Biomedical subjects
Publications and source records attributed to G L Moore.
Approval of the new anticoagulant preservative, CPDA-1, was based in part on data from human clinical trials of CPDA-1 performed by six cooperating laboratories and two blood bag manufacturers. Biochemical and red blood cell survival properties of 123 units of blood stored for 28 to 35 days in CPDA-1 (as reported by cooperating laboratories) are briefly summarized.
The modified CPD-adenine anticoagulants CPDA-2 and CPDA-3 were developed to improve red blood cell storage to 35 days, since CPDA-1 was found marginal at 35 days in high hematocrit samples. In this study red blood cell storage was extended to 56 days. In vitro correlates of viability were monitored to determine the feasibility of evaluating in vivo the ability of CPDA-2 and CPDA-3 to extend storage past 35 days. The data suggest that red blood cells stored up to 56 days may have acceptable viability, providing the possibility of extended storage for the military and certain special civilian situations.
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.
Experiments with citrate-phosphate-dextrose supplemented with adenine, 0.5 mM final concentration (CPD-A2), provided an opportunity for study of red blood cells (RBC) membrane storage lesions which could limit extension of storage. Impairment in drug-induced endocytosis in intact RBC occurred with storage, but the changes produced did not quantitatively predict or reflect the 24-hour in vivo 51Cr-labelled RBC survival. Abnormalities in RBC membrane protein did appear after storage, but these alterations neither paralleled nor predicted the in vivo 24-hour 51Cr-labelled RBC survival. The RBC membrance protein changes were not reminiscent of those produced by either acute adenosine triphosphate depletion, oxidative attack, or calcium accumulation. Therefore, while storage produces significant alterations in RBC membrane protein and function, the changes detected were not those that determined in vivo RBC survival.
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The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.
The effects of an 8-hour hold at 22 degrees C prior to component preparation were evaluated in a split-bag study using nine units of blood preserved in citrate-phosphate-dextrose-adenine (CPDA-2). Each unit was divided in half, platelet-rich plasma removed at 0 or 8 hours, respectively, and the half units of red blood cells stored at 4 degrees C for 42 days. The only red blood cell metabolic differences seen in the bags held 8 hours (compared to those not held) were a 21 percent rise in adenosine triphosphate, which was not significant after 14 days of storage, and a 33 percent loss of 2,3-diphosphoglycerate which resulted in a loss curve similar to that seen with acid-citrate-dextrose blood. The logistic advantages seem to warrant an 8-hour holding period for red blood cells drawn in CPDA-2.
An additive solution containing adenine, ascorbate-2-phosphate, sodium phosphate, dextrose, and saline was developed for packed red cell preservation. The combination of all components was simultaneously optimized so that the resulting solution produced the maximum retention of both red cell adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG) concentrations. Fourteen nutrient combinations were tested; each combination was evaluated for 42 days of storage using cells from three donors. The nutrient combinations were chosen with the aid of a computerized experimental design process. Results of the experiments were modeled by regression analysis, and the model was optimized to produce the "best" formulation for simultaneous maintenance of ATP and 2,3 DPG. The resulting mathematically optimal formulation was tested in the laboratory using 10 units of red cells. With this solution, it was possible to store red cells for 42 days with retention of 45 to 55 percent of the initial ATP and 85 to 150 percent of the initial 2,3 DPG. Red cell lysis was low (0.8 percent), and most of the cells were biconcave discs (by scanning electron microscopy) at the end of storage. The studies were carried out in an efficient manner by using computer-optimized experimental design techniques coupled with multiple regression modeling and subsequent computer optimization of the models. This experimental approach has potential application to many current blood banking procedures. This additive solution should maintain viable red cells for 42 days. In addition, the solution will maintain red cell 2,3 DPG throughout storage.
Immunosuppressed patients who require red cell transfusions receive irradiated (1500-3000 rad) packed red cells. These cells are irradiated immediately before infusion. If a large group of patients become immunosuppressed due to exposure to radiation or chemicals, the ability to supply large volumes of irradiated blood at the time of use might not be possible. An alternate solution to providing quantities of irradiated blood is to irradiate the units prior to storage. This study presents in vitro data comparing storage of paired packed red cell units either irradiated or not irradiated. Five units of fresh blood drawn into citrate-phosphate-dextrose-adenine (CPDA-1) were packed to a hematocrit of 75 +/- 1 percent, and then each unit was divided in two equal parts. One of each pair was irradiated (4000 rads), and both parts of each unit were stored for 35 days at 4 degrees C. Samples were analyzed every 7 days. Irradiation caused a slight drop in red cell adenosine triphosphate and 2,3 diphosphoglycerate and a slight increase in plasma hemoglobin compared to controls. Methemoglobin, pH, and glucose consumption were identical to the controls. The evidence indicates that irradiation did not cause biochemical or metabolic changes in the red cells that would lead us to suspect a difference between irradiated and nonirradiated stored red cells in function or viability. These negative findings require in vivo confirmation.
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Fresh human blood was collected in citrate-phosphate-dextrose, frozen by a high-glycerol technique, and stored at -80 degrees C. The red cells were thawed, deglycerolized, and resuspended in a final wash solution, ADSOL (Fenwal Laboratories), or an additive solution (AS) containing glucose, adenine, mannitol, and phosphate. The cells were then stored at 4 to 6 degrees C for 21 days and assayed weekly for adenosine triphosphate and 2,3 diphosphoglycerate, pH, glucose use, and lysis. AS and, to a lesser extent, ADSOL produced metabolic profiles similar to or better than profiles of cells not frozen and stored in commercially available additive solutions. AS offers a potential post-thaw preservative solution for red cells that would greatly increase the flexibility and reduce the expense of using frozen blood. A sterile post-thaw storage capability will make the stockpiling of frozen red cells a practical concept for both military and civilian blood banks.
The 2,3 diphosphoglycerate (2,3 DPG) content of red cells stored in current anticoagulant-preservative products decreases rapidly after the first few days of storage, and by 3 weeks the red cells are essentially depleted of 2,3 DPG. Because ascorbic acid and ascorbate-2-phosphate (A-2-P) are effective in maintaining erythrocyte 2,3 DPG during liquid preservation, ascorbate was stabilized through autoclaving and subsequent storage by adding it as the trisodium salt of A-2-P to a phosphate-adenine-saline solution at a pH of 8.5 to 9.0. Red cell concentrates prepared from blood drawn into citrate-phosphate-double-dextrose were supplemented with the A-2-P additive solution (AS-4) and studied in vitro and in vivo. Mean 2,3 DPG values for 22 units were 147.6, 113.5, and 82.3 percent of initial value after storage for 3, 4, and 5 weeks, respectively. Maintenance of 2,3 DPG was at the expense of adenosine triphosphate (ATP), which fell to as low as 22.2 percent of initial value after 5 weeks. Despite the low ATP values, the 24 hour 51Cr-labeled red cell recoveries averaged 80.8 and 74.1 percent after 4 and 5 weeks of storage, respectively. The AS-4 system provides a red cell product with acceptable viability and improved oxygen off-loading function.
A red cell additive solution (AS-005) containing ascorbate-2-phosphate (AsP) to maintain 2,3-diphosphoglycerate, plus adenine, phosphate, and mannitol to retain viability and reduce hemolysis, was evaluated by human clinical trials. A crossover design was used with another additive solution (Nutricel AS-3, Cutter Laboratories) serving as the control for each donor. Each additive solution was evaluated at 35 and 42 days of storage. There was no significant difference between the red cell viability of the two storage solutions at either time period. Split-bag, AS-005 in vitro studies at two temperatures (2.5 and 5.5 degrees C), both within the range of 1 to 6 degrees C approved by the American Association of Blood Banks and the Food and Drug Administration, resulted in dramatically different in vitro parameters, including a threefold difference in 2,3-diphosphoglycerate (2,3-DPG), a fivefold difference in glucose, and significant differences in pH and adenosine triphosphate. High-pressure liquid chromatography data confirmed the preliminary report that 1 to 2 percent (wt/wt) oxalate was present in preparations of AsP. In vitro storage data confirmed that oxalate is the active component of AsP that preserves 2,3-DPG during storage.
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