ABERRANT SECRETION OF SALIVARY A, B, AND H GROUP
SUBSTANCES IN HUMAN BEINGS
CRICHTON McNEIL, M.D., ELMER F. TRENTELMAN, B.S., VIRGINIA 0.
KREUTZER, B.A., AND CYRIL D. FULLMER, M.D.
Holy Cross Hospital Research Foundation, and Intermountain Regional Blood Center of the
American Red Cross, Salt Lake City, Utah
The present concept of secretion of blood group antigens is based on the theory
that 2 genes, Se and se, govern the ability to secrete aqueous soluble A and B
and O (H) substance in saliva. According to the work of Schiff and Sasaki,15 <Se
is inherited as a dominant trait, so that homozygous or heterozygous secreting
genes produce the characteristic. A nonsecretor is, therefore, a homozygous se/
se. The substance secreted depends, of course, upon the person's blood group—
with A, B, O, or a combination of these factors. Almost all human tissues (perhaps brain excepted) contain the A, B, or O (H) factors, 5,13 and, in one who
secretes, a large amount of these substances is released daily into the gastrointestinal tract. The term secretor actually refers to the person's ability to secrete water-soluble group substances, inasmuch as it has been found that all
human beings produce the alcohol-soluble form. Chemically speaking, the nonsecretors produce a substance of similar mucoprotein nature, but lacking in its
ability to neutralize anti-A, B, or H.17 Of the other blood group factors, only the
Lewis group is known to be secreted, and that without relation to the secretion
of A, B, and O (H) substance.3
The determination of the secretion of A or B substance has been a relatively
simple laboratory procedure of neutralizing a homologous antiserum with clear,
boiled saliva. The recognition of secretion of O (H) factor is a somewhat more
difficult problem. This has been accomplished by the use of so-called anti-0 (H)
serums, the most common of which have been derived from bovine,17 rabbit, 11 and
eel6 serums or certain plant extracts. 1 ' 2 , 7 , 1 0 The results consistently indicate
that approximately 80 per cent of human beings secrete A, B, or H substance,
whereas 20 per cent do not.
We became interested in the salivary secretion of H factor in 1953, when we
were able to produce an adequate amount of a potent and reliable anti-H reagent from the seeds of Lotus tetragonolobotus}" Although the literature includes
little reference to this problem, we find that most human secretors (regardless
of blood group) produce H substance as well as A orB. This was originally mentioned by Sasaki14 and recently reaffirmed by Boyd.2 In a larger survey, however,
Received, January 25, 1957; revision received, February 25; accepted for publication
April 1.
Dr. McNeil is Pathologist and Director of Laboratories, Holy Cross Hospital. Mr.
Trentelman is Chief Serologist, Holy Cross Hospital Laboratory. Mrs. Kreutzer is Chief
Technologist, Intermountain Regional Blood Center of American Red Cross. Dr. Fullmer
is Associate Pathologist, Holy Cross Hospital Laboratories.
This work was supported by funds made available from the Hoi}' Cross Hospital Research
Foundation and the American Red Cross Blood Program.
145
146
MCNEIL ET
AL.
Vol. 28
we found that this is not a 100 per cent rule, and that there is a respectable
number of human beings of blood groups A and B who secrete H, but not A or
B, and sometimes vice versa. This irregularity of secretion immediately conflicts
with the original theory and compels us to reconsider the genetic theories of
secretion.
The authors' purpose in this paper is to present new data that illustrate the
irregularity of salivary secretion of A, B, O (H) secretion, which we term aberrant secretion, and to extend the work we previously reported.9
METHODS AND
REAGENTS
Reagents. The anti-H reagent used in all of the tests was prepared as previously described,10 and it is a saline extract of the seeds of Lotus tetragonolobotus.
This sort of extract was originally tested by Krupe, 7 who first described its antiH activity. Our modification of the extract was compared with an anti-H eel
serum sent to us by Morgan and Watkins,12 and the 2 extracts were entirely
similar in activity.
The anti-A and anti-B serums were obtained from immunized donors of blood
groups A and B whose native titers manifested levels of 1:1000 or more.
Titrations with 2-fold dilutions of these 3 reagents were performed in order to
determine a suitable dilution as a working solution. The last 4-plus (solid cellbutton) reaction was used as an indication of the proper dilution to be used in
the titration system described in the next section.
Specimens. Specimens of blood and saliva were obtained (1) from men who
volunteered to donate blood at the Utah State Prison and (2) from families and
patients reporting for blood group tests at the Holy Cross Hospital Laboratory.
Specimens of blood were tested forAi, A2, B, Rh, and occasionally Le a antigens.
Specimens of saliva were immediately placed in a boiling water-bath for 15
min., centrifuged, and the clear supernatant tested for inhibition of anti-A, antiB, and anti-H reagent. If not tested immediately, specimens of saliva were
heated and the clear fluid was frozen until tested.
Saliva Inhibition Tests
The tests were performed at room temperature.
1. Two-fold dilutions of saliva were made in 0.2-ml. amounts in 0.9 per cent
solution of sodium chloride. Ten tubes were usually sufficient.
2. Two-tenths milliliter of the anti-H working reagent, diluted in 10 per cent
PVP (buffered to pH 7.0), were added to the diluted saliva. When testing for
A and B substances, 0.2 ml. of anti-A or anti-B working reagent, diluted in 0.9
per cent solution of sodium chloride, were added to saliva.
3. The contents of the tubes were mixed and permitted to stand 30 min.
(Almost all neutralization occurs prior to this time, and additional time does
not enhance the reaction.)
4. Four per cent suspensions of group O cells in 10 per cent PVP were added
in 0.1-ml. amounts to each tube in the rack with anti-H reagent, and a 4 per
cent suspension of Ai or B cells in physiologic saline solution was added to re-
Aug. 1957
147
A, B, AND H SECKETOKS
spective A- and B-inhibition tubes. Ai cells were used for the determination of
activity of A antigen, inasmuch as A2 cells vary considerably in their content of
A antigen and a more potent anti-A reagent is required in order to detect the
same level of inhibition.
5. The contents of the tubes were again mixed thoroughly and permitted to
stand 10 min., after which the tubes were centrifuged at 2000 r.p.m. for 1 min.
6. Readings from zero to 4 + were made and the last 3 + reading was regarded as the end point of inhibitory activity of the saliva.
Interpretation. Persons were classified as nonsecretors of H, A, or B if their
saliva manifested a lack of inhibition of the antiserums or if the titer was from
zero to 1:4. Secretors of H, A, or B factor were indicated by saliva that had an
inhibition titer of 1:64 or more. In some instances an intermediate titer, that is
one from 1:4 to 1:32, was obtained even after duplicate determinations, and
persons with such titers were termed partial secretors. Aberrant secretors were
defined as those who secrete 1 substance at a level of 1:64 or more, and another
at 1:4 or less. All instances of irregularities that were regarded as aberrant secretion were studied again with proper controls, in order to confirm the results.
KESULTS
Table 1 consists of the data from a group of control persons who were selected
from voluntary blood donors at the Utah State Prison. Deliberate attempts were
made to select men of group A or B, thus accounting for their preponderance in
this study. Most persons secreted A or B, and H, or none of the factors, but
certain exceptions were noted. A few in group A, as well as group B, clearly
secreted H, but not A, according to tests with Ai cells. These aberrant secretors
were persons in blood groups A2 and B; at first, it seemed that this irregularity
TABLE 1
CONTROL S E R I E S OF A-, B-, AND H - S B C R E T O R STATUS OF 100 SELECTED P E R S O N S
Secretors* of
A, B, and H
Aberrant Secretors of H, A, or B
H
per cent
per cent
per cent
per cent
per cent
A,
A2
B
MAX
11
38
11
15
None
0
0
2
2
0
2
0
—
0
—
—
0
1
11
4
3
Totals§
75
4
2
0
19
Of
B
Secreted Neither
A, B, nor H
A
* F o r purposes of simplification, partial secretors are included with secretors. Read the
text. All persons were tested q u a n t i t a t i v e l y .
f Owing to the fact t h a t only 1 substance (H) is secreted in this group, the detection of
aberrant secretion is impossible a t present.
| Although none of this group occurred in the first 100, we have since tested persons of
blood group AB and found t h a t t h e y usually secrete all 3 substances.
§ Totals represent percentage.
148
MCNEIL ET
Vol. 28
AL.
was confined to members of blood group A2, but subsequently we found some in
group Ai who did not conform to the usual pattern of all-or-none secretion. Of
the 100 tested, it may be noted that the control group included only 6 who fit
the pattern of aberrant secretion. This is a low frequency in comparison with
that of the test group.
Data from the test group are compiled in Table 2, representing specially selected persons who presented themselves for routine studies of blood groups,
owing to the fact that they had perinatal pathologic conditions. Here we encountered a considerable number of aberrant secretors, and a group AB person who secreted B and H, but not A, is included in this group. Six in this group
TABLE 2
D I F F E R E N T I A L SECRETION OF A, B, AND H FACTORS IN T H E SALIVA O F
A B E R R A N T SECRETORS
Salivary Secretion Titer
Case
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Group
W. E .
R . A.
S. T .
A. N .
B . R.
N.E.
L. 0 .
A. T .
S. T .
B . A.
M. E.
G. I.
R. T .
Rh
+
+
+
+
+
+
+
+
+
+
+
+
+
A,
A,
A,
A,
A2
A2
A2
A2
A2
A2
B
B
A,B
H
A
1:4
1:2
1:64
1:2
0
0
1:512
1:256*
1:128
1:64
1:64
1:2
1:128
1:64
1:32,000
0
1:128
1:128
1:512
1:2
0
0
0
1:2
B
1:4
1:64
1:512
* See Table 3. Actually, 3 separate specimens were analyzed: (1) 1:256, (2) 1:256, (3)
1:4096 for H substance. All of t h e specimens were completely negative for A.
TABLE 3
T I T R A T I O N R E C O R D O F AN A B E R R A N T S E C R E T O R ( C A S E 8, P A T I E N T A. T . , B L O O D G R O U I '
A2, R H - P 0 S I T I V E ,
Titer of Saliva from an Aberrant Secretor
Inhibited
Anti-H
Anti-A
Anti-Ai*
Le a -NEGATIVE)
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
0
4
4
0
4
4
0
4
4
0
4
4
0
4
4
0
4
4
1
4
4
3
4
4
4
4
4
4
4
4
H-Secretor
A-Nonsecretor
A-Nonsecretor
1
0
2
2
4
3
H-Secretor
A-Secretor
Control Secretor
Anti-H
Anti-A
0
0
0
0
0
0
0
0
0
0
* Dolichos bilflorus Anti-A, (Bird 1 ).
0
0
0
0
Aug. 1957
A, B, AND H SECRETORS
149
(Cases 3, 5, 6, 8, 9, and 10) totally lacked secretion of 1 antigen, although the
other was present in significant quantity. Especially noteworthy is Case 8; this
person secreted H antigen to a level of 1:256 or more on 3 separate occasions,
without any detectable secretion of A. An unusually high frequency of persons
in group A2 is noted in our series of aberrant secretors. This frequency of 54 per
cent of the group A persons listed in Table 2 is probably beyond the possibility
of chance selection. Although the data in the table indicate that all of the persons were Rh-positive, the total was not sufficient to justify significant comment
at this time.
Table 3 represents the results of a typical titration with saliva from an aberrant secretor, as compared with those from persons who were normal secretors
of A and H factors.
DISCUSSION
The results of this study indicate that the simple theory of secretion and
nonsecretion as controlled by genes Se and se is not wholly tenable. Exceptions
occur with sufficient frequency that it may be necessary to reconsider the genetic
basis for secretion. To our knowledge, the only reference in regard to irregularity
of secretion was mentioned by Simmons,16 who noted an irregular secretion in a
person of blood group AB. Although deductions based upon Lewis typing are
still uncertain, because of the lack of complete definition of the Lewis system,
we have used Grube's 4 serum "Gli" to test several aberrant secretors and we
find that (according to the secretion) they do not behave as they should. For
example, an aberrant secretor who fails to secrete A is Le a -negative, whereas
data from all of Lewis' papers indicate that nonsecretors of A should be Le n positive. Further work is required in order to confirm the situation.
Levine's8 paper on suppression of certain characteristics of the B group offers
a possible source of explanation. Perhaps nonsecretors represent the suppression
of a gene during 1 generation. It may be noted in his paper that there was an
irregularity of secretion in the offspring of the propositus, and this might represent the same phenomenon we observed.
We wish to emphasize that it is necessary to perform quantitative titrations
of saliva in order to detect secretion of A, B, and H substances. The simple
qualitative test is too often inadequate for accurate determination of secretion.
SUMMARY
1. The secretion of A, B, and O (H) substances is not as simple as originally
postulated by Schiff and Sasaki.
2. Whereas we formerly believed that a person was a secretor or nonsecretor
on the basis of a single test for A, B, or H, it now seems that irregularities in
such secretion are fairly frequent when 2 antibodies, i.e., anti-A and anti-H or
anti-B and anti-H, are used for the testing. We regard such persons as aberrant
secretors.
3. Persons in blood group A2 were unusually frequent among the aberrant
secretors in our study series to date.
150
MCNEIL ET
Ah.
Vol. 28
4. Le"-typing of aberrant secretors does not yield results that conform to the
established patterns.
5. The explanation for the secretion of 1 substance, i.e., for example, H and
not A, may be based on the failure of gene penetrance or reduced expression.
Suppression of the action of a gene for 1 generation provides another possible
explanation. This phenomenon should be studied more thoroughly after the
accumulation of significant data.
6. The occurrence of aberrant secretors of A, B, and H factors has a higher
than normal rate among habitual aborters. This observation will be demonstrated
and discussed in a subsequent communication.
SUMMAHIO I N INTERLINGTJA
1. Le secretion del substantias A, B, e O (H) es minus simple que lo que esseva
originalmente postulate per Schiff e Sasaki.
2. Durante que in le passato nos credeva que un individuo esseva secretor o
nonsecretor super le base de un sol test pro A, B, o H, il nunc appare que irregularitates in le secretion es satis frequente quando 2 anticorpores, i.e., anti-A e
anti-H o anti-B e anti-H, es usate in le tests. Nos designa tal individuos como
secretores aberrante.
3. In le series studiate per nos usque al tempore presente, individuos del
gruppo sanguinee A2 constitueva un inusualmente alte procentage del secretores
aberrante.
4. Typation a Le a produce, in secretores aberrante, resultatos non conforme
al regulas establite.
5. Le explication del secretion de 1 substantia, i.e., per exemplo, H e non A,
pote basar se super non-penetrantia 0 dysexpression del gen. II es etiam possibile
que nonsecretores representa le suppression de un gen durante 1 generation.
Iste phenomeno deberea esser studiate plus meticulosemente post le accumulation
additional de datos significative.
6. Le occurrentia de secretores aberrante de factores A, B, e H monstra un
incidentia plus que normal inter abortrices habitual. Iste observation va esser
demonstrate e discutite in un communication futur.
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A, B, AND H SECRETORS
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