Studies on the Preservation of Red Blood Cells

Utilizing an automated antiglobulin test, we have investigated the presence of the third and fourth components of human complement on normal red blood cells (RBCs). Only negligible amounts of the fourth component, C4, could be detected on either freshly collected or stored RBCs. The fragment C3d of the third component, C3, was detectable on both freshly collected and stored normal RBCs. A product derived from C3 and reacting with anti-C3c antibody was only barely detectable on freshly collected normal RBCs. During storage of blood at 4 degrees C, increasing quantities of this material were detected on the RBC membrane. Bromelin treatment rendered stored RBCs completely nonreactive with anit-C3c antibody, whereas only partial loss of reactivity was observed following incubation with heated plasma. In contrast, incubation of EC43 with heated plasma completely abolished their ability to react with anti-C3c antibody. We suggest that the presence of this C3 fragment on stored RBCs may contribute to the development of "preservation injury".     

Studies in Red Blood Cell Preservation. 8. Liquid Storage of Red Cells in a Glycerol-Containing Additive Solution

The purpose of the present study was to determine whether a hypotonic additive containing a low concentration of glycerol as a membrane permeable solute would improve the liquid storage of red blood cells (RBCs). Packed RBCs were stored either with 200 ml of an experimental additive solution, EAS 25, containing (m M ): glycerol 150, adenine 2, glucose 110, mannitol 55, and NaCl 50, or with 100 ml/unit of a conventional additive solution Adsol®. The results show that the adenosine triphosphate values, hemolysis, potassium leakage, and the morphology scores of RBCs were significantly better with EAS 25 than with Adsol up to 84 days of storage. The ATP values were significantly different only after the first 42 days of storage. The mean corpuscular volumes (MCVs) of the RBCs were significantly higher throughout in the experimental additive accompanied by decreased microvesiculation as compared to Adsol. The total microvesicle membrane protein shed by 100 ml of RBCs was 47.92±12.31 mg in Adsol and 18.96±5.49 mg in EAS 25 (p<0.001). The larger MCVs of the RBCs in EAS 25 may have a favorable effect on maintaining membrane integrity by decreasing the loss of membrane by microvesiculation.     

Effect on Red Cells of a Small Rise in Temperature: in Vitro Studies

An increased destruction of red cells was previously demonstrated during experimental fever. The present study deals with the changes in red cells produced by extended exposure in vitro to temperatures in the biological range of fever.
By elevating the temperature only a few degrees above normal body temperature a distinct increase in spontaneous haemolysis and osmotic fragility of rabbit red cells was observed. By combination of exposure to heat in vitro and survival-studies in vivo , using red cells labelled with ⁵¹Cr or ⁵⁹Fe, it was confirmed that even small elevations of temperature greatly reduced the survival of the red cells in the circulation. In a supplementary investigation in rabbits a significant increase in osmotic fragility was observed during experimentally produced fever using injections of bacterial pyrogen or external heating.
No difference in glucose consumption during incubation of red cells in vitro at normal and elevated temperature could be demonstrated and inactivation of vital enzymes seems not to be responsible for the injury to the cells. Other mechanisms, especially the possibility of changes in the lipoprotein structure of the cell membranes, are discussed.
In a single study, human red cells exhibited the same vulnerability as rabbit red cells to small elevations of temperature, although their sensitivity was less.     

Protection of Lethally Irradiated Mice by Spleen Cells from Neonatally Thymectomized Mice

Long-lived, immunologically vigorous (C3H(f) x A(f))F(1) hybrids were produced after lethal irradiation by administration of spleen cells from C3H(f) or syngeneic donors. Further, neonatally thymectomized C3H(f) or A(f) strain donors reconstituted irradiated C3H(f) or (C3H(f) x A(f))F(1) hosts. In addition, C3H(f) spleen cells from nonthymectomized 10- to 15-day-old donors protected irradiated hybrid mice, but A(f) cells of young mice as well as of older mice produced graft-versus-host reaction and early death in irradiated C3H(f) or (C3H(f) x A(f))F(1) hybrids.Abrogation of secondary disease by treatment of irradiated mice with spleen cells from allogeneic neonatally thymectomized mice is possibly attributable to diminished immunologic competence of the cells grafted, followed by the development of immunological tolerance of the donor cells. Donor cells, receiving thymus influence in the recipient host after transplantation, could explain the long-lived immunologically vigorous radiation chimeras that did not experience graft-versus-host reactions. The findings of this study help to understand the differential susceptibility of A(f) and C3H(f) mice to development of tolerance to one another's antigens observed in prior investigations.It appears that, in these mice, the host thymus influences the maturation of the spleen cells from young mice or from neonatally thymectomized mice. However, this influence was often greater in mice given 767 rads than in those given 1046 rads. This differential influence is possibly attributable to irradiation damage to the thymus produced by the higher dose of irradiation. Spleen cells from neonatally thymectomized mice can be differentiated and expanded by the thymus of the host. The differential susceptibility of T(1), early differentiation stages, of thymus-dependent lymphocytes and T(2), late differentiation stages, of thymus-dependent lymphocytes to tolerance induction and immunostimulation, respectively, are proposed as the bases for these otherwise paradoxical influences.     

Enzyme Abnormalities in Red Cells

Hereditary deficiencies involving eight enzymes of the Embden-Meyer-hof pathway, at least five involving the hexosemonophosphate shunt, and GSH metabolism, and at least two involving nucleotide metabolism, are known to be associated with haemolytic syndromes in man. Lactate dehydrogenase deficiency is also recognized, but is not associated with haemolysis. In addition, markedly aberrant enzyme activity ratios characterize a variety of dyserythropoietic disorders, both hereditary and acquired. In some disorders, haemolysis is the sole clinical manifestation; in others, dysfunction of the nervous system, the heart, the skeletal muscle, and the leucocytes may occur. The red cell may also mirror enzymopathies having expression in non-haematopoietic tissues without a haemolytic syndrome being present. While double heterozygosity for two separate enzymopathies or for an enzymopathy and hereditary spherocytosis or elliptocytosis is recognized in a number of cases, clinical manifestations have thus far appeared minimal or non-existent. In a recently described haemolytic syndrome uniquely large accumulations of cytidine and uridine nucleotides have been noted secondary to a severe deficiency of a pyrimidine specific 5'nucleotidase.     

Studies of Incomplete Antibodies: I. Effect of Papain on Red Cells

A variety of typical agglutinating G antibodies agglutinated papain-treatedcells in a uniform manner in about 16-fold higher dilutions than nontreatedcells. With M antibodies the corresponding titer increase was on the average4-fold. The papain-treatment of cells also increased the titers of cold agglutinins, phytohemagglutinins and preparations of PVP. Antiglobulin consumption experiments revealed that no more antibody was fixed from the samevolume of antiserum onto papain-treated than onto nontreated cells. Possiblemechanisms underlying enhanced agglutination of enzyme-treated cells werediscussed. It is suggested that the same type of antibody may react with papaintreated and nontreated cells but that a smaller number of antibody moleculesare needed for agglutination of papain-treated cells because of altered surfaceproperties of treated erythrocytes.

Submitted on June 15, 1965 Accepted on September 8, 1965     

Preparation of Granulocyte-Poor Red Blood Cells by Microaggregate Filtration

Abstract. 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 × 10⁸/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.     

Increased Potassium Permeability by Calcium in Hypochromic Red Blood Cells

The red blood cell (RBC) content of Na⁺ and K⁺ were measured both on fresh cells from normal, heterozygous β-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37° C, either in presence or in absence of Calcium (Ca²⁺). Ca²⁺ did not increase membrane permeability to Na⁺, but increased the K⁺ loss, both from normal cells and to a greater degree much more from hypochromic cells. Glucose largely prevented the K⁺ loss from hypochromic cells incubated either in absence or in presence of Ca²⁺, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K⁺ in hypochromic cells incubated for 24 h at 37° C, possibly removing Ca²⁺ bound to the cell membrane. The results suggest that Ca²⁺ does not represent the primary cause of K⁺ leak in hypochromic cells, but it is able to enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down.     

Studies on the in vivo Elution of 51Cr from Baboon Red Blood Cells1,2,3

Abstract. Four anticoagulant solutions were added to baboon red blood cells prior to labeling with ⁵¹Cr to determine how each would influence the distribution of ⁵¹Cr within the red blood cells, the loss of ⁵¹Cr from the red blood cells after transfusion, and the calculated red cell survival value. The ⁵¹Cr label was detected in the hemoglobin and in the low molecular weight compounds within the red blood cells. The elution of ⁵¹Cr from labeled baboon red blood cells following transfusion could not be explained by the distribution of ⁵¹Cr between hemoglobin and low molecular weight compounds within the red blood cells.     

The Inhibition of Anaerobic Glycolysis in Red Cells by Leukaemic Leucocytes

Rates of anaerobic glycolysis were determined simultaneously in the red cells and leucocytes of patients with malignant haematologic disorders. The disappearance of glucose and the evolution of CO₂ from bicarbonate were measured in the same Warburg flasks over the same period of time.
In four patients with lymphogenous leukaemia and in one with acute leukaemia of disputed type but probably myelogenous, the leucocytes suppressed the glycolytic activity of the red cells. This abnormal finding appeared with exacerbation of the disease and disappeared with remission. It was present during the terminal phase in four patients, but could not be attributed to the presence of blast cells in the peripheral blood.
The leukaeinic leucocytes exerted the same inhibitory effect on normal donor cells as on those of the patient. The plasma and the supernatant fluid from strongly inhibited reactions were without effect, as were aqueous and hypertonic extracts of the leucocytes. In all Warburg reactions the rate was constant, with no evidence of a cumulative effect. The inhibition was reversed by removal of the leucocytes.
The characteristics of this phenomenon resemble molecular behaviour and suggest an equilibrium of some kind. Its kinetic properties closely resenible those of stoichiometric inhibition of an enzyme system. It is conjectured that an intercellular stress, perhaps physicochemical in nature, may have been exerted between the leucocytes and red cells, temporarily interfering with some event in thc glycolytic cycle of the latter.     

Preservation of Red Blood Cells: Studies of Erythrocyte Adenine Nucleotides Using Luminescence Analysis

The levels of ATP and total adenine nucleotides (ATP + ADP + AMP) were determined by firefly luciferase assay in red blood cells during storage for 5 weeks at 4 degrees C. With few exceptions, no significant differences in nucleotide levels were found between whole blood stored in CPD-adenine and various preparations of red blood cells in CPD-adenine or CPD with saline-adenine-glucose (SAG) as additive. The levels of ATP and total adenine nucleotides during storage are discussed in relation to glucose levels, extracellular pH and shelf life of the red blood cells.     

Metabolic Studies on Red Cells from Patients with Chronic Renal Disease on Haemodialysis

S ummary . Studies were performed which suggest intracorpuscular metabolic alterations in red cells from patients with chronic renal disease. In a buffered salt solution, the red cell metabolic rate of uraemic subjects was significantly greater that that of normal subjects. Furthermore, the metabolic rate of uraemic red cells was not diminished when incubated in normal plasma. Finally, incubation of control and uraemic red cells in a buffered salt medium with 10 mM phosphate did not obliterate the differences in metabolic rate between control and uraemic cells. When the activities of several rate-limiting glycolytic enzymes were measured, red cells from the uraemic subjects had a normal level of phosphoglycerate kinase and a significant increase in the activities of hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase and glutamic-oxaloacetic transaminase compared to control red cells. These results indicate that red cells from patients with chronic renal disease are intrinsically hypermetabolic, and the pattern of increased enzyme activities suggests the presence of a red cell population with a younger mean cell age due to preferential loss of older cells.     

Studies on Naturally Occurring Warm Haemolysins and Haemagglutinins Active against Trypsinized Red Cells

Abstract. A normal serum with haemolytic and agglutinating activity against trypsintreated red blood cells at 37°C has been studied. The properties of this serum appear to be very similar, if not identical, to those of the warm haemolysin described by Heistø et al. [1].     

Studies on the Agglutinin Accompanying the Warm Haemolysin Active against Trypsinized Red Cells

Abstract. The titres of warm haemolysin and its accompanying agglutinin were determined (1) in sera stored at -30 and -72°C for about 3 months, (2) in sera obtained with short intervals during 1 month, (3) after treatment of red cells with various concentrations of trypsins, (4) after adding soybean trypsin inhibitor (STI) to the trypsin solution, (5) after exposure of the trypsin solutions to 55 °C for 30 min, and (6) with red cells to which bovine albumen had been added before adding the trypsin solution.
Relatively small variations in the agglutinin titre were observed when testing the deep frozen sera in contrast to much greater variations when testing fresh samples drawn on different days. Variations in agglutinin titre were poorly related to the trypsin activity expressed in Anson units. The factor in the trypsin preparation which sensitizes the red cells for haemolysis is more temperature labile than the factor responsible for the agglutination. STI and albumen inhibit both haemolysis and agglutination.     

Filterability of ACD-Stored Red Cells

The deformability of stored erythrocytes was studied using the Myrenne filtrometer MF4. Blood was stored in ACD under usual blood banking conditions and filterability studies were done at various intervals during a 3-week period. Marked reductions in stored erythrocyte filterability were noted as early as day 5 of storage. These results show that ACD is not a good "rheological' storage medium and indicate that further studies are required, using different storage media to assess the importance and significance of this rheological lesion.