Studies on Stored Whole Blood II. Use of Packed Red Blood Cells

Whole blood collected in ACD or ACD with adenine was studied as units stored with or without plasma over 14 to 28 days. Comparisons were made using various tests, with posttransfusion survival in vivo being the primary guideline. The adverse effects which resulted from storage appeared more slowly in units with plasma than in units without plasma. The inclusion of adenine to either packed or nonpacked units was associated with even less evidence of storage damage. Using the criterion of over 70 per cent recovery in 24 hours as signifying adequate preservation, the average survival value for each group was better when the plasma was present, and even higher when adenine had been added. All tested groups had average values exceeding 70 per cent recovery after 21 days of storage. Blood stored for 28 days with adenine had over 70 per cent recovery with or without plasma. Though the storage damage became more apparent in units without plasma, the average survival value would suggest that the packed units were still useful after 21 days of storage, with extension to 28 days when ACD with adenine was used.     

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.     

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.     

Effect of Adenine on Stored Erythrocytes Evaluated by Autologous and Homologous Transfusions

Blood obtained from normal volunteers and stored in ACD with or without adenine was evaluated by both tests in vitro and by 24-hour recovery following transfusion. Significant differences between autologous and homologous transfusions were not detected. The changes that did occur were limited to evidence of lesser erythrocyte breakdown in ACD units fortified with adenine. These units were associated with higher survival values at all storage periods evaluated and displayed over 70 per cent 24-hour posttransfusion survival even after 42 days of storage.     

The in vivo survival of red blood cells stored in modified CPD with adenine: report of a multi-institutional cooperative effort

In order to provide data in support of licensure applications for citrate-phosphate-dextrose (CPD) supplemented with adenine, a multi- institutional cooperative effort was organized to determine survivability of red blood cells subjected to prolonged liquid storage. Two manufacturers supplied plastic multiple bag blood storage containers prefilled with modified CPD (glucose 25/ greater than the normal concentration) supplemented with adenine (17.0 to 17.3 mg per 63 ml of anticoagulant; 0.25 millimolar approximate final concentration when diluted with 450 ml of whole blood for 35 days showed a mean survival of 80.53 +/- 6.44 per cent (1 SD). Both red blood cell and supernatant plasma biochemical characteristics were comparable to those reported for whole blood stored for 21 days in either acid-citrate- dextrose (ACD) or CPD. Red blood cells from 19 units stored as concentrates for 35 days (Hct 75.03 +/− 3.74%) had a mean survival of 71.38 +/− 10.3 per cent with considerable interdonor variation in survival and interlaboratory variation in some biochemical characteristics. Red blood cells from eight units stored as concentrates (Hct 75.38 +/− 4.30%) for 28 days showed a mean survival of 83.97 +/− 6.10 per cent and biochemical characteristics comparable to those reported for red blood cell concentrates stored in CPD or ACD for 21 days. Modified CPD with adenine as formulated offers an improved anticoagulant for blood banking by extending the permissible red blood cell storage period.     

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.     

Adenine in Blood Preservation: Posttransfusion Viability and Biochemical Changes

Posttransfusion viability was studied in red blood cells stored for 21 days in ACD solution and for 35 days in ACD solution supplemented with adenine to a final concentration of 0.5 mM. The survival of radio-chromium-labeled red cells was determined after transfusion of 10 ml. of autologous blood and 350–400 ml. homologous blood. The viability values were about the same for the two transfusion procedures. The mean posttransfusion viability was 80 per cent for erythrocytes stored for 35 days in the medium containing adenine and 79 per cent for cells preserved in ACD solution for 21 days.
The concentration in the erythrocytes of ATP, ADP, AMP, reduced and oxidized glutathione, 2,3-diphosphoglycerate, and four glycolytic enzymes was measured before and after storage in the two media. The ATP and the total adenine nucleotide concentrations were much higher in the red cells stored in the adenine-containing solution. Of the enzymes tested, only phosphofructokinase decreased in activity during 35 days of storage. The decrease was about 50 per cent and was not dependent on the storage solution.
This study supports the theory that decreased adenine supply is an important cause of damage to erythrocytes in ACD solution.     

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.     

Transfusion of Long Stored Whole Blood or Washed Red Blood Cells Incubated with Adenine and Inosine

Full unit autotransfusions of long stored ACD blood, incubated with adenine and inosine at 37 C., were given to healthy young male volunteers. Red cells of blood stored for 35 days showed, after regeneration, a significant increase in ATP and a 24-hour posttransfusion survival of 78.8 per cent (70.9–85.9%); red cells of blood stored for 42 days, thus regenerated, showed a similar increase in ATP and a 24-hour posttransfusion survival of 75.6 per cent (71.5–80.6%). These results were not significantly different from those obtained with 10-ml token autotransfusions of blood similarly treated, the posttransfusion survival of red cells in token transfusions being 78.8 per cent for blood stored 35 days prior to regeneration with adenineinosine and 74 per cent for blood stored for 42 days prior to regeneration. Available data on toxicity of adenine and inosine have been critically reviewed: Chance of direct toxic effects with the small amounts involved may be dismissed when few transfusions are involved; however, uric acid overload must be considered when multiple transfusions are required within a short period of time. A single washing with saline-glucose solution reduces by 90 per cent the concentration of un-metabolized adenine and inosine, and of the product of their metabolism, hypoxanthine. The washing procedure involves a loss of only 0.55 per cent of the total red blood cell population; washing additionally reduces the amount of free hemoglobin. Washing has no effect on the ATP or red blood cell viability, and is recommended when multiple transfusions of cells treated with adenine and inosine are required in a short period of time.     

Antigen Reactivity in Pilot Tube Blood Specimens: Clotted Blood vs. Blood Stored in Acid-Citrate-Dextrose

Present blood bank practices of using either clotted blood or ACD pilot tubes stored at 4 C. give equally satisfactory antigen preservation up to 21 days of storage. Thereafter, there is a distinct difference, in that the pilot tube blood sample stored in ACD retains antigen reactivity for a longer period of time than does the blood stored as a clot. The added difficulty of the repeated washings of the ACD stored sample required to remove traces of plasma and anticoagulant must be considered in evaluating the advantages of either system. The anticipated use of storage formulae capable of maintaining better preservation of the stored blood for periods up to 42 days requires that the problem of the "proper" pilot tube be reconsidered. In this light, the preliminary serum screening of all recipients, using fresh pools of red cells bearing all the important known blood factors, becomes valuable. It is therefore recommended that the screening test be adopted by all blood banks and that particularly careful attention be directed to crossmatching tests on stored blood approaching the expiration date.     

The distribution and utilization of adenine in red blood cells during 42 days of 4 C storage

The fate of adenine in CPD whole blood (17.3 mg/500 ml, or 0.25 mM) was evaluated during 42 days of 4 C storage. In whole blood, 95 per cent of the adenine was removed from the plasma by 42 days while the cellular adenosine triphosphate (ATP) levels remained above 60 per cent of the initial concentration. Packed red blood cells (concentrates) were stored with the same relative quantity of adenine (0.1 mg/ml red blood cells) used in whole blood units by the addition of adenine after packing and were shown to take up adenine in a similar manner. Calculations of the initial adenine distribution indicated higher intracellular adenine concentrations than predicted from distribution equilibrium based on volume considerations. The presence of inorganic phosphate has marginal effects on adenine incorporation but does elevate ATP levels, while contributing to the reduction of 2,3-diphosphoglycerate (2,3-DPG) content. The free adenine equilibrium between plasma and red blood cells favors the red blood cells, suggesting adenine binding by red blood cell membranes as shown by initial distribution studies with ¹⁴C-adenine and equilibrium dialysis.     

Further studies on the effect of adenine in blood preservation

Clinical and experimental studies on the effect of adenine supplementation to ACD blood are reported. The frequency and type of transfusion reactions in 5,595 transfusions of ACD-adenine blood (storage time 0–35 days) was the same as in 4,519 transfusions of ACD blood (storage time 0–21 days). There was no change in the plasma haptoglobin level in 10 recipients during and after transfusion of 29 to 35 day old ACD-adenine blood. The relation between posttransfusion viability, in vitro hemolysis, erythrocyte ATP content and duration of storage were studied. Supplementation with adenine increased the posttransfusion viability and the erythrocyte ATP content; these effects were noticeable after two weeks of storage. A negative correlation was found with respect to posttransfusion viability and the concentration of total lipids in the medium. A significant covariation between the concentrations of the three nucleotides ATP, ADP and AMP was found. This indicates that erythrocyte adenylate kinase dominates in the regulation of the relative concentrations of these nucleotides.     

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.     

Di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylexyl phthalate (MEHP) accumulation in whole blood and red cell concentrates

Plasma DEHP concentrations were measured weekly in whole blood and red cell concentrates (RCC) during 21 days of storage in standard CPD within PL-130 blood bags. In addition, DEHP and MEHP accumulation patterns were investigated in blood stored for 42 days in modified CPD with adenine within PL-146 and BB-69 storage containers. Total per-unit plasma DEHP of RCC units was 49 to 71 per cent of the total in plasma of whole blood units (PL-130). From 28 to 42 days, mean DEHP levels were 12 to 19 per cent higher in whole blood stored in PL-146 than in BB-69. Although MEHP was not found in any blood bag plastic, MEHP accumulated in plasma during whole blood storage. MEHP concentrations were 2.8 to 3.8 times higher in plasma stored in BB-69 than in PL-146. It is postulated that MEHP arises from hydrolysis of DEHP by plasma lipase, even in frozen plasma sample, and that the rate of this reaction is influenced by blood bag plastic surface characteristics.     

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.     

The Rhesus Monkey as a Model for Evaluation of the Preservation of Stored Whole Blood

Blood was collected into Acid Citrate Dextrose solution (ACD) from a group of 20 large, adult rhesus monkeys. In ten of the experiments, the blood was labeled immediately with radioisotopes and reinfused into the donor monkeys. The other ten blood units were stored at 4 C for 21 days, then tabled and infused into the donor monkeys. In vitro determinations were made immediately after collection and at the end of the storage period and compared with the results obtained in stored human blood.
The ten fresh blood infusions had a mean survival of 101.3 per cent with an average half time of 10.4 days. The 21 (lay‐old bloods had a mean survival of 73.7 per cent (S.D. = 5.1%) with an average half time of 11.3 days. The average red blood cell survival of 123 human blood units stored 21 days and reinfused into their donors had an average red cell survival of 81.0 per cent (S.D. = 8.6%). The determinations in rhesus blood and human blood were comparable.
These data strongly suggest that in vivo studies done in the rhesus monkey may be substituted for those performed in man to evaluate red cell preservation.     

Rejuvenation of Human Red Blood Cells During Liquid Storage

Blood in ACD was stored under blood banking conditions. At 21 days of storage, aliquots of blood were rejuvenated by incubation at 37 C for one hour, with a solution containing pyruvate, inosine, phosphate, glucose, and adenine, at pH 7.2. The rejuvenated erythrocytes were washed with a solution of NaCl-Na phosphate and resuspended in an artificial medium or in the freshly drawn plasma from the same donor. These suspensions were stored in liquid form and analyzed at predetermined intervals. The cellular ATP and 2,3-DPG levels of rejuvenated cells were higher than those in the control cells and were maintained higher throughout the storage intervals investigated. The metabolic integrity of the rejuvenated cells was also improved, as evidenced by greater osmotic resistance, decreased spontaneous lysis, and greater uptake of methylene blue when compared to control cells.     

Comparative Viability of Blood Stored in ACD and CPD

The normal distribution of red blood cell viability for ACD and CPD units stored for 21 days was studied. For ACD and CPD units, respectively (n=41,37), the means and standard deviations were as follows: 24-hour survival, 75.7 + 6.2 and 79.4 + 6.4; early recovery, 91.6 + 3.6 and 94.1 + 3.5; t/2, 29.4 + 3.0 and 27.9 + 4.3. Early recovery and survival were significantly higher for CPD, but more important than the difference in mean survival is that by current standards of acceptability, the incidence of donors who will be deemed undesirable is approximately 6 per cent for CPD, as opposed to 20 per cent for ACD. Neither early recovery, 24-hour survival, nor t/2 could be shown to correlate with pH, plasma potassium, plasma sodium, per cent hemolysis, and osmotic fragility. The mean and standard deviation of survival for 18 units of 28-day-old CPD blood was 70.7 + 11. Since the standard error was large, the frequency distribution could not be determined, and the number of units with survivals that would fall below the minimum standard could not be ascertained. Nevertheless, comparison with 21-day old ACD did not show a significant difference in the mean survival, although the range observed was much wider. The results also point out the need for greater number of observations with increasing duration of storage for adequate appraisal of blood preservative solutions.     

Accumulation of DI-2-Ethylhexyl Phthalate (DEHP) in Whole Blood, Platelet Concentrates, and Platelet-Poor Plasma

The concentration of the plasticizer, di-2-ethylhexyl phthalate (DEHP), in the plasma was measured after storage of the whole blood in polyvinylchloride plastic bags at 4 C for up to 38 days in either ACD or CPD. The plasma from ACD-stored whole blood contained more DEHP than that from CPD-stored whole blood. The continuous-flow centrifugation washing procedures removed about 98 per cent of the DEHP from ACD whole blood stored for 33 days at 4 C.
DEHP was assayed in the platelet-rich plasma, platelet-poor plasma, supernatant of the platelet concentrate, washed platelets, and washed red blood cells prepared from CPD whole blood. Very little DEHP was found in the washed red blood cells and platelets, a small amount was found in the platelet-poor plasma, and a large amount in the supernatant of the platelet concentrate. A greater amount of DEHP accumulated in the platelet concentrates that were stored at 22 C than in those stored at 4 C. When platelet concentrates from CPD whole blood were stored at 22 C for 72 hours, the amount of DEHP was about four times that observed after 4 C storage for the same length of time.     

Storage of saline-adenine-glucose-mannitol-suspended red cells in a new plastic container: polyvinylchloride plasticized with butyryl-n- trihexyl-citrate

Blood collection and component preparation have been performed in integrally connected multiple plastic containers made with a new plastic. This polyvinylchloride (PVC) container plasticized with butyryl-n-trihexyl-citrate (BTHC) is a new material for blood storage; it contains no di(2-ethylhexyl)phthalate (DEHP). After removal of plasma and buffy coat, the red cells were suspended in saline-adenine-glucose-mannitol (SAGM) medium. After 42-day refrigerator storage, the total adenine nucleotide concentration remained the same as the initial concentration in the red cells, whereas ATP levels had decreased to 61 percent of the initial value. The 2,3 DPG concentration was 62 percent of normal on Day 7 and 21 percent on Day 14. Glucose consumption, lactate production, potassium leakage from red cells, and pH levels were similar to those found after storage in DEHP-plasticized containers under the same conditions. After 42 days, hemolysis levels were 0.56 +/- 0.21 percent and 0.42 +/- 0.17 percent in two series of units mixed weekly and 0.70 +/- 0.27 percent in units stored unmixed. Although even higher levels of hemolysis were observed in the units stored unmixed and used for 24-hour posttransfusion survival, the autologous red cell recovery results were excellent (83.2 +/- 5.1%, n = 8). BTHC-plasticized PVC is found to be a suitable material for 42-day storage of red cells in SAGM solution.