Quality determinants of erythrocyte destined for transfusion.

An overview is given of a series of standard assays to evaluate the quality of red cell concentrates for transfusion. These are visual inspection, assessment of hemolysis, quantitation of 2,3-DPG and nucleotide levels (especially ATP) and evaluation of morphology. These parameters, relatively easy to measure, are main determinants of in vivo recovery after transfusion. In addition, some other assays are described, which should give more information about the function of red blood cells after transfusion. These assays include plasma-induced hemolysis, binding of annexin-V, deformability measurements and a rat model to judge oxygen delivery by human red blood cells (RBC). Especially in judging new protocols for the preparation of red cell products, involving e.g. improved additive solutions or pathogen inactivation methods, these quality parameters should not be compromised.     

Footstrike is the major cause of hemolysis during running.

There is a wide body of literature reporting red cell hemolysis as occurring after various forms of exercise. Whereas the trauma associated with footstrike is thought to be the major cause of hemolysis after running, its significance compared with hemolysis that results from other circulatory stresses on the red blood cell has not been thoroughly addressed. To investigate the significance of footstrike, we measured the degree of hemolysis after 1 h of running. To control for the potential effects of oxidative and circulatory stresses on the red blood cell, the same subjects cycled for 1 h at equivalent oxygen uptake. Our subjects were 10 male triathletes, who each completed two separate 1-h sessions of running and cycling at 75% peak oxygen uptake, which were performed in random order 1 wk apart. Plasma free hemoglobin and serum haptoglobin concentrations were measured as indicators of hemolysis. We also measured methemoglobin as a percentage of total hemoglobin immediately postexercise as an indicator of red cell oxidative stress. Plasma free hemoglobin increased after both running (P < 0.01) and cycling (P < 0.01), but the increase was fourfold greater after running (P < 0.01). This was reflected by a significant fall in haptoglobin 1 h after the running trials, whereas no significant changes occurred after cycling at any sample point. Methemoglobin increased twofold after both running and cycling (P < 0.01), with no significant differences between modes of exercise. The present data indicate that, whereas general circulatory trauma to the red blood cells associated with 1 h of exercise at 75% maximal oxygen uptake may result in some exercise-induced hemolysis, footstrike is the major contributor to hemolysis during running.     

The use of ELISA to monitor amplified hemolysis by the combined action of osmotic stress and radiation: potential applications

A new assay has been developed to study the osmotic fragility of red blood cells (RBCs) and the involvement of oxygen-derived free radicals and other oxidant species in causing human red blood cell hemolysis. The amount of hemoglobin released into the supernatant, which is a measure of human red blood cell hemolysis, is monitored using an ELISA reader. This ELISA-based osmotic fragility test compared well with the established osmotic fragility test, with the added advantage of significantly reduced time and the requirement of only 60 mul of blood. This small amount of blood was collected fresh by finger puncture and was immediately diluted 50 times with PBS, thus eliminating the use of anticoagulants and the subsequent washings. Since exposure of RBCs to 400 Gy gamma radiation caused less than 5% hemolysis 24 h after irradiation, the RBC hemolysis induced by gamma radiation was amplified by irradiating the cell in hypotonic saline. The method was validated by examining the protective effect of Trolox, an analog of vitamin E and reduced glutathione (GSH), a well-known radioprotector, against human RBC hemolysis caused by the combined action of radiation and osmotic stress. Trolox, a known membrane stabilizer and an antioxidant, and GSH offered significant protection. This new method, which is simple and requires significantly less time and fewer RBCs, may offer the ability to study the effects of antioxidants and membrane stabilizers on human red blood cell hemolysis induced by radiation and oxidative stress and assess the osmotic fragility of erythrocytes.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.     

Differences between cation-osmotic hemolysis and filterability in exaprolol- and glutaraldehyde-treated human red blood cells

The changes in human red blood cell microrheology in different glutaraldehyde (3.0 and 5.0 x 10(-6) mol x l(-1)) and exaprolol (2.5 and 5.0 x 10(-4) mol x l(-1)) concentrations were studied. The method of millipore filtration was compared with the method of cation-osmotic hemolysis. Both drugs prolonged the filtration time. Cation-osmotic hemolysis in glutaraldehyde-treated cells was significantly lower in comparison with the control group. On the other hand, there was a significant increase in cation-osmotic hemolysis in exaprolol-treated cells. Besides cation-osmotic hemolysis and filterability of erythrocytes, we evaluated the medium cell volume (MCV) and the medium cell hemoglobin concentration (MCHC). No changes in MCV and MCHC in glutaraldehyde-treated cells were observed. However, the MCV was significantly lower and the MCHC was significantly higher in exaprolol-treated cells. In conclusion, we suggest that the method of cation-osmotic hemolysis is more sensitive than the filtration method for determination of red blood cell microrheology.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 +/- 1.3 x 10(7) per unit). The system is recommended for specialized institutions with a high demand of blood components.     

Improved preservation of human red blood cells by lyophilization

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.     

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.     

不同全血过滤方法用于去白细胞血液制备的临床对照研究

目的:观察不同全血过滤方法用于去白细胞血液制备的效果。方法:采用两种全血过滤方法进行对比研究,对照组采用常规法,将采集后全血混匀后直接与白细胞滤器连接直接过滤;实验组采用湿润滤盘法,血液采集完成混匀后静置,先用上层血清10～20 m L湿润滤盘,再混匀与白细胞滤器连接后进行过滤。比较两组制备方法所用的过滤时间、血液回收率、过滤前后血液指标情况及24小时内溶血的发生情况。结果:两组全血过滤方法过滤前后白细胞、红细胞、血红蛋白、血小板及血浆游离血红蛋白水平比较差异均无明显统计学意义(P0.05)。而实验组过滤时间短于对照组,血液回收率高于对照组,且24小时内溶血比例明显低于对照组(P0.05)。结论:常规法与湿润滤盘法均能达到去白细胞血液标准,但湿润滤盘法较常规法能有效的降低过滤时间、增加血液回收率,减少去白细胞悬浮红细胞因溶血造成的血液不合格率,值得临床推广应用。     

Hemoglobin and Escherichia coli, a Lethal Intraperitoneal Combination

Intraperitoneal injection into mice of approximately 8 x 10(6) washed cells of Escherichia coli suspended in a lysate of washed human red blood cells or an aqueous solution of crystalline hemoglobin was lethal. E. coli suspended in washed intact erythrocytes, whole blood, plasma, or saline was innocuous. Fractionation of non-hemoglobin proteins from hemoglobin in lysates showed that only hemoglobin promoted a lethal infection. Overwhelming intraperitoneal growth of E. coli was attained in about 12 hr in lethal infections. The polymorphonuclear leukocytic response was ineffective against this rapid growth. The lethal mechanism is hypothesized to center on a unique role for free hemoglobin in inhibiting peritoneal absorption and stimulating an intraperitoneal exudate which supports luxuriant bacterial growth. Death is attributed to a lethal intoxication from bacterial endotoxins. This role for hemoglobin involves neither enhanced bacterial virulence nor lowered host resistance, and it would be of importance not only in peritonitis but also in problems where hemolysis and infection coexist.     

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway

Molecular and Cellular Biochemistry - Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells,...     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Mechanisms of Hemolysis Induced by Copper

An excess of copper is the cause of hemolysis in a number of clinical conditions. Incubation of human erythrocyte (RBC) suspensions with copper (II) causes the formation of methemoglobin, lipid peroxidation and hemolysis.

A new variant of the thiobarbituric acid (TBA) method, which minimizes the formation of interfering chromophores, was used to detect lipid peroxidation. Lipid peroxidation precedes hemolysis and the antioxidant vitamins C and E, which inhibit lipid peroxidation, also inhibit hemolysis. Consequently lipid peroxidation appears to be the cause of RBC destruction. Lipid peroxidation arises mostly from the oxidation of oxyhemoglobin by copper as it is inhibited in RBCs with carbon monoxyhemoglobin or methemoglobin. A direct interaction of copper with the red cell membrane seems to play only a minor role. Copper effects depend on the presence of free SH groups. Lipid peroxidation is probably initiated by activated forms of oxygen as it is increased by an inhibitor of catalase and reduced by hydroxyl radical scavengers. With higher copper concentrations hemolysis is greater: its mechanism appears different as lipid peroxidation is smaller but hemoglobin alterations, namely precipitation, are more pronounced.     

Mechanisms of Hemolysis Induced by Copper

An excess of copper is the cause of hemolysis in a number of clinical conditions. Incubation of human erythrocyte (RBC) suspensions with copper (II) causes the formation of methemoglobin, lipid peroxidation and hemolysis.

A new variant of the thiobarbituric acid (TBA) method, which minimizes the formation of interfering chromophores, was used to detect lipid peroxidation. Lipid peroxidation precedes hemolysis and the antioxidant vitamins C and E, which inhibit lipid peroxidation, also inhibit hemolysis. Consequently lipid peroxidation appears to be the cause of RBC destruction. Lipid peroxidation arises mostly from the oxidation of oxyhemoglobin by copper as it is inhibited in RBCs with carbon monoxyhemoglobin or methemoglobin. A direct interaction of copper with the red cell membrane seems to play only a minor role. Copper effects depend on the presence of free SH groups. Lipid peroxidation is probably initiated by activated forms of oxygen as it is increased by an inhibitor of catalase and reduced by hydroxyl radical scavengers. With higher copper concentrations hemolysis is greater: its mechanism appears different as lipid peroxidation is smaller but hemoglobin alterations, namely precipitation, are more pronounced.