Preparation Of Red Blood Cells Research Articles

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.

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.

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.

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.

On the mechanism of the oxidation of human and rat hemoglobin by propylene glycol dinitrate

The kinetics and stoichiometry of the oxidation of human and rat oxy-hemoglobin (O 2Hb) by propylene glycol dinitrate (PGDN) have been investigated in vitro in both hemolysate and in intact red blood cell preparations. In hemolysate fractions from both these species, the rate constant for oxidation is nearly constant between pH 7.0 and 9.0, but it increases dramatically at lower pH values. The reaction is molecular and approximately first-order in both [PGDN] and in [O 2Hb]. The rate of oxidation is related complexly to the O 2 concentration. No oxidation occurs at zero O 2 concentration or at very high O 2 concentrations. Maximal rates are observed at O 2 concentrations where the hemoglobin is only partially saturated with O 2. The stoichiometry appears to be 1.5 hemes oxidized per ester bond broken. In whole cells, the reaction is still molecular, is approximately first-order in both reactants, and has a stoichiometry of 1.9 to 2.3 moles heme oxidized per mole of reacted ester.

A red blood cell freezing program for Massachusetts.

A regular service for freezing, thawing and washing red blood cells was established at the Massachusetts Red Cross Blood Center in 1970. This was the culmination of many years of investigation by the American National Red Cross and was part of a nation‐wide program to introduce frozen, thawed, washed red blood cells. Frozen red blood cells have the advantages of a prolonged dating period, freedom from major hepatitis risks, and negligible leukocyte, platelet and plasma content. The major disadvantages are the complexity of solutions and equipment and the high degree of quality control required. This leads to a five‐to‐tenfold increase in cost when compared to standard red blood cell preparations. If better recruitment methods and better liquid blood processing procedures are developed, the first two advantages of frozen red blood cells over standard refrigerated red blood cells would be lost. There are some patients who cannot tolerate ordinary red blood cells and can only be transfused with the frozen, thawed, washed product. In Massachusetts, the obligatory requirement for frozen red blood cells is approximately 100 units per day or less. Until better liquid blood processing methods and recruitment procedures are developed, the Massachusetts Red Cross Blood Program will need to continue expanding the output of frozen, thawed, washed red blood cells.

Burr cells, hemolytic anemia and cirrhosis

A prolonged hemolytic disorder characterized by sustained burr poikilocytosis, reticulocytosis, thrombocytopenia and high serum levels of unconjugated bilirubin is described in ten patients, of whom nine had alcoholic cirrhosis and one cardiac cirrhosis. Six patients with the disorder died within five months of its recognition. As a readily identifiable and constant feature of the syndrome, the detection of burr cells in the blood film of a patient with cirrhosis serves to indicate the presence of the disorder. Patients with liver disease had an increased incidence in wet preparations of another variety of morphologically abnormal erythrocytes resembling acanthocytes (acanthroid). Of forty-seven of 103 patients with severe alcoholic liver injury, acanthroid cells comprised more than 10 per cent of the cells in wet preparations. In wet preparations of red blood cells from fifty-six additional patients with alcoholic liver injury and fifteen patients with nonalcoholic liver disease, less than 10 per cent of the cells were acanthroid. In alcoholic patients having only acanthroid cells, a decrease in the percentage of acanthroid cells appeared to be related temporally to improvement in hepatic function. In vitro, the serum of patients with burr or acanthroid cells induced acanthroid transformation of normal erythrocytes. Conversely, normal serum restored acanthroid, but not burr cells, to a normal morphologic appearance. A serum factor related in some way to serum albumin appeared to be associated with acanthroid transformation. An additional factor acting in vivo is posulated to be necessary for the formation of burr cells.

Lipid synthesis by red blood cell preparations in Wolman's disease (a familial lipidosis)

Red blood cell preparations of known reticulocyte content from an infant affected with Wolman's disease and from two other infants of the same age were incubated with [ 14C]glycerol and [ 14C]mevalonic acid. The incorporation of [ 14C]-glycerol into lipids (calculated as radioactivity per unit volume of reticulocytes) was nine times greater for the Wolman's disease cells than those of the controls; most of the increased incorporation was into triglyceride. The incorporation of [ 14C]mevalonic acid into cholesterol and cholesterol esters was five times greater for the Wolman's disease cells than those of the controls; most of this incorporation was into the cholesterol esters. This demonstration that the blood cells share the metabolic defect of Wolman's disease offers an increased opportunity to elucidate the mechanism of the defect.

Studies of Avian Sarcoma and Erythroblastosis (Strain 13). VI. Response in Birds to Two Routes of Induction and Pathology of a Virus-Induced Tumor

The pathological alterations in chickens with avian sarcoma and erythroblastosis Strain 13 (S13) were described in 1935 by Stubbs and Furth (8). All of the chickens in that study were inoculated intramuscularly or intravenously with tumor cell mince or heparinized whole blood from birds with erythroblastosis. After intramuscular inoculation, tumors appeared at the site of injection. Intravenous inoculation resulted in diffuse sarcomatosis of the spleen, gonads, bone marrow, liver, kidney, and heart. Filtered tumor mince or filtered blood also produced tumors, establishing the agent as a filterable virus. Erythroblastosis was frequently an associated finding, and the view was proposed that a single oncogenic virus might stimulate two different types of cells to neoplastic growth, or that two viruses were present. Studies of S13 were resumed after 20 years employing a lyophilized preparation of tumor mince and red blood cells that had been stored in a refrigerator (9). Minced tumor inoculated into the bursa of Fabricius was used in transmission experiments to determine whether bursa-inoculated cells would produce any kind of immunity in the chicken. Neither tumors nor immunity were produced by these bursal inoculations (10). The same type of experiment was conducted by vent-drop and vent-brush application of tumor mince. Bursa-exposed, vent-exposed, and control chickens

Inhibition of cholinesterases by fluoride in vitro.

1. Series of colorimetric dynamic assays allowed the study of the inhibition of cholinesterases by F(-) ions in vitro, by using, as sources of enzyme, whole human blood, human serum, homogenized rat brain and two preparations of red blood cells (human and bovine) whose enzymic purity was ascertained. 2. The first evidence of inhibition of human serum pseudocholinesterase by fluoride was noticed at 15-25mum-fluoride. Ten times as much fluoride was needed to start inhibition of acetylcholinesterase of the red blood cells. 3. The action of fluoride on the enzymic reaction was immediate. The reversibility of the inhibition was shown by dialysis and dilution. 4. Kinetic measurements showed that the inhibition under study was not dependent on the substrate concentration and was of the uncompetitive type, similar to that observed in the presence of a heavy metal (cadmium). 5. The activity of serum cholinesterase did not change in the absence of Mg(2+) and Ca(2+) ions. Fluoride was shown to inhibit the enzyme in the absence of these ions as well as of phosphate. 6. Fluoride could inhibit cholinesterases in the presence of three different substrates and had no action on the non-enzymic hydrolysis. 7. It is thought that the halide is bound reversibly to the enzyme molecule, with the probable exclusion of the active site, but no firm conclusion could be reached on this point.

Studies on Lysolecithin Formation in Immune Hemolysis

Summary The possibility that the hemolytic step of immune hemolysis is mediated through lysolecithin formed from lecithin through the action of a lecithinase activated by the reaction of antigen, antibody and complement was studied in a system of sheep red blood cells, rabbit antisheep red blood cell serum and guinea pig serum complement. The presence of a hemolysin in the supernatant following centrifugation after immune hemolysis could not be demonstrated, even when lecithin had been included in the system as a substrate. The inhibitory effect of albumin, which inhibits lysolecithin hemolysis, on immune hemolysis could be explained by its osmotic effect, as dextran similarly inhibited immune hemolysis without a comparable effect on lysolecithin hemolysis. Cobra venom, which contains a lecithinase, did not produce hemolysis of the sheep red blood cells, even when the amount of complement was present as was needed for complete immune hemolysis of 0.015 ml of sheep red blood cells; nor did the venom lyse an EAC′1,4,2,3ab preparation of sheep red blood cells. Direct lipid analysis of the pellet and supernatant following centrifugation of the immune hemolysate showed no formation of lysolecithin or decrease of lecithin. Complement fixation of human serum by heat-aggregated γ-globulin resulted in no lysolecithin formation or change in the concentration of the other phospholipids. The small amount of lysolecithin present in the immune hemolysis system was not enough to account for the hemolysis and appeared to be a normal constituent of the red blood cells. Moreover, the lecithin present in the system was not enough to account for the hemolysis, even if it had been converted entirely to lysolecithin. An EAC′1,4,2,3ab preparation of sheep red blood cells showed no increase in susceptibility to lysolecithin lysis. On the basis of these findings, lysolecithin did not appear to be responsible for the hemolysis in the immune hemolysis system studied.

Photo-Hemolysis Associated with Protoporphyrinemia: Preliminary Report

Since 1961 when Magnus, Jarrett, Prankard, and Rimington (1) first described a new disease entity, viz. erythropoietic protoporphyria, there have been reports of similar cases by several investigators (2, 3, 4, 5, 6). In all the reported cases, there were markedly elevated erythrocyte protoporphyrin levels associated with cutaneous photosensitivity. This form of photosensitivity may express itself in diverse cutaneous lesions including erythema, vesicles, wheals and eczema. We wish to report the preliminary results of studies concerning the in vitro hemolysis of red blood cells by ultraviolet light using cells from a patient having erythropoietic protoporphyrinemia. The patient is a seven year old girl who has vesicular and crusted skin lesions on the light exposed areas on and off since the age of 4 and who has been under our observation for the past 2 years. Erythrocyte protoporphyrin blood levels (7) were assayed on 4 occasions and were found to be significantly elevated. Preparation of Red Blood Cells for Hemolysis Studies: In each test run, the effects of ultraviolet light on the patient's red blood cells and on the red blood cells of a normal control subject were compared. Prior to irradiation, the cells were washed three times and resuspended in a 1:400 dilution of a 0.9% NaC1 phosphate buffer. Light Source: The red blood cells were irradiated with light from a bank of 4 Westinghouse F20 T12 BL blacklight fluorescent tubes. The emission spectrum of these tubes is mainly between 3200A° and 4500A°. In order to minimize the infrared radiation and the minute amount of radiation below 3200A the light was directed through a Corning 1-69 filter and through a window glass filter, 3 mm thick. The red blood cells were enclosed in quartz receptacles, and irradi* From the Department of Dermatology of New York University Schools of Medicine, New York, New York. This study was supported by Grant Number DA-49-193-MD-2275 from the U.S. Army Medical Research and Development Command. Technical Assistance given by Mary E. Moragne, B.S. Received for publication October 30, 1963. ated at a target distance of 6.25 centimeters. The intensity of the incident radiation was approximately 1100 microwatts per centimeter square. Measurement of Hemolytis was carried out by two methods: 1) direct cell counts: aliquots of the red blood cell suspensions were counted in a No. 500 Levy Counting Chamber (hemocytometer), at 10 minute intervals. Hemolysis was evident in the reduction of the number of red cells counted; 2) spectrophotometrically: the concentration of hemoglobin in the red blood cell supernatant was measured in a Coleman junior spectrophotometer.

Methylglyoxal Formation from Aminoacetone by Ox Plasma

IT was previously reported1–3 that washed suspensions of Staphylococcus aureus synthesize aminoacetone from either threonine or glycine and the new amino-ketone was isolated in crystalline form. Laver et al. 4 and Kikuchi et al. 5 have independently demonstrated the formation of aminoacetone by preparations of red blood cells and have shown that the system which forms δ-aminolaevulinic acid is responsible for this activity.