with their viability and resistance to hemolysis ,19

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1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, V o l. 1, N o. 2 C o p y r i g h t , I n s t i t u t e f o r C l i n i c a l S c i e n c e In Vitro Parameters of the Integrity of the Preserved Erythrocyte JOHN B. DERRICK, Ph.D. a n d RITA M cconn, Ph.D. The N ew York Blood Center and A lbert Einstein College of Medicine, N ew York, NY The ultimate aim in devising methods of preserving red blood cells is to maintain them in their normal in vivo state during storage. Currently the main criterion of satisfactory red blood cell preservation is a normal post-transfusion survival time. It is, however, primarily an index of viability and thus of only limited value as an expression of the functional integrity of the preserved cells. To be comprehensive, evaluation of the efficiency of red blood cell preservation should also include criteria that are indicative of cell function, in particular of oxygen transport. Because, at the present time, there are no in vivo criteria which specifically characterize this aspect, this must be attempted through the use of in vitro parameters. Our experience indicates that measurement of the param eters listed below should, along with the post-transfusion survival time, constitute the absolute minimum requirement for the comprehensive evaluation of m ostf m ethods of red blood cell preservation: the cell content of adenosine triphosphate (A T P), 2-3 diphosphoglyceric acid (2-3 D PG ), sodium and potassium and the oxygen dissociation curve. Our choice of these particular indices was based on the following considerations. The ATP content of red blood cells has long been known to b e closely correlated This work supported by N IH grants, H E and RR fw here there is a possibility of physical damage or dénaturation, such as m ight occur in methods of preservation involving freezing, in vitro incubation studies are also indicated. with their viability and resistance to hemolysis ,19 and with their capability to reinitiate glycolysis, to maintain shape18,21 and to actively transport sodium and potassium.5'12 23 More recently, statements by the Benesches1 concerning the effects of ATP on the oxygen affinity of hemoglobin suggest that the intracellular concentration of this organic phosphate compound may also have a direct effect on oxygen transport. The method of Adam2 is used in our laboratory for the determination of ATP. The normal concentration in freshf red blood cells collected in acid-citrate dextrose (ACD) solution is 3.65+:.73 /xmatp/g. Hb. It also has been established that red blood cells are still capable of reinitiating glycolysis under in vitro incubation conditions at 37 C with concentrations of ATP in the range of 2.15±,2^M /g.hb. The biological role of 2-3 DPG has been defined by the studies of Chanutin and Curnish4 and the Benesches1 as being closely associated with the ability of hemoglobin to transport oxygen. Studies by the authors on patients receiving massive transfusions have extended this observation to the red blood cell.15,18 Other evidence7,13 has implicated the intracellular content of this metabolite with the regulation of the potassium, and probably sodium, content of red blood cells. Indeed, at the present state of knowledge, it is tem pting to hypothesize that part of the effect of 2-3 DPG on the affinity of hemoglobin for oxygen may be attributable to effects on i < 24 hours old. Presented at the A pplied Seminar on Chemical Hematology, Novem ber,

2 IN VITRO PARAMETERS OF TH E INTEGRITY OF THE PRESERVED ERYTHROCYTE 135 the sodium-potassium content of the red cell. The method of Krimsky2 is eminently satisfactory for the determination of erythrocyte 2-3 DPG content. Normal concentrations in red blood cells freshly collected* in ACD from non-smokers have been established as falling in the range of 0.79 ±.09 M /M Hb. The use of the sodium and potassium content of red blood cells as indices of their state of preservation was prompted initially by the necessity to measure the effects on the intracellular concentrations of these ions of cell washing procedures used in cryopreservation methods. Subsequently, it became apparent that measurement of the passive and active flux of these ions during storage and also during experimental incubation was of considerable value as an index of the effects not only of cryopreservation but also of various other methods of preservation on both the physical and functional integrity of the cell membrane. The possibility of an interrelationship between changes in the concentrations of sodium and potassium and changes in the 2, 3 DPG content of red blood cells was noted earlier in this discussion. The intracellular concentrations of sodium and potassium are determined using a modification of the method of Funder and W ieth.8 10 The normal concentration of sodium in red blood cells freshly collectedf in ACD has been established as 13.2± 1.5 m Eq/K g RBC and that of potassium 84.3± 3.8 m Eq/K g RBC. The changes which occur within erythrocytes during storage at 4 C result in an increase of the affinity of the hemoglobin for oxygen and the extent to which they have affected the respiratory function of the cells can best be characterized by the * < 8 hours old. f < 24 hours old. position of the oxygen dissociation curve. This value is defined by the partial pressure of oxygen required to produce half saturation (reduced hemoglobin concentration = oxyhemoglobin concentration) at constant ph and temperature and is commonly written as the P50. The normal value for man is mm Hg, ph c 7.10, 36.5 C. An increase in P50 is denoted by a shift to the right, i.e., a decreased affinity of hemoglobin for oxygen, whereas a decrease in P50 is denoted by a shift in the reverse direction. The dissociation curves for blood are determined by the method of Duvelleroy, et al9 using a hemoglobin dissociation curve analyzer. The mean P50 has been established from studies on non-smoking volunteers to be 25.2 ± 1.1 mm Hg. The proposal that these criteria constitute the minimum requirement for the comprehensive evaluation of preserved red blood cells is borne out by the data presented in figures 1 and 2. These data were accumulated in studies in which these criteria were used to evaluate the effects of the prestorage addition of adenine (Im M ) and/or inosine (lomm) to ACD blood.7'15,16 Using ATP as the criterion and taking the point at 21 days of storage as an example, inosine alone would appear to best maintain the ATP content of the preserved erythrocytes (figure 1). Upon examining the 2-3 DPG content of these preserved cells after the same period of storage, it appears that adenine with inosine is slightly more effective in delaying the loss of 2-3 DPG. T he oxygen dissociation curves of the variously preserved red cells indicate th at the prestorage addition of adenine and inosine is decidedly the most effective means of delaying the shift to the left which occurs with storage at 4 C. The effects of the various methods of preservation on the sodium and potassium content of the stored red blood cells are shown in figure 2. When the prestorage

3 136 DERRICK AND M C CONN CHANGES IN PsqPPG A N D ATP LEVELS DURING LONG TERM STORAGE OF A C D BLOOD WITH PRE-STORAGE A D D IT IO N OF ADENINE A N D /O R INOSINE F ig u r e 1.

4 IN VITRO PARAMETERS OF THE INTEGRITY OF THE PRESERVED ERYTHROCYTE 137 additions involve inosine, there is, concomitant with the favorable effects on oxygen affinity, a marked acceleration in the rate of deterioration of the sodiumpotassium gradient which occurs in red cells stored at 4 C. F ig u r e 2.

5 138 DERRICK AND M C CONN These results show that prestorage addition of adenine and inosine, while effectively improving the maintenance of the P50 and the ATP and 2-3 DPG content in the stored erythrocytes, do so at the expense of maintaining the gradient of intracellular electrolytes. Until it becomes possible to define to what extent the composition of the red blood cell may change during storage before therapeutic efficacy is significantly affected, it is suggested that evaluation of methods of red blood cell preservation should include measurement of parameters indicative of their effectiveness in maintaining a normal intracellular environment. The data presented here demonstrate that these four criteria, along w ith the obvious factor of viability, constitute an absolute minimum requirement for the comprehensive assessment of the state of preservation of red blood cells. References 1. B e n e s c h, R. a n d B e n e s c h, R. E.: The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem. Biophys. Res. Commun. 26: 162, B e r g m e y e r, H. U.: Methods of Enzymatic Analysis, p. 539, Academic Press, N ew York Ibid, p C h a n u t in, A. a n d C u r n is h, R. R.: Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121: 96, D a n o w s k i, T. S.: The transfer of potassium across the human blood cell membrane. J. Biol. Chem. 139: 693, D e r n, R. J., B r e w e r, G. J., a n d W io r k o w s k i, J. J.: Studies on the preservation of human blood. II. The relationship of erythrocyte adenosine triphosphate levels and other in vitro measurements to red cell storageability. J. Lab. Clin. Med. 69: 968, D e r r ic k, J. B.: Effects of inosine metabolism on the ionic equilibrium of the red blood cell stored at 4 C. Fed. Proc. 29:424, D e r r ic k, J. B., L in d, M., a n d R o w e, A. W.: Studies of the Metabolic Integrity of Human Red Blood Cells after Cryopreservation. I. Effects of low-glycerol-rapid-freeze preservation on energy status and intracellular sodium and potassium. Transfusion 9: 317, D u v e l l e r o y, M. A., B u c k l e s, D. G., R o s e n - k a im e r, S., T u n g, C., a n d L a v e r s, M. D.: An oxyhemoglobin dissociation analyzer, J. Appl. Physiol. 28: 227, F u n d e r, J. a n d W i e t h, J. O.: Determination of sodium, potassium and water in human red blood cells. Scand. J. Clin. Lab. Invest. 18: 151, C a b r io, B. W., F i n c h, C. A., a n d H u e n - n e k e n s, F. M.: Erythrocyte preservation: A topic in molecular biochemistry. Blood 11: 103, G a r d o s, G.: Akkumulation der Kaliummionen durch menschilde Blutkörperchen. Acta Physiol. Acad. Sei. Hung. 6 : 191, Ibid., The mechanism of ion transport in human erythocytes. Acta Biochim. Biophys. Acad. Sei. Hung. 1: 139, K r e b s, H. H. a n d K o r n b e r g, H. H.: A survey of the energy transformation in living matter. Ergb Physiol. 49: 212, M c C o n n, R., a n d D e r r ic k, J. B.: The respiratory function of blood: Tranfusion and blood storage. ( In press.) Anesthesiology. 16. M c C o n n, R., D e l G u e r c io, R. L. M., R o w e, A. W., a n d D e r r ic k, J. B.: Massive blood transfusion and the transport and release of oxygen. Fed. Proc. 29: 787, M o l l is o n, P. L. a n d R o b in s o n, M. A.: Observation on the effects of purine nucleotides on red cell preservation. Brit. J. Haemat. 5: 331, N a k a o, M., N a k a o, T., T a t ib a n a, M., Yosh ik a w a, H., a n d A b e, T.: Effect of inosine and adenine on adenosine triphosphate regeneration and shape transformation in long stored erythrocytes. Biochem. Biophys. Acta 32: 564, R a p a p o r t, S.: Dimensional, osmotic and chemical changes of erythrocytes in stored blood. I. Blood preserved in sodium citrate, neutral and acid citrate glucose (A C D ) mixtures. J. Clin. Invest. 26: 591, T su bo i, K. K.: Limiting role of adenine nucleotides in the glycolysis of the human erythrocyte. J. Biol. Chem : 582, W e e d, R. I. a n d L a C e l l e, P. L.: ATP dependence of erythrocyte deformability: Relation to in vivo survival and blood storage. American National Red Cross Second Annual Science Symposium, Washington, D C, W h i t t a m, R.: Potassium movement and ATP in human red cells. J. Physiol 140: 479, Ibid., The asymmetrical stimulation of a membrane adenosine triphosphatase in relation to active cation transport. Biochem. J. 8 4 : 110, 1962.