The in vitro evaluation of modifications in CPD-adenine anticoagulated- preserved blood at various hematocrits

Erythrocytes stored in the new CPD-adenine anticoagulant (CPDA-1) barely met the 70 per cent 24-hour postinfusion 51Cr recoveries on day 35 when stored at hematocrit greater than or equal to 75 per cent. CPDA- 1 differs from CPD in that it has 1.25 times the glucose concentration plus 17.3 mg adenine/63 ml. In an effort to improve the survivability (or viability) of red blood cells following extended storage (35+ days), two new CPD-adenine anticoagulants have been tested in vitro. CPDA-2 and CPDA-3 (both of which contain 34.6 mg/63 ml of anticoagulant or 0.50 mM adenine [final blood concentration], and either 1.75 times or 2.0 times respectively the amount of glucose used in CPD) have been tested for whole blood or red blood cell storage to 42 days. Red blood cell ATP concentrations were better maintained throughout 42 days of storage in both of these formulations than in CPDA-1 at hematocrits that ranged from 40 to 85. Other biochemical parameters (2,3-DPG, pH, plasma hemoglobin) were similar to those of blood stored in CPD or CPDA- 1.     

Red cell storage for 56 days in modified DPD-adenine. An in vitro evaluation

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.     

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.     

Survival and biochemical characteristics of stored red cells preserved with citrate-phosphate-dextrose-adenine-one and two and prepared from whole blood maintained at 20 to 24 degrees C for eight hours following phlebotomy

Studies were conducted to evaluate the characteristics of red cells stored for 35 days following preparation from units of whole blood anticoagulated with citrate-phosphate-dextrose-adenine-one and two (CPDA-1 and CPDA-2) and maintained at 20 to 24 degrees C for 8 hours after phlebotomy. The mean (+/− 1 SD) 24-hour survival for transfused CPDA-1 autologous red cells with hematocrit levels of 78.1 +/− 2.3 percent was 78.0 +/− 8.1 percent (n = 9). The 24-hour survival of red cells from units preserved with CPDA-2 with hematocrit levels of 79.3 +/− 4.5 percent was 74.8 +/− 8.6 percent (n = 15). This difference in survival was not significant. Red cells from control units stored for 1 day showed a 24-hour survival of 91.9 +/− 4.2 percent (n = 7). During the 8-hour holding, red cell adenosine triphosphate levels increased by 15 to 25 percent in units drawn into both CPDA-1 and CPDA-2. After the initial 8-hour period, the red cell 2,3 diphosphoglycerate levels were 54 +/− 12 percent (mean +/− 1 SD) of initial levels in units drawn into CPDA-1 and 58 +/− 8 percent of initial levels in units drawn into CPDA- 2. Following 35 days of storage, units of red cells prepared from whole blood drawn into CPDA-1 and CPDA-2 had comparable plasma cation and ammonia levels and similar amounts of cell-free hemoglobin. These data indicate that red cells can be stored satisfactorily for 35 days when prepared from whole blood held at 20 to 24 degrees C for 8 hours.     

The extension of 4 degrees C storage time of frozen-thawed red cells

Blood was drawn from volunteer donors and frozen using the high glycerin, mechanical freezing procedure accepted by the United States Navy. Subsequently, the units of blood were thawed and washed. Various anticoagulants were added, and the red cells were stored in a refrigerator at 4 degrees C for periods of up to 28 days. Chemical analyses were performed periodically. These showed that the addition of the anticoagulants ACD, CPD and CPDA-1 caused the red cells to be preserved better than the currently accepted 0.9-percent NaCl, 0.2-percent glucose solution. In vivo 51Cr viability studies performed on blood stored with CPDA-1 for 14 days showed a 24-hour viability of 78.8 +/- 8.4 percent. In a subsequent study, the blood was stored for 21 days prior to freezing and then was rejuvenated and frozen. The cells were thawed, washed, and stored at 4 degrees C with CPDA-1 for an additional 14 days. The 24-hour viability of these cells was determined to be 74.0 +/- 5.1 percent. These findings show that the postthaw storage time of red cells can be increased greatly over the now-accepted 24 hours, if bacterial sterility can be assured.     

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.     

Effects of 4000 rad irradiation on the in vitro storage properties of packed red cells

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.     

Measurement of the viability of stored red cells by the single-isotope technique using 51Cr. Analysis of validity

A single-isotope 51Cr method often is used to evaluate the viability of stored red cells. In this technique, the red cell mass is measured by back-extrapolation to time zero (t0) of the radioactivity of the blood between 5 and 20 minutes after infusion of the sample. If there is early destruction of stored cells, this method provides an overestimate of the red cell mass and, hence, of the viability of the stored cells. Freshly drawn red cells from normal donors were labeled with 99mTc, and cells from the same donor which had been stored in citrate-phosphate-dextrose-adenine-one (CPDA-1) for periods ranging from 7 to 49 days were labeled with 51Cr. A comparison of the "true red cell mass" as determined with 99mTc with the back-extrapolated red cell mass from stored 51Cr-labeled cells has made it possible to define the magnitude of error introduced by early loss of red cells. The overestimation of red cell mass and viability was diminished if only the 51Cr radioactivity between 5 and 15 minutes after infusion was used in back-extrapolating to t0. The degree of overestimation of red cell mass was greatest when the red cell viability had declined to very low levels. However, in the entire range of 10 to 80 percent viability, the overestimate of viability was usually less than 4 percent. The overestimate of viability proved to be quite similar for all samples and may be taken into account when using the single-isotope technique for measurement of red cell viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Circulation and function of human platelets isolated from units of CPDA- 1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2–3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 ± 17 percent (mean ± one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 ± 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 ± 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin- prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.     

The biochemical effects of CPDA-2-drawn red blood cells of delayed refrigeration prior to component preparation

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.     

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.     

Some properties of blood stored in anticoagulant CPDA-1 solution. A brief summary

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.     

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.     

Detection of cytomegalovirus antibody in stored blood products using latex agglutination

The detection of antibody to cytomegalovirus (CMV) in donor sera is one effective method for the prevention of posttransfusion CMV infection in seronegative recipients. A passive latex agglutination test (CMV Scan, Hynson, Wescott & Dunning, Baltimore, MD) has been shown to be an acceptable method of screening sera from the blood donor population. The procedure for this test, however, does not permit the testing of stored donor blood products. To evaluate the feasibility of testing blood components at various times during storage, the authors examined 25 CMV-positive and 25-CMV negative samples of CPDA-1 plasma from 50 units each of platelets and red cells. Plasma samples from platelets stored at 22 degrees C were tested on each of the 5 days of storage. Samples from red cell units stored at 2 to 6 degrees C were tested on storage Days 1, 2, 4, 6, 8, 10, 12, and 14. Of 250 tests done on plasma from platelet units, there were 123 true-positive and 123 true-negative results (sensitivity and specificity, 98.4%). Of 400 tests on plasma from red cell units, there were 200 true-positive and 198 true-negative results (sensitivity, 100%; specificity, 99.5%). These data show that the CMV Scan test can be used reliably to test segments of CPDA-1 plasma from platelets stored for up to 5 days and from red cells stored for up to 14 days.     

Radiolabeled red cell viability. I. Comparison of 51Cr, 99mTc, and 111In for measuring the viability of autologous stored red cells

The simultaneous determination of autologous 99mTc red cell (RBC) and 51Cr RBC viability at 24 hours was measured in 19 normal volunteers whose RBCs had been stored in additive media (Nutracel) for 42 or 49 days. The ratio of the 51Cr:99mTc value was 1.23. In this experiment we also calculated 51Cr RBC viability by both the single-isotope method (extrapolation) and the double-isotope method (using 125I human serum albumin for an independent plasma volume) in the same volunteers. The corresponding viability values were not significantly different. The simultaneous determination of autologous 111In-oxine RBC and 51Cr RBC viability at 24 hours was measured in 19 other normal volunteers whose RBCs had been stored in citrate-phosphate-dextrose-adenine (CPDA-1) for 1 or 15 days. The ratio of the 51Cr:111In value was 1.1. Use of these 24-hour viability ratios as conversion factors permits direct comparison of 99mTc or 111In RBC viability with a 51Cr standard, and therefore expands the application of these newer RBC radiolabels.     

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.     

An in vivo comparison of CPD and CPDA-2 preserved platelet concentrates after an 8-hour preprocess hold of whole blood

To see if citrate-phosphate-dextrose-adenine-two (CPDA-2) anticoagulant- preservative had an effect on the viability of platelets, we studied autologous in vivo recovery and survival in humans for platelet concentrates prepared from six units of blood drawn into CPDA-2 and compared them to six units drawn into citrate-phosphate-dextrose (CPD). These units were prepared from whole blood held at room temperature for 8 hours after collection and were then stored for 3 days at 22 ± 2 degrees C. The recovery for platelets preserved in CPD was 39.0 +/0 4.8 percent and for platelets preserved in CPDA-2, 32.5 ± 4.4 percent. The difference was not significant (p greater than 0.10). In order to estimate population differences, in vitro effects on in vivo viability were also evaluated. Six in vitro variables were studied but only pH at 72 hours (r = 0.77), platelet count (r = 0.64), and morphology score (r = 0.66) correlated to recovery. Only pH at 72 hours significantly influenced recovery (p = 0.007). By adjusting for individual pH differences, mean recovery for platelets stored in CPD was 37.5 percent, and for platelets stored in CPDA-2, 34.0 percent. The mean lifespan was 6.7 ± 0.7 days for platelets preserved in CPD and 6.1 ± 1.0 days for those preserved in CPDA-2. Although hemostatic function was not studied, these data support in vitro observations that platelets preserved with CPDA-2 are not different from platelets preserved with CPD, even after 8-hours of storage of whole blood at room temperature prior to platelet concentrate preparation.     

Reduction of plasma volume after storage of platelets in CP2D

BACKGROUND: There has been concern that further deterioration might occur if stored platelets are centrifuged to reduce their volume. Although such centrifugation appears to have minimal effect on platelets in CPDA-1 (osmolarity, 470 mOsm) there is no information on the situation with CP2D (580 mOsm). STUDY DESIGN AND METHODS: Platelet concentrates from CP2D packs were sampled at 1 and 5 days and after centrifugation was used to reduce the plasma volume to 10 mL. The aggregation, hypotonic shock response, morphology, pH, and lactate, glucose, pCO2 and pO2 levels were assessed, and values were compared to those seen with CPDA-1. In addition, blood was collected from the same donors into both CP2D and standard sodium citrate anticoagulant in an anticoagulant-to-blood ratio of 1:8 and the aggregation response of the fresh platelets was measured. RESULTS: Collection of blood into CP2D results in an immediate reduction of the platelet aggregation response when compared to that found after collection of blood into sodium citrate or CPDA-1. Aggregation is further decreased after storage; however, these changes and those for hypotonic shock, pH, lactate, glucose, and pCO2 are similar to those seen for CPDA-1. Additional centrifugation did not cause further change. CONCLUSION: Platelets stored in CP2D have reduced in vitro function after 5 days of storage, but subsequent centrifugation to reduce the plasma volume does not further alter these platelets.     

Adequate survival of red cells from units "undercollected" in citrate- phosphate-dextrose-adenine-one

The lower limit for the volume of whole blood that may be collected for transfusion into standard blood recipient sets is 405 ml. Each year it is estimated that 82,500 to 161,700 units are drawn which contain between 275 and 405 ml. This study evaluated whether these "undercollected" units would be suitable for transfusion. Twenty normal adults donated both a "standard" unit (450 ml) and an "undercollected" unit (275 ml) in 63 ml of citrate-phosphate-dextrose-adenine-one (CPDA- 1). The units were packed within 4 hours (mean Hct 71%) and stored undisturbed at 4 degrees C for 35 days. Aliquots of 1 to 2 ml of red cells from each unit were then labeled with 51chromium (51Cr) and reinfused into the original donor. The mean 24-hour survival of the 450- ml units was 78.8 percent (SD 12.2, SEM 2.7), while the mean 24-hour survival of the 275-ml units was 87.7 percent (SD 10.7, SEM 2.4; p less than .01). Seven inadvertently undercollected units (mean vol: 295 ml) had a mean 24-hour survival of 91.9 percent. The higher concentration of dextrose and adenine in the undercollected units may improve posttransfusion red cell viability. These data suggest that 275 ml is the minimum acceptable volume for blood donated into CPDA-1.