CHAPTER XII
The Bovine and Human Albumin Programs
Part I. Bovine Albumin
In the spring of 1940, when medical resources first began tobe mobilized with the realization that the United States would eventually enterWorld War II, the use of blood serum for shock and other conditions was limitedto a few pioneer workers in a few medical centers. Serum albumin, which was toprove the mainstay of the Navy, had not yet been developed. Work with bovinealbumin was limited to a few pilot studies, chiefly by Wangensteen at theUniversity of Minnesota (1).
The bovine albumin program in World War II began in late 1940and progressed in a series of highly encouraging developments until July 1942,when the first real setback was encountered, in the form of a fatal case ofapparent serum sickness. Because the potentialities of this form of therapy werebelieved to outweigh the risks, the program was continued cautiously. A numberof serious reactions, however, occurred in volunteers used for testing purposes,and, when a second fatal case of serum sickness of an unusual type wasencountered in February 1943, further work with bovine albumin was regarded asunjustified, and the program was officially discontinued the following month. Itwas a truly discouraging end to a highly promising project.
DEVELOPMENT OF PROGRAM
One of the questions raised at the first meeting of theCommittee on Transfusions, Division of Medical Sciences, 31 May 1940 (2), wasthe possible development of a substitute, preferably synthetic, for humanplasma. At this same meeting, "in the interest of clear thinking," itwas proposed that protein chemists be brought into the work, and the assistanceof Dr. Edwin J. Cohn (fig. 73), Department of Physical Chemistry, HarvardMedical School, was obtained (p. 336). A synthetic substitute was not developedduring World War II, but, as just indicated, a great deal of time and effortwent into the development of a bovine substitute for human serum albumin.
The first step in the program was a report, at the firstmeeting of the Subcommittee on Blood Substitutes on 30 November 1940 (3), ofprevious work with bovine and human plasma by Dr. Owen H. Wangensteen,Department of Surgery, University of Minnesota Medical School. His presentation,like his first publication on the subject (1), made it clear that theintravenous clinical administration of bovine plasma had not yet beenestablished as a safe routine hospital procedure. His work, however, hadindicated that this agent could be
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FIGURE 73.-Edwin J. Cohn, Ph. D.
given by this route to some patients in fairly largequantities. It was Dr. Wangensteen's opinion that, when the possibilities andlimitations of this method became clear, it was likely that it would become apractical hospital procedure "useful in civil as well as in war surgery forvarious purposes having to do with contracted blood volumes and proteinstores."
The first decision in the bovine albumin program concernedthe agent to be used; namely, serum, plasma, or a purified fraction of one orthe other. As the experiments in the Harvard laboratory progressed (4),it became clear that the albumin fraction had many desirable physiologic andchemical properties and that it was more stable, less viscous, and lessantigenic than whole plasma. It also had a larger relative osmotic effect.Chemically, Dr. Cohn reported, there appeared to be no difference between humanand bovine albumin. They were the same as to solubility, isoelectric point,electrical charge, mobility, electrophoretic pattern, sedimentation constant,and shape. The difference between them could be detected only by precipitintests.
Preparation - Bovine serum albumin was first prepared bya large-scale ethanol-water fractionation method, with purification byisoelectric precipitation. The chief advantage of this technique, which had beendeveloped in the Harvard laboratory, was that at the end of the process, thematerial could be passed through a Seitz filter. The globulin content wasundesirably high, 1 percent, and the single commercial firm (Armour LaboratoriesDivision of Armour & Co.) attempting to produce bovine albumin was having agreat deal
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of difficulty in reducing it. This firm, incidentally, clearly understoodthat if an acceptable product was finally accomplished, it would have nomonopoly on the process.
Beginning with the meeting of the Subcommittee on Blood Substitutes on 19April 1941 (4), successive reports were made on the clinical testing ofbovine albumin. It was Dr. Cohn's opinion then, and continued to be hisopinion until almost the end of the project, that reactions observed were moreprobably the result of globulin, which might be present in such small amounts asnot to be demonstrable by present techniques, than the result of albumin per se.
Dr. Cohn listed as immediate objectives in clinical tests:
1. Further proof that beef albumin would replace blood lost inacute hemorrhage.
2. Proof that it would replace plasma in shock. At this time,Dr. Cohn could not recommend it for this purpose.
3. Statistical studies on such matters as the incidence ofserum disease and the safety of multiple injections.
It was the opinion of the group at Harvard, based on their immunologicstudies, that beef albumin was remarkably inert in the circulation, in which itremained detectable for long periods of time, and was apparently a fairly poorantigen in the human bloodstream.
PROGRESS OF PROGRAM
Since bovine albumin does not appear in human urine, animal experiments werenecessary to determine its ultimate fate in the body. Studies conducted by Dr.Orville T. Bailey, Harvard Medical School, on rabbits indicated that such tissuechanges as occurred were apparently reversible and were of biologic rather thanclinical interest. Nothing resembling amyloid was observed(5, 6).
By July 1941, the crystallized bovine albumin originally produced had beengreatly improved in purity. The coloration always present in all albuminpreparations disappeared upon recrystallization, a phenomenon which suggestedthat, in both human and bovine products, the coloration was chiefly dependentupon concentrations of globulins, especially beta globulin, in them.
By April 1942, progress had been so satisfactory in all respects that adetailed report on the crystallization of bovine albumin was made to NRC(National Research Council), acting for the Committee on Medical Research,Office of Scientific Research and Development (7). This report includedinstructions for the preparation of crystallized bovine albumin, a tabulation ofits physical constants in solution, a complete report of its molecularproperties in comparison with those of human serum albumin, a report on theexperimental histologic effects of the crystalline preparation, and the courseand progress of commercial preparation of the material at the ArmourLaboratories. A progress report on the clinical experience to date was alsoincluded.
When no detectable globulin was found in the available preparations bychemical tests, Dr. Charles A. Janeway undertook a study of bovine albumin
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by immunologic methods. His results led him to conclude, for two reasons,that he was measuring a residual impurity:
1. The impurity diminished with each successiverecrystallization. This was strikingly illustrated in one preparation, which wasrecrystallized four times, each time with a decrease in the precipitablesubstance.
2. The same results were always obtained, no matter whatpreparation of crystallized bovine albumin was used in adsorbing the antiserum.
The practical application of these results, in Dr. Janeway's opinion,involved the solution of two problems:
1. The development of a more potent antiserum to provide formore readily detectable precipitation. It was thought that an alum-precipitatedantigen might be useful.
2. The development of a more accurate method of quantitatingthe amount of precipitation. It was thought that nephelometry would be simplerthan the Kjeldahl nitrogen technique then in use.
Plans were made for further testing by immunologic methods.
At the conference on 16 July 1942 (8), plans were made to study anumber of points concerning bovine albumin, but it was agreed that, important aswere these matters, none of them should be permitted to divert attention fromsecuring answers to two questions of primary importance to the Armed Forces, (1)the safety of repeated large clinical doses of bovine albumin and (2) itseffectiveness in shock.
At this conference, the first instance of serious serum sickness was reported(p. 330), following the use of material that had been recrystallized four times.In discussing the case, Dr. Cohn said that when bovine albumin of satisfactorystability had been obtained, the globulin component had been reduced to lessthan 2 percent, but preliminary work had shown that no such amount of globulincould be permitted. Crystallization was therefore undertaken, and clinical testswere begun with the new product in November 1941.
Since serum sickness had not been eliminated by the use of evenrecrystallized bovine albumin, material was being sent to a number ofinvestigators who thought that they could despeciate the molecule, and similarstudies were being conducted in Dr. Cohn's laboratory.1
Three months later (in October 1942), Armour Laboratories was producingbovine albumin with 0.01 percent globulin, and the Harvard laboratory was makinga product with 0.001 percent (9).
CLINICAL TESTING
April 1941-June 1942
The development of the bovine albumin program in respect to clinical testingwas reported at various meetings of the Subcommittee on Blood Substitutes and atvarious conferences, and is most conveniently discussed chrono-
1The clinicians testing bovine albumin reported to Dr. Janeway, who summarized their data and passed it on to Dr. Cohn for further evaluation.
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logically. Dr. Wangensteen, who was continuing his personalstudies with this agent (10), also reported at each meeting.
Encouraging reports were made at the meetings on 19 April1941 (table 9) (4); 8 May 1941 (11); 10 March 1942 (12); 12May 1942 (13); and 23 June 1942 (14). At the May 1942 meeting, theresults seemed so promising that it was agreed to expand the testing program,heretofore confined to Peter Bent Brigham Hospital and Dr. Wangensteen'sclinic, to certain other selected hospitals as supplies permitted.2
TABLE 9.-Results ofclinical testing with bovine albumin to 19 April 1941
| Preparation | Persons injected | Amount | Solution | Reactions |
|
| | | |
|
| Armstrong No. 3 | 4 | 0.4 | 4 | None. |
| Armour's IV W | 4 | 0.4 | 4 | None. |
| Armour's IV W | 1 | 8 | 4 | None. |
| Armour's VII | 1 | 4 | 20 | ??Slight, 2 hr. later. |
| Armour's VII | 2 | 6 | 4 | None. |
| Armour's VII | 1 | 12 | 4 | Serum sickness after 10 days. |
| Armour's VII | 1 | 16 | 4 | ??Slight, immediate, when |
| Armour's VII W |
|
|
|
|
| 1 | 5 | 10 | None. |
| 1 | 22.5 | 5 | None. |
July 1942
7 July.-At a special meeting of the Committee onMedical Research on 7 July 1942 (15), there was a full discussion of thecriteria to be employed in the recommendation of bovine albumin for militaryuse. Capt. C. S. Stephenson, MC, USN, thought that 50-60 persons should first beinjected with doses of shock size, repeated within 1 or 2 weeks, and followed byreinjections of 25 to 50 gm. after the blood was free of circulating antigen.Dr. Robert F. Loeb deplored the selection of any special number of testsubjects. Dr. Wangensteen thought it would be safe to recommend bovine albuminafter it had been used successfully in all conditions for which plasma was used.The meeting was
2Through error, this promising news was released to the press, and it had an immediately adverse effect on Red Cross solicitation of blood donors.
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reminded by the representative of The Surgeon General that noblood substitute would be practical under combat conditions if a skin test wasnecessary before it was used. No action was taken.
16 July -At the Conference on Bovine Albumin on16 July 1942 (8), the outlook, with one exception, seemed veryencouraging. Only four reactions had been encountered in 86 injections ofcrystalline bovine albumin, in amounts ranging from 10 to 50 gm., against thesame number in 35 injections of amorphous albumin. Three of the four reactionswith the crystalline substance were pyrogenic, all from the same preparation andto be explained by allowing the solution to stand too long at room temperaturebetween solution and sterilization.
The fourth reaction introduced a disturbing note, for it wasa case of possible serum sickness. The summarized case history follows:
Case 1 -A 62-year-old Italian,casually selected for testing during an uneventful convalescence fromherniorrhaphy and orchiectomy (and with an overlooked previous history of a20-pound weight loss and mild epigastric pain during the preceding 6 months),was given 12.5 gm. of crystallized bovine albumin twice,at a 6-day interval. The first injection was with a preparation suspected,because of results of the thermal rabbit test, of being pyrogenic. On the 10thday after this injection (the 4th day after the second injection), he developedfever, anemia, arthralgia, edema, purpura due to capillary fragility, urticaria,hypoproteinemia, and nitrogen retention. The fact that bovine albumindisappeared more rapidly than usual from his bloodstream, and that he had astrongly positive skin test as soon as it disappeared, favored the diagnosis ofserum sickness. On the other hand, three of seven blood cultures were positivefor Bacillus pyocyaneus. Contamination was suspected, and it isimpossible to avoid the suspicion that this patient had a primary bloodstreaminfection.
In the discussion that followed the presentation of this casereport, the Navy representative stated that he was not too much disturbed by asingle instance of serum sickness, especially in a patient with so many othercomplications. In Dr. Loeb's opinion, in which he was joined by others, unlesstesting was proceeded with more rapidly, and with the use of larger doses, theproblem of bovine albumin, from the military point of view, would becomeacademic.
There was a considerable discussion at this conference of therisks run by subjects and investigators in this kind of work. Dr. Wangensteenthought that the administration of bovine albumin was a justifiable therapeuticprocedure, which carried no greater risks than transfusion. Others took theposition that reinjections into healthy subjects carried definite risks and mustusually be on a voluntary basis. It was the opinion of the meeting that signedreleases were not binding and simply emphasized the risks. Dr. Alfred Blalockthought that medical students should no longer be used as subjects.
The conference ended, the minutes relate, "on anoptimistic note." All present believed that any risks involved in clinicaltesting were justified by the encouraging results obtained to date and by theurgency of the needs of the Armed Forces.
At this conference, Dr. Wangensteen reported that 40 to 60percent of 120 of his patients injected with whole bovine albumin developedserum sickness,
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usually about 4 days after the injection had been concluded. He believed thecase reported at this meeting to be an instance of serum sickness, thoughmodified by some other factor. He had had no reactions in about 60 patientstreated with the amorphous and crystalline preparations of serum albuminprepared by Dr. Cohn and his group, which had been injected in amounts of 25 to50 gm. in 200 cc. of physiologic salt solution. Six of these patients hadpreviously reacted to injections of bovine plasma.
October 1942
When a Conference on Albumin Testing was convened on 19 October 1942 (16),part of the picture was extremely encouraging. There had been only oneimmediate anaphylactoid reaction in 170 first injections, and none in over 100reinjections. Of 36 injections made with a purified, globulin-free, well-testedbovine albumin, 25, in which no reactions had been experienced, were second,third, or fourth injections.
The rest of the picture, Dr. Janeway reported, was highly discouraging. Thepatient with serum sickness reported on at the 7 July meeting still had severerenal damage. There had been a high incidence of serum disease in the volunteersbeing studied at Welfare Island, the Norfolk Prison Colony, and theMassachusetts Department of Correction. Although the testing group had beendirected to proceed with all possible speed and to inject as many persons aspossible three times each over a 3-month period, Dr. Janeway considered that therisks had become too great, and, on 18 September, he had telegraphed theinvestigators to discontinue all clinical testing until further notice.
Dr. Wangensteen expressed himself as having been surprised and disappointedwhen he had been asked to discontinue testing. He regarded his results with thenew preparation as most encouraging. He had given 126 injections to 80 patientswith eight immediate reactions (one anaphylactoid) and four delayed reactions.One of the latter occurred 21 days after the injection and was an instance ofsevere serum sickness; the situation was confused by the fact that the patienthad also had tetanus antitoxin and that her skin test, which was positive tohorse serum, was negative to bovine albumin during the latter part of herillness.
Two other reports were made:
1. Dr. Blalock, Johns Hopkins Hospital, reported the treatment of fourpatients in traumatic shock, with good clinical responses to 25-50 gm. of bovinealbumin. One patient, with multiple fractures, had died of fat embolism. At theend of a month, the other three had negative skin tests, but two showedleukopenia.
2. Dr. James T. Heyl reported on his work in Boston. Up to this time, therehad been no significant reactions in 25 patients and medical students in whomthe emphasis had been on reinjections. When 200 prisoner volunteers wereobtained, the risk of serum sickness was one case in 180 injections and
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reinjections. There were, however, 21 delayed reactions in 66 injections inthis group, 1 of which was fatal and 20 of which were severe enough to requirehospitalization.3 A followup revealed threeadditional delayed reactions.
Case 2 -In the case which ended fatally, thepresenting symptom, which was also the most prominent symptom, was acutearthralgia, which appeared in the hip 19 days after the injection. Fever,myalgia, and malaise were moderate. Chills and visceral pain were not present.The patient died in his sleep suddenly and unpredictably on the eighth day aftersymptoms appeared, 2 hours after he had waked spontaneously for 20 minutes. Whenhis bed was changed at this time, he seemed perfectly normal.
Autopsy, performed by Dr. Bailey, revealed significantfindings only in the heart and lungs. The heart, which weighed 400 gm., wasflabby, and the myocardium was soft. Histologic examination showed extensiveinterstitial edema of the myocardium, with infiltration by mononuclear andpolymorphonuclear leukocytes. In focal areas, especially in the interventricularseptum, coagulation necrosis of myocardial fibers was present, in associationwith more extensive cellular infiltration.
The right lung weighed 790 gm. and the left, 730 gm. There wasextensive pulmonary edema.
The combined changes in the heart and lung were considered tobe the cause of death, but the pathologist stated that, if he had performed theautopsy in ignorance of the situation, he could not have determined that aforeign protein had been injected.
The kind of delayed reaction observed in these recent cases resembled thesyndrome in the case of serum sickness that had occurred earlier (p. 330). Itoccurred, on the average, about 14 days after the injection, which most oftenwas the first. It did not resemble the type of reaction previously observedafter injections of amorphous bovine albumin.
A vigorous attempt to determine the component of the albumin preparationsresponsible for the recent reactions was already underway. Dr. Cohn was surethat stable crystallized bovine albumin could be prepared by careful control ofthe conditions of laboratory testing. It was essential to have tests that couldpick up alterations in the product. Until some satisfactory animal test wasdeveloped, nephelometry could be used to detect the lack of heat stabilitycharacteristic of preparations containing denatured albumin.
In spite of the discouraging data presented at this meeting, it was theconsensus that the military situation justified the continuation of bothexperimental studies and clinical testing. Dr. Cohn and the group working withhim were authorized to take all the time necessary to develop tests todistinguish between good and bad preparations of bovine albumin. When theyconsidered it safe, clinical testing could be cautiously resumed.
When the Subcommittee on Blood Substitutes met the day after the Conferenceon Albumin Testing (9), it accepted this recommendation, and the proposedspecifications for further testing were outlined in an addendum to the minutes.It is significant and prophetic, however, that Lt. Col. (later Col.) Douglas B.Kendrick, MC, wrote in the margin of his copy of the conference minutes,"Bovine albumin is now dead as a dodo."
3Dr. Heyl and others connected with the project paid tribute to the courage and unselfishness of the volunteers from the Commonwealth of Massachusetts Department of Correction and the Norfolk State Prison Colony. They clearly understood the risk involved before they volunteered, and their cooperation continued in spite of the high incidence of reactions and the fatality that followed one of them.
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December 1942
At a meeting of the Subcommittee on Blood Substitutes on 15 December 1942 (17),Dr. Cohn reported a marked improvement (10 to 20 times) in the stability ofbovine albumin preparations. Immunologic studies, however, had still failed todevelop satisfactory tests to distinguish lots of albumin that had producedclinical reactions from those that had not. Dr. Wangensteen was extremelydesirous of resuming clinical testing, on the basis of his previous goodresults, and its cautious resumption was authorized, on the ground that furthercriteria of safety could be developed only by such observations.
TERMINATION OF THE PROGRAM
The official bovine albumin program ended at the meeting of the AlbuminTesting Group on 22 March 1943 (18), with the report of a seriousreaction in the first patient who had been injected with the new preparation ofbovine albumin. The stability of this product was 20 times that of thepreparations which had caused the previous serious reactions. The globulincontent was estimated at 0.008 percent.
Case 3.-The patient, a 55-year-old white hemiplegic,had had a practically stationary course for the last 3-4 years. He was aphasic,and his spinal fluid Wassermann reaction was positive. He was injected with 25gm. of the new bovine serum albumin on 20 February 1943. Fifteen days later,without previous symptoms, he suddenly developed a purpuric rash on both legs.He had a chill 3 days later followed by 11 days of fever, during which therewere two other chills. He had migratory pains in the limbs, chest, precordium;urticarial as well as purpuric lesions over the legs and lower trunk; andgeneral malaise. For the 10 days before the 22 March meeting, he had felt well.
Laboratory tests showed an increased sedimentation rate; decreased red bloodcell counts; decreased hemoglobin values; signs of impaired liver function; asharp decrease in serum albumin, with no change in the serum globulin; asignificant decrease in excretion by the phenolsulfonphthalein test; and nochange in the nonprotein nitrogen of the blood.
It was generally agreed that this reaction, whatever its cause, was entirelysimilar to those previously observed. As soon as Dr. Cohn had heard of it, hehad written to Dr. Alfred N. Richards, chairman of the Committee on MedicalResearch, NRC, informing him of the reaction to a preparation which, likeanother prepared at the same time, he described as "beautiful." Dr.Wangensteen and Dr. Blalock had used the same preparation without any reaction,immediate or delayed, and a patient on whom Dr. Heyl had used it wasasymptomatic at the end of 3 weeks.
In view of the reaction which had occurred with such a greatly improvedproduct, Dr. Cohn had no choice but to consider that his earlier workinghypothesis, that the first reactions with bovine albumin were due to theinstability of the product, was no longer tenable. He also believed that he hadno choice but to recommend that all further chemical and clinical efforts todevelop bovine albumin as a project by the Committee on Medical Research
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should be discontinued. The Albumin Testing Group approved his action andrecommendation, although Dr. Wangensteen continued to be puzzled by thedifference between his own results and those of others. To date, he had injected90 patients with crystalline bovine albumin with only one mild immediateanaphylactoid reaction and only eight delayed reactions, seven of which weremild.
Dr. Cohn regretted that the early promise of bovine albumin had not beenborne out. Regardless of the success or failure of future attempts to developit, he pointed out that if Dr. Walter B. Cannon had not suggested the originalplan of developing a blood substitute from animal sources (p. 76), the presentmethod of plasma fractionation, which was yielding human serum albumin, antibodyglobulins, fibrinogen, and thrombin, might not have been devised.
The action of the Albumin Testing Group was approved at the 23 March 1943meeting of the Committee on Medical Research, Office of Scientific Research andDevelopment (19), and steps were taken to dissolve the contracts inexistence with the various testing groups.
Dr. Cohn emphasized that 177 patients had received injections of crystallinebovine albumin before a severe reaction was encountered. This fact was achallenge which called for an explanation, but solution would be a long termproject. Although the hope was expressed that independent efforts would be madeto continue testing of bovine albumin, it was realized that this would not bepractical without the official sanction of the Committee on Medical Research,because of the risks to patients.
LATER DEVELOPMENTS
Although all intensive work on bovine albumin ended in March 1943, certainother developments might be mentioned to complete the record.
Antimicrobial agents - All the reactions caused by bovine albumin hadfollowed injections of preparations containing Merthiolate (thimerosal), and Dr.Cohn thought that its presence might play some part in them (20). Clinicalresults were not conclusive, and animal experiments were undertaken to determinethe possible influence of Merthiolate upon antigenicity, especially upon theArthus phenomenon. Early experiments were suggestive, and it was decided for thefuture to release bovine albumin without Merthiolate for all clinical testing.
Despeciation - All work on the despeciation of bovine albumin had beenextremely disappointing. In some instances, it had not been accomplished at all,and, in others, extremely toxic substances had been produced.4
Clinical testing -The final report of Dr. Wangensteen's contract withthe Office of Scientific Research and Development, dated 10 September 1943 (22),
4Nine months after the U.S. program had been abandoned, a group of English workers reported the successful injection of despeciated bovine albumin (21).
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covered 139 patients and included a series of 25 subjects injected with thesame preparation from the Harvard laboratory with no reactions. His experienceindicated to him that bovine albumin would be a satisfactory blood substitute.
At the 2 June 1944 meeting of the Subcommittee on Blood Substitutes (23), Dr.Wangensteen reported on 15 patients who had been injected with the latestpreparation of bovine albumin prepared in the Harvard laboratory and all of whomhad received injections from previous lots. Two patients who had had severereactions on their initial injections had mild purpuric reactions after thesecond injections. There were no other reactions in this series.
In April 1944, Dr. Wangensteen (7) reported the injection of 83additional patients with solutions of bovine albumin, with three pyrogenicreactions, all susceptible of explanation by technical errors in preparation ofthe equipment. There were eight delayed reactions, with incubation periodsranging from 14 to 24 days, all of the type seen in mild serum sickness. TheRumpel-Leede test was positive, but there was no purpura. The incidence of serumdisease in his experience thus remained at about 10 percent, as in his originalreport.
A comparative study on human and bovine albumin by Heyl, Gibson, and Janeway (24),published in November 1943, showed no essential difference in the ability ofthese agents to draw fluid into the circulation after acute blood loss. Nosignificant immediate or delayed harmful effects were observed during the3-month period the subjects of the test were followed, but the investigatorswould not commit themselves, without more extensive clinical tests, as to thesafety of using a protein of animal origin in man.
Offers and suggestions.-During the war, occasional letters werereceived from lay persons suggesting that the blood of beef cattle be used inthe treatment of wounded men. It was pointed out in the replies that anextensive experience had revealed no way of making animal blood safe for humanuse.
On occasion, suggestions of the same kind were made from foreign countries,some purporting to have solved all the problems connected with the use of bovineplasma. These suggestions were similarly answered.
No serious attempt was made to revive bovine albumin as a substitute forhuman serum albumin after the Office of Scientific Research and Developmentabandoned the idea in 1943.
Part II. Human Serum Albumin
HISTORICAL NOTE
The first significant work on blood serum was done in 1918 by Mann (25), whoobserved that the parenteral injection of homologous serum in surgical shock,particularly when large doses were used, produced results as good as, or betterthan, were obtained by any other method. He therefore suggested that this agentmight be of value in conditions in which it could be stored and whole bloodcould not be obtained.
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When Dr. Max M. Strumia and his group at the Bryn Mawr Hospital (26) beganto use fresh and preserved serum in the treatment of severe infections, theyalso encountered frequent severe reactions, even when the serum was homologousand caused no agglutination of the recipient erythrocytes. In 1936, Elliott (27)proposed that untyped serum and plasma be used in obstetric shock (p. 266),his reasoning being that the maintenance of osmotic pressure is a function ofthe plasma proteins and that the need for replacing lost blood volume is moreimportant than the need for replacing red blood cells. Meantime, Stokes, Mudd,Flosdorf, and their associates at the University of Pennsylvania (28-30) wereusing lyophilized human serum5 prophylacticallyand therapeutically in various infectious diseases.
In 1937, Fantus (31) advocated serum in burns and in shock withouthemorrhage, because of its therapeutic and natural immunizing properties andalso because these conditions were usually associated with an excess of redblood cells. In 1940, Strumia, Wagner and Monaghan (26) reported theirresults with fresh and preserved plasma in a variety of diseases, but foundthemselves handicapped by frequent, severe reactions, which they attributed tothe changes induced in the serum by the lyophile process. In 1938, after aconsiderable experimental experience, Bond and Wright (32) suggested theuse of regenerated lyophilized serum in hemorrhage and traumatic shock. Mahoney (33)reported similar experimental results the same year.
It is typical of the status of human serum albumin at the beginning of U.S.participation in World War II that, at the organization meeting of the Committeeon Transfusions on 31 May 1940 (2), only whole blood and dried and liquidplasma were discussed. Serum albumin was not mentioned at all, though the hopewas expressed that possible substitutes for human plasma would be found,especially a substitute that could be prepared by synthesis.
LABORATORY DEVELOPMENT
The development of serum albumin in the treatment of shock really began atthe first meeting of the Subcommittee on Blood Substitutes on 30 November 1940 (3),with the suggestion, already mentioned, of the chairman, Dr. Cannon, that"it would be in the interest of clear thinking" if protein chemistswere brought into the picture (p. 325). In response to the suggestion,Dr. Cohn, whose work on plasma fractionation was already outstanding, undertookhis work on serum albumin. Albumin was selected rather than any other componentof plasma for several reasons: that it constitutes 65 percent of the proteins ofplasma, that it exerts 85 percent of the osmotic pressure of
5Reichel was the first to apply the term "lyophilization" (copyrighted by Sharp & Dohme) to the process by which serum was dehydrated (30). The root word, which means solvent-loving, was convenient and well chosen because it emphasized the noteworthy characteristic of the product, its remarkable solubility, which is a result of both the unaltered lyophilic properties of the serum proteins and the physical structure of the porous solid. The antibodies and complement of the serum suffered no detectable loss in processing, and the rate of subsequent deterioration was reduced to a small fraction of the losses in the liquid state.
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plasma, and that it was hoped that it could be packaged in small kits andused in preference to plasma whenever space limitations indicated.
At the 19 April 1941 meeting of the subcommittee (4),after a discussion of the best methods of collecting and dispensing both plasmaand serum, the following recommendation was adopted: "* * * the consensusof the committee is that either serum or plasma reduced to either a frozen or adried state is acceptable and that production should proceed at once with theunderstanding that in time other recommendations may be made."
The Red Cross had already begun to supply Dr. Cohn with 15bloods per week, and, at this meeting, he presented three exhibits of albuminprepared by plasma fractionation. The first was a tube containing 10 gm. ofserum albumin in 11 cc. of water. The solution appeared slightly discoloredbecause it contained traces of globulin, but it was described as a very stableliquid. It had a high viscosity, like that of heavy machine oil. The second tubecontained albumin in a 25-percent solution; this was a clear monogenous fluid.The third tube contained 10 gm. of crystalline albumin. Dr. Milton V. Veldeepreferred the 25-percent solution but Dr. Cohn thought the crystallinepreparation would be more satisfactory for shipment or for storage in bulk.
CLINICAL TESTING
At the meeting of the Subcommittee for the Standardization ofDispensing Equipment (11), a number of experimental studies werereported, and Captain Kendrick reported the first clinical use of human albuminin traumatic shock:
Case 4.-A 20-year-old man wasadmitted to Walter Reed General Hospital, Washington, D.C., in May 1941, 16hours after he had sustained bilateral compound comminuted fractures of thetibia and fibula, fractures of five ribs; and associated pleural damage,pneumothorax, and subcutaneous emphysema. He was confused and irrational, with ablood pressure of 76/30 mm. Hg. After he had been given two units of albumin(each approximately 25 gm.), over a 30-minute period, the pressure rose to106/70 mm. Hg, and two hours later, after insertion of a Kirschner wire,reduction of one of the fractures, and application of a cast, it was 130/80 mm.Hg. Over the next 12 hours, the patient received 1,250 cc. of fluid by mouth and1,000 cc. of physiologic salt solution subcutaneously. The systolic pressureremained above 130 mm. Hg during this period, with occasional elevations to 150mm. Hg. There was no evidence of circulatory failure at any time after theadministration of the albumin.
The red blood cell count on admission was 4.1million per cu. mm., the hemoglobin 14.5 gm. percent, and the hematocrit 44.Twelve hours after the administration of albumin, the red cell count was3,690,000. Forty-eight hours later, it was 3,780,000 per cu. mm., and thehematocrit was 35 percent. The hemoglobin level was between 12 and 12.5 gm.percent. There were no urinary abnormalities at any time.
At the meeting of the Committee on Blood Transfusions and theSubcommittee on Blood Substitutes on 23 May 1941 (5), it was recommendedthat the albumin produced in Dr. Cohn's laboratory be tested as rapidly and asextensively as possible. If the clinical tests proved satisfactory, Col. (laterBrig. Gen.) Charles C. Hillman, MC, for the Army and Captain Stephenson for theNavy stated that serum albumin would be accepted by the services.
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Production of serum albumin in the Harvard laboratory rose to500-800 gm. per week in July (6). At the 19 September 1941 meeting of thesubcommittee (34), Dr. Cohn announced that 3,407 gm. had been prepared orwas in preparation; that 447 grams had been used clinically on 11 patients, oneof whom had been reinjected after 13 days and another after 2 months; that thedosages had ranged from 7.6 gm. to 61.2 gm.; and that there had been noreactions of any kind.
Further reports were equally good. Up to 31 October 1941 (35), 50patients had been injected with 788.4 gm. of albumin with no reactions. By 11February 1942 (36), 125 patients had been injected with 4,247 gm., withonly four mild reactions and one moderately severe reaction. The latter followedthe use of albumin prepared from dried plasma secured from broken containers.When it was used, it was noted that it was of darker than usual coloration. Thisreaction occurred after 60 gm. of the albumin had been given to a patient whohad lost 1,080 cc. of blood.
The Pearl Harbor experience - At the Conference on Albumin on 5 January1942 (37), Dr. (later Brig. Gen.) Isidor S. Ravdin, who had just returnedfrom Hawaii, reported the administration of albumin to seven very severelyburned patients injured 10 days earlier at Pearl Harbor. All were edematous andall were losing plasma at the site of their burns. Some of them were soedematous, in fact, that albumin had to be injected into the femoral veinbecause other veins could not be located. The Naval Hospital had had only 40units of dried plasma for all its casualties, and some of these patients, in theemergency, had received too much salt solution.
All seven patients were given albumin, and all showed prompt clinicalimprovement, including one whose state was so critical that the administrationof albumin to him was debatable. There was no question as to his response: Hewas unconscious in the morning when he was given 250 gm. of albumin. In theafternoon, he was talking but was disoriented. The following morning, he wasgiven the same amount of albumin. Twenty-four hours later, the edema haddisappeared and he was taking food by mouth.
Most of these patients had hypoproteinemia and had very low hemoglobinreadings and red blood cell counts when they were first seen. Some had as littleas 8 gm. percent of hemoglobin, 3.5 mg. percent of protein, and 2? million redblood cells per cc. After the second injection of albumin, all showedhemodilution. Urinary outputs were not recorded, since most of the patients wereincontinent, but the rapid disappearance of edema in all cases suggested theexcretion of large quantities of urine.
The single reaction occurred in a patient who had had reactions after eachearlier injection of plasma. He had a chill after the first injection of albuminbut no reaction after the second.
The injections had been accomplished with some difficulty because of the poorquality of the gum-rubber tubing and the small diameter of the rubber latextubing which had been sent to Hawaii with the albumin because of the emergency.
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From his experience at Pearl Harbor, Dr. Ravdin concluded:
1. Albumin accomplished osmotically everything that normal human plasma could accomplish.
2. It produced hemodilution
3. Laboratory studies showed that it caused a rise in the albumin fraction of the blood and often in the globulin fraction as well. The fibrinogen component was not investigated.
4. Albumin was a very satisfactory agent in patients who needed protein and who had not had too great a red blood cell loss.
The formal testing program continued into early 1943. By the end of Februaryof that year (38), the total number of injections had reached 550, ofwhich 72 were for hemorrhage and for surgical or posttraumatic shock and 48 forburns. Reactions continued insignificant and mild.
Testing Procedures
The following criteria were established after clinical testing had been inprogress long enough to indicate what direction they should take (35):
1. Serum albumin should be given to patients in shock, who, ifpossible, should receive only albumin. Normal subjects should be tested forpossible reactions. In all cases, attention should be paid to whether or notfluids were withdrawn from the tissues.
2. The initial dose should usually be 100 cc. of 25-percentsolution (1 unit), which is roughly the equivalent of 500 cc. of citrated plasma(39). Administration should take about 20 minutes. The original injectionshould be repeated in 15 to 30 minutes if the desired response is not obtained.Not more than 10 units (250 gm.) of serum albumin should be given to any patientin 48 hours.
3. Vital signs should be recorded at specified intervals: Inexperimental subjects, at 30-minute intervals for 2 hoursand then at 4-hour intervals for 24 hours; in shocked subjects, at 30-minuteintervals as long as shock persists, then at 4-hour intervals for another 24hours. Hematocrit determinations should also be made before the albumin isadministered and at regular intervals thereafter in relation to the patient'sprogress.
4. Concealed hemorrhage is a possibility to be borne in mind,for it may occur or recur as the blood pressure returns to normal. This risk wasevident in a patient at Grady Hospital, with a stab wound of the chest, whoresponded well to albumin but collapsed suddenly when the blood pressure hadreached normal, from hemorrhage from a severed mammary artery undetected untilalbumin had become effective.
5. In extremely dehydrated patients, additional fluid andelectrolytes must be given, since albumin draws fluid into the blood at theexpense of other body tissues.
6. Serum albumin is not a satisfactory agent in severe anemia,since it simply increases the circulating blood volume without adding red bloodcells.
RECOMMENDATION OF SERUM ALBUMIN
TO THE ARMED FORCES
The first formal action on the use of serum albumin in the Armed Forces wastaken at the Conference on Albumin on 5 January 1942 (37), when itwas recommended to the Surgeons General of the Army and the Navy that, in
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addition to continuation of the plasma program, serum albumin be immediatelyadopted for clinical use for the following reasons:
1. Albumin can be packaged and stored in less than a tenth of the spacerequired for the standard Army-Navy package of dried plasma.
2. It is ready for immediate emergency use, without regeneration.
3. It is stable in solution in temperatures as high as 113? F. (45? C.) forprotracted periods.
4. Its adoption will accelerate and supplement the procurement ofsatisfactory blood substitutes for military use.
It was further recommended at this conference:
1. That the Surgeons General request the Red Cross to secure voluntary blooddonors for serum albumin, as part of the total national program.
2. That Dr. Cohn be asked to assume general supervisory direction of theprocessing of albumin in commercial laboratories.
The Navy, which was primarily interested in serum albumin rather than inplasma, took the necessary steps to implement this recommendation(40).
Further recommendations were made at the Conference on Albumin on 11 February1942 (36), as follows:
1. That the specifications for the preparation and packaging of human serumalbumin prepared by Major Kendrick and Cdr. (later Capt.) Lloyd R. Newhouser,MC, USN, be accepted with certain modifications, and that future modificationsbe made at the discretion of these officers and Dr. Veldee.
2. That after specifications for the preparation of serum albumin werecomplete, Major Kendrick and Commander Newhouser meet with representatives ofsupply and equipment firms to determine the availability of equipment and thetime required to supply the component parts for the Standard Package of HumanSerum Albumin.
It was unanimously agreed, at the meeting of the Subcommittee on BloodSubstitutes on 12 May 1942 (13), that the members reaffirm their earlierrecommendation that serum albumin be considered as an established bloodsubstitute of great importance and with many practical advantages. It wasfurther recommended that clinical testing be discontinued and that theapproximately 1,000 units of serum albumin in Dr. Cohn's laboratory be turnedover to the Navy for clinical use.
At the third Conference on Albumin and By-Products on 26 May 1942 (40), thechairman thought it necessary, in the interests of clearness, that themembership reaffirm its recommendations and limitations by signing the followingstatements (which were duly agreed to and signed):6
Human serum albumin in 25% solution has been recommended tothe Army and the Navy to fulfill their specific requirements for a bloodsubstitute which can be transported and administered in small volumes with greatfacility and with safety. It is clearly understood, as has been etched on thecontainers and as is indicated in the directive to the Navy,
6This second action was taken at Dr. Cohn's insistence, because of the reluctance of some medical officers in high places to accept serum albumin.
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that this concentrated solution should not beadministered in severe dehydration without the simultaneous administration offluids of such types as to maintain normal salt and water equilibria. The fluidsshould be given intravenously, orally if tolerated or by any otheravailable route. The above statements apply only to the use of hypertonicalbumin solution.
Human serum albumin has been established asbeing a blood substitute of proven value in that it causes hemodilution andraises blood pressure in a manner similar to blood plasma or serum. On the basisof clinical tests human albumin produces no more reactions than does plasma.
The stability of the human albumin solution aspackaged has been established over a period of one year at room temperature intemperate climates. Such material has been carried at sea in the sick bay of acruiser for three months at a temperature reported to average 37? C. (98? F.)without deteriorations as demonstrated by inspection and human injection.Stability has also been established by inspection at 45? C. (113? F.) for aperiod of 3 months and at 50? C. (122? F.) for a period of two weeks in thelaboratory.
Training
It was decided early in the albumin program that no specialindoctrination of medical officers concerning serum albumin would be necessary (16).They had had no experience in its use, but numerous articles had beenpublished on it, and the directions and warnings etched on the can wereconsidered adequate. Informal teaching emphasized the fact that serum albuminmust not be used in severe dehydration unless other fluids could be supplied tomaintain the normal salt and water equilibrium. Later, a somewhat more intensiveeducational program was undertaken, to encourage the use of serum albumin in theNavy (7), and the Army made a filmstrip on the subject.
COMMERCIAL PRODUCTION
Although it was not legally possible to enter into contracts forthe commercial production of albumin until it had become an accepted product,discussions concerning this phase of the program were recorded at variousmeetings of the Subcommittee on Blood Substitutes and at albumin and otherconferences. Dr. Veldee took the position that the same licensed laboratoriesshould not prepare both human plasma and albumin, but this policy was notadopted. Dr. Cohn believed it would be unfortunate to process albumin in anumber of small plants and similarly unfortunate to have manufacturers buildtheir plants on the basis of the amounts specified in the first contracts, sincethe Navy had promptly indicated its plans for larger amounts.
At the 18 July 1941 meeting of the Subcommittee on BloodSubstitutes (6), Dr. Veldee thought that serum albumin had been proved tobe of sufficient worth to be licensed as a biologic product. Dr. Cohn said that,while further improvements were to be expected, there were at this time noserious difficulties concerning the stability and sterility of serum albumin orits handling, and he foresaw no obstacles to its commercial production. Althoughit was against the laws of the Commonwealth of Massachusetts to preparebiologicals directly for use outside the state, the State Attorney General hadruled that
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the first 4 kilos of human albumin could be thus distributed,and a bill was being prepared for liberalization of the existing law.
At this same meeting, the subcommittee recommended that allphases of commercial production be under Dr. Cohn's direct supervision. Dr.Cohn believed that the present operation at Harvard should be continued, toavert the lag which might occur when commercial houses without previousexperience began to make albumin and also because the many byproducts obtainedby fractionation of plasma should be developed.
All possible preliminary steps were taken as soon as theConference on Albumin on 5 January 1942 (37) recommended serum albumin tothe Armed Forces, and, at the next meeting of the Subcommittee on BloodSubstitutes (12), it was reported that six commercial firms had indicatedtheir willingness, in response to the letters of intent they had received, toprocess 8,500 packages of albumin each before 1 October 1942. This would meanthat 180,000 additional bleedings must be absorbed by the bleeding centers. TheNavy's intention to contract for 250,000 additional packages of serum albuminin the next fiscal year7 would require 750,000additional bleedings, in addition to the 900,000 bleedings necessary for theplasma already contracted for.
Nothing came of the recommendation made at the secondConference on Albumin and By-Products on 15 April 1942 (41) that a pilotplant be established by the Army or the Navy for the processing of human andbiologic products for research purposes and clinical use.
In January 1942 (37), Dr. Cohn's laboratory, ArmourLaboratories, and Lederle Laboratories were considered capable of producing1,300 units of serum albumin (25 gm. each) per week. Dr. Cohn stated that anycommercial firm could participate in the program if it had large coldrooms andSharples centrifuges and would send personnel to be trained in his laboratoryfor at least a month.
A number of important steps were taken after this meeting (40).Specifications for commercial production were drawn up (42). Lettersof intent to purchase were sent to the various processing laboratories. Dr. Cohnreceived official Navy authorization to receive personnel from these firms forinstruction in the preparation of serum albumin, and representatives from sixproducers were thus trained.
There were two chief problems connected with the program. Onewas the steady increase in the Armed Forces requirements for serum albumin; therequirements for fiscal year 1942-43 were set at 110,000 units for the Army and250,000 units for the Navy. Each unit of processed albumin required 3.5 units ofblood. The second problem was the delay in obtaining Sharples centrifuges. Theprocessing of albumin, while complex, offered no particular difficulties, butthe procurement of equipment to process it threatened to delay the program formany months. Dr. Cohn doubted that the amount
7Contrary to the plasma procurement policy, by which all supplies were purchased by the Army Medical Procurement Agency, Brooklyn, all serum albumin was purchased on contracts made by the Navy with processing firms. The smaller amounts of serum albumin which the Army used were purchased from the Navy.
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desired could be produced within the specified time and warned that unduehaste might be dangerous.
When the Conference on the Preparation of Normal Human Plasma was held at thePlasma Fractionation Laboratory, Department of Physical Chemistry, HarvardMedical School (43), the agenda included discussions and demonstrationsof the size of a single batch of plasma, its electrophoretic analysis,bacteriologic filtration, preservatives, temperature stability test, coloration,hemolysis, sterility, separation of red cells, pyrogens, contamination, andplasma fractionation and processing of fractions. Clinical testing of serumalbumin was no longer considered necessary. Present problems concerned quantityproduction and distribution. To date, there had been 41 runs at the PlasmaFractionation Laboratory, 18 at Armour Laboratories, and 6 at LederleLaboratories.
A number of obstacles to the rapid implementation of the program werediscussed, including the joint problems of securing the additional bloods (1?million) required by the program; their cost (about $3 million per millionbloods); the shortages of equipment, particularly rubber tubing; the futility ofpriority ratings; the possible substitution of cup centrifuges for Sharplescentrifuges, which continued to be in very short supply; and the criticalmaterials in the completed serum albumin package, which was demonstrated at theconference. It was a very different matter, Dr. Veldee remarked, to makediphtheria antitoxin from horses in the backyard and to process serum albuminfrom blood secured from millions of donors scattered all over the country. Dr.Cohn would not recommend that the Red Cross turn over any bloods to anyprocessing firms until sample and practice runs could be completed with the useof hemolyzed blood or contaminated plasma, which most of these firms had onhand.
At the 23 June 1942 meeting of the Subcommittee on Blood Substitutes (14),the directive prepared by Commander Newhouser for the complete package ofserum albumin was approved with the deletion of the footnote dealing withfemoral injection (p. 338). At his suggestion, the label was changed to read"Standard Army and Navy Package of Normal Serum Albumin (Human,Concentrated)," the designation "serum albumin" being retainedbecause it was customary in the chemical literature and by general usage.
A special Conference on Albumin on 9 July 1942 (44) dealt with variousproduction matters, including procurement of materials; economy of production;length of processing; deferment of trained personnel; the risk of too greathaste; and yields with various types of centrifuges, all of which were still inshort supply. It was decided that the question of costs was beyond thecompetence of the committee, though it was brought out that they would becontingent on the rate at which the initial expenses of equipment could bewritten off.
At the Conference on Albumin Testing on 19 October 1942 (16), whilethe quantity production of concentrated human serum albumin still seemed far inthe future, prospects were considerably more encouraging. The War
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Production Board had expedited the procurement of necessary equipment.Practice runs with contaminated plasma had been carried out at four of the sixfirms to which contracts had been granted. In every instance, the chemicalpurity of albumin had been acceptable, less than 2 percent of globulin beingpresent. Three firms had begun to process serum albumin from blood supplied bythe Red Cross, and, after some initial difficulties from contamination, theywere producing satisfactory material, which would be released after clinicaltesting. Personnel from Dr. Cohn's laboratory had worked in several commerciallaboratories, to aid in the beginning of production. Production at the Harvardlaboratory was continuing.
Up to this time (October 1942), there had been:
78 first injections of crystalline albumin with no reactions of any kind in 61.
76 second injections, with no reactions in 69.
28 third injections, with no reactions in 26.
3 fourth injections, with no reactions.
At this conference, Dr. Cohn outlined the testing of commercial serum albuminas follows:
Navy specifications called for a 25-percent solution, to save space, and forstability at 122? F. (50? C.) to avoid spoilage in tropical temperatures. Assoon as a lot of albumin was filtered, six bottles were forwarded to the Harvardlaboratory. To date, no reaction had occurred in patients who had received humanalbumin that had passed the rabbit thermal test then in use.
After Captain Stephenson and Colonel Kendrick had expressed themselves asmore anxious about rubber tubing than about albumin, it was formally recommendedthat testing thereafter be conducted on the final package. Also, in deference tothose present who thought three tests insufficient, it was recommended thatclinical testing thereafter include five tests, carried out in Boston and at theArmy and the Navy Medical Schools. Since a standard batch of albumin would notusually exceed 380 bottles (38,000 cc.), the revised tests would require 8bottles, just over 2 percent, of each lot.
Progress thereafter was much faster. By the middle of December (17), fourlaboratories were using Red Cross blood in production, and two other firms weremaking practice runs. Difficulties with pyrogens had been overcome; they hadbeen traced to filter pads and distilled water. Dr. Cohn suggested that troublewould be avoided, and valuable plasma conserved, if, from time to time, testingwere carried out on distilled water, new lots of supplies, and equipment,including the intravenous equipment placed in the final Army-Navy containers.
Accelerated tests were being used, with assays carried out by electrophoreticanalysis. Studies indicated that in all samples of albumin tested to date therewas less globulin than the amount permitted in the specifications. Progress withall the first lots, however, was cautious, it being considered more advisable toestablish correct standards than to make haste.
Requests from the Armed Forces for serum albumin continued to increase. InMarch 1943 (18), the Navy, which had already contracted for 360,000 units
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(100,000 for the Army), asked for an additional 350,000 units, to bedelivered by July 1944. Since this goal was obviously impossible with presentfacilities, the problem was solved by opening a new processing plant (ArmourLaboratories) at Fort Worth on 1 November 1943; Fort Worth, Dallas, and Houstonwere the last untapped blood donor centers in the country.
At the Conference on Albumin on 22 March 1943 (18), Dr. Cohn discussedvarious aspects of the quantity production of serum albumin:
1. Achievement of chemical purity presented little difficulty.In all, 100 of 113 preparations examined to date had been found more than 99percent pure, and only 1 had contained more than 1.5 percent of globulin. Inview of these observations, Dr. Cohn intended to discontinue routineelectrophoretic analysis of every lot.
2. Studies by Dr. George Scatchard indicated that theinstability of some serum albumin preparations was caused less by the albuminthan by the impurities in it. Studies on bovine albumin had contributed greatlyto the stabilization of human albumin. The flocculation and haziness in certainpreparations after heating remained a problem to be solved.
3. Most preparations had passed the rabbit thermal testwithout difficulty. After a discussion of the number of clinical tests thatshould be required-some observers thought as many as 12 should be used-itwas agreed that the same criteria should be applied to human albumin renderedpyrogen-free by heating as would be applied to standard albumin preparations.Dr. Veldee had one reservation: Heating itself might do damage, and, for thisreason, he thought that the use of heated preparations should be restricted.
4. Albumin made from contaminated plasma was apparently safeand satisfactory for use, but more caution should be used in accepting it, andmore careful clinical testing carried out with it, than were employed in albuminmade from uncontaminated plasma. Similar precautions should be employed in serumalbumin made from plasma in broken bottles.
5. The production figures with the use of the DeLavalcentrifuge were attractive, but the introduction of new methods at this stagewould probably delay the program. If the program was to be expanded, and if itwere certain that these centrifuges could be obtained, then it might be well toconsider their use.
6. Dr. Cohn was now ready to perform only stability tests onthe bulk product, and to perform all other tests in the final containers. Thiswould save both time and material. Of the 328 clinical pyrogen tests performedbetween 10 June 1941 and 19 March 1943, only one preparation that hadsuccessfully passed NIH (National Institute of Health) rabbit thermal test hadgiven any febrile reaction in man.
7. One of the commercial laboratories had been informed that adonor had developed mumps 48 hours after his donation. There was no knowninstance of transmission of mumps by transfusion, and it was agreed that itwould not be practical to follow donors after they had given blood.
At the 9 April 1943 meeting of the Subcommittee on Blood Substitutes (45),Dr. Cohn reported that the program was proceeding satisfactorilyquantitatively and so well qualitatively that a revision of the test schedulemight be considered; in particular, the number of heated specimens tested couldbe reduced, which would save 10 days. The subcommittee authorized Dr. Cohn toestablish the number and type of tests to be employed.
At the 13 May 1943 meeting (20), it was reported that 700 bottles ofcontaminated plasma processed into albumin by the Cutter Laboratories had beenfound satisfactory chemically. One of two lots tested for pyrogens had caused noreactions. The other lot had given three reactions. The present policy was torequest three additional containers for examination if a reaction occurred
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in any of the five routine clinical tests. If any of thethree additional clinical tests was positive, the entire lot was discarded.
Delivery of serum albumin was at first disappointingly slow (table 10). Only 26,119 packages had been delivered by the end of July 1943, against an expected 150,000 (46). Then, the situation improved. Within the next 2 weeks, the number rose to 45,000 (the figures are cumulative), by the first of October (47) to more than 80,000, and by the middle of November (48) to almost 125,000. As of 2 June 1944 (23), 325,838 units of human serum albumin had been authorized for shipment to the U.S. Naval Medical Supply Depot; of this amount, 36,699 units had been produced during May. Quality continued to improve. All the albumin made at the Harvard pilot plant over the past several months had been held in the dry state, in anticipation of experimental needs.
TABLE 10.-Accepted production of units of normal humanserum albumin, June 1942-November 19431
| Year and month | Production laboratory | |||||||
| Cutter | Harvard | Lederle | Lilly | Sharp & Dohme | Squibb | Upjohn | Total | |
1942 | ||||||||
| June | --- | 83 | --- | --- | --- | --- | --- | 83 |
| July | --- | 241 | --- | --- | --- | --- | --- | 241 |
| August | --- | 370 | --- | --- | --- | --- | --- | 370 |
| December | --- | 611 | --- | 88 | --- | 71 | --- | 770 |
1943 | ||||||||
| January | 31 | 220 | 168 | --- | --- | --- | --- | 419 |
| February | 380 | --- | --- | 2,042 | --- | 272 | --- | 2,694 |
| March | --- | --- | 87 | 506 | --- | --- | --- | 593 |
| April | --- | 400 | 522 | 2,330 | 575 | 45 | --- | 3,872 |
| May | 1,671 | 408 | 914 | 2,233 | 1,245 | 179 | 2,066 | 8,716 |
| June | 2,146 | --- | 1,106 | 4,503 | 1,437 | 1,869 | --- | 11,061 |
| July | 3,652 | 126 | 607 | 3,033 | 228 | 4,473 | --- | 12,119 |
| August | 3,629 | --- | 1,820 | 2,619 | 1,409 | 5,325 | 792 | 15,594 |
| September | 4,505 | --- | 4,371 | 3,981 | 2,635 | 7,441 | 2,957 | 25,890 |
| October | 4,841 | --- | 4,264 | 6,113 | 964 | 9,235 | 2,513 | 27,930 |
| November | 2,168 | --- | 678 | 3,471 | --- | 7,051 | 546 | 13,914 |
|
|
|
|
|
|
|
|
|
1The production of the Harvard laboratory was shipped to the Bethesda NavalHospital; that of the commercial laboratories went to the U.S. Naval MedicalSupply Depot.
There had been a striking decrease in the amount of material requiring reworking because of failure to meet specifications of either sterility or stability. The yield of albumin from plasma had also increased. With modifications in
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processing methods, it rose from 27.5 gm. per liter to 29.3gm., and then to 30.2 gm. per liter. Allowing for losses, the final yield wasabout 25.4 gm. per liter.
After extensive testing and the ironing out of certaininitial difficulties, albumin made from contaminated plasma had proved safe andeffective, and sizable amounts were secured from this source. In addition, firmsnot making albumin were turning their contaminated stocks over to firms thatdid, and great savings were thus being effected.
In April 1944 (7), the Canadian National Research Counciloffered to the United States about 10,000 liters of contaminated serum, all ofwhich it proposed to destroy if U.S. authorities could not reclaim it asalbumin. It would be given to the Army and the Navy with no financial or otherobligations. Eli Lilly and Co. were able to work the contaminated serum intoacceptable, pyrogen-free serum albumin, and the Canadian offer was gladlyaccepted.
Some anxiety was originally felt that the development ofbyproducts other than albumin might slow up the albumin program. This fear wasdiscounted. In fact, when the Navy made contracts for byproducts with firmsholding contracts for serum albumin, Dr. Cohn approved the plan, on the groundthat plasma fractionation was an integrated process (40).
The report of the American Red Cross Blood Donor Service to 1September 1945 (table 11) showed that the seven commercial firms eventuallyinvolved in the albumin program had processed 2,329,175 donations into 569,014packages, all of which had been delivered to the Navy except for 1,704 packageswhich one firm was holding, awaiting shipping instructions (49).
ARMY REQUISITIONS FOR SERUM ALBUMIN
When serum albumin was in process of development, the smallsize of the package made it seem of great potential usefulness during landingoperations and for airborne troops and such ground forces as pack-drawn mountaintroops. The Army requisition for fiscal year 1942-43 was 110,000 units, but,up to 1 January 1944, because of production delays, only 36,000 units had beenreceived. In November 1943, the requirements for calendar year 1944 weretentatively set at 150,000 units, including the 74,000 units undelivered in1943. This requisition was later reduced to 60,000 units (50).
The reason for the reduction in the requisition for serumalbumin was a revision of the premises on which the original requirements hadbeen made (50). The small size of the package lost some of itsattractiveness to the Army in the face of the necessity for making intravenousfluids available along with the albumin; unless fluids were available, albumincould not be used in dehydrated casualties. Also, experience had shown thatthere were almost no circumstances, including combined landing operations, inwhich plasma could not be supplied in adequate quantities. Albumin, of course,continued to be used according to indications in head injuries and in burns.
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TABLE 11.-Summarized report of albumin production to 1September 1945
Production laboratory | Bloods | Packages delivered to U.S. Naval Medical Supply Depot |
|
|
|
Lederle | 285, 809 | 91, 022 |
|
|
|
Lilly1 | 413, 588 | 101, 916 |
|
|
|
Squibb | 551, 143 | 116, 395 |
|
|
|
Cutter | 321, 464 | 79, 719 |
|
|
|
Sharp & Dohme | 136, 906 | 26, 675 |
|
|
|
Upjohn | 324, 766 | 78, 913 |
Armour | 295, 499 | 74, 374 |
| 2,329,175 | 569, 014 |
1This firm had on hand 1,704 completed packages being held for shipping instructions.
TECHNIQUES OF PLASMA FRACTIONATION
In February 1942, when Dr. Cohn was requested to preparespecifications for the commercial production of serum albumin, he described fourmethods for the fractionation of plasma (40). The first technique wasimpractical for industry. The third, in which the supernatant of fraction IV wasconcentrated in a still, had to be given up because of the high incidence ofpyrogens and the time required to dialyze away the accumulated salts. The fourthmethod, crystallization, occupied too much time. The second technique, with somemodifications, was used by the processing firms. The technique is too complex tobe described in detail here, but its essential steps were as follows:
1. Separation of fraction I, separation of fractions II andIII, and precipitation of fraction IV.
2. Sedimentation of fraction IV.
3. Clarification of the supernatant from fraction IV,precipitation of fraction V, and reprecipitation of fraction V
More than one step could be accomplished at a time, and eachcould be completed within 48 hours. The complete processing of one batch wouldthus require a total time of 6 days. The method was the same as that employed inDr. Cohn's laboratory; expansion of production involved no changes in thebasic principles but merely changes in the mechanical equipment for handling thevarious steps, as well as changes in certain details, of the process. Thus, therecovery of tax-free alcohol used in the small-scale operations at the Harvardlaboratory did not pay. It became an immediate problem in commercial production,and care had to be taken to provide against the distillation of volatilecontaminants. There was some delay when one commercial firm was permitted tochange from dialysis to capillary methods of adding alcohol. All the firstmaterial thus treated had to be reworked, but there were no difficulties insucceeding runs.
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REFRIGERATION
It was originally thought that albumin would require refrigeration (37). Then,it was found that it could be stored at room temperature without deterioration.8Since, however, it was intended for use of the Armed Forces in all partsof the world, samples were tested for stability at 113? F. (45? C.) for 1month and at 98.6? F. (37? C.) for 2 months. It was found that albumin wouldnot remain stable at these temperatures if more than a very small amount ofglobulin was present. Later (34), in view of military requirements, itwas decided to extend the temperature range from -58? F. to +122? F. (-50?C. to +50? C.).
ADDITIVES
Sodium Chloride
One of the first observations made about serum albumin in the Subcommittee onBlood Substitutes (34) was that it was more stable when it was made upwith sodium chloride; without it, early preparations became cloudy at roomtemperature. The matter was to come up in other meetings of the subcommittee andat various conferences on albumin.
An ad hoc committee (Dr. Veldee, Dr. Earl S. Taylor, and Lt. William G.Workman, MC) was appointed to study the problem in May 1942 (13), afterDr. Cohn stated that the addition of sodium chloride to the blood intended forplasma fractionation greatly complicated the processing of serum albumin. Thehigher the salt content, the larger was the amount of globulin passing intofraction V, and the larger the amount in fraction VI. The ad hoc committee foundthe plasma yield to vary by less than 1 percent with and without salt. Theseobservations paralleled those made by Eli Lilly and Co. It was also found thatthe total osmotic pressure of 25-percent albumin could beconsiderably increased by adding more sodium chloride and considerably decreasedby reducing the amount used if either change were desired(16).
In January 1943, at a meeting of the Subcommittee on Albumin and By-Products (51),it was pointed out that the clinician's preference for isotonic solutionsof serum albumin might be on unsound grounds from the standpoint of physicalchemistry; a hypertonic solution would increase the stability of the product. Itwas proposed that the sodium chloride content of serum albumin be increased to 2percent. The amount of solution injected was so small that there could be novalid objection to this increase. Potassium salts, however, should not be added,because some of the clinical conditions encountered would be complicated byhyperpotassemia.
8At this time (1962), albumin is stored at 4? to 6? C., and the datingperiod has been correspondingly increased.
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In March 1943, the salt content of serum albumin was alteredfrom 0.15 to 0.3 molar. The change at least doubled the stability of the productat 135? F. (57? C.) and also increased its stability at 122? F. (50? C.),though less strikingly (48). Samples from each of the processing firmskept for 100 days at 122? F. (50? C.) looked clear enough at the end of theperiod to be used clinically (table 12). Dr. Cohn would have expected a twofoldimprovement in the stability of the product with the doubling of the saltcomponent, not the sixfold increase that had occurred and that might reflectincreased skill on the part of the producers.
When 0.3 molar sodium chloride was first used, authorization was given for making as much as 0.09 of this component sodium acetate. Later (52), Dr. J. Murray Luck and his associates at Stanford University demonstrated that a further increase in the length of the paraffin chain of the anion would increase for this purpose the stability of the serum albumin. Sodium butyrate was tested and discarded because of the undesirable odor. Albumin was then prepared with 0.05 molar sodium phenylacetate plus 0.25 molar sodium chloride. Still later, sodium mandelate was substituted for phenylacetate because of its therapeutic properties. Tests showed that whichever of these agents was used would make possible the heating of albumin for hours at temperatures close to 168? F. (70? C.), a step which would also reduce the dangers of bacterial or virus contamination (tables 12 and 13).
TABLE 12.-Effect of storage at various temperatureson stability at 57? C. of crystalline human albumin (lot HA-64)
|
|
|
| |||||||
| NaC1 | 0.15 | 0.15 | 0.30 | 0.30 | 0.15 | 0.15 | 0.30 | 0.30 | |
| pH | 6.8 | 7.0 | 6.8 | 7.0 | 6.8 | 7.0 | 6.8 | 7.0 | |
| Storage days | Storage temperature |
|
|
|
|
|
|
|
|
|
0 | Degrees C. |
|
|
|
|
|
|
|
|
| 100 | 0 | 105 | 81 | 183 | 167 | 58 | 40 | 117 | 93 |
| 200 | 0 | 98 | 83 | 182 | 184 | 55 | 45 | 117 | 94 |
| 100 | 25 | 63 | 55 | 153 | 117 | 39 | 28 | 94 | 68 |
| 200 | 25 | 39 | 45 | 103 | 117 | 24 | 27 | 71 | 60 |
| 100 | 37 | 63 | 47 | 110 | 124 | 33 | 23 | 85 | 87 |
| 200 | 37 | 30 | 32 | 81 | 108 | 18 | 30 | 65 | 61 |
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TABLE 13.-Effect of storage at various temperatureson viscosity of crystalline human albumin (lot HA-64)
| Ratio of viscosity after storage to initial viscosity | ||||
| 0.15 | 0.15 | 0.30 | 0.30 | |
| 6.8 | 7.0 | 6.8 | 7.0 | |
Storage days | Storage temperature |
|
|
|
|
| Degrees C. | |||||
0 | --- | 1.00 | 1.00 | 1.00 | 1.00 |
100 | 0 | 0.99 | 1.01 | 1.01 | 0.99 |
200 | 0 | 0.99 | 1.00 | 1.01 | 1.00 |
100 | 25 | 0.99 | 1.01 | 1.01 | 0.99 |
200 | 25 | 1.00 | 1.01 | 1.01 | 1.00 |
100 | 37 | 1.00 | 1.02 | 1.03 | 1.01 |
200 | 37 | 1.02 | 1.03 | 1.03 | 1.02 |
In a supplementary report, Dr. Luck and his associates (53)recommended that a large-scale clinical experiment be conducted with 25-percenthuman serum albumin solution, of pH 6.6 to 7.0, containing 0.04 molar sodiummandelate and 0.26 molar sodium chloride. Such solutions would probably be ofgreat stability and, if clinical trials revealed no adverse effects, should beemployed in place of the present serum albumin solution. Dr. Hans Clarke hadsuggested acetyl phenylalanine, and it was thought that it might be possible toreplace mercurials with it in both albumin and dried plasma. Studies based onthese proposals were limited to trial runs.
Meantime, thermal stability was generally improving, and bythe spring of 1944 (52), it was unusual to find more than an occasionalsample with stability of less than 50 hours at 135? F. (57? C.). Muellernephelometers had been distributed to the processing houses, and comparativestudies of their results and those of the Harvard laboratory showed satisfactoryagreement. Electrophoretic and ultracentrifugal controls had long since beendiscontinued.
In October 1944, Captain Newhouser directed all laboratoriesfractionating plasma under Navy contracts to prepare serum albumin of low saltcontent "if such a change is commercially feasible, will supply a productwith satisfactory stability and does not increase the contract cost of the serumalbumin."
Isoelectric albumin could be prepared practically free ofsodium ions, but it was stable only in the dry state and could not be dispensedin solution. If there were sufficient advantage in preparing completelysalt-free albumin, it could be dispensed in the dry state, with glucose in thediluent bottle, but this
352
would require new contract specifications and a new package. No clinicalevidence existed, however, of the value of reducing the sodium ion concentrationbelow a certain level.
A preliminary report of this investigation was made on 16 January 1945 (54),and a summary report was made to the Subcommittee on Blood Substitutes on 16March 1945, by Dr. Cohn (55), as follows:
1. Accumulated evidence to date indicates no significant behavioraldifferences between the new salt-poor albumin and the standard salt-containingpreparation in shocked or in normal individuals. In shock, injection of25-percent albumin, whether salt-poor or salt-containing, always increases theblood volume. In dehydration, the injection of additional fluids is stillrecommended.
2. Preliminary evidence indicates that salt-poor albumin is an effectivediuretic agent in the nephrotic syndrome and in hepatic cirrhosis associatedwith severe edema.
3. In a small group of surgical patients with hypoproteinemia and edema,concentrated albumin has been found to mobilize water from the interstitialspaces into the plasma. Used in a few patients after trauma, it produced asignificant rise in plasma protein concentration.
4. Salt-poor albumin, stabilized with 0.04 molar acetyl tryptophan or 0.04molar mandelic acid, retains its stability better than standard serum albuminpreparations when heated at 140? F. (60? C.) for 10 hours. This procedure, orsome comparable heating procedure, would destroy most bacteria in vegetativeforms as well as such viruses as that of infectious hepatitis. It has beenrecommended that studies be undertaken as soon as possible of the time requiredto destroy the agent of hepatitis at a given temperature.9
5. The original opinion of serum albumin as to its convenience, rapidity andease of administration, and effectiveness in increasing plasma volume in injuryand shock has been fully confirmed by this investigation.
As a result of the evidence secured in this investigation, it was recommendedthat salt-poor albumin be substituted for the current preparation, whichcontained 0.3 molar sodium chloride. It should be prepared without a mercurialpreservative (p. 354); should be stabilized with 0.04 molar acetyl tryptophan, aderivative of a natural amino acid; and should be heated for such periods and atsuch temperatures as might be necessary for the destruction of viruses.
At this meeting it was also recommended that products of plasma fractionation, including human serum albumin, be included in the "United States Pharmacopeia." This recommendation was not made effective until November 1950, with the 14th edition. Plasma had first appeared in the 1942 edition.
Antibacterial Agents
At the January 1942 Conference on Albumin (37), Dr. Cohn reported thatthe experience gained at one of the processing companies had indicated that itwas highly dangerous to add Merthiolate to albumin. Experimental studies were tothe same effect. The production of albumin was delayed at least 6 weeks by thenecessary investigations. A proposal that preservatives be eliminated entirelywas made at the 19 July 1943 conference (56), but if this was
9The menace of hepatitis was just beginning to be appreciated (p. 647).
353
done, it was pointed out in the discussion, at least a third of the totalproduct would have to be tested. This plan would be workable as far asindividual bulk containers were concerned but totally impractical in the finalcontainers.
The problem, as Dr. Cohn pointed out at this conference, fell into two parts, (1) filtration to achieve a sterile product and (2) the use of preservatives. With a 30-percent solution, the viscosity was too great for convenience. Later (16), Dr. Cohn reported an observation not previously made, that 25-percent normal human serum albumin is essentially isoviscous with respect to blood.
Serial studies by Col. Elliott S. Robinson, MC, showed that the more rapidly the albumin was filtered, the better the results from the standpoint of sterility. Using Seitz filters, he found that contamination was not a particularly troublesome problem. He attributed the good results partly to good luck and partly to attempts to expedite the procedure. Filtration became easier as the globulin content of preparations was reduced.
Studies reported by Dr. Janeway at the 19 July 1943 meeting (56) hadbeen undertaken on the assumption that alcohol was a good bacteriostatic agent.In four complete runs, and an almost completed fifth run, he had taken bacterialcounts at every fractionation and found contamination low; the average, figuredin terms of the original plasma to take care of dilution, was 30 colonies percc. Although the counts were very low, sterility was not achieved at any pointuntil fraction V, which carried down bacteria, was taken off. The supernatantwas free from bacteria, but the precipitate was not; it usually contained asmaller number of bacteria than might have been anticipated, but the countsmight run up to about 100 colonies per cc. of redissolved fraction V. Sampleswere taken in quadruplicate. The results were the same whether they were frozenor left at ice box temperatures, but there was occasionally a considerabledifference between cultures made at room temperature and at 98.6? F. (37? C.).
It was concluded that alcohol did not act as a sterilizing agent, at least atthe temperatures at which these studies were conducted. It was also concludedthat bacteria were collected at most of the steps in the process, from theoperators, filters, and other sources, but that the number was probably notgreat enough to have any influence on pyrogens, though if the organisms were ofthe gram-negative variety, a few would be sufficient to cause trouble.
Dr. Veldee agreed that, assuming dried serum albumin to be relativelysterile, the important points would be the speed of getting the material intosolution, the temperature of the solution, and the speed with which it wentthrough the filter. Colonel Robinson's experience had been limited torelatively small lots. The NIH experience had included 75-liter lots. ColonelRobinson had used a very simple type of Seitz filter, which could be cleaned andinspected for leaks. Dr. Veldee doubted that such filters would be used in thelarger commercial houses and thought that in certain circumstances the techniqueof filtration might well be a source of contamination. If there was a smalldefect in the filter, there was more chance of its showing up with 75- than with25-liter lots.
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As to preservatives, Merthiolate in 1:15,000 concentrationhad always been acceptable to NIH, but if 1:10,000 had been proposed whenthe minimum requirements for human plasma were being drawn up, it would havebeen equally acceptable. The problem was this: In all other biologics excepthuman plasma and serum, the dosage was small, usually 1 or 2 cc., or under 10cc., and the effective bactericidal dose per patient was equally small.Therefore, any one of a number of preservatives could be used to kill any livingorganism. With albumin, plasma, or serum, however, such large doses had to begiven that preservatives could not be used in the same concentration as insmaller dosages because of the risk of toxicity.
FURTHER CLINICAL STUDIES
At the Conference of the Albumin and By-Products Group on 14December 1943 (57), Dr. Cohn pointed out that the development of serum albuminhad undergone two phases. In the first, all the material produced was devoted toan appraisal of its efficiency in shock. In the second, all the material wasdelivered to the Armed Forces for use overseas. At this time, with fullcommercial production underway, the output of the pilot plant at the HarvardMedical School constituted less than 1 percent of the total production, and hebelieved that a third phase might be considered, in which the output at thisplant could profitably be devoted to the study of diseases and conditions otherthan shock. He agreed with those who pointed out that, while the amount of serumalbumin thus used would be small in comparison with the total amount, greatquantities would be required in the treatment of nephrotic states, cirrhosis,and nutritional edema, and the needs of the Armed Forces must not be jeopardizedin any way by the proposed research program.
In December 1944, the Committee on Medical Research, NRC, wasrequested by the Navy to provide additional clinical data on serum albumin, withspecial reference to the increase in plasma volume accomplished by its use withand without the additional administration of crystalloid solution. An additionalpurpose of the investigation was the testing of the new salt-poor albuminpreparation just developed. The conditions studied included trauma of variouskinds, with and without shock and with and without active fluid loss at the timeof the investigation; postoperative states, with and without shock; and medicalconditions. Normal subjects were also studied.
Considerable scattering was found in the increase in plasmavolume per gram of albumin injected, which was to be expected, even in normalsubjects, as the result of differences in tissue hydration, circulatory state,renal activity, and other variants. Injured subjects, especially those withhemorrhage, burns, or peritonitis, who were losing fluid actively, sometimesheld little or none of the injected albumin and fluid and showed onlyinsignificant increases in plasma volume.
355
The following conclusions were considered warranted:
1. As an overall average, the injection of concentrated human serum albumin was associated with a rapid, and sometimes immediate, increase in plasma volume of 12-14 cc. per gram of injected albumin. The same average held per gram of albumin retained.
2. Administration of a fixed amount of saline solution intravenously with the serum albumin (800 cc. with 200 cc. of 25-percent albumin) resulted in an appreciable increase in plasma volume as compared with the increase that followed the administration of albumin alone. Experimental studies on dogs, in which dehydration was produced by withholding fluids or by inducing diuresis with glucose, had shown that the administration of albumin alone did not restore the circulating blood volume and blood pressure to normal and that the survival rate was low.
3. Patients in severe shock, presumably with continuing losses of blood or plasma, showed much smaller increases in plasma volume per gram of albumin given (an average of 8 cc. in shock against 14 cc. without shock). Thus, in severe shock, the administration of 25 gm. of serum albumin without additional fluid would correspond to a plasma infusion of 200 cc. Maj. (later Lt. Col.) Henry K. Beecher, MC, reached approximately the same conclusion in his study of battle casualties in Italy, although the only criterion of improvement available to him was arterial blood pressure (p. 40).
4. In several instances, the administration of additional intravenous salt solution after the administration of albumin produced further increases in plasma volume.
5. No differences were noted in the results of salt-poor and salt-containing solutions.
TERMINATION OF PROGRAM
The production of serum albumin consumed an increasing numberof blood donations until February 1944, when about 30,000 bloods a week werebeing supplied for this program. Then, with the needs of the Navy and the farsmaller needs of the Army well provided for and supply simply a maintenancematter, production was gradually reduced until 15 October 1944, when it was cutsharply. The Armour Laboratories plant in Fort Worth was closed and the blooddonor centers which had supplied it, in Fort Worth, Dallas, and New Orleans,were also closed. Four other laboratories ceased receiving blood for serumalbumin at this time, but other processing laboratories continued to operate, ona curtailed scale, until the end of the war.
References
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8. Minutes, Conference on Bovine Albumin, Division of Medical Sciences, NRC,16 July 1942.
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10. Kremen, A. J., Hall, H., Koschnitzke, H. K., Stevens, B., andWangensteen, O. H.: Studies on the Intravenous Administration of Whole Bovine Plasma and Serum toMan. Surgery 11: 333-355, March 1942.
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21. Edwards, F. R.: Despeciated Bovine Serum (D.B.S.): A Substitute for Human Plasma. Brit. M. J. 1: 73-76, 15 Jan. 1944.
22. Wangensteen, O. H., Kremen, A. J., and State, D.: The Experience of the Surgical Clinic of the University of Minnesota Medical School in the Use of Bovine Albumin as a Blood Substitute. Committee on Medical Research, Office of Scientific Research and Development, 10 Sept. 1943.
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24. Heyl, J. T., Gibson, J. G., 2d, and Janeway, C. A.: Studies on the Plasma Proteins. V. The Effect of Concentrated Solutions of Human and Bovine Serum Albumin on Blood Volume After Acute Blood Loss in Man. J. Clin. Invest. 22: 763-773, November 1943.
25. Mann, F. C.: Further Experimental Study of Surgical Shock. J.A.M.A. 71: 1184-1188, 12 Oct. 1918.
26. Strumia, M. M., Wagner, J. A., and Monaghan, J. F.: The Intravenous Use of Serum and Plasma, Fresh and Preserved. Ann. Surg. 111: 623-629, April 1940.
27. Elliott, J.: Preliminary Report of a New Method of Blood Transfusion. South. Med. & Surg. 98: 643-645, December 1936.
28. Stokes, J., Jr., Mudd, S., Roddy, R. L., Eagle, H., Flosdorf, E. W., and Lucchesi, P.: The Use of Lyophile Human Serums for Prevention and Treatment of Infectious Diseases. Am. J. Dis. Child. 48: 1428-1429, December 1934.
29. Flosdorf, E. W., and Mudd, S.: Procedure and Apparatus for Preservation in "Lyophile" Form of Serum and Other Biological Substances. J. Immunol. 29: 389-425, November 1935.
30. Flosdorf, E. W., and Mudd, S.: An Improved Procedure and Apparatus for Preservation of Sera, Micro?rganisms and Other Substances-the Cryochem-Process. J. Immunol. 34: 469-490, June 1938.
31. Fantus, B.: The Therapy of the Cook County Hospital. Blood Preservation. J.A.M.A. 109: 128-131, 10 July 1937.
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32. Bond, D. B., and Wright, D. G.: Treatment of Hemorrhage and Traumatic Shock by the Intravenous Use of Lyophile Serum. Ann. Surg. 107: 500-510, April 1938.
33. Mahoney, E. B.: A Study of Experimental and Clinical Shock With Special Reference to Its Treatment by the Intravenous Injection of Preserved Plasma. Ann. Surg. 108: 178-193, August 1938.
34. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 19 Sept. 1941.
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36. Minutes, Conference on Albumin, Division of Medical Sciences, NRC, 11 Feb. 1942.
37. Minutes, Conference on Albumin, Division of Medical Sciences, NRC, 5 Jan. 1942.
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42. Specifications for a Complete Package of Serum Albumin (Human)Concentrated for the Army and Navy, n. d.
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53. Luck, J. M., Ballou, G. A., Bassett, D., Boyer, P. D., Lum, F. G., andRice, R.: The Stabilization of Standard Human Serum Albumin With Sodium Mandelate andSodium Phenylactetate. Blood Substitutes Report No. 15, Subcommittee on BloodSubstitutes, Division of Medical Sciences, NRC, 15 Mar. 1944.
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57. Minutes, Conference of the Albumin and By-Products Group, Division ofMedical Sciences, NRC, 14 Dec. 1943.
