What quality of red blood cells shall we offer the transfused patient?

Individual units of red blood cells (RBCs) can vary widely in their benefit to recipients depending on how the unit is prepared and the duration and quality of pretransfusion storage. The haemoglobin (Hb) concentration of the donor blood, the volume of the collection, leucocyte depletion procedures and the composition of the additive solution are all factors that will influence the final product. It may not be generally appreciated that, as red cell preparation is currently practiced, the haemoglobin content of viable RBCs in an individual blood unit collected within current standards may range from as little as 30 g to as much as 90 g, a variation of which the transfusing physician is generally unaware and which should no longer be considered acceptable. The transfusion community is currently in the untenable position of attempting to balance the quality of transfused blood against perceived cost, a balance that needs to be re‐examined in the light of recent developments. A number of measures are currently available that can readily eliminate many of these shortcomings. There are four obvious ways by which the quality and uniformity of RBC units can be improved: (1) The volume of blood collected from the donor should be adjusted depending on the donor's haemoglobin concentration. (2) Red cell losses during preparation should be minimized and accounted for in the collection (3) The subsequent preservation procedure should both maintain RBC viability during storage and minimize the loss of 2,3‐DPG and of membrane flexibility. (4) The haemoglobin content of a unit of red cells should be standardized. We recommend 50 g ± 5 g of haemoglobin as the amount of blood that should be collected from a donor. For all of these measures the technology is currently available and may be readily introduced, especially through the use of automated collection systems. It is the responsibility of the transfusion organisations to redefine their goals and, in collaboration with industry, develop routine procedures that do not unnecessarily sacrifice to economics the best interests of blood recipients and their prescribing physicians.     

Phenotypic differences of CD4+ T cells in response to red blood cell immunization in transfused sickle cell disease patients

Alloimmunization against red blood cells (RBCs) is the main immunological risk associated with transfusion in patients with sickle cell disease (SCD). However, about 50–70% of SCD patients never get immunized despite frequent transfusion. In murine models, CD4⁺ T cells play a key role in RBC alloimmunization. We therefore explored and compared the CD4⁺ T‐cell phenotypes and functions between a group of SCD patients (n = 11) who never became immunized despite a high transfusion regimen and a group of SCD patients (n = 10) who had become immunized (at least against Kidd antigen b) after a low transfusion regimen. We studied markers of CD4⁺ T‐cell function, including TLR, that directly control lymphocyte function, and their spontaneous cytokine production. We also tested responders for the cytokine profile in response to Kidd antigen b peptides. Low TLR2/TLR3 expression and, unexpectedly, strong expression of CD40 on CD4⁺ T cells were associated with the nonresponder status, whereas spontaneous expression of IL‐10 by CD4⁺ T cells and weak Tbet expression were associated with the responder status. A Th17 profile was predominant in responders when stimulated by Jb^k. These findings implicate CD4⁺ T cells in alloimmunization in humans and suggest that they may be exploited to differentiate responders from nonresponders.     

Galactitol and galactonate in red blood cells of galactosemic patients

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[13C]galactitol and UL[13C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98+/-1.2 microM (M+/-SD) with a range of 3.54-8.81 microM. The mean in non-galactosemic patients was 0.73+/-0.31 microM with a range of 0.29-1.29 microM. The mean of RBC galactonate in the same galactosemic patients was 4.16+/-1.32 microM (M+/-SD) with a range of 0.68-6.47, while the mean in non-galactosemic subjects was 1.94+/-0.96 (M+/-SD) with a range of 0.69-3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.     

Mast cell phagocytosis of red blood cells.

The prevalence of mast cells infiltrating bone marrow of different rats varied widely, as did the staining properties and size of their cytoplasmic granules. Bone marrow mast cells from several rats revealed large membrane-limited inclusions which stained metachromatically or orthochromatically and resembled inclusions in some macrophages. Ultrastructurally, mast cells varied widely in content of uniform dense granules or enlarged granules with less dense, fine grained content. Some of the large inclusions observed ultrastructurally in mast cells were heterophagic vacuoles which contained erythrocytes or reticulocytes, or remnants from other phagocytized cells, possibly neutrophils or unidentified homogeneous material. Smaller bodies, interpreted as fragments of erythrocytes, lay extracellularly near mast cells and occupied small, membrane-limited, heterophagic vacuoles in some mast cells. In other mast cells, communal vacuoles enclosed several specific cytoplasmic granules in various stages of disruption. The communal vacuoles occasionally opened to the extracellular space. A few large indeterminate vacuoles in mast cells contained amorphous flocculent matter which apparently derived either from coalescence of cytoplasmic granules through fusion of granule membranes or from endocytosis.     

Irradiation effect on aging red blood cells.

Irradiation of stored red blood cells (RBC) is increasingly utilized for patients who are immunosuppressed or on chemotherapeutic regimens. With the growing demand for irradiated cellular blood products, there has been an increasing need for transfusion services to store previously irradiated blood until needed for transfusion. The effect of irradiation on aging stored RBC has not been studied to date. Five units each of group A, RBC collected in CPD-Adsol (AS-1) with a prior shelf-life of 10, 20, 30, and 40 days, respectively, were divided equally utilizing a sterile docking device and stored at 1 to 6 degrees C. Baseline samples from each bag were obtained for the measurement of extracellular potassium (K+), plasma free hemoglobin (PFH), total lactate dehydrogenase (LD), and erythrocyte 2,3-DPG activity. One of each pair received 2,000 rads of gamma irradiation. Samples were obtained at 3 and 7 days post-irradiation, and multiples of 7 days until expiration. All irradiated units reached a state of K+ equilibrium at 60 to 70 mmol per L irrespective of the length of previous storage with an inverse relationship of RBC age at irradiation and the time required to reach the state of equilibrium. Increased K+ leakage from irradiated aging RBC suggests the need for including in vivo studies of cell survival to establish a post-irradiation storage life. Length of storage prior to irradiation had no effect on PFH, LD activity, and 2,3-diphosphoglycerate (2,3-DPG) activity compared to paired controls.     

Serum erythropoietin in regularly transfused thalassemic patients.

Serum erythropoietin levels were measured in 67 regularly transfused thalassemic patients with pre-transfusion hematocrit ranging from 25-32% and in 40 normal individuals. In patients, mean erythropoietin levels were slightly increased (mean 91.5 miu/ml) as compared to normal individuals (mean 42 miu/ml). The distribution of erythropoietin (Ep) was wide in thalassemic patients. 40% had normal or decreased and 60% increased Ep levels. A reverse relation between pretransfusion Hct and erythropoietin activity was observed only among patients with normal erythropoietin levels and splenectomized patients with high erythropoietin titers suggesting that a normal feedback between tissue hypoxia and erythropoietin activity occurs in these groups. The effect of regular blood transfusions in reversing tissue hypoxia resulting from anemia in the majority of regularly transfused thalassemic patients seems to be satisfactory, as it is assessed by serum erythropoietin levels.     

DMO-transport in human red blood cells

The DMO (5,5-dimethyloxazolidine-2,4-dione) transport was studied in human red blood cells by measuring the¹⁴C-DMO back exchange under self exchange conditions from¹⁴C-DMO labeled cells at 0°C. The unidirectional DMO-flux was linearly related to the DMO concentration up to approx. 100 mM. The unidirectional DMO-flux increases as pH is reduced. The DMO transport is not inhibited by anion transport inhibitors like DIDS, SITS, dipyridamole, phlorhizin, salicylate or butanol. A close correlation between the unidirectional DMO-flux and DMOH, the unionized form of DMO, has been observed suggesting that DMO is transported predominantly by nonionic diffusion. The permeability of DMOH is 9.5·10^–6 cm/s (0°C) while the permeability of DMO^– cannot be assessed from our data.     

Lack of increased bleeding after liver biopsy in patients with mild hemostatic abnormalities

Prophylactic transfusions of fresh frozen plasma and platelets are sometimes given to patients with mild elevations in prothrombin time (PT) and partial thromboplastin time (PTT) and mild thrombocytopenia before percutaneous liver biopsy. To determine whether PTs and PTTs 1.1-1.5 times midrange normal levels and platelet counts 50-99 x 10(9)/L are associated with increased bleeding complications, hospital records of all patients who underwent percutaneous liver biopsy during 56 consecutive months (n = 291) were reviewed. Complete information was available for 177 inpatient procedures (155 standard, 22 fine needle). Overall, the frequency of bleeding complications in patients with platelet counts greater than or equal to 50 X 10(9)/L was 3.4% (6 of 175), with no significant difference between patients with mild hemostatic abnormalities and patients with normal parameters. These data suggest that prophylactic transfusions may not be necessary. One factor was highly associated with bleeding complications: a patient diagnosis of malignancy, 14% (7 of 50) compared with 0.8% (1 of 127) among other patients (P less than 0.001). These patients should be monitored closely after biopsy.     

An antibody against anemia-inducing substance inhibits cation influx in red blood cells from cancer patients.

Resealed ghost of red blood cells (RBCs) from advanced cancer patients includes proteins with antigenicities common to an anemia-inducing substance that we separated from plasma of patients with advanced malignant neoplasms. Although cation influx in vitro in RBCs from a cancer patient is larger than that from a normal healthy volunteer, an antibody against anemia-inducing substance inhibited the in vitro cation influx in RBCs from the cancer patient. Activation of the cation influx with N-ethylmaleimide after reaction with the antibody reversed the effect to create a greater cation influx in RBCs from the patient, as compared with that from the healthy volunteer.     

Cow red blood cells. II. Stimulation of bovine red cell glycolysis by plasma.

Cow red cell glycolysis, which can be stimulated by a variety of purines and pyrimidines, was also found to be elevated by its own plasma. Dialyzed or charcoal-treated plasma could no longer stimulate glycolysis, suggesting that the stimulating factors may be purines or pyrimidines. Determination of purines or pyrimidines in plasma revealed the presence of xanthine (0.31 muM), hypoxanthine (0.60 muM), and adenosine (0.05 muM), as well as unknown compounds. A physiologic level of hypoxanthine, with or without xanthine and adenosine approximating their concentrations in plasma, resulted in the stimulation of cow red cell glycolytic rate by 16% (P less than 0.01). These findings suggest that plasma-borne purines may act on cow red cells in concert with as yet unidentified factors. Moreover, exchanging calf and cow plasmas produced no stimulatory effect on either calf or cow red cell glycolysis, suggesting that a) calf red cells lack some of the cellular components that respond to this stimulator and, b) only cow plasma contains this specific stimulator. In other species, including dog, cat, rabbit, rat, guinea pig, and human, stimulation of glycolysis by plasma was not observed.     

TT virus (TTV) genotyping in blood donors and multiple transfused patients in Brazil

TT virus (TTV) is widely distributed in the general population. The objective of the present study was to investigate the prevalence and distribution of TTV genotypes among blood donor candidates and multiple transfused patients in the Southeast region of the state of São Paulo, Brazil. TTV-DNA detection by amplification of a segment of the ORF-1 region, presented a prevalence of 11.9% in 270 serum samples from blood donors, of 46.2% in 18 samples from patients with coagulopathies, and of 31.8% in 15 samples from patients with hemoglobinopathies. When specific primers for the non-coding (UTR) region of the TTV genome were used the prevalences were 50.5%, 95.0%, and 82.0% for blood donors, patients with coagulopathies and patients with hemoglobinopathies, respectively. Positive samples from 49 individuals were sequenced and partial segments of 230 base pairs referring to the ORF-1 region of the TTV genome were used for the determination of their genotypes with the aid of phylogenetic analysis. The most frequent genotype was 1 (74.0%), followed by genotype 2 (26.0%). These data indicate a high prevalence of this virus in the populations of blood donors and transfused patients, providing further evidence for the role of transfusions as an efficient pathway in the transmission chain.     

Refractoriness of red blood cells and platelets

The effect of proteolytic enzymes and of arachidonic acid on aggregation of red blood cells and platelets was studied. These substances were found to stimulate aggregation of the blood cells. Preliminary incubation of fibrinolysin, trypsin, and arachidonic acid with suspensions of blood cells, however, is followed by a marked decrease in their ability to aggregate, i.e., by the development of a refractory state. The possible mechanism of this phenomenon is discussed.Research Institute of Traumatology and Orthopedics, Gor'kii. (Presented by Academician of the Academy of Medical Sciences of the USSR B. A. Korolev.) Translated from Byulletin' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 11, pp. 524–526, November, 1977.     

Effect of red blood cells and red blood cell ghosts on reticuloendothelial system function

Previous studies have shown that hepatic phagocytosis of red blood cell (RBC) stroma can depress reticuloendothelial system (RES) phagocytic function and increase susceptibility to shock. Since the RBC stroma used in these experiments contained substantial amounts of adherent hemoglobin, the present study was carried out to evaluate the role of the hepatic uptake of RBC membrane material on RES phagocytic function and susceptibility to endotoxin shock in rats. Neuraminidase-treated RBC which contained normal amounts of hemoglobin and RBC ghosts which were hemoglobin-free were used. Both preparations were removed from the circulation primarily by the liver. RES phagocytic function was depressed following the hepatic uptake of 29 X 10(8) neuraminidase-treated RBC and 26 X 10(8) RBC ghosts. RES uptake of neuraminidase-treated RBC was associated with an increase in susceptibility to endotoxin shock, but RBC ghosts did not affect shock susceptibility. Thus, RBC ghosts and intact RBC are equally effective in depressing RES phagocytic function, but RBC ghosts did not affect susceptibility to endotoxin shock.     

Calcium transport in human red blood cells under hypertonic conditions.

Calcium accumulation in intact human erythrocytes is enhanced by incubation in hypertonic solutions. Hypertonicity produces an increased permeability of the membrane to calcium that is time-dependent and occurs in the presence or absence of calcium. When hypertonically treated cells are incubated for more than 30 min in 300 mosmol/kg solutions the permeability of the membrane to calcium returns to basal values. Oligomycin inhibits the effect of hypertonicity on calcium uptake. The inhibitory action of oligomycin diminishes as the external sodium increases and can only be observed when the external concentration of potassium is at or below 3 mM. Low intracellular sodium and high intracellular potassium concentrations increase the uptake of calcium. It is concluded for human erythrocytes that 1) the increased permeability of the membrane to calcium produced by hypertonicity is a time-dependent, reversible phenomenon and is independent of calcium, 2) the increase in intracellular potassium concentration associated with hypertonic exposure is an important factor contributing to this response, and 3) interactions between calcium and components of the sodium-potassium transport system may account for the enhanced uptake of calcium produced by hypertonicity.     

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.     

Organ distribution of sheep red blood cells in ALS-treated rats.

The inter-organ distribution of radioactivity in rats injected with 51Cr-labelled SRBC is altered after treatment with ALS absorbed with this antigen. The alteration is due to the presence of soluble SRBC antigens in the serum and subsequent immunization of the tested animals. The 51Cr distribution does not correspond to the uptake of antigenic material in immunized rats.