CHAPTER X
Laboratory Techniques and Special Laboratory
Studies
BLOOD TYPING OF MILITARY PERSONNEL
Implementation of Policy
After considerable discussion of the wisdom of, and necessityfor, the blood typing of military personnel, instructions were issued for thisaction in War Department Circular No. 123, 24 June 1941 (1), by theaddition of changes in AR (Army Regulations) 40-1715, 15 August 1932, and AR600-40, 22 June 1931, as follows:
1. AR 40-1715-Paragraph 7 is addedas follows:
Determination and recording of blood types ofall military personnel.-A. The blood group of each individual on active dutyin the military service will be determined, using the International (orLandsteiner) classification. The proper performance of the tests in eachorganization will be a responsibility of the surgeon. The results will berecorded, using the symbols "A", "B", "AB", or"O", as indicated.
b. The surgeon willbe responsible for the proper recording of the blood group on the identificationtag of each individual tested. An additional record will be made in the case ofenlisted personnel on W.D., A.G.O. Form No. 24 (Service Record) and in the caseof officers or other personnel, on W.D., M.D. Form No. 81 (ImmunizationRegister).
In conformity with this War Department circular, CircularLetter No. 70, Office of The Surgeon General, War Department, was issued on 14July 1941, with the subject: The determination and recording of the blood groupsof all individuals in the military service (2). It dealt with thefollowing points:
1. Materials for blood typing with methods oftheir procurement. The reagents required consisted of mixtures of sucrose anddried sera from rabbits that had been immunized, with human erythrocytes ofgroups A and B, respectively. Medical Department specifications required thatthe potency of the sera be such that when they were used as directed specificmacroscopic agglutination of A, B, and AB human red blood cells, respectively,would occur within a period not to exceed 60 seconds.
2. Interpretation of results.
3. Recording of results. The blood type was tobe recorded on the man's identification tag only after it had been checked bya medical officer against the individual's completed blood grouping test.
4. Performance of the test (a) in small postsor isolated detachments by a surgeon with a few enlisted assistants and (b) itsperformance in larger organizations, on an assembly line basis, with the help ofone or more teams of Medical Department personnel. Details were given for eachstep of the procedure.
The letter pointed out that an error in the technique of thetest or in the recording of the result could be extremely serious and might evenresult in a fatality.
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Supplemental Instructions
4 September 1941-Only a brief experience with thetyping of military personnel brought to light difficulties and errors. CircularLetter No. 88, Office of The Surgeon General, War Department, was issued on 4September 1941, with the subject: Supplemental information concerningdetermination of blood type in accordance with SGO (Surgeon General's Office)Circular Letter No. 70 (3). It covered the following points:
1. Variations in temperature and concentrationof the red cell suspension in tests run in the field necessitated some latitudein the time allowed for agglutination (specified in the original directions asnot to exceed 1 minute). This variation was to be determined by the medicalofficer in charge of the team.
2. All normal saline solution used in typingmust be prepared from sodium chloride, A.C.S. (Item No. 14290, MedicalDepartment supply catalog), thoroughly desiccated and accurately weighted.Tablets intended for the preparation of normal saline solution (Item No. 13020),apparently due to some inhibiting substance in their content, had been found todelay or inhibit the test.
3. The precise amount of serum indicated wasto be used; an excess delayed agglutination.
4. If dried sera became so tightly packed inthe original containers that difficulty was experienced in measuring out thesmall amounts required for individual tests, it could all be put into solutionand a specified amount of the solution used.
26 November 1941-Circular Letter No. 112, Office ofThe Surgeon General, War Department, issued on 26 November 1941, subject:Supplemental information, concerning determination of blood type in accordancewith SGO Circular Letters Nos. 70 and 88 (4), reflected additionalexperience. In this letter, the objective of blood typing of military personnelwas defined-it had apparently not been clearly understood before-as makingpossible the calling of voluntary donors of a specific blood type and securingthem on very short notice. The following points were also covered:
1. The possibility of error in mass bloodgrouping was recognized, but it was postulated that crossmatching would be donebefore transfusion, and errors would thus be recognized. This precaution,however, was no reason for relaxing efforts to be absolutely accurate in testingand recording.
2. Insufficient time was sometimes beingallowed for proper agglutination. As a result, blood types other than O,particularly the AB group, were being recorded as O. Such errors could beprevented if all tests in which agglutination did not occur promptly were heldfor observation for 20 minutes or longer.
3. Any validated evidence of apparentlyunsatisfactory sera should be reported to the Army Medical School, so that itcould be determined whether the sera were really unsatisfactory or technicalerrors were responsible for the poor results.
2 December 1942.-Still further clarification wasattempted in Circular Letter No. 170, Office of The Surgeon General, WarDepartment, Services of Supply, 2 December 1942, subject: The purpose of bloodgrouping Army personnel (5). In this letter, it was pointed out that thepolicy of blood grouping of military personnel and its purpose seemed to havebeen misunderstood by some medical officers. Some reports of alleged errorssuggested that
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the writers believed the errors would inevitably lead toserious reactions if the persons whose blood was wrongly classified should beused as either donors or recipients. The assumption that the first typing testwould be the only one performed was based on a false premise. To correct themisconception, it was pointed out again that the purpose of the program was tosimplify assembling donors whose blood would probably crossmatch with that ofintended recipients. It was never intended, when the mass grouping program wasset up, that crossmatching should be omitted.1
Errors would be reduced, the letter continued, by care in allsteps of the grouping process and in recording of results. Some stations werepreparing their own grouping sera from tested human donors with high titerserum, but this was not recommended, at least as a routine.2
The following precautions were also emphasized:
Slides should be observed long enough for slower reactions todevelop, though no arbitrary period of observation could be specified because ofvariations in temperature, serum titer, degree of mixing of serum and cells, andthe relative agglutinating ability of the cells. Various expedients to provide arough check had been tried: The test could be done on groups of 20 to 25 men ata time and no results recorded until half of the slides showed agglutinationwith anti-A serum. Or no results except in the AB group should be recorded untilthe slides had been under observation for 20 minutes, even though mostagglutinations were evident within a minute or two. Observations up to 30minutes were desirable if excessive drying could be avoided.
Some blood grouping teams observed all slides microscopicallyunless macroscopic agglutination was evident. Other teams repeated the tests onall blood that gave no agglutination with anti-A serum; that is, they repeatedthe tests on all persons originally grouped as B or O. This was regardedofficially as an unnecessary precaution, not worth the effort if the originaltests had been carefully performed.
Mass Methods of Typing
Necessary as it was, the blood grouping program was one morething to interfere with the training of troops. Many installations thereforedevised their own methods of expediting the procedure. In some, unfortunately,the haste led to confusion, and the confusion led to errors, a certainproportion of which could unquestionably be explained in this way. In otherinstallations, the short cuts were really efficient.
1In spite of errors in the laboratory, the blood grouping program was highly practical. If 100 group O donors were desired, only men whose identification tags were so marked would report. The chances were that, after retyping, 85 to 90 would prove to be group O. Without the preliminary screening, it would have been necessary to call at least 200 prospective donors to find approximately 100 group O donors.
2For some reason, the responsibility for the original typing program was assumed by the Preventive Medicine Division, Office of The Surgeon General, and was retained by it throughout the war. It would obviously have been more efficient for the personnel directly in charge of the blood program to have supervised this part of it.
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National Research Council
The matter of typing military personnel was brought up at themeeting of the Subcommittee on Blood Substitutes on 23 May 1941 (6), whenthe policy was still under consideration. The experience of the BloodTransfusion Association in New York (p. 13) had indicated that the rabbit serapresently in use were not so potent as they should be, but it was expected thatmore avid material would shortly be available.
When the subcommittee met on 18 July 1941 (7), typingof all military personnel on active service had been authorized (p. 233), andsubstantial contracts for rabbit serum had been let with the only firm thenprocessing it. In view of the circumstances, the subcommittee considered thatany action in the matter was outside of its jurisdiction. A year and a halflater, in January 1943 (8), it completely reversed this attitude andinterested itself in the development of new and more avid sera.
DEVELOPMENT OF TYPING SERA
Types of Sera
Rabbit sera.-The anti-A and anti-B rabbit seraavailable at the beginning of the war were, as just indicated, not so avid asthey should have been. Tests by the DeGowin technique (9) showed that Atiter agglutinated cells in dilutions up to 1,000 but not in dilutions up to2,000. The B titer was strong; agglutination could be obtained in dilutions upto 1:4,000 though not up to 1:8,000. These sera had two advantages, that theydid not need refrigeration and that the titer was so high that expert skill wasnot necessary to use them. They also had a serious disadvantage, that they werehygroscopic and, when exposed to air, took up moisture.
In the beginning, when material could be secured only from asingle laboratory, there was not very much that could be done. As time passed,the rabbit sera improved, and, even when globulin fractions were available toprepare more avid sera, rabbit sera were still considered satisfactory and theArmy continued to use them throughout the war. Each lot purchased was tested atthe Army Medical School, which was a relatively simple matter: Techniques forpreparation of sera produced relatively large lots, all units of which couldreasonably be expected to be identical. Tests on a single unit therefore gaveinformation which could be applied to all units in the lot. As a practicalmatter, it was recommended that samples of each pool be submitted forexamination before the material was packaged, so that, if they did not meetspecifications, they could be fortified with more active material and submittedagain for appraisal. The production of satisfactory sera depended upon rigidadherence to all details of processing, including temperature controls, proteinconcentration, and salt content.
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Work at the Army Medical School showed that an increase inthe salt component of the serum-cell suspension mixture resulted in more rapidagglutination. After many tests, it was found that a 1.4-percent concentrationof sodium chloride in the final mixture was optimum, and the original directionspacked with dried rabbit sera were altered to include this information.
Human sera-One of the early activities of the Divisionof Surgical Physiology, Army Medical School, was an attempt to improve theavidity of the grouping sera then in use. Its personnel, working with theChemistry Division of the school, demonstrated that very avid and very hightiter grouping material could be prepared from plasma by separation andconcentration of the globulin fraction containing the isoagglutinins. Anti-A andanti-B globulins were produced as byproducts of the fractionation process andwere available in large quantities because of the contracts for albumin let bythe Navy.
The original work on plasma fractionation had been done inDr. Edwin J. Cohn's laboratory at the Harvard Medical School, and, in December1942, at the request of Col. George R. Callender, MC, and Lt. Col. Douglas B.Kendrick, MC, Capt. John Elliott, SnC, and Lt. Louis Pillemer, SnC, were placedon temporary duty there, to develop a new technique for preparing typing serafrom human plasma. The method developed called for preparation of albumin andits byproducts from pools of plasma made up exclusively of A bloods or B bloods,it having been found that the appropriate globulin fractions from such poolsordinarily contained highly potent blood grouping substances.
At the meeting of the Albumin and By-Products Group on 22January 1943 (8), Dr. Cohn reported on Lieutenant Pillemer's work. Thetechnique he had developed was closely related to the fractionation processdevised at Harvard, but it employed methyl alcohol, since the low temperaturesrequired with ethanol were not required with this precipitant. The demonstrationthat isoagglutinins could be prepared from both type A and type B bloods showedthat they could be prepared as byproducts of the large-scale industrialpreparation of albumin. They were concentrated in fraction II+III, and if theeuglobins were separated from the pseudoglobulins, the isoagglutinins would befound in the euglobin fraction. With this technique, sufficient typing solutionto carry out an enormous number of typing tests could be prepared from arelatively small amount of plasma.
The separated, concentrated material prepared by the Pillemertechnique lent itself well to blood grouping purposes. While it was not soviscous as whole serum, it possessed sufficient surface tension to formwell-rounded droplets on a glass slide. The addition of Merthiolate to a finalconcentration of 1:1,000 did not interfere with the interactions ofisoagglutinins and red cells and eliminated the necessity for filtering outbacteria. The isoagglutinating activity of the separated globulins stored as aliquid at room temperature (77? F., 25? C.) remained unimpaired for 4 weeks.
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When the macroscopic slide technique was used, theconcentration of this serum could be so adjusted that agglutination withincompatible erythrocytes occurred visibly in 5 seconds and was complete at 60seconds.
DeGowin Technique
At the 23 March 1943 Conference on Blood Grouping (9),Dr. Elmer L. DeGowin recommended the following technique, which he had employedsatisfactorily on more than 4,000 bloods:
The slides used were made of double-thickness window glass,with the edges ground smooth by a suitable stone. Areas 1 by 3 inches weremarked out on the slides with a glass cutter or wax pencil, or by sprayinglacquer over mask paper. Each of the 20 such areas on each slide was marked withthe number of the blood specimen to be examined.
Three medicine droppers of similar bore were used, two forthe typing sera and the third for cell suspensions. The third dropper was washedin physiologic salt solution after each use. Typing sera could be either rabbitor human but must have a titer of at least 1:128.
Drops of sera were placed in the proper areas on each plateand the cell suspensions added. The time of the test was recorded on the platewhich was set aside to be read in 30 minutes. In the interim, the plate wastilted a few times, to disturb the sedimented films, and it was also checked forTyndall's phenomenon.
Two independent tests were made of the same bloods and theresults of the two series were compared. Additional tests were made on thebloods in which discrepancies were found.
By this technique, which was generally the technique followedin the Army, one worker, with a minimum amount of laboratory equipment, couldset up and read over 100 tests an hour.
Establishment of Criteria
At the Conference on Transfusion Equipment and Procedure on 25August 1942 (10), it had been unanimously recommended to the American RedCross and to the Surgeons General of the Army and the Navy that when largenumbers of bloods were to be tested, the following precautions be taken:
1. Two independent series of typings should beperformed on the same bloods. If discrepancies were discovered when results werecompared, the affected subjects should be reexamined.
2. Only typing sera of high titer should beused.
3. Only freshly prepared red cell suspensionsshould be used.
4. The utmost care should be exercised in therecording of results.
5. The slide method, with reading in 30minutes at room temperature, was faster than the centrifuge method for masstyping and was quite as accurate if sera were potent.
These recommendations were accepted and put into practice.
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At the Conference on Blood Grouping on 23 March 1943 (9),Lieutenant Pillemer reported continuing increases in the potency of the seraprepared by his technique; a number of other observers confirmed his statement.
The following standards for typing sera, both human andrabbit, were suggested, but no formal action was taken on them:
1. Sera should have a macroscopic titer of atleast 1:100.
2. Clumping should begin within 20 seconds,and agglutination should be complete within 60 seconds.
3. Anti-A sera should be sensitive for A2and A2B cells andshould react definitely with other rare subgroups of A.
4. Negative reactions should be clear cut.
5. Keeping qualities of the sera, whichpreferably should be dried, should be defined.
6. If possible, a central authority shouldpass upon the quality of sera intended for distribution.
At this conference, special emphasis was placed upon theimportance of accepting rabbit antisera for typing only if it met the samestandards as those laid down for human serum. A good deal of rabbit sera thathad been examined had not been satisfactory. In some lots, the anti-B serum hadnot exceeded the titer of average human serum and absorption had beeninadequate. One lot had been too weak to produce any agglutination at all.Another lot, evidently because of unsatisfactory absorption, had shown crossreactions with group A cells. Dr. Philip Levine, Beth Israel Hospital, Newark,N.J., thought that if rabbit sera were used, a stipulation must be made thatthey must be absorbed with group A or group B cells rather than group O cells.Dr. Ernest Witebsky, who had been able to immunize rabbits with salivacontaining A or B specific substances, pointed out that this technique had theadvantage of not producing the species-specific agglutinins which requiresubsequent absorption.
In 750 bloods examined by the technique described by themanufacturers of rabbit sera, there had been an error of 10 percent, but therewere no errors at all when the same sera were used by the DeGowin technique. Itwas concluded that a considerable number of errors could be explained by thefact that the manufacturer's directions were simply not specific enough.
Universal Donors
It was tentatively proposed at the 23 March 1943 Conference onBlood Grouping (9) that universal donors be employed for the ArmedForces, with the following specifications:
1. Group O blood should be used only when titration indicatedthat the agglutinins in the plasma were weak.
2. Crossmatching must never be omitted, because someagglutinins act on O cells.
3. A biologic test should be employed; that is, 50 to 100 cc.of donor blood should be injected into the recipient, and his plasma before thetransfusion should be compared with his plasma 1? hours afterward for evidenceof
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hemolysis. (This specification was quickly discarded when itwas pointed out by the chairman of the conference that biologic tests would beentirely impractical in frontline hospitals.)
The vigorous discussion that followed the proposal torecommend group O blood for the Armed Forces covered the following points:
1. The determination of blood groups byidentification of the agglutinogens with known sera must be further checked bymatching unknown sera against known cells.
2. The proposed checks on cells and plasmashould be made with venous blood. This proposal was considered unnecessary,since enough blood for testing could be secured from the lobe of the ear.
3. There was a wide difference of opinion asto the most common typing errors and which bloods they concerned. After severalproposals to avoid these errors had been made, Dr. DeGowin pointed out that themost expeditious procedure would be to make two independent determinations ofthe bloods. This plan, he believed, would have the further advantage of makingit unnecessary to deal with agglutinins of weak titer in unknown sera.
4. It was agreed that the centrifuge techniquewas preferable when only a few bloods were to be examined but that it wasimpractical with large numbers, when it would impose too great a burden inrespect to both time and equipment. It was granted that rouleaux formation wassometimes confusing when the slide technique was used.
5. Captain Elliott had observed that a periodof more than 20 minutes, with constant agitation (which was practical onlymechanically) was necessary to secure agglutination in some rare bloods. Dr.William Thalhimer used plate glass slides, which were agitated constantly, andread at the end of 30 minutes. Dr. DeGowin had found 30 minutes withoutagitation sufficient if the sera employed had a titer of at least 1:128. Othersagreed that the time required depended upon the avidity of the sera used.
6. There was no agreement as to the advantagesof macroscopic versus microscopic observation. Some workers considered both werenecessary. Others considered microscopic observations necessary only whenpersonnel were relatively untrained. Still others thought that, in traininglaboratory technicians, more errors were committed with the microscope thanwithout it.
7. Dr. Alexander S. Wiener reported compositeobservations to the effect that transfusions with incompatible blood resulted inan increase in the titer of the natural agglutinins of the recipient. Bloodstroma (cells and fibrinogen) had proved nonantigenic on injection. Aftertransfusion of 250 cc. of pooled plasma (Sharp & Dohme), the increase in therecipient's agglutinin titer had been from 5- to 10-fold. To obtain themaximum titer, the serum must be withdrawn no sooner than 10 to 14 days afterthe transfusion; otherwise, potency was lost rather quickly.
Testing and Acceptance
At a meeting of the Albumin and By-Products Committee on 10May 1943 (11), Dr. Cohn pointed out that continuous improvements had beenmade in the concentration of isohemagglutinins, and presumably more could bemade, but the question, for practical purposes, was how much more improvementwas necessary. He suggested that Dr. DeGowin and Colonel Kendrick act as acommittee to examine the various products and report on their relative values.
At the meeting of the Subcommittee on Blood Substitutes on 13May 1943 (12), Dr. DeGowin reported results of titration of variousglobulin preparations to be at wide variance. He thought it plain that allcooperating workers must adopt uniform techniques and suggested thatmimeographed
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instructions be prepared and sent to all workers, specifying methods to beused and criteria to be employed in evaluating agglutination tests.This was done.
At the 10 August 1943 meeting of the subcommittee (13), Dr. DeGowinannounced that the various workers who had been evaluating Lieutenant Pillemer'sserum had agreed on criteria of potency. New preparations had been sent to themfor evaluation by these criteria. It had also been agreed by these workers thata large amount of the isoagglutinin preparation should be processed and, ifaccepted as satisfactory, should be dried, packaged, and used as"reference" serum. Other sera could be compared with it directly, thuseliminating many of the variables of sensitivity of test erythrocytes anddifferences in titration techniques.
At the meeting of the subcommittee on 24 September 1943 (14), Dr. Cohnreported that reference sera had been prepared in quantity and were being heldin bulk awaiting the report of the evaluating workers, as well as instructionsconcerning packaging. Six lots had already been accepted as approximately equalin potency.
When the subcommittee met on 17 November 1943 (15), Colonel Kendrickreported that all necessary steps in the development of the new sera had beencarried out. Both the Lederle Laboratories and Eli Lilly and Co. were producingthem; Lieutenant Pillemer had been sent to the Lederle Laboratories and CaptainElliott to both firms to instruct their personnel in the new technique. Initialdifficulties had been eliminated. Dr. Cohn pointed out that, while the presentmethod of preparation was not entirely satisfactory, it was the best available,and that uniformity of a product was seldom attained in the first few runs.Captain Elliott emphasized the value of testing the material at each stepin the process, so that, if any change in avidity occurred, it could be detectedat once.
Dr. Cohn had emphasized, at an earlier meeting (14), that commercialfirms must be as rigidly supervised in the production of sera as of otherbyproducts of plasma fractionation. At still another meeting (16), he hademphasized the practical importance of making sure that the method currentlyused in the preparation of isoagglutinins would give a reproducible product. Hewould consider the responsibility of his own laboratory ended in regard totyping sera when satisfactory reference material had been prepared.
The Army Medical School and the Navy used these sera throughout the remainderof the war. The Army did not utilize them until after the war had ended.
TYPING ERRORS
The Red Cross Experience
The original plan to type donors at the processing firms, at the expense ofthe American Red Cross, had been adopted with the full realization that it wouldbe costly (17). It was discontinued as of 1 November 1942, because
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the great expansion of the program made it impossible to secure thetechnical help necessary to carry out the tests accurately (18).
Shortly after the institution of the typing program, errors began to bediscovered, and at the meeting of the Subcommittee on Blood Substitutes on 12May 1942 (19), Dr. G. Canby Robinson asked for guidance on the course tobe followed at the blood donor centers. It was recommended that a statement beadded to the cards given to the donors after each donation to indicate that theblood should be regrouped and crossmatched before all transfusions. Thisrecommendation was put into effect.
Controlled Studies
At the next meeting of the subcommittee on 23 June 1942 (20), too fewdata had been collected concerning techniques at the various processinglaboratories (as had been recommended at the previous meeting) to be useful. Dr.DeGowin, however, outlined a very promising experiment, undertaken shortlybefore, whereby the blood groups of cadets at the U.S. Naval Preflight School atIowa City were being checked by two workers independently, in lots of 40 to 93specimens. In his opinion, the personal equation was such that no technician waslikely to perform large numbers of tests without making occasional errors. Apharmacist's mate, well trained in laboratory work, was making one seriesof tests and Dr. DeGowin was making the other. His technique (p. 238) was usedin both series.
The final report on this study, which concerned 3,876 bloods, was made at theConference on Blood Grouping on 23 March 1943 (9) (table 6). The errorsin 24 tests were unassigned because there was no opportunity to check the bloodsinvolved. In the remaining tests, one worker made 40 errors (1.0 percent) andthe other, 110 (2.8 percent). "None of the errors," said the finalreport, "could have been detected except by comparison of the results oftwo independent tests on the same blood."
At this conference and at previous meetings (9, 20), at which preliminary reports of this study had been made, the sources of these errors were discussed. Both workers made more errors (double the number) when larger numbers of bloods were examined. Many errors were made when the two workers used the same typing sera; they could be attributed to minor variations in technique; transposition of specimens; errors in transcription; confusion in reading results, such as failure to detect agglutination or reporting false agglutination; the use of too thin cell suspensions; or the addition of insufficient serum to the suspension.
An analysis of the errors threw considerable light upon the reasons some of them had been made. All of the transpositions were made by one worker, who had apparently developed habits that facilitated the selection of the wrong tube from the rack or who had neglected opportunities for checking the labels. The same worker made most of the mistakes in the identification of A and B blood. Most of the false agglutinations were reported by the worker who used
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TABLE 6.-Errors in blood grouping determinations made bytwo independent workers on identical blood specimens
| Date 1942-43 | Number of bloods | Author (DeGowin) | Navy | |||||||||
| Missed aggluti-nation | False positive | Trans-position | Clerical | Total | Missed aggluti- | False positive | Trans-position | Clerical | Total | Un-assigned errors | ||
| 6/9 | 140 | 1 | --- | --- | --- | 1 | 2 | --- | --- | --- | 2 | --- |
| 6/10 | 135 | --- | --- | --- | 2 | 2 | --- | --- | --- | --- | --- | --- |
| 6/11 | 135 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 6/12 | 1192 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | 3 |
| 6/26 | 1286 | --- | 7 | --- | --- | 7 | 1 | 1 | 7 | --- | 9 | 1 |
6/29 | 139 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 6/30 | 137 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 7/3 | 138 | --- | --- | --- | --- | --- | --- | --- | --- | 2 | 2 | --- |
| 7/7 | 143 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 7/8 | 129 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 7/10 | 1164 | 4 | 1 | --- | --- | 5 | --- | --- | --- | --- | --- | --- |
| 7/24 | 1163 | --- | --- | --- | --- | --- | 2 | 4 | 1 | --- | 7 | --- |
| 8/9 | 2185 | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | 8 |
| 8/22 | 2186 | 12 | --- | --- | --- | 12 | 1 | --- | --- | --- | 1 | --- |
| 9/5 | 2184 | 1 | --- | --- | --- | 1 | 5 | --- | --- | --- | 5 | --- |
| 9/19 | 2202 | --- | --- | --- | --- | --- | 1 | --- | 2 | --- | 3 | --- |
| 10/3 | 2198 | --- | --- | --- | --- | --- | 3 | --- | --- | --- | 3 | 3 |
| 10/17 | 2432 | --- | 1 | --- | --- | 1 | 12 | --- | --- | --- | 12 | 9 |
| 1/11 | 1335 | 1 | --- | --- | --- | 1 | 2 | 1 | --- | --- | 3 | --- |
| 1/23 | 1300 | --- | --- | 1 | 7 | 8 | 2 | 1 | --- | --- | 3 | --- |
| 1/31 | 130 | --- | --- | --- | --- | --- | 1 | --- | 1 | --- | 2 | --- |
| 2/12 | 1212 | --- | --- | --- | --- | --- | 3 | --- | --- | --- | 3 | --- |
| 2/18 | 1146 | --- | --- | 1 | --- | 1 | 8 | --- | --- | --- | 8 | --- |
| 3/2 | 1365 | --- | 1 | --- | --- | 1 | 27 | 18 | 2 | --- | 47 | --- |
| Total | 3,876 | 19 | 10 | 2 | 9 | 40 | 70 | 25 | 13 | 2 | 110 | 24 |
1Same typing sera employed by both workers.
2Rabbit sera employed by author, human sera by Navy.
KEY: Missed agglutination-errors due to failure to note orto demonstrate agglutination with either anti-A or anti-B sera, or both. Falsepositive-errors due to reporting of agglutination that could not bedemonstrated on recheck. Transposition-errors due to reporting of group A forgroup B or vice versa, usually as the result of confusing the right and leftsides of the slide. Clerical-errors committed in transcription of results.Unassigned-errors apparent as discrepancies in reports of the two series butnot rechecked because second blood specimens could not be obtained. Bloodsreported in all assigned errors were rechecked by potent typing sera from atleast two human sources and frequently by rabbit sera as well.
sedimented cell suspensions. The use of 3-percent physiologic salt solution,as recommended by Hans Sachs, tended to produce pseudoagglutination insedimented cells, particularly when microscopic readings were attempted.
The slide and centrifuge techniques proved equally accurate, but thecentrifuge technique, while faster for single examinations, consumed more time
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than could be spent in mass typing. It took more time tomanipulate the tubes and centrifuge, required more glassware, and required timeto clean the glassware. With strong sera, it was useful to use the slide methodat room temperature, reading the results at the end of 30 minutes with a minimumof agitation.
This type of study did not reveal errors due to defective orweak sera; errors in labeling of the original cell suspensions; or errors intranscribing results from laboratory reports to notification cards, formalreports, and identification tags.
The errors detected were also, of course, entirely unrelatedto later errors made in releasing incorrect bottles of group-specific blood fromthe storage refrigerators and administering the wrong bottles of blood torecipients. These administrative errors seldom occurred in blood banks operatedunder the control of physicians and other workers well trained in laboratorytechniques and interested in blood banking as a medical specialty.
The Subcommittee on Blood Substitutes found Dr. DeGowin'sreport extremely significant. Probably both workers made fewer errors than theywould have made ordinarily since the planned comparision of results served as aconstant stimulus to careful work, a factor which would be absent if there wasno parallel grouping. Dr. DeGowin was eminently skilled in this work. Thewell-trained pharmacist's mate was undoubtedly supercareful during the periodin question.
In view of the errors made in this controlled study by askilled physician and an extremely skilled worker, the members of thesubcommittee found it alarming to consider the percentage of errors thatprobably had been made in the mass typing performed on Army and Navy personnel.By this time (March 1943), there must have been, by conservative estimate, atleast 100,000 men in the Army alone who were erroneously typed. The situationwas not dangerous if all medical officers who gave transfusions clearlyunderstood that the blood type stamped on the identification tag was simplytentative. In the pressure of an emergency, it was feared that some might omitcrossmatching.
The Armed Forces Experience
Reports from various military organizations could be cited toindicate the percentage of error in recorded blood groups of military personneland also, fortunately, the realization of those in authority that such errorsexisted.
On 4 March 1943, for instance, Maj. Gen. Paul R. Hawley wasinformed of grouping errors (6 percent) that had been discovered in the 10thStation Hospital, then in Northern Ireland (21). If the margin of errorwas as high in other units, the commanding officer wrote General Hawley, thematter required attention because of the risk of placing undue reliance on theinformation on the casualty's identification tag if emergency transfusionshould be required.
General Hawley immediately ordered sample checks of otherinstallations.
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As a result, checks were made of practically all the patientsthen in four general hospitals, four station hospitals, and one evacuationhospital, the numbers ranging from 810 at a general hospital to 83 at a stationhospital. It was found that 154 of the 2,340 bloods examined had beenincorrectly typed. In addition, 179 patients had no identification tags, andthere was no record of the blood group on 33 tags. The investigation thusrevealed 366 unsatisfactory records, 14.34 percent. The largest observed errorwas in group B, but the total error was not related to any separate blood group.
Lt. Col. (later Col.) John E. Gordon, MC, Chief, PreventiveMedicine Division, Office of the Theater Chief Surgeon, who had conducted theinvestigation, recommended to General Hawley that the Office of The SurgeonGeneral be advised of the error that had been discovered, that all medicalofficers in the command be advised of it and be warned that direct crossmatchingmust be carried out before every transfusion, that command action be taken toassure the completion of required data on identification tags, and that measuresbe instituted to insure the constant wearing of tags by all members of thecommand. All of these recommendations were implemented.
The experience just described was typical of that of otherhospitals in the theater and in other theaters, but, once the error was realizedand bloods were regrouped routinely before transfusion, it did no real harm.
The 5- to 10-percent error in blood grouping was unfortunateand undesirable, but it might have been expected for a number of reasons: thelack of avidity of the typing serum, the utilization of antibody from rabbitserum that was not always as good as it might have been, and the inexperienceof the personnel who did the typing. One source of errors has already beenintimated, the fact that in many camps and posts during the war, personnelresponsible for mass typing, through a mistaken sense of values, placed high ontheir priority list the speed with which the typing was done. Speed led toconfusion, and confusion produced errors, which were compounded by the lack ofexperience of those doing the typing.
THE RH FACTOR
Historical Note
It is an interesting commentary, on what was known of bloodat the beginning of World War II, that the book on blood banks and transfusionsby Kilduffe and DeBakey (22), which was published in 1942 and was asauthoritative as such a text could be in changing times, contains no entry inthe index for the Rh factor.
It was not until 1940 that the cause was found for theoccasional hemolytic reactions that occurred when blood was properly classifiedand crossmatched. In that year, Landsteiner and Wiener (23) demonstratedthat incompatibility could occur within the same blood group in whichcompatibility had been
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proved. By injecting rabbits or guinea pigs with red bloodcells obtained from the Macaca rhesus monkey, they obtained a serum whichwould agglutinate 100 percent of rhesus monkey cells and with which theydemonstrated what they named, for obvious reasons, the Rh factor.
Further studies showed that a large proportion of all humanbeings, regardless of their basic A, B, AB or O blood groups, have Rhagglutinogen in their red blood cells. It is inherited as a mendeliandominant in about 87 percent of white persons, 95 percent of Negroes, and 100percent of Chinese. Those who do not have it naturally occasionally becomeisoimmunized with it if they are transfused with Rh-positive blood. It was soonrealized that the important aspect of this new discovery was not the group whichhad the Rh factor in their red cells but the smaller group who did not, and whomight become immunized when it was introduced into the bloodstream.
Two other discoveries were also made. The first was that whenan Rh-negative woman becomes pregnant with a fetus whose cells are Rh-positive,she will probably become immunized against the positive cells. The seconddiscovery was that when Rh-negative recipients received multiple transfusions ofRh-positive blood, even though it was group-compatible, hemolytic reactions ofincreasing severity might occur.
Military Significance
The second discovery concerning the Rh factor was of greatmilitary importance, since, as time passed, it became more and more the practiceto give multiple transfusions to combat casualties after injuries and topatients with chronic sepsis. The prospects were that this practice wouldincrease. From the military standpoint, the chief significance of the formationof anti-Rh agglutinins by an Rh-negative individual was that subsequenttransfusions of Rh-positive bloods might lead to increasingly severe, andeventually fatal, reactions.
By 1943, enough such instances of Rh immunization hadoccurred at the Walter Reed General Hospital, Washington, D.C., where thepatients could be observed directly by the transfusion personnel at the ArmyMedical School, to make the potential seriousness of this threat very clear. Bythe end of 1944, certain conclusions were possible (24):
1. Minor reactions usually preceded serious reactions andwere likely to be mistaken for ordinary pyrogenic reactions because they werecharacterized only by chills and fever. They might occur before very much bloodhad been given and thus serve as an indication for stopping the transfusion. Inmilitary practice, this necessity was extremely unfortunate because transfusionswere so often lifesaving.
2. The in vivo survival of transfused Rh incompatible cellswas poor.
3. Reactions to Rh incompatibility did not occur innonimmunized patients. Therefore, the first transfusion, or even a series ofclosely spaced
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transfusions, could often be given without untoward results.After the initial transfusion of incompatible Rh blood, antibodies which hadformed in the recipient's circulation in small amounts might react immediatelywith the specific Rh (or Hr) antigen in the transfused cells and become soneutralized that none remained in the circulation. For this reason, although therecipient of the incompatible blood had become immunized, reactions in latertransfusions would not occur until there had been a sufficient interval betweentransfusions to permit the removal of the antigen (that is, the transfusedcells) and the accumulation of sufficient antibody to cause incompatibility.
4. The severity of the reaction would depend upon the amountof antibody in the recipient's blood. There might not be enough to cause evena mild reaction but there also might be enough present, or enough might developlater, to cause a fatal reaction, because of lysis of the transfused cells overa period of a few days to a few weeks. While little lasting benefit would beattained by the transfusion, the major important result would be the fatalhemolytic reaction that might occur in an Rh-negative individual because anti-Rhagglutinins were present in his bloodstream.
When Captain Elliott visited the Continental Blood Bank inParis in January 1945, he was particularly impressed with the low rate ofreported transfusion reactions (25). Maj. Robert C. Hardin, MC, hereported, did not agree with those who feared reactions on the basis of the Rhfactor. From reports available to him, Major Hardin thought that the incidencein multiple transfusions was not more than 0.6 per thousand. In his opinion, itwas far better to continue to put the emphasis on indications for transfusionand to attack the problem of pyrogenic reactions and hemolytic reactionsresulting from the A and B agglutinin system, which were at least three timesmore common. He did not mean that the Rh factor should be ignored, merely thatit should be kept in the proper perspective, which it would not be if emphasison it were permitted to obscure the primary transfusion problems in the Europeantheater.
In his report on this trip, Captain Elliott discussed the Rhfactor in military medicine as follows:
The natural incidence of Rh agglutinogen in the general whitepopulation meant that one out of every eight white recipients of Rh-positiveblood might become isoimmunized. Since, however, donors were selected at random,it could be assumed that 13 percent would be Rh-negative, which would make theproportion of Rh-incompatible donors 1:10 instead of 1:8. Since approximately 7out of every 8 random recipients of random bloods would be Rh positive and 1 outof every 8 random donors would be Rh negative, approximately 1 of every 10positive recipients would be isoimmunized by transfusion of blood fromRh-negative donors.
In the event that Rh-negative recipients and donors were notidentified and specific Rh blood was transfused, it was entirelypossible, Captain Elliott continued, that some 10 percent of Rh-positiverecipients and an equal propor-
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tion of Rh-negative recipients would become isoimmunized.This did not mean, however, as on first glance it might seem to mean, that 20percent of recipients would have reactions caused by Rh incompatibility. What itdid mean was that, at some time interval after the first transfusion or after aclosely spaced series of transfusions, subsequent transfusions might causenumerous mild reactions, some serious reactions, and a few fatal reactions.Moreover, because of the rapid destruction of transfused Rh-incompatible redcells after the formation of Rh or Hr antibody, such recipients might requiremany more transfusions than if Rh-compatible blood had been used. The warning oftrouble would be reactions of progressively more serious nature with everysubsequent transfusion.
Provision of Rh Testing Serum
Multiple spaced transfusions were usually given in rearhospitals and in hospitals in the Zone of Interior. It would be necessary,therefore, to provide every such hospital with enough potent Rh testing serum toprevent such reactions by the use of Rh-negative blood for Rh-negativerecipients, this being the only sure way of avoiding them. Rh testing was alsoessential in military hospitals that operated an obstetric service, since allwomen who gave birth to erythroblastotic children were liable to fataltransfusion reactions if they were transfused with Rh-positive blood. The use ofsuch serum would also help to prevent or modify erythroblastosis in infants andpermit its correct treatment when it occurred.
When the military significance of the Rh factor began to beappreciated in 1943, there was not enough Rh testing serum available to supplyeven a small proportion of the Army hospitals that should have it. Attention wastherefore directed toward making supplies of it available, and towarddetermining when it should be used.
Indications for Rh testing-The Rh factor was firstdiscussed by NRC (National Research Council) personnel at the Conference onBlood Grouping on 23 March 1943 (9). The matter arose in connection withthe technical manual then in preparation and how much concerning the Rh factorshould be included in it. The scarcity of potent serum at this time made therecommendation that testing should be carried out routinely little more thanacademic, except, perhaps, in base hospitals. The meeting concluded withouttaking formal action.
The matter came up again at a conference held on 24 June 1943(26) to revise the Army manual on blood grouping, which was not, however,issued until 1946. It was pointed out that, at this time (1943), so muchconfusion existed concerning the military importance of the Rh factor that adefinitive statement by the conference would serve to throw the problem intoproper perspective. Except for Dr. Louis K. Diamond, of the Children'sHospital, Boston, who
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presented a minority report,3 the followingstatement was agreed to by all present:
The problem of isoimmunization with the Rh factor ispredominantly encountered in Rh-negative women who have become immunized as aresult of one or more pregnancies with Rh-positive children. It is possible toimmunize some of the 15% of males and females who are Rh-negative by repeatedtransfusions with Rh-positive blood. To effect this, multiple transfusionscarried out over a period of weeks, or even months, are necessary to induce asufficient degree of immunity [titer of antibodies] so that subsequenttransfusions with Rh-positive blood may cause reactions. It is common experiencethat reactions in individuals so sensitized by transfusion begin with mildreactions which progress in severity with subsequent transfusions withRh-positive blood. Accordingly, there is usually adequate warning before adangerous or fatal reaction develops.
Since only a small percentage of Rh-negative individuals canbe sensitized with the Rh factor, this group feels that there is noindication to embark on a large scale routine Rh testing of the personnel of thearmed forces. It would also seem more practical to this group to recommendthat anemias in members of the armed forces who have apparently become immunizedshould be treated by means other than blood transfusions.
In view of the occasional sensitized patient in continentalmilitary establishments in whom further blood transfusions seem essential, andin view of the much greater importance of Rh sensitization in obstetrical andgynecological practice, it is recommended to the Subcommittee on BloodSubstitutes that all means be furthered for the collection and proper distributionof potent anti-Rh serum for both the armed forces and for the civilianpopulation.
In view of the current low supply of potent human anti-Rhserum, it is recommended that it be employed only by experienced and skilledworkers and only then when specific indications arise.
On the basis of these recommendations, the conference saw noreason for including a section on Rh testing in the Army Laboratory Manual (TM 8-227)at this time. This recommendation was reversed at the 10 August 1943 meeting ofthe Subcommittee on Blood Substitutes (13), when it was recommended thatthe section covering the Rh factor drawn up by the Conference on Blood Groupingheld 23 March 1943 (9) should be included in the manual.
Testing difficulties continued throughout the war. In hismemorandum of 11 December 1944 to Col. B. Noland Carter, MC, Captain Elliottmentioned the scarcity of testing serum and the unsuitability of the test thenin use in frontline installations (24). This test took an hour to run andrequired the use of an 81? F.- (27? C.-) waterbath, a centrifuge, and amicroscope. Later, when the wounded casualties were moved back to rearinstallations, where additional transfusions might be necessary and where theirRh group could be determined, there might already be so many transfused redcells in the circulation that Rh-negative recipients might be erroneouslygrouped as Rh positive and given
3At the Conference on Blood Grouping on 23 March 1943(9), Dr. Diamond reported a study he was directing in over 8,000 transfusions, 90 percent of the reactions in which hethought were on the basis of Rh incompatibility. These reactions werecharacterized by chills, jaundice, anuria, transitory bilirubinemia, or failureof the recipient's red blood cells to increase to the expected amount aftertransfusion. Dr. Diamond also called attention to the multiple transfusionswhich had been necessary in the victims of the Cocoanut Grove disaster.
At the Conference on Blood Preservation and Red CellSuspension on 6 December 1945, he reported that he had examined 300 servicemenwho had received transfusions in which the Rh type was unknown. He had foundthat 30 of these were Rh negative and that about a quarter of the 30 had anti-Rhagglutinins in their blood.
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transfusions of Rh-positive blood, possibly with fatalconsequences. Captain Elliott saw only one way to eliminate this risk; namely,to determine the Rh group before the first transfusion. If this was notpossible, it was still desirable to make the test later, so that subsequenttransfusions would be Rh compatible.
Procurement of serum.-Late in 1943, Captain Elliottdemonstrated that naturally immunized individuals could be stimulated to producehigh-titer, anti-Rh serum by the injection of very small amounts of Rh-positiveblood. These persons could be safely bled at frequent intervals if the bloodwithdrawn was replaced by transfusions of Rh-negative blood. Unfortunately, thispromising plan to enlist cooperative persons with Rh-negative blood in a programfor the production of Rh testing serum was not carried out.
Until the middle of 1945, which means practically until theend of the war, most of the Rh testing serum used in Army hospitals wasprepared at the Army Medical School or purchased from Dr. Diamond.
Late in 1944, Lederle Laboratories developed a very potentanti-Rh serum, prepared from animals, which could be used by a slide techniqueat room temperature, with macroscopic reading of the result (24). Thetest could be completed within 10 minutes and required no equipment other thanglass microslides. The reaction was clear, and the technique so simple thatlaboratory technicians could easily learn it.
The test seemed admirably adapted for use in frontlineinstallations in which primary transfusions were given. If the transfusion hadto be given before Rh grouping could be done, a suspension of the recipient'scells could be prepared before the blood was given and his Rh group determinedlater. Determination of the blood group and the transfusion of Rh-compatibleblood later would prevent isoimmunization and assure longer in vivo survival oftransfused red cells.
If this test were put into use, it would permit theidentification and maintenance of a panel of Rh-positive donors. Bloodcollected in donor centers in the Zone of Interior for shipment overseas couldbe properly grouped and the Rh factor marked on the bottle.
At this time (December 1944), the Lederle Laboratories had onhand for immediate shipment material for about 1.5 million tests and would havemore within the next 3 months. The total supply would be more than enough fordistribution for all Army installations in which it could be used.
Captain Elliott recommended that the Lederle serum beprocured and distributed, that laboratory officers and technicians be taught thetechnique, and that the importance of transfusing Rh-compatible blood wheneverit was possible be called to the attention of all concerned.
Promising as the outlook seemed, it was found, on furtherinvestigation, that the Lederle serum produced false positive results if thetests were not very carefully performed and were not read within a definite timeafter they were set up. The allowable margin of error was so small that it wasreluctantly concluded that the serum could not safely be used routinely in Armylaboratories.
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At a meeting of an ad hoc committee of the Subcommittee onBlood Substitutes on 2 June 1944 (28), it was announced that Dr. Wienerhad been able to produce a high-titer anti-Rh agglutinin in women witherythroblastotic fetuses, whose blood originally showed a low titer. This wasaccomplished by the intravenous injection into them of 50 cc. of Rh-positiveblood. A proposal for a contract was being submitted, but no action was taken onit during the war.
Early in 1945, Dr. Joseph M. Hill, Baylor University, beganto produce large quantities of Rh testing serum by stimulating naturallyimmunized individuals. The serum was dried from the frozen state, vacuum sealedin ampules, and offered for sale to the Army with the assurance that all Armyneeds could thus be met. The serum was thoroughly tested and was found to beboth potent and stable. Specifications were therefore written, and it wasrecommended that the serum be included in the medical supply catalog as astandard item. This was done.
Shipments of Rh-negative blood to Europe began on 12 February1945, in the amount of 240 pints; 625 pints were shipped in March and 576 pintsin June, after hostilities had ended in that theater.
Technique.-The test for Rh compatibility used at theDivision of Surgical Physiology, Army Medical School, was carried out as follows(29):
1. A drop of Rh antiserum was placed in a test tube, and 1drop of fresh 2-percent blood suspension in saline solution was added.
2. The tube was shaken, then placed in a water bath at 37?C. or in an air incubator for an hour.
3. After incubation, the sedimented cells were very gentlyresuspended and inspected for macroscopic agglutination. If agglutination wasnot observed, the tubes were centrifuged at 750-1,000 r.p.m. for 1 minute. Thepacked cells were then gently resuspended and observed for macroscopicagglutination.
4. If macroscopic agglutination was not evident, the slidewas examined microscopically.
5. Absence of agglutination denoted that the blood was Rhnegative (fig. 58). Any degree of agglutination indicated that it was Rhpositive.
SCREENING TEST FOR O BLOOD
When the airlift of group O blood was planned, there wasimmediate need for a rapid screening test to select group O donors. CaptainElliott devised a simple, effective method:
Avid, proved high-titer group O serum was dried, in singletest doses, in small glass shell vials. Individual grouping sets were made up,consisting of a vial of serum and a vial of salt solution. One drop of the donor'sblood was added to the salt solution and two drops of the suspension weretransferred to the dried serum. The serum agglutinated cells of groups A, B, andAB, but did not agglutinate group O cells.
This technique effectively identified about 98 percent ofgroup O donors. A second check, to reduce the possibility of error to a minimum,was carried out at the donor centers by checking the blood of each apparentgroup O donor with red blood cells of known A and B groups before the blood wasshipped.
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The screening sets were quickly put into production by theReichel Division of Wyeth Laboratories and later by Cutter Laboratories. Theywere used in all the Red Cross centers in which blood was collected for shipmentoverseas, the donors thus identified being bled into the special bottlesprovided for oversea shipment.
HEMATOCRIT DETERMINATIONS
Copper Sulfate Falling Drop Test
A most important consideration in the management of shock wasits early recognition. The degree of reduction in the blood volume was usuallythe initial, and often the only, determining factor. With this informationavailable, therapy could be precise, and, if the supply of blood or plasma werelimited, these agents could be most usefully distributed among the casualties.
No means of making this determination existed when the UnitedStates entered the war, nor was any developed until almost 2 years had passed.At the Conference for the Revision of the Army Manual on Blood Grouping on 24June 1943 (26), part of the discussion concerned the pressing need forsome
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method for the rapid measurement of blood concentration under field conditions. Certain specifications had to be met: The method mustrequire little blood, little time, and apparatus that would not be disturbed bytemperature ranges from Guadalcanal to Alaska. The method must be applicable onshipboard, on the unstable base of a rolling vessel, and must be simple enoughto be mastered readily by an enlisted man. The problem of devising such a methodwas assigned to the group working on shock at the Rockefeller Institute forMedical Research.
At the meeting of the Subcommittee on Shock on 1 December1943 (27), Dr. Donald D. Van Slyke, of the institute, described the techniquejointly devised by himself; Lt. Robert A. Phillips, Lt. Vincent P. Dole, and Lt.Kendall Emerson, Jr., all MC, USN; Dr. Paul B. Hamilton, and Dr. Reginald M.Archibald (30). Specific gravity measurements, Dr. Van Slyke stated, werethe only ones that would meet the specifications. The Barbour falling droptechnique was excellently adapted to civilian needs, but it required specialapparatus, a stable base, and organic liquids with a high temperaturecoefficient. His group therefore decided to initiate its search for a suitabletest for field use on the old principle of dropping blood into standardsolutions of known gravity. They further decided that, in order to avoid thetemperature coefficients of organic liquids, which expand about five times asmuch as aqueous solutions, aqueous solutions must be used as the standard.
When salt and glycerol solutions were used for this purpose,the drop of blood or plasma being tested dissolved so rapidly that results wereonly approximate. To prevent this phenomenon, a protein precipitant in the formof a mixture of salt and picric acid was added to the solution, to form a filmabout the drop of blood or plasma and hold it together.
This test had been in use only a single day when LieutenantPhillips walked into the laboratory with a copper sulfate solution which, initself, met all the requirements. It was easily prepared by dilution of a coppersulfate solution made up by shaking a pound of copper sulfate crystals in a pintof water for 5 minutes. The temperature coefficient of the standardsolution was the same as that of blood, which made a temperature controlunnecessary.
When a drop of blood or plasma was dropped into this solution(fig. 59), a layer of copper proteinate formed about it and held it together ina sac that did not change its gravity for about 20 seconds, which was longenough to determine whether the drop rose or fell in the solution. At the end ofthis time, the drop absorbed the copper and fell to the bottom. Another testcould be run at once and about a hundred tests could be run with the samesolution. A convenient portable kit (fig. 60) contained everything necessary forthe test.
Accuracy was surprisingly high; it was possible to obtainblood and plasma gravities precise to 1 in the fourth decimal place, which wasseveral times as accurate as was needed (27). With accuracy so easilyobtained, the method could be used not only to estimate plasma proteins but alsoto estimate hemoglobin, by determining the difference between the gravities ofwhole blood and plasma. In 20 bloods tested by this method, hemoglobin
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FIGURE 59.-Demonstration of copper sulfate (falling drop) method ofmeasuring specific gravities of whole
blood and plasma (30).
estimates had varied only 0.7 percent from the results ofdeterminations by a particularly precise form of the oxygen capacity method.Hematocrit values could also be determined from the blood and plasma gravities;they agreed, within an average of 2 percent, with determinations by the standardcentrifuge method.
The test had been employed under a variety of circumstances.A medical officer on a hospital ship in the Pacific had seen a preliminarydescription of it in BUMED and had made a test study of it on medicalpersonnel. The results had not been affected by the motion of the ship or bytemperature changes within the range of 60? to 106? F. (15? to 41? C.).Later, the same officer had used the test on some 800 sick and wounded broughtaboard in 2 hours. Under the circumstances, he wrote Dr. Van Slyke and hisassociates, there was not much time for anything but a laboratory test as simpleas this.
The line charts (charts 4 and 5) devised by LieutenantDole for calculations of plasma proteins, hematocrit, and hemoglobin greatlysimplified the test. When a straight edge was laid across a set of scales, allof these values could be obtained, and the entire test completed, within 2 minutes.The possible variations of hemorrhage, seepage of plasma, and dehydration wereso numerous that any quick means of making the diagnosis and furnishing
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FIGURE 60.-Portable kit in portable typewriter case 13 inches by 12 inches by 6 inches for copper sulfate (falling drop) method of measuring specific gravities of whole blood and plasma (30). Line chart for calculating plasma proteins and hemoglobin (a), metal centrifuge cups (b), removable centrifuge head (c), 12 oxalated tubes with rubber caps (d), 3/8 inch plywood partitions (e), twelve 10-cc. syringes and needles in sterile packs (f), medicine droppers (g), handle for centrifuge (h), tourniquet (i), 2-oz. bottle with alcohol sponges (j), portable hand centrifuge (k), and 2-oz. bottles containing copper sulfate solution (1).
a guide to treatment was useful, for the fluid needs of thecasualty in shock depended upon the category into which he fell. Hematocrits of25 to 30 indicated a blood loss of 2,000 cc. or more, which was common incasualties with multiple or extensive wounds.
The copper sulfate (falling drop) test does not indicate thetotal circulating blood volume, as it is sometimes stated that it does. It doesprovide a method of determining the relative quantity of circulating red bloodcells plus information concerning the hematocrit, hemoglobin, and plasma proteinlevels. It not only indicates the need for whole blood and the approximate
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CHART 4.-Line chart for calculating plasma proteins,hemoglobin and hematocrit from specific gravities of plasma and blood
Source: Phillips, R. A., Van Slyke, D. D., Dole, V. P., Emerson, K., Jr., Hamilton, P. B., and Archibald, R. M.: Copper Sulfate Method of Measuring Specific Gravities of Whole Blood and Plasma with Line Charts for Calculating Plasma Proteins, Hemoglobin and Hematocrit from Plasma and Whole Blood Gravities. From the U.S. Navy Research Unit at the Hospital of the Rockefeller Institute for Medical Research, n.d.
amounts needed (about 500 cc. for each three points ofdesired increase in the hematocrit), but also indicates the absence ofindications for blood transfusion and thus prevents the waste of this valuableand scarce substance.
This test was used with great satisfaction in all theaters ofoperations, one reason being that it required neither elaborate equipment nortrained personnel. When the tubes for the test were kept on the ward, with (dry)syringes, the test could be made on the spot and the information needed could beacted upon without delay.
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CHART 5.-Line chart for calculating percentages of normal plasma proteins and hemoglobin from specific gravities of plasma and blood
Source: Phillips, R. A., Van Slyke, D. D., Dole, V. P., Emerson, K., Jr., Hamilton, P. B., and Archibald, R. M.: Copper Sulfate Method of Measuring Specific Gravities of Whole Blood and Plasma with Line Charts for Calculating Plasma Proteins, Hemoglobin and Hematocrit from Plasma and Whole Blood Gravities. From the U.S. Navy Research Unit at the Hospital of the Rockefeller Institute for Medical Research, n.d.
Thalhimer modification of the Van Slyke et al. test-Shortlyafter the test devised by Van Slyke and his group was described, the Thalhimer modification of the test, which had the approval of the workers at theRockefeller Institute, was officially adopted for use in American Red Crossblood donor centers (31). This modification was the use of a singlesolution of copper sulfate, of appropriate specific gravity, to register thecritical level of hemoglobin agreed on for the selection or rejection of donors.A drop of blood
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was collected from a needle or knife prick in an expendableglass capillary tube, from which it was expelled into the solution by pressureon a rubber bulb of the type used in vaccination. The level of acceptance ofdonors was 12.3 gm. percent, and the specific gravity of the solution was setfor that level. If the drop of blood floated, the donor was rejected.
DYE MEASUREMENT OF BLOOD VOLUME
Dr. Magnus I. Gregersen, Columbia University College of Physicians and Surgeons, at the Conference on Shock on 1 December 1943 (27), described a dye technique for the determination of the total plasma. Technical difficulties hampered the production of the materials in quantity and it was not possible to develop the method further during the war.
TITRATION OF BLOOD
Shortly after the blood bank was established in theMediterranean theater, certain difficulties arose (p. 424) that led to thepractice of titering all O blood and reserving blood with an anti-A or anti-Bagglutinogen titer of 1:250 or higher for group O recipients.
Titration was not practiced at the beginning of the airliftof blood to Europe, and, though it was directed by The Surgeon General in earlyOctober that it should be carried out, it was not begun until February 1945,because of the delay in collecting the necessary equipment and securing andtraining personnel (32). Except for the first few shipments, all bloodflown to the Pacific areas was titrated. The practice in both airlifts was todesignate blood with high titer as suitable for group O recipients only.
Special Investigations
Naval Medical Research Institute.-Several special studieson the titer of blood and plasma were made in the course of the war.
Two studies of the titer of pooled plasma at the NavalMedical Research Institute, National Naval Medical Center, gave essentiallythe same results (33). The first study covered 300 titrations and thesecond 1,000, with 100 monovalent controls. The investigations produced noevidence of any harmful effects as a result of the titer of isohemagglutininspresent in pooled plasma and no justification for preliminary crossmatching ofplasma before it was administered. The data also tended to discredit reportsthat undue reactions after the infusion of pooled plasma were caused byagglutinins in it.
American Red Cross Blood Donor Service-Dr. Thalhimer,Associate Technical Director of the Red Cross Blood Donor Service, and Maj. EarlS. Taylor, MC, Technical Director (34), reported on the anti-A and anti-Bagglutinin titers in 1,354 pools of human plasma, in which the number ofindividual plasmas in the pools varied from 6 to 60. The pools were eitherrandom
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samples or were secured from every 5th or every 10th specimenencountered in routine processing. In 99.7 percent of the pools, macroscopicagglutinin titers were less than 1:40, and in 97 percent they were less than1:20. In 355 small pools, made up of 6 to 10 individual plasmas, the percentageof relatively low titers (1:20 to 1:40) was somewhat higher than in largerpools, but the proportion of titers of 1:40 or higher was no greater than in thelarger pools, which indicated the safety of the smaller pools.
A special study was made of pools of type O plasma withtiters up to 1:256. Clinical administration of these plasmas caused noreactions, nor were there any signs or symptoms of intravascular agglutination.
These observers concluded that pooled plasma prepared insmall pools at hospital blood banks or in larger pools at processinglaboratories, in which the plasmas entered the pools by chance and withoutselection, could be safely administered to all individuals, regardless of theirblood type.
Whole Blood Procurement Service-The Army Whole BloodProcurement Program processed some 1,500 bloods daily, which made it practicalto obtain enough high titer bloods for a comprehensive study of the effects oftransfusion of such blood to incompatible recipients. The study was undertakenby Maj. Leslie H. Tisdall, MC, Coordinator, Army Whole Blood ProcurementService, and his associates, with the cooperation of volunteers at the ColoradoState Penitentiary (35).
The study covered the titration of 1,650 group O bloods, 376of which (22.7 percent) were found to have anti-A or anti-B agglutinins, orboth, of 1:640 or higher. Infusions in the amount of 250 cc. of high-titer groupO plasma were given to 39 volunteers representing other than O blood groups.Plasma was used instead of blood to make sure that any subsequent hemolyticreactions would be the result of hemolysis of recipient, not donor, cells. Theinvestigators considered the criterion of a hemolytic reaction to be thedemonstration of hemolysis of the recipient's red blood cells while the donorcells remained intact. Posttransfusion observation lasted for a minimum of 8hours and was personal and careful.
The isoagglutinin titer of the 39 incompatible transfusionsranged from 1:400 to 1:4,000. Two volunteers, given plasma with a titer of 1:500, had no reactions. Three, given plasma with titers of 1:1,000 to 1:3,000,had chills and fever but no evidence of hemolysis. The remaining 34 volunteers,given plasma with titers from 1:400 up to 1:4,000, all had hemolytic reactions;full recovery ensued in all cases in from several hours to 4 days.
Major Tisdall and his associates concluded that O blood wassafe to use for universal donations when the antibody titer was no higher than1:1-200 by the centrifuge technique which they had devised. They believed thata standardized technique of this kind should be adopted and that Rh-negative,low-titer O blood should be kept available in every hospital blood bank.
The experience with high-titer group O blood in theMediterranean theater was substantiated by the studies made at Walter ReedGeneral Hospital in 1945 by Maj. John J. McGraw, Jr., MC. They showed asubclinical increase in
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bilirubin after the use of high-titer blood and pointed tothe desirability of using low-titer group O blood whenever possible in combatcasualties. Major McGraw's studies also showed the importance of Rh testing toreduce the risk of severe and fatal transfusion reactions in Rh-negativerecipients.
STUDIES ON A AND B SUBSTANCES
A number of studies were made before and during the war onthe use of the A and B substances developed by Witebsky to neutralize theagglutinins in group O blood and condition it, so to speak, for universaldonation. These substances were procurable commercially. Witebsky developed themoriginally from human gastric juice and later from saliva, and he questionedthe safety of the products produced by commercial firms from hog (A substance)and horse (B substance) stomachs.
A conference on Group-Specific Substances A and B was held on19 March 1945, with an extensive agenda (36). It was the sense of themeeting that these preparations were now sufficiently safe to be recommended foraddition to human blood employed for transfusion in the Armed Forces; that asubcommittee under the chairmanship of Dr. Witebsky be appointed to assumeresponsibility for their standardization, control, safety, and manufacturingimprovement; and that fundamental research be undertaken in the field.
At the Conference on Resuspended Blood Cells, combined with ameeting of the Subcommittee on Blood Substitutes, on 18 May 1945 (37), thead hoc committee appointed at the 19 March meeting made its report onstandardization of A and B substances and on directions for their use.Studies were then in progress to determine techniques of sterilization,incorporation in ACD (acid-citrate-dextrose) solution, storage without loss ofantibody-neutralizing power, and other details. When the data listed were athand, the subcommittee would be asked to vote on recommending that thesesubstances be used to neutralize anti-A and anti-B agglutinins in group O wholeblood distributed to the Armed Forces.
The war ended before these studies were concluded, and A andB substances were not used in the Korean War.
STUDIES OF CELL SURVIVAL AFTER TRANSFUSION
Ashby, in 1919, was the first to publish studies ondifferential cellular agglutination as a method of estimating cell survivalrates after transfusion (38). During World War II, his method wasconsidered more reliable than even the ingenious technique described by Ross andChapin (39) in 1943 of radioactive iron tagging of red blood cellsbecause the Ashby technique, unlike the Ross and Chapin technique, permitted theinvestigator to follow the transfused red cells throughout their entirelifespan.
Gibson's studies (39) by the Ashby techniqueindicated that the type of preservative used and the conditions under which theblood was stored had
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much to do with the lifespan of the red cells. Humanerythrocytes transfused shortly after their collection in sodium citrateremained intact after transfusion and disintegrated at the rate of about 1percent per day. Cells that were nonviable at transfusion disappeared from thebloodstream within 24 hours. Viable cells completed the normal 120-day lifecycle.
SEROLOGIC TESTING FOR SYPHILIS
The original Red Cross donor regulations prohibited the useof serologically positive blood (p. 142), and this restriction continuedthroughout the war though all the evidence was to the effect that this was anunnecessary precaution when the blood was not used for immediate transfusion (40).
Details of losses from this cause are stated elsewhere (p.568). By the end of August 1942, the eight commercial laboratories processingblood for plasma had received a total of 559,767 units of which 32,812 (5.8percent) had been lost for various reasons, including 3,094 units (0.55 percent)discarded as serologically positive (41). This proportion remainedessentially the same whether blood was collected at the donor centers oroverseas.
The matter of serologically positive blood was discussed indetail at the meeting of the Subcommittee on Blood Substitutes on 15 December1942 (42), in connection with the work being done by Dr. Cohn on plasmafractionation. Dr. Cohn made the following points:
1. The minimum requirements for plasma and albumin laid downby the National Institute of Health did not prohibit the use of serologicallypositive blood.
2. A search of the literature by Drs. William C. Boyd, JohnF. Enders, and Charles A. Janeway revealed no evidence that spirochetes surviveoutside of the body for as long as 48 hours.
3. To fortify the evidence, these observers conductedstudies that showed that Spirochaeta pallidum was rendered noninfectious,if not killed, by the lyophilizing process used in the processing of plasma. Thestudies included inoculation, transfer, and adequate controls.
In Dr. Cohn's opinion, there was no question that thesereports and studies proved the inability of lyophilized plasma to transmitsyphilis. Since the processing of serum albumin involved both freezing anddrying, he saw no reason to exclude serologically positive blood from use aslong as the processing included these two phases. A letter from Dr. Milton V.Veldee, read at this meeting, stated that he was "unable to get very muchdisturbed over the chance introduction of 1 serologically positive bleeding intoa pool intended for processing to serum albumin." He considered that thisrandom happening had "absolutely no significance from the standpoint oftransmission of syphilis * * * or from the aesthetic point either,whatever the latter is." Dr. Veldee did object, however, to the proposalthat all serologically positive bloods be concentrated and used for albumin, andthis was not done.
Blood collected at the Red Cross blood donor centers forplasma and albumin was tested serologically at the processing plants. When theairlift to Europe began in August 1944, equipment and personnel were supplied to
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permit each donor center to perform its own serologic tests.It was thus possible for each center to ship out each day's collection at theend of the day and for all the bloods collected to be sent overseas withoutfurther delay for processing. The same plan was used with the Pacific airlift.
Syphilitic Donors
At the 15 December 1942 meeting of the Subcommittee on BloodSubstitutes (42), it was agreed that nothing could be done to exclude theblood of syphilitic donors who applied to the centers in the very early stagesof the disease, before serologic tests were positive. Blood was never taken frompersons with a history of genital sores or discharges within the last 6 months,and, in deference to public opinion (probably the "aesthetic point"mentioned in Dr. Veldee's letter just quoted), donors who were known to havesyphilis or to have had syphilis were not permitted to give blood. A Kahn orKolmer test was always performed on bloods to be used for transfusion.
In oversea military hospitals, if no nonsyphilitic donor wasavailable in an emergency, it was the practice to use a donor with a history ofsyphilis if he had no clinical evidence of the disease, if he had beenadequately treated in accordance with current directives, and if serologic testshad been negative for not less than a year.
As time passed, large numbers of soldiers on leave orstationed near blood donor centers began to give blood, and an occasionalpositive serologic reaction was turned up among them. The problem of reportingthese donors was complicated by the fact that many of them were onfurlough, or were moved, or soon to be moved, from the posts to which they werethen attached. On 21 September 1943, Dr. Robinson wrote to The Surgeon Generalfor instructions in these cases (43). Although it might add somewhat tothe responsibilities of the donor centers, Dr. Robinson thought it would bepractical to obtain the serial number of each donor from the Armed Forces.
The reply from The Surgeon General (44) asked that the name,Army serial number, and station of each donor from the Armed Forces be obtainedat the time of the donation. If the blood proved serologically positive, theinformation should be sent at once to the commanding general (attention: chief,medical branch) of the army service area in which the donor center was located,with the request that the information be relayed to the surgeon of the Armyinstallation in which the soldier was stationed. This procedure would insurethat the information reached the responsible medical officer in the mostexpeditious manner possible and would simplify tracing the soldier in the eventthat he had changed stations.
On 16 March 1944 (45), these instructions were furthermodified in the light of information recently obtained to the effect that, afterrepeated blood donations, a certain proportion of donors would show falsepositive tests.4 It was
4It was also discovered that false positive testsmight occur shortly after the smallpox vaccination required of all militarypersonnel.
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therefore requested that positive serologic tests on soldiers who had madetwo or more donations should not be reported as directed unless it were possibleto recheck the tests and they were still positive 2 months later, without bloodhaving been given in the interim.
References
1. War Department Circular No. 123, 24 June 1941, paragraph I, Determinationand Recording of Blood Types-Changes in AR 40-1715 and 600-40.
2. Circular Letter No. 70, Office of The Surgeon General, War Department, 14July 1941, subject: The Determination and Recording of the Blood Groups of AllIndividuals in the Military Service.
3. Circular Letter No. 88, Office of The Surgeon General, War Department, 4Sept. 1941, subject: Supplemental Information Concerning Determination of BloodType in Accordance with SGO Circular Letter No. 70.
4. Circular Letter No. 112, Office of The Surgeon General, War Department, 26Nov. 1941, subject: Supplemental Information Concerning Determination of BloodType in Accordance with SGO Circular Letters No. 70 and No. 88.
5. Circular Letter No. 170, Office of The Surgeon General, Services ofSupply, War Department, 2 Dec. 1942, subject: The Purpose of Blood Grouping ArmyPersonnel.
6. Minutes, meeting of Subcommittee on Blood Substitutes, Division of MedicalSciences, NRC, 23 May 1941.
7. Minutes, meeting of Subcommittee on Blood Substitutes, Division of MedicalSciences, NRC, 18 July 1941.
8. Minutes, meeting of Subcommittee on Albumin and By-Products, Division ofMedical Sciences, NRC, 22 Jan. 1943.
9. Minutes, Conference on Blood Grouping, Division of Medical Sciences, NRC,23 Mar. 1943.
10. Minutes, Conference on Transfusion Equipment and Procedure, Division ofMedical Sciences, NRC, 25 Aug. 1942.
11. Minutes, meeting of the Albumin and By-Products Group, Division ofMedical Sciences, NRC, 10 May 1943.
12. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 13 May 1943.
13. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 10 Aug. 1943.
14. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 24 Sept. 1943.
15. Minutes, Conference of Albumin and By-Products Group,Division of Medical Sciences, NRC, 17 Nov. 1943.
16. Minutes, Conference of Albumin and By-Products Group,Division of Medical Sciences, NRC, 28 July 1943.
17. Minutes, meeting of Subcommittee on Blood Procurement,Division of Medical Sciences, NRC, 18 Aug. 1941.
18. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 10 Nov. 1942.
19. Minutes, meeting of Subcommittee on BloodSubstitutes, Division of Medical Sciences, NRC, 12 May 1942.
20. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 23June 1942.
21. Letter, Lt. Col. Thomas E. Lanman, MC, to Chief Surgeon,European Theater of Operations, U.S. Army, 4 Mar. 1943,subject: Incorrect Recording of Blood Type on Identification Tags.
22. Kilduffe, Robert A., and DeBakey, Michael: The Blood Bank and the Technique and Therapeutics of Transfusions. St. Louis: C. V. Mosby, 1942.
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23. Landsteiner, K., and Wiener, A. S.: Studies on anAgglutinogen (Rh) in Human Blood Reacting with Anti-Rhesus Sera and HumanIsoantibodies. J. Exper. Med. 74: 309-320, 1 Oct. 1941.
24. Memorandum, Capt. John Elliott, SnC, for Division ofSurgery, Office of The Surgeon General (attention: Col. B. N. Carter, MC),through Director, Army Medical School, 11 Dec. 1944, subject: Rh BloodGrouping.
25. Memorandum, Capt. John Elliott, SnC, to Chief, SurgicalConsultants Division, Office of The Surgeon General, through Director, ArmyMedical School, 1 Feb. 1945, subject: Transportation of Blood from the U.S. tothe ETO Blood Bank in Paris.
26. Minutes, Conference for the Revision of the Army Manualon Blood Grouping, Division of Medical Sciences, NRC, 24 June 1943.
27. Minutes, Conference on Shock, Subcommittee on Shock,Division of Medical Sciences, NRC, 1 Dec. 1943.
28. Minutes, Meeting of the Group Appointed by theSubcommittee on Blood Substitutes to Consider Methods of Using Normal HumanSerum Albumin in the Army Air Forces, Division of Medical Sciences, NRC, 2 June1944.
29. Clinical Significance of the Rh Factor. Bull. U.S. ArmyM. Dept. No. 78, July 1944, pp. 50-52.
30. Phillips, R. A., Van Slyke, D. D., Dole, V. P., Emerson,K., Jr., Hamilton, P. B., and Archibald, R. M.: Copper Sulfate Method ofMeasuring Specific Gravities of Whole Blood and Plasma with Line Charts forCalculating Plasma Proteins, Hemoglobin and Hematocrit from Plasma and WholeBlood Gravities. From the U.S. Navy Research Unit at the Hospital of theRockefeller Institute for Medical Research, n.d.
31. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 2 Mar. 1944.
32. Memorandum, Maj. F. N. Schwartz, MAC, to Chief, PersonnelService, Office of The Surgeon General, 11 Oct. 1944, subject: Allotment of SixAdditional Enlisted Technicians.
33. Lozner, Lt. Eugene L., MC, USN: Studies on AgglutininTiters of Pooled Plasma. Research Project X-104, Report No. 2. Naval MedicalResearch Institute, National Naval Medical Center, Bethesda, Md., 19 Aug. 1943.
34. Thalhimer, W., and Taylor, E. S.: The Low AgglutininTiter of Both Small and Large Pools of Plasma. J.A.M.A. 128: 277-279, 26 May1945.
35. Tisdall, L. H., Garland, D. M., Szanto, P. B., Hand, A.M., and Barnett, J. C.: The Effects of the Transfusion of Group O Blood of High Iso-Titerinto Recipients of Other Blood Groups. Am. J. Clin. Path. 16: 193-206, March1946.
36. Minutes, Conference on Blood Group Specific Substances A& B, Division of Medical Sciences, NRC, 19 Mar. 1945.
37. Minutes, Conference on Resuspended Blood Cells andmeeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC,18 May 1945.
38. Ashby, W.: The Determination of the Length of Life ofTransfused Blood Corpuscles in Man. J. Exper. Med. 29: 267-281, 1 Mar. 1919.
39. Minutes, Conference on Preserved Blood, Division ofMedical Sciences, NRC, 25 May 1943.
40. Taylor, E. S.: Procurement of Blood for the Armed Forces.J.A.M.A. 120: 119-123, 12 Sept. 1942.
41. Report on Loss of Blood due to Syphilis From EightProcessing Laboratories, n.d.
42. Minutes, meeting of Subcommittee on Blood Substitutes,Division of Medical Sciences, NRC, 15 Dec. 1942.
43. Letter, Dr. G. Canby Robinson to Maj. Gen. Norman T.Kirk, attention V. D. Control Division, 21 Sept. 1943.
44. Letter, Lt. Col. Thomas B. Turner, MC, (for The SurgeonGeneral) to Dr. G. Canby Robinson, 24 Sept. 1943.
45. Letter, Lt. Col. Douglas B. Kendrick, MC, to Maj. Earl S.Taylor, MC, 16 Mar. 1944.
