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HISTORY OF THE OFFICE OF MEDICAL HISTORY
The Plasma Program
World War I
This whole chapter on the plasma program should be read with the recollection that for all practical purposes, the clinical use of plasma for shock and hemorrhage was a development of World War II, just as the concept of hemorrhagic shock was a development of that war.
Reviews of the literature indicate (1) that the clinical use of blood plasma and serum was first suggested by Bowditch in 1871 and Luciana in 1872. Ehrlich was the first to point out that the most stable method of preserving the total solids of plasma or serum was removal of the water component. The basis of the enormous plasma and serum albumin programs, as well as of the clinical use of these agents in World War II, is inherent in these two suggestions.
The military use of blood plasma as a substitute for whole blood in combat casualties was proposed in March 1918, in the correspondence columns of the British Medical Journal, by Gordon R. Ward (2), as a way of eliminating the risk, in transfusions given at casualty clearing stations, that the donor red blood cells might be hemolyzed in the recipient bloodstream (p. 7). Citrated plasma would be easy to store and administer, and its use was rational: Wounded men did not die from lack of hemoglobin but from loss of fluid, with resulting devitalization and low blood pressure. Ward's suggestion of a controlled study of plasma, whole blood, and gum acacia was apparently not followed up, nor was his idea put to clinical use in combat casualties in World War I.
Also in 1918, Rous and Wilson (3) concluded from experimental studies on dogs that loss of blood volume, not loss of red blood cells, was the important consideration in hemorrhage. Even after gross hemorrhage, these workers were able to restore the blood pressure to normal, and maintain it at the normal level, by replacing the blood they had removed with an equal quantity of plasma. Plasma, they pointed out, had only half the viscosity of blood, and it seemed to them, in order that the diminished number of red cells might carry on the work of the body, that a brisker circulation, secured by a less viscid fluid, would be desirable.
Mann (4), also in 1918, reported that parenteral injections of homologous serum were fully as effective in experimental surgical shock as any other method (p. 335). Serum might therefore be a valuable agent in the treatment of shock if whole blood were not available.
The Interval Between the Wars
In 1927, Strumia and his group (5) at the Bryn Mawr Hospital began to use plasma in the treatment of severe infections because it was simpler to prepare, and had a larger yield, than homologous serum, and also because serum frequently caused severe reactions, which even heterologous plasma, given intravenously, did not. As early as 1900, Brodie (6) had called attention to the differences in the behavior of serum and plasma. He thought, though the hypothesis is still unproved, that the untoward reactions from serum were caused by the fibrin precipitation which occurred when serum was separated from clotting blood.
By 1931, the Bryn Mawr group were using plasma routinely in the treatment of certain hemorrhagic diseases as well as infectious diseases. Their policy of using it within 24 hours after the blood was drawn deprived them of one of its chief advantages; namely, its safe storage.
The first use of plasma as a hemostatic agent was apparently by Filatov and Kartasevskij (7) in 1935. In the same year, Heinatz and Sokolow (8) used it in the treatment of hemolytic shock.
The following year, Elliott (9) proposed that both plasma and serum be used in the treatment of surgical, obstetric, or traumatic shock whenever transfusion was indicated. His reasoning, like Ward's, was that the replacement of lost blood volume was more important than the replacement of red blood cells, because the maintenance of osmotic pressure is a function of the plasma proteins. Elliott also advanced two other ideas: (1) that liquid plasma could be stored for long periods without deterioration, and (2) that if plasma was pooled (he used up to eight donors), neither typing nor crossmatching would be necessary because the antibody titer would be neutralized.
Elliott's observations were quickly confirmed by a number of other observers. Then, in 1939, he, Tatum, and Nesset (10) recommended stored plasma as "an ideal substitute for whole blood in the emergency treatment of shock and hemorrhage for war wounds." Elliott's earlier recommendation that plasma could be safely used without typing or crossmatching was now supported by their experience in 191 transfusions. The technique of collecting the blood in "a sealed vacuum transfusion set" was described, with the separation of the plasma from the blood in a completely closed system that prevented contamination.
In 1940-the year before the United States entered World War II-Strumia and his associates (11) recommended the use of citrated blood plasma, without crossmatching, in the treatment of burns and shock, their results paralleling those of Mahoney (12), Elkinton (13), and McClure (14). Best and Solandt (15) reported encouraging results with plasma and serum in the prevention of experimental shock. In 1941, Kekwick and his associates (16) reported the treatment of shock and hemorrhage in air raid casualties and concluded that plasma was as effective as whole blood in restoring blood volume
in injuries of this type.1 By this time, Strumia and McGraw (5) were using plasma in large amounts, up to 950 cc. in a single injection. One of their burned patients received 7,300 cc. of plasma over an 11-day period.
All of these early experimental and clinical studies were made with liquid plasma, prepared in small amounts in hospital laboratories.
The experience with plasma in the Blood for Britain project is described elsewhere under that heading (p. 13).
The procurement of plasma on the major scale required by the Armed Forces in World War II was possible only because of the cooperation of many persons and agencies, including:
1. Volunteer donors; that is, the general public. The alternative to their donations would have been the outright purchase of blood, which would have been very expensive, probably not practical in the amounts required, and undesirable from other aspects.
2. The Army Medical School, whose activities and functions have already been described (p. 61).
3. The Army and the Navy, whose cooperation, which is described under appropriate headings, eliminated duplications, cut through bottlenecks, and added greatly to the efficiency and success of the project. By agreement, the Army handled all contracts for plasma and the Navy, all contracts for serum albumin and the other plasma fractions.
4. The American Red Cross, with its national scope, hundreds of well-organized chapters, and thousands of volunteer workers.
5. The NRC (National Research Council), acting chiefly through the Subcommittee on Blood Substitutes of the Committee on Blood Transfusions.
6. The Army Medical Procurement Agency, which, to avoid confusion and the writing of multiple contracts, acted as purchasing agent for all plasma, albumin, and byproducts supplied commercially to the Army and the Navy.
7. Commercial biologic firms, selected according to their geographic location in respect to blood donor centers and their actual and potential facilities for processing blood.
8. The manufacturing firms which developed and supplied the equipment necessary for the collection and processing of blood.
Plasma is the supernatant fluid that separates from the cellular elements when an anticoagulant is added to blood. Serum is the liquid portion that separates during the process of clotting. Plasma contains fibrinogen. Serum
does not. The distinction in nomenclature should be carefully observed, for the plasma and serum albumin programs in World War II were separate projects, though the serum program was a development of the plasma program. The "technical paradox" by which dried blood plasma was listed as serum in the Army supply catalog was not corrected until well into 1944 (17).
FORMS OF PLASMA
Background of Selection of Plasma for the Armed Forces
An earlier chapter in this volume, dealing with the evolution of the whole blood program contains an extended discussion of the reasons that led up to the selection of plasma for use of the Armed Forces by the Subcommittee on Blood Substitutes on 19 April 1941 (18) (p. 51). Briefly, they were as follows:
By the time World War II broke out in Europe, which was more than 2 years before the United States entered the war, experimental studies and clinical testing had advanced sufficiently far to make it clear that either serum or plasma would be the most desirable agent for the management of shock in battlefield casualties and in forward hospitals. There was practically no difference in their clinical effect. The only biochemical difference between them was that serum contained no fibrinogen, its removal having occurred during the clotting process. Reactions with both agents were insignificant. Liquid plasma, if properly handled, could be safely stored for months. Frozen plasma could also be kept for indefinite periods. A dried form of plasma could be produced, though methods of drying were not yet entirely satisfactory. The chief advantage of plasma was that the yield was 15-20 percent greater per pint of blood than the yield of serum.
When the subcommittee recommended that either frozen or dried plasma be employed in lieu of blood in the treatment of shock, there were a number of reasons why the Armed Forces had little choice but to accept the decision:
1. Supplying whole blood to the Armed Forces in the now imminent war, in the quantities likely to be needed, together with its safe storage and transportation, presented logistic problems of enormous proportions that simply could not be solved in the light of either the knowledge possessed or the facilities available in 1940-41. Preservative solutions that would permit long storage periods of blood were just being developed (p. 221). Investigations on thoroughly dependable, avid grouping sera were in their very early stages (p. 236). The development of suitable equipment for the collection, storage, and dispensing of whole blood was in its infancy (p. 163). Refrigeration equipment for use in the field under varying conditions of heat, cold, and humidity had not yet been manufactured (p. 206). Finally, an airlift capable of delivering blood to the far reaches of the battlefront was still almost 3 years away.
2. Frozen plasma was obviously unsuitable for use under battlefield conditions. While liquid plasma could have been used, dried plasma had greater
advantages It could be dried from the frozen state to less than 1-percent moisture content. In this state, it could be packaged under vacuum and preserved for years without refrigeration and without being affected by extremes of heat and cold. From the standpoint of logistics, the equipment necessary for its reconstitution and intravenous administration could be incorporated in a small kit, which could be made available under almost any conditions of war.
3. No matter in what form it was used, plasma could be administered without typing or crossmatching.
4. The administration of plasma was attended with a negligible incidence of reactions.
5. Most important of all, in the light of immediate needs, dried plasma could be easily, safely, and quickly produced commercially in the large quantities likely to be needed.
The inherent organic and other characteristics of plasma, particularly the ease with which it could be manufactured, stored, and transported, clearly made it a practical and desirable agent. The reasons for its selection in 1941, while they do not fully explain lack of attempts to supply whole blood to field units at this time, did take cognizance of obstacles that went far toward discouraging even the most ardent advocates of whole blood as a feasible replacement fluid in Zone of Interior hospitals. These reasons were even more valid in the recommendation at this time of plasma as a feasible and practical agent in oversea hospitals.
The 1941 decision of the subcommittee was, of course, colored by the position of the Office of The Surgeon General almost a year earlier, to the effect that when blood could not be collected locally, plasma, "either plain or dried," would have to be used (19). At this meeting (Committee on Transfusions, 31 May 1940), arrangements were also made for Dr. Max M. Strumia to be provided with blood, collected by the southwestern Pennsylvania Chapter of the American Red Cross, for the production of dried plasma to be tested by the Army and the Navy Medical Schools and by members of the Subcommittee on Blood Substitutes.
The several hundred lots of plasma prepared and distributed by Dr. Strumia from the blood secured by the Red Cross, as arranged at the 31 May 1940 meeting of the Committee on Transfusions, were reported on at the 18 July 1941 meeting of the Subcommittee on Blood Substitutes (20, 21). Each lot contained from 17.5 to 18 gm. of plasma dried by sublimation from the frozen state by a technique employing water vapor condensation by low temperature in flame-sealed ampules (vacuoles). The containers were large enough to permit reconstitution with water and were suitable for direct administration of the solution. The difficulties, all minor, which were reported by the workers who used the plasma, chiefly Army and Navy medical officers, were assumed to be those that would be experienced on the average hospital service. The only reactions were urticarial, and they were few and mild.
The following conclusions were drawn from this experience:
1. Although plasma concentrated four times can be given without untoward reactions, normal reconstituted plasma is usually superior to the concentrated variety.
2. Without additional supplemental electrolytes, serum albumin cannot be depended upon to restore circulating blood volume in acute peripheral circulatory failure, especially in dehydrated patients.
3. The investigation was regarded as entirely successful from the standpoint of practical production of dried plasma which was safe for human administration.
The dating periods for all varieties of plasma, which eventually proved surprisingly long, were originally little more than guesswork, because of lack of previous experience.
When liquid plasma was first prepared, NIH (National Institute of Health) set the dating period at a year. Studies by Dr. F. H. L. Taylor, with Capt. Lloyd R. Newhouser, MC, USN, and Lt. Cdr. Eugene L. Lozner, MC, USN, to determine whether this limit was justified, showed that it could safely be extended. Clinical administration of 2-year-old plasma to burned patients, with normal controls, showed it to be an excellent agent to combat shock. It is true that this plasma was devoid of most active globulin components, complement, fibrinogen, and prothrombin, and had no power of coagulation on recalcification. Nonetheless, the administration of 2,000 cc. did not increase the coagulation time of the recipient's blood nor did it decrease its concentration of prothrombin.
The U.S. Public Health Service first set 2 years as the dating period for dried plasma (with the specification that the cutoff date should appear on the container). Later, the date was extended to 36 months, and, finally, all limitations were removed.
Development of Use
30 November 1940-At the 30 November 1940 meeting of the Subcommittee on Blood Substitutes (22), Dr. Strumia reported on the technique of drying plasma from the frozen state and recommended the standardization of apparatus and technique, the location of Red Cross centers for collecting and drying plasma, and the establishment of a distribution chain. It was at this meeting that the Red Cross was asked to assume the responsibility of collecting blood for the plasma program (p. 102).
Also at this meeting, in a report of the Blood for Britain project, it was pointed out that liquid plasma was frequently contaminated, but that there was apparently no multiplication of bacteria in dried plasma.
19 April 1941-At the 19 April 1941 meeting of the Subcommittee (18), Dr. Strumia reversed his previous position and stated that he now thought
that in many ways frozen plasma was superior to both liquid and dried plasma. The method developed in his laboratory for the preservation of frozen plasma was "very simple, very economical and * * * likely to apply to the needs of both civil and military nature in the greatest majority of cases."
The only disadvantages Dr. Strumia could see to his proposals were that it would be more difficult to transport frozen plasma under adverse conditions and that this form could not be reconstituted into concentrated plasma if it were needed in that form.
All of Dr. Strumia's remarks were predicated on the correct processing of the plasma; that is, it must be collected under aseptic precautions; in a closed system; and with as brief a timelag as possible between the collection of the blood, its centrifugation, and the fixation of the plasma by freezing (fig. 61). Care also had to be taken that thawing did not occur accidentally, from frequent opening of the storage cabinet, or from transient failure of current. In these circumstances, labile constituents could deteriorate.
In view of the advantages he had listed, Dr. Strumia considered the frozen variety of plasma to be the product of choice for those communities in which the exact time or the exact need could not be predetermined, but in which unexpected needs might be extremely heavy, as in large industrial areas or communitywide catastrophes. Processing should be done with the technical advice, and under the supervision, of some such authority as NRC. The bulk of the plasma processed from the blood procured by the Red Cross should be processed and distributed in this form.
These views were not generally accepted. Dr. Milton V. Veldee stated that only in grave emergencies would he favor the freezing of plasma or serum by hospitals, though he agreed with Capt. (later Col.) Douglas B. Kendrick's point, in which Dr. Edwin J. Cohn concurred, that plasma might be preserved, as a matter of expediency, in the frozen state while the capacity of commercial plants for desiccation was being increased.
18 July 1941-At the 18 July meeting of the subcommittee (20), certain facts were frankly faced. The Navy had impelling needs and would require most of the 200,000 units of dried plasma now on order. This would leave no blood substitutes on hand for emergencies likely to occur in the training maneuvers underway and to continue during the summer. Base hospitals had no blood banks, since The Surgeon General had refused to permit the storage of either blood or plasma. In addition, medical officers had to be trained in the handling of plasma and in an understanding of its potentialities and limitations.
Captain Kendrick therefore proposed, with Commander Newhouser concurring, that a limited number of bleeding units be set up to supply frozen plasma for the Armed Forces in the Zone of Interior and that personnel in charge of these units be trained in Washington or at some other suitable center. For the past year, the Naval Medical School in Washington had been supplying adjacent naval hospitals with limited amounts of frozen plasma.
FIGURE 61.-Preparation of liquid plasma at 8th Service Command Laboratory, Fort Sam Houston, Tex., September 1942. A. Centrifugation of blood to separate plasma from red blood cells. B. Pooling of plasma after centrifugation of blood in 2,000-cc. bottle containing 200 cc. of glucose solution. C. Display showing bleeding bottles, pooled liquid plasma, liquid plasma in 600-cc. bottles ready for dispensing, frozen plasma, and cultures to check sterility of plasma. D. Pooled liquid plasma held in storage until cultures are reported negative; then, it will be frozen. E. Freezer loaded with frozen plasma, which will be kept in this state until it is removed and thawed for shipment. Toward end of war it was found that this technique, adopted to preserve plasma proteins, also preserved virus of serum hepatitis.
This recommendation was conveyed to the Surgeons General of the Army and the Navy and was put into effect, with certain modifications.
Conclusions Concerning Frozen Plasma
At the annual meeting of the American Society of Refrigerating Engineers on 7 December 1943, Colonel Kendrick summarized the experience of the Medical Department with frozen plasma as follows(23):
1. Plasma, properly prepared in a closed system which excludes contamination, can be preserved at room temperature and safely administered after 24 months' storage.
2. Plasma can be safely prepared from blood kept at room temperature. This method yields a very clear product.
3. If plasma is stored as a liquid, it should be maintained at room temperature rather than 39° F. (4° C.) because at lower temperatures fibrin precipitates readily, and the result is an undesirable product from an esthetic standpoint.
4. Liquid plasma stored at room temperature loses its labile components (complement and prothrombin), which are useful in wound healing, but retains intact its albumin and globulins, which are essential in the treatment of shock and burns.
5. If plasma is stored in the frozen state, there are almost no changes in its constituents.
6. While plasma can be either shell frozen (p. 281) or frozen without rotation, there is no advantage to the former technique if it is merely to be stored frozen.
7. Commercially available ice cream cabinets are well suited for both freezing and storing plasma. The position of the bottle during the freezing process is of little importance.
8. The time required for freezing should not exceed 6 hours, but the complete process can be accomplished routinely in 3-4 hours. When the 6-hour limit is exceeded, the plasma tends to look turbid when it is thawed. Its efficacy is not affected, but the clinician is likely to regard it as contaminated.
9. Frozen plasma should be kept at a constant temperature, preferably between 14° to -4° F. (-10° to -20° C.). If the temperature is allowed to rise slowly, up to 32° F. (0° C.), the minute thawing which occurs may result in the precipitation of fibrin.
10. If plasma is thawed rapidly in a water bath at 98.6° F. (37° C.), fibrin will not be precipitated. Plasma thawed by this method can be refrozen, rethawed, and refrozen again, and the cycle can be repeated many times, without any apparent changes it its properties. These facts carry an important practical implication: If power failure occurs, it is better to remove the plasma from the icebox, thaw it, and refreeze it when power has been restored.
These various observations made it possible to draw up the following rules for the preparation and storage of frozen plasma in hospitals:
1. Blood must be collected in a completely closed system.
2. The blood can be kept at room temperature or at 39° F. (4° C.), but the supernatant plasma must be recovered within 72 hours after the blood is collected.
3. The plasma should be stored in a well insulated, low temperature cabinet, maintained at 14° to -4° F. (-10° to -20° C.), capable of freezing the plasma within 4 hours.
4. When plasma is required for use, it can be thawed in a water bath at 98.6° F. (37° C.) in 20-25 minutes. It is good practice to keep three or four bottles of plasma thawed out and available for immediate use in the operating room or emergency room.
5. Pools of 1,800 cc. of plasma should be aspirated into receptacles containing 200 cc. of glucose, which gives a final solution of 5-percent glucose in each 500 cc. of plasma. With this dilution, fibrin precipitation does not occur when plasma is stored at room temperature after thawing.
Refrigeration was at first considered necessary for the preservation of liquid plasma simply because it was the universal custom to refrigerate the blood from which it was prepared. When it was refrigerated at 43° to 46° F. (6° to 8° C.) by Strumia and McGraw (5), there was progressive flocculation of the more unstable proteins, and bacterial contamination was also a risk. Control series at the Army and the Navy Medical Schools later showed that liquid plasma stored without refrigeration was just as satisfactory as refrigerated plasma from the standpoint of sterility, hemoglobin content, and plasma protein content, and was far more satisfactory from the standpoint of clarity.
Supply of Liquid Plasma for Zone of Interior Hospitals
When liquid plasma was first introduced, it was thought best to use it in the hospital in which it was prepared, or in hospitals in the community. The results of the mass collection of liquid plasma in the Blood for Britain project, with its high rate of contamination (p. 14), had been discouraging. The explanation of the difficulties in the British program was simple: If the mass processing of plasma was to prove safe and practical, collection and processing must be carried out by a completely closed, completely aseptic technique.
While the Blood for Britain project was underway, the Blood Research Division in the Army Medical School was investigating the possibility of supplying liquid plasma to hospitals in the Zone of Interior. Names of possible donors were provided in groups of 10 by the American Red Cross, and from the 10, a daily average of 6 donors was secured, who were bled at a small center set up in the school. The first plasma processed for this purpose was distributed in December 1940. At the same time, by a similar project, the Naval Medical School, U.S. Naval Medical Center, made liquid plasma available to all naval hospitals in the continental United States. The plasma was kept in the frozen state until just before shipment; then it was thawed at 98.6° F. (37° C.) and shipped in the liquid state.
In June 1941, the Army and the Navy Medical Schools combined their efforts, and a collection center was opened in Washington, D.C., as a joint Army-Navy project. The Red Cross procured the donors by public solicitation, and helped in the operation of the center. The technical help was furnished by the Army and the Navy Medical Schools. The blood thus secured was divided between the Army and the Navy, and the Army share was processed into liquid plasma at the Army Medical School.
From these small beginnings came the project by which liquid plasma was supplied to military hospitals in the Zone of Interior as an easy, quick, and economical way of supplying them with plasma (p. 95). The Division of Surgical Physiology at the Army Medical School supplied liquid plasma for
the hospitals in the First, Second, Third, and Fourth Service Commands. It also trained personnel who operated the plasma processing laboratories in the other service commands (fig. 61), and supervised the installation and operation of these laboratories. After 22 March 1944, all liquid plasma for Zone of Interior hospitals, whether for emergency use or routine replacement, was obtained from the Army Medical School.
Concentrated plasma was discussed at several of the meetings of the Subcommittee on Blood Substitutes in 1941 (20, 24, 25), and several tests were made with it. The basic of the proposal was twofold: (1) that any casualty who really required plasma would need at least two units, and (2) that the adoption of the plan would reduce the container volume by half, with a corresponding saving in supplies and shipping space. It was concluded that concentrated plasma was not so safe an agent as isotonic plasma (26).
In the spring of 1943, at the request of The Surgeon General, E. G. Pickels (27), from the International Health Division of the Rockefeller Foundation, visited the nine biologic firms then processing dried plasma for the Armed Forces. He was accompanied by representatives of a firm of engineering consultants engaged by the Government to conduct a survey in connection with the renegotiation of contracts. On his return, Dr. Pickels felt justified in quoting from a publication by Franz Oppenheimer in the New York State Journal of Medicine dealing with techniques of drying plasma:
It is a truly remarkable achievement that the American biologic industry has taken a laboratory process, which is complicated and meticulous, and has rapidly developed production methods so efficient that they were now supplying several million units of plasma per year to the armed forces.
It was a well-deserved tribute, which was repeated many times, in one form or another, in the course of the war.
The processing of whole blood into dried plasma for the Armed Forces became a function of the large commercial biologic and pharmaceutical laboratories. When the need arose, in 1940, no individual and no private or commercial organization had had any extensive experience with the production of dried plasma. The Sharp & Dohme experience, while considerable, had been on a small scale. Some consideration was given to the formation of a large, nonprofit plasma processing plant in cooperation with some large hospital, but the idea promptly gave way to the more realistic decision that commercial laboratories would be better suited for the task. Their performance, as just indicated, was remarkably satisfactory.
After the blood for plasma had been collected at the Red Cross blood donor centers, it was refrigerated and shipped to the commercial firms which would process it and which had been selected for the work for two reasons: their nearness to the bleeding centers and their facilities.
Plasma was processed in the following steps:
1. When the blood reached the processing firms, the required serologic studies were performed on each donation.
2. Then the plasma was separated from the cellular elements in centrifuges, after which varying numbers of bloods were pooled and bacteriologic and toxicity tests were performed.
3. The plasma was shell frozen in individual bottles by rotating them in a properly cooled medium. It was important that the freezing process be carried out in as short a time as possible.
4. When necessary-when, for instance, the supply of blood exceeded the processing facilities-the plasma could be stored indefinitely in the frozen state. Otherwise, it was desiccated from the frozen state under high vacuum, after which the bottles of dried plasma were evacuated, stoppered, and placed in evacuated metal containers.
The description just given makes the process of drying plasma sound reasonably simple. The manual published in November 1942 by the Office of Civilian Defense (28) pointed out that, contrary to the prevailing opinion among the uninformed, the preparation of dried plasma of U.S.P. quality or better was an exacting task, requiring expensive equipment and trained personnel. A small, inexpensive drying machine, suitable for use in individual hospitals or small communities, and meeting the specifications for processing dried plasma, did not exist. The preparation of dried plasma was an undertaking for a large laboratory, with financial resources and an adequate staff, and, even then, it was practical only if the distribution area extended over a very large territory.
Historically, the development of desiccated plasma falls into three distinct periods (28):
1. Desiccation was used on a very small scale, for a limited amount of research and teaching.
2. Research and teaching were expanded, and desiccation was applied to the preservation of convalescent serum in a few large medical centers.
3. The growing appreciation of the value and necessity of supporting blood protein levels and the administration of concentrated plasma for its hypertonic effects foreshadowed the mass preservation and use of desiccated plasma in various concentrations.
Development of special techniques-Ehrlich, as already pointed out, was the first to observe that the most stable method of preserving the total solids of plasma or serum was to remove the water. The process was carried out successfully in serum by Rosenau in 1895, Martin in 1896, and Noguchi in 1907. It was not until 1909, however, that any practical application was made of these observations. In that year, Shackell (29) described the basic principle
of vacuum desiccation from the frozen state, a process essential to the production of a highly soluble product which would retain its original properties.2
Shackell also solved another basic problem, how to deal with the huge volume of water vapor released from only a few cubic centimeters of frozen plasma under the high vacuum conditions required for the drying process. The volume was far too great for any pump then available to handle. By the use of the principle of chemical adsorption, Shackell provided a lead which many subsequent investigators followed, though his choice of sulfuric acid for a desiccant, as well as his design of the machine, limited the usefulness of his method. In addition to his use of a chemical adsorbent, which prevented saturation of the exposed surface, the type of pump Shackell used produced an extreme vacuum very rapidly. Moreover, freezing the material before desiccation eliminated possible concentration of substances and, to a lesser degree, prevented shrinkage and hardening (30).
In 1935, Elser, Thomas, and Steffen (31) improved the design of the original drying machine by the use of a manifold system with attached glass containers and a cold trap for collecting the moisture. They found themselves severely restricted, however, by the characteristics of the chemicals used. Greaves and Adair (32) had the same experience the following year. These difficulties, including insufficient speed of adsorption, dilution, scum formation, and other changes which retarded the pickup of water vapor as the process proceeded, increased as the size of the apparatus was increased. Qualitatively, a highly successful product could be obtained, but the difficulties mentioned, together with the expense of using a new desiccant for each lot of plasma, limited the use of the method devised by Elser and his associates.
Later, this group (33) used carbon dioxide snow, which produced a temperature of -94° F. (-70° C.), and still later they used mechanical refrigeration, which produced a temperature of -29° F. (-34° C.).
Within a closed system of manifolds and tanks hooked up to a vacuum pump, water vapor escapes from the plasma by sublimation. Between the plasma bottle and the pump, there is a cold chamber, refrigerated externally mechanically or by carbon dioxide and alcohol, to provide temperatures of -58° to -101° F. (-50° to -74° C.). The water vapor is thus condensed and frozen on the inner wall of the chamber, and the remaining water is thus removed from the line and the vacuum is, in turn, adequately maintained.
Elser must be credited with the first processing of large quantities of biologicals in their original containers. He also accomplished vacuum sealing directly from the machine.
In 1934 and 1935, Mudd, Flosdorf, and their associates (34, 35) continued the work done by Elser and his group and placed the large-scale desiccation of frozen sera on a practical basis, by attaching ampules of prefrozen material
to a manifold in the open air and employing solid CO2 to refrigerate the cold trap (fig. 62). They also used alcohol and Methyl Cellosolve for the freezing mixture.
In 1939, Flosdorf and Mudd (36) introduced what they termed the Cryochem process, using a specially prepared anhydrous calcium sulfate (Drierite), which had the property of regeneration. It was an ingenious idea, but it did not prove practical for large-scale production.
In 1939, Greaves and Adair (33) reported a technique which utilized a cold-trap condenser. They placed the serum in a desiccating chamber provided with electrical heaters and arranged for the moisture to be collected on mechanically refrigerated coils. Their report included a comprehensive discussion of the temperature relations to be considered in drying large volumes of sera.
In 1940, Hill and Pfeiffer (37) reported the Adtevac technique, which used cooled silica gel as an adsorbent for the water vapor. The gel could be regenerated by heating and the adhered moisture drained off. This technique, the report noted, was entirely satisfactory for use in connection with a hospital blood bank but was not suitable for large-scale production.
A variety of other techniques and modifications of older techniques were introduced during 1940 (1, 36, 38-40).
At a meeting of the Blood Plasma Producers Association on 27 October 1942 (41), Dr. Sidney O. Levinson and Dr. Franz Oppenheimer of the Michael Reese Hospital described a technique for shortening the desiccation process by
the use of a sort of cage of copper and aluminum sheeting around each bottle, with an individual gold reflector which provided infrared heat. A new type of compressor was also used. The technique was not accepted for a number of reasons, including the expense ($15,000 per unit) and the requirement for critical materials in the construction of the apparatus. If the requirements for blood plasma were increased and expansion of the present program became necessary, it was the sense of the meeting that the installation of the Levinson-Oppenheimer method might then be considered.
Although, for practical reasons, the Levinson-Oppenheimer technique could not be adopted at this time (1942), it was an important contribution, for it permitted complete control of the amount of heat imparted to all areas of the shell-frozen plasma, in keeping with the variations in the vacuum gage readings. Dr. Levinson's additional suggestion, that drying could be facilitated by the techniques then in use if a widemouthed bottle were substituted for the constricted-neck bottle employed, was protested by Dr. Veldee because of the danger of contamination. The action of the conference, based, as it was, on wartime conditions, was entirely justified. Nonetheless, this was superb, highly efficient equipment because of its correct application and control of heat exchange. It was installed later at Hyland Laboratories and was put to good use during the Korean War.
Conclusions-In concluding his historical review of techniques for the desiccation of plasma, which was prepared after commercial production had been on a practical basis for almost 3 years, Pickels made the following points:
1. When a cold surface of sufficiently low temperature is employed for collecting water vapor, the maximum possible rate of sublimation from frozen material in a given container will be obtained if the conduits from the container to the trap are sufficiently wide and short.
2. This important theoretical condition was taken advantage of by Oppenheimer and Levinson in their experiments with combined drying and condensing chambers which depended on mechanical refrigeration. The same condition was also taken advantage of by Wyckoff in the design of his multiteated, CO2-cooled "pigs" (p. 280).
3. These methods began to approach the optimum solution from the standpoint of theoretically desirable features and practical performance, especially when speed of drying was a prime consideration.
4. The steam ejector technique offered a decided advantage only for very large-scale production, and even then only when circumstances were peculiarly favorable.
Criteria of Acceptable Dried Plasma
At the meeting of the Subcommittee on Blood Substitutes on 8 May 1941 (24), the following criteria of acceptable commercially dried plasma were stated:
1. The moisture content of the final product must be less than 1 percent.
2. The hemoglobin content must not exceed 25 mg. percent.
3. The sterility standards must be equivalent to those required by the National Institute of Health (42).
4. The product must be soluble within 10 minutes when reconstituted to its original volume.
5. The reconstituted material must be no more turbid than the product from which it was made.
At this time (May 1941), the equipment devised by Dr. Strumia was proving highly efficient for hospital and laboratory preparation of dried plasma, though it was not suitable for commercial production. The Sharp & Dohme method and the Reichel modification of this method had been approved by the National Institute of Health. The Hill and Pfeiffer Adtevac method had not yet been approved, but it was thought that the changes being made in it would make it acceptable. The Wyckoff-Lagsden "pig" method was under development at Lederle Laboratories, and it was thought that it would eventually be capable of handling large quantities of plasma. When it had been fully developed, it proved a most efficient technique.
COMMERCIAL PROCESSING OF DRIED PLASMA
By December 1943, nine commercial laboratories were engaged in the production of dried plasma. As they entered the program separately, with little liaison with one another in the initial phases, they used widely different types of equipment, though the principles of desiccation which each employed were practically identical.
Shell Freezing Technique
There was general agreement that shell freezing of liquid plasma was necessary to obtain the best dried product. By this process, the plasma was frozen to the inner aspect of the bottle in a shell or layer of uniform thickness, with an empty cone or circular channel in the long axis of the bottle extending from the bottom to the neck (fig. 63). Since the volume of plasma frozen did not ordinarily exceed three-quarters of the volume of the container, the diameter of the circular channel was equal to, or greater than, the thickness of the shell.
Shell freezing (fig. 64) was essential before the plasma was dried, to provide for proper evaporation of the moisture from it. When the shell was formed, the surface area from which evaporation could take place was increased. The dried plasma had a flaky appearance and the interstices present increased the speed of reconstitution when distilled water was introduced into the container. A shell of uniform thickness was essential; otherwise, the rate of evaporation might be irregular, and melting and fusion of the plasma would result.
Methods.-Shell freezing and drying were accomplished in a variety of ways (figs. 65-71). Most often, the stoppered bottles containing the liquid plasma (300 cc. of plasma in 400-cc. bottles) were rotated horizontally at 2-8 r.p.m. in a cold bath ranging in temperature from -58° to -101° F. (-50° to -74° C.). It was essential that the bottles make contact with the bath for depths of no more than one-eighth to one-fourth of an inch, to prevent freezing of plasma in the neck of the bottle. When plasma froze in this location, the diameter of the orifice was reduced and drying time was increased, since the escape of water vapor from the bottle was partly a function of the diameter of the outlet. It was found that horizontal rotation produced a better shell than rotation at a 45°- or 60°-angle. When the bottles were rotated at an angle, plasma froze in the bottom of the container and formed a button which was thicker than the remainder of the shell and which frequently melted slightly during the drying process. The result was fusion or gumming of the plasma,
and the further result was that the plasma became denatured and would not go into solution normally.
Ethyl alcohol and Methyl Cellosolve were usually used to secure the desired temperatures. The alcohol bath was considered better because the fumes from the latter agent, particularly when the shelling was carried out in a closed room, might produce symptoms of methyl alcohol poisoning in the workers.
The original idea that biologics must be shell frozen at -101° F. (-74° C.) arose from the practice of using Dry Ice in alcohol, which produced temperatures at that level. Long after mechanical refrigeration, which permitted graded temperature levels, was available, the idea persisted that for the best results, shell freezing was necessary at the level mentioned. The extensive experimental work on freezing plasma by Strumia and his group (5), which was published in 1941, disproved this contention and established the feasibility of shell freezing within the temperature range of -58° to -76° F. (-50° to -60° C.).
Refrigeration machines first developed for maintaining shell freezing units at -101° F. (-74° C.) consisted of two high-stage compressors with propane on the low side and ethane on the high side; each compressor operated with a 2-hp. motor. This equipment did not prove satisfactory: The refrigeration capacity was insufficient. The ethane compressor frequently became fouled with oil. The expansion valve on the high side frequently became clogged with ice from water in the ethane gas.
The possibility of using higher temperature ranges for shell freezing greatly simplified the use of mechanical refrigeration for this purpose. In several commercial laboratories, shell freezing was accomplished routinely at -58° to -76° F. (-50° to -60° C.). The machines were operated by one-stage compressors which used F-22 gas or two-stage compressors which used F-12 gas. At such ranges, plasma could be shell frozen in 15-25 minutes.
Equipment for Shell Freezing and Drying Plasma
Space does not permit the detailed description of the equipment used for shell freezing of plasma (fig. 72) and for drying it, but the whole process is described in an article published in 1944 (23).
Preservatives-The National Institute of Health minimum requirements for normal human plasma, issued on 20 February 1941, provided that after the sample of pooled plasma had been withdrawn for the sterility test, a sufficient amount of a "suitable preservative" should be added, "except that phenol or a similar compound" should not be regarded as suitable.
The Subcommittee on Blood Substitutes discussed this matter on several occasions. It was brought out, at the first discussion (22), that the Blood Transfusion Association of New York had found Merthiolate unsatisfactory and Zephiran too toxic for use. Some of the subcommittee membership thought that plasma was best stored without any preservative at all (25). A recommendation to this effect was waived when it was found that commercial firms were not inclined to process plasma without one (43).
Merthiolate (1:10,000) was originally employed for this purpose. At the 19 September 1941 meeting of the subcommittee (43), the question was raised whether, when massive doses of plasma were necessary, there was danger that the patient might receive too much of the mercurial preservative and might incur renal damage. A motion setting a limit to the amount of plasma containing a mercurial preservative which might be given was lost after it was pointed out that the amount of mercury administered with plasma did not compare with that used in antisyphilitic treatment or given in the form of mercurial diuretics.
FIGURE 67.-Equipment (Cutter Laboratories) for Emery technique of shell-freezing plasma: cold alcohol (a), low temperature compressor (b), circulating pump (c), end view of apparatus (d), and top view (e).
It was agreed that a preservative was an added protection during the time which elapsed between the opening of the container of plasma and its infusion, but no action was taken, though some members of the subcommittee thought-and action was later taken to that effect-that the lapsed time should not be more than 3 hours. The NIH minimum requirements simply stated that plasma "should be used promptly after restoration."
At the 3 November 1941 meeting of the subcommittee (44), Dr. Veldee reported for himself and Dr. Soma Weiss on the review of the literature which they had been appointed to make at the previous meeting: Merthiolate apparently had some bacteriostatic value, and possibly some bactericidal value. The presently employed concentration of phenylmercuric nitrate in plasma (1:50,000) was not considered toxic. Dr. Weiss, however, was willing to accept Merthiolate as a preservative only if a definite limitation were set on
the dosage of plasma and if the symptoms of mercurial poisoning were published on the label of the can. Other members suggested a limit of 3-6 liters of plasma in 24 hours. Dr. Strumia suggested putting the preservative in the distilled water, the amount of which could be regulated as desired. It was finally passed that phenylmercuric nitrate, 1:50,000, or Merthiolate, 1:35,000, be used in plasma, with the maximal dose to be 4 liters in 24 hours. This recommendation was later adopted. On 10 April 1942, Dr. Veldee authorized the change, with the concurrence of others concerned, from phenylmercuric
nitrate to phenylmercuric borate, the proportions (1:50,000) to remain the same.
On two occasions, the subcommittee decided that sulfonamide derivatives should not be used in plasma (43, 45). Dr. Veldee's studies confirmed the observations of others, including the British, that they produced no significant bacteriostatic effects.
Sodium citrate-An attempt on the part of one of the processing firms to substitute 25 cc. of sodium citrate for the usual 50 cc. of 4-percent solution, in order to reduce the amount of fluid in the large plasma bottle, began hopefully, as did the control series conducted at the Army Medical School. The high percentage of clotting, however, forced a return to the original practice.
Sodium chloride-The question of omitting sodium chloride from the citrate solution used as a preservative came up when Dr. Cohn reported that processing of serum albumin was simpler when it was not used (45). When
its omission was studied from the standpoint of plasma yield, the difference was found to be only about 1 percent (46). The plasma yield in 1,500 liters of blood processed without salt by Eli Lilly and Co., averaged 55.3 percent after centrifugation (47).
Dextrose.-In June 1942, Commander Newhouser reported that he had begun to make pools of plasma diluted in the proportion of 2,000 cc. of plasma to 200 cc. of 50-percent dextrose, by the sedimentation technique, with results that were so far very satisfactory. Two opinions were expressed, that the addition of glucose would have no effect on flocculation and that there was more flocculation without glucose than with it. At the Naval Medical School, dilute plasma had been kept in the plasma bank for 24 months without precipitation, and there had been no flocculation over a 14-month period in plasma to which dextrose had been added.
Citric acid-Studies by Dr. Strumia (45) on the reconstitution of dried plasma with 0.1-percent citric acid solution instead of distilled water showed that the pH of 7.4-7.6 thus secured preserved much of the complement and prothrombin, labile elements which were lost in considerable amounts on storage. It was also found that a citric acid solution with a pH of 2.8 kept much better in glass than did distilled water. These observations were confirmed by Commander Newhouser, Colonel Kendrick, Dr. F. H. L. Taylor,
and others, and it was therefore recommended on 15 December 1942 that 0.1 percent citric acid solution be substituted for 1 .0 percent sodium chloride in the reconstitution of dried plasma (48). This recommendation was later put into practice.
National Institutes of Health requirements specified that each package of dried plasma contain a warning as to the danger of injecting plasma intravenously without the use of a filter in the tube leading from the plasma reservoir to the recipient.
The amount of plasma which could be filtered through the ordinary Seitz filter before the filter became clogged and closed was so small that this method was not practical until the pads were saturated with sodium citrate or some other solution. Five-inch pads permitted the filtration of 18,000 cc. of plasma at 98.6° F. (37° C.) in less than 3 hours.
In February 1942, Dr. Strumia reported a series of experiments intended to determine the effects on citrated plasma of filtration through a Seitz sterilizing filter (49). Filtration through pads saturated, respectively, with 0.85-percent saline solution and 4-percent sodium citrate-saline solution apparently caused a loss of some or all of the fibrinogen. When the material was left standing at 39° F. (4° C.), both lots developed a heavy precipitate, resembling fibrin, and containing, respectively, 0.11 gm. and 0.13 gm. of fibrinogen.
A second experiment indicated that filtration through a Whatman filter No. 1 under the conditions of the experiment caused an appreciable loss of fibrinogen, a sharp increase in pH, an apparent loss of prothrombin, but no appreciable loss of other proteins and no changes in the complement titration.
When the material was kept in a liquid state, flocculation was extremely annoying.
Dr. Strumia did not consider filtration of properly prepared plasma necessary for purposes of sterilization, since bacterial contamination in it was extremely small. In 105 pools made from 885 individual bloods, cultures made by the National Institute of Health technique had shown only two pools to be contaminated.
Occasional complaints were received that some bottles of dried plasma contained an excess of fibrin or fibrinogen particles, which interfered with the infusion. The complainants were always informed that there was no risk from the use of such plasma if it were filtered during administration, as all plasma should be.
Dr. Leo Rane, at the Lederle Laboratories, conducted studies with special clarifying and bacteria-absorbing filter pads almost free of calcium. They were not yet available commercially when this plant was inspected in January 1945, but Dr. Rane sent a few to the Army Medical School, to be tested for sterility, postfiltration, precipitation, fibrinogen, and other things. The war ended before the study could be implemented.
MASS PRODUCTION OF DRIED PLASMA
Establishment of Program
The first contract for dried plasma, for 15,000 packages, in February 1941, was made with Sharp & Dohme because of their previous experience in this field (19). By April (18), this firm had processed 1,140 units of 250 cc. each, with a loss of 126 units, 76 by breakage and the remainder for other reasons. By July (20), it had received 5,902 bloods, processed 5,496, and released 2,976 for distribution.
Before the declaration of war on 8 December 1941, three other contracts had been made. A small amount of plasma (750 packages) was available at Pearl Harbor, but the Navy, whose immediate needs were greater than those of other services, had received most of the other production.
Eventually, eight commercial firms were processing plasma, as follows (50):
Sharp & Dohme, beginning on 4 February 1941.
In the opinion of some members of the Subcommittee on Blood Substitutes, the multiplicity of contractors introduced elements of danger. Others thought it safer to spread out the contracts, in case of unforeseen emergencies. The
original contracts were difficult to write because the time factor was unknown and because the performance of the companies would be dependent upon the supply of blood from the Red Cross. All through the war, it was a major problem to establish a satisfactory correlation between the full utilization of the processing capacities of the laboratories and the procurement of blood by the Red Cross blood donor centers.
Other laboratories that came into the blood program later processed only serum albumin. Although the Subcommittee on Blood Substitutes had ruled that plasma or serum, frozen or dried, would be acceptable for use in wounded casualties, it was Dr. Veldee's opinion that the same laboratory should not produce both serum and plasma (24).
The subcommittee, at the April 1941 meeting (18), had accepted the "Minimum Requirements for Filtered Normal Human Plasma or Serum," issued by the National Institute of Health on 25 February 1941 (p. 279). It also recommended that the Armed Forces purchase dried plasma from any processing firm that employed methods acceptable to the U.S. Public Health Service.
There was never any objection to holding plasma in a shell-frozen state until drying apparatus was installed and ready for use. The purpose of this plan was threefold: (1) to provide a reserve against future augmented demands for plasma; (2) to enable the Red Cross donor centers to step up blood deliveries as rapidly as possible, without regard to present limitations in commercial drying capacity; and (3) to stockpile plasma for use when the albumin program would make additional demands on blood supplies.
The story of the processing of dried plasma throughout the war was one of continued increases in the requirements of the Armed Forces, continued expansion of the processing laboratories to meet these demands, and ingenious solution of the various problems that arose, including the shortages of trained personnel and of equipment needed for production. There would be little profit in going into details of these matters, but a few special points might be mentioned, beginning with the fact, already stated, that to avoid confusion and the writing of multiple contracts, arrangements were made for the Army Medical Procurement Agency, in Brooklyn, to act as purchasing agent for all plasma prepared commercially for the Army and the Navy.3
An early investigation showed that contracts entered into by any firm with the Army and the Navy precluded the taking out of an injunction against that firm because of infringement of patent rights (24).
EXPANSION OF REQUIREMENTS
The first contract for dried plasma, 15,000 packages from Sharp & Dohme in February 1941, was deliberately small, partly because the resources of the firm were then quite limited, partly because this was frankly a trial contract (19).
At the 23 May 1941 meeting of the subcommittee (25), Col. (later Brig. Gen.) Charles C. Hillman, MC, reported that letters had been sent to a number of processing laboratories asking each for bids on 25,000-lots of dried plasma. The requirements for fiscal year 1941-42 had been set at 100,000 units (18). By the 18 July meeting (20), 6 months before the United States entered the war, they had risen to 200,000 units. In July 1942, 6 months after the United States entered the war, the estimates for fiscal year 1942-43 were for 1,640,000 units of dried plasma, and the processing firms were being requested to bid on production up to 350,000 units each. These estimates were all for the small (250-cc.) package of plasma.
In March 1945, when it was estimated that the war in Europe might end in August, the estimated requirements for the Army and the Navy for the remainder of 1945 (March-December) were for 1,010,000 large packages of plasma which would require 2,222,000 bloods. This would have made the total Army-Navy requirements for 1945, counting what they had already received, 1,020,000 large packages or 2,244,000 bloods.
By the end of the war, of the more than 13 million pints of blood collected by the American Red Cross, 10,299,470 pints had been processed into dried plasma, put up in 3,147,744 250-cc. packages and 3,049,636 500-cc. packages.
Throughout the war, the increase in requirements of the Armed Forces, the capacity of the blood donor centers, and the capacity of plasma processing laboratories had to be kept in balance. The proper distribution of donations from the centers to the processing laboratories involved the geographic grouping of the centers in relation to single laboratories, the provision of transportation facilities, and the rapid readjustment of shipping occasionally necessary to move bloods to another laboratory because the laboratory to which they were usually allotted could not handle them.
Weekly reports of the distribution of blood donations were carefully studied by the Red Cross, by personnel in the Office of The Surgeon General, and by other interested parties. If donations were in excess, blood was likely to be wasted. If they did not come up to the capacity of the processing laboratory, equipment was wasted, and, even more important, so was personnel. The distribution of blood donations from Red Cross collection centers for the week ending 22 July 1944 to commercial laboratories was as follows:
The essential factor in meeting these conditions was the assignment of correct weekly quotas to each of the bleeding centers. The quotas were set, and changes were made in them, only by the National Director, American Red Cross, after he had taken into consideration (1) the total requirements of the Army and the Navy; (2) the distribution of these requirements among the contracting laboratories; and (3) the number of bloods necessary to enable each laboratory to fulfill its Army or Navy contract by the date specified in the contract. In deciding upon possible increases in quotas, it was also necessary to consider the professional staff of the center and the possibility of
expanding it, the facilities of the center, the size of its physical plant, the number of mobile units which it could operate, and the facilities of the processing plant which the center supplied.
EQUIPMENT FOR THE PLASMA PROGRAM
When the plasma program was set up, the chief bottleneck was the shortage of equipment for commercial processing (18). The laboratories were unwilling to provide equipment, or to expand what they already possessed, without formal contracts. The most essential item in the program, centrifuges, was the item in shortest supply, since those which would take the large American Red Cross horse bottles were then made by only one firm in the country, the International Equipment Co., Boston.
A number of suggestions were made to overcome these difficulties:
1. That the Army and the Navy should purchase the necessary equipment, and rent it or lease it to the processing firms.
2. That the equipment be developed under the auspices of the National Research Council and be popularized under its name and auspices. The Medical Research Council of Great Britain had established this precedent.
3. That sedimentation techniques be considered as a substitute for centrifugation.
4. That centrifuges of different sizes be used. Thus, 450-cc. bottles could be spun with No. 1 centrifuges, which were in ample supply. If No. 3 centrifuges, which were in short supply, could be used, each of them could handle 32 bottles every 9 hours. The yield, which would be greater than with any other centrifuge, would shortly compensate for their additional cost.
5. That representatives of the Army and the Navy be authorized to visit firms making equipment for the program, in an endeavor to expedite production. This recommendation was carried out.
Confusion continued to mark the procurement of equipment for the plasma processing program for the first year of its existence. On 16 September 1942, a meeting of all concerned with the program was held in the Office of The Surgeon General (51), and the following plan of organization was decided on:
1. By the express desire of The Surgeon General, the responsibility for the whole plasma program would remain entirely within the Armed Forces. Manufacturers would obtain extensions of preference ratings for materials and supplies, except for expansion projects, through the Supply Division, Office of The Surgeon General.
2. All expansion projects and all requests for preference ratings for replacement equipment would be cleared through the War Production Board. The Army-Navy Munitions Board would pass on all such requests by the customary procedure requiring concurrence of this Board, which would keep in close contact with the requirements of the Medical Department in acting upon the applications. Also, in view of the fact that supplies of blood represented the essential raw material in the plasma and albumin program, the Board would keep informed on the bleeding program.
3. The Army-Navy Munitions Board would maintain a compilation of requirements of all manufacturers of materials other than blood needed in the preparation of plasma.
This listing would be based on estimated production for periods of approximately 4 months. Calculations would be on the basis of anticipated blood deliveries and plant capacities. These data would be submitted to the Office of The Surgeon General for guidance in scheduling production and delivery of equipment to individual processing laboratories. The performance of each processing firm would be evaluated through review of periodic reports to be submitted at the request of the Legal Division, Office of The Surgeon General.
4. To avoid confusion, it was suggested (a) that the Office of The Surgeon General instruct processing laboratories regarding the procedures they should follow in procurement of equipment; and (b) that, except on expansion projects or replacement of equipment, contacts with the processing laboratories be limited to representatives of the Armed Forces, the National Institute of Health, and the Red Cross.
When these arrangements were put into effect, the procurement and replacement of equipment in the plasma program were both greatly simplified, and the changeover to the larger package, which was effected in July 1943 (p. 172), was carried out with remarkable smoothness. Problems continued to arise, of course, but very often it was found that they had been caused by failure of the processing laboratory to follow the directions laid down.
The part of the plasma program concerned with supplies was stabilized during fiscal year 1943-44. By this time, most of the material required by the processing laboratories had been standardized, and the various concerns which supplied the equipment had scheduled their production to conform with the needs of the program. Centrifuges, mobile refrigerating chests, and other items whose lack had seriously curtailed the plasma program for an extended period were no longer limiting factors. The War Production Board made spare parts available for centrifuges, so that repairs could be effected with a minimum of delay.
Equipment for Red Cross Blood Donor Centers
Shortages of equipment and supplies and delays in their procurement plagued the Red Cross blood donor centers during the entire war. The opening of new centers was frequently delayed for this reason. The lack of such small items as gauze, adhesive, needles, syringes, and thermometers could delay the operation of a center until they were supplied. Many times, since the quantities were not excessive, manufacturers and jobbers supplied enough material to permit continued operation, and repeated requests were made of the proper agencies of the War Production Board.
On 3 March 1943, a meeting to determine the best method of securing supplies and equipment for the bleeding centers was attended by Dr. G. Canby Robinson, National Director of the Blood Donor Service, American Red Cross; Dr. (later Major) Earl S. Taylor, Technical Director of the Service; Col. Charles F. Shook, MC, Special Representative of The Surgeon General for the Blood-Plasma Program; and representatives of the Procurement Division, American Red Cross, and the Priorities Division, War Production Board. A special expediter was to be appointed from the War Production Board for requests from the Red Cross, and all requests for priority ratings would be
expedited. It was expected that, for the usual items, clearance would not require over 48 hours. The centers were requested to anticipate their requirements for 4 months in advance so that contracts could be placed accordingly.
In general, the plan decided on at this meeting worked out very well, but there were still many delays. The procurement of paper cups is an illustration, which could be multiplied many times over, of the difficulty of securing essential items during the war. A steady supply of paper cups was necessary at all blood centers, to provide fluids for the donors, as there were no facilities for washing and sterilizing nonexpendable cups. About 35,000 cups of three sizes were needed each month. When they could not be secured through regular channels, a special endeavor was made to expedite them, but it took 3 weeks and a dozen letters and memorandums, as well as multiple phone calls, to set in motion the action that finally led to their procurement. Meantime, some chapters were securing their cups by daily purchases of small numbers wherever they could be bought.
OTHER PRODUCTION DIFFICULTIES
In spite of the amicable relations that existed between the processing laboratories and those concerned with the plasma program in the Army, the Navy, and the American Red Cross, there were numerous arguments and misunderstandings of various kinds. This was inevitable. The drying of plasma was a new process, not yet reduced to strict formulas, for which equipment was being devised as the program developed.
There were mechanical difficulties of various kinds to be ironed out, particularly after production was stopped for any reason. An extended shutdown was usually followed by trouble on the first run afterward, and it was found to be economical to keep all desiccation units running at a fairly steady rate.
In March 1943 and again in May 1944, labor troubles led to impending strike threats. In each instance, The Surgeon General wrote the employees of the processing laboratories of the vital importance of the plasma program and the imperative necessity of avoiding any work stoppage. In both instances, the difficulties were settled without any loss of time, but in both, arrangements had been made to transfer the involved firm's quotas of blood to the nearest unaffected firm if the threatened strikes had actually occurred.
Sabotage, as already intimated, was a theoretical possibility during the entire war in the whole program-whole blood, plasma, albumin, byproducts, and intravenous fluids. Fortunately, it never was anything but theoretical. The usual precautions against it were practiced throughout the war, including fencing of all plants, 24-hour-a-day guards, and screening of employees as far as was practical. Empty bleeding bottles were kept under lock and key. Blood was transported from the blood centers to the processing plants by prearranged express schedules, and all containers used were sealed in transportation.
Army Medical School
The first extensive testing in the plasma program was conducted by Dr. Strumia (p. 269). This was a clinical study of plasma prepared in his laboratory from blood provided by the American Red Cross. He was assisted in it by Captain Kendrick, Commander Newhouser, and members of the Subcommittee on Blood Substitutes.
When the first supplies of plasma became available commercially, the practice was adopted of testing each lot chemically and clinically in the Blood Research Division, Army Medical School, before it was released for use from the medical depots. Originally, one package in each thousand was tested (table 7). In March 1943, in order to reduce the heavy workload, the number of samples was reduced to 1 in each 5,000. Since individual lots of plasma were made up of only 15-25 bottles, it was neither practical nor economical to test one package per lot. When it was proposed by a hospital commander in January 1944 that this be done, it was pointed out that his plan would mean that 5 percent of all plasma produced would be tested, which would be a waste of valuable material as well as an unnecessary precaution: During the last 5 months, only two packages had been rejected out of 350 examined, this number representing a production of approximately 1¾ million packages. A little later, testing was limited to one package per month selected at random from each laboratory.
When the samples were received at the Army Medical School, the package was first studied for possible external defects. The plasma was examined in the Chemistry Division to determine moisture content, hemoglobin content,
and plasma protein content. It was then tested for solubility, with a note of the time required for complete solution. Finally, the plasma was administered to patients who were carefully observed for any adverse reactions, particularly chills and fever.
Each laboratory was required to maintain complete records on each lot of plasma processed, the reports showing serologic tests; cultures; toxicity tests and bulk sterility tests on each pool; time required for processing; time required for drying and the temperature levels during the process; and final sterility and residual moisture determination on the dried product. Monthly reports were also required.
In view of the completeness of these records and their availability for inspection, it was decided, in the spring of 1944, to discontinue testing at the Army Medical School. The change was considered entirely safe since by this time all the laboratories under contract had had extensive experience and were turning out satisfactory products.
Analysis of Questionnaires
Materials and methods-An analysis of 1,407 of the questionnaires included in the packages of dried plasma and returned after material had been used was carried out at the Naval Medical Research Institute in December 1943 (52, 53). The 1,407 infusions of dried plasma were compared with a previous study of 1,751 infusions of liquid plasma.
Of the 1,407 infusions analyzed, 300 had been given at the National Naval Medical Center and 119 were given between midnight and 8 a.m., the time of administration suggesting that they were given for emergency reasons. The 1,407 infusions had been given to 734 patients, 457 of whom had received 1 injection each, 2 of whom had received 22 injections each, and 34 of whom had received 9 injections each. Eighty percent of the material injected was produced by three laboratories; there was no essential difference in clinical results or incidence of reactions.
The distribution of clinical indications and therapeutic results was essentially the same for both dried and liquid plasma. Hypoproteinemia, including jaundice and infection, was the chief indication, followed by shock and hemorrhage. The peak month of administration, July 1942, coincided with the peak month of the postvaccination hepatitis epidemic. In 22 of the 25 fatal cases in the dried plasma series, the indications for the infusion included shock, with or without hemorrhage, and burns. Although hypoproteinemia and jaundice accounted for more than 40 percent of the indications in the total series, only 2 of the 25 deaths were associated with these conditions. In the remaining death, the indications for plasma administration were not stated.
The average amount of dried plasma given to each patient was 526 cc. and of liquid plasma, 711 cc. The fact that the reported average for all patients exceeded 500 cc. clearly justified the recent adoption of 500-cc. packages.
Results-The response to plasma was considered favorable in 96 percent of the patients in each series. There were no hemolytic reactions in either series, and the reaction rate in both was essentially the same, about 5 percent. Patients in shock had fewer reactions than those not in shock. Only one patient had more than 2 reactions; this man had 4 reactions in 18 infusions, 2 of which occurred on the same day, after consecutive infusions. Of the eight other patients with multiple reactions, five sustained both on the same day after consecutive infusions.
Of the 74 reactions in the dried plasma series, 25 were urticarial, a larger proportion than in the liquid plasma series. Two explanations were advanced:
1. The lability of the allergen, a theory for which no proof could be demonstrated.
2. Something in the diet of the donors. All of the liquid plasma used in the series was prepared at the Naval Medical School, from blood drawn at a single, closely supervised center, whose donors had eaten little in the 6 hours before the donation. It was conceivable that at centers at which blood was procured for dried plasma this regulation was not strictly enforced.
The reaction rate for liquid plasma stored for less than 4 months was 5.1 percent, and for plasma stored beyond this limit, 3.0 percent. These rates further suggested that some mildly toxic labile constituent of fresh plasma might be stored in the dry state.
Studies on blood pressure and pulse rates-As a part of this general study, the effect of plasma administration on blood pressure was investigated in 392 infusions given to 378 patients, and on the pulse rate in 513 infusions given to 442 patients. The data were as follows:
1. The average increase in systolic blood pressure produced by the administration of plasma, over a period not exceeding 24 hours, to patients with initial systolic pressures under 100 mm. Hg was 28 mm. Hg in those in shock and 14 mm. Hg in those with hypoproteinemia.
2. The average decrease in pulse rate in patients with initial rates over 100 per minute was 13 per minute in those in shock and 6 per minute in those with hypoproteinemia.
3. In general, the increase in systolic blood pressure was much greater than the decrease in pulse rate. In patients in shock, the coefficient of correlation between the two responses was extremely low and not statistically significant.
4. There were no statistically significant differences between the results of dried and liquid plasma.
INSPECTION OF PROCESSING LABORATORIES
After all laboratories were in production, periodic inspections were made, in compliance with a directive from the Office of The Surgeon General on 9 July 1942. Reports were sent to his office, to the Army Medical Purchasing Officer, and to others concerned in the program (54-58).
The original purpose of these inspections was to make certain that the best possible production techniques were being employed, and that all possible precautions were being taken to provide a safe and effective product. Variations from standard techniques were detected and corrected. Any improvement in procedure observed at one laboratory was promptly made known to other processing firms.
These frequent contacts made it possible to reduce contamination rates and losses from other causes. One of their most useful functions was the correction of the rumors, false impressions, and misunderstandings that invariably rose in a project of such magnitude. The relations between the Division of Surgical Physiology, Army Medical School, and the commercial laboratories had been excellent before large-scale production of plasma began, and this liaison, which also existed with the Navy, was maintained by these visits, which were usually made by Colonel Kendrick and Captain Newhouser.
YIELDS OF PLASMA FROM BLOOD
One of the subjects discussed at the Blood Plasma Conference in Chicago on 24 March 1943 was the volume of plasma recovered from each bleeding bottle (59). The monthly reports indicated wide variations. In January, the yield for the processing laboratories ranged from 275 to 309 cc., with yields for individual firms varying from 260 to 314 cc.
A number of reasons might account for these discrepancies:
1. The volume of the anticoagulant solution in the bleeding bottle varied, though the intent was that it should be 50 cc., less whatever amount was lost by evaporation during, and subsequent to, sterilization. Measurements made in 12 bleeding centers showed the maximum range from 47 to 70 cc., the minimum from 16 to 50 cc., and the average from 44 to 58 cc. It was urgent that each laboratory overhaul its technique so that the employee charged with introducing the anticoagulant could not vary it from bottle to bottle. Dr. Taylor, who had directed this investigation, considered it probable that sterilization techniques should also be checked.
2. The volume of blood collected varied. To study this discrepancy, 2,475 bleeding bottles were selected at random from the Church containers as they were received at one laboratory. The volume of blood was determined by measurements on a marked bottle, in 50-cc. graduations from 550 to 250 cc. Percentage variations ranged from 50.0 and 29.5 to 0.9 and 0.2. These variations had been called to the attention of all bleeding centers.
3. Wide differences were found in revolutions per minute and the time factor with various models of centrifuges. When a No. 13 model was used, the revolutions per minute varied from 1,300 to 2,300 and the time interval of four motor speeds from 20 to 60 minutes. When a No. 3 model was used, the corresponding figures were 2,000-2,500 r.p.m. and 25-45 minutes. Unless it were assumed that the lowest number of revolutions per minute, or the shortest interval, was optimum, it had to be assumed that centrifugation at some laboratories was not adequate. One laboratory reported a yield of plasma of 285.5 cc. when a No. 13 centrifuge was used at 1,800 r.p.m. for 50 minutes and of 297 cc. when the time was extended to 65 minutes. It was planned to supply each processing laboratory with a table showing the relation of revolutions per minute to the time factor necessary to secure a full plasma yield.
4. A study of two processing laboratories that received blood from the same bleeding centers showed, over a 5-month period, that the yield of plasma was the same in both on one occasion but that there was a maximum variation of 45 cc. and an average variation of 12 cc.
5. Variations were also found in the amount of plasma drawn off after centrifugation. The closed technique, investigation showed, had been well standardized in all processing laboratories. Any variation, therefore, was evidently the fault of the individual who performed the operation. Variations were least when supervision was most careful.
6. The temperature of the blood at the time the plasma was drawn off apparently influenced the yield. Commander Newhouser reported that the Upjohn Co. had found that, if the blood was chilled after centrifugation, it was possible to draw off all but 4 cc. of plasma without distributing the red blood cell layer. The high hemoglobin content of the last bit of plasma drawn off did not interfere with the manufacture of albumin, which this firm was making.
One laboratory made a check of 25 bloods received from five centers, spinning them for 50 minutes at 1,800 r.p.m. After carefully removing all the plasma possible without drawing off red blood cells, the maximum blood-tinged residual plasma recovered varied from 1 to 20 cc. and the average from 4.0 to 8.5 cc. for the individual centers. Based on the total bleedings in all centers for January 1943, the residual yield of plasma would have ranged from 747,000 to 1,589,000 cc., very considerable amounts. If the residual could not be used in dried plasma, it was thought that methods could be devised for salvaging it for use for serum albumin.
The analysis clearly indicated that if optimum conditions were ever approached, the yield of plasma could be materially increased, perhaps by as much as 100,000 bottles per year.
At a conference of the Albumin Testing Group on 22 March 1943 (60), Dr. Veldee again called attention to the very considerable amount of plasma then going to waste because of breakage and because of the amount left in the layer over the packed red cells. An average of 8.5 cc. could be recovered in this layer in every bottle, and the total amount would be about a million cubic centimeters every month. This residual could not be used for plasma because of its red cell content, but it should be processed in some stable form, so that it could be held without loss. These losses were never fully corrected.
Since donated blood immediately became the property of the U.S. Government, it was essential that a precise record be kept of its disposition and of losses of it from all causes. Legally, this was required by Army regulations. Because of the usual source of the raw product, such an accounting was also morally obligatory, and the responsibility for it was keenly felt by all connected in any way with the program.
At an early meeting of the Subcommittee on Blood Substitutes, it was agreed that if contamination, breakage, insufficient samples for serologic
testing, and any other losses and errors were found at the processing laboratories, the information should be telegraphed immediately to the bleeding center responsible and a copy of the wire sent to Dr. Robinson. Gross errors would therefore be investigated and corrected as soon as they occurred.
Accounting practices were fully discussed at a meeting on revision of plasma contracts held at the Purchasing and Contracting Office of the New York Medical Depot on 28 July 1942 and attended by representatives of that office and of two processing laboratories (61). The following agreements were reached: All contracts would contain a provision for complete accounting to the Army of all blood received from the Red Cross and of all its byproducts. The report would start with an inventory of the blood, frozen plasma, and dried plasma on hand when the new contract commenced; would add to the inventory the blood received during the month; would deduct from it losses due to contamination, ordinary breakage, and other causes; would make the proper conversion from amounts of blood to comparable quantities of plasma; and would convert frozen plasma to appropriate quantities of dried plasma, with due regard to shrinkage due to further breakage, possible contamination, and other causes. Credit would be taken for deliveries of dried plasma during the month, and an inventory of blood, frozen plasma, and dried plasma on hand at the end of each month would be shown.
The Army also wished each laboratory to prepare a certified statement accounting for all the blood received from the beginning of the project up to the first report by the new accounting system.
The laboratories were quite willing to prepare the desired accounting. It was thought, however, that the standard Red Cross form was not adequate for this purpose, while the very elaborate statistical report which the Army-Navy Munitions Board had recently requested was rather cumbersome. A single form was later developed which served the Army-Navy Munitions Board requirements and a copy of which was sent to the Red Cross for information.
The laboratories also requested at this meeting that the Red Cross be instructed each month by the Army and the Navy as to the quantity of total blood to be used for plasma and for albumin. If shortages in either program should develop, the laboratories would be in an awkward position if they had made the allocations, and they would probably be criticized by both services.
Criticisms of Original Practices
On 14 September 1943, the Renegotiation Division, Office of The Surgeon General, called to the attention of the Director, Procurement Division, a number of errors in the accounts submitted by the processing laboratories, emphasizing that there was no implication whatsoever of any bad faith on the part of any firms concerned with the program (62). The reports in question were designed to afford protection to the laboratories as well as to the Government and the personnel concerned with letting the contracts. If there were errors in them,
this protection was not being afforded. Moreover, the maintenance of adequate records was related to the renegotiation program in the sense that the relative operating efficiency of the company was always taken into consideration in determining the amount of excess profits to be recaptured. Any figures compiled on the basis of the reports in question would also be in error and subject to criticism by the companies concerned when settlement agreements were entered into.
The chief errors were listed as follows:
1. The form used was not adequate to account for contaminated material that could not be used for plasma but was suitable for albumin.
2. There was no uniformity in the reports of the various laboratories, particularly in respect to contaminated plasma. Some companies wrote it off. Others carried it in their inventory. Still others dropped it and picked it up again, from time to time, to effect so-called recalcification. Some companies reported quantities converted to albumin as if they had been lost in freezing or for other reasons. Some reported high hemoglobin losses as miscellaneous. One company reported large miscellaneous losses as the difference between actual and estimated plasma volume.
3. The present forms did not definitely segregate losses subject to the penalty clause from those not subject to it, though whether this was important would depend upon whether a penalty was to be imposed for negligence, deviation from expected or average performance, or other causes.
4. Some errors originated on the companies' reports. Some were errors in transcription and columnization from the companies' reports to the stock movement record. Other discrepancies might exist which could be disclosed only by a complete audit of records.
To correct these errors the following suggestions were made:
1. Some revision in the form in use was necessary, to allow for contaminated material, which should be recorded uniformly by all companies.
2. The mathematical accuracy of each monthly report should be established on its receipt.
3. Amounts listed as miscellaneous should be analyzed to show causes of loss before the figures were recorded on the stock account.
4. A formal monthly columnarized stock record should be maintained in binder form and balanced and reconciled each month, as was apparently required by paragraph 10 of Army Regulations No. 38-6520. The record should account for the disposition of the blood from the time it was delivered by the American Red Cross until it was received and accepted by an Army depot or other Army installation. The record should be supported by shipping tickets for quantities delivered to the processing companies, receiving reports for quantities delivered to Army depots, monthly reports from each company to show the movement of the blood, and forms to cover quantities charged out. The report should also summarize losses by causes and according to whether or not liability was attached to them.
Justification of Original Reports
As Captain Taylor pointed out to Capt. Frederic N. Schwartz, MAC, in a memorandum commenting on this letter from the Renegotiation Division (63), a number of points had to be taken into consideration in analyzing current accounting practices:
1. The processing firms had never before handled so large a volume of human biologic material. In the initial stages of the operation, many innovations and improvisations were
necessary, and the resulting delays and losses were reflected in conflicting and inaccurate inventory reports.
Some of the early records were quite complex. One firm, for instance, made restitution, of its own accord, for material lost in processing and adjusted its subsequent reports in conformity with the replacement. Another laboratory also made restitution for blood lost through a truck accident on its property.
2. The program was underestimated in its potential size by all concerned with it. Rapid expansion took place before facilities were available, and makeshift arrangements were often necessary to handle the enormous increases in blood delivered to the laboratories.
3. As a result of these various factors, initial losses were excessive in the light of present operations, and errors in bookkeeping occurred that could not be adjusted until there was time for a less hurried appraisal of stocks on hand and other matters.
4. In the first year of the program, contaminated and fused (denatured) material was looked upon as highly dangerous. Some companies charged it off as a complete loss, but others kept it on their shelves, hoping that some rise might be developed for it in the future. Some plasma was discarded on the ground that it had lost its original properties. A good deal was used experimentally, in attempts to devise methods of salvaging it, such as recalcification and chloroform extraction. Between October 1942 and February 1943, many large batches of contaminated plasma were used in trial runs at plants preparing to process albumin, so that good material would not be wasted getting the initial difficulties of production ironed out. It was not until July 1943 that the Harvard pilot plant considered it safe to use contaminated plasma in the serum program. Any diversion of such plasma to this use before that date was entirely experimental, and the material used was of no value according to the criteria in the National Institute of Health regulations and those implied in the plasma contracts.
For these various reasons, the forms then in use provided no method of accounting for plasma that, for contamination and other reasons, was charged off on the report but was physically retained by the laboratory for possible conversion to other uses. The conversion of contaminated plasma was a development of the past few months. It was not provided for on the report forms previously in use because this development did not then exist.
National Institute of Health rulings permitted the processing of liquid plasma into dry plasma if contamination was limited to 200 organisms per cubic centimeter. The rationale was twofold, the dilution accomplished by pooling the bloods, and the freezing and drying processes, which killed organisms in this number.
5. In dealing with biologic substances of this nature, and in such volume, it was not practical to interrupt the process to secure measurements exact to the cubic centimeter at various stages of production. Originally filling and sample losses were simply estimated (so-called stick measurements). At Dr. Veldee's request, in October 1942, uniform measurements were made in all laboratories, though it was still impractical to account accurately for every 10 cc., or even every 100 cc.
6. The blood donor centers had the same difficulties as the laboratories. At the beginning of the program, donations probably averaged 20-30 cc. less than present donations. Even at the time of this report, however, the amount of potentially available plasma was no better than an estimated average determined by comparing the amount in individual bottles as they arrived with the average yield of plasma per bleeding as determined by the laboratory. Each bleeding ideally yielded 300+ cc. of plasma, the equivalent of one finished standard package. This ideal 1:1 ratio was based on the assumption that each bottle of blood was completely filled (which, for the reasons already stated, it was not). It also made no allowance for breakage, positive serology, filling losses, or contamination.
Standards of performance and checks of efficiency of operation of the processing laboratories by the American Red Cross took into consideration the realistic factors just listed. In planning for deliveries of blood to the processing
laboratories to meet Army contracts, the Red Cross, the National Institute of Health, and the Office of The Surgeon General decided that a ratio of 1:1.2 finished packages of plasma to bleeding would represent excellent performance. As of 31 August 1943, the ratio was 1:1.079.
A further check on laboratory performance, and indirectly on the overall accuracy of the laboratory records, was the breakdown sheet for mechanical and contamination losses prepared each month by Dr. Veldee. Over the last 2 months, these losses, exclusive of serology losses of 0.30 percent, amounted to 2.47 percent. Thus, without taking into consideration filling and sample losses, there was a known and recorded loss of 2.77 percent. In terms of the ratio of bleedings to finished packages, this was 1.03 percent, which compared favorably with the 1:1.079 ratio obtained by the other method of checking.
There were, therefore, five bleedings per hundred, or 1,500 cc. of plasma per hundred bloods, not accounted for statistically. Sterility samples and similar amounts required by National Institute of Health regulations amounted to about 160 cc. per hundred bloods. A fair estimate of the variation of the ideal 550 cc. of citrated blood per donation would account for 2,000 cc. per each hundred bloods or 1,000 cc. of plasma. This left 340 cc. of plasma, or something over one bleeding per hundred, to cover filling and other incidental production losses that could not be measured.
It was Captain Taylor's opinion that those examining the reports submitted to date did not completely understand the background of the plasma production program and the early difficulties it encountered. Also they did not possess the necessary medical knowledge to assess phases of operation and production which did not lend themselves to mathematical calculations. For these reasons, Captain Taylor questioned the justification for the somewhat sweeping condemnation of the present system of recording. By far the largest numbers of errors were clerical, and simple auditing of the monthly reports would easily correct that situation.4
Changes in Reporting Practices
At a meeting of representatives of the various components of the plasma program on 1 October 1943, reporting practices were discussed in detail (64). It was not believed that the correction of the errors complained of by the Renegotiation Division would present great difficulties or require basic changes in present policies.
Colonel Kendrick and Captain Taylor pointed out that, at the present time, there was no known method of salvaging contaminated blood, and dropping it from accountability represented no problem at all; it would never
be dropped on one report and picked up on another. They also emphasized the difficulties that would face laboratories if they tried to take precise inventories of material which they had retained on their own initiative, in the hope that it might eventually be useful, and which was frequently stored in containers of various shapes and sizes. They thought that an approximate report in liters should be permitted. Also, to take cognizance of the storage problem faced by some processing laboratories, these officers proposed, with Dr. Veldee's concurrence, that contaminated material on hand before 1 August 1943 should be destroyed.
Auditing arrangements for the correction of mathematical errors were suggested, and it was also proposed that the same auditor who performed this task, and who thus became familiar with the form and content of the reports and the problems involved, should make a monthly audit of all reports in the future.
On 6 November 1943, in accordance with suggestions made at the 1 October 1943 meeting, a preliminary letter was sent to the processing laboratories from the Army Medical Purchasing Office in New York incorporating the following information:
1. Recent developments indicated the possibility that blood or plasma hitherto regarded as unsuitable for use because of contamination or for other reasons could be salvaged for certain purposes.
2. At the request of the Army, several laboratories had been retaining this material and had sometimes overloaded their storage facilities with it. Permission to discard all such material on hand before 1 August 1943 was therefore granted all laboratories.
3. Hereafter, no such material would be discarded without first obtaining, through the contracting officer, Army Medical Purchasing Office, permission of the chief, Laboratory of Biologics Control, National Institute of Health.
4. Hereafter, a supplement should be filed with each monthly report indicating the material charged off in all stages of processing, the volume to be reported in liters. If exact quantities were not readily obtainable, as nearly accurate estimates as possible should be used.
Detailed instructions for the reports to be required in the future were sent to all processing laboratories from the Army Medical Purchasing Office on 17 May 1944 (65).
In the Journal of the American Medical Association for 12 September 1942, Dr. Taylor summarized the losses which had occurred in 320,442 bloods collected up to 1 May 1942 as follows (66):
Breakage, 0.345 percent, including 126 bottles broken in transit and 842 cracked during centrifugation.
Hemolysis, 0.569 percent. All but 406 of the 1,591 units lost in this category had to be discarded because the blood froze when inadequate shipping arrangements allowed it to be exposed for considerable periods to subzero weather. Some early losses occurred because of failure to remove the Dry Ice used for precooling shipping containers.
Contamination, 2.26 percent. The loss of 6,260 bloods from this cause was considered small in view of the 16 bleeding centers and 4 processing laboratories which had entered the project without any previous experience in this field.
Miscellaneous, 0.284 percent. In all, 794 units in this category had to be discarded. Railroad breakdowns resulting from weather conditions caused some bloods to be held beyond the time permitted by National Institute of Health specifications. One pool of 36 bloods had to be discarded because it contained blood from a donor who developed typhus fever.
Most blood that was discarded was made into typing sera or was used in pilot bottles to test moisture content.
The final report of the American Red Cross (50) shows a loss of 204,848 bloods (1.6 percent) of a total of 12,589,034 delivered to the processing laboratories (table 8). It will be noted that, as in the earlier report, bacterial contamination was the major cause of losses but the percentage had been materially reduced, as had that of all the other causes listed in the first report in 1942.
Losses From Contamination
The risk of contamination was first discussed at the meeting of the Subcommittee on Blood Substitutes on 23 May 1941 (25), when it was learned that a pool of 40 bloods being processed in a commercial laboratory had been found to be contaminated. It was recommended, to prevent wastage from this cause, that pools should consist of not more than 12 bloods. Later, when it was found possible to process contaminated plasma into albumin, the limits were successively lifted to 25 bloods, and then to 50 and more (67).
Two special experiences with losses of liquid plasma from contamination are sufficiently instructive to be reported in some detail.
First experience-When the liquid plasma center was inspected on 30 July 1943, the chief problem was the gradual increase in contaminated plasma which had occurred over the last 4 months, always with Staphylococcus albus. Contamination was occurring in the pools retained in the center as well as in those shipped for testing to the Army Medical School. The circumstances were always the same: the primary culture was negative. Cloudiness began to appear in the pools on the 7th or 8th day, and culture of the pilot bottle on the 10th day revealed the contaminant. It was concluded that contamination probably occurred either when the primary culture was taken or when Merthiolate was added.
Investigation of the technique employed at the center made clear the prime cause of contamination, that the bottles were being entered at the free hole (p. 386), where the stoppers were only 1½ to 1 mm. thick, instead of at the X-mark, where the diaphragm was 7 mm. thick and sealed itself when it had been penetrated, as the thinner diaphragm did not. The result of this technique was that the closed system, essential to the preservation of sterility, ceased to exist.
It was directed that this practice be stopped immediately. It was also directed that individual syringes and needles be used to introduce Merthiolate into the pools; that the practice of covering the plunger of the syringe with glycerin be discontinued; and that as few technicians as possible be assigned to aspirate and dispense the plasma, so that responsibility could be specified if contamination continued to occur.
The contamination rate at this center promptly fell to an acceptable level (less than 1 percent) and continued at this level until the middle of December 1943. Then, it again rose sharply. When the liquid plasma center and the blood donor center which supplied it were visited on 16-17 January 1944, the cause was immediately evident, that the combined bleedings of the fixed donor center and the mobile unit were amounting to 500-580 per day, considerably in excess of the processing capacities of the liquid plasma center. All past experience, in both hospital and commercial laboratories, had shown that the contamination rate always increased in direct proportion to the amount of blood processed beyond the capacities of the laboratories to handle it. The current policies had been instituted by the director of the bleeding center, a civilian, against the wishes of the physician in charge of the center and the chief of the laboratory service at the Army liquid plasma center. Unlike most donor centers, this center was operating as an Army, rather than as a Red Cross, donor center.
Arrangements were made to operate the center in the future as all other bleeding centers were operated, with the technical representative of the Red Cross responsible for personnel, blood quotas, technical procedures, and other policies. Daily bleedings were to be reduced to 300 per day. With these changes enforced, the contamination rate again dropped to an acceptable level.
Second experience.-When the liquid plasma center was inspected on 12 August 1943, another problem in contamination, chiefly from Staphylococcus aureus, was encountered. Several explanations for the 3-percent rate were promptly found.
1. Responsibility could not be individualized because of the number of technicians aspirating, dispensing, and culturing the plasma.
It was recommended that the actual preparation of the plasma be limited to two technicians, and that no new technicians be trained in these special procedures until the contamination rate had fallen to an acceptable minimum.
2. Similarly, it was impossible to place the responsibility for autoclaving bleeding and aspirating sets. Untrained technicians were being permitted to operate the autoclave, and undesirable techniques were employed.
It was recommended that a trained technician be made responsible for the sterilization of all sets; that the autoclave be operated at 15 pounds' pressure for 45 minutes; and that a vacuum be pulled for at least 20 minutes, to insure dry sets.
3. Plasma was aspirated under a hood in a cubicle that, because of the heat, could not be closed tightly. Windows in the room also had to be kept partly open.
Nothing could be done at the time to improve these physical conditions except to urge all possible care to counteract them.
4. Inspection of presumably sterile sets showed that the muslin wrappers were completely saturated with glycerin; the humidity was high, and the sets therefore remained continuously wet. When they were opened, excessive amounts of glycerin were found on the aspirating needles, and the valves, the Penrose drains, and other parts of the set were all bathed in it.
It was recommended that the use of glycerin on aspirating needles be discontinued at once.
5. The Penrose drains used in the sets were so short that they did not completely cover the aspirating needles. Also, the glass funnel tips supposed to protect the tips of the needles during aspiration were frequently broken, so that the rubber on the end of the drain came into contact with the needle each time a plasma bottle was penetrated.
It was recommended that all aspirating sets be completely reworked; that new muslin wrappers be used; that the Penrose drains used be long enough to cover the needles; and that new, intact glass tips be supplied.
The prompt fall in the contamination rate at this liquid plasma center proved once again the extreme importance of strict attention to all details of the procedure if a safe and effective product were to be secured.
Processing laboratories-The rate of contamination at processing laboratories was generally very low. When it rose above accepted levels, the explanation was usually evident. In October 1944, for instance, the high rate at one firm was explained by a break in technique; namely, permitting rubber tubing to lie around for 3 or 4 days before it was sterilized. Directions were given that this period must be reduced to 3 hours. It was arranged that before further release of material from this laboratory, at least 100 packages must be tested, half at the Army and half at the Navy Medical Schools.
At another processing firm, a high rate of contamination was explained by crowded conditions compounded by construction work immediately adjacent to the laboratory. The physical setup continued to be undesirable but the rate fell when special precautions were instituted, and it reached an acceptably low level when the construction work was finished.
Losses From Clotting
Losses from clotting were small in the total plasma program, but the possibility was the occasion for a number of heated discussions and several special investigations in 1942. The question first arose (45) in a letter circulated by Dr. Strumia to the Subcommittee on Blood Substitutes on 8 April 1942, in which he incorporated material from a letter he had written on the subject to the Journal of the American Medical Association. In these communications, he stated:
1. That the technique employed by the American Red Cross in collecting blood for the Armed Forces was unsafe.
2. That clotting occurred as the result of this technique in from 12 to 30 percent of all bleedings and that the clots varied in weight from a few grams to 150 gm.
3. That if the larger (850-cc.) bottles used by Sharp & Dohme were employed, instead of the 750-cc. bottles then used by the Red Cross, and if the bottles were agitated during the collection, the rate of clotting would be 12 percent.
In his reply, Dr. Taylor pointed out that agitation of the bottle during bleeding had never been a part of Red Cross technique. In his opinion, Dr. Strumia's statement gave rise to three questions:
1. Did clotting occur in Red Cross donations and if so, did it occur to the extent stated?
2. If clotting did occur, could it be overcome by any special technique, such as agitation of the bottle during the withdrawal of blood?
3. If a minimal degree of clotting occurred, was there any evidence, experimental or clinical, to prove that it was of clinical significance?
A number of investigations were undertaken in the seven processing plants then engaged in the production of dried plasma to settle these questions:
1. Statistical analysis of approximately 200,000 bloods processed up to 1 April 1942 showed that only 97 had been discarded because of gross clotting. The explanation was usually small, unnoticed cracks in the bottom of the bottles, which had permitted the citrate to leak out slowly and resulted in the collection of blood without any citrate solution or with an ineffective amount.
2. A total of 9,164 bloods were strained through coarse- and fine-mesh screens and the solid material thus secured was carefully examined, in many instances by Dr. Taylor, Major Kendrick, or Commander Newhouser. Results varied from firm to firm but were difficult to state comparatively because of the different techniques of reporting. Except in a single laboratory, the rate of clotting was always low, and, with very few exceptions, all of the clots were small. The explanation of the high rate of clotting at the single laboratory just mentioned was that the investigation coincided with the employment of five part-time women physicians, who had had no previous experience in the field, at the blood center supplying much of the blood.
3. To compare the collection of blood with and without agitation, 100 bloods were carefully agitated during collection at the Red Cross Donor Center in New York. No clots were found. On the same day, with the same personnel, another 100 bloods were collected without agitation. One clot, 1½ inches in diameter, was found.
4. In 100 bottles picked at random from collections at the blood donor centers feeding Sharp & Dohme, four clots were found, the largest three-fourths of an inch in diameter. At the Sharp & Dohme laboratory, 100 bottles of blood were collected in the 850-cc. bottle in which it had been stated clotting would be minimal, with and without agitation. In all, seven clots were found, four, 1 inch in diameter and three, 2 inches in diameter. A second
hundred bloods, collected from the same source and in the same manner the following day, revealed six clots, ranging from 1½ to 2½ inches in length and three-fourths of an inch in diameter.
It was concluded from these various investigations that the degree of clotting at the donor centers across the country was of no real significance; that a certain amount would occur, no matter what system, within reason, was employed to collect the blood; and that considerably more conclusive evidence must be produced before the Red Cross technique of collecting blood for conversion to plasma could be considered unsafe. No such proof was ever forthcoming.
DISPOSITION OF SURPLUS PLASMA
The accepted potential of plasma in the management of shock, hemorrhage, burns, and special diseases is evident in the thesis written by a student at the George Washington University School of Business Administration on 11 January 1944, entitled "Potential Post-War Market for Dried Blood Plasma" (68). At that time, there was no indication of the risks of serum hepatitis introduced by its use, a risk which was to complicate the disposition of surplus stocks and lead to the replacement of plasma in the Korean War by serum albumin (p. 782).
Among the earliest plans for the disposition of surplus stocks of plasma at the end of the war was Colonel Kendrick's recommendation after his visit to the Mediterranean theater in October 1944. At that time, the ratio of blood to plasma in forward areas was about 1:1, and it was thought that it might approach 2:1. Since whole blood was then available in adequate quantities, Colonel Kendrick thought that consideration should be given to reducing plasma contracts by two plans:
1. All smaller Red Cross bleeding centers should be closed and the larger centers (New York, Washington, Philadelphia, Detroit, Chicago, San Francisco, and Los Angeles) should be operated at full capacity.
2. All surplus stocks of plasma in oversea theaters should be returned to the United States at once, preferably on hospital ships, to avoid the postwar difficulties of returning surplus material. The plasma thus returned could be used for years to come (again, it must be emphasized that the danger of infectious hepatitis from the use of pooled plasma had not yet been realized). When this recommendation was made, recent correspondence had shown that the European theater had an excess of plasma on hand and would need no more for at least 6 months. Inquiries to other theaters also revealed large stocks.
As a matter of fact, by this time, tentative plans had already been made to cut back production of plasma so that contracts could be terminated promptly when the war ended. All producers were asked to limit their purchases of equipment, and contracts were made for the minimum practical level.
Long before the war ended, requests began to be received from various civilian agencies and institutions for unused stores of plasma and its byproducts,
for use therapeutically, prophylactically, and experimentally. The first official steps in the disposition of surplus plasma were not taken, however, until after the war with Japan ended.
On 25 September 1945, Mr. DeWitt Smith, Vice Chairman of the American Red Cross (69), inquired of The Surgeon General whether there would be a surplus of plasma produced from blood obtained by the American Red Cross over and above the amount the Army could use before it became outdated. His interest in the matter arose from the responsibility of the Red Cross to the people who had contributed the blood and who had the right to insist that it be utilized to the best advantage and not wasted by deterioration. If there were a surplus, Mr. Smith wished to know whether the Army would release it without charge for appropriate civilian use, also without charge.
On 1 October 1945, Maj. Gen. Norman T. Kirk (70) replied that, while stocks from the Pacific had not yet been reported, there were already available overseas, in excess of Medical Department demands for the next 14 months, 387,385 large packages of plasma and 258,560 small packages. Available in the Zone of Interior were 515,749 large packages and 346,670 small packages. All of the small packages and all but 75,000 of the large packages could be transferred to the American Red Cross.
On 5 November 1945, General Kirk (71) notified Mr. Smith that the excess stocks of plasma and other byproducts described in his 1 October letter were now available for transfer to the Red Cross. The Army would be glad to store the material in its depots until the Red Cross could assume ownership. As to material overseas, the Army, on request, would bring designated amounts to the Zone of Interior or deliver them to Red Cross depots in the various theaters. The actual ownership of the material was to remain with the Government until the supplies were delivered to the Red Cross at the designated depots, to which they would be shipped at Government expense.
In this letter, which was to constitute the terms of the final transfer of excess plasma and byproducts by the Army to the Red Cross, it was emphasized that these materials would be used on a nonprofit basis for public and other appropriate use, "consistent with the terms and spirit of the donations," with the distribution left "to the wise discretion and judgment of the Red Cross." It was also noted:
1. That the Red Cross would be given information concerning the reactions observed in certain groups of products.
2. That the dating on each individual package made it quite clear as to the period in which the plasma could safely be used.
3. That it was assumed that all safeguards would be employed in the distribution of this material.
General Kirk's letter was formally acknowledged on 21 November 1945 by Mr. Smith (72). All proposals in it were accepted. It was requested that all oversea supplies be transferred to the American Red Cross in the Zone of Interior on shipping instructions to follow later.
The remainder of the story of the disposal of surplus plasma is best told in connection with the story of hepatitis (p. 674).
OFFERS AND PROPOSALS
As soon as it became public knowledge that plasma was being processed for use in the Armed Forces, proposals to manufacture it were received from individual physicians and scientists, university and other research laboratories, and commercial laboratories not already engaged in the program.
It was the policy, so far as practical, to inspect the facilities in which it was proposed that the plasma should be processed. In no instance did they prove adequate for large-scale production. The majority of smaller commercial firms, once they learned how delicate and complicated a process large-scale production of plasma (and albumin) was, wanted nothing to do with it.
The reply to proposals to alter methods of production was also usually the same: No matter how excellent the proposed change might be, changes in an established process were simply not practical in wartime because of the difficulty of procuring materials and also because a change in any step of the process meant changes all along the line, which meant delays that could not be permitted in what amounted to a crash operation. Most clinical proposals were completely impractical in the circumstances in which plasma was used.
RED BLOOD CELL RESIDUA
When Oswald H. Robertson (73), in 1917, performed the first transfusion with banked blood (p. 5), he was really using a suspension of red blood cells and not whole blood. Two years earlier, Rous and Turner (74) had reported the successful experimental use of the same method. It is surprising, in view of the good experimental and clinical results, that, except for work by Castellanos and his group (75, 76), this method was not used again until World War II. In 1940, McQuaide and Mollison (77) reported its use in 61 cases of anemia, with 8-percent dextrose in isotonic salt solution as the suspension fluid. The reaction rate was 6.5 percent.
The first use of red blood cells in the United States was during the Blood for Britain project (p. 13). When the plasma, which was sent to England, was separated, a large supply of red blood cells was left, and Scudder and his group (78), at the Presbyterian Hospital in New York, used them for transfusion in 227 cases. As a rule, 500 cc. of the cell residual was used in 500 cc. of physiologic salt solution. The transfusions were type-specific, and the reaction rate was comparable to that at the hospital for transfusions of whole blood. Other civilian hospitals also took up the method, but it did not come into general military use immediately because of the primary necessity for concentrating all efforts on plasma production.
As the red blood cell program finally developed, it was an outgrowth of the preparation of peptone by Parke, Davis and Co., from the blood sludge previously discarded.
Organization of Red Cross Program
The distribution of red blood cells to hospitals began on 1 January 1943 at the Red Cross Blood Donor Center in Detroit (79), but it was not until November of that year that the formal program was set up, with the following arrangements (80):
1. The service was conducted by the technical staff of the American Red Cross Blood Donor Service, under the supervision of the Division of Medical Sciences, NRC, through the Subcommittee on Blood Substitutes. Locally, the service was operated through the donor centers and under the control of the technical supervisors of the centers.
2. The cellular residue was released by the Army and the Navy, which had title to it, to the Red Cross Blood Donor Service, for distribution for therapeutic purposes.
3. Since the cells had to be used within 5 days after the blood was collected, the method was available only to the eight or nine hospitals immediately adjacent to processing laboratories. This restriction was based on the practical consideration that the blood had to be transported to a processing laboratory from the bleeding center; transported back to the center after red cells and plasma had been separated; and, finally, transported to the hospital at which the cells were to be used.
4. The service was designed primarily for military hospitals but it was extended, as was practical, to hospitals organized and equipped for such a service. At the meeting of the Subcommittee on Blood Substitutes on 16 March 1945 (81), when Dr. Robinson requested permission to extend the service beyond the teaching hospitals, to which it had been chiefly limited up to this time, it was recommended that the selection of additional hospitals be left to the discretion of the Red Cross Blood Donor Service.
5. All procedures in each hospital from the time the blood was obtained until it was dispensed were under the control of a single responsible physician. Both physicians and hospitals had to agree in writing to carry out the prescribed methods and techniques for the use of suspended red blood cells and had to assume final responsibility for their administration.
6. The service was to be conducted without cost to those served and without financial profit to any person or institution connected with it. All expenses were borne by the National Red Cross Blood Donor Service, by the mechanisms already in operation.
Technique of Collection, Distribution, and Administration
The following technique was specified for the use of red blood cells, the procedure up through the withdrawal of the plasma being the standard procedure for plasma processing:
1. After centrifugation of the blood and withdrawal of the plasma, a sterile solid rubber stopper is placed in the bottle containing the cells. Only type O cells are used for this purpose. The original white tag is left on the bottle.
2. The cells are resuspended in a dustproof room, with a filling burette, in a pyrogen-free physiologic salt solution (or other solution approved by NRC). The diluent is added as soon as possible after centrifugation. Another sterile solid rubber stopper is inserted.
3. The resuspended cells are returned in refrigerated containers to the blood donor center, where the tags on the bottles are checked with the original list. Pilot tubes are not returned.
4. The resuspended cells are stored in the icebox, at temperatures between 39° and 50° F. (4° and 10° C.). Before they are distributed, the cells are inspected for hemolysis and possible color changes.
5. The dispensing laboratory or blood donor center must ascertain the sterility of all cell suspensions. Fifty negative cultures, by the technique required, must be obtained before any cell suspension is distributed. Thereafter, every fifth bottle must be tested until 300 negative cultures are obtained. Then one bottle is tested by random selection every day of operation.
6. If any contamination is detected, all red blood cell activities must be stopped until an adequate explanation is obtained by investigation of all possible causative factors. Sterility tests must be reinstituted by the required techniques before cell suspensions are again released for distribution.
7. After distribution, the cell suspensions must be stored at the temperatures specified. If there is any possibility that the temperature has fallen below the freezing point, the suspensions must be discarded.
8. The bottles are observed at intervals for hemolysis or for color changes in the supernatant fluid. If a violaceous or blackish-red coloration is apparent, or there is any question as to the condition of the suspension, or any unusual odor is detected, the cells must be discarded.
9. The cells must be used within 5 days of the date of bleeding.
10. The suspension must not be dispensed from the original container but must not be removed from it until just before it is to be used. As it is emptied into the dispensing flask, it is carefully observed, so that gross clotting, unusual odors, or other changes will be detected. Retyping and crossmatching are done immediately before the cells are used. The suspension must be given within 5 hours of the time the bottle is opened.
11. The suspension is filtered through four layers of a 44 by 40 bandage roll or through the 100-mesh stainless steel filter in the blood transfusion set. It is not warmed. If the entire contents of a bottle are not used, the unused portion is discarded.
12. Bottles in which cells were delivered must be returned to the blood donor center whence they were dispensed, each bottle accompanied by a properly executed report of the transfusion.
These reports became the property of the Red Cross Blood Donor Service, and its approval before publication of any data or other material concerning the experience was one of the conditions under which cell suspensions were furnished to hospitals and physicians.
The handling of the red blood cells by three separate groups of persons offered chances for breaks in technique because of the divided responsibility, as well as for errors in transcription and for other reasons. It was therefore imperative that the regulations laid down be followed without any deviations.
Hospitals were cautioned not to use red blood cell suspensions for pregnant women or women in the postpartum period without an investigation of the Rh factor. In cases of doubt, only Rh-negative cells were used. The same precautions were observed when repeated transfusions were given with red cell suspensions.
The Detroit Experience
The Detroit Red Cross Blood Donor Center had the first experience with red blood cell transfusions as well as the most extensive; before the war ended, Dr. Warren B. Cooksey, the technical supervisor, had supervised almost 18,000 transfusions with resuspended red cells in 14 local hospitals.
The first report by Dr. Cooksey and Lt. William H. Horwitz, MC, published in the Journal of the American Medical Association on 1 April 1944,
covered 4,050 of the 7,864 cell suspensions delivered to the Detroit hospitals to date(82).
Materials and methods-As a rule, 500 cc. of saline-suspended red cells was given. When large amounts of blood were required, two or three transfusions a day were given, though on a few occasions two to three bottles of diluted and undiluted cells were given as a single transfusion.
The cells were prepared by the immediate resuspension technique, which made it possible to administer them by the gravity method through a standard 18-gage needle. Earlier investigators had shown the difficulty of administering undiluted (packed) red cells and the undesirable pressure needed to accomplish it. Moreover, resuspension at the end of 5 days' storage was accompanied by greater hemolysis and more fragility than when resuspension was carried out as soon as the plasma was withdrawn.
Extensive studies carried out before the formal program was instituted showed no contamination in any sample, and later studies also showed none. When suspensions were deliberately contaminated for experimental purposes, it was found that occasionally within 24 hours, and almost invariably after 48 hours, the affected cells turned dark red and the supernatant fluid showed a purplish-red discoloration that at once distinguished these bottles from the others. The center employed a method of distribution which made it impossible for any hospital to receive the suspensions earlier than 48 hours after the blood was drawn, and this macroscopic observation was therefore employed in lieu of culture of each bottle, which would have been an impossible task. All bottles which showed any discoloration, as well as all bottles not used by the fifth day after bleeding, were discarded.
Before large-scale distribution of these cell suspensions was permitted, the effects of transfusion were studied in 200 patients, with recollection of the demonstration by Denstedt and his associates (83) and by Mollison and Young (84) that the fate of stored blood in vitro does not parallel its fate after transfusion.
Hemoglobin determinations were made by the Haden-Hauser technique (16 gm. hemoglobin=102 percent). All determinations were made 2 hours before the transfusion and were repeated serially 24 hours after it. Urinalysis was carried out before the transfusion and for several days afterward, to investigate the presence of hemoglobin or any of its end products. The icteric index was also determined before the transfusion and for several days afterward. The single abnormality in the series, an increase in the icteric index and hemoglobinuria, was found in a woman with Rh-positive blood and a grave anemia of pregnancy. She had the same reaction after transfusions of stored whole blood.
Suspension media-At first, resuspension was accomplished in salt solution (0.85 percent) adjusted to a pH of 7.2. It was then found that, unless diluents other than physiologic salt solution were used, cells returned to the center after high speed centrifugation showed considerable hemolysis or alterations in the fragility index. Five-percent glucose solution in distilled
1Number of bottles with clear supernatant fluid or fluid
that showed only a trace of hemolysis are shown in percentages by days from day
water produced complete hemolysis within a short time (chart 6), and 5-percent glucose in physiologic salt solution, 2-percent glucose, and 2- and 5-percent sucrose often had the same effect. Alsever's solution and Denstedt's solution preserved the red cells for much longer periods of time. The fragility index was initially higher with both, but it remained at a more constant level after the fifth day than did the index of saline-diluted cells. Hemolysis and fragility index were not significantly altered when the amount of diluent added to the packed cells was so varied that its volume was a quarter of, a half of, or equal to, the volume of the cells.
Results.-Typical results of red blood cell transfusions were the hemoglobin elevations in 629 transfusions at three hospitals, which ranged, per 500 cc. of suspension administered, from 0.46 gm. in malignant disease to 1.3 gm. in obstetric cases. The red blood cell increase in the same series ranged from 123,157 per cubic centimeter in malignancy to 497,000 in obstetric cases.
In another series of 67 transfusions given to 25 patients, the average hemoglobin elevation per 500 cc. of cellular suspension was 0.56 gm. and the average red blood cell increase 206,700 per cubic centimeter.
Statistics in this study bore out the observations of others that the percentage of reactions was less with resuspended red cells than with stored blood. In one series of 413 red blood cell transfusions in 139 patients, there were nine reactions, 2.1 percent. The definition of a reaction was a chill followed by a temperature elevation. When 342 whole blood transfusions were given to the same group of patients, there were 12 reactions, 3.5 percent. There was 3 percent of reactions in the 629 transfusions just mentioned.
The New York Experience
The New York experience was reported in 1945 by Dr. William Thalhimer, Associate Technical Director, American Red Cross Blood Donor Service, and Major Taylor, Technical Director (85).
Materials and methods-This experience was based on 761 transfusions of centrifuged type O cells resuspended and stored in 10-percent corn syrup for periods up to 60 days. (By the time the report was published, 3,000 such transfusions had been given.) The transfusions were given to 437 patients, many of whom received repeated injections, sometimes daily, sometimes several times weekly. They suffered from a variety of chronic diseases, such as arthritis; Hodgkin's disease; leukemia of several types; nephritis; anemias; pulmonary tuberculosis; inoperable malignancies; a few acute conditions; and, in one instance, massive hemorrhage from a gastric ulcer.
At the beginning of this investigation, type-specific cells were used, but as time passed, transfusions were limited to type O cells. There was thus much less wastage of resuspended cells, and the possibility of transcription errors was reduced.
At first, only small amounts of cells resuspended in corn syrup were given. Later, as no harmful effects were evident, the amounts were gradually increased from 50 to 75 cc., and then to 500 cc., per transfusion. Still later, a number of patients were given 1,000 cc. in single injections, and several received 1,000 cc. per day over a 3-day period. The patient with a bleeding gastric ulcer received 3,500 cc. in 7 days.
Experimental studies-Before cells suspended in corn syrup were used clinically, a long series of in vitro and animal studies were carried out. The suspension used was 250 cc. of prechilled (41° F., 5° C.) of 10-percent corn syrup (Corn Products Refining Co.) in sterile, pyrogen-free distilled water. The composition of the syrup before dilution was 17.7-percent dextrose; 16.8-percent dextrin (pro-sugars); and 19.7-percent moisture.
It was consistently demonstrated that cells thus resuspended were more stable and less fragile at the end of 21 days' storage at 41° F. (5° C.) than were cells suspended in physiologic salt solution, Alsever's solution, or Denstedt's solution at the end of 5 days. The amount of hemoglobin in the supernatant fluid averaged from 30 to 40 mg. percent at the end of 21 days in corn syrup against 100 mg. percent at the end of 5 days in saline solution.
No deleterious effects were evident in rabbits which received repeated injections of large amounts of 10-percent corn syrup, sometimes as many as 20 injections in 40 days. There was also no evident deleterious effect on rabbit cells preserved in corn syrup for 7 to 28 days. Finally, histologic examinations of animal tissues showed no pathologic changes and no deposits of iron pigment.
The freezing point of the corn syrup was the same as that of 0.85-percent sodium chloride solution. In behavior, the syrup appeared to be isotonic, or very slightly hypertonic, for blood cells. It was speculated that the dextrins
in it, because of their molecular size, might function somewhat as the original plasma in maintaining the stability of the stored, resuspended cells.
The length of survival of transfused cells was studied by the Ashby technique (p. 260), which was followed closely, since it was found that deviations from it gave inconsistent and inaccurate results. Counts were always made in duplicate, and unless the two counts were reasonably close, the whole procedure was repeated. In the 253 consecutive nonagglutinable cell counts done in duplicate on patients of A and B groups who had been transfused with group O cells, the average difference between the counts was 14,000 nonagglutinable cells per cubic centimeter. The unavoidable error-which is present in all red cell counting by even the most competent technicians-did not exceed 10 percent.
Results.-Clinical results in patients treated with red blood cells resuspended in corn syrup were what might have been expected from the administration of the same amounts of whole blood of the same age. There were no evident deleterious effects, and there was a complete absence of hemoglobinuria, hemoglobinemia, and jaundice. A number of patients showed prolonged beneficial effects under truly adverse conditions. Some cases suggested that adequate amounts of blood given over short periods of time had a more generally beneficial effect, and a more sparing effect on the bone marrow, than the same total amount given over a period of several weeks, a plan often necessary because of the difficulty of securing donors.
The greatest field of usefulness of resuspended red blood cells was in chronic secondary anemias of various origins. The cells were available in enormous quantities, and experience soon showed that large amounts could be injected within relatively short periods. Whole blood, because of its greater viscosity, more effective osmotic pressure, high cost, and relative scarcity, was seldom used in this type of anemia. Another advantage of the cell resuspension technique was the reduction in the volume of fluid injected, which was of considerable benefit in such conditions as cardiac failure.
The New York experience furnished significant information about the safety of injecting older blood. Some of the 382 transfusions given to 125 patients at Montefiore Hospital were given with red blood cells only 3 days old, but in many instances the cells were 7, 14, 22, and 24 days old. In two instances, they were 31 days old, and in two other cases they were 38 and 41 days old, respectively. One patient received transfusions with cells that were 50 and 60 days old, respectively.
The survival of transfused cells dropped off sharply after storage periods of more than 24 days. The survival of cells stored for 30, 40, 50, and 60 days was essentially the same as the survival of transfused whole blood stored for the same intervals. Although from 20 to 40 percent of these cells survived in the recipient circulation from 2 to 10 days, it was concluded that it would not be advisable to transfuse cells stored for these periods unless an emergency existed or fresher cells were not available. The results with cells stored in corn syrup for 21 days were just as satisfactory as those obtained with blood
stored in ACD (acid-citrate-dextrose) solution for the same interval, but, after the desired studies had been made, a 14-day expiration period was established.
The experience at Mount Sinai Hospital, 192 transfusions in 150 patients, paralleled that just described for the Montefiore Hospital. On the strength of these results, cells resuspended in 10-percent corn syrup came to be preferred, because of their longer life, to those resuspended in physiologic salt solution. When only red blood cells were needed, clinical results indicated that a transfusion of centrifuged cells resuspended in 10-percent corn syrup gave as satisfactory results as a transfusion of whole blood.
Extension of Service
Experiences at other civilian and military hospitals paralleled those just described at Detroit and New York. The first red blood cell service set up at a military hospital was established at Walter Reed General Hospital, Washington, D.C., in 1942. By the spring of 1944, the use of red blood cell transfusions was standard at all Army general hospitals in the Zone of Interior near enough to processing laboratories for the material to be delivered to them by automobile. This policy resulted in a great saving in the use of whole blood.
Among the suggestions made by medical officers not directly connected with the blood program was one for the use of red blood cells in pooled plasma in forward hospitals and on the battlefield. The pooled plasma, this particular officer's argument ran, was already available in these areas, and the red blood cells could be sent overseas, by plane, preserved in glucose for 14 days. It was his opinion that the morale of wounded men would be raised if they knew they would receive whole blood and not plasma. He also recommended the method for Zone of Interior hospitals.
The Transfusion Branch, Surgical Consultants Division, Office of The Surgeon General, explained to the writer that his plan was not necessary in the Zone of Interior, where a modification of it was already employed, and was not feasible in forward areas overseas, where present plans did not provide for typing in the field, since blood grouping was done before whole blood was released. It was also pointed out that the mechanical mixing of blood cells and plasma would require equipment not then supplied. It would be hazardous from the standpoint of possible contamination even in a well-controlled laboratory, and extremely dangerous in forward areas and under field conditions. Not included in the letter to the writer was the fact that at the time of the correspondence (March and April 1944), it was entirely feasible to fly whole blood overseas-as was done 5 months later-but that The Surgeon General had rejected the suggestion when it was made to him in November 1943 (p. 465).
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17. Memorandum, Maj. Karl Bambach, SnC, to Catalog and Equipment List Branch, Supply Planning Division. 1 Feb. 1944, subject: Items 16088 and 16089, Serum, Normal Human Plasma, Dried.
18. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 19 Apr. 1941.
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42. Minimum Requirements for Unfiltered Normal Human Plasma, National Institute of Health, 20 Feb. 1941.
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44. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 3 Nov. 1941.
45. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 12 May 1942.
46. Minutes, Conference on the Preparation of Normal Human Serum Albumin, Division of Medical Sciences, NRC, 5-6 June 1942.
47. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 23 June 1942.
48. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 15 Dec. 1942.
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53. Dochterman, Ens. Elsie, W-V(S) (H), USNR, and Lozner, Lt. Eugene L., MC-V(S), USNR. Research Project X-179. Final Report. Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md., 23 Feb. 1944.
54. Memorandum, Lt. Col. Douglas B. Kendrick, MC, for Brig. Gen. Fred W. Rankin, n.d., subject: Reports of Visits to Parke Davis and Company, Abbott Laboratories, and Ben Venue Laboratories, 23-26 August 1943.
55. Memorandum, Maj. F. N. Schwartz, MAC, for Brig. Gen. Fred W. Rankin, 4 Aug. 1944, subject: Inspection Trip of American Red Cross Blood Donor Centers.
56. Memorandum, Lt. Col. Douglas B. Kendrick, MC, for Brig. Gen. Fred W. Rankin, n.d., subject: Report of Visit to the Miller Rubber Division of the B. F. Goodrich Company, Akron, Ohio-23 August 1943.
57. Memorandum, Lt. Col. Douglas B. Kendrick, MC, for The Surgeon General, 13 Aug. 1943, subject: Report of Inspections of Army Liquid Plasma Processing Centers.
58. Memorandum, Lt. Col. Douglas B. Kendrick, MC, for The Surgeon General, 29 Jan. 1944, subject: Report of Visit to Denver, Colorado, 16 and 17 January 1944.
59. Minutes, Blood Plasma Conference, Division of Medical Sciences, NRC, 24 Mar. 1943.
60. Minutes, Conference of the Albumin Testing Group, Division of Medical Sciences, NRC, 22 Mar. 1943.
61. Memorandum, Col. Tracy S. Voorhees, JAGD, 28 July 1942, subject: Meeting at the Purchasing and Contracting Office of the New York Medical Depot on July 28th Concerning Revision of Plasma Contracts.
62. Memorandum, Capt. W. G. Patten, SnC, for Lt. Col. Lee I. Park, JAGD, 14 Sept. 1943, subject: Accounting for Donated Blood and Processing Losses in Relation to the Renegotiation Program.
63. Memorandum, Capt. Earl S. Taylor, MC, to Capt. Frederic N. Schwartz, MAC, n.d., subject: Memorandum on Accounting for Donated Blood and Processing Losses in Relation to the Renegotiation Program.
64. Memorandum, Capt. F. N. Schwartz, MAC, for Director, Procurement Division, 2 Oct. 1943, subject: Plasma Accounting.
65. Letters, Lt. Col. H. T. Marshall, MC, to all laboratories processing plasma, 17 May 1944, subject: Instructions for Preparation of Monthly Blood Plasma Report.
66. Taylor, E. S.: Procurement of Blood for the Armed Forces. J.A.M.A. 120: 119-123, 12 Sept. 1942.
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69. Letter, DeWitt Smith, Vice Chairman, American Red Cross, to Maj. Gen. Norman T. Kirk, 25 Sept. 1945.
70. Letter, Maj. Gen. Norman T. Kirk to Mr. DeWitt Smith, 1 Oct. 1945.
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