Improved red blood cell storage using optional additive systems (OAS) containing adenine, glucose and ascorbate-2-phosphate

Red blood cells were treated with optional additive system (OAS) solutions to provide component-specific metabolic enhancement for improved storage. Red blood cell viability, as monitored by ATP concentrations, was maintained by use of adenine and extra glucose. Red blood cell oxygen offloading characteristics were improved by maintenance of red blood cell 2,3-DPG concentrations with ascorbate-2- phosphate (AsP). The use of CPD-collected red blood cells with an OAS containing adenine, glucose, and AsP, or CPD-adenine collected red blood cells with an OAS containing AsP demonstrates the potential to store red blood cells at least 42 days and to maintain red blood cell 2,3-DPG.     

Hemoglobin function in stored blood. IX. Preservative with pH to maintain red blood cell 2,3-DPG (function) and ATP (viability)

An optimal pH was sought to maintain hemoglobin function, ATP, and red blood cell viability during liquid storage under blood banking conditions. Ten units of blood from normal volunteers were subjected to an automated analytical system for determining concentrations of 2,3-DPG and ATP. Each unit was split during donation into five parts containing citrate—dextrose solutions of pH 5.0, 5.5, 6.0, 6.5, and 7.0. Significant differences at the 95 per cent level were based on the paired t-test. In addition, osmotic fragility and methylene blue uptake were determined to assess their possible usefulness as indicators of either red blood cell viability or ATP. With pH 5.0 preservative 2,3-DPG fell from day 0 to day 3, with pH 5.0 and 5.5 preservatives from day 3 to day 7, and from day 7 to day 14 in all pH groups. A plot of 2,3-DPG versus hydrogen ion concentration showed that in excess of 1 × 10^?7hydrogen ion, corresponding to pH 7.0, 2,3-DPG concentration falls at a rapid rate. From 2,3-DPG and ATP data, a preservative with pH higher than 5.5 would seem to be optimal for maintaining hemoglobin function and red blood cell viability, but adenine may be needed to maintain adequate ATP levels.     

Depletion and Regeneration of 2,3-diphosphoglyceric Acid in Stored Red Blood Cells

2,3-diphosphoglyceric acid appears to be an important regulator of the oxygen dissociation curve of hemoglobin in intact red blood cells. The rate of loss of 2,3-DPG under various storage conditions therefore was investigated. 2,3-DPG disappeared rapidly from conventional preservative media, CPD, and ACD solutions. After only two weeks' storage, 65 per cent to 85 per cent of erythrocyte 2,3-DPG had been lost from ACD-stored blood and slightly less from CPD-stored blood. Although the addition of adenine to ACD solution aided in the maintenance of ATP levels, it hastened the rate of loss of 2,3-DPG. The rate of 2,3-DPG depletion was strongly dependent on pH. In more alkaline storage media, levels of this compound were relatively well maintained for as long as two, or even three weeks. However, under these circumstances ATP maintenance was less satisfactory. The levels of 2,3-DPG and ATP in red blood cells incubated in fresh plasma at 37 C, pH 7.4, to simulate the conditions after reinfusion of stored cells also was investigated. ATP levels remained relatively stable under these circumstances and 2,3-DPG levels were restored gradually. However, the repletion of 2,3-DPG was sufficiently slow so that even after eight hours only approximately one third of the 2,3-DPG which had been lost was regenerated. Thus, stored blood may fail to transport oxygen efficiently for many hours after reinfusion.     

Reversal of the storage lesion of CPD bank blood: a problem in clinical medicine

The effect of phosphate buffer on the course of pH, ATP, and 2,3-PDG of CPD red blood cells stored at three temperatures was observed. Basic phosphate at an equilibrated level of 10 mM (as iP) maintained pH above 7.00 and ATP and 2,3-DPG above 70 per cent of initial value in cells stored at 37 C for 24 hours. In contrast however, at 25 and 4 C no buffering was obtained with basic phosphate concentrations up to 50 mM, but values for both ATP and 2,3-DPG were higher in phosphate treated aliquots than in controls throughout storage. When the pH of blood stored at 4 C was adjusted into the range 7.15 to 7.25 with tromethamine and the level of iP raised to 10 mM by addition of Na2HPO4 on day seven, it was found that ATP and 2,3-DPG levels were maintained at 90 and 120 per cent, while control levels fell to 60 and 12 per cent, respectively at 21 days. The process described parallels the normal repair of damaged red blood cells of bank blood that occurs in vivo following transfusion.     

The effect of pre-storage cooling on 2,3-DPG levels in red cells stored in SAG-M.

BACKGROUND: The concentration of red cell 2,3-DPG (2,3-diphosphoglycerate) rapidly decreases during storage. A favourable effect on red cell 2,3-DPG has been demonstrated by rapid cooling of whole blood prior to storage. In our study we have investigated how different methods of cooling whole blood immediately after donation effect 2,3-DPG levels during storage. STUDY DESIGN AND METHODS: Thirty-six whole blood units (in 6 groups) of 450 ml were collected in 63 ml CPD. SAG-M was used as preservative solution for red cell concentrates (RCC). The units in one group were cooled down at ambient temperature, while units in the other groups were cooled down rapidly by different ways immediately after bleeding. Samples from the whole blood units were collected at various days during storage for 2,3-DPG measurements. RESULTS: The decline in 2,3-DPG during the first two weeks of storage was significantly slower in the groups which were cooled down rapidly to 17-18 degrees C within 1h after bleeding (all p相似文献   

Blood preservation XXVII. Fructose and mannose maintain ATP and 2,3-DPG

Mannose and fructose as well as glucose have been shown to be effective for maintaining ATP and thus viability of stored red blood cells. Normal 2,3-DPG levels are desirable in stored red blood cells to provide the needed oxygen transport upon transfusion. ATP levels in sotred concentrated red blood cells in the new preservative, CPD- adenine (citrate-phosphate-dextrose-adenine) become critically low in the 5th week. In this study two hexoses and two pentoses are compared with dextrose in their ability to maintain ATP and 2,3-DPG. ATP levels were best maintained by fructose, then dextrose and mannose. ATP levels had fallen to critically low levels by four weeks with ribose and xylose. Red blood cell 2,3-DPG concentrations were also maintained by hexoses, with mannose being best, dextrose and fructose being similar. When ribose was used in addition to dextrose in CPD-adenine, ATP maintenance was improved and under the same conditions xylose improved 2,3-DPG maintenance. Fructose and mannose may be as useful as dextrose in citrate-phosphate preservatives for maintaining ATP and 2,3-DPG levels. Also, ribose and xylose may help the maintenance of ATP and 2,3- DPG, respectively, in CPD-adenine.     

Blood preservation XLIV. 2,3-DPG maintenance by dehydroascorbate better than D-ascorbic acid

A study was designed to compare the effects of D-ascorbate and dehydroascorbate on red blood cell metabolism during blood storage. Dehydroascorbate increased red blood cell concentrations of 2,3-DPG such that the levels are above normal for four weeks and normal at six weeks of storage. In contrast, there is a gradual decrease in 2,3-DPG levels with D-ascorbate such that the levels are approximately 80 per cent of normal after six weeks. ATP levels were adversely effected such that the worst levels were produced by 10 and 5 mM dehydroascorbate, with 10 mM having a more adversive effect than 5 mM. Intermediate levels of ATP were produced by D-ascorbate, with the 10 mM concentration. The control CPD-adenine preservative maintained near normal ATP levels for the entire six-week storage period. pH values were initially slightly lower with dehydroascorbate compared to the other preservatives early in storage, the difference being slightly over 0.1 pH units.     

Blood storage XXII. Improvement in red blood cell 2,3-DPG levels at six weeks by 20 mM PO4 in CPD-adenine-inosine

Inorganic phosphate has been known to assist red blood cell maintenance of ATP and in the presence of inosine to assist in the maintenance of 2,3-DPG. High concentrations of phosphate, while helping ATP maintenance, were found to be deleterious to 2,3-DPG maintenance in CPD- adenine preservatives. However, in the presence of inosine, concentrations of phosphate as high as 10 mM were advantageous to 2,3- DPG maintenance. The present study extends the observations on ATP and 2,3-DPG maintenance in CPD-adenine-inosine preservatives from the previous 10 mM to 20 mM phosphate. A high phosphate (20 mM) effect has been seen as improved maintenance of 2,3-DPG levels during the fifth and sixth weeks of storage of whole blood at 4C. This supports the previously reported observation of improved maintenance of 2,3-DPG in a 10 mM phosphate preservative. This is ten times the 2 mM phosphate concentration in CPD-adenine. In the low phosphate preservative (2 mM), 2,3-DPG maintenance is less than that in all of the higher phosphate preservatives after the second week of storage. ATP concentrations in this experiment show good maintenance throughout six weeks of storage.     

Storage of saline-adenine-glucose-mannitol suspended red cells in diethylhexyl phtalate and butyryl-n-trihexyl-citrate plasticized polyvinyl chloride containers. An in vitro comparative study

Units of whole blood collected into butyryl-n-trihexyl-citrate (BTHC) and diethylhexyl phtalate (DEHP) plasticized polyvinylchloride (PVC) blood storage containers and processed by means of an 'Optipress', which allows automated removal of the buffy coat, were compared. Units collected into standard PVC containers processed by the traditional method (no buffy coat removal) were used as a control group. The red cell concentrates were suspended in saline-adenine-glucose-mannitol (SAGM) and stored 42 days at 2-6 degrees C. Comparison of the buffy coat depleted red cell concentrates showed that red cell energy and oxygen delivery capacity, as evidenced by ATP and 2,3-DPG values, were slightly better preserved in the BTHC plasticized container, compared to the DEHP container. The red cell membrane, however, was slightly less well preserved, (the hemolysis at day 42 with BTHC ww 0.39%; with DEHP, 0.20%) in this container. The higher ATP levels might lead to a better in vivo recovery of stored red cells. In vivo studies comparing both plastic containers, therefore are indicated in order to determine if these differences have practical significance. A longer holding time of the whole blood at room temperature before processing reduced the hemolysis (42 days stored RCC as 0.26%). Slightly more fibrinopeptide A (FPA) generation and marginally lower pH and 2,3-DPG values were observed in this situation. This finding suggests an effect of higher plasticizer levels on the red cell membrane.     

Blood preservation. XXIX. Pyruvate maintains normal red cell 2,3-DPG for six weeks of storage in CPD-adenine

Pyruvate was placed in experimental CPD-adenine (0.25 mM) blood preservative mixtures in four concentrations ranging from 40 to 320 mM. In the 320 mM pyruvate preservative, 2,3-DPG levels were elevated above normal for six weeks of whole blood storage at 4 C. The lower pyruvate concentrations maintained elevated or normal 2,3-DPG levels for less time: four weeks with 160 mM, two weeks with 80 mM, and one week or less with 40 mM or the control. ATP values were best maintained in the control. The higher pyruvate concentrations resulted in the most rapid decreases at ATP. However, even the 320 mM pyruvate did not cause ATP to fall below 2 microM/gm of Hb. The higher pyruvate concentrations produced and maintained a higher pH during storage. On the other hand, 2,3-DPG levels increased with pyruvate during the first week of storage when the pH was decreasing rapidly. This could be the result of its oxidation of NADH to NAD. The high pyruvate concentration which maintained elevated 2,3-DPG levels throughout the six weeks might be simulating the effect reported in pyruvate kinase-deficient red blood cells, in which blockage of glycolysis at that step is preventing 2,3- DPG catabolism.     

Effect of hydrocortisone on the synthesis of 2,3-diphosphoglycerate in human erythrocytes

Studies were carried out on the mechanism of action of glucocorticoids on erythrocyte glycolysis. Inosine increased the levels of 2,3-DPG, lactate, and ATP in red blood cell samples with very low glucose concentrations in which the glycolytic system was unable to utilize glucose. With inosine and hydrocortisone added to red blood cells, 2,3- DPG and lactate increased more than in red blood cells supplemented with inosine alone. There was no significant increase in the level of ATP when glucose and inosine were added compared to the addition of inosine only. There were no changes in the activity of enzymes such as PFK and PGK. From these findings, it is suggested that glucocorticoids increase the level of red blood cell 2,3-DPG, by activating the conversion of 1,3-DPG to 2,3-DPG during glycolysis, probably by stimulating 2,3-DPG mutase.     

Blood preservation. XXVIII. Galactose and maltose maintain red blood cell 2,3-DPG and ATP

Because there may be inadequate dextrose in the newly licensed CPD- adenine for five or six weeks storage of high hematocrit red blood cells, this laboratory has examined some alternate sugars for their ability to maintain red blood cell metabolism during storage. In the current study, dextrose and fructose were studied as model or prototype nutrients. A third six carbon monosacharide, galactose, three dissacharides, lactose, maltose, and sucrose were studied in the same experiment. Of these, fructose best maintained ATP and 2,3-DPG during the fourth to sixth week of whole blood storage at 4 C. Dextrose was next best during this time and was nearly equivalent to fructose in the first three weeks of storage. Galactose and maltose both maintained ATP and 2,3-DPG, but not nearly so well as did fructose and dextrose. Sucrose and lactose were associated with the most rapid deterioration of ATP and DPG levels and they failed to maintain the progressive fall in pH which is usually associated with continuing, useful metabolism.     

Improved red cell quality after erythroplasmapheresis with MCS-3P

The cell separator MCS-3P is an apheresis system offering the flexibility to collect standardized red blood cells, plasma, and/or platelets from one donor. Two different programs were used for the red cell apheresis'RBCP (collection of one unit of red cells and two units of plasma) and RBCPS (one unit of red cells and one unit of plasma). The quality of the red cell concentrates (RCC resuspended in SAG-Mannitol) during the storage time of 42 days was measured by biochemical (ATP, 2,3-DPG, pH, free Hb, free potassium, glucose, lactose, p⁵⁰, hemoglobin derivatives) and Theological (morphological index, filtration/rigidity index) parameters. The donation time with 53 donors was 20 min for 355 ml RCC-SAGM and 440 ml plasma and 7 min for 335 ml RCC-SAGM and 239 ml plasma. The donor tolerance was analogous to plateletpheresis or plasmapheresis. Twenty units of the RCC-SAGM were in-line filtered within 6 or 24 hours after donation. The results obtained for red blood cell storage are at least as good as with standard collection (free hemoglobin, free potassium, glucose, lactose, hemolysis) or better (ATP, 2,3-DPG, p⁵⁰, hemoglobin derivatives, filtration/rigidity index) owing to prevention of collection lesion. All blood preparations were sterile after storage (red cells 42 days, plasma after freezing). The erythroplasmapheresis with MCS-3P can be especially recommended for application in an autologous blood program because the application of autologous blood donation in hospitals is often limited by the preconditions of component separation. The erythroplasmapheresis data with MCS-3P are encouraging for the development of a new blood collection methodology.     

Hemoglobin function in stored blood

Serial oxygen dissociation curves were performed on blood units preserved in acid-citrate-dextrose (ACD), ACD-adenine, and ACD-adenine-inosine. Dividing blood from a single donor into two or more bags allowed direct comparison between preservatives. During the 1st wk of storage in ACD, a progressive increase in oxygen affinity was observed. Thereafter, little further change was noted. Oxygen affinity increased even more rapidly during initial storage in ACD-adenine. However, with the inclusion of inosine as a preservative, oxygen affinity remained unaltered during the first 2 wk. Increases in oxygen affinity correlated well with falling levels of red cell 2,3-diphosphoglycerate (2,3-DPG) during storage. No significant changes in glutathione, reduced form (GSH), or A3 (A(I)) hemoglobin levels were noted during the first 3 wk of storage. No significant accumulation of ferrihemoglobin was detected. When blood stored 20 days in ACD or ACD-adenine was incubated with inosine for 60 min at 37 degrees C, 2,3-DPG and adenosine triphosphate (ATP) were resynthesized, and oxygen affinity was decreased. The distribution of 2,3-DPG in fresh and stored red cells appeared to influence experimental values for Hill's n, a measure of heme-heme interaction.     

The effect of delayed refrigeration on red blood cells, platelet concentrates and cryoprecipitable AHF

The effect of delaying blood processing for six hours while maintaining it at ambient temperature was investigated. Blood drawn from volunteers on two occasions was processed immediately (I) or after a six-hour delay (D). The effects of the delay on the efficacy and safety of red blood cells, platelet concentrates and cryoprecipitable AHF were studied. There was a more rapid decrease in 2,3-DPG in the delayed group during 21 days of refrigerated storage. ATP levels declined at similar rates. 24-hour survival of 51CR-labeled autologous cells was slightly better (p = .05) in the (I) group but excellent for both. Total platelets, per cent recovery and pH at 72 hours were identical for both groups. All cultures were sterile. There was no difference in total AHF recovered or per cent recovery between the two groups. To increase the availability of blood components, a six-hour processing delay seems warranted.     

Blood preservation. XLIII. Studies on the ascorbate mechanisms of maintaining red cell 2,3-DPG

Our previous experiments on the mechanisms of ascorbate's effect on the red blood cell failed to show an effect of iodoacetate (IA), a sulfydryl inhibitor. In this study, in contrast to the previous, iodoacetate (85 micromolar) was seen to prevent continued red blood cell metabolism. During the first weeks there was an absence of a continual fall in pH; ATP levels were depressed below half normal; and 2,3-DPG levels fell to very low values within the first week. ATP was best maintained in the control preservative and next best maintained, at adequate levels, with ascorbate, 5 mM, with and without glutathione, 5 mM. 2,3-DPG levels were well maintained with ascorbate and ascorbate with glutathione. Poor ATP maintenance and rapid decreases in 2,3-DPG were observed with iodoacetate, IA plus ascorbate, and IA plus ascorbate and glutathione.     

Changes of blood oxygen affinity in different CPD solutions during liquid storage

Soft and hard-packed red blood cells in four different CPD anticoagulant-preservative solutions were stored with and without added glucose, adenine, and electrolytes. The hemoglobin-oxygen affinity of the red blood cell concentrates was tested over a six-week storage period. No single solution conferred better protection than any other against an expected increase in oxygen affinity due to loss of 2,3-DPG during storage. In all solutions, P50 at pH 7.4 decreased linearly when measured in a physiological system using CO2. After six weeks' storage at 4 C, the normal oxygen-binding properties of red blood cells could be restored in all instances following incubation for one hour in a rejuvenation solution. By contrast, red blood cell ATP levels were highest when resuspending solutions contained adenine and added glucose, but did not significantly compensate the allosteric role of 2,3-DPG in regulating oxygen affinity when the latter became depleted.     

The Effect of Ascorbate and Dihydroxyacetone on the 2,3-Diphosphoglycerate and ATP Levels of Stored Human Red Cells

Fifty ml aliquots of blood were stored in modified CPD-adenine preservative solutions at pH 4.8, 5.6, and 7.0 containing either dihydroxyacetone alone, ascorbic acid alone, or a combination of both. Red blood cell ATP and 2,3-DPG determinations showed that the effect of dihydroxyacetone and ascorbic acid were synergistic at all pH levels, and that even at the lowest pH levels excellent 23-DPG maintenance was observed. A reciprocal relationship existed between 2,3-DPG and ATP maintenance. Studies in 500 ml units of blood containing both dihydroxyacetone and ascorbate gave similar results to those in 50 ml aliquots. There was excellent maintenance of 2,3-DPG levels throughout the 28-day storage period.     

The effect of refreezing previously frozen-deglycerolized red blood cells

Frozen red blood cells, once thawed and washed, must be used within 24 hours or discarded. In this study, three units of blood underwent two freeze-thaw cycles. It was shown that red blood cell survival, ATP, and 2,3-DPG were not adversely effected. The data suggest that previously frozen red blood cells should be refrozen, rather than discarded, if not used within 24 hours of deglycerolization.     

Blood preservation 33. Phosphate enhancement of ribose maintenance of 2,3-DPG and ATP

Blood storage in CPD-adenine supplemented with 25 mM inosine and 10 mM phosphate gave 2,3-DPG levels as high as 140 per cent of normal for six weeks of blood storage at 4 C. Lower but normal 2,3-DPG levels were maintained throughout six weeks with inosine or inosine plus ribose. Ribose alone provided marginally increased DPG maintenance over the control, but ribose with phosphate maintained 2,3-DPG levels above 70 per cent of normal for five weeks of storage and two weeks longer than the control preservative. ATP levels were maintained at normal or above for six weeks with phosphate plus ribose or inosine. 2,3-DPG maintenance has previously been shown to be impaired by phosphate, unless inosine is also present. The ribose and inosine effects on 2,3-DPG maintenance are not additive. Phosphate also has an enhancement effect on ATP maintenance in the presence of either ribose or inosine.