Clinical application of recombinant erythropoietin in myelodysplasia.

This article attempts to summarize the current information regarding the clinical application of recombinant human erythropoietin in patients with myelodysplastic syndromes (MDS). To date, more than 300 MDS patients have been reported to be treated with recombinant human erythropoietin. The response patterns have been variable, but in general they range from 20% to 30%. The hormone has been shown to be safe and nontoxic.

Elevated urinary levels of thromboxane and prostacyclin metabolities in sickle cell disease reflects activated platelets in the circulation.

There is evidence for increased factor VII turnover and the associated increased thrombin generation and fibrinolytic activities in sickle cell disease (SCD) that may affect in vivo platelet and endothelial cell reactivity. We studied the release of specific eicosanoids that are indicative of in vivo platelet activation and endothelial cell injury. The circulating and urinary levels of 2,3-dinor thromboxane B2(2,3-dinor-TxB2),TxB2,PGI2 [as 6-keto-PGF1 alpha], and PGE2 were measured in 15HbSS patients, eight HbAA non-haemolytic anaemic individuals and 12 healthy HbAA controls using specific RIAs. The mean urinary 2,3-dinor-TxB2 in the HbSS patients was significantly higher than in both the healthy HbAA and the anaemic controls. 6-keto-PGF1 alpha was undetected in the urines of the healthy HbAA controls, but was measured insignificant amounts in the HbSS and the HbAA anaemic patients. The urinary concentrations of PGE2 and TxB2 in HbSS patients' samples were also significantly higher than those of both control groups (P < 0.05). PGE2 and TxB2 levels were below the detection limit in the plasmas of the HbAA subjects, but were measurable in the HbSS and HbAA anaemic plasmas. The plasma level of 6-keto-PGF1 alpha in the HbSS patients was also significantly higher than in the control groups. The data indicates a persistent inflammatory process in the HbSS patients, and is consistent with the hypothesis that there is platelet and endothelial cell activation in SCD.

Assessing clinical severity in children with sickle cell disease. Preliminary results from a cooperative study.

PURPOSE: Although it is clear that sickle cell disease is curable with bone marrow transplantation, there are few objective criteria that are helpful in the identification of suitable candidates for this aggressive and potentially life-threatening procedure. This disease is characterized by a highly variable clinical course, and there is a need to intervene with marrow transplant before the onset of disease-mediated chronic organ damage. These factors high-light the need for a clinical severity index that can prospectively identify patients who are at high risk for a turbulent clinical course and a poor prognosis. PATIENTS AND METHODS: We used the Cooperative Study of Sickle Cell Disease data base to identify features of the disease in early childhood (i.e., < 2 years of age) that are associated either with significant morbidity later in childhood or early mortality. Our study population includes the 1,944 children who entered the study before 12 years of age. Univariate analysis showed that factors associated with the occurrence of cerebrovascular accident (51 patients) include hematocrit, rate of change of pocked red cell count, and polymer fraction at 40% oxygen saturation (PF40). Only low hematocrit was predictive of death in this pediatric cohort (45 disease-related deaths). RESULTS: Our ability to identify other potential factors that correlate with these outcome measures is limited by their small numbers. Hence, it was necessary to designate a different endpoint whose relationship with various clinical and laboratory parameters could be assessed. To accomplish this, a distribution of acute events, which were defined as any episode of pain or acute chest syndrome, was calculated. Also, the age-specific "expected" event rate, defined as the mean number of events per patient-year of observation, was determined. CONCLUSIONS: The relationship between various aspects of sickle cell disease and high positive deviance from the expected event rate will be assessed in a cohort of 519 children who entered the study prior to 7 months of age and were followed beyond their second birthday.

Plasma and urinary leukotrienes in sickle cell disease: possible role in the inflammatory process.

Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady-state and age-matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean +/- SEM, ng ml-1) were: LTB4, 8.95 +/- 0.26; LTC4, 7.24 +/- 0.21; LTD4, 11.42 +/- 0.40; and LTE4, 14.51 +/- 0.50. Corresponding values for HbSS patients were: LTB4, 6.15 +/- 0.42; LTC4, 13.61 +/- 1.45; LTD4, 6.44 +/- 0.51 and LTE4, 4.97 +/- 0.37. The differences were significant at P < 0.05. Urine levels (mean +/- SEM, ng mmol-1 creatinine), for controls were: LTB4, 10.60 +/- 0.35; LTC4, 360.0 +/- 9.82. Values for HbSS urine were: LTB4, 27.50 +/- 3.33; LTC4, 356.0 +/- 17.87; LTD4, 69.90 +/- 14.51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.

High Ia (HLA-DR) and low CD11b (Mo1) expression may predict early conversion to leukemia in myelodysplastic syndromes.

The FAB classification of myelodysplastic syndromes (MDS) has been useful in predicting prognosis; however, additional methods are required to detect patients at high risk for early conversion to acute nonlymphoblastic leukemia (ANLL). Using a panel of monoclonal antibodies to myelomonocytic surface antigens (MMSA) and flow cytometry, we studied bone marrow cells from 26 patients with MDS of all five FAB subtypes. The MMSA studied included Ia (HLA-DR), CD11b (Mo1), CD14 (Mo2, My4), CD13 (My7), and CD33 (My9). Marrows were considered "positive" for a given MMSA if the percentage of reactive cells exceeded the upper limit of the normal range. Twenty-four of twenty-six patients (92.3%) were CD13 (My7)+, suggesting that CD13 may serve as a diagnostic marker for MDS. Ten of twelve patients who developed ANLL during a median follow-up of 44 weeks were Ia(HLA-DR)+. The Kaplan-Meier estimated median time to leukemia (TTL) was 16 weeks for Ia+ patients and 88 weeks for Ia- patients (P = 0.004). All six patients who developed ANLL before 16 weeks from diagnosis were Ia+, while none of the Ia- patients converted to ANLL before 24 weeks. Nine of thirteen patients with low CD11b (Mo1) expression (< 53% reactive cells) developed ANLL, compared with only two of 11 patients with high CD11b expression (> 53% reactive cells). Kaplan-Meier estimated TTL was 29 weeks for patients with low CD11b, compared to 160 weeks for patients with high CD11b (P < 0.05). Patients who met both criteria, Ia+ and low CD11b, represented the poorest prognostic subgroup, with median TTL of 13 weeks compared with 88 weeks for the others (P = 0.017). Ia and CD11b patterns were not specific for MDS subtype, and their expression did not correlate with blast count. These data suggest that MDS patients whose bone marrow cells demonstrate high Ia (HLA-DR) and low CD11b (Mo1) expression represent a poor prognostic subgroup with short TTL. These patients may be candidates for early aggressive or investigational treatment.

Treatment of myelodysplastic syndromes with daily oral idarubicin. A phase I-II study.

BACKGROUND: Idarubicin, a new anthracycline analogue, is available in an oral preparation, and responses have been observed using relatively aggressive therapy in patients with myelodysplastic syndromes (MDS). The authors studied whether a chronic low-dose schedule would be effective but less myelotoxic. METHODS: Forty-two patients with MDS received daily low-dose oral idarubicin in 5-week courses that included 3 weeks of treatment, followed by a 2-week rest period. Doses were escalated when possible after the second course, and each patient was to receive six courses. RESULTS: Only one partial response was observed. Although no patient had fatal bone marrow toxicity, only eight patients received the full six courses, primarily because of myelosuppression. CONCLUSIONS: This schedule of oral idarubicin is relatively safe but produces fewer responses than are reported with the high-dose pulse regimens.

Plasma factor VII and thrombin-antithrombin III levels indicate increased tissue factor activity in sickle cell patients.

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasma in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin-antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D-dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme-linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0.70 +/- 0.19 U/ml versus 1.16 +/- 0.41 U/ml), whereas F(VII)z in the patients and controls were 0.47 +/- 0.15 U/ml and 1.15 +/- 0.33 U/ml respectively, P < 0.001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 +/- 44 pM versus 42 +/- 2 pM) (P < 0.001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12.85 +/- 1.96 ng/ml versus 4.22 +/- 0.37 ng/ml) (P < 0.001). The D-dimer levels were also higher in the HbSS than control plasmas (1029.6 +/- 58.6 ng/ml versus 224.3 +/- 27.6 g/ml) (P < 0.001), with the patients' values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso-occlusive processes associated with sickle cell painful episodes.

Drug-associated "bite cell" hemolytic anemia.

PURPOSE: "Bite cell" hemolytic anemia is a variant of drug-related hemolysis usually associated with methemoglobinemia and Heinz body inclusions in red blood cells secondary to oxidant drug injury. Bite cells are morphologically characterized as poikilocytes with one or more semicircular portions removed from the cell margin. The purpose of this report is to emphasize the importance of peripheral smear examination in patients with possible drug-associated hemolytic anemia. The morphologic characteristics of bite cells by light microscopy and scanning electron microscopy are detailed in this study, and the pathophysiologic mechanism is discussed. PATIENTS AND METHODS: Clinical and laboratory data were retrospectively studied on eight patients (two men and six women, aged 29 to 85 years) who showed evidence of bite cell hemolytic anemia associated with drug exposure. Multiple standard hematologic laboratory evaluations for hemolytic anemia were performed. Five hundred red blood cells were counted from randomly selected peripheral smear fields for the calculation of bite cell percentage. RESULTS: Peripheral smears showed predominantly normochromic normocytic red cells with prominent bite cells and occasional blister cells. Bite cell counts ranged from 5.5% to 13.6% (mean, 8.7 +/- 3.0% SD) associated with a hematocrit reduction of 3.0% to 13.2% (mean, 8.5 +/- 3.8% SD) and concomitant reticulocytosis of varying degree from 2.3% to 15.4% (mean, 7.3 +/- 4.9% SD). Withdrawal of the offending drug(s) and treatment of underlying diseases resulted in improvement of hemolytic anemia and eventual disappearance of bite cells. A close correlation between hematocrit reduction, reticulocyte response, and bite cell percentage increase was seen. The usual biochemical markers of hemolysis were not consistently observed. Scanning electron microscopy confirmed the light microscopic evidence of bite cell morphology and revealed a keratocytic variant. CONCLUSION: This study emphasizes the importance of peripheral smear examination for early diagnosis and management of bite cell hemolytic anemia. Withdrawal of the putative offending drug(s) and treatment of underlying disorders should result in improvement of this form of drug-associated hemolysis.

Indium-111 oxine labeled erythrocytes: cellular distribution and efflux kinetics of the label.

Indium-111 oxine label erythrocytes are useful in scintigraphic studies of splenic function because of the high yield of gamma-photons [172(90%) and 247(94%) keV] of indium-111. However, the effects of indium-111 oxine on the structural and functional integrity of erythrocytes which might influence their reticulo-endothelial (RE) sequestration are unknown. We examined the morphology of human and rat indium-111 labeled erythrocytes by SEM, the distribution of the label within the cell by analysis of the membrane and cytosol (hemoglobin solution) and the kinetics of efflux of indium-111 from erythrocytes incubated at 37 degrees C in plasma or physiological buffer. Indium-111 oxine labeled red cells retain their discocytic morphology and the cell indices, and density characteristics on phthalate ester are similar to those of the control cells. The efficiency of labeling may be as high as 97%. Human or rat erythrocyte membranes retain 33 and 41% of indium-111, and the cytosol contains 67 and 59%, respectively. About 98% of the indium-111 is bound to the membrane proteins and 1% to the lipid bilayer. Efflux of indium-111 from cells in autologous plasma showed a multiphasic release resulting in about 4-5% release of the label in 2 h and 11.5% in 20 h. Cells in PBS showed 1-5% release of the label during the incubation period. These findings suggest that indium-111 oxine labeling of erythrocytes does not grossly alter the structural and deformability integrity of the cells to induce selective RE sequestration, unless the cells have been damaged prior to or during the labeling procedure, or the spleen is hyperactive.

Irreversible erythrocyte volume expansion induced by tellurite.

Tellurite (K2TeO3) has been suggested as a potential anti-sickling compound because it causes a selective increase in the water content of RBC. To investigate the conditions underlying the increase in RBC volume due to tellurite, normal RBCs were incubated with the compound in a physiological medium and the cells washed with a 10-fold volume of the medium. The washed cells were then incubated at 24 degrees C for periods up to 4 h and the following parameters were determined: MCV, MCH, MCHC and supernatant haemoglobin concentration by standard methods, the density distribution profile by phthalate esters and cell morphology by scanning electron microscopy (SEM). The effect of hypertonic PBS on the tellurite-treated cells was also tested. K2TeO3 induced concentration and time dependent increases in MCV and decreases in MCHC without any apparent change in MCH. The median density and the transitional 60% density range of the cell distribution profile respectively decreased and increased in proportion to [K2TeO3] and time. Hypertonic PBS did not inhibit or reverse the tellurite-induced changes in MCV and MCHC. SEM and photovolumetric measurements demonstrated tellurite-induced large vesicles ranging in size from 24 to 32 micron 3. The proportion of these vesicles increased with time and K2TeO3 concentration. Since tellurite is an oxidant, these findings suggest that its influx into the red cell results in irreversible reactions that disrupt the ion and water regulatory properties of the membrane.

Alcohol-induced vacuolization in bone marrow cells: ultrastructure and mechanism of formation.

Vacuolization has been known for two decades to occur in the cytoplasm and over the nuclei of the erythroid and myeloid precursors in bone marrows of patients with acute alcoholism. Electron microscopic examination of the marrows from four acute alcohol-intoxicated subjects disclosed that the vacuoles are present only in the cytoplasm and free of organized structure. Surface invagination of the cell membrane of erythroblasts leads to endocytosis and consequent vacuole formation. Cytoplasmic vacuolization of bone marrow cells was reproduced in vitro in 8 of 12 bone marrows from normal individuals when incubated for 6 hours or more in nutrient medium containing alcohol. The critical alcohol concentration for vacuolization was 62.5 mg/dl. The proportion of cells developing vacuoles appeared to correlate with the concentration of alcohol particularly above levels of 250 mg/dl.

Vaso-occlusion in sickle cell disease: pathophysiology of the microvascular circulation.

Microvascular dysfunction accounts for the major morbidity and contributes to the mortality among patients with sickle cell hemoglobinopathies. We summarize the microcirculatory dynamics of red cells in sickle cell disease. An overview of the physiological attributes of the microcirculation is presented. The microcirculatory module is a unique organic entity within the tissue domain, which is concerned with the functional exchange of substances between the blood and the tissue environment. The impairment in deformability of sickle red cells and their heterogeneity cause them to show abnormal microvascular flow dynamics that, in turn, contribute to derangement of the microvascular bed. Studies of experimental models in animals have employed the microcirculation of the mesentery, the cremaster muscle, and the mesoappendix. These studies showed the rheological disequilibrium that results as sickle cells course through successive segments of the arterioles, capillaries, and venules. Direct in vivo microscopic observations in human subjects, with analysis and quantitation of the nailfold and bulbar conjunctival capillaries, have also provided useful information as to the adverse effects of sickling on the microcirculation. Sickle cell vaso-occlusion has three phases--initiation, propagation, and resolution. This framework provides a basis for testable hypotheses for verification in appropriately designed experiments. In this context, the determinants of the microvascular flow of erythrocytes in sickle cell disease are emphasized.

Flow dynamics of human sickle erythrocytes in the mesenteric microcirculation of the exchange-transfused rat.

To analyze the microvascular rheology of sickle cells in an intact animal model, rats were isovolemically exchange transfused with human normal (hemoglobin AA) or sickle (hemoglobin SS) erythrocytes (blood group O) or autologous red cells under ambient conditions, and the effects of the heterologous or autologous cells on (a) hemodynamics and respiration, (b) blood gases, and (c) acid-base status of the recipients were determined. Exchange transfusion of rats with autologous red cells or hemoglobin AA or hemoglobin SS erythrocytes was associated with stable mean arterial blood pressure, central venous pressure, respiration rate, blood pH, pCO2, and pO2 during the experimental period, except for tachycardia among the group of rats that received HbSS cells. Arteriovenous oxygen content varied among the three groups of animals, but, nonetheless, suggested adequate tissue oxygen supply under the conditions of the study. Acid-base status also was similar in the three groups of rats. The exchange-transfused rats were utilized to investigate the flow dynamics of red cells in the mesenteric microcirculation by applying intravital microscopy. Time-averaged velocities of the autologous red cells in 16- to 30-microns (id) vessels ranged from 1.07 to 1.25 mm/sec in single unbranched arterioles with varying flux and wall shear rates. Time-averaged velocities of the HbAA cells in single 15- to 35-microns arterioles ranged from 1.16 to 1.24 mm/sec with wall shear rates similar to the estimates for the autologous cells. For both rat and human HbAA RBCs, the flow dynamics were indicative of normal shear-dependent and deformability characteristics of the cells under the flow conditions. Sickle cells exhibited time-averaged velocities of 0.384 to 0.452 mm/sec, lower wall shear rates in 10- to 35-microns single unbranched arterioles, and three times less volumetric flux. In some arterioles, sickle cells with high axial ratio and low deformability showed definite adhesion to the endothelial surface, residing at such sites for several seconds until dislodged by the force of flow. Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and high axial ratio obstructed flow and exhibited residence times of 2 to 88 sec, thereby causing stasis. These data illustrate the microvascular flow behavior of sickle cells and demonstrate the rheological disequilibrium state that can result as sickle cells course through successive segments of the microcirculation.

Assessment of the hydration state of sickle cells by phthalate ester density distribution.

Intracellular hemoglobin S (Hb SS) concentration, a function of cell hydration, has a major influence on the rate of Hb SS polymerization and, therefore, cellular sickling. To determine the density distribution of homozygous sickle hemoglobin cells as a function of cell hydration, cells were incubated in autologous plasma buffer mixtures with final osmolalities ranging from 195 to 490 mosm/kg at ambient Po2. The density distribution of the cells was determined by differential flotation on 20 mixtures of di-n-butyl and dimethyl phthalates with specific gravities of 1.062 to 1.142. Mean cell hemoglobin concentration (MCHC) and mean cell volume (MCV) were determined by standard manual procedures. Cell shape was assessed by scanning electron microscopy (SEM), and the axial ratio (L/W) of the elliptical dense cell fraction measured by an image analyzer interfaced with a computer. The density distribution of normal red blood cells lies within a narrow 1.090 to 1.118 gm/ml density band with the middle or transitional 60% (T60) of the cells occupying a density range of 0.0067 +/- 0.0007 gm/ml (+/- SD). The density distribution of sickle cells shows a broader density band of 1.064 to 1.134 gm/ml, and the T60 was 0.0139 +/- 0.0022 gm/ml. The mean T60 did not change with osmotic variation but the mean T60 of Hb SS cells was significantly greater (P less than 0.005). MCHC and 1/MCV varied directly with the median density of the density distribution. By linear regression analysis and Ponder's osmotic equation, it is evident that sickle cells exhibit restricted volume increases in hypotonic media.(ABSTRACT TRUNCATED AT 250 WORDS)

Sickle cell vaso-occlusion in an animal model; intravital microscopy and radionuclide imaging of selective sequestration of dense cells.

The impaired deformability and heterogeneity of erythrocytes in sickle cell disease endows them with abnormal microvascular flow dynamics. In the sickle cell exchange-transfused rat model, sickle cells exhibit lower volumetric flux and shear rates compared to normal (HbAA) or autologous rat cells. A subpopulation of dense sickle cells and irreversibly sickled cells show a propensity to induce occlusion at precapillary arterioles, residing at such sites for several seconds and causing local rheological disequilibrium. Radionuclide studies with indium-111 demonstrate preferential uptake of labeled HbSS cells in pulmonary microcirculation. These data are relevant to the factors that are involved in the initiation and propagatin of vaso-occlusion resulting in derangement of homeostasis in certain microvascular beds and perhaps painful crisis and selective organ injury.

Cellular and rheological factors contributing to sickle cell microvascular occlusion.

Sickle (HbSS) erythrocytes contain subpopulations that are heterogeneous in shape, size, and density and exhibit abnormal microcirculatory behavior. Their phthalate esters density distributions quantitatively distinguish subpopulations of HbSS cells from density profiles of normal (HbAA) erythrocytes. Filtration of HbSS cell suspensions, devoid of leukocytes, through 5-microns Nucleopore filters at constant flow rate (29.5 microliters/s) yields pressure-time curves that demonstrate deformability of the sickle cells to be several-fold less than equivalent suspensions of normal (HbAA) cells. For a cell flux of 6.43 X 10(5) cells/s, the rate of the rise of the pressure (Pi/t) following 1-2 s of the initial pressure reading indicates occlusion of the filter pores by the dense cell fraction. Rats exchange-transfused with human sickle (HbSS), normal (HbAA), or autologous rat erythrocytes were used to investigate the flow dynamics of these cells in the mesenteric microcirculation by intravital videomicroscopy. Time-averaged velocities of the autologous rat red cells in 16-30 microns (i.d.) arterioles ranged from 1.10 to 1.25 mm/s with varying flux and wall shear rates. Time-averaged velocities of the HbAA cells in single 15-35-microns arterioles ranged from 1.16 to 1.24 mm/s with wall shear rates similar to the estimates for the autologous cells. In contrast, sickle cells exhibited time-averaged velocities of 0.38-0.45 mm/s with lower wall shear rates in 10-35 microns single unbranched arterioles with three times less volumetric flux. In some arterioles, sickle RBCs with a high axial ratio of 3-4 and low deformability showed apparent adhesion to endothelial surfaces and occluded precapillary junctions or entry points for several seconds until dislodged by the higher flow velocity. Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and axial ratios of 3-4 obstructed flow and exhibited residence times of 6-75 s at the sites of occlusion, thereby causing stasis and increasing the local apparent viscosity. Thus, both the in vitro and in vivo data demonstrate the rheological disequilibrium state induced by HbSS cells as they traverse artificial micropores or course through successive segments of the microcirculation. The specific tendency of dense cells with high axial ratio (ISCs) to manifest precapillary junctional blockade and prolonged residence times implicates this cell fraction in the initiation of microvascular occlusion.

Freeze-etching and biochemical analysis of human fetal erythrocyte membranes.

In order to test the hypothesis that there are ultrastructural and supramolecular differences between fetal and adult erythrocyte membranes that are manifested in their functional characteristics, the cells were studied by freeze-etching and transmission microscopy and biochemical methods. Freeze-etching and transmission electron microscopy of fetal erythrocyte membranes showed that the protoplasmic and exoplasmic fracture faces have 24% and 45% greater intramembrane particles respectively compared to adult cells (p less than 0.01). The apparent diameters of the intramembrane particles estimated on the exoplasmic fracture face averaged as follows: 4.84, 7.74, 11.42, and 15.64 nm, which are similar to estimates in adult cell membranes, suggesting similar dimensions for the presumptive glycoprotein structures in the fluid mosaic complex of the cell membranes. The average total cholesterol, phospholipid, and protein content per fetal erythrocyte ghost as well as ratios of protein/lipid, protein/cholesterol, and protein/phospholipids were all significantly greater than in the adult ghost (p less than 0.01). Analysis of fetal and adult ghost proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed similar qualitative and quantitative polypeptide and glycopeptide bands except for the intense appearance of bands 4.5 and 8 in the fetal samples. Polypeptide chains per ghost membrane were significantly greater in fetal ghosts than in adult ghosts. However, the molar ratios of the major polypeptides relative to band 3, the predominant protein in the ghost membrane, are comparable for the two cell types except for bands 4.5 and 8. These findings suggest that the molecular characteristics of the erythroid plasma membrane vary with the developmental age.

Erythrocyte density distribution in sickle cell anemia.

Density distributions were determined for sickle cell erythrocytes from 27 patients with HbSS genotype using the phthalate ester microcapillary differential flotation method of Danon and Marikovsky. Mean density distribution curves showed HbSS erythrocytes to have trimodal populations with significant increases in both dense and light cell fractions when compared to 20 normal controls of HbAA genotype (p less than 0.05). Irreversibly sickled cell (ISC) and reticulocyte counts were compared with density distributions. Integrated unit areas under the dense cell population curves correlated with ISC percentages, while corresponding unit areas for light populations correlated to a lesser extent with reticulocyte percentages. Mean cell density, D50, varied widely among patients and correlated poorly with the ISC or reticulocyte percentage; however, D50 did correlate with the net change in integrated unit areas. Several patients had repeated density distributions over a 2-year time period. All HbSS patients showed increased but variable dense cell fractions which could not be definitively correlated with the clinical state of the patient. Transfusion reproducibly resulted in a lowering of the dense cell fraction. Erythrocytes from the HbSC patients showed a uniform increase in density and absence of the large dense cell fraction seen in most HbSS patients. This method provides a simple means for quantitation of the light and dense cell fraction in blood of patients with sickling disorders and displays the profile of erythrocyte density heterogeneity for the individual sickle cell patient.