The Distribution of Buoyant Density of Human Erythrocytes in Bovine Albumin Solutions

Leif, Robert C.; Vinograd, Jerome

doi:10.1073/pnas.51.3.520

Cited by 157 publications

(65 citation statements)

References 15 publications

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“…However, those studies either involved other gradient media such as Percoll with 5 % BSA (171, or high-speed centrifugation 39,000 g at 30°C for l h (l), reported average buoyant density greater than that observed with BSA, and did not report an equivalent separation by age. Leif and Vinograd (8) did find a decrease in cellular volume with buoyant density but did not find any significant change in mean cellular hemoglobin concentration across the gradient.…”

Section: Discussionmentioning

confidence: 98%

“…It was demonstrated by Leif and Vinograd (8) and Leif (10) that there is a significant change in shape of the erythrocyte, and differences in deformability have been reported by Me1 and Yee (15) and by Schmid-Schonbeim and Gaehtgeus (18). It is therefore quite possible that the effect of changes in shape could cancel or at least partially mask a true volume change.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, it has been demonstrated that buoyant density, as determined in linear gradients of bovine serum albumin, was directly related to rabbit erythrocyte age (8,16). Having demonstrated that opacity increases with increasing buoyant density, and since buoyant density is directly related to erythrocyte age, opacity is thus directly related to erythrocyte age.…”

Section: Discussionmentioning

confidence: 99%

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Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

Leif

Schwartz

Rodriguez³

et al. 1985

Cytometry

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Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.Key terms: Coulter electronic cell volume, opacity, erythrocytes, impedance, radio frequency, buoyant density, bovine serum albumin (BSA), density gradient centrifugation Leif and co-workers (9,131 proposed that automated cell analysis should be based on the assumption that there are many descriptors available that permit the easy identification of each type of cell present in a heterodisperse population. However, the use of a combination of two impedance measurements (13,5) together with multidimensional fluorescence and light scattering data on a cell-by-cell basis should allow adequate quantification of cellular parameters for specific identification of cells, For flow analysis to maintain a high level of accuracy, it will be necessary to include redundant information by increasing the dimensionality of data in order to prevent errors.The Coulter volume measurement, which was one of the first parameters to be used for the automated counting and analysis of biological cells (2,3), is an impedance measurement at zero frequency (dc) or a measure of resistance. Besides the abovementioned dc resistance (electronic cell volume) measurements, it is possible to perform simultaneously ac impedance measurements. An instrument for this purpose has been developed by Coulter and Hogg (4). The highest resolution in a twodimensional distribution is achieved by producing orthogonal parameters. In the present case, this was accomplished by dividing the radio frequency ac impedance by the dc resistance, which is defined as the opacity.Hoffman and Britt (6) developed a flow-system technique that simultaneously detected the dc Coulter volume and ac impedance. As pointed out by the authors, the use of the low ac frequency of 1 MHz resulted in "a high correlation between the ac and dc parameters for CHO cells." These authors performed their measurements at the reactance maximum in order to maximize the difference between cell types (Britt, personal communication) rather than at the higher frequency range.The ac impedance changes slowly with frequency above approximately 20 MHz, where, according to the bulk measurements of erythrocyte suspensions by Jenin et al.(71, the conductance of intact erythrocytes plateaus. According to these authors, the erythrocyte membrane appears to be practically short-circuited at frequencies above 50 MHz.The purpose of this study was to determine the relationship of the electrical parameters of Co...

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

Leif

Schwartz

Rodriguez³

et al. 1985

Cytometry

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“…[44,45] Red blood cells with a normal morphology have a maximum density of roughly 1.10 g/cm 3 . [45,46] In SCD, sickling and dehydration can lead to the formation of cells (ρ > 1.12 g/cm 3 ) that are denser than the most dense red blood cells in a healthy individual. [44,45,47,48] We developed aqueous multiphase systems (AMPS)-mixtures of polymers in water that spontaneously separate into immiscible liquid phases-to separate and visually identify the presence of dense cells characteristic of SCD.…”

Section: The Testmentioning

confidence: 99%

From the Bench to the Field in Low‐Cost Diagnostics: Two Case Studies

et al. 2015

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Despite the growth of research in universities on point‐of‐care (POC) diagnostics for global health, most devices never leave the laboratory. The processes that move diagnostic technology from the laboratory to the field—the processes intended to evaluate operation and performance under realistic conditions—are more complicated than they might seem. Two case studies illustrate this process: the development of a paper‐based device to measure liver function, and the development of a device to identify sickle cell disease based on aqueous multiphase systems (AMPS) and differences in the densities of normal and sickled cells. Details of developing these devices provide strategies for forming partnerships, prototyping devices, designing studies, and evaluating POC diagnostics. Technical and procedural lessons drawn from these experiences may be useful to those designing diagnostic tests for developing countries, and more generally, technologies for use in resource‐limited environments.

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“…These cells exhibit a marked acceleration of RNA synthesis, demonstrable by radioautography, within 1 Several methods of cell separation dependent upon physical properties of developing erythroblasts have been explored in this and other laboratories. Sedimentation methods, including density gradients (14) and unit-gravity sedimentation (15) have proved unsuccessful, in our hands, to obtain pure populations of nonhemoglobinized erythroid precursors. Exploitation of antigenic differences between immature and mature erythroid cells was suggested by the observations of Borsook and coworkers (4,5) and Minio-Paluello et al (6).…”

Section: Discussionmentioning

confidence: 99%

Purification of Erythropoietin-Responsive Cells by Immune Hemolysis

Cantor

Morris

Marks

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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Immature erythroid cell precursors from 12-and 13-day fetal mouse livers were concentrated by differential antibody-mediated hemolysis. The effect of erythropoietin on these purified erythroid cell precursors was studied in vitro. Addition of erythropoietin stimulates erythroid cells to proliferate, and to differentiate to cells that actively synthesize hemoglobin. This erythropoietininduced erythropoiesis is sustained in culture for at least 48 hr.Elucidation of the cellular and molecular effects of erythropoietin has been aided by the development of several in vitro systems, including the short-term culture of bone marrow (1) and fetal hepatic erythroid cells (2, 3). However, interpretation of these studies is hindered by the presence of large numbers of differentiated hemoglobin-synthesizing cells in the culture. Although such cells constitute about 70% of the total cell population in the 13-day fetal mouse liver, previous studies (2) indicate that in this system the primary target cell responsive to erythropoietin is a large, nonhemoglobinized precursor, morphologically indistinguishable from the cells called proerythroblasts, that constitutes less than 10% of the population. The present paper describes a method that uses immunological hemolysis (4-6) to concentrate immature erythroid cell precursors from the total erythropoietic population of fetal mouse liver. This immature cell population responds to erythropoietin by proliferation of erythroid cell precursors, which differentiate to hemoglobin-synthesizing cells. MATERIALS AND METHODSThe present procedures for the preparation of antibody to mature mouse erythrocytes are modified from earlier studies of Borsook et al. (4, 5) with antiserum to rabbit erythrocytes and of Minio-Paluello et al. (6) with antiserum to mouse erythrocytes. Rabbits were immunized with erythrocytes obtained from adult C57BL/6J female mice bled by cardiac puncture under ether anesthesia. Heparinized mouse blood was pooled, washed three times with saline, packed, and diluted 1:1 with saline; penicillin was then added. 1-1.5 ml Of the erythrocyte suspension was injected intravenously into the rabbits, via the ear vein, three times a week for 4 weeks. 7 Days after the last injection, the rabbits were bled by heart puncture and the serum was sterilized by Millipore filtration (0.45,um pore size). The sera were tested for complement-dependent hemolysin titer (1: 4000 or greater in each of three antisera prepared, all of which proved satisfactory for differential cell lysis). Rabbit sera were not subjected to heat inactivation.Timed pregnancies were obtained in hormonally primed C57BL/6J female mice (7); fetal hepatic hemopoietic cells were obtained from 12-or 13-day embryos by mechanical disaggregation of the dissected livers (3). Cells were collected in ice-cold Waymouth MB752/1 medium (GIBCO Grand Island, N.Y.), supplemented with 10% fetal-calf serum (Microbiological Associates, Bethesda, Md.), 3% chickembryo extract (GIBCO), and 1% penicillin-streptomycin (5000 units of peni...

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The Distribution of Buoyant Density of Human Erythrocytes in Bovine Albumin Solutions

Abstract: raise from delocali~ation, among several BChl molecules, of the charge associated with the oxidized BChl. A delocalization of this sort can be used as the conduction system for separation of charge in the primary quantum conversion act.

Cited by 157 publications

References 15 publications

Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

From the Bench to the Field in Low‐Cost Diagnostics: Two Case Studies

Purification of Erythropoietin-Responsive Cells by Immune Hemolysis

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