Study of a kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) and compensated hemolysis.

A kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) was studied. The propositus presented with indirect hyperbilirubinemia, normal hemoglobin (15.8 g/dl), and elevated reticulocyte count (4.6%). The red cell 51Cr survival was decreased (tau1/2 16 days). Incubated osmotic fragility was normal; autohemolysis was increased and corrected with glucose and ATP. The P50 was 18.5 mm Hg (normal 25.5 +/- 3), but the stability, electrophoresis, and fingerprinting of hemoglobin were normal. The concentration of 2,3-diphosphoglycerate (2,3-DPG) was reduced to 43% of normal. Red cell 2,3-DPGM was decreased to 59% of normal; 2,3-DPG phosphatase was similarly decreased. All red cell glycolytic and hexose monophosphate shunt enzymes, glycolytic intermediates other than 2,3-DPG, and glucose consumption and lactate production were normal. Five family members showed similar hematologic findings. The deficiency appears to be secondary to decreased enzyme synthesis and to be inherited as an autosomal dominant trait in this family. Partial deficiency of 2,3-DPGM should now be considered in the differential diagnosis of compensated hemolysis associated with increased oxygen affinity.     

Changes in 2,3-DPG Content and Oxygen Affinity in Erythrocytes Stored at 4°C

The 2,3-DPG content of red blood cells increased within the first 24 h when fresh erythrocytes or whole blood were stored 4 degrees C. This phenomenon was strongly pH dependent. The temporary increase in 2,3-DPG was scarcely observed below pH 7.4 or above pH 7.8. In the case of whole blood, the increase was observed in CPD blood out not in ACD blood. Similar results were obtained with erythrocytes suspended in saline, when its pH was adjusted to approximately 7.6. Plasma proteins were not essential for the increase in 2,3-DPG content. Extracellular oxygen levels were continually measured in erythrocytes suspensions in order to check the changes in oxygen affinity of hemoglobin without damaging the cells. Both extracellular oxygen levels and 2,3-DPG contents were simultaneously increased by keeping fresh erythrocytes at 4 degrees C. Inhibition of glycolysis with sodium fluoride and monoidoacetic acid indicated that the in vivo steady state of glycosis in erythrocytes might be altered by chilling to make the rate of 2,3-DPG synthesis faster than that of 2,3-DPG decomposition.     

mPEG修饰对保存期内红细胞结构和功能的影响

目的：用mPEG-SPA（20kD、2mmol／L）修饰红细胞悬液,对修饰前后的红细胞结构功能和保存期进行分析、研究,以验证是否符合临床用血的要求。方法：分别将4种血型的红细胞悬液,用mPEG-SPA修饰,将修饰前和修饰后红细胞标本按成分血储存要求储存,并分别在保存期的1、7、14、21、28、35d检测红细胞的PH值、K^＋浓度、Na^＋浓度、渗透脆性、血浆游离血红蛋白、乙酰胆碱酯酶活性、ATP含量、2,3DPG含量、氧饱和度（P50）含量。结果：修饰对保存红细胞乙酰胆碱酯酶活性的含量无影响,修饰后红细胞悬液PH值、2,3-DPG含量在14d内正常、ATP含量在21d内正常,修饰后红细胞悬液的K^＋浓度保持低值达35d,修饰后红细胞悬液的Na^＋浓度、红细胞携氧能力、红细胞渗透脆性28d内正常,但血浆游离血红蛋白的含量在保存过程中增高。结论：mPEG修饰红细胞的大部分结构、功能指标在一定的保存期内可维持正常,但血浆游离血红蛋白含量超较高,这为进一步研究mPEG修饰红细胞用于临床打下了基础。     

Biochemistry of whole blood in poly(ethylene-co-ethylacrylate) experimental blood containers

The biochemical status of whole blood stored in containers fabricated of ethylene ethylacrylate (EEA) film was monitored at several times during 4 weeks of storage at 4 degrees C. Fifteen biochemical indicators were studied to reflect on erythrocyte integrity, cellular metabolism, plasma protein stability, and microaggregate formation. Comparison to storage in polyvinyl chloride (PVC) containers was made by distributing aliquots from each unit of blood among the containers being compared. Whole blood in EEA developed significantly higher levels of plasma hemoglobin, erythrocyte osmotic fragility, and D-glycerate-2,3-diphosphate (2,3-DPG), and somewhat greater glucose utilization, lactate production, and pH. These biochemical differences were not of great magnitude and the data suggest that EEA containers are compatible with the storage of whole blood.     

Red cell osmotic fragility studies in hemoglobin C-beta thalassemia: osmotically resistant microspherocytes

Typically certain features of red cell morphology predict the results of osmotic fragility testing. Microspherocytes generally have increased and target cells decreased fragility. Blood smears in homozygous hemoglobin C disease show an interesting admixture of microspherocytes and target cells. Yet osmotic fragility studies generally show only reduced fragility and no population of fragile cells to correspond with the spherocytes. The present study demonstrates that the red cells of patients with hemoglobin C-beta thalassemia share many characteristics with hemoglobin C red cells, including the decreased osmotic fragility of all cells despite the presence of both spherocytes and target cells. These paradoxically osmotically resistant spherocytes probably arise because of cellular dehydration due to a K-Cl transport system which may be activated by binding of hemoglobin C to the red cell membrane.     

In vitro Interactions of 51r in Human Red Blood Cells and Hemolysates1,2

Abstract. The intracellular distribution of radioactivity was studied in normal and sickle erythrocytes labeled with sodium ⁵¹ Cr chromate. Both types of cells had a higher fraction of ⁵¹ Cr bound to hemoglobin when labeled in the presence of ACD at a pH of 7.09 than when labeled in the presence of CPD at a pH of 5.96. Citrate at a pH of 5.96 or less entered the red blood cells and decreased the ⁵¹Cr binding hemoglobin. Binding of ⁵¹Cr to hemoglobin within the red cells was also reduced when the ATP and DPG levels in the red blood cells were elevated. Studies with hemoglobin solution showed that ⁵¹Cr binding to hemoglobin was influenced by 2,3-DPG, ATP and citrate.     

Red cell osmotic fragility studies in hemoglobin C‐β thalassemia: osmotically resistant microspherocytes

Typically certain features of red cell morphology predict the results of osmotic fragility testing. Microspherocytes generally have increased and target cells decreased fragility. Blood smears in homozygous hemoglobin C disease show an interesting admixture of microspherocytes and target cells. Yet osmotic fragility studies generally show only reduced fragility and no population of fragile cells to correspond with the spherocytes. The present study demonstrates that the red cells of patients with hemoglobin C‐β thalassemia share many characteristics with hemoglobin C red cells, including the decreased osmotic fragility of all cells despite the presence of both spherocytes and target cells. These paradoxically osmotically resistant spherocytes probably arise because of cellular dehydration due to a K‐Cl transport system which may be activated by binding of hemoglobin C to the red cell membrane.     

Preservation of Human Erythrocytes in the Liquid State: Biological Results with a New Medium

Abstract. Red blood cells (RBC) were collected with citrate-phosphate-dextrose (CPD) in a blood-pack optimal additive system. After concentration to 90% hematocrit they were diluted with saline-adenine-glucose medium (SAG-RBC), and stored for 35 days. In this work the RBC were stored in the presence of leukocytes. The SAG medium allows RBC conservation during 35 days at +4°C. The adenosine triphosphate (ATP) level of RBC is compatible with their survival. During the first 2 weeks, hemolysis of SAG-RBC was not greater than in CPD blood. Nevertheless, hemolysis reached 1.49% on day 35, and there was a marked increase in RBC osmotic fragility. Scanning electron-microscopic studies of 35-day RBC showed that the majority of them became echinocytes. After incubation in fresh frozen plasma, the RBC recovered satisfactory osmotic resistance and normal disc shape. The post-transfusion viability was normal with >70% recovery after 48 h. The in vivo restoration of 2,3-diphosphoglycerate (2,3-DPG) was rapid in the transfused SAG-RBC, 50% of the initial 2,3-DPG level being restored in 1 h. The in vivo studies proved that the functional quality of these RBC was compatible with their use in transfusion. The most important problem concerns the supernatant hemoglobin level of the SAG-RBC to be used for massive transfusion.     

2,3-Diphosphoglycerate Metabolism in Hereditary Spherocytosis

The red cell 2,3-DPG content of unsplenectomized patients with hereditary spherocytosis (HS) was found to be markedly lower than that of other haemolytic disorders with similar reticulocyte values. However, there were no differences in 2,3-DPG between splenectomized HS patients and healthy subjects.
The cause of this decreased 2,3-DPG in unsplenectomized patients was studied. When heparinized blood both of unsplenectomized and splectomized HS individuals was incubated with excess of glucose, there was a significantly higher splitting of 2,3-DPG than in the normal subjects.
Since the activity of the 2,3-DPG splitting enzyme, 2,3-DPGase, was shown to be increased by decreasing pH, the increased 2,3-DPG splitting during incubation of HS blood could be explained by the increased fall in pH during incubation of the blood, presumably due to increased lactate formation. This assumption was further supported by the finding that 2,3-DPG splitting both in normal and HS blood was completely inhibited when the pH of blood was raised to 7.8–7.9 and maintained in this range during the incubation.
In vitro incubation of blood simulates in some ways in vivo erythrostasis in the spleen and it is suggested that the decreased 2,3-DPG content found in the fresh red cells of unsplenectomized HS patients is due to increased 2,3-DPG splitting in these cells during their erythrostasis in the spleen.     

The Effect of Ascorbate on the Maintenance of 2,3-Diphosphoglycerate (2,3-DPG) in Stored Red Cells

S ummary . The addition of adenine and ascorbate to citrate-phosphate-dextrose solution has been shown to prolong the maintenance of 2,3-DPG levels of stored blood. The final concentrations of adenine and sodium ascorbate in the blood-preservative mixture were 0.5 and 5.05 m m , respectively. In most cases, red cells stored in this preservative maintained levels of 2,3-DPG greater than 50% of fresh values for 28 days. Red-cell viability was not adversely affected by ascorbate. The mechanism by which ascorbate produced this effect is unknown.     

Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.     

Effect of oxalate and malonate on red cell metabolism

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.     

Investigation of the haemolytic effects of Ancylostoma ceylanicum: observations on infected dogs in vivo and human and dog blood in vitro

Investigations into the probable role of haemolysins in the causation of hookworm anaemia have been undertaken in living infected dogs. Secondly, the effects of living hookworms and various worm products on erythrocytes in vitro have been assessed. In dogs infected with varying numbers of A. ceylanicum, severe microcytic anaemia developed in the most heavily infected animals six weeks after infection. Erythrocytes from the latter animals showed significantly greater autohaemolysis in the presence of added glucose. When serum bilirubin and methaemalbumin, plasma haemoglobin, urinary urobilinogin and osmotic fragility of their red cells were measured, however, no evidence of haemolysis was detected. Erythrocytes from these animals appeared normal under scanning electron microscopy. In in vitro studies varying concentrations of adult worm extract had no effect on the haemolysis of either dog or human erythrocytes in the presence or absence of glucose nor on their mechanical fragility. There was no increase in 51Cr release from dog or human labelled red cells when incubated with either adult worm extract or excretory/secretory products of worms. Living adult worms caused an increase in 51Cr release from human but not dog labelled erythrocytes. Thus, the role of haemolysins in the genesis of hookworm anaemia is minimal.     

Stabilization of Red Blood Cells by the Plasticizer,Diethylhexylphthalate1

Abstract. The red blood cells of blood stored in containers made of polyvinylchloride (PVC) film are osmotically more stable and lose on average about 1/3 less hemoglobin than when blood is stored in another plastic [poly-(ethylene-co-ethyl acrylate); EEA]. The stability of uniform volumes of stored red blood cells varies directly with PVC surface area, whereas changes in EEA surface area have comparatively little or no effect. PVC contains high concentrations of the plasticizer, diethylhexylphthalate (DEHP), known to migrate into blood and to have a high potential for toxicity. To determine if DEHP could be the red cell stabilizing agent in PVC, whole blood was stored in containers made from EEA into which was incorporated varying amounts of DEHP. Incorporation of DEHP into EEA significantly reduced erythrocyte osmotic fragility (p = 0.01). The degree of reduced fragility correlated with the level of DEHP in the cell phase implicating DEHP in PVC containers as the stabilizing agent for red cells.     

Extended storage of red blood cells under anaerobic conditions

BACKGROUND: Red blood cells (RBC) are subject to oxidative stress by reactive oxygen species during refrigerated storage. Near-complete removal of oxygen from red cells during storage should eliminate this contributor to the red cell 'storage lesion'. The in vitro effects of storing red cells under oxygen-depleted conditions for extended periods were investigated, and these were correlated with the observed recoveries after reinfusion. STUDY DESIGN AND METHODS: Units of red cells, obtained after 'soft spin', were placed in a double volume of AS-3 additive solution and subdivided. Oxygen in the test units was depleted by repeated exposure to Ar gas (to O(2) saturation < 4%), and units were stored in anaerobic canisters for up to 15 weeks. Samples were taken weekly to monitor adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), cell-free haemoglobin, and vesicle production. In a parallel experiment, six units of red cells was depleted of oxygen in a similar manner, stored for 8, 9 and 10 weeks, and reinfused autologously to determine the 24 h post-transfusion recovery via (51)Cr/(99m)Tc radiolabelling. A similar study was also carried out using EAS61 additive solution, which by itself, had shown the ability to support 9-week storage, comparing biochemical profiles and in vivo recovery after aerobic vs. anaerobic storage. RESULTS: Oxygen-depleted AS-3 units had significantly elevated ATP levels compared to controls. They also had significantly lower cell free haemoglobin and vesicle production when RBCs were stored for more than 9 weeks. An average of over 75% post-transfusion survival was observed after 9 weeks of anaerobic storage with less than 0.43% haemolysis. However, no further extension of storage was achieved with EAS61 additive. CONCLUSION: Anaerobic conditions permit acceptable 9-week storage of RBCs using double-volume AS-3 additive solution. It did not synergize with the alkaline, 9-week additive, EAS61, to further lengthen the acceptable storage time. These studies indicate that anaerobic storage may allow reduction in the effect of the storage lesion, but suggest that other factors contribute to limitations of RBC storage as well.     

Influence of hypercholesterolemia on morphological and rheological characteristics of erythrocytes

The changes in shape and rheological parameters of erythrocytes in cholesterol-fed rabbits (fed with normal diet plus 0.5% cholesterol for a duration of 1 year) and in vitro cholesterol enrichment of erythrocytes in cholesterol enriched plasma were studied. This process affects the shape of erythrocytes. In rabbits the actual shape change begins after 6 weeks of cholesterol feeding. The shape changes in rabbits and in vitro cholesterol-enrichment are similar. The extent of shape changes depends on the amount of cholesterol incorporated into the membrane. The rheological parameters such as blood viscosity, osmotic fragility and deformability are measured. The whole blood viscosity is increased and deformability decreased. The cells become osmotically more fragile in cholesterol-fed rabbits but are resistant to osmotic lysis in in vitro cholesterol enrichment. The altered lipid composition may be responsible for the observed changes in these parameters.     

Post-thaw storage at 4 degrees C of previously frozen red cells with retention of 2,3-DPG

Fresh human blood was collected in CPD, frozen by either the Meryman or the Valeri high glycerol technique, and stored at -80 degrees C. Later the red cells were thawed, deglycerolized by the appropriate technique and resuspended in either saline-glucose wash solution or an additive solution containing ascorbate-2-phosphate, adenine, glucose (dextrose), mannitol and sodium phosphate. The cells were stored at 4-6 degrees C for 21 days and assayed weekly for ATP, 2,3-DPG, pH, P50, glucose utilization and lysis. The additive solution maintained red cell 2,3-DPG at fresh blood levels for 3 weeks and maintained ATP levels sufficiently well to suggest good red cell viability for 21 days. There was no difference in results between the Meryman or the Valeri freezing methods if sodium phosphate was used with the saline-glucose wash solution in the Valeri method. If this additive solution is coupled with sterile deglycerolization techniques, 3 weeks of post-thaw red cell preservation would be practical. Using this additive solution would make frozen blood a reasonable source of red cells for emergency needs in both military and civilian blood banking.     

Evaluation of Adenosine Triphosphate and 2,3-Diphosphoglycerate after Twenty-Four Hours in Red Cells Washed for Neonatal Transfusion

Adenosine triphosphate and 2,3-diphosphoglycerate concentrations in 14 U of CPDA-1 stored red cells (PRBC) and washed red cells (WRBC) were measured to assess indirectly the quality of WRBC for neonatal transfusion after 24 h. The results indicate that there is no difference in red cell ATP and 2,3-DPG concentrations between PRBC and WRBC after a 24-hour period.     

Linolenoyl Sorbitol and the Fragility of Hereditary Spherocytes

S ummary . Linolenoyl sorbitol decreased uniformly the osmotic fragility of normal human red cells. When added to red cells of patients with hereditary spherocytosis (HS), the synthetic lipid afforded the most fragile cells the greatest shift in osmotic stability. The greater the osmotic fragility of red cells from different patients, the greater the protective effect afforded by the added lipid. Linolenoyl sorbitol fully reversed the increased fragility of vinblastin-treated red cells. The results indicate a causal relationship between the lipid composition and the increased fragility of red cells in hereditary spherocytosis.     

Improved post-transfusion quality of density separated AS-3 red cells after extended storage

A centrifugal method of red cell density separation was utilized for unit processing in these studies to determine the quality of the lighter fraction (neocytes) after storage for up to 42 d and to evaluate whether the heavier fraction (gerocytes) deteriorated more rapidly than neocytes during storage. Each unit was passed through a Leukotrap filter to remove white cells prior to density separation. Red cell recovery and survival were evaluated using double label technetium-99m with either chromium-51 or nonradioisotopic chromium which permitted concurrent paired analysis. In vivo neocyte red cell recovery, as tested on the same 11 donors on days 1, 7 and 42 of storage, was effectively unchanged. Recovery and survival half-life (that is, the number of days after transfusion at which half of the cells infused remain in the circulation) of 42 d stored gerocytes were significantly lower than similarly stored neocytes (75.5 +/- 7.2% and 20.1 +/- 6.5 d for gerocytes versus 84.4 +/- 4.9% and 39.0 +/- 9.0 d for neocytes). One-day stored neocytes showed a 16.5% increase in red cell availability over the combined average for 42 d stored neocytes and gerocytes. Statistically, while there were significantly higher ATP, 2,3-DPG, and lactate levels pre-storage by paired t-test for neocytes compared to gerocytes, by day 42 there were no significant differences detected between the two red cell fractions by any of the in vitro variables measured. These studies suggest that this simple separation technique for leucocyte-poor red cell units provides a neocyte population with improved viability and the potential for increased transfusion intervals in chronically transfused patients.