Red blood cell deformability in iron deficiency anaemia

In patients with iron deficiency anaemia (IDA) it has been suggested that the shortened erythrocyte lifespan may be in part due to decreased erythrocyte deformability. In order to know whether erythrocyte deformability is decreased in IDA patients, we have determined the erythrocyte Elongation Index (EI) by means of ektacytometric techniques (Rheodyn SSD, Myrenne Gmbh, Germany), in 50 IDA patients and 100 well age and sex matched healthy controls. At the three shear stresses tested, 12, 30 and 60 Pa, IDA patients show statistically lower EI than controls (37.4+/-6.7 vs 48.6+/-2.9; 45.0+/-6.0 vs 54.5+/-2.8; 48.7+/-5.8 vs 57.0+/-2.9 mPa.s, respectively; p<0.001). A statistically significant correlation was found between EI at 12, 30, and 60 Pa and the hematimetric indices (MCV, MCH and MCHC), suggesting that the alteration in surface/volume ratio (shape) which characterizes this kind of microcytic hypocromic anaemia, accounts in part for the decreased EI. Rheodyn SSD, as an ektacytometric technique, is very sensitive to alterations in red blood cell geometry, for what seems to be a useful tool for detecting diminished erythrocyte deformability in IDA patients.     

Red cell superoxide dismutase is increased in iron deficiency anemia

Red blood cells (RBC) from iron-deficient rats were found to generate more malonyldialdehyde after in vitro incubation with H2O2 than RBC from control rats (p less than 0.001). The iron-deficient RBC, however, had a higher content of superoxide dismutase (SOD) than control RBC (p less than 0.02). This finding suggests an increased formation of SOD compensatory to an increased oxidant stress.     

Red cell lipid peroxidation and antioxidant enzymes in iron deficiency

Whether iron deficient RBC in humans have a reduced, or an increased, susceptibility to lipid peroxidation was studied in the iron deficiency states of primary proliferative polycythaemia and iron deficiency anaemia and related to changes in the activities of iron-dependent and non-iron dependent antioxidant enzymes. Susceptibility of RBCs to lipid peroxidation was increased when expressed per g Hb. However, this was a result of the low RBC Hb giving an increased membrane lipid: Hb ratio in the incubations. Results were normal when expressed either per cell, or per ml, RBC. Glutathione reductase was normal. Increased RBC superoxide dismutase activity in iron deficiency may be explained by the younger RBC population and reductions in glutathione peroxidase and catalase activities by the microcytic hypochromic changes and the lack of availability of iron, respectively. There is no evidence of an increased susceptibility of RBC to lipid peroxidation in iron deficiency.     

Red cell membrane and cation deficiency in Rh null syndrome

A 52-yr-old multiparous white female was found to have Rh null blood type. She had macrocytic anemia, with reticulocytosis (15%-20%), of long duration. Although stomatocytes in peripheral blood were numerous and osmotic fragility was increased, suggesting increased cell water, the RBC cation content, and thus cell water, was decreased. Cell dehydration was confirmed by an increased proportion of high density RBC on Stractan density gradients. The deformability of RBC from four gradient subpopulations was measured in the ektacytometer as a function of suspending medium osmolality. Analysis of these measurements showed an abnormal reduction in cell surface area with increasing cell density, thus explaining the increased osmotic fragility of whole blood. This was confirmed by a density-dependent reduction in cell cholesterol content, suggesting membrane instability in vivo. Rh null subpopulations showed a twofold increase in both ouabain-sensitive and - insensitive Na-K ATPase activity and 86Rb transport, even in the dense fraction with the fewest reticulocytes. No membrane protein or glycoprotein abnormality was detected by SDS-PAGE. The associated deficiencies of both membrane surface area and cation content in Rh null cells, as well as increased Na-K pump activity, suggest a pleiotropic functional interrelationship among Rh antigen, membrane stability, and cation regulation.     

Red cell nucleotide levels in patients with thalassaemia, iron deficiency, and renal failure

S ummary . Red cell nucleotides, predominantly adenine nucleotides, were measured spectrophotometrically in normal individuals and in patients with iron deficiency, β thalassaemia and renal failure following dialysis or transplantation. There was a significant correlation between the mean red cell haemoglobin (MCH) or the mean cell volume (MCV) and the nucleotide levels in red cells ( P < 0·001). Patients with β thalassaemia did not have increased nucleotide levels compared to red cells having the same MCH or MCV. Patients with renal failure had significantly increased levels of nucleotides compared to normal individuals, and this level remained high after renal dialysis but returned to normal following renal transplantation.     

Red blood cell microcytosis and hypochromia in the differential diagnosis of iron deficiency and beta-thalassaemia trait

Iron deficiency anaemia (IDA) and beta-thalassaemia are the most common causes of microcytic anaemia. Some indices have been defined to quickly discriminate this diseases based on red cell parameters obtained from automated blood cell analyzers, and can be effective for use as a preliminary screening tool to allow the reflex HbA(2) analysis, when a proper cut-off is chosen. Advia 2120 (Siemens Medical Solutions Diagnostics) directly measures volume and haemoglobin concentration of individual red cells, and quantifies the percentage of microcytic, normocytic, macrocytic, hypochromic, normochromic and hyperchromic red cells. Because of the inverse behaviour of the % microcytic and % hypochromic red cells in beta-thalassaemia trait and in IDA the ratio between these two values was computed and its discriminant efficiency assessed. The aim of the study was to assess the predictive value of the new index % microcytic/% hypochromic ratio in the differential diagnosis of beta-thalassaemia compared with Mentzer index, currently used in our Laboratory. Sensitivity, specificity and total efficiency of both indices were calculated for a set of 110 IDA patients and 150 beta-thalassaemia carriers. Discriminant efficiency was similar for both indices.     

Red blood cell membrane disorders

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).     

Red cell membrane transport abnormalities

PURPOSE OF REVIEW: The present review describes the red cell transport abnormalities of proteins of the band 3 macrocomplex. The macrocomplex is involved in red cell gas exchange and recent findings have furthered our understanding of this process. RECENT FINDINGS: Study of a novel band 3 hereditary spherocytosis variant suggests that expression of mutant band 3 protein can be rescued by wild-type band 3. Other studies show that some mutant band 3 protein can mediate a cation conductance. Recent work suggests both Rh-associated glycoprotein and aquaporin act as gas channels confirming the integrated function of the macrocomplex and the importance of its role in red cell gas transport. SUMMARY: The most recent studies on band 3-induced hereditary spherocytosis are reviewed and an explanation for the mild phenotype of heterozygous hereditary spherocytosis is discussed. A number of red cell conditions (hereditary stomatocytosis, south-east Asian ovalocytosis, distal renal tubular acidosis, Rhnull), associated with both stomatocytosis and a cation leak, are described. The evidence that Rh-associated glycoprotein forms a gas channel that transports CO2 and/or NH3 is reviewed and discussed, together with recent studies that show that aquaporin 1 transports both CO2 and O2.     

Red cell iron uptake in hereditary microcytic anemia

The iron uptake in vitro of red cells from mice with hereditary microcytic anemia (gene symbol mk) was studied to examine the hypothesis of a generalized impairment of cellular iron uptake in this conidition. Reticulocyte-rich red cells from anemic (mk/mk) and acutely bled normal (+/+) mice were incubated in 59Fe-labeled mouse plasma and the radioiron uptake measured. The 59Fe uptake of the mk/mk and +/+ cells was related in the same way to the reticulocyte concentration, the duration of incubation, and the percentage saturation of the plasma iron-binding capacity. However, under the same conditions, the iron uptake of red cells from normal (+/+) mice was greater than that by red cells from anemic (mk/mk) mice. Furthermore, the cellular loss of radioiron on exposure to EDTA was greater for the mk/mk red cells, although the proportion of the radioiron taken up that was incorporated into heme was the same for mk/mk and +/+ red cells. These results support the hypothesis of a generalized impairment of cellular iron uptake in hereditary microcytic anemia and suggest that there might be a defect in red cell receptor sites for transferrin in this condition.     

Red cell distribution width, mean corpuscular volume, and transferrin saturation in the diagnosis of iron deficiency

The usefulness of the red cell distribution width, mean corpuscular volume, and the transferrin saturation in diagnosing iron deficiency anemia were evaluated in a retrospective study of 247 anemic hospitalized patients, many of whom had chronic liver disease. A red cell distribution width greater than 15% had a sensitivity of 71% and a specificity of 54% for iron deficiency as diagnosed by a low serum ferritin or bone marrow examination. A mean corpuscular volume less than 80 femtoliters had a sensitivity of 53% and a specificity of 84%. Transferrin saturation less than 16% had a sensitivity of 61% and a specificity of 86%. Because the sensitivities and specificities of these tests are less than reported in studies of healthier populations, they cannot be relied on for screening for iron deficiency in sick hospitalized patients.     

Red cell ferritin and iron overload in heterozygous beta-thalassemia

Red cell ferritin was evaluated in 101 individuals with heterozygous beta-thalassemia to determine its clinical utility as an index for iron deficiency or overload in these subjects. The mean red cell ferritin for the total population was elevated threefold and showed a significant correlation with transferrin saturation, plasma ferritin, and HbA2 levels. Five of six subjects with reduced red cell ferritin had associated iron deficiency; a further five had iron deficiency and normal red cell ferritin. Normal red cell ferritin occurred in 51 subjects, and 44 had increased values. In the elevated red cell ferritin group, 21 individuals had associated normal plasma ferritin, and 23 had increased plasma ferritin. Only in the latter group was red cell ferritin significantly correlated with transferrin saturation and plasma ferritin. Ten individuals had a red cell ferritin greater than or equal to 150 attogram/cell, and liver biopsy performed in four showed grades II to IV iron overload. A clinical feature of subjects with both increased red cell and plasma ferritin levels was a high incidence of inappropriate iron administration. These findings suggest that red cell ferritin, particularly when combined with plasma ferritin, is a useful parameter for determining potential iron overload in individuals with heterozygous beta-thalassemia.     

Red cell membrane deformability: new data

Red cells arrested by fine fibers and subjected to drag forces in a moving stream of fluid can be stressed beyond the point of rupture. The technique, which is basically an in vitro analogue of the microangiopathic hemolytic state, thus permits a study of red cell behavior over the entire range of stress that the membrane can withstand. Measurements made by this approach show an exponential relationship between the velocity of fluid flow and the resultant membrane deformation. Such exponential membrane deformation is consistent with the morphology of red cell fragments and appears to explain their mechanism of formation.     

Red cell membrane and erythropoiesis genetic defects

In recent years, the frontline of red cell membrane research has shifted. Seeking new mutations in known genes has been taken over by the quest of new genes. It remains that many natural mutations, both in human and mice, have an irreplaceable heuristic value. For example, it is some of these mutations that eventually paved the way to the demonstration that the band 3 and the Rh complexes form a macrocomplex fulfilling, as one may assume, the function of a gas transport metabolon. Mapping and individualization of novel genes have been successful in inherited disorders of the membrane permeability to monovalent cations, such as dehydrated hereditary stomatocytosis and pseudohyperkalemia, and congenital dyserythropoietic anemias (CDAs). We herein included CDAs because the red cell membrane is abnormal in at least CDA I and II. A major breakthrough was the identification of the gene, the mutations of which cause CDA I. It encodes codanin-1. The occurrence of lipid rafts in the red cell starts being documented. GPI-anchored proteins and a number of minor proteins associated with the rafts allow foreseeing a chapter of great novelty in red cell membrane physiology.