The influence of oxygen tension, temperature, and hemoglobin concentration on the rheologic properties of sickle erythrocytes.

With the use of micromanipulation techniques, the shear modulus or "rigidity" mu, the recovery time tc, and the unfolding time tf for individual sickle cells have been measured at different oxygen tensions, temperatures, and cell densities. In these experiments, the partial pressure of oxygen was varied from 156 to 40 mm Hg and the temperature was controlled at 25 degrees C or 37 degrees C. Three mean cellular hemoglobin concentrations were studied: 29 g/dL, 33 g/dL, and 46 g/dL. The lighter cells (29 and 33 g/dL) exhibited at most a threefold increase in rigidity as the pO2 was decreased from 156 to 40 mm Hg. At 25 degrees C, the densest cells (46 g/dL) also exhibited a threefold increase. However, at 37 degrees C, the rigidity of these cells increased eightfold between 156 to 40 mm Hg. Compared with normal cells, this gives a rigidity that is 18 times larger. In contrast to the values for mu, the values for tc and tf remained essentially unchanged (within the accuracy of the experiments) for the lighter cells and could not be measured for the densest cells.

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis.

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis.

Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.

Splenic phagocytic function in children with sickle cell anemia receiving long-term hypertransfusion therapy.

To determine the effects of blood transfusions on splenic function in older patients with sickle cell anemia, we investigated splenic function in 12 patients who had had cerebrovascular accidents and who were being treated at two collaborating centers using different transfusion protocols. Splenic function was assessed by radionuclide scan and pocked erythrocyte count. Patients were 6 to 18 years of age and had been receiving transfusions for 7 months to 10 years (median 4.2 years). Of the 12 children, five had normal or increased splenic size and function (normal scan and normal or minimally elevated pocked erythrocyte count). All were receiving intensive transfusion therapy, with the aim of maintaining the hemoglobin S level at less than 20%. The other seven patients had abnormal splenic function (absent radionuclide uptake and elevated pocked erythrocyte count); each was receiving less intensive transfusion therapy, with the pretransfusion hemoglobin S level usually at 30% to 40%. No patient developed bacterial septicemia while receiving hypertransfusion therapy. We conclude that splenic function during a long-term transfusion program is variable, depending in part on the "intensity" of transfusion therapy. Apparent splenic involution and fibrosis may be a reversible event in some patients.

Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement and perforin.

Previous reports have suggested that a 65 kDa membrane protein, termed homologous restriction factor (HRF), in addition to protecting erythrocytes (E) against lysis by homologous complement (C), may also be involved in protecting cytolytic lymphocytes against lysis mediated by a pore-forming protein (PFP/perforin), one of their own lytic mediators. Here, we used HRF-deficient type III E of patients with paroxysmal nocturnal hemoglobinuria (PNH) to study their susceptibility to lysis mediated by homologous C and perforin, and compared it with lysis of HRF-bearing control or PNH type I E. We show that type III E of PNH patients are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E. In addition, all human E (type I or III) tested here are equally susceptible to lysis mediated by either human (homologous) or murine (heterologous) perforin. By immunoblot analysis, we confirm that type III E, in contrast to type I E, were deficient in the 65 kDa HRF. These results support the notion that homologous species restriction is seen in the C- but not in the lymphocyte perforin-system and argue against an active participation of HRF in protecting cells from perforin-mediated lysis.

Hereditary stomatocytosis: consistent association with an integral membrane protein deficiency.

We studied the RBC membrane proteins of four patients, including a mother and daughter, with hereditary stomatocytosis. One- and two-dimensional gel electrophoresis revealed that a 28 kDa integral protein, present in normal RBC membranes, was absent in all four patients. This abnormality, reported once previously (Lande et al, 1982), appears to be a characteristic feature of hereditary stomatocytosis, and may be related to the underlying permeability defect in this disorder.

Vacuoles in red cells from splenectomized subjects originate during cell life: association with glycosylated haemoglobin?

Whole blood from splenectomized subjects (n = 8) contained a substantial percentage of vacuolated ('pitted') red cells (34.7 +/- 8.3%), while blood from controls revealed none. The percentage of haemoglobin A1 (HbA1) had increased significantly compared with controls (p less than 0.01). Fractionation on cell density revealed that the number of 'pitted' cells and the HbA1 percentage were associated with increased cell density. Fractionation on cell volume showed that 'pitted' cells are equally distributed in fractions with varying mean cell volume (MCV) and that decreasing MCV was associated with a linear rise of HbA1. It appeared that, shortly after splenectomy (n = 4), 'pits' develop early in cell life and that also older cells, after previous splenic contact, are capable of pit formation. A positive correlation found between the number of 'pitted' cells and the percentage of HbA1 might point to an impaired release of HbA1, manifest in the presence of 'pits'. The increased percentage of HbA1 in whole blood from splenectomized subjects may thus be explained.

Blood group antigen deficiencies associated with abnormal red cell shape.

Rare individuals are known with erythrocytes which show an inherited deficiency of certain blood group antigens and also have abnormal red cell shape. Studies of these cells can give an insight into the functional role of blood group active components in maintaining the shape and membrane properties of the normal erythrocyte. The biochemical characterisation of the red cell membrane alterations occurring in two such rare erythrocyte phenotypes--the Leach phenotype and the Rhnull phenotype are reviewed here.

Erythrocytes in Hb SC disease are microcytic and hyperchromic.

We have used the new Technicon H.1 Hematology Analyzer to determine the indices of erythrocytes obtained from 18 nonthalassemic (alpha alpha/alpha alpha genotype) adult patients with Hb SC disease. Controls were 14 normal black adults and 29 white adults. The data showed that SC erythrocytes are significantly smaller than normal RBC (P less than .001) with a significantly higher MCHC value (P less than .001) than controls. These features of SC RBC could not be reproduced by an Ortho ELT-8 electronic counter. Hb SC erythrocytes have unique indices which are best demonstrated either by semimanual methods or by the H.1 system.

Decay-accelerating factor (DAF) on the blood cell membranes in patients with paroxysmal nocturnal haemoglobinuria (PNH): measurement by enzyme-linked immunosorbent assay (ELISA).

We developed a quantitative enzyme-linked immunosorbent assay (ELISA) for decay-accelerating factor (DAF) on blood cell membranes using monoclonal anti-DAF antibodies. DAF is an integral membrane protein of several blood cells. It regulates the C3 and C5 convertase of the classical and alternative pathways of complement activation. It is partially or completely deficient in the membranes of blood cells of patients with paroxysmal nocturnal haemoglobinuria (PNH). The ELISA we developed for DAF measurement indicated a reliable range of measurement from 2.25 to 11.25 ng of DAF. In particular, ELISA proved to be a technically simple method and its sensitivity was enhanced by using avidin-biotin complex. In this study, DAF levels in extracts of erythrocytes from 30 healthy donors and in extracts of PMN and platelets from 15 healthy donors were measured by ELISA. The DAF content of blood cells from eight patients with PNH and erythrocytes from 13 patients with anaemia were also measured. The DAF levels of normal erythrocytes, PMN and platelets were 3110 +/- 960, 28,000 +/- 13,900 and 3100 +/- 1370 (mean +/- SD) molecules/cell, respectively. In general, the DAF content of PNH cells was below the normal range, although it was within the normal range in some cases of PNH. The DAF levels of PNH-I and -II erythrocytes were estimated from the ratio of PNH-I, -II and -III erythrocytes. And, in four cases of PNH, the DAF levels of PNH-I erythrocytes separated by acidified serum lysis were measured by ELISA. In most cases of PNH, DAF was found to be partially deficient in PNH-I and PNH-II erythrocytes.

Red-cell-volume distribution curves in diagnosis of glomerular and non-glomerular haematuria.

The distribution curves of urinary red-blood-cell (RBC) size were obtained from automated blood-cell analysis in 146 patients with definite causes of haematuria. In 65 of 67 patients (97%) with haematuria and glomerulonephritis demonstrated by renal biopsy, urinary RBC had an irregular and asymmetrical distribution with RBC size showing a much smaller volume than that of venous RBC. This "glomerular" distribution contrasted with the "non-glomerular" normal distribution when the peak for RBC was at a larger volume than that for peripheral RBC. In 46 of 47 patients with haematuria who had lower urinary tract lesions other than infection, a non-glomerular distribution was obtained; 30 of these cases also showed glomerular distribution, and were classified as "mixed". All 32 patients with urinary tract infection had either a glomerular or mixed distribution, suggesting that they excreted distorted and dysmorphic urinary RBC. After excluding infections, this simple, rapid, reproducible, and non-invasive technique provides reliable information in distinguishing glomerular bleeding from other causes of haematuria.

Density distribution of red cells and prognostic significance in 50 patients with homozygous sickle-cell disease.

The sickle cell disease is characterized by a heterogeneous clinical and biological expression. In order to evaluate the prognostic significance of the red blood cell density distribution: D50 (median cell density of the distribution), R60 (middle density range in which 60% of the cells can be found), F4 and F5 (proportion of cells with density higher than 1.110 and 1.120 g/ml, respectively) have been determined in 50 patients with homozygous sickle cell disease. The alpha gene status was determined in 27 patients. All patients have been included in an original score of severity fitted to infancy and childhood. A positive correlation has been found between D50 and the clinical score. This result illustrates the potential clinical importance of this parameter as well as other biological indices such as the haemoglobin F level, the alpha gene status and the haplotypes of the beta-like gene cluster.

Clinical disorders of the erythrocyte membrane skeleton.

With advances in understanding of the biochemistry of the erythrocyte membrane skeleton, the molecular basis of some of the hereditary anemias has been determined. Distinct spectrin abnormalities have been identified in spherocytosis and elliptocytosis. Protein 4.1 variants may result in elliptocytosis or may be clinically silent. While abnormalities of other proteins have not yet been identified, it is likely that variants of ankyrin and perhaps other proteins will eventually be identified as defects in these biochemically heterogeneous disorders of blood. Proteins related to spectrin, ankyrin, and protein 4.1 have been found in multiple other tissues, including brain, lymphocytes, and connective tissue, and the role of these proteins is being studied. Identification of the functional abnormalities and molecular biology of abnormal erythrocyte membrane proteins is therefore likely to have direct relevance to understanding of other cell types and diseases.

The effect of deoxygenation rate on the formation of irreversibly sickled cells.

The effects of the deoxygenation rate on the formation of irreversibly sickled cells (ISCs) were investigated by using metabolically replete sickle cells (SS cells). We found that the formation of ISCs required Ca2+ and that the amount formed depended on the rate of deoxygenation. When less dense SS discocytes were deoxygenated slowly by flushing with 95% N2 and 5% CO2 at a rate of 3 mL/min, the percentage of ISCs increased from 5% to 26.5% after 24 hours. In contrast, upon rapid deoxygenation (10, 35 mL/min) ISC formation was reduced significantly. The difference may be related to fact that more sickle-shaped cells were formed upon slow deoxygenation than upon the rapid deoxygenation that resulted in the formation of star-shaped and granulated cells. So-called ISCs were formed more easily from sickle-shaped cells. To express the shape of sickled cells numerically, we calculated the mean maximum cell length (MCL) after cells were incubated under various deoxygenation conditions. The MCL of slowly deoxygenated SS cells after 24 hours of incubation was about twice (20.0 +/- 7.0 micron) that of quickly deoxygenated (35 mL/min) SS cells (12.5 +/- 5.0 microns) (initial MCL, 8.0 +/- 1.0 micron). The decrease in potassium content was greater with slow deoxygenation than with rapid deoxygenation. Because the increase in sodium influx was less than that of potassium efflux under slow deoxygenation, SS cells became more dense than those rapidly deoxygenated. In the absence of Ca2+, morphological changes were the same as in the presence of Ca2+; however, under this condition there was no change in density, and no ISCs were formed regardless of the rate of deoxygenation. These results demonstrate that the number of ISCs formed correlates with the MCL. The length of fibers of sickle hemoglobin may be a determinant of the length of sickled cells. This suggests that membrane stretching plays an important role in cell density and irreversible membrane deformation.

Single cell laser light scattering spectroscopy in a flow cell: repeated sickling of sickle red blood cells.

We performed dynamic laser light scattering measurements of hemoglobin aggregates in single, sickle erythrocytes. Sickle erythrocytes were attached to the poly-(L-lysine)-coated surface of a flow cell. They were exposed to several oxygenation-deoxygenation cycles by repeatedly changing the flowing solution. The rate of cycling was found to be a determining factor for the formation of irreversible morphologic alterations as well as irreversible hemoglobin aggregates. In slow cycling, the sickle erythrocytes took an irreversible, irregular, rounded shape, and hemoglobin aggregates were observed even in the oxygenated state after 20 cycles. In the fast cycling, however, these changes did not take place even after 60 cycles.

Normal membrane function of abnormal beta-related erythrocyte sialoglycoproteins.

Red cells totally deficient in beta and gamma sialoglycoproteins (the Leach type of Gerbich-negative) are elliptocytic and have altered membrane physical properties as evidenced by marked decreases in both membrane mechanical stability and membrane deformability. Red cells from individuals who are of the Gerbich and Yus phenotypes of Gerbich-negative are also deficient in beta and gamma sialoglycoproteins, but possess abnormal beta-related sialoglycoproteins. In order to determine if these beta-related sialoglycoproteins can functionally substitute for normal sialoglycoproteins, we measured membrane deformability and stability of red cells of the Gerbich and Yus phenotypes. In contrast to the red cells of the Leach phenotype, cells of Gerbich and Yus phenotypes were found to have normal membrane deformability and stability. Moreover, flow cytometric analysis using a monoclonal anti-beta antibody revealed that the Gerbich and Yus phenotype red cells expressed the beta-related sialoglycoprotein to the same extent as its normal counterpart on normal cells. Based on these data, we suggest that the abnormal beta-related sialoglycoproteins can functionally substitute for normal beta and gamma sialoglycoproteins.

Duchenne muscular dystrophy: do both parents contribute genetically to the disease?

By observing the effect of 0.08 and 0.02 mg/ml linoleic acid (LA on the electrophoretic mobility of fresh red blood cells derived from the parents of a subject with DMD, it has been shown that all of 16 fathers as well as 15 mothers, consistently differ from normal. In normal subjects, whilst 0.08 mg/ml LA causes increased mobility, 0.02 mg/ml causes reduced mobility (P less than 0.001). In both parents this reversal is abolished and 0.02 mg/ml LA gives the same increased electrophoretic mobility of the RBC as does 0.08 mg/ml. This indicates that there is qualitative alteration of the RBC membrane in both, apparently genetically determined. Only when such an abnormal male mates with an abnormal female can DMD occur in a male offspring; other children appear either normal or showing the same abnormality as do the parents, thus continuing the production of DMD parents, the chance mating of whom produces further DMD. The evidence supports the hypothesis that in DMD there is a widespread membrane disturbance. Other relatives have also been explored. It would appear that the common DMD of children is of autosomal recessive origin with strong limitation to the male.

Cytosolic free calcium levels in sickle red blood cells.

In this study, we used a recently developed nuclear magnetic resonance (NMR) technique to measure ionized calcium in sickle erythrocytes. The NMR technique, which involves 19F NMR studies of a fluorinated calcium chelator quinMF, [2-(2-amino-4-methyl-5-fluorophenoxy)methyl-8-aminoquinoline-N,N,N',N'- tetraacetic acid] provides a novel approach to the study of ionized calcium in erythrocytes since the presence of hemoglobin precludes the use of fluorescent calcium indicators. The mean value for ionized calcium in oxygenated sickle erythrocytes was 18 +/- 2 nmol/L (SE). Experiments with normal RBCs gave a mean value of 21 +/- 2 nmol/L (SE). After 1 hour of deoxygenation, mean values for ionized calcium in sickle erythrocytes did not increase as compared with values obtained under oxygen. To investigate whether deoxygenation stimulated endocytosis, sickle erythrocytes were deoxygenated for 1 hour in the presence of impermeant FBAPTA (1,2 bis-(2-amino-5-fluorophenoxy) ethane N,N,N',N'-tetraacetic acid). Cells were then separated from the extracellular medium and assayed for the presence of FBAPTA; they had incorporated significant quantities of the extracellular FBAPTA. This incorporation was not observed with normal erythrocytes. These data are consistent with at least a portion of the elevation in total cell calcium in sickle erythrocytes arising as a consequence of an endocytotic process in which extracellular calcium ions are incorporated into vesicles. Additional experiments show that these intracellular vesicles accumulate Ca2+ on further deoxygenation, consistent with a transient increase in ionized cell calcium. These studies represent the first use of NMR spectroscopy to evaluate endocytotic processes.