Escolar Documentos
Profissional Documentos
Cultura Documentos
AND
DISTRIBUTION SECTIONS
OF
WATER USING
BLOOD
GROUP
IN TISSUE EXTRACT OF
SIDNEY
ULEX
P. KENT Medical
EUROPEUS
Department
of
Pathology,
University
of
Alabama
Center,
Birmingham
33,
Alabama
Received
SUMMARY
for publication
August
15,
1963,
revised
April
A fluorescein seed
ing
labelled
H
extract
antigen
of Ulex
in formalin
europeus
agglutination technique, hibition technique and, cent antibody the technique ABO (H) distributed methods
influoresthese
shown
was
water
found
soluble
to be satisfactory tissue
typing
for demonstratfixed
paraffin
available
embedded
anti-A
sections.
serum
to be widely
How-
fluorescein A antigen
tissues.
and used to demonstrate water soluble in formalin fixed paraffin embedded The reactivity of A and H antigen after buffered formalin fixation was superior observed reactivity
tissues Autolysis of blood were
ever, the study of H antigen in normal and diseased human tissues by labelled antibody techniques has been limited by the availability of suitable anti-H serum. extracts of that reacts Cazal
Ulex
and
europeus
Lalaurie
have
after
Zenkers, in paraffin
equal to not antigen hut
Hellys
or Bouins
with
blood
did
group tissues
antigen and to a less extent with A and B antigens (3). In the following study the use of a fluorescein labelled extract of Ulex europeu.s seed for demonstrating
sections was
in fixed
water evaluated.
soluble
group
H antigen
in tissue
was in
associated formalin
of the
the
blood
more
cells,
abundant
vascular
blood
substances
group
distributed whereas
confined to
in the in nonsecretors
the deep
epithehial the
portion Brunnets
of the are
found in epithelial mucins (5, 17). In previous studies of the distribution of blood group antigens by the fluorescent antibody techniquse frozen sections using well antigens with but the water
As
of suich That
the
stomach of
and A and
were the
used of
By as
alcohol
tribution
antigen
group antigens of
of tissues
soluble
H auitigen; antigen:
Thie pueviously significance
contained contained
of this
does
deunonstration antigens. been Recently, demonstrated tissues studying suitability water The of autolysis antigens were soluble effects on
both
mosaic described
H antigen.
discussed.
water
soluble the
not
been
INTRODUCTION
paraffin snaterial,
embedded
The
distribution
of
blood stus(lie(l
group using
in
human
tissues
has
been
and material
surgical
was
* This investigation was supported Grant AI-03188 fronss the National Healths, U.S. Public Health Service. f Presented in part at the Annual thie American Society for Experimental Atlantic City, N. J., April, 1963.
by Research
Institutes
and
A antigens
591
592
SIDNEY
P.
KENT
TABLE
Agglutination
Titer
I
Extract
8 16
Titers
of
Fluorescein
2 4
Labelled
of
Ulex
32
europeus
64
Seed*
128 256
A1 Cells
BCells
Le
+ + + +
+ was
+ + + +
+
+ + + +
+
+ + + +
-
+ + +
-
+ +
-
+ +
-
* A reactioti batioti at
considered
positive
when
agglutination
was
grossly
visible
after
a two
hour
incu-
22#{176}C.
sulfate solution. Sufficient saline was added to the precipitate to restore the original volume. The serum was then dialyzed against buffered saline Extract of UIex Europeus Seed (Anti-H-F). for three days to remove the residual dye. After Ulex europeus seed* were ground in a coffee removing the dye the serum was labelled with grinder. One thoumsand grams of the ground seed fluorescein isothiocyanate and absorbed with were extracted with 2000 cc of 0.85% saline at 4#{176}C mouse liver powder as previously described (10). overnight. The suipernsatamit fluid was collected Determination of the Titer of Anti-H-F after centrifugation at 2000 r.p.m. for 15 minutes. and Anti-A-F Solutions. Employing the test Sufficient cold absolute ethianol was added to make tube hemagglutination test the anti-A, B and H a 20% ethanol solution (v/v) After 18 hours at titers of the anti-H-F solution were determined 4#{176}Cwhite a precipitate had formed which was col(2). Double dilutions of the anti-H-F solution lected by centrifugation of 2000 r.p.m. for 30 minwere used. The titer reported is the reciprocal of utes. The precipitate was extracted for 2 hours the highest dilution of the anti-H-F solution in with 100 cc of phosphate (0.01 Al, pH 7.1) buffered which grossly visible agglutination of 0 cells was physiological saline. The supernatant fluid was seen after incubation at 22#{176}C for two hours collected after centrifugation at 2000 r.p.m. for (Table I). Employing A1 cells the titer of the anti30 minutes amid placed in a dialysis bag. The A-F serum was also determined by the same techsupernatanit fluid was concentrated to 20 cc by nique. placing the dialysis hag in front of a fan at 4#{176}C. Determination of Secretory Status. Saliva After dialysis against 500 cc of phosphate buffered was collected at autopsy from patients of known saline for 2 hourrs the concentrate was centrifuged blood type (Table II). Material was obtained at 2000 r.p.m. for 15 minutes and the supernatant from patients with type A, B, and 0 blood. When fluid was collected. the subgroups of type A were determined anti-A1 Five cc of the above concentrate was labelled serums was used (1). The presence of H, A and/or with fluorescein isothiocyanate as previously deB antigen in the saliva was determined by the scribed (10). The labelled extract was dialyzed for slide hemagglutination inhibition technique (7). 4 days against three changes of buffered saline per Preparation of Tissue. At autopsy samples of day. The labelled extract was then absorbed with tissue were obtained from various organs (Tables mouse liver powder (100 mg/cc) on two occasions II and III). A block of tissue from each organ was before use. fixed in 4% neutral buffered formalin, dehydrated, Preparation of Fluorescein Labelled Anti-A and embedded in paraffin. Sections were cut at 4 Serum (Anti-A-F). To satisfactorily label anti-A and the sections were brought to water before serum with fluorescein isothiocyanate the blueuse. Surgically resected duodenal mucosa from four green dye which has been added to the commerindividuals with type A blood was also studied using cially available serumt niust be removed. Comfrozen sections as well as paraffin embedded tissue. mercially available amiti-A serum was precipitated One section of each tissue was stained by the alcian by adding an equal volume of saturated amblue-periodic acid Schiff method (AB-PAS) to monium sulfate solution. The precipitate was demonstrate epithelial mucins (16). Tissues that washed once with one-half saturated ammonium did not contain epithelial mucin either because of * Obtained from F. W. Schumacker, Horticulturist, Sandwich, Mass. Hyland Laboratories, Los Angeles, Calit Ortho Pharmaceutical Corp., Raritan, New Jersey. fornia.
MATERIALS
ANt)
METHODS
The
Preparation
of
Fituorescein
Labelled
DEMONSTRATION
OF
BLOOD
GROUP
(H)
ANTIGEN
IN
TISSUE
593
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594
autolysis satisfactory or or for on the patients type tissue this selectiosi study. were of various of considered fixatives A and
SIDNEY
P.
KENT
unamid H anti-
with
intensity
0 cells
of
was intensity
0 to
=
less
than
of
staining 4+ the
To
autolysis
evaluate fresh
four blood of
the surgically
effect
When
mated titerofl:128
the
from
of
1:128
desnomistration
results
as
follows:
gens,
from
were
Blocks
resected stomach (pylorus) was obtained. Two patients 0 amid two were blood type A.
taken from each specimen
were
buffered fornialin, Bouins, fluid for comuparison with (13). A portion of each
at. 22#{176}C amid samples 48 hours after paraffin of tisthe
sections
fixed
of unfixed
tissues
Employed.
the labelled solut.ions in a moist chamber (Petri dish with moist filter paper isi the top) for 10, 30 or 6() minutes. Unless otherwise stated thie data reported are based on a 30 nsinut.e incubation. The sections were washed its three changes of buffered saline for a total of 15 misinuites and sisounted in phosphate buiffered glycerisi (pH 7.1). A Zeiss fluorescence unit with a UG-5 and a BG-12 excitor filter and a Wratten-2A barrier filter was employed. Color photographs were made using Kodak E-135 film. Black and white prints are nsade from the colored slides using a Kodak 58 green filter (10). Controls. I)ne sect i(Iti (If each tissue was mounted in buffered glycerin to evaluate autofluorescence. Sections were also incubated with anti-H-F soluntion which had beets absorbed for 30 minutes with type 0 cells or with anti-A-F serum to whiich 10 mg of A antigen per cc had been added. Other sections were incubated with anti-H or asit.i-A solution for 30 misiutes, washed for 15 mninutes iui buffered saline amid exposed to anti-HF or anti-A-F solution for 15 minutes, washed in buffered saline and mounted in buffered glycerin.
Additional bothi A amid sections H antigesi which were were known to to contain exposed unlabelled
2+;titerof 1:32 = ; titer of 1 : 16 or less = 0. Similar results were obtained using labelled anti-A serum. Epithelial mucins were found in the glands and/ (I epithelial cells of all of the tissues studied. Its the paraffin embedded tissues used in this study water soluble A and H antigen were foumid in the epithelial mucins but not in the other elements of glands. Regardless of the A and H antigen content the mucous acini were PAS but not always AB positive. For example, the mucous acini in submaxillary glands were AB-PAS positive and the acini in Brunners glands were PAS positive but AB negative even though some acini its each type of gland contained A antigen, H antigen, or neither A nor H antigen. The Distribution of Water Soluble H Antigeur in Secretors and in Nonsecretors. Patients with type 0, A or B blood who secreted blood group antigens in their saliva showed H antigen in most of the epithelial mucims studied (Table II). The amount of H antigen demonstrable varied
4+;titerofl:64 from tissue to tissue. It was most abundant in the
upper digestive tract and respiratory tract (Figure 1). Patients with type 0 blood had a greater abundance of H antigen than patients with type A or B blood. The blood type of the patient was related to the pattern of distribution of H antigen within some types of glands. H antigen was
relatively evenly distributed among patients the mucous
acinii
blood.
of most individuals
glands with
antigen
from type
was
with or B
type
0 the
2
In
distribution
blood,
(Figures
of H
irregular
serum for 30 minutes, washed in buffered for 15 minutes and exposed to labelled antiH solution for 30 misiurtes or were exposed to unlabelled anti-H solution for 30 nsinutes, washed and exposed to labelled anti-A serum for 15 minutes before examination with ultraviolet light.
saline
RESULTS
anti-A
titer of the concentrated extract of Ulex europeus seed was 1:1024. The titer of the anti-H-F solution used in studying tissue sections is given in Table I. The solurtion agglutissated A1, B and 0 cells. The anti-H titer however was highier than the amiti-A or anti-B titers. If the titer
The
anti-H
and 3). That is, mucous acini in some areas were rich in H antigen yet closely associated mucous acini with similar AB-PAS staining characteristics were devoid of H antigen. This was particularly evident its Brunners glands but was also noted in the salivary glands and submucosal glands in the trachea. This pattern was observed in paraffin sections of autopsy and surgical specimens and in frozen sections of surgical specimens (Table III). In nonsecretors water soluble H antigen was largely confined to the deep portion of the pyloric mucosa and Brunners glands. The irregular distribution of H antigen described above for secretors also was found in Brunners glands of nonsecretors. The gen in Distribution Secretors of and Water in Soluble Nonsecretors. A AntiThe
J)EMONSTHATION
OF
BLOOD
GROUP
(H)
ANTIGEN
IN
TISSUE
595
6
showed yellow-green iluoresceusce withi urltraviolet are white. lhie snagnification for :tll Figitres 2 throurghi Ii are frossi ami A secretor (.&-6545). Fm G . 1 . St ( )stiachr fr )t11 :n ii ( ) se(ret ( )r exposed t ) nut i -H -F . The surface as well as t lie deeper )rt i 5 II (If the glauids exhibit :t l)rilliant. yellow-greets fluorescemice. FIG. 2. Subtnaxill:try glanid exposed to assti-H-F. Note thre iuitense fluorescence of thie sisucin in most acini . Soune aciuii (10 n( )t finn )resce (arrow) Fm G. 3. Bruuiner s glands after exposumre to ant i-H-F. Massy of t lie aci ni sh )W :nn i sit ense yellow -greemi flumorescence. Souire show a few flumorescent granusles whsile (It hiers are dark. FIG. 4. Brususners glasids after exposure to anti-A-F. A clurster of acini rich isr A antigen is seen. Ihse dark acisri fluoresced after exposure to atrt i-H-F. Fs. 5. Brunners glands after exposure to anti-A-F. Aur occasiotial cell wit irisi an acimius is rich mi A antigen. The dark areas fluroresced after exposuire to atiti-A-F. Fuo. 6. t ssst:(iuie(l suhssiaxillary gland as seems with ultraviolet.. Fhie mucous aciuti are (lark its contrast (.1 t hie aut ofluorescesice sf t lie st rouna and serous acini.
figures is
Xi45.
The
areas
that
seeur
for
F!
aurt igetr
austigen
III)
(ontaine(I
( lasids
H
acini
antigen
were
rich
tnatiy
its
A asit igeui,
to lIe
others
com-
A
tire glands,
and
appeared
(list
ribnmt (If
A glatsds
devoid H of
4). The
glands A was
distrijust antigen.
glands glands
tioussecretors of secretors
salivary
amid
sumbusiumcosal
Brunssers
in A antigen
hiad no demosr-
596
SIDNEY
P.
KENT
1+ a ;o
0
++
C)
++
++
C)
C)
a
a a1 ++ ++ +1 ++ II -a
a
C)
1)
)aI
++
-.
+1
II
II
-a
.0
C)
J
-C)
0 0
C)
I I
-1--I-
a
1.
._
0
01-ll
++
++
++
++
II
-C)
C)
-H
++
tl
++
++
++
++
++
++
++
++
a
a
I)
:
.c
a
0
CI)
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I) C)
++
++
+1
++
I I
++
I I
C)
a a
1-
a a -
5)
(li
Eo
______
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++
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.0
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CI)
++
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++
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a
.
cI
++
++
++
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++
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a a
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++++
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C)
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a
a
-
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0
bIll
++
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C)
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C)
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C1
DEMONSTRATION
OF
BLOOD
GROUP
(H)
ANTIGEN
IN
TISSUE
597
on intensity Water of
strable
some antigen
H antigen.
demonstrable were rich
Those
H antigen. in H antigen.
poor
in A antigen
Those For devoid example, exposed
had
of A when to 5-10%
Effect Soltuble
of A
Fixatiour and H
and Antigen.
Autolysis The
a section anti-H-F
yellow-greesi
of duodenum 90-95% of
fluorescence. either did acini
the
not was
specific Zenkers
anti-H-F
a specific
all or con-
in the 3).
to
same
tissue
fixed in
seen in tissues fixed in Hellys, fluid followed by exposure to was miot as bright as that seen fixed in neutral buffered formaHellys or Zenkers fluid must
of thse tamed
When anti-A-F
lims. Tissue
only
such
a few
a section
fluorescent the
gramsules
subsequently
(Figure
exposed
with iodine potassiuns iodide to remove mercury salts; otherwise the antibody
on the fixed frozen A sections. The intensity of
serums
acini previously nonreactive yellow-green fluorescence, sugof A antigen. If such a section initially exposed to anti-A-F
acini whereas showed a brilliant yellow-
specific
buffered noted was Water a more
fluorescence
formalin in fresh distinct. soluble
of mucous
tissue was and antigens sections and H
acini
equal the
in
neurt.ral
to that
90-95%
exposure
of the
of
acini
such
did
Subsequent
strable
section to anti-H-F resulted in a yellow-green flurorescence of the acini that were previously nonreactive. In the teus patients with type A blood studied thie percentage of acini in Brunners glands reacting with anti-A-F varied from 5 to 50%. The acinii containing A asitigen tended to be in clusters. However, within a group of acini rich in A antigets an occasional isolated cell rich in H antigen was seen (Figure 4). Also in groups of acini rich in H antigess ann occasional cell with A antigen was sometimes noted (Figure 5). In the stonsach the areas that stained with antiA-F also stained with anti-H-F. After exposure to anti-A-F souse of the epithelial cells ins the stomachs did not show specific fluorescence, however I could not be sure that these cells contained H antigen. Controls. exposed
tissues allowed to autolyze up to 48 hours at 22#{176}C provided they were then fixed in neutral buffered formalin and embedded in paraffin. However, progressive lysis of the cells was associated with diffusion of the antigens and some loss of the antigens in the 24 and 48 hour specimens. Fresh frozen sections of the autolyzed tissue were much less satisfactory. By 8 hiours some loss of antigen was evident ansd in the 24 and 48 hour samples these losses were striking. This was due at least in part to fragmentation and loss of the outer portion of the niucosa in the process of sectioning and staining.
DISCUSSION
in
The extract
agglutination of Ulex
europeus
of 0,
by question
the
that were not to either labelled solution showed considautofluoresceusce when examined with ultraThis autofluorescence which was bluesilver and occasionally orange was particevident in the stronsa and blood vessels.
The sectiosss of
tissue
tract is lower than that of the H titer. Further, the A and B titer of the extract used was so low that A and B ant.igens would not be expected to be fact
antigen
were
(Figure A or or anti-H
6).
by mucous
the
by a
employed. to contain
with anti-A-F
I he A
to contain
H anti-
some
(evidenced
serum) extract
that
not
Ulex
react
is not
with
the
seed
fluorescein
further A
labelled
suggests antigen
europeus
extract
demonstrating
of it.. Absorption
in
gens
tissue.
the
reacting
body solution with appropriate antigen or red blood cells comsipletely ehimimsated the reaction of antiserum with the tissue antigens. Incubation of the tissue known to contaimi both A and H antigen with unlabelled anti-A followed by exposure to
labelled specific anti-H fluorescence. resulted in Exposure a slight of similar decrease sections in
europeuc
be ruled the to
labelled The
in nonsecretors reaction
for the with titer
seems
unlikely of
solution followed by exposure serum did not result in any of staining by the anti-A-F
the
That
extract
is, type the
of the
exto
Le
A cells
is too
low
598 produce (Table I). group etc., iaraffin a positive reaction in tissue
SIDNEY
P.
KENT
sections
additional
polysaccharide
evidence
antigens
of
stability
despite
of
exposure
many
to
The alcohol soluble blood in blood vessels, erythrocytes, removed in the process of
factor present apparently is embedding: group factors are preserved. H antigen de-
formalin use of
freedom
embedding. permits
and surgical
The greater
mate-
autopsy
however, the water soluble blood associated with epithelial mucins The distribution of water soluble
scribed
Further,
sections
the
is
localization
sharper
of in fixed
the
antigen preparations
in
in frozen
sections. REFERENCES
tribution
exposed
in the present report is similar to the disof this antigen noted by Szulman who
frozen
sections
with
1. BOORMAN,
dnLction
K. E., to Blood
AND
DODD,
Group
B. E. Serology,
An
Intro-
2nd
Little, a
Brown
C.
& Co.,
Fundamentals
Boston,
of
1961.
Immunology.
some not
gland
in other
2. BoYD, W. Interscience
1956.
Publishers,
Inc., M.
Acta
New
distribution
and
tolysis,
is l)robably
or enshedding
not
of auobserved tissues
only
in
l)araffin
ensbedded
3. CAzAL, P., AND LALAURIE, quelques phytoagglutinines groupes sanguins ABO. 73-80, 1952. 4. CEPELLINI, R. Physiological blood factors. In: Ciba
posium on Human Relation to the Naples, Little, 263, 1959. GLYNN, L. E., HoLBoRow,
Recherches specifiques
Haemat.
genetics
of human
specimens in formalin
5.
Foundation SymBiochemical Genetics in Problem of Gene Action, Brown, Boston, pp. 242E.
)rior to paraffin embedding. This distribution of A and H antigen necessitates a re-evaluation of the hypothesis conicerning the origin of A and
B substance fornsuilated
J.,
AND JOHNSON,
by (18).
serves substances
(4),
Mor-
to this substrate
by a
U. D. The distribution of blood group substances in human gastric and duodenal mucosa. Lancet 2: 1083-1088, 1957. 6. HoLBoRow, E. J., BROWN, P. C., GLYNN, L. E., HAWES, M. D., GRESHAM, U. A.,
OBRIEN, T. distribution human tissues.
F.,
AND
CooMBs,
R. Path.
R.
A. 41:
The
of
blood
Brit. J.
group
Exp.
A antigen
in
430-
is usually
of H correct
incomplete
specificity one would
thus
to
allowing
remain. expect If to
437,
7.
KABAT,
1960. E. A.
and New
M. H.,
Chemistry
Their Academic
hypothesis
find
the
A and
methods
H antigen
employed
ins the
in this
same
study
cells.
most
Yet,
of
by
the
Press, 8. KAPLAN,
acini
antigen
contain
and a
only
few
H
both
antigen,
A and H
others
antigen.
only
The
A 9.
H., AND DEANE, H. W. Localization of antigemi in tissue cells. III. Cellular distribution of pneumococcal polysaccharides types II and III in the mouse. J. Exp. Med. 91: 15-30, 1950. KENT, S. P. A study of mucins in tissue sec-
obsetved be explained
hypothesis
of A and
H antigen
could
the framework of the above at a given time synthesizes subsequently converts the before it again synthesizes three
and would
10.
Histochem.
Thus
ri(h,
the
A
types
H
of cells
represent
antigen
and
stages
A The
antigen
in
rich,
11.
S. P. Study of tissue mucins using the fluorescent antibody technique. II. The preparation and specificity of human submaxillary gland mucin antibody. J. Histochess. Cytochem. 11: 273-282, 1963. KENT, S. P. A study of mucisis in tissue sections by the fluorescent antibody technique. III. The specificity of antibody to salivary
gland nsucins and the effect of chemical alterations of mucins on the specificity of the antibody. Ann. New York Acad. Sci. 106:
pneuimococcal has
antibody previounsly
antibody embedded
paraffin reported
389-401,
LANDSTEINER,
1963.
K.,
AND LEVINE,
P.
On
group
specific
J.
13.
substances
in human
spermatozoa.
R. W. StainHistochemical,
The
tlse fixed
to demonstrate
H antigen
in sections
MCMANLJS,
12: 415-418, 1926. J. F. A. AND MOWRY, ing Methods: Histologic and Hoeber, New York, 1960.
Imnnunol.
DEMONSTRATION
OF
BLOOD
GROUP
(H)
ANTIGEN
IN
TISSUE
599
acid-Schiff Sci.
106:
14.
MoRGAN,
pects
immunochemical
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