Effect of Storage of Blood in ACD-Adenine-Inorganic Phosphate Plus Nucleosides on Metabolic Intermediates of Human Red Cells

Bloods collected in ACD-adenine-inorganic phosphate (ACD-Ad,P,) preservatives, supplemented with varying amounts of inosine, adenosine and guanosine, were incubated at 37 C for four hours and then stored at 4 C for six weeks. At two-week intervals, phosphorylated intermediates of washed red cells were assayed by ion exchange chromatography. The 0-day 2,3-diphosphoglycerate concentrations of the incubated ACD-Ad, P₁ bloods were below the normal range and decreased rapidly during storage; in the presence of inosine or adenosine, the initial values were elevated and remained high during the first two weeks before decreasing. Following incubation the 0-day values of ATP were markedly increased in the red cells of bloods supplemented with adenine and P₁ and during storage the rate of decrease was dependent on the pH of the blood and the type and amount of supplement. The red cells of blood collected in the ACD-Ad, P₁ (pH 6.5) preservative maintained the ATP concentrations within the normal range after six weeks storage as effectively as bloods collected in preservatives supplemented with inosine, adenosine or guanosine. After a small initial increase, the ADP levels remained fairly constant. The AMP values increased as the ATP decreased and in most cases, the total adenylate concentration remained fairly constant throughout the storage period. The changes in other phosphorylated compounds were negligible. The significance of these results is discussed.     

The post-thaw viability of red blood cells of ACD and CPD blood preserved in the frozen state with and without added adenine

Bloods collected in ACD and CPD were frozen by the Blood Research Institute Glycerol method using the Latham Processor. Washed, thawed red blood cells were suspended in autologous plasma with and without added adenine and stored at 4 C. Post-transfusion survival studies by ¹²⁵I, ⁵¹Cr methods had values of 70 per cent or better for seven and 14 days for ACD and CPD bloods, and 14 and 21 days for ACD-adenine and CPD-adenine bloods, respectively. Integration of a frozen blood program in conventional blood banks is discussed.     

Hemoglobin Function of Blood Stored at 4 C in ACD and CPD with Adenine and Inosine

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound that is poorly maintained during blood bank storage in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained at the higher pH afforded by citrate‐phosphate‐dextrose (CPD), hemoglobin function was compared during storage in CPD and ACD. Further, hemoglobin function was studied in CPD blood containing adenine and inosine, compounds that provide metabolic energy and thus prolong the shelf‐life of blood, because they also effect the levels of 2,3‐DPG during storage. Hemoglobin function, expressed as the P₅₀ (the P₀₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity) is considerably better maintained during storage in CPD than in ACD. The hemoglobin function or P₅₀ of blood stored in CPD‐adenine is not maintained as well as blood stored in CPD without adenine, but the oxyhemoglobin dissociation curves show only a small difference when compared to the difference between ACD and CPD. Blood stored in CPD‐adenine with inosine, present initially or added at day 25, allows higher P₅₀ values late in storage, thus providing better hemoglobin function for more of the storage period.     

Comparison Studies of Whole Blood Stored in ACD and CPD and with Adenine

Using 70% 24-hour posttransfusion survival as one of the criteria of preservation, ACD and CPD anticoagulant solutions with and without adenine were tested after storage for 28, 35, or 42 days. At 28 days, all solutions had average survivals of 70% or better. The average survival values for the ACD and CPD solution groups were less than 70% at 35 and 42 days. However, both solutions with adenine had a group average value over 70% even after 35 or 42 days of storage. The average survival values between the two anticoagulants alone, were not significantly different at each time period. The two anticoagulants when adenine was included had increased survival and there was no statistical difference between the levels. In comparing two units obtained from the same subject, survival percentages were significantly higher in almost every recipient when the adenine-supplemented stored blood was used. Other chemical determinations did not show significant alterations and no toxic effects were observed in the recipients.
Since all units containing adenine had survival values greater than 70% in the 28- and 35-day periods, these units would appear to have been effectively preserved and blood stored under these conditions could be used in routine transfusions, reserving units stored 42 days for emergency use.     

Phosphorylated Carbohydrate Intermediates of the Human Erythrocyte during Storage in Acid Citrate Dextrose:

Blood was stored at 4 C. in ACD without additive or supplemented with adenine, inosine, inosine-adenine, or adenosine. After six weeks of storage the carbohydrate intermediates of the erythrocytes were studied by column chromatography. Adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) had disappeared from blood stored without additive or with adenine. Both ATP and DPG remained in erythrocytes stored with the other additives. Concentration of ATP was highest in blood stored with inosine-adenine, and the level of DPG was highest with the addition of adenosine to the storage solution.     

Oxygen Equilibria and Biochemical Changes of Whole Blood Stored in Different Preservation Media

Human blood was stored in its oxygenated and in its deoxygenated forms for a period of 14 to 56 days at 4 C in six different preservation media, namely, ACD and CPD, ACD and CPD supplemented with adenine, ACD and CPD supplemented with adenine and with tris (hydroxymethyl) amino-methane at pH 7.2. At regular intervals determinations were made of the pH and the oxygen affinities of the total blood, of the intracellular concentrations of ATP, 2,3-DPG and inorganic phosphate, of plasma electrolytes and lactic add concentrations, and of the minor hemoglobin components. The investigations have shown: 1) Storage of blood in the deoxygenated form results in a rapid decrease of the blood pH, an increased lactic acid production, and a less rapid change in the oxygen affinities and 2,3-DPG levels; 2) Storage of blood at a more neutral pH prevents the changes in 2,3-DPG level and oxygen affinity; 3) Supplementation of the media with adenine may increase to some extent the ATP level of the red blood cells; 4) No significant changes are observed in the percentages of the minor hemoglobins. A striking correlation was observed between the oxygen affinity of the blood and the intracellular 2,3-DPG level; no such correlation was present between the ATP level of the red blood cells and the oxygen affinity of the blood. The 2,3-DPG level (and the oxygen affinity) was also dependent on the pH of the blood-preservation media. A rapid disappearance of 2,3-DPG was noted below rather fixed pH values; these pH values were consistently lower when blood was stored in its deoxygenated form than in its oxygenated form.     

Plasma adenine and cellular ATP in red cell concentrates collected and stored in modified CPD at 4 C

Eight units of blood were drawn into modified CPD containing 25 per cent higher glucose and 17.3 mg adenine (0.25 mM in blood). Red blood cell concentrates (RCC) were prepared to a mean hematocrit (Hct) of 70, the cells stored at 4 C, and plasma adenine and red blood cell adenosine triphosphate (ATP) were measured weekly for 42 days. The removal of plasma in the preparation of RCC reduced by 39 per cent the available adenine. As a result measurable plasma adenine was depleted by 21 days. The loss of ATP in RCC occurs at a significantly faster rate than in whole blood stored under the same conditions. When red blood cells are stored at higher HCT or for periods longer than 35 days, increased anticoagulant adenine levels are recommended.     

In vitro evaluation of platelets stored in CDP-adenine formulations

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.     

Characterization of biochemical changes occurring during storage of red cells. Comparative studies with CPD and CPDA-1 anticoagulant- preservative solutions

Citrate-phosphate-dextrose-adenine (CPDA-1), containing 0.25 mM adenine (final concentration) and 25 percent more glucose than citrate-phosphate-dextrose (CPD), has extended the allowable storage time for red cells to 35 days. Studies were conducted to understand better the characteristics of stored CPDA-1 red cells in relation to the properties of stored CPD red cells. Units with hematocrits near 80 percent showed the following: First, adenosine triphosphate (ATP) and total adenine nucleotide levels of red cells stored with CPDA-1 remained essentially constant during the first 3 weeks of storage after which the levels decreased; with red cells stored with CPD, ATP, and adenine nucleotide, levels were decreased even after 1 week of storage. Second, the pattern of the fall in 2,3-diphosphoglycerate was similar in red cells stored with CPD and CPDA-1. Third, changes in plasma and red cell levels of sodium and potassium, and in plasma ammonia levels, were comparable in CPD and CPDA-1 units; changes in cation levels were most pronounced during the initial 2 weeks of storage. Fourth, hemolysis was much greater in units stored in CPDA-1 for 35 days than in units stored in CPD for 21 days. Fifth, residual glucose concentrations were adequate in units drawn in CPDA-1 and stored for 35 days. We conclude that the changes in the biochemical characteristics of units of red cells stored with CPD and CPDA-1 are similar in most instances with the notable exception of the better maintenance of adenosine triphosphate levels in red cells stored with CPDA-1.     

In vitro metabolism of packed erythrocytes stored in CPD-adenine

In vitro metabolism of erythrocytes packed at 70 and 90 per cent hematocrits and stored in various CPD-adenine preservatives was studied. It was found that maintenance of acceptable levels of adenosine triphosphate (ATP) for the full 42 days of storage could be accomplished only if glucose levels were doubled from the standard 138.7 mM concentration to 277.5 mM level. If glucose levels were doubled, the amount of adenine could be decreased from 4.07 mM (0.50 mM final concentration) to 2.04 mM (0.25 mM final concentration) with maintenance of ATP at greater than 2.0 mumoles/g Hb. 2,3- diphosphoglycerate concentrations were essentially absent by 21 days in the various media studies. Thus, in vitro levels of ATP appear to be maintained at acceptable levels in a CPD media modified to contain 2 times glucose and 2.04 mM adenine.     

Hemoglobin Function and 2,3‐DPG Levels of Blood Stored at 4 C in ACD and CPD pH Effect

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound which is poorly maintained in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained in citrate‐phosphate‐dextrose (CPD) and this preservative has a higher pH (5.5) than ACD (pH = 5.0), these preservatives were prepared at each pH and studied. The CPD preservatives (pH 5.0, 5.5) had similar amounts of phosphate so the differences between them, obtained by altering the buffer ratio, should relate to pH. The ACD solutions (pH 5.0, 5.5) contained no phosphate. Hemoglobin function, expressed as P₅₀ (the Po₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity), and 2,3‐DPG were better maintained in ACD and CPD of pH 5.5. The lower pH (5.0) preservatives, whether ACD or CPD, showed rapidly declining hemoglobin function and 2,3‐DPG levels. The values at the higher pH remained close to normal for two weeks and above those of the lower pH preservatives for most of the four‐week storage period.     

Comparison of Blood Stored in ACD and in a Solution Containing Heparin, Phosphate, Glucose and Adenine

Duplicate blood samples were obtained from six healthy young males, one aliquot was collected in ACD and one aliquot in a solution of heparin-glucose-phosphate-adenine (HGPA). All were stored under blood bank conditions for a total of four weeks. The HGPA samples maintained their ATP and nucleotide adenine levels much better than the ACD samples and had generally higher p H's and faster rates of glycolysis. Their osmotic fragility and plasma K⁺ levels were similar to those of the ACD samples. Because there was considerably more hemolysis in the HGPA samples than in the ACD samples and in the absence of red cell survival data, the HGPA system is not recommended at this time as a substitute for the ACD system but as a comparative system to reveal metabolic shortcomings of the ACD storage system.     

Some in Vitro Effects of Adenine Added to Stored Blood

When adenine was added to freshly collected ACD blood and the blood incubated at 37 C. for one or two days, the concentration of ATP and adenine nucleotides (AMP + ADP + ATP) was higher than in the same blood without adenine. The addition of inosine with adenine was even more effective. Adenine was used by the red cells and the effective amounts of adenine to be added varied with the experimental conditions, for example, length of incubation. When adenine or adenine and inosine were incubated with outdated ACD blood, the ATP and adenine nucleotide concentrations rose, indicating that synthetic abilities were still present in red cells which had deteriorated energetically as a result of storage in citrate in the cold. When outdated red cells were returned to a more normal p H, the ATP levels increased. When adenine addition was included along with neutralization, the effects were remarkable, the ATP and adenine nucleotide levels going even above normal fresh blood levels. These experiments suggest that added adenine enhances ATP synthesis and allows formulation of a scheme for the participation of adenine in the metabolic reactions of stored cells.     

Effect of Plasma Removal on Blood Stored in ACD with Adenine

An evaluation of the effects of plasma in blood preservation was carried out on units of blood from which the plasma had been removed at different times during storage and on other units in which the plasma volume had been replaced by saline. Blood was either collected in ACD and stored for 14 or 21 days, or collected in ACD supplemented with adenine and stored for 21 or 42 days. These units were given in homologous transfusions to human volunteers. Autologous transfusions were used for separate groups studied at the same time.
Stored units reflected progressive chemical deterioration with increasing storage. The elevation of plasma hemoglobin concentration was observed to be greater in units stored as packed cells.
Average posttransfusion survival values for blood stored for 21 or 42 days were higher for adenine-supplemented blood than for blood in ACD alone, Autologous transfusions with ACD-adenine blood stored for 21 or 42 days survived for the same length of time as did homologous transfusions. Packed cells with plasma removed shortly after collection had the poorest survival.
The similarity of values obtained for cells without plasma suggested that plasma was not particularly involved with the preservation of blood for up to 21 days of storage as tested by red blood cell survival. Improved preservation obtained with adenine was observed even when plasma was removed. Hence, the usefulness of plasma for component therapy may well outweigh its role in blood storage.     

Storage of Erythrocytes in Artificial Media

The storage of red blood cells (RBC) for extended periods in artificial media without plasma has been studied. Blood was collected in either heparin or ACD solution; the plasma was removed; and one or two volumes of a solution containing 2 to 3 mM adenine, 5 to 60 mM Na₂HPO₄ 55 mM glucose, and 120 to 140 mM NaCl was added to the packed RBC. Control samples were stored in ACD plasma containing an equivalent concentration of adenine (ACD-ad). Viability studies were done on three consecutive days in each of 22 subjects, using the subject's own blood stored in various preservatives for 41 to 57 days. After this period of storage, the mean viability of ACD-ad stored blood was 73.5 per cent and of erythrocytes stored in artificial media, 74.4 per cent. Cells stored for longer periods had diminished viability, but the viability of cells stored in artificial media was equivalent to or superior to that of controls. After 42 days' storage, 2,3 DPG levels of RBC stored in artificial media were higher than those of controls, and in some instances, the 2,3 DPG content was one-third to two-thirds that of fresh blood. Some of the potential advantages of this system for blood preservation are:
1. The suspending medium is discarded prior to infusion so that less potentially toxic substances are administered.
2. Plasma is removed at the beginning of storage so that labile factors are available for fractionation.
3. 2,3 DPG levels are higher, so that, theoretically, the oxygen-delivering capacity of the transfused cells is greater.     

Effects of Adenine on Clotting Factors in Fresh Blood, Stored Blood, and Stored Fresh Frozen Plasma

The addition of small quantities of adenine to whole blood may prolong the useful shelf life of bank blood. The present study was undertaken to evaluate the effects of adenine on the clotting factors in blood containing ACD and CPD. Units of whole blood were collected in ACD, ACD-adenine, CPD and CPD-adenine, and each was stored 42 days under standard blood bank conditions. Samples of fresh frozen plasma containing these anticoagulants were stored three to four months at -30 C. Assays of Factors V, VIII (AHF), IX (PTC), X (Stuart Factor), fibrinogen, and prothrombin were performed on fresh blood, stored blood and stored fresh frozen plasma. The presence of small quantities of adenine did not appear to produce any appreciable alteration in the activity of the clotting factors in fresh blood. Further, adenine did not appear either to improve or worsen the survival of the procoagulants in whole blood stored 42 days or in fresh frozen plasma stored three to four months. There was significant deterioration of Factors V and VIII in whole blood stored 42 days in ACD, ACD-adenine, CPD, and CPD-adenine, but the degree of storage loss was independent of the anticoagulant employed. Factor X, fibrinogen, and prothrombin remained stable in blood stored 42 days regardless of the anticoagulant used, but Factor IX activity increased during storage possibly as the result of contact activation. Fresh frozen plasma stored three to four months showed a uniform slight loss of Factor VIII in all four anticoagulants, but Factors V, IX, X, fibrinogen, and prothrombin remained stable in stored fresh frozen plasma regardless of the anticoagulant employed.     

Correlation between adenylate metabolizing enzymes and adenine nucleotide levels of erythrocytes during blood storage in various media

Blood from normal donors and from heterozy-gotes for adenine phosphoribosyltransferase was stored in ACD, ACD-adenine, and ACD-adenine-phosphate media. The levels of AMP, ADP and ATP along with the enzymes involved in adenine nucleotide metabolism; adenine phosphoribosyl-transferase, ribose-5-P pyrophosphokinase and AMP-deaminase, were determined at various stages of blood storage in various media. One of the heterozygotes for adenine phosphoribosyltransferase maintained ATP levels poorly in all the media, but another heterozygote maintained ATP levels in a normal fashion. No significant correlation was established between the activities of various enzymes of adenine nucleotide metabolism and the levels of adenine nucleotides at any stage of blood storage in any medium studied. A population survey showed that the frequency of adenine phosphoribosyltransferase deficiency is of the order of 1 per cent. The heterozygote defect is not accompanied by any obvious abnormalities.     

Clinical Evaluation of Transfused Blood after Long-term Storage in ACD with Adenine

Whole blood collected in ACD with or without adenine was transfused into patients with various forms of blood loss. The units collected in ACD alone had been stored 14 to 28 days, while those units collected in ACD with adenine had been stored 28 to 42 days. Of the 771 units in the study, 545 were transfused into 295 patients. A total of 276 units were collected in ACD with adenine and were given to 130 patients. Little or no reaction was detected from clinical observation and selected laboratory tests related to the transfusion and/or solution. Isotope labeling of some of the transfused units given to more than 80 patients provided an estimate of the 24-hour posttransfusion survival and the results were similar to the average survival obtained in normal subjects after single-unit transfusions. Based on clinical laboratory findings, apparent lack of toxicity combined with adequate support of the bleeding patient suggest that blood stored in adenine is useful after longer storage periods than is blood stored in plain ACD. The 70 per cent 24-hour survival guideline suggests satisfactory survival after 35 days of storage, and possibly after 42 days.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

Preservation of Red Blood Cell 2,3‐Diphosphoglycerate in Stored Blood Containing Dihydroxyacetone

In a search for red blood cell metabolites which would preserve 2,3‐DPG during storage of blood, it was discovered that dihydroxyacetone (DHA) prolonged the maintenance of 2,3‐DPG levels for up to four weeks of storage, compared to about one week for presently used preservatives such as CPD. Four‐week preservation of 2,3‐DPG at normal levels was desired. CPD‐adenine was used as the starting point and a formulation having a pH of 7.0 and a DHA concentration of 20 millimoles per liter of blood was developed. The 2,3‐DPG level at four weeks of storage was proportional to DHA concentration in the 5 to 20 mM range. The osmotic fragility, red blood cell ATP levels, and plasma sodium, potassium, and hemoglobin during four weeks of storage in CPD‐adenine‐DHA were similar to those in blood stored in CPD‐adenine.