Evaluation of a new blood cell counter with sheath flow system

A blood cell counter with a sheath flow system has been developed to eliminate the drawback of the Coulter type blood cell counter, namely, signal distortion caused by the cells passing through the electric aperture gate. With the new system, signal distortion was low and a near-normal distribution curve for erythrocytes and resin particles was obtained. The counter has a computer program for determining the red cell distribution width, which represents an actual size difference at 20% of the relative frequency of the distribution curve. This is independent of mean cell volume, and is considered to be of clinical importance. We examined the values in 2,300 healthy subjects and patients with various hematological disorders.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

High mean platelet volume after myocardial infarction: is it due to consumption of small platelets?

Seventy nine men surviving after sustaining a myocardial infarction in 1982, and who had at that time had raised mean platelet volumes compared with controls, were followed up after 18 months. The shape of each man''s platelet distribution curve was calculated from the mean platelet volume, platelet count, and platelet distribution width. The calculated curves were in close agreement with the curves plotted by the Coulter counter from the raw data. These curves did not differ significantly from those of a current control group, but the curves plotted from the variables measured at the time of myocardial infarction in 1982 showed a deficit of platelets in the volume range 5-12 fl amounting at maximum to 30% (p less than 0.0001); there were no significant differences above 12 fl. The deficit of small platelets became more appreciable during initial admission, was less at one month''s follow up, and had disappeared at one year. The deficit of small platelets is probably an effect rather than a cause of infarction.     

Age dependent change in size distribution of blood cells in fetal and young mice

The mean corpuscular volume (MCV) and the size distribution of circulating blood cells were determined in the fetal mice of C3H/He strain by using a new electric cell size analyser, Coulter Channelyzer C-256. On the 12th day of gestation, the volume of circulating blood cells was distributed between approximately 240-820 fl (mode at 470 fl), and the MCV was 534.9 +/- 30.1 fl, i. e., ten to eleven times that of adult one. On the 14th day, two types of cell population were observed; one with smaller cell volume and another with larger one corresponded to that of the blood cells on day 12. Therefore, two peaks were observed to be at 140 and 501 fl in the size distribution curve. The cell population with large volume observed on day 12-14 had been almost disappeared by the 16th day of gestation, and the small blood cells became dominant. The MCV of blood cells was then decreased with the development of fetus, from 188.2 +/- 19.3 fl on day 16, to 135.1 +/- 7.3 fl on day 18, and 117.5 +/- 7.2 fl on day 20. The size of blood cells continued to decrease gradually after birth, and became adult range by 8 weeks after birth. The MCV values of the blood cells were 120.9 +/- 8.6 fl, 87.5 +/- 6.2 fl, and 48.7 +/- 0.8 fl for the newborns of 1 day and 7 days old, and the adult mice, respectively. White blood cells were not separated from the blood samples in this study. However, the size distribution and MCV presented above were appeared to be related essentially to the red blood cells, since the number of white blood cells are negligible small compared with that of the red cells.     

Possible sequential transformation of a series of acidophils in the pituitary autografts in the renal capsule of male rats in association with ACTH cell.

In our electron microscopy, acidiphils in the pituitary autografts placed in the renal capsules of immature male rats underwent a sequential transformation with the lapse of time: Within 3 and 6 days, all the somaotrophs packed with the large granules of about 350 mmu diameter dispersed. The size and number of the granules in somatotrophs were quickly and markedly reduced with severe modification of cell shape. There was evidence during this time course that Siperstein's or Moriarty's corticotrophs might be synonymous with the stellate shape of acidophils with the arrangement of small granules 150-200 mmu in diameter along the cell acidophils. The "acidophils of the small granule type" possibly related to ACTH production according to Yoshimura et al. (1974) were frequently detected in the grafts as elongated or irregularly shaped cells. Their minute granules 100-150 mmu in diameter were also distributed in row in the cytoplasmic peripheral area. Gradual loss of the minute granules below 100 mmu in diameter eventually made the acidophils to transform into agranular cells. Our own idea that ACTH secretion might correlate with a series of cells transforming along the acidophil-axis was indirectly supported by the present observation on pituitary grafts. On the other hand, basophils rapidly degenerated and died away. Ten and 20 days after autografting, the graft cells which might be principally composed of the cells of acidophil origin enormously proliferated through mitotic division, showing the homologous fine structure, without the normal cell individuality. They always contained three different size and shape of granules simulataneously. Significance of such a rapid and strong response of acidophils to the ectopic replacement in the immature male rats was discussed from the view-point of hypothalamic regulation to simple protein hormones.     

Constancy of cell volume during shape change of erythrocytes induced by increasing ATP content

Erythrocytes in long-preserved blood are spherical, but when the cells are incubated with inosine and adenine, the resulting increase in ATP content is accompanied by a shape change of the cells to discoidal form via a crenated form. The cells incubated with adenine alone or with no addition remain almost unchanged in shape. When incubated with inosine alone, the elevation in ATP level is less than that with both inosine and adenine, and the cell shape remains unchanged or changes partially into a crenated form. These phenomena occur in the presence of EDTA as well as in the absence of serum protein in the media. The cell volumes are measured as packed cell volume after centrifugation, by means of a Coulter counter (model S), and by determination of the intercellular space by the use of¹³¹I-labeled bovine serum albumin. The results show that no alteration in cell volume occurs during the shape changes. Accordingly, the surface area of the cell must increase with increase in the ATP content. This suggests that both the lipid bimolecular layer and the undermembrane structure are altered during the shape change.     

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

Formation and contraction of a microfilamentous shell in saponin- permeabilized platelets

To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin-stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.     

Platelets, endothelial cells and macrophages in the spleen. An ultrastructural study on perfusion-fixed organs.

Rabbit spleens have been examined after perfusion fixation with and without prior washing with various fluids. The platelets were stored in the splenic sinuses and in the cord spaces as single platelets, or in loosely packed aggregates which appeared to be anchored to the endothelium by one or a few platelets. After washing prior to fixation most of the platelets disaggregated and regained their normal shape. Some platelets adhered to morphologically normal endothelium even after prolonged perfusion. Occasionally, platelets were observed inside splenic endothelial cells. Others were closely associated with macrophages, many of which also contained engulfed platelets. There was no morphological evidence of a particular platelet population being retained in the spleen after washing. In the sinuses special granule-rich cytoplasmic structures were observed. They were interposed between ordinary endothelial cells and contained a large number of small lysosome-like granules. Nuclei were never observed in these structures, probably because they consisted of pseudopod-like protrusions. Their origin and function are discussed. They may represent actively phagocytizing elements.     

Inhibitory effects of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the ADP-stimulated aggregation of gel-filtered bovine blood platelets

The effect of DIDS, a specific inhibitor of anion transport in the erythrocyte membrane, on the ADP-stimulated aggregation of gel-filtered bovine blood platelets was examined. Marked inhibition of aggregation was observed at concentrations of more than 5 x 10(-5)M DIDS. On preincubation with platelets for 30 min, DIDS was more potent and significant inhibition was observed at concentrations of over 2 x 10(-7)M. Since ADP-stimulated aggregation of bovine gel-filtered platelets precedes the release reaction, these results suggest that an anion transport system in the plasma membrane is involved in platelet aggregation.     

The kinetics of cellular aggregation induced by turbulent flow

A theoretical treatment for the aggregation of cellular dispersions in a turbulent fluid is proposed based on the work of Saffman &; Turner (1956). The use of an expression for the rate of collisions between cells and cell aggregates which is dependent on the size of the colliding cell particles gives theoretical results which markedly reflect many of the features of cellular aggregation as found experimentally by pulse height analysis using a Coulter counter and particle size discriminator. In particular the shape of the distribution curves, the rate of change of single cell population and the attainment of an equilibrium state as well as the occurrence of cell aggregate redistribution during aggregation are shown to be consistent with aggregation in a turbulent field.It is also shown that the nature of the initial cell aggregate distribution has a very significant effect on subsequent aggregation kinetics.The theory has been applied to the aggregation of two Chinese hamster cell lines and gives a satisfactory explanation of the experimental results.     

PRECISE MEASUREMENT OF THE CHANGE OF STATISTICAL DISTRIBUTION OF CELL SIZE OCCURRING DURING THE SYNCHRONOUS CULTURE OF CHLORELLA

Change of statistical distribution of cell size occurring inthe synchronous culture of Chlorella ellipsoidea was followedby using a COULTER counter. The culture was started using apopulation of D-cells which showed a sharp distribution of cellsize. During the growth phase, there occurred characteristicchanges in the height and shape of the distribution curve; namely,the height first decreased with broadening of the distributionand then increased to the level even higher than the originalpeak. The broadening of the curves, which indicates the loweringof degree of homogeneity of population, occurred during theperiod of most active growth, and the homogeneity was restoredat later stages of ripening where the growth became sluggish.When the ripened cells (L₃) were transferred to the darknesstheir volume decreased to some extent before the occurrenceof cellular division. It was assumed that the shrinkage of cellsobserved may be partly due to exudation of water from the cellsand partly to the consumption of fuel material caused by enhancedrespiration, both having been shown to occur at the stage ofcell maturation. (Received June 12, 1964; )     

Measurement of the size distribution of human erythrocytes by a sedimentation method

The distribution of sedimentation velocities was determined, by a photoelectric method, for human erythrocytes at low concentrations in Ringer solution. The light absorption at 414 nm was measured, as a function of time, 10 mm below the top of the column. From the frequency distribution of cell velocities that of R_s √ρ-σ was found; R_s being the Stokes' radius, ρ the cell density and σ the density of the solution. Cell density was measured by the phthalate method and the mean Stokes' radius was found to be 2.58 μm. The size distributions showed some skewness but were in good general agreement with those measured by Celloscope counter, and with reported measurements from photomicrographs of cells in hanging drop suspensions. The skewness was much less than that encountered with electric sensing zone instruments (e.g. Celloscope) and the sedimentation method, being based on entirely different premises, provides an important check on such data. The skewness is due to a bias in the orientation of human erythrocytes during sedimentation. This bias may be a characteristic of biconcave cells; it could be absent in many species and reliable measurements of size distribution would then be obtained.     

肺炎支原体感染对儿童血常规参数及C反应蛋白水平的影响

摘要 目的：探讨肺炎支原体（Mycoplasma pneumoniae, MP）感染对儿童血常规数及C反应蛋白（C reactive protein, CRP）水平的影响。方法：以60例肺炎支原体抗体（MP-IgM）阳性患儿作为观察组,选取同期60名MP-IgM阴性儿童作为对照组,对两组患儿血常规参数和CRP水平进行回顾性分析。结果：与对照组比较,观察组红细胞压积、血红蛋白、单核细胞比例、单核细胞计数、中性粒细胞比例、中性粒细胞计数、红细胞计数均数或中位数升高,嗜碱性粒细胞比例、嗜碱性粒细胞计数、嗜酸性粒细胞比例、嗜酸性粒细胞计数、淋巴细胞比例（LY）、淋巴细胞比计数（LY#）、平均红细胞血红蛋白量、红细胞平均体积、平均血小板体积、血小板压积、血小板分布宽度、血小板计数、红细胞分布宽度均数或中位数降低;观察组患儿的CRP水平中位数显著高于对照组,以上差异有统计学意义（P<0.05）;Logistic多元回归分析结果显示,MP感染与淋巴细胞比例、淋巴细胞比计数降低具有正相关性（P<0.05）。结论：MP感染患儿的血常规参数和CRP水平均发生变化,临床医生应对这些参数给予关注和连续监测,从而提高诊疗效果。     

Profile of hematological values in hybrid hairless dogs

The purpose of this paper is to describe some fundamental physiological data in F 1 hybrids bred from a Mexican hairless dog and beagle cross. These F 1 hybrids numbered 5 hairless dogs and 12 haired dogs. The hematological profile of these offspring was assessed via an automated cell counter and compared with those of healthy beagles. In hairless dogs, red blood cell count, hemoglobin concentration and packed cell volume tended to be higher than in beagles. White cell distribution curves in hairless dogs and beagles yielded a single peak, while in haired dogs one or two peaks were present. Red blood cell and platelet distribution curves revealed few differences among the 3 kinds of dogs.     

Autoradiographic studies of the synthesis of RNA and protein as a function of cell volume in Streptococcus faecium

Mid-exponential-phase cultures were either labeled continuously with tritiated leucine and uracil or pulse-labeled with tritiated leucine. The amount of leucine and uracil incorporated into protein or RNA per cell was determined by grain counts of autoradiographs of cells seen in electron micrographs; the volume of each cell was determined by three-dimensional reconstruction. The average number of autoradiographic grains around cells continuously labeled with uracil and leucine increased linearly with cell volume. In contrast, while the average grain count around cells pulse-labeled with leucine increased in a near-linear fashion over most of the volume classes, less than the expected number of grains were seen around cells in large- and small-size classes. The distribution of grains around cells from both the continuously and pulse-labeled populations could be fit at the 5% confidence level with a Poisson distribution modified to take into consideration the volume distribution of each population of cells analyzed. These findings suggested that large changes in the density of RNA and protein do not occur in most cells as they increase in size; however, there may be decreases in the rate of protein synthesis in some large and small cells. The decrease in the rate of protein synthesis appears consistent with the hypothesis that new sites of envelope growth must be introduced into cells that are close to the division event to restore rapid growth.     

Organic synthesis in algal cells separated into age groups by fractional centrifugation

Summary Synthesis of organic matter was studied in cells of the green, high-temperature alga Chlorella 7-11-05 separated from a nonsynchronized population into fractions of predominantly small or large cells by centrifugation. Rates of synthetic activity were determined as changes in optical density, dry weight, and packed volume of cells in several suspending fluids and under various light intensity conditions.It was found that synthetic activity of the smaller (younger) cell fraction was invariably higher than that of the larger (older) cell fraction, provided a reasonably good separation of cells into size fractions was achieved during centrifugation and the difference between size composition of these fractions was maintained throughout observation.An occasional lack of difference in the performance of the small- and large-cell fractions, or even a higher synthetic activity of the originally large-cell fraction, was traced to cell division taking place during observation. Under conditions favorable to cell division, the large-cell fraction was progressively enriched with smaller (younger) cells, the average age composition of the originally large-cell fraction was shifted toward younger age, and the metabolic activity of the large-cell fraction increased to the extent that, in some experiments, it surpassed that of the originally small-cell fraction.The decline in synthetic activity of cells in the course of cell development previously observed on synchronized cells was thus substantiated on nonsynchronized populations in the absence of the light: dark synchronizing agent and is, therefore, characteristic of normal cell development.     

Structural changes in platelets in alloxan diabetes as parameters of their activation in the vascular bed and morphologic features]

The present study shows that a stable level of blood platelet number persists in diabetic rats (following 15 and 60 days after experimental alloxan injection) Unusual platelet megaforms are seen accumulating (up to 36%), having no size analogs in control animals. A quantitative electron-microscopic analysis demonstrated that the enlargements of diabetic platelets was accompanied by their rounding in shape and by a more frequent (by 2.0-2.6 times) generation of surface pseudopodia. The share of platelets increased in two independent fractions, with considerable deflexion over the normal range limit variations in the number, size and general volume of their alpha-granules (cutting down) or dense (increase) bodies. At the same time a common decrease in these indexes for different types of grains in platelets of various dimensions occurred. The integral picture of the platelet granular apparatus (image of granular apparatus), that demonstrates quantitative proportions between the number and size indexes of different grain types, is found distorted in diabetic rats.     

Size distribution of luteal cells from pregnant and non-pregnant marmoset monkeys and a comparison of the morphology of marmoset luteal cells with those from the human corpus luteum

The size distribution of marmoset luteal cells was determined on Days 6, 14 and 20 after ovulation in non-pregnant cycles and in early pregnancy. Image analysis was used to estimate the cell diameter of dispersed cells prepared from the marmoset corpus luteum (CL). Steroidogenic cells showed a size distribution consistent with one population of cells. There was a significant increase in mean cell diameter (P less than 0.05) from Day 6 to Day 14 in pregnant and non-pregnant animals with no further increase on Day 20. Micrographs of marmoset luteal tissue showed cells of greater than 10 micron containing the organelles typical of steroid-producing cells, and smaller non-steroidogenic cells surrounding the steroid-producing cells. On the basis of microscopy, there were no areas within the CL where cell composition was noticeably different. In contrast, micrographs of human luteal tissue showed two types of steroidogenic cell; most cells were similar to those in the marmoset CL but a smaller population of smaller cells could be distinguished around the periphery and along vascular septa. It is likely that these smaller and larger types of steroidogenic cells are of theca and granulosa cell origin respectively, the two cell populations differing in the degree of electron density and amount of rough endoplasmic reticulum. A distinguishing feature between marmoset and human luteal cells was the shape of the mitochondrian which were considerably rounder in marmoset luteal cells. The origin of steroidogenic cells in the marmoset CL is unclear, although in marmosets and man the luteal cell types display morphological characteristics distinct from the large and small luteal cells described for CL of the domestic ungulates.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.