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The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1).

The preservation of red cells "hard packed" to a hematocrit of over 80% from blood collected in citrate-phosphate-dextrose (CPD) or CPD-adenine (CPDA-1) has been investigated. After 21 days of storage, cells that had been collected in CPD solution had consumed most or all of the available glucose and manifested markedly impaired viability after reinfusion into the normal donor. In contrast, red cells prepared from blood collected in CPDA-1, a medium containing supplementary adenine and an increased amount of glucose, maintained higher glucose and adenosine triphosphate levels and, in most instances, manifested satisfactory posttransfusion viability. We emphasize that in addition to providing longer shelf life of stored blood, CPDA-1 provides a better hard-packed red cell concentrate for transfusion at 21 days.

Adenine

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.

Adenine

Complement components detected on normal red blood cells taken into EDTA and CPD.

Normal red blood cells (RBC) from fresh EDTA and CPD blood and from stored CPD blood were examined for the presence of bound subcomponents of C3 and C4. By serologic agglutination tests, only C3d was detectable on the cells. Incubation in compatible fresh normal serum (FNS) at 37 degrees C appeared to increase the amount of 3Cd on the RBC. C3b was serologically detectable only on stored CPD cells and only after incubation in compatible FNS. No. C4 components were detected on the cell surfaces in agglutination tests. Using an indirect labeling technique, small, but significant, amounts of C3d and C4d were found on all three types of untreated cells. C3b was present on stored CPD cells only. The indirect labeling technique showed a significant increase in C3d and C4d on all cells following incubation i- compatible FNS, whereas bound C3b was significantly increased only with stored CPD cells. There was no increase in bound C4b following serum incubation. The average number of C3d molecules per cell on normal EDTA cells was 557 and average Ko was 3.6 x 10(7) l/mol.

Animals

Oxygenation properties and intraerythrocytic constituents of human blood when stored in different media of ACD and CPD.

In the blood stored in acid-citrate-dextrose solution (ACD blood), the oxygen affinity and red cell 2, 3-diphosphoglycerate (2, 3-DPG) content showed parallel exponential decays with half-lives of 3 to 4 days. In the blood stored in citrate-phosphate-dextrose solution (CPD blood), the two parameters increased during the first 4 days before showing the same decay as that seen in the ACD blood. There was no significant change in the transmembrane pH gradient of the red cells, and thus the intracellular pH at the plasma pH of 7.40 was always in the range of 7.17 +/- 0.02 throughout the period of storage in ACD medium. In both ACD and CPD blood, the Hill exponent n was always normal (approximately 2.8) while the Bohr coefficient (delta log P50/delta pH) rose along with the lapse of time for preservation. The oxygen affinity of the CPD blood was less influenced by the red cell 2, 3-DPG than was that of the ACD blood. This phenomenon was thought to derive from higher concentration of salts within the CPD-stored red cells. The efficiency of blood oxygen transport in ACD and CPD blood was compared.

Blood Preservation

Blood storage XXIII: 2,3-DPG maintenance for six weeks in a CPD-adenine-inosine preservative with and without methylene blue.

In a pilot study the optimal concentration of inosine for 2,3-DPG maintenance in a CPD-adenine (0.25 mM) preservative was confirmed to be at lease 10 mM. In these experiments, 2,3-DPG maintenance was nearly normal for six weeks of storage in CPD-adenine-inosine (10 mM) preservative with or without methylene blue. The control preservative lacking inosine showed a statistically significant decrease in 2,3-DPG concentrations after the 3rd week. Finally, 2,3-DPG levels were significantly better maintained in CPD-adenine preservatives that contained 15 mM concentrations of inosine, whether methylene blue was present or not (10(-6)M), compared to CPD-adenine-inosine preservatives that contained 5 mM inosine, with or without methylene blue. The methylene blue effect, while it can be demonstrated in most experiments to help the red blood cell maintain 2,3-DPG during prolonged blood storage, is judges to be a slight value. However, inosine is of great value in maintaining 2,3-DPG for prolonged (five to six weeks) liquid storage.

Adenine

Blood preservation XXVI, CPD-adenine packed cells: benefits of increasing the glucose.

In searching for the optimal glucose concentration, this lab has monitored ATP, 2,3-DPG, pH, and glucose levels of samples taken from full blood units stored for 6 weeks at 4 C. The blood was collected into CPD-adenine containing 100, 125, 150, 175, and 200 per cent of the glucose present in CPD. The units were stored as whole blood, soft packed (50 to 70% Hct), or hard packed units (80 to 95% Hct). ATP values in general did not decrease very greatly in whole blood units and only moderately in soft packed units. However, in hard packed units a steady progressive decrease in the ATP values was seen to begin at day 14. In these hard-packed units the only improvement with extra glucose was seen beginning at day 14 when ATP maintenance was better with 200 per cent glucose, but the improvement was not significant until day 42. However, at 35 days the ATP values for 200 and 175 per cent glucose were noticeably better than for the other preservatives. Therefore, it appears from this study that the glucose concentration in CPD-adenine for hard-packed cells should be at least 175 per cent of that in regularly formulated CPD. Also, there would appear to be an advantage of having 200 per cent glucose in those units of blood that may be stored beyond 35 days for emergency blood shortage times.

Acid-Base Equilibrium

Studies on citrate-phosphate-dextrose (CPD) blood supplemented with adenine.

The effect of varying adenine concentrations in citrate-phosphate-dextrose (CPD) blood was studied in an attempt to optimize the storage conditions for human erythrocytes with regard to posttransfusion viability and oxygen release function. The maintenance of diphosphoglycerate (DPG) was impaired by adenine supplementation; this effect was closely related to the adenine concentration. A 0.25 mM adenine concentration in CPD blood improved the adenosine triphosphate (ATP) levels and the posttransfusion viability markedly, without appreciably impairing the DPG maintenance. The results suggest that CPD solution supplemented with adenine to give a 0.25 mM concentration in the blood is a better preservative for human erythrocytes than the commonly used acid-citrate-dextrose (ACD), CPD, and ACD-adenine solutions with regard to posttransfusion viability and oxygen release function. Adenine addition to this low concentration is not expected to cause renal damage even after massive transfusion.

Adenine

[Changes in ionized calcium and citrate levels in dogs during mechanical autotransfusion with heparin, ACD and CPD].

The control of ionized calcium (Ca++), total calcium, and citrate levels in serum were determined in dogs during autotransfusion (AT) of blood stabilized with heparin, ACD (formula B) and CPD. Blood samples were taken according to the changes of aortic pressure (AOP), which was continuously monitored. Taking the values during the stable phase of AOP preceding the AT as baseline, Ca++ dropped by 27% with ACD and by 34% with CPD at the maximum decrease of AOP immediately after the AT. The corresponding increase of citrate was 174% with ACD and 521% with CPD, while total calcium remained stable. Thus cardiac depression after AT of citrated blood seems to be mainly caused by the drop of Ca++, which is significantly more pronounced with CPD, corresponding to the higher content of citrate.

Animals

Hemoglobin function in stored blood, XVII. Maintenance of red cell 2,3 DPG (function) and ATP (viability) for six weeks in ACD or CPD-adenine-inosine-methylene blue.

Blood preservatives containing adenine for six week storage have been prepared with inosine and methylene blue at various pH levels in order to maintain, 23-DPG levels for immediate oxygen transport upon transfusion. In one experiment, the adverse effect of a high pH on ATP maintenance was demonstrated in the presence of methylene blue and inosine. In this and other experiments it was clear that ATP was better maintained in low pH preservatives and DPG better maintained in higher pH preservatives. However, 2,3-DPG levels were kept from falling with CPD-adenine-inosine over a wide range of pH values. A CPD-adenine-inosine preservative at a pH 5.8 maintained normal DPG levels for three weeks of storage. A similar preservative but with a pH of 6.6 maintained normal DPG levels for 35 days of storage. It is suggested that if all blood bank units are going to have normal DPG levels for optimal oxygen transport at the time of transfusion then a CPD preservative with a higher pH and/or metabolic nutrients and regulators such as inosine or methylene blue would be required.

Adenine

The effect of agitation on in vitro metabolism of erythrocytes stored in CPD-adenine.

Agitation of blood stored in plastic containers has been reported to lead to improved posttransfusion survival and it has been found that, in some media, agitation has improved erythrocyte 2,3-diphosphoglycerate (2,3-DPG) levels. Using CPD II media (CPD with 277.5 mM glucose and 2.04 mM adenine), we were not able to identify any improvement in levels of adenosine triphosphate, 2,3-DPG or glucose in whole blood under various agitation conditions when compared with nonagitated control. The 2,3-DPG level was moderately improved through 28 days in the 90 per cent hematocrit packed erythrocytes but the results were not considered to be significantly beneficial to warrant agitation. Thus, the application of agitation to the CPD II blood storage system was of no great benefit in improving metabolic intermediate levels.

Adenine

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.

Adenine

Blood storage XXIV: red blood cell 2,3-DPG and ATP maintenance for six weeks in CPD-adenine with higher phosphate, pyruvate, and dihydroxyacetone.

The individual and collective effects of various phosphate, pyruvate and dihydroxyacetone concentrations on 2,3-DPG and ATP maintenance during blood storage with CPD-adenine (0.25 mM), were studied. Phosphate concentrations ranged from 2 to 100 mM. Low concentations were best for 2,3-DPG maintenance during the first three weeks, after which there was no difference. ATP concentrations were better maintained by the highest phosphate concentrations in the first week. After the second week the lower concentrations of phosphate were better. With pyruvate 40 and 60 mM were the best for 2,3-DPG levels through six weeks of storage. ATP concentrations were poorest with high pyruvate. Maintenance of 2,3-DPG was above half normal for six weeks of storage in the 60, 80 and 100 mM DHA preservatives. ATP concentrations were best maintained in the preservative lacking DHA. Combinations of phosphate, pyruvate and DHA in concentrations which had been found to be effective when used individually were studied. Best maintenance of 2,3-DPG (above half normal levels) for six weeks was afforded by pyruvate, phosphate and DHA, and by pyruvate and DHA. ATP maintenance was best afforded by CPD-adenine alone and CPD-adenine with pyruvate and phosphate. Pyruvate alone maintained ATP less well and the pyruvate-DHA was worst. Intermediate in maintenance of ATP was the preservative containing pyruvate, phosphate and DHA.

Adenine

Oxygen dissociation after transfusion of blood stored in ACD or CPD solution.

In 20 patients undergoing open-heart surgery, 2,3-diphosphoglycerate (2,3-DPG) concentrations, oxygen affinity of hemoglobin (Po2 at half saturation of hemoglobin with oxygen [P50]), hemoglobin concentration, and pH were measured repeatedly. Measurements were made before and at various times after open-heart surgery and replacement of blood loss with blood stored in acid-citrate-dextrose (ACD) or citrate-phosphate-dextrose (CPD) solutions for less than 72 hours (10 cases per group). Infusion of ACD blood caused P50 and 2,3-DPG concentration to decrease significantly after the operation. The infusion of blood stored in CPD did not significantly increase the oxygen affinity. No significant changes in hemoglobin concentration or pH were observed immediately after the operation in either group. To compensate for the increased oxygen affinity, there must be a rise in cardiac output or more likely a decrease in venous Po2. The transfusion of CPD blood, therefore, is more favorable in terms of oxygen supply, particularly in patients who have had cardiac surgery.

Adult

[Studies of thrombocyte function in CPD blood].

The CPD stabilizer according to Gibson with an addition of 1.25 mMol adeninesulfate and 2.50 mMol guanosine is used in blood storage for better preserving 2.3-bis-phosphoglycerate of erythrocytes. Here platelet-rich CPD plasma was investigated before and during a 3 days storage at 4 degrees C or room temperature with regard to preserving the global thrombocyte function. The latter consists in the ability to seal blood vessels and is tested by means of pressure registration in combined thrombocyte-aggregation-adhesion (DKTA method) as an ability to close the pores of a sieve by adding 10(-5) mM/l of ADP. At room temperature this thrombocyte function is approximately 0 following 3 days of storage in CPD plasma excess without shaking. When stored at 4 degrees C it is preserved to a slight degree. Loss of thrombocyte function will depend on pH, thus being particularly evident at room temperature.

Blood Platelets

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.

Adenine

Chemical and hematological changes in stored CPD blood.

Blood was drawn from ten healthy volunteer donors into citrate-phosphate-dextrose (CPD) anticoagulant and placed on the quarantine shelf of the blood bank refrigerator. Plasma dextrose, sodium, potassium, chloride, bicarbonate, GOT, LDH, and hemoglobin as well as WBC, hematocrit, MCV, MCHC, whole blood pH, and ammonia were measured on all samples initially and at one, two, seven, 14, 21, and 28 days of storage at 4 C. Whole blood lactate also was analyzed serially on five of the units. An additional 27 units of CPD bank blood (two to 21 days of age), routinely processed, handled, and stored by the blood bank, were submitted to the same analyses on the day of administration to the patient. Five of these processed units, 21 days old, were resampled at 28 days. Results of the analyses are presented and discussed. The most pronounced changes were seen for dextrose, potassium, bicarbonate, lactate, LDH, ammonia, and hemoglobin. Plasma dextrose and bicarbonate declined in concentration while potassium, lactate, LDH, ammonia, and hemoglobin rose with storage. In general, changes in the regularly processed, singly sampled bank units were greater than those observed in the specially processed, quarantined units sampled serially. This study indicates that routine transportation, processing, and handling of bank blood may lead to increased biochemical alteration.

Ammonia