An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.     

Variability of erythrocyte size and hemoglobin content observed in man and four selected mammals

A new measurement technique employing light scattering at different angles has been developed for analysis of blood cells. This method which is part of the Technicon H*1 system, a hematology analyzer designed for routine processing of human blood samples, allows the independent measurement of cell volume and hemoglobin content of isovolumetrically sphered red blood cells. Analysis with this instrument of the blood of humans, dogs, rabbits, rats and mice demonstrates that in addition to the expected differences in hematologic parameters, the intrasample distribution of cell hemoglobin is species dependent. In general, cell hemoglobin content is more tightly controlled for the other mammals when compared to humans. In particular, the dogs tested showed the least variability in cell hemoglobin content both within species and within sample.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Acellular hemoglobin solution enters compressed lung capillaries more readily than red blood cells.

High lung inflation pressures compress alveolar septal capillaries, impede red cell transit, and interfere with oxygenation. However, recently introduced acellular hemoglobin solutions may enter compressed lung capillaries more easily than red blood cells. To test this hypothesis, we perfused isolated rat lungs with fluorescently labeled diaspirin cross-linked hemoglobin (DCLHb; 10%) and/ or autologous red cells (hematocrit, 20). Septal capillaries were compressed by setting lung inflation pressure above vascular pressures (zone 1). Examination by confocal microscopy showed that DCLHb was distributed throughout alveolar septa. Furthermore, this distribution was not affected by adding red blood cells to the perfusate. We estimated the maximum acellular hemoglobin mass within septa to be equivalent to that of 15 red blood cells. By comparison, we found an average of 2.7 +/- 4.6 red cells per septum in zone 1. These values increased to 30.4 +/- 25.8 and 50.4 +/- 22.1 cells per septum in zones 2 and 3, respectively. We conclude that perfusion in zone 1 with a 10% acellular hemoglobin solution may increase the hemoglobin concentration per septum up to fivefold compared with red cell perfusion.     

对地贫红细胞的显微激光散射和图象分析

应用显微准弹性激光散射(MQLS)技术与显微生物医学图象分析技术对地中海贫血红细胞及胞内血红蛋白动态特性进行了研究.在实验中,比较了正常人及地贫患者红细胞胞内血红蛋白聚集体的平均流体力学半径、平均平动扩散系数及红细胞膜的搏动频率等动态特性参数,以及细胞的截面积、规化形状因子、长径、短径、灰度等图象分析数据,发现地贫红细胞的血红蛋白聚合物平均流体力学半径远远大于正常人红细胞的,其大小变异亦较正常人大,且其膜搏动频率也较为缓慢,细胞的截面积也变小.这反映了地贫红细胞内有较大的蛋白质聚合物存在和红细胞变形能力差的特性.研究还表明,显微准弹性激光散射技术结合图象分析技术,可使测量的可比性和准确性大大提高,预期可广泛适用于各种活细胞动态特性的研究.     

Crosslinking of isolated cytoskeletal proteins with hemoglobin: a possible damage inflicted to the red cell membrane

Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility.     

Alterations in chloride transport during differentiation of Friend virus-transformed cells.

Friend erythroleukemic cells can be induced by a variety of agents to synthesize hemoglobin and to exhibit other characteristics suggesting erythroid maturation. Upon induction of hemoglobin synthesis with dimethylsulfoxide (DMSO), the chloride flux in Friend cells gradually increases, until after five days of exposure to DMSO (when the hemoglobin content of the cells approaches that of the mature erythrocyte) the flux is three times the value in non-induced cells. A similar flux increase is observed in the presence of a different type of inducer, hypoxanthine, but no increase in flux is seen in the mutant cell line, TG-13, which does not synthesize hemoglobin after DMSO treatment. Thus, the flux increase seems to be associate d with the induction process, rather than being a direct effect of the inducing agent. After DMSO treatment, the sulphate flux decreases and the chloride/sulphate selectivity increases, aswould be expected if the cells were becoming more like red cells. On the other hand, the sensitivity of the chloride flux to the inhibitor, furosemide, and to temperature is the same in the induced as in the non-induced Friend cells, and different from that of the mature red cell. Thus, the anion transport properties of the induced Friend cell are different from those of both the non-induced Friend cell and the mature erythrocyte. Either the system in the induced cell represents an intermediate stage in the development of the mature red cell characteristics, or else the maturation of transport function in the Friend cell differs from that in normal erythrocyte precursors.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

The accumulation of basic nonhemoglobin proteins during the differentiation of primitive (embryonic) red cells of amphibia

The preferential accumulation of hemoglobin is a characteristic of the differentiation of definitive (adult) red blood cells. Since primitive (embryonic or larval) red blood cells have many properties which contrast with definitive red cells, the accumulation of red cell proteins was analyzed during the differentiation of primtive red cells to determine whether or not hemoglobin was the only protein which showed a substantial increase in amount. Primitive red cells of amphibia were used because the mature circulating cell retains large amounts (13%) of specific, well characterized, nonhemoglobin proteins (CP). Preparations of primitive red cells enriched in immature cells were obtained from the circulation of bullfrog tadpoles recovering from phenylhydrazine-induced anemia. The amount of CP, determined by electrophoresis, imunodiffusion, and ion-exchange chromatography, was compared for red cells from normal animals and anemic animals. Mature cells contained three to six times the amount of CP and three times the amount of hemoglobin found in the population of red cells enriched in immature cells. The accumulation of CP during maturation of primitive red cells indicates that differentiation of primitive red cells is less restrictive than differentiation of definitive red cells. Since the primitive red cell is less specialized than the definitive red cell, it is possible that primitive red cells have several roles in the developing animal, in contrast to the single role of synthesizing and maintaining hemoglobin in the adult animal.     

A Color Image Analysis Method for Assessment of Germination Based on Differential Fluorescence Staining of Bacterial Spores and Vegetative Cells Using Acridine Orange

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.     

X-ray microanalysis investigation of the changes in Na, K, and hemoglobin concentration in plasmodium falciparum-infected red blood cells

Plasmodium falciparum is responsible for severe malaria. During the ∼48 h duration of its asexual reproduction cycle in human red blood cells, the parasite causes profound alterations in the homeostasis of the host red cell, with reversal of the normal Na and K gradients across the host cell membrane, and a drastic fall in hemoglobin content. A question critical to our understanding of how the host cell retains its integrity for the duration of the cycle had been previously addressed by modeling the homeostasis of infected cells. The model predicted a critical contribution of excess hemoglobin consumption to cell integrity (the colloidosmotic hypothesis). Here we tested this prediction with the use of electron-probe x-ray microanalysis to measure the stage-related changes in Na, K, and Fe contents in single infected red cells and in uninfected controls. The results document a decrease in Fe signal with increased Na/K ratio. Interpreted in terms of concentrations, the results point to a sustained fall in host cell hemoglobin concentration with parasite maturation, supporting a colloidosmotic role of excess hemoglobin digestion. The results also provide, for the first time to our knowledge, comprehensive maps of the elemental distributions of Na, K, and Fe in falciparum-infected red blood cells.     

Sickle hemoglobin instability: a mechanism for malarial protection

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

Density distribution and cation composition of red blood cells in newborn puppies

The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr⁵¹ and neonatal cells with Fe⁵⁹. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.     

The Homeostasis of Plasmodium falciparum-Infected Red Blood Cells

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15–32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before ~48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.