Acarbose Mitigates Age-Dependent Alterations in Erythrocyte Membrane Transporters During Aging in Rats.

Acarbose (ACA), a well-studied and effective inhibitor of α-amylase and α-glucosidase, is a postprandial-acting antidiabetic medicine. The membrane of the erythrocyte is an excellent tool for analyzing different physiological and biochemical activities since it experiences a range of metabolic alterations throughout aging. It is uncertain if ACA modulates erythrocyte membrane activities in an age-dependent manner. As a result, the current study was conducted to explore the influence of ACA on age-dependent deteriorated functions of transporters/exchangers, disrupted levels of various biomarkers such as lipid hydroperoxides (LHs), protein carbonyl (PCO), sialic acid (SA), total thiol (-SH), and erythrocyte membrane osmotic fragility. In addition to a concurrent increase in Na+/H+ exchanger activity and concentration of LH, PCO, and osmotic fragility, we also detected a considerable decrease in membrane-linked activities of Ca2+-ATPase (PMCA) and Na+/K+-ATPase (NKA), as well as concentrations of SA and -SH in old-aged rats. The aging-induced impairment of the activities of membrane-bound ATPases and the changed levels of redox biomarkers were shown to be effectively restored by ACA treatment.

Antisickling effect of chrysin is associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of functional chemistry and metabolic pathways of human sickle erythrocytes.

Sickle cell disease (SCD) treatment and management remain a challenging puzzle especially among developing Nations. Chrysin's sickling-suppressive properties in human sickle (SS) erythrocytes in addition to its effect on AA-genotype erythrocytes were evaluated. Sickling was induced (76%) with 2% sodium metabisulphite at 3 h. Chrysin prevented (81.19%) the sickling and reversed same (84.63%) with strong IC50s (0.0257 µM and 0.00275 µM, respectively). The levels of oxygenated haemoglobin in the two groups (before and after induction approaches) were similar but significantly (P < 0.05) higher than that of SS erythrocytes (the 'induced' control), with chrysin-treated AA-genotype showing no effects relative to the untreated. The level of deoxygenated haemoglobin in the 'induced' control group was significantly (P < 0.05) higher than those of the chrysin-treated SS erythrocytes. Normal and chrysin-untreated erythrocytes (AA-untreated) were significantly more resistant to osmotic fragility than the SS-untreated. However, treatment with chrysin significantly reduced the osmotic fragility of the cells relative to the untreated cells. Furthermore, chrysin treatment significantly lowers the high level of 2,3-diphosphoglycerate (2,3-DPG) observed in the sickle erythrocytes, with no effects on AA-genotype erythrocytes. Based on functional chemistry, chrysin treatment alters the functional groups in favour of its antisickling effects judging from the observed bends and shifts. From metabolomics analysis, it was observed that chrysin treatment favors fatty acid alkyl monoesters (FAMEs) production with concomitant shutting down-effects on selenocompound metabolism. Thus, sickling-suppressive effects of chrysin could potentially be associated with modulation of oxygenated and deoxygenated haemoglobin via alteration of human sickle erythrocyte's functional chemistry and metabolic pathways implicated in SCD crisis.

Osmotic tolerance of rabbit spermatozoa is affected by extender composition and temperature.

Sperm osmotic adaptability to anisosmotic conditions is important for sperm epididymal maturation, motility activation at ejaculation, and female tract colonization, or for conducting technological procedures such as cryopreservation. Several factors affect this adaptability, including the fluid composition that contributes to water flow dynamics, and the temperature at which osmotic stress is initiated. This study was designed to investigate the effect of medium composition (electrolyte- or sugar-based extender) and temperature (25 and 5 °C) on rabbit sperm adaptability to anisosmotic conditions. Rabbit spermatozoa, therefore, were diluted at both temperatures (25 and 5 °C) in electrolyte- or sugar-based media at increasing osmotic conditions (100 to 1,000 mOsm/kg), and values for sperm variables (sperm kinetics, membrane integrity, mitochondrial membrane potential) were estimated as endpoints. Sperm kinetics seemed to be more sensitive to osmotic stress than membrane integrity or mitochondrial function. The effect of moderate hypoosmotic stress did not differ when there was use of sugar- and electrolyte-based extenders at 25 °C (P > 0.05). In hyper-tonic conditions at 25 °C, the sugar-based extender was more effective in protecting sperm membrane integrity and mitochondrial function (P < 0.05). The lesser temperature made the differences more relevant because of the detrimental effect of hyperosmotic stress was more evident in the electrolyte-based extender at 5 °C (P < 0.05). The results from this study indicated rabbit spermatozoa have different adaptability to anisosmotic conditions induced by sugar- and electrolyte-based media and that the temperature at which the osmotic stress is initiated affects the cellular response.

Mechanism of CuSO4 cytotoxicity in goat erythrocytes after high-level in vitro exposure to isotonic media.

Copper (Cu) is a common environmental pollutant in nature. Cu-poisoning can cause liver damage and erythrocytes hemolysis. To evaluate the effect of CuSO4 poisoning on the morphological and functional characteristics of goat red blood cells. Five 10-14-month-old goats were selected for jugular vein blood sampling to obtain erythrocytes, and then the erythrocytes were processed with different concentrations (0, 10, 20, 30, 40 and 50 µmol/L) of CuSO4 for 48 h, and 40 µmol/L doses CuSO4 incubated for different time (12, 24, 36, 48 and 60 h) to process erythrocytes. We observed the changes in erythrocyte morphology through scanning electron microscopy, and detected the antioxidant function and activities of three ATPases. Additionally, biological properties were examined from the perspectives of phospholipids and membrane protein components, permeability fragility, and fluidity in erythrocytes. We found that after CuSO4 treatment, the antioxidant capacity of erythrocytes decreased, which was manifested as increased MDA content and decreased CuZn-SOD and GSH-Px activities (p < 0.05). In addition, we also found that erythrocyte fluidity decreased, osmotic fragility increased, membrane phospholipid percentage and protein composition changes abnormally, and Na+/K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activities decreased (p < 0.05). From the results, it can be concluded that CuSO4 exposure causes hemolysis of goat erythrocytes through oxidative stress to the structure and function of erythrocytes, showing a dose-time effect.

Viper toxins affect membrane characteristics of human erythrocytes.

Elucidating electrokinetic stability by which surface charges regulate toxins interaction with erythrocytes is crucial for understanding the cell functionality. Electrokinetic properties of human erythrocytes upon treatment of Vipoxin, phospholipase A2 (PLA2) and Vipoxin acidic component (VAC), isolated from Vipera ammodytes meridionalis venom were studied using particle microelectrophoresis. PLA2 and Vipoxin treatments alter the osmotic fragility of erythrocyte membranes. The increased stability of cells upon viper toxins is presented by the increased zeta potential of erythrocytes before sedimentation of cells during electric field applied preventing the aggregation of cells. Lipid peroxidation of low dose toxin-treated erythrocytes shows reduced LP products compared to untreated cells. The apparent proton efflux and conductivity assays are performed and the effectiveness PLA2 > Vipoxin>VAC is discussed. The reported results open perspectives to a further investigation of the electrokinetic properties of the membrane after viper toxins treatment to shed light on the molecular mechanisms driving the mechanisms of inflammation and neurodegenerative diseases.

Oxidative stress assessment in sickle cell anemia patients treated with hydroxyurea.

Hydroxyurea (HU) is used as a therapy in sickle cell anemia (SCA). Many studies have established that HU improves patient quality of life by reducing symptoms. However, the effect of HU on erythrocytes is not well-described. We evaluated several parameters related to oxidative stress and total lipid content of erythrocytes in patients with SCA. The patient cohort consisted of 7 SCA patients treated with HU, 17 untreated SCA patients, and 15 healthy subjects. Erythrocytes from patients with SCA displayed increased oxidative stress relative to the control group, including higher thiobarbituric acid reactive substances (TBARS), Fe3+ content, and osmotic fragility, and decreased total cholesterol. We observed that treatment of SCA patients with HU increased Fe3+ content and activity of glutathione peroxidase, and decreased glutathione reductase activity, glutathione levels, total cholesterol, and phospholipid content comaperaded to patients untreated with HU. Thus, HU alters biochemical characteristics of erythrocytes; future studies will determine whether they are beneficial or not.

The Predictive Value of Salt Sensitivity and Osmotic Fragility in the Development of Cerebral Small Vessel Disease.

Increased salt intake in food probably affects the progression of cerebral small vessel disease (CSVD), which justifies the study of disturbances in sodium homeostasis associated with the development of CSVD. We aimed to clarify the role of salt sensitivity and osmotic fragility in the development of CSVD. Erythrocyte salt sensitivity was measured using the modified salt blood test, and osmotic fragility was measured using the classic osmotic fragility test in 73 patients with CSVD (48 women; 60.1 ± 6.5 years) and 19 healthy volunteers (14 women; 56.9 ± 6.4 years). Salt sensitivity and osmotic fragility exhibited a predictive value in relation to CSVD. These parameters were associated with an increase in white matter hyperintensities (P = 0.019 and 0.004, respectively). Their simultaneous use increased their predictive ability for CSVD (P < 0.000001; AUC (95% CI), 0.824 (0.724-0.923)). The possibility of predicting CSVD using erythrocyte salt sensitivity and osmotic fragility indicates the value of the individual glycocalyx buffer capacity in relation to sodium and the activity of sodium channels in the development of CSVD. Increased salt sensitivity and osmotic fragility seem to be risk factors for CSVD.

Effects of L-carnitine on the erythrocytes of stored human blood.

OBJECTIVES: This study aimed to assess the effects of L-carnitine on oxidative stress in human erythrocytes during storage. BACKGROUND: Using antioxidants as components of blood storage solutions may combat the effects of storage-induced oxidative stress on erythrocytes. METHODS: Blood from male adults was stored at 4 °C for 55 days in citrate phosphate dextrose adenine solution, without L-carnitine (Control) and with L-carnitine as an additive (at concentrations of 10, 30 and 60 mM - Experiments). Every fifth day, erythrocyte markers (morphology, count, haemoglobin, haemolysis and osmotic fragility), antioxidant defences (antioxidant enzymes and total antioxidant capacity) and oxidative stress markers (superoxides, lipid peroxidation and protein oxidation products) were analysed. RESULTS: Oxidative damage was observed in controls (day 25 onwards) and in experiments (day 35 onwards). L-carnitine (10 and 30 mM) protected erythrocytes from damage up to day 35 by maintaining haemoglobin and lipid peroxidation, assisting antioxidant enzymes and increasing antioxidant capacity by elevating sulfhydryls and ascorbic acid. L-carnitine was beneficial in prolonging storage up to 55 days but could not prevent oxidative damage completely in terms of haemolysis and osmotic fragility. CONCLUSIONS: L-carnitine ameliorated oxidative stress, but combinations with other antioxidants may provide comprehensive protection to erythrocytes during storage.

Triglyceride-lowering effect of the aldose reductase inhibitor cemtirestat-another factor that may contribute to attenuation of symptoms of peripheral neuropathy in STZ-diabetic rats.

Hyperglycemia is considered a key risk factor for development of diabetic complications including neuropathy. There is strong scientific evidence showing a primary role of aldose reductase, the first enzyme of the polyol pathway, in the cascade of metabolic imbalances responsible for the detrimental effects of hyperglycemia. Aldose reductase is thus considered a significant drug target. We investigated the effects of cemtirestat, a novel aldose reductase inhibitor, in the streptozotocin-induced rat model of uncontrolled type 1 diabetes in a 4-month experiment. Markedly increased sorbitol levels were recorded in the erythrocytes and the sciatic nerve of diabetic animals. Osmotic fragility of red blood cells was increased in diabetic animals. Indices of thermal hypoalgesia were significantly increased in diabetic rats. Tactile allodynia, recorded in diabetic animals in the early stages, turned to mechanical hypoalgesia by the end of the experiment. Treatment of diabetic animals with cemtirestat (i) reduced plasma triglycerides and TBAR levels; (ii) did not affect the values of HbA1c and body weights; (iii) reversed erythrocyte sorbitol accumulation to near control values, while sorbitol in the sciatic nerve was not affected; (iv) ameliorated indices of the erythrocyte osmotic fragility; and (v) attenuated the symptoms of peripheral neuropathy more significantly in the middle of the experiment than at the end of the treatment. Taking into account the lipid metabolism as an interesting molecular target for prevention or treatment of diabetic peripheral neuropathy, the triglyceride-lowering effect of cemtirestat should be considered in future studies. The most feasible mechanisms of triglyceride-lowering action of cemtirestat were suggested.

Sickling-suppressive effects of chrysin may be associated with sequestration of deoxy-haemoglobin, 2,3-bisphosphoglycerate mutase, alteration of redox homeostasis and functional chemistry of sickle erythrocytes.

Sickle cell disease (SCD) is a medical condition caused by mutation in a single nucleotide in the ß-globin gene. It is a health problem for people in sub-Saharan Africa, the Middle East and India. Orthodox drugs developed so far for SCD focus largely on symptomatic respite of pain and crisis mitigation. We investigated the antisickling effects of chrysin via modulation of deoxy-haemoglobin, 2,3-bisphosphoglycerate mutase, redox homeostasis and alteration of functional chemistry in human sickle erythrocytes. In silico and in vitro methods were adopted for the studies. Chrysin was docked against deoxy-haemoglobin and 2,3-bisphosphoglycerate mutase, with binding energies (-24.064 and -18.171 kcal/mol) and inhibition constant (K i) of 0.990 µM and 0.993 µM at their active sites through strong hydrophobic and hydrogen bond interactions. Sickling was induced with 2% metabisulphite at 3 h. Chrysin was able to prevent sickling maximally at 2.5 µg/mL and reversed the same at 12.5 µg/mL, by 66.5% and 69.6%, respectively. Treatment with chrysin significantly (p < 0.05) re-established the integrity of erythrocytes membrane as evident from the observed percentage of haemolysis relative to induced erythrocytes. Chrysin also significantly (p < 0.05) prevented and reversed lipid peroxidation. Similarly, glutathione and catalase levels were observed to significantly (p < 0.05) increase with concomitant significant (p < 0.05) decrease in superoxide dismutase activity relative to untreated. From Fourier-transform infrared results, treatment with chrysin was able to favourably alter the functional chemistry, judging from the shifts and functional groups observed. Sickling-suppressive effects of chrysin may therefore be associated with sequestration of deoxy-haemoglobin, 2,3-bisphosphoglycerate mutase, alteration of redox homeostasis and functional chemistry of sickle erythrocytes.

Antioxidant potentials and effects on the hematology and osmotic fragility scores of a polyherbal formulation used in Southeast Nigeria.

Background In this study, the hematological and antioxidant potential as well as the osmotic fragility effects of a Nigerian polyherbal formulation were evaluated. Materials and methods A total of 40 fats were divided into four groups of 10 rats each. Group 1 served as the control group, and the rest were assigned increasing daily oral administration of the extract for 28 days. At the end of treatment, blood was collected for hematological and osmotic fragility studies. The free radical scavenging effect of the extract was investigated via different in vitro models as well. Results Results showed that the nitric oxide scavenging and 2,2-diphenyl-1-picrylhydrazyl (DPPH) activities of the extract were significant (p < 0.05) and compared favorably with that of vitamin C. At 200 and 400 µg/mL, the nitric oxide scavenging activities for Ajumbise Polyherbal Extract (APE) were 60.71 ± 0.25% and 59.49 ± 0.98%, respectively, whereas for the same concentrations of vitamin C, 74.60 ± 0.25% and 85.24 ± 0.14 scavenging activities were obtained. The (DPPH) activity at 100 µg/mL was 81.24 ± 0.02% for the extract and 96.22 ± 0.18% for vitamin C. However, at all concentrations, the extract had significantly lower Ferric Reducing Antioxidant Power (FRAP) activity than vitamin C. Red blood cell counts (RBCC), hemoglobin and packed cell volume values (PCV) were significantly lowered only in groups treated with 400 and 800 mg/kg of the extract (p < 0.05), whereas other RBCC parameters and white blood cell counts (WBCC) were not significantly affected (p < 0.05). Platelet (PLT) count was also significantly lowered in all extract-treated groups. The extract also significantly reduced RBCC percentage hemolysis (p < 0.05). Conclusions Ajumbise polyherbal may be free of hematoxicity and may improve the integrity of the RBC membrane due to its appreciable antioxidant activity.

Effect of fisetin and probiotic supplementation on erythrocyte osmotic fragility, malondialdehyde concentration and superoxide dismutase activity in broiler chickens exposed to heat stress.

The aim of the study was to evaluate effects of fisetin and probiotic on erythrocyte osmotic fragility (EOF), malondialdehyde (MDA) and superoxide dismutase (SOD) in broiler chickens exposed to heat stress. Sixty day-old broilers were divided into: Group I (control) given distilled water; Group II, fisetin (5 mg/kg); Group III, probiotic Saccharomyces cerevisiae (4.125 × 106 cfu/100 ml); and Group IV, fisetin (5 mg/kg) + probiotic (4.125 × 106 cfu/100 ml) orally for 7 days. Blood samples collected from 42-day-old birds were evaluated for EOF, serum MDA concentration and SOD activity. Percentage EOF at 0.5% NaCl was lower (P<0.05) in fisetin, probiotic and fisetin + probiotic groups (34.26 ± 0.98%, 35.65 ± 0.81% and 34.25 ± 1.98%, respectively) than in controls (48.42 ± 0.40%). The MDA concentrations in broiler chickens administered with fisetin (14.37 ± 1.15 nmol/l), probiotic (5.66 ± 1.06 nmol/l) and fisetin + probiotic (4.136 ± 0.58 nmol/l) were lower (P<0.05) than in controls (22.64 ± 2.95 nmol/l). Activities of SOD were higher (P<0.05) in fisetin, probiotic and fisetin + probiotic broiler chickens (6.34 ± 0.24 IU/l, 5.67 ± 0.09 IU/l and 5.93 ± 0.13 IU/l, respectively) than in controls (5.37 ± 0.09 IU/l). Fisetin + probiotic ameliorated oxidative stress changes in broiler chickens better than fisetin or probiotic alone. In conclusion, administration of fisetin or probiotic and, especially their combination, decreased EOF, lipoperoxidation and increased superoxide dismutase activity in broiler chickens exposed to heat stress.

Flow Cytometric Analysis of Erythrocytes Osmotic Fragility in Hereditary Spherocytosis: A Case-Controlled Study Evaluating the Best Anticoagulant, Sample Pre-Treatment and NaCl Concentration for Reliable Screening of this Red Blood Cell Membrane Disorder.

BACKGROUND: The cytometric flow osmotic fragility test (FC-OFT) was recently introduced. However, the test is still under development and some variables have not yet been fully tested. METHODS: The osmotic fragility of hereditary spherocytosis (HS) cases and healthy controls were evaluated by FC-OFT using a series of tubes containing decreasing concentrations of NaCl. The analyses were executed in fresh and incubated (37°C for 24 h) blood samples anticoagulated with EDTA and heparin. The percentages of residual red blood cells were used to plot the osmotic fragility curves. The OF curves of each tested condition were compared using the median corpuscular fragility (MCF). ROC curve analyses identified the most accurate NaCl concentrations for differentiation between HS cases and healthy controls. RESULTS: FC-OFT curves assumed a sigmoidal dose-response shape and the MCF of cases and controls were different in all instances. MCF comparisons revealed that incubation and anticoagulant have major and minor effects on the FC-OFT, respectively. One hundred percent of sensitivity and specificity was obtained from 5.5 to 6.0 g/L of NaCl in EDTA-treated fresh blood, from 6.0 to 8.0 g/L of NaCl in EDTA-treated incubated blood, and in none of the tested NaCl concentration in heparinized blood. CONCLUSIONS: EDTA is the anticoagulant of choice for the assay. Incubation at 37°C for 24 h increased its diagnostic capability. The most reliable NaCl concentration for the discrimination of HS case from controls was 6.0 g/L of NaCL in fresh EDTA-treated blood, and was 7.5 g/L of NaCl in incubated EDTA-treated blood. © 2018 International Clinical Cytometry Society.

Cell Swelling Induced by the Antimalarial KAE609 (Cipargamin) and Other PfATP4-Associated Antimalarials.

For an increasing number of antimalarial agents identified in high-throughput phenotypic screens, there is evidence that they target PfATP4, a putative Na+ efflux transporter on the plasma membrane of the human malaria parasite Plasmodium falciparum For several such "PfATP4-associated" compounds, it has been noted that their addition to parasitized erythrocytes results in cell swelling. Here we show that six structurally diverse PfATP4-associated compounds, including the clinical candidate KAE609 (cipargamin), induce swelling of both isolated blood-stage parasites and intact parasitized erythrocytes. The swelling of isolated parasites is dependent on the presence of Na+ in the external environment and may be attributed to the osmotic consequences of Na+ uptake. The swelling of the parasitized erythrocyte results in an increase in its osmotic fragility. Countering cell swelling by increasing the osmolarity of the extracellular medium reduces the antiplasmodial efficacy of PfATP4-associated compounds, consistent with cell swelling playing a role in the antimalarial activity of this class of compounds.

Rapamycin mitigates erythrocyte membrane transport functions and oxidative stress during aging in rats.

Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (-SH), sialic acid (SA) and intracellular calcium ion [Ca2+]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na+/K+-ATPase (NKA) and Ca2+-ATPase (PMCA), and levels of -SH and SA was observed along with a simultaneous induction in Na+/H+ exchanger (NHE) activity and levels of [Ca2+]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.

Effects of L-glutamine on rectal temperature and some markers of oxidative stress in Red Sokoto goats during the hot-dry season.

The experiment investigated the ameliorative effects of L-glutamine administration on rectal temperature (RT), erythrocyte osmotic fragility (EOF), serum antioxidant enzyme activities and malondialdehyde (MDA) concentration in Red Sokoto goats during the hot-dry season. Twenty eight healthy Red Sokoto goats, comprising 14 experimental (administered 0.2 g/kg of L-glutamine dissolved in 10 mL of distilled water, once daily for 21 days) and 14 control (administered equivalent of distilled water) goats served as subjects. Rectal temperature (measured at 6:00, 13:00 and 18:00 h) and blood samples (taken at 8:00 h) were obtained from all subjects weekly, before, during and after L-glutamine administration. Data obtained were compared using one-way repeated-measures ANOVA, followed by Tukey's post-hoc test. The dry-bulb temperature, relative humidity and temperature-humidity index for the study period ranged between 24.0 and 37.5 °C, 26.0 and 84.0% and 73.0 and 86.3, respectively. L-glutamine administration decreased (P < 0.05) RT, EOF and MDA and increased superoxide dismutase (SOD) activity in experimental group, compared to controls during weeks 1, 2 and 3. Glutathione peroxidase (GPx) and catalase activities were higher (P < 0.05) in the experimental group than in the controls only during week 1 of L-glutamine administration. In conclusion, L-glutamine administration mitigated increases in RT, EOF and serum MDA concentration and enhanced serum SOD, GPx and catalase activities and may be beneficial in heat-stressed goats during the hot-dry season.

Evaluation of OSMOCELLS, a new semi-automatic device for osmotic fragility assessment.

INTRODUCTION: The osmotic fragility (OF) test was a central test for the diagnosis of hereditary red blood cell (RBC) disorders (mostly hereditary spherocytosis (HS), but thalassaemia as well). Nowadays although the traditional multitubes method has lost a prominent place, many laboratories still perform such a laboured test, despite the lack of standardization. In fact, the evaluation of OF may offer an inexpensive screening for RBC disorders. We present a new semi-automatic device, allowing the continuous recording of OF, by an updated dialysis method. METHODS: Repeatability, stability over time, influence of the anticoagulant were evaluated among a population of healthy blood donors. The test was then performed among patients presenting inherited RBC disorders (HS or haemoglobinopathies) where OF is typically altered. RESULTS: Repeatability was excellent; the parameters were greatly influenced by the nature of the anticoagulant and interestingly appeared stable for 48 h. Patients with RBC disorders displayed the expected profile in regard with their disease: patients with HS all presented an increased OF while patients with haemoglobinopathy displayed resistant profiles. CONCLUSION: The device offers a substantial improvement in terms of standardization and consistency of the results and may offer a considerable gain for general laboratories.

Pathophysiological aspects of red blood cells in end-stage renal disease patients resistant to recombinant human erythropoietin therapy.

OBJECTIVE: Modified, bioreactive red blood cells (RBCs) and RBC-derived microvesicles (MVs) likely contribute to the hematological and cardiovascular complications in end-stage renal disease (ESRD). This study assesses the physiological profile of RBCs in patients with ESRD receiving standard or high doses of recombinant human erythropoietin (rhEPO). METHOD: Blood samples from twenty-eight patients under sustained hemodialysis, responsive, or not to standard rhEPO administration were examined for RBC morphology, fragility, hemolysis, redox status, removal signaling, membrane protein composition, and microvesiculation before and after dialysis. Acute effects of uremic plasma on RBC features were examined in vitro through reconstitution experiments. RESULTS: Overall, the ESRD RBCs were characterized by pathological levels of shape distortions, surface removal signaling, and membrane exovesiculation, but reduced fragility compared to healthy RBCs. Irreversible transformation of RBCs was found to be a function of baseline Hb concentration. The more toxic uremic context in non-responsive patients compared to rhEPO responders was blunted in part by the antioxidant, antihemolytic, and anti-apoptotic effects of high rhEPO doses, and probably, of serum uric acid. A selective lower expression of RBC membrane in complement regulators (CD59, clusterin) and of CD47 "marker-of-self" was detected in non-responders and responders, respectively. Evidence for different short-term dialysis effects and probably for a different erythrocyte vesiculation mechanism in rhEPO responsive compared to non-responsive patients was also revealed. CONCLUSION: Deregulation of RBC homeostasis might involve diverse molecular pathways driving erythrocyte signaling and removal in rhEPO non-responders compared to responsive patients.

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.