THE PRESENT STATUS OF THE RH FACTOR*
PHILIP LEVINE, M.D.
From the Blood Typing Laboratory of Orlho Research Foundation, Linden, N. J.
The fundamental basis for the recognition of the Rh factor was laid in 1900
when three important discoveries were.made. First, the Mendelian laws of
heredity, published 35 years previously, were independently rediscovered by
Correns, Tchermak, and de Vries. Second, in the same year, Landsteiner discovered the phenomenon of normal isoagglutination as the basis of the four blood
groups and in the next year he mentioned their significance in the selection of
donors for transfusion. Third, and of more immediate importance, Ehrlich and
Morgenroth discovered the phenomenon of isoimmunization which they demonstrated in goat blood and which other workers later demonstrated in many
species.
Ehrlich and Morgenroth injected hemolyzed goat blood into other goats. Although the blood of the recipient was compatible with the first injection of blood,
subsequent injection revealed the presence of immune antibodies which differentiated the blood of various individuals of the same species. Isoimmunization
has since been induced in many animal species by the simple procedure of crosstransfusions. Presumably, the red blood cells of animals contain a sufficiently
large number of agglutinable substances so that by permutation these substances
may be found in combinations which render to each member of a species a characteristic individuality of his red blood cells. An excellent description of the
early findings on the individuality of blood is given in Landsteiner's Nobel Prize
lecture.24
Until recently this remarkable phenomenon was considered to be merely of
theoretical interest to a small number of serologists and some geneticists, with
its importance in man limited to occasional recipients of numerous transfusions.
Some patients who developed post-transfusion reactions were suspected to have
become immunized to factors other than A and B, but until 1940, actual proof of
isoimmunization in man by repeated transfusions was limited to a few instances.
It is of historical interest that up to 1928, individual human blood differentiation was limited to the four blood groups and to the subgroups of A and AB.
In that year, Landsteiner and Levine26 discovered the factors M, N and P, but
these factors almost entirely lacked the capacity to induce isoimmunization in
man, and consequently, were not clinically important in selection of donors for
transfusions. Landsteiner, Levine, and Janes 28 were the first to employ the
specific M and N substances in a study of the longevity of blood of a donor of
type M in a recipient of type N.
* Presented at the Twenty-Fourth Annual Meeting of the American Society of Clinical
Pathologists, San Francisco, California, June 28, 1946. Received for publication, August
2, 1946.
This paper and the one entitled, "Recent Developments in the Knowledge of the Rh-Hr
Blood Types; Tests for Rh Sensitization" by A. S. Wiener, published in the August, 1946.
issue of the American Journal of Clinical Pathology (pp. 477-497), constitute the Ward!
Burdick Lecture.
597
598
PHILIP LEVIKE
When, in 1940, the relationship of a factor in rhesus blood to a property in
human blood was found with the aid of rabbit anti-rhesus blood immune serums,
Landsteiner and Weiner29 had no way of knowing that their factor was important
clinically. Later in the same year, Wiener and Peters 83 showed that patients
whose blood failed to react with their experimental serum, after a series of transfusions developed intolerance to further injection of Rh-positive blood. The
immune isoantibodies observed which, curiously enough, were not active at 37°C,
or at a low room-temperature, could not be used as routine diagnostic reagents
because of lack of potency and because of their limited supply. It is now
established that the Rh factor is of great importance because of its capacity
to induce isoimmunization across the placenta, rather than by repeated transfusion.47' 49 ' 51 Aside from the clinical implications of this factor, immunized
mothers are a source of large quantities of potent anti-Rh serum, particularly if
their blood is replaced by transfusions of Rh-negative blood.
In the light of these facts, the observation of Levine and Stetson51 in 1939,
that a serious and almost fatal reaction from a first transfusion in a pregnant
woman, was attributed to isoimmunization by a dominant hereditary property
in fetal blood that was not present in the mother's blood, is historically significant. A complete description had been given of a new blood factor independent
of the factors M, N and P. Attempts had been made in 1937 to produce in an
experimental animal a serum of similar or identical specificity, but while these
experiments met with failure, it is not generally appreciated that in 1937 (Rh)positive bloods were on hand for injection and (Rh)-negative bloods for their
absorption. Several years later (1942-1943), anti-Rh serums were produced
in rabbits by injection of human blood, but the absorbed serums were not sufficiently potent for routine testing. 37
In the latter part of 1940 and early in 1941, it could be shown that blood of
the patient studied in 1937 was Rh-negative and that atypical agglutinins which
it contained corresponded with anti-Rh in specificity. Furthermore, all of the
patient's compatible donors, who uneventfully gave blood to the patient in 1937
during the patient's convalescence, were shown to belong to the Rh-negative
list prepared for the Blood Transfusion Association.46 That Levine and Stetson
were already dealing with the Rh factor was suspected by Wiener and Peters 83
who wrote as follows: "In the case reported by Levine and Stetson, the incidence
of bloods agglutinable by the patient's serum (based on 104 tests) was about 80
per cent, which is not significantly different from the frequency of Rh-plus blood
(about 85 per cent), but the antibodies gave just as intense reactions at 37°C.
as at low temperatures."*
* While intragroup transfusion reactions a t the first transfusion were previously reported
by other workers, no progress could be made without a suitable explanation for the origin
of the atypical agglutinin. In the light of our present knowledge, it is certain t h a t the Rh
blood factor was involved in these early cases.
The early observations are fully described by Boyd in an excellent review. 2 A different
version of the historical development of the Rh factor and erythroblastosis is given elsewhere .74
T H E E H FACTOR
599
Later in 1940, Levine, Katzin and Burnham, 47 ' 49 studied other instances of
intragroup reactions from the first transfusion, all of which occurred in women
with recent pregnancies. In the majority of these cases, it could be demonstrated
that: (1) the patients were Rh-negative, (2) their anti-Rh agglutinins were
mainly of the "warm" variety, and (3) these women showed a high incidence of
fetal and neonatal morbidity. Accordingly, it was stated that the two phenomena, isoimmunization of the Rh-negative mother by Rh-positive fetal blood and
the fetal and neonatal deaths, were correlated. The nature of the condition in
the infants could be readily determined on clinical and pathologic grounds to be
erythroblastosis fetalis. On the basis of the findings is seven cases, it was suggested, as had been long suspected, that maternal anti-Rh agglutinins passed
through the placental barrier and reacted with Rh-positive fetal blood causing
blood destruction.60
The complete evidence was presented in an analysis of 153 cases and from this study and
subsequent studies by Levine and his co-workers,46 the following facts emerged:
1. With the aid of a particular anti-Rh serum (now termed either anti-Rh 0 or anti-D)
derived from a mother of an erythroblastotic infant, more than 90 per cent of such mothers
were Rh-negative.
2. In the remaining cases, which comprised from 7 to 8 per cent of such mothers, immunization was attributed to finer differences of the Rh factor, to a new factor called Hr32 (genetically related to Rh), and probably to the factors A and B.
3. The Rh factor was presumably limited to red blood cells.48' 82
Certainly in the
affected infants the Rh factor could not be demonstrated in a water-soluble form in the
body fluids.1'86
4. Curiously enough, only about 50 per cent of the Rh-negative mothers of erythroblastotic infants had anti-Rh agglutinins. In the remaining cases it was assumed that the
hemolytic process in the fetus was brought about by the action of an antibody of another
form not detectable by routine procedures. (In 1944 and 1945, Race,61 Wiener,73 and Diamond14 independently described the blocking antibody).
5. Intragroup transfusion accidents in this group of women could be prevented by the
administration of Rh-negative blood only.46' 50
6. Anti-Rh serums differed in specificity, two of them now called anti-Rh„ (anti-D)
and anti-Rh' (anti-C) gave four types of reactions, while a third variety, now called antiRh, (anti-D, anti-C), had both antibodies. The first statistical study revealed the far
greater importance of anti-Rh 0 serums for routine diagnostic purposes.36' 38>46
7. Human anti-Rh serums were far superior to the experimental serums first produced
in rabbits or guinea pigs with either rhesus blood or human blood. It is of interest that
the use of the experimental serum was abandoned at an early date when large quantities
of potent human anti-Rh serums became available.
8. The hemolytic process in affected infants could be treated more favorably by transfusion of Rh-negative blood since the transfused Rh-positive, as well as the infant's own
Rh-positive blood, were still subject to hemolysis in the early neonatal period.3' 46
9. A concept wes proposed for a mechanism of transfer of fetal blood in one form or
another to the mother's circulation in the absence of grossly detectable lesions.38
10. Several factors were listed which were responsible for the low incidence of erythroblastosis fetalis in spite of a comparatively high frequency of incompatible matings (85
per cent X 15 per cent or 13 per cent). Among these factors were: the current tendency to
raise small families; a certain incidence of heterozygous fathers (somewhat more than half of
the Rh-positive individuals); and the failure on the part of many Rh-negative women to
respond to isoimmunization. It was suggested that the capacity to produce antibodies was
itself, determined by one or more genetic factors.35- 3S' 43
600
PHILIP
LEVINE
11. The incidence of erythroblastosis fetalis in any given population was shown to be
directly proportional to its incidence in individuals whose blood is negative with anti-Rh„
or anti-D. 84 ' 64
Soon after these observations were made, extensive Rh studies were carried
out, principally in this country and in England. Hundreds of publications on
this subject have appeared annually and a number of significant contributions
have been made, some of which will be described below. These deal mainly with
finer methods for detection of sensitization and theories on genetics of the Rh-Hr
blood factors and their implications. The influence of indiscriminate blood
transfusion in inducing isoimmunization prior to pregnancy so that the first-born
may be affected is now well established.13 •41 •63 Because isoimmunization may
seriously affect the health of mother and infant, several recommendations will
be proposed for adoption by state public health authorities.
In 1943 and 1944, a theory was proposed to explain how fetal blood may pass
into the maternal circulation in sufficient quantity to induce isoimmunization.36 • 38
Since this transfer of fetal blood across the placental barrier serves as the initial
step in the pathogenesis of erythroblastosis fetalis, it will be discussed first.
MECHANISM OP
ISOIMMUNIZATION
As an explanation for the origin of atypical agglutinins which were held to
be the cause of severe intragroup transfusion reactions in pregnant women, it
was suggested, in 1939, that fetal blood cells and/or tissue cells find their way
into maternal circulation.61 When the pathogenesis of erythroblastosis fetalis
was later described, it had already been shown that the Rh factor, in contrast
to the factors A and B, was not present in a water-soluble form.48 Accordingly,
the question arose as to the mechanism which permitted formed elements, the
size of red blood cells, to penetrate the placental barrier in sufficient quantity
to induce isoimmunization in the mother. If the Rh factor were also present
in body fluids, its mode of entry into the maternal circulation would present no
difficulty. Were this the case, the maternal anti-Rh antibodies, after passage
into the fetus, would be so completely neutralized by the excess of the watersoluble antigenic material that the red cells of the fetus would be spared.
The hemolytic nature of the disease substantiates the view that the Rh factor,
at least in the affected infant,* is limited to red cells, and perhaps also to tissue
cells. It does not seem necessary to assume the existence in the placental villi
of grossly detectable defects which have to recur in each succeeding pregnancy
with an Rh-positive fetus, but not with an Rh-negative fetus. In the vast
majority of the immunized mothers, the course of the pregnancy and the delivery is entirely normal. In spite of the absence of direct proof of passage of
fetal red cells across the placental barrier, the statistical data on the pathogenesis
of erythroblastosis fetalis permit no other conclusion.
* Witebsky86 produced evidence to show that Rh soluble material may be present in verysmall quantities in amniotic fluid of some, but not all, Rh-positive fetuses. But it is
significant that each of the affected infants behaved like a "non-secretor." (The secretor
type implies the presence of a factor in a water-soluble form; in the non-secretor, the factor
is limited to red cells or perhaps tissue cells).
THE BH FACTOR
601
The proposed theory is based on the well-established fact that extremely
minute amounts of antigenic material (soluble proteins, suspensions of bacteria,
or red blood cells) suffice to induce antibody production in an experimental
animal. In recent experiments in rabbits, 38 distinct increases in agglutinin
content were observed after fourteen daily injections of 2 cc. of 1:5000 suspension of human blood, the total volume of which was equivalent to 0.0056 cc. of
whole blood. For a woman weighing 120 pounds, the corresponding value of
injected blood would be only 0.13 cc.
It will be recalled that in mid-pregnancy certain structural changes occur in
the villi. The Langhans cells tend to disappear and the blood vessels, at first
small and centrally located, gradually become more prominent and approach
the maternal sinuses. In the latter part of pregnancy, only a single layer of
syncytial cells separates the fetal blood vessels from the maternal sinuses. According to Dodds,17 in the human placenta at term, the total area of fetal villi
exposed to maternal sinuses is about 70 square feet and the total lengtiti of these
villi, if laid end to end, would measure 11.4 miles. Furthermore, at least onefourth of the fetal blood is outside of the fetus and in the placental circulation.
Blood formation can be observed in the yolk sac of the four week old fetus
and agglutinable properties in the blood cells can be demonstrated in the two to
three month old fetus. It is probable that the more fundamental property of
antigenicity may be inherent even in the embryologic forerunners of red blood
cells. Nevertheless, conditions favorable for intimate contact of fetal blood
vessels over an ever increasing area of maternal sinuses are not present until the
latter half of pregnancy. These views are compatible with the clinical observation that the pathogenic effects of isoimmunization by the Rh factor are seen
almost exclusively in the fully, or almost fully, developed fetus.
Intrauterine fetal death in the seventh or eighth month is never observed in
the first born unless the patient has been immunized by previous administration
of Rh-positive blood. With increasing degree of isoimmunization, fetal death
may occur, but scarcely before the sixth or seventh month. More recent data
do not support the view that isoimmunization resulting from incompatibilities
of the Rh factor cause abortions or early miscarriages. The increased incidence
of abortions and miscarriages in Rh-negative mothers of erythroblastotic infants
may possibly result from the action of antibodies produced in a preceding fullterm pregnancy. In any event, this subject merits further investigation.
This concept of the mechanism of isoimmunization is compatible with the
clinical observation that once an Rh-negative mother is immunized, the condition
is likely to recur in all succeeding pregnancies in which the fetus is Rh-positive.
Apparently, the isoimmunization is renewed even if an interval of several years
elapses between pregnancies. Furthermore, the erythroblastosis is likely to be
increasingly severe in successive pregnancies.
Without referring to the views expressed above, an alternative theory of the
mechanism of inducing isoimmunization, recently suggested by Wiener,74 assumes that fetal blood enters the maternal circulation only during labor and
delivery. In support of his view, the fact that erythroblastosis fetalis rarely
occurs in the first born unless the mother has been previously sensitized by trans-
602
PHILIP LEVINE
fusion, is cited. However, in some of these cases an interval of many years has
elapsed between the transfusion and the pregnancy. In somewhat less than
one-half of the cases of erythroblastosis fetalis in the first born, a transfusion
history could not be elicited.63 Assuming that each of these patients received
an intramuscular injection of small quantities of Rh-positive blood a number of
years earlier, it is not conceivable that this alone served as the sole antigenic
stimulus. Undoubtedly, the transfer of fetal blood in the course of the pregnancy is the determining factor in the production of antibodies. There is no
other explanation for the appearance, in the latter half of any particular pregnancy, of antibodies which were not demonstrable several weeks or months
previously. If fetal blood is transferred only during labor and delivery, periodic
tests for detection of antibodies in the course of subsequent pregnancies have
no meaning.*
The increase of antibody content during puerperium reported by Davidsohn11
indicates an additional antigenic stimulus during delivery and/or labor. So
far as the pathogenesis of erythroblastosis fetalis is concerned, this factor plays
a rdle which is secondary to the silent process of immunization during the latter
half of pregnancy. In this connection it is significant that pregnancy offers two
conditions most favorable for antibody production, viz., slow administration of
the antigen, and action of the antigen over a long period.
According to my own views,38 there is a slight transfer of fetal blood in some
form in every normal pregnancy, but antibody production will occur in only a
small percentage of Rh-negative women bearing Rh-positive fetuses. With a
sufficiently large number of pregnancies, it is probable that each of these Rhnegative women eventually would be immunized and would deliver erythroblastotic infants. As is true in experimental animals, it is probable that genetic
factors determine the capacity for antibody production.
At present, no measures are available which will prevent the silent process of
transplacental transfer. It is conceivable that after numerous pregnancies the
cells of the reticulo-endothelial system could lose their capacity to produce antibodies. The suggestion has recently been made that the injection of typhoid
vaccine early in the course of the pregnancy might prevent or delay formation
of anti-Rh antibodies.76 However, there is considerable evidence to indicate
that experimental animals, when injected with a mixture of numerous antigens,
respond by the production of a corresponding multiplicity of antibodies. It is
also conceivable that administration of a nonspecific antigen may stimulate
production of specific antibody. Nevertheless, in the absence of other therapeutic measures, the effect of the injection of typhoid vaccine should be given
a fair trial. It may be suggested, however, that women be injected with antigens, such as tetanus and diphtheria toxoid, and pertussis vaccine, so that while
the prevention of isoimmunization is tested, the mothers are, at the same time,
producing antibodies which are useful for their newborn infants.7
* Because of the occurrence of erythroblastosis fetalis in the first born, Wiener" altered
his initial views by assuming transplacental isoimmunization limited to this group of cases.
For all other cases he assumes that the transfer of fetal blood occurs only at labor and
delivery.
T H E RH
FACTOR
603
The possibility of specific preventive measures must await the outcome of the
experiments on the extraction of substances (haptens) which have the capacity
of neutralizing the activity of anti-Rh antibodies without stimulating their
production. A step in this direction was taken by Witebsky86 when he found
small amounts of water-soluble Rh substances in amniotic fluid. More recently,
Calvin and his co-workers6 succeeded in extracting from stroma, a preparation
called elinin which has the property of neutralizing the action of anti-Rh agglutinins. A concentration of this substance was found in ether extracts of
elinin.
Homburger23 recently reported the inhibiting effect of sodium salicylates on the experimental production of anti-Rh agglutinins following the injection of rhesus blood cells.
T E S T S FOR D E T E C T I O N O F ISOIMMUNIZATION
In the first study on the pathogenesis of erythroblastosis fetalis anti-Rh agglutinins were observed in about 50 per cent of Rh-negative mothers of erythroblastotic infants.46 It is obvious that the remaining mothers were actively
immunized as indicated by (1) the presence of hemolysis in their infants, and
(2) the occurrence of severe and fatal intragroup transfusion accidents in the
absence of anti-Rh agglutinins. In 1941, it was felt "that antibodies capable
of reacting in vivo cannot be demonstrated because of limitations in the sensitivity of the technique involved."33
For the demonstration of anti-Rh agglutinins washed red cells suspended in a
medium of physiological saline were employed. The first indication of antibodies, other than agglutinins, was observed in anti-Rh serums which exhibited
the so-called prozone phenomenon. Interpretation, however, was not clarified
until Race,61 Wiener,73 and Diamond14 demonstrated the presence of antibodies
which are capable of uniting specifically with Rh-positive red blood cells without
producing the visible effect of agglutination. They have variously been called
"blocking", "incomplete", or "inhibiting" antibodies. Presumably, the surface
of these cells is coated with a specific antibody and consequently they can no
longer be agglutinated by potent anti-Rh agglutinins. Subsequently, Coombs,
Mourant, and Race 8 ' 9 succeeded in demonstrating a direct reaction of Rh-positive cells specifically coated with blocking antibodies on the addition of antihuman precipitin for globulin.* This test is especially useful for demonstration
of the presence of both antigen (Rh-positive cells) and the passively transferred
maternal antibodies in the affected infant's circulation. It has the added advantage that only minute quantities of blood are required. Actually, only a
dilute cell suspension will suffice. In the presence of potent blocking antibodies,
the affected infant's Rh-positive cells fail to react with anti-Rh 0 agglutinins.
The specific coating of such cells, however, renders them susceptible to agglutination on the addition of anti-human globulin precipitin. This test which is
remarkably sensitive has been successfully employed by Hill and Haberman 22
who designate this phenomenon as the "developing test."
* Recently Coombs and Race10 have demonstrated that Rh-positive red cells treated
with either anti-Rh agglutinins or blocking antibodies, show specific changes in mobility
in an electric field.
604
PHILIP LEVINE
In several recently studied cases of Rh-negative mothers of group 0 it was not possible
to differentiate isoimmunization due to the Rh factor from that due to the factor A (orB).
The affected infant's blood behaved like Rh-negative, but the maternal anti-Rh blocking
antibodies demonstrable in the infant's blood could have been the residual antibodies from
the preceding pregnancy when the mother had anti-Rh blocking antibodies and the infant
had a severe form of erythroblastosis fetalis. The results of tests with anti-human precipitin would have been decisive. It so happens that in any event the type of blood recommended for transfusing the affected infant is Rh-negative blood of group O.
In 1944, Diamond15 observed that blocking antibodies which failed to react
with a saline suspension of cells would agglutinate heavy suspensions of fresh
blood. That the presence of serum was essential was indicated by the failure
to demonstrate the effect with heavy suspensions of washed blood (Wiener76
and Levine62). Wiener, who demonstrated the importance of serum as a suspending medium instead of saline, designated the phenomenon as "conglutinin
test." Since this term has been applied previously to an entirely different and
thoroughly studied phenomenon in beef blood, it is preferable to refer to this
TABLE 1
COMPARISON OF BLOCKING TEST AND TEST WITH SERUM AND ALBUMIN SUSPENDED CELLS
SERUMS OF Rh-NEGATIVE WOMEN
l
Blocking
Serum -sus- [Rhj.
pended <
cells
[Rh2.
Albumin
suspended cells
2
+ + ± ++
3
4
5
0
0
0
++
0
0
0
?
0
0
0
0
0
0
+++
0
6
7
8
9
+++ ++± ++± +±
10
0
indirect*
direct
0
0
0
0
+
+±
0
0
0
0
+±
direct
+++
0
0
0
0
+++
direct
* In the indirect test, the readings were made after the addition of potent anti-Rh
agglutinins in a suitable dilution.
effect as "the test with serum suspended cells." Subsequently, Diamond and
Denton16 made the significant observation that human or bovine albumin in a
20 per cent solution could also be used as a suspending medium.
The observation of Levine and Bernstein46 indicated that bovine albumin was
preferable to human serum for a qualitative test to detect the presence of blocking
antibodies. For a quantitative study, however, satisfactory results were obtained if dilutions of the blocking antibody were made with pooled male serum
and the test cells were suspended in bovine albumin. This routine was adopted
because serum from healthy male individuals was cheaper and more readily
available than bovine albumin.
Some typical findings are given in table 1 which shows the varying behavior
of the blocking test as first performed by Race and Wiener, and the direct tests
with serum and albumin suspended cells.
This experiment was selected to show the variable behavior of serum and,
perhaps cells, as a suspending medium in contrast to the striking specificity of
bovine albumin. Similar but less striking discrepancies were frequently ob-
605
T H E R H FACTOR
served. Because potent anti-Rh agglutinins were required as a third reagent
for the original blocking test, this technic is no longer recommended as a routine
procedure.
Subsequently, quantitative studies on the direct reaction of the blocking
antibody revealed, in certain instances, the presence of a prozone phenomenon.
In some instances the high titers found were most unexpected, particularly since
the reactions in the qualitative test with undiluted serum were minimal. A
similar instance was observed in one case studied in 1944 in which both serumsuspended cells and the slide reaction of Diamond were used. A typical experiment is given below.
This observation raised the question of two varieties of blocking antibodies
just as the prozone phenomenon observed in the titration of agglutinins in
saline-suspended cells indicated the presence of an additional antibody, the
blocking antibody. Although comprehensive confirmatory studies are still
required, it is of interest that Hill and Haberman 22 arrived at a similar conclusion
based on discrepancies in their quantitative studies on titers of agglutinins,
TABLE 2
THE PROZONE EFFECT
Dilutions in normal male serum; test cells suspended in 20 per cent bovine albumin.
SERUM DILUTION 1:
1
Mother
Cord blood
Mother
(-07
\-08
-09
2
4
8
16
32
64
128
256
512
+± +± + ++ ++ ++ + +++ +++ + + +± ++ ++ +±
+
+ +± + ++ ++ + ++ + + ++ +± +
tr.
tr + ± + + + + + + + + + + + + + + + + + +
+
blocking antibodies, and the precipitin reaction of Coombs and Race on the
affected infant's cells. However, proof for the existence of other varieties of
blocking antibodies depends upon the result of specific absorption which are, as
yet, not conclusive.
Soon after Diamond and Denton16 made their important observation on the
direct reaction of blocking serums with albumin-suspended cells, attempts were
made to find other media which would serve as substitutes for bovine albumin.
The purpose of these tests was two-fold: (1) to study the mechanism of the
reaction involved and (2) to find a more readily available substitute for a highly
purified and, therefore, expensive product.
Numerous substances, such as gums, proteins, polypeptides, amino acids,
polyvinyl alcohol, and inorganic salts, were tested in suitable dilutions, made
isotonic, and used as suspending media for the sediment of saline-washed Rhpositive cells. These suspensions were tested with potent blocking and normal
serums which served as controls. Gum acacia, certain varieties of gelatin,
polyvinyl alcohol, and still other substances were shown to elicit a specific direct
agglutination with blocking serums.46* The complete details of the procedures
*Previously, Diamond16 reported negative results, but more recently he found several
substances which could be used successfully.12
606
PHILIP LEVINE
are still to be supplied. Levine and Bernstein found that certain dilutions of
Le Page's mucilage # 7 may also be used as a substitute for bovine albumin.
However, the manufacturer states that the composition base of this material is a
very fine grade of gum arabic (or acacia).
The usefulness of acacia in quantitative tests is shown in table 3 which also
indicates the importance of the titration studies of the blocking antibody in the
serums of Rh-negative mothers and their affected infants.
On the basis of the titration values, a fatal outcome could have been expected
in the infant having serum 16 in contrast to the infant having serum 14 (table 3)
who recovered with the aid of several transfusions. Perhaps, the striking
difference between the qualitative and quantitative tests in serums 15 and 16 is
due to the presence of another variety of antibody or a nonspecific substanee
which must have passed the placental barrier.
The successful application of bovine albumin, acacia and other substances
raises the validity of Wiener's claim74 that the reaction is brought about by
"a colloidal constituent (conglutinin) in the plasma probably identical with the
TABLE 3
ACACIA AND BOVINE ALBUMIN AS SUSPENDING MEDIUMS
TESTS OF SERUMS OF FOUR IMMUNIZED MOTHERS AND TWO AFFECTED INFANTS
11
Bovine albumin
12
Mother Cord Mother
Blood
Cord
Blood
14
16
13
15
++ +++ ++ +± ++ ++
+
+ +± +++ 0
0
512
8
64
256
4
512
Normal
17
18
19
0
0
0
0
0
0
tr
0
0
so-called X-protein, which is a large molecular complex of albumin, globulin,
and phospholipid." Wiener, more recently has stated his belief77 that fibrinogen
is another important constituent of X-protein.
In this connection, human cord serum is certainly not much less sensitive than
pooled male serum for suspending Rh-positive blood, and certain specimens of
cord serum have been found preferable to selected male serum. In fact, the
active material, albumin, is present in relatively greater concentration in cord
than in adult serum.67 As indicated above, bovine albumin is preferable to
human serum.
The results of quantitative studies of several varieties of maternal antibodies
present in infant's circulation are useful in formulating a prognosis of the condition in the infant. With the availability of sensitive tests for detection of isoimmunization in both mother and her affected infant, it becomes increasingly
important to study the infant's blood in the neonatal period. Unfortunately,
the cord blood in these cases is frequently not submitted for examination.
In one case recently studied the mother's serum showed only a weak reaction in the test
with albumin-suspended cells, and yet the infant was Rh-negative. Titration of the
THE RH FACTOR
607
mother's serum showed the presence of both agglutinins (titer 1:256) and blocking antibodies (titer 1:64). A zone effect was also observed on titration of both mother's and
infant's serum for blocking antibodies and the end points were respectively 1:64 and 1:32.
It is of interest that on the twenty-fifth day of life the infant was still Rh-negative and
maternal antibodies were still present, but in low concentration. After numerous transfusions of Rh-negative blood, the blood of the infant, on the thirty-fourth day of life, contained about one-third Rh-positive blood, which represented his own blood, while the remainder was obviously the surviving transfused Rh-negative blood. The infant made an
uneventful recovery although the hemoglobin was only about 60 per cent. Thereafter it
would matter little if Rh-positive blood was used for further transfusions. In severe cases
characterized by the persistence of maternal antibodies, transfusion of Rh-negative blood
is certainly preferable to Rh-positive blood. Even in mild cases Rh-negative blood is
preferred and it can be expected that careful studies will reveal a more rapid rate of recovery in these infants than in those receiving Rh-positive blood.
Recent claims were made by Wiener" on the correlation of the symptoms in
the infant and the type of antibody, agglutinin or blocking, in the mother's
serum. In connection with these views, he suggested that the molecular size
of the blocking antibody is smaller than the agglutinin so that the latter does
not cross the placenta. Thus, purely hemolytic symptoms were associated
with blocking antibodies and agglutinins with toxic symptoms, severe jaundice,
and kernicterus. Kernicterus was claimed to be the result of thrombi of specifically agglutinated cells in the arterioles of liver, causing severe icterus (not
associated with the degree of anemia) which supplies the characteristic coloring
to certain nuclear brain centers whose arterioles are likewise plugged with
agglutinative thrombi. 81
Unfortunately, these claims cannot be fully supported because of numerous
exceptions, especially in regard to the occurrence of purely hemolytic symptoms
in the presence of strong anti-Rh agglutinins and the absence of blocking antibodies. In regard to kernicterus, it is curious that most affected infants, whose
mothers have strong anti-Rh agglutinins, remain entirely free from symptoms
referable to lesions in the central nervous system. Kernicterus also has been
observed by Diamond12 and Levine44 in instances where the mother has had
blocking antibodies. Furthermore, it has been observed in the absence of
erythroblastosis fetalis.18
It is also remarkable that the agglutinative thrombi, which were reported to
have been observed in many organs, exert harmful effects only in the liver, brain,
and bone marrow. If these thrombi represent lesions, one should expect complications due to their presence in the arterioles of the kidney where there is no
collateral circulation. As indicated above, the so-called deficiencies in cord
serum, claimed by Wiener in connection with his recent theories, cannot be
substantiated since cord serum rich in albumin can be used to elicit the direct
agglutination given by blocking serums. While bovine albumin is preferable
for the qualitative test, cord serum was found to be satisfactory as a diluent for
the titration of blocking antibodies with albumin suspended cells.
Although there are striking differences between agglutinins and blocking
antibodies demonstrable in vitro, no mention has yet been made of the fact that
608
PHILIP LEVINE
in vivo some of these differentiating features lose their significance because under
these conditions both sorts of antibodies react with fetal blood in a medium of
plasma. The fact that blocking antibodies may be more frequently observed in
the infant's circulation than are agglutinins need not, in the absence of physicochemical measurements, constitute proof that the blocking antibody is of smaller
molecular size. As one alternative explanation, the suggestion is made that
agglutinins may be more readily destroyed than blocking antibodies.
According to Wiener's hypothesis, erythroblastosis fetalis associated with the
presence of anti-Rh agglutinins in the mother, is not the result of intrauterine
blood destruction. There are many exceptions and it is difficult to accept the
view that a concentration of maternal antibodies sufficient to induce severe
symptoms can result from the process of parturition and delivery. It certainly
cannot be expected in instances where delivery is by cesarean section.
In conclusion, new procedures are now available for detection of sensitization
and for quantitative determination of several antibodies. Preliminary data
TABLE 4
THE CROSS-ROADS EXPERIMENT (after Levine")
B A S E D ON T E S T S W I T H 334
R A N D O M B L O O D S ( W H I T E ) C A R B I E D OUT IN A P R I L , M A Y
.
TERMINOLOGY OP
WIENER AND
LANDSTEINER
Rhi
Rh2
Rh'
Rh-
JUNE,
MRS. M . F . A N T I - R h o
MRS. M.S. A N T I - R h '
MRS. K.B. A N T I - H r
+
+
+
0 or ±
0
+
0
0
+
0
AND
1941*
0 or ±
+
INCIDENCE OP
TYPE
(PER CENT)
71
14
2
13
* At the request of D r . Wiener, the scheme of the reactions indicated was made available
to him for inclusion in the third edition of his book, "Blood Groups and Transfusion,"
p p . 253-254 (C. C Thomas).
indicate that the severity of the condition in the infant could now be more
readily correlated with quantitative studies of maternal antibodies persisting
in the affected infant's circulation. Intensive studies on the properties of the
several varieties of agglutinins and blocking antibodies which are essential for a
full understanding of the finer details of erythroblastosis are still to be carried out.
GENETICS OF THE R h - H r BLOOD TYPES
Reference already has been made to large quantities of two varieties of anti-Rh
serums and of one poorly active anti-Hr serum available to the author in 1941.32 •46
The behavior of the two qualitatively different varieties of Rh serums and of the
anti-Hr serum served as the starting point for theories on the heredity of the Rh
factor. Because of the historical significance of these facts and their bearing
on the now generally accepted theory of Fisher, the table showing the relationships of these three serums is herewith reproduced (Table 4).
I t will be observed that the two anti-Rh serums give four types of reactions
THE RH
FACTOR
609
much like that in the scheme of the four blood groups.* But the anti-Rh' serum
and the anti-Hr (now called anti-Hr') give only three types of reactions and,
just as in the case of the M and N factors, a type of blood failing to react with
both antibodies has not been observed.
These two contrasting facts are most significant because they served as the
focus for different theories, that of multiple alleles for the variants of the Rh
factor proposed by Wiener71' 72> 8 °. 85 as opposed to Levine's view 40 that two
allelic genes, analogous to M and N, determine the two factors which give the
above mentioned three types of reaction. As will be shown below, Wiener's
views no longer appear to be tenable and the evidence points to three closely
linked Rh genes with their corresponding Hr alleles, as postulated by Fisher
and Race.19 Reference will be made below to the necessity of adopting the new
British terminology for these three sets of genes, i.e., D, C, and E and d, c, and e.
With the description of the Rh" factor by Race64 and Wiener,84 the latter
postulated a series of at least six genes at a certain locus in a particular chromosome, any two of which determine the genotype. Subsequently, it became
necessary to include two genes, Rhz66 and Rhy65, and in general, this or any theory
must take into account still additional variants of the Rh factor such as these
observed by several workers. 4,63 ' 69
At first Wiener72 justified the omission of the Hr factor on the ground that this
type of serum, like anti-0 serum, was rare and always of weak activity. It was
subsequently shown by Levine40 that anti-Hr serum must be rare because 92 per
cent of immunized mothers are Rh-negative (Hr-positive) and, therefore, cannot
produce anti-Hr antibodies. They are usually weak because only a portion of
the 8 per cent of Rh-positive mothers of erythroblastotic infants (about 2 per
cent) are Hr-negative and only these are the source of anti-Hr antibodies.
Accordingly, the ratio of chances for finding potent anti-Hr and potent anti-Rh
-agglutinins are 1:46.
It is true that Levine's original Hr' serum was of weak activity, but nevertheless, its incidence of positive and negative reactions could have been calculated
on the basis of the M and N analogy by taking into account the incidence of
negative reactions with anti-Rh' serum, which was maximally active.
As indicated in table 4, made available to Wiener at his request, the first
anti-Hr' serum did react also on some bloods of types Rhi and Rh'. The serum
obviously failed to detect many bloods of the heterozygous form. In an unpublished series of tests, carried out in 1941 the first anti-Hr serum gave positive
reactions in 44 per cent of the white subjects tested and in 86 per cent of the
Negroes tested.44 In a subsequent series of tests carried out with a potent serum,
the incidence of positive reactions in the white group was 81 per cent and 99 per
cent in the Negro group, as was to be expected from the theory.42
* Bloods corresponding to the qualitatively different subtypes Rh2 and Rh' and the
essential human anti-Rh serums were supplied to the late Dr. Landsteiner who confirmed
these findings.43 From a number of discrepant reactions in tests with the experimental
serum and several human anti-Rh serums,30 one of these bloods was subsequently (1942)
selected by Wiener70 as representing the type Rh'.
610
PHILIP LEVINE
It is not generally appreciated that in 1943 when Race and Taylor64 described
their more potent anti-Hr serum (called St.), its relationship was studied only
with regard to anti-Rh 0 and anti-Rh" serums. In tests with either one of these
two agglutinins and the anti-Hr' antibody, four types of reactions were observed. It so happened that in the early investigations of Race and Taylor,
anti-Rh' agglutinins had not yet been observed in England. Accordingly, they
were then not in a position to confirm Levine's observations on the peculiar
genetic relationship of the two blood factors described by the two antibodies
anti-Rh' and anti-Hr'. Two facts, (1) the absence of bloods which failed to
react with both anti-Rh' and anti-Hr' antibodies, and (2) the stronger reactions
given by Rh2 and Rh-negative bloods, led Levine in 1941, to suspect a close
genetic relationship analogous to the factors M and N. It is for this reason that
the two letters "Rh" were reversed to supply the term "Hr" for the new blood
factors.32
The genetics of the Rh-Hr system is now much clarified, due to the excellent
work of Fisher and Race,19 who confirmed Levine's initial views that the relationship of one of the Rh genes to its corresponding Hr gene is far closer than
that of the same Rh gene to the other Rh genes. In order to simplify matters,
the genetics of the M and N system will now be briefly reviewed. Thus there
are two genes M and N (m) and three genotypes, each corresponding to a
particular phenotype. These facts along with the essential statistical proof are
indicated below:
REACTION WITH
GENE
M
N
GENOTYPE
M N heterozygous
M M homozygous
N N homozygous
anti-M
anti-N
+
+
+
0
+
0
MN
M
N
The frequency of gene M or m = s/M. type, and the frequency of gene N or n =
\ / N type. The values of m + n = 10 and m2 + 2 mn + n2 = 100. Thus, in a
population of American Indians with 4.9 per cent N, application of the formula
gives an expected value of 60.8 for M, which is in excellent agreement with the
observed value of 60.0 per cent.26 • 27
Similarly given anti-Rh' serum with 73 per cent positive reactions and 27 per
cent negative reactions the incidence of corresponding reactions with the antiHr' serum can readily be calculated. For convenience, the British terminology
will be given in which anti-Rh' = anti-C and anti-Hr' = anti-c. Application
of the formulae, C + c = 10 and C2 + 2 Cc + c2 = 100, gives the following:
incidence of gene c = V27 = 5.2
incidence of gene C = 10 — 5.2 = 4.8
incidence of CC (homozygous) = (4.8)2 = 23
incidence of 2 Cc (heterozygous) = 2 X 4.8 X 5.2 = 50
incidence of cc (homozygous) = 100 — (23 + 50) = 27
derived incidence of positive reaction with anti-c = 50 + 27 = 77
observed incidence (Race62, Levine40'42) =. 80.
T H E RH
611
FACTOR
It is a great tribute to Fisher and Race19 that they predicted two remaining
varieties of Hr serums, corresponding to anti-RhD (anti-Hr 0 ) and anti-Rh" (antiHr"). Subsequently, one of these, anti-Hr", was found by Mourant 69 . Diamond12 has recently found a serum which seems to correspond to anti-Hr 0 . By
similar calculation, the incidence of positive and negative reactions given by the
anti-Hr" serum (anti-e) could be predicted on the basis of these simple calculations, and the observed value was found to correspond exactly with the derived
value of 97 per cent.
Before these theories are graphically represented, it may have become obvious
to the reader why the current complicated terminology will eventually have to
be abandoned in favor of the British terminology which takes into account the
TABLE 5
D E T E R M I N A T I O N OP G E N E F R E Q U E N C I E S FOR T H E R h - H r SYSTEMS ( C - C , D-d,
AND E-e)
X = Incidence one dominant gene
x = Incidence of the other dominant gene
X2 + 2Xx + x! =100
X + x = 10
0 + 10
1 + 9
2 + 8
3 + 7
4 + 6
5 + 5
6 + 4
7 + 3
8 + 2
9 + 1
10 + 0
0
1
4
9
16
25
36
49
64
81
100
0
19
32
42
48
50
48
42
32
18
0
100
81
64
49
36
25
16
9
4
1
0
true genetic relationship to the Hr factors. The contrasting theories of Wiener
and the British workers are illustrated below:
Wiener
RiR2r°|
II
f R'R"r
Fisher
O
c
El ie
The tendency at present is to delete the letter "h" from the genes for Rh.
With regard to the theory of linked genes, the latter are arbitrarily arranged
on the length of the chromosome, but Fisher believes that the genes C-c may be
located between D-d and E-e.
A table which permits the calculation of the expected values of positive reactions given by anti-d (anti-Hr 0 ) or anti-e (anti-Hr") is given above. It may also
be used in all instances in which there are only two genes which determine three
varieties (phenotypes) as in the case of M and N and in accordance with Levine's
original concept of the relationship of anti-Rh' and anti-Hr'.
The British workers at first believed that Levine employed an anti-Hr 0 serum
in his statistical studies on Negro and white individuals referred to above.44
612
PHILIP LEVINB
These studies were mentioned in the course of another paper dealing with the
incidence of the Rh factor in Japanese. 68 Unfortunately, a clerical error, subsequently corrected,42 misled Fisher19 and Race62 and it is now clear that the
serum employed was anti-Hr' in specificity.
Actually, there are additional variants of the Rh factor, one of which was
described by Waller, Levine and Garrow,69 and another called anti-C w , which was
described by Race.63 Indeed, there are hints in the literature that further
study will show still other variants. 4 • "4 • 58 Furthermore, Wiener72 himself,
described so-called intermediate factors. It is difficult to visualize such a complicated series of events at a particular locus so crowded with numerous allelomorphs, any two of which determine the genotype. The complex antigenic
structure of the Rh factor can be more readily explained by assuming the existence of closely linked genes at several loci on the length of the chromosome.
Wiener's objection80 that this would be unique in genetics is perhaps more applicable to his own theory, particularly since there is a wealth of genetic precedence in the literature on linkage.
Comparatively little is known of human heredity, in general, so that it is not
a simple matter to predict the probability of multiple alleles rather than linkage.
The same choice is frequently difficult for geneticists to make even when sufficient experimental data can be readily obtained as in Drosophila. Nevertheless, the concept of linkage makes it possible to apply gene frequencies for any
one set of alleles at a time as well as the linked genes.
The objection to the use of capital letters for the one gene, e.g. "C", and small
letters for the other gene, e.g. "c", does not imply dominance of the one over the
other. It simply represents established genetic usage. Indeed, geneticists
object to the use of the letters M and N as genes but prefer to use M for one,
and m, instead of N, for the other. The results of recent surveys of prominent
geneticists indicate a preference for British terminology because the available
data can be better understood on the basis of close linkage rather than on the :
basis of multiple alleles. The geneticist hesitates to accept a theory which fails
to take into account the Hr factors which have such an obvious genetic relationship to their corresponding Rh factors.
So far as terminology is concerned, it will be necessary, at least for the present,
to employ Wiener's nomenclature and gradually to introduce the British terminology. The British terminology for the six genes and Wiener's corresponding
brief symbols are given below.
1
2
3
4
5
6
BRIEF SYMBOL FOR GENE
FULL SYMBOL FOR GENE
B'
R2
CDe
cDE
cDe
Cde
cdE
cde
,.0
B'
R"
r
REACTING W I I H SERUM
C
c
c
C
c
c
D e
D E
D e
d e
d E
d e
613
T H E R H FACTOR
The clinically important serum is anti-D (anti-Rh 0 ) and the first three genes
will determine the presence of the Rh factor in conjunction with any one of the
others. The genotype can be written as fractions; thus, an individual homozyCDe
gous for Rhi can be represented as ^f^~, while the genotype of an individual
heterozygous for Wiener's RhiRh 2 can be represented as one of several types, for
. x
CDe CDe ,
instance, -^TFT, ?TTF;> e t c cdE CdE
Unfortunately, four of the serums are quite rare and it is doubtful if any of
the qualified workers in this field have all of these reagents at all times. Until
large quantities of potent specimens of the rare serums become available, further
progress in this work will be delayed. The clinician, however, needs only anti-D
(anti-Rh 0 ), and must consult experts for further analysis.39
SOME A P P L I C A T I O N O F T H E G E N E T I C
THEORIES
It is of interest that almost identical rules of heredity follow from either of the
two contrasting theories. With the use.of potent serums, a particular factor in
the blood of a child may be shown to be present, only if the same factor is found
in the blood of one or both parents. Because of the M and N types of relationship of the three Rh-Hr factors, a parent homozygous for any Rh factor C, D,
or E cannot have a child homozygous for the corresponding Hr factor (c, d,
ore). 78,85
These tests are useful in determining the genotype of fathers of erythroblastotic
infants. The important consideration is the differentiation of individuals whose
genotypes are such that Rh-negative offspring may or may not be excluded. In
selected cases, characterized by intense isoimmunization and a history of severe
erythroblastosis in more than one infant, termination of an early accidental
pregnancy may be recommended. Such Rh-negative women should be advised
to undergo artificial insemination, or to adopt children. Whether or not they
should become pregnant several years after the disappearance of antibodies is
problematic. Possibly, the degree of isoimmunization in the following pregnancy
will not be too intense and premature delivery may result in an infant not too
severely affected.
The following illustrative case is cited. A woman, 34 years of age, had had 11
induced abortions, five pregnancies which had ended in late fetal or neonatal
death, apparently due to erythroblastosis fetalis, and was the mother of three
living children. She was in the third month of her 20th pregnancy when she
requested its termination. Before the blood specimens were drawn, she volunteered the information that her husband was not the father of her first child.
This could be verified by the results of the tests (table 6), which at the same
time also showed that he was not the father of her other two children.
With anti-E (anti-Rh") the first child could be excluded because the property
Rh" or E present in the blood of the first child was not present in the blood of
the mother or the husband. On the basis of M and N tests, the other two
614
PHILIP LEVINE
children could be excluded because the husband was homozygous for N, while
the two children were homozygous for M.
In any event, only rigid criteria should be applied in recommending termination of an early accidental pregnancy in an Rh-negative mother still immunized
from a previous pregnancy. This is illustrated in the following case in which the
termination of the pregnancy was fully justified. This Rh-negative patient
had four full term pregnancies, the first-born being mildly erythroblastotic, even
though the mother had not been immunized by previous transfusions. At no
time did the patient lose her anti-Rh agglutinins which were demonstrable for
the first time in 1942. The second infant had severe erythroblastosis with
residual symptoms of spasticity presumably due to kernicterus. After this
pregnancy, the patient was warned not to become pregnant until an interval of
four years had elapsed. A third child, born in 1943, died of severe erythroblastosis. She then became pregnant again early in 1945. Tests showed her
husband's blood to be homozygous for the Rh factor, since his blood failed to
react with anti-Hr' serum. On the basis of these facts, the pregnancy was
terminated.
TABLE 6
T E S T S WITH A N T I - S E R U M
Husband
Mother
Child 1
Child 2
Child 3
ON
OMN
OMN
OM
OM
Rh„
D
Rh'
C
Rh'
E
Hr'
c
+
+
0
0
0
0
0
+
+
+
+
+
+
+
+
+
+
+
0
0
A PUBLIC HEALTH PROGRAM
In 1941, after the role of the Rh factor in the pathogenesis of erythroblastosis
fetalis was established, the writer drafted the following two rules for the benefit
of appropriate public health authorities:
1. In all individuals receiving repeated transfusions in whom untoward (intra group) transfusion reactions have been noted, tests for the Rh factor shall be
performed before any subsequent transfusion. No subsequent transfusion
shall be given to any such recipient found to be Rh-negative except from an
Rh-negative donor whose red blood cells are shown to be compatible with the
recipient's serum at 37°C.
2. No woman with an obstetrical history characterized by habitual abortion,
stillbirth, macerated fetus or erythroblastosis fetalis, shall receive a transfusion
unless tests for the Rh factor have been made; and then, if her blood shall prove
to be Rh-negative, such transfusions shall be made only from an Rh-negative
donor whose red blood cells shall have been shown to be compatible with the
recipient's serum at 37°C.
THE EH FACTOR
615
Although these preventive measures were generally approved, no official action
could be taken because no assurance could be given at the time that sufficient
quantities of potent anti-Rh serum would be available.
It will be noted that, at first, the emphasis was placed on the prevention of
intragroup transfusion reactions in the two groups indicated above. In the
light of more recent findings on the long duration of the immunized state, it now
becomes necessary to prevent isoimmunization in all Rh-negative individuals
who may be candidates for transfusion by the exclusive use of Rh-negative blood.
Obviously, this precaution is far more important for females of all ages than for
males. Since only minute amounts of fetal blood during the course of a pregnancy suffice to induce isoimmunization, the above mentioned measure should
apply also to the intramuscular administration of blood. Accordingly, it now
becomes necessary to modify my second proposal so that it may perhaps read as
follows:
"No transfusions in a female of any age, from infancy on, may be carried out
unless she has been tested for the Rh factor. If found to be Rh-negative in tests
with potent standard diagnostic anti-Rh 0 (or anti-D) serums, she must receive
only Rh-negative blood, whether it be given intravenously, subcutaneously or
by intramuscular injection."
General adoption of this rule will have the following results:
1. The incidence of erythroblastosis fetalis in the first born will be greatly
diminished. This will be especially applicable to transfusion in Rh-negative
female infants and children, or in Rh-negative women prior to their pregnancies.
2. The incidence of erythroblastosis fetalis in general will be diminished if
all Rh-negative women who may require transfusions during the child-bearing
age will receive only Rh-negative blood. Under these conditions, the silent
process of isoimmunization by pregnancy will be so delayed that with a limited
number of properly spaced pregnancies, all Rh-positive children may be either
entirely normal or have only mild symptoms of hemolytic disease.
3. The incidence of serious intragroup transfusion reactions will be lowered by
more than 90 per cent. This is applicable not only to severe reactions, those in
which anuria develops, but also to the milder, and even the delayed reactions.
In other words, every transfusion should result in a normal increment of
erythrocytes and hemoglobin, except in those patients suffering from blood
dysc'rasias.
It is obvious that there is no need ever to resort to the so-called "biologic
test" 74 since the amounts of blood recommended for the test will serve to immunize.
If sufficient quantities of diagnostic anti-Rh serum become available, the same
rules should be applied to all male patients prior to transfusions.
The writer has been encouraging health authorities to adopt comprehensive Rh
testing programs arid in several states the matter is now under consideration. 20 ' 31' M ' 6 0 For example, there is the successful experience reported by
Lee, Van Saun and Brown31 in Passaic County, New Jersey. Since many states
616
PHILIP LEVINE
already have compulsory premarital and prenatal tests, no statutory authority
is required except to obtain financial support for the program. All workers in
the field are agreed that Rh tests should not be done premaritally, but only on
specimens submitted when a woman is in her early pregnancy. There is almost
never any indication for a couple to break an engagement solely because of Rh
incompatibility. A possible reason might be that a Rh-negative girl had, previous to her engagement, become immunized by transfusion with presumably Rhpositive blood. In the absence of a history of transfusion, or intramuscular
administration of blood, erythroblastosis fetalis in the first born is remarkably
rare. I do not know of any cases of fetal death due to erythroblastosis fetalis
in the first pregnancy of an Rh-negative woman who had not been previouslyimmunized.
There is reason to believe that sufficient quantities of potent diagnostic antiRh 0 serum will soon become available. At present, there is not yet on hand an
experimental anti-Rh serum which satisfies the several criteria of potency and
specificity.67 Even if such an anti-Rh 0 serum were to become available, it
would still be necessary to collect and store large quantities of the three human
anti-Rh serums, the three varieties of anti-Hr serums, and any other of the very
rare serums of unusual specificities.
Physicians should encourage all Rh-negative women who have potent agglutinins or blocking antibodies to submit to periodic blood lettings. The yield
of such blood can be considerably increased if each bleeding is followed by a
replacement transfusion with other Rh-negative blood. In selected women
who do not plan further pregnancies, Hill and Haberman 21 have shown that the
potency of the anti-Rh antibodies can be maintained by the intravenous injection of minute quantities of Rh-positive blood. The amounts of the blood
required to maintain high levels of antibody content are remarkably low and
are insufficient to induce unpleasant reactions. More recently, normal male
Rh-negative individuals have been immunized on a voluntary basis by repeated
administration of small quantities of Rh-positive blood.79 In this manner, large
quantities of potent diagnostic anti-Rh serums may be collected and stored.
Obviously, more intensive treatment of Rh-negative male individuals is required
in contrast to the group of Rh-negative women already immunized by fetal
blood.
Another important source of diagnostic anti-Rh serum may be derived from
those Rh-negative women who have the so-called blocking antibody. Under
certain conditions (use of normal human serum, plasma or bovine albumin or
other suitable media for suspension of the red blood cells, such as acacia or
polyvinyl alcohol) blocking serums will give direct agglutination reactions which
differentiate sharply Rh-positive and Rh-negative individuals. 6,12 > 16 ' 45 ' 65 ' 76
Occasionally, immunized Rh-negative mothers will have the two antibodies
(agglutinin and blocking) in such concentrations that titration will show the
prozone effect. Levine and Waller62 have demonstrated that the blocking antibody can be specifically absorbed without much diminution of the agglutinin.
THE HH FACTOR
617
In addition to routine diagnostic screening tests for the selection of Rh-positive and Rh-negative mothers, tests for the presence of active isoimmunization
should be carried out. While these tests should also be performed in a statewide program, all hospital laboratories, especially those with large obstetrical
services, also should be prepared to carry out these comparatively simple
procedures. Unfortunately, there are altogether too few individuals with sufficient experience or background in this new and clinically important field. When
such trained workers become available, it will become essential for larger
hospitals to add one or more to their staff. It is recommended that smaller
hospitals pool their interests in organizing a properly equipped laboratory
to serve the interests of their local community. This program can readily be
organized in conjunction with a central blood bank.
In urging a public health program in connection with the Rh problem, it is
pertinent to mention that in the white population the morbid effects of isoimmunization; i.e., erythroblastosis fetalis and intragroup transfusion accidents,
are observed far more frequently than those resulting from syphilis. Obviously,
the element of contagion is not present in the case of isoimmunization.
I wish to pay tribute to my teacher, the late Dr. Karl Landsteiner, with whom
I was associated at Rockefeller Institute from 1925 to 1932. Our research,
which dealt mainly with his concept of the individuality of human blood, served
as the basis for my later studies on isoimmunization across the placenta.
It was a rare privilege to collaborate with this inspired genius, who devoted
all his efforts toward extending our knowledge in problems which were exclusively
fundamental in nature.
Scornful of windy generalities, he was constitutionally incapable of resorting
to that popular method of suppressing or minimizing the contribution of other
workers in order to lend greater prominence to his own. Landsteiner had a
passionate devotion to the sanctity of his own printed words and he was quick
to detect in others any deviation from the high and rigid standards which he
set for himself.
How many scientists can view their careers objectively and can honestly state
that all of their observations and interpretations have successfully passed the
acid test of time? Consider his numerous accomplishments: the discovery of
the blood groups and their application; the cause of paroxysmal hemoglobinuria;
the darkfield illumination for demonstration of spirochetes; the use of alcoholic
extracts of normal organs in the complement-fixation test for syphilis; the viral
nature of poliomyelitis; the long and tedious studies on the chemical basis of
serologic specificity; the mechanism of experimental allergy; and finally, his
concept of the individuality of human blood.
It was my privilege to hear on many occasions Landsteiner's reply to a technical question, "I don't know. We shall have to devise ways and means to test
it." We would do well to heed his wise counsel and submit ourselves to his
restraining influence.
618
PHILIP LEVINE
REFERENCES
1. BOORMAN, K . E . , AND D O D D , B . E . : Group-specific substances A, B , M , N a n d R h ;
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