T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 35, No. 1, pp. 31-35
January, 1961
Copyright © 1961 by The Williams & Wilkins Co.
Printed in U.S.A.
ANTI-M ANTIBODY AS A CAUSE OF INTRAUTERINE DEATH
REPORT OF Two
STILLBIRTHS RESULTING FROM A N T I - M ANTIBODY
C. R. MACPHERSON, M.D., MARA J. CHRISTIANSEN, M.T. (ASCP),
WILLIAM A. NEWTON, JR., M.D., WARREN E. WHEELER, M.D.,
AND EDWIN R. ZARTMAN, M.D.
Departments of Pathology, Pediatrics, and Obstetrics, Ohio State University, College of Medicine,
Columbus, Ohio
Erythroblastosis fetalis resulting from
anti-M antibody seems to be an extremely
rare phenomenon. We know of only 1
published case, that of Bomchil, in 1951.2
Levine and his associates, in 1956,8 mentioned
an unpublished case of their own, in referring
to Bomchil's report, and we have also
heard of 2 affected babies being born of the
same mother.1 It seems of sufficient rarity
and interest to report on 2 other infants, observed by us and born to the same mother,
both of whom we believe to have suffered
from fetal-maternal incompatibility involving the factor M.
The rarity of this phenomenon is rather
surprising, in view of the fact that the
presence of anti-M antibody during pregnancy is well documented, 4 ' 6i *•9 but, in all
of these instances, the child was reported to
be entirely healthy. It was believed that in
each instance the anti-M was of natural
occurrence and coincidental, although it is
known that anti-M can cross the placenta,6
and nearly all of the children tested were
reported to possess the factor M.
Although it is known that anti-M can be
formed in response to antigenic stimulation,
for example, in the volunteers reported by
Wiener and co-workers (1948),10 the examples of anti-M reported above were all
assumed by the authors to be naturally
occurring. The immune form of anti-M has
not been reported previously in pregnancy,
to our knowledge, except possibly
Bomchil's case, mentioned above.
in
REPORT OF CASE
The patient, Mrs. P. N., was first seen in
February 1957, at which time she was 2
months pregnant. She was 22 years of age,
and had a history of a miscarriage 18 months
previously. This was at approximately 5 ^
months' gestation, and was apparently the
result of an abruptio placentae.
There was no clinical abnormality and all
laboratory investigations were within normal
limits. Pregnancy progressed uneventfully
for the next 5 months, but, on July 23, 1957,
the patient said she had gained 5 lb. in
weight during the previous 2 weeks. She
manifested slight pitting edema of the feet,
ankles, and pretibial region, but was normotensive, and her urine contained no protein.
Her condition was thought to be related to a
period of unusually hot and humid weather,
and she was treated with a low-salt diet, and
Acetazoleamide, 250 mg. per day, was prescribed.
One week later she had gained only one
half pound in weight, and her edema had
disappeared. Her abdomen was distended,
however, suggestive of polyhydramnios, and
the fetal heart could no longer be heard.
Three days later she spontaneously delivered
a macerated, edematous female child. The
placenta was large, bulky, and friable.
Urinalysis at the time of admission to the
hospital revealed no sugar, protein, or
formed elements. Blood sent for typing and
crossmatching revealed an antibody, identified as anti-M by means of its reactions
with a panel of cells of known antigenic
composition (Panocell, Knickerbocker Laboratories, New York). The mother's red
blood cells were demonstrated to be Group
Received, April 1, 1960; revision received,
July 28; accepted for publication September 1.
Dr. Macpherson is Associate Director, Clinical
Laboratories, Ohio State University Medical
Center; Miss Christiansen is Supervisor, Transfusion Service; Dr. Newton is Director of Laboratories, Children's Hospital; Dr. Wheeler is Professor
of Pediatrics; Dr. Zartman is Clinical Instructor, Department of Obstetrics and Gynecology.
31
32
MACPHERSON ET
0 , Rh-positive, and were strongly positive
for factor N, but negative for M. The
antibody will be further discussed later.
At autopsy (Dr. Chi, Children's Hospital),
the weight of the body was 2000 Gm, and its
length 43 cm. There was a great degree of
maceration and edema, but jaundice was
not detectable. The peritoneum contained
50 ml. of dark, red-brown fluid, the pleural
cavities 15 and 20 ml. of pink fluid, and the
pericardial sac 6 ml. of clear yellow fluid.
Examination of the internal organs revealed
no abnormality other than maceration,
grossly or histologically, except for sections
of the liver, which contained large amounts
of pigment that produced the staining reactions of iron.
The placenta (540 Gm.) was strikingly
enlarged for the size of the child, and it was
19 cm. in its largest diameter. The thickness
varied between 2.7 and 3.0 cm. No infarcts
were noted, but, histologically, there was
edema and hypertrophy of the fetal villi.
The puerperium was uneventful, and a
specimen of blood collected 6 months after
delivery demonstrated that the anti-M antibody was still present. In August 1958, just
12 months after delivery, the patient reported that she thought she was again
pregnant, and this was confirmed by means
of a positive Xenopus test. Her course again
was initially uneventful. Both clinical and
laboratory investigations revealed no abnormality other than her anti-M antibody
{vide infra). In December 1958, however,
when she was approximately 53^ months
pregnant, she again reported some peripheral
edema, although this had disappeared by
the time she was examined. She was normotensive and had no albuminuria or excessive
gain in weight, but the fetal heart was heard
with difficulty, and 1 week later could not
be heard at all. Another week later she
spontaneously gave birth to a slightly macerated male fetus, which, at autopsy, manifested no gross abnormality of internal
organs. There was slight icterus, but little
edema, and study of sections of the liver
again revealed a heavy deposit of iron
pigment. The placenta manifested no
striking change, either grossly or microscopically.
Vol. 35
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A small quantity of blood was collected
with great difficulty from the fetus at
autopsy, and was divided into 3 aliquots in
an attempt to obtain as much information
as possible. The first 2 aliquots yielded unequivocally positive results with anti-M and
anti-N typing serum, but when an attempt
was made to perform a direct Coombs test
on the third aliquot, it was found to be
composed of cell debris with too few intact
cells to be of use. By this time, it was too
late to obtain another specimen. Even
though the lack of this information is regrettable, we still think that, under the
circumstances, the decision to try to get the
maximal amount of information from the
available material was a correct one.
SEROLOGIC
STUDIES
Because of the lack of information on the
manner in which anti-M antibody might be
expected to behave during pregnancy, and
particularly in anti-M erythroblastosis, a
variety of technics was used. Tests were
run at 4, 20, and 37 C , using cells suspended
in physiologic saline solution, AB serum, and
22 per cent bovine albumin. After reading
the results, the tubes of the first 2 tests were
converted to an antiglobulin test, performed
at 4 and also at 37 C. In each instance, the
serum was diluted in the medium used to
suspend the cells, and the AB serum throughout was from the same donor, a point of
great technical importance, as emphasized
by Boorman and Dodd,3 who originally
described this technic.
In order to be assured of uniformity, the
same cells (Group O, M-homozygous) were
used throughout the studies, and were used
within 18 hr. of being removed from the
donor (M. J. C ) . This was done because (1)
of the well-known loss of reactivity of factor
M during storage, and also because (2) the
husband's cells (on the only occasion on
which we were able to obtain a specimen)
reacted much more avidly with his wife's
serum when fresh than after 48 hr. storage
at 4 C. All of the serums were stored at —15
C , and, at the end of the second pregnancy,
they were thawed and the tests were repeated in parallel.
It became evident that the tests per-
Jan.1961
formed at 20 C. resulted in little significant
information; therefore, they are not reported. The complete results of tests at 4 C.
and 37 C. are summarized in Tables 1 and 2,
but the most striking variations are those
observed in the tests with cells suspended in
physiologic saline solution, in AB serum,
and in AB serum followed by a test with the
Coombs technic. These results are illustrated
in Figures 1 and 2.
The fact that the titer of antibody is
enhanced by means of the Coombs technic,
and by means of AB serum, is a strong point
in favor of the antibody being of immune
type rather than naturally occurring. The
titers of 1:1024 and 1:2048 obtained repeatedly are conspicuously higher than those
reported by most authors for the naturally
occurring isoagglutinin. Indeed, it is rare to
find any activity in such cases at 37 C.6 The
only comparable 37-C. titer we have observed in the pertinent literature is that of
1:256 in the case briefly mentioned by
Wiener and co-workers.10
If, as we believe, this represents an
immune antibody, the further point arises
as to whether or not there is also a naturallyoccurring antibody of the same specificity.
One point is in favor of this hypothesis, i.e.,
TABLE 1
TITRATIONS OF P A T I E N T ' S SERUM W I T H G R O U P O,
MM C E L L S ( T E S T S INCUBATED FOR 1 H R . AT 4 C.)*
Technic
Date of Specimen
Aug. 10, 1957
Sept. 8, 1957
Feb. 1, 1958
June 1, 1958
Aug. 6, 1958
Sept. 3, 1958
Oct. 2, 1958
Nov. 4, 1958
Dec. 2, 1958
June 3, 1959
33
ANTI-M ANTIBODY
Saline
solution
32
128
128
256
128
64
32
32
16
128
Saline
serum
AB ABplus
Albu- solution
min plus AHG serum
AHG
64
256
512
256
256
128
64
64
128
64
64
128
256
256
256
128
64
64
t
256
64
256
1024
1024
512
256
256
256
256
512
t
256
1024
1024
1024
256
2048
1024
512
1024
* Tube method. Tests read macroscopically.
Results recorded as t h e reciprocal of the last tube
yielding a definite 1 + reaction.
f Not done.
TABLE 2
T I T R A T I O N S OF P A T I E N T ' S SERUM WITH G R O U P O,
M M C E L L S ( T E S T S INCUBATED FOR 1 H R .
A T 37 C.)*
Technic
Date of Specimen
Aug. 10, 1957
Sept. 8, 1957
Feb. 1, 1958
J u n e 1, 1958
Aug. 6, 1958
Sept. 3, 1958
Oct. 2, 1958
Nov. 4, 1958
Dec. 2, 1958
J u n e 3, 1959
Saline
solution
16
16
16
16
16
8
16
8
8
16
Saline
serum
AB ABplus
Albu- solution
min plus AHG serum
AHG
16
16
64
32
16
8
8
8
8
4
32
32
64
64
32
16
16
8
8
256
32
64
32
32
32
16
16
16
8
64
t
64
64
64
128
64
32
128
64
1024
* Tube method. Tests read macroscopically.
Results recorded as t h e reciprocal of t h e last tube
yielding a definite 1 + reaction. Tests read in
parallel from frozen specimens except for specimen of Aug. 10, 1957.
f Not done.
the strong reaction with saline-suspended
cells at 4 C , but not enough is known of
the possible variations in the behavior of
this antibody to provide a definite answer.
One point that should be stressed in
establishing the diagnosis of maternal-fetal
incompatibility is the presence of iron pigment in the liver. The livers of 9 other
stillborn infants were examined by means of
similar technics. Five of these had no evidence of hemolytic anemia, and only trace
amounts of iron were found. Two of them
had erythroblastosis on the basis of the Rh
factor and massive deposition of iron was
noted. The last 2 were also erythroblastotic,
but on the basis of ABO incompatibility,
and they revealed moderate deposition of
iron, similar in amount to that observed in
the 2 children described in this paper.
This finding, the positive Coombs test in
the first child, the finding of MN cells in the
second child, and the serologic changes observed in the mother's blood, all lead us to
believe that this represents erythroblastosis
fetalis caused by factor M. The final proof,
of course, would be the production of a
34
MACPHERSON ET
Vol. 35
AL.
/2048S
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CK
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Id
V)
w
;
'
TV V
M28-
2
''16-1
CO
z
o
Pregnancy
Delivery
Aug. 1957
Feb. 1958
June 1958
Delivery
1
Jan. 1959
i
June 1959
TIME IN MONTHS
/
§ I024£C
LLI
CO
-2 W
<
Q.
o
16-
i-
a
Delivery
—i
1
Aug. 1957
Feb. 1958
Pregnancy
1
June 1958
Delivery
TT
Jan. 1959
June 1959
TIME IN MONTHS
FIG. 1 (upper). The titers recorded are the last dilutions yielding a 1+ reaction macroscopically.
One-tube differences not confirmed in subsequent tests are not illustrated. For full details, examine
Table 1.
#
• Tests performed in saline medium; #
# Tests performed in AB-serum medium;
•
# Tests performed in AB-serum medium, followed by anti-human globulin.
FIG. 2 (lower). The titers recorded are the last dilutions yielding a 1+ reaction macroscopically.
One-tube differences not confirmed in subsequent tests are not illustrated. For full details, examine
Table 2.
•
• Tests performed in saline medium; #
# Tests performed in AB-serum medium;
•
# Tests performed in AB-serum medium, followed by anti-human globulin.
normal child, homozygous for factor N, but
the parents seem unwilling to undergo a
further "ordeal by gestation," although it is
possible that they may change their minds
at a later date.
to 2 successive stillborn fetuses, believed to
have resulted from maternal-fetal incompatibility involving the blood factor M. The
serologic findings are reported in some detail.
STJMMARIO I N
SUMMARY
This paper deals with the findings in an
instance where a young woman gave birth
INTERLINGUA
Iste reporto presenta le constationes in le
caso de un juvene femina qui parturiva successivemente 2 morte fetos in consequential,
Jan. 1961
ANTI-M ANTIBODY
35
presumibilemente, de incompatibilitate fetomaterne in terminos concernente le factor
sanguinee M . Le observationes serologic es
reportate in detalio.
5. C R O W L E Y , L. V., R I C E , J. D . , J R . , AND B R E E N ,
REFERENCES
7. JAKOBOWICZ, R., AND B R Y C E , L. M . : A n o t e on
1. B E N Z , E . J . : Personal communication, 1958.
2. BOMCHIL, G.: Isoimmunicacion por el antigeno
M. Nueva cause de enfermedad hemolitic
neonatal. Hematologia y Hemoterapia, 3:
104-109, 1951.
3. BOOHMAN, K . E . , AND D O D D , B . E . T h e a c t i v a -
tion of haemagglutinins by human serum.
P a t h . & Bact., 59: 95-104, 1947.
J.
M . : High-titered anti-M isoagglutinins in
human blood. Am. J. Clin Path., 28: 481488, 1957.
6. H A M I L T O N - P A T E R S O N , J . L., R A C E , R . R., AND
TAYLOR, G. L.: A case of human isoagglutinin anti-M. Brit. M. J., 2: 37-38, 1942.
placenta-permeating
anti-M
agglutinin.
M. J . Australia, 1: 365-367, 1951.
8. L E V I N E , P . , R O B I N S O N , E . , S T R O U P , M . ,
M C G E E , R., AND B U S H N E L L , L. N . : A s u m -
mary of atypical antibodies, rare genotypes,
and ABO hemolytic disease encountered in a
one-year survey. Blood, 11:1097-1117,1956.
9. M C F A R L A N E , M . N . : Anti-M agglutinins in
human serum. Brit. M. J., 2: 883-884, 1945.
4. B O W L E T , C. C., AND D U N S P O R D , I . : T h e agglu-
10. W I E N E R , A. S., GORDON, E . B . , AND MAZZA-
tinin anti-M associated with pregnancy. Report on two cases. B r i t . M . J . , 2: 681-682,
1949.
RINO, C. A.: Observations on isosensitization
t o t h e M agglutinogen in man. Proc. Soc.
Exper. Biol. & Med., 69: 8-11, 1948.