RARE HUMAN ISOAGGLUTININS AND THEIR
IDENTIFICATION
PHILIP LEVINE, M.D., ELIZABETH A. KOCH, M.S., ROBERT T. McGEE,
B.A., AND GLEN H. HILL
Rh Consultation Service, Ortho Research Foundation, Rarilan, New Jersey
Following the discovery of the causal relationship of hemolytic disease and
transplacental isoimmunization, numerous new blood factors were described
with the aid of atypical isoagglutinins other than anti-D. These findings were
made possible by the elaboration of several methods for detection of isoimmunization resulting from the antigenic stimuli of pregnancy or transfusion or both.
Clinically, the identification of the atypical antibodies is essential for the selection
of compatible blood in the therapy of the affected infant or the immunized patient, or both. From a theoretical standpoint, identification of the antibodies
provides reagents for essential studies on the hereditary nature of the corresponding blood factors and their incidence in various racial groups. The discussion to
follow is limited mainly to Rh-positive persons who produce antibodies other than
anti-D; but one must take into account Rh-negative persons whose serum may
contain additional antibodies such as anti-K or anti-Fy a .
A list of known blood factors other than A or B, antibodies for which may be
found in human serum, is given in Table 1.
Not all blood factors have equal significance clinically, and a number of them
have thus far not been implicated in hemolytic transfusion reactions or in hemolytic disease of the newborn. These include P, Lu a , Le b , and with rare exceptions,
Len, M, N and S.14 By and large, their antibodies have a low titer at room temperature and little, if any, activity at 37 C. From their occurrence in presumably
normal persons not exposed to antigenic stimuli, it may be concluded that they
are physiologic or normal for the individual, much like anti-A or anti-B. Under
the antigenic stimulus of transfusion or pregnancy, however, they may increase
somewhat in titer and in range of temperature within which they are detectable.
Under these conditions, they are likely to occur as secondary antibodies in
conjunction with warm immune isoagglutinins such as anti-D, anti-E and antiK, which are of themselves responsible for destruction of the blood of the newborn or the transfused incompatible blood. It is only rarely that a naturally
occurring anti-S, anti-M or anti-Le a , under specific antigenic stimulus, develops
immune properties and sufficient titer to become of itself important clinically.17
In view of these exceptional cases, a clear-cut serologic differentiation of
atypical agglutinins active at 20-25 C. could not be made from warm immune
Received for publication September 3, 1953.
Presented as part of the Symposium on Blood Bank Methodology at the joint meeting
of the College of American Pathologists and the American Society of Clinical Pathologists,
in Chicago, October 13, 1953.
Dr. Levine is Director of Rh Consultation Service, Miss Koch and Mr. McGee are Assistant Serologists and Mr. Hill is Research Associate.
292
MAR. 1954
293
RARE ISOAGGLUTININS
isoagglutinins, and it became necessary to include another group with serologic
properties intermediate between the two.14
Excluding ABO incompatibility, it is probable that anti-D is responsible for
about 97 per cent of all potential intragroup transfusion reactions and hemolytic
disease, and in the remaining 3 per cent any one of numerous blood factors may
induce the isoimmunization. In order of their frequency, blood factors whose
antibodies by themselves are known to cause hemolytic disease, and by the same
token transfusion reactions, are listed below in order of their statistical importance.
TABLE 1
H U M A N BLOOD FACTORS D E M O N S T R A B L E BY I S O A N T I B O D I E S
GENETIC
SYSTEM
1
FACTORS
ALTERNATE NAMES
PER CENT OF POSITIVE REACTIONS
IN WHITE POPULATION*
D-d
Rho-Hro
85
63
C-c
C*
rh'-hr'
rh w
73
1
80
E-c
rh"-hr"
30
97
2
K-k
Kell-Cellano
3
Fy»-Fy>>
Duffy
66
82
4
5
Jk»-Jk b
S-s
Kidd
77
56
73
88
6
7
M-N
Tj»
Le"-Le b
Jay
Lewis
8
Lu»
Lutheran
0
P
S
99.8
79
71
Slightly less t h a n 100
72
18
S
75
COMMENTS
Anti-d is very r a r e , " and its
existence has been questioned. 30
Anti-C produced by R h + individuals is very rare product of an allele a t C-c locus.
A n t i - C " by itself occurs very
rarely.
Anti-E is more frequently
produced.
K-k are becoming increasingly i m p o r t a n t clinically:
6 examples of anti-k.
Only one example 1 3 of antiFyb
Only one example 2 2 of anti-Jk 1 '
Only one example of anti-s 1 9
Numerous examples of antiH u m a n anti-N is very rare.
8 examples 1 6 of a n t i - T j "
Le"-Le b are associated with
secretor s t a t u s of ABO
system.
Probably not i m p o r t a n t clinicallv.
N o t i m p o r t a n t clinically.
ABO system and the Ai-A2 variants are not included, b u t mention must be made of
anti-H in serums of 3 individuals living in Bombay which acted on all group O bloods
except their own red cells presumably of genotype OcOc.^ T h e " p r i v a t e " or famity factors of low-incidence are listed in Table 3.
* In each case in which the factors are paired, e.g., D-d, the 2 values give the incidence of reactions for D and d, respectively.
294
LEVINB ET
c
E
K
k
Jk»
cS
M
s
. Fy»
Numerous cases
Numerous cases
Numerous cases
6 cases
Several
Several
Several
1 case
2 cases
1 case
VOL. 2 4
Ah.
" P r i v a t e " factors
Mi" 1 case
Be" 1 case
Becker 1 case
Diego 1 case
In a number of cases antibodies for Fy" and e were found to cause transfusion
reactions but have not yet been shown to be associated with hemolytic disease,
although these may be considered as potentially capable of inducing this condition. In addition, there are 8 examples of the high incidence factor, Tj a , whose
antibody is potentially responsible for transfusion accidents and possibly hemolytic disease. 16,16 In 4 women of child-bearing age, there were no full-term
pregnancies and a total of 12 pregnancies ended in miscarriages. From this it may
be concluded that anti-Tj a induces early fetal death before the fetus is sufficiently
developed to exhibit symptoms of blood destruction.
Although actually belonging to the scheme of the ABO groups, one may include 3 remarkable group 0 individuals whose serum contained anti-H which
hemolyzed or agglutinated all other group 0 bloods except their own.2 The activity of anti-H in each of the 3 cases was of the same order as their normal anti-A
and anti-B.
ROUTINE TESTS OF ALL RED BLOOD CELLS FOR R H - H R ANTIGENS
The identification of Rh-Hr antibodies other than anti-D is simplified by
noting the contrast of antigens in the blood of husband and wife. In general,
antibodies are likely to be specific for factors in the husband's blood that are not
present in the antigenic make-up of his wife's red cells. For example, a woman
of genotype DCe/DCe may produce anti-c or anti-E, or both, while in a genotype
of DCe/dce one may suspect the presence of anti-E provided that the incompatible factors are present in her husband's blood.
With emphasis on rare atypical antibodies, the exclusion of the very frequent
anti-D (along with anti-C and/or anti-E) is simplified by testing the red blood
cells for their Rh-Hr factors. With very rare exceptions, as in the case of Argall
and his associates,1 anti-D will be produced by Rh-negative persons.* For example, the possibility of the presence of anti-K, anti-Fy a , or others in addition
to anti-D is determined in special screening tests with selected test cells in
batches of 100 or more serums. When indicated on the basis of the history, as in
transfusion cases, these serums are immediately tested along with other selected
serums with the panel of 6 test cells as described below.
Technic of Rh-Hr tests on numerous bloods. In the daily handling of numerous
* In this remarkable case, a D u -positive patient produced a n t i - D . In a second exceptional
instance, reported by Weineret al. (Brit. M . J., 2 : 125-128, 1953), the patient suffering from
acquired hemolytic anemia was of genotype DCe/DCe and yet he produced anti-e.
MAR. 1954
WARE
295
ISOAGGLUTININS
blood specimens submitted for special studies, it is necessary to adopt rapid and
efficient methods for determination of the antigenic structure of the red blood
cells. The test-tube technic is employed with washed saline suspensions that are
tested for their ABO groups (at room temperature) and with complete anti-D,
anti-C, anti-E and anti-c (in a water bath at 37 C.) In the absence of complete
antibodies, as in the case of anti-c, specific serums are selected that contain both
complete and incomplete antibodies. When diluted with 30 per cent bovine
albumin, such reagents give distinct and clear-cut reactions with saline suspensions. With this uniform procedure of test-tube technic and saline suspended
cells, 100 or more bloods can be tested and read by 2 trained technicians in a
period not exceeding 2 or 3 hours. (While these tests are incubating, screening of
the serums for detection of atypical agglutinins is carried out in accordance with
procedure given below.) Readings are made at the end of 1 hour with the aid of a
hand lens (3X, 3.5X or 6X, Bausch & Lomb). All bloods failing to react with
anti-D are subjected to the test for D".
I N I T I A L S C R E E N I N G T E S T F O R S E L E C T I O N O P ATYPICAL
ANTIBODIES
All serums including those of husbands and members of families submitted are
tested with:
a. Groups A and B, Rh-negative blood for "reverse" blood grouping at room
temperature.
b. A mixture of 2 specially selected group 0 , Rh-positive blood, containing all
the Rh-Hr antigens, and if possible, Kell and F y \ These test cells are used in
triplicate in the form of (1) saline suspensions, (2) trypsinized suspensions and
(3) albumin (20 per cent) suspensions.
The 3 different series of tests are incubated in a water bath at 37 C. After the
tests for detection of "saline" or "complete" antibodies have been read at the
end of 1 hour's incubation, this set of tests is subjected to the Coombs antiglobulin technic. The series of cells treated with trypsin is generally read after
30 to 45 minutes, while the tests with albumin-suspended cells are read after
2 hours' incubation.
With rare exceptions, the results with the 3 differently treated test cells will be
in complete agreement. Reference to the antigenic structure of the red cells will
confirm that in the vast majority of the cases the Rh-negative person produces
anti-D. Unless it is indicated by the history, no further tests with these serums
are carried out at this time except for a titration with trypsin-treated red blood
cells. If strong complete agglutinins are present, the particular serum is studied
thoroughly for titer and specificity, in order to determine whether or not it is
useful as a diagnostic reagent specific for D alone, C alone or E alone. It is now
common knowledge that serums containing anti-D incomplete antibodies together with anti-C or anti-E complete antibodies may prove to be useful diagnostic reagents for C orE, respectively. Asmentioned above, all other serums containing anti-D are stored and tested periodically in large batches for the presence
of other antibodies outside of the Rh-Hr system.
296
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AL.
VOL.
24
IDENTIFICATION OF ANTIBODIES WITH A PANEL OF 6 SELECTED TEST CELLS
Excluding most serums presumed to contain anti-D, all other serums containing atypical antibodies detectable by the method described above are studied
with a panel of carefully selected red blood cells in an attempt to identify the
antibody. Specimens from immunized Rh-negative patients are also included if
they are to receive further transfusions, or if there are other unusual features.
In addition, other serums from Rh-negative or Rh-positive patients, although
failing to react in the initial screening, are included if the obstetric or transfusion
history is suggestive of isoimmunization.
AVith a limited technical staff, it is not feasible, although it is highly desirable,
to test all serums presumed to contain anti-D. If, in the future, all Rh-negative
patients receive exclusively Rh-negative blood, they may be immunized to the
same antigens outside of the Rh-Hr system as the Rh-positive patient who
receives Rh-positive blood. In a recent case, an Rh-negative patient transfused
3 times with Rh-negative blood produced anti-K only. In all cases, it is assumed
that in the event of further transfusion therapy, any incompatibility will be
detected by suitable crossmatching test.
• In the daily run there may be as few as 5 or as many as 20 serums selected for
specificity tests. With only 1 worker available to handle this specialized procedure, the single method chosen as yielding the greatest number of serums containing "warm" immune isoantibodies is the antiglobulin technic. Although this
requires repeated washings of numerous mixtures of test cells and serums, the
results obtained justify the retention of this routine procedure because a number
of antibodies, anti-Fy a , anti-K and still others, which may fail to react with
trypsin-treated or albumin-suspended test cells, can be detected in the antiglobulin test.*
With the use of this procedure only, there is the risk of failing to detect a
number of antibodies that have little or no clinical significance, i.e., antibodies
of Ai, 0 (H), P, Le b , and some instances of Le a , S, M and N. Occasionally,
however, weak direct reactions may be observed in the first stage of the antiglobulin test, i.e., after incubation at 37 C , and if the result of the second stage
after the addition of the antihuman serum, is negative, the patient's serum can
be retested with the same panel or an extended panel at 18 C. Furthermore, there
is a factor of safety, since some examples of anti-Le a and anti-S will be detected
by the antiglobulin test if the history reveals antigenic stimulation.
The panel of test cells for identification of antibodies. In order to obtain the
maximum yield with minimum effort, each serum is tested with 6 bloods carefully
selected for their antigenic structure as shown in Table 2. If compatible blood of
the husband's group is available, it is always included as the seventh blood in the
particular series. In cases of intragroup transfusion difficulties, blood from one or
more donors is tested with the appropriate serum.
The choice of test cells selected for the panel is not binding and depends in
large measure upon available bloods, preferably from personnel of the institution
* Exceptionally, anti-K or anti-Py a will react with saline suspended cells.8, 24
MAR. 1 9 5 4
297
RARE ISOAGGLUTININS
TABLE 2
A N T I G E N S IN P A N E L OK G R O U P 0
T E S T C E L L S FOR I D E N T I F I C A T I O N
OF A N T I B O D I E S
R h ANTIGENS
Most Likely Genotype
1.
2.
3.
4.
5.
6.
1
R'R DCe/DCe
R"°r DCe/dce
R'R2 DcE/DcE
r'r
dCe/dce
r"r
dcE/dce
rr
dee/dee
OTHER ANTIGENS
MN
Kk
Ss
MN
N
N
M
MN
M
0+
0+
0+
0+
0+
++
0
0
0
0
0
0+
0+
+0
+
+
+
+
+
+
+
+
+
+
++
0+
0
0
0
0
++
Jk»
0
+
0
0
Fy'
0
Le"
p
0
0
Lu°
0
+
0
0
Saline suspensions are employed and after 1 hour's incubation, reactions specific for
complete antibodies are recorded. This is followed by the second stage of the antiglobulin reaction for detection of incomplete antibodies.
or neighboring institutions. AVorkers affiliated with large blood banks should
have no difficulty in finding group 0 donors with representation of antigenic
structures similar, if not preferable, to those given in Table 2. There is considerable advantage in the selection of the 3 Rh-negative bloods (Table 2, bloods 4, 5
and 6) which contain antigens C, E and K, respectively. By happy coincidence,
these 3 bloods define also the 3 genotypes of the S-s system. Two of them (bloods
4 and 5) contain the Fy" factor while 1 (blood 4) also has .Ik".
In any event, the donors of these selected bloods should be available for weekly
specimens, which may be drawn under sterile conditions and preserved in Alsever's solution.! It is advisable also to prepare in advance of requirements a list
of alternate donors with similar, if not identical, antigenic properties.
Experience has shown that in the vast majority of cases the atypical antibody
if occurring by itself, can readily be identified. The specificity can then be confirmed in a larger panel with parallel tests with the known corresponding antibody. If a particular serum contains potent specific antibodies giving clear-cut
definition of positive and negative reactions, arrangements are made to purchase
the blood and store the serum for future use.
Tests with this limited panel of 6 test bloods will reveal the presence of anti-e
which acts on 97 per cent of all bloods because a nonreacting blood homozygous
for E, i.e., DcE/DcE (Table 2, blood 3) is represented in the panel. Anti-Kell may
be identified by the presence of reactions with the Rh-negative blood containing
the Kell factor (blood 6 in the panel). The presence of both anti-D and anti-K in
the same serum may be suspected by the reactions, perhaps in different titers,
on the first 3 bloods all containing D and the sixth blood specific for K. The
combined presence of anti-E and anti-c, which may be produced by individuals
of genotype DCe/DCe, may be determined if one of the antibodies reacts with
| I t is now possible to preserve red blood cells for longer periods by freezing. T h e method
of thawing and washing with graded doses of glycerine-citrate mixture is given by Chaplin
and Mollison (Lancet, 1 : 215-21S, 1953).
298
L E V I N E ET
AL.
VOL. 24
saline-suspended cells and the other with the antiglobulin technic. Otherwise,
one may have to resort either to differential titrations or specific absorption with
the rare blood of genotype DCE/DCe (RzRl) to remove anti-E and with dee/dee
to remove anti-c.7
Fortunately, one member of our staff has the property O ' (Table 2, blood 2),
which has an incidence of only 2 per cent in the general population. With the aid
of this blood it was possible to identify anti-C"' as the only warm immune atypical
antibody in the serum of a patient of genotype DcE/DCe, who was immunized
after receiving 19 blood transfusions in a period of 6 months.* It is remarkable
that in spite of the likelihood of numerous antigenic differences in the red cells of
the patient and her 19 donors, the only incompatibility involved 1 blood factor.
Race and Sanger23 cite 2 cases of anti-C w as the only antibody produced as a result
of transplacental isoimmunization.
The importance oj very rare bloods not represented in the panel. Tests with the
limited panel of 6 bloods will fail to identify a number of antibodies either because
the antibody with which to select suitable test cells is not available locally or the
required test cells have exceedingly low incidences in the general population. The
factors involved and their main characteristics are listed below:
1. Fy b : only 1 example of anti-Fy b discovered in Germany, but its activity has
rapidly deteriorated.13
2. Jk b : only 1 example of anti-Jk b discovered in England22 in a serum that also
contained a more powerful anti-Fy".
3. k: nonreacting or compatible blood of genotype KK has an incidence of
0.2 per cent.2'
4. Tj": as an example of a high-incidence factor with only 8 compatible bloods
scattered over 4 continents. 16, 16 ' 31
5. Two or more antibodies producing the same effect as that for a high-incidence factor.
6. "Private" or low-incidence factors—detected by action on husband's
blood such as Mi", Be a , Becker and several others.
Anti-Fy b , which gives 83 per cent positive reactions, should react on all bloods
except those of genotype FyaFya. Since anti-Fy b is not available, this type of
blood could perhaps be selected on the basis of the so-called double-dose effect,
i.e., stronger reactions of anti-Fy° with Fy*Fy"- than with FyaFyh. Since many
anti-Fy a serums exhibit this phenomenon, these titrations will be carried out in
the near future, and the proper blood will be substituted or added to the panel.
In any event, anti-Fy b should be suspected if it splits the 65 per cent Fy a -positive
reactions into 2 unequal classes of 17 per cent negatives (Fy°-Fya) and 48 per cent
positive (FyaFyb).13- 1Z-24 The general procedure for establishing the serologic and
genetic relationship in a 2-gene system with equal dominance has previously been
described for the K-k and S-s blood factors. 19 ' 21
Similarly, anti-.Ikb (73 per cent positives) should be suspected if it splits the 77
per cent Jk a positives into 2 unequal groups of 27 per cent nonreactors (.//ca,//ca)
* The specimens in this case were submitted by Miss Frances Rohrbaugh, Maryland
General Hospital, Baltimore, Md.
MAR. 1954
RARE ISOAGGLUTININS
299
and 50 per cent positive reactions (,//c"«/fcb) as shown by Plaut and his associates.22
For identification of anti-k, which reacts on 99.8 per cent of all bloods, the
essential nonreacting blood of genotype KK is not represented in the panel
because of its low incidence, i.e., 0.2 per cent of all bloods or 0.08 per cent for
group 0 bloods (1:1250). Since only individuals of genotype KK can produce
anti-k, the identification of anti-k is almost certain if each of the patient's parents
reacts with anti-K. This procedure was recently employed as an aid in the
demonstration of anti-k, which was responsible for a severe transfusion reaction.*
In the absence of the patient's parents as in another recent case,f the diagnosis
of anti-k is most conveniently established if the patient's cells do not react with a
standard anti-k and if the patient's serum fails to react with the rare blood, KK.
In the course of genetic studies of the K-k system, 14 bloods of genotype 7v/v
were found, and it was not difficult to obtain 3 group O bloods from the same
family that failed to react with the 2 antibodies now established as the fifth and
sixth examples of anti-k. 6 ' 18 ' 26
There are now 8 examples of anti-Tj", which is specific for a factor having a
high incidence. In more than 5000 random bloods tested not one blood was found
to be compatible with this antibody, which in several instances was also a hemolysin. Six of these were found in 3 pairs of siblings, 2 in the United States and .1.
in Australia. 15, 16 ' 29 The other 2 examples of anti-Tj" were found in South
Africa31 and Poland.12 Anti-Tj" should be suspected if a particular serum hemolyzes or agglutinates almost all bloods tested. As a rule, the titer is not higher
in the antiglobulin test. If this antibody is suspected, a compatible blood presumably of genotype TjhTjb is essential in order to establish the diagnosis.
Of the 4 living in the United States, 2 in Virginia, and 2 in Michigan, 3 are in
group O and 1 in group A. Anti-Tj" in each of the Michigan siblings was identified, since the serums acted on hundreds of bloods tested except the 2 Virginian
siblings,J and their red cells failed to react with the original anti-Tj", the Autralian and Polish serums.
In the Michigan cases of anti-Tj", the parents of the 2 siblings are related, so
that each of the parents contributed the rare Tjh gene and 25 per cent of the
offspring may produce anti-Tj". A history of cousin relationship in the patients'
parents was elicited in the r'r' case and in each of the 3 D—/D-- cases discussed
below.
Antibodies for factors of high incidence must be differentiated from anti-e,
* This patient's blood was referred by the Central Florida Blood Bank, and it was also
tested by Dr. J. J. Griffitts.
f D r . T. R. Helmbold, Elizabeth Steel Magee Hospital, P i t t s b u r g h , Pennsylvania, submitted the specimens in this ease in which the isoimmunization, induced by a pregnancy
and a transfusion, caused hemolytic disease of the hydrops variety.
| The first antibody was found in a Virginia patient (Mrs. Jay) whose serum was compatible with her sister's blood only. The Michigan case was referred for diagnosis by Miss
B e t t y Pryor, Hillsdale H e a l t h Center, Hillsdale, Michigan. T h e findings in the 8 cases were
recently summarized in tabular form and will be described in greater detail in a forthcoming
paper.
300
LEVINE ET
AL.
VOL. 24
anti-k, anti-Tj tt and particularly from serums giving reactions on all or almost all
bloods tested by virtue of a combination of 2 or more antibodies. As an example
one may cite the group 0, Rh-negative patient of genotype KK, who produced
as a result of transfusions both anti-D and anti-k as subsequently confirmed by
absorption.18 The only group 0 blood that was compatible with both antibodies
has an incidence of 1:7407. Each of the 3 such bloods selected from the genetic
studies of the K-k system was shown to be compatible. In a similar case of anti-D
plus anti-k that ended in fatal transfusion anuria, Crawford6 found it necessary
to resort to elution from strongly agglutinated Rh-negative blood in order to
demonstrate anti-k, because the very rare Rh-negative compatible blood was not
available.
Another unusual combination of antibodies was readily identified in a patient
immunized by transfusion and pregnancies by taking into account the antigenic
components in her red cells and the history.* This group 0, Rh-negative patient
was of the rare genotype r'r' (dCe/dCe) and her husband was also Rh-negative,
rr. Thus, the mating was compatible for D but not for c (hr'), so that anti-c was
suspected. Her serum, however, acted on each one of several hundred random
group O bloods including bloods of genotype RlRl (DCe/DCe). Because of the
transfusion with presumably Rh-positive blood, the production of anti-D could
be anticipated. The presence of both anti-c and anti-D was readily established
by cross-absorption experiments. The only compatible blood, obtained from one
of her siblings, was of group O, Rh-negative, genotype r'r', which has an incidence of 1:25,000 (1:2.5 X 1:10,000).
Perhaps the most unexpected of any combinations of antibodies was described
by Race, Sanger and Selwyn25 in a case of possible chromosome deletion characterized by the absence of C-c and E-e factors. The serum agglutinated all
random bloods tested by virtue of simultaneous production of anti-C, anti-c and
also anti-e. A similar case was reported by Waller and his associates,28 who confirmed that with the absence of C-c and E-e factors there was a compensatory
increase of D antigen. This is indicated by the capacity of saline-suspended red
cells to react with incomplete anti-D. In a more recent case,f the D--/D— effect
was suspected because the high incidence of positive reactions could not be
attributed to anti-k or anti-Tj" and the red cells failed to react with pure antibodies for the C-c and E-e factors. Although absorption experiments have not
yet yielded clear-cut results, there are indications for the anomalous presence of
both anti-E and anti-e.
The solution of other cases of multiple sensitization, mainly due to repeated
transfusion, is far more difficult and, in a number of cases pending, the several
antibodies have not yet been identified. The reader is referred to 3 remarkable
cases published recently in which the identified antibodies are listed below in
terms of their blood factors. In each case the clinically important factors are given
* This case referred byM. B. Cooper, General Hospital of Syracuse, Syracuse, New York,
will be described more fully in a forthcoming paper.
f The bloods of this patient were submitted by Dr. Joseph Rini, Providence Hospital,
Holyoke, Massachusetts.
MAR. 1954
RARE ISOAGGLUTININS
301
in the upper line while the incompatibilities with the factors listed in the lower
line are probably not significant clinically because of poor antigenicity or weak
or no reactivity at 37 C.
Patient I
Callendar and Race3
c, O
Lu, Levay, N
Patient II
Collins, Sanger el al.4
E, K, S, O
N, Le", Le b
Patient III
Waller and Race27
C, E, S, F y
M
In general, the first step in the study is an analysis of the antigens in the recipient. Thus, the patient studied by Waller and Race who received 11 transfusions was of group B, had N, E°r, and was S-negative and Fy°-negative; and
he theoretically could, and actually did, produce antibodies for M, C, E, S and
Fy". Aside from anti-Fy", the 4 antibodies were demonstrated by direct tests with
selected cells at several temperatures, but for the demonstration of anti-Fy" it
was necessary to resort to specific absorption with selected Fy"-negative blood.
If one disregards the antibodies that are inactive or poorly reactive at 37 C ,
there should be no difficulty in selection of compatible donors for patients I and
II. For patient III who produced 4 warm immune antibodies with incidence of
positive reactions of 70, 30, 58 and 66 per cent, respectively, a long and diligent
search may be required. The problems of selection of compatible donors, however,
is far more acute for patients whose serums contain anti-k, anti-Tj", anti-H
(as in the 3 patients in Bombay) or combinations of anti-D plus anti-k, or anti-D
plus anti-c; or for immunized patients of genotype D--/D-. In these cases the
incidence of compatible donors is so low that it may be impossible to transfuse
these patients. Occasionally, compatible donors may be found in 1 or more of the
patient's relatives. Perhaps a cooperative project on an international level can be
organized for preparation of lists of individuals of rare genotypes to serve as
donors exclusively for immunized patients of identical genotypes.
The specificity of antibodies for the "private" or family blood factors is such
that the proper test cell cannot be included in the panel unless the husband's
or donor's blood is submitted. In several such cases studied, the antibodies
reacted on the blood of the husband or donor and members of their immediate
families, but failed to react with numerous random bloods.
The essential details in the more clearly defined cases are outlined in Table 3.
Aside from anti-Graydon and anti-Becker, the 4 remaining antibodies appear
to be individually specific. Thus, serums 5 and 6 fail to react with the Mi" factor
and serums 3 and 5 fail to react with the Diego factor. Furthermore, anti-Mi"
failed to react with Levay.20 A systematic study of these antibodies can be
carried out only by an exchange of the preserved serums among the several workers. Possibly many more cases of this sort exist because the studies presented
above are necessarily limited to ABO compatible matings. In an ABO incompatible mating with hemolytic disease it is difficult to exclude the possibility that
the blood destruction may be caused by an antibody for a factor of low incidence
limited to the husband and immediate members of his family, rather than by the
result of the action of a much higher-titered anti-A or anti-B.
302
LEVINE ET AL.
VOL. 2 4
TABLE 3
" P R I V A T E " OR FAMILY BLOOD FACTORS
REACTION WITH
NO.
NAME OF ANTIGEN
LOCATION
ANTIGENIC STIMULUS
Random
Bloods
Negative
Familial Bloods
Positive*
SIGNIFICANCE
1
Levay3
England
Transfusion
350
3 of 7
2
Australia
None
191
5 of 8
United States
Pregnancy
320
4 of 9
4
5
Graydon' 0
(Gr)
Miltenberger20
(Mi*>)
Becker9
Berrens 6 (Be")
Probably
none
Probably
none
H.d. J
Germany
United States
272
44S
3 of 4
4 of 7
H.d.
H.d.
6
Diegof
Pregnancy
Transfusion and
pregnane}'
Pregnancy
200
Only husband
tested
H.d.
3
Venezuela
United States
* N u m b e r of specimens positive out of total tested in family,
f Currently under investigation at the Ortho Research F o u n d a t i o n .
\ H.d. indicates hemolytic disease of the newborn.
Another example with hemolytic disease was published recently by Wiener and Brancato (Am. J. H u m a n Genetics, 5 : 350-355, 1953).
As a result of these studies, 2 suggestions may be made for the physician and
laboratory worker.
1. A female patient should never be transfused with her husband's or her
children's blood.
2. In hemolytic disease caused by unusual antibodies, the father's red cells
should always be tested when making tests of the mother's serum.
CONCLUDING REMARKS
In many hospitals it is the responsibility of the clinical pathologist to direct
the activities of the blood bank and to supervise all blood-grouping tests in
addition to his many other duties. It is physically impossible for each hospital
laboratory to be self-sufficient in the proper handling of this highly specialized
and complex material. At present, it is a routine policy in all hospitals to transfuse Rh-negative patients, particularly female patients, with Rh-negative blood.
In many hospitals proper methods are employed for detection of immunized
Rh-negative women and for the immediate transfusion therapy of infants with
hemolytic disease. These constitute the minimum requirements in the hospitals'
obligation to their patients.
It must be borne in mind that all other cases discussed in this paper constitute
highly exceptional material. Nevertheless, steps should be taken in each hospital
laboratory to train one or more of their workers so that the more important of
the rare antibodies can be detected, i.e., antibodies for c, E and K. For this, a
panel of test cells is essential, and this is now available commercially. There is
MAR. 1 9 5 4
RARE ISOAGGLUTININS
303
always the possibility of discovering a new blood factor, and this should prove to
be a powerful stimulus. In any event, the laboratory personnel should be trained
to select cases of potential isoimmunization on the basis of the history and to
submit duplicate specimens to workers who are fortunate in having the opportunity to devote all their efforts to this highly rewarding field of activity. Finally,
reference should be made to the vital and often astonishing contributions to
clinical medicine, genetics and anthropology that have resulted from studies of
human blood factors.
REFERENCES
1. ARGALL, B . A., B A L L , J . M . , AND T R E N T E L M A N , B . S.: Presence of a n t i - D antibody in
the serum of a I > patient. J . L a b . & Clin. Med., 41: 895-89S, 1953.
2. B H E N D E , Y . M . , D B S H P A N D E , C. K., B H A T I A , H . M . , SANGER, R., R A C E , R . R., M O R G A N ,
W. T . J., AND WATKINS, W. M . : A " n e w " blood-group character related t o the ABO
system. Lancet, 1: 903-906, 1952.
3. CALLENDAR, S. T . , AND R A C E , R . R.: A serological and genetical study of multiple antibodies formed in response t o blood transfusion by patient with lupus erythematosus
diffusus. Ann. Eugenics, 13: 102-117, 1946.
4. C O L L I N S , J . 0 . , SANGER, R., A L L E N , F . H . , J R . , AND R A C E , R. R . : N i n e blood-group
antibodies in a single serum after multiple transfusions. Brit. M . J., 1: 1297-1299,
1950.
5. CRAWFORD, H . : A third example of t h e blood group antibody anti-k. J . Clin. P a t h . , 6:
52-53, 1953.
6. DAVIDSOHN, I., S T E R N , K., S T R A U S E R , E . R., AND S P U R R I E R , W . : Be, a new " p r i v a t e "
blood factor. Blood, 8: 747-754, 1953.
7. D U N N E , C. J . , J R . , AND INNELLA, F . P . : D u a l sensitization to t h e r h " (E) and h r ' (c)
factors detected antenatally; a case report. Blood, 7: 526-532, 1952.
8. DUNSFORD, I . : A saline agglutinating Kell antibody. N a t u r e , 163: 962, 1949.
9. E L B E L , H . , AND PROKOP, 0 . : Ein neues erbliches Antigen als Ursache gehauftcr Fehlgeburten. Ztschr. H y g . , 132: 120-130, 1951.
10. GRAYDON, J . J . : A rare iso-haemagglutinogen. M. J . Australia, 2 : 9-10, 1946.
11. H I L L , J . M . , H A B E R M A N , S., G U Y , R., AND D A V E N P O R T , J . W., J R . : Additional evidence
for t h e anti-d: statistical studies. Blood, 5: 1049-105S, 1950.
12. HIRSZFELD, L.. AND GRABOWSKA, M . : Ueber individuelle Bluteigenschaften. Experentia,
8: 355-357, 1952.
13. I K I N , E . W., M O U R A N T , A. E . , P E T T E N K O F F E R , H . J . , AND B L U M E N T H A L , G . : Discovery
of the expected hemagglutinin, anti-Fy b . N a t u r e , 168: 1077-1080, 1951.
14. L E V I N E , P . : A brief review of t h e newer blood factors. T r . New York Acad. S c , 13:
205-209, 1951.
15. L E V I N E , P . : Genetics of t h e Newer Blood Factors. I n Advances in Genetics. New
Y o r k : Academic Press, Inc., in press.
16. L E V I N E , P., B O H B I T T , O. B . , W A L L E R , R . K., AND K U H M I C H E L , A. B . : I s o i m m u n i z a -
tion by a new blood factor in tumor cells. Proc. Soc. Exper. Biol. & Med., 77: 403-405,
1951.
17. L E V I N E . P . , F E R R A R O , L. R., AND K O C H , E . : Hemolytic disease of t h e newborn due to
anti-S. Blood, 7: 1030-1037, 1952.
18. L E V I N E , P . , KUHMICHEL, A. B . , AND W I G O D , M . : A second example of anti-Cellano
(anti-k). Blood, 7: 251-254, 1952.
19. L E V I N E , P . , KUHMICHEL, A. B . , W I G O D , M., AND K O C H , E . : A new blood factor, s, allelic
to S. Proc. Soc. Exper. Biol. & Med., 78: 218-220, 1951.
20. L E V I N E , P . , STOCK, A. H . , K U H M I C H E L , A. B . , AND BRONIKOVSKY, N . : A new h u m a n
blood factor of rare incidence in t h e general population. Proc. Soc. Exper. Biol. &
Med., 77: 402-403, 1951.
21. L E V I N E , P., W I G O D , M . , BACKER, A. M . , AND P O N D E R , R . : T h e Kell-Cellano
(K-k)
genetic system of human blood factors. Blood, 4: S69-872, 1949.
22. P L A U T , G., I K I N , E . W., M O U R A N T , A. E . , SANGER, R., AND R A C E , R . R . : A new blood-
group antibody, anti-Jk b . N a t u r e , 171: 431, 1953.
23. RACE, R . R., AND SANGER, R . : Blood Groups in M a n . Springfield, Illinois: C. C Thomas,
1950,290 p p .
24. R A C E , R . R., SANGER, R., AND L E H A N E , D . : Q u a n t i t a t i v e aspects of t h e blood-group
antigen Fy". Ann. Eugenics, 17: 255-266, 1953.
304
LEVINE ET AL.
VOL. 2 4
25. 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. B r i t . J . Exper. P a t h . , 32: 124-135, 1951.
26. SHAPIRO, M . : Observations on the Kell-Cellano (K-k) blood group system. So. African
M . J., 26:951-955,1952.
27. WALLER, R. K., AND R A C E , R. R . : Six blood-group antibodies in t h e serum of a t r a n s fused patient. Brit. M . J . , 1: 225-226, 1951.
28. W A L L E R , R. K., S A N G E R , R., AND B O B B I T T , 0 . B . : Two examples of t h e
D--/D--geno-
t y p e in an American family. Brit. M . J., 1: 198-201, 1953.
29. WALSH, R. J . , AND KOOPTZOPP, 0 . : Cited by Zoutendyk and Levine, 31 1952.
30. W I E N E R , A. S.: Letter t o the E d i t o r s . Blood, 5:693-694,1950.
31. ZOUTENDYK, A., AND L E V I N E , P . : A second example of t h e rare serum anti-Jav (Tj").
Am. J . Clin. P a t h . , 22: 630-633, 1952.