Preparation Of Red Cells Research Articles

Study on the dehydrating effect of the red cell Na +/K +-pump in nystatin-treated cells with varying Na + and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na + concentrations and water content. Such cells provided an experimental model to study the effect of Na +/K + pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na +/K + pump by elevated intracellular Na + caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na out + : 2 K in + stoichiometry of the Na +/K + pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.

Preparation of granulocyte-poor red blood cells by microaggregate filtration: a simplified method to minimize febrile transfusion reactions.

A simple, effective method for removing granulocytes from stored blood is described. Microaggregate filtration removes approximately 95% of the granulocytes from blood which has been stored for 2 weeks, centrifuged and filtered. The mean number of remaining leukocytes is 8 +/- 3.7 x 10(8)/unit. The residual white cell population, which is composed almost entirely of lymphocytes, is substantially less than the average number of cells previously associated with febrile reactions. 45 patients were selected for the study. All had significant febrile transfusion reaction histories, and averaged one reaction for every 3.6 U of conventional red cell product transfused. Administration of 212 units of microaggregate filtered granulocyte poor red cells caused a 95% reduction in the incidence of fibrile reactions. The technique is inexpensive, easily incorporated into the routine of the clinical blood bank, and does not require "open-system' processing. These considerations make microaggregate filtration a logical first choice method for the preparation of granulocyte-poor red blood cells.

Interdependence of hemoglobin, catalase and the hexose monophosphate shunt in red blood cells exposed to oxidative agents

Hydrogen peroxide, 1, 4-naphthoquinone-2-sulfonic acid and 6-hydroxydopamine were used as biochemical probes to study the interdependence of hemoglobin, catalase and the hexose monophosphate shunt in protection of red blood cell (red cell) function against Superoxide, hydrogen peroxide and organic free radicals. It was shown that catalase may remove hydrogen peroxide both catalatically and peroxidatically in the red cell and that glucose metabolism supplies electron donors for the peroxidatic function of catalase. The hexose monophosphate shunt is known to participate in removal of hydrogen peroxide by supplying electrons for the action of glutathione reductase and glutathione peroxidase. Experiments in which red cell catalase was irreversibly inhibited by interaction with hydrogen peroxide and 3-amino-1, 2, 4-triazole showed that both catalase and the hexose monophosphate shunt share in the removal of hydrogen peroxide from red cells. The effect of the hemoglobin oxidation state on the interaction of red cells and oxidative agents was studied using red cell preparations containing hemoglobin, carbonmonoxyhemoglobin or methemoglobin. Oxyhemoglobin was able to accelerate the production of Superoxide and hydrogen peroxide with agents like 1, 4-naphthoquinone-2-sulfonic acid, whereas oxyhemoglobin had little effect with 6-hydroxydopamine. In experiments with red cells containing oxyhemoglobin, the accumulation of catalase in the form of Compound II, with resulting loss of available catalatic activity, was directly proportional to formation of methemoglobin. The close relationship between loss of catalatic activity and formation of methemoglobin may indicate that catalase has a protective effect on hemoglobin. Experiments with red cells containing methemoglobin indicated that once methemoglobin is formed it protects the red cell from further loss of the catalatic activity of catalase. In some circumstances oxyhemoglobin was formed from methemoglobin as a by-product of the protective effect of methemoglobin on red cell function. Formation of oxyhemoglobin by this mechanism was many times faster than oxyhemoglobin formation by the methemoglobin reductase system.

Preliminary characterization of vasocontractile activities in erythrocytes.

The vasocontractile activities of washed red cell preparations hemolyzed by various methods were studied in vitro using isolated canine basilar arteries. Significant contractions were induced by each preparation. The maximum strength of contraction attained by the various preparations was similar. The contractile activity appeared to be dose-related, and molecular exclusion chromatography demonstrated that the activity migrated with the fraction of approximately 40,000 to 45,000 molecular weight. The vasocontractile effect of the active fraction was sustained in vitro when tested against basilar artery, but was inactive in peripheral arterial preparations. Preliminary biochemical characterization indicates that the contractile activity resides in a protein. Enzymatic digestion of the crude fraction appears to enhance the contractile activity significantly, and this observation suggests a possible mechanism for the delayed onset of ischemic symptoms encountered in the clinical situation.

Evaluation of three procedures for the preparation of leukocyte-poor and leukocyte-free red blood cells for transfusion.

Abstract. Three methods of leukocyte deprivation of blood for transfusion have been evaluated. Filtration of relatively fresh packed red cells (up to 5 days old) through Erypur filters appears to be the method of choice for the preparation of leukocyte‐free red cells, used to prevent the production of antileukocyte antibodies in nonimmunized, nontransfused patients undergoing repeated blood transfusions. Double dextran sedimentation plus saline washings also yields leukocyte‐free red cells, although with a lower frequency, and represents an alternative to Erypur filtration, especially because of the lower cost and no need of special equipment. Imugard filtration appears to be a simple and effective way for preparing leukocyte‐poor red blood cells. For all the three procedures, results are definitely better when buffy coat‐free packed red cell units are processed. In a given hospital the choice of the technique(s) for the preparation of leukocyte‐poor (free) red blood cells for transfusion depends on a number of factors, such as type of patients to be transfused (immunized or nonimmunized, small children or boys and adults), aim of the procedure (prevention of the febrile transfusion reaction or prevention of antileukocyte antibody production), continuous availability of materials, cost, length and easiness of the procedure; these three latter parameters are in turn related to a number of local situations and facilities.

The potential use of dihydroxyacetone for improved 2,3-DPG maintenance in red blood cell storage: solution stability and use in packed cell storage.

Dihydroxyacetone (DHA) is effective in maintaining 2,3-diphosphoglycerate (2,3-DPG) concentrations in stored red blood cells. One limitation to the use of DHA is its instability when added to anticoagulant solutions during blood bag manufacture. The stability of DHA solutions have been evaluated. Solutions of DHA are stable at 25 C in water or isotonic saline, with or without the addition of glucose or adenine. DHA is stable to autoclaving; 99 + per cent surviving at 150 mM, and 89 per cent surviving at 1.9 M concentrations. DHA can be incorporated into a satellite addition pouch attached to the main blood drawing bag, and be added to the blood-anticoagulant mixture after phlebotomy or the preparation of red blood cells. Addition of the DHA solution, containing adenine and extra glucose, to packed cells causes significantly improved maintenance of 2,3-DPG during 42 days of 4 C storage, while maintaining adequate concentrations of red blood cell ATP. The use of DHA, adenine, and glucose in extended storage of packed cells, using either zero or seven day addition of the nutrient solution, produces similar efficacious results.

Evaluation of Three Procedures for the Preparation of Leukocyte-Poor and Leukocyte-Free Red Blood Cells for Transfusion

Evaluation of Three Procedures for the Preparation of Leukocyte-Poor and Leukocyte-Free Red Blood Cells for Transfusion

A comparison of positron-emitting blood pool imaging agents

Several agents are currently available for blood-pool imaging with positron scintigraphy. Haemoglobin may be labelled with 11 C(T 1 2 20 min) either by administration of 11CCO by inhalation or by intravenous administration of a sample of the subject's whole blood previously labelled by exposure to 11CCO. Since labelling with 11C requires a particle accelerator, alternative agents labelled with generator-produced 68 Ga (T 1 2 68 min) would be advantageous. Blood-pool images may be obtained with 68Ga-transferrin following intravenous administration of 68Ga-chloride at acid pH. Recently a method for the preparation of red cells labelled with 68Ga-oxine has been reported. We have compared the three agents, 11C-carboxyhaemoglobin, 68Ga-transferrin and 68Ga-labelled red cells in dogs to assess their relative merits for blood-pool imaging. For 1 h following administration of each agent, periodic blood samples were withdrawn for counting in a NaI (Tl) well counter while conventional two-dimensional images were obtained simultaneously on the Massachusetts General Hospital positron camera. Count rates in regions about the heart, liver and spleen were obtained for each image. The disappearance of blood activity as shown from the results of counting the blood samples and from the counting rate in regions about the heart was found to be identical within experimental error for the three agents. In the liver and spleen regions, the highest count rates were obtained with 68Ga-transferrin and the lowest with 68Ga-labelled red cells; count rates in these regions with labelled red cells were virtually constant throughout the 1 h study. It may be concluded that with the exceptions noted above, the three agents are approximately equivalent for blood-pool imaging.

Preparation of stable erythrocyte target cells suitable for detection of the antibody response to the haptens azobenezenearsonate, azophenyltrimethylammonium and azophenylphosphorylcholine by plaque-forming cell assay

The preparation of sheep red blood cells (SRBC) which have been sensitized with the haptens azobenzenearsonate, phosphorylcholine and azophenyltrimethylammonium, and are suitable for use in a plaque-forming cell assay, is described. Each hapten is first coupled to a phenolic imidoester (methyl- p-hydroxybenzimidate) and the substituted imidoesters are employed to prepare target SRBC. Details are presented concerning synthesis and purification of the imidoesters, as well as conditions for coupling to SRBC. The hapten-SRBC prepared by this method are very stable and offer significant advantages over other procedures for coupling these haptens to red blood cells.

Preparation of Antibody-Coated Red Cells by the Haemonetics Cell Separator or Cell Washer

Preparation of Antibody-Coated Red Cells by the Haemonetics Cell Separator or Cell Washer

Preparation of leukocyte-poor red blood cells using the IBM 2991 blood cell processor.

Leukocyte‐poor red blood cells (RBCs) were prepared from one‐to‐seven‐day‐old whole blood using the IBM 2991 Blood Cell Processor. Blood was centrifuged at 3,000 rpm for 5 minutes on the initial processing cycle and for 1.5 minutes each subsequent cycle. The red blood cell override (RCO) control was used to remove portions of the leukocyte‐rich red blood cell layer at the cell/liquid interface after separation. RBC loss was directly proportional to RCO time and averaged 5.4, 10.6, 15.6, 21.2, and 26.7 ml with RCO settings of 1.5, 2.5, 3.5, 4.5, and 5.5 seconds, respectively. A single RCO cycle removed 54.7, 66.0, 70.7, and 69.7 per cent of total leukocytes at RCO settings of 2.5, 3.5, 4.5, and 5.5 seconds, respectively. With double cycle processing at the same RCO times, 78.6, 88.9, 90.3, and 90.7 per cent of white blood cells were removed with loss of 12.4, 16.7, 22.4, and 25.4 per cent of RBCs respectively. Four‐cycle processing with an initial 2.5‐to‐5.5‐second RCO followed by three 1.5‐second RCO cycles removed the greatest number of leukocytes (86.9, 93.3, 93.3, and 91.7 per cent, respectively) with corresponding RBC losses of 11.9, 17.2, 20.2, and 23.1 per cent. Total leukocytes contaminating the processed RBCs averaged 0.37, 0.18, 0.18, and 0.22 × 109, respectively. The processing cycle with the 3.5‐second initial RCO provided maximal leukocyte removal and minimal erythrocyte loss.

Is very fresh blood needed in preparing leukocyte-poor red blood cells by inverted centrifugation?

It is recommended that units selected for inverted centrifugal preparation of leukocyte-poor red blood cells be less than 24 hours old. The reason for this is unclear and the recommendation poses practical problems. We evaluated the effectiveness of inverted centrifugation in reducing the WBC:RBC ratio in 31 units stored up to six days at 4 C. Leukocyte removal was at least as effective with six-day stored units as with less than one day old, averaging 77 per cent reduction with 76 per cent recovery of red blood cells. No reduction in leukocytes occurred with six days storage alone. It appears to be unnecessary to restrict leukocyte removal by inverted centrifugation to units less than 24 hours old.

Preliminary Observations with an Electromagnetic Method for the Noninvasive Analysis of Cell Suspension Physiology and Induced Pathophysiology

A technique for the electromagnetic analysis of physiological and patho-physiological states in cell suspensions is presented. The technique is based upon high speed automatic network analysis in the HF band for measurement of complex permittivity. The results demonstrate that changes in HF-band permittivity dispersion may be related to physiological and drug-induced patho-physiological states of the cell membrane. Mechanical disruption of the cell membrane by sonication obliterates the HF-band dispersion of permittivity that is present in undisrupted cells. The effect of species, suspending medium, and temperature were systematically analyzed in erythrocyte suspension in order to aid comparison between published studies of red cell preparations.

Red blood cell preservation in protein-poor media. I. Leukocyte enzymes as a cause of hemolysis.

Red blood cells were prepared from CPD whole blood concentrated to 90 per cent hematocrit, diluted with saline‐adenine‐glucose (SAG) media and then stored for 35 days. The dilution was undertaken to improve the flow properties of the blood and to provide the cells with glucose and adenine allowing prolonged storage. Several different compositions were tested. ATP could be maintained at about 70 per cent of the initial level and the 24 hour red blood cell posttransfusion survival was 82 per cent. The leakage of potassium was less than in CPD‐adenine whole blood if the dilution volume was half or less of the red blood cell volume. The only problem was that the hemolysis was greater in SAG‐stored blood than in CPD whole blood or undiluted CPD red blood cell concentrate. Hemolysis could be reduced by removal of huffy coat cells or by storage of blood in the presence of synthetic enzyme inhibitors. A chymotrypsin‐like enzyme isolated from human leukocytes had a potent hemolytic effect. The hypothesis is presented that red blood cells stored in an electrolyte medium in the presence of leukocytes undergo increased hemolysis because they lack the protecting effect of plasma enzyme inhibitors which normally inhibit any hemolytic enzymes leaking from damaged leukocytes. The study has practical implications for the prolonged storage of buffy‐poor red blood cell preparations to be used in transfusion therapy.

KININ, KININOGEN AND KININASE LEVELS DURING ACUTE Babesia bovis (=B. argentina) INFECTION OF CATTLE

1. Kinin levels began rising on day 3 after infection of cattle with Babesia bovis (= B. argentina) and attained a maximum value of 98% above preinfection levels by day 7. 2. Kininogen levels began falling on day 3 and reached minimum levels of 83% below preinfection levels on day 8. 3. Changes in both kinin and kininogen levels on day 3 coincided with the detection of low levels of parasites, and with a fall in packed cell volume. 4. Plasma kininase levels rose significantly 6 to 9 days after infection. Preparations of lysed and sonicated uninfected and infected red cells contained kininase activity, the respective red cell preparations being 23.9 and 11.4 times more active per mg protein than uninfected red cell preparations. The effect of pH, and the inhibitors disodium edetate, 1,10 phenanthroline and aprotinin on normal and infected plasma and on the various red cell preparations suggested that the rise in plasma kininase levels during infection was probably at least partly due to parasite products. 5. These results are discussed in relation to previous data showing that both kallikrein activation and the onset of hypotension also occur on or about day 3.

Development and evaluation of a rapid and efficient electrolytic preparation of 99mTc-labeled red blood cells

This paper reports a new, simple and reproducible method for routine preparation of 99 m Tc-labeled red blood cells (RBC). This method employs electrolytic generation of minute quantities of stannous ion to serve as the reducing agent for 99 m Tc-pertechnetate. The RBC are incubated with Sn(II), washed with saline, incubated with Na 99 m TcO 4, and washed again with saline. The entire procedure requires about 45 min and consistently provides labeling efficiencies above 90%. The half-clearance time in the blood in humans is approximately 24 hr. Activity released from the labeled RBC in vivo is largely in chelate form which is rapidly excreted in the urine. High quality blood pool images can be obtained for many hours after injection. In a damaged form, 99 m Tc-RBC provide spleen images of diagnostic quality. This 99 m Tc-RBC preparation appears to have good potential for routine use.

Preparation of red cells coated with C4 and C3 subcomponents and production of anti-C4d and anti-C3d.

Methods of preparing red cells coated only with C4b, C3b or C3d have previously been described by others; the present observations supply further information about the optimal conditions for preparing such cells and also describe conditions for the operation of cells carrying only C4d. In addition, the preparation of specific anti-C4d and anti-C3d sera is described.

Preparation of Red Cells Coated with C4 and C3 Subcomponents and Production of Anti-C4d and Anti-C3d

Preparation of Red Cells Coated with C4 and C3 Subcomponents and Production of Anti-C4d and Anti-C3d

Cyclic AMP-dependent protein kinases and binding sites for cyclic AMP in rat erythrocytes

In red cell preparations from reticulocyte-poor (untreated animals; approximately 2% reticulocytes) and reticulocyte-rich blood (animals pretreated with acetylphenylhydrazide; approximately 60% reticulocytes) of rats, cAMP binding sites and cAMP-dependent protein kinase activities were determined. High affinity binding sites for cAMP were present both in membrane and cytoplasmic preparations; while the apparent binding constants determined in both cell fractions (approximately 3 x 10(-9) M for membrane, approximately 2 x 10(-8) M for cytoplasmic fractions) were independent of the reticulocyte content of the preparations, the respective numbers of sites were about twice as high in the reticulocyte-rich as in the reticulocyte-poor preparations. In membrane preparations, significant cAMP-dependent protein kinase activity could be detected only in membrane fractions from reticulocyte-rich blood which were considerably contaminated by intracellular components ("haemoglobin-containing membranes') while in washed ("haemoglobin-free') membranes no cAMP-dependent protein kinase activity was found. In cytoplasmic preparations both from reticulocyte-poor and reticulocyte-rich blood, two different protein kinases, a low and a high Ka enzyme, were tentatively differentiated by kinetic data; the apparent activation constant for the high Ka enzyme (approximately less than 5 x 10(-8) M) was in the concentration range of the binding constants determined on cytoplasmic preparations. The activity of the high Ka protein kinase was several fold higher in reticulocyte-rich than in reticulocyte-poor cytoplasmic fractions, while the activity of the low Ka enzyme was obviously independent of the reticulocyte content. From the results obtained, it is concluded that in premature rat erythrocytes, membrane protein(s) may serve as protein substrates for cAMP-dependent protein kinase(s) located in the cytoplasm. This assumption was supported by experiments with intact erythrocytes (prelabelled with inorganic 32P-phosphate) from reticulocyte-rich blood: isoprenaline, theophylline, and also dibutyryl-cAMP significantly increased phosphorylation of membrane protein of these cells. From the results presented (and others previously reported) it becomes evident that only premature rat erythrocytes, i.e. reticulocytes, are equipped with a beta-adrenergic receptor-effector system consisting of a beta-adrenergically stimulated adenyl cyclase and cAMP-dependent protein kinase(s). Obviously, the adrenergic receptor system and also part of the effector system is lost during the process of red cell maturation.

Effects of centrifugation on erythrocytes.

In order to determine the effects of centrifugation on stored erythrocytes, survival studies were performed on units which were centrifuged either once on the day of collection, twice on the day of collection, or once on day 21. No meaningful differences existed between the study groups and a control group. These findings indicate that single centrifugation at any time throughout storage is not harmful, and that double centrifugation on the day of collection does not significantly alter erythrocyte survival. Regulations requiring sedimentation rather than centrifugation for the preparation of Red Blood Cells (Human) after the sixth day of storage should be re‐evaluated.