红细胞的前向光散射

本文从红细胞前向光散射的观点出发对红细胞的尺寸、红细胞的变形性研究以及红细胞容积、血红蛋白浓度等参数的测定作了系统的思考。提出在红细胞与周围悬浮介质折射车相差不大时,反常衍射比夫朗和费衍射更适合用于红细胞前向光散射的研究。利用两组不同角间隔的前向散射光来同时测量红细胞容积和血红蛋白浓度。同时其它一些标识红细胞的参数如平均红细胞容量（ＭＣＶ）、平均血红蛋白量（ＭＣＨ）等均可直接或间接由这两项参数导出。最后还将红细胞的光散射与Ｍｉｅ理论作了对比．     

CONDUCTIVITY AS A MEASURE OF VITALITY AND DEATH

The conductance of Laminaria, Saccharomyces, Bacillus coli and Bacillus butyricus, Chlorella, and of red blood cells has been studied by the writer''s method, and Laminaria by that of Osterhout. For the material studied it has been found that: 1. The conductance of living tissue is closely proportionate to, and determined by that of the surrounding fluid with which it is apparently in equilibrium. Changes in the conductance of the fluid are quickly followed by compensatory changes in that of the tissue. 2. A quantity is defined which is independent of the conductivity of the fluid bathing the tissues. This is called the "net conductance." 3. All the tissues studied, even when dead, offer a resistance to the passage of current greater than that of the surrounding solution. Exceptions which occur under certain conditions will be discussed in a later paper. 4. In view of the wide variety of material studied it seems admissible, in the absence of any evidence to the contrary, to suppose that these conclusions are generally applicable.     

Filterability of blood cells: methods and clinical applications

It could be claimed that expansion in clinical aspects of haemorheology has largely been fueled by the development of a simple test of blood flow properties, i.e., analysis of filterability. With time, the level of sophistication in equipment, theory and sample preparation has increased. Theories for the development of flow in the filter have been described and these enable cellular parameters, such as transit times, to be calculated from experimental data. These theories can be quite general, and applied to filtration of red or white cells. Ideally, experimental design requires an understanding of cell behaviour at the filter and of the effects of factors such as the sample concentration and volume, and the presence of different types of cells or subpopulations. Otherwise, results are susceptible to misinterpretation, particularly if impurities or mixed populations of cells are present. It is thus very important to know accurately the constituents of the test suspension. In clinical applications the trend has been to move away from whole blood filtration, toward use of relatively pure suspension of separated red cells and white cells. In the area of red cell filtration this has led to reappraisal of some previously reported abnormalities. The relatively new study of white cell filtration should benefit from previous experience, but there is the added problem of the reactivity of the cells under test, and their mixed nature. In any case, critical evaluation of the meaning and clinical relevance of results is necessary.     

RAPID ALDOSTERONE-INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na⁺/H⁺exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na⁺/H⁺exchanger.     

REGULATION OF HAEMOPOIESIS IN ALTERED GRAVITY

(1) The aetiology of one of the most striking physiological changes occurring during space-flight, the loss of red blood cells, remains unknown, and its precise time-pattern in flight has not yet been studied. (2) It is suggested that the changes during space-flight responsible for loss of red blood cells in man are (a) loss of plasma volume resulting from disappearance of hydrostatic pressure in the circulation during weightlessness and (b) reduced energy expended in maintenance of form, posture and locomotion resulting from elimination of the usual gravitational load on the muscles. Quadrupeds, like rats, would be expected to suffer minimal blood shifts in weightlessness and therefore have an unchanged plasma volume. However, since in weightlessness the activity-related energy expenditure by the muscles is reduced, the accompanying reduced oxygen demand by the tissues would cause a reduction in erythropoietin levels and so in the production of red blood cells, and a progressive lowering of the total red blood cell mass toward a new steady-state level. (3) Loss of plasma volume alone does not explain the observed loss of red blood cells in astronauts because, in the three manned Skylab missions, as the duration of the missions increased, loss of red blood cell mass decreased, whereas loss of plasma volume increased. This discrepancy is, however, well accounted for by the above hypothesis by taking into consideration the increased level of exercise of the astronauts as the duration of the mission increased. (4) Though water submersion of human subjects does mimic the effects of weightlessness, such effects were overriden in sea mammals because of adaptation to other factors associated with a life in the sea. (5) From the presented analysis of haemopoietic changes observed in spaceflight, an experiment can be designed for a future flight to uncover the causes and mechanisms of these changes and provide a basis for developing protective measures. Thus, the space environment can be used as an investigative tool to enhance the knowledge of the function of the haemopoietic system, which is a major homeostatic system of man and other vertebrates.     

Effect of high osmotic media on blood viscosity and red blood cell deformability

Effects of high osmotic media on the shape and deformability of RBC were examined for determining increasing factors of blood viscosity. Dog blood and Urographin (a hypertonic contrast medium) were used; the plasma osmolality was changed by Urografin suspended in blood. The viscosity was measured for normal RBC and glutaraldehyde-treated RBC suspensions with a cell volume concentration. The RBC deformability was evaluated from the difference in viscosity between the two suspensions. It was shown that normal RBC suspension increased the viscosity with increase in osmolality at high shear rate; hardened RBC suspension decreased the viscosity with increase in osmolality. It was concluded that the RBC deformability decreased with increasing osmolality.     

NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Red blood cell motion and hematocrit distribution in a deforming capillary

Based on the relation between the red blood cell velocity and the blood velocity of the Fahraeus effect, we analyze the motion of a red blood cell in a deforming capillary to investigate how the ventilatory induced deformation affects the transit of the red blood cell through the pulmonary capillary and changes the hematocrit discharged from the capillary. An analytical solution is also obtained for the case of an infinitesimal deformation. The numerical and analytic solutions demonstrate that the variation in discharge hematocrit is proportional to the change of pulmonary capillary blood volume between the time that the red cell enters and the time that it exits the capillary. We also find that this hematocrit variation could be regarded, in terms of transport, to originate from the mid-section of the capillary.     

Effects of sedimentation of small red blood cell aggregates on blood flow in narrow horizontal tubes

The flow properties of aggregating red cell suspensions flowing at low flow rates through horizontal tubes are analyzed using a theoretical model. The effects of sedimentation of small aggregates, which will be formed at comparatively high flow rates, on the relative apparent viscosity are considered. In the case in which a large number of small aggregates are formed in a suspension flowing through a horizontal tube, it seems that red cells are transported as a concentrated suspension through the bottom part of the tube because of sedimentation of aggregates. A two-layer flow model is used for the distribution of red cells. It consists of plasma in the upper part and a concentrated red cell suspension in the bottom part of the tube divided by a smooth and horizontal interface. It is assumed that the suspension is a Newtonian fluid whose viscosity increases exponentially with hematocrit. The velocity distribution, the relative apparent viscosity and the flux of red cells are calculated as functions of width of plasma layer for a different discharge hematocrit. The theoretical results are compared with the results obtained from experimental data. The relative apparent viscosity increases rapidly with an increasing degree of sedimentation over a wide range of plasma layer widths.     

Rosette-forming ability of virus-treated human leukocytes (V-rosettes)

Rosette-formation with auto- and allogeneic red blood cells was applied to detection of human leucocyte subpopulations interacting with Sendai virus (V-rosettes). It was shown that the majority of V-rosette-forming cells appeared to be monocytes. T lymphocytes did not take part in V-rosette-formation since selective elimination of T cells from the mononuclear cells population did not lead to reduction of but increased the number of V-rosettes. Enrichment of cell suspension with B lymphocytes was followed by a rise in the number of V-rosettes thereby allowing the attribution of B lymphocytes along with monocytes to the cell population interacting with virus. The results suggest that ability of virus-exposed immunocompetent cells to react with their own red blood cells may lie at the basis of the development of autoimmune hemolytic anemia and other autoimmune diseases.     

Separation of red blood cells from G6PD-deficient patients in dextran density gradients

Red blood cells of 30 patients with G6PD deficiency were separated and characterized by means of isopyknic dextran density gradient centrifugation. The simultaneous determination of G6PD activity and the percentage of NADPH deficiency cells in relation to the maturation parameters of density, reticulocyte share, GOT and PK activity made it possible to recognize differences in the maturation of red blood cells with G6PD deficiency in normal persons as well as within a group of patients. In each case the more or less diminished enzyme activity of the cell suspension was accompanied by a marked enzyme deficiency of the youngest fraction. It is possible that NADPH defect cells are being eliminated at first. In many cases a direct correlation between the percentage of "empty cells" and the in vitro stability tests with and without NADP+ addition could be identified. Decreased maximal speed, changed kinetic behaviour, and instability of these variants are stressed as being the decisive parameters for the life expectation of red blood cells in patients with G6PD deficiency.     

THE EFFECT OF VALENCY OF CATIONS AND ANIONS ON NEGATIVELY AND POSITIVELY CHARGED RED BLOOD CELLS

1. Under comparable conditions, valency effect may be demonstrated with a suspension of red blood cells and the cations and anions of salts. 2. The valency of the cation determines the degree of the effect on negatively charged cells, the valency of the anion, the effect on positively charged cells. 3. Anomalies in valency effects with different salts and red cell suspensions are in part due to variations in H ion concentration, depending on the degree of hydrolysis of the salt.     

Heterogeneity among dog red blood cells

A phthalate density-separation technique has been used to study the heterogeneity of dog red blood cells that becomes manifest when they are suspended in KCl media. It is demonstrated that the proportions of cells that separate into light and dense fractions can be varied by altering the tonicity of the KCl medium. This results from the fact that the Na and K permeabilities of each cell are continuous functions of cell volume. It was found that quinidine inhibits selectively the volume dependence of Na permeability. In the presence of this drug, the heterogeneity demonstrated by KCl incubation disappears. The notion that dog red blood cells are heterogeneous in their permeabilities to Na and K is thus upheld, but the heterogeneity is not an abruptly discontinuous one, as has been claimed. A sample of dog blood does not contain two discrete populations of red cells.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

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.     

Mesoscale simulation of blood flow in small vessels

Computational modeling of blood flow in microvessels with internal diameter 20-500 microm is a major challenge. It is because blood in such vessels behaves as a multiphase suspension of deformable particles. A continuum model of blood is not adequate if the motion of individual red blood cells in the suspension is of interest. At the same time, multiple cells, often a few thousands in number, must also be considered to account for cell-cell hydrodynamic interaction. Moreover, the red blood cells (RBCs) are highly deformable. Deformation of the cells must also be considered in the model, as it is a major determinant of many physiologically significant phenomena, such as formation of a cell-free layer, and the Fahraeus-Lindqvist effect. In this article, we present two-dimensional computational simulation of blood flow in vessels of size 20-300 microm at discharge hematocrit of 10-60%, taking into consideration the particulate nature of blood and cell deformation. The numerical model is based on the immersed boundary method, and the red blood cells are modeled as liquid capsules. A large RBC population comprising of as many as 2500 cells are simulated. Migration of the cells normal to the wall of the vessel and the formation of the cell-free layer are studied. Results on the trajectory and velocity traces of the RBCs, and their fluctuations are presented. Also presented are the results on the plug-flow velocity profile of blood, the apparent viscosity, and the Fahraeus-Lindqvist effect. The numerical results also allow us to investigate the variation of apparent blood viscosity along the cross-section of a vessel. The computational results are compared with the experimental results. To the best of our knowledge, this article presents the first simulation to simultaneously consider a large ensemble of red blood cells and the cell deformation.     

Rheological behaviors of bovine blood forming artificial rouleaux

A purpose of the present study is to make an artificial rouleau of bovine red blood cells which is not capable of rouleau formation under physiological condition. Rheological behaviors of bovine blood forming artificial rouleaux were examined. The modification of cell surface by enzyme trypsin induced rouleau formation, whereas the modification of cell surface by neuraminidase did not cause any aggregate formation. The drastic elevation of the fibrinogen content in bovine red blood cells suspension also brought about the formation of rouleau. The value of dynamic rigidity modulus G' of bovine red blood cells in saline solution containing high concentration of fibrinogen is somewhat smaller than that of trypsin treated bovine red blood cells in plasma. The value of G' of trypsin treated bovine red blood cells in plasma first increased to a maximum value and then decreased with the time. It is supposed that the removal of macro-molecules from the cell surface facilitates the mutual approach of cells and causes the formation of rouleau which seems to be the same as that of human and horse bloods.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : III. VISCOSITY.

1. Gelatin solutions have a high viscosity which in the case of freshly prepared solutions varies under the influence of the hydrogen ion concentration in a similar way as the swelling, the osmotic pressure, and the electromotive forces. Solutions of crystalline egg albumin have under the same conditions a comparatively low viscosity which is practically independent of the pH (above 1.0). This difference in the viscosities of solutions of the two proteins seems to be connected with the fact that solutions of gelatin have a tendency to set to a Jelly while solutions of crystalline egg albumin show no such tendency at low temperature and pH above 1.0. 2. The formulæ for viscosity demand that the difference in the order of magnitude of the viscosity of the two proteins should correspond to a difference in the relative volume occupied by equal masses of the two proteins in the same volume of solution. It is generally assumed that these variations of volume of dissolved proteins are due to the hydration of the isolated protein ions, but if this view were correct the influence of pH on viscosity should be the same in the case of solutions of gelatin, of amino-acids, and of crystalline egg albumin, which, however, is not true. 3. Suspensions of powdered gelatin in water were prepared and it was found, first, that the viscosity of these suspensions is a little higher than that of gelatin solutions of the same concentration, second, that the pH influences the viscosity of these suspensions similarly as the viscosity of freshly prepared gelatin solutions, and third, that the volume occupied by the gelatin in the suspension varies similarly as the viscosity which agrees with the theories of viscosity. It is shown that this influence of the pH on the volume occupied by the gelatin granules in suspension is due to the existence of a Donnan equilibrium between the granules and the surrounding solution.     

Physical properties of flowing blood

The changes of viscosity, optical reflection and electrical resistivity of blood due to flow are dependent on the orientation and deformation of red cells. From electrical point of view, it can be assumed that blood is suspension of small insulating particles (red cells) in conductive fluid (plasma) when the frequency of supplied voltage is lower than several hundreds KHz. When blood flows, red cells deform and orient in flow direction. Therefore, flowing blood shows anisotropic electrical and optical properties. In steady flow, blood resistivity longitudinal to flow decrease with flow rate, and transverse one increases. Blood flow in living body is not steady but pulsatile. We measured both longitudinal and transverse resistivity changes, optical reflection change and viscosity change of sinusoidally flowing blood in a rectangular conduit. The results are 1) during one period of sinusoidal flow the longitudinal resistivity change is opposite to that of transverse one, 2) the waveform of reflection light change is similar to that of resistance change, and 3) minimum points of both longitudinal resistivity and viscosity changes do not appear at the moment when flow is zero but are delayed. When the amplitude of sinusoidal flow is small and oscillation frequency is high, the phase difference between the zero crossing period of flow and the period of minimum change in resistivity, increases up to 90 degrees. Viscosity of blood decreases with increase of amplitude and frequency of sinusoidal flow.     

CONTROL OF GRANULOCYTE PRODUCTION: SEPARATION AND CHEMICAL IDENTIFICATION OF A SPECIFIC INHIBITOR (CHALONE)

Abstract. It has previously been shown by others that blood serum contains inhibitors of blood cell production acting on the proliferation of granulocy tic and erythrocytic precursor cells in the bone marrow. It is now shown that the active extract from calf blood serum can be further subfractionated into six different components, all of them exhibiting inhibitory effects on the proliferation of rat bone marrow cells in vitro. Ascitic fluid from rats treated intraperitoneally with polyvinylpyrrolidone contains inhibitors which apparently are the same as those found in calf serum.
It was further possible to demonstrate that only one of these inhibitors is contained in mature granulocytes where it is actively synthesized from amino acids and subsequently released into the surrounding medium. By chromatography on Sephadex G-25 of this conditioned medium the inhibiting substance could be obtained in relatively pure form being contaminated only by low amounts of two ninyhdrin-positive substances. the experiments allow the granulocytic inhibitor to be identified as a polypeptide with a molecular weight below 5000. the results suggest that this substance is the granulocytic chalone.