BLOOD GENOTYPES - D - / - D - AND
CDE/-D-
TuAiVSPUsrOiV T H E R A P Y AND S O M E E F F E C T S O F M U L T I P L E P R E G N A N C Y
DONALD I. BUCHANAN, M.B.
Canadian lied Cross Blood Transfusion, Service, Edmonton, Alberta, Canada
In 1950 and 1951, Race, Sanger, and Selwyn described a remarkable blood
sample having the antigenic formula - D - / - D - . 7 ' 8 They regarded the absence of
Cc and Ee as representing a deletion in the Rh chromosomes. That this patient
was homozygous for the deletion was undoubtedly due to the fact that her
parents were half second cousins, each presumably having the - D - chromosomes.
In 1953 two similar cases were reported in which the genotypes could also be
interpreted as
-D-/-D-.n
By 1954 a relatively large series of Canadian Metis living in central Alberta
were found to be either homozygous (-D-/-D-) or heterozygous ( C D e / - D - or
cDe/-D-). Consanguinity was a prominent feature.1 As soon as the first case in
the series had been detected, the probability that cousins or second cousins had
mated was predicted by Levine who was then engaged in the investigation of
another similar case of Rh chromosomal deletion.3
Thus it is thought that pathologists and those engaged in the various aspects
of immunohematology will have a particular interest in the historical background and in the subsequent developments relating to some of the members of
the Canadian group, for not only is the genetic study of value, but these individuals present an unusual problem when it comes to providing them with crossmatched blood.
HISTORICAL NOTE
More than 30,000 persons in the four Western provinces of Canada are officially classified as Metis, and the majority of these have "by nature and historic
environment, trended more to the culture of their Indian forebears."" The
Indian families (or, more correctly, Metis families) now described occupy a reserve near old Fort Edmonton. Although the total population of the Reserve is
only 117, including children, other relatives are to be found in small family
groups throughout central and northern Alberta—in small settlements from
Embarras Portage, Lake Athabasca (in the extreme northeastern area of the
province), to Lesser Slave Lake; and upstream along the Athabasca river to Fort
Assiniboine and westward towards the Rocky Mountains beyond Edson.
A point of historic interest is that they still occupy places near the sites of old
fur-trading posts and early European settlements along the banks of both the
Athabasca and North Saskatchewan rivers, and near the lakes of the province
such as Cold Lake, Lac Ste Anne, Lake Wabumum, and Lac La Nonne. This
Tteecivcd, June 6, 1955; revision received, September 29; accepted for publication September 29.
Dr. Buchanan is Provincial Medical Director.
21
22
BUCHANAN
Vol. 26
close contact with the original fur-trading posts of the West, and therefore with
the Indian hunters and French-Canadian voyageurs of the Hudson's Bay Company and their rivals of the North West Company, gives a clue to the racial
origins of the Metis, for their forebears were the product of marriage or casual
association of the western Cree Indians and those working for the fur traders.
It has been possible to trace their ancestry back to the days when the canoe
brigades of the trading companies brought in a number of Iroquois to act as
hunters for the posts. Because of the abundance of beaver and fur-bearing animals between the North Saskatchewan and the Athabasca rivers, the first houses
at Fort Augustus and Fort Edmonton were established in 1794. "within a musket
shot of each other." 5 The intense rivalry of the two trading companies brought
about many changes in the way of life of the local bands of Cree Indians. Voyageurs of French-Canadian, Iroquois, and European descent were encouraged to
live with the local family bands during the winters, returning with them to the
posts in early spring with their harvest of furs. The rapid depletion of fur-bearing
animals caused the companies to move their posts, Fort Edmonton and Fort
Augustus, four times in all, while new forts were created at Rocky Mountain
House, Fort Assiniboine, and near Jasper in the Rocky Mountains in the early
years of the nineteenth century.
In common with all the other Indians of the Western prairies and Northern
forests, the family group in whom we have special interest was devastated by
recurring epidemics of smallpox, and many infectious diseases unwittingly introduced by the white man and his half-breed followers.6
This large family, descended principally from Cree, Iroquois, and French
Metis, settled at Lac Ste Anne about 1850, later moving a few miles to the
Michel Reserve under the guidance of Father Lacombe, O.M.I., a well-beloved
missionary of the Canadian West. Although they remained true nomads, the
excellent records kept at the mission houses make it possible to trace their lineage
back for some 150 years.
Thus, with the coming of the whites, intermarriage began; interbreeding with
the Europeans and their followers was succeeded by a serious and protracted
phase of inbreeding, which is still common.
The present-day families, including those possessing an Rh chromosomal deletion, have a high incidence of active tuberculosis.
TYPICAL ANTIGEN-ANTIBODY REACTIONS
Race and his associates8 have established that persons possessing Rh genotypes
- D - / - D - have certain red cell characteristics: (1) Their erythrocytes are readily
agglutinated in saline suspensions by incomplete anti-D serums. Those investigated by us, including those heterozygous for the deletion ( C D e / - D - or
cDe/-D-) varied in the degree of agglutination from 2 + to 4 + (complete) with
several different incomplete anti-D serums.1 In general, cells that are homozygous
for the deletion ( - D - / - D - ) are more intensely agglutinated than are heterozygous cells such as C D e / - D - in saline. (2) Because of this peculiarity, pure Rh
testing serums must be used. For example, - D - / - D - cells are not agglutinated
Jan.
1956
BLOOD GENOTYPES - D - / - I > - ANL> C D E / - O -
23
TABUS 1
CASK 1. R E S U L T S OF T I T K A T I O N S O F SEHUM IN 20 P E R C E N T ALBUMIN
Thcr
Test Cells
0,
0,
0,
0,
0,
0,
Sister (V-14) 0,
Fnthcr (IV-7) 0,
Cusc 1 (V-ll) 0,
Brother (V-S) 0,
Sister (V-6)
0,
CDe/eDE
cdF/cdc
Cde/cdo
cde/ede
cDe/odc
cDE/cDE
CDc/-DcDe/-D-D-/-D-D-/-D-D-/-D-
Score*
1:4
1:4
1:2
1:16
1:S to 1:10
1:S
1:4
1:S
Negative
Negative
Negative
25
25
20
34
33
31
23
30
0
0. Indirect Coombs test negative
0. Indirect Coombs test negative
* T h e score in the titrations was obtained by assessing the intensity of agglutination
in each agglutinin tube and adding t h e total obtained. + + + = 10, + + = S, + = 5, ( + ) =
3, w = 2, - = 0.
by anti-C, anti-E, anti-c, and anti-e, but if these serums contain traces of anti-D,
agglutination will occur.
KEPOKTS OF 3 CASES
In 2 of the 3 cases reported here, hyperimmune Rh antibodies were discovered
2 or 3 years after these individuals had received apparently compatible crossmatched blood. At that time they had not been recognized as having Rh chromosomal deletions; there were no immediate difficulties in crossmatching and no
apparent hemolytic transfusion reactions ensued. However, the presence of incomplete antibodies now makes it impossible to crossmatch their blood from the
usual blood bank sources. Particular care has to be taken in finding a suitable
donor of the correct Rh genotype, et cetera.
Cascl
(V-ll*)
This young Mctisse, born in 1932, is group O, MN ss kk F y " + Lu n - Jk a -,
-D-/-D-.
She has only 2 living children, despite 5 pregnancies. The first child,
born when the mother was 16 years old, died at the age of 2 months. The second
child, age 4 years, is alive and well. The third pregnancy terminated in October
1952 in an abortion at 4 months, at which time the mother received 2 transfusions of group O, Rh-positive blood. The fourth child, stillborn in February
1954, probably suffered from erythroblastosis foetalis. Birth occurred en route
to hospital and there was no postmortem examination. The fifth infant (VT-15),
born in February 1955, proved to belong to group O, MN, -D-/-D-.
Shortly
before her last confinement, blocking antibodies were detected in the mother's
serum. On February 24, 1955, titrations in 20 per cent'albumin gave the results
shown in Table 1.
* Code numbers refer to Figure 1
24
BUCHANAN
Vol. 26
After absorption with appropriate cells (such as cDE/cDE), anti-e was detected. The patient's absorbed serum was weakly reacting, but of the probable
anti-e and anti-c present, anti-e was the more active. An antibody reacting
strongly with two available cDE/cDE bloods was detected in her freshly absorbed serum by the indirect Coombs technic.2
In view of the incomplete antibodies present, it was anticipated that the infant
would be affected. This impression was strengthened by the history of a stillborn
infant in the previous year. The only reservation made in discussing the findings
in this patient with her obstetrician was the slight possibility that the unknown
father possessed a - D - chromosome which might descend to the child. This last
thought was based on a growing knowledge of the marked consanguinity in the
family. Nevertheless, labor was induced about 10 days before full term.
The rare chance of mating with a consanguineous male proved to be the most
probable answer, since the infant at birth was found to be unaffected. Had the
infant been heterozygous, like the only other living child, it is possible that the
incomplete antibodies would have been of greater intensity, and their isolation
would then have been simplified. The infant is group 0 , MN - D - / - D - . Cord
hemoglobin was 16.25 Gm. per 100 ml., erythrocytes 4,560,000 per cu. mm.,
nucleated red cells 3 per 100 leukocytes; cord bilirubin 0.7 mg. per 100 ml.,
direct Coombs test negative.
The problem of transfusion for the mother or infant. From past experience, it was
realized that the best hope was to take blood from a near relative. Her brother
(V-8; O MN Ss kk F y a + , -D-/-D-)
was selected, and his fresh citrated blood
kept on hand. He had received several transfusions in 1953 without immediate
complications. His blood (screened by the usual tests) also possessed incomplete
Rh antibodies. 9 The supernatant plasma was removed from his blood a few hours
before the birth of the infant. The packed cells were washed and resuspended in
normal group AB plasma and administered to the mother shortly after confinement. There was no reaction.
Irregular, naturally occurring antibodies have not been detected in other
members of the band possessing -D-/-D- genotypes. Naturally occurring anti-A
and anti-B are present in those of group O, but incomplete agglutinins have only
been found in 3 persons who had previous transfusions.
Case 2 {IV-12)
This patient, aged 47, group O, MN Ss kk F y + Lu a - Jk"-, C D e / - D - , is the
mother of V-8 and V-ll (Case 1). She had been hospitalized for over a year and
recently required blood during operation. Since many of her relatives who are
group 0, - D - / - D - , or group O, C D e / - D - , are either suffering from active tuberculosis or are too young to act as donors, it was decided to crossmatch with
blood from 2 unrelated white persons of genotype CDe/CDe. The patient had
had no previous transfusions and there was no indication of irregular or Rh
antibodies in her serum. There was no difficulty in crossmatching. It is suggested
that in this particular case, bank blood is as safe and perhaps more desirable than
the blood of relatives, provided the Rh genotype of the bank blood is selected
with care.
0871)
cde/cde
cdt/tde
TO
(I860
S ,£
3>->
(1886)
5
4L-1
(1945)
CDc/cde CDe/cdc
cOEAde C D f / c 0 e c ^ f c , , .
2'—'
(1917)
66
(1941)
(1943)
(1945)
Ab . STULBORN
• . ABORTION or MISCARRIAGE
t - DIED
(dote). YEAR of BIRTH
COe/O-
(1940)
C0e/-0-
(1948)
cOE/ed*
8<i
C0e/O- J
/
9*+
(1953)
ce/D-
(1954)
CDe/-D-
10 L.
l*r*
-3
COeAO-
COa&e
(1880)
II U
f
rS
CDe/-D-
\
(1936)
cD«/-D-
•
•
Q
Q
O
D
•
•
-
isi
0832)
"A
l 7
b
,-o
(1861)
CCe/CO-
ieO
18^
7P
BSLCOURT
HOMOZYGOUS Rh DELETION
-D-/-DHETEROZYGOUS Rh DELETION
CDe/-D- etc.
FEMALE (Rh genotype normal
MALE
or unknown)
-07-0-
13 •
(1955)
I
COe/cOE
C0«/-0-
A J &
a6
,-o
(1872) 087S) (1882) (1883)
j:De/cOE
COe/cOE
,
CDe/fc a
C0e/C0e
i s i ,*6
(1824)
.£
(died 1846)
LOUIS L IROQUOIS
<^ Ash
CQe^de
'3
|2<^ 13 14153
(1951) ('52) (1954)
COe/.O-
I2L] 13O 14® 6 ™ 16
cOe/cde
a
rO
,oD l O - n C B r Q
10'"H I I P - 1
JL ,
1
'
]cde/cde
O loO 11 •
(1952)
c e/-D-
(1848)
75
8 (WfTJ
0869)
cOE/cde
9^1
aidi
PROPOSITUS
\pgoi
~^ 1 4T5^
7y a
c0e/-O-
7 i~1
(1902)1
09O2)
(1870)
T ^
[O
»D-
CHALIFOUX
F I G . 1. Diagram of consanguinity and Rh chromosomal deletions
CDe/-0-
(1949)
£
6
(1876)
55 " r s 6w
cOE/cde
(1893)
vr ,i ,i ,4 .i ,i .i unu
0941)
cde/cde
,0~
(196)
(1907)
lO Z6 36~
i
0 - TO
T6
.0-
(1838)
,£
0834)
(died 1854)
rO
L' HIRONDELLE
(100)
iD-
CAMPION
26
BUCHANAN
Vol. 26
Although this woman has 7 living children, it would appear that she has
escaped sensitization during her 12 pregnancies. Her husband (IV-7) possesses
the Rh genotype c D e / - D - .
Case S (IV-18)
This woman, aged 57, group 0 , MN, CDe/cde, was admitted to the Aberhart
Memorial Sanitorium, Edmonton, in April 1951, suffering from active-tuberculosis involving the right lung. On May 25, 1952, after a rather large refill of a
pneumoperitoneum, she began to cough up bright red blood. Hemorrhage soon
became profuse, and blood transfusions were ordered. At the time we did not
know of her relationship to the previously mentioned patients, but eventually it
was discovered that she was the sister of IV-12 (Case 2).
This patient had massive hemoptysis, one of the most alarming complications
of pulmonary tuberculosis which reaches dangerous proportions in only about
2 per cent of cases.10 She received 16 transfusions of 500 ml. each of whole blood.
In 2 of these the bank blood was group 0, Rh-negative cde/cde, in 14 group 0 ,
Rh-positive. A successful right pneumonectomy was performed with the aid of
these transfusions, the majority of which were administered within a 24-hour
period. Owing to the nature of the emergency, several transfusions of unmatched
group 0 "universal donor" blood were given. There were no complications from
these multiple massive transfusions, and the patient is presently in fair health.
Tests of her serum performed after operation and repeatedly in 1954 and 1955
have failed to reveal any immune antibody response; the findings were negative
in tests with panels of group-0 cells in saline, albumin, and by the indirect
Coombs method.
I t has not been possible to retrace all the donors involved, but it is remarkable
that this woman has not, apparently, become sensitized to one of the rarer antigens. Had the potentially incompatible transfusions been spaced over some days
or weeks, the situation might have been different.
DISCUSSION
The chief emphasis in this paper has been placed on transfusion therapy in
rare cases of Rh chromosomal deletion. While these individuals present a problem
as recipients of transfusions, they are sometimes of great value as blood donors.
Recently 2 members of this large family who are group 0 , MN, - D - / - D - ,
donated blood for a broadly similar case in a California hospital. The recipient,
a newborn infant, was suffering from hemolytic disease of the newborn, the
mother's red cells having a unique Rh chromosomal deletion, while her serum
contained an uncommon combination of incomplete Rh antibodies, anti-c and
anti-e.4 I t is important that special donors of this and other rare types should be
classified and registered on an international basis at specified centers.
Although the exact cause of "extrusion" or "deletion" within the Rh complex
is not yet known, consanguinity plays an important role, since the stage is then
set for a multiplicity of similar cases (Fig. 1). It will be noted that the 21 cases of
- D - shown stem from a closely related group of ancestors (III-6, -7, -8, -9, -11,
and -12; III-5 and IV-5), while the known cases of the heterozygous Rh deletion
Jan.
1956
BLOOD GENOTYPES - D - / - D - AND C D E / - D -
27
occur in certain of their immediate descendants (IV-7, IV-12, IV-15, and V-5).
As a result of subsequent consanguineous matings, the heterozygous deletions
discovered in generation IV led to either a homozygous - D - / - D - or heterozygous
- D - series in generation V; extending further by a process of close descent to all
the young living offspring of generation VI.
CONCLUSIONS A N D SUMMARY
1. Some unusual cases are discussed of Rh chromosomal deletion in a group of
Metis in western Canada. I t is the author's belief that this group of Metis provide
a wealth of material for a study in the genetics of the Rh system and that additional similar cases will be detected among the Metis of Alberta and elsewhere.
2. High incidences in this group of infections such as tuberculosis and of
multiple pregnancies automatically create the risk that blood transfusions may
be required. This, in turn, demands special care in donor selection where the
recipient possesses the - D - chromosome, indicating absence of C-c, and E-e
on that chromosome, and the probability of sensitization to the e, c, C, and/or E
antigens present in the usual donors' erythrocytes in normal combinations, but
lacking in the cells of the recipients.
3. In this same family group one individual having a normal genotype
CDe/cde did not become sensitized despite 16 transfusions of 500 ml. of blood
each, given within a 24-hour period.
4. I t has been shown that repeated pregnancies plus transfusions led to considerable difficulty in the case of a - D - / - D - mother. Her last child, also
-D-/-D-, escaped any damage or danger from the mother's pre-existing incomplete antibodies, anti-e being specifically detected.
5. Individuals possessing the Rh genotype - D - / - D - should be registered as
potential blood donors at internationally recognized laboratories. A specific case
is mentioned where blood of this type was of great value.
CONCLUSIONES
E Sl/MMAKIO I N INTJDRLINGUA
1. Es discutite un serie de casos inusual de deletion chromosomal de Rh in un
gruppo de mesticios in West-Canada. Le autor crede que iste gruppo de mesticios
provide un abundantia de materiales pro le studio del genetica del systema Rh e
que simile casos va etiam esser detegite inter le mesticios de Alberta e alterubi.
2. Le alte frequentia de infectiones (como per exemplo tuberculosis) e de
multiple pregnantias in iste gruppo resulta automaticamente in le frequente
necessitate de transfusiones de sanguine. Isto, de su parte, require un attention
special in le selection del donator quando le recipiente possede le chromosoma
- D - (indicante le absentia de C—c) e E-e super ille chromosoma con le resultante probabilitate de sensibilisation al antigenos e, c, C, e/o E que se trova in
normal combinationes in le erythrocytes del donatores usual sed que es absente
del cellulas del recipiente.
3. In iste mesme gruppo familial, un individuo con un normal genotypo
CDe/cde non deveniva sensibilisate in despecto de 16 transfusiones de 500 ml de
sanguine cata un, administrate intra un periodo de 24 horas.
4. Es monstrate que repetite pregnantias in combination con transfusiones
28
BUCHANAN
Vol. 26
duceva a considerable difficultates in le caso de un matre a - D - / - D - . Su ultime
infante, etiam a -D-/-T)-, escappava a omne damno o periculo ab le pre-existente
incomplete anticorpores del matre. In iste caso anti-e esseva specificamente
detegite.
5. Individuos qui ha le genotypo - D - / - D - deberea esser iminatriculate como
potential donatores de sanguine in laboratories que es internationalmente recognoscite. Es mentionate un caso specific in que sanguine de iste typo esseva de
grande valor.
REFERENCES
1. BUCHANAN, D. I., AND MCINTYHB, J . : Consanguinity and two rare matings; - D - / - D - X
C D e / - D - , and C D e / - D - X c D e / - D - . Brit. J. Haemat., 1 : 304-307, 1955.
2. CHOWN, B . , AND L E W I S , M . : Personal communications t o t h e author, 1955.
3. L B V I N E , P . : Personal communication to t h e author, 1954.
4. L E V I N E , P . , AND R A C E , R. R . : Personal communications t o t h e author, 1955.
5. MORTON, A. S.: A History of t h e Canadian West. T o r o n t o : Thomas Nelson and Sons.,
1945, p p . 354, 463.
6. P r i v a t e Church Records of t h e Missions of Saint Albert, and Lac Stc Anne, Alberta,
Canada.
7. R A C E , R . R., SANGER, R., AND SELWYN, J. G.: A probable deletion in a h u m a n R h
chromosome. N a t u r e , London, 166: 520-521, 1950.
8. R A C E , R. R., SANGER, R., AND SELWYN, J. G.: A possible deletion in a human R h chromosome: A serological and genetical study. Brit. J. Exper. P a t h . , 32: 124-128, 1951.
9. R A C E , R . R . : Personal communication to t h e author, 1955.
10. R o s s , C. A.: Emergency pulmonary resection for massive hemoptysis in tuberculosis.
Am. J . Thoracic Surg., 26: 435-438, 1953.
11. SYMINGTON, D . F . : Metis rehabilitation. Canad. Geograph. J . , 46: 128-132. 1953.
12. W A L L E R , R . K . , SANGER, R., AND B O B B I T T , O. B . : Two examples of t h e - D - / - D - geno-
t y p e in an American family. Brit. M . J., 1: 19S-199, 1953.