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—A method combining phase-sensitive optical coherence microscopy (OCM) and optical tweezers for studying mechanical properties of microobjects including biological cells is implemented. The process of microbead trapping was studied using the developed method. The response of the red blood cell (dyscocyte and spherocyte) membrane to the optical tweezer excitation is measured.

Aim. To assess the morphology of circulating red blood cells in patients with stage III ovarian cancer. Methods. The object of the study was the peripheral blood erythrocytes of primary patients with ovarian cancer (n=25) who had stage III according to International Federation of Gynecology and Obstetrics. Patients were examined in the gynecological department of Ulyanovsk Regional Clinical Oncology Center. The control group included 25 somatically healthy women. Morphological studies were performed using light microscopy. The number of red blood cells with an unchanged and altered shape was expressed as a percentage. By the method of atomic force microscopy, the topology and rigidity of red blood cells was studied. Results. A statistically significant decrease in the number of circulating blood erythrocytes was found in patients with ovarian cancer compared to somatically healthy women. At the same time, the number of discocytes is markedly reduced while the number of morphologically altered forms: echinocytes, stomatocytes, spherocytes and erythrocyte rigidity are increased. Conclusion. With the appearance of altered forms of red blood cells and increase of the transformation index and erythrocyte rigidity in patients with stage III ovarian cancer, total number of red blood cells decreases in circulating blood compared to somatically healthy women.

Aims Intraoperative blood salvage (IBS) is associated with shortened lifespan of red blood cells (RBCs). This study aims to examine how salvaged RBCs are compromised during IBS. Main methods Thirty patients who underwent vertebra surgery with IBS were included in the study. To examine possible mechanisms of IBS-induced injury, both fresh and salvaged RBCs from each patient were mixed with plasma, the Ca2+ ionophore ionomycin or mannitol-adenine-phosphate (MAP) solution (n = 10 patients per condition). Binding of Fluo-3 and/or Annexin V by RBCs was measured. Key findings The percentage of Fluo-3-binding RBCs in salvaged samples was 2.83 ± 0.76%, which increased to 15.34 ± 5.99% after 48-h incubation in plasma. These percentages were significantly higher than those observed with fresh RBCs (P Significance Our results suggest that IBS induces a postponed RBC damage by inducing spherocyte formation, which likely reflects Ca2+ entry induced by energy exhaustion, as well as by exposing phosphatidylserine on the RBC surface, which likely occurs via Ca2+ entry and via Ca2+-independent pathways.

Hereditary spherocytosis (HS), an erythrocyte membranopathy, is a heterogeneous disease, even at the level of the erythrocyte population. The paper aims at studying the mechanical properties (the Young’s modulus, median and RMS roughness of friction force maps; fractal dimension, lacunarity and spatial distribution parameters of lateral force maps) of the cell surface layer of the erythrocytes of two different morphologies (discocytes and spherocytes) in HS using atomic force microscopy. The results of spatial-spectral and fractal analysis showed that the mechanical property maps of the HS spherocyte surface were more structurally homogeneous compared to the maps of HS discocytes. HS spherocytes also had a reduced RMS roughness and lacunarity of the mechanical property maps. The Young’s modulus and averaged friction forces over the microscale HS spherocyte surface regions were approximately 20% higher than that of HS discocytes. The revealed significant difference at the nano- and microscales in the structural and mechanical properties of main (discoidal and spheroidal) morphological types of HS erythrocytes can potentially cause blood flow disturbance in the vascular system in HS.

Background: Immune-mediated hemolytic anemia (IMHA) is characterized by an autoimmune response with production of auto-antibodies and destruction of erythrocytes resulting in anemia. Primary IMHA is referred to a condition when the cause is unknown (idiopathic), whereas secondary IMHA involves changes in red blood cells caused by the action of drugs, neoplasms, or infectious diseases. The diagnosis can be made through changes in the blood count, usually of a regenerative character, Coombs test, and autoagglutination test. The present study aimed to report a case of drug-induced hemolytic anemia, with emphasis on the clinical signs, diagnostic methods, and treatment, in a female dog. Case: A 9-year-old mixed-breed bitch weighing 29.6 kg was referred to the Veterinary Medical Teaching Hospital (HCVUFRGS) with a previous diagnosis of gallbladder mucocele that was unresponsive to clinical treatment. After laboratory tests, cholecystectomy was performed, and the procedure required conversion from laparoscopic to open cholecystectomy. Therapy included administration of amoxicillin, dipyrone, tramadol hydrochloride, and meloxicam. Three days after surgery, the dog presented with apathy, lethargy, hyporexia, and a pale and subicteric mucosa. The patient developed hypochromic macrocytic anemia with reticulocytosis, spherocytosis, anisocytosis, and leukocytosis with neutrophilia. The result of the autoagglutination test was positive, confirming the diagnosis. All medications were suspended, and immunosuppressive treatment with dexamethasone was included, with a subsequent switch to prednisolone. After 10 days of treatment, the patient experienced significant improvement, and therapy was discontinued. Discussion: Based on the patient’s history, the cause of the IMHA was secondary to drug administration, and it is not possible to distinguish if it was due to one or a combination of drugs, as they were all started and stopped simultaneously. The patient had hypothyroidism, which may have contributed to the production of antibodies against TSH receptors, blocking the hormone’s action, thereby causing tissue damage due to T cell-mediated cytotoxicity and the effect of cytokines. The pale and subicteric mucosa, apathy, weakness, lethargy, exercise intolerance, and dyspnea resulted from extravascular hemolysis and bilirubin released from erythrocyte rupture with a subsequent decrease in the number of red blood cells, leading to oxygen transport deficiency. The diagnosis is based on the blood count and results of autoagglutination supported by the response to immunosuppressive therapy. Anemia results in increased production and release of precursor cells from the bone marrow, accompanied by reticulocytosis and increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). The treatment of IMHA consists of supportive care and immunosuppressive therapy with corticosteroids to ensure suppression of the immune system, preventing response against erythrocytes. Initially, tramadol hydrochloride, dipyrone, and amoxicillin with potassium clavulanate were suspended to interrupt the cause of IMHA, and administration of dexamethasone in an immunosuppressive dose was started. Therefore, it is important to include drug-induced IMHA in the differential diagnosis of patients who present with anemia after using medications. Early diagnosis, initiation of therapy, and adequate care were important factors for the recovery of the animal.

In the current investigation, peripheral blood films of 15 COVID-19 patients (44.78±16.55 years), proven by computed tomographic imaging and RT-PCR for coronavirus SARS-CoV-2, were analyzed at the moment of hospital admission. Blood tests showed raised inflammatory markers (C-reactive protein 58.2±61.2 mg/L) with normal values for hemoglobin (126.2±2.6 g/L), WBC (6.8±18.74 109/L) RBC (4.55±0.99 1012/L) platelets (262.4±141.8, 109/L) MCV (79.84±8.2 fL) MCH (28±3.31 pg) and MCHC (350.3±1.15 g/L). The results revealed the presence of hypersegmented neutrophils in 66.66%% of the patients. The percentages of neutrophils with 4 and 5 lobes were 46.25 ± 4.83% and 31.5 ± 14.84%, respectively. Three major red blood cells morphological alteration were observed: (1) erythrocytes in “rouleaux” formation represented by linear erythrocytes aggregation, (2) spherocytes with the disappearance of the usual biconcave disk, and (3) echinocytes showing spiky projections. Apparent reorganization of hemoglobin is found in the majority of the analyzed erythrocytes. Rouleaux formation is observed in 33.33% of patients and spherocytes and echinocytes are present at variable levels in the all analyzed patients. The current results revealed erythrocytes injuries in COVID-19 peripheral blood, in association with hypersegmented neutrophils, alterations that could be involved in the respiratory syndrome.

Background Hypothermic storage transforms red blood cells (RBC) from smooth biconcave discocytes into increasingly spherical spiculated echinocytes and, ultimately, fragile spherocytes (S). Individual cells undergo this transformation at different rates, producing a heterogeneous mixture of RBCs at all stages of echinocytosis in each unit of stored blood. Here we investigated how washing (known to positively affect RBC properties) changes morphology of individual RBCs at the single-cell level. Study design and methods We tracked the change in shape of individual RBCs (n = 2870; drawn from six 4- to 6-week-old RBC units) that were confined in an array of microfluidic wells during washing in saline (n = 1095), 1% human serum albumin (1% HSA) solution (n = 999), and the autologous storage supernatant (control, n = 776). Results Shape recovery proceeded through the disappearance of spicules followed by the progressive smoothening of the RBC contour, with the majority of changes occurring within the initial 10 minutes of being exposed to the washing solution. Approximately 57% of all echinocytes recovered by at least one morphologic class when washed in 1% HSA (36% for normal saline), with 3% of cells in late-stage echinocytosis restoring their discoid shape completely. Approximately one-third of all spherocytic cells were lysed in either washing solution. Cells washed in their autologous storage supernatant continued to deteriorate during washing. Conclusion Our findings suggest that the replacement of storage supernatant with a washing solution during washing induces actual shape recovery for RBCs in all stages of echinocytosis, except for S that undergo lysis instead.

Objetivo: desarrollar una nueva metodología que evalúe la forma del eritrocito normal y el esferocito a partir de los espacios ocupados por cada uno de ellos, medido con el método de Box Counting. Materiales y métodos: se analizaron 20 imágenes de extendido de sangre periférica, en las cuales se observaron un gran número de eritrocitos normales y esferocitos. Se superpusieron dos rejillas Kp de 5 x 5 pixeles y Kg de 10 x 10 pixeles, para calcular el espacio ocupado por dos regiones del eritrocito normal las cuales son, el disco y centro de este visto de manera frontal, así como el espacio ocupado por el esferocito mediante el método de Box Counting. Resultados: los espacios ocupados por el exterior e interior del eritrocito normal con la rejilla Kp variaron entre 47 y 55, y entre 8 y 16, respectivamente. En cambio, los espacios ocupados por los esferocitos variaron entre 25 y 36. Las proporciones entre la superficie y los valores de la rejilla Kp, de las regiones del eritrocito normal variaron entre 9,250 y 32.24, y para los esferocitos variaron entre 34,63 y 71.39. Conclusiones: las alteraciones en la forma del eritrocito normal pueden ser diferenciadas mediante los rangos hallados para el espacio ocupado por la superficie y las proporciones S/Kp de cada una de las regiones del eritrocito a partir de las nociones de la geometría fractal y euclidiana.

The morphology of erythrocyte Packed Red Cells (PRC) is subject to changes during storage at 2 o –8 o C. In maintaining their viability, erythrocytes require adenosine triphosphate (ATP). The decrease in intracellular ATP level results in damaged membrane lipid, accumulation of intracellular Sodium and Calcium, decreased intracellular potassium and water, cell dehydration, rigidity of membrane and changes in its shape from a disc into crenated red cells, spherocytes and bite cells. This study was aimed to observe the presentation of erythrocyte crenated cells, spherocytes and bite cells of stored PRC at day 3 (control), day 7, day 14 and day 21 from the blood collection date of the blood bag. An observational study with cohort study approach was conducted in August 2015 at the Blood Bank of Dr. Wahidin Sudirohusodo Hospital Makassar. The total sample was 30 blood bag that resulted in 120 slide blood smears. From 30 samples of blood bags comprising blood group A 26.6%, B 13.3%, AB 16.6% and O 43.3%, the increased percentages of crenated cells, spherocytes and bite cells were observed at day 3, day 7, day 14 and day 21. Day 3 of storage was taken as control. Data were processed by Fiedman test and Wilcoxon test with significance level at p

Background Storage lesion may explain the rapid clearance of up to 25% of transfused red blood cells (RBCs) in recipients. Several alterations affect stored RBC but a quantitative, whole cell-based predictor of transfusion yield is lacking. Because RBCs with reduced surface area are retained by the spleen, we quantified changes in RBC dimensions during storage. Study design and methods Using imaging flow cytometry we observed the dimension and morphology of RBCs upon storage, along with that of conventional biochemical and mechanical markers of storage lesion. We then validated these findings using differential interference contrast (DIC) microscopy and quantified the accumulation of microparticles (MPs). Results Mean projected surface area of the whole RBC population decreased from 72.4 to 68.4 µm2 , a change resulting from the appearance of a well-demarcated subpopulation of RBCs with reduced mean projected surface (58 µm2 , 15.2%-19.9% reduction). These "small RBCs" accounted for 4.9 and 23.6% of all RBCs on Days 3 and 42 of storage, respectively. DIC microscopy confirmed that small RBCs had shifted upon storage from discocytes to echinocytes III, spheroechinocytes, and spherocytes. Glycophorin A-positive MPs and small RBCs appeared after similar kinetics. Conclusion The reduction in surface area of small RBCs is expected to induce their retention by the spleen. We propose that small RBCs generated by MP-induced membrane loss are preferentially cleared from the circulation shortly after transfusion of long-stored blood. Their operator-independent quantification using imaging flow cytometry may provide a marker of storage lesion potentially predictive of transfusion yield.

The toxic effect of Al2O3, SiО2 and ZrО2 nanoparticles on red blood cells of Wistar rats was studied in vitro using the atomic force microscopy and the fluorescence analysis. Transformation of discocytes into echinocytes and spherocytes caused by the metal oxide nanoparticles was revealed. It was shown that only extremely high concentration of the nanoparticles (2mg/ml) allows correct estimating of their effect on the cell morphology. Besides, it was found out that the microviscosity changes of red blood cell membranes treated with nanoparticles began long before morphological modifications of the cells. On the contrary, the negatively charged ZrO2 and SiO2 nanoparticles did not affect ghost microviscosity up to concentrations of 1μg/ml and 0.1mg/ml, correspondingly. In its turn, the positively charged Al2O3 nanoparticles induced structural changes in the lipid bilayer of the red blood cells already at a concentration of 0.05μg/ml. A decrease in microviscosity of the erythrocyte ghosts treated with Al2O3 and SiO2 nanoparticles was shown. It was detected that the interaction of ZrO2 nanoparticles with the cells led to an increase in the membrane microviscosity and cracking of swollen erythrocytes.

Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were\ud written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability\ud before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.

BACKGROUND The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). STUDY DESIGN AND METHODS Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. RESULTS The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. CONCLUSION The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.

Red blood cells (RBC) ability to circulate is closely related to their surface area-to-volume ratio. A decrease in this ratio induces a decrease in RBC deformability that can lead to their retention and elimination in the spleen. We recently showed that a subpopulation of "small RBC" with reduced projected surface area accumulated upon storage in blood bank concentrates, but data on the volume of these altered RBC are lacking. So far, single cell measurement of RBC volume has remained a challenging task achieved by a few sophisticated methods some being subject to potential artifacts. We aimed to develop a reproducible and ergonomic method to assess simultaneously RBC volume and morphology at the single cell level. We adapted the fluorescence exclusion measurement of volume in nucleated cells to the measurement of RBC volume. This method requires no pre-treatment of the cell and can be performed in physiological or experimental buffer. In addition to RBC volume assessment, brightfield images enabling a precise definition of the morphology and the measurement of projected surface area can be generated simultaneously. We first verified that fluorescence exclusion is precise, reproducible and can quantify volume modifications following morphological changes induced by heating or incubation in non-physiological medium. We then used the method to characterize RBC stored for 42 days in SAG-M in blood bank conditions. Simultaneous determination of the volume, projected surface area and morphology allowed to evaluate the surface area-to-volume ratio of individual RBC upon storage. We observed a similar surface area-to-volume ratio in discocytes (D) and echinocytes I (EI), which decreased in EII (7%) and EIII (24%), sphero-echinocytes (SE; 41%) and spherocytes (S; 47%). If RBC dimensions determine indeed the ability of RBC to cross the spleen, these modifications are expected to induce the rapid splenic entrapment of the most morphologically altered RBC (EIII, SE, and S) and further support the hypothesis of a rapid clearance of the "small RBC" subpopulation by the spleen following transfusion.

Using low-angle light scattering technique, the features of erythrocyte transformation dynamics (osmotic fragility, the hemolysis level and ratio of different cell forms determined by the spherical index) were revealed in hetero- (golden hamsters Mesocricetus auratus) and homoiothermic (white rats Rattus norvegicus) animals under natural (hibernation) and artificial (suspended animation) hypometabolic states, craniocerebral and immersion hypothermia. In control, golden hamsters exhibited higher levels of erythrocyte osmotic fragility and hemolysis than rats, with transformed erythrocytes (especially stomatocytes) being prevalent. Under artificial hypothermia, regardless of the way of its attaining, depth and duration, the changes were observed that were similar in their directionality but dissimilar in the degree of manifestation: increased osmotic fragility and hemolysis, reduced portion of discocytes (particularly sharp in golden hamsters under suspended animation), increased number of transformed erythrocytes. In contrast, under hibernation the osmotic fragility, hemolysis and number of stomatocytes decreased, the portion of discocytes increased, while the number of prehemolytic forms (spherocytes) also increased. In 24 h, both the osmotic fragility (stronger in hamsters after suspended animation) and the level of hemolysis (particularly after immersion hypothermia) decreased, the portion of discocytes recovered (even exceeding the control level in hamsters after suspended animation and in rats after immersion hypothermia), spherocytes were not detectable in hamsters but elevated in rats. It is likely that the qualitative changes in the erythrocyte population towards its homogeneity detected 24 hours after hypothermia may be mediated not only by the elimination of old and defective cells, erythropoiesis activation and production of high-resistant erythrocytes but also by the fine membrane-stabilizing mechanisms.

OBJECTIVE: We have studied dense erythrocytes separated on Arabinogalactan (Stractan) ultracentrifuged gradients in flame-burned patients and in normal individuals. In each case, the percentage of erythrocytes in the densest layers was increased when compared to age and sex matched controls. METHODS AND RESULTS: Using an in vitro system, we showed that as human whole blood is warmed to 48.6°C, the number of dense erythrocytes increases. In addition, the reduced glutathionine (GSH) content of the densest red blood cells is decreased compared to those in lighter fractions on the same gradient or to dense erythrocytes separated from blood incubated at room temperature. These dense red cells were largely composed of spherocytes and spheroechynocytes, two forms of erythrocytes which have been shown by others to have markedly abnormal flow characteristics in vitro. CONCLUSIONS: We have demonstrated that in vivo dense erythrocytes can be generated in the setting of flame burns. Thus, the underlying reason may be oxidant injury as represented by the reduced level of GSH that we found in association with the generation of dense erythrocytes.

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.

Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC(50) = 3.4 μM), leukocytes (EC(50) = 19.5 μM) and erythroleukemia K562 cells (EC(50) = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.

We review our recent dielectric spectroscopy (DS) studies of rabbit erythrocytes of various shapes (spherocytes, echinocytes, enlarged erythrocytes, and discocytes) and present additional results obtained through a new analysis of the previous data. First, a numerical method to accurately obtain the specific cell membrane capacitance Cm and the cytoplasm conductivity κcp for non-spherical and non-ellipsoidal cells is demonstrated. This method uses numerical electromagnetic simulation with three-dimensional cellular modeling. The value of Cm for the discocyte agrees well with the reported values for swollen erythrocytes. In contrast to the numerical method, the analytical method, in which the discocyte must be assumed to have a spheroidal shape, rather than a more realistic biconcave shape, leads to significant overestimation of Cm. Second, DS is applied to monitor the dielectric response of preserved rabbit blood over 37 days. Microscope observation revealed that ∼90% of the erythrocytes were echinocytes at the beginning of preservation and that the erythrocytes started to transform into microspherocytes around the 20th day. The dielectric response also changed at the same critical point of the 20th day. Therefore, DS is revealed to be a sensitive tool for monitoring the deterioration of preserved blood.

Summary. The fluidity gradient and sulphydryl groups in erythrocyte ghost membranes from healthy subjects and from seven patients with hereditary spherocytosis (HS) Lvere studied by spin label techniques. The mobility of the stearic acid spin labels was lower in the spherocyte membranes, indicating greater stability of the phospholipid layer in these membranes than in those of matched controls. This stabilization was most distinct in the intermediate and deep membrane layers. No clear differences were observed in the protein sulphydryl groups. It is suggested that the decreased cellular deformability characterizing spherocytes reflects altered lipid organization or lipid-protein interaction in the interior of the cell membrane.

Haemoglobin H disease, also known as the alpha-thalassaemia is characterized by the presence of HbH inclusions in red blood cells, detectable on supra-vital stain. We present a case of a previously asymptomatic 31-year-old male, who insidiously developed anaemia and had prominent splenomegaly. Peripheral smear examination revealed microcytic hypochromic anaemia with numerous spherocytes and moderate polychromasia. In reticulocyte preparation with Brilliant cresyl blue, HbH inclusions were mistakenly identified as granulofilamentous reticulum of reticulocytes, giving a spuriously high reticulocyte percentage. After the literature review, repeat assessment was performed and with the aid of high performance liquid chromatography result, it was possible to delineate the HbH inclusions.

An erythrocyte and a spherocyte are subjected to aspiration pressure with a micropipette and analyzed by the finite element method (FEM). The comparison of the erythrocyte and the spherocyte indicates that the y-direction displacement of the erythrocyte was larger than that of the spherocyte under the same aspiration pressure. The x-direction stress distributions show that it is easier to change the shape of the erythrocyte model than that of the spherocyte model because a force in the opposite direction appears in the erythrocyte. This force seems to move the erythrocyte membrane to its center. The results indicate that the shape of the erythrocyte membrane changes partially under aspiration pressure at the point of contact with the micropipette and aspiration pressure. The results also indicate that the shape of the spherocyte membrane changes under aspiration pressure, but not only at a point of contact with the micropipette and the area subject to aspiration pressure; the entire spherocyte membrane seem to change the shape.

Summary The human erythrocyte adopts a distinctive biconcave disc form in vivo. The question as to why the red blood cell should have this particular profile remains unresolved. It has been suggested that this shape maximises the surface area to volume ratio and thus expedites diffusion. This hypothesis, however does not stand up to examination. Maximal diffusion occurs in the small vessels. In order to pass through the microvasculature the erythrocyte becomes distorted and deviates from the biconcave disc shape [Branemark PI, Lindstrom J. The shape of circulating blood corpuscles. Biorheology 1963;1:139; Guest MM, Bond TP, Cooper RG, Derrick JR. Red blood cells: change in capillaries. Science 1963;142:1319–21]. Here, it is suggested the haemodynamic factors have dictated the peculiar shape of the discocyte. The deleterious nature of turbulent flow on the cardiovascular system suggests that the biconcave disc form has evolved out of a necessity to maximise laminar flow, minimise platelet scatter which in turn suppress atherogenic activity in the large vessels [Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses: athero-protective effect of laminar fluid shear stress in endothelial cells: possible and mitogen-activated protein kinases. J Pharmacol Sci 2003;9:172–6]. The biconcave profile of the discocyte means that much of the mass is distributed in the periphery. This increases the moment of inertia of the cell and subsequently renders the erythrocyte less prone to rotation during flow in the large vessels. Here it is suggest that this reduction in rotation promotes laminar flow and discourages platelet scattering by minimising the "Eddy currents" and it thus anti-atherogenic. A number of pathological mutations result in the red blood cell adopting a spherical shape as opposed to the biconcave disc profile. The sphere has a smaller moment of inertia when compared to the discocyte, as much of the mass is distributed round the centre. The spherocyte is hence much more prone to rotation during flow in the large vessels. There is evidence to suggest that asplenic individuals suffering from spherocytosis are at increased risk of atherogenic cerebrovascular and cardiovascular large-vessel disease, when compared with their asplenic haematological normal counterparts [Schilling RF. Spherocytosis, splenectomy, strokes and heart attacks. Lancet 1997;350:1677–8; Robinnette CD, Fraumeni Jr JF. Splenectomy and subsequent mortality in veterans of the 1939–1945 war. Lancet 1977;ii:127–9].

The aim of the present study was to investigate poikilocytosis in Labeo rohita (an important food fish) as an early indicator of stress due to an azo dye, Basic Violet-1 (CI: 42535). This dye was observed to be very toxic to test fish (96 h LC50 as0.45 mg/L dye). Fish were given short-term (96 h) and subchronic (150 days) exposures to the dye, and poikilocytosis was recorded under light and scanning electron microscopy (SEM). Light microscopy helped in identification of micronuclei along with irregularities, notches, blebs, lobes, crenation, clumps, chains, spherocytes, vacuolation, and necrosis in erythrocytes. However, SEM indicated shrinkage, oozing of cytoplasm, and several new abnormal shapes including marginal foldings, discocytes, keratocytes, dacrocytes, degmacytes, acanthocytes, echinocytes, protuberances, stomatocytes, drepanocytes, holes in the membrane, stippling/spicules, crescent-shaped cells, triangular cells, and pentagonal cells. Earlier studies speculated changes in the membrane to be responsible for clumping and chaining of erythrocytes, whereas the present SEM study clearly indicates that oozing out of cytoplasm is also responsible for the formation of chains and clumps. This study also shows that erythrocytes exhibit pathological symptoms before the appearance of other external symptoms such as abnormal behavior or mortality of fish. There was a dose- and duration-dependent increase; therefore, poikilocytosis, especially echinocytes, spherocytes, and clumps, can act as a biomarker for the stress caused by azo dyes.

The diagnosis of hereditary spherocytosis (HS) in a newborn infant is generally made on the basis of a positive family history, spherocytes on blood film and Coombs-negative hemolytic jaundice of variable severity with an elevated mean corpuscular hemoglobin concentration (MCHC) and a low mean corpuscular volume (MCV). In general, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) quantification of erythrocyte membrane proteins is not needed to make the clinical diagnosis of HS. However, we observed that a neonate with no family history of HS, but with abundant spherocytosis on repeated blood films, Coombs-negative hemolytic jaundice and normal MCHC and MCV measurements, where SDS-PAGE revealed alpha-spectrin deficiency, a rare autosomal-recessive variety of HS that generally has a severe clinical phenotype.

Summary: Using the classical cryolysis test suggested by Rosas and coworkers, it is shown that hyperchromic erythrocytes correspond to spherocytes destined for rapid elimination by lysis in the reticulo-endothelial cells. The percentage of hyperchromioc erythrocytes is determined without further cost by the H*-1, H*-2, and ADVIA 120 haematological analysers. An increased percentage of hyperchromic red blood cells thus emphasises increased haemolysis. It is shown that approximately 80 % of adult men with a percentage of hyperchromic erythrocytes exceeding 9.4 % have serious iron overload evidenced by high ferritin. The percentage in women with hyperchromia is much lower, eventually due to menstrual blood loss. Significant hyperchromia in adult men represents a marker of iron overload, justifying further investigation.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.

More stomatocytes, spherocytes, and erythrocytes with spicules and protrusions are found in blood from newborn infants than in blood from adults. The objective of this study was to measure volume, surface area, membrane rigidity, and viscosity of red blood cells (RBC) with irregular shapes in preterm and full-term neonates. RBC from 13 full-term and 12 preterm (25-36 wk of gestation) infants were studied by means of a micropipette system. In full-term neonates and in preterm infants, 19 and 26% of RBC, respectively, showed shapes different from those of discocytes. Keratocytes, spherocytes, akanthocytes (in full-term infants), and elliptocytes (in preterm infants) had smaller volumes than did discocytes (p < 0.05). Spherocytes showed decreased and elliptocytes had increased excess surface area and swelling capacity compared with discocytes, indicating both volume and membrane loss for spherocytes and only volume loss for elliptocytes. Immature RBC and knizocytes had similar large volumes and surface areas, suggesting that knizocytes in neonates are relatively young RBC. Increased membrane rigidity was found only in stomatocytes. Membrane viscosity was decreased in immature RBC and knizocytes and increased in stomatocytes, keratocytes, akanthocytes, and elliptocytes when compared with discocytes. There were inverse relationships between RBC volume and membrane rigidity (r=-0.42) and between RBC volume and membrane viscosity (r=-0.72). This suggests that volume loss of neonatal RBC is associated with impaired membrane deformability. Moreover, we conclude that the geometry and membrane deformability of some of the irregularly shaped neonatal RBC resemble properties of RBC in hemolytic anemias (e.g. spherocytosis).

The investigation of red cell anion exchange protein, band 3 has been at the center of two fundamental questions on functions of membrane proteins : how they regulate cell morphology, and how they regulate homeostasis in the cell and throughout the whole organism. Our recent studies on bovine hereditary band 3 deficiency, a novel disorder inherited in an autosomal dominant mode in which homozygous animals totally lack band 3 due to a nonsense mutation Arg664 → stop, have addressed the importance of band 3 in red cell morphology and homeostasis. The animals with total lack of band 3 had red cells that were also deficient in other major membrane skeletal proteins, resulting in a distorted and disrupted membrane skeletal network. Their red cell membranes, therefore, were extremely unstable and showed a continuous loss of surface area, leading to spherocyte formation and anemia. The decreased 4.1a/4.1b ratio implicated a high rate of hematopoiesis compensatory to survive. Rapid anion exchange sensitive to stilbene disulfonate was completely absent in band 3-deficient cells. The animals had slight acidosis and their blood bicarbonate concentration and total CO2 were lower than control levels but within a normal range, suggesting that there are alternate or multiple strategies for CO2 and pH homeostasis. These observations demonstrate that band 3 indeed contributes to red cell membrane organization, CO2 transport, and acid-base homeostasis, but it is not always essential. A putative dominant negative effect of the mutant band 3 protein on expression of functional band 3 is also discussed.

International audience; q-Space plots obtained experimentally using pulsed field-gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) spectroscopy from water diffusing in red blood cells (RBCs) of different canonical (distinct variant) morphologies have "signature" features. The experimental q-space plots from suspensions of stomatocytes, echinocytes and spherocytes generated chemically had no diffraction features; in contrast a sample of blood from a patient with hereditary spherocytosis showed diffraction minima. To understand the forms of q-space plots, mathematical/geometrical models of discocytes, stomatocytes, echinocytes and spherocytes were used as restricting boundaries in simulations of water diffusion with Monte Carlo random walks. These simulations indicated that diffusion-diffraction minima are expected for each of the cell shapes considered. The absence of diffusion-diffraction minima in stomatocytes generated by dithiothreitol treatment was surmised to be due to non-alignment of the cells with the magnetic field of the NMR spectrometer. Differential interference contrast microscopy images of the chemically generated spherocyte and echinocyte suspensions showed them to be heterogeneous in cell shape. Therefore, we concluded that the shape heterogeneity caused the loss of the diffusion-diffraction features, which were observed in the more homogeneous sample from a patient with hereditary spherocytosis, and in the simulations of homogeneous cell suspensions. This understanding of factors that affect q-space plots from RBC suspensions will assist morphological studies of other cell and tissue types.

In this article, the transmembrane voltage induced on erythrocyte, codocyte, ovalocyte and spherocyte cell models exposed to a linearly polarised electromagnetic plane wave of frequency 1800 MHz is calculated. For this purpose, a finite element (FE) numerical technique with adaptive meshing is used. The results show that the value of the induced voltage on the original erythrocyte shape is higher than the one observed on the rest of the altered cell geometries studied. The erythrocyte shape and the membrane electric permittivity are shown to play a fundamental role on the values of the induced transmembrane voltage.

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

We report two patients with prominent spherocytosis in the peripheral blood who later went on to develop myelodysplasia. There was no evidence of a myelodysplastic syndrome at the time of presentation. Morphological abnormalities of the peripheral blood along with possible explanations of spherocytosis are discussed.

Mechanical measurements of intrinsic membrane material properties are used to characterize the defect in hereditary spherocyte membrane at a continuum level. The value of the surface elastic shear modulus is two-thirds as large as normal values, and the value of the yield shear resultant is one-third as large as normal values. The viscosity of the surface above the elastic-plastic transition appears normal. Under similar geometric conditions, the force required to fragment a hereditary spherocyte is about one-third as large as the force required to fragment a normal cell.

We present the measurement of red blood cell (RBC) volume change induced by Ca 2+ for a live cell imaging with full field quantitative phase microscopy (FFQPM). FFQPM is based on the Mach-Zehnder interferometer combined with an inverted microscopy system. We present the effective method to obtain a clear image and an accurate volume of the cells. An edge detection technique is used to accurately resolve the boundary between the cell line and the suspension medium. The measurement of the polystyrene bead diameter and volume has been demonstrated the validity of our proposed method. The measured phase profile can be easily converted into thickness profile. The measured polystyrene bead volume and the simulated result are about 14.74 μm 3 and 14.14 μm 3 , respectively. The experimental results of our proposed method agree well with the simulated results within less than 4 %. We have also measured the volume variation of a single RBC on a millisecond time scale. Its mean volume is 54.02 μm 3 and its standard deviation is 0.52 μm 3 . With the proposed system, the shape and volume changes of RBC induced by the increased intracellular Ca 2+ are measured after adding ionophore A23187. A discocyte RBC is deformed to a spherocyte due to the increased intracellular Ca 2+ in RBC. The volume of the spherocyte is 47.88 μm 3 and its standard deviation is 0.19 μm 3 . We have demonstrated that the volume measurement technique is easy, accurate, and robust method with high volume sensitivity ( 3 ) and this provides the ability to study a biological phenomenon in Hematology.

A new red cell preservation solution is described in which the red cells may be stored for 42 and 49 days. An in vivo and vitro study of 42 and 49 days stored red cells with saline, adenine, glucose, mannitol (SAGM) preservation solution (OPTISOL, Terumo Corporation) was conducted using 20 healthy volunteers to document in vivo efficacy and analyze the validity of the 51Cr technique. Standard in vitro parameters of OPTISOL red cell concentrates were well preserved with low levels of hemolysis and high levels of red cell ATP which is compatible with their survival after 42 and 49 days storage. Osmotic pressure at hemolysis ending point of 42 and 49 days stored red cells did not change, while osmotic pressure at hemolysis starting point increased on 49 days. The red cell viscosity increased and scanning electron-microscopic studies showed that the majority of them became echinocyte and spherocyte on 49 days. In vivo autologous post-transfusion recovery was measured by using a 51Cr-labeled red cells. After 42 days of storage, post-transfusion 24-hour recovery averaged 82.3±8.4% and 48-hour recovery was 79.1±9.1%, and after 49 days, 24-hour recovery was 75.9±3.6%, but 48-hour recovery, which averaged 69.9±5.2%, was significantly lower.The present study reports in vivo and in vitro evaluation of a new red cell storage medium which allows high levels of post-transfusion recovery and permits without significant hemolysis. The results suggested that 42 days if a more suitable shelf life than 49 days.

A new theoretical five-compartmental model (5CM) was developed for analysis of the clearance of heat-damaged erythroctes (HDE) labelled with chromium 51. Besides the HDE-spleen interaction, this new model also takes into account the interaction between extrasplenic reticuloendothelial (RES) sites and HDE, i.e. the hepatic clearance of fragmented erythrocytes (FE). Accordingly, HIDE clearance curves are analysed into three exponential components, the fastest of which describes the RES-FE interaction, whereas the others describe the splenic clearance of spherocytes. Therefore, an estimation of the effective liver blood flow for HDE (ELBF) was achieved, along with a series of parameters describing splenic function. The 5CM proved to be more efficient than a previously proposed three-compartmental model (3CM) in the mathematical description of HDE clearance. Comparison was made by applying both models to 37 experimental curves obtained from 20 patients with congenital hemolytic anemias. The values for the splenic function parameters calculated by 5CM analysis and the strong correlations observed among them offer evidence that this model provides an adequate approximation to the real conditions under which HDE clearance takes place. Furthermore, a detailed quantitative analysis of the pooling of spherocytes within the spleen was attempted in this work, and this phenomenon was found to compete with splenic irreversible spherocyte trapping. The ELBF proved to be closely correlated with the hemodynamic splenic parameters, following first-order kinetics, as do low-dose colloids.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane–skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

Quantitative phase information obtained by digital holographic microscopy (DHM) can provide new insight into the functions and morphology of single red blood cells (RBCs). Since the functionality of a RBC is related to its three-dimensional (3-D) shape, quantitative 3-D geometric changes induced by storage time can help hematologists realize its optimal functionality period. We quantitatively investigate RBC 3-D geometric changes in the storage lesion using DHM. Our experimental results show that the substantial geometric transformation of the biconcave-shaped RBCs to the spherocyte occurs due to RBC storage lesion. This transformation leads to progressive loss of cell surface area, surface-to-volume ratio, and functionality of RBCs. Furthermore, our quantitative analysis shows that there are significant correlations between chemical and morphological properties of RBCs.

In order to quantitatively investigate their ultramorphological characteristics, normally‐ and abnormally‐shaped human erythrocytes have been imaged by atomic force microscopy. Various fixation and staining methods have been examined for sample preparation. Roughness of the external membrane appeared to be independent of the overall cell shape (discocyte or spherocyte) and sample preparation; very stable in time up to one year and, most important, coupled with the meshwork of the membrane skeleton.