Acrolein Induces Changes in Cell Membrane and Cytosol Proteins of Erythrocytes.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

Modification of deglycerolization procedure improves processing and post-thaw quality of cryopreserved sickle trait red cell concentrates.

Red blood cell (RBC) transfusion is a critical therapy for those with sickle cell disease (SCD). Alloimmunization is frequent for those with SCD and may limit the availability of matched RBC. Cryopreserved RBCs, from family members or donors with a similar RBC antigen profile could provide a viable alternative to avoid further alloimmunization and prevent hemolytic transfusion-related events. However, cryopreserved SCD and Sickle Cell trait (S-trait) donor RBC units suffer from reduced recovery following deglycerolization. This study proposes and tests a modified deglycerolization protocol using an automated cell processor to mitigate RBC loss. Six red cell concentrates (RCC) from donors with S-trait and six control RCCs were glycerolized, frozen (<-65 °C) and deglycerolized on the ACP 215 using modified parameters (decreased hypertonic solution flow rate (100 mL/min) and hypertonic equilibration delay (120 s), and increased NaCl dilution volumes (500 mL). Quality testing included: hematocrit (HCT), hemolysis, indices, extracellular potassium, morphology, osmotic fragility, osmotic gradient ektacytometry, hemoglobin (HGB), and recovery. Canadian standards (CS) indicate that acceptable deglycerolized units for transfusion require a HCT ≤0.80 L/L, HGB ≥35 g/unit, and hemolysis <0.8 % in 90 % of units tested. No significant differences in HGB or RBC recovery were observed between study groups. Significant differences between study groups were identified in osmotic fragility and osmotic gradient ektacytometry parameters. Of the 6 S-trait RCCs, 3/6 units were within the HCT, HGB and hemolysis thresholds set by the CS. The modified deglycerolization protocol provides a path for the routine cryopreservation of S-trait RBCs.

Membrane-Bound Ferric Hemoglobin in Nucleated Erythrocytes of the Black Scorpionfish Scorpaena porcus, Linnaeus 1758.

The content of membrane-bound methemoglobin (MtHb) in nucleated erythrocytes was studied in the black scorpionfish Scorpaena porcus (Linnaeus, 1758) in vitro. Spectral characteristics were determined for a whole hemolysate, a hemolysate obtained by stroma precipitation (a clarified hemolysate), and a resuspended stroma. The MtHb proportion in the erythrocyte stroma was found to exceed 80% (6.20 ± 0.59 µM). Clarified hemolysates were nearly free of MtHb (0.5 ± 0.2 µM). Membrane-bound ferric hemoglobin did not affect the erythrocyte resistance to osmotic shock. The osmotic fragility range was determined using a LaSca-TM laser microparticle analyzer (BioMedSystems, Russia) to be 102-136 mOsm/kg, much the same as in other bony fish species. A nitrite load (10 mg/L) significantly increased the MtHb content in the blood. However, the membrane-bound ferric hemoglobin content did not change significantly, amounting to 6.34 ± 1.09 µM (approximately 95%). The finding suggested a functional importance for MtHb present in the plasma membrane of nucleated erythrocytes. Membrane-bound MtHb was assumed to neutralize the external oxidative load and the toxic effect of hydrogen sulfide in bottom water layers, where the species lives.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

Letter to the Editor: Comparing the Novel Parameter 'Slope of the Steepest Part of Hemolysis Curve' to Standard Median Corpuscular Fragility for Evaluating Erythrocyte Stability during Prolonged Storage.

OBJECTIVE: Several ways for presenting the results of osmotic fragility test have been described in the literature. Our aim was to compare the utility of a novel parameter for assessment of erythrocyte osmotic properties, i.e., 'Slope of the steepest part of hemolysis curve' with the most frequently used parameter 'Median corpuscular fragility' in order to assess the stability of erythrocytes in a blood sample during prolonged storage. METHODS: Ten whole blood samples were obtained from healthy donors. The osmotic fragility test was initially conducted on the day of venipuncture, and subsequent analyses were carried out on days 1, 2, 4, 7, 9, 11, and 14 after the venipuncture. Mean hemolysis percentage values were used to construct hemolysis curves. The steepest parts of hemolysis curves were estimated to be linear, and lines that overlapped those parts of the curves were created. The slope of these lines was calculated, and the resulting mean values are presented. RESULTS: A significant increase in Median corpuscular fragility values was observed, starting from day of venipuncture. We compared the average values for each day of analysis. The first significant difference in Median corpuscular fragility values was observed on day 4 compared to the day of venipuncture (p=0.006), with values 0.53±0.030 % and 0.41±0.014% respectively. Meanwhile, differences in the values of the slopes of the steepest parts of hemolysis curves were observed as early as day 2 when compared to the day of venipuncture (p=0.046), with values of -966.23±233.07 and -588.01±222.85, respectively. CONCLUSION: Prolonged storage of whole blood samples leads to an increase in osmotic fragility and alters the shape of the hemolysis curve. These changes suggest that postponing the osmotic fragility test could lead to diagnostic inaccuracies. These findings suggest that slope value is a more accurate parameter for evaluating erythrocyte stability during storage, compared to commonly used Median corpuscular fragility value. Hence, it has potential importance and can be complementary to the laboratory result of the OFT. Therefore, it can be useful to provide these results jointly with the results of the OFT test.

Role of Goldenberry (Fruits with Husk) Extract in Ameliorating the Architecture and Osmotic Fragility of Red Blood Cells in Obese Rats.

Goldenberry (GB) is a promising fruit that can be a constituent in many possible nourishments. No notifications were obtained regarding the impact of exposure to goldenberry extract in the viewpoint of blood rheological properties as well as erythrocyte osmotic fragility of red blood cells (RBCs) in obese rats. A substantial reduction in plasma triglyceride, total cholesterol, and LDL, with a considerable increment in HDL levels relative to the obese group (p ≤ 0.05), was observed in rats receiving low and high doses of GB, accompanied by restoration of SOD activity and GSH levels. Rheological parameters of rats' blood have been studied over a wide range of shear rates (225-1875 s-1). A significant decrease in blood viscosity in rats who received low and high doses of GB extract was compatible with every shear rate compared to the control group. The shear stress values of the obese rats reduced appreciably (p ≤ 0.05) in all values of shear rate (from 75 to 500 s-1) proportional to the control group, while in the groups that received low and high doses of GB extract, shear stress was restored to the control values. Finally, administration of GB extract significantly decreased yield stress and indices of whole blood aggregation, with an extremely substantial increment in flow rate, in rats given low or high doses of GB compared to obese ones. The result also showed a decrease in both the average raised osmotic fragility and the hemolysis rate in rats after supplementation with low and high doses of GB extract.

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.

Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Clinical Performance Study of a New Fully Automated Red Blood Cell Permeability Fragility Analyzer.

In order to verify the applicability of the erythrocyte fragility test (EFT) carried out by the new fully automatic erythrocyte permeability fragility analyzer RA-800 for thalassemia screening, a total of 100 cases of suspected thalassemia patients who underwent pregnancy examinations at Luohu District People's Hospital are included. The results of a new automatic erythrocyte permeability fragility analyzer RA-800 are compared with the results of the detection system composing of the KOFA erythrocyte fragility test kit currently used in clinical laboratories. The diagnosis confirmed by genetic testing is used as the gold standard to evaluate the applicability of RA-800. The sensitivity, specificity, and accuracy of the new automatic erythrocyte permeability fragility analyzer RA-800 screening for thalassemia were 66.67%, 92.86%, and 85.00%. The KOFA direct colorimetries are 76.67%, 81.43%, and 80.00%. The kappa value for the screening of thalassemia was 0.558, which concludes that the consistency was moderate. The ROC curve indicates that both two methods had diagnostic significance for the diagnostic value of thalassemia. The new automatic erythrocyte permeability fragility analyzer RA-800 is suitable for thalassemia screening, and the performance indexes meet the clinical requirements.

Clasificación y métodos de diagnóstico de las membranopatías / Classification and diagnostic methods of membranopathies

Introducción: Las membranopatías son anemias hemolíticas hereditarias debidas a anomalías cualitativas o deficiencias cuantitativas de las proteínas del citoesqueleto del glóbulo rojo. Objetivo: Actualizar el diagnóstico de las membranopatías con la inclusión de las últimas recomendaciones del comité de grupos de expertos a nivel nacional e internacional. Métodos: Se realizó una revisión de la literatura en inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico, de artículos publicados en los últimos cinco años. Análisis y síntesis de la información: Las enfermedades de mayor interés clínico son: la esferocitosis, la eliptocitosis y la estomatocitosis hereditaria. Estas en general se heredan con carácter autosómico dominante pero existen formas que se transmiten con carácter recesivo, sin descartar posible mutación de novo. Para su diagnóstico se utilizan pruebas que incluyen el estudio de la morfología de los glóbulos rojos, la fragilidad osmótica, la lisis de glicerol acidificado, la criohemólisis hipertónica, la prueba de unión a la eosina-5'-maleimida por citometría de flujo, la electroforesis en gel de poliacrilamida con dodecilsulfato sódico y la ectacitometría. Conclusiones: Las membranopatías pueden sospecharse de manera preliminar teniendo en cuenta algunas alteraciones de la morfología eritrocitaria, aunque el diagnóstico se basa en estudios familiares y otros de carácter confirmatorio de la enfermedad, como los estudios moleculares. Los profesionales de la salud que atienden a pacientes jóvenes con anemia deben considerar la posibilidad de una anemia hemolítica por trastornos de la membrana eritrocitaria(AU)

ABSTRACT Introduction: Membranopathies are inherited hemolytic anemias due to qualitative abnormalities or quantitative deficiencies of red blood cell cytoskeletal proteins. Objective: to update the diagnosis of membranopathies with the inclusion of the latest recommendations from the committee of expert groups at the national and international level. Methods: A review of the literature in English and Spanish was carried out, through the PubMed website and the academic search engine Google, in articles published in the last five years. Analysis and synthesis of information: The diseases of greatest clinical interest are: spherocytocis, elliptocytosis and hereditary stomatocytosis. These are generally inherited with an autosomal dominant character but there are forms that are transmitted recessively, without ruling out a possible de novo mutation. For its diagnosis, tests are used that include the study of red blood cell morphology, osmotic fragility, acidified glycerol lysis, hypertonic cryohemolysis, eosin-5'-maleimide binding test by flow cytometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ectacytometry. Conclusions: Membranopathies can be preliminarily suspected taking into account some alterations in erythrocyte morphology, although the diagnosis is based on family studies and others confirming the disease, such as molecular studies. Healthcare professionals caring for young patients with anemia should consider the possibility of hemolytic anemia due to red cell membrane disorders(AU)

Cord and peripheral blood erythrocyte analysis by scanning electron microscopy and flow cytometry.

INTRODUCTION: Human umbilical cord blood is rich in hematopoietic cells. We aimed to focus on the morphological, biochemical, membrane protein profile and surface protein expression differences of erythrocytes, isolated from cord and adult peripheral blood using techniques such as high-resolution scanning electron microscopy (SEM), gel electrophoresis (SDS-PAGE) and flow cytometry. METHODS: Adult peripheral blood was collected from consenting adults, and umbilical cord blood was procured from consenting mothers, post-delivery at Medical College, Kolkata. We emphasized on cord and adult peripheral blood erythrocytes' morphological variations using SEM images and protein expression by flow cytometric analysis. Some conventional biochemical analyses such as osmotic fragility of the cell membrane, haemoglobin co-oxidation study and lipid peroxidation assay were done for supporting evidence along with membrane protein content using gel electrophoresis. RESULTS: Our SEM images indicated clear morphological variations in cord erythrocyte with a higher degree of cellular deformities and difference in membrane texture. Flow cytometric analysis of cord erythrocyte showed a significant difference in CD235a expression than adults. We observed an overexpression of GLUT1 and decreased expression of Band 3 in cord erythrocyte membrane. Our results also showed cord erythrocytes have low osmotic fragility, a slower rate of co-oxidation of cord haemoglobin and a lesser lipid peroxidation level than that of adults. CONCLUSION: Cord blood erythrocytes have deeper indentations leading to higher flexibility, more oxygen-carrying capacity and less osmotic fragility in comparison with adult erythrocytes. The expression of CD235a and Band 4.5 (GLUT 1) was significantly higher in cord erythrocytes than peripheral adult erythrocytes.

Conclusions about osmotically inactive volume and osmotic fragility from a detailed erythrocyte model.

This paper develops a detailed model of human and bovine erythrocytes quantifying the dependence of total cell volume upon composition of an aqueous solution in which it is immersed. The cytoplasm is represented as an aqueous solution of hemoglobin and salt (KCl or NaCl). Model-based analysis of literature data on human erythrocytes, and of new experiments with bovine erythrocytes, leads to two findings. First, the Boyle-van't Hoff plot for human erythrocytes is found to be well described based on a solid volume fraction of âˆ¼0.3 in complete agreement with desiccation experiments. The linear portion of the calculated curve turns out to be numerically indistinguishable from the commonly used ideal model parameterized with an apparent osmotically inactive volume fraction of âˆ¼0.5. This mathematical outcome explains the longstanding perceived (but actually nonexistent) disconnect between the aforementioned fractions âˆ¼0.3 and âˆ¼0.5. A corollarial implication is that the actual volume fraction of osmotically nonparticipant (vicinal) water is very small (∼0.035). Second, an initial crenation of bovine erythrocytes (which occurs in classical techniques for measuring membrane permeability) is found to increase their fragility to an extent which correlates well with the crenated cell volume, and would affect the permeability determination.

A Systematic review on diagnostic methods of red cell membrane disorders in Asia.

Membranopathies are a group of inherited blood disorders where the diagnosis could form a challenge due to phenotype-genotype heterogeneity. In this review, the usage and limitations of diagnostic methods for membranopathies in Asian countries were evaluated. A systematic review was done using articles from PubMed, Google Scholar, and EBSCO from 2000 to 2020. Thirty-six studies conducted in seven Asian countries had used different diagnostic methods to confirm membranopathies. In 58.3% of studies, full blood count (FBC), reticulocyte count, and peripheral blood smear (PBS) were used in preliminary diagnosis. The combination of the above three with osmotic fragility (OF) test was used in 38.8%. The flowcytometric osmotic fragility (FC-OF) test was used in 27.7% where it showed high sensitivity (92%-100%) and specificity (96%-98%). The eosin-5-maleimide (EMA) assay was used in 68.1% with high sensitivity (95%-100%) and specificity (93%-99.6%). About 36.1% of studies had used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a further diagnostic method to detect defective proteins. Genetic analysis to identify mutations was done using Sanger sequencing, next-generation sequencing (NGS), and whole-exome sequencing (WES) in 33.3%, 22.2%, and 13.8% of studies, respectively. The diagnostic yield of NGS ranged from 63% to 100%. Proteomics was used in 5.5% of studies to support the diagnosis of membranopathies. A single method could not diagnose all membranopathies. Next-generation sequencing, Sanger sequencing, and proteomics will supplement the well-established screening and confirmatory methods, but not replace them in hereditary hemolytic anemia assessment.

Effect of microaggregate filter passage on feline whole blood stored for 35 days.

OBJECTIVES: The aim of this study was to compare the characteristics of fresh and stored feline red blood cells (RBCs) after passage through an 18 µm microaggregate filter. METHODS: Nine cats were recruited for a single blood donation using an open collection system. A simulated transfusion using a syringe driver and microaggregate filter was performed over 2 h with half the blood on the day of donation and the other half after 35 days of storage. Differences in haematological parameters, haemolysis percentage and osmotic fragility (OF) were compared on the day of donation pre-filter passage (D0-) vs day of donation post-filter (D0+) or day 35 storage pre-filter (D35-) and post-filter (D35+). Blood was cultured at D0+ and D35+. RESULTS: There were no statistically significant differences in the D0- vs D0+ comparisons. There were statistically significant (P <0.05) increases in haemolysis percentage, red cell distribution width (RDW) percentage and mean OF, and decreases in packed cell volume (PCV), RBC count, haemoglobin and haematocrit for D0- vs D35-. The same was found for D0- vs D35+ with the addition of a significant increase in mean cell haemoglobin (MCH). For D35- vs D35+ only MCH significantly increased. At day 35, 6/9 units had haemolysis percentages that exceeded 1%. This increased to 8/9 of stored units post-filter passage. All blood units cultured negative. CONCLUSIONS AND RELEVANCE: Fresh RBCs exhibited no in vitro evidence of injury following passage through an 18 µm microaggregate filter. Increased MCH was observed in the stored blood and may represent haemolysis induced by the filter. All other changes can be explained by storage lesion rather than filter passage. The findings highlight the importance of blood banking quality controls and the need for further research to assess the effects of transfusion technique, specifically filter passage, on storage lesion-affected feline blood.

Comparison of a modified flow cytometry osmotic fragility test with the classical method for the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. The flow cytometric test using eosin-5'maleimide (EMA) is a well-established diagnostic method. However, in order to improve HS detection, it is recommended that EMA and an osmotic fragility test (OFT) both be performed. OFT is time consuming and labor intensive. We used a flow cytometric (FOFT) adaptation of the classical OFT reported by Yamamoto. We compare the FOFT to the classical OFT including practical data and propose options for simplifying this method. METHODS: Suspected and known HS patients and controls were tested by the following methods: EMA, OFT, and FOFT including some modifications. RESULTS: The FOFT method is robust and correlates to loss of red blood cells. OFT and FOFT gave similar results in healthy controls and four HS patients. Normal range for FOFT in 70 adults is shown and can be used as a reference value. Neonates should have their own normal range defined. Overnight sample incubation at 37°C did not add information to the FOFT results. CONCLUSION: Our modified Yamomoto FOFT can replace the classic OFT as the addition to EMA for the diagnosis of HS. The use of flow cytometry in both these methods requires small sample volume, is reproducible, simpler, and produces results more rapidly.

Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility-A Comparative GP.Mur Erythrocyte Study.

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3-AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3-AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural-functional roles of band 3.

Análise qualitativa do teste de fragilidade osmótica para amostras processadas a fresco ou após 24 horas de incubação a 37ºC / Qualitative analysis of the osmotic fragility test for samples processed fresh or after 24 hours of incubation at 37ºC

Objetivo: Analisar qualitativamente o teste de fragilidade osmótica (F.O.) para amostras a fresco ou após incubação a 37°C. Métodos: Foram processadas 20 amostras de sangue periférico, coletadas em duplicata com 5mL em cada tubo com heparina, de pacientes com solicitação de F.O. como exame de rotina para processamento a fresco e após incubação por 24 horas em banho-maria a 37°C, em 13 tubos com concentrações variáveis de 0,1% a 0,9% de NaCl. Resultados: Foram analisadas 20 amostras de pacientes em sua maioria do gênero feminino 17/20 (85%), com idades entre 3 meses a 75 anos, para realização do teste de F.O. A análise qualitativa dos resultados mostrou que 9/20 (45%) amostras tiveram resultado concordante entre os testes de F.O. para amostras a fresco e após incubação a 37°C. Dos resultados discordantes, 8/11 (72,7%) resultados mostram fragilidade dos eritrócitos à hemólise nas amostras a fresco e curva normal (sem hemólise) após incubação da amostra a 37°C. Outros 3/11 (22,3%) resultados apresentaram curva normal (sem hemólise) no teste com amostra à fresco e resistência à hemólise no teste com a amostra após incubação a 37°C. Com o teste de Extato de Fisher não mostrou diferença estatística (p=0,5743) para as amostras processadas a fresco ou após incubação a 37°C. Conclusão: O teste de F.O. se mostrou mais eficiente quando a amostra testada foi analisada após incubação por 24 horas a 37°C em banho-maria, contudo não houve diferença estatística para resultados processados a fresco ou após incubação a 37°C.

Objective: Qualitatively analyze the osmotic fragility test (O.F.) for samples fresh or after incubation at 37°C. Methods: Twenty peripheral blood samples were processed, collected in duplicate with 5 ml in each tube with heparin, from patients with O.F. request. as a routine examination for fresh processing and after incubation for 24 hours in a water bath at 37°C, in 13 tubes with varying concentrations of 0.1% to 0.9% NaCl. Results: Twenty samples of patients were analyzed, mostly female, 17/20 (85%), aged between 3 months to 75 years, for the O.F. test. The qualitative analysis of the results showed that only 9/20 (45%) samples had a consistent result between the F.O. tests for fresh samples and after incubation at 37°C. From the discordant results, 8/11 (72.7%) results show fragility of erythrocytes to hemolysis in fresh samples and normal curve (without hemolysis) after sample incubation at 37o C. Other 3/11 (22.3%) results showed normal curve (without hemolysis) in the test with fresh sample and resistance to hemolysis in the test with the sample after incubation at 37o C. With the Fisher Extact test showing no statistical difference (p=0.5743) for samples processed fresh or after incubation at 37°C. Conclusion: The O.F. proved to be more efficient when the tested sample was analyzed after incubation for 24h at 37°C in a water bath, however, there was no statistical difference for results processed fresh or after incubation at 37°C.

Minor Changes in Erythrocyte Osmotic Fragility in Trace Amine-Associated Receptor 5 (TAAR5) Knockout Mice.

Trace amine-associated receptors (TAARs) are a group of G protein-coupled receptors that are expressed in the olfactory epithelium, central nervous system, and periphery. TAAR family generally consists of nine types of receptors (TAAR1-9), which can detect biogenic amines. During the last 5 years, the TAAR5 receptor became one of the most intriguing receptors in this subfamily. Recent studies revealed that TAAR5 is involved not only in sensing socially relevant odors but also in the regulation of dopamine and serotonin transmission, emotional regulation, and adult neurogenesis by providing significant input from the olfactory system to the limbic brain areas. Such results indicate that future antagonistic TAAR5-based therapies may have high pharmacological potential in the field of neuropsychiatric disorders. TAAR5 is known to be expressed in leucocytes as well. To evaluate potential hematological side effects of such future treatments we analyzed several hematological parameters in mice lacking TAAR5. In these mutants, we observed minor but significant changes in the osmotic fragility test of erythrocytes and hematocrit levels. At the same time, analysis of other parameters including complete blood count and reticulocyte levels showed no significant alterations in TAAR5 knockout mice. Thus, TAAR5 gene knockout leads to minor negative changes in the erythropoiesis or eryptosis processes, and further research in that field is needed. The impact of TAAR5 deficiency on other hematological parameters seems minimal. Such negative, albeit minor, effects of TAAR5 deficiency should be taken into account during future TAAR5-based therapy development.

Changes in Human Erythrocyte Membrane Exposed to Aqueous and Ethanolic Extracts from Uncaria tomentosa.

Uncaria tomentosa (Willd.) DC is a woody climber species originating from South and Central America that has been used in the therapy of asthma, rheumatism, hypertension, and blood purification. Our previous study showed that U. tomentosa extracts altered human erythrocyte shape, which could be due to incorporation of the compounds contained in extracts into the erythrocyte membrane. The aim of the present study was to determine how the compounds contained in U. tomentosa extracts incorporate into the human erythrocyte membrane. The study has assessed the effect of aqueous and ethanolic extracts from leaves and bark of U. tomentosa on the osmotic resistance of the human erythrocyte, the viscosity of erythrocyte interior, and the fluidity of erythrocyte plasma membrane. Human erythrocytes were incubated with the studied extracts in the concentrations of 100, 250, and 500 µg/mL for 2, 5, and 24 h. All extracts tested caused a decrease in erythrocyte membrane fluidity and increased erythrocyte osmotic sensitivity. The ethanolic extracts from the bark and leaves increased viscosity of the erythrocytes. The largest changes in the studied parameters were observed in the cells incubated with bark ethanolic extract. We consider that the compounds from U. tomentosa extracts mainly build into the outer, hydrophilic monolayer of the erythrocyte membrane, thus protecting the erythrocytes against the adverse effects of oxidative stress.