R. B. Dawson

The Use of Ion-Exchange Resins as a Blood Preservation System

A system using ion‐exchange resins for the storage of whole blood was investigated. Blood was collected into CPD, thoroughly mixed and then divided into equal amounts. To one half of the split units, four grams of phosphate charged Amberlite IR‐45 resin were added, while no additions were made to the other split units. Both aliquots were stored at 4 C, adhering to routine blood banking, standards. The blood stored with the resin for up to 28 days maintained high levels of 2,3‐DPG and adequate levels of ATP. This new resin system has several advantages over the present blood preservation technology. There is initial increase in pH that promotes greater glucose utilization. Inorganic phosphate is provided to maintain both ATP and 2,3‐DPG levels. The resin buffers the blood and maintains a narrower pH range for red blood cell enzyme activity. The resin is totally inert, completely filterable and has the ability to adsorb bacteria from the suspending solution.

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.

Blood preservation XXVII. Fructose and mannose maintain ATP and 2,3-DPG.

Mannose and fructose as well as glucose have been shown to be effective for maintaining ATP and thus viability of stored red blood cells. Normal 2,3‐DPG levels are desirable in stored red blood cells to provide the needed oxygen transport upon transfusion. ATP levels in stored concentrated red blood cells in the new preservative, CPD‐adenine (citrate‐phosphate‐dextrose‐adenine) become critically low in the 5th week. In this study two hexoses and two pentoses are compared with dextrose in their ability to maintain ATP and 2,3‐DPG. ATP levels were best maintained by fructose, then dextrose and mannose. ATP levels had fallen to critically low levels by four weeks with ribose and xylose. Red blood cell 2,3‐DPG concentrations were also maintained by hexoses, with mannose being best, dextrose and fructose being similar. When ribose was used in addition to dextrose in CPD‐adenine, ATP maintenance was improved and under the same conditions xylose improved 2,3‐DPG maintenance. Fructose and mannose may be as useful as dextrose in citrate‐phosphate preservatives for maintaining ATP and 2,3‐DPG levels. Also, ribose and xylose may help the maintenance of ATP and 2,3‐DPG, respectively, in CPD‐adenine.

Blood preservation 35. Red cell 2,3-DPG and ATP maintained by DHA-ascorbate-phosphate.

DHA (dihydroxyacetone, 60 mM) with ascorbic acid (d‐ascorbate, 10 mM) kept 2,3‐DPG concentrations above normal for six weeks. Levels of 2,3‐ DPG were below normal after four weeks with DHA alone and after two weeks with DHA‐ascorbate‐phosphate. As in previous studies, high phosphate concentrations decreased 2,3‐DPG maintenance. ATP maintenance was best achieved with the following (in order of performance): DHA‐ phosphate (20 mM); DHA‐phosphate (10 mM); the control, CPD‐adenine preservative; Phosphate 20 mM; and DHA. DHA with ascorbate provides normal 2,3‐DPG for six weeks. The adverse effects of DHA and DHA with ascorbate on ATP levels are modified by 10 mM phosphate.

Blood preservation XLIV. 2,3-DPG maintenance by dehydroascorbate better than D-ascorbic acid

A study was designed to compare the effects of D‐ascorbate and dehydroascorbate on red blood cell metabolism during blood storage. Dehydroascorbate increased red blood cell concentrations of 2,3‐DPG such that the levels are above normal for four weeks and normal at six weeks of storage. In contrast, there is a gradual decrease in 2,3‐DPG levels with D‐ascorbate such that the levels are approximately 80 per cent of normal after six weeks. ATP levels were adversely effected such that the worst levels were produced by 10 and 5 mM dehydroascorbate, with 10 mM having a more adversive effect than 5 mM. Intermediate levels of ATP were produced by D‐ascorbate, with the 10 mM concentration. The control CPD‐adenine preservative maintained near normal ATP levels for the entire six‐week storage period. pH values were initially slightly lower with dehydroascorbate compared to the other preservatives early in storage, the difference being slightly over 0.1 pH units.

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.

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.

Blood storage XXV: Ascorbic acid (vitamin C) and dihydroxyacetone (DHA) maintenance of 2,3-DPG for six weeks in CPD-adenine

In experiments in which ascorbate was included in CPD‐adenine preservatives, 2,3‐DPG levels were maintained above normal for 28 days with an ascorbate concentration of 10 mM or higher and concentrations of 20 to 80 had no greater effect on 2,3‐DPG maintenance. Less ascorbate (5 mM) provided better 2,3‐DPG maintenance than was obtained with no ascorbate throughout six weeks of storage but was not as good as the higher concentrations after the third week. ATP concentrations were adversely affected by the presence of ascorbate. The highest ATP concentrations were without ascorbate, next highest with 5 mM, and the worst ATP was with 80 mM. The pH values did not differ from one preservative to another.

Blood Preservation XXIX. Pyruvate Maintains Normal Red Cell 2,3-DPG for Six Weeks of Storage in CPD-Adenine

Pyruvate was placed in experimental CPD‐adenine (0.25 mM) blood preservative mixtures in four concentrations ranging from 40 to 320 mM. In the 320 mM pyruvate preservative, 2,3‐DPG levels were elevated above normal for six weeks of whole blood storage at 4 C. The lower pyruvate concentrations maintained elevated or normal 2,3‐DPG levels for less time: four weeks with 160 mM, two weeks with 80 mM, and one week or less with 40 mM or the control. ATP values were best maintained in the control. The higher pyruvate concentrations resulted in the most rapid decreases at ATP. However, even the 320 mM pyruvate did not cause ATP to fall below 2 μM/gm of Hb. The higher pyruvate concentrations produced and maintained a higher pH during storage. On the other hand, 2,3‐DPG levels increased with pyruvate during the first week of storage when the pH was decreasing rapidly. This could be the result of its oxidation of NADH to NAD. The high pyruvate concentration which maintained elevated 2,3‐DPG levels throughout the six weeks might be simulating the effect reported in pyruvate kinase‐deficient red blood cells, in which blockage of glycolysis at that step is preventing 23‐DPG catabolism.

Hemoglobin function in stored blood. XIX. Inosine maintenance of 2,3-DPG for 35 days in a CPD-adenine preservative.

This study establishes that 10 mM inosine is a sufficient additive to maintain 2,3‐DPG levels in blood for five weeks of storage in CPD‐adenine. No previous experiments were done with CPD‐adenine (0.25 mM) using a design which would give statistical proof of the optimal concentration of inosine needed for maintenance of normal hemoglobin function (2,3‐DPG) for five weeks of blood bank storage.