Sickle cell disease: selected aspects of pathophysiology.

Sickle cell disease (SCD), a genetically-determined pathology due to an amino acid substitution (i.e., valine for glutamic acid) on the beta-chain of hemoglobin, is characterized by abnormal blood rheology and periods of painful vascular occlusive crises. Sickle cell trait (SCT) is a typically benign variant in which only one beta chain is affected by the mutation. Although both SCD and SCT have been the subject of numerous studies, information related to neurological function and transfusion therapy is still incomplete: an overview of these areas is presented. An initial section provides pertinent background information on the pathology and clinical significance of these diseases. The roles of three factors in the clinical manifestations of the diseases are then discussed: hypoxia, autonomic nervous system regulation and blood rheology. The possibility of a causal relationship between these three factors and sudden death is also examined. It is concluded that further studies in these specific areas are warranted. It is anticipated that the outcome of such research is likely to provide valuable insights into the pathophysiology of SCD and SCT and will lead to improved clinical management and enhanced quality of life.

The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation.

The effects of nonionic polymers on human red blood cell (RBC) aggregation were investigated. The hydrodynamic radius (Rh) of individual samples of dextran, polyvinylpyrrolidone, and polyoxyethylene over a range of molecular weights (1,500-2,000,000) were calculated from their intrinsic viscosities using the Einstein viscosity relation and directly measured by quasi-elastic light scattering, and the effect of each polymer sample on RBC aggregation was studied by nephelometry and low-shear viscometry. For all three polymers, despite their different structures, samples with Rh <4 nm were found to inhibit aggregation, whereas those with Rh >4 nm enhanced aggregation. Inhibition increased with Rh and was maximal at approximately 3 nm; above 4 nm the pro-aggregant effect increased with Rh. For comparison, the Rh of 12 plasma proteins were calculated from literature values of intrinsic viscosity or diffusion coefficient. Each protein known to promote RBC aggregation had Rh >4 nm, whereas those with Rh <4 nm either inhibited or had no effect on aggregation. These results suggest that the influence of a nonionic polymer or plasma protein on RBC aggregation is simply a consequence of its size in an aqueous environment, and that the specific type of macromolecule is of minor importance.

A new, pluronic-based, bone hemostatic agent that does not impair osteogenesis.

OBJECTIVE: Intraoperative bone hemostasis can be accomplished using surgical beeswax (bone wax). However, bone wax locally interferes with osteogenesis, and its use is avoided when bone fusion is critical. We describe the use of a Pluronic copolymer blend as a biocompatible, absorbable, hemostatic agent. METHODS: A rat femur defect model and a femur gap nonunion model were used. For each surgical model, 24 rats were divided into three treatment groups, i.e., those receiving bone wax implants, Pluronic (90% Pluronic P85/10% Pluronic F88) implants, or no implants (control group). After 10, 21, or 42 days, animals were killed and femora were removed for radiographic analysis and hematoxylin and eosin staining. RESULTS: In the femur defect model, no differences were observed between the Pluronic-treated and control groups; hematoxylin and eosin staining demonstrated bone formation and osteocytes within the defect. In the femur gap nonunion model, no fusions occurred in any group. Development of an osseous callus at the gap site was observed for the control and Pluronic-treated groups. In both models, rats that received bone wax implants exhibited no osseous growth. CONCLUSION: The Pluronic blend exhibits handling properties similar to those of bone wax, readily achieves hemostasis, and does not inhibit bone regrowth. Pluronic compounds may serve as effective absorbable hemostatic agents for the treatment of bone bleeding in sites where fusion is critical. In addition, this copolymer blend may find use as a vehicle for the short-term release of pharmacological agents, which may further reduce the incidence of infections, reduce inflammation, and improve fusion rates.

Short-term exposure to sub-lethal doses of lindane affects developmental parameters in Chironomus riparius Meigen, but has no effect on larval glutathione-S-transferase activity.

Chironomus riparius Meigen were exposed to 0, 0.01, 0.1, 0.5, 0.75 and 1.0 ppm lindane for 48 h as fourth instar larvae. Exposure had no effect on glutathione-S-transferase (GST) activity in larvae snap-frozen immediately following exposure. In contrast, exposure had longer-term consequences affecting developmental parameters. Concentrations above 0.5 ppm lindane affected larval behaviour, reduced adult body size and fecundity and delayed emergence times. The lack of significant change in GST activity when life history characters were affected by high concentrations of lindane, suggests that in C. riparius, GST is not a sensitive biomarker of pesticide exposure or effect.

Aggregation of human RBC in binary dextran-PEG polymer mixtures.

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.

Modulation of red blood cell aggregation and blood viscosity by the covalent attachment of Pluronic copolymers.

Despite many years of research, the physiologic or possible pathologic significance of RBC aggregation remains to be clearly determined. As a new approach to address an old question, we have recently developed a technique to vary the aggregation tendency of RBCs in a predictable and reproducible fashion by the covalent attachment of nonionic polymers to the RBC membrane. A reactive derivative of each polymer of interest is prepared by substitution of the terminal hydroxyl group with a reactive moiety, dichlorotriazine (DT), which covalently bonds the polymer molecule to membrane proteins. Pluronics are block copolymers of particular interest as these copolymers can enhance or inhibit RBC aggregation. Pluronics exhibit a critical micellization temperature (CMT): a phase transition from predominantly single, fully hydrated copolymer chains to micelle-like structures. The CMT is a function of both copolymer molecular mass and concentration. This micellization property of Pluronics has been utilized to enhance or inhibit RBC aggregation and hence to vary low-shear blood viscosity. Pluronic-coated RBCs were prepared using reactive DT derivatives of a range of Pluronics (F68, F88, F98 and F108) and resuspended in autologous plasma at 40% hematocrit. Blood viscosity was measured at a range of shear rates (0.1-94.5 s(-1)) and at 25 and 37 degrees C using a Contraves LS-30 couette low shear viscometer. RBC aggregation and whole blood viscosity was modified in a predictable manner depending upon the CMT of the attached Pluronic and the measurement temperature: below the CMT, RBC aggregation was diminished; above the CMT it was enhanced. This technique provides a novel tool to probe some basic research questions. While certainly of value for in vitro mechanistic studies, perhaps the most interesting application may be for in vivo studies: typically, intravital experiments designed to examine the role of RBC aggregation in microvascular flow require perturbation of the suspending plasma to promote or reduce aggregation (e.g., by the addition of dextran). By binding specific Pluronics to the surface, we can produce RBCs that intrinsically have any desired degree of increased or decreased aggregation when suspended in normal plasma, thereby eliminating many potential artifacts for in vivo studies. The copolymer coating technique is simple and reproducible, and we believe it will prove to be a useful tool to help address some of the longstanding questions in the field of hemorheology.

Polyethylene glycol-coated red blood cells fail to bind glycophorin A-specific antibodies and are impervious to invasion by the Plasmodium falciparum malaria parasite.

This study was designed to assess the binding of glycophorin A-specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion by Plasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A-specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A-specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol).

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.

Increased polymorphonuclear leucocyte rigidity in HIV infected individuals.

AIM: Individuals with human immunodeficiency virus (HIV) infection were evaluated for evidence of abnormal polymorphonuclear leucocyte (PMN) rigidity, which can alter capillary blood flow. METHODS: The transit time of individual PMN through 8 microm pores in a cell transit analyser was used as a measure of cell rigidity. PMN transit time was compared between HIV infected individuals (n=45) with and without CMV retinitis and HIV negative controls (n=17). RESULTS: Transit times were longer for PMN from HIV infected individuals than for PMN from controls (p<0.001). PMN from HIV infected individuals with CMV retinitis (n=13) had longer transit times than PMN from those without CMV retinitis (n=32, p<0.001). Transit times were longer in HIV infected individuals with lower CD4+ T lymphocyte counts (p<0.001). Regression analysis indicated that the relation between transit times and the presence of CMV retinitis could not be explained solely on the basis of low CD4+ T lymphocytes. In HIV infected individuals, mean transit time was not correlated with age, blood pressure, or serum creatinine, cholesterol, or triglycerides. CONCLUSIONS: HIV infected individuals appear to have increased PMN rigidity, a cellular change that might be involved in the pathogenesis of HIV related retinal microvasculopathy. PMN rigidity appears to be related to severity of immune dysfunction. PMN rigidity may remain high in patients with CMV retinitis after elevations of CD4+ T lymphocyte counts that result from potent antiretroviral therapy.

Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage.

Beta-thalassemia major is characterized by ineffective erythropoiesis, although it is difficult to define the dynamics of this process from the static information revealed by analysis of bone marrow (BM) aspirates. We aimed to study the kinetics of sequential erythroid differentiation in beta-thalassemia major. We isolated the progenitor cells (CD34(+) and CD34(+)CD38(-) cells) from BM of thalassemia major patients and studied in vitro erythropoiesis. This is the first report of an in vitro study in human beta-thalassemia major from purified BM CD34(+) progenitor cells, using erythroid culture conditions, which allow unilineage differentiation to mature enucleated red blood cells. In contrast to normal donors, a high proportion of BM CD34(+) and CD34(+)CD38(-) progenitors from beta-thalassemia major coexpressed the late erythroid lineage-specific protein glycophorin A and generated a higher proportion of erythroid colonies. However, despite the marked increase in erythroid clonogenicity of the progenitor population, erythroid cultures initiated from beta-thalassemia major BM CD34(+) cells expanded 10- to 20-fold less than from normal BM. There were less viable cells during differentiation, specifically after the polychromatophilic normoblast stage. There was a progressive increase in the apoptotic erythroid progeny with differentiation, and apoptosis occurred predominantly at the polychromatophilic normoblast stage. In thalassemia major, BM progenitor cells show increased erythroid clonogenicity, increased expression of late erythroid lineage-specific proteins, and accelerated erythroid differentiation. However, despite the apparent increased erythroid commitment, ineffective erythropoiesis occurs due to apoptosis at the polychromatophil stage. Identification of the differentiation stage at which apoptosis occurs will permit further studies of the underlying mechanisms and target therapeutic strategies to improve red cell production.

Increased rigidity and priming of polymorphonuclear leukocytes in sepsis.

It has been proposed that abnormal mechanical properties may contribute to capillary retention of polymorphonuclear leukocytes (PMN) in sepsis, leading to the development of organ dysfunction. The present study was designed to determine whether PMN rigidity is increased in severe sepsis, and whether changes in the rheologic behavior of PMN correlate with the clinical course in sepsis. Eighteen adults with severe sepsis were studied over a period of 14 d; 11 survived and seven died. PMN deformation behavior was investigated via micropore filtration, using the cell transit analyzer. On Day 0, PMN rigidity was 2.5-fold greater for sepsis patients than for five normal controls (p < 0.001). PMN rigidity progressively improved over the 14 d study period for patients who recovered, but not for those who died; clinical indicators correlated with PMN rigidity. Patient PMN also exhibited a 5-fold greater increase in rigidity in response to formyl-methionylleucylphenylalanine (fMLP) than did control PMN. Both the increased rigidity and enhanced response to fMLP could be simulated in vitro by incubation of normal PMN with tumor necrosis factor-alpha (TNF-alpha). We conclude that circulating PMN are more rigid in severe sepsis, and are "primed" for an augmented response to chemotactic stimuli. These findings support the hypothesis that cytokine-mediated increases of PMN rigidity may lead to sequestration of these cells in capillaries and to the consequent impairment of microvascular perfusion in sepsis.

An in vitro model of human red blood cell production from hematopoietic progenitor cells.

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

Covalent binding of poly(ethylene glycol) (PEG) to the surface of red blood cells inhibits aggregation and reduces low shear blood viscosity.

A simple method to coat human red blood cells (RBC) with PEG is described. Using a reactive derivative, monomethoxy-PEG (mPEG) was covalently attached to the surface of RBC in aqueous media under mild conditions. The PEG coating dramatically reduced aggregation and low shear viscosity of RBC resuspended in autologous plasma, and inhibited RBC agglutination by blood group-specific antibodies. Morphology and deformability of the PEG-treated cells were unaltered. The PEG coating of the RBC surface may be of significant benefit in the treatment of a variety of diseases characterized by vasoocclusion or impaired blood flow, e.g., myocardial infarction, sickle cell disease.

Determination of particle sedimentation rate by ultrasonic interferometry: role of particle size, density and volume fraction.

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25 degrees C using latex spheres of different diameters (7 to 20 micron) and densities (1.062 to 1.190 g/cm3) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.

Decreased deformability of polymorphonuclear leukocytes in diabetic cats.

OBJECTIVE: Polymorphonuclear leukocytes (PMN) have been found to be less deformable in humans with non-insulin-dependent diabetes. It has therefore been hypothesized that white blood cells (WBC) may affect the development of diabetic microangiopathy. This study was designed to determine whether PMN or small and large lymphocytes were less deformable in a large animal model of diabetes-chronically hyperglycemic pancreatectomized cats. METHODS: Venous blood was withdrawn from 13 normal and 14 diabetic cats. The diabetic cats had been kept in poor metabolic control since their pancreatectomy (7-113 months before this study). Blood cell counts, hemoglobin concentration, hematocrit, erythrocyte volume, and WBC differential counts were obtained from the blood samples. Purified mononuclear WBC and PMN fractions were obtained by separating the blood on a discontinuous Percoll gradient. The deformability of each cell fraction was determined using a Cell Transit Analyzer (ABX, Montpellier, France) that measures the transit time of cells through 7.5-microns pores. By varying the sampling rate of the CTA and the pressure difference across the filter, the subpopulations of the mononuclear fractions (small and large lymphocytes) could be identified and each subpopulation analyzed separately. RESULTS: Median transit times for the PMN and small lymphocytes were significantly greater for the diabetic cats, but no difference was found in the large lymphocyte fractions. CONCLUSIONS: These results are in accordance with the finding that PMN are less deformable in humans with diabetes. We also showed that the small lymphocytes from diabetic cats have prolonged transit times. The results suggest that PMN may contribute to the development of microangiopathies like diabetic retinopathy. Diabetic cats may prove useful for testing potential therapies to improve WBC deformability.

Mean red cell volume as a correlate of blood pressure.

BACKGROUND: Clinical studies suggest that hypertensives have lower mean corpuscular volume (MCVs) than do normotensives. Epidemiological studies show no relation or higher MCVs. In the present study of elderly men (71 to 93 years of age) of the Honolulu Heart Program, elements of both findings are confirmed. METHODS AND RESULTS: Three groups are identified: (1) those receiving no hypertension treatment, (2) those receiving treatment with any diuretic, and (3) those receiving treatment with nondiuretics only. MCV is lower in group 3 than in group 1 (-0.85 fL, P<.001) but the same in groups 1 and 2. Within groups 1 and 3, inverse relations of -0.22 and -0.09 mm Hg/fL (P<.05) are noted for systolic (SBP) and diastolic (DBP) blood pressures. No relations are observed in group 2. MCV and red blood cell count (RBC) are inversely correlated (r=-.45). In group 2, adjustment for RBC unmasks a direct relation between MCV and SBP (0.5 mm Hg/fL, P=.02) and DBP (0.3 mm Hg/fL, P=.02). In groups 1 and 3, relations between SBP and MCV are lost after adjustment for RBC (0.005 mm Hg/fL). For DBP, adding RBC plus an MCV x RBC interaction is significant (P<.001). DBP is 5 mm Hg greater in the highest RBC quartile than in the lowest. A +3 mm Hg difference between extreme MCV quartiles is noted only at high RBC levels. CONCLUSIONS: The relation between blood pressure and red cell measures is probably mediated by whole blood viscosity. Hematocrit is a determinant of whole blood viscosity. Viscosity affects peripheral resistance to blood flow, and peripheral resistance affects DBP. At high RBC levels, MCV may be "downregulated." This may lower whole blood viscosity and partially reduce DBP without compromising flow.

The influence of suspending phase viscosity on the passage of red blood cells through capillary-size micropores.

Much attention has been paid to the study of blood flow in long, narrow tubes. While the influence of tube diameter and driving pressure have been examined in detail, the influence of suspending phase viscosity has generally been assumed only to affect the blood viscosity in a linearly proportional manner, hence the practice of normalizing apparent blood viscosity values by the suspending phase viscosity to give a relative viscosity (e.g., Pries et al., 1992). While this assumption is probably valid for long tubes, it apparently does not hold for blood flow in short tubes (and by extension also for flow in short or branching capillary segments in vivo) in which RBC deformation plays a more significant role. In this paper we present a series of experiments using the Cell Transit Analyzer (CTA) in which the influence of driving pressure and suspending phase viscosity on RBC passage through short, narrow tubes has been systematically evaluated. Over the range studied (1 to 10 cm water), the influence of driving pressure was found to be unremarkable, in that RBC velocity scaled directly and linearly with pressure. This finding is consistent with previous studies. However, a distinct intercept was observed in the linear relationship between RBC pore transit time and suspending phase viscosity, which presumably arises as a consequence of RBC deformation either at the pore entrance or within the pore. Two simple mathematical models for the suspending phase-viscosity/transit-time relationship were considered. The results show that making CTA measurements over a range of suspending medium viscosities is a simple and practical way to obtain additional information about RBC mechanical properties.