OBSERVATIONS ON T H E Rh AGGLUTINOGEN OF HUMAN BLOOD

ROY T. FISK AND ALVIN G. FOORD

From the Collis P. and Howard Huntington Memorial Hospital, Pasadena, California and the
Departments of Bacteriology and Pathology, School of Medicine, University of Southern
California, Los Angeles

A new agglutinogen of human blood, designated as Rh, was recently demon-

strated by Landsteiner and Wiener1-2 by means of immune serums prepared
against rhesus (Macaca mulatto) monkey blood. This factor, inherited as a
simple Mendelian not sex-linked dominant, was present in about 85 per cent of
the bloods tested irrespective of agglutinogens A, B, M, N, and P. Observa-
tions by Wiener and Peters 3 and by Wiener4-6-12 associated the Rh factor with
certain untoward transfusion reactions and revealed that this agglutinogen is
capable of causing isoimmunization. This occurred in Rh-negative patients
as the result of repeated transfusions of blood from Rh positive donors, and in
several instances atypical (i.e., intragroup) agglutinins were found which paral-
leled the specificity of anti-rhesus immune serums.
Transfusion reactions of a similar nature but following a single injection of
blood were reported by Levine and Stetson 6 , Levine and Katzin 7 and Levine and
Polayes 8 to occur in association with pregnancy, usually in cases where some
complication was evident. Atypical agglutinins were found in the serums of
these women which the authors suggested could be formed by isoimmunization
of the mother through the absorption of antigenic substances from fetal tissues.
Because the cells of the father were sensitive to the isoagglutinins in question it
was presumed that the immunizing substance of the fetus was inherited from
this source. Isohemagglutinins formed in this fashion, like those resulting from
the repeated transfusion of Rh-negative patients with Rh-positive blood, often
paralleled the specificity of anti-rhesus sera. Levine, Katzin and Burnham 9
used these observations to explain the pathogenesis of erythroblastosis fetalis
as resulting from the destructive action of isoantibodies formed by the mother
during pregnancy on the susceptible erythrocytes of the fetus. This attractive
theory, in which the immunizing antigen is most frequently the Rh aggluinogen
inherited by the fetus from the father, was supported in reports by Levine, Vogel,
Katzin and Burnham 10 and Levine, Burnham, Katzin and Vogel11, of tests on
153 mothers of infants with erythroblastosis in which about 90 per cent were
found to be Rh-negative. The husbands of the 141 Rh-negative mothers were
typed in 89 instances and all proved to be Rh-positive, which was likewise true
for 76 affected infants on whom tesus were run. Anti-Rh agglutinins were
demonstrated in 50 per cent of the mothers when tests were made within
2 months after delivery and, although a decrease in incidence was found after
greater periods of time had elapsed, antibodies were still detectable in 2 cases
after 2 years. Agglutinins of different specificity were found in one of the 11 Rh-
positive mothers, indicating that isoimmunization with agglutinogens other than
Rh may occur in relation to hemolytic anemias of the newborn.
545
546 ROY T. FISK AND ALVIN G. FOORD

The present report is concerned with the results of Rh tests on a series of 1239
individuals (927 adults and 312 infants) selected at random and 9 cases
of erythroblastosis fetalis. Its confirmatory nature together with certain original
observations justify its presentation.

METHODS
The reports of other investigators have emphasized that methods usually employed for
blood grouping are not sufficiently sensitive for Rh tests and several procedures, differing
particularly in the temperature of incubation, were used by the authors.
In the present study tests were run with either or both anti-rhesus and human anti-Rh
serums. The preparation and method of use of the two different products will be consid-
ered separately. The anti-rhesus serums were prepared by immunizing guinea pigs with
rhesus monkey (Macaco, mullata) blood according to the procedure of Landsteiner and
Wiener2. These serums varied greatly in their suitability for Rh typing tests, several being
unsatisfactory because of weak antibody content and others were discarded because of
failure to differentiate between Rh-positive and Rh-negative bloods. These difficulties
were overcome in a few animals by giving additional injections of rhesus blood but the
production of a good testing serum was largely fortuitous. The antiserums were employed
without adsorption in a few experiments but adsorbed preparations which could be used in
lower dilutions gave more definite reactions and were used in all regular typing tests.
The serums were adsorbed once in a dilution of TV with a half volume of Rh-negative washed
blood sediment.
The human anti-Rh serums were obtained from Rh-negative women who had delivered
infants affected with erythroblastosis. These serums were diluted before use but were not
adsorbed to remove anti-A or anti-B agglutinins and were therefore used on compatible
blood groups only.
The bloods to be tested were collected in dry tubes and allowed to clot, but citrated blood
was sometimes used when tests were to be made immediately. Test suspensions were made
to approximate the opacity of a 2 per cent suspension of washed blood sediment. It was
found that lighter suspensions gave results which were difficult to read macroscopically and
that heavier suspensions were likely to produce false negative reactions.
Tests with guinea pig antiserums were run by adding 1 drop of freshly prepared cell
suspension to 2 drops of adsorbed serum diluted -&. In early tests the tubes were incubated
at 37°C. for 10 minutes and centrifuged at 500-1000 r.pm. before reading, but this method
was later replaced by the following which proved to be preferable for guinea pig serums.
The tubes were mixed and allowed to stand on the laboratory bench for 30 minutes or an
hour at which time the pattern of blood sediments was examined for some indication of
agglutination. Negative reactions gave smooth circular sediments by this method while
tubes in which agglutination had occurred showed a larger pattern in which varying
amounts of roughness or striations could be seen. After 2 hours the sediments were resus-
pended by gently shaking the tubes and examined for gross agglutination. Tubes showing
no visible clumping were examined microscopically before being recorded as negative.
Tests with human serums were run for the most part at room temperature by the method
outlined above. More recently, to conserve material, tests with these sera were incubated
for 30 minutes at 37 C. and the tubes centrifuged at 1000 r.p.m. for 1 minute to facilitate
clumping. By this method higher dilutions of serum could be used with satisfactory
results.
Blood specimens from members* of our laboratory personnel were used as standard con-
trols for the study. It is important that Rh tests be adequately controlled with both
positive and negative bloods.

* These were typed for us at the beginning of this study by Dr. Alexander Wiener of
Brooklyn, New York, whose courtesy is hereby gratefully acknowledged.
 STUDIES ON THE M l FACTOR 547

RESULTS
The reactions produced by suitable guinea pig preparations were sufficiently definite
that with experience the majority of tests could be interpreted with little difficulty. It
should be pointed out, however, that the results were of a quantitative nature in that the
degree of agglutination shown by blood suspensions from different persons was not constant.
Reactions were for the most part of moderate intensity but strong agglutination occurred
with certain bloods and in other instances only slight clumping could be demonstrated.
With the methods outlined above questionable reactions were not common and sediment
readings were particularly helpful in determining weak reactions. The outcome of tests
with anti-rhesus serums on specimens from adults and newborn infants were so different
that they will be presented separately.
The results of typing adult bloods with these antiserums are summarized in table 1
where it will be seen that the incidence of Rh-negative persons was 15 per cent in a random
series of 927 individuals. This is in agreement with the findings of Landsteiner and Wiener2
who reported 15.4 per cent among 448 persons. Our results are also in accord with those
of the above authors in that they suggest no relationship between the presence of the Rh
factor and agglutinogens A and B.

TABLE 1
SUMMARY OP RESULTS OBTAINED IN TYPING 927 ADULT PERSONS FOR THE Rh AGGLUTINOGEN
USING GUINEA PIG ANTI-RHESUS SERUMS

GROUP NUMBER PER CENT R h - P O S I T I V E PER CENT Rh-NEGATIVE

0 425 83.8 16.2

A 316 84.8 15.2
B 77 90.9 9.1
AB 27 81.5 18.5
Not typed 82 87.8 12.2

Total 927 85.0 15.0

Reactions with potent human anti-Rh serums on adult bloods were for the most part
similar to those produced by guinea pig preparations but were somewhat more definite and
therefore easier to interpret. Serum specimens from 9 Rh-negative women who had re-
cently delivered infants affected with erythroblastosis fetalis were available for study.
Six of these showed antibodies corresponding to Rh agglutinins and 4 were sufficiently
potent to be employed in typing tests. These gave the following results in tests on un-
selected blood specimens:
Serum "Sh" (group 0),* 272 group O bloods tested, 84.9 per cent R h + and 15.1 per
cent Rh—
Serum " L e " (group A), 100 group O and A bloods tested, 83.0 per cent Rh-f- and 17.0
per cent Rh—
Serum " C o " (group B), 47 group O and B bloods tested, 87.2 per cent R h + and 12.8
per cent Rh—
Serum " R o " (group O), 100 group O bloods tested, 86.0 per cent R h + and 14.0 per
cent Rh—.
* Sera " S h " and " R o " were generously supplied by Dr. Angus Wright of the Childrens
Hospital, Los Angeles, and by Dr. John Hughes of the Los Angeles County Hospital.

In comparative tests with group O bloods the reactions of serums " C o " (38 bloods) and
" R o " (100 bloods) were parallel with serum "Sh". Serums " L e " and "Sh" also gave paral-
lel reactions with 35 bloods but reacted differently with 2 specimens. Similar results were
548 ROY T. FISK AND ALVIN G. FOORD

obtained, as shown in table 2, when two h u m a n serums were compared with guinea pig
preparations on a series of adult bloods b u t greater differences occurred with bloods from
newborn infants as will be discussed presently. Conflicting results with different serums
make i t difficult to interpret t h e typing of occasional bloods. For example, one of our
erythroblastosis mothers belonging to group O was Rh-negative with guinea pig serum and
with serum " L e " b u t Rh-positive with serum " S h " , even though her serum contained R h
agglutinins which gave parallel reactions with serum " S h " on 18 bloods (15 R h + and 3
R h — ) . Such differences are strongly suggestive of subtypes to the R h agglutinogen and
have been noted by other investigators.
Our results with cord blood specimens from newborn infants are of special interest
since, compared with adult bloods, they showed a consistent difference in reactivity to
guinea pig antiserums. I t will be seen in table 3 t h a t all of 312 cord bloods reacted posi-
tively t o these serums, whereas serums " S h " a n d " L e " gave a percentage of negative reac-
tions. Altogether 191 (147 group O and 44 group A) different infants were typed with the
human anti-Rh serums, t h e incidence of Rh-negative bloods being 10.5 per cent. T h e
specificity of the two kinds of serum was therefore not parallel in t h e case of these sus-
pensions although a high degree of correlation resulted with adult bloods. I t is also note-
worthy t h a t the degree of agglutination in guinea pig antiserums was in every instance much
stronger with cord bloods t h a n with adult cells. I t was a t first suspected t h a t these strong

TABLE 2
COMPARISON o r R E S U L T S O B T A I N E D IN T Y P I N G A D U L T BLOODS W I T H A N T I - R H E S U S IMMUNE
SERUMS AND T W O H U M A N A N T I - R I I S E R U M S

REACTIONS WITH HITMAN A N T I - R h SERUMS

REACTIONS WITH GUINEA PIG

SERUMS Serum "Sh" Serum "Le"

Positive Negative Positive Negative

Positive 142 0 51 3
Negative 5 50 1 28

Agreement 192 out of 197 79 out of 83

reactions were due t o some property of blood collected from t h e cord which was not altered
by repeated washings in saline, b u t this seemed unlikely since blood collected by heel punc-
ture from 9 babies on the 10th day of life reacted in t h e same way.
Differences between newborn infant and adult bloods were also shown in titration experi-
ments such as the one summarized in table 4 which was r u n with unabsorbed anti-rhesus
serum number 19. I t will be seen t h a t this serum, which was sufficiently specific to differen-
tiate between Rh-positive a n d Rh-negative adult bloods, reacted in higher dilutions with
infant bloods and agglutinated these suspensions as well or somewhat better t h a n it did
rhesus monkey blood.
Experiments t o determine the activity of potent h u m a n anti-Rh serums for rhesus mon-
key cells gave results of some interest. T h e tests were r u n a t room temperature or a t 37°C.
by t h e methods used for typing human bloods. Two different anti-Rh serums (group O)
were employed and experiments were run with cell suspensions from 8 monkeys. These
serums gave definite reactions with Rh-positive human bloods in a dilution of 1:40 when
tested a t room temperature and produced visible clumping in a dilution of 1:160 by the more
sensitive centrifugation method. With monkey cells visible clumping occurred for the
most p a r t only in dilutions lower t h a n 1:20 although weak agglutination was noted a t 1:20
with bloods from 2 animals. Some of the normal control serums (groups O and A) used in
these tests gave as strong or stronger reactions hence t h e results with anti-Rh serums were
not necessarily related to their R h antibody content. I t might be added t h a t several of
 STUDIES ON THE M l FACTOR 549
the monkey bloods used in these experiments were employed successfully in guinea pig
immunization for anti-rhesus testing serums.

TABLE 3
COMPARISON OF R E S U L T S O B T A I N E D W I T H A N T I - R H E S U S IMMUNE S E E U M S AND H U M A N
ANTi-Rh S E R U M S IN T Y P I N G C O R D B L O O D S U S P E N S I O N S FROM N E W B O R N INFANTS

REACTIONS WITH GUINEA REACTIONS WITH REACTIONS WITH

PIG SERUMS HUMAN SERUM, " S h " HUMAN SERUM, " L e "
BLOOD GROUP

Rh + Rh- Rh + Rb- Rh + Rh-

0 147 0 134 13 20 2
A 119 0 37 7
B 34 0
AB 7 0
Not typed 5 0

Total number 312 0 134 13 57 9

Per cent 100 0 91.2 8.8 86.4 13.6

TABLE 4
T I T R A T I O N OF UNABSORBED A N T I - R H E S U S G U I N E A P I G S E R U M N O . 19 W I T H
VARIOUS BLOOD SUSPENSIONS

DEGREE OP CLUMPING I N LOW

BLOOD SUSPENSION TITER*
DILUTIONS

1. Infant O 160 Strong

2. Infant O 320 Strong
3. Infantf A 320 Strong
4. Infant A 320 Strong
5. Infant A 320 Strong
6. Rhesus monkey 160 Strong
7. Rhesus monkey 160 Strong
8. Adult, R h + . . . A 80 Moderate
9. Adult, R h + . . . A 80 Moderate
10. Adult, R h + . . . O 80 Moderate
11. Adult, R h + . . . 0 80 Moderate
12. Adult, R h - . . . o 10 Slight
13. Adult, R h - . . . A 10 Slight

* Highest serum dilution (2 drops) causing visible clumping of blood suspension (1 drop)
after 2 hours a t room temperature. Control tubes with normal guinea pig serum (1:10 t o
1:40) were negative with all suspensions.
t This infant was 11 days old. Blood from other infants was obtained from the cord
a t birth.

COMMENT
The results of the present study obtained with reagents prepared by ourselves
are in accord with the observations of other investigators and suggest that the
methods of preparation and use of Rh typing serums are sufficiently well estab-
550 EOY T. FISK AND ALVIN G. FOOED

lished to permit the practical application of this procedure in clinicallaboratories.

I t should be emphasized, however, that in the light of present knowledge these
tests must be regarded as a specialized procedure requiring a certain amount of
experience and the use of known control blood specimens. This we feel is par-
ticularly true when guinea pig serums are being employed, since in our experience
these produce reactions likely to be more difficult to interpret than anti-Rh
human serums. The question of the relative suitability of the two preparations
for routine typing naturally arises but in view of certain unanswered problems
pertaining to the Rh factor, especially in respect to apparent but as yet un-
classified subtypes, it would be impossible to make final comparisons at this time.
Satisfactory guinea pig antiserums present some difficulty in their preparation
but nevertheless afford a universal source of testing material which is more or
less standard, inasmuch as results obtained with a given serum are reproducible
by other serums. It is also possible that future refinements in the preparation
of animal testing serums may render these products more suitable for general
use and additional study is clearly indicated along these lines.
The relationship between rhesus monkey antigen and the Rh factor of human
blood is interesting but has not been explained. Differences between Rh-posi-
tive and negative bloods as detected by anti-rhesus serums are of a quantitative
rather than qualitative nature. The quantitative action of these antiserums
is still apparent after absorption with Rh-negative blood and according to
Landsteiner and Wiener2 absorption with Rh-positive blood has about the
same effect, viz. a nonspecific lowering of agglutinin potency. Even so, the
absorption of guinea pig antiserums with Rh-negative blood (similar treatment
with Rh-positive blood was not tried), led to sharper differentiation of positive
and negative reactions in the present study.
It would appear that if anti-rhesus serums agglutinate human cells because of
a specific antigen-antibody reaction, then Rh agglutinins of human origin might
be expected to show a similar activity for rhesus blood. Attempts made with
two human anti-Rh serums to support this premise did not give conclusive
results since these serums failed to agglutinate rhesus blood in dilutions com-
parable to their potency for Rh-positive bloods, and since some normal human
serums agglutinated rhesus cells in dilutions equal to or greater than the im-
mune serums. This finding does not of itself disprove the identity of the Rh
factor of human blood and rhesus antigen but is of some interest and suggests
further study with a larger series of human anti-Rh sera.
The universal capacity of bloods from newborn infants to react strongly with
anti-rhesus serums raises a question in regard to the specificity of these prepara-
tions for the Rh agglutinogen. The same bloods could be differentiated into
Rh-positive and negative groups by means of human anti-Rh serums which
indicates, as might be expected, that all newborn infants are not Rh-positive.
Whatever the explanation for these differences might be, it is evident from our
findings that the anti-rhesus serums prepared in this study, although satisfactory
for typing adult bloods, were not suitable for those of the newborn.
Although human antiserums give definite Rh reactions certain factors appear
 STUDIES ON THE M l FACTOR 551

to limit their usefulness in practical work. Patients with suitable antibody

content are not always available and the presence of agglutinins anti-A, and
anti-B may limit the number of bloods which can be typed with each serum.
Absorption with Rh-negative blood of the proper group to remove these anti-
bodies is not altogether satisfactory because this procedure may interfere with
the Rh agglutinin content. This difficulty may be overcome according to
Levine et al.n by neutralization with specific substances A and B or by a similar
method indicated by Wiener12 in which "purified" saliva preparations from
group A and B persons known to be secretors are used. It has already been
mentioned that the antiserums from different persons do not always give parallel
reactions and this possibility must be taken into consideration in interpreting
results. Although conflicting results occurred infrequently with the material
employed in the present study it is apparent from the reports of other workers
that certain serums are particularly atypical. I t is possible that future in-
vestigation may clarify this problem by classifying the Rh agglutinogen and cor-
responding antibodies into definite subtypes.

SUMMARY AND CONCLUSIONS

1. Satisfactory results were obtained in Rh tests with suitable anti-rhesus
immune serums. These preparations gave an incidence of 85.0 per cent Rh-
positive and 15.0 per cent Rh-negative persons in a random series of 927 adults.
2. The differential action of anti-rhesus serums for Rh-positive and negative
bloods is of a quantitative nature and a certain amount of experience is required
to assure consistent results with these products.
3. Anti-rhesus serums, while giving satisfactory results with adult bloods, did
not differentiate infant bloods into positive and negative groups. Reactions
with these bloods were much stronger and all of 312 samples were Rh-positive.
4. The specificity of human anti-Rh serums was for the most part parallel
with that of the guinea pig preparations in tests on adult bloods and these serums
were also suitable for typing infant bloods.
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