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Chapter X

Contents

CHAPTER X

Laboratory Techniques and Special Laboratory
Studies

BLOOD TYPING OF MILITARY PERSONNEL

Implementation of Policy

After considerable discussion of the wisdom of, and necessity for, the blood typing of military personnel, instructions were issued for this action in War Department Circular No. 123, 24 June 1941 (1), by the addition of changes in AR (Army Regulations) 40-1715, 15 August 1932, and AR 600-40, 22 June 1931, as follows:

1. AR 40-1715-Paragraph 7 is added as follows:

Determination and recording of blood types of all military personnel.-A. The blood group of each individual on active duty in the military service will be determined, using the International (or Landsteiner) classification. The proper performance of the tests in each organization will be a responsibility of the surgeon. The results will be recorded, using the symbols "A", "B", "AB", or "O", as indicated.

b. The surgeon will be responsible for the proper recording of the blood group on the identification tag of each individual tested. An additional record will be made in the case of enlisted personnel on W.D., A.G.O. Form No. 24 (Service Record) and in the case of officers or other personnel, on W.D., M.D. Form No. 81 (Immunization Register).

In conformity with this War Department circular, Circular Letter No. 70, Office of The Surgeon General, War Department, was issued on 14 July 1941, with the subject: The determination and recording of the blood groups of all individuals in the military service (2). It dealt with the following points:

1. Materials for blood typing with methods of their procurement. The reagents required consisted of mixtures of sucrose and dried sera from rabbits that had been immunized, with human erythrocytes of groups A and B, respectively. Medical Department specifications required that the potency of the sera be such that when they were used as directed specific macroscopic 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 to be recorded on the man's identification tag only after it had been checked by a medical officer against the individual's completed blood grouping test.

4. Performance of the test (a) in small posts or isolated detachments by a surgeon with a few enlisted assistants and (b) its performance in larger organizations, on an assembly line basis, with the help of one or more teams of Medical Department personnel. Details were given for each step of the procedure.

The letter pointed out that an error in the technique of the test or in the recording of the result could be extremely serious and might even result in a fatality.


234

Supplemental Instructions

4 September 1941-Only a brief experience with the typing of military personnel brought to light difficulties and errors. Circular Letter No. 88, Office of The Surgeon General, War Department, was issued on 4 September 1941, with the subject: Supplemental information concerning determination 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 concentration of the red cell suspension in tests run in the field necessitated some latitude in the time allowed for agglutination (specified in the original directions as not to exceed 1 minute). This variation was to be determined by the medical officer in charge of the team.

2. All normal saline solution used in typing must be prepared from sodium chloride, A.C.S. (Item No. 14290, Medical Department 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 to delay or inhibit the test.

3. The precise amount of serum indicated was to be used; an excess delayed agglutination.

4. If dried sera became so tightly packed in the original containers that difficulty was experienced in measuring out the small amounts required for individual tests, it could all be put into solution and a specified amount of the solution used.

26 November 1941-Circular Letter No. 112, Office of The Surgeon General, War Department, issued on 26 November 1941, subject: Supplemental information, concerning determination of blood type in accordance with SGO Circular Letters Nos. 70 and 88 (4), reflected additional experience. In this letter, the objective of blood typing of military personnel was defined-it had apparently not been clearly understood before-as making possible the calling of voluntary donors of a specific blood type and securing them on very short notice. The following points were also covered:

1. The possibility of error in mass blood grouping was recognized, but it was postulated that crossmatching would be done before transfusion, and errors would thus be recognized. This precaution, however, was no reason for relaxing efforts to be absolutely accurate in testing and recording.

2. Insufficient time was sometimes being allowed for proper agglutination. As a result, blood types other than O, particularly the AB group, were being recorded as O. Such errors could be prevented if all tests in which agglutination did not occur promptly were held for observation for 20 minutes or longer.

3. Any validated evidence of apparently unsatisfactory sera should be reported to the Army Medical School, so that it could be determined whether the sera were really unsatisfactory or technical errors were responsible for the poor results.

2 December 1942.-Still further clarification was attempted in Circular Letter No. 170, Office of The Surgeon General, War Department, Services of Supply, 2 December 1942, subject: The purpose of blood grouping Army personnel (5). In this letter, it was pointed out that the policy of blood grouping of military personnel and its purpose seemed to have been misunderstood by some medical officers. Some reports of alleged errors suggested that


235

the writers believed the errors would inevitably lead to serious reactions if the persons whose blood was wrongly classified should be used as either donors or recipients. The assumption that the first typing test would be the only one performed was based on a false premise. To correct the misconception, it was pointed out again that the purpose of the program was to simplify assembling donors whose blood would probably crossmatch with that of intended recipients. It was never intended, when the mass grouping program was set up, that crossmatching should be omitted.1

Errors would be reduced, the letter continued, by care in all steps of the grouping process and in recording of results. Some stations were preparing their own grouping sera from tested human donors with high titer serum, 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 to develop, though no arbitrary period of observation could be specified because of variations in temperature, serum titer, degree of mixing of serum and cells, and the relative agglutinating ability of the cells. Various expedients to provide a rough check had been tried: The test could be done on groups of 20 to 25 men at a time and no results recorded until half of the slides showed agglutination with anti-A serum. Or no results except in the AB group should be recorded until the slides had been under observation for 20 minutes, even though most agglutinations were evident within a minute or two. Observations up to 30 minutes were desirable if excessive drying could be avoided.

Some blood grouping teams observed all slides microscopically unless macroscopic agglutination was evident. Other teams repeated the tests on all blood that gave no agglutination with anti-A serum; that is, they repeated the tests on all persons originally grouped as B or O. This was regarded officially as an unnecessary precaution, not worth the effort if the original tests had been carefully performed.

Mass Methods of Typing

Necessary as it was, the blood grouping program was one more thing to interfere with the training of troops. Many installations therefore devised their own methods of expediting the procedure. In some, unfortunately, the haste led to confusion, and the confusion led to errors, a certain proportion of which could unquestionably be explained in this way. In other installations, 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.


236

National Research Council

The matter of typing military personnel was brought up at the meeting of the Subcommittee on Blood Substitutes on 23 May 1941 (6), when the policy was still under consideration. The experience of the Blood Transfusion Association in New York (p. 13) had indicated that the rabbit sera presently in use were not so potent as they should be, but it was expected that more avid material would shortly be available.

When the subcommittee met on 18 July 1941 (7), typing of all military personnel on active service had been authorized (p. 233), and substantial contracts for rabbit serum had been let with the only firm then processing it. In view of the circumstances, the subcommittee considered that any action in the matter was outside of its jurisdiction. A year and a half later, in January 1943 (8), it completely reversed this attitude and interested 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 sera available at the beginning of the war were, as just indicated, not so avid as they should have been. Tests by the DeGowin technique (9) showed that A titer agglutinated cells in dilutions up to 1,000 but not in dilutions up to 2,000. The B titer was strong; agglutination could be obtained in dilutions up to 1:4,000 though not up to 1:8,000. These sera had two advantages, that they did not need refrigeration and that the titer was so high that expert skill was not necessary to use them. They also had a serious disadvantage, that they were hygroscopic and, when exposed to air, took up moisture.

In the beginning, when material could be secured only from a single laboratory, there was not very much that could be done. As time passed, the rabbit sera improved, and, even when globulin fractions were available to prepare more avid sera, rabbit sera were still considered satisfactory and the Army continued to use them throughout the war. Each lot purchased was tested at the Army Medical School, which was a relatively simple matter: Techniques for preparation of sera produced relatively large lots, all units of which could reasonably be expected to be identical. Tests on a single unit therefore gave information which could be applied to all units in the lot. As a practical matter, it was recommended that samples of each pool be submitted for examination before the material was packaged, so that, if they did not meet specifications, they could be fortified with more active material and submitted again for appraisal. The production of satisfactory sera depended upon rigid adherence to all details of processing, including temperature controls, protein concentration, and salt content.


237

Work at the Army Medical School showed that an increase in the salt component of the serum-cell suspension mixture resulted in more rapid agglutination. After many tests, it was found that a 1.4-percent concentration of sodium chloride in the final mixture was optimum, and the original directions packed with dried rabbit sera were altered to include this information.

Human sera-One of the early activities of the Division of Surgical Physiology, Army Medical School, was an attempt to improve the avidity of the grouping sera then in use. Its personnel, working with the Chemistry Division of the school, demonstrated that very avid and very high titer grouping material could be prepared from plasma by separation and concentration of the globulin fraction containing the isoagglutinins. Anti-A and anti-B globulins were produced as byproducts of the fractionation process and were available in large quantities because of the contracts for albumin let by the Navy.

The original work on plasma fractionation had been done in Dr. Edwin J. Cohn's laboratory at the Harvard Medical School, and, in December 1942, 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 placed on temporary duty there, to develop a new technique for preparing typing sera from human plasma. The method developed called for preparation of albumin and its 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 pools ordinarily contained highly potent blood grouping substances.

At the meeting of the Albumin and By-Products Group on 22 January 1943 (8), Dr. Cohn reported on Lieutenant Pillemer's work. The technique he had developed was closely related to the fractionation process devised at Harvard, but it employed methyl alcohol, since the low temperatures required with ethanol were not required with this precipitant. The demonstration that isoagglutinins could be prepared from both type A and type B bloods showed that they could be prepared as byproducts of the large-scale industrial preparation of albumin. They were concentrated in fraction II+III, and if the euglobins were separated from the pseudoglobulins, the isoagglutinins would be found in the euglobin fraction. With this technique, sufficient typing solution to carry out an enormous number of typing tests could be prepared from a relatively small amount of plasma.

The separated, concentrated material prepared by the Pillemer technique lent itself well to blood grouping purposes. While it was not so viscous as whole serum, it possessed sufficient surface tension to form well-rounded droplets on a glass slide. The addition of Merthiolate to a final concentration of 1:1,000 did not interfere with the interactions of isoagglutinins and red cells and eliminated the necessity for filtering out bacteria. The isoagglutinating activity of the separated globulins stored as a liquid at room temperature (77 F., 25 C.) remained unimpaired for 4 weeks.


238

When the macroscopic slide technique was used, the concentration of this serum could be so adjusted that agglutination with incompatible erythrocytes occurred visibly in 5 seconds and was complete at 60 seconds.

DeGowin Technique

At the 23 March 1943 Conference on Blood Grouping (9), Dr. Elmer L. DeGowin recommended the following technique, which he had employed satisfactorily 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 were marked out on the slides with a glass cutter or wax pencil, or by spraying lacquer over mask paper. Each of the 20 such areas on each slide was marked with the number of the blood specimen to be examined.

Three medicine droppers of similar bore were used, two for the typing sera and the third for cell suspensions. The third dropper was washed in physiologic salt solution after each use. Typing sera could be either rabbit or human but must have a titer of at least 1:128.

Drops of sera were placed in the proper areas on each plate and the cell suspensions added. The time of the test was recorded on the plate which was set aside to be read in 30 minutes. In the interim, the plate was tilted a few times, to disturb the sedimented films, and it was also checked for Tyndall's phenomenon.

Two independent tests were made of the same bloods and the results of the two series were compared. Additional tests were made on the bloods in which discrepancies were found.

By this technique, which was generally the technique followed in the Army, one worker, with a minimum amount of laboratory equipment, could set up and read over 100 tests an hour.

Establishment of Criteria

At the Conference on Transfusion Equipment and Procedure on 25 August 1942 (10), it had been unanimously recommended to the American Red Cross and to the Surgeons General of the Army and the Navy that when large numbers of bloods were to be tested, the following precautions be taken:

1. Two independent series of typings should be performed on the same bloods. If discrepancies were discovered when results were compared, the affected subjects should be reexamined.

2. Only typing sera of high titer should be used.

3. Only freshly prepared red cell suspensions should be used.

4. The utmost care should be exercised in the recording of results.

5. The slide method, with reading in 30 minutes at room temperature, was faster than the centrifuge method for mass typing and was quite as accurate if sera were potent.

These recommendations were accepted and put into practice.


239

At the Conference on Blood Grouping on 23 March 1943 (9), Lieutenant Pillemer reported continuing increases in the potency of the sera prepared by his technique; a number of other observers confirmed his statement.

The following standards for typing sera, both human and rabbit, were suggested, but no formal action was taken on them:

1. Sera should have a macroscopic titer of at least 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 A2 and A2B cells and should react definitely with other rare subgroups of A.

4. Negative reactions should be clear cut.

5. Keeping qualities of the sera, which preferably should be dried, should be defined.

6. If possible, a central authority should pass upon the quality of sera intended for distribution.

At this conference, special emphasis was placed upon the importance of accepting rabbit antisera for typing only if it met the same standards as those laid down for human serum. A good deal of rabbit sera that had been examined had not been satisfactory. In some lots, the anti-B serum had not exceeded the titer of average human serum and absorption had been inadequate. One lot had been too weak to produce any agglutination at all. Another lot, evidently because of unsatisfactory absorption, had shown cross reactions 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 that they 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 saliva containing A or B specific substances, pointed out that this technique had the advantage of not producing the species-specific agglutinins which require subsequent absorption.

In 750 bloods examined by the technique described by the manufacturers of rabbit sera, there had been an error of 10 percent, but there were no errors at all when the same sera were used by the DeGowin technique. It was concluded that a considerable number of errors could be explained by the fact that the manufacturer's directions were simply not specific enough.

Universal Donors

It was tentatively proposed at the 23 March 1943 Conference on Blood Grouping (9) that universal donors be employed for the Armed Forces, with the following specifications:

1. Group O blood should be used only when titration indicated that the agglutinins in the plasma were weak.

2. Crossmatching must never be omitted, because some agglutinins 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 the transfusion should be compared with his plasma 1 hours afterward for evidence of


240

hemolysis. (This specification was quickly discarded when it was pointed out by the chairman of the conference that biologic tests would be entirely impractical in frontline hospitals.)

The vigorous discussion that followed the proposal to recommend group O blood for the Armed Forces covered the following points:

1. The determination of blood groups by identification of the agglutinogens with known sera must be further checked by matching unknown sera against known cells.

2. The proposed checks on cells and plasma should 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 as to the most common typing errors and which bloods they concerned. After several proposals to avoid these errors had been made, Dr. DeGowin pointed out that the most expeditious procedure would be to make two independent determinations of the bloods. This plan, he believed, would have the further advantage of making it unnecessary to deal with agglutinins of weak titer in unknown sera.

4. It was agreed that the centrifuge technique was preferable when only a few bloods were to be examined but that it was impractical with large numbers, when it would impose too great a burden in respect to both time and equipment. It was granted that rouleaux formation was sometimes confusing when the slide technique was used.

5. Captain Elliott had observed that a period of more than 20 minutes, with constant agitation (which was practical only mechanically) was necessary to secure agglutination in some rare bloods. Dr. William Thalhimer used plate glass slides, which were agitated constantly, and read at the end of 30 minutes. Dr. DeGowin had found 30 minutes without agitation sufficient if the sera employed had a titer of at least 1:128. Others agreed that the time required depended upon the avidity of the sera used.

6. There was no agreement as to the advantages of macroscopic versus microscopic observation. Some workers considered both were necessary. Others considered microscopic observations necessary only when personnel were relatively untrained. Still others thought that, in training laboratory technicians, more errors were committed with the microscope than without it.

7. Dr. Alexander S. Wiener reported composite observations to the effect that transfusions with incompatible blood resulted in an increase in the titer of the natural agglutinins of the recipient. Blood stroma (cells and fibrinogen) had proved nonantigenic on injection. After transfusion of 250 cc. of pooled plasma (Sharp & Dohme), the increase in the recipient's agglutinin titer had been from 5- to 10-fold. To obtain the maximum titer, the serum must be withdrawn no sooner than 10 to 14 days after the transfusion; otherwise, potency was lost rather quickly.

Testing and Acceptance

At a meeting of the Albumin and By-Products Committee on 10 May 1943 (11), Dr. Cohn pointed out that continuous improvements had been made in the concentration of isohemagglutinins, and presumably more could be made, but the question, for practical purposes, was how much more improvement was necessary. He suggested that Dr. DeGowin and Colonel Kendrick act as a committee to examine the various products and report on their relative values.

At the meeting of the Subcommittee on Blood Substitutes on 13 May 1943 (12), Dr. DeGowin reported results of titration of various globulin preparations to be at wide variance. He thought it plain that all cooperating workers must adopt uniform techniques and suggested that mimeographed


241

instructions be prepared and sent to all workers, specifying methods to be used and criteria to be employed in evaluating agglutination tests. This was done.

At the 10 August 1943 meeting of the subcommittee (13), Dr. DeGowin announced that the various workers who had been evaluating Lieutenant Pillemer's serum had agreed on criteria of potency. New preparations had been sent to them for evaluation by these criteria. It had also been agreed by these workers that a large amount of the isoagglutinin preparation should be processed and, if accepted as satisfactory, should be dried, packaged, and used as "reference" serum. Other sera could be compared with it directly, thus eliminating many of the variables of sensitivity of test erythrocytes and differences in titration techniques.

At the meeting of the subcommittee on 24 September 1943 (14), Dr. Cohn reported that reference sera had been prepared in quantity and were being held in bulk awaiting the report of the evaluating workers, as well as instructions concerning packaging. Six lots had already been accepted as approximately equal in potency.

When the subcommittee met on 17 November 1943 (15), Colonel Kendrick reported that all necessary steps in the development of the new sera had been carried out. Both the Lederle Laboratories and Eli Lilly and Co. were producing them; Lieutenant Pillemer had been sent to the Lederle Laboratories and Captain Elliott to both firms to instruct their personnel in the new technique. Initial difficulties had been eliminated. Dr. Cohn pointed out that, while the present method 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 step in the process, so that, if any change in avidity occurred, it could be detected at once.

Dr. Cohn had emphasized, at an earlier meeting (14), that commercial firms must be as rigidly supervised in the production of sera as of other byproducts of plasma fractionation. At still another meeting (16), he had emphasized the practical importance of making sure that the method currently used in the preparation of isoagglutinins would give a reproducible product. He would consider the responsibility of his own laboratory ended in regard to typing sera when satisfactory reference material had been prepared.

The Army Medical School and the Navy used these sera throughout the remainder of 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 of the American Red Cross, had been adopted with the full realization that it would be costly (17). It was discontinued as of 1 November 1942, because


242

the great expansion of the program made it impossible to secure the technical help necessary to carry out the tests accurately (18).

Shortly after the institution of the typing program, errors began to be discovered, and at the meeting of the Subcommittee on Blood Substitutes on 12 May 1942 (19), Dr. G. Canby Robinson asked for guidance on the course to be followed at the blood donor centers. It was recommended that a statement be added to the cards given to the donors after each donation to indicate that the blood should be regrouped and crossmatched before all transfusions. This recommendation was put into effect.

Controlled Studies

At the next meeting of the subcommittee on 23 June 1942 (20), too few data had been collected concerning techniques at the various processing laboratories (as had been recommended at the previous meeting) to be useful. Dr. DeGowin, however, outlined a very promising experiment, undertaken shortly before, whereby the blood groups of cadets at the U.S. Naval Preflight School at Iowa City were being checked by two workers independently, in lots of 40 to 93 specimens. In his opinion, the personal equation was such that no technician was likely to perform large numbers of tests without making occasional errors. A pharmacist's mate, well trained in laboratory work, was making one series of tests and Dr. DeGowin was making the other. His technique (p. 238) was used in both series.

The final report on this study, which concerned 3,876 bloods, was made at the Conference on Blood Grouping on 23 March 1943 (9) (table 6). The errors in 24 tests were unassigned because there was no opportunity to check the bloods involved. In the remaining tests, one worker made 40 errors (1.0 percent) and the other, 110 (2.8 percent). "None of the errors," said the final report, "could have been detected except by comparison of the results of two 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


243

TABLE 6.-Errors in blood grouping determinations made by two 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-
nation

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
(1.0%)

70

25

13

2

110
(2.8%)

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 or to demonstrate agglutination with either anti-A or anti-B sera, or both. False positive-errors due to reporting of agglutination that could not be demonstrated on recheck. Transposition-errors due to reporting of group A for group B or vice versa, usually as the result of confusing the right and left sides of the slide. Clerical-errors committed in transcription of results. Unassigned-errors apparent as discrepancies in reports of the two series but not rechecked because second blood specimens could not be obtained. Bloods reported in all assigned errors were rechecked by potent typing sera from at least 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 in sedimented cells, particularly when microscopic readings were attempted.

The slide and centrifuge techniques proved equally accurate, but the centrifuge technique, while faster for single examinations, consumed more time


244

than could be spent in mass typing. It took more time to manipulate the tubes and centrifuge, required more glassware, and required time to clean the glassware. With strong sera, it was useful to use the slide method at room temperature, reading the results at the end of 30 minutes with a minimum of agitation.

This type of study did not reveal errors due to defective or weak sera; errors in labeling of the original cell suspensions; or errors in transcribing results from laboratory reports to notification cards, formal reports, and identification tags.

The errors detected were also, of course, entirely unrelated to later errors made in releasing incorrect bottles of group-specific blood from the storage refrigerators and administering the wrong bottles of blood to recipients. These administrative errors seldom occurred in blood banks operated under the control of physicians and other workers well trained in laboratory techniques and interested in blood banking as a medical specialty.

The Subcommittee on Blood Substitutes found Dr. DeGowin's report extremely significant. Probably both workers made fewer errors than they would have made ordinarily since the planned comparision of results served as a constant stimulus to careful work, a factor which would be absent if there was no parallel grouping. Dr. DeGowin was eminently skilled in this work. The well-trained pharmacist's mate was undoubtedly supercareful during the period in question.

In view of the errors made in this controlled study by a skilled physician and an extremely skilled worker, the members of the subcommittee found it alarming to consider the percentage of errors that probably 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, at least 100,000 men in the Army alone who were erroneously typed. The situation was not dangerous if all medical officers who gave transfusions clearly understood that the blood type stamped on the identification tag was simply tentative. In the pressure of an emergency, it was feared that some might omit crossmatching.

The Armed Forces Experience

Reports from various military organizations could be cited to indicate the percentage of error in recorded blood groups of military personnel and also, fortunately, the realization of those in authority that such errors existed.

On 4 March 1943, for instance, Maj. Gen. Paul R. Hawley was informed of grouping errors (6 percent) that had been discovered in the 10th Station Hospital, then in Northern Ireland (21). If the margin of error was as high in other units, the commanding officer wrote General Hawley, the matter required attention because of the risk of placing undue reliance on the information on the casualty's identification tag if emergency transfusion should be required.

General Hawley immediately ordered sample checks of other installations.


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As a result, checks were made of practically all the patients then in four general hospitals, four station hospitals, and one evacuation hospital, the numbers ranging from 810 at a general hospital to 83 at a station hospital. It was found that 154 of the 2,340 bloods examined had been incorrectly typed. In addition, 179 patients had no identification tags, and there was no record of the blood group on 33 tags. The investigation thus revealed 366 unsatisfactory records, 14.34 percent. The largest observed error was in group B, but the total error was not related to any separate blood group.

Lt. Col. (later Col.) John E. Gordon, MC, Chief, Preventive Medicine Division, Office of the Theater Chief Surgeon, who had conducted the investigation, recommended to General Hawley that the Office of The Surgeon General be advised of the error that had been discovered, that all medical officers in the command be advised of it and be warned that direct crossmatching must be carried out before every transfusion, that command action be taken to assure the completion of required data on identification tags, and that measures be instituted to insure the constant wearing of tags by all members of the command. All of these recommendations were implemented.

The experience just described was typical of that of other hospitals in the theater and in other theaters, but, once the error was realized and bloods were regrouped routinely before transfusion, it did no real harm.

The 5- to 10-percent error in blood grouping was unfortunate and undesirable, but it might have been expected for a number of reasons: the lack of avidity of the typing serum, the utilization of antibody from rabbit serum that was not always as good as it might have been, and the inexperience of the personnel who did the typing. One source of errors has already been intimated, the fact that in many camps and posts during the war, personnel responsible for mass typing, through a mistaken sense of values, placed high on their priority list the speed with which the typing was done. Speed led to confusion, and confusion produced errors, which were compounded by the lack of experience of those doing the typing.

THE RH FACTOR

Historical Note

It is an interesting commentary, on what was known of blood at the beginning of World War II, that the book on blood banks and transfusions by Kilduffe and DeBakey (22), which was published in 1942 and was as authoritative as such a text could be in changing times, contains no entry in the index for the Rh factor.

It was not until 1940 that the cause was found for the occasional hemolytic reactions that occurred when blood was properly classified and crossmatched. In that year, Landsteiner and Wiener (23) demonstrated that incompatibility could occur within the same blood group in which compatibility had been


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proved. By injecting rabbits or guinea pigs with red blood cells obtained from the Macaca rhesus monkey, they obtained a serum which would agglutinate 100 percent of rhesus monkey cells and with which they demonstrated what they named, for obvious reasons, the Rh factor.

Further studies showed that a large proportion of all human beings, regardless of their basic A, B, AB or O blood groups, have Rh agglutinogen in their red blood cells. It is inherited as a mendelian dominant in about 87 percent of white persons, 95 percent of Negroes, and 100 percent of Chinese. Those who do not have it naturally occasionally become isoimmunized with it if they are transfused with Rh-positive blood. It was soon realized that the important aspect of this new discovery was not the group which had the Rh factor in their red cells but the smaller group who did not, and who might become immunized when it was introduced into the bloodstream.

Two other discoveries were also made. The first was that when an Rh-negative woman becomes pregnant with a fetus whose cells are Rh-positive, she will probably become immunized against the positive cells. The second discovery was that when Rh-negative recipients received multiple transfusions of Rh-positive blood, even though it was group-compatible, hemolytic reactions of increasing severity might occur.

Military Significance

The second discovery concerning the Rh factor was of great military importance, since, as time passed, it became more and more the practice to give multiple transfusions to combat casualties after injuries and to patients with chronic sepsis. The prospects were that this practice would increase. From the military standpoint, the chief significance of the formation of anti-Rh agglutinins by an Rh-negative individual was that subsequent transfusions of Rh-positive bloods might lead to increasingly severe, and eventually fatal, reactions.

By 1943, enough such instances of Rh immunization had occurred at the Walter Reed General Hospital, Washington, D.C., where the patients could be observed directly by the transfusion personnel at the Army Medical School, to make the potential seriousness of this threat very clear. By the end of 1944, certain conclusions were possible (24):

1. Minor reactions usually preceded serious reactions and were likely to be mistaken for ordinary pyrogenic reactions because they were characterized only by chills and fever. They might occur before very much blood had been given and thus serve as an indication for stopping the transfusion. In military practice, this necessity was extremely unfortunate because transfusions were so often lifesaving.

2. The in vivo survival of transfused Rh incompatible cells was poor.

3. Reactions to Rh incompatibility did not occur in nonimmunized patients. Therefore, the first transfusion, or even a series of closely spaced


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transfusions, could often be given without untoward results. After the initial transfusion of incompatible Rh blood, antibodies which had formed in the recipient's circulation in small amounts might react immediately with the specific Rh (or Hr) antigen in the transfused cells and become so neutralized that none remained in the circulation. For this reason, although the recipient of the incompatible blood had become immunized, reactions in later transfusions would not occur until there had been a sufficient interval between transfusions to permit the removal of the antigen (that is, the transfused cells) and the accumulation of sufficient antibody to cause incompatibility.

4. The severity of the reaction would depend upon the amount of antibody in the recipient's blood. There might not be enough to cause even a mild reaction but there also might be enough present, or enough might develop later, to cause a fatal reaction, because of lysis of the transfused cells over a period of a few days to a few weeks. While little lasting benefit would be attained by the transfusion, the major important result would be the fatal hemolytic reaction that might occur in an Rh-negative individual because anti-Rh agglutinins were present in his bloodstream.

When Captain Elliott visited the Continental Blood Bank in Paris in January 1945, he was particularly impressed with the low rate of reported transfusion reactions (25). Maj. Robert C. Hardin, MC, he reported, did not agree with those who feared reactions on the basis of the Rh factor. From reports available to him, Major Hardin thought that the incidence in multiple transfusions was not more than 0.6 per thousand. In his opinion, it was far better to continue to put the emphasis on indications for transfusion and to attack the problem of pyrogenic reactions and hemolytic reactions resulting from the A and B agglutinin system, which were at least three times more common. He did not mean that the Rh factor should be ignored, merely that it should be kept in the proper perspective, which it would not be if emphasis on it were permitted to obscure the primary transfusion problems in the European theater.

In his report on this trip, Captain Elliott discussed the Rh factor in military medicine as follows:

The natural incidence of Rh agglutinogen in the general white population meant that one out of every eight white recipients of Rh-positive blood might become isoimmunized. Since, however, donors were selected at random, it could be assumed that 13 percent would be Rh-negative, which would make the proportion of Rh-incompatible donors 1:10 instead of 1:8. Since approximately 7 out of every 8 random recipients of random bloods would be Rh positive and 1 out of every 8 random donors would be Rh negative, approximately 1 of every 10 positive recipients would be isoimmunized by transfusion of blood from Rh-negative donors.

In the event that Rh-negative recipients and donors were not identified and specific Rh blood was transfused, it was entirely possible, Captain Elliott continued, that some 10 percent of Rh-positive recipients 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 20 percent of recipients would have reactions caused by Rh incompatibility. What it did mean was that, at some time interval after the first transfusion or after a closely spaced series of transfusions, subsequent transfusions might cause numerous mild reactions, some serious reactions, and a few fatal reactions. Moreover, because of the rapid destruction of transfused Rh-incompatible red cells after the formation of Rh or Hr antibody, such recipients might require many more transfusions than if Rh-compatible blood had been used. The warning of trouble would be reactions of progressively more serious nature with every subsequent transfusion.

Provision of Rh Testing Serum

Multiple spaced transfusions were usually given in rear hospitals and in hospitals in the Zone of Interior. It would be necessary, therefore, to provide every such hospital with enough potent Rh testing serum to prevent such reactions by the use of Rh-negative blood for Rh-negative recipients, this being the only sure way of avoiding them. Rh testing was also essential in military hospitals that operated an obstetric service, since all women who gave birth to erythroblastotic children were liable to fatal transfusion reactions if they were transfused with Rh-positive blood. The use of such serum would also help to prevent or modify erythroblastosis in infants and permit its correct treatment when it occurred.

When the military significance of the Rh factor began to be appreciated in 1943, there was not enough Rh testing serum available to supply even a small proportion of the Army hospitals that should have it. Attention was therefore directed toward making supplies of it available, and toward determining when it should be used.

Indications for Rh testing-The Rh factor was first discussed by NRC (National Research Council) personnel at the Conference on Blood Grouping on 23 March 1943 (9). The matter arose in connection with the technical manual then in preparation and how much concerning the Rh factor should be included in it. The scarcity of potent serum at this time made the recommendation that testing should be carried out routinely little more than academic, except, perhaps, in base hospitals. The meeting concluded without taking 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 much confusion existed concerning the military importance of the Rh factor that a definitive statement by the conference would serve to throw the problem into proper perspective. Except for Dr. Louis K. Diamond, of the Children's Hospital, Boston, who


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presented a minority report,3 the following statement was agreed to by all present:

The problem of isoimmunization with the Rh factor is predominantly encountered in Rh-negative women who have become immunized as a result of one or more pregnancies with Rh-positive children. It is possible to immunize some of the 15% of males and females who are Rh-negative by repeated transfusions with Rh-positive blood. To effect this, multiple transfusions carried out over a period of weeks, or even months, are necessary to induce a sufficient degree of immunity [titer of antibodies] so that subsequent transfusions with Rh-positive blood may cause reactions. It is common experience that reactions in individuals so sensitized by transfusion begin with mild reactions which progress in severity with subsequent transfusions with Rh-positive blood. Accordingly, there is usually adequate warning before a dangerous or fatal reaction develops.

Since only a small percentage of Rh-negative individuals can be sensitized with the Rh factor, this group feels that there is no indication to embark on a large scale routine Rh testing of the personnel of the armed forces. It would also seem more practical to this group to recommend that anemias in members of the armed forces who have apparently become immunized should be treated by means other than blood transfusions.

In view of the occasional sensitized patient in continental military establishments in whom further blood transfusions seem essential, and in view of the much greater importance of Rh sensitization in obstetrical and gynecological practice, it is recommended to the Subcommittee on Blood Substitutes that all means be furthered for the collection and proper distribution of potent anti-Rh serum for both the armed forces and for the civilian population.

In view of the current low supply of potent human anti-Rh serum, it is recommended that it be employed only by experienced and skilled workers and only then when specific indications arise.

On the basis of these recommendations, the conference saw no reason 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 of the Subcommittee on Blood Substitutes (13), when it was recommended that the section covering the Rh factor drawn up by the Conference on Blood Grouping held 23 March 1943 (9) should be included in the manual.

Testing difficulties continued throughout the war. In his memorandum of 11 December 1944 to Col. B. Noland Carter, MC, Captain Elliott mentioned the scarcity of testing serum and the unsuitability of the test then in use in frontline installations (24). This test took an hour to run and required the use of an 81 F.- (27 C.-) waterbath, a centrifuge, and a microscope. Later, when the wounded casualties were moved back to rear installations, where additional transfusions might be necessary and where their Rh group could be determined, there might already be so many transfused red cells in the circulation that Rh-negative recipients might be erroneously grouped 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 he thought were on the basis of Rh incompatibility. These reactions were characterized by chills, jaundice, anuria, transitory bilirubinemia, or failure of the recipient's red blood cells to increase to the expected amount after transfusion. Dr. Diamond also called attention to the multiple transfusions which had been necessary in the victims of the Cocoanut Grove disaster.

At the Conference on Blood Preservation and Red Cell Suspension on 6 December 1945, he reported that he had examined 300 servicemen who had received transfusions in which the Rh type was unknown. He had found that 30 of these were Rh negative and that about a quarter of the 30 had anti-Rh agglutinins in their blood.


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transfusions of Rh-positive blood, possibly with fatal consequences. Captain Elliott saw only one way to eliminate this risk; namely, to determine the Rh group before the first transfusion. If this was not possible, it was still desirable to make the test later, so that subsequent transfusions would be Rh compatible.

Procurement of serum.-Late in 1943, Captain Elliott demonstrated that naturally immunized individuals could be stimulated to produce high-titer, anti-Rh serum by the injection of very small amounts of Rh-positive blood. These persons could be safely bled at frequent intervals if the blood withdrawn was replaced by transfusions of Rh-negative blood. Unfortunately, this promising plan to enlist cooperative persons with Rh-negative blood in a program for the production of Rh testing serum was not carried out.

Until the middle of 1945, which means practically until the end of the war, most of the Rh testing serum used in Army hospitals was prepared at the Army Medical School or purchased from Dr. Diamond.

Late in 1944, Lederle Laboratories developed a very potent anti-Rh serum, prepared from animals, which could be used by a slide technique at room temperature, with macroscopic reading of the result (24). The test could be completed within 10 minutes and required no equipment other than glass microslides. The reaction was clear, and the technique so simple that laboratory technicians could easily learn it.

The test seemed admirably adapted for use in frontline installations in which primary transfusions were given. If the transfusion had to be given before Rh grouping could be done, a suspension of the recipient's cells could be prepared before the blood was given and his Rh group determined later. Determination of the blood group and the transfusion of Rh-compatible blood later would prevent isoimmunization and assure longer in vivo survival of transfused red cells.

If this test were put into use, it would permit the identification and maintenance of a panel of Rh-positive donors. Blood collected in donor centers in the Zone of Interior for shipment overseas could be properly grouped and the Rh factor marked on the bottle.

At this time (December 1944), the Lederle Laboratories had on hand for immediate shipment material for about 1.5 million tests and would have more within the next 3 months. The total supply would be more than enough for distribution for all Army installations in which it could be used.

Captain Elliott recommended that the Lederle serum be procured and distributed, that laboratory officers and technicians be taught the technique, and that the importance of transfusing Rh-compatible blood whenever it was possible be called to the attention of all concerned.

Promising as the outlook seemed, it was found, on further investigation, that the Lederle serum produced false positive results if the tests were not very carefully performed and were not read within a definite time after they were set up. The allowable margin of error was so small that it was reluctantly concluded that the serum could not safely be used routinely in Army laboratories.


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At a meeting of an ad hoc committee of the Subcommittee on Blood Substitutes on 2 June 1944 (28), it was announced that Dr. Wiener had been able to produce a high-titer anti-Rh agglutinin in women with erythroblastotic fetuses, whose blood originally showed a low titer. This was accomplished by the intravenous injection into them of 50 cc. of Rh-positive blood. A proposal for a contract was being submitted, but no action was taken on it during the war.

Early in 1945, Dr. Joseph M. Hill, Baylor University, began to produce large quantities of Rh testing serum by stimulating naturally immunized individuals. The serum was dried from the frozen state, vacuum sealed in ampules, and offered for sale to the Army with the assurance that all Army needs could thus be met. The serum was thoroughly tested and was found to be both potent and stable. Specifications were therefore written, and it was recommended that the serum be included in the medical supply catalog as a standard item. This was done.

Shipments of Rh-negative blood to Europe began on 12 February 1945, in the amount of 240 pints; 625 pints were shipped in March and 576 pints in June, after hostilities had ended in that theater.

Technique.-The test for Rh compatibility used at the Division 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 1 drop 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 gently resuspended and inspected for macroscopic agglutination. If agglutination was not observed, the tubes were centrifuged at 750-1,000 r.p.m. for 1 minute. The packed cells were then gently resuspended and observed for macroscopic agglutination.

4. If macroscopic agglutination was not evident, the slide was examined microscopically.

5. Absence of agglutination denoted that the blood was Rh negative (fig. 58). Any degree of agglutination indicated that it was Rh positive.

SCREENING TEST FOR O BLOOD

When the airlift of group O blood was planned, there was immediate need for a rapid screening test to select group O donors. Captain Elliott devised a simple, effective method:

Avid, proved high-titer group O serum was dried, in single test 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's blood was added to the salt solution and two drops of the suspension were transferred to the dried serum. The serum agglutinated cells of groups A, B, and AB, but did not agglutinate group O cells.

This technique effectively identified about 98 percent of group 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 apparent group O donor with red blood cells of known A and B groups before the blood was shipped.


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FIGURE 58.-Sedimentive technique of testing for Rh factor (23). This illustration shows the red cell sediment (magnification 1:2) in agglutination tubes examined from below with hand lens: negative reactions (a and b); the inner light disk in fig. b is explained by a slight convexity in the bottom of the tube; faintly positive reaction (c); weakly positive reaction (d); and typical positive reactions (e and f).

The screening sets were quickly put into production by the Reichel Division of Wyeth Laboratories and later by Cutter Laboratories. They were used in all the Red Cross centers in which blood was collected for shipment overseas, the donors thus identified being bled into the special bottles provided for oversea shipment.

HEMATOCRIT DETERMINATIONS

Copper Sulfate Falling Drop Test

A most important consideration in the management of shock was its early recognition. The degree of reduction in the blood volume was usually the initial, and often the only, determining factor. With this information available, therapy could be precise, and, if the supply of blood or plasma were limited, these agents could be most usefully distributed among the casualties.

No means of making this determination existed when the United States 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 24 June 1943 (26), part of the discussion concerned the pressing need for some


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method for the rapid measurement of blood concentration under field conditions. Certain specifications had to be met: The method must require little blood, little time, and apparatus that would not be disturbed by temperature ranges from Guadalcanal to Alaska. The method must be applicable on shipboard, on the unstable base of a rolling vessel, and must be simple enough to be mastered readily by an enlisted man. The problem of devising such a method was assigned to the group working on shock at the Rockefeller Institute for Medical Research.

At the meeting of the Subcommittee on Shock on 1 December 1943 (27), Dr. Donald D. Van Slyke, of the institute, described the technique jointly 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, were the only ones that would meet the specifications. The Barbour falling drop technique was excellently adapted to civilian needs, but it required special apparatus, a stable base, and organic liquids with a high temperature coefficient. His group therefore decided to initiate its search for a suitable test for field use on the old principle of dropping blood into standard solutions of known gravity. They further decided that, in order to avoid the temperature coefficients of organic liquids, which expand about five times as much 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 were only approximate. To prevent this phenomenon, a protein precipitant in the form of a mixture of salt and picric acid was added to the solution, to form a film about the drop of blood or plasma and hold it together.

This test had been in use only a single day when Lieutenant Phillips walked into the laboratory with a copper sulfate solution which, in itself, met all the requirements. It was easily prepared by dilution of a copper sulfate solution made up by shaking a pound of copper sulfate crystals in a pint of water for 5 minutes. The temperature coefficient of the standard solution was the same as that of blood, which made a temperature control unnecessary.

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 in a sac that did not change its gravity for about 20 seconds, which was long enough to determine whether the drop rose or fell in the solution. At the end of this time, the drop absorbed the copper and fell to the bottom. Another test could be run at once and about a hundred tests could be run with the same solution. A convenient portable kit (fig. 60) contained everything necessary for the test.

Accuracy was surprisingly high; it was possible to obtain blood and plasma gravities precise to 1 in the fourth decimal place, which was several times as accurate as was needed (27). With accuracy so easily obtained, the method could be used not only to estimate plasma proteins but also to estimate hemoglobin, by determining the difference between the gravities of whole blood and plasma. In 20 bloods tested by this method, hemoglobin


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FIGURE 59.-Demonstration of copper sulfate (falling drop) method of measuring specific gravities of whole 
blood and plasma
(30).

estimates had varied only 0.7 percent from the results of determinations 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 standard centrifuge method.

The test had been employed under a variety of circumstances. A medical officer on a hospital ship in the Pacific had seen a preliminary description of it in BUMED and had made a test study of it on medical personnel. The results had not been affected by the motion of the ship or by temperature 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 brought aboard in 2 hours. Under the circumstances, he wrote Dr. Van Slyke and his associates, there was not much time for anything but a laboratory test as simple as this.

The line charts (charts 4 and 5) devised by Lieutenant Dole for calculations of plasma proteins, hematocrit, and hemoglobin greatly simplified the test. When a straight edge was laid across a set of scales, all of these values could be obtained, and the entire test completed, within 2 minutes. The possible variations of hemorrhage, seepage of plasma, and dehydration were so 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 the casualty in shock depended upon the category into which he fell. Hematocrits of 25 to 30 indicated a blood loss of 2,000 cc. or more, which was common in casualties with multiple or extensive wounds.

The copper sulfate (falling drop) test does not indicate the total circulating blood volume, as it is sometimes stated that it does. It does provide a method of determining the relative quantity of circulating red blood cells plus information concerning the hematocrit, hemoglobin, and plasma protein levels. 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 of desired increase in the hematocrit), but also indicates the absence of indications for blood transfusion and thus prevents the waste of this valuable and scarce substance.

This test was used with great satisfaction in all theaters of operations, one reason being that it required neither elaborate equipment nor trained 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 be acted 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-Shortly after 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 the Rockefeller Institute, was officially adopted for use in American Red Cross blood donor centers (31). This modification was the use of a single solution of copper sulfate, of appropriate specific gravity, to register the critical 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 expendable glass capillary tube, from which it was expelled into the solution by pressure on a rubber bulb of the type used in vaccination. The level of acceptance of donors was 12.3 gm. percent, and the specific gravity of the solution was set for 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 the Mediterranean theater, certain difficulties arose (p. 424) that led to the practice of titering all O blood and reserving blood with an anti-A or anti-B agglutinogen titer of 1:250 or higher for group O recipients.

Titration was not practiced at the beginning of the airlift of blood to Europe, and, though it was directed by The Surgeon General in early October that it should be carried out, it was not begun until February 1945, because of the delay in collecting the necessary equipment and securing and training personnel (32). Except for the first few shipments, all blood flown to the Pacific areas was titrated. The practice in both airlifts was to designate blood with high titer as suitable for group O recipients only.

Special Investigations

Naval Medical Research Institute.-Several special studies on the titer of blood and plasma were made in the course of the war.

Two studies of the titer of pooled plasma at the Naval Medical Research Institute, National Naval Medical Center, gave essentially the same results (33). The first study covered 300 titrations and the second 1,000, with 100 monovalent controls. The investigations produced no evidence of any harmful effects as a result of the titer of isohemagglutinins present in pooled plasma and no justification for preliminary crossmatching of plasma before it was administered. The data also tended to discredit reports that undue reactions after the infusion of pooled plasma were caused by agglutinins in it.

American Red Cross Blood Donor Service-Dr. Thalhimer, Associate Technical Director of the Red Cross Blood Donor Service, and Maj. Earl S. Taylor, MC, Technical Director (34), reported on the anti-A and anti-B agglutinin titers in 1,354 pools of human plasma, in which the number of individual plasmas in the pools varied from 6 to 60. The pools were either random


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samples or were secured from every 5th or every 10th specimen encountered in routine processing. In 99.7 percent of the pools, macroscopic agglutinin titers were less than 1:40, and in 97 percent they were less than 1:20. In 355 small pools, made up of 6 to 10 individual plasmas, the percentage of relatively low titers (1:20 to 1:40) was somewhat higher than in larger pools, but the proportion of titers of 1:40 or higher was no greater than in the larger pools, which indicated the safety of the smaller pools.

A special study was made of pools of type O plasma with titers up to 1:256. Clinical administration of these plasmas caused no reactions, nor were there any signs or symptoms of intravascular agglutination.

These observers concluded that pooled plasma prepared in small pools at hospital blood banks or in larger pools at processing laboratories, in which the plasmas entered the pools by chance and without selection, could be safely administered to all individuals, regardless of their blood type.

Whole Blood Procurement Service-The Army Whole Blood Procurement Program processed some 1,500 bloods daily, which made it practical to obtain enough high titer bloods for a comprehensive study of the effects of transfusion of such blood to incompatible recipients. The study was undertaken by Maj. Leslie H. Tisdall, MC, Coordinator, Army Whole Blood Procurement Service, and his associates, with the cooperation of volunteers at the Colorado State Penitentiary (35).

The study covered the titration of 1,650 group O bloods, 376 of which (22.7 percent) were found to have anti-A or anti-B agglutinins, or both, of 1:640 or higher. Infusions in the amount of 250 cc. of high-titer group O plasma were given to 39 volunteers representing other than O blood groups. Plasma was used instead of blood to make sure that any subsequent hemolytic reactions would be the result of hemolysis of recipient, not donor, cells. The investigators considered the criterion of a hemolytic reaction to be the demonstration of hemolysis of the recipient's red blood cells while the donor cells remained intact. Posttransfusion observation lasted for a minimum of 8 hours and was personal and careful.

The isoagglutinin titer of the 39 incompatible transfusions ranged 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 was safe to use for universal donations when the antibody titer was no higher than 1:1-200 by the centrifuge technique which they had devised. They believed that a 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 the Mediterranean theater was substantiated by the studies made at Walter Reed General Hospital in 1945 by Maj. John J. McGraw, Jr., MC. They showed a subclinical increase in


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bilirubin after the use of high-titer blood and pointed to the desirability of using low-titer group O blood whenever possible in combat casualties. Major McGraw's studies also showed the importance of Rh testing to reduce the risk of severe and fatal transfusion reactions in Rh-negative recipients.

STUDIES ON A AND B SUBSTANCES

A number of studies were made before and during the war on the use of the A and B substances developed by Witebsky to neutralize the agglutinins in group O blood and condition it, so to speak, for universal donation. These substances were procurable commercially. Witebsky developed them originally from human gastric juice and later from saliva, and he questioned the 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 on 19 March 1945, with an extensive agenda (36). It was the sense of the meeting that these preparations were now sufficiently safe to be recommended for addition to human blood employed for transfusion in the Armed Forces; that a subcommittee under the chairmanship of Dr. Witebsky be appointed to assume responsibility for their standardization, control, safety, and manufacturing improvement; and that fundamental research be undertaken in the field.

At the Conference on Resuspended Blood Cells, combined with a meeting of the Subcommittee on Blood Substitutes, on 18 May 1945 (37), the ad hoc committee appointed at the 19 March meeting made its report on standardization 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 of antibody-neutralizing power, and other details. When the data listed were at hand, the subcommittee would be asked to vote on recommending that these substances be used to neutralize anti-A and anti-B agglutinins in group O whole blood distributed to the Armed Forces.

The war ended before these studies were concluded, and A and B substances were not used in the Korean War.

STUDIES OF CELL SURVIVAL AFTER TRANSFUSION

Ashby, in 1919, was the first to publish studies on differential cellular agglutination as a method of estimating cell survival rates after transfusion (38). During World War II, his method was considered more reliable than even the ingenious technique described by Ross and Chapin (39) in 1943 of radioactive iron tagging of red blood cells because the Ashby technique, unlike the Ross and Chapin technique, permitted the investigator to follow the transfused red cells throughout their entire lifespan.

Gibson's studies (39) by the Ashby technique indicated that the type of preservative used and the conditions under which the blood was stored had


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much to do with the lifespan of the red cells. Human erythrocytes transfused shortly after their collection in sodium citrate remained intact after transfusion and disintegrated at the rate of about 1 percent per day. Cells that were nonviable at transfusion disappeared from the bloodstream within 24 hours. Viable cells completed the normal 120-day life cycle.

SEROLOGIC TESTING FOR SYPHILIS

The original Red Cross donor regulations prohibited the use of serologically positive blood (p. 142), and this restriction continued throughout the war though all the evidence was to the effect that this was an unnecessary 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 processing blood for plasma had received a total of 559,767 units of which 32,812 (5.8 percent) had been lost for various reasons, including 3,094 units (0.55 percent) discarded as serologically positive (41). This proportion remained essentially the same whether blood was collected at the donor centers or overseas.

The matter of serologically positive blood was discussed in detail at the meeting of the Subcommittee on Blood Substitutes on 15 December 1942 (42), in connection with the work being done by Dr. Cohn on plasma fractionation. Dr. Cohn made the following points:

1. The minimum requirements for plasma and albumin laid down by the National Institute of Health did not prohibit the use of serologically positive blood.

2. A search of the literature by Drs. William C. Boyd, John F. Enders, and Charles A. Janeway revealed no evidence that spirochetes survive outside of the body for as long as 48 hours.

3. To fortify the evidence, these observers conducted studies that showed that Spirochaeta pallidum was rendered noninfectious, if not killed, by the lyophilizing process used in the processing of plasma. The studies included inoculation, transfer, and adequate controls.

In Dr. Cohn's opinion, there was no question that these reports and studies proved the inability of lyophilized plasma to transmit syphilis. Since the processing of serum albumin involved both freezing and drying, he saw no reason to exclude serologically positive blood from use as long 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 much disturbed over the chance introduction of 1 serologically positive bleeding into a pool intended for processing to serum albumin." He considered that this random happening had "absolutely no significance from the standpoint of transmission of syphilis * * * or from the aesthetic point either, whatever the latter is." Dr. Veldee did object, however, to the proposal that all serologically positive bloods be concentrated and used for albumin, and this was not done.

Blood collected at the Red Cross blood donor centers for plasma and albumin was tested serologically at the processing plants. When the airlift 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 the end of the day and for all the bloods collected to be sent overseas without further delay for processing. The same plan was used with the Pacific airlift.

Syphilitic Donors

At the 15 December 1942 meeting of the Subcommittee on Blood Substitutes (42), it was agreed that nothing could be done to exclude the blood of syphilitic donors who applied to the centers in the very early stages of the disease, before serologic tests were positive. Blood was never taken from persons 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 have syphilis or to have had syphilis were not permitted to give blood. A Kahn or Kolmer test was always performed on bloods to be used for transfusion.

In oversea military hospitals, if no nonsyphilitic donor was available in an emergency, it was the practice to use a donor with a history of syphilis if he had no clinical evidence of the disease, if he had been adequately treated in accordance with current directives, and if serologic tests had been negative for not less than a year.

As time passed, large numbers of soldiers on leave or stationed near blood donor centers began to give blood, and an occasional positive serologic reaction was turned up among them. The problem of reporting these donors was complicated by the fact that many of them were on furlough, or were moved, or soon to be moved, from the posts to which they were then attached. On 21 September 1943, Dr. Robinson wrote to The Surgeon General for instructions in these cases (43). Although it might add somewhat to the responsibilities of the donor centers, Dr. Robinson thought it would be practical 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 obtained at the time of the donation. If the blood proved serologically positive, the information 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 Army installation in which the soldier was stationed. This procedure would insure that the information reached the responsible medical officer in the most expeditious manner possible and would simplify tracing the soldier in the event that he had changed stations.

On 16 March 1944 (45), these instructions were further modified in the light of information recently obtained to the effect that, after repeated blood donations, a certain proportion of donors would show false positive tests.4 It was

4It was also discovered that false positive tests might occur shortly after the smallpox vaccination required of all military personnel.


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therefore requested that positive serologic tests on soldiers who had made two or more donations should not be reported as directed unless it were possible to recheck the tests and they were still positive 2 months later, without blood having been given in the interim.

References

1. War Department Circular No. 123, 24 June 1941, paragraph I, Determination and Recording of Blood Types-Changes in AR 40-1715 and 600-40.

2. Circular Letter No. 70, Office of The Surgeon General, War Department, 14 July 1941, subject: The Determination and Recording of the Blood Groups of All Individuals in the Military Service.

3. Circular Letter No. 88, Office of The Surgeon General, War Department, 4 Sept. 1941, subject: Supplemental Information Concerning Determination of Blood Type in Accordance with SGO Circular Letter No. 70.

4. Circular Letter No. 112, Office of The Surgeon General, War Department, 26 Nov. 1941, subject: Supplemental Information Concerning Determination of Blood Type in Accordance with SGO Circular Letters No. 70 and No. 88.

5. Circular Letter No. 170, Office of The Surgeon General, Services of Supply, War Department, 2 Dec. 1942, subject: The Purpose of Blood Grouping Army Personnel.

6. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 23 May 1941.

7. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 18 July 1941.

8. Minutes, meeting of Subcommittee on Albumin and By-Products, Division of Medical 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 of Medical Sciences, NRC, 25 Aug. 1942.

11. Minutes, meeting of the Albumin and By-Products Group, Division of Medical 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 Blood Substitutes, Division of Medical Sciences, NRC, 12 May 1942.

20. Minutes, meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 23 June 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 an Agglutinogen (Rh) in Human Blood Reacting with Anti-Rhesus Sera and Human Isoantibodies. J. Exper. Med. 74: 309-320, 1 Oct. 1941.

24. Memorandum, Capt. John Elliott, SnC, for Division of Surgery, Office of The Surgeon General (attention: Col. B. N. Carter, MC), through Director, Army Medical School, 11 Dec. 1944, subject: Rh Blood Grouping.

25. Memorandum, Capt. John Elliott, SnC, to Chief, Surgical Consultants Division, Office of The Surgeon General, through Director, Army Medical School, 1 Feb. 1945, subject: Transportation of Blood from the U.S. to the ETO Blood Bank in Paris.

26. Minutes, Conference for the Revision of the Army Manual on 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 the Subcommittee on Blood Substitutes to Consider Methods of Using Normal Human Serum Albumin in the Army Air Forces, Division of Medical Sciences, NRC, 2 June 1944.

29. Clinical Significance of the Rh Factor. Bull. U.S. Army M. 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 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.

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, Personnel Service, Office of The Surgeon General, 11 Oct. 1944, subject: Allotment of Six Additional Enlisted Technicians.

33. Lozner, Lt. Eugene L., MC, USN: Studies on Agglutinin Titers of Pooled Plasma. Research Project X-104, Report No. 2. Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md., 19 Aug. 1943.

34. Thalhimer, W., and Taylor, E. S.: The Low Agglutinin Titer of Both Small and Large Pools of Plasma. J.A.M.A. 128: 277-279, 26 May 1945.

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-Titer into Recipients of Other Blood Groups. Am. J. Clin. Path. 16: 193-206, March 1946.

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 and meeting of Subcommittee on Blood Substitutes, Division of Medical Sciences, NRC, 18 May 1945.

38. Ashby, W.: The Determination of the Length of Life of Transfused Blood Corpuscles in Man. J. Exper. Med. 29: 267-281, 1 Mar. 1919.

39. Minutes, Conference on Preserved Blood, Division of Medical 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 Eight Processing 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 Surgeon General) 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.

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