The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

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.

Assessment of the red blood cell proteome in a dog with unexplained hemolytic anemia.

A 7-year-old female neutered Jack Russell Terrier was presented to Langford Vets, the University of Bristol, with a history of chronic intermittent lethargy. Investigations and clinical course were compatible with hereditary hemolysis due to a red blood cell membrane defect. Proteomics was used to explore protein alterations in the presence of a hypothesized red blood cell membrane protein deficiency. Proteomic analysis revealed downregulation of the band 3, and alpha- and beta-adducin proteins, and alterations in the red blood cell proteome consistent with previous reports of changes due to the presence of reticulocytosis and ongoing hemolysis. The spectrum of protein alterations identified in the affected dog may be homologous to a band 3 protein deficiency secondary to hereditary spherocytosis, as described in people.

In vitro assessment of quality of citrate-phosphate-dextrose-adenine-1 preserved feline blood collected by a commercial closed system.

BACKGROUND: Optimal procedure for storage of feline blood is needed. Open-collection systems have been employed in feline medicine, thus limiting the possibility for storage. OBJECTIVES: To evaluate indicators of quality of feline blood stored for 35 days at +4°C in a closed-collection system specifically designed for cats. ANIMALS: Eight healthy adult European domestic shorthair cats with a weight of 5-6.8 kg. METHODS: This is a case series study. A bacteriological test, CBC, blood smear, pH, osmotic fragility, 2,3-diphosphoglycerate (2,3-DPG), and adenosine triphosphate (ATP) measurement were performed weekly on whole blood (WB) units from day 1 to day 35 after donation. The hemolysis index, lactate and potassium concentrations, prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen were measured on plasma aliquots. RESULTS: One out of eight blood units (BUs) had bacterial growth (Serratia marcescens) at day 35. No significant differences were found regarding CBC, morphology, pH, and osmotic fragility. Despite high inter-individual variability and low starting levels, significant decreases in the mean concentrations of 2,3-DPG (T0 1.99 mmol/g Hb, SD 0.52, T35 1.25 mmol/g Hb, SD 1.43; P = .003) and ATP (T0 1.45 mmol/g Hb, SD 0.71, T35 0.62 mmol/g Hb, SD 0.51; P < .001) were detected during the study, as opposed to an increase in hemolysis (T0 0.11 mmol/L, SD 0.07, T35 0.84 mmol/L, SD 0.19; P < .001), lactate (T0 3.30 mmol/L, SD 0.86, T35 13.36 mmol/L, SD 2.90; P < .001), and potassium (T0 3.10 mmol/L, SD 0.21, T35 4.12 mmol/L, SD 0.35; P < .001) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The commercial BU kit is appropriate for blood collection and conservation of WB in cats. The maintenance of WB quality indicators during storage is essential for future improvements of feline transfusion medicine.

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.

Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM.

Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2-6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy.

Sensitivity and specificity of single-tube osmotic fragility test and its different methods as screening test for thalassemia trait: an alternative to expensive laboratory tests for resource-limited countries.

OBJECTIVE: To determine the sensitivity and specificity of single-tube osmotic fragility (SOFT) and its different methods as screening test for thalassemia trait. METHODS: A cross-sectional study was conducted at Omair Sana Foundation. A total of 400 participants were included in the study. Three hundred were known thalassemia carriers (parents with at least one child with thalassemia major), while 100 were healthy blood donors. SOFT was performed on all 400 participants. Serum iron, ferritin, and DNA tests were performed on 100 participants (donors). ARMS technique was used for detecting thalassemia mutations. RESULTS: Sensitivity and specificity of SOFT (venous method) were found to be 99.6% and 86%, respectively, while with EDTA method, sensitivity was 95% and specificity was 96%. For venous and EDTA methods, positive predictive values were 95.5% and 98.6%, respectively, while negative predictive values were 98.8% and 86.6%, respectively. Use of EDTA and storage had an effect on the results. Sensitivity of SOFT was 95% at 5 min, while it decreased to 87% with EDTA method at 240 min. Sensitivity of SOFT for iron deficiency anemia was found to be 14%. CONCLUSION: SOFT can be used as screening test for thalassemia trait in a cost-effective way. Moreover, we also found that SOFT should be performed on venous blood without adding preservatives (EDTA) that can interfere with the results.

Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen.

Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.

Effect of toluene on erythrocyte membrane stability under in vivo and in vitro conditions with assessment of oxidant/antioxidant status.

Toluene, an organic solvent used widely in the industry, is highly lipophilic and accumulates in the cell membrane impeding transport through it. Its metabolites cause oxygen radical formation that react with unsaturated fatty acids and proteins in erythrocytes leading to lipid peroxidation and protein breakdown. In this study, we aimed to investigate the membrane stabilizing and the oxidative stress-inducing effects of toluene in human erythrocytes. Measurements of osmotic fragility, mean corpuscular volume (MCV), oxidative stress parameters and antioxidant enzyme activities were performed simultaneously both in individuals exposed to toluene professionally (in vivo) and human erythrocytes treated with toluene (in vitro). To measure osmotic fragility, erythrocytes were placed in NaCl solutions at various concentrations (0.1% [blank], 0.38%, 0.40%, 0.42%, 0.44%, 0.46%, 0.48% and 1% [stock]). Percentage of haemolysis in each solution was calculated with respect to the 100% haemolysis in the blank solution. The erythrocyte packs prepared at the day of the above-mentioned measurements were kept at -80 degrees C until the time for determination of malonyldialdehyde and protein carbonyl levels, and catalase (CAT) and glutathione peroxidase activities as indicators of oxidative stress. Toluene increased oxidative stress parameters significantly both in vivo and in vitro; it also caused a significant decrease in the activities of antioxidant enzymes. Osmotic fragility was altered only in the case of in vitro exposure. In conclusion, toluene exposure resulted in increased lipid peroxidation and protein damage both in vivo and in vitro. Although, it is natural to expect increased osmotic fragility due to oxidative properties of toluene, its membrane-stabilizing effect overcame the oxidative properties leading to decreased osmotic fragility or preventing its deterioration in vitro and in vivo toluene exposures, respectively, in the present study.

Flow cytometric detection of erythrocyte osmotic fragility.

BACKGROUND: Hereditary spherocytosis (HS) is the most common cause of inherited chronic hemolysis. The various tests developed for HS screening have many shortcomings. The purpose of this study was to develop a new, simple, reliable test using flow cytometry. METHODS: In this new test, deionized water, a hemolysis-inducing agent, is spiked to a red cell suspension during acquisition, and the count of red cells is measured sequentially in real-time using flow cytometry. In this study, the optimal parameters for this test were established, and the discriminatory power of the adopted protocol was verified among several groups with various degrees of osmotic fragility (OF). RESULTS: The established protocol successfully discriminated between blood analyzed immediately and blood stored for 24 h (P < 0.00001). With healthy individuals as a reference, the OF measured by this protocol was significantly decreased in patients with iron deficiency anemia (P = 0.030) and was significantly increased in HS patients (P = 1.1 x 10(-9)). This new test found all 11 HS patients to have increased OF. CONCLUSIONS: This new test was verified to be simple, quantitative, objective, and cost-effective for the measurement of OF. We suggest this test as another effective approach for HS screening, although its assay performance will require further verification through more clinical trials.

Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E.

OBJECTIVE: To evaluate hemoglobin E screening tests in a large scale of cases. MATERIAL AND METHOD: A cross-sectional descriptive study was conducted Whole blood obtained from subjects was evaluated for CBC, OF, DCIP, and hemoglobin typing. RESULTS: Five hundred twenty seven hemoglobin E and 280 reference subjects participated. DCIP's sensitivity, specificity, positive predictive value, and negative predictive value were 97.16%, 98.93%, 99.42%, and 95.19%, respectively. These values of OF were 69.12%, 80.00%, 86.67%, and 57.88%, respectively. In the combination of DCIP and OF gave rise to these values of 99.43%, 79.29%, 90.03%, and 96.67%, respectively. Finally the combination of DCIP and MCV < 80 fL resulted in these values to be 99.43%, 98.93%, 99.43%, and 98.93%, respectively. False positive and false negative rate were 1.07% and 0.57%, respectively. CONCLUSION: Combination of DCIP and MCVwas better than that of DCIP and OF in hemoglobin E screening.

Assessment of effects of a formula used in the traditional Chinese medicine (Buzhong Yi Qi Wan) on the morphologic and osmotic fragility of red blood cells

Buzhong Yi Qi Wan (BYQW) is a combination of some medicinal herbs widely used in traditional Chinese medicine to treat blood, spleen and stomach disorders. Morphometric analysis and osmotic fragility assay have been used to evaluate changes on membrane integrity of red blood cells. The aim of this work was to evaluate the effect of an aqueous BYQW extract on the morphology and osmotic fragility of red blood cells. Blood samples were treated with BYQW extract, quantitative/qualitative morphological analysis and osmotic fragility assay were carried out against control groups treated with saline. The data obtained indicated no modification on morphology but osmotic fragility assay suggested a significant (p<0.05) increasing of hemolysis in red blood cells isolated from blood treated with aqueous BYQW extract. In conclusion, the aqueous BYQW extract could affect the membrane integrity decreasing the osmotic resistance but without altering the shape of red blood cells.

Buzhong Yi Qi Wan (BYQW) é uma combinação de algumas ervas medicinais amplamente usada na medicina tradicional chinesa para tratar o sangue, baço e desordens do estômago. A análise morfométrica e o ensaio de fragilidade osmótica têm sido usados para avaliar alterações na integridade da membrana de hemácias. O objetivo deste trabalho foi avaliar os efeitos de um extrato aquoso de BYQW na morfologia e na fragilidade osmótica de hemácias. Amostras sangüíneas foram tratadas com o extrato de BYQW, análise morfológica quantitativa/qualitativa e o ensaio de fragilidade osmótica foram realizados e comparados com grupo controle tratado com salina. Os dados obtidos indicaram ausência de modificações na morfologia, mas o ensaio de fragilidade osmótica sugeriu um aumento significativo (p<0,05) da hemólise em hemácias isoladas de sangue tratado com extrato aquoso de BYQW. Em conclusão, o extrato aquoso de BYQW poderia afetar a integridade da membrana diminuindo a resistência osmótica sem alterar a forma das hemácias.

Screening for beta-thalassaemia carriers in Egypt: significance of the osmotic fragility test.

To estimate beta-thalassaemia carrier rate and to determine an accurate mass screening test, we tested 1000 randomly selected children aged 5-16 years from different geographical areas of Egypt. Microcytosis was present in 412 participants. The osmotic fragility test was positive in 81.1% of the 90 beta-thalassaemia carriers; in the indeterminate group (12 participants), the test was positive in 83.3%; in the 310 who were iron deficient, the test was positive in 63.9%. beta-thalassaemia carrier rate was > or = 9%. Serum iron, microcytosis, HbA2 level and transferrin saturation were accurate tests for detecting carriers. For the one-tube osmotic fragility test, sensitivity was 87.0% and specificity 34.1%; the test has limited use for a mass screening programme in Egypt, where iron deficiency is prevalent.

Evaluation of single-tube osmotic fragility as a screening test for thalassemia.

A single-tube osmotic fragility test has been proposed for thalassemia screening with a range of different concentrations of saline having been employed. We have compared the sensitivity and specificity of 0.32%, 0.34%, and 0.36% buffered saline, and on the basis of our findings, recommend the use of 0.36% saline. This gave definitely positive or equivocal results in 81 of 85 patients with beta thalassemia trait and in 4 of 4 with alpha(0) thalassemia trait. There were 14% false positive results in hematologically normal patients and 81% of the samples from patients with various variant hemoglobins gave positive results. The sensitivity was 95% and specificity 86%. The single-tube osmotic fragility test is potentially useful in under-resourced laboratories although it cannot replace automated red cell indices using electronic counters.

Comparative assessment of the erythrocyte osmotic fragility and of haematological and plasma biochemical values in the Nigerian white Fulani and N'dama breeds of cattle.

The blood profiles of the Nigerian White Fulani and N'dama breeds of cattle were compared. The White Fulani cattle had a significantly higher haemoglobin concentration (p < 0.001), mean corpuscular haemoglobin (p < 0.05), plasma sodium (p < 0.02), total protein (p < 0.01), albumin (p < 0.01) and globulin (p < 0.02), but lower neutrophil counts (p < 0.01) and osmotic fragility of erythrocytes than the N'dama cattle. The plasma potassium, calcium, bicarbonate, inorganic phosphate, albumin/globulin ratio, urea, creatinine, packed cell volume and mean corpuscular volume, and the erythrocyte, leukocyte, lymphocyte, eosinophil and monocyte counts were similar in the two breeds.

Assessment of qualitative functional parameters of stored red blood cells from donors with sickle cell trait (AS) or with heterozygote (AC) status.

We have compared the storage quality of three groups of 10 red blood cell concentrates of the AA, AS and AC haemoglobin phenotype. The 30 samples were drawn on CPD on day 0, and were centrifuged 6 hours later. Leukocytes were removed on D1 and the samples were kept at +4 degrees C for 42 days in SAG-mannitol. Viability tests were performed using a series of in vitro tests, including: ATP, 2,3-DPG, K+, glucose, lactic acid, pH, osmotic fragility tests and erythrocytic morphology determination. Results demonstrated a good functional quality for AS and AC haemoglobin RBCs. Higher 2,3-DPG levels were found in Hb AS and Hb AC. A lower osmotic fragility in Hb AC and AS RBCs versus Hb AA was observed; no significant difference was found in terms of ATP levels and other parameters. In addition no variation in S and C haemoglobin levels and no sickling were observed. In conclusion, these results indicate an overall good quality for haemoglobin AS and AC RBCs. Further in vivo studies must now be performed in order to confirm the transfusional quality of haemoglobin AS and AC RBCs.

The assessment of endothelial integrity by scanning electron microscopy and fluorescein diacetate staining following treatment with cryoprotective additives.

As part of the development of methods of corneal cryostorage for transplantation, a toxicity study was carried out on the rabbit corneal endothelium using four cryoprotective additives (CPA's) 1) dimethyl sulphoxide (Me2SO), 2) propane-1,2-diol (PG), 3) glycerol (GLY), 4) polyvinylpyrrolidone (PVP). A fifth group, based upon a CPA combination of Me2SO and PVP, was used to characterize both the assays, and the response of the endothelial layer to osmotic stress. The effect upon the cell membrane was assessed using scanning electron microscopy (SEM) and fluorescein diacetate with ethidium bromide staining (FDA/EB). Two sampling points were used, one immediately after treatment and the other following an incubation period. Calculations were performed to predict the maximum relative volume of cells during CPA exchange. Immediately following serial addition and removal of 2 or 3 mol/L (M) PG or GLY, the cells exhibited adverse morphological changes shown with SEM, and the proportion of intact cells judged by FDA/EB staining was significantly reduced when CPA equilibration was performed at 37 degrees C rather than at 20 degrees C. A 3M Me2SO concentration gave less morphological change than 3M PG or GLY, but even after treatment with 4M Me2SO more than 95% cells were judged intact by FDA/EB staining. PVP at 40% w/v showed minimal damage with both assays, and the fifth experimental group suggested that PVP may protect from injury during hypotonic stress. With all groups, the integrity of the cell layer recovered during incubation, so that for each sample the percentage of intact cells was high. However, although confluency was often restored following incubation, total cell density was usually reduced. The results indicate that serial addition and removal of 3M Me2SO is tolerated by the cornea, whereas PG or GLY cannot be used at 2 or 3M without inducing osmotic damage. There was low toxicity to PVP, and it was an effective osmotic buffer.

Evaluación de 3 pruebas de laboratorio para el diagnóstico de la microesferocitosis / Assessment of 3 laboratory tests for the diagnosis of mycrospherocytosis

Se comparan 3 pruebas de laboratorio para el diagnóstico de la microesferocitosis, la prueba cuantitativa de hemólisis a 3 concentraciones de NaCl, la denominada "prueba rosada" y la prueba del glicerol acidificado. Los resultados que se obtuvieron en 62 contoles y en 40 casos de microesferocitosis indican que la prueba más confiable, específica, simple y barata, es la prueba del glicerol acidificado

Assessment of membrane permeability in primary cultures of neurons and glia in response to osmotic perturbation.

In primary cultures of neurons and astrocytes from rat brain, the cellular contents of K+ and Na+ were used to assess plasma membrane permeability in response to osmotic shock. In contrast to trypan blue exclusion, the measure of ionic content reflected both transient and permanent membrane damage, and was also applicable to aggregates of cells. In steady state, the neurons and glia exhibited a K+ to Na+ ratio of 3-5, and the mean cellular contents (mu Eq/mg protein) of K+ and Na+ were 0.72 and 0.17 for astrocytes and 0.78 and 0.23 for neurons. Both hypo- and hypertonicity resulted in marked efflux of cell K+, but elevation of cellular Na+ occurred only under severe hypertonic conditions. Relative to neurons, astrocytes displayed considerably higher resistance to osmotic shock. During subsequent isotonic incubation, these cells were able to completely recover from transient membrane damage caused by a 10-min exposure to fourfold hypertonicity. Permanent changes in glial permeability were obtained only after a 20-min hypertonic shock. In contrast, 5 min hypertonic treatment of neurons decreased the ratio of cellular K+ to Na+ from 4.5 to 1. This ratio was restored twofold by isotonic incubation, but decreased permanently to below 1 after 10 min of hypertonic shock. The results describe marked differences in the osmotic fragility of neurons and glia and demonstrate that the determination of cellular K+ and Na+ provides a sensitive and accurate indicator of membrane permeability in neural cells propagated as surface-growing cultures. The approach has wide-ranging applicability.