CHAPTER IX
Preservative Solutions
DEFINITIONS AND CRITERIA
As pointed out elsewhere, there is a distinction betweenstored blood and preserved blood that is not always observed but that alwaysshould be. Muether and Andrews (1), whose extensive studies in this fieldwill be mentioned later, were among the first to point it out. Stored blood iscitrated blood which has been kept for hours or days, at temperatures between36? and 46? F. (2? and 8? C.), and to which nothing has been added toinhibit deterioration of the erythrocytes; sodium citrate is an anticoagulant,not a preservative. Preserved blood is blood to which some additional substancehas been added to check disintegration of the erythrocytes. It was the solutionof the problem of halting this disintegration that made possible the use inoversea theaters of whole blood flown from the United States.
While varying criteria have been advanced for safe andeffective preservation of blood, the following met essential militaryrequirements:
1. The blood must be collected in a closed system and handledas little as possible at every step of processing to avoid contamination.
2. It must be kept at a low, constant temperature fromcollection to administration.
3. The technique of preservation must be simple andinexpensive.
4. The preserved blood must maintain to a high degree theproperties of fresh blood if it is to serve its therapeutic purpose. Thispurpose can be accomplished only if the blood shows a low rate of hemolysis anda high rate of red blood cell survival.
5. The ultimate fate of transfused red blood cells determinesthe effect of the transfusion upon the recipient.
Red blood cell survival has been studied in detail by anumber of observers, beginning in 1919 with Ashby (2), whosedifferentiation agglutination technique is still regarded by many observers asmore efficient than the modern radioactive cell-tagging technique (p. 221)because the Ashby technique permits the observer to follow the red cells throughtheir 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 hadbeen able to find only a single
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report on this or any other related study except thedemonstration, in 1914, by Abel, Rowntree, and Turner (5), that onlyformed elements of the blood need to be replaced in healthy animals depleted byhemorrhage.
The results of the Rous-Turner studies were as follows:
1. The rapidity of hemolysis was generallyincreased when only electrolytes were added to the blood. When sodium citratewas used, human red blood cells tended to break down rather rapidly;disintegration occurred relatively early even when the smallest quantity thatwould prevent clotting was used.
2. Hemolysis was greatly diminished whensaccharose or dextrose was added to the citrated blood. Human red blood cellsremained intact for about 4 weeks when a solution was used consisting of twovolumes of 3.8-percent sodium citrate solution and five volumes of 5.4-percentdextrose solution for every three volumes of blood.
3. The most effective preservative solutionswere approximately isotonic with blood serum.
Robertson (6), in 1917, applied the technique of Rousand 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 inWorld War II battle casualties was the successful use of citrated blood in 1914at Mount Sinai Hospital, New York City(7-9).
In a retrospective view of his experiences, published in1958, the late Dr. Richard Lewisohn (10), who is generally credited withthe introduction of the method, pointed out that the famous Britishobstetrician, Braxton Hicks, used sodium citrate as an anticoagulant in 1869 butthat a number of fatalities forced him to abandon it.
Later experiments by other observers with hirudin, sodiumoxalate, and peptone showed all of these agents to be too toxic for clinicaluse. Sodium citrate, usually in 1-percent concentration, had long been used asan anticoagulant in blood collected for laboratory purposes, but thisconcentration, which was assumed to be necessary to prevent coagulation, wasalso assumed to be too toxic for human administration. By a careful series ofanimal experiments, Lewisohn showed that a concentration of 0.2-percent sodiumcitrate 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 safelyintroduced into adults intravenously, though, paradoxically, its introductioncaused a temporary shortening of the recipient's own coagulation time. It wasthus 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 hisoriginal report in January 1915 (7), Lewisohn pointed out that resultsidentical to his own had been published by Agote in Buenos Aires in the samemonth his report was published.
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In his 1915 communication, he also acknowledged the work oftwo other observers: In March 1914, Hustin, in Brussels, had treated a patientwith blood mixed with glucose and sodium citrate. In January 1915, Weil (11) publishedthe report of an earlier report before the New York Academy of Medicine dealingwith the use of citrated blood at General Memorial Hospital. He used a10-percent citrate solution, in the proportion of 1 cc. of solution to 10 cc. ofblood, and gave transfusions of as much as 350 cc. from 3 to 5 days after theblood had been collected.
Over forty years later (10), Lewisohn's philosophicreaction to these various reports about the same time as his own was that whenan 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 citratedblood might destroy the usefulness of the method. It was evident, however, thatcitrate played no part in the chills when Lewisohn and Rosenthal (12), alsoat Mount Sinai Hospital, demonstrated that reactions were not caused by sodiumcitrate but by pyrogens present in carelessly cleaned transfusion equipment.When a special department was created to handle the equipment, the incidence ofposttransfusion chills fell from 12 percent to 1 percent. Many years later, whenthe 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 Statesbetween the World Wars, but it was difficult to prepare; the bulk of the finalsolution was undesirably large; and the concentration of plasma was sodangerously high that the plasma had to be discarded (13, 14). Thesolutions introduced by Perry (15), at the Moscow Institute of Hematology(M.I.H. solution) (16), and by Gnoinski of Warsaw all had veryundesirable features.
DeGowin, Harris, Plass, and their associates used amodification of the Rous-Turner solution, consisting of 3.2-percent trisodiumcitrate 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-diluentratio of 1.5:1. The solution preserved red cells in vitro quite as well as theRous-Turner solution; produced satisfactory clinical results; and had theadvantage of conserving the plasma, which had to be discarded when the Rous-Turnersolution was used.
From their reviews of the literature and their ownexperience, DeGowin and his associates drew the following conclusions(13,14, 17):
1. Progressive hemolysis occurred in humanblood in all the preservatives studied and was much greater when the blood wasstored at 68? F. (20? C.) rather than at 41? F. (5? C.).
2. The addition of large quantities ofisotonic dextrose solution slowed the rate of hemolysis considerably as comparedwith the rate in blood stored with little or no added dextrose.
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3. Hemolysis was much less in blood stored insealed 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 citratealone or in saline. These observers used blood stored in their own solution upto 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 ofthe Subcommittee on Blood Substitutes on 30 November 1940 (19), much ofthe discussion concerned improvement of methods of preserving whole blood, bothto increase the safe period of its use and to make it safely transportable. Aswas pointed out at a later conference (20), the decision to use preservedblood in wartime would make a great deal of difference in its availability atmany points at which the wounded would be treated; the question was not one ofadvisability but of feasibility.
In 1940, there was very little authoritative information onthe 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 takenit by plane to a patient in another city. Blood had also been transported by theBritish Medical Service in World War I (p. 6).
At both of the meetings just mentioned, work carried out atthe State University of Iowa, by Drs. Everett D. Plass, Elmer L. DeGowin, RobertC. Hardin, and their group, was summarized; it was reported in the literaturethe following year (14). Between 1 September 1938 and 17 November 1940,2,123 transfusions had been given with blood preserved in the DeGowinmodification of the Rous-Turner solution, with 4.1 percent of reactions and withhighly satisfactory clinical results. Optimum inhibition of hemolysis duringstorage required a dextrose concentration of at least 3 percent, though, asBritish workers had shown, concentrations of as low as 0.3 percent permittedlonger red blood cell survival in the recipient's blood than was achieved withcitrate alone.
The prevailing belief that red blood cells would notwithstand transportation and would rupture with slight trauma did not seemreasonable to the Iowa group, in view of the trauma such cells ordinarilywithstand as they are forced through the capillary circulation. Rigid tests oftransportability were therefore undertaken on blood put up in Baxter bottles incitrate-glucose (3 percent) solution after it had been kept in a refrigerator upto 18 days. To avoid shaking, the contents of each bottle had been brought up to1,250 cc. The bottles were transported in ordinary milk cans, covered withquilts and re-iced at least once every 24 hours. Twenty bloods were shipped byplane to San Francisco and back, and 20 other samples were taken by ambulance tooutlying districts of Iowa City, to test the effect of rough roads.
There was no appreciable increase in hemoglobin levels in thecentrifuged plasma either before or immediately after transportation in 20bloods. In
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the remainder, the increases were insignificant, the largestbeing from 8.4 to 18.3 mg. percent.
All these bloods were then used for transfusion, onappropriate indications, from 3 to 45 days after they had been collected. Theonly transfusion reaction, which was limited to chills and fever, occurred in apatient who had undergone thoracoplasty.
Blood was also carried by car for 30 hours, includingstopovers, for a distance of 720 miles. It was then flown from Iowa City toOakland and back, a distance of 3,539 miles and 24 hours of flying time (46hours including stopovers). When the flasks were examined 30 minutes after theplane had landed, the erythrocytes in many of them had almost completelysedimented during the trip.
DEVELOPMENT OF PRESERVATIVE SOLUTIONS
No action was taken on preservative solutions until theConference on Transfusion Equipment and Procedure on 25 August 1942 (20), atwhich the chairman, Dr. Robert F. Loeb, requested Dr. DeGowin to draw up astatement of the problem and propose a plan to be submitted for consideration tothe Surgeons General of the Army and the Navy. At the meeting of theSubcommittee on Blood Substitutes on 20 October 1942 (21), the proposalwas made to recommend to the Armed Forces the use of preserved blood wheneverthis was feasible and fresh blood could not be used. There was no discussion ofthe preservative, and action on the recommendation was deferred.
At the meeting of the subcommittee on 13 May 1943 (22), aletter was read from Dr. P. L. Mollison, suggesting the use of what he termed a"slough preservative," incorporating citrate, citric acid, anddextrose, the constituents used in the preservative solution later adopted forblood flown overseas. Dr. DeGowin was authorized to prepare a summary statement,incorporating all experimental and clinical data available on preservativesolutions and physical equipment for whole blood preservation and shipment.
The Conference on Preserved Blood recommended by thesubcommittee 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 transfusedpreserved red cells, and Dr. Joseph F. Ross, Evans Memorial Hospital, Boston,reported, with Dr. Milan A. Chapin, on studies on the hemoglobin molecule madewith radioactive isotopes of iron.
It was brought out in the discussion that thevalue of a preservative must be judged by its effectiveness in prolonging the invivo survival of transfused erythrocytes as well as by its ability to prevent invitro hemolysis, changes in osmotic fragility and cellular potassium content,and changes in other components. The radioactive tagged cell technique reportedby Drs. Ross and Chapin was so sensitive that 0.005 cc. of transfused cellscould be detected in 1.0 cc. of the recipient's blood with an accuracy of ?10percent, and the total volume of labeled cells which had to be injected into therecipient was so small that it produced no hemodynamic or hematopoieticdisturbance. The results of this study also indicated that the breakdownproducts of hemoglobin were rapidly reutilized for the synthesis of newhemoglobin. It was therefore impossible to trace the survival of the injectedcells 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 thegreatest importance. It could be concluded from these observations that eventhough transfused cells were destroyed rapidly, they were of distinct, iftemporary, value in promoting blood formation.
2. Dr. Denstedt discussed the modifications ofthe DeGowin modification of the Rous-Turner solution which he and his associateshad devised at McGill University (and which he later requested be called theMcGill 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-percentdextrose. These quantities constituted 600 cc. of solution, with a blood-diluentratio of 2:1. With this dilution, most bloods could be stored up to 6-8 weeksat 39? F. (4? C.), with less than 1-percent loss by hemolysis.
The second solution consisted of 400 cc. ofblood, 80 cc. of 3.2-percent sodium citrate, 80 cc. of 5.4-percent dextrose, and40 cc. of an isotonic buffer solution. The resulting solution consisted of 1,000cc. 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. ofwater. These quantities also produced 600 cc. of solution with a blood-diluentratio of 2:1. The second solution was recommended on the ground that itmaintained the citrate concentration above 0.34 percent and also maintained aneffective level of dextrose. The buffered solution retarded cell swelling duringstorage, retarded changes in organic phosphates, and reduced cohesion of cellson sedimentation. When buffered bloods were stored at 50? F. (10? C.), thechanges were no greater than when they were stored at 39? F. (4? C.). Withunbuffered bloods, the changes at the higher temperature were more rapid.
At the 10 August 1943 meeting of the subcommittee (25), thediscussion of preservatives was continued. Dr. Edwin J. Cohn introduced arecommendation that glucose be added whenever blood was stored at lowtemperatures. Dr. Max M. Strumia objected to the additive; his own work withurobilin levels in blood had not shown any superior red blood cell survival invivo when it was used; in fact, cellular fragility seemed to be increased. Healso saw no advantage to preserving blood for more than 5 days, theaverage time for which blood was then banked. Dr. DeGowin pointed out thatmilitary demands were irregular and that the attempt to operate a bank withblood that could be stored only 5 days would result in excessive losses ofblood, which could be used if longer periods of storage were possible. In viewof the differences of opinion, Dr. Cohn withdrew his recommendation.
Meantime, Dr. John B. Alsever, then in the U.S. Public HealthService, 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 mixturehad to be used with each 500 cc. of blood (fig. 57).
Alsever's solution had proved satisfactory with the usualcriteria for studying red blood cell preservation; that is, rate of spontaneoushemolysis, fragility tests, hemoglobin levels, and incidence of reactions. Atthe 24 September 1943 meeting of the Subcommittee on Blood Substitutes (26), itwas recommended to the Armed Forces that, if blood was to be stored for morethan 5 days, refrigeration and the addition of glucose was essential. In thelight of present knowledge, Alsever's, DeGowin's and Denstedt's solutionswould be equally effective.
With the recommendation at the subcommittee meeting on 17November 1943 (27), that The Surgeon General of the Army giveconsideration to the
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FIGURE 57.-Preservationof blood with Alsever's solution. A. One-liter bottle containing 500 cc. ofAlsever's solution. B. Bottle ready for collection of blood. C. Blood put upin Alsever's solution in use in postoperative ward, 10th Field Hospital,Granovillers, France, November 1944. Note suspension of bottles from cord overbeds.
transportation of whole blood by airlift to certain theatersof operations (p. 465), the question of preservatives became more urgent, thoughit was not discussed further at this time. Maj. Gen. Norman T. Kirk'sacknowledgment of the recommendation about the airlift of blood was read at the5 January 1944 meeting (24). In the meantime, he had rejected it.
Dr. DeGowin reported at the Second Conference on BloodStorage on 2 March 1944 (28) that the modified Rous-Turner solution hadbeen used in
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more than 13,000 transfusions given over the past 5 years atthe State University of Iowa College of Medicine. The upper storage limit was 30days, and the incidence of reactions was low and no greater with aging bloodthan with blood used earlier. No definitive action was taken in regard topreservatives at this conference or at the meeting of the subcommittee on 3March 1944 (29), at which the Loutit-Mollison solution (30) wasfirst mentioned.
Selection of Alsever's Solution for Oversea Airlift
When the Third Conference on Blood Storage was convened on 30August 1944 (31), blood put up in Alsever's solution and flown from theUnited States was already being administered in field hospitals in France. Whenthe decision concerning a preservative had to be made without delay, because ofthe critical situation in Europe, Alsever's solution, in the absence of adefinitive recommendation from the Subcommittee on Blood Substitutes, seemed tobe the wisest choice for a number of reasons. They were stated (32) atthe 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 on24 September 1943 (26) that Alsever's solution was at least as good asDeGowin's and Denstedt's solutions; up to that time, the experience withDenstedt's solution had been limited.
2. Tests with these solutions had been carried out at theArmy Medical School since 1939. In 1943, a blood transfusion and intravenousfluid 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 offibrin, particularly when blood that had been stored for more than 7 days wasused, was so great that the steel filters in the giving sets had frequentlybecome completely blocked.
4. The more dilute Alsever's solution, which had thedisadvantage of requiring 500 cc. of solution to each 500 cc. of blood, had beenused in more than 2,000 transfusions. The added crystalloid content minimizedfibrinogen precipitation, and blood stored in this solution was much easier toadminister 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 byplane to the west coast without refrigeration and returned to the Army MedicalSchool in good condition. With continuous refrigeration, the blood could be usedfrom 25 to 30 days after it was collected; the free hemoglobin was no more than25 mg. percent. When refrigeration was interrupted for 24 hours, the datingperiod was reduced to 18-21 days. When the blood was taken from the refrigeratorand left at room temperature for 48-72 hours, spontaneous hemolysis reduced thedating period to 14-16 days.
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Blood put up in Alsever's solution was also tested in otherways. During 28 days of storage, it was shaken every day. It was transported intrucks for 8- to 24-hour periods two or three times a week, in temperaturesranging from just above freezing to 50? to 60? F. (10? to 15? C.). Themethod of testing was therefore extremely severe, and the fact that the bloodstood up well under the conditions-most of them far more severe than bloodhandled under controlled conditions would be subjected to-made it evident thatit would be practical to supply preserved blood to oversea theaters.
The reaction rate with blood preserved in Alsever'ssolution was about 1 percent. No jaundice had followed any transfusion. Eventhough blood preserved in this solution had been given in quantities up to 3,000and 4,000 cc. over a 12-hour period, none of the patients who had received thesequantities had developed pulmonary edema. Kilduffe and DeBakey's (33) reviewof the literature did not support the contention that if 4,000 to 5,000 cc. ofblood 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 itsdevelopment. It was understood, of course, that these generalizations did notapply to casualties with blast injuries or to patients with organic heartdisease and myocardial insufficiency.
5. A final and very practical reason for selecting Alsever'ssolution rather than ACD (acid-citrate-dextrose) solution as the preservativefor blood to be airlifted to Europe was that the containers for it were alreadyin production in August 1944 when General Kirk reversed his decision not tosupply blood to the European theater, as many working in this field had alwaysbelieved that it inevitably would be reversed. As it was, their procurement ofthese containers in the necessary quantities was a crash operation. With wartimeshortages and priorities, it would have taken at least 3 to 4 months to providethe 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 readerswho were not participants in the events to understand why ACD solution was notused when the airlift of blood to Europe began in August 1944, or at least whyit was not substituted for Alsever's solution in the winter of 1944-45, whenthe Navy had already proved its safety and efficiency in the airlift of blood tothe Pacific. There are a number of explanations, though perhaps no real excuses.One was the late development of the Loutit-Mollison solution; it was firstdescribed in December 1943 (30). Another was Major Hardin'sunwillingness to accept the changeover in the European theater before the newsolution had been adequately tested. The third was procurement of the smallerbottles necessary when a smaller amount of preservative solution was used.
Testing of ACD solution-The Loutit-Mollisonsolution was first mentioned in a meeting of the Subcommittee on BloodSubstitutes on 3 March
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1944 (29), when Dr. G. M. Guest, who was representingthe Canadian Committee on Medical Research, recommended that it be investigated,on the ground that the British were rapidly coming to use it since itsdescription in December 1943 (30).
On the basis of Dr. Guest's suggestion, a number ofcomparative investigations were undertaken, including clinical testing at theArmy Medical School. Preliminary reports were made at the 2 June 1944 meeting ofthe Subcommittee on Blood Substitutes (34), 4 days before D-day, and atthe Third Conference on Blood Storage on 30 August 1944 (31), after theairlift to Europe had already begun. They showed that ACD solution preservedblood satisfactorily for at least 21 days. An investigation at the Children'sHospital in Cincinnati showed that, after 38 days' storage in it, the redblood cells were in the same state of preservation as after 10 days' storagein simple citrate solution and after 22 days' storage in neutralcitrate-glucose solution.
In addition to the reduced bulk (the initial blood-diluentratio 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 fromother solutions containing only sodium citrate and physiologic salt solution.
2. The addition of citric acid to the solutionlowered the pH sufficiently (to about 5) to eliminate the tendency tofibrin-clot formation evident in blood kept longer than 14 days in othersolutions.
3. When blood was preserved in the naturalenvironment of this solution, under constant refrigeration, tests showed thatthe red blood cells were less fragile, and tolerated handling and transportationmuch better, than when they were preserved in other solutions.
At the conference on 30 August 1944 (31), too late forimplementation in the airlift already underway, it was passed, Dr. DeGowindissenting, that ACD solution be recommended as the best available solution forthe preservation of whole blood; the optimum dilution factor was considered tobe 20 percent solution to 80 percent blood. Dr. DeGowin did not consider thatsufficient evidence had been presented to date for evaluation of Alsever'ssolution, nor did he consider that ACD solution had been tested sufficientlyclinically to permit an opinion concerning fibrin precipitation with it.
It was further recommended at this conference that the ArmedForces transport whole blood at temperatures between 39? and 50? F. (4? and10? C.) from the time of collection to the point of final delivery. There wasno discussion of how this recommendation should be implemented.
Further studies on ACD solution were reported at theConferences on Blood Preservation on 19 January 1945 (35) and on 8February 1945 (36), as well as in Weekly Newsletter No. 136, Division ofMedical Sciences, National Research Council, 7 October 1944. Among the datareported were the following:
1. ACD solution was much simpler to prepareand autoclave than Alsever's solution.
2. Fibrin formation was minimal. There hadbeen no difficulties of this kind in 200
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transfusions of blood stored in this solutionand used in routine blood bank operations at the Massachusetts MemorialHospital.
3. When the survival of transfused cellstagged with radioactive isotopes in the recipient circulation was used as acriterion, there was little difference between ACD and Alsever's solutionsduring the first 15 days of storage; 70 percent of the cells survived during thefirst 48 hours after transfusion. Later, there was a considerable difference,survival being 20 days in Alsever's solution and 30 days in ACD solution. Thiswas a discrepancy of practical military importance.
4. A report by Dr. Strumia, whose conclusionswere of value because of his association with the blood-plasma project from itsinception in 1940, indicated that ACD was the most effective preservativesolution tested to date.
All reports emphasized that the data were obtained underconstant refrigeration. Deterioration of the red blood cells began during shortperiods of storage at room temperature and was not halted by subsequentrefrigeration.
Procurement of new bottles-The substitution of 600-cc.bottles for collection of the blood in ACD solution for the 1,000-cc. bottlesused to collect it in Alsever's solution required changes of orders tocontractors, who had to continue to produce the larger bottles to meet currentneeds while preparing for the changeover to the smaller bottles. By carefulplanning, the changeover to the smaller bottles was made without delay andwithout undue wastage of the larger bottles.
Acceptance of ACD solution in the European theater-WhenCapt. John Elliott, SnC, returned from the European theater after his January1945 visit (37), he brought word that Major Hardin was unwilling to haveACD solution substituted for Alsever's solution until there was overwhelmingevidence of its superiority. He wished comparative tests of the two solutions tobe conducted in the theater, on the reasonable ground that it was not wise todiscard an agent that had proved satisfactory for one that he did not considerhad been adequately tested.
The necessary tests were carried out by flying successiveshipments of blood in ACD solution to the European theater, as describedelsewhere (p. 215). The results proved entirely satisfactory clinically, thoughperhaps less impressive numerically. Major Hardin was informed, however, of thesatisfactory results obtained with ACD solution in the massive airlift of bloodto the Pacific, as well as of the fact that the use of smaller bottles wouldpermit refrigeration of the blood during the airlift across the Atlantic. He wasalso told of an improvement in the bottles; the stoppers were now hollowed outinside, so that the tip of the filter housing projected a short distance intothe bottle and any clots that might form fell around, rather than into, itsadapter.
When ACD solution began to be used on 1 April 1945, the 50cc. of 4-percent sodium citrate solution formerly placed in a 750-cc. bottle wasreplaced by 100 cc. (later 70 cc.) of a solution consisting of 2.0 gm. of citricacid, 8.0 gm. of sodium citrate, 27.0 gm. of dextrose (all U.S.P.), with waterto 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 finalconcentration of sodium citrate was 0.46 percent, of dextrose 0.45 percent, andof citric acid 0.03 percent.
Critique of Preservatives
There was, of course, never any argument about thesuperiority of fresh whole blood over the most efficiently preserved wholeblood. But fresh whole blood was not practical from any standpoint for frontlineuse. It was impractical to collect it locally, and its life was too short tofulfill requirements imposed by shipping it from the Zone of Interior to theEuropean theater. Some preservative therefore had to be used.
Since preserved blood had to be used in forward areas, theimportant point was that when the decision not to airlift blood to Europe,recommended in November 1943, was necessarily reversed in August 1944, nodecision had been reached by the Subcommittee on Blood Substitutes as to thebest preservative to use (26, 38).
When the airlift to Europe was authorized, the decision as tothe preservative to use therefore had to be made in the Blood Research Division,Army Medical School, and made without delay. Alsever's solution was selectedfor a number of reasons: It contained a satisfactory concentration ofelectrolytes and dextrose. Its dilution was less than the 750-cc. dilution ofthe DeGowin solution, though its 500-cc. bulk was undesirably large. Its pH wasdesirable. 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 testedadequately when the selection of a preservative for oversea use became necessaryin August 1944. It is also regrettable that the changeover from Alsever's toACD solution was not made as soon as the Navy experience in the Pacific provedthe safety and efficiency of the latter solution. On the other hand, as has beenpointed out in connection with flying blood to Europe without refrigerationduring the period of the flight, there were many thousands of lives savedbecause blood was provided in Alsever's solution. Many of these lives wouldhave been lost if the airlift had been deferred until a more efficientpreservative 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'ssolution in the field transfusion set should be replaced by the 600-cc. bottlecontaining ACD solution as soon as the next contracts became effective andshould thereafter be used for blood sent to the European theater. He consideredthe recommendation justified because, by 12 October 1944, Capt. Lloyd R.Newhouser, MC, USN, had received reports on trial runs to the Pacific thatindicated 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 25May 1943 (23) that the basic criterion of the value of a preservative wasthe in vivo survival of transfused erythrocytes (p. 217). At the ThirdConference on Blood Storage on 30 August 1944 (31), it was formallyagreed that the conference favored, as an optimal criterion of whole bloodpreservation, the survival of 90 percent of the transfused red blood cells for48 hours and, as a satisfactory criterion, the survival of 70 percent for 48hours. Any specimen of blood should be rejected which contained in excess of 50mg. percent of free hemoglobin.
At this same conference, it was agreed that tests fordetermining the in vivo preservation of red blood cells should includeagglutination techniques, the radioactive isotope method, studies on bloodbilirubin, and determination of the total urobilinogen output. In vitro testingshould include spontaneous hemolysis, osmotic resistance of the red cells,escape of potassium and other components from the cells, glycolysis, fibrinformation, and enzyme systems. The osmotic fragility test was not considered asatisfactory determination of in vivo survival.
Bushby and his group (40), studying various bloodpreservatives, showed, from the bilirubinemia associated with the transfusion ofstored blood, that the older the blood, the more rapidly were its corpusclesdestroyed in the recipient bloodstream. The iron pigment thus set free, however,was phagocytosed by the reticuloendothelial system and assisted in bloodregeneration during recovery from hemorrhage. These observers believed that ifthe blood was not so old that a dangerous quantity of pigment would be suddenlyliberated, the administration of even quite old blood to an exsanguinatedpatient had much to recommend it. Their theory seemed to be proved by thesuccessful use of blood well beyond its dating period during the fighting inFrance in May 1940 (p. 20).
To complete the record, a final study on posttransfusionsurvival of erythrocytes might be mentioned, which was reported at theConference on Blood Preservation and Red Cell Resuspension on 6 December 1945 byCapt. John B. Ross, MC (41). It was based on a study made with ACDsolution in the original volume (120 cc. per 480 cc. of blood) and in a reducedvolume (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, whichwere determined by the radioactive isotope technique, showed the in vivosurvival of erythrocytes to be the same in both solutions. To exclude possiblevariations in the donor blood, the bloods stored in each solution were obtainedfrom the same donors.
SPECIAL STUDIES
It has not been possible, in the compass of this chapter, todescribe all of the various studies on blood preservatives made by workers undercontracts set up by the Committee on Medical Research, National ResearchCouncil,
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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 onBlood Substitutes and other committees and subcommittees.
It is not the function of this history to review theliterature 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 itsfunctions. Attention is called particularly, in addition to the studiesmentioned 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" bloodwere particularly useful. They endeavored to meet the objection that, as theyput it, much of the literature on changes in stored blood was lacking incontrols and colored by preconceived ideas on the subject.
6. Ross, Finch, Peacock and Sammons, whichwere concluded in 1947 (51). Their extensive studies on in vitropreservation amid posttransfusion survival of stored blood, made at theMassachusetts Memorial Hospital and the Massachusetts Institute of Technology,included 16 solutions. Their chief conclusion was that, from a practicalstandpoint, blood stored in ACD solution, or one of its modifications, for 7 to10 days is as satisfactory for transfusion as fresh blood. Blood stored for 3weeks and providing cells of 70-percent viability is satisfactory for emergencytransfusions but is not so good as whole blood or blood stored for shorterperiods of time.
References
1. Muether, R. O., and Andrews, K. R.: Studiesand Uses of Stored Blood and Plasma. South. M. J. 34: 453-462, May 1941.
2. Ashby, W.: The Determination of the Lengthof Life of Transfused Blood Corpuscles in Man. J. Exper. Med. 29: 267-281, 1Mar. 1919.
3. Rous, P., and Turner, J. R.: ThePreservation 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.: ThePreservation of Living Red Blood Cells in Vitro. II. The Transfusion of KeptCells. 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 ofLiving Animals by Dialysis. J. Pharmacol. & Exper. Therap. 5: 275-316,January 1914.
6. Robertson, O. H.: Transfusion WithPreserved Red Blood Cells. Brit. M. J. 1: 691-695, 22 June1918.
7. Lewisohn, R.: A New and Greatly SimplifiedMethod of Blood Transfusion. A Preliminary Report. M. Rec. 87: 141-142, 23 Jan.1915.
8. Lewisohn, R.: The Development of theTechnique of Blood Transfusion Since 1907: With SpecialReference 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 YearsAgo and Today. Surg. Gynec. & Obst. 101: 362-368,September 1955.
10. Lewisohn, R.: The Citrate Method of BloodTransfusion in Retrospect. Surgery 43: 325-327, February1958.
11. Weil, R.: Sodium Citrate in theTransfusion 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 InfluencingHemolysis. 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. WarMed. 1: 326-341, May 1941.
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16. Elliott, G. A., MacFarlane, R. G., and Vaughan, J. M.: TheUse of Stored Blood for Transfusion. Lancet 1: 384-387, 18 Feb. 1939.
17. DeGowin, E. L., Hardin, H. C., and Alsever, J. B.: BloodTransfusion. Philadelphia and London: W. B. Saunders Co., 1949.
18. Minutes, meeting of Committee on Transfusions, Division ofMedical 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 andProcedure, 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 ofMedical 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 ofMedical 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 aDisodium-Citrate-Glucose Mixture as a Blood Preservative. Brit. M. J. 2:744-745, 11 Dec. 1943.
31. Minutes, Third Conference on Blood Storage, Division ofMedical Sciences, NRC, 30 Aug. 1944.
32. Memorandum, Lt. Col. D. B. Kendrick, MC, for Brig. Gen.Fred W. Rankin, 23 Aug. 1944, subject: Conference onSupply of Whole Blood for the ETO.
33. Kilduffe, Robert A., and DeBakey, Michael: The Blood Bankand 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 ofMedical Sciences, NRC, 19 Jan. 1945.
36. Minutes, Conference on Blood Preservation, Division ofMedical Sciences, NRC, 8 Feb. 1945.
37. Memorandum, Capt. John Elliott, SnC, to Chief, SurgicalConsultants Division, Office of The Surgeon General, through Director, ArmyMedical School, 1 Feb. 1945, subject: Transportation of Blood From the U.S. tothe ETO Blood Bank in Paris.
38. Minutes, meeting of ad hoc Committee on Transfusion andEquipment, 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 toPacific.
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, 5Oct. 1940.
41. Minutes, Conference on Blood Preservation and Red CellResuspension, 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 theTransfused Erythrocytes of Stored Blood. Lancet 2: 420-421, 5 Oct. 1940.
44. Mollison, P. L., and Young, I. M.: Failure of in VitroTests 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 Bloodafter 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: APractical 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 StoredBlood. J. Clin. Invest. 26: 687-703, July 1947.
