic regression analyses, followed by the evaluation of prediction models using receiver operating characteristic curves, calibration curves, and decision curve analysis (DCA). RESULTS: Overall, 81 neonatal patients were transfused with RBCs, and the variables of gestational age at birth, age < 1 month, receipt of mechanical ventilation, and infant anemia were included in the final prediction model. The area under the curve of the prediction model was 0.936 (0.921 - 0.949), which was significantly higher than that of the individual indicators of gestational age at birth, age at admission < 1 month, receipt of mechanical ventilation, and infant anemia (p < 0.001). DCA showed a standardized net benefit for the possible risk of infant RBC transfusion at 0.1 - 1.0. CONCLUSIONS: We developed a risk assessment model to predict the risk of RBC transfusion in neonatal patients that can effectively assess the risk of RBC transfusion in children.

Decreasing alloimmunization-specific mortality in sickle cell disease in the United States: Cost-effectiveness of a shared transfusion resource.

Red blood cell alloimmunization and consequent delayed hemolytic transfusion reaction (DHTR) incidence and mortality in patients with sickle cell disease (SCD) are high. A shared transfusion resource has decreased both in other countries, while in the United States cost concerns persist. We conducted a Markov cohort simulation of a birth cohort of alloimmunized patients with SCD to estimate lifetime DHTR incidence, DHTR-specific mortality, quality-adjusted life expectancy (QALE), and costs with the implementation of a shared transfusion resource to identify antibody history versus without (i.e., status quo). We conducted our analysis using a lifetime analytic time horizon and from a United States health system perspective. Implementation of shared transfusion resource projects to decrease cumulative DHTR-specific mortality by 26% for alloimmunized patients with SCD in the United States, relative to the status quo. For an average patient population of 32 000, this intervention would generate a discounted increment of 4000 QALYs at an incremental discounted cost of $0.3 billion, resulting in an incremental cost-effectiveness ratio of $75 600/QALY [95% credible interval $70 200-81 400/QALY]. The results are most sensitive to the baseline lifetime medical expenditure of patients with SCD. Alloantibody data exchange is cost-effective in 100% of 10 000 Monte Carlo simulations. The resource would theoretically need a minimum patient population of 1819 patients or cost no more than $5.29 million annually to be cost-effective. By reducing DHTR-specific mortality, a shared transfusion resource in the United States projects to be a life-saving and cost-effective intervention for patients with SCD in the United States.

A comparative study on chromium-induced micronuclei assessment in the peripheral blood of Hsd:ICR mice.

This study investigated the genotoxic effects of chromium (Cr) in Hsd:ICR mice, considering factors such as oxidative state, apoptosis, exposure pathway, duration, pregnancy, and transplacental exposure. Genotoxicity was assessed using the erythrocytes' micronucleus (MN) assay, while apoptosis was evaluated in nucleated blood cells. The results showed that Cr(III) (CrK(SO4 )2 and CrCl3 ) did not induce any marked genotoxic damage. However, Cr(VI) (CrO3 , K2 Cr2 O7 , Na2 Cr2 O7 , and K2 CrO4 ) produced varying degrees of genotoxicity, with CrO3 being the most potent. MN frequencies increased following 24-h exposure, with a greater effect in male mice. Administering 20 mg/kg of CrO3 via gavage did not lead to significant effects compared to intraperitoneal administration. Short-term oral treatment with a daily dose of 8.5 mg/kg for 49 days elevated MN levels only on day 14 after treatment. Pregnant female mice exposed to CrO3 on day 15 of pregnancy exhibited reduced genotoxic effects compared to nonpregnant animals. However, significant increases in MN levels were found in their fetuses starting 48 h after exposure. In summary, data indicate the potential genotoxic effects of Cr, with Cr(VI) forms inducing higher genotoxicity than Cr(III). These findings indicate that gender, exposure route, and pregnancy status might influence genotoxic responses to Cr.

Novel bioanalytical strategy using isotope pattern deconvolution and ICP-QMS for the study of iron incorporation in erythrocytes: An insight to better assessment.

Iron is an essential element for human life and its nutritional status in the human body is directly linked to human health. More than 1015 atoms of iron per second are necessary for the maintenance of haemoglobin formation. To predict iron bioavailability three approaches are normally employed: (a) faecal recovery; (b) plasma appearance; and (c) erythrocyte incorporation (the most used). Isotope Pattern Deconvolution (IPD) is a mathematical tool that allows the isolation of distinct isotope signatures from mixtures of natural abundance and enriched tracers. In this work we propose a novel strategy to assess erythrocyte iron incorporation, based on the use of an iron stable isotope (57Fe) and the IPD concept. This strategy allows direct calculation of the exogenous concentration of 57Fe incorporated into RBCs after supplementation. In this way, to determine the mass of iron incorporated into erythrocytes, the unique prediction that must be made is the blood volume, estimate to reproduce the natural dilution of the tracer (57Fe) in the blood. This novel bioanalytical approach was applied for the measurements of iron incorporation and further iron absorption studies in humans, using a group of twelve healthy participants, that should be further evaluated for the assessment of other chemical elements that could be of health concerns and directly impact society.

Comparative assessment of erythrocyte sphingolipid levels as potential cardiovascular health markers in women from Libya and Serbia: a small-scale study.

Aim: Cardiovascular diseases (CVDs) represent the major cause of morbidity and mortality worldwide including Libya, where they account for 43% of all deaths. Sphingolipids are involved in the pathology of numerous diseases including cardiovascular diseases and are proposed as potential biomarkers of cardiovascular health that could be more effective compared to traditional clinical biomarkers. The aim of this study was to determine the sphingolipid content in the erythrocyte membrane of Libyan migrant and Serbian resident women. In addition, to examine if sphingolipid levels could be used as a novel indicator of cardiovascular risk, we evaluated possible correlations with some well-established biomarkers of cardiovascular health.Materials and Methods: A total of 13 Libyan and 15 Serbian healthy women participated in the study. The high-performance version thin-layer chromatography (HPTLC) using the image analysis tool JustTLC was applied for quantification of erythrocytes' sphingolipids.Results: Lower mean values of erythrocytes' sphingolipids and cholesterol concentrations were found in the group of Libyan emigrants compared to Serbian resident women. Besides, in this group of apparently healthy women (n = 28), the sphingolipid content of erythrocytes was inversely related to the Omega-3 index (r =-0.492, p = 0.008) and directly linked to vitamin D status (r = 0.433, p = 0.021) and membrane cholesterol levels (r = 0.474, p = 0.011).Conclusion: The erythrocytes' sphingolipid levels should be measured/assessed as an additional biomarker of CV health, by applying a simple and routine method. Still, further investigation in a larger population-specific context is warranted.

The assessment of haematologic and serum chemistry parameters in canine pyometra: a systematic review and meta-analysis.

OBJECTIVES: Pyometra is common in non-spayed adult female dogs requiring early diagnosis and treatment to increase the survival rate. The diagnosis of pyometra is mainly based on clinical examination and anamnesis. Radiography, ultrasonography and laboratory analyses are recommended to support a diagnosis. The aim of this study was to assess blood parameters associated with pyometra by performing a systematic review and meta-analysis. MATERIALS AND METHODS: The review was conducted in accordance with PRISMA guidelines. A search of three databases (PubMed, Google Scholar and CAB abstracts) was performed in July 2022. Studies providing information about laboratory parameters for both the pyometra group and healthy control group dogs were eligible for inclusion. Data extraction included the first author's name, publication year, country, number of participants in both groups, mean values of selected studies, standard deviation values, and blood parameters. The risk of bias for each study was evaluated, and a random-effects meta-analysis was performed. RESULTS: We included 44 studies which investigated 12 blood parameters. High heterogeneity was detected in all parameters in these meta-analyses. The following blood parameters were increased in dogs with pyometra: white blood cell (mean=27.75×109 L-1 , mean difference (MD)=17.16, 95% confidence interval (CI) 14.85 to 19.47), monocytes (mean=2.06×1012 /L, MD=1.37, 95% CI 0.99 to 1.74), blood urea nitrogen (mean=41.42 mg/dL MD=18.06, 95% CI 12.26 to 23.85), alkaline phosphatase (mean=212.78 IU/L, MD=137.51, 95% CI 81.81 to 88.62), and aspartate aminotransferase (mean=48.31 IU/L, MD=16.96, 95% CI 10.61 to 23.30). The following parameters were reduced: red blood cell (mean=5.42 1012 /L, MD=-1.37, 95% CI -1.68 to -1.05), haemoglobin (mean=121.20 g/L, MD=-30.57, 95% CI -39.70 to 21.45), albumin (mean=23.71 g/L, MD = -8.16, 95% CI -11.46 to -4.86). Lymphocyte, creatinine, urea, and alanine transaminase parameters were increased in some studies and decreased in others. CLINICAL SIGNIFICANCE: In canine pyometra, some blood parameters are consistently increased, some consistently decreased, and some increased or decreased depending on the study.

Management of perioperative iron deficiency anemia as part of patient blood management in France: A budget impact model-based analysis based on real world data.

OBJECTIVES: Patient Blood Management (PBM) is defined as a patient-centered, systematic, evidence-based approach to improve patient outcomes by managing and preserving a patient's own blood, while promoting patient safety and empowerment. As a corollary, it also reduces the utilization of allogeneic blood components. However, demonstrating cost-effectiveness depends on the health insurance system considered. This analysis aims to estimate the one-year budget impact of PBM in four elective surgical areas, from French National Health Insurance and hospital perspectives. METHODS: A budget impact model was developed to estimate the difference in the cost of care between scenarios with and without PBM. The impact of hematopoiesis optimization (first pillar of PBM) was studied throughout the management of preoperative anemia and iron deficiency in four types of surgeries: orthopedic, cardiac & cardiovascular, vascular & thoracic, and urologic & visceral surgery. Estimation of model's parameters was based on data collected in 10 French hospitals, literature, and on data from the French national medico-administrative database. RESULTS: A total of 980,125 patients were modeled for all four therapeutic areas. Results shows that implementation of a PBM program could generate annual savings up to €1079 M from the French National Health Insurance perspective (€1018 M from the hospital perspective), and the sparing of 181,451 red blood cells units per year. The deterministic sensitivity analysis showed that PBM generates savings for both perspectives in most parameters tested. CONCLUSION: Implementing PBM programs could result in important savings for the health care system in France.

Immunohematological evaluation of red cell alloimmunization and statistical assessment of various adsorption techniques in warm autoimmune hemolytic anemia.

Adsorption techniques are widely applied to detect underlying masked alloantibodies in warm autoimmune hemolytic anemia (WAIHA). We established various adsorption techniques with an aim to detect alloimmunization in WAIHA This study conducted over a period of nine years included 298 patients of WAIHA. Complete immunohematological evaluation was performed on these 298 samples following departmental protocols. Clinical and laboratory details of patients were obtained from patient files. Various adsorption methods were performed and statistically evaluated in the study. Out of 479 cases of autoimmune hemolytic anemia, WAIHA comprised of 62.2 % (N = 298). A total of 139 (46.6 %) serum samples revealed autoantibodies. Adsorption study was performed in 101 (72.7 %) indicated samples and 24 (23.8 %) of these showed 26 alloantibodies. Among the patients subjected to adsorption study hemolytic marker were significantly deranged in the alloimmunization group (p < 0.01). Polyethylene glycol (PEG) adsorption was the quickest (52.2-54.6 min) of all adsorption techniques with minimum (1.3-1.5) numbers of adsorptions needing for complete removal of serum antibodies. The LISS-papain (LP) technique was found to be more sensitive and specific compared to the other two techniques. The agreement between PEG adsorption and LP adsorption was found to be 'perfect' (96.4 %) with a Cohen's kappa (k) value of 0.9. We conclude that identification of alloantibody specificities underlying a warm autoantibody is critical for a safe and effective transfusion. All WAIHA patients with history of blood transfusion, pregnancy or both should be subjected to adsorption study. Selection of a suitable adsorption technique depends on multiple important factors.

A critical assessment of the cellular defences of the avian respiratory system: are birds in general and poultry in particular relatively more susceptible to pulmonary infections/afflictions?

In commercial poultry farming, respiratory diseases cause high morbidities and mortalities, begetting colossal economic losses. Without empirical evidence, early observations led to the supposition that birds in general, and poultry in particular, have weak innate and adaptive pulmonary defences and are therefore highly susceptible to injury by pathogens. Recent findings have, however, shown that birds possess notably efficient pulmonary defences that include: (i) a structurally complex three-tiered airway arrangement with aerodynamically intricate air-flow dynamics that provide efficient filtration of inhaled air; (ii) a specialised airway mucosal lining that comprises air-filtering (ciliated) cells and various resident phagocytic cells such as surface and tissue macrophages, dendritic cells and lymphocytes; (iii) an exceptionally efficient mucociliary escalator system that efficiently removes trapped foreign agents; (iv) phagocytotic atrial and infundibular epithelial cells; (v) phagocytically competent surface macrophages that destroy pathogens and injurious particulates; (vi) pulmonary intravascular macrophages that protect the lung from the vascular side; and (vii) proficiently phagocytic pulmonary extravasated erythrocytes. Additionally, the avian respiratory system rapidly translocates phagocytic cells onto the respiratory surface, ostensibly from the subepithelial space and the circulatory system: the mobilised cells complement the surface macrophages in destroying foreign agents. Further studies are needed to determine whether the posited weak defence of the avian respiratory system is a global avian feature or is exclusive to poultry. This review argues that any inadequacies of pulmonary defences in poultry may have derived from exacting genetic manipulation(s) for traits such as rapid weight gain from efficient conversion of food into meat and eggs and the harsh environmental conditions and severe husbandry operations in modern poultry farming. To reduce pulmonary diseases and their severity, greater effort must be directed at establishment of optimal poultry housing conditions and use of more humane husbandry practices.

Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

[Predictive role of erythrocytes in assessment of COVID-19 outcomes].

INTRODUCTION: The search for affordable and accurate predictors of the outcome of COVID-19 is extremely important, as it provides the possibility to effectively correct the patient treatment tactics. AIM OF THE STUDY: To develop simple and accurate criteria based on the dynamics of red blood counts that predict the outcome of COVID-19. MATERIALS AND METHODS: Observations were carried out in 125 patients with severe and extremely severe COVID-19, in whom indicators characterizing the state of red blood were determined in dynamics on days 1, 5, 7, 10, 14 and 21 after the hospitalization. ROC analysis was performed to calculate the threshold predictive values for survival and mortality. RESULTS: The total number of erythrocytes and the level of hemoglobin in severe and extremely severe patients did not go beyond the acceptable limits, although showed a tendency to decrease in the group of fatal cases. On the 1st and 21st days, the number of MacroR in the deceased patients was reduced compared to those in group of survivors. It has been established that the RDW-CV test can predict the outcome of the COVID-19 with a high degree of probability at a relatively early stage of disease. RDW-SD test can be an additional predictive criterion of COVID-19 outcome. CONCLUSION: The RDW-CV test can be used as an effective predictor of disease outcome in patients with severe COVID-19.

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

Assessment of precision in growth inhibition assay (GIA) using human anti-PfRH5 antibodies.

BACKGROUND: For blood-stage malaria vaccine development, the in vitro growth inhibition assay (GIA) has been widely used to evaluate functionality of vaccine-induced antibodies (Ab), and Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage antigen. However, precision, also called "error of assay (EoA)", in GIA readouts and the source of EoA has not been evaluated systematically. METHODS: In the Main GIA experiment, 4 different cultures of P. falciparum 3D7 parasites were prepared with red blood cells (RBC) collected from 4 different donors. For each culture, 7 different anti-RH5 Ab (either monoclonal or polyclonal Ab) were tested by GIA at two concentrations on three different days (168 data points). To evaluate sources of EoA in % inhibition in GIA (%GIA), a linear model fit was conducted including donor (source of RBC) and day of GIA as independent variables. In addition, 180 human anti-RH5 polyclonal Ab were tested in a Clinical GIA experiment, where each Ab was tested at multiple concentrations in at least 3 independent GIAs using different RBCs (5,093 data points). The standard deviation (sd) in %GIA and in GIA50 (Ab concentration that gave 50%GIA) readouts, and impact of repeat assays on 95% confidence interval (95%CI) of these readouts was estimated. RESULTS: The Main GIA experiment revealed that the RBC donor effect was much larger than the day effect, and an obvious donor effect was also observed in the Clinical GIA experiment. Both %GIA and log-transformed GIA50 data reasonably fit a constant sd model, and sd of %GIA and log-transformed GIA50 measurements were calculated as 7.54 and 0.206, respectively. Taking the average of three repeat assays (using three different RBCs) reduces the 95%CI width in %GIA or in GIA50 measurements by ~ half compared to a single assay. CONCLUSIONS: The RBC donor effect (donor-to-donor variance on the same day) in GIA was much bigger than the day effect (day-to-day variance using the same donor's RBC) at least for the RH5 Ab evaluated in this study; thus, future GIA studies should consider the donor effect. In addition, the 95%CI for %GIA and GIA50 shown here help when comparing GIA results from different samples/groups/studies; therefore, this study supports future malaria blood-stage vaccine development.

Observing temporal variation in hemolysis through photoacoustics with a low cost LASER diode based system.

Patients under hemolytic condition need continuous monitoring of lysis as depletion of Red Blood Cells (RBC) and the presence of antioxidant free hemoglobin (Hb) in excess amount due to hemolysis lead to severe deterioration of their health. Out of many modalities, Photoacoustics (PA) offers real time information noninvasively from deep lying blood vessels since Hb is the strongest chromophore in mammalian blood and the PA response of blood varies with the amount of Hb present. During hemolysis, total Hb content in blood however remains unchanged, thus, questions the use of PA in hemolysis detection. In this report, a hypothesis that the amplitude of the PA signal would not change with the amount of lysis is framed and tested by applying osmotic shock to the RBCs in hypotonic environment and the PA response is recorded over time using a low cost NIR based PA system. The experimental outcome indicates that PA amplitude falls off as lysis progresses in course of time consequently rejecting the hypothesis. The decaying PA response also carries the signature of RBC swelling during the early phase of lysis. The PA measurement can detect hemolysis as low as 1.7%. These findings further advocate transforming this NIR-PA system into a portable, noninvasive patient care device to monitor hemolysis in-vivo.

[Bioactive compounds anthocyanins as a factor in the nutritional recovery of the body's adaptive potential after intense physical activity in the experiment: assessment of immunological and hematological indicators of adaptation].

Restoring the adaptive potential of an athlete is of paramount importance not only for the implementation of his training and competitive activities, but also for maintaining health. One of the leading place in complex recovery programs in sports is given to full-fledged optimal nutrition, which provides for meeting the body's requirements not only in energy, macro- and micronutrients, but also in minor bioactive compounds. The use of anthocyanin-containing products is a promising strategy for the normalization of metabolic and immune disorders that develop as a result of intense physical and neuro-emotional stress not only in athletes, but also in other groups of people exposed to these factors, including military personnel undergoing training in conditions close to combat. This determines the relevance of this study. The aim of the research was to study the effect of an anthocyanin-enriched diet on hematological profile and cellular immunity in rats after intense physical activity. Material and methods. The experiment was carried out for 4 weeks on 4 groups of male Wistar rats with an initial body weight of ~300 g. The motor activity of the animals of the 1st (control) and 2nd groups was limited by the standard keeping animals in the vivarium, while physically active rats of the 3rd and 4th groups received additional physical activity - training on a treadmill. Before the end of the experiment, the animals of 3rd and 4th groups were given debilitating physical activity on a treadmill (until the rats refused to continue the exercise). Rats of all 4 groups received a standard semi-synthetic diet, water ad libitum. Animals in 2nd and 4th groups were additionally fed blueberry and blackcurrant extract (30% anthocyanins) as part of the diet at a daily dose of 15 mg anthocyanins/kg body weight. Hematological parameters were determined on a Coulter ACT TM 5 diff OV hematological analyzer. Expression of CD45R, CD3, CD4, CD8a, CD161 receptors on rat peripheral blood lymphocytes was determined by direct immunofluorescent staining of whole blood cells using a panel of monoclonal antibodies conjugated with fluorescent dyes: APC, FITC, PE. The measurements were carried out on an FC-500 flow cytometer. Results. Intense physical activity in rats of the 3rd group did not lead to a significant change in erythrocyte parameters compared with the control group. Enrichment of the diet with blueberry and black currant extract (the 2nd and the 4th groups) provided a significant (p<0.05) increase in blood content of hemoglobin (Hb) (150.7±0.9 and 154.4±2.0 vs 145.4±0.9 g/l in control), hematocrit (44.95±0.21 and 46.18±0.64 vs 43.78±0.32%) and the average content of Hb in erythrocytes (18.00±0.20 and 18.03±0.24 vs 17.35±0.24 pg). The absolute content of leukocytes and other cellular elements of the leukocyte formula, as well as leukocyte indices in rats of the experimental groups didn't significantly differ from those of the control rats, which confirms the absence of an inflammatory process. Intense physical activity and anthocyanin enrichment of the diet didn't have a significant effect on rat platelet parameters. Enrichment of the diet of rats of the 4th group with blueberry and black currant extract led to the activation of cellular immunity, as evidenced by a significant (p<0.01) increase in the percentage (from the total content of T-lymphocytes) of T-helpers (70.13 ±1.34 vs 63.75±0.99%) and a decrease in the relative content of cytotoxic T-lymphocytes (28.65±1.38 vs 34.71±0.95%) in comparison with those in rats of the 3rd group and at the level of the trend (р<0.1) - from the 1st group indexes (66.87±1.20 and 31.87±1.26%, accordingly). Intense physical activity led to a decrease in immunoregulatory index in rats of the 3rd group (1.86±0.07) compared with the control (2.13±0.12) (p<0.1), and in animals of the 4th group this indicator was significantly higher (2.50±0.14, p<0.05). In animals of the 3rd group a statistically significant (p<0.05) decrease in the relative content of NK cells in peripheral blood was found compared to the control. Enrichment of the diet of physically active rats with blueberry and black currant extract led to a significant (p<0.05) increase in the percentage of NK cells compared to this indicator in rats of the 3rd group (4.87±0.75 vs 2.08±0.18%) and had no significant difference with the indicator in rats of the control group (4.32±0.98%). Conclusion. The enrichment of the rats' diet with blueberry and blackcurrant extract containing a daily dose of 15 mg of anthocyanins per kg of body weight provides an increase in blood Hb content, hematocrit and the average content Hb in erythrocytes. It has been established that intense physical activity induces the cellular immunity suppression. The activating effect of anthocyanins on adaptive cellular immunity and NK cells, which are lymphocytes of innate immunity, was revealed. The data obtained indicate the effectiveness of the use of bioactive compounds (anthocyanins) to increase the adaptive potential of the organism.

Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity.

In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

A report on a modified protocol for flow cytometry-based assessment of blood group erythrocyte antigens potentially suitable for analysis of weak ABO subgroups.

BACKGROUND: Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non-negligible levels. STUDY DESIGN AND METHODS: We sought to establish an FC method that could prevent antibody-induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC-FC analysis of AB expression. We optimized fixatives, multicolor-staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, Bm and Ael , were analyzed with the established protocol. RESULTS: The optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co-staining of leukocyte- and erythrocyte-markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the Bm sample a small RBC population exhibiting weak B antigen expression. The assay also proved it feasible to identify a small population (0.04%) of RBCs weakly expressing the A antigen in the Ael sample confirmed as harboring a rare c.816dupG ABO variant allele. CONCLUSION: The RBC-FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.

Protocol and application of basal erythrocyte transketolase activity to improve assessment of thiamine status.

Thiamine (vitamin B1) is an essential micronutrient required as a cofactor in many metabolic processes. Clinical symptoms of thiamine deficiency are poorly defined, hence biomarkers of thiamine status are important. The erythrocyte transketolase activity coefficient (ETKac) is a sensitive measure of thiamine status, but its interpretation may be confounded where the availability of the transketolase enzyme is limited. Basal ETK activity per gram of hemoglobin provides a complementary biomarker of thiamine status; however, its measurement and calculation are poorly described. Here, we describe in detail the assessment of basal ETK activity, including the calculation of path length in microplates and the molar absorption coefficient of NADH specific to the assay, and the measurement of hemoglobin in sample hemolysates. To illustrate the application of the methods, we present ETKac and basal ETK activity from women in The Gambia and UK. In conclusion, we present a clear protocol for the measurement of basal ETK activity that will permit the harmonization of methods to improve replication between laboratories.