CHAPTER XI
The Plasma Program
HISTORICAL NOTE
World War I
This whole chapter on the plasma program should be read withthe recollection that for all practical purposes, the clinical use of plasma forshock and hemorrhage was a development of World War II, just as the concept ofhemorrhagic shock was a development of that war.
Reviews of the literature indicate (1) that theclinical use of blood plasma and serum was first suggested by Bowditch in 1871and Luciana in 1872. Ehrlich was the first to point out that the most stablemethod of preserving the total solids of plasma or serum was removal of thewater component. The basis of the enormous plasma and serum albumin programs, aswell as of the clinical use of these agents in World War II, is inherent inthese two suggestions.
The military use of blood plasma as a substitute for wholeblood in combat casualties was proposed in March 1918, in the correspondencecolumns of the British Medical Journal, by Gordon R. Ward (2), asa way of eliminating the risk, in transfusions given at casualty clearingstations, that the donor red blood cells might be hemolyzed in the recipientbloodstream (p. 7). Citrated plasma would be easy to store and administer, andits use was rational: Wounded men did not die from lack of hemoglobin but fromloss of fluid, with resulting devitalization and low blood pressure. Ward'ssuggestion of a controlled study of plasma, whole blood, and gum acacia wasapparently not followed up, nor was his idea put to clinical use in combatcasualties in World War I.
Also in 1918, Rous and Wilson (3) concluded fromexperimental studies on dogs that loss of blood volume, not loss of red bloodcells, was the important consideration in hemorrhage. Even after grosshemorrhage, these workers were able to restore the blood pressure to normal, andmaintain it at the normal level, by replacing the blood they had removed with anequal quantity of plasma. Plasma, they pointed out, had only half the viscosityof blood, and it seemed to them, in order that the diminished number of redcells might carry on the work of the body, that a brisker circulation, securedby a less viscid fluid, would be desirable.
Mann (4), also in 1918, reported that parenteral injectionsof homologous serum were fully as effective in experimental surgical shock asany other method (p. 335). Serum might therefore be a valuable agent in thetreatment of shock if whole blood were not available.
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The Interval Between the Wars
In 1927, Strumia and his group (5) at the Bryn MawrHospital began to use plasma in the treatment of severe infections because itwas simpler to prepare, and had a larger yield, than homologous serum, and alsobecause serum frequently caused severe reactions, which even heterologousplasma, given intravenously, did not. As early as 1900, Brodie (6) hadcalled attention to the differences in the behavior of serum and plasma. Hethought, though the hypothesis is still unproved, that the untoward reactionsfrom serum were caused by the fibrin precipitation which occurred when serum wasseparated from clotting blood.
By 1931, the Bryn Mawr group were using plasma routinely inthe treatment of certain hemorrhagic diseases as well as infectious diseases.Their policy of using it within 24 hours after the blood was drawn deprived themof one of its chief advantages; namely, its safe storage.
The first use of plasma as a hemostatic agent was apparentlyby Filatov and Kartasevskij (7) in 1935. In the same year, Heinatz andSokolow (8) used it in the treatment of hemolytic shock.
The following year, Elliott (9) proposed that bothplasma and serum be used in the treatment of surgical, obstetric, or traumaticshock whenever transfusion was indicated. His reasoning, like Ward's, was thatthe replacement of lost blood volume was more important than the replacement ofred blood cells, because the maintenance of osmotic pressure is a function ofthe plasma proteins. Elliott also advanced two other ideas: (1) that liquidplasma could be stored for long periods without deterioration, and (2) that ifplasma was pooled (he used up to eight donors), neither typing nor crossmatchingwould be necessary because the antibody titer would be neutralized.
Elliott's observations were quickly confirmed by a numberof other observers. Then, in 1939, he, Tatum, and Nesset (10) recommendedstored plasma as "an ideal substitute for whole blood in the emergencytreatment of shock and hemorrhage for war wounds." Elliott's earlierrecommendation that plasma could be safely used without typing or crossmatchingwas now supported by their experience in 191 transfusions. The technique ofcollecting the blood in "a sealed vacuum transfusion set" wasdescribed, with the separation of the plasma from the blood in a completelyclosed system that prevented contamination.
In 1940-the year before the United States entered World WarII-Strumia and his associates (11) recommended the use of citratedblood plasma, without crossmatching, in the treatment of burns and shock, theirresults paralleling those of Mahoney (12), Elkinton (13), andMcClure (14). Best and Solandt (15) reported encouraging resultswith plasma and serum in the prevention of experimental shock. In 1941, Kekwickand his associates (16) reported the treatment of shock and hemorrhage inair raid casualties and concluded that plasma was as effective as whole blood inrestoring blood volume
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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 singleinjection. One of their burned patients received 7,300 cc. of plasma over an11-day period.
All of these early experimental and clinical studies weremade with liquid plasma, prepared in small amounts in hospital laboratories.
The experience with plasma in the Blood for Britain projectis described elsewhere under that heading (p. 13).
GENERAL CONSIDERATIONS
The procurement of plasma on the major scale required by theArmed Forces in World War II was possible only because of the cooperation ofmany persons and agencies, including:
1. Volunteer donors; that is, the general public. Thealternative to their donations would have been the outright purchase of blood,which would have been very expensive, probably not practical in the amountsrequired, and undesirable from other aspects.
2. The Army Medical School, whose activities and functionshave already been described (p. 61).
3. The Army and the Navy, whose cooperation, which isdescribed under appropriate headings, eliminated duplications, cut throughbottlenecks, and added greatly to the efficiency and success of the project. Byagreement, the Army handled all contracts for plasma and the Navy, all contractsfor serum albumin and the other plasma fractions.
4. The American Red Cross, with its national scope, hundredsof well-organized chapters, and thousands of volunteer workers.
5. The NRC (National Research Council), acting chieflythrough the Subcommittee on Blood Substitutes of the Committee on BloodTransfusions.
6. The Army Medical Procurement Agency, which, to avoidconfusion and the writing of multiple contracts, acted as purchasing agent forall plasma, albumin, and byproducts supplied commercially to the Army and theNavy.
7. Commercial biologic firms, selected according to theirgeographic location in respect to blood donor centers and their actual andpotential facilities for processing blood.
8. The manufacturing firms which developed and supplied theequipment necessary for the collection and processing of blood.
Definitions
Plasma is the supernatant fluid that separates from thecellular elements when an anticoagulant is added to blood. Serum is the liquidportion that separates during the process of clotting. Plasma containsfibrinogen. Serum
1These observations support the concept that if grosshemorrhage is not a factor, plasma is adequate in the management of shock.Victims of air raids had been struck by flying debris or had been crushed orburied; they did not have the multiple wounds caused by high-velocity missilesand associated with hemorrhage which require whole blood.
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does not. The distinction in nomenclature should be carefully observed, forthe 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 inthe 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 wholeblood program contains an extended discussion of the reasons that led up to theselection of plasma for use of the Armed Forces by the Subcommittee on BloodSubstitutes 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 yearsbefore the United States entered the war, experimental studies and clinicaltesting had advanced sufficiently far to make it clear that either serum orplasma would be the most desirable agent for the management of shock inbattlefield casualties and in forward hospitals. There was practically nodifference in their clinical effect. The only biochemical difference betweenthem was that serum contained no fibrinogen, its removal having occurred duringthe clotting process. Reactions with both agents were insignificant. Liquidplasma, if properly handled, could be safely stored for months. Frozen plasmacould also be kept for indefinite periods. A dried form of plasma could beproduced, though methods of drying were not yet entirely satisfactory. The chiefadvantage of plasma was that the yield was 15-20 percent greater per pint ofblood than the yield of serum.
When the subcommittee recommended that either frozen or dried plasma beemployed in lieu of blood in the treatment of shock, there were a number ofreasons 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 thequantities likely to be needed, together with its safe storage andtransportation, presented logistic problems of enormous proportions that simplycould not be solved in the light of either the knowledge possessed or thefacilities available in 1940-41. Preservative solutions that would permit longstorage periods of blood were just being developed (p. 221). Investigations onthoroughly dependable, avid grouping sera were in their very early stages (p.236). The development of suitable equipment for the collection, storage, anddispensing of whole blood was in its infancy (p. 163). Refrigeration equipmentfor use in the field under varying conditions of heat, cold, and humidity hadnot yet been manufactured (p. 206). Finally, an airlift capable of deliveringblood to the far reaches of the battlefront was still almost 3 years away.
2. Frozen plasma was obviously unsuitable for use under battlefieldconditions. While liquid plasma could have been used, dried plasma had greater
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advantages It could be dried from the frozen state to lessthan 1-percent moisture content. In this state, it could be packaged undervacuum and preserved for years without refrigeration and without being affectedby extremes of heat and cold. From the standpoint of logistics, the equipmentnecessary for its reconstitution and intravenous administration could beincorporated in a small kit, which could be made available under almost anyconditions of war.
3. No matter in what form it was used, plasma could beadministered without typing or crossmatching.
4. The administration of plasma was attended with anegligible 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 thelarge quantities likely to be needed.
The inherent organic and other characteristics of plasma,particularly the ease with which it could be manufactured, stored, andtransported, clearly made it a practical and desirable agent. The reasons forits selection in 1941, while they do not fully explain lack of attempts tosupply whole blood to field units at this time, did take cognizance of obstaclesthat went far toward discouraging even the most ardent advocates of whole bloodas a feasible replacement fluid in Zone of Interior hospitals. These reasonswere even more valid in the recommendation at this time of plasma as a feasibleand practical agent in oversea hospitals.
The 1941 decision of the subcommittee was, of course, coloredby the position of the Office of The Surgeon General almost a year earlier, tothe effect that when blood could not be collected locally, plasma, "eitherplain 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 southwesternPennsylvania Chapter of the American Red Cross, for the production of driedplasma to be tested by the Army and the Navy Medical Schools and by members ofthe Subcommittee on Blood Substitutes.
The several hundred lots of plasma prepared and distributedby Dr. Strumia from the blood secured by the Red Cross, as arranged at the 31May 1940 meeting of the Committee on Transfusions, were reported on at the 18July 1941 meeting of the Subcommittee on Blood Substitutes (20, 21). Eachlot contained from 17.5 to 18 gm. of plasma dried by sublimation from the frozenstate by a technique employing water vapor condensation by low temperature inflame-sealed ampules (vacuoles). The containers were large enough to permitreconstitution with water and were suitable for direct administration of thesolution. The difficulties, all minor, which were reported by the workers whoused the plasma, chiefly Army and Navy medical officers, were assumed to bethose that would be experienced on the average hospital service. The onlyreactions were urticarial, and they were few and mild.
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The following conclusions were drawn from this experience:
1. Although plasma concentrated four times can be given without untowardreactions, normal reconstituted plasma is usually superior to the concentratedvariety.
2. Without additional supplemental electrolytes, serum albumin cannot bedepended upon to restore circulating blood volume in acute peripheralcirculatory failure, especially in dehydrated patients.
3. The investigation was regarded as entirely successful from the standpointof practical production of dried plasma which was safe for human administration.
Dating Period
The dating periods for all varieties of plasma, which eventually provedsurprisingly long, were originally little more than guesswork, because of lackof previous experience.
When liquid plasma was first prepared, NIH (National Institute of Health) setthe 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 whetherthis limit was justified, showed that it could safely be extended. Clinicaladministration 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 plasmawas devoid of most active globulin components, complement, fibrinogen, andprothrombin, and had no power of coagulation on recalcification. Nonetheless,the administration of 2,000 cc. did not increase the coagulation time of therecipient's blood nor did it decrease its concentration of prothrombin.
The U.S. Public Health Service first set 2 years as the dating period fordried plasma (with the specification that the cutoff date should appear on thecontainer). Later, the date was extended to 36 months, and, finally, alllimitations were removed.
FROZEN PLASMA
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 waspointed out that liquid plasma was frequently contaminated, but that there wasapparently 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 nowthought
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that in many ways frozen plasma was superior to both liquidand dried plasma. The method developed in his laboratory for the preservation offrozen plasma was "very simple, very economical and * * * likely to applyto the needs of both civil and military nature in the greatest majority ofcases."
The only disadvantages Dr. Strumia could see to his proposalswere that it would be more difficult to transport frozen plasma under adverseconditions and that this form could not be reconstituted into concentratedplasma if it were needed in that form.
All of Dr. Strumia's remarks were predicated on the correctprocessing of the plasma; that is, it must be collected under asepticprecautions; in a closed system; and with as brief a timelag as possible betweenthe collection of the blood, its centrifugation, and the fixation of the plasmaby freezing (fig. 61). Care also had to be taken that thawing did not occuraccidentally, from frequent opening of the storage cabinet, or from transientfailure of current. In these circumstances, labile constituents coulddeteriorate.
In view of the advantages he had listed, Dr. Strumiaconsidered the frozen variety of plasma to be the product of choice for thosecommunities in which the exact time or the exact need could not bepredetermined, but in which unexpected needs might be extremely heavy, as inlarge industrial areas or communitywide catastrophes. Processing should be donewith the technical advice, and under the supervision, of some such authority asNRC. The bulk of the plasma processed from the blood procured by the Red Crossshould be processed and distributed in this form.
These views were not generally accepted. Dr. Milton V. Veldeestated that only in grave emergencies would he favor the freezing of plasma orserum by hospitals, though he agreed with Capt. (later Col.) Douglas B. Kendrick'spoint, in which Dr. Edwin J. Cohn concurred, that plasma might be preserved, asa matter of expediency, in the frozen state while the capacity of commercialplants for desiccation was being increased.
18 July 1941-At the 18 July meeting of thesubcommittee (20), certain facts were frankly faced. The Navy hadimpelling needs and would require most of the 200,000 units of dried plasma nowon order. This would leave no blood substitutes on hand for emergencies likelyto occur in the training maneuvers underway and to continue during the summer.Base hospitals had no blood banks, since The Surgeon General had refused topermit the storage of either blood or plasma. In addition, medical officers hadto be trained in the handling of plasma and in an understanding of itspotentialities and limitations.
Captain Kendrick therefore proposed, with Commander Newhouserconcurring, that a limited number of bleeding units be set up to supply frozenplasma for the Armed Forces in the Zone of Interior and that personnel in chargeof these units be trained in Washington or at some other suitable center. Forthe past year, the Naval Medical School in Washington had been supplyingadjacent naval hospitals with limited amounts of frozen plasma.
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This recommendation was conveyed to the Surgeons General of the Army and theNavy and was put into effect, with certain modifications.
Conclusions Concerning Frozen Plasma
At the annual meeting of the American Society of Refrigerating Engineers on 7December 1943, Colonel Kendrick summarized the experience of the MedicalDepartment 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. Thismethod yields a very clear product.
3. If plasma is stored as a liquid, it should be maintained at roomtemperature rather than 39? F. (4? C.) because at lower temperatures fibrinprecipitates readily, and the result is an undesirable product from an estheticstandpoint.
4. Liquid plasma stored at room temperature loses its labile components(complement and prothrombin), which are useful in wound healing, but retainsintact its albumin and globulins, which are essential in the treatment of shockand burns.
5. If plasma is stored in the frozen state, there are almost no changes inits constituents.
6. While plasma can be either shell frozen (p. 281) or frozen withoutrotation, there is no advantage to the former technique if it is merely to bestored frozen.
7. Commercially available ice cream cabinets are well suited for bothfreezing and storing plasma. The position of the bottle during the freezingprocess is of little importance.
8. The time required for freezing should not exceed 6 hours, but the completeprocess can be accomplished routinely in 3-4 hours. When the 6-hour limit isexceeded, the plasma tends to look turbid when it is thawed. Its efficacy is notaffected, but the clinician is likely to regard it as contaminated.
9. Frozen plasma should be kept at a constant temperature, preferably between14? to -4? F. (-10? to -20? C.). If the temperature is allowed to riseslowly, up to 32? F. (0? C.), the minute thawing which occurs may result inthe 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, withoutany apparent changes it its properties. These facts carry an important practicalimplication: If power failure occurs, it is better to remove the plasma from theicebox, thaw it, and refreeze it when power has been restored.
These various observations made it possible to draw up the following rulesfor 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 thesupernatant plasma must be recovered within 72 hours after the blood iscollected.
3. The plasma should be stored in a well insulated, low temperature cabinet,maintained at 14? to -4? F. (-10? to -20? C.), capableof freezing the plasma within 4 hours.
4. When plasma is required for use, it can be thawed in a water bath at98.6? F. (37? C.) in 20-25 minutes. It is good practice to keep three orfour bottles of plasma thawed out and available for immediate use in theoperating room or emergency room.
5. Pools of 1,800 cc. of plasma should be aspirated into receptaclescontaining 200 cc. of glucose, which gives a final solution of 5-percent glucosein each 500 cc. of plasma. With this dilution, fibrin precipitation does notoccur when plasma is stored at room temperature after thawing.
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LIQUID PLASMA
Refrigeration
Refrigeration was at first considered necessary for thepreservation of liquid plasma simply because it was the universal custom torefrigerate the blood from which it was prepared. When it was refrigerated at43? to 46? F. (6? to 8? C.) by Strumia and McGraw (5), there wasprogressive flocculation of the more unstable proteins, and bacterialcontamination was also a risk. Control series at the Army and the Navy MedicalSchools later showed that liquid plasma stored without refrigeration was just assatisfactory as refrigerated plasma from the standpoint of sterility, hemoglobincontent, and plasma protein content, and was far more satisfactory from thestandpoint of clarity.
Supply of Liquid Plasma for Zone of Interior Hospitals
When liquid plasma was first introduced, it was thought bestto use it in the hospital in which it was prepared, or in hospitals in thecommunity. The results of the mass collection of liquid plasma in the Blood forBritain project, with its high rate of contamination (p. 14), had beendiscouraging. The explanation of the difficulties in the British program wassimple: If the mass processing of plasma was to prove safe and practical,collection and processing must be carried out by a completely closed, completelyaseptic technique.
While the Blood for Britain project was underway, the BloodResearch Division in the Army Medical School was investigating the possibilityof supplying liquid plasma to hospitals in the Zone of Interior. Names ofpossible donors were provided in groups of 10 by the American Red Cross, andfrom the 10, a daily average of 6 donors was secured, who were bled at a smallcenter set up in the school. The first plasma processed for this purpose wasdistributed in December 1940. At the same time, by a similar project, the NavalMedical School, U.S. Naval Medical Center, made liquid plasma available to allnaval hospitals in the continental United States. The plasma was kept in thefrozen 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 combinedtheir efforts, and a collection center was opened in Washington, D.C., as ajoint Army-Navy project. The Red Cross procured the donors by publicsolicitation, and helped in the operation of the center. The technical help wasfurnished by the Army and the Navy Medical Schools. The blood thus secured wasdivided between the Army and the Navy, and the Army share was processed intoliquid plasma at the Army Medical School.
From these small beginnings came the project by which liquidplasma 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 ofSurgical Physiology at the Army Medical School supplied liquid plasma for
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the hospitals in the First, Second, Third, and Fourth ServiceCommands. It also trained personnel who operated the plasma processinglaboratories in the other service commands (fig. 61), and supervised theinstallation and operation of these laboratories. After 22 March 1944, allliquid plasma for Zone of Interior hospitals, whether for emergency use orroutine replacement, was obtained from the Army Medical School.
CONCENTRATED PLASMA
Concentrated plasma was discussed at several of the meetingsof the Subcommittee on Blood Substitutes in 1941 (20, 24, 25), andseveral tests were made with it. The basic of the proposal was twofold: (1) thatany 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 acorresponding saving in supplies and shipping space. It was concluded thatconcentrated plasma was not so safe an agent as isotonic plasma (26).
DRIED PLASMA
General Considerations
In the spring of 1943, at the request of The Surgeon General,E. G. Pickels (27), from the International Health Division of theRockefeller Foundation, visited the nine biologic firms then processing driedplasma for the Armed Forces. He was accompanied by representatives of a firm ofengineering consultants engaged by the Government to conduct a survey inconnection with the renegotiation of contracts. On his return, Dr. Pickels feltjustified in quoting from a publication by Franz Oppenheimer in the New YorkState Journal of Medicine dealing with techniques of drying plasma:
It is a truly remarkable achievement that the Americanbiologic industry has taken a laboratory process, which is complicated andmeticulous, and has rapidly developed production methods so efficient that theywere now supplying several million units of plasma per year to the armed forces.
It was a well-deserved tribute, which was repeated manytimes, in one form or another, in the course of the war.
The processing of whole blood into dried plasma for the ArmedForces became a function of the large commercial biologic and pharmaceuticallaboratories. When the need arose, in 1940, no individual and no private orcommercial organization had had any extensive experience with the production ofdried plasma. The Sharp & Dohme experience, while considerable, had been ona small scale. Some consideration was given to the formation of a large,nonprofit plasma processing plant in cooperation with some large hospital, butthe idea promptly gave way to the more realistic decision that commerciallaboratories would be better suited for the task. Their performance, as justindicated, was remarkably satisfactory.
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After the blood for plasma had been collected at the RedCross blood donor centers, it was refrigerated and shipped to the commercialfirms which would process it and which had been selected for the work for tworeasons: 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 requiredserologic studies were performed on each donation.
2. Then the plasma was separated from the cellular elementsin centrifuges, after which varying numbers of bloods were pooled andbacteriologic and toxicity tests were performed.
3. The plasma was shell frozen in individual bottles byrotating them in a properly cooled medium. It was important that the freezingprocess be carried out in as short a time as possible.
4. When necessary-when, for instance, the supply of bloodexceeded the processing facilities-the plasma could be stored indefinitely inthe frozen state. Otherwise, it was desiccated from the frozen state under highvacuum, after which the bottles of dried plasma were evacuated, stoppered, andplaced in evacuated metal containers.
The description just given makes the process of drying plasmasound reasonably simple. The manual published in November 1942 by the Office ofCivilian Defense (28) pointed out that, contrary to the prevailingopinion among the uninformed, the preparation of dried plasma of U.S.P. qualityor better was an exacting task, requiring expensive equipment and trainedpersonnel. A small, inexpensive drying machine, suitable for use inindividual hospitals or small communities, and meeting the specifications forprocessing dried plasma, did not exist. The preparation of dried plasma was anundertaking for a large laboratory, with financial resources and an adequatestaff, and, even then, it was practical only if the distribution area extendedover a very large territory.
Historical Note
Historically, the development of desiccated plasma fallsinto three distinct periods (28):
1. Desiccation was used on a very small scale, for a limitedamount of research and teaching.
2. Research and teaching were expanded, and desiccation wasapplied to the preservation of convalescent serum in a few large medicalcenters.
3. The growing appreciation of the value and necessity ofsupporting blood protein levels and the administration of concentrated plasmafor its hypertonic effects foreshadowed the mass preservation and use ofdesiccated plasma in various concentrations.
Development of special techniques-Ehrlich, as alreadypointed out, was the first to observe that the most stable method of preservingthe total solids of plasma or serum was to remove the water. The process wascarried out successfully in serum by Rosenau in 1895, Martin in 1896, andNoguchi in 1907. It was not until 1909, however, that any practical application was made of these observations. In that year, Shackell (29) described thebasic principle
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of vacuum desiccation from the frozen state, a processessential to the production of a highly soluble product which would retain itsoriginal properties.2
Shackell also solved another basic problem, how to deal withthe huge volume of water vapor released from only a few cubic centimeters offrozen 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 useof the principle of chemical adsorption, Shackell provided a lead which manysubsequent investigators followed, though his choice of sulfuric acid for adesiccant, as well as his design of the machine, limited the usefulness of hismethod. In addition to his use of a chemical adsorbent, which preventedsaturation of the exposed surface, the type of pump Shackell used produced anextreme vacuum very rapidly. Moreover, freezing the material before desiccationeliminated possible concentration of substances and, to a lesser degree,prevented shrinkage and hardening (30).
In 1935, Elser, Thomas, and Steffen (31) improved thedesign of the original drying machine by the use of a manifold system withattached glass containers and a cold trap for collecting the moisture. Theyfound themselves severely restricted, however, by the characteristics of thechemicals used. Greaves and Adair (32) had the same experience thefollowing year. These difficulties, including insufficient speed of adsorption,dilution, scum formation, and other changes which retarded the pickup of watervapor as the process proceeded, increased as the size of the apparatus wasincreased. Qualitatively, a highly successful product could be obtained, but thedifficulties mentioned, together with the expense of using a new desiccant foreach lot of plasma, limited the use of the method devised by Elser and hisassociates.
Later, this group (33) used carbon dioxide snow, whichproduced a temperature of -94? F. (-70? C.), and still later they usedmechanical refrigeration, which produced a temperature of -29? F. (-34?C.).
Within a closed system of manifolds and tanks hooked up to avacuum pump, water vapor escapes from the plasma by sublimation. Between theplasma bottle and the pump, there is a cold chamber, refrigerated externallymechanically or by carbon dioxide and alcohol, to provide temperatures of -58? to-101? F. (-50? to -74? C.). The watervapor is thus condensed and frozen on the inner wall of the chamber, and theremaining water is thus removed from the line and the vacuum is, in turn,adequately maintained.
Elser must be credited with the first processing of largequantities of biologicals in their original containers. He also accomplishedvacuum 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 thelarge-scale desiccation of frozen sera on a practical basis, by attachingampules of prefrozen material
2E. G. Pickels (27), in his comprehensive reporton the desiccation of plasma from the frozen state, noted that Shackell wasapparently not familiar with the desiccation experiments of Altmann, reported in1890. Although Altmann was interested only in fixing and preserving thestructure of tissue, he was the first to dry biologic material kept in a frozenstate. Pickels' report has an excellent, usefully annotated list ofreferences, many of which themselves contain comprehensive lists of references.
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to a manifold in the open air and employing solid CO2 torefrigerate the cold trap (fig. 62). They also used alcohol and MethylCellosolve for the freezing mixture.
In 1939, Flosdorf and Mudd (36) introduced what theytermed the Cryochem process, using a specially prepared anhydrous calciumsulfate (Drierite), which had the property of regeneration. It was an ingeniousidea, but it did not prove practical for large-scale production.
In 1939, Greaves and Adair (33) reported a techniquewhich utilized a cold-trap condenser. They placed the serum in a desiccatingchamber provided with electrical heaters and arranged for the moisture to becollected on mechanically refrigerated coils. Their report included acomprehensive discussion of the temperature relations to be considered in dryinglarge volumes of sera.
In 1940, Hill and Pfeiffer (37) reported the Adtevactechnique, which used cooled silica gel as an adsorbent for the water vapor. Thegel could be regenerated by heating and the adhered moisture drained off. Thistechnique, the report noted, was entirely satisfactory for use in connectionwith a hospital blood bank but was not suitable for large-scale production.
A variety of other techniques and modifications of oldertechniques were introduced during 1940 (1, 36, 38-40).
At a meeting of the Blood Plasma Producers Association on 27October 1942 (41), Dr. Sidney O. Levinson and Dr. Franz Oppenheimer ofthe Michael Reese Hospital described a technique for shortening the desiccationprocess by
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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 ofcompressor was also used. The technique was not accepted for a number ofreasons, including the expense ($15,000 per unit) and the requirement forcritical materials in the construction of the apparatus. If the requirements forblood plasma were increased and expansion of the present program becamenecessary, it was the sense of the meeting that the installation of theLevinson-Oppenheimer method might then be considered.
Although, for practical reasons, the Levinson-Oppenheimer technique could notbe adopted at this time (1942), it was an important contribution, for itpermitted complete control of the amount of heat imparted to all areas of theshell-frozen plasma, in keeping with the variations in the vacuum gage readings.Dr. Levinson's additional suggestion, that drying could be facilitated by thetechniques then in use if a widemouthed bottle were substituted for theconstricted-neck bottle employed, was protested by Dr. Veldee because of thedanger of contamination. The action of the conference, based, as it was, onwartime conditions, was entirely justified. Nonetheless, this was superb, highlyefficient equipment because of its correct application and control of heatexchange. It was installed later at Hyland Laboratories and was put to good useduring the Korean War.
Conclusions-In concluding his historical review of techniques for thedesiccation of plasma, which was prepared after commercial production had beenon a practical basis for almost 3 years, Pickels made the following points:
1. When a cold surface of sufficiently low temperature is employed forcollecting water vapor, the maximum possible rate of sublimation from frozenmaterial in a given container will be obtained if the conduits from thecontainer to the trap are sufficiently wide and short.
2. This important theoretical condition was taken advantage of by Oppenheimerand Levinson in their experiments with combined drying and condensing chamberswhich depended on mechanical refrigeration. The same condition was also takenadvantage 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 standpointof theoretically desirable features and practical performance, especially whenspeed of drying was a prime consideration.
4. The steam ejector technique offered a decided advantage only for verylarge-scale production, and even then only when circumstances were peculiarlyfavorable.
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 theNational Institute of Health (42).
4. The product must be soluble within 10 minutes when reconstituted to itsoriginal volume.
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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. Strumiawas proving highly efficient for hospital and laboratory preparation of driedplasma, though it was not suitable for commercial production. The Sharp &Dohme method and the Reichel modification of this method had been approved bythe National Institute of Health. The Hill and Pfeiffer Adtevac method had notyet been approved, but it was thought that the changes being made in it wouldmake it acceptable. The Wyckoff-Lagsden "pig" method was underdevelopment at Lederle Laboratories, and it was thought that it would eventuallybe capable of handling large quantities of plasma. When it had been fullydeveloped, it proved a most efficient technique.
COMMERCIAL PROCESSING OF DRIED PLASMA
By December 1943, nine commercial laboratories were engagedin the production of dried plasma. As they entered the program separately, withlittle liaison with one another in the initial phases, they used widelydifferent types of equipment, though the principles of desiccation which eachemployed were practically identical.
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Shell Freezing Technique
There was general agreement that shell freezing of liquidplasma was necessary to obtain the best dried product. By this process, theplasma was frozen to the inner aspect of the bottle in a shell or layer ofuniform thickness, with an empty cone or circular channel in the long axis ofthe bottle extending from the bottom to the neck (fig. 63). Since the volume ofplasma frozen did not ordinarily exceed three-quarters of the volume of thecontainer, 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,
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and the further result was that the plasma became denatured and would not gointo 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 producedtemperatures at that level. Long after mechanical refrigeration, whichpermitted graded temperature levels, was available, the idea persisted that forthe best results, shell freezing was necessary at the level mentioned. Theextensive experimental work on freezing plasma by Strumia and his group (5), whichwas published in 1941, disproved this contention and established the feasibilityof shell freezing within the temperature range of -58? to -76? F. (-50?to -60? C.).
Refrigeration machines first developed for maintaining shell freezing unitsat -101? F. (-74? C.) consisted of two high-stage compressors withpropane on the low side and ethane on the high side; each compressor operatedwith a 2-hp. motor. This equipment did not prove satisfactory: The refrigerationcapacity was insufficient. The ethane compressor frequently became fouled withoil. The expansion valve on the high side frequently became clogged with icefrom water in the ethane gas.
The possibility of using higher temperature ranges for shell freezing greatlysimplified the use of mechanical refrigeration for this purpose. In severalcommercial laboratories, shell freezing was accomplished routinely at -58? to -76? F. (-50? to-60? C.). The machines were operated by one-stagecompressors which used F-22 gas or two-stage compressors which used F-12gas. At such ranges, plasma could be shell frozen in 15-25 minutes.
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Equipment for Shell Freezing and Drying Plasma
Space does not permit the detailed description of the equipment used forshell freezing of plasma (fig. 72) and for drying it, but the whole process isdescribed in an article published in 1944 (23).
ADDITIVES
Preservatives-The National Institute of Healthminimum 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 matteron several occasions. It was brought out, at the first discussion (22), thatthe Blood Transfusion Association of New York had found Merthiolateunsatisfactory and Zephiran too toxic for use. Some of the subcommitteemembership thought that plasma was best stored without any preservative at all (25).A recommendation to this effect was waived when it was found thatcommercial firms were not inclined to process plasma without one (43).
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Merthiolate (1:10,000) was originally employed for thispurpose. At the 19 September 1941 meeting of the subcommittee (43), thequestion was raised whether, when massive doses of plasma were necessary, therewas danger that the patient might receive too much of the mercurial preservativeand might incur renal damage. A motion setting a limit to the amount of plasmacontaining a mercurial preservative which might be given was lost after it waspointed out that the amount of mercury administered with plasma did not compare with that used in antisyphilitic treatment or givenin the form of mercurial diuretics.
It was agreed that a preservative was an added protectionduring the time which elapsed between the opening of the container of plasma andits infusion, but no action was taken, though some members of the subcommitteethought-and action was later taken to that effect-that the lapsed timeshould not be more than 3 hours. The NIH minimum requirements simply stated thatplasma "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 literaturewhich they had been appointed to make at the previous meeting: Merthiolateapparently 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 acceptMerthiolate as a preservative only if a definite limitation were set on
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the dosage of plasma and if the symptoms of mercurialpoisoning were published on the label of the can. Other members suggested alimit of 3-6 liters of plasma in 24 hours. Dr. Strumia suggested putting thepreservative in the distilled water, the amount of which could be regulated asdesired. It was finally passed that phenylmercuric nitrate, 1:50,000, orMerthiolate, 1:35,000, be used in plasma, with the maximal dose to be 4 litersin 24 hours. This recommendation was later adopted. On 10 April 1942, Dr. Veldeeauthorized the change, with the concurrence of others concerned, fromphenylmercuric
FIGURE 68.-Wyckoff-Lagsden cans (Lederle pigs) for cold surface condensation technique of drying plasma. A. Lateral view. B. Top view: container for Dry Ice and alcohol (a).
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
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FIGURE 69.-Wyckoff-Lagsden pigs used to dry plasma at Blood Research Division, Army Medical School.
its omission was studied from the standpoint of plasma yield,the difference was found to be only about 1 percent (46). The plasmayield 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 thathe had begun to make pools of plasma diluted in the proportion of 2,000 cc. ofplasma to 200 cc. of 50-percent dextrose, by the sedimentation technique, withresults that were so far very satisfactory. Two opinions were expressed, thatthe addition of glucose would have no effect on flocculation and that there wasmore flocculation without glucose than with it. At the Naval Medical School,dilute plasma had been kept in the plasma bank for 24 months withoutprecipitation, and there had been no flocculation over a 14-month periodin plasma to which dextrose had been added.
Citric acid-Studies by Dr. Strumia (45) on thereconstitution of dried plasma with 0.1-percent citric acid solution instead ofdistilled water showed that the pH of 7.4-7.6 thus secured preserved much ofthe complement and prothrombin, labile elements which were lost in considerableamounts on storage. It was also found that a citric acid solution with a pH of2.8 kept much better in glass than did distilled water. These observations wereconfirmed by Commander Newhouser, Colonel Kendrick, Dr. F. H. L. Taylor,
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and others, and it was therefore recommended on 15December 1942 that 0.1 percent citric acid solution be substituted for 1 .0percent sodium chloride in the reconstitution of dried plasma (48). Thisrecommendation was later put into practice.
FILTERS
National Institutes of Health requirements specified thateach package of dried plasma contain a warning as to the danger of injectingplasma intravenously without the use of a filter in the tube leading from theplasma reservoir to the recipient.
The amount of plasma which could be filtered through theordinary Seitz filter before the filter became clogged and closed was so smallthat this method was not practical until the pads were saturated with sodiumcitrate or some other solution. Five-inch pads permitted the filtration of18,000 cc. of plasma at 98.6? F. (37? C.) in less than 3 hours.
In February 1942, Dr. Strumia reported a series ofexperiments intended to determine the effects on citrated plasma offiltration through a Seitz sterilizing filter (49). Filtration throughpads saturated, respectively, with 0.85-percent saline solution and 4-percentsodium citrate-saline solution apparently caused a loss of some or all of thefibrinogen. When the material was left standing at 39? F. (4? C.), bothlots 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 aWhatman filter No. 1 under the conditions of the experiment caused anappreciable loss of fibrinogen, a sharp increase in pH, an apparent loss ofprothrombin, but no appreciable loss of other proteins and no changes in thecomplement titration.
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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 ofdried plasma contained an excess of fibrin or fibrinogen particles, whichinterfered with the infusion. The complainants were always informed thatthere was no risk from the use of such plasma if it were filtered duringadministration, as all plasma should be.
Dr. Leo Rane, at the Lederle Laboratories, conducted studieswith special clarifying and bacteria-absorbing filter pads almost free ofcalcium. They were not yet available commercially when this plant was inspectedin January 1945, but Dr. Rane sent a few to the Army Medical School, to betested for sterility, postfiltration, precipitation, fibrinogen, and otherthings. The war ended before the study could be implemented.
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FIGURE 72.-Schematic drawing of blood plasma freeze drying equipment.
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 thisfield (19). By April (18), this firm had processed 1,140 units of250 cc. each, with a loss of 126 units, 76 by breakage and the remainder forother reasons. By July (20), it had received 5,902 bloods, processed5,496, and released 2,976 for distribution.
Before the declaration of war on 8 December 1941, three other contracts hadbeen made. A small amount of plasma (750 packages) was available at PearlHarbor, but the Navy, whose immediate needs were greater than those of otherservices, had received most of the other production.
Eventually, eight commercial firms were processing plasma, as follows (50):
Sharp & Dohme, beginning on 4 February 1941.
Eli Lilly and Co., beginning on 1 October 1941.
Lederle Laboratories (Division of American Cyanamid Co.), beginning on 14 October 1941.
Reichel Laboratories, Inc. (later Reichel Division of Wyeth, Inc.), beginning on 18 November 1941.
Ben Venue Laboratories, beginning on 10 January 1942.
Cutter Laboratories, beginning on 12 January 1942.
Hyland Laboratories, beginning on 13 May 1942.
Parke, Davis and Co., beginning on 29 June 1942.
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
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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 untildrying apparatus was installed and ready for use. The purpose of this plan wasthreefold: (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 rapidlyas possible, without regard to present limitations in commercial dryingcapacity; and (3) to stockpile plasma for use when the albumin program wouldmake additional demands on blood supplies.
The story of the processing of dried plasma throughout the war was one ofcontinued increases in the requirements of the Armed Forces, continued expansionof the processing laboratories to meet these demands, and ingenious solution ofthe various problems that arose, including the shortages of trained personneland of equipment needed for production. There would be little profit in goinginto details of these matters, but a few special points might be mentioned,beginning with the fact, already stated, that to avoid confusion and the writingof multiple contracts, arrangements were made for the Army Medical ProcurementAgency, in Brooklyn, to act as purchasing agent for all plasma preparedcommercially for the Army and the Navy.3
An early investigation showed that contracts entered into by any firm withthe Army and the Navy precluded the taking out of an injunction against thatfirm because of infringement of patent rights (24).
EXPANSION OF REQUIREMENTS
The first contract for dried plasma, 15,000 packages from Sharp & Dohmein February 1941, was deliberately small, partly because the resources of thefirm were then quite limited, partly because this was frankly a trial contract (19).
3Unless otherwise specified, the data in the remainder of this chapter arederived from Dr. G. Canby Robinson's final report of the Red Cross Blood DonorProgram (50).
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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 numberof 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 Statesentered the war, they had risen to 200,000 units. In July 1942, 6 months afterthe United States entered the war, the estimates for fiscal year 1942-43 werefor 1,640,000 units of dried plasma, and the processing firms were beingrequested to bid on production up to 350,000 units each. These estimates wereall for the small (250-cc.) package of plasma.
In March 1945, when it was estimated that the war in Europe might end inAugust, the estimated requirements for the Army and the Navy for the remainderof 1945 (March-December) were for 1,010,000 large packages of plasma whichwould require 2,222,000 bloods. This would have made the total Army-Navyrequirements for 1945, counting what they had already received, 1,020,000 largepackages or 2,244,000 bloods.
By the end of the war, of the more than 13 million pints of blood collectedby the American Red Cross, 10,299,470 pints had been processed into driedplasma, 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, thecapacity of the blood donor centers, and the capacity of plasma processinglaboratories had to be kept in balance. The proper distribution of donationsfrom the centers to the processing laboratories involved the geographicgrouping of the centers in relation to single laboratories, the provision oftransportation facilities, and the rapid readjustment of shipping occasionallynecessary to move bloods to another laboratory because the laboratory to whichthey were usually allotted could not handle them.
Weekly reports of the distribution of blood donations were carefully studiedby the Red Cross, by personnel in the Office of The Surgeon General, and byother interested parties. If donations were in excess, blood was likely to bewasted. If they did not come up to the capacity of the processing laboratory,equipment was wasted, and, even more important, so was personnel. Thedistribution of blood donations from Red Cross collection centers for the weekending 22 July 1944 to commercial laboratories was as follows:
Lederle: |
| Lilly: |
|
| 18,500 |
| 17,000 |
|
|
|
|
New York | 9,204 | St. Louis | 3,855 |
Brooklyn | 3,639 | Indianapolis | 2,026 |
Rochester | 1,319 | Cincinnati | 2,777 |
Schenectady | 1,616 | Atlanta | 1,381 |
Buffalo | 2,175 | Louisville | 1,907 |
| 17,953 | Columbus | 2,675 |
Milwaukee | 3,068 | ||
| 17,689 | ||
Sharpe & Dohme: |
| Hyland: |
|
| 12,500 |
| 5,000 |
|
|
|
|
| 4,853 |
| 3,933 |
| 5,300 |
| 1,570 |
| 1,604 |
| 5,503 |
| 11,757 | Abbott: |
|
Reichel: |
|
| 6,750 |
| 7,500 |
|
|
|
|
| 2,468 |
| 4,400 |
| 2,198 |
| 3,550 |
| 1,814 |
| 7,950 |
| 6,480 |
Squibb: |
| Upjohn: |
|
| 5,000 |
| 5,000 |
|
|
|
|
| 2,632 |
| 4,551 |
| 1,997 | Armour: |
|
| 4,629 |
| 7,000 |
Ben Venue: |
|
|
|
| 4,000 |
| 1,898 |
|
|
| 1,890 |
| 3,800 |
| 1,450 |
Parke, Davis: |
|
| 1,681 |
| 5,000 |
| 6,919 |
|
| Army, Navy Laboratories: |
|
| 4,584 |
| 2,250 |
Cutter: |
|
|
|
| 13,500 |
| 1,587 |
|
|
| 372 |
| 4,819 |
| 1,959 |
| 4,067 | Whole Blood (for U.S. Military Hospitals): |
|
| 1,763 |
|
|
| 2,458 |
| 10 |
| 13,107 | Total Bleedings | 106,891 |
Maximum capacity of laboratories | 109,000 |
The essential factor in meeting these conditions was theassignment of correct weekly quotas to each of the bleeding centers. The quotaswere set, and changes were made in them, only by the National Director, AmericanRed Cross, after he had taken into consideration (1) the total requirements ofthe Army and the Navy; (2) the distribution of these requirements among thecontracting laboratories; and (3) the number of bloods necessary to enable eachlaboratory to fulfill its Army or Navy contract by the date specified in thecontract. In deciding upon possible increases in quotas, it was also necessaryto consider the professional staff of the center and the possibility of
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expanding it, the facilities of the center, the size of itsphysical plant, the number of mobile units which it could operate, and thefacilities of the processing plant which the center supplied.
EQUIPMENT FOR THE PLASMA PROGRAM
Commercial Production
When the plasma program was set up, the chief bottleneck wasthe shortage of equipment for commercial processing (18). Thelaboratories were unwilling to provide equipment, or to expand what they alreadypossessed, without formal contracts. The most essential item in the program,centrifuges, was the item in shortest supply, since those which would take thelarge American Red Cross horse bottles were then made by only one firm in thecountry, the International Equipment Co., Boston.
A number of suggestions were made to overcome thesedifficulties:
1. That the Army and the Navy should purchase the necessaryequipment, and rent it or lease it to the processing firms.
2. That the equipment be developed under the auspices of theNational Research Council and be popularized under its name and auspices. TheMedical Research Council of Great Britain had established this precedent.
3. That sedimentation techniques be considered as asubstitute 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 couldhandle 32 bottles every 9 hours. The yield, which would be greater than with anyother centrifuge, would shortly compensate for their additional cost.
5. That representatives of the Army and the Navy beauthorized to visit firms making equipment for the program, in an endeavor toexpedite production. This recommendation was carried out.
Confusion continued to mark the procurement of equipment forthe plasma processing program for the first year of its existence. On 16September 1942, a meeting of all concerned with the program was held in theOffice of The Surgeon General (51), and the following plan oforganization was decided on:
1. By the express desire of The Surgeon General, theresponsibility for the whole plasma program would remain entirely within theArmed Forces. Manufacturers would obtain extensions of preference ratings formaterials and supplies, except for expansion projects, through the SupplyDivision, Office of The Surgeon General.
2. All expansion projects and all requests for preferenceratings for replacement equipment would be cleared through the War ProductionBoard. The Army-Navy Munitions Board would pass on all such requests by thecustomary procedure requiring concurrence of this Board, which would keep inclose contact with the requirements of the Medical Department in acting upon theapplications. Also, in view of the fact that supplies of blood represented theessential raw material in the plasma and albumin program, the Board would keepinformed on the bleeding program.
3. The Army-Navy Munitions Board would maintain a compilationof requirements of all manufacturers of materials other than blood needed in thepreparation of plasma.
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This listing would be based on estimated production forperiods of approximately 4 months. Calculations would be on the basis ofanticipated blood deliveries and plant capacities. These data would be submittedto the Office of The Surgeon General for guidance in scheduling production anddelivery of equipment to individual processing laboratories. The performance ofeach processing firm would be evaluated through review of periodic reports to besubmitted at the request of the Legal Division, Office of The Surgeon General.
4. To avoid confusion, it was suggested (a) that the Officeof The Surgeon General instruct processing laboratories regarding the proceduresthey should follow in procurement of equipment; and (b) that, except onexpansion projects or replacement of equipment, contacts with the processinglaboratories be limited to representatives of the Armed Forces, the NationalInstitute of Health, and the Red Cross.
When these arrangements were put into effect, theprocurement and replacement of equipment in the plasma program were bothgreatly simplified, and the changeover to the larger package, which waseffected in July 1943 (p. 172), was carried out with remarkable smoothness.Problems continued to arise, of course, but very often it was found that theyhad been caused by failure of the processing laboratory to follow the directionslaid down.
The part of the plasma program concerned with supplies wasstabilized during fiscal year 1943-44. By this time, most of the materialrequired by the processing laboratories had been standardized, and the variousconcerns which supplied the equipment had scheduled their production to conformwith the needs of the program. Centrifuges, mobile refrigerating chests, andother items whose lack had seriously curtailed the plasma program for anextended period were no longer limiting factors. The War Production Board madespare parts available for centrifuges, so that repairs could be effected with aminimum of delay.
Equipment for Red Cross Blood Donor Centers
Shortages of equipment and supplies and delays in theirprocurement plagued the Red Cross blood donor centers during the entire war. The opening of new centers was frequently delayed for this reason. The lack ofsuch small items as gauze, adhesive, needles, syringes, and thermometers coulddelay the operation of a center until they were supplied. Many times, since thequantities were not excessive, manufacturers and jobbers supplied enough materialto permit continued operation, and repeated requests were made of the properagencies of the War Production Board.
On 3 March 1943, a meeting to determine the best method ofsecuring supplies and equipment for the bleeding centers was attended by Dr. G.Canby Robinson, National Director of the Blood Donor Service, American RedCross; Dr. (later Major) Earl S. Taylor, Technical Director of the Service; Col.Charles F. Shook, MC, Special Representative of The Surgeon General for theBlood-Plasma Program; and representatives of the Procurement Division, American Red Cross, andthe Priorities Division, War Production Board. Aspecial expediter was to be appointed from the War Production Board for requestsfrom the Red Cross, and all requests for priority ratings would be
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expedited. It was expected that, for the usual items,clearance would not require over 48 hours. The centers were requested toanticipate their requirements for 4 months in advance so that contracts couldbe placed accordingly.
In general, the plan decided on at this meeting worked outvery well, but there were still many delays. The procurement of paper cups isan illustration, which could be multiplied many times over, of the difficultyof securing essential items during the war. A steady supply of paper cups wasnecessary at all blood centers, to provide fluids for the donors, as there wereno facilities for washing and sterilizing nonexpendable cups. About 35,000 cupsof three sizes were needed each month. When they could not be secured throughregular channels, a special endeavor was made to expedite them, but it took 3weeks 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 numberswherever they could be bought.
OTHER PRODUCTION DIFFICULTIES
In spite of the amicable relations that existed between the processing laboratories andthose concerned with the plasma program in theArmy, the Navy, and the American Red Cross, there were numerous argumentsand misunderstandings of various kinds. This was inevitable. The drying ofplasma was a new process, not yet reduced to strict formulas, for whichequipment was being devised as the program developed.
There were mechanical difficulties of various kinds to beironed out, particularly after production was stopped for any reason. Anextended 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 afairly steady rate.
In March 1943 and again in May 1944, labor troubles led toimpending strike threats. In each instance, The Surgeon General wrote theemployees of the processing laboratories of the vital importance of theplasma program and the imperative necessity of avoiding any work stoppage.In both instances, the difficulties were settled without any loss of time, butin both, arrangements had been made to transfer the involved firm's quotasof blood to the nearest unaffected firm if the threatened strikes had actuallyoccurred.
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.
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TESTING
Army Medical School
The first extensive testing in the plasma program wasconducted by Dr. Strumia (p. 269). This was a clinical study of plasma preparedin his laboratory from blood provided by the American Red Cross. He was assistedin it by Captain Kendrick, Commander Newhouser, and members of the Subcommitteeon Blood Substitutes.
When the first supplies of plasma became availablecommercially, the practice was adopted of testing each lot chemically andclinically in the Blood Research Division, Army Medical School, before it wasreleased for use from the medical depots. Originally, one package in eachthousand was tested (table 7). In March 1943, in order to reduce the heavyworkload, the number of samples was reduced to 1 in each 5,000. Since individuallots of plasma were made up of only 15-25 bottles, it was neither practicalnor economical to test one package per lot. When it was proposed by a hospitalcommander in January 1944 that this be done, it was pointed out that his planwould mean that 5 percent of all plasma produced would be tested, whichwould 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 350examined, this number representing a production of approximately 1? millionpackages. A little later, testing was limited to one package per month selectedat 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 wasexamined in the Chemistry Division to determine moisture content, hemoglobincontent,
TABLE 7.-Results of testing of 1,008 lots of commerciallyproduced plasma in Army Medical School
Producer | Lots | |||
Tested | Retested | Accepted | Rejected | |
A | 177 | 5 | 170 | 7 |
B | 100 | 0 | 99 | 1 |
C | 83 | 0 | 83 | 0 |
D | 131 | 0 | 131 | 0 |
E | 43 | 3 | 43 | 0 |
F | 159 | 3 | 158 | 11 |
G | 88 | 1 | 84 | 4 |
H | 154 | 4 | 154 | 0 |
I | 73 | 0 | 73 | 0 |
| 1,008 | 16 | 995 | 13 |
1Partial.
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and plasma protein content. It was then tested forsolubility,with a note of the time required for complete solution. Finally, the plasma wasadministered to patients who were carefully observed for any adverse reactions,particularly chills and fever.
Processing Laboratories
Each laboratory was required to maintain complete records oneach lot of plasma processed, the reports showing serologic tests; cultures;toxicity tests and bulk sterility tests on each pool; time required forprocessing; time required for drying and the temperature levels during theprocess; and final sterility and residual moisture determination on the driedproduct. Monthly reports were also required.
In view of the completeness of these records and theiravailability for inspection, it was decided, in the spring of 1944, todiscontinue testing at the Army Medical School. The change was consideredentirely safe since by this time all the laboratories under contract had hadextensive experience and were turning out satisfactory products.
Analysis of Questionnaires
Materials and methods-An analysis of 1,407 of thequestionnaires included in the packages of dried plasma and returned aftermaterial had been used was carried out at the Naval Medical Research Institutein December 1943 (52, 53). The 1,407 infusions of dried plasma werecompared with a previous study of 1,751 infusions of liquid plasma.
Of the 1,407 infusions analyzed, 300 had been given at theNational Naval Medical Center and 119 were given between midnight and 8 a.m.,the time of administration suggesting that they were given for emergencyreasons. The 1,407 infusions had been given to 734 patients, 457 of whom hadreceived 1 injection each, 2 of whom had received 22 injections each, and 34 ofwhom had received 9 injections each. Eighty percent of the material injectedwas produced by three laboratories; there was no essential difference inclinical results or incidence of reactions.
The distribution of clinical indications and therapeuticresults 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 22of the 25 fatal cases in the dried plasma series, the indications for theinfusion included shock, with or without hemorrhage, and burns. Althoughhypoproteinemia and jaundice accounted for more than 40 percent of theindications in the total series, only 2 of the 25 deaths were associated withthese conditions. In the remaining death, the indications for plasmaadministration were not stated.
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The average amount of dried plasma given to each patient was 526 cc. and ofliquid plasma, 711 cc. The fact that the reported average for all patientsexceeded 500 cc. clearly justified the recent adoption of 500-cc. packages.
Results-The response to plasma was considered favorable in 96 percent ofthe patients in each series. There were no hemolytic reactions in eitherseries, and the reaction rate in both was essentially the same, about 5percent. Patients in shock had fewer reactions than those not in shock. Only onepatient had more than 2 reactions; this man had 4 reactions in 18 infusions, 2of which occurred on the same day, after consecutive infusions. Of the eightother patients with multiple reactions, five sustained both on the same dayafter consecutive infusions.
Of the 74 reactions in the dried plasma series, 25 were urticarial, a largerproportion 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 theseries 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 beforethe donation. It was conceivable that at centers at which blood was procured fordried plasma this regulation was not strictly enforced.
The reaction rate for liquid plasma stored for less than 4 months was 5.1percent, and for plasma stored beyond this limit, 3.0 percent. These ratesfurther suggested that some mildly toxic labile constituent of fresh plasmamight be stored in the dry state.
Studies on blood pressure and pulse rates-As a part of this generalstudy, the effect of plasma administration on blood pressure was investigated in392 infusions given to 378 patients, and on the pulse rate in 513 infusionsgiven to 442 patients. The data were as follows:
1. The average increase in systolic blood pressure produced by theadministration of plasma, over a period not exceeding 24 hours, to patientswith initial systolic pressures under 100 mm. Hg was 28 mm. Hg in those in shockand 14 mm. Hg in those with hypoproteinemia.
2. The average decrease in pulse rate in patients with initial rates over 100per minute was 13 per minute in those in shock and 6 per minute in those withhypoproteinemia.
3. In general, the increase in systolic blood pressure was much greater thanthe decrease in pulse rate. In patients in shock, the coefficient of correlationbetween the two responses was extremely low and not statistically significant.
4. There were no statistically significant differences between the results ofdried 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 July1942. Reports were sent to his office, to the Army Medical Purchasing Officer,and to others concerned in the program (54-58).
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The original purpose of these inspections was to makecertain that the best possible production techniques were being employed, andthat all possible precautions were being taken to provide a safe and effectiveproduct. Variations from standard techniques were detected and corrected. Anyimprovement in procedure observed at one laboratory was promptly made knownto other processing firms.
These frequent contacts made it possible to reducecontamination rates and losses from other causes. One of their most usefulfunctions was the correction of the rumors, false impressions, andmisunderstandings that invariably rose in a project of such magnitude. Therelations between the Division of Surgical Physiology, Army Medical School,and the commercial laboratories had been excellent before large-scaleproduction of plasma began, and this liaison, which also existed with the Navy,was maintained by these visits, which were usually made by Colonel Kendrick andCaptain Newhouser.
YIELDS OF PLASMA FROM BLOOD
One of the subjects discussed at the Blood Plasma Conferencein Chicago on 24 March 1943 was the volume of plasma recovered from eachbleeding 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 bleedingbottle varied, though the intent was that it should be 50 cc., less whateveramount was lost by evaporation during, and subsequent to, sterilization.Measurements made in 12 bleeding centers showed the maximum range from 47 to 70cc., the minimum from 16 to 50 cc., and the average from 44 to 58 cc. It wasurgent that each laboratory overhaul its technique so that the employee chargedwith introducing the anticoagulant could not vary it from bottle to bottle. Dr.Taylor, who had directed this investigation, considered it probable thatsterilization techniques should also be checked.
2. The volume of blood collected varied. To study thisdiscrepancy, 2,475 bleeding bottles were selected at random from the Churchcontainers as they were received at one laboratory. The volume of blood wasdetermined by measurements on a marked bottle, in 50-cc. graduations from 550 to250 cc. Percentage variations ranged from 50.0 and 29.5 to 0.9 and 0.2. Thesevariations had been called to the attention of all bleeding centers.
3. Wide differences were found in revolutions per minute andthe time factor with various models of centrifuges. When a No. 13 model wasused, the revolutions per minute varied from 1,300 to 2,300 and the timeinterval of four motor speeds from 20 to 60 minutes. When a No. 3 model wasused, 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 theshortest interval, was optimum, it had to be assumed that centrifugation at somelaboratories was not adequate. One laboratory reported a yield of plasma of285.5 cc. when a No. 13 centrifuge was used at 1,800 r.p.m. for 50 minutes andof 297 cc. when the time was extended to 65 minutes. It was planned to supplyeach processing laboratory with a table showing the relation of revolutions perminute to the time factor necessary to secure a full plasma yield.
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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 aftercentrifugation. The closed technique, investigation showed, had been wellstandardized in all processing laboratories. Any variation, therefore, wasevidently the fault of the individual who performed the operation. Variationswere least when supervision was most careful.
6. The temperature of the blood at the time the plasma was drawn offapparently influenced the yield. Commander Newhouser reported that the UpjohnCo. had found that, if the blood was chilled after centrifugation, it waspossible to draw off all but 4 cc. of plasma without distributing the red bloodcell layer. The high hemoglobin content of the last bit of plasma drawn off didnot interfere with the manufacture of albumin, which this firm was making.
One laboratory made a check of 25 bloods received from five centers, spinningthem for 50 minutes at 1,800 r.p.m. After carefully removing all the plasmapossible without drawing off red blood cells, the maximum blood-tingedresidual plasma recovered varied from 1 to 20 cc. and the average from 4.0 to8.5 cc. for the individual centers. Based on the total bleedings in all centersfor January 1943, the residual yield of plasma would have ranged from 747,000 to1,589,000 cc., very considerable amounts. If the residual could not be used indried plasma, it was thought that methods could be devised for salvaging itfor use for serum albumin.
The analysis clearly indicated that if optimum conditions were everapproached, the yield of plasma could be materially increased, perhaps by asmuch 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 thengoing to waste because of breakage and because of the amount left in the layerover the packed red cells. An average of 8.5 cc. could be recovered in thislayer in every bottle, and the total amount would be about a million cubiccentimeters every month. This residual could not be used for plasma because ofits red cell content, but it should be processed in some stable form, so that itcould be held without loss. These losses were never fully corrected.
ACCOUNTING PRACTICES
Original Plans
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 lossesof it from all causes. Legally, this was required by Army regulations. Becauseof the usual source of the raw product, such an accounting was also morallyobligatory, and the responsibility for it was keenly felt by all connected inany way with the program.
At an early meeting of the Subcommittee on Blood Substitutes, it was agreedthat if contamination, breakage, insufficient samples for serologic
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testing, and any other losses and errors were found at the processinglaboratories, the information should be telegraphed immediately to the bleedingcenter responsible and a copy of the wire sent to Dr. Robinson. Gross errorswould therefore be investigated and corrected as soon as they occurred.
Accounting practices were fully discussed at a meeting on revision of plasmacontracts held at the Purchasing and Contracting Office of the New York MedicalDepot on 28 July 1942 and attended by representatives of that office and of twoprocessing laboratories (61). The following agreements were reached: Allcontracts would contain a provision for complete accounting to the Army of allblood received from the Red Cross and of all its byproducts. The report wouldstart with an inventory of the blood, frozen plasma, and dried plasma on handwhen the new contract commenced; would add to the inventory the blood receivedduring the month; would deduct from it losses due to contamination, ordinarybreakage, and other causes; would make the proper conversion from amounts ofblood to comparable quantities of plasma; and would convert frozen plasma toappropriate quantities of dried plasma, with due regard to shrinkage due tofurther breakage, possible contamination, and other causes. Credit would betaken for deliveries of dried plasma during the month, and an inventory ofblood, frozen plasma, and dried plasma on hand at the end of each month wouldbe shown.
The Army also wished each laboratory to prepare a certified statementaccounting for all the blood received from the beginning of the project up tothe first report by the new accounting system.
The laboratories were quite willing to prepare the desired accounting. It wasthought, however, that the standard Red Cross form was not adequate for thispurpose, while the very elaborate statistical report which the Army-NavyMunitions Board had recently requested was rather cumbersome. A single form waslater developed which served the Army-Navy Munitions Board requirements and acopy of which was sent to the Red Cross for information.
The laboratories also requested at this meeting that the Red Cross beinstructed each month by the Army and the Navy as to the quantity of totalblood to be used for plasma and for albumin. If shortages in either programshould develop, the laboratories would be in an awkward position if they hadmade 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 SurgeonGeneral, 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 thepart of any firms concerned with the program (62). The reports inquestion were designed to afford protection to the laboratories as well as tothe Government and the personnel concerned with letting the contracts. Ifthere were errors in them,
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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 thatcould 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 upagain, from time to time, to effect so-called recalcification. Some companiesreported quantities converted to albumin as if they had been lost in freezing orfor other reasons. Some reported high hemoglobin losses as miscellaneous. Onecompany reported large miscellaneous losses as the difference between actual andestimated plasma volume.
3. The present forms did not definitely segregate losses subject to thepenalty clause from those not subject to it, though whether this was importantwould depend upon whether a penalty was to be imposed for negligence, deviationfrom expected or average performance, or other causes.
4. Some errors originated on the companies' reports. Some were errors intranscription and columnization from the companies' reports to the stockmovement record. Other discrepancies might exist which could be disclosed onlyby 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 contaminatedmaterial, which should be recorded uniformly by all companies.
2. The mathematical accuracy of each monthly report should be established onits receipt.
3. Amounts listed as miscellaneous should be analyzed to show causes of lossbefore the figures were recorded on the stock account.
4. A formal monthly columnarized stock record should be maintained in binderform and balanced and reconciled each month, as was apparently required byparagraph 10 of Army Regulations No. 38-6520. The record should account forthe disposition of the blood from the time it was delivered by the American RedCross until it was received and accepted by an Army depot or other Armyinstallation. The record should be supported by shipping tickets for quantitiesdelivered to the processing companies, receiving reports for quantitiesdelivered to Army depots, monthly reports from each company to show the movement of the blood, and forms to cover quantities charged out. The reportshould also summarize losses by causes and according to whether or notliability was attached to them.
Justification of Original Reports
As Captain Taylor pointed out to Capt. Frederic N. Schwartz, MAC, in amemorandum commenting on this letter from the Renegotiation Division (63), anumber of points had to be taken into consideration in analyzing currentaccounting practices:
1. The processing firms had never before handled so large a volume ofhumanbiologic material. In the initial stages of the operation, many innovations andimprovisations were
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necessary, and the resulting delays and losses were reflected in conflictingand inaccurate inventory reports.
Some of the early records were quite complex. One firm, for instance, maderestitution, of its own accord, for material lost in processing and adjusted itssubsequent reports in conformity with the replacement. Another laboratory alsomade restitution for blood lost through a truck accident on its property.
2. The program was underestimated in its potential size by all concerned withit. Rapid expansion took place before facilities were available, and makeshiftarrangements were often necessary to handle the enormous increases in blooddelivered to the laboratories.
3. As a result of these various factors, initial losses were excessive in thelight of present operations, and errors in bookkeeping occurred that could notbe adjusted until there was time for a less hurried appraisal of stocks on handand 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 acomplete loss, but others kept it on their shelves, hoping that some rise mightbe developed for it in the future. Some plasma was discarded on the ground thatit had lost its original properties. A good deal was used experimentally, inattempts to devise methods of salvaging it, such as recalcification andchloroform extraction. Between October 1942 and February 1943, many largebatches of contaminated plasma were used in trial runs at plants preparing toprocess albumin, so that good material would not be wasted getting the initialdifficulties of production ironed out. It was not until July 1943 that theHarvard pilot plant considered it safe to use contaminated plasma in the serumprogram. Any diversion of such plasma to this use before that date was entirelyexperimental, and the material used was of no value according to the criteriain the National Institute of Health regulations and those implied in theplasma contracts.
For these various reasons, the forms then in use provided no method ofaccounting for plasma that, for contamination and other reasons, was charged offon the report but was physically retained by the laboratory for possibleconversion to other uses. The conversion of contaminated plasma was adevelopment of the past few months. It was not provided for on the report formspreviously in use because this development did not then exist.
National Institute of Health rulings permitted the processing of liquidplasma into dry plasma if contamination was limited to 200 organisms per cubiccentimeter. The rationale was twofold, the dilution accomplished by pooling thebloods, and the freezing and drying processes, which killed organisms in thisnumber.
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 tothe cubic centimeter at various stages of production. Originally filling andsample losses were simply estimated (so-called stick measurements). At Dr.Veldee's request, in October 1942, uniform measurements were made in alllaboratories, though it was still impractical to account accurately for every10 cc., or even every 100 cc.
6. The blood donor centers had the same difficulties as the laboratories. Atthe beginning of the program, donations probably averaged 20-30 cc. less thanpresent donations. Even at the time of this report, however, the amount ofpotentially available plasma was no better than an estimated average determinedby comparing the amount in individual bottles as they arrived with the averageyield of plasma per bleeding as determined by the laboratory. Each bleedingideally yielded 300+ cc. of plasma, the equivalent of one finished standardpackage. This ideal 1:1 ratio was based on the assumption that each bottle ofblood was completely filled (which, for the reasons already stated, it was not).It also made no allowance for breakage, positive serology, filling losses, orcontamination.
Standards of performance and checks of efficiency of operation of theprocessing laboratories by the American Red Cross took into consideration therealistic factors just listed. In planning for deliveries of blood to theprocessing
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laboratories to meet Army contracts, the Red Cross, the National Institute ofHealth, and the Office of The Surgeon General decided that a ratio of 1:1.2finished 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 accuracyof the laboratory records, was the breakdown sheet for mechanical andcontamination losses prepared each month by Dr. Veldee. Over the last 2 months,these losses, exclusive of serology losses of 0.30 percent, amounted to 2.47percent. 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 ofbleedings to finished packages, this was 1.03 percent, which compared favorablywith 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 perhundred bloods, not accounted for statistically. Sterility samples and similaramounts required by National Institute of Health regulations amounted to about160 cc. per hundred bloods. A fair estimate of the variation of the ideal 550cc. of citrated blood per donation would account for 2,000 cc. per each hundredbloods or 1,000 cc. of plasma. This left 340 cc. of plasma, or something overone bleeding per hundred, to cover filling and other incidental productionlosses that could not be measured.
It was Captain Taylor's opinion that those examining the reports submittedto date did not completely understand the background of the plasma productionprogram and the early difficulties it encountered. Also they did not possess thenecessary medical knowledge to assess phases of operation and production whichdid not lend themselves to mathematical calculations. For these reasons, CaptainTaylor questioned the justification for the somewhat sweeping condemnation ofthe present system of recording. By far the largest numbers of errors wereclerical, and simple auditing of the monthly reports would easily correct thatsituation.4
Changes in Reporting Practices
At a meeting of representatives of the various components of the plasmaprogram on 1 October 1943, reporting practices were discussed in detail (64). Itwas not believed that the correction of the errors complained of by theRenegotiation Division would present great difficulties or require basic changesin 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 fromaccountability represented no problem at all; it would never
4Captain Taylor also pointed out in this memorandum that the penaltyclause in the contracts, particularly its invocation, should be clarified. Thelegal mechanism for imposition of penalties was not within the competence ofinterested medical officers. On the other hand, if negligence or deviation fromexpected or average performance were to be the basis for its invocation, theopinion of those who had intimate knowledge of plasma processing-that is,representatives of the National Institute of Health, the Blood ResearchDivision, Army Medical School, and other agencies-should be sought beforeaction was taken.
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be dropped on one report and picked up on another. They also emphasized thedifficulties that would face laboratories if they tried to take preciseinventories of material which they had retained on their own initiative, in thehope that it might eventually be useful, and which was frequently stored incontainers of various shapes and sizes. They thought that an approximate reportin liters should be permitted. Also, to take cognizance of the storage problemfaced by some processing laboratories, these officers proposed, with Dr.Veldee's concurrence, that contaminated material on hand before 1 August1943 should be destroyed.
Auditing arrangements for the correction of mathematical errors weresuggested, and it was also proposed that the same auditor who performed thistask, and who thus became familiar with the form and content of the reports andthe problems involved, should make a monthly audit of all reports in thefuture.
On 6 November 1943, in accordance with suggestions made at the 1October 1943 meeting, a preliminary letter was sent to the processinglaboratories from the Army Medical Purchasing Office in New York incorporatingthe following information:
1. Recent developments indicated the possibility that blood or plasmahitherto regarded as unsuitable for use because of contamination or for otherreasons could be salvaged for certain purposes.
2. At the request of the Army, several laboratories had been retaining thismaterial and had sometimes overloaded their storage facilities with it.Permission to discard all such material on hand before 1 August 1943 wastherefore 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 sentto all processing laboratories from the Army Medical Purchasing Office on 17 May1944 (65).
LOSSES
Total Losses
In the Journal of the American Medical Association for 12 September1942, Dr. Taylor summarized the losses which had occurred in 320,442 bloodscollected up to 1 May 1942 as follows (66):
Breakage, 0.345 percent, including 126 bottles broken in transit and 842cracked during centrifugation.
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Hemolysis, 0.569 percent. All but 406 of the 1,591 units lostin this category had to be discarded because the blood froze when inadequateshipping arrangements allowed it to be exposed for considerable periods tosubzero weather. Some early losses occurred because of failure to remove the DryIce used for precooling shipping containers.
Contamination, 2.26 percent. The loss of 6,260 bloods fromthis cause was considered small in view of the 16 bleeding centers and 4processing laboratories which had entered the project without any previousexperience in this field.
Miscellaneous, 0.284 percent. In all, 794 units in thiscategory had to be discarded. Railroad breakdowns resulting from weatherconditions caused some bloods to be held beyond the time permitted by NationalInstitute of Health specifications. One pool of 36 bloods had to be discardedbecause it contained blood from a donor who developed typhus fever.
Most blood that was discarded was made into typing sera orwas used in pilot bottles to test moisture content.
The final report of the American Red Cross (50) showsa loss of 204,848 bloods (1.6 percent) of a total of 12,589,034 delivered to theprocessing laboratories (table 8). It will be noted that, as in the earlierreport, bacterial contamination was the major cause of losses but the percentagehad been materially reduced, as had that of all the other causes listed in thefirst report in 1942.
Losses From Contamination
The risk of contamination was first discussed at the meetingof the Subcommittee on Blood Substitutes on 23 May 1941 (25), when it waslearned that a pool of 40 bloods being processed in a commercial laboratory hadbeen found to be contaminated. It was recommended, to prevent wastage from thiscause, that pools should consist of not more than 12 bloods. Later, when it wasfound possible to process contaminated plasma into albumin, the limits weresuccessively lifted to 25 bloods, and then to 50 and more (67).
Two special experiences with losses of liquid plasma fromcontamination are sufficiently instructive to be reported in some detail.
TABLE 8.-Percentage distribution of causes of 204,848 losses in 12,589,034 bloods delivered to plasma processing laboratories in World War II
Causes of losses | Number of bottles | Percentage of total collections |
Bacterial contamination | 125,748 | 0.99 |
Positive serology | 28,364 | .22 |
Breakage in centrifuge | 17,048 | .13 |
Breakage in transit | 1,660 | .01 |
Hemolysis | 7,567 | .06 |
Clotting | 3,727 | .02 |
Miscellaneous | 20,734 | .16 |
| 204,848 | 1.6 |
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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 madeclear the prime cause of contamination, that the bottles were being entered atthe free hole (p. 386), where the stoppers were only 1? to 1 mm. thick, insteadof at the X-mark, where the diaphragm was 7 mm. thick and sealed itselfwhen it had been penetrated, as the thinner diaphragm did not. The result ofthis technique was that the closed system, essential to the preservation ofsterility, ceased to exist.
It was directed that this practice be stopped immediately. Itwas also directed that individual syringes and needles be used to introduceMerthiolate into the pools; that the practice of covering the plunger of thesyringe with glycerin be discontinued; and that as few technicians as possiblebe assigned to aspirate and dispense the plasma, so that responsibility could bespecified if contamination continued to occur.
The contamination rate at this center promptly fell to anacceptable level (less than 1 percent) and continued at this level until themiddle of December 1943. Then, it again rose sharply. When the liquid plasmacenter and the blood donor center which supplied it were visited on 16-17January 1944, the cause was immediately evident, that the combined bleedings ofthe 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 shownthat the contamination rate always increased in direct proportion to the amountof blood processed beyond the capacities of the laboratories to handle it. Thecurrent policies had been instituted by the director of the bleeding center, acivilian, against the wishes of the physician in charge of the center and thechief of the laboratory service at the Army liquid plasma center. Unlike mostdonor centers, this center was operating as an Army, rather than as a RedCross, donor center.
Arrangements were made to operate the center in the future asall other bleeding centers were operated, with the technical representative ofthe Red Cross responsible for personnel, blood quotas, technical procedures, andother policies. Daily bleedings were to be reduced to 300 per day. With thesechanges enforced, the contamination rate again dropped to an acceptable level.
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Second experience.-When the liquid plasma center wasinspected on 12 August 1943, another problem in contamination, chiefly from Staphylococcusaureus, was encountered. Several explanations for the 3-percent rate werepromptly found.
1. Responsibility could not be individualized because of thenumber of technicians aspirating, dispensing, and culturing the plasma.
It was recommended that the actual preparation of the plasmabe limited to two technicians, and that no new technicians be trained in thesespecial procedures until the contamination rate had fallen to an acceptableminimum.
2. Similarly, it was impossible to place the responsibilityfor autoclaving bleeding and aspirating sets. Untrained technicians were beingpermitted to operate the autoclave, and undesirable techniques were employed.
It was recommended that a trained technician be maderesponsible for the sterilization of all sets; that the autoclave be operated at15 pounds' pressure for 45 minutes; and that a vacuum be pulled for at least20 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 hadto be kept partly open.
Nothing could be done at the time to improve these physicalconditions except to urge all possible care to counteract them.
4. Inspection of presumably sterile sets showed that themuslin 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 thevalves, the Penrose drains, and other parts of the set were all bathed in it.
It was recommended that the use of glycerin on aspiratingneedles be discontinued at once.
5. The Penrose drains used in the sets were so short thatthey did not completely cover the aspirating needles. Also, the glass funneltips supposed to protect the tips of the needles during aspiration werefrequently broken, so that the rubber on the end of the drain came into contactwith the needle each time a plasma bottle was penetrated.
It was recommended that all aspirating sets be completelyreworked; that new muslin wrappers be used; that the Penrose drains used be longenough to cover the needles; and that new, intact glass tips be supplied.
The prompt fall in the contamination rate at this liquidplasma center proved once again the extreme importance of strict attention toall details of the procedure if a safe and effective product were to be secured.
Processing laboratories-The rate of contamination atprocessing laboratories was generally very low. When it rose above acceptedlevels, the explanation was usually evident. In October 1944, for instance, thehigh rate at one firm was explained by a break in technique; namely, permittingrubber tubing to lie around for 3 or 4 days before it was sterilized. Directionswere given that this period must be reduced to 3 hours. It was arranged thatbefore further release of material from this laboratory, at least 100 packagesmust be tested, half at the Army and half at the Navy Medical Schools.
At another processing firm, a high rate of contaminationwas explained by crowded conditions compounded by construction work immediatelyadjacent to the laboratory. The physical setup continued to be undesirable butthe rate fell when special precautions were instituted, and it reached anacceptably low level when the construction work was finished.
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Losses From Clotting
Losses from clotting were small in the total plasma program, but thepossibility was the occasion for a number of heated discussions and severalspecial investigations in 1942. The question first arose (45) in a lettercirculated by Dr. Strumia to the Subcommittee on Blood Substitutes on 8 April1942, in which he incorporated material from a letter he had written on thesubject to the Journal of the American Medical Association. In thesecommunications, he stated:
1. That the technique employed by the American Red Cross in collecting bloodfor the Armed Forces was unsafe.
2. That clotting occurred as the result of this technique in from 12 to 30percent of all bleedings and that the clots varied in weight from a few grams to150 gm.
3. That if the larger (850-cc.) bottles used by Sharp & Dohme wereemployed, instead of the 750-cc. bottles then used by the Red Cross, and if thebottles were agitated during the collection, the rate of clotting would be 12percent.
In his reply, Dr. Taylor pointed out that agitation of the bottle duringbleeding 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 theextent stated?
2. If clotting did occur, could it be overcome by any special technique, suchas 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 plantsthen engaged in the production of dried plasma to settle these questions:
1. Statistical analysis of approximately 200,000 bloods processed up to 1April 1942 showed that only 97 had been discarded because of gross clotting. Theexplanation was usually small, unnoticed cracks in the bottom of the bottles,which had permitted the citrate to leak out slowly and resulted in thecollection of blood without any citrate solution or with an ineffective amount.
2. A total of 9,164 bloods were strained through coarse- and fine-meshscreens and the solid material thus secured was carefully examined, in manyinstances by Dr. Taylor, Major Kendrick, or Commander Newhouser. Results variedfrom firm to firm but were difficult to state comparatively because of thedifferent techniques of reporting. Except in a single laboratory, the rate ofclotting was always low, and, with very few exceptions, all of the clots weresmall. The explanation of the high rate of clotting at the single laboratoryjust mentioned was that the investigation coincided with the employment of fivepart-time women physicians, who had had no previous experience in the field, atthe blood center supplying much of the blood.
3. To compare the collection of blood with and without agitation, 100 bloodswere carefully agitated during collection at the Red Cross Donor Center in NewYork. No clots were found. On the same day, with the same personnel, another 100bloods were collected without agitation. One clot, 1? inches in diameter, wasfound.
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
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hundred bloods, collected from the same source and in the same manner the following day, revealed six clots, ranging from 1? to 2? inches in length and three-fourths of an inch in diameter.
It was concluded from these various investigations that the degree ofclotting at the donor centers across the country was of no real significance;that a certain amount would occur, no matter what system, within reason, wasemployed to collect the blood; and that considerably more conclusive evidencemust be produced before the Red Cross technique of collecting blood forconversion to plasma could be considered unsafe. No such proof was everforthcoming.
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 theGeorge Washington University School of Business Administration on 11 January1944, entitled "Potential Post-War Market for Dried Blood Plasma" (68).At that time, there was no indication of the risks of serum hepatitisintroduced by its use, a risk which was to complicate the disposition of surplusstocks 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 atthe end of the war was Colonel Kendrick's recommendation after his visit tothe Mediterranean theater in October 1944. At that time, the ratio of blood toplasma in forward areas was about 1:1, and it was thought that it might approach2:1. Since whole blood was then available in adequate quantities, ColonelKendrick thought that consideration should be given to reducing plasma contractsby two plans:
1. All smaller Red Cross bleeding centers should be closed and the largercenters (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 theUnited States at once, preferably on hospital ships, to avoid the postwardifficulties of returning surplus material. The plasma thus returned could beused for years to come (again, it must be emphasized that the danger ofinfectious hepatitis from the use of pooled plasma had not yet been realized).When this recommendation was made, recent correspondence had shown that theEuropean theater had an excess of plasma on hand and would need no more for atleast 6 months. Inquiries to other theaters also revealed large stocks.
As a matter of fact, by this time, tentative plans had already been made tocut back production of plasma so that contracts could be terminated promptlywhen the war ended. All producers were asked to limit their purchases ofequipment, and contracts were made for the minimum practical level.
Long before the war ended, requests began to be received from variouscivilian agencies and institutions for unused stores of plasma and itsbyproducts,
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for use therapeutically, prophylactically, and experimentally. The firstofficial 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 RedCross (69), inquired of The Surgeon General whether there would be asurplus of plasma produced from blood obtained by the American Red Cross overand above the amount the Army could use before it became outdated. His interestin the matter arose from the responsibility of the Red Cross to the people whohad contributed the blood and who had the right to insist that it be utilizedto 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 forappropriate civilian use, also without charge.
On 1 October 1945, Maj. Gen. Norman T. Kirk (70) replied that, whilestocks from the Pacific had not yet been reported, there were already availableoverseas, in excess of Medical Department demands for the next 14 months,387,385 large packages of plasma and 258,560 small packages. Available in theZone of Interior were 515,749 large packages and 346,670 small packages. All ofthe small packages and all but 75,000 of the large packages could betransferred to the American Red Cross.
On 5 November 1945, General Kirk (71) notified Mr. Smith that theexcess stocks of plasma and other byproducts described in his 1 October letterwere now available for transfer to the Red Cross. The Army would be glad tostore the material in its depots until the Red Cross could assume ownership. Asto material overseas, the Army, on request, would bring designated amounts tothe Zone of Interior or deliver them to Red Cross depots in the varioustheaters. The actual ownership of the material was to remain with theGovernment until the supplies were delivered to the Red Cross at the designateddepots, to which they would be shipped at Government expense.
In this letter, which was to constitute the terms of the final transfer ofexcess plasma and byproducts by the Army to the Red Cross, it was emphasizedthat these materials would be used on a nonprofit basis for public and otherappropriate use, "consistent with the terms and spirit of thedonations," with the distribution left "to the wise discretion andjudgment of the Red Cross." It was also noted:
1. That the Red Cross would be given information concerning the reactionsobserved in certain groups of products.
2. That the dating on each individual package made it quite clear as to theperiod in which the plasma could safely be used.
3. That it was assumed that all safeguards would be employed in thedistribution 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 alloversea supplies be transferred to the American Red Cross in the Zone ofInterior on shipping instructions to follow later.
The remainder of the story of the disposal of surplus plasma is best told inconnection with the story of hepatitis (p. 674).
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OFFERS AND PROPOSALS
As soon as it became public knowledge that plasma was being processed for usein the Armed Forces, proposals to manufacture it were received from individualphysicians and scientists, university and other research laboratories, andcommercial laboratories not already engaged in the program.
It was the policy, so far as practical, to inspect the facilities in which itwas proposed that the plasma should be processed. In no instance did they proveadequate for large-scale production. The majority of smaller commercial firms,once they learned how delicate and complicated a process large-scale productionof plasma (and albumin) was, wanted nothing to do with it.
The reply to proposals to alter methods of production was also usually thesame: No matter how excellent the proposed change might be, changes in anestablished process were simply not practical in wartime because of thedifficulty of procuring materials and also because a change in any step of theprocess meant changes all along the line, which meant delays that could not bepermitted in what amounted to a crash operation. Most clinical proposals werecompletely impractical in the circumstances in which plasma was used.
RED BLOOD CELL RESIDUA
Historical Note
When Oswald H. Robertson (73), in 1917, performed the firsttransfusion with banked blood (p. 5), he was really using a suspension of redblood cells and not whole blood. Two years earlier, Rous and Turner (74) hadreported the successful experimental use of the same method. It is surprising,in view of the good experimental and clinical results, that, except for work byCastellanos and his group (75, 76), this method was not used again untilWorld War II. In 1940, McQuaide and Mollison (77) reported its use in 61 casesof anemia, with 8-percent dextrose in isotonic salt solution as the suspensionfluid. The reaction rate was 6.5 percent.
The first use of red blood cells in the United States was during the Bloodfor Britain project (p. 13). When the plasma, which was sent to England, wasseparated, 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 transfusionin 227 cases. As a rule, 500 cc. of the cell residual was used in 500 cc. ofphysiologic salt solution. The transfusions were type-specific, and the reactionrate 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 intogeneral military use immediately because of the primary necessity forconcentrating all efforts on plasma production.
As the red blood cell program finally developed, it was an outgrowth of thepreparation of peptone by Parke, Davis and Co., from the blood sludge previouslydiscarded.
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Organization of Red Cross Program
The distribution of red blood cells to hospitals began on 1January 1943 at the Red Cross Blood Donor Center in Detroit (79), but it wasnot until November of that year that the formal program was set up, with thefollowing arrangements (80):
1. The service was conducted by the technical staff of theAmerican Red Cross Blood Donor Service, under the supervision of the Division ofMedical Sciences, NRC, through the Subcommittee on Blood Substitutes. Locally,the service was operated through the donor centers and under the control of thetechnical supervisors of the centers.
2. The cellular residue was released by the Army and theNavy, which had title to it, to the Red Cross Blood Donor Service, fordistribution for therapeutic purposes.
3. Since the cells had to be used within 5 days after theblood was collected, the method was available only to the eight or ninehospitals immediately adjacent to processing laboratories. This restriction wasbased on the practical consideration that the blood had to be transported to aprocessing laboratory from the bleeding center; transported back to the centerafter red cells and plasma had been separated; and, finally, transported to thehospital at which the cells were to be used.
4. The service was designed primarily for military hospitalsbut it was extended, as was practical, to hospitals organized and equipped forsuch a service. At the meeting of the Subcommittee on Blood Substitutes on 16March 1945 (81), when Dr. Robinson requested permission to extend theservice beyond the teaching hospitals, to which it had been chiefly limited upto this time, it was recommended that the selection of additional hospitals beleft to the discretion of the Red Cross Blood Donor Service.
5. All procedures in each hospital from the time the bloodwas obtained until it was dispensed were under the control of a singleresponsible physician. Both physicians and hospitals had to agree in writing tocarry out the prescribed methods and techniques for the use of suspended redblood cells and had to assume final responsibility for their administration.
6. The service was to be conducted without cost to thoseserved and without financial profit to any person or institution connected withit. All expenses were borne by the National Red Cross Blood Donor Service, bythe mechanisms already in operation.
Technique of Collection, Distribution, and Administration
The following technique was specified for the use of redblood cells, the procedure up through the withdrawal of the plasma being thestandard procedure for plasma processing:
1. After centrifugation of the blood and withdrawal of theplasma, a sterile solid rubber stopper is placed in the bottle containing thecells. Only type O cells are used for this purpose. The original white tag isleft on the bottle.
2. The cells are resuspended in a dustproof room, with afilling burette, in a pyrogen-free physiologic salt solution (or other solutionapproved 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.
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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 cellactivities must be stopped until an adequate explanation is obtained byinvestigation of all possible causative factors. Sterility tests must bereinstituted by the required techniques before cell suspensions are againreleased for distribution.
7. After distribution, the cell suspensions must be stored atthe temperatures specified. If there is any possibility that the temperature hasfallen below the freezing point, the suspensions must be discarded.
8. The bottles are observed at intervals for hemolysis or forcolor changes in the supernatant fluid. If a violaceous or blackish-redcoloration is apparent, or there is any question as to the condition of thesuspension, or any unusual odor is detected, the cells must be discarded.
9. The cells must be used within 5 days of the date ofbleeding.
10. The suspension must not be dispensed from the originalcontainer but must not be removed from it until just before it is to be used. Asit is emptied into the dispensing flask, it is carefully observed, so that grossclotting, unusual odors, or other changes will be detected. Retyping andcrossmatching are done immediately before the cells are used. The suspensionmust be given within 5 hours of the time the bottle is opened.
11. The suspension is filtered through four layers of a 44 by40 bandage roll or through the 100-mesh stainless steel filter in the bloodtransfusion set. It is not warmed. If the entire contents of a bottle are notused, the unused portion is discarded.
12. Bottles in which cells were delivered must be returned tothe blood donor center whence they were dispensed, each bottle accompanied by aproperly executed report of the transfusion.
These reports became the property of the Red Cross BloodDonor Service, and its approval before publication of any data or othermaterial concerning the experience was one of the conditions under which cellsuspensions were furnished to hospitals and physicians.
The handling of the red blood cells by three separate groupsof persons offered chances for breaks in technique because of the dividedresponsibility, as well as for errors in transcription and for other reasons. Itwas therefore imperative that the regulations laid down be followed without anydeviations.
Hospitals were cautioned not to use red blood cellsuspensions for pregnant women or women in the postpartum period without aninvestigation of the Rh factor. In cases of doubt, only Rh-negative cells wereused. The same precautions were observed when repeated transfusions were givenwith red cell suspensions.
The Detroit Experience
The Detroit Red Cross Blood Donor Center had the firstexperience with red blood cell transfusions as well as the most extensive;before the war ended, Dr. Warren B. Cooksey, the technical supervisor, hadsupervised almost 18,000 transfusions with resuspended red cells in 14 localhospitals.
The first report by Dr. Cooksey and Lt. William H. Horwitz,MC, published in the Journal of the American Medical Association on 1April 1944,
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covered 4,050 of the 7,864 cell suspensions delivered to theDetroit hospitals to date(82).
Materials and methods-As a rule, 500 cc. ofsaline-suspended red cells was given. When large amounts of blood were required, two orthree transfusions a day were given, though on a few occasions two to threebottles of diluted and undiluted cells were given as a single transfusion.
The cells were prepared by the immediate resuspensiontechnique, which made it possible to administer them by the gravity methodthrough a standard 18-gage needle. Earlier investigators had shown thedifficulty of administering undiluted (packed) red cells and the undesirablepressure needed to accomplish it. Moreover, resuspension at the end of 5 days' storagewas accompanied by greater hemolysis and more fragility than when resuspensionwas carried out as soon as the plasma was withdrawn.
Extensive studies carried out before the formal program wasinstituted showed no contamination in any sample, and later studies also showednone. When suspensions were deliberately contaminated for experimental purposes,it was found that occasionally within 24 hours, and almost invariably after 48hours, the affected cells turned dark red and the supernatant fluid showed apurplish-red discoloration that at once distinguished these bottles from theothers. The center employed a method of distribution which made it impossiblefor any hospital to receive the suspensions earlier than 48 hours after theblood was drawn, and this macroscopic observation was therefore employed in lieuof culture of each bottle, which would have been an impossible task. All bottleswhich showed any discoloration, as well as all bottles not used by the fifth dayafter bleeding, were discarded.
Before large-scale distribution of these cell suspensions waspermitted, the effects of transfusion were studied in 200 patients, withrecollection of the demonstration by Denstedt and his associates (83) andby Mollison and Young (84) that the fate of stored blood in vitro doesnot parallel its fate after transfusion.
Hemoglobin determinations were made by the Haden-Hausertechnique (16 gm. hemoglobin=102 percent). All determinations were made 2hours before the transfusion and were repeated serially 24 hours after it.Urinalysis was carried out before the transfusion and for several daysafterward, to investigate the presence of hemoglobin or any of its end products.The icteric index was also determined before the transfusion and for severaldays afterward. The single abnormality in the series, an increase in the ictericindex and hemoglobinuria, was found in a woman with Rh-positive blood and agrave anemia of pregnancy. She had the same reaction after transfusions ofstored whole blood.
Suspension media-At first, resuspension wasaccomplished in salt solution (0.85 percent) adjusted to a pH of 7.2. It wasthen found that, unless diluents other than physiologic salt solution were used,cells returned to the center after high speed centrifugation showed considerablehemolysis or alterations in the fragility index. Five-percent glucose solutionin distilled
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CHART 6.-Hemolytic effect of various diluting solutions1,2
1Number of bottles with clear supernatant fluid or fluidthat showed only a trace of hemolysis are shown in percentages by days from dayof bleeding.
2 Each point on the graph represents observations on atleast 12 bottles: Dextrose in distilled water (a), isotonic solution of NaC1(b), Alsever's solution (c), undiluted cells (hemolysis tested by dilutionwith physiologic salt solution on day of testing only) (d), dextrose in salinesolution (e), and Denstedt's solution (f).
water produced complete hemolysis within a short time (chart6), and 5-percent glucose in physiologic salt solution, 2-percent glucose, and2- and 5-percent sucrose often had the same effect. Alsever's solution andDenstedt'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 moreconstant level after the fifth day than did the index of saline-diluted cells.Hemolysis and fragility index were not significantly altered when the amount ofdiluent added to the packed cells was so varied that its volume was a quarterof, a half of, or equal to, the volume of the cells.
Results.-Typical results of red blood cell transfusionswere the hemoglobin elevations in 629 transfusions at three hospitals, whichranged, per 500 cc. of suspension administered, from 0.46 gm. in malignantdisease to 1.3 gm. in obstetric cases. The red blood cell increase in the sameseries ranged from 123,157 per cubic centimeter in malignancy to 497,000 inobstetric 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 othersthat the percentage of reactions was less with resuspended red cells than withstored 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 chillfollowed by a temperature elevation. When 342 whole blood transfusions weregiven to the same group of patients, there were 12 reactions, 3.5 percent. Therewas 3 percent of reactions in the 629 transfusions just mentioned.
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The New York Experience
The New York experience was reported in 1945 by Dr. WilliamThalhimer, Associate Technical Director, American Red Cross Blood Donor Service,and Major Taylor, Technical Director (85).
Materials and methods-This experience was based on 761transfusions of centrifuged type O cells resuspended and stored in10-percent corn syrup for periods up to 60 days. (By the time the report waspublished, 3,000 such transfusions had been given.) The transfusions were givento 437 patients, many of whom received repeated injections, sometimes daily,sometimes several times weekly. They suffered from a variety of chronicdiseases, such as arthritis; Hodgkin's disease; leukemia of several types;nephritis; anemias; pulmonary tuberculosis; inoperable malignancies; a few acuteconditions; and, in one instance, massive hemorrhage from a gastric ulcer.
At the beginning of this investigation, type-specific cellswere used, but as time passed, transfusions were limited to type O cells.There was thus much less wastage of resuspended cells, and the possibility oftranscription errors was reduced.
At first, only small amounts of cells resuspended in cornsyrup were given. Later, as no harmful effects were evident, the amounts weregradually 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 ableeding gastric ulcer received 3,500 cc. in 7 days.
Experimental studies-Before cells suspended in cornsyrup were used clinically, a long series of in vitro and animal studies werecarried out. The suspension used was 250 cc. of prechilled (41? F., 5? C.) of10-percent corn syrup (Corn Products Refining Co.) in sterile, pyrogen-freedistilled water. The composition of the syrup before dilution was 17.7-percentdextrose; 16.8-percent dextrin (pro-sugars); and 19.7-percent moisture.
It was consistently demonstrated that cells thus resuspendedwere 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'ssolution, or Denstedt's solution at the end of 5 days. The amount ofhemoglobin in the supernatant fluid averaged from 30 to 40 mg. percent at theend of 21 days in corn syrup against 100 mg. percent at the end of 5 days insaline solution.
No deleterious effects were evident in rabbits which receivedrepeated injections of large amounts of 10-percent corn syrup, sometimes as manyas 20 injections in 40 days. There was also no evident deleterious effect onrabbit cells preserved in corn syrup for 7 to 28 days. Finally, histologicexaminations of animal tissues showed no pathologic changes and no deposits ofiron pigment.
The freezing point of the corn syrup was the same as that of0.85-percent sodium chloride solution. In behavior, the syrup appeared to beisotonic, or very slightly hypertonic, for blood cells. It was speculated thatthe dextrins
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in it, because of their molecular size, might functionsomewhat as the original plasma in maintaining the stability of the stored,resuspended cells.
The length of survival of transfused cells was studied by theAshby technique (p. 260), which was followed closely, since it was found thatdeviations from it gave inconsistent and inaccurate results. Counts were alwaysmade in duplicate, and unless the two counts were reasonably close, the wholeprocedure was repeated. In the 253 consecutive nonagglutinable cell counts donein duplicate on patients of A and B groups who had been transfused with group Ocells, the average difference between the counts was 14,000 nonagglutinablecells per cubic centimeter. The unavoidable error-which is present in all redcell counting by even the most competent technicians-did not exceed 10percent.
Results.-Clinical results in patients treated with redblood cells resuspended in corn syrup were what might have been expected fromthe administration of the same amounts of whole blood of the same age. Therewere no evident deleterious effects, and there was a complete absence ofhemoglobinuria, hemoglobinemia, and jaundice. A number of patients showedprolonged beneficial effects under truly adverse conditions. Some casessuggested that adequate amounts of blood given over short periods of time had amore 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 oftennecessary because of the difficulty of securing donors.
The greatest field of usefulness of resuspended red bloodcells was in chronic secondary anemias of various origins. The cells wereavailable in enormous quantities, and experience soon showed that large amountscould be injected within relatively short periods. Whole blood, because of itsgreater viscosity, more effective osmotic pressure, high cost, and relativescarcity, was seldom used in this type of anemia. Another advantage of the cellresuspension technique was the reduction in the volume of fluid injected, whichwas of considerable benefit in such conditions as cardiac failure.
The New York experience furnished significant informationabout the safety of injecting older blood. Some of the 382 transfusions given to125 patients at Montefiore Hospital were given with red blood cells only 3 daysold, but in many instances the cells were 7, 14, 22, and 24 days old. In twoinstances, they were 31 days old, and in two other cases they were 38 and 41days old, respectively. One patient received transfusions with cells that were50 and 60 days old, respectively.
The survival of transfused cells dropped off sharply afterstorage 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 wholeblood stored for the same intervals. Although from 20 to 40 percent of thesecells survived in the recipient circulation from 2 to 10 days, it was concludedthat it would not be advisable to transfuse cells stored for these periodsunless an emergency existed or fresher cells were not available. The resultswith cells stored in corn syrup for 21 days were just as satisfactory as thoseobtained with blood
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stored in ACD (acid-citrate-dextrose) solution for the sameinterval, but, after the desired studies had been made, a 14-day expirationperiod was established.
The experience at Mount Sinai Hospital, 192 transfusions in150 patients, paralleled that just described for the Montefiore Hospital. On thestrength of these results, cells resuspended in 10-percent corn syrup came to bepreferred, because of their longer life, to those resuspended in physiologicsalt solution. When only red blood cells were needed, clinical results indicatedthat a transfusion of centrifuged cells resuspended in 10-percent corn syrupgave as satisfactory results as a transfusion of whole blood.
Extension of Service
Experiences at other civilian and military hospitalsparalleled those just described at Detroit and New York. The first red bloodcell service set up at a military hospital was established at Walter ReedGeneral Hospital, Washington, D.C., in 1942. By the spring of 1944, the use ofred blood cell transfusions was standard at all Army general hospitals in theZone of Interior near enough to processing laboratories for the material to bedelivered to them by automobile. This policy resulted in a great saving in theuse of whole blood.
Proposals
Among the suggestions made by medical officers not directlyconnected with the blood program was one for the use of red blood cells inpooled 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 glucosefor 14 days. It was his opinion that the morale of wounded men would be raisedif they knew they would receive whole blood and not plasma. He also recommendedthe method for Zone of Interior hospitals.
The Transfusion Branch, Surgical Consultants Division, Officeof The Surgeon General, explained to the writer that his plan was not necessaryin the Zone of Interior, where a modification of it was already employed, andwas not feasible in forward areas overseas, where present plans did not providefor typing in the field, since blood grouping was done before whole blood wasreleased. It was also pointed out that the mechanical mixing of blood cells andplasma would require equipment not then supplied. It would be hazardous from thestandpoint of possible contamination even in a well-controlled laboratory, andextremely dangerous in forward areas and under field conditions. Not included inthe 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-aswas done 5 months later-but that The Surgeon General had rejected thesuggestion when it was made to him in November 1943 (p. 465).
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4. Mann, F. C.: Further Experimental Study of Surgical Shock. J.A.M.A. 71:1184-1188, 12 Oct. 1918.
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13. Elkinton, J. R.: The Systemic Disturbances in Severe Burns and TheirTreatment. Bull. Ayer Clin. Lab., Pennsylvania Hosp. 3: 279-291, December1939.
14. McClure, R. D.: The Treatment of the Patient With Severe Burns.J.A.M.A.113: 1808-1812, 11 Nov. 1939.
15. Best, C. H., and Solandt, D. Y.: Concentrated Serum in Treatment ofTraumatic and Histamine Shock in Experimental Animals. Brit. M.J. 1: 799-802,18 May 1940.
16. Kekwick, A., Marriott, H. L., Maycock, W. d'A., and Whitby, L. E. H.:Diagnosis and Treatment of Secondary Shock. A Study of 24 Cases. Lancet 1: 99-103,25 Jan. 1941.
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 ofMedical Sciences, NRC, 19 Apr. 1941.
19. Minutes, meeting of Committee on Transfusions, Division of MedicalSciences, NRC, 31 May 1940.
20. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 18 July 1941.
21. Strumia, M. M.: Disposition Made of Blood Obtained Through the AmericanRed Cross From September 9, 1940 to April 7, 1941. Bull. Blood Substitutes,Division of Medical Sciences, NRC, pp. 189-204.
22. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 30 Nov. 1940.
23. Kendrick, D. B., Jr.: Refrigeration Applications for Blood Plasma andBiologicals. Refrigerating Engineering 47: 33-39, January 1944.
24. Report, Subcommittee for the Standardization of Dispensing Equipment,Committee on Blood Substitutes, Division of Medical Sciences, NRC, 8 May 1941.
321
25. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 23 May 1941.
26. Minutes, Conference on Albumin, Division of Medical Sciences, NRC, 5 Jan.1942.
27. Pickels, E. G.: The Desiccation of Plasma From the FrozenState. NewYork: The International Health Division of The Rockefeller Foundation, 1943.
28. A Technical Manual on Citrated Human Blood Plasma Detailing ItsProcurement, Processing, and Use. Washington: U.S. Office of Civilian Defense,November 1942.
29. Shackell, L. D.: An Improved Method of Desiccation, With SomeApplications to Biological Problems. Am. J. Physiol. 24: 325-340, 1 June 1909.
30. Stokes, J. S., McGuinness, A., and Mudd, S.: Clinical Applications of theLyophile Process. Am. J. Dis. Child. 50: 535-536, August 1935.
31. Elser, W. J., Thomas, R. A., and Steffen, G. I.: The Desiccation of Seraand Other Biological Products (Including Micro?rganisms) in the Frozen StateWith the Preservation of the Original Qualities of Products So Treated. J.Immunol. 28: 433-473, June 1935.
32. Greaves, R. I. N., and Adair, M. E.: A Simple Method for the Preservationof Sera by Desiccation in the Frozen State Without the Use of Refrigerants. J.Hyg. 36: 507-513, October 1936.
33. Greaves, R. I. N., and Adair, M. E.: High-Vacuum Condensation Drying ofProteins From the Frozen State. J. Hyg. 39: 413-445, July 1939.
34. Mudd, S., Reichel, J., Flosdorf, E. W., and Eagle, H.: The Preparation,Properties and Applications of Lyophile Serum Proteins and Complement. J.Immunol. 26: 341-342, April 1934.
35. Flosdorf, E. W., and Mudd, S.: Procedure and Apparatus for Preservationin "Lyophile" Form of Serum and Other Biological Substances. J.Immunol. 29: 389-425, November 1935.
36. Flosdorf, E. W., Stokes, F. J., and Mudd, S.: The Desivac Process forDrying From the Frozen State. J.A.M.A. 115: 1095-1097, 28 Sept. 1940.
37. Hill, J. M., and Pfeiffer, D. C.: A New and Economical DesiccatingProcess Particularly Suitable for the Preparation of Concentrated Plasma orSerum for Intravenous Use: The ADTEVAC Process. Ann. Int. Med. 14: 201-214, August 1940.
38. Hartman, F. W.: Use of Cellophane Cylinders for Desiccating Blood Plasma.J.A.M.A. 115: 1989-1990, 7 Dec. 1940.
39. Edwards, F. R., Kay, J., and Davie, T. B.: The Preparation and Use ofDried Plasma for Transfusion. Brit. M.J. 1: 377-381, 9 Mar. 1940.
40. Aylward, F. X., Mainwaring, B. R. S., and Wilkinson, J. F.: TheConcentration and Drying of Plasma. Brit. M.J. 2: 583-586, 2 Nov. 1940.
41. Memorandum, Maj. Howard Hogan, MC, for Col. Charles F. Shook, MC, 2 Nov.1942, subject: Meeting Blood Plasma Producers, St. Louis, Mo., 10-27-42.
42. Minimum Requirements for Unfiltered Normal Human Plasma, NationalInstitute of Health, 20 Feb. 1941.
43. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 19 Sept. 1941.
44. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 3 Nov. 1941.
45. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical 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 ofMedical Sciences, NRC, 23 June 1942.
48. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 15 Dec. 1942.
49. Strumia, M. M.: Effect of filtration on plasma. Report 9 Feb. 1942,prepared for Bull. Blood Substitutes.
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50. Robinson, G. C.: American Red Cross Blood Donor Service During World WarII. Its Organization and Operation. Washington: The American Red Cross, 1 July1946.
51. Memorandum, Capt. John A. Purinton, Jr., SnC, to Col. Clifford V. Morgan,MC, 16 Sept. 1942, subject: Human Blood Plasma, Dried.
52. Lozner, Lt. Eugene L., MC, USN: An Investigation and Report of 1,407Administrations of Dried Plasma in the Standard Package. Research Project X-1279.Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md.,14 Dec. 1943.
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, AbbottLaboratories, and Ben Venue Laboratories, 23-26 August 1943.
55. Memorandum, Maj. F. N. Schwartz, MAC, for Brig. Gen. Fred W. Rankin, 4Aug. 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, 13Aug. 1943, subject: Report of Inspections of Army Liquid Plasma ProcessingCenters.
58. Memorandum, Lt. Col. Douglas B. Kendrick, MC, for The Surgeon General, 29Jan. 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 MedicalSciences, NRC, 22 Mar. 1943.
61. Memorandum, Col. Tracy S. Voorhees, JAGD, 28 July 1942, subject: Meetingat the Purchasing and Contracting Office of the New York Medical Depot on July28th Concerning Revision of Plasma Contracts.
62. Memorandum, Capt. W. G. Patten, SnC, for Lt. Col. Lee I. Park, JAGD, 14Sept. 1943, subject: Accounting for Donated Blood and Processing Losses in Relationto 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 Lossesin Relation to the Renegotiation Program.
64. Memorandum, Capt. F. N. Schwartz, MAC, for Director, ProcurementDivision, 2 Oct. 1943, subject: Plasma Accounting.
65. Letters, Lt. Col. H. T. Marshall, MC, to all laboratories processingplasma, 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.
67. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 9 Apr. 1943.
68. Sweezy, E. E.: Potential Post-War Market for Dried BloodPlasma. Thesissubmitted to George Washington University, Business Administration 151,Marketing, 11 Jan. 1944.
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.
71. Letter, Maj. Gen. Norman T. Kirk to Chairman, American Red Cross, 5 Nov.1945.
72. Letter, DeWitt Smith, Vice Chairman, American Red Cross, to Maj. Gen.Norman T. Kirk, 21 Nov. 1945.
73. Robertson, O. H.: Transfusion With Preserved Red Blood Cells. Brit. M. J.1: 691-695, 22 June 1918.
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74. Rous, P., and Turner, J. R.: The Preservation of Living Red Blood Cellsin Vitro. II. The Transfusion of Kept Cells. J. Exper. Med. 23: 239-248,February 1916.
75. Castellanos, A., and Riera, R.: Sobre la transfusion de gl?bulos y latransfusion de plasma; sus tecnicas e indicaciones. Bol. Soc. cubana de pediat.9: 234-270, June 1937.
76. Castellanos, A.: La transfusi?n de gl?bulos. Arch. med. inf. 6: 319-333,July-September 1937.
77. McQuaide, D. H. G., and Mollison, P. L.: Treatment of Anaemia byTransfusion of Concentrated Suspensions of Red Cells. Brit. M. J. 2: 555-556,26 Oct. 1940.
78. Scudder, J., Drew, C. R., Corcoran, D. R., and Bull, D. C.: Studies inBlood Preservation. J.A.M.A. 112: 2263-2271, 3 June 1939.
79. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 13 May 1943.
80. Letter, Capt. Earl S. Taylor, MC, to Col. Tracy S. Voorhees, JAGD, 22Nov. 1943, subject: The Red Blood Cell Transfusion Service of the American RedCross Blood Donor Service.
81. Minutes, meeting of Subcommittee on Blood Substitutes, Division ofMedical Sciences, NRC, 16 Mar. 1945.
82. Cooksey, W. B., and Horwitz, W. H.: Use of Salvaged Red Cells. J.A.M.A.124: 961-964, 1 Apr. 1944.
83. Denstedt, O. F., Osborne, D. E., Stansfield, H., and Rochlin, I.: TheSurvival of Preserved Erythrocytes After Transfusion. Canad. M.A.J. 48: 477-486,June 1943.
84. Mollison, P. L.. and Young, I. M.: Failure of In Vitro Tests as a Guideto the Value of Stored Blood. Brit. M. J. 2: 797-800, 6 Dec. 1941.
85. Thalhimer, W., and Taylor, E. S.: The Transfusion of Centrifuged HumanType O Cells Resuspended and Stored in 10% Corn Syrup. J.A.M.A. 127: 1096-1101, 28 Apr. 1945.
