Washed Blood Cells Research Articles

Evaluation of the contribution of red and white cells to the flow of suspensions of washed blood cells through 3 micron Nuclepore membranes.

This study was designed to investigate a method of analysis which can quantitate the contribution of white cells to the flow of washed suspensions of blood cells. Such an analysis would obviate the need to remove white cells when studying the filterability of normal and abnormal red cells. The flow of suspensions of washed blood cells through a 3 micron Nuclepore membrane declines continually due to the occlusion of pores and the degree of pore occlusion is reduced significantly by the removal of the 'buffy coat' during the preparation of the suspension. These findings are in complete agreement with many other reports. However, a detailed kinetic analysis of the results suggests strongly that the white cell population makes a small contribution to the degree of pore occlusion which is caused largely by approximately 1% of the total red cell population. Despite the phenomenon and extent of pore occlusion, a kinetic analysis of the complete non-linear flow profile allows a measure of the deformability of red cells from filtration studies of washed but unfractionated blood cells.

Margination of leukocytes in blood flow through small tubes

Leukocyte margination in the vessels of the microcirculation has been attributed to a flow-dependent interaction with red cells. To determine the extent of this effect, experiments with human blood were done in 100- to 180-μm tubes to detect changes in cell distribution as a function of hematocrit and flow rate. Using a flow visualization technique, the leukocyte concentration distribution was determined in 45% ghost cell suspensions. Migration of cells toward the wall was observed at centerline velocities > 1 mm sec −1 and increased with increasing flow rate. The effect was probably due to a more rapid inward migration of ghosts than leukocytes because of fluid inertia and cell density differences. Experiments were therefore carried out in whole blood at hematocrits from 20 to 60%, measuring the number concentration of leukocytes and erythrocytes within the tube, n t, and comparing it to that in the infusing reservoir, n 0, (Fahraeus effect). At mean tube shear rates G < 100 sec −1, n t n 0 < 1 for both leukocytes and erythrocytes showing net migration of cells away from the wall, although at nearly all hematocrits there was an enrichment of leukocytes relative to erythrocytes in the tubes. At G < 50 sec −1, n t n 0 remained < 1 for erythrocytes but increased to > 1 for leukocytes showing migration toward the wall, the increase being greatest at 20% hematocrit in the 100-μm tubes. The nature of the effect was revealed by cine films which showed that, as the flow rate decreased, erythrocytes formed rouleaux which migrated inward creating a core and displacing leukocytes to the periphery. In control experiments using washed blood cells in phosphate buffer-albumin, n t n 0 < 1 for both leukocytes and erythrocytes at all G and hematocrits, and leukocytes were now depleted relative to erythrocytes in the tubes, i.e., the leukocytes were more axially distributed. Cine films of washed blood confirmed that, in the absence of rouleaux, no significant inward migration of erythrocytes occurred.

Both protein and blood cells reduce coronary microvascular permeability to macromolecules.

A number of studies report that isolated Krebs-perfused hearts deteriorate with time, develop edema, and demonstrate a progressive increase in coronary vascular resistance. However, hearts perfused with filtered blood are more stable with regard to cardiac function and coronary resistance. The changes observed in the Krebs hearts may be due to a "permeability"-type edema. The purpose of this study was to systematically determine whether adding protein and blood cells to a Krebs perfusate affected coronary microvascular permeability to macromolecules. The rat heart preparation employed allowed direct visualization and quantification of transcoronary macromolecular leakage. We observed severe transcoronary leakage of fluorescent albumin (FITC-BSA) when FITC-BSA was later added after 20 min of perfusion with Krebs. Leakage was decreased by including 2 g/100 ml albumin (BSA) in the initial perfusate but was not further reduced by increasing the BSA concentration to 5 g/100 ml. However, adding washed blood cells to the initial perfusate did further reduce FITC-BSA leakage. The index of FITC-BSA exchange, the O/I ratio, was 0.70 +/- 0.02 (+/- SE) for Krebs perfusate, 0.55 +/- 0.03 for Krebs-BSA, and 0.45 +/- 0.02 for Krebs-BSA-blood cells, indicating significant effects for both protein and blood cells (P less than 0.05). The results suggest that both protein and blood cells are necessary to maintain the semipermeable characteristics of the coronary exchange vessels.

In vitro biotransformations of [ 14C]captopril in the blood of rats, dogs and humans

The biotransformations of captopril (CP), an orally effective antihypertensive agent, were studied in vitro in whole blood, plasma, and washed blood cells of rats, dogs and humans, using 14C-labeled CP. Thin-layer chromatographic profiles of the radioactivity extracted into methanol showed that, in plasma and whole blood, [ 14C]CP was biotransformed to its disulfide dimer, [ 14C]CP-CP, and to two other compounds identified as the [ 14C]CP-glutathione and [ 14C]CP- l-cysteine mixed disulfides. After incubation with either washed blood cells or a saline control, most of the [ 14C]CP remained unchanged. Since not all of the radioactivity in the biological samples could be extracted, the nature of the unextracted radioactivity was investigated. Results obtained using methanol extraction, ultrafiltration and acid precipitation techniques indicated that [ 14C]CP was extensively and covalently bound to plasma proteins. In human plasma, [ 14C]CP was shown to be bound primarily to albumin through covalent disulfide bonds. In the plasma of all three species, these covalent disulfide bonds could be cleaved by glutathione or l-cysteine, resulting in the formation of mixed disulfides, or by dithiothreitol, resulting in the release of [ 14C]CP. The behavior of CP in vitro in blood was qualitatively similar to that reported for other endogenous and xenobiotic sulfhydryl compounds and was consistent with the results obtained from in vivo studies with CP.

In vitro evaluation of porcine lymphocyte response to phytohaemagglutinin using a modified “whole blood” technique

The purpose of these investigations was to develop a modified whole blood technique for measuring quantitatively the responsiveness of pig peripheral blood lymphocytes to phytohaemagglutinin (PHA) in vitro. Washed blood cells from a fixed volume of blood were suspended in culture medium supplemented with foetal calf serum and stimulated with a pure mitogenic PHA preparation. The stimulation was measured by the incorporation of 3H-thymidine. Data are presented to show the effect of different variables on the culture system. The transformation response was measured at different levels of PHA and the reproducibility of the different responses from repeated investigations was used to evaluate the usefulness of the test. The best reproducibilities of the stimulation response were found at the high PHA concentrations. Also the calculated PHA concentration giving maximum stimulation response had a relatively high reproducibility and indicates that this value is a convenient and reliable alternative measure of the functional capacity of the lymphocytes.

Radiometric Detection of Haemophilus in Simulated Blood Cultures

The radiometric detection of various Haemophilus species was studied in simulated blood cultures and in blood culture media without added blood, but supplemented with nicotinamide-adenine dinucleotide (NAD) or hemin, or both. Haemophilus aphrophilus was the only species studied that was radiometrically detectable without added blood or NAD. All other species studied (Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus) required either added NAD, whole blood, or washed blood cells for growth and radiometric detection. The results of this study and extensive experience with clinical specimens show that the radiometric system is an effective means of rapidly detecting Haemophilus in blood cultures, but it is essential that it be used in conjunction with a subculture three to five days after inoculation.

Plasmapheresis using the IBM 2991 blood cell processor.

The IBM 2991 Blood Cell Processor, normally used to wash or deglycerolize red blood cells, has been modified to permit serial plasmapheresis using a small roller pump, an adapter line and the disposable processing set usually used for cell washing. Blood is withdrawn from the donor, pumped into the plastic centrifuge bag and the plasma removed after centrifugation. Donor erythrocytes are pumped from the processing bag to a reservoir bag from which they are returned to the donor by gravity. Multi-unit plasmapheresis to harvest plasma for transfusion or to remove plasma as a therapeutic measure is possible using the modified system. A four-unit plasmapheresis can be done in 65 to 75 minutes at a cost of $27.61, including disposables and technologist labor. This modification allows the IBM 2991 to function for red blood cell washing and as an efficient instrument for plasmapheresis.

Distribution of pentobarbital and diphenylhydantoin between plasma and cells in blood: effect of salicylic acid, temperature and total drug concentration.

The binding of pentobarbital, diphenylhydantoin and salicylic acid to cells in blood was found to be independent of total drug concentration within therapeutic levels. Salicylic acid displaced pentobarbital and diphenylhydantoin from plasma protein binding sites, but high levels of salicylic acid had no effect on the distribution of the other two drugs to washed blood cells. Thus, in whole blood the presence of salicylic acid decreased the fraction of pentobarbital or diphenylhydantoin bound to plasma proteins and increased the fraction of the drug in plasma water and in blood cells. Diphenylhydantoin was shown not to be bound irreversibly to blood cells and equilibration in between the inside and outside of the cells was found to be rapid (within 5 min), even at high concentrations. Binding to washed blood cells was the same at 37 degress C and 25 degrees C, in contrast to plasma protein binding. It is pointed out that these effects may cause certain analytical errors, resulting in changes in plasma concentration if plasma is separated at a low temperature.

Simultaneous identification of T and B cells in blood smears using antibody coated bacteria

A new method is described which allows separate or simultaneous identification of T and B cells in stained blood smears using as markers morphologically distinguishable antibody-coated bacteria. Glutaraldehyde was used to coat Escherichia coli and itGaffkya tetragena with purified antibody, namely anti-b5Ig allotype (Ifk chains) or rabbit anti-guinea pig IgG, both made in b4 rabbits. Blood was collected from b5 rabbits, the cells were washed, treated with guinea pig anti-rabbit T cell antiserum and washed again. E. coli coated with anti-GpIg and G. tetragena coated with anti-b5Ig were added individually or together to the washed blood cells and centrifuged to promote binding of E. coli to T cells and G. tetragena to B cells. The cells were smeared and stained with Wright's stain. We found that: a) the binding of bacteria to T and B cells depended only on the reaction between the antibody molecules on bacteria and the antigen (surface Ig or guinea pig anti-T cell Ig) on the cell membrane; b) lymphocytes were surrounded by either E. coli or G. tetragena, but never by both; c) of the total blood lymphocytes, 43–48% were T cells and 49–51% were b5Ig bearing B cells; d) about 6% of the lymphocytes were unlabeled (probably B cells bearing λ chain Ig); and e) all monocytes and basophils had surface Ig (cytophilic). Thus, differential counting of T and B cells can be easily performed in stained blood smears using antibody-coated bacteria.

Internal Structure of the Chicken Erythrocyte after Exposure to Hydrogen Plasma as Seen With Sem

Smears were made of either whole chicken blood or washed blood cells. The smears were exposed to hydrogen plasma for varying periods of time and examined with the scanning electron microscope. The pitted surface of the nucleus was clearly seen along with the granular cytoplasm. After prolonged etching nuclear material appeared homogeneous and smooth.

Pathogenesis of Acute Avian Malaria

In a study of antigens and antibodies found in malarious chicken blood, antigen activity was found in plasma, and in hypertonic saline eluates of blood cells. A soluble antigen was extracted from parasites liberated from erythrocytes. Two classes of antigen were differentiated, one a globulin associated "serum antigen" which was found to show identity with a serum antigen from blood of rats with acute Babesia rodhaini infection, and another that was associated with the Plasmodium gallinaceum parasite. The plasma also contained antibody to both classes of antigen. Study of blood cells from normal chickens made anemic by injections of autohemagglutinin-free malarious plasma indicated that both serum antigen and its antibody were present in eluates of the washed blood cells. Direct immunofluorescent tests of these cells with conjugated antibody to serum antigen, and with conjugated antibody to P. gallinaceum parasite antigen showed that they reacted with the antibody to serum antigen but gave no reaction with antibody to parasite antigen. From this information it is suggested that soluble complexes of serum antigen and its antibody combined with the erythrocytes, and that these complexes acted as opsonin to cause the cells to be sequestered and destroyed in the spleen, or may have fixed complement to cause intravascular hemolysis. The serologic identity of serum antigen from malarious chickens and from rats with babesiosis, and its distinction from parasite antigen, led to the speculation that it might be an autoantigenic macroglobulin.

Pathogenesis of Acute Avian Malaria

Anemia, splenomegaly and glomerulonephritis in acute Plasmodium gallinaceum infections of chickens were studied in association with cold-active hemagglutinin for trypsinized human “O” erythrocytes, serum antigen, and the concurrent presence of antibody to serum antigen, as well as with the parasitemia of acute infection. Anemia with splenic enlargement was produced in normal chickens within 24 hours by intravenous injection of malarious chicken plasma that contained high titers of hemagglutinin, serum antigen, and antibody. A similar anemia and splenic engorgement resulted in normal birds injected with eluates from the washed blood cells of malarious chickens. Within 24 hours, the malarious plasma produced an acute glomerulonephritis similar to that which had been observed on the 5th day of infection. The concurrent presence of the hemagglutinin, serum antigen, and its antibody in the injected plasma, and the failure of the plasma to produce nephritis in immunized chickens, suggested that immune substances might be causal in acute malarial anemia and nephritis.

赤血球再浮遊液について

1) General review of red blood cell suspended solution and experience with LMWD preparations used as suspension media are described.2) Recently transfusion of red blood cell suspended solution turns to be re-appreciated as adequate and excellent transfusion therapy. The use of red blood cell suspended solution is supported by correct method of blood transfusion meeting the needs of whole blood, plasma and blood cells, by clinical use of frozen blood and by clinical usefulness of the washed blood cells.3) Studies on the red blood cell suspended solution have given too much importance to method of separation and preservation of the red blood cells. It is important, however, to examine suspension media of the red blood cells also. Therefore, each preparation for fluid transfusion should be examined whether it is available to suspension media of the red blood cells or not.4) Low molecular weight dextran solutions added by Ringer's solution, lactate Ringer's solution and xilitol respectively (DI-15, DI-25, DI-26) are studied on suitability as suspension media of the red blood cells and clinical experiences of using these red blood cells suspended solutions are examined.5) All of DI-15, DI-25, DI-26 solutions are adequate suspension media of the red blood cell. All of these red blood cell suspended solutions may be used clinically even two days after preparation. Suspension of the red blood cells is excellent in all of these solutions. Thus the red blood cells are transfused easily even under condition of packed red blood cells. Consequently patients recovered from anemia well by transfusion of these red blood cell suspended solutions. Injuries to the red blood cells are more slight in DI-15 solution on examination of red cell fragibility test and hemolysis, more mild in DI-25 solution on examination of pH.6) It is necessary to prepare several kinds of solution for transfusion which are available to suspension media on properties of protection and suspension to the red blood cells, In future, optional concentration of the red blood cells, collected from frozen blood, are suspended in one of these solutions according to patient's condition.

The role of adenine nucleotides in the peripheral vasodilating effect of thrombin.

AbstractThe experiments were designed to study the mediating mechanism for the previously demonstrated vasodilating effect of thrombin when small amounts are infused into the femoral artery of the dog. Washed blood cells, suspended in physiological saline, were incubated for a short time with small amounts of thrombin. The supernatant from such a cell suspension exerted a highly vasodilating effect when infused into the femoral artery. This vasodilating effect was compared to that of serotonin, histamine, adenosine and adenine nucleotides. Histamine, adenosine and adenine nucleotides caused the same type of reactions as that of vasoactive supernatants. By inactivation experiments, where these substances, as well as vasoactive super‐natants, were heated or incubated with plasma or ADP‐ase–ATP‐ase (Apyrase), it was concluded that the vasodilating agents in the latter are mainly ADP and to some extent ATP.

Free fatty acid titration of human plasma and erythrocytes

Free fatty acids (FFA) have been determined in human plasma and compared with the values of the heptane-soluble fraction in the whole blood and in washed erythrocytes. The acidity of the washed blood cells did not vary with plasma FFA, but showed a rather wide-range variation between subjects. No attempt was made to identify the acids of the blood cells. An equation has been presented that describes the relation between the titre of heptane-soluble acidity in whole blood, plasma and blood cells, including a figure of the haematocrit. Any of these four variables can be calculated if the other three are known. Since plasma FFA represent the fraction of FFA in the whole blood that is accessible to the tissues, the exchange of FFA between blood and tissues can be calculated from the plasma FFA and the haematocrit.

Erythropoietic activity of saline washings of blood cells subjected to low atmospheric pressure in vitro. II. Effect of partial hemolysis of the blood.

AbstractIn order to test the effect of initial hemolysis on the formation of erythropoietins in vitro, saline suspensions of partially hemolysed and non‐hemolysed washed blood cells were subjected to low atmospheric pressure, and the erythropoietic activity tested by injecting samples of the saline into test rabbits. The recipient animals in the“non‐hemolysed group reacted to the injection with an increase in the erythrocyte and reticulocyte count, while in the ”hemolysed group“ the injection was ineffective save for a transient reticulocytosis. It is concluded that the active agent or its precursor is lost from the blood during initial hemolysis.

Action of plasma and of plasma and albumin on palmitate-1-C-14 oxidation by blood cells.

SummaryWashed blood cells from normal, fasting adult rats were incubated with palmitate-1-C14 in the presence of increasing dilutions of homologous blood plasma, or in the presence of various concentrations of bovine plasma albumin. It has been observed that (a) blood plasma, even when diluted several hundred times, has an inhibitory action on palmitate oxidation by washed blood cells; (b) plasma albumin is one of the substances contributing to this inhibitory action of blood plasma; (c) under certain experimental conditions involving very high dilutions of blood plasma, its action is stimulatory rather than inhibitory. The data from several consecutive periods of incubation were used for construction of skewed bell-shaped curves of instantaneous C14O2 evolution; they are steep and high in the absence of plasma albumin, but flatten out in its presence. The results are discussed in relation to the action of plasma albumin on cell metabolism and to interactions between fatty acids, plasma albumin, and red b...

Acquired tolerance of turkeys to Rous sarcoma agent.

Five- to eight-weeks old A.M.B. turkeys cannot be infected by intramuscular injection with cell-free Rous tumour agent unless they have been made tolerant by intravenous inoculation at 1 day old with whole blood or with washed blood cells from young normal chickens of the strain in which the sarcoma is usually propagated. The duration of the immunologically null period in the turkey poult has been investigated together with the specificity and stability of the tolerance-producing antigens and their relationship to skin transplantation antigens. The results afford strong support for the existence of at least one immunologically distinctive host component for the Rous agent, and this method of investigation, i.e. the production of virus tolerance in another species with identifiable antigens (possible sources of which are discussed), appears to offer an opportunity for identifying such a component or components.

An Agglutination Reaction Observed with Some Human Bloods, Chiefly Among Negroes

Abstract In an attempt to produce immune sera for the human blood property (1) designated as P two batches of 12 rabbits each were injected with the blood of individuals known to give strong reactions for P. The first batch received blood of the type O, M, N of a white man L. F., the second that of a Negro C. H. (O, N), the latter having been used for the preparation of anti-P-serum once before. The animals received several courses of six daily injections of 0.1 to 0.4 cc. washed blood cells in a volume of 1 cc. The courses were separated by rest periods of eight days; a fourth course of injections was given ten weeks after the third. From the first lot of rabbits, after three courses of injections, one P serum of moderate strength and two rather weak sera were obtained.

Observations Upon the Nature of the Virus of Hog Cholera.

Although hog cholera has been the subject of extensive investigation by immunological methods, little has appeared in the literature concerning the nature of the specific virus, aside from the discovery of DeSchweinitz and Dorset that the causal agent is filtrable through Berkefeldt and Chamberlain candles. This paper records certain observations made with the virus in the course of an experimental study. The virus in the blood of hog cholera withstands rapid desiccation over sulphuric acid, unslacked lime or other hygroscopic agents in vacuo at O° C., without its infectiousness and antigenic property being apparently affected. The resulting virus-powder may be kept in sealed glass containers for years and probably indefinitely without loss in viability, virulence and antigenicity. Though the virus in the powdered form keeps indefinitely, after it is redissolved in a suitable solvent such as normal physiological salt solution, it loses in viability and antigenic property at the same rate and under like conditions as is the case with the undesiccated virus-blood. The desiccated virus-serum can be utilized in conjunction with immune serum (desiccated or fresh) to produce an active immunity in the hog. The virus-powder is first thoroughly dissolved in the fresh immune serum or in the dissolved desiccated immune serum in the proportion of one part virus to 2 parts serum. This in vitro admixture of virus and immune sera in no way prevents the production of an active immunization of the host following its subcutaneous injection, and gives rise to no objective symptoms of infection. It has been determined by animal experiment that the virus of hog cholera at no period during the infection is intracellular, in so far as the blood cells are concerned. Repeated injections into young non-immune hogs of large quantities of the freshly washed blood cells have failed to infect.