The biochemical processes that occur during blood preservation or storage contribute to the diminution of the post-transfusion viability, i.e. survival of stored rec cells after transfusion to a recipient, and oxygen off-loading capacity of red cells, both of which are statistically correlated with the duration of storage period. Previous studies of stored red blood cells have indicated that intracellular levels of ATP (adenosine triphosphate) and 2,3-DPG (2,3-diphosphoglycerate) largely or entirely determine the post-transfusion viability and oxygen off-loading capabilities respectively of the red cell. It is well-established that both of these substances are products of glycolysis, a biochemical pathway that degrades glucose.
Maintenance of post-transfusion viability of stored red cells is closely correlated with the levels of cellular ATP, a high-energy compound. Glycolysis is the only ATP generated pathway in the red cell. It appears that ATP preserves membrane integrity by maintaining proper ionic gradients across the red cell membrane, adequate lipid turnover rate, hemoglobin in a functional state and normal equilibrium of oxidized and reduced glutathione, along with synthesis of adequate amounts of NAD+and NADP+, i.e. Nicotinamide adenine dinucleotide and its phosphate.
It is well-established that oxygen off-loading ability of red cells is determined by another glycolytic intermediate, 2,3-DPG. This compound declines as a function of time during storage, most likely secondary to both a decreased rate of synthesis and an increased rate of degradation. The red cells with low 2,3-DPG show increased oxygen affinity or decreased ability to release oxygen at the tissue level. Thus, the stored red cells are less efficient vehicles of oxygen transport, the most important function of the red cells--stated otherwise, this means that stored and therefore 2,3-DPG depleted red cells are of poor quality with regard to their function.
It follows that it would be highly desirable to be able to maintain near-normal red cell 2,3-DPG and ATP as sucn a result would have profound effects in terms of both red cell function and post-transfusion survivability in vivo. Thus, adequate maintenance of both of these compounds would permit the storage of red cells for a longer period of time, which would alleviate problems of shortage of blood for transfusion and low quality of stored blood.
Previous studies of stored whole blood and stored red cell concentrates have indicated that intracellular levels of ATP and 2,3-DPG are important in extending storage capabilities. To this end, several studies have been conducted which have incorporated various chemical additives along with CPD (citrate-phosphate-dextrose) anticoagulant to stimulate glycolysis, yielding a net increase in red cell ATP level. One of the commercial additives that has recently been studied is adenine. The incorporation of adenine along with CPD anticoagulant into stored blood appears to increase ADP (adenine diphosphate) levels, thereby driving the glycolytic equilibrium toward the synthesis of ATP. However, adenine has an adverse effect on the maintenance of the level of 2,3-DPG, i.e. it lowers 2,3-DPG level with concomitant poor function of the stored red cells.
Recent concern over the levels of ATP and 2,3-DPG has become a controversial subject. Because the main objective of transfusing patients is to provide or improve the oxygen delivery to the tissues, the blood oxygen affinity, directly determined by 2,3-DPG, is of critical importance. Therefore, in providing patents with suitable blood for transfusion, one must now consider not only red cell viability in vivo but also hemoglobin oxygen affinity for adequate oxygen transport function, the ultimate goal of red cell transfusion. As a result, research has also been geared towards incorporation of chemicals into the CPD and other preservative media to increase 2,3-DPG and ATP levels.
The significance of near-normal 2,3-DPG-containing red cells becomes self-evident when one examines various clinical conditions such as congestive heart failure, right to left cardiac shunts, and hypoxemia due to pulmonary disease, where patients singularly require the oxygen transport function of the transfused red cells. The transfused red cell, totally depleted of 2,3-DPG, is said to regain half the normal level of this substance within about 24 hours, but this increase may not be rapid enough to be effective in severely ill patients. Furthermore, it is not known whether the rate of resynthesis of 2,3-DPG in the donor cells given to a critically ill patient is comparable to that observed in normal recipients. Dennis et al. (Surgery 77 (6):741-747, June 1975) has reported a direct correlation between the ability to compensate for low 2,3-DPG levels and the severity of the illness of the patient. Blood with nearly normal hemoglobin-oxygen affinity is thus preferable for use in massive transfusions, particularly in infants, older patients, and patients with complicating cardiovascular and pulmonary disease.
The physiological effects of high oxygen-affinity (2,3-DPG depleted) red cells on the myocardial, cerebral, hepatic, and renal functions have not yet been fully evaluated, although patients requiring massive transfusions seem to be most susceptible to the adverse effects due to very low levels of 2,3-DPG; see Beutler et al., Vox Sang. 20:403-413 (1970).
Although numerous investigations indicate that the levels of ATP and 2,3-DPG can be better maintained when the two chief preservative solutions ACD (acid citrate dextrose) and CPD (citrate phosphate dextrose) are supplemented with adenine, inosine, or both during storage at 4.degree. C., this conclusion must be approached with some caution. As has been reported by Bunn et al. in New England Journal of Medicine 282:1414-1421 (1970), a patient receiving three or four units of thus-supplemented blood may develop hyperuricemia, which persists for approximately 24 hours. As reported by Valeri in J. Med. (Basel) 5(5):278-291 (1974), a further cause for concern is the possible renal toxicity of 2,8-dioxyadenine, a metabolite of adenine. Moreover, additives that maintain ATP level, i.e. adenine, tend to lower 2,3-DPG level and those that maintain 2,3-DPG tend to lower ATP level, thus making the maintenance of both of these compounds a currently unrealizable goal. No matter which chemical is used with an ACD or CPD preservative solution, it appears that only a combination of various chemical additives will maintain 2,3DPG levels in blood under refrigeration conditions for an acceptable period of time.
Dieindoerfer et al, in U.S. Pat. No. 3,795,581, disclose a method of storing and preserving whole blood using an aqueous solution of dihydroxyacetone to increase the 2,3-DPG content. In a later patent, 3,874,384, Deindoerfer et al disclose the use of a combination of dihydroxyacetone and ascorbic acid to maintain DPG levels in stored blood. Estep, No. 4,386,069, discloses the use of a fatty ester having at least two ester linkages comprising fatty hydrocarbon groups of from four to twelve carbon atoms each to enhance the preservation of normal red blood cell morphology during storage. Harmening, No. 4,112,070, discloses a process for extending the useful shelf life of red cells by maintaining adequate levels of ATP and 2,3-DPG by adding an insoluble polymer material as a source of inorganic phosphate ions during the storage period. Harmening-Pettiglion, No. 4,390,619, discloses a method for extending the shelf life of blood platelets by maintaining both the pH and ATP levels suitable for transfusion. This is accomplished by providing to the platelets a water-insoluble polymer containing releasable weakly basic buffer ions capable of continuously supplying buffer to the platelets.