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Chapter IX

Contents

CHAPTER IX

Preservative Solutions

DEFINITIONS AND CRITERIA

As pointed out elsewhere, there is a distinction between stored blood and preserved blood that is not always observed but that always should be. Muether and Andrews (1), whose extensive studies in this field will be mentioned later, were among the first to point it out. Stored blood is citrated blood which has been kept for hours or days, at temperatures between 36 and 46 F. (2 and 8 C.), and to which nothing has been added to inhibit deterioration of the erythrocytes; sodium citrate is an anticoagulant, not a preservative. Preserved blood is blood to which some additional substance has been added to check disintegration of the erythrocytes. It was the solution of the problem of halting this disintegration that made possible the use in oversea theaters of whole blood flown from the United States.

While varying criteria have been advanced for safe and effective preservation of blood, the following met essential military requirements:

1. The blood must be collected in a closed system and handled as little as possible at every step of processing to avoid contamination.

2. It must be kept at a low, constant temperature from collection to administration.

3. The technique of preservation must be simple and inexpensive.

4. The preserved blood must maintain to a high degree the properties of fresh blood if it is to serve its therapeutic purpose. This purpose can be accomplished only if the blood shows a low rate of hemolysis and a high rate of red blood cell survival.

5. The ultimate fate of transfused red blood cells determines the effect of the transfusion upon the recipient.

Red blood cell survival has been studied in detail by a number of observers, beginning in 1919 with Ashby (2), whose differentiation agglutination technique is still regarded by many observers as more efficient than the modern radioactive cell-tagging technique (p. 221) because the Ashby technique permits the observer to follow the red cells through their lifespan.

HISTORICAL NOTE

First Studies on Hemolysis

When Rous and Turner (3, 4), in 1916, published their work on the preservation of living red cells in vitro, they had been able to find only a single


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report on this or any other related study except the demonstration, in 1914, by Abel, Rowntree, and Turner (5), that only formed elements of the blood need to be replaced in healthy animals depleted by hemorrhage.

The results of the Rous-Turner studies were as follows:

1. The rapidity of hemolysis was generally increased when only electrolytes were added to the blood. When sodium citrate was used, human red blood cells tended to break down rather rapidly; disintegration occurred relatively early even when the smallest quantity that would prevent clotting was used.

2. Hemolysis was greatly diminished when saccharose or dextrose was added to the citrated blood. Human red blood cells remained intact for about 4 weeks when a solution was used consisting of two volumes of 3.8-percent sodium citrate solution and five volumes of 5.4-percent dextrose solution for every three volumes of blood.

3. The most effective preservative solutions were approximately isotonic with blood serum.

Robertson (6), in 1917, applied the technique of Rous and Turner in what amounted to the operation of the world's first blood bank, in casualty clearing stations of the British Army (p. 5).

Introduction of Sodium Citrate

The first step on the road to the therapeutic use of blood in World War II battle casualties was the successful use of citrated blood in 1914 at Mount Sinai Hospital, New York City(7-9).

In a retrospective view of his experiences, published in 1958, the late Dr. Richard Lewisohn (10), who is generally credited with the introduction of the method, pointed out that the famous British obstetrician, Braxton Hicks, used sodium citrate as an anticoagulant in 1869 but that a number of fatalities forced him to abandon it.

Later experiments by other observers with hirudin, sodium oxalate, and peptone showed all of these agents to be too toxic for clinical use. Sodium citrate, usually in 1-percent concentration, had long been used as an anticoagulant in blood collected for laboratory purposes, but this concentration, which was assumed to be necessary to prevent coagulation, was also assumed to be too toxic for human administration. By a careful series of animal experiments, Lewisohn showed that a concentration of 0.2-percent sodium citrate was sufficient to prevent coagulation of blood in vitro for 2 or 3 days. His clinical studies showed that up to 5 gm. of sodium citrate could be safely introduced into adults intravenously, though, paradoxically, its introduction caused a temporary shortening of the recipient's own coagulation time. It was thus possible to transfuse as much as 2,500 cc. of blood at a time-and that, added Lewisohn, quite unprophetically, "is more than anybody ever wants * * * to introduce into the recipient" (10).

In his 1958 communication (10), as well as in his original report in January 1915 (7), Lewisohn pointed out that results identical to his own had been published by Agote in Buenos Aires in the same month his report was published.


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In his 1915 communication, he also acknowledged the work of two other observers: In March 1914, Hustin, in Brussels, had treated a patient with blood mixed with glucose and sodium citrate. In January 1915, Weil (11) published the report of an earlier report before the New York Academy of Medicine dealing with the use of citrated blood at General Memorial Hospital. He used a 10-percent citrate solution, in the proportion of 1 cc. of solution to 10 cc. of blood, and gave transfusions of as much as 350 cc. from 3 to 5 days after the blood had been collected.

Over forty years later (10), Lewisohn's philosophic reaction to these various reports about the same time as his own was that when an idea is ripe, it occurs to a number of persons at the same time.

For a time, it seemed that reactions to the use of citrated blood might destroy the usefulness of the method. It was evident, however, that citrate played no part in the chills when Lewisohn and Rosenthal (12), also at Mount Sinai Hospital, demonstrated that reactions were not caused by sodium citrate but by pyrogens present in carelessly cleaned transfusion equipment. When a special department was created to handle the equipment, the incidence of posttransfusion chills fell from 12 percent to 1 percent. Many years later, when the Mount Sinai technique was introduced in a hospital in Novgorod, by Satunov, the incidence of reactions there fell from 53 percent to 2 percent.

Preservatives Between the World Wars

The Rous-Turner solution was widely used in the United States between the World Wars, but it was difficult to prepare; the bulk of the final solution was undesirably large; and the concentration of plasma was so dangerously high that the plasma had to be discarded (13, 14). The solutions introduced by Perry (15), at the Moscow Institute of Hematology (M.I.H. solution) (16), and by Gnoinski of Warsaw all had very undesirable features.

DeGowin, Harris, Plass, and their associates used a modification of the Rous-Turner solution, consisting of 3.2-percent trisodium citrate in 100 cc. of water; 5.4-percent anhydrous dextrose in 650 cc. of water; and 500 cc. of blood. This gave a total volume of 1,250 cc., and a blood-diluent ratio of 1.5:1. The solution preserved red cells in vitro quite as well as the Rous-Turner solution; produced satisfactory clinical results; and had the advantage of conserving the plasma, which had to be discarded when the Rous-Turner solution was used.

From their reviews of the literature and their own experience, DeGowin and his associates drew the following conclusions(13, 14, 17):

1. Progressive hemolysis occurred in human blood in all the preservatives studied and was much greater when the blood was stored at 68 F. (20 C.) rather than at 41 F. (5 C.).

2. The addition of large quantities of isotonic dextrose solution slowed the rate of hemolysis considerably as compared with the rate in blood stored with little or no added dextrose.


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3. Hemolysis was much less in blood stored in sealed flasks from which air was completely excluded.

4. Erythrocytes stored in the DeGowin et al. solution resisted destruction by shaking better than those stored in citrate alone or in saline. These observers used blood stored in their own solution up to 38 days with no reactions.

TRANSPORTABILITY OF WHOLE BLOOD

At the first meeting of the Committee on Transfusions, NRC (National Research Council) on 31 May 1940 (18), and the first meeting of the Subcommittee on Blood Substitutes on 30 November 1940 (19), much of the discussion concerned improvement of methods of preserving whole blood, both to increase the safe period of its use and to make it safely transportable. As was pointed out at a later conference (20), the decision to use preserved blood in wartime would make a great deal of difference in its availability at many points at which the wounded would be treated; the question was not one of advisability but of feasibility.

In 1940, there was very little authoritative information on the transportability of whole blood, though as Lewisohn pointed out in 1958 (10), in 1916 Brem had collected blood in citrate solution in his office and taken it by plane to a patient in another city. Blood had also been transported by the British Medical Service in World War I (p. 6).

At both of the meetings just mentioned, work carried out at the State University of Iowa, by Drs. Everett D. Plass, Elmer L. DeGowin, Robert C. Hardin, and their group, was summarized; it was reported in the literature the following year (14). Between 1 September 1938 and 17 November 1940, 2,123 transfusions had been given with blood preserved in the DeGowin modification of the Rous-Turner solution, with 4.1 percent of reactions and with highly satisfactory clinical results. Optimum inhibition of hemolysis during storage required a dextrose concentration of at least 3 percent, though, as British workers had shown, concentrations of as low as 0.3 percent permitted longer red blood cell survival in the recipient's blood than was achieved with citrate alone.

The prevailing belief that red blood cells would not withstand transportation and would rupture with slight trauma did not seem reasonable to the Iowa group, in view of the trauma such cells ordinarily withstand as they are forced through the capillary circulation. Rigid tests of transportability were therefore undertaken on blood put up in Baxter bottles in citrate-glucose (3 percent) solution after it had been kept in a refrigerator up to 18 days. To avoid shaking, the contents of each bottle had been brought up to 1,250 cc. The bottles were transported in ordinary milk cans, covered with quilts and re-iced at least once every 24 hours. Twenty bloods were shipped by plane to San Francisco and back, and 20 other samples were taken by ambulance to outlying districts of Iowa City, to test the effect of rough roads.

There was no appreciable increase in hemoglobin levels in the centrifuged plasma either before or immediately after transportation in 20 bloods. In


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the remainder, the increases were insignificant, the largest being from 8.4 to 18.3 mg. percent.

All these bloods were then used for transfusion, on appropriate indications, from 3 to 45 days after they had been collected. The only transfusion reaction, which was limited to chills and fever, occurred in a patient who had undergone thoracoplasty.

Blood was also carried by car for 30 hours, including stopovers, for a distance of 720 miles. It was then flown from Iowa City to Oakland and back, a distance of 3,539 miles and 24 hours of flying time (46 hours including stopovers). When the flasks were examined 30 minutes after the plane had landed, the erythrocytes in many of them had almost completely sedimented during the trip.

DEVELOPMENT OF PRESERVATIVE SOLUTIONS

No action was taken on preservative solutions until the Conference on Transfusion Equipment and Procedure on 25 August 1942 (20), at which the chairman, Dr. Robert F. Loeb, requested Dr. DeGowin to draw up a statement of the problem and propose a plan to be submitted for consideration to the Surgeons General of the Army and the Navy. At the meeting of the Subcommittee on Blood Substitutes on 20 October 1942 (21), the proposal was made to recommend to the Armed Forces the use of preserved blood whenever this was feasible and fresh blood could not be used. There was no discussion of the preservative, and action on the recommendation was deferred.

At the meeting of the subcommittee on 13 May 1943 (22), a letter was read from Dr. P. L. Mollison, suggesting the use of what he termed a "slough preservative," incorporating citrate, citric acid, and dextrose, the constituents used in the preservative solution later adopted for blood flown overseas. Dr. DeGowin was authorized to prepare a summary statement, incorporating all experimental and clinical data available on preservative solutions and physical equipment for whole blood preservation and shipment.

The Conference on Preserved Blood recommended by the subcommittee convened on 25 May 1943 and heard a number of reports, as follows (23):

1. Dr. O. F. Denstedt, of McGill University, reported on survival experiments with the Ashby technique, on transfused preserved red cells, and Dr. Joseph F. Ross, Evans Memorial Hospital, Boston, reported, with Dr. Milan A. Chapin, on studies on the hemoglobin molecule made with radioactive isotopes of iron.

It was brought out in the discussion that the value of a preservative must be judged by its effectiveness in prolonging the in vivo survival of transfused erythrocytes as well as by its ability to prevent in vitro hemolysis, changes in osmotic fragility and cellular potassium content, and changes in other components. The radioactive tagged cell technique reported by Drs. Ross and Chapin was so sensitive that 0.005 cc. of transfused cells could be detected in 1.0 cc. of the recipient's blood with an accuracy of 10 percent, and the total volume of labeled cells which had to be injected into the recipient was so small that it produced no hemodynamic or hematopoietic disturbance. The results of this study also indicated that the breakdown products of hemoglobin were rapidly reutilized for the synthesis of new hemoglobin. It was therefore impossible to trace the survival of the injected cells for more than


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24-48 hours, but this made little difference, since it is in the period immediately after transfusion that survival is of the greatest importance. It could be concluded from these observations that even though transfused cells were destroyed rapidly, they were of distinct, if temporary, value in promoting blood formation.

2. Dr. Denstedt discussed the modifications of the DeGowin modification of the Rous-Turner solution which he and his associates had devised at McGill University (and which he later requested be called the McGill solution (24)). The first of these solutions consisted of 400 cc. of blood, 80 cc. of 3.2-percent sodium citrate, and 120 cc. of 5.4-percent dextrose. These quantities constituted 600 cc. of solution, with a blood-diluent ratio of 2:1. With this dilution, most bloods could be stored up to 6-8 weeks at 39 F. (4 C.), with less than 1-percent loss by hemolysis.

The second solution consisted of 400 cc. of blood, 80 cc. of 3.2-percent sodium citrate, 80 cc. of 5.4-percent dextrose, and 40 cc. of an isotonic buffer solution. The resulting solution consisted of 1,000 cc. of 0.3 molar (4.14 percent) monobasic sodium phosphate (NaH2PO4.H2O), 925 cc. of 0.3 molar (1:2 percent) sodium hydroxide (NaOH), and 480 cc. of water. These quantities also produced 600 cc. of solution with a blood-diluent ratio of 2:1. The second solution was recommended on the ground that it maintained the citrate concentration above 0.34 percent and also maintained an effective level of dextrose. The buffered solution retarded cell swelling during storage, retarded changes in organic phosphates, and reduced cohesion of cells on sedimentation. When buffered bloods were stored at 50 F. (10 C.), the changes were no greater than when they were stored at 39 F. (4 C.). With unbuffered bloods, the changes at the higher temperature were more rapid.

At the 10 August 1943 meeting of the subcommittee (25), the discussion of preservatives was continued. Dr. Edwin J. Cohn introduced a recommendation that glucose be added whenever blood was stored at low temperatures. Dr. Max M. Strumia objected to the additive; his own work with urobilin levels in blood had not shown any superior red blood cell survival in vivo when it was used; in fact, cellular fragility seemed to be increased. He also saw no advantage to preserving blood for more than 5 days, the average time for which blood was then banked. Dr. DeGowin pointed out that military demands were irregular and that the attempt to operate a bank with blood that could be stored only 5 days would result in excessive losses of blood, which could be used if longer periods of storage were possible. In view of the differences of opinion, Dr. Cohn withdrew his recommendation.

Meantime, Dr. John B. Alsever, then in the U.S. Public Health Service, had devised a solution composed of 0.42-percent sodium chloride, 0.8-percent sodium citrate, and 2.05-percent dextrose; 500 cc. of this mixture had to be used with each 500 cc. of blood (fig. 57).

Alsever's solution had proved satisfactory with the usual criteria for studying red blood cell preservation; that is, rate of spontaneous hemolysis, fragility tests, hemoglobin levels, and incidence of reactions. At the 24 September 1943 meeting of the Subcommittee on Blood Substitutes (26), it was recommended to the Armed Forces that, if blood was to be stored for more than 5 days, refrigeration and the addition of glucose was essential. In the light of present knowledge, Alsever's, DeGowin's and Denstedt's solutions would be equally effective.

With the recommendation at the subcommittee meeting on 17 November 1943 (27), that The Surgeon General of the Army give consideration to the


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FIGURE 57.-Preservation of blood with Alsever's solution. A. One-liter bottle containing 500 cc. of Alsever's solution. B. Bottle ready for collection of blood. C. Blood put up in Alsever's solution in use in postoperative ward, 10th Field Hospital, Granovillers, France, November 1944. Note suspension of bottles from cord over beds.

transportation of whole blood by airlift to certain theaters of operations (p. 465), the question of preservatives became more urgent, though it was not discussed further at this time. Maj. Gen. Norman T. Kirk's acknowledgment of the recommendation about the airlift of blood was read at the 5 January 1944 meeting (24). In the meantime, he had rejected it.

Dr. DeGowin reported at the Second Conference on Blood Storage on 2 March 1944 (28) that the modified Rous-Turner solution had been used in


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more than 13,000 transfusions given over the past 5 years at the State University of Iowa College of Medicine. The upper storage limit was 30 days, and the incidence of reactions was low and no greater with aging blood than with blood used earlier. No definitive action was taken in regard to preservatives at this conference or at the meeting of the subcommittee on 3 March 1944 (29), at which the Loutit-Mollison solution (30) was first mentioned.

Selection of Alsever's Solution for Oversea Airlift

When the Third Conference on Blood Storage was convened on 30 August 1944 (31), blood put up in Alsever's solution and flown from the United States was already being administered in field hospitals in France. When the decision concerning a preservative had to be made without delay, because of the critical situation in Europe, Alsever's solution, in the absence of a definitive recommendation from the Subcommittee on Blood Substitutes, seemed to be the wisest choice for a number of reasons. They were stated (32) at the conference held in the Office of The Surgeon General on 15 August 1944 (p. 209). In substance, they were as follows:

1. The Subcommittee on Blood Substitutes, NRC, had agreed on 24 September 1943 (26) that Alsever's solution was at least as good as DeGowin's and Denstedt's solutions; up to that time, the experience with Denstedt's solution had been limited.

2. Tests with these solutions had been carried out at the Army Medical School since 1939. In 1943, a blood transfusion and intravenous fluid service had been set up at Walter Reed General Hospital, Washington, D.C., and operated by the Blood Research Division of the school.

3. When the McGill solution was tested, the precipitation of fibrin, particularly when blood that had been stored for more than 7 days was used, was so great that the steel filters in the giving sets had frequently become completely blocked.

4. The more dilute Alsever's solution, which had the disadvantage of requiring 500 cc. of solution to each 500 cc. of blood, had been used in more than 2,000 transfusions. The added crystalloid content minimized fibrinogen precipitation, and blood stored in this solution was much easier to administer through standard Army filters.

Alsever's solution had been submitted to rigorous testing. Only proved O blood had been used, up to 30 days old. It had been shipped by plane to the west coast without refrigeration and returned to the Army Medical School in good condition. With continuous refrigeration, the blood could be used from 25 to 30 days after it was collected; the free hemoglobin was no more than 25 mg. percent. When refrigeration was interrupted for 24 hours, the dating period was reduced to 18-21 days. When the blood was taken from the refrigerator and left at room temperature for 48-72 hours, spontaneous hemolysis reduced the dating period to 14-16 days.


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Blood put up in Alsever's solution was also tested in other ways. During 28 days of storage, it was shaken every day. It was transported in trucks for 8- to 24-hour periods two or three times a week, in temperatures ranging from just above freezing to 50 to 60 F. (10 to 15 C.). The method of testing was therefore extremely severe, and the fact that the blood stood up well under the conditions-most of them far more severe than blood handled under controlled conditions would be subjected to-made it evident that it would be practical to supply preserved blood to oversea theaters.

The reaction rate with blood preserved in Alsever's solution was about 1 percent. No jaundice had followed any transfusion. Even though blood preserved in this solution had been given in quantities up to 3,000 and 4,000 cc. over a 12-hour period, none of the patients who had received these quantities had developed pulmonary edema. Kilduffe and DeBakey's (33) review of the literature did not support the contention that if 4,000 to 5,000 cc. of blood were given over a 24-hour period, pulmonary edema would necessarily occur. DeBakey had personally given as much as 9,000 cc. in 12 hours without its development. It was understood, of course, that these generalizations did not apply to casualties with blast injuries or to patients with organic heart disease and myocardial insufficiency.

5. A final and very practical reason for selecting Alsever's solution rather than ACD (acid-citrate-dextrose) solution as the preservative for blood to be airlifted to Europe was that the containers for it were already in production in August 1944 when General Kirk reversed his decision not to supply blood to the European theater, as many working in this field had always believed that it inevitably would be reversed. As it was, their procurement of these containers in the necessary quantities was a crash operation. With wartime shortages and priorities, it would have taken at least 3 to 4 months to provide the necessary containers for the Loutit-Mollison acid-citrate-dextrose solution.

Substitution of ACD Solution for Alsever's Solution

Many years after the war, it is perhaps difficult for readers who were not participants in the events to understand why ACD solution was not used when the airlift of blood to Europe began in August 1944, or at least why it was not substituted for Alsever's solution in the winter of 1944-45, when the Navy had already proved its safety and efficiency in the airlift of blood to the Pacific. There are a number of explanations, though perhaps no real excuses. One was the late development of the Loutit-Mollison solution; it was first described in December 1943 (30). Another was Major Hardin's unwillingness to accept the changeover in the European theater before the new solution had been adequately tested. The third was procurement of the smaller bottles necessary when a smaller amount of preservative solution was used.

Testing of ACD solution-The Loutit-Mollison solution was first mentioned in a meeting of the Subcommittee on Blood Substitutes on 3 March


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1944 (29), when Dr. G. M. Guest, who was representing the Canadian Committee on Medical Research, recommended that it be investigated, on the ground that the British were rapidly coming to use it since its description in December 1943 (30).

On the basis of Dr. Guest's suggestion, a number of comparative investigations were undertaken, including clinical testing at the Army Medical School. Preliminary reports were made at the 2 June 1944 meeting of the Subcommittee on Blood Substitutes (34), 4 days before D-day, and at the Third Conference on Blood Storage on 30 August 1944 (31), after the airlift to Europe had already begun. They showed that ACD solution preserved blood satisfactorily for at least 21 days. An investigation at the Children's Hospital in Cincinnati showed that, after 38 days' storage in it, the red blood cells were in the same state of preservation as after 10 days' storage in simple citrate solution and after 22 days' storage in neutral citrate-glucose solution.

In addition to the reduced bulk (the initial blood-diluent ratio of 4:1 was later reduced to 6:1, 70 cc. of solution to 450 cc. of blood), ACD solution was considered to have the following advantages:

1. With an enriched solution of dextrose, nutrition was provided for the red blood cells that they did not receive from other solutions containing only sodium citrate and physiologic salt solution.

2. The addition of citric acid to the solution lowered the pH sufficiently (to about 5) to eliminate the tendency to fibrin-clot formation evident in blood kept longer than 14 days in other solutions.

3. When blood was preserved in the natural environment of this solution, under constant refrigeration, tests showed that the red blood cells were less fragile, and tolerated handling and transportation much better, than when they were preserved in other solutions.

At the conference on 30 August 1944 (31), too late for implementation in the airlift already underway, it was passed, Dr. DeGowin dissenting, that ACD solution be recommended as the best available solution for the preservation of whole blood; the optimum dilution factor was considered to be 20 percent solution to 80 percent blood. Dr. DeGowin did not consider that sufficient evidence had been presented to date for evaluation of Alsever's solution, nor did he consider that ACD solution had been tested sufficiently clinically to permit an opinion concerning fibrin precipitation with it.

It was further recommended at this conference that the Armed Forces transport whole blood at temperatures between 39 and 50 F. (4 and 10 C.) from the time of collection to the point of final delivery. There was no discussion of how this recommendation should be implemented.

Further studies on ACD solution were reported at the Conferences on Blood Preservation on 19 January 1945 (35) and on 8 February 1945 (36), as well as in Weekly Newsletter No. 136, Division of Medical Sciences, National Research Council, 7 October 1944. Among the data reported were the following:

1. ACD solution was much simpler to prepare and autoclave than Alsever's solution.

2. Fibrin formation was minimal. There had been no difficulties of this kind in 200


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transfusions of blood stored in this solution and used in routine blood bank operations at the Massachusetts Memorial Hospital.

3. When the survival of transfused cells tagged with radioactive isotopes in the recipient circulation was used as a criterion, there was little difference between ACD and Alsever's solutions during the first 15 days of storage; 70 percent of the cells survived during the first 48 hours after transfusion. Later, there was a considerable difference, survival being 20 days in Alsever's solution and 30 days in ACD solution. This was a discrepancy of practical military importance.

4. A report by Dr. Strumia, whose conclusions were of value because of his association with the blood-plasma project from its inception in 1940, indicated that ACD was the most effective preservative solution tested to date.

All reports emphasized that the data were obtained under constant refrigeration. Deterioration of the red blood cells began during short periods of storage at room temperature and was not halted by subsequent refrigeration.

Procurement of new bottles-The substitution of 600-cc. bottles for collection of the blood in ACD solution for the 1,000-cc. bottles used to collect it in Alsever's solution required changes of orders to contractors, who had to continue to produce the larger bottles to meet current needs while preparing for the changeover to the smaller bottles. By careful planning, the changeover to the smaller bottles was made without delay and without undue wastage of the larger bottles.

Acceptance of ACD solution in the European theater-When Capt. John Elliott, SnC, returned from the European theater after his January 1945 visit (37), he brought word that Major Hardin was unwilling to have ACD solution substituted for Alsever's solution until there was overwhelming evidence of its superiority. He wished comparative tests of the two solutions to be conducted in the theater, on the reasonable ground that it was not wise to discard an agent that had proved satisfactory for one that he did not consider had been adequately tested.

The necessary tests were carried out by flying successive shipments of blood in ACD solution to the European theater, as described elsewhere (p. 215). The results proved entirely satisfactory clinically, though perhaps less impressive numerically. Major Hardin was informed, however, of the satisfactory results obtained with ACD solution in the massive airlift of blood to the Pacific, as well as of the fact that the use of smaller bottles would permit refrigeration of the blood during the airlift across the Atlantic. He was also told of an improvement in the bottles; the stoppers were now hollowed out inside, so that the tip of the filter housing projected a short distance into the bottle and any clots that might form fell around, rather than into, its adapter.

When ACD solution began to be used on 1 April 1945, the 50 cc. of 4-percent sodium citrate solution formerly placed in a 750-cc. bottle was replaced by 100 cc. (later 70 cc.) of a solution consisting of 2.0 gm. of citric acid, 8.0 gm. of sodium citrate, 27.0 gm. of dextrose (all U.S.P.), with water to make 1,000 cc. After 100 cc. of this solution was placed in a 600-cc. bottle, the vacuum in the bottle was checked and corrected with a vacuum pump to 720


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mm. Hg. The pH of the ensuing mixture was 5-5.6. When 500 cc. of blood was added, the pH of the contents was 6.8-7; and the final concentration of sodium citrate was 0.46 percent, of dextrose 0.45 percent, and of citric acid 0.03 percent.

Critique of Preservatives

There was, of course, never any argument about the superiority of fresh whole blood over the most efficiently preserved whole blood. But fresh whole blood was not practical from any standpoint for frontline use. It was impractical to collect it locally, and its life was too short to fulfill requirements imposed by shipping it from the Zone of Interior to the European theater. Some preservative therefore had to be used.

Since preserved blood had to be used in forward areas, the important point was that when the decision not to airlift blood to Europe, recommended in November 1943, was necessarily reversed in August 1944, no decision had been reached by the Subcommittee on Blood Substitutes as to the best preservative to use (26, 38).

When the airlift to Europe was authorized, the decision as to the preservative to use therefore had to be made in the Blood Research Division, Army Medical School, and made without delay. Alsever's solution was selected for a number of reasons: It contained a satisfactory concentration of electrolytes and dextrose. Its dilution was less than the 750-cc. dilution of the DeGowin solution, though its 500-cc. bulk was undesirably large. Its pH was desirable. Fibrin formation was minimal. Finally, with the Alsever solution, preservation of the blood for 21 days was possible.

It is regrettable that ACD solution had not been tested adequately when the selection of a preservative for oversea use became necessary in August 1944. It is also regrettable that the changeover from Alsever's to ACD solution was not made as soon as the Navy experience in the Pacific proved the safety and efficiency of the latter solution. On the other hand, as has been pointed out in connection with flying blood to Europe without refrigeration during the period of the flight, there were many thousands of lives saved because blood was provided in Alsever's solution. Many of these lives would have been lost if the airlift had been deferred until a more efficient preservative had been tested.

In his report on his Pacific tour to Col. George R. Callender, MC, in December 1944 (39) (p. 590), Lt. Col. Douglas B. Kendrick, MC, repeated his earlier recommendation that the 1,000-cc. bottle containing Alsever's solution in the field transfusion set should be replaced by the 600-cc. bottle containing ACD solution as soon as the next contracts became effective and should thereafter be used for blood sent to the European theater. He considered the recommendation justified because, by 12 October 1944, Capt. Lloyd R. Newhouser, MC, USN, had received reports on trial runs to the Pacific that indicated that both bottle and solution were entirely satisfactory.


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TESTS OF EFFICIENCY OF PRESERVATIVE SOLUTIONS

It was brought out at the Conference on Preserved Blood on 25 May 1943 (23) that the basic criterion of the value of a preservative was the in vivo survival of transfused erythrocytes (p. 217). At the Third Conference on Blood Storage on 30 August 1944 (31), it was formally agreed that the conference favored, as an optimal criterion of whole blood preservation, the survival of 90 percent of the transfused red blood cells for 48 hours and, as a satisfactory criterion, the survival of 70 percent for 48 hours. Any specimen of blood should be rejected which contained in excess of 50 mg. percent of free hemoglobin.

At this same conference, it was agreed that tests for determining the in vivo preservation of red blood cells should include agglutination techniques, the radioactive isotope method, studies on blood bilirubin, and determination of the total urobilinogen output. In vitro testing should include spontaneous hemolysis, osmotic resistance of the red cells, escape of potassium and other components from the cells, glycolysis, fibrin formation, and enzyme systems. The osmotic fragility test was not considered a satisfactory determination of in vivo survival.

Bushby and his group (40), studying various blood preservatives, showed, from the bilirubinemia associated with the transfusion of stored blood, that the older the blood, the more rapidly were its corpuscles destroyed in the recipient bloodstream. The iron pigment thus set free, however, was phagocytosed by the reticuloendothelial system and assisted in blood regeneration during recovery from hemorrhage. These observers believed that if the blood was not so old that a dangerous quantity of pigment would be suddenly liberated, the administration of even quite old blood to an exsanguinated patient had much to recommend it. Their theory seemed to be proved by the successful use of blood well beyond its dating period during the fighting in France in May 1940 (p. 20).

To complete the record, a final study on posttransfusion survival of erythrocytes might be mentioned, which was reported at the Conference on Blood Preservation and Red Cell Resuspension on 6 December 1945 by Capt. John B. Ross, MC (41). It was based on a study made with ACD solution in the original volume (120 cc. per 480 cc. of blood) and in a reduced volume (50 cc. per 450 cc. of blood). The solution, which had a pH of 5.0, contained 2.5 gm of disodium citrate and 3.0 gm. of dextrose. The results, which were determined by the radioactive isotope technique, showed the in vivo survival of erythrocytes to be the same in both solutions. To exclude possible variations in the donor blood, the bloods stored in each solution were obtained from the same donors.

SPECIAL STUDIES

It has not been possible, in the compass of this chapter, to describe all of the various studies on blood preservatives made by workers under contracts set up by the Committee on Medical Research, National Research Council,


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nor has it been possible to describe any of them in detail. The reports are available in the minutes and conferences of the Subcommittee on Blood Substitutes and other committees and subcommittees.

It is not the function of this history to review the literature on the subject, though much of it was pertinent to, and useful in, the work of the Subcommittee on Blood Substitutes in the fulfillment of its functions. Attention is called particularly, in addition to the studies mentioned in the text, to the studies of the following workers:

1. Scudder and his associates in 1939 (42).

2. Mollison and Young in 1940 and 1941 (43, 44).

3. Maizels and Paterson in 1940 (45).

4. Mainwaring, Aylward, and Wilkinson in 1940 (46).

5. Muether and Andrews in 1940 and 1941 (1, 47-50). The series of studies by these observers on "stored" blood were particularly useful. They endeavored to meet the objection that, as they put it, much of the literature on changes in stored blood was lacking in controls and colored by preconceived ideas on the subject.

6. Ross, Finch, Peacock and Sammons, which were concluded in 1947 (51). Their extensive studies on in vitro preservation amid posttransfusion survival of stored blood, made at the Massachusetts Memorial Hospital and the Massachusetts Institute of Technology, included 16 solutions. Their chief conclusion was that, from a practical standpoint, blood stored in ACD solution, or one of its modifications, for 7 to 10 days is as satisfactory for transfusion as fresh blood. Blood stored for 3 weeks and providing cells of 70-percent viability is satisfactory for emergency transfusions but is not so good as whole blood or blood stored for shorter periods of time.

References

1. Muether, R. O., and Andrews, K. R.: Studies and Uses of Stored Blood and Plasma. South. M. J. 34: 453-462, May 1941.

2. Ashby, W.: The Determination of the Length of Life of Transfused Blood Corpuscles in Man. J. Exper. Med. 29: 267-281, 1 Mar. 1919.

3. Rous, P., and Turner, J. R.: The Preservation of Living Red Blood Cells in Vitro. I. Methods of Preservation. J. Exper. Med. 23: 219-237, February 1916.

4. Rous, P., and Turner, J. R.: The Preservation of Living Red Blood Cells in Vitro. II. The Transfusion of Kept Cells. J. Exper. Med. 23: 239-248, February 1916.

5. Abel, J. J., Rowntree, L. G., and Turner, B. B.: On the Removal of Diffusible Substances From the Circulating Blood of Living Animals by Dialysis. J. Pharmacol. & Exper. Therap. 5: 275-316, January 1914.

6. Robertson, O. H.: Transfusion With Preserved Red Blood Cells. Brit. M. J. 1: 691-695, 22 June 1918.

7. Lewisohn, R.: A New and Greatly Simplified Method of Blood Transfusion. A Preliminary Report. M. Rec. 87: 141-142, 23 Jan. 1915.

8. Lewisohn, R.: The Development of the Technique of Blood Transfusion Since 1907: With Special Reference to Contributions by Members of the Staff of the Mount Sinai Hospital. J. Mt. Sinai Hosp. 10: 605-622, January-February 1944.

9. Lewisohn, R.: Blood Transfusion: 50 Years Ago and Today. Surg. Gynec. & Obst. 101: 362-368, September 1955.

10. Lewisohn, R.: The Citrate Method of Blood Transfusion in Retrospect. Surgery 43: 325-327, February 1958.

11. Weil, R.: Sodium Citrate in the Transfusion of Blood. J.A.M.A. 64: 425-426, 30 Jan. 1915.

12. Lewisohn, R., and Rosenthal, N.: Prevention of Chills Following Transfusion of Citrated Blood. J.A.M.A. 100: 466-469, 18 Feb. 1933.


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13. DeGowin, E. L., Harris, J. E., and Plass, E. D.: Studies on Preserved Human Blood. I. Various Factors Influencing Hemolysis. J.A.M.A. 114: 850-855, 9 Mar. 1940.

14. DeGowin, E. L., and Hardin, R.: A Plan for Collection, Transportation and Administration of Whole Blood and of Plasma in Warfare. War Med. 1: 326-341, May 1941.

15. Perry, M. C.: Preservation of Blood for Transfusion. Wisconsin M.J. 25: 123-127, March 1926.

16. Elliott, G. A., MacFarlane, R. G., and Vaughan, J. M.: The Use of Stored Blood for Transfusion. Lancet 1: 384-387, 18 Feb. 1939.

17. DeGowin, E. L., Hardin, H. C., and Alsever, J. B.: Blood Transfusion. Philadelphia and London: W. B. Saunders Co., 1949.

18. Minutes, meeting of Committee on Transfusions, Division of Medical Sciences, NRC, 31 May 1940.

19. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 30 Nov. 1940.

20. Minutes, Conference on Transfusion Equipment and Procedure, Division of Medical Sciences, NRC, 25 Aug. 1942.

21. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 20 Oct. 1942.

22. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 13 May 1943.

23. Minutes, Conference on Preserved Blood, Division of Medical Sciences, NRC, 25 May 1943.

24. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 5 Jan. 1944.

25. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 10 Aug. 1943.

26. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 24 Sept. 1943.

27. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 17 Nov. 1943.

28. Minutes, Second Conference on Blood Storage, Division of Medical Sciences, NRC, 2 Mar. 1944.

29. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 3 Mar. 1944.

30. Loutit, J. F., and Mollison, P. L.: Advantages of a Disodium-Citrate-Glucose Mixture as a Blood Preservative. Brit. M. J. 2: 744-745, 11 Dec. 1943.

31. Minutes, Third Conference on Blood Storage, Division of Medical Sciences, NRC, 30 Aug. 1944.

32. Memorandum, Lt. Col. D. B. Kendrick, MC, for Brig. Gen. Fred W. Rankin, 23 Aug. 1944, subject: Conference on Supply of Whole Blood for the ETO.

33. Kilduffe, Robert A., and DeBakey, Michael: The Blood Bank and the Technique and Therapeutics of Transfusions. St. Louis: C. V. Mosby Co., 1942.

34. Minutes, meeting of the Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 2 June 1944.

35. Minutes, Conference on Blood Preservation, Division of Medical Sciences, NRC, 19 Jan. 1945.

36. Minutes, Conference on Blood Preservation, Division of Medical Sciences, NRC, 8 Feb. 1945.

37. Memorandum, Capt. John Elliott, SnC, to Chief, Surgical Consultants Division, Office of The Surgeon General, through Director, Army Medical School, 1 Feb. 1945, subject: Transportation of Blood From the U.S. to the ETO Blood Bank in Paris.

38. Minutes, meeting of ad hoc Committee on Transfusion and Equipment, Division of Medical Sciences, NRC, 12 May 1943.


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39. Letter, Lt. Col. D. B. Kendrick, MC, to Col. George R. Callender, MC, 28 Dec. 1944, subject: Blood Supply to Pacific.

40. Bushby, S. R. M., Kekwick, A., Marriott, H. L., and Whitby, L. E. H.: Survival of Stored Red Cells After Transfusion. Lancet 2: 414-417, 5 Oct. 1940.

41. Minutes, Conference on Blood Preservation and Red Cell Resuspension, Division of Medical Sciences, NRC, 6 Dec. 1945.

42. Scudder, J., Drew, C. R., Corcoran, D. R., and Bull, D. C.: Studies in Blood Preservation. J.A.M.A. 112: 2263-2271, 3 June 1939.

43. Mollison, P. L., and Young, I. M.: Survival of the Transfused Erythrocytes of Stored Blood. Lancet 2: 420-421, 5 Oct. 1940.

44. Mollison, P. L., and Young, I. M.: Failure of in Vitro Tests as a Guide to the Value of Stored Blood. Brit. M. J. 2: 797-800, 6 Dec. 1941.

45. Maizels, M., and Paterson, J. H.: Survival of Stored Blood after Transfusion. Lancet 2: 417-420, 5 Oct. 1940.

46. Mainwaring, B. R. S., Aylward, F. X., and Wilkinson, J. F.: Potassium and Phosphate Content of Plasma From Stored Blood. Lancet 2: 385-387, 28 Sept. 1940.

47. Muether, R. O., and Andrews, K. R.: Use of Stored Blood: A Practical Application. Hosp. Progress 21: 246-250, July 1940.

48. Muether, R. O., and Andrews, K. R.: Studies on "Stored Blood." I. Technic for Storage of Blood. Am. J. Clin. Path. 11: 307-313, April 1941.

49. Muether, R. O., and Andrews, K. R.: Studies on "Stored Blood." II. Effect of Storage on Human Blood. Am. J. Clin. Path. 11: 314-320, April 1941.

50. Muether, R. O., and Andrews, K. R.: Studies on "Stored Blood." III. Effects of Stored Blood on the Recipient. Am. J. Clin. Path. 11: 321-328, April 1941.

51. Ross, J. F., Finch, C. A., Peacock, W. C., and Sammons, M. E.: The in Vitro Preservation and Post-Transfusion Survival of Stored Blood. J. Clin. Invest. 26: 687-703, July 1947.

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