CHAPTER XIII
Byproducts of Plasma Fractionation
GENERAL CONSIDERATIONS
One of the truly important achievements of theplasma-blood program in World War II-and, indeed, one of the importantscientific contributions of the century-was the development by Dr. Edwin J.Cohn and his group at the Department of Physical Chemistry, Harvard MedicalSchool, of a practical physicochemical technique by which plasma could beseparated into clinically usable fractions. The wartime work was an extension ofprevious work done in this laboratory on plasma fractionation, and was directlystimulated by the endeavor to find a purified preparation of bovine albumin. Thefractions separated, particularly albumin, proved of great value during the warand have since affected many areas of medicine. The work that Dr. Cohn began iscontinuing and expanding under the auspices of the Protein Foundation, which wasestablished in 1953.
By the Cohn technique, each plasma fraction isprecipitated in ethyl alcohol, under specific conditions of temperature, pH,ionic strength, and protein concentration, in a coldroom, at a temperature of23? F. (-5? C.). Since the processing is carried out below the freezing pointof water, denaturation of the plasma proteins by alcohol does not occur.Bacterial growth is also inhibited. The dried fractions can be stored for anindefinite period before they are used.
The six major fractions of plasma weredescribed in 1947 by Dr. Cohn as follows (1):
Fraction I contains most of the fibrinogen andthe antihemophilic globulin.
Fraction II, obtained by subfractionation ofII+III, contains the gamma-globulin antibodies of proven value in theprophylaxis of measles and probably also of infectious hepatitis.
Fraction III-1 contains other antibodies,including those to typhoid 0. The isoagglutinins, including the anti-Rhantibodies of value in blood typing, are also concentrated in this fraction.1
Fraction III-2 contains prothrombin and one ofthe components of complement. Prothrombin converted by thromboplastin tothrombin has proved of value in conjunction with fibrin foam or some otherpledget as an hemostatic agent and, in conjunction with fibrinogen, in theformation of clots, films as dural substitutes, and tubes for other surgicaluses.
Fraction III-3 contains plasminogen, theprecursor of plasmin, which has sometimes been called the fibrinolytic enzyme.
Fraction III-0 is rich in lipoprotein,including the so-called X-protein of McFarlane, which interacts in the plasma insuch a way as to suggest that the molecular weight varies with concentration. ** *
1Isoagglutinins and the Rh factor are discussed in the chapter on laboratory tests.
360
CHART 7-Fractional distribution of variouscomponents of plasma, proportion separated in eachfraction, and uses for each to 1947
Source: Cohn, E. J.: The Separation ofBlood into Fractions of Therapeutic Value. Ann. Int. Med. 26: 341-352, March1947.
Fraction IV-1 is lipoprotein in nature. * * *
Fraction V contains the human serum albuminthat has been made available in such large amounts to the Armed Forces for usein the treatment of shock, hypoproteinemia and edema. As at present released fordistribution under conditions that have been specified by George Scatchard,Laurence E. Strong and Walter L. Hughes, Jr., it is poor in salt and is sostable in the presence of nonpolar anions, developed for this purpose largely byJ. Murray Luck, that it is heated in the final container for 10 hours at 60? C.That these conditions suffice for the destruction of the virus of infectioushepatitis has been demonstrated by Joseph Stokes, Jr.
Fraction VI consists of the large amount ofsalts, especially citrates, and the small amount of protein left in the motherliquors following the removal of these various precipitates. * * * Fraction VIdeserves further exploration, as do fractions of proved therapeutic value.
361
The distribution of the various components ofplasma into fractions was indicated graphically in this same presentation (chart7).
Aside from the specific value of the variousplasma fractions, the plasma fractionation program had three general advantages:
1. Whole blood was conserved, since only thespecial component required in the special case was used.
2. Great economic savings were effected in theblood program.
3. Since plasma fractions were of humanderivation, they possessed all the advantages of homologous substances. Therewas therefore no fear of sensitivity reactions.
The size of the plasma fractionation programis an indication of its importance. Up to May 1945, 1,218,531 units of plasmafractions had been produced, the largest production of a single fraction being576,996 units of human serum albumin(2).
The Conference on Plasma Fractionation on 14March 1945 (3) summarized the previous experience with plasmafractionation and indicated future trends. Reference to the minutes of thisconference and to the minutes and appended reports of various meetings of theSubcommittee on Blood Substitutes is recommended for readers who desire moredetailed information concerning this program than limitations of space permithere.
HEMOGLOBIN
Several research projects on hemoglobin solutionsin replacement therapy were undertaken during the war, under the auspices of theSubcommittee on Blood Substitutes (4, 5), but their use was neverseriously considered. Some studies were encouraging, but the careful work ofLamson and his associates (6) showed that such solutions would supportlife in shocked animals for only a few hours and that, to be effective at all,they must be given before severe shock developed. There were also otherobjections: The rise in blood pressure which they produced was extremely high inrelation to the volume of fluid replacement. The metabolic rate rose more than100 percent, an extremely undesirable reaction in patients in shock. Theincrease in pulse pressure was also considerable. Clinically, moderately severereactions resulted even when only small testing amounts were injected. All ofthese phenomena were related to the established facts concerning the toxicity ofhemoglobin.
GLOBIN
Although extensive studies were carried outwith globin (3, 7, 8), the protein component that makes up about96 percent of hemoglobin, nothing of practical value came of them during thewar.
The first publication on globin, by Schulz in1898 (9), which concerned its preparation and properties, was laterinvalidated when it was found that he was dealing with a denatured protein. Mostsubsequent investigations were
362
concerned with modifications of his method inan attempt to obtain so-called native globin. The attempt was successful, butthe material obtained was either toxic or antigenic.
Dr. Max M. Strumia's work with globin, whichwas begun in the spring of 1941, was first brought up in the Subcommittee onBlood Substitutes at the meeting of 13 May 1943 (8). The project heproposed-the derivation of globin from the hemoglobin of discardederythrocytes and its use as a blood substitute-was recommended to theCommittee on Medical Research, with the stipulation that Dr. Strumia use thefacilities and advice of Dr. Cohn and Dr. Linus Pauling.
Reports on the progress of this project weremade at various meetings of the Subcommittee on Blood Substitutes (2, 10-13),and at a special conference on globin on 21 April 1944 (14).
Globin production proved highly practical (15),for 700 gm. could be prepared with relative ease from about 3,000 cc. ofpacked red cells; this was the equivalent of 13,000 cc. of plasma or 53 blooddonations. Globin, however, was never put to military use.
IMMUNE SERUM GLOBULIN
A conference on immune globulins was held on 8February 1943 (16) to discuss possible uses for the globulin fractionscontaining immune bodies prepared by Dr. Cohn's group at Harvard, to evaluatethe immunologic data already obtained on various preparations, and to considercriteria for further assay. At an earlier meeting (of the Albumin andBy-Products Group), Dr. Cohn had emphasized the desirability of so fractionatinghuman plasma that the greatest possible use might be made of its byproducts (7).
The data presented to, and the significantconclusions of, the conference may be summarized as follows:
1. Fraction II+III, obtained as a byproduct ofprocessing human plasma and containing most of the beta and gamma globulins, wasshown to contain antibodies that reacted with a variety of infectious agents andwith the isoantibodies of the human blood groups.
2. Fraction II, obtained by furtherfractionation of fraction II+ III, and thus freed of prothrombin, thrombin, andmuch of the undesirable isoantibody, was shown to contain certain antibodiesthat were protective against the viruses of mumps and influenza A, as well as anantibody that inhibited the agglutination of chicken red cells by influenza Avirus and another antibody that reacted with the H antigen of Eberthellatyphosa.
3. The antibody content of fraction IIrepresented a concentration of at least 14 to 16 times that of plasma, thoughnot all the antibody of the plasma had yet been recovered.
4. Further investigations would include: (1)tests of the intravenous use of fraction II, to be run by Drs. Charles A.Janeway and Stokes; (2) tests to determine the cause of the immediate, painfulreactions occasionally encountered, after modification of the final product inrespect to salt concentration; and (3) further experiments on the effect of timeand temperature on the activity of suitably resistant viruses or bacteriophagein the presence of albumin and globulin fractions, to determine whetherinactivation by mild heating was practicable.
5. Preliminary clinical trials of theprophylactic and modifying effect of fraction II + III and of fraction IIstrongly suggested that both would be of value in the prevention or
363
modification of measles, and further trialswith fraction II among military personnel were considered warranted, especiallyfor the protection of troops about to be sent overseas. The risk of inoculatingsoldiers with an unknown virus and producing infectious jaundice was recognized,but it was considered justified in order to achieve mass protection againstmeasles.
For this purpose, 10,000 vials containing 5cc. each of fraction II, prepared according to specifications already decidedupon, would be produced as soon as possible and kept ready for immediate use bythe Army and the Navy. On the authorization of the Acting Surgeon General, Navy,certain commercial firms were already proceeding with the preparation offraction II from stocks of fraction II+III then available on their shelves.These preparations would be tested at the Harvard laboratory and would not bereleased for clinical use until physicochemical tests and tests for immunebodies had been satisfactory. The stocks of fraction II+III at the Harvardlaboratory would also be converted to fraction II.
6. The possible value of fraction II againstmumps, scarlatina, influenza, poliomyelitis and other diseases was discussed butdefinite conclusions were considered justified only in respect to measles.
The commercial production of immune globulinsteadily improved. Some preparations, Dr. Cohn reported on 21 April 1944 (13),contained over 99 percent of gamma globulin, against 85 percent in some ofthe earlier preparations. Because of the improvement, the protein concentrationwas decreased from 20 to 16.5 percent in preparations containing over 96 percentgamma globulin, thus standardizing it at a concentration 25 times that of pooledplasma. After clinical tests proved satisfactory, 0.3 molar solution of glycinewas employed as a diluent.
At the end of the war, gamma globulin wasconsidered effective in preventing measles or in decreasing its severity (17-20).There had been enough experience with it in infectious hepatitis to warrantits consideration as a therapeutic agent (p. 679), but its preventive effect inthis condition and in homologous serum jaundice had not been established. It wasnot successful as a preventive or therapeutic agent in mumps, scarlet fever, orother communicable diseases with the possible exception of anteriorpoliomyelitis.
FIBRIN FOAM AND FIBRIN FILM
The development of fibrin foam and fibrin filmrepresented an extremely important neurosurgical advance, for these materialshelped to solve two major problems outstanding in the field at the beginning ofWorld War II, one connected with hemostasis and the other with the prevention ofmeningocerebral adhesions.
Prothrombin, which was obtained in thefractionation of plasma as a byproduct of fraction II, rapidly loses itsactivity. When it was converted into thrombin, the only form in which its potentcoagulating properties could be exerted, it could be filtered and stored in thefrozen state until facilities were available for preparing it in dry form (21,22). When contracts were let for the preparation of immune globulin, aprovision to this effect was included and thrombin was thus available in amplequantities.
364
Preliminary studies by Dr. Orville T. Bailey,to determine the effect of thrombin on bleeding from the cut surface of theliver in guinea pigs, showed that oozing was reduced by 50 percent (7). Thematerial was then tested as a hemostatic spray at Peter Bent Brigham Hospital,the Boston Children's Hospital, the Hospital of the University ofPennsylvania, and several other institutions, in neurosurgery, surgery of thespleen and gallbladder, tonsillectomy, and other operations. All reports (23)indicated that thrombin was a most effective agent in controlling oozingthat could not be controlled by sutures. The experience of Lt. Col. (later Col.)R. Glen Spurling, MC, at Walter Reed General Hospital, Washington, D.C., was,however, generally confirmed, that neither human nor bovine thrombin gave morethan temporary hemostatic results unless it was supported on some sort of matrix(17, 24).
At the 22 January 1943 meeting of the Albuminand By-Products Group (7), it was agreed that thrombin was now ready forextensive clinical testing. It was also proposed, on the basis of experimentalevidence, that films of fibrinogen and thrombin might prove useful in themanagement of burns in the field because of the simplicity and speed of thetechnique and the small bulk of the material.
It was a great disappointment to find, shortlyafter the war, that thrombin harbored the virus of hepatitis and that thepromising use of fibrin foam therefore had to be discontinued.
Fibrin Foam
At the 13 May 1943 meeting of the Subcommitteeon Blood Substitutes (8), it was reportedthat soluble cellulose manufactured by the Eastman Kodak Co. had been saturated with thrombin, bythe method developed by Dr. Tracy Putnam, and had proved satisfactoryin a small number of clinical cases.
When thrombin first became available, Lt.Edgar A. Bering, Jr., MC, USN (25), and Dr. Bailey had applied it insolution to bleeding points in several cranial and spinal operations. It did noharm, even when it reached the lateral ventricle, but its effect was entirelytransient. Lieutenant Bering then conceived the idea of using fibrinogen,converted into fibrin foam, as a matrix. The dry foam was a light, porous,slightly brittle material, in which the air spaces could easily be seen with thenaked eye. When it was wet with thrombin solution, it became soft, pliable, andsomewhat resilient.
In vitro testing of pledgets of fibrin foamand of soluble cellulose soaked in thrombin solution showed, on the basis ofclotting of fibrinogen solution, that the foam was a much more effective matrixthan cellulose. Solutions of only 10 thrombic units per cc. were necessary withit, against solutions of at least 40 thrombic units with cellulose.
Using monkeys (Macaca mulatta), theseobservers placed fibrin foam saturated with thrombin on traumatized anduntraumatized areas of the cortex and into the cortical substance. Sulfadiazineand penicillin were used locally
365
in some of the animals. Soluble cellulose wasused in one control series and muscle in another, smaller series. The animalswere sacrificed at intervals of 24 hours to 3 months.
The local reaction of the tissues to solublecellulose and to fibrin film was insignificant. Most of the foam had disappearedat the end of a week, and no fragments of it could be identified at the end of 3weeks. The speed of absorption and the nature of the tissue reaction were notinfluenced by the presence of antimicrobial agents. The reaction of the tissuesto muscle was considerably greater.
The first applications of fibrin foam weremade in cases in which bleeding was difficult to control and the application ofmuscle was not feasible. The hemostatic effect was evident even when largevenous channels were opened. The technique was next extended to simple oozingfrom the cerebral surface or the outer surface of the dura. Finally, the foamwas left in place. No traces of it were found on histologic examination orautopsy from 9 to 81 days after it was used.
At a conference of the Albumin and By-ProductsGroup on 17 November 1943 (24), it was reported that fibrin foam had beenused successfully on 60 neurosurgical patients at Walter Reed General Hospitaland on the same number at Peter Bent Brigham Hospital, as well as in severalsmaller series. It was agreed that the material was of extraordinary value as ahemostatic agent in neurosurgery.
By the end of 1943, it had been used in wellover 500 neurosurgical cases. It had also been used experimentally to controlbleeding from the kidney, liver, spleen, lung, and heart, and in a few clinicalcases. It had proved of great value in hemophiliacs, in controlling bleedingfrom traumatic lacerations, and in maintaining hemostasis during minor surgicalprocedures such as tooth extractions.
The demand for thrombin foam was not exceedingthe supply in Dr. Cohn's laboratory, and the conference agreed that the nextimportant step was to produce it on a scale sufficient to permit its widespreaduse. These arrangements were duly made (5). The first contracts, forappraisal purposes, were let by the Committee on Medical Research. Thesubsequent contracts were made by the Navy.
Demands for fibrin foam increased throughoutthe remainder of the war. When Lederle Laboratories reported that it had beensuccessful in filtering contaminated plasma and expected, as a result, to reduceplasma losses from contamination, some anxiety was expressed that there wouldnot be enough substandard plasma available for the production of fibrin foam andthrombin, which were being made from it. The war ended before any such shortagesdeveloped.
In February 1945, it was recommended thatfibrin foam and thrombin, presently in use as nonstandard items, should bestandardized. The length of the dating period had not been determined, but itwas evident that no deterioration would occur as long as sterility wasmaintained.
366
Fibrin foam was packaged with a small vialcontaining dried human thrombin, which, at the operating table, was dissolved in50 cc. of physiologic salt solution (fig. 74). The solution was complete in lessthan a minute if the mixture was vigorously stirred. Portions of fibrin foam,cut in the desired shapes and sizes, were placed in it. As the foam becamesaturated with thrombin, a moderate amount of spontaneous shrinkage occurred.The porosity of the fibrin matrix permitted swift penetration of the thrombinsolution into all parts of the mass.
The following case history is an illustrationof the prompt and effective hemostasis accomplished by fibrin foam.
Case 1.-This patient was received at the45th General Hospital after exploration for a thoracoabdominal wound. Severaldays later, when the large gauze pack in the liver was removed, a hemorrhageoccurred. After several episodes of bleeding, the abdomen was reopened and anumber of clots were removed, along with a necrotic portion of the liver. Thewound, which was on the superior surface of the right hepatic lobe, had to berepacked to check bleeding.
Several additional episodes of bleedingoccurred over the next several days, the blood losses ranging from 200 to 800cc. each time. The longest period without bleeding was the 10 days immediatelyafter the first laparotomy.
The patient gradually lost ground in spite of38 blood transfusions. Attempts to pack the bleeding tract with dried bloodplasma were not successful. Then, after some fibrin foam had been obtained, athird laparotomy was done. After clots and additional necrotic tissue had beenremoved, the wound in the liver was packed with the fibrin foam.
The gauze pack over the foam was removedwithout incident on the fourth postoperative day. The single hemorrhage afterthis operation, on the 10th day, was so slight that there
367
was no change in pulse or blood pressure. Withthis exception, recovery was smooth. The patient was given 4 additionaltransfusions, making 42 in all, with a total of 21,000 cc. of blood.
Fibrin Film
Techniques developed at the Harvard laboratorymade it possible to process fibrin clots into films of any desired size, shape,and thickness (26). These films were translucent, flexible, and elastic,and possessed of considerable tensile strength. Changes in preparation made itpossible to vary the time required for absorption in situ from a few days toseveral months. The films were made in various weights and were of the followingtypes:
P, plain fibrin film.
F, a fibrin film with a fabric backing.
W and WF, types P or F with a waterproofbacking.
The fibrin film used in both clinical andexperimental studies was prepared in flat sheets of various sizes and thickness.The films were sufficiently strong to be sutured without tearing. They could betrimmed to fit the defect and were so elastic that they could easily be fittedover rounded or irregular surfaces, whose contours they assumed. They werecomposed of two parts. The protein part, which made up 20 to 60 percent of thefilms by weight, was at least 90 percent fibrin. When the films were prepared,the remainder was composed of glycerol, but when they were immersed in water orphysiologic salt solution, the glycerol was removed and the water taken up wasregarded as the final plasticizer under these conditions.
The process of dipping fibrin film in hotglycerol required special handling by operators skilled in sterile techniques.In November 1944 (27), an alternative technique was worked out in theHarvard laboratory, by which the material was packaged in a flame-sealed glasstube and sterilized by steam. The final moisture content was not more than 10percent. This method was suitable for large-scale production and yielded aproduct which could be given a much longer dating period.
Before fibrin film was used clinically as acerebral covering for dural defects caused by either accident or surgery, Drs.F. D. Ingraham and Bailey (27) studied its use in monkeys (M. mulatta),applying it to replace the dura mater over both traumatized anduntraumatized cerebral cortex, under bone flaps; after removal of the bone; andwith and without the local application of sulfadiazine and penicillin. Theanimals were sacrificed at intervals ranging from 1 day to 6 months. There wasno physiologic evidence of cortical irritation during any of these periods.Detailed histologic studies revealed no essential tissue changes and noadhesions. As time passed, the film was first surrounded by a small amount offibrous tissue, from which it could easily be separated, and was then replacedby a layer of fibrous tissue about the thickness of the original film. Neitherhealing nor tissue reaction was influenced by the use of sulfadiazine orpenicillin.
368
Fibrin film was first used as a clinical duralsubstitute in a patient with lead encephalitis, who required two operations. Theoriginal film was replaced at the second operation. Nine months afterward, thepatient was in excellent condition.
The original policy was to use fibrin filmonly in such cases as the one just described, in which multiple surgery waslikely to be necessary, and in relatively hopeless conditions, such as braintumors (26-28). Later, it was used in any condition in which dura hadto be removed or the cortex was left unprotected because of the retraction ofnormal dura, as in decompression operations. The film was cut slightly largerthan the defect, and the edges were passed underneath the cut dural margin.
By the end of 1943, Drs. Ingraham and Baileyhad used fibrin film in 44 cases, including 25 intracranial and intraspinaltumors; 8 congenital anomalies; 6 lead encephalopathies; 3 cases of Jacksonianepilepsy; and 2 compound fractures. They had recovered the film for examination18 times, at secondary surgery or autopsy, at intervals of 14 hours to 81 daysafter implantation. In no instance was the slightest evidence of an inflammatoryreaction seen grossly, and there was a striking absence of adhesions. In 10 ofthese cases, the films were examined histologically. The tissue reactions, bothin extent and character, were similar to those already described forexperimental animals.
In 1945, Dr. Ingraham and his associates (28)reported a total of 94 cases in which fibrin film was used; glycerol-treatedmaterial was used in 59 cases, and steam-sterilized material in the remainder.No abnormalities of any kind were evident in the 33 patients followed 6 monthsor more, nor were there any instances of tissue reactions, adhesions, orretarded healing in 19 specimens recovered at a second operation or at autopsy.
Fibrin film was also used successfully in thetreatment of a small number of second and third degree burns. Healing was rapidas in control areas covered with petrolatum-impregnated gauze.
Fibrin film was occasionally used inperipheral nerve suture but gave rise to foreign body reactions in practicallyevery instance.
References1. Cohn, E. J.: The Separation of Blood intoFractions of Therapeutic Value. Ann. Int. Med. 26: 341-352, March 1947.
2. Minutes, Conference on Resuspended BloodCells, and meeting of Subcommittee on Blood Substitutes, Division of MedicalSciences, NRC, 18 May 1945.
3. Minutes, Conference on PlasmaFractionation, Division of Medical Sciences, NRC, 14 Mar. 1945.
4. Minutes, Conference on Albumin andBy-Products, Division of Medical Sciences, NRC, 24 June 1942.
5. Minutes, meeting of Subcommittee on BloodSubstitutes, Division of Medical Sciences, NRC, 2 June 1944.
6. Lamson, P. D., Robbins, B. H., and Greig,M. E.: Studies on Shock Induced by Hemorrhage. X. Hemoglobin Solutions as BloodSubstitutes. J. Pharmacol. & Exper. Therap. 83: 225-234, March 1945.
369
7. Minutes, meeting of Subcommittee on Albumin andBy-Products, Division of Medical Sciences, NRC, 22 Jan. 1943.
8. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 13 May 1943.
9. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 5 Jan. 1944.
10. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 16 Mar. 1945.
11. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 3 Mar. 1944.
12. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 17 Nov. 1943.
13. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 21 Apr. 1944.
14. Minutes, Conference on Globin, Division of MedicalSciences, NRC, 21 Apr. 1944.
15. DeGowin, E. L., Hardin, R. C., and Alsever, J. B.: BloodTransfusion. Philadelphia and London: W. B. Saunders Co., 1949.
16. Minutes, Conference on Immune Globulins, Division ofMedical Sciences, NRC, 8 Feb. 1943.
17. Minutes, Conference of Albumin and By-Products Group;meeting of Subcommittee on Blood Substitutes with Subcommittee on Neurosurgery,Division of Medical Sciences, NRC, 5 Oct. 1943.
18. Minutes, Conference on Production of Normal Human SerumAlbumin and Its By-Products, Division of Medical Sciences, NRC, 19 July 1943.
19. Janeway, C. A.: Clinical Use of Products of Human PlasmaFractionation. I. Albumin in Shock and Hypoproteinemia. II. Gamma-Globulin inMeasles. J.A.M.A. 126: 674-680, 11 Nov. 1944.
20. Blood Substitutes Report No. 18, Committee on MedicalResearch, Office of Scientific Research and Development, subject: Testing ofImmune Serum Globulin (Human) in the Prophylaxis of Measles, 21 Apr. 1944.
21. Minutes, Conference of Albumin and By-Products Group,Division of Medical Sciences, NRC, 28 July 1943.
22. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 24 Sept. 1943.
23. Minutes, Conference on Albumin and By-Products, Divisionof Medical Sciences, NRC, 15 Apr. 1942.
24. Minutes, Conference of Albumin and By-Products Group,Division of Medical Sciences, NRC, 17 Nov. 1943.
25. Bering, E. A., Jr.: Clinical Uses of Products Made FromHuman Fibrinogen and Human Thrombin. Work carried out between Office ofScientific Research and Development and Harvard University on products developedin the Department of Physical Chemistry, Harvard Medical School, from bloodcollected by the American Red Cross. 1943.
26. Ingraham, F. D., and Bailey, O. T.: The Use of ProductsPrepared From Human Fibrinogen and Human Thrombin in Neurosurgery. FibrinFoamsas Hemostatic Agents; Fibrin Films in Repair of Dural Defects and in Prevention ofMeningocerebral Adhesions. J. Neurosurg. 1: 23-44, January 1944.
27. Ingraham, F. D., and Bailey, O. T.: Clinical Use ofProducts of Human Fractionation. III. The Use of Products of Fibrinogen andThrombin in Surgery. J.A.M.A. 126: 680-685, 11 Nov. 1944.
28. Ingraham, F. D., Bailey, O. T., and Cobb, C. A.: The Useof Fibrin Film as a Dural Substitute. Further Studies and Clinical Results.J.A.M.A. 128: 1088-1091. 11 Aug. 1945.
