Acoustophoretic Orientation of Red Blood Cells for Diagnosis of Red Cell Health and Pathology.

Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.

Protein 4.2: a complex linker.

The peripheral membrane protein, protein 4.2, is one of the most abundant protein components of the erythrocyte membrane. Protein 4.2 has an important role in red cell membrane structure, its absence due to natural mutations in humans or gene knockout in mice has a detrimental effect on membrane stability and results in hereditary spherocytosis. It is known to be a point of connection between the band 3 complex and the Rhesus protein complex, through its associations with band 3 and CD47 and also via interactions with the cytoskeletal protein ankyrin. Considering its relatively high abundance and importance in stability of the erythrocyte membrane, protein 4.2 has proved a somewhat neglected protein in recent years. In this review we will summarize our current understanding of protein 4.2, discuss its known interactions and describe the effects and implications of protein 4.2 deficiency. Based on protein 4.2's close homology with transglutaminase family proteins, we propose a new speculative "open" homology structure for protein 4.2 that may represent the active, membrane associated protein 4.2 molecule in red blood cells and also explain the dependence of protein 4.2 on band 3 binding for stability.

Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

Artificially induced unusual shape of erythrocytes: an atomic force microscopy study.

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.

Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton.

Band 3 is the most abundant integral protein of the red blood cell membrane. It performs two critical biological functions: maintaining ionic homeostasis, by transporting Cl- and HCO3-ions, and providing mechanical stability to the erythroid membrane. Erythroid band 3 (AE1) is one of three anion exchangers that are encoded by separate genes. The AE1 gene is transcribed by two promoters: the upstream promoter produces erythroid band 3, whereas the downstream promoter initiates transcription of the band 3 isoform in kidney. To assess the biological consequences of band 3 deficiency, we have selectively inactivated erythroid but not kidney band 3 by gene targeting in mice. Although no death in utero occurred, the majority of homozygous mice die within two weeks after birth. The erythroid band 3 null mice show retarded growth, spherocytic red blood cell morphology and severe haemolytic anaemia. Remarkably, the band 3-/- red blood cells assembled normal membrane skeleton thus challenging the notion that the presence of band 3 is required for the stable biogenesis of membrane skeleton. The availability of band 3-/- mice offers a unique opportunity to investigate the role of erythroid band 3 in the regulation of membrane-skeletal interactions, anion transport and the invasion and growth of malaria parasite into red blood cells.

Decreased content of protein 4.2 in ankyrin-deficient normoblastosis (nb/nb) mouse red blood cells: evidence for ankyrin enhancement of protein 4.2 membrane binding.

Homozygous normoblastosis (nb/nb) mice, whose red blood cell (RBC) membranes are nearly completely deficient in full-length 210-kD ankyrin, were used to study interactions between ankyrin and protein 4.2 (P4.2). Although it is unclear whether or not these proteins interact in the membrane, both ankyrin and P4.2 bind to the cytoplasmic domain of band 3 (cdb3). In addition to the complete deficiency of full-length ankyrin, nb/nb RBC membranes are also partially spectrin deficient, resulting in morphologically spherocytic and mechanically fragile cells. A new finding was that nb/nb RBC membranes are severely (approximately 73%) P4.2 deficient compared with wild type (+/+) or high reticulocyte mouse RBC membranes. Metabolic labeling of nb/nb reticulocytes showed active P4.2 synthesis at levels comparable with high reticulocyte controls suggesting that the nb/nb P4.2 deficiency was not the result of defective P4.2 synthesis. Reconstitution of nb/nb inside-out vesicles (IOVs) with human RBC ankyrin restored ankyrin levels to approximately 80% of +/+ IOV levels and increased binding of exogenously added human RBC P4.2 by approximately 60%. These results suggest that ankyrin is required for normal associations of P4.2 with the RBC membrane.

Hereditary spherocytosis (HS) due to loss of anion exchange transporter.

BACKGROUND: Hereditary spherocytosis encompasses a heterogenous group of inherited disorders due to alteration of r.b.c. surface/volume ratio. Spectrin deficiency is the most common observed defect. We analyzed a case of HS associated with band 3 deficiency without spectrin reduction. METHODS: In the study of a family originating from southern Italy, we show that a 20% deficiency of band 3 with normal spectrin content may be responsible for dominantly inherited hereditary spherocytosis (HS). The proband is a 12 years old girl consulting for jaundice, chronic anaemia and splenomegaly. Her mother had a similar haematologic phenotype. RESULTS: Electrophoretic analysis of erythrocyte membrane proteins showed a deficiency in band 3 protein. Band 3 protein chymotryptic fragments, deglycosylated band 3, and its isolated cytoplasmic domain, all displayed normal electrophoretic migrations. Furthermore, the tryptic peptides profile of the cytoplasmic domain of the protein did not demonstrate any abnormality, nor did the amino acid composition of the peptides. Analysis of the membrane proteins during erythrocyte ageing, evaluated in density-fractionated red cells, showed that band 3 content was normal in the lighter fraction, whereas in the denser fraction band 3 deficiency was more pronounced than in membranes from non fractionated red blood cells. CONCLUSIONS: This case describes HS due to anion exchange transporter deficiency. Our results on fractioned red cells support the hypothesis that the defect was probably due to a band 3 protein loss during cell ageing and not to a primitive quantitative defect.

Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes.

A novel method for the preparation of spheroids containing two types of cells (hetero-spheroid) has been successfully developed by utilizing a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), as a cell substratum. PNIPAAm solidifies above its lower critical solution temperature (LCST, about 30 degrees C), and instantly dissolves into the culture medium below its LCST. We firstly seeded and cultured human dermal fibroblasts on the substratum up to a confluent state and then seeded rat primary hepatocytes onto the fibroblast monolayer. The heterospheroid was prepared by detaching the hepatocyte-attached fibroblast monolayer at a temperature below LCST and culturing it on the non-adhesive substratum. The surface area of the substratum and the seeding population ratio of each cell precisely and reproducibly regulated the size and the cell composition of the resulting hetero-spheroid, respectively. Histological and immuno-cytochemical observations of spheroids revealed characteristic organizations of fibroblasts and hepatocytes within a spheroid because the latter cells expressed albumin for up to at least 3 weeks. TEM study of the hetero-spheroid showed the presence of structures morphologically similar to the Disse's space and the bile canaliculus, which are features characteristic of liver. These findings suggest that the method described above is useful for making a hetero-spheroid that morphologically and functionally resembles tissues or organs in vivo, i.e. an organoid.

Transformation of erythrocytes to spherocytes following incubation with malignant cells.

Incubation of erythrocytes from controls and patients with colorectal carcinoma with tumor cells, or their metabolites, caused transformation of the normally-shaped cells to spherocytes. It is assumed that abnormal metabolites produced by malignant cells affect the cell membrane inducing the subsequent appearance of spherocytes. The increased destruction of these cells by the reticuloendothelial system may serve as an additional factor for the explanation of anemia accompanying malignant diseases.

Emission of microvesicles and erythrocytic spherocytation.

We have examined mathematically the "cause-effect" relationship between the phenomenon of emission of microvesicles and that of erythrocytic sphericitation, especially when the age of the cell is the main factor involved.

Adenosine triphosphate restoration and discocytic transformation of stored human erythrocytes.

Erythrocytes in human blood stored for 120 days were collected by centrifugation after dispersion in buffered physiological saline. The aged erythrocytes thus collected were incubated with inosine, adenine, glucose or other media, and their shapes and ATP levels were studied by scanning electron microscopy and a luciferine-luciferase method. The aged erythrocytes incubated in a mixture of adenine and inosine markedly regained their ATP levels, and also showed a marked transformation from spiked spherocytes to normal discocytes. Incubation with inosine alone restored ATP levels of the aged erythrocytes to some extent, but did not result in morphological rejuvenation. Incubation in a mixture of citrate and glucose caused morphological rejuvenation, though it restored ATP levels less effectively than incubation in inosine alone. Incubation with adenine alone neither restored ATP levels nor resulted in morphological rejuvenation of the stored erythrocytes.

Morphology and fatty acid composition of erythrocytes from monkeys exposed to ozone for one year.

Monkeys (Macaca radiata) were exposed to a low dose (0.64 ppm) of ozone (O3) for 8 hr each day over a 1-year period. Control monkeys were exposed to filtered air. The morphology of the red blood cell (RBC) from these monkeys was analyzed by scanning electron microscopy. Red cells from control monkeys had normal morphology with 69% having biconcave shape (discocytes). In O3-exposed animals, blood contained only 35% discocytes. Knizocytes, stomatocytes, and spherocytes each have defined shape which are different from discocytes. The number of knizocytes and stomatocytes in O3-exposed monkeys was twice that of controls. In addition, significant levels of spherocytes were observed while they were absent in the blood of controls. The fatty acid composition of RBC from control and O3-exposed monkeys was the same. These were similar to that of human RBC. However, earlier reports of monkey RBC fatty acid composition differ from our results. These differences are discussed. We conclude that low-dose O3 exposure changes the morphology, but not the fatty acid composition, of erythrocytes in vivo.

Red cell fragmentation in the dog: an editorial review.

Red blood cell fragments in blood smears from dogs are described morphologically and pathogenetically. Categories include microangiopathic fragmentation, spherocytic fragmentation. Heinz body fragmentation, metabolic fragmentation associated with systemic disease, and artifactual fragmentation. Microangiopathic fragmentation is associated with direct physical damage to normal circulating red blood cells as they pass through abnormal capillary beds. Spherocytic fragmentation is a common feature of immune-mediated hemolytic anemia and results from the removal of portions of antibody-coated erythrocyte plasma membranes by phagocytes of the reticuloendothelial system. Heinz body fragmentation occurs when rigid particles of oxidized hemoglobin are torn from affected red cells as they circulate through the spleen. Metabolic fragmentation is an ill-defined syndrome most commonly associated with cholesterol loading of red cell membranes caused by lipid metabolism abnormalities. Resulting spiculated red cells are more susceptible to traumatic disruption. All the types of red cell fragmentation described in dogs have been observed and documented in man.