Hemolysis Time Research Articles

Evaluation of Antioxidant, Antihemolytic Activity, Analgesic and Anti-Inflammatory Potential of Aristolochia clematitis Extracts

There is an increasing interest in medicinal plants to find natural remedies relatively safer than synthetic alternatives. The present study reports the evaluation of the in vitro and in vivo biological properties of crude extract (CrE), hexane (HeE), ethyl acetate (EAE) and aqueous (AqE) fractions of Aristolochia clematitis. The polyphenol and flavonoid content were determined and varied from 15.77 ± 0.28 to 93.62 ± 1.63 mg EGA/g, and 5.71 ± 0.1 to 13.53 ± 0.15 µg EQ/mg, respectively. Crude extract (CrE) and ethyl acetate (EaE) showed the highest scavenging activity of DPPH radicals (IC50 = 50.66 ± 1.41 and 69.57 ± 0.38 µg/mL) and hydroxyl radicals (IC50 = 0.894 ± 0.042 and 0.905 ± 0.890 mg/mL), reducing power (113.85 ± 0.5 and 82.45 ± 0.13 mg/mL) and the ability to inhibit β-carotene oxidation (76.13 ± 0.93, 69.18 ± .95 %). All extracts have the ability to protect red blood cells from AAPH peroxyl radicals and significantly increase hemolysis time compared to vitamin C. Tested on normal human plasma at a concentration of 50 and 100 mg/mL, these extract prolonged prothrombin (PT) and partial thromboplastin (aPTT) times. In addition, CrE tested at doses of 150 and 300 mg/kg, showedpotent antinociceptive effect (40.68 ± 3.84 and 66.61 ± 4.71%) and anti-inflammatory activity (65.71 ± 1.41 and 63.89 ± 2.17%). The results support the use of this plant in traditional medicine and suggest that it may contain phytochemicals that have the potential to be active agents as antioxidants, anticoagulants and analgesics.&nbsp

IN VITRO PROTECTIVE EFFECT OF LEAF EXTRACT OF FICUS DELTOIDEA JACK (MORACEAE) ON BIOMARKERS OF OXIDATIVE STRESS IN THE HUMAN ERYTHROCYTES

Purpose: to investigate the antioxidant properties of the aqueous extracts derived from leaves of Ficus deltoidea using the model of human blood. For this purpose, oxidative stress biomarkers [2-thiobarbituric acid reactive substances (TBARS), content of aldehydic and ketonic derivatives of oxidatively modified proteins, total antioxidant capacity (TAC)] in the human erythrocytes after in vitro incubation with aqueous extracts derived from leaves of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL) were used. Resistance of human erythrocytes after in vitro treatment by aqueous extracts derived from leaves of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL) was evaluated by HCl-induced hemolysis using a percentage of hemolyzed erythrocytes per each 30 sec. and a total time of hemolysis. Methodology. The leaves of F. deltoidea were collected in M.M. Gryshko National Botanic Garden (Kyiv, Ukraine). Blood (10-20 mL) was obtained from normal volunteers via venipuncture (4 males and 5 females aged 28-53 years old). An erythrocyte suspension at 1% hematocrit was incubated with 4 mM phosphate buffer (pH 7.4) (control) and pre-incubated with the extract of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL, respectively) at 37 °C for 60 min. The level of lipid peroxidation was determined by quantifying the concentration of 2-thiobarbituric acid reacting substances (TBARS). The rate of protein oxidative destruction was estimated from the reaction of the resultant carbonyl derivatives of amino acid reaction with 2,4-dinitrophenylhydrazine (DNFH). The total antioxidant capacity (TAC) in the samples was estimated by measuring the TBARS level after Tween 80 oxidation. The HCl-induced resistance of erythrocytes was measured spectrophotometrically with 0.1M HCl. The significance of differences (significance level, p < 0.05) was examined using the Mann-Whitney U test. Scientific novelty. The treatment of human erythrocytes by extract derived from leaves of F. deltoidea at the final concentration of 5 mg/mL resulted in an increase of TBARS as biomarkers of lipid peroxidation and aldehydic derivatives of oxidatively modified proteins with a simultaneous decrease in the total antioxidant capacity compared to the untreated samples. On the other hand, in vitro treatment of human erythrocytes with an extract derived from leaves of F. deltoidea at the final concentration of 0.5 mg/mL resulted in the same values of TBARS level, aldehydic and ketonic derivatives of OMP, and total antioxidant capacity as the untreated samples. Extract of F. deltoidea at the final concentrations of 5 mg/mL and 0.5 mg/mL possess hemolytic properties to human erythrocyte suspension after 1-h incubation in vitro. Conclusions. The changes in the oxidative stress biomarkers using the in vitro model of human erythrocytes to evaluate the antioxidant activities of the aqueous extract derived from the leaves of F. deltoidea at the two final concentrations (5 mg/mL and 0.5 mg/mL) revealed that high concentration of extract (5 mg/mL) resulted in the increase of lipid peroxidation and protein oxidation with a simultaneous decline in the total antioxidant capacity. HCl-induced hemolysis was activated after the treatment by extract derived from the leaves of F. deltoidea at the two final concentrations. More studies are warranted in the future, to illustrate the potential and mechanisms of F. deltoidea in preventing oxidative stress using different cell models in vitro and different final concentrations of the extract. Also, further studies are warranted to identify the bioactive components that contribute to this protective effect.

A hemolysis verified to be induced by anti-meropenem

Incompatibility of blood groups or unexpected antibodies are primary considerations when acute hemolysis occurs during or after transfusion. However, less attention is paid to drug-induced immune hemolytic anemia (DIIHA), which is a rare but potentially life-threatening autoimmune disease. We present the case of a 34-year-old woman (group A, RhD+) who was treated with multiple antibiotics after meningioma resection. As her hemoglobin (Hb) decreased significantly from 109 g/L to 52 g/L without obvious bleeding, a blood transfusion was conducted soon after the medication, during which acute hemolysis occurred. An unexpected antibody, anti-M (MNS blood group system), was identified in the patient. It was confirmed that both the recipient and donor were group A, M antigen negative (M−) with CCDee phenotype, and no agglutination reactivity was observed in major crossmatch by testing the specimens before and after transfusion. Meanwhile, the results of the direct antiglobulin test (DAT) changed from negative to positive. Anti-meropenem, a drug-dependent antibody of meropenem, was detected, and hemolysis resolved after cessation. Anti-meropenem may mainly act through an "immune complex-type" reaction that induces intravascular hemolysis. Notably, the peculiarity of this case was dependent on the occurrence time of the acute hemolysis. Hence, it is necessary to raise awareness of DIIHA in clinical antibiotic treatments.

STUDY OF ASPIRIN EFFECT ON STABILITY OF MEMBRANES OF RAT BLOOD ERYTHROCYTES BY ACIDIC HEMOLYSIS METHOD

In this work the effect of aspirin on stability of rat blood erythrocyte membranes was studied, using acidic hemolysis method. Hemolysis rate of erythrocytes was shown to depend on preservation time of rat erythrocyte suspension. With increasing of preservation time the original value of optic density of rat erythrocyte suspension decreases and hemolysis time reduces. It was also shown that in the presence of aspirin the membrane stability of low-stable erythrocytes increases, moreover, hemolysis duration enhances, while aspirin leads to decreasing of membrane stability of both high-stable and enhanced-stable erythrocytes of rats.

Glucose-6-Phosphate dehydrogenase deficiency associated hemolysis in a cohort of new onset type 1 diabetes children in Guangdong province, China

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human genetic abnormalities, with a high prevalence in Guangdong, China. The purpose of this study was to explore the characteristics of newly diagnosed type 1 diabetes (T1D) patients with G6PD deficiency in a cohort of Chinese children and to investigate the relationship between the diabetic ketoacidosis (DKA) and hemolysis due to G6PD deficiency in these patients.MethodsA total of 503 newly diagnosed T1D children aged 6 months–18 years were collected and their G6PD enzyme activity were measured. Fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), and G6PD gene were analysed. The pH, HCO3, and plasma osmotic pressure between DKA patients with and without hemolysis at the presentation were compared.ResultsIn the present study, G6PD deficiency accounted for 5.3% of newly diagnosed T1D children. There were no significant differences in FPG/HbA1c and HbA1c levels between T1D children alone and T1D children with G6PD deficiency. Hemolysis appeared in five of the twenty-two DKA patients with G6PD deficiency. Two patients had fever at onset and were given ibuprofen and cefazolin. The other three patients did not have infection or ingestion of hemolytic drugs. There were no significant difference in pH, HCO3, and osmotic pressure between the children with DKA with and without hemolysis at the presentation. The hemolysis occurred between 2 and 7 days after admission and the hyperglycaemia had been corrected by the time hemolysis occurs. Four G6PD gene mutations were found in the diabetes with G6PD deficiency patients: c.1376G > T, c.1388G > A, c.95A > G, and c.871G > A, all of which were genes with high frequency of G6PD deficiency in Guangdong Province. No correlation between genotype and hemolysis was found.ConclusionIn the present study, we found the frequency of G6PD deficiency among newly diagnosed T1D children was similar to that of the general population. However, DKA children with G6PD deficiency are prone to occur hemolytic anemia, and these hemolysis usually occurs when DKA is corrected and blood glucose is in homeostatic state, which is easy to be ignored. To reduce the risk of this complication, especially in areas with high incidence of G6PD deficiency, screening for G6PD activity in people with newly diagnosed diabetes should be considered.

The Participation of the Organism Adaptation Mechanisms to the Lack of Oxygen According to the Assessment of the Fethemoglobin Content in the Peripheral Blood of Patients with Community-acquired Pneumonia

Introduction. Сommunity-acquired pneumonia (COP) is a global socio-medical problem. At emergence of pneumonia by any genesis, hypoxia develops. Oxygen homeostasis of the body is provided by the coordinated interaction of external respiration, circulatory system and oxygen-transport system of the blood. Hypoxia, due to the malfunction of the external respiratory system, causes the formation of compensatory changes, in the implementation of which involved components of the oxygen transport system. Molecular genetic mechanisms play an important role in the body's adaptation to oxygen deficiency. Fetal hemoglobin (FetHb), having an increased affinity for oxygen, makes a significant contribution to the body's adaptation to new conditions with altered gaseous environment in the presence of pathological processes occurring with hypoxia. In this regard, it is interest to determine FetHb in adults with COP to study its effect on the diagnosis, prognosis and outcome of the disease. The aim of the study. To determinate the participation of the organism adaptation mechanisms to the lack of oxygen according to the assessment of the content of fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia. Materials and methods. We examined 34 adult patients (18 women and 16 men) with COP, aged 18 to 80 years, who were in the therapeutic department of the City Clinical Hospital № 25 in Kharkiv. The control group was formed of 20 healthy individuals. Spirography was performed on the diagnostic complex "Valenta"; hematological examinations – on the analyzer "ADVIA 60"; measurement of pO2 and pCO2, oxygen saturation, content of fetal hemoglobin – on the device "RAPIDLAB865". Results. In patients with community-acquired pneumonia, there was a decrease of the ventilatory function of external respiration, which is confirmed by a marked decrease in partial oxygen pressure. Oxygen saturation of blood was reduced in the group of patients with COP, but the difference was not statistically significant 94.8 ± 1.0 %. This indicates the presence of compensatory mechanisms aimed at maintaining adequate blood oxygen saturation. Significant increase in pH (from 7.40 to 7.53) and decrease in standard bicarbonate (from 1.27 to 0.68 mmol/l) resulting from violation of the gas composition of the blood can be regarded as a manifestation of partially compensated respiratory alkalosis. In patients with COP, there was a reduction in the total time of hemolysis, a shift of the maximum erythrogram to the left and an increase in the maximum itself, indicating a sharp decline in erythrocyte resistance. The proportion of erythrocytes with reduced resistance was twice as large as similar forms in the control group and the number of highly resistant cells in patients with COP sharply decreased. Obviously, oxygen starvation-mediated stress erythropoiesis is accompanied by the entry into the circulation of functionally defective erythrocytes. They are subject to accelerated elimination from the vascular bed, which causes a decrease in the quantitative indicators of red blood (erythrocyte content, hemoglobin) while maintaining corpuscular parameters (Mean Corpuscular Volume, Mean Cell Hemoglobin Concentration). At the same time, the analysis of individual hemoglobin fractions revealed an increase in the proportion of fetal hemoglobin (from 2.90 ± 0.31 % in the group of healthy individuals to 5.43 ± 1.05 % in patients with COP) (p less than 0.05). Conclusions. Changes in the parameters of acid hemolysis, fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia with impaired pulmonary ventilation function indicate their participation in the mechanisms of adaptation to oxygen deficiency and they have informative potential. Elevated fetal hemoglobin in peripheral blood in these patients can be used as an indicator of hypoxia, accompanied by impaired oxygen delivery to tissues, which should be used as an additional criterion for diagnosing tissue hypoxia and justify the timely appointment of antihypoxia drugs. Keywords: hypoxia, community-acquired pneumonia, red blood cells, fetal hemoglobin.

Influence of ultraviolet radiation on kinetic characteristics in erythrocytes

Erythrocytes were irradiated with an ultraviolet lamp of the VMT type (λ = 253.7 nm). The kinetic characteristics in irradiated and non-irradiated blood samples were compared by the acid (chemical) erythrogram method and by the counter transport method. Obtained:
 - by the method of acidic erythrograms it was found that in ultraviolet-irradiated blood samples there is a decrease in the time of half hemolysis.
 - ultraviolet radiation increases the speed of counter movement of ions through erythrocyte membranes.
 - a decrease in the time of half hemolysis and an increase in the rate of counter transfer of ions, irradiated blood samples, is due to a decrease in the "effective" thickness of the near-membrane diffusion layer - an immiscible layer of water adjacent to the erythrocyte membrane.
 A decrease in the "effective thickness" of the near-membrane water layer changes the rate of metabolic processes in the "cell - intercellular medium" system, changing the mode of cell functioning. The altered mode of functioning is a biological response to light radiation. Red blood cells with an altered mode of functioning are stimulus signals that force the body to mobilize resources to fight pathology. These circumstances can predict the creation of a universal phototherapeutic equipment for extracorporeal blood irradiation based on LEDs with certain exposure parameters. Figs.: 4. Tabl.: 3. Refs.: 12 titles.

Influence of cardiopulmonary bypass on the erythrocyte membranes and the method of its protection

The damage to erythrocytes during cardiopulmonary bypass (CPB) remains a recent problem. The aim of this research was to study the effect of fructose-1,6-diphosphate on the state of the erythrocyte membrane during CPB and the level of phosphorus in blood as a marker of the energy potential in the cell. Patients were divided into two groups. The control group 1 (Gr 1) consisted of 75 individuals. The group 2 (Gr 2) included patients to whom fructose-1,6-diphosphate (FDP) was administrated according to the developed scheme as follows 10 g of the drug was diluted in 50 ml of a solvent, 5 g of the drug was injected intravenously with the use of perfusor immediately before initiation of CPB at a rate of 10 ml/min and 5 g at the 30th minute of CPB (before the stage of warming) the same way. When comparing two groups the best results in hemolysis (p<0.01), mechanical (p<0.01). osmotic resistance of erythrocytes (p<0.01), the time of acid hemolysis (p<0.01) and the permeability of the erythrocyte membrane in postperfusion period were in Gr 2. Вefore cardiac surgery hypophosphatemia was detected in 18% out of 150 and in 32% out of 150 patients – a lower limit of normal phosphorus content in the blood. After CPB in Gr 1 phosphorus content in blood was 0.85±0.32 mmol/l and hypophosphatemia was in 53% out of 75 patients. This indicates a pronounced energy deficit in this group. In Gr 2 phosphorus level was 1.7±0.31 mmol/l and there was no hypophosphatemia. As a result, FDP as an endogenous high-energy intermediate metabolite of the glycolytic pathway leads to resistance to hemolysis, protects the erythrocyte membrane from damage and increases the energy potential of the cell during CPB.

Hematological parameters and protein metabolism in the blood of pregnant rats under the effect of vanadium citrate

Dose-dependent changes in protein metabolism in the blood and hematological parameters of pregnant rats under the effect of vanadium citrate are presented in the article. The animals were divided into five groups: group I – non-pregnant females, II – pregnant females consuming pure water without additives, III, IV, V – females which during the mating and pregnancy period received the solution of vanadium citrate at concentrations of 0.03, 0.125 and 0.50 μg V/mL water. The research findings show that in pregnant animals of group II, the level of urea and alkaline phosphatase activity increased, meanwhile aspartate aminotransferase activity decreased, as compared to the non-pregnant females of group І. The levels of total protein and albumin decreased; however, the content of β-globulins increased in the pregnant animals of group II, as compared with that in group I. Also, in the rats of group II, there was a decrease in hemolysis time, total content of erythrocytes and hemoglobin, the content of old and mature erythrocytes, while the content of young erythrocytes increased, as compared to group I. The platelet content and thrombocrit in rats of group II increased in comparison with group I. The content of leukocytes and lymphocytes in pregnant animals of group II decreased, while the content of granulocytes increased, in contrast to non-pregnant rats. Under the effect of vanadium citrate at concentrations of 0.03–0.50 μg V/mL, there was a significant increase in the maximum number of prohemolized erythrocytes, the time of maximum hemolysis was delayed by 0.4–0.6 min, as compared with the pregnant rats of group II. This did not affect the time of total hemolysis in rats of groups III and V, as compared with the pregnant animals in group II. Under the effect of vanadium citrate, an increase in the content of young erythrocytes was observed, as compared with group II. The hemoglobin content decreased at the concentration of 0.125 μg V/mL, while at the concentration of 0.50 μg V/mL it increased, as compared to the pregnant animals of group II. Also, under the effect of vanadium citrate there was a decrease in the mean hemoglobin concentration in the erythrocyte. In pregnant animals fed with vanadium citrate solutions, the platelet content and thrombocrit, the relative width of platelet distribution by volume decreased, as compared with the pregnant rats of group II. The content of leukocytes, lymphocytes and granulocytes under the effect of vanadium citrate increased, as compared with the pregnant animals in group II. Under the effect of vanadium citrate at the concentration of 0.03 μg V/mL, the level of albumin, creatinine and aspartate aminotransferase activity increased in blood plasma in comparison with group II. Meanwhile, at the concentration of 0.125 μg V/mL, the relative content of γ-globulins and aspartate aminotransferase activity increased, alkaline phosphatase activity and urea level decreased in comparison with group II. However at the concentration of 0.50 μg V/mL, the relative α- and γ-globulins content and aspartate aminotransferase activity increased, at the same time, the relative β-globulins content and urea level decreased in comparison with group II. Therefore, vanadium citrate normalizes the indicators of protein metabolism during pregnancy, thus it can be considered as a potential dietary drug for the pregnant.

Когерентне та монохроматичне випромінювання змінює час гемолізу еритроцитів, а також швидкість зустрічного переміщення іонів крізь еритроцитарну мембрану

The erythrocytes of human blood were sequentially irradiated with a low-intensity laser (λ = 640 nm), a violet LED (λ = 400 nm), a green LED (λ = 540 nm), and a yellow LED (λ = 592 nm). The method of acid (chemical) erythrograms and the method of counter ion transport compared the kinetic characteristics in irradiated and unirradiated blood samples. Received: - by the method of acid erythrograms it was found that in the irradiated blood samples there is a decrease in the time of hemolysis; - low intensity laser radiation, as well as the emission of LEDs, increase the rate of counter ion transport through red blood cells. - a decrease in the time of hemolysis and an increase in the rate of counter ion transport of irradiated blood samples is due to a decrease in the "effective" thickness of the near-membrane diffusion layer - an immiscible layer of water adjoined to the erythrocyte membrane. A decrease in the "effective” thickness of the near-membrane water layer (minimal in the wavelength range of 570-590 nm and 630-640 nm) changes the rate of metabolic processes in the "cell - intercellular medium" system, changing the mode of cell functioning. The altered mode of functioning is a biological response to light radiation. Red blood cells with an altered mode of functioning are signals - stimuli that cause the body to mobilize resources to fight pathology. These circumstances can predict the creation of a universal phototherapeutic equipment for extracorporeal blood irradiation based on light-emitting diodes with certain exposure parameters.

Intake of dietary advanced glycation end products influences inflammatory markers, immune phenotypes, and antiradical capacity of healthy elderly in a little-studied population.

Dietary advanced glycation end products (dAGE) have profound negative effects on overall health, and their intake must be assessed. In this cross‐sectional study, we investigated dAGE intake of 337 adult participants (180/157:M/F; age range 50–73 years). Data were collected on anthropometrics, body composition, dietary intake, selected blood biochemistry, immunological parameters, and antiradical capacity (50% hemolysis time; HT50). From the dietary data, dAGEs and phytochemical index (PI) were calculated. Mean BMI, % body fat (%BF), and fasting plasma glucose were all within the accepted normal range. Subjects with high dAGE intake had higher %BF, higher energy intake, and lower PI. They tended to have lower CD4/CD8 ratios and higher proportions of B cells and NK cells, but had significantly higher hs‐CRP levels and lower HT50 values. Results on HT50 suggested that being >60 years of age enhanced dAGE‐associated impairment of defense capacity in both those with low and high HT50 compared with those <60 years of age. Thus, overall dAGE consumption was high, but elderly participants had lower dAGE intake than younger adults. Indicators of nutritional status and immunological parameters of the subjects were found to be associated with dAGE intake, suggesting a potential impact on health.

Implementation of the method of acidic erythrograms to reveal the membranotropic effect of biologically active peptides

The method of acidic erythrograms was used to study the membranotropic effect of biologically active peptides (BAP). The air exposure of erythrocyte suspension in isotonic sodium chloride solution caused a decrease in their resistance to acid hemolysis: the time of hemolysis decreased, the integral indicator of erythrocyte resistance decreased too. The air exposure of erythrocyte suspension with the BAP solutions (10-8M) changed the hemolytic resistance of erythrocytes. It was found out that a non-opiate analogue of leu-enkephalin (NALE – Phe-D-Ala-Glu-Phe-Leu-Arg), Selank (Thr-Lys-Pro-Arg-ProGly-Pro) and Semax (Met-Glu -His-Phe-Pro-Gly-Pro) increase the resistance of erythrocyte membranes to acid hemolysis, and the Arg-Gly-Arg-Pro-Gly-Pro peptide decreases the resistance of erythrocyte membranes to acid hemolysis. Membranotropic action can determine the nonspecific effects of BAP and the range of their clinical use. Acidic erythrograms can be used as a screening method for the selection of BAP with membranotropic effects.

Nutritional, immunological and antioxidant defense status of outpatients diagnosed with colorectal cancer – a case–control study of the little-studied population

This cross-sectional study was conducted to investigate nutritional and immunological status of colorectal cancer (CRC) patients in a little-studied population from developing country, Pakistan. Data on 81 CRC patients and 37 healthy controls (HCs) were collected on nutritional status, nutrient intake, percent body fat (%BF), selected immunological parameters, phytochemical index (PI), healthy eating index (HEI), and prognostic nutrition index (PNI). Blood samples were used for immunological and antiradical defense potential (expressed as 50% hemolysis time; HT50). Results show 40/81 (49.4%) patients reported weight loss in past 3–6 mo, Significant differences were found in HEI values between patients vs. HCs, and between patients in low vs. high PNI groups (P, for all trends <0.05). Patients in the higher PNI group were heavier, had higher % BF, higher energy intake, and higher PI score as compared to patients in the low PNI group (P < 0.05). Low PNI was positively associated with non-significantly lower CD4:CD8 ratios, higher B-cells and NK cells (P, for all trends >0.05), but with significantly higher hs-CRP levels, and lower HT50 values (P, for all trends <0.001). In conclusion, CRC patients in a little-studied population have compromised nutritional and immunological health with lower HEI and PNI scores.

Intradonor reproducibility and changes in hemolytic variables during red blood cell storage: results of recall phase of the REDS-III RBC-Omics study.

Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study. RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency. A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001). Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.

Parameters of erythrocytopoiesis, acid resistance and population composition of erythrocytes of cows with chronic hematuria

Clinically, chronic hematuria is a disease characterized mainly by symptoms of massive cystogamia, which can be constant or periodic and is accompanied by common phenomena of anemia. The object of the study was the blood of clinically healthy and chronic hematuria cows of the Brown Carpathian breed. Based on clinical studies, there are two stages of the disease in cows: subclinical and clinically pronounced. In turn, the clinically expressed stage of the disease is divided into medium and severe. The last stage was accompanied by massive hematuria. To study the pathogenesis of chronic hematuria of cattle, data of erythrocytopoiesis, acid resistance and population composition of erythrocytes in their blood are of great importance. Anemia in cows with an average degree of hematuria is characterized by oligocythemia and oligochroma and was mainly hyperchromatic macrocytic. Anemia due to severe course of the disease is hyperchromic and macrocytic, both indicators (MCH and MCV) are significantly higher than in cows for subclinical and moderate hematuria. Analysis of the population composition of blood erythrocytes indicates that the response of cellular populations is ambiguous. It was found that in cows with chronic hematuria, the relative number of “ old ” populations of erythrocytes that actively participate in oxygenation processes is decreased. The picture was similar with the population of “ mature ” , most functionally active red blood cells. In particular, the number of “ mature ” cells in comparison with healthy cows was decreased by 7.0%. When studying the “ young ” forms of red blood cells, it was found that in the blood of cows suffering from chronic hematuria, they were growing, depending on the stage of the disease course. It is established, that the number of “ young ” populations of erythrocytes in the blood of cows in chronic hematuria was increased by 11.1% compared with clinically healthy animals. A significant decrease in the number of “ old ” and increasing “ young ” erythrocytes in the blood of cows suffering from chronic hematuria is a compensatory phenomenon in the development of hypoxia. Oxygen starvation due to anemia causes bone marrow irritation. Erythrogram in sick cows was characterized by a longer time of hemolysis of erythrocytes up to 6 – 7 minutes (in clinically healthy cows is completed by 5.5 min.). Erythrogram of cows in subclinical course of chronic hematuria peak of hemolysis accounted for 3 min (22.9%), whereas in cows with an average course of chronic hematuria, the acid hemolysis of erythrocytes was 3.5 minutes and its height was 15.5% of the hemolysed cells. Sick cows with a massive degree of course of chronic hematuria, the erythrogram had two peaks: for 3.5 min and 4.5 min, its height was respectively 12.9 and 11.7% of hemolyzed cells.

P 206 - Quercetin supplementation decreases erythrocytes oxidative damage at resting and after an acute bout of eccentric exercise in humans

Quercetin (Q) functions as antioxidant in vitro, but its effect have been minimally examined in combination with exercise in humans. The purpose of this investigation was to determine the effects of a diet supplemented with 1 g per day of Q for 2 weeks on the erythrocytes oxidative balance before and after an acute bout of eccentric exercise (EE). Fourteen volunteer males were randomly assigned, in a double-blind crossover design, to a placebo or experimental supplemented groups. Blood samples were taken before and after 2 weeks of supplementation under resting and post-exercise conditions. Erythrocytes glutathione (GSH, GSSG, GSH/GSSG), malonaldehyde (TBARs), enzymes antioxidant activities as well as time of hemolysis were evaluated in ex vivo. Quercertin per se did not affect redox homeostasis but increased the time of hemolysis and decreased TBARs levels. Following the EE the Q group displayed a higher GSH/GSSG ratio and a less pronounced increase in TBARs, compared to placebo group. Moreover, we found that GPx enzyme activity were induced after EE only in Q group, while any significant modification of this parameter was detected in placebo group. In conclusion, the Q supplementation may be used as a countermeasure against oxidative stress inducing, in erythrocytes, a cellular adaptation allowing subjects to better cope with the oxidative stress induced by an acute exercise.

A microplate assay to measure classical and alternative complement activity.

We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Individualized correction of insulin measurement in hemolyzed serum samples.

Insulin measurement plays a key role in the investigation of patients with hypoglycemia, subtype classification of diabetes mellitus, insulin resistance, and impaired beta cell function. However, even slight hemolysis can negatively affect insulin measurement due to RBC insulin-degrading enzyme (IDE). Here, we derived and validated an individualized correction equation in an attempt to eliminate the effects of hemolysis on insulin measurement. The effects of hemolysis on insulin measurement were studied by adding lysed self-RBCs to serum. A correction equation was derived, accounting for both percentage and exposure time of hemolysis. The performance of this individualized correction was evaluated in intentionally hemolyzed samples. Insulin concentration decreased with increasing percentage and exposure time of hemolysis. Based on the effects of hemolysis on insulin measurement of 17 donors (baseline insulin concentrations ranged from 156 to 2119pmol/L), the individualized hemolysis correction equation was derived: INScorr=INSmeas/(0.705lgHbplasma/Hbserum-0.001Time-0.612). This equation can revert insulin concentrations of the intentionally hemolyzed samples to values that were statistically not different from the corresponding insulin baseline concentrations (p=0.1564). Hemolysis could lead to a negative interference on insulin measurement; by individualized hemolysis correction equation for insulin measurement, we can correct and report reliable serum insulin results for a wide range of degrees of sample hemolysis. This correction would increase diagnostic accuracy, reduce inappropriate therapeutic decisions, and improve patient satisfaction with care.

Hemolysis Affects C‐Peptide Immunoassay

C-peptide is used widely as a marker of insulin secretion, and it participates in the inflammatory response and contributes to the development of coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM). Previous studies have reported that C-peptide measurement was unaffected by hemolysis. However, we found that hemolysis negatively affected C-peptide assay in routine laboratory practice. We further established and validated an individualized hemolysis correction equation to correct and report accurate serum C-peptide results for hemolyzed samples. We studied the effects of hemolysis on C-peptide assay by adding lysed self red blood cells (self-RBCs) to serum. An individualized correction equation was derived. Further, we evaluated the performance of this individualized correction equation by artificially hemolyzed samples. C-peptide concentration decreased with increasing degree and exposure time of hemolysis. The individualized hemolysis correction equation derived: C-Pcorr = C-Pmeas /(0.969-1.5Hbserum/plasma -5.394 ×10-5 Time), which can correct bias in C-peptide measurement caused by hemolysis. Hemolysis negatively affects C-peptide measurement. We can correct and report accurate serum C-peptide results for a wide range of degrees of sample hemolysis by individualized hemolysis correction equation for C-peptide assay. This correction would improve diagnostic accuracy and reduce inappropriate therapeutic decisions.

The study on the influence of blood specimen collection to the hospital inspection results

Objective To investigate the blood specimen collection site, inspection time and hemolysis influence on biochemical test results. Methods The specimens of 2586 cases outpatients and hospitalized patients in our hospital who accepted blood routine and blood coagulation four check were analyzed.The hemolysis and qualified specimens of biochemical test results were compared. Results 2 586 specimens found unqualified blood samples in 18 cases, and the incidence rate was 0.70% (18/2 586). Samples of different section and source were compared and found there were no significant difference (F=0.36, P>0.05). In 18 cases of unqualified blood samples due to technical problems and unqualified sample accounted for 88.89% (16 / 18). The technical factors that lead to unqualified samples was significantly higher than that of the instrument factor or fixed factors, and the difference was statistically significant(χ2=152.63, 98.52, all P<0.05). In addition, hemolysis caused the most of the unqualified samples for the technical factors.To choose potassium infusion on the side ipsilateral to the site or from the opposite side of the site collected blood samples.Sodium, chlorine, creatinine, uric acid, glucose detection found in effect of blood on the same side of the infusion of the biochemical test results in varying degrees.Potassium, chlorine, sodium, creatinine decreased significantly, and uric acid, glucose and other indicators were significantly increased (t=4.51, 4.98, 5.50, 3.25, 10.18, 15.25, all P<0.05). Gamma transpeptidase, creatine kinase, sodium and uric acid and other index of hemolytic samples were significantly lower than those of normal samples, and alanine aminotransferase (ALT), direct bilirubin, potassium, phosphorus, serum creatinine and creatine kinase were significantly higher than those of the normal samples (t=14.85, 21.21, 7.15, 4.86, 10.33, 4.02, 3.11, 8.20, 7.54, 5.11, all P<0.05). This may cause the depth in erythrocytes and some related substances.The time of collecting blood to sent to the room had obvious influence on the biochemical test results, alanine aminotransferase, aspartate amino transferase, glucose, creatine kinase and hydroxybutyrate and project them 1 h after submission were significantly lower than those of the standard inspection, and 1 hour after the submission of lactate dehydrogenase and creatine kinase isoenzyme index were significantly higher than those of the standardize inspection means (t=2.95, 4.82, 2.88, 5.44, 4.05, 3.98, 4.89, all P<0.05). Conclusion Sampling sites, hemolysis and inspection time had effect on blood specimen, so for the collection of blood samples must be strictly in accordance with the requirements of the operation. Key words: Blood specimen; Collection; Biochemical test