No. 9] Posttransfusion Survival of the Stored Red Blood Cells 619

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1 618 [Vol. 36, 147. Posttrans f usion Survival o f the Red Blood Cells Stored in a Medium Containing Adenine and Inosine By Takehisa WADA,*' Fumimaro TAKAKU,*' Kiku NAKAO,*' Makoto NAKAO,**' Toshiko NAKAo,**' and Haruhisa YosHIKAwA***' (Comm. by T. FORUHATA,, M.J.A., Nov. 12, 1960) Since Gabrio et a1.1' reported that the addition of inosine could prolong the survival time of human red blood cells when stored in acid citrate-dextrose (ACD) solution at 4 C, cooperative studies2'3' have been carried out in which the posttransfusion survival of the red blood cells stored in ACD alone and ACD containing inosine (ACDI) was compared. On the basis of the results of biochemical studies on the phosphorus metabolism in human red blood cells, M. Nakao et al, have proposed a theory explaining the process of adenosine triphosphate (ATP) degradation encountered in the human red blood cells during preservation,4' and have reported briefly that the addition of adenine and inosine suppressed markedly not only the ATP degradation in red blood cells, but also the decrease in the glycolytic activity, the increase in osmotic fragility and the shape-change of the cells.5' This note is concerned with the results of studies on the posttransfusion survival of the human red blood cells which were preserved in ACD solution containing both adenine and inosine (ACDIA) for 3 weeks. Materials and methods. About 200 ml of whole blood was collected from a professional donor, divided into three portions and placed in sterile siliconized glass bottles containing ACD solution of one-fifth volume of the whole blood. Either one-tenth volume of the saline of adenine and inosine or inosine alone, or saline alone, was added to each bottle containing ACD blood. The final concentration of adenine and inosine in the bottle was 1.25 mmol/ l and 2.5 mmol/l, respectively. After preservation, 150 micro-curies of radioactive sodium chromate (Na2Cr5104) was added to every 50 ml of the whole blood, and allowed to stand at room temperature for 30 to 60 minutes with gentle mixing every five minutes. Then the whole blood was centrifuged at low speed to remove the supernatant, and red cells were washed once with the saline and resuspended in the saline to the original volume. With *) Department of Medicine, School of Medicine, Gunma University, Maebashi. **) Department of Biochemistry, School of Medicine, Gunma University, Maebashi. ***) Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Tokyo.

2 No. 9] Posttransfusion Survival of the Stored Red Blood Cells 619 this method the red blood cell usually absorbed over 90 per cent of the added Cr51. Then 15 to 20 ml of the red blood cell suspension was transfused into ABO system-compatible recipients, who were apparently healthy or without any hematological disorders. All these procedures were carried out with the usual aseptic precautions. The first blood sample was taken from the cubital vein opposite to that used for the transfusion 5 or 15 minutes after the transfusion and the following blood samples were collected at suitable time intervals. The radioactivity of the blood samples was measured by using a well-type scintillation counter, apparent posttransf usion survival of the stored red blood cells was estimated by the chromium method, and "apparent circulating blood volume" was calculated by dividing the total counts of radioactivity of the infused red blood cells by the counts of radioactivity of the venous blood taken 15 minutes after transfusion. Through this observation period any noticeable side effects of the transfusion was not observed. Results. a) Decay rate of radioactivity in circulating blood. A decay of radioactivity for the 24 hours after transfusion of the preserved human red blood cells was determined. Representing the radioactivity of circulating blood 15 minutes after the transfusion as 100 per cent, the blood cells stored in ACDIA (13 cases) solution Fig, 1. Fig. 2 Decay of radioactivity in circulating blood after transfusion of red blood cells which had been stored for 8 weeks and labeled with Cr51 x - in ACDIA sol., in ACDI sol., o in ACD sol. Radioactivity of blood 5 min after the transfusion was represented as 100 per cent.

3 620 T. WADA et al. [Vol. 36, showed radioactivity as high as 39.5 per cent on an average (range from 10 to 66%), while the blood stored in ACDI (3 cases) and ACD (7 cases) solutions, dropped to 3.3 per cent (0 to 7%) and to 18.3 per cent (0 to 46%), respectively. Following this, the posttransf usion disappearance rates of radioactivity from the circulating blood were investigated using the red blood cells taken from a single donor, divided into 3 portions and stored in each of 3 solutions, respectively. In this case, the radioactivity of the circulating blood 5 minutes after the transfusion was settled as 100 per cent. As indicated on the left of Fig. 1, the red blood cells stored in ACD solution showed a steep drop in radioactivity, reaching 25 and 35 per cent 15 minutes after transfusion, and there was no longer detectable radioactivity 16 hours after transfusion. The blood stored in ACDI solution showed 90 per cent 15 minutes after transfusion, the further disappearance rate was surprisingly rapid and fell to less than 2 per cent 16 hours later. On the other hand, the blood stored in ACDIA solution showed a high activity of 96 per cent in both of two cases 15 minutes after transfusion, and still retained a radioactivity of 22.5 per cent on the average (19 and 26 /, respectively) even 24 hours after transfusion. Other similar experiments revealed the same trend, as shown on the right in Fig. 2. This fact clearly indicates that the red blood cells stored in ACDIA solution for 8 weeks showed markedly high posttransfusion viability, compared with that of the red blood cells preserved in either ACD alone or ACDI solution. b) "Apparent circulating blood volume". "Apparent circulating blood volume" was calculated by the method described before, and was represented as ml per kg of body weight and summarized in Table I. A normal value of the circulating blood volume measured by the Cr51 method is widely accepted as ranging from 80 to 85 ml/kg body weight. In our experiments, the apparent circulating blood volume, which was measured in healthy persons by using the Cr51- labeled red blood cells stored in ACDIA solution for 8 weeks, showed almost normal values ranging from 67.3 to 94.3 ml/ kg (mean value 78.1±9.4 ml/kg) and the value measured by using the blood stored in ACDI solution in the same way was slightly increased to 92, 90.5, and 80.3 ml/kg (mean value 87.6±9.0 ml/kg), respectively. However, the apparent circulating blood values which were measured by using the blood stored in ACD alone showed strikingly large values ranging from 221 to 940 ml /kg (mean value 456±269), as shown in Table I. c) Posttransf usion survival of the stored red blood cells. Posttransfusion survival time was investigated by the Cr51 method applied to each blood sample stored in one of the three different preservation

4 No. 9] Posttransfusion Survival of the Stored Red Blood Cells 621 solutions. zero time, observed. counts of In this case, the 48 hours posttransfusion was settled as and the decay of radioactivity in the circulating blood was From the decay curve drawn by plotting the number of radioactivity against time on semilogarithmic paper, the Table I Table II

5 622 T. WADA et al. [Vol. 36, apparent half survival time was obtained. The normal value of apparent half survival times of red blood cells obtained in our laboratory ranges from 27 to 32 days. As shown in Table II, the posttransf usion half survival times ranged between 20 and 32 days (mean value : 24.3±4.1 days). It is noteworthy that the more rapid the decay of radioactivity of circulating blood during the first 24 hours posttransfusion, the shorter was the apparent half survival time. It is also important to note that measuring the posttransf usion survival time later than 48 hours after transfusion was impossible in the majority of the subjects into whom the red blood cells stored either in ACD or ACDI solution for 8 weeks was transfused. The reason for this fact is that the red blood cells stored in these solutions had too little viability to remain in the circulating blood for a period of more than 48 hours following transfusion. Discussion. The posttransfusion survival of the stored red blood cells mainly depends both upon a length of preservation period and a chemical composition of the preservation solution. In order to preserve in vitro the human blood cells retaining high viability for as long as possible, it is necessary to maintain a low metabolic rate in the red cells and to provide the inevitable materials for their essential metabolism. From the viewpoint of biochemistry, M. Nakao et al.5' have emphasized that the addition of both of adenine and inosine to ACD solution is very effective in preventing human red blood cells from undergoing ATP deterioration. In carrying out this series of experiments the present authors attempted to apply this principle to clinical medicine. For this purpose the following three types of observation were made: 1) The decay rate of radioactivity in circulating blood within 24 hours posttransfusion, 2) the apparent circulating blood volume, and 3) the posttransf usion half survival time. The data obtained from these observations showed clear evidence that the human red blood cells stored in ACDIA solution for 8 weeks maintained higher posttransfusional viability compared with the blood stored either in ACDI or ACD alone. However, there was somewhat marked difference in posttransf usion viability between individual samples of the blood units stored in ACDIA solution. The highest posttransf usion survival in our experiments was 66 per cent at 24 hours posttransf usion and the lowest was 10 per cent. The reason for this diversity of results obtained from our observations is not clear at the present time. One of the possible explanations of this fact, however, is that the red blood cells population could be changed to contain much more immatured cells, because the blood was collected

6 No. 9] Posttransf usion Survival of the Stored Red Blood Cells 623 from professional donors without any blood examination at that time. It is generally accepted that the immature red blood cells are more fragile during preservation than the mature ones. Conclusion. In conclusion we might say that the presence of both adenine and inosine in acid-citrate-dextrose solution provides a suitable preservation media to maintain in human red blood cells a high viability for a long time. This work was aided by a Grant from the Scientific Research Fund of the Ministry of Education. The authors wish to thank Dr. Hiroshi Toyama, Blood Transfusion Center, the University of Tokyo Hospital, and Dr. Masaji Miyamoto, the President of Plasma Institute, Tokyo, for making available facilities for the preservation of the blood samples. References 1) Gabrio, B. W., Donohue, D. M., Huennekens, F. M., and Finch, C. A.,: J. Clin. Invest., 35, 657 (1956). 2) Lange, R. D., Crosby, W. H., Donohue, D. M., Finch, C. A., Gibson, J. G., II., McManus, T. J., and Strumia, M. M.,: J. Clin. Invest., 37, 1485 (1958). 3) Schmidt, H. A. E., Schmidt, H., Keiderling, W., Matthes, M., and Feiser, W.,: Acta Heamt., 23, 208 (1960). 4) Nakao, M., Tatibana, M., and Yoshikawa, H.,: J. Biochem., 46, 672 (1960). 5) Nakao, M., Nakao, T., Arimatsu, Y., and Yoshikawa, H.,: Proc. Japan Acad., 36, 43 (1960).