THE

DEMONSTRATION 0 (H) ANTIGEN LABELLED

AND

DISTRIBUTION SECTIONS

OF

WATER USING

SOLUBLE A FLUORESCEIN SEED*t

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

2, 1964 the more antigens agglutination recently, the By have

been

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

(5, 6, 12, 17). in normal tissues.

paraffin
available

embedded
anti-A

sections.
serum

Commercially was labelled with

to be widely

How-

fluorescein A antigen
tissues.

neutral to that fixation; lin fixed

sections. stration

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

shown that tain a protein

with

blood

seed congroup 0 (H)

sections of formathat of fresh frozen

prevent A or the demon-

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

paraffin embedded with some diffusion

was in

associated formalin

of the

antigens. for patients

was were studied. widely body

The distribution of H antigen fixed paraffin embedded tissue section

with In type secret(Irs A, B blood amid

The tissues found

etc. and

ABO (H) blood in two forms: in red

the

the

antigens occur in alcohol soluble form endotheliurn,

soluble form water

blood
more

cells,
abundant

vascular

blood
substances

also mucins antigens

group

distributed whereas
confined to

in the in nonsecretors
the deep

epithehial the
portion Brunnets

of the are

largely mucosa The nlisa gland, no no A

which

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

of the pyloric glands. within

of fresh fresh as the were ethanol not frozen water

tissue sections soluble the

were the

used of

(5, 6, 17). soluble blood

By as

alcohol

tribution

antigen

forms Pretreatnnent alcohol the has group

group antigens of

as Brunners glands, was is, some acini contained others a few

has

not homogeneous. A antigen but H antigen but A and

distribution was

identified. removes interfere blood A antigen paraffin fixed greater with

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 fixed use permit of

soluble the

not

been

in formahin would topsy such

of

emnbedded freedomis in the

(11). tissues auof H of the also

INTRODUCTION

paraffin snaterial,

embedded

The

distribution

of

blood stus(lie(l

group using

antigens the mixed

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

for demonstrating investigated. and and A H used fixatives of

and

A antigens

commonly Meeting Pathology, of demonstration

evaluated.

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

OCellsLe OCellsLeb A1CellsLe

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
I-

II
I I

1+

1+

C) a

iLiiLi
.

+++

++

+++I++I

C)

C)

a
a
C)

III

1+

I+IIII

s
0

&)

I +

++

+++

-C)
.

&

a +++ +++ ++ ++ ++ I + ++++ I +++

a

C)

a
C)

I
.

!
.
CI)

a C)

1+

1+

I++II+

a
a

a
CI)

++

+++II

a
. a

a
+
.0

+++
I4

++ I

++ 1+

+++I++++
.

C)

III

1+111

I
+

o
C)
a
a

a iii II 1+ 1+11
a
C)

a
0

+++ +++

++ 1+ ++

a ++II+
C)

E- .
H H

+++I++++
.

o
a ..a

:
. C)

a +++ I + ++ ++ I
,-

a
C)

a -C). SC)

H
.

H H H
.

-5 +++

+
I I

++
+

+++I

C)

.-.

+++

++I

1+
-

C)

.oa
a
0

+++

1+

++

++l

I++I
#{176}.

ofi
C) C)

_C)

>)

a1.

a
C)% .2C)

5z
a
.
. . . .

a
. .
#{149} . #{149} . . . . . . .

: :
.

..

.a

.a
.

: :
.

a::

:
.

a a

.a
aa
C)

: aa...S)...aC)-C)-S
.

:
. .

s.

.a

.#{176}+ .

. .

a a
a ri *
rJ

:-ua

. ;3 -u

I
a

cI:
---

_
a
0

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

a decrease in the was noted. fluorescence was estiantigen were

=

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

and fixed in neuitral Hellys, and Zenkers fresh frozen sections

stoniachi was incubated

buffered fornialin, Bouins, fluid for comuparison with (13). A portion of each
at. 22#{176}C amid samples 48 hours after paraffin of tisthe

sues taken tut I , 2, 4, 8, 24, and specimnen was resected. Frozen

tissue embedded Fluoresceuu Tissue sect iosis as well as sections were
t

sections
fixed

of unfixed

of formalin studied. Method to

tissues

Antibody were exposed

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

pattern of distribution and nonsecretors was

of A antigen essentially the

in secretors same as that

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

(Table also jour of

III)
(ontaine(I

( lasids
H

that tue the

omuse had pletely butious the That

suiucous less of reverse is, the

acini
antigen

were

rich
tnatiy

its

A asit igeui,
to lIe

others
com-

cositained However, Brumminers

A
tire glands,

amitigen. iusto iui trachea irregular.

and

appeared

(list

ribnmt (If

A glatsds

amitigeni assd its tue was

devoid H of

of A atitigen asstigeui the :rcini rich its the pattersr

(Figure sanse sects with

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)

H
I) C)

++

++

+1

++

I I

++

I I

C)

a a

1-

a a -

5)

(li

Eo

______
1

++

++

++

++

+1

++

a
.0

0
CI)

++

++

+1

++

I I

LI

0 C)

.H
a

.
cI

++

++

++

++

++

I I

a
a a

a a

n-I

___ .
L)

++

+1

++++

a
C)

a ++ ++ ++ ++ ++ I I
a

a
-

.
0
bIll

++

++

++

++

++

I I

a
C)

LI
C)

CI)

E-ci

++

++

++

++

++

I I

._0
.-.

I0

.ll

a
aC) a

CI)

ci:

c,

c,

a
XCI) 0
. -

C)

Ci

c#{176}o

I,-

I,

.C)

S
_________ _________________________________

a
LI aO

z
0 Cs -i C C
-

aa aaa II-a
*+-++

CI)
I .

Ctd

CI

CI

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

(A-6545) was acini showed

Approximately fluoresce at

specific Zenkers
anti-H-F

fluorescence and Bouins

or anti-A-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

be treated the excess

precipitates

with iodine potassiuns iodide to remove mercury salts; otherwise the antibody
on the fixed frozen A sections. The intensity of

serums

showed a brilliant gesting the presence of duodenum was

serum green not 5-10% fluorescence (Figurre 4). of the

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

localization were demon-

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,

A and seed raises

B cells the of It titer

by question

the

erable violet. gray, ularly

The Sections gen solution ing the plete

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

of the specificity with H antigen noted that the

of the reaction in tissue sections. anti-A and anti-B

the extract should be of the ex-

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

mucin of when labelled specific absensce

containing tissue exposed followed solution yellow-green known to by

areas unlabelled exposunre showed

were

dark anti-A to the

(Figure A or or anti-H

6).

demonstrable that did of

the

by mucous

the
by a

method acini known

reaction

employed. to contain
with anti-A-F

I he A

to contain

H anti-

some
(evidenced

corresponddecrease or labelled a comantiin

serum) extract
that

not
Ulex

react
is not

with

the
seed

fluorescein
further A

labelled
suggests antigen

a striking fluorescence of the

europeus

extract

demonstrating

of it.. Absorption

in
gens

tissue.

Couild seed be as the gland Lea

also

the
reacting

labelled with The

extract Le reaction extract (lid

of or Leb of Lea not with

sanse A and

Ulex antican stain known Le reason

B

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

in tissue out salivary contain

sections? mucins antigen.

be ruled the to

labelled The

in nonsecretors reaction
for the with titer

antigen that antigens tract with the

seems

unlikely of

to unlabelled to labelled detectable serum.

anti-H anti-A decrease

solution followed by exposure serum did not result in any of staining by the anti-A-F

reaction is unlikely. Le and

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

the paraffin material

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

fixation and fixed embedded

in studying

embedding. permits
and surgical

The greater
mate-

autopsy

however, the water soluble blood associated with epithelial mucins The distribution of water soluble
scribed

na!. tissue than

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

Bombay The given same

viously

serum finding of and

to fluorescein a high anti-H in has an

as

labelled titer (17). acini acini been of of the preof A

1. BOORMAN,
dnLction

K. E., to Blood

AND

DODD,

Group

B. E. Serology,

An

Intro-

2nd

ed., York, sur des

8:

Little, a

Brown
C.

& Co.,
Fundamentals

Boston,
of

1961.
Immunology.

A antigen H antigen here

mosaic

some not

gland

in other

2. BoYD, W. Interscience
1956.

Publishers,

Inc., M.
Acta

New

gland as reported described. The H antigen

fixation,

distribution

and
tolysis,

is l)robably
or enshedding

not

artefact it was autopsy

of auobserved tissues

not but and

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

also in frozen sections of surgical surgical specimens promptly fixed

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

Cepellini According as the

originate

(4),

Mor-

gan (14, hypothesis

from process a this certain which

15) and Watkins H substance

A and B which degree is

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

Blood Group Substances; Immunochemistry.

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-

York, 1956. CooNs, A.

obsetved be explained
hypothesis

distmibuition within if a cell

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.

tions nique. bovine

J.
KENT,

using the fluorescent I. The preparation submaxillary gland

Cytochem.

antibody techand specificity of mucin antibody.

9: 491-497, 1961.

Histochem.

largely H antigen and H antigen to A asitigen H antigen.

H antigen

Thus
ri(h,

the
A

types
H

of cells
represent

observed, containing, transient mucin with fluoem(8,

formais
12.

antigen

and
stages

A The

antigen
in

rich,

11.

the synthesis densonstration labelled tissues ability

fluorescent paraffin

of A antigen. of cow submaxillary polysaccharides in fixed been A and

technique tissue

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:

and rescein bedded 9). by

lin

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

W. T. J. Sonse of the products

immunochemical

as17.

consbinations

group genies. In: Ciba on Human Biochemical the Problem of Gene

of the hunian blood Foundation Symposium Genetics in Relation to Action, Naples, Little,

Brown, 15.
MORGAN,

Boston,
W.

biochsemical blood-group 16.

Proc. 347, MowEy, Roy.

pp. 194-216, 1959. A contribution to human genetics: the chemical basis of specificity, (Croonian lectuire). Soc.; London, Series B 151: 308T. J.

18.

reaction. Ann. 423, 1963. SZULMAN, A. E. of the blood disclosed by H antigen and gens. J. Exp. WATKINS, W. M.

with New

tine York

periodic Acad.

402-

The histological group substances insnnunofluorescence.

its relatioss

distribution its mssan as II. The to A an(l B anti-

Med. 115: 977-995, 1962. Some genetical aspects of the

that
groups

1960. F!. W. The special value of methods color both acidic and vicinal hydroxyl
ins the hsistochsemisical

biosynthesis of hunssan blood group substances, its Biochemistry of Human Genetics, Ciba Foundation Symposium jointly with inlernational Union of Biological Sciences, (G.
E. W.

With
stain,

revised
thie use

(lirectionss
of alciaus

for

study of nnucins. tine colloidal iron

Wolstenholme
London, J Little, Brown

and

C. M.

blue

80X

and

their

editors), Boston,

& A Churchill and Co., 1959,

OConnon, Ltd., 217.