From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Cell
Antigens
I. A Study
of Blood
By
T
HERE
ARE
man.
cytes.
All
homogenous
by
of the
of the same
It is the
cell
specific
Nucleated
and
present
cells
cells
bright
of the
in mixed
population
system
ABO
to exist
bone
They
were
Visualization
populations
was
method,7
Coombs’
technic,#{176} and
direct
realized
the
same
whether
cells
fluorescent
presence
species.
or not
are
present
derived.
use
the
agglutination
been
receptors
the
of phase
receptors
applying
also
cells,3
and
et al.,5 using
showed
are
by
antigen
by
mixed
cells
identified
of
Coons’
have
specialized
adult
in
erythro-
antigen
further
adult
the
known
human
epidermal
Coombs
cultures
from
to determine
on
which
marrow
the
species-specific
method.
from
microscopy.”
agglutination
the
demonstrated
known
primitive
antigens
on
on dividing
cells of tissue
of the present
investigation
isoantigens
on dividing
of
group
described
human
leukocytes,2
human
of isologous
individuals.4
agglutination
antigen
purpose
YUNIS
blood
been
receptors
on Normoblasts
EDMOND
of
have
populations,
mixed
AND
SYSTEMS
antigen
Specialization
Antigens
YuNIs
isoantigens
platelets,’
secretions
cell
means
J.
JORGE
the
on human
tissues
and
Cell
Group
TWELVE
these
However,
found
some
and
on
principles
of:
method,3
antibody
contrast
normoblasts
direct
Jones’
minor
technic.
MATERIALS
Preparation
marrow
of
nucleated
(1-3
samples
volumnteers
and
cythemia
patients
and
30
and
75
in
60
minutes.
cell
ml.)
with
lymphocytosis.
of age
The
and
fresh
by
human
sternal
anemia,
individuals,
an erythrocyte
was
collected
marrow:
bone
aspiration
hypochromic
The
showed
marrow
of
obtained
affected
benign
years
suspension
were
from
from
blood
Versenate
bone
normal
loss,
both
sedimentation
in
Twenty-five
adult
human
secondary
sexes,
poly-
ranged
rate
greater
Disodium
(1
between
than
15 mm.
rng.
per
ml.
of bone
marrow),
tile particles
removed
and the remaining
material
allowed
to sediment
for 2 to 3 hours
in Wintrobe
hernatocrit
tubes
at 25 C. At the end of this period
the marrow
fat and remaining
bone marrow
particles
were
removed,
and the marrow
plasma
rich in
nucleated
cells was separated.
Only those
specimens
free of microscopic
agglutinates
and
containing
15 or more normoblasts
but not more than 15 contaminating
reticulocytes
per 100 nucleated
cells were used in the present
study.
rich in nucleated
cells was tile basic
material
of all tests.
All individuals
sified
according
Antisera
From
School,
to their
and
the
blood
blood
into
specific
groop
Department
type
of
Laboratory
A1,
A2,
B and
0
The
substances:
Medicine,
erythrocytes
Fresh
and/or
marrow
studied
plasma
were
anti-A,
University
anti-B,
of
and
anti-A1
Minnesota,
The
study
Division
has
of
antisera
Medical
Minn.
Minneapolis,
in part
by a training
grant
number
5TIGM-628
Sciences,
and in part by a grant from the American
Society,
Minnesota
Division,
Fluid
Fund.
Preliminary
communication
presented
at the IX Congress
of the
International
of Hematology,
September
9, 1962,
Mexico
City,
Mexico.
Submitted
Dec. 11, 1962; accepted
for publication
Mar.
1, 1963.
This
the
clas-
groups.
been
General
supported
Medical
from
Cancer
Society
53
BLOOD,
VOL.
22,
No.
1
(JuLY),
1963
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
54
YUNI5
and
the
H
specific
specific
substancesf
following
the
laboratory
after
substance
The
method
from
of
et
adult
of the blood
obtained
commercially0
( alex
used
Boyd
healthy
collection
were
lectins
al.9
donors.
and then
and
as
dolichos
)
were
and
AB
The
groups
0
The
serum
was
inactivated
were
A
prepared
sera
and
in
were
separated
by heating
the
AND
YTJNIS
B
our
group
laboratory
prepared
in
our
clot 24 hours
30 minutes
before
from
the
at 56 C. for
use.
Epidermal
patients
and
skins
used
cell
suspension:
also
from
were
obtained
12-15
cc.
A
saline
was
of
sterile
the
with
The
mixture
moved
from
of epidermal
further
washed
cent
cells
scraping
thus
and
by
pensions
were
then
minuites.
The
supernatant
resuspended
and
capillary
preparations
were
in
10
containing
and
at 35
C. After
free
out
a
in
1 ml.
of
mm.
was
removed
with
with
placed
and
cell
and
containing
with
antibiotics.
( 200
units/mI.)
kept
in a Petri
at 4 C.
Before
placed
in
then
All
stored
suspensions
jar
preserved
kept
was
a fine
of
dish
trypsinizaa Petri
of
isotonic
saline
pipette.
and
spun
a capillary
and
dish
the
individual
drops
cell
by
cell
sus-
1000
rpm
for
The
cells
were
drawn
off
of saline.
Only
suspensions
re-
sheets
broken
The
at
supernatant
was
small
and
pipette.
in a few
dermis
The
capillary
tubes
saline
the
policeman.
bore
with
resuspended
preponderance
incubation,
rubber
siliconized
isotonic
button
a
saline
surgical
were
a sterile
penicillin-streptomycin
specimens
were
saline
x 75
saline
once
specimens
in
it was
isotonic
hour
it
in
cell
The
one
scraping
drawing
washed
pipette.
in isotonic
adult
post-mortem.
epidermal
placed
used,
The
The
Individual
was
from
6 hours
solution.
obtained
placed
use.
) and
sterile
than
individuals.
skin
storage.
out
for
by
0
The
obtained
less
immediately
with
incubated
epidermis
B and
sg./ml.
trypsin
were
obtained
before
before
wrung
per
was
the
not
(2
was
specimens
Medawar.’#{176}
When
squares
a 0.5
A1,
specimen
gauze
specimen
covered
group
fungizone
the
skin
patients
trypsinized
by
saline.
to wash
between
tion,
isotonic
split
adult
and
described
solution
used
from
1 week
as
of
of
obtained
at 4 C. for less than
were
Ten
skin
were
2
with
a
those
chosen
for
study.
Preparation
were
of cell suspensions
made
of
washed
packed
isotonic
saline
mixtures
and
tures
human
and
population
tween
1:10,000
other
cells
of each
the
suspensions
each
cell
0(0),
for the minor population
method:
Two
per cent
suspensions
and 0 Rh negative
red cells by mixing
0.2 ml. of the thricetype with 9.8 ml. of EDTA
saline
( 1 mg. of EDTA
per ml. of
detector
In
marrow
minor
B
These
.
used.
0(B)
A1,
cells
)
for
were
0
group
were
cell
case
the
the
letter
of the
bone
It
(one
components
was
found
normoblast
marrow).
the
preparation
were
The
that
per
actual
of
mixture:
a safe
concentration
mixture
by
mixture,
steps
repeated
further
with
the
Preparation
who
had
inversion
of
the
flasks
mixtures
could
be made
by
2 per cent group
0 suspension.
and labeling
of antisera:
Anti-A
recently
delivered
ABO
erythroblastotic
for
at
least
0 erythrocytes
normoblasts
in
diluting
and
the
anti-B
infants.
3 minutes.
initial
From
mixture
sera
were
The
original
B
the
this
obtained
from
of
be-
and
populawere
of the
was
population
in twofold
titers
and
0( A),
of the
ranged
group
tion mixture
was determined
by direct
cell counts
under
phase
contrast.
The
mixtures
prepared
by mixing
0.1 ml. of the marrow
nucleated
cell rich plasma
with 0.1 ml.
suspension
of group
0
erythrocytes.
Complete
dispersion
of the
minor
population
achieved
mix-
A1,
mixtures
as:
the antigen
proportion
of the
population
population
group
these
indicates
to 50,000
10,000
the
Three
respectively
It is convenient
to identify
in parenthesis
in each case
(nornioblasts).
to 1:50,000
for
Population
minor
suspensions.
where
used
suspensions.
both
serial
mothers
anti-A
and anti-B
were
1:512
in saline
and 1:1,024
in Coombs.
These
sera
were
fractionated
by
DEAE
cellulose
chromatography
following
the method
of Levy and Sober.11
The
fraction
used was that containing
the agglutinating
antibody
(usually
the fourth
peak).
This was
evaporated
in a dialysis
bag to 1:10 the original
volume
and dialysed
five times
against
physiologic
saline
(250
ml. each).
#{176}Ortho Foundation,
Knickerbocker,
Raritan,
New
York,
J.
N.
N.
Y.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
OF
STUDY
The
the
BLOOD
GROUP
fractions
method
ANTIGENS
of anti-A
of
Riggs
as
and
ON
anti-B
modified
were
by
55
NORMOBLASTS
conjugated
Marshall
et
with
al.12
and
titer
of both labeled
antisera
was at least
1 :64.
Neutralized
were also prepared.#{176}
Optical apparatus:
A G.F.L.
Zeiss
microscope
with
bright
and a IIIZ-6 condenser
was
used.
The
condenser
contains
field annular
diaphragms
fitted
with
a dual
interchangeable
the illumination
can come alternately
from a tungsten
bulb
sure
mercury
vapor
lamp.
for fluorescence
used
A
BG
12
ultra-violet
pass
fluorescein
kept
isothiocyanate
frozen
at
labeled
and
light
and
anti-B
objectives
and
arranged
dark
so
a 200-watt
a 5,000A
final
and
contrast
source
or from
The
contrast
phase
by
C.
anti-A
phase
bright,
filter
-60
that
high
pres-
barrier
filter
were
method
of
microscopy.
METHODS
Direct
agglutination
The
method:
slide
test
for ABO
grouping
was
the
choice.
In general,
each
sample
of marrow
plasma
rich
in nucleated
cells
was
treated
with
anti-A
serum,
neutralized
antiA,*
anti-A1
serum,
Dolichos
biflorus,
Ulex
europeus,
neutralized
ulex,#{176}
anti-B serum, neutralized
anti-B,#{176}group 0 serum and group AB serum.
One 0.1 ml.
drop of marrow
plasma rich in nucleated
cells was placed
on a clean
glass
slide
and,
at a
short distance,
an equal
volume of antiserum.
The two were mixed
with
a wood
applicator
and the mixture
tipped
back
and
forth
on the slide
for 3 to 15 seconds.
A coverglass
was
then
placed
over
applied
on the
Kroening
wax
ence
of
and
as
part
of
and
with
examined
was
under
differential
cells
counted
removed
were
of
gentle
pressure
were
reactions
and
counts
by
preparations
Positive
erythrocytes
for
was
The
were
sealed
read
with
as
pres-
reticulocytes.
Each
one
the
cells
present
cells,
normoblasts
nucleated
grouped
into
white
of
the
free
erythrocytes.
In
one
stages
step
of
further,
we
development
Wright-Ciemsa
attempted
to
staining
the
by
method.
For
this,
and one drop of the specific
smear made
by drawing
this
end of a disposable
Pasteur
Wright-Giemsa
positive
method.
reactions
Mixed
exposed
(10
x 75
added
cells
(20-25
taken
original
cells
for
for
to avoid
rpm
were
completely
under
sisted
of
*The
H
and
equal
of
tile
of
and
marrow
normoblasts
positive
plasma
in
different
reactions
rich
in
with
the
nucleated
cells
were placed
at the edge of the slide,
mixed,
and a
along the slide with tile lateral
aspect
of the capillary
The smear
was then
air-dried
and stained
with
the
counts
of
1000
normoblasts
of both
controls
and
anti-B
cells
sera.
volumes
interspersed
centrifuged
at
resuspended
slide
drop
counts
control
obtained
Each
from
group
experiment
A1,
included
B
a
and
set
0
of
persons
six
tubes
suspension.
To three
of tile tubes
were
three,
0.1 ml. of anti-B.
The epidermal
and
incubated
at room
temperature
1 hour.
After
incubation,
the cells were
deposited
by centrifugation
at
2 minutes
and washed
three
times
with
isotonic
saline,
care having
been
loss of cells. Finally,
the suspensions
were
restored
approximately
to their
thoroughly
pensions
of
0.1 of epidermal
cell
serum,
and to tile other
the antisera
by agitation
containing
volume
were
differential
smears
Epidermal
and
ml. of anti-A
mixed
with
C.)
1000
make
performed.
anti-A
mm.)
0.1
were
antisera
mixture
pipette.
method:
to
one
Differential
were
agglutination
were
and
fluid
paper.
microscopy.
with
The
excess
of blotter
phase
examined
agglutinates.
any
a piece
agglutinated
reactions
and
preparation
normoblasts
positive
or
the
coverslide
by
a coverglass
and
neutralization
5 minutes
incubated
respective
erythrocytes.7
1
shaking.
erythrocytes
ml. of the
at 25 C. and
for
shaking.
examined
procedure
to
nucleated
rpm
gentle
and
normoblasts
substances
1000
of
by
cell
The
1 minute
One
under
was
carried
drop
were
containing
of this
by
corresponding
serum
found
to be completely
or
the
suspension
A
agglutinates
adding
added.
the
which
microscopy.
mixed
out
plasma
following
phase
forming
rich
tubes
0.2
uiex.
neutralized
ml.
cell
added
sus-
deposit
was
positive
with
The
The
composite
placed
was
on
reaction
con-
epidermal
cells.
of concentrated
serum
was
when
tested
a
A, B
used
with
after
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
56
YUNIS
population
Minor
0.2
ml.
were
of
the
added
and
to
left
pensions
to
room
in
all
avoid
were
added
mixture
The
to
suspension
was
excess
fluid
blotter
paper.
those
Direct
the
technic
of
0
supernatant
the
contents
period
the
pipettes
anti-A
in
to
anti-B
Following
each
separate
wells.
rpm
) at
presence
the
morphologic
reaction
each
well
were
last
wash
and
was
read
the
population
anti-B
minor
population
stained
A
for
rpm
10
for
wells
were
30
minutes.
for
transferred
to
buffered
then
test
(0.1
M
10
correlation
halo
of
green
of
the
fluorescence
a
end
of
x 75
mm.
phosphate
cells
an
C.
The
one
of
and
moist
the
chamber
the
incubation
)
with
buffer
under
around
up
Pasteur
with
pH
their original
volume.
covered
with 22 x 40
were
examined
with
phase
microscopy
for
the
a clear
25
Each
in
the
(
tubes
saline
set
with
anti-serum
placed
At
was
at
minutes.
labeled
A1,
anti-B,
treated
minutes
2
following
groups
test
was
of
cells.
labeled
blocking
any
differentiated
from
mixture
the
and
wells
anti-A,
in
of
surrounded
we
in
1
a piece
cell
),
corresponding
the
it,
with
nucleated
sera.
1000
at
over
other
for
process
deposit
drop
formation
labeled
incubated
the
cell
One
mixtures
with
rpm
The
the
cent.
a sensitized
or
labeled
C.
with
of
population
wells
and
All
immunochemical
as
suspensions
2500
coverglass
were
Minor
at
covergiass
the
preparations
with
25
a
on
( rosette
separate
mixed
times
placing
the
centrifugation
then
three
the
sustaken
one-half
cent
mixing.
cells were restored
to approximately
One drop of each suspension
was placed
on different
glass slides,
mm.
coverslips,
and
sealed
with
melted
paraffin.
All specimens
parallel
observations
of a given
field with
both
fluorescence
and
7.2).
shaking,
been
in
gentle
1 per
erythrocyte
serum
180
(
of
of
after
was
as
All
neutralized
and
washed
2 per
serum
cell
approximately
cell
resuspending
of
by
Evans.13
treated
removed
contents
and
by
the
having
centrifuged
by
of agglutinates
and
and
out
central
a volume
shaker
to
0
by
applied
read
technic:
anti-A
transferred
on a rotatory
mixed
care
each
AB
period,
interspersed
concentration
spread
was
Zuelzer
cells
containing
group
were
restored
were
pressure
scoring
were
was
sedimented
and
gentle
antibody
of
and
saline,
resuspended
carried
a
normoblast,
tubes:
volume
tubes
tubes
thoroughly
was
was
slide
the
Cohen,
separate
equal
a
central
labeled
two
test
anti-B,
incubation
B and
suspensions
produce
reaction
normoblasts
neutralized
to
by
During
a
all
isotonic
A,
were
deposit
Reading
on
fluorescent
and
composite
removed
cells.
in
group
cells
cell
times.
placed
containing
this
were
of
added
the
A positive
by detector
of
After
suspensions
the
saline
was
contents
times
volumes
The
which
physiologic
the
anti-A,
YUNIS
inversion.
containing
three
three
equal
tube.
the
three
of
5 minutes.
washed
Finally,
and
included
volumes
and
for
and
each
repeated
sufficient
in
cells.
following
was
respectively,
were
slinking
tubes
minute
tube
of
experiment
Equal
temperature
volume
by
Each
mixture.
tubes
loss
original
B
each
at
their
technic:
population
AND
the
study.
cell
A
positive
membrane.
RESULTS
Direct
in
agglutination
nucleated
Five
method:
cells
group
were
A1, two
group
tralized
antisera
actions
example
and
a typical
as part
AB
group
They
0
human
obviously
erythrocytes
tive
reactions
and
not
for the
and
Table
cells
and
serum.
that
counted)
specific
white
cells.
three
from
the
results
cells.
corresponding
Furthermore,
group
as
B. The
rich
follows:
nucleated
to their
ABO
blood
group
the controls
with
the neu-
Table
of normoblasts,
the
plasmas
grouped
1 summarizes
give
these
agglutination
exre-
of the same
ABO
blood
group.
An
is shown
in figure
1, where
group
or as free
for
marrow
were
normoblasts
reticulocytes
2 shows
of agglutinates
are
of 25 bone
classified
according
specific
antisera
and
quite
agglutinate.
nucleated
present
with
out
study.
similar
to those
of the erythrocytes
of a positive
agglutination
reaction
A1 normoblasts,
(1000
and
shows
for
A2, five
cell rich plasma
from
subjects
were
treated
with
different
periments
Fifteen
suitable
it shows
a group
A1 individual
of
differential
the
white
cells
and
It demonstrates
normoblasts
that
the
erythrocytes
that
and
proportion
form
counts
the
posi-
erythrocytes
of normo-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
STUDY
OF
Table
BLOOD
GROUP
ANTIGENS
ON
57
NORMOBLASTS
1.-Qualitative
Demonstration
of A, B and H Receptors
on Human
Normoblasts
(Positive
Reactions
by Phas2
Microscopy
as Presence
of Agglutination
of Normoblasts
with
Reticulocytes
and
Erythrocytes)
Normoblasts
from
Subjects
of
A1
Antisera
5/5
(+)
0/2
(+)
0/2
(-)
(-)
0/5
5/5
0/5
H)
0/5
(-)
2/2
(+)
5/5
(+)
0/3
(-)
0/5
(-)
0/2
(-)
0/5
(-)
0/3
(-)
0/5
(-)
0/2
(-)
0/2
(-)
3/3
(+)
0/5
(-)
0/2
(-)
0/2
0/3
(-)
5/5
(+)
5/5
(+)
0/5
3/3
(+)
0/5
(-)
0/5
(-)
0/5
(-)
(-)
(-)
0/5
(-)
to
the
proportion
2/2
(+)
0/5
with
A-GSS
0/5
(-)
0/2
(-)
0/5
Anti-A1
(absorbed
anti-A
serum)
(anti
Ulex
neutralized
with
H substance
Anti-B
Anti-B
neutralized
Group
0
Group
AB
with
serum
positive/number
blasts
to erythrocytes
thcse
cells
glutinates
blasts,
seen
show
(fig.
2).
typical
The
of
seen
in the
in the
control
agglutinates
material.
is similar
WTrightGiemsa
basophilic
normoblasts,
occasionally
one
results
of differential
experiments
#{176}
are
0/3
(-)
(-)
0/3
(-)
0/3
(-)
0/3
samples.
pronormoblasts,
orthochromatic
glutinates;
two
B-GSS
serum
#{176}Number
B
biflorus
(+
neutralized
Ulex
Group
(-)
(-)
(-)
(-)
)
5/5
Anti-A
Anti-A
Dolichos
Blood
0
can
and
see
shown
Fig. 1.-Specific
positive
agglutination
cytes and erythrocytes.
Phase
contrast
normoblasts,
erythrocytes
mitotic
counts
in
stains
of different
table
reaction
microscopy.
3. Similar
of group
X500.
of positive
polychromatic
forming
normoblasts
stages
results
the
agnormo-
part
in
of
of
the
ag-
agglutinates
of normoblasts
were
obtained
A1 normoblasts,
reticuloPrint
=
2.000X.
on
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
58
YUNIS
c10
0
OO
z
‘.
a
1..
a
0
..
C)01
0
5
.
o
Z
-
00C’l.-
0
.9!?
o
.i
.
.
1I)1(C1)
..
I.
010000
C)
-
.l
Z
,.
cioov-ci
-‘
.l
a
#{149}.
00
01
C’)
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t-
01
C)c’)
0
0,,
.
a
O
C
I-
,.0
a
.s
.4l
“
a
a,....
,
AND
YUNIS
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
OF
STUDY
BLOOD
GROUP
Fig. 2.-Wright-Giemsa
A1 normobiasts
and
normoblasts
and
Print
=
with
group
also
two
the
in the
Mixed
B and
presence
that
present
group
of the
the
relative
control
experiments
cells
obtained
corresponding
Table
0
normoblasts.
A, B and
of
is kept
ABO
group
of group
three
pro-
Xl
experiments
on
different
closely
human
kinds
in the
antigens.
3.-Differential
Count
agglutination
A1 and
In
Group
our
.250.
further
normoblasts.
of
counts
Free
polychromatic
Free
orthochromatic
1.3
8.8
normoblasts
normoblasts
Clumped
pronormoblasts
Clumped
basophilic
normoblasts
(per
Controls
+ neutralized
Casel
pronormoblasts
hasophilic
normoblasts
reaction:
B have
experiments,
of Normoblasts
(Wright-Giemsa
A1 ceHs
Free
These
H receptors
proportion
cell
from
Counted)
Free
reaction
including
microscopy.
normoblasts
of the
normo-
in agglutinates.
nornioblas’ts-ejyidermal
dermal
the
59
NOBMOBLASTS
stain
of a specific
positive
agglutination
erythrocytes.
All stages
of normoblasts
dividing
normoblasts
are present.
Bright
A2, group
shows
counted
blasts
ON
1 .250X.
demonstrate
It
ANTIGENS
been
Sensitized
shown
a positive
cent
Stain)
in 1000
mixed
ag-
Normoblasts
Positive
anti-A
Case2
epi-
to contain
A1 cells
Casel
Agglutination
+ anti-A
Case2
1.6
0.0
0.0
8.2
0.1
0.4
0.0
1.5
0.5
0.9
49.8
54.1
40.1
36.1
0
0
0.8
0
0
5.9
13.0
1.4
Clumped
polychromatic
normobiasts
0
0
48.2
47.8
Clumped
orthochromatic
normoblasts
0
0
44.1
35.4
0
0
1.8
3.2
Clumped
WBC
“contaminants”
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
YUNIS
60
Fig.
calls’
3.-Mixed
blasts
and
X500.
Print
epidermal
tion
skin
agglutination
of
group
from
a group
of an
epidermal
Normoblasts
of
obtained
reaction
casionally
from
B
consists
(fig.
some
4 depicts
3).
mitotic
B donor.
Central
Phase
specifiby
surrounded
contrast
normo-
microscopy.
the
normoblasts,
results
cell
all
were
of the
mixed
With
this
surrounded
stages
seen
of
in
by
normoblasts
development,
the
and
mixed
oc-
agglutinates.
normoblast-epidermal
cell
agglutina-
reaction.
Minor
population
method:
ence
of a normoblast
erythrocytes
are seen
summarized
Table
forming
around
in table
5.
of
the
4.-Results
the
it
Mixed
(fig.
technic
center
4).
of
The
a positive
Epidermal
blood
group
Normobkzst-Epiderrnal
Cell
+0
anti-A
-
anti-A
-
anti-B
-
B
anti-B
-
0
anti-B
-
by
normoblasts
pres-
Reaction
Cell Suap
of donor
ension)
-
anti-A
mixed
is the
Agglutination
C ella (Nucleated
Blood
group
A
Sensitization
A1
B
0
A1
#{176}A
positive
surrounded
Cells
of donor
reaction
a rosette
in which
the detector
results
of these
experiments
are
Detector
ABO
reaction.
donor
YUNIS
2.000X.
=
erythrocytes
Table
cell
erythrocytes
glutination
and
cell
normoblast-epidermal
sensitized
AND
normoblast-epidermal
cell
and
(see
erythrocytes
agglutination
figure
3).
B
0
-
-
-
-
-
consists
of
epidermal
cells
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
OF
STUDY
Fig.
with
trast
BLOOD
GROUP
4.-Minor
population
in mixed
anti-B
microscopy.
Direct
halo
X500.
fluorescent
cells
of
seen
the
bone
The
results
of
population
method
that
normoblasts
responding
antigen
Table
5.-Results
technic:
A positive
cell
membrane.
by
the
nucleus
Figure
mixture
with
experiments
distinct
cytoplasm
of
a positive
phase
is shown
was
was
and
in table
direct
fluorescence
from
group
A and
group
receptors.
cell
antibody
B individuals
of Detection
of A and B Isoantigen
Minor
Population
Mixture
Method
0
(
)
denotes
nucleated
fA
minor
most
on
the
non-
nucleated
one
normoThe
agglutination,
minor
technic
demonstrate
possess
the
cor-
Receptors
by
the
Mixture
0(B)
0
anti-A
+1
-
-
anti-B
-
+
-
-
-
group
A1 or B
as a clear
from
microscope.
0(A1)
Sensitization
A1
B
coIl-
6.
normoblast-epidermal
and
Cells
group
Phase
read
reaction
fluorescent
Population
Detector
ABO blood
sensitized
ervthrocvtes.
reaction
This
and
5 shows
observation
mixed
population
B detector
the
shown
the
(B)
group
2.000X.
=
marrow.
of these
method.
with
antibody
in parallel
summary
Print
61
NORMOBLASTS
mixture
around
fluorescence
blast
ON
agglutination
of fluorescence
specific
ANTIGENS
AB serum
population
of
the
mixture
composed
of
suspension
-
of
bone
marrow
cells.
positive
of a central
reaction
normoblast
consists
surrounded
of
the
by
presence
erythrocytes
of
typical
(rosette
spherical
formation).
agglutinates
See
made
figure
4.
up
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62
YUNIS
5.-(Lcft)
The same
Fig.
(Right)
by
seen
Labeled
minor
normoblast
fluorescent
microscopy.
the
labeled
Both
X500.
DiscussIoN
The
antigens
on the
However,
on human
sues
of all
erythrocytes
isologous
called
individuals4
“secretors.”
only
found
on the erythrocytes
on platelets
and
have
found
in
been
the
secretions
The
findings
described
isoantigen
receptors
on
Table
_______
groups
6.-Demonstration
Normoblasts
in
antigens
been
found
substances
of man
of the
the
with
leukocytes
of some
have
other
the
and
of
blood
serum
serum
phenotypes
in this paper
demonstrate
human
normoblasts.
By
of the
the
B Isoantigen
Antibody
Fluorescence
of the
membrane
-
-
-
of
the
normoblasts
0
-
-
consists
donor
-
serum
reaction
Normoblasts
+
serum
#{176}A
positive
on Human
-
anti-B
anti-B
system.
A, B and H
and
experi-
-
anti-A
anti-A
and Tja
which
+0
labeled
labeled
inhave
B
labeled
labeled
group.
systems
Lewis
of
group
described
isologous
Receptors
Technic
Neutralized
Blocking,
the
the group
procedures
Neutralized
Blocking,
as
found
to exist
as in most
tis-
group
A1
anti-A
(A1)
blood
exception
of the MN
the Lewis
substances
Blood
anti-B
been
have also been
cells3
as well
Specific
Labeled
0
particular
secretions
of the
of Group
A and Group
by Direct
Fluorescent
Labeled
microsopv.
mixture
2.000X.
=
individuals
and
The
Phase
YUNIS
CONCLUSIONS
blood
isologous
(A1).
population
Prints
AND
different
0
minor
A and B receptors
of the ABO system
platelets,1
leukocytes,2
and epidermal
of the
dividuals
the
of the
mixture
population
of
AND
-
of the
(see
figure
presence
5).
of specific
green
fluorescence
around
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
STUDY
OF
ments
BLOOD
GROUP
described,
into
group
A2, B and
exactly
marrow
mental
to
ABO
conditions
to the
reticulocytes
or
receptors
are
The
relative
polychromatic
and
direct
agglutination
cells
present
the
others”2
and
from
of the
tions
described
of antigen
avid
on
antigen
in
sites
we
series
this
cells.
relatively
pure
number
failed
to
bone
This
In
white
of red
cell
cell
in
have
been
can
B or
stages
H
of
de-
normoblasts,
the
those
on
the
to
they
compete
can
the
and
sufficient
platelets
and
to
condi-
lesser
number
with
only
suspensions,
obtained
experimental
related
when
by
leukocytes
receptors
under
be
demonstrated
adult
B antigens
platelet
A,
all
basophilic
is probably
or
avail-
as compared
present
in positive
agglutinates
the relative
percentages
of
marrow
elements
in the
normoblasts
and
show
A1 and
the
experi-
samples.
elements
fact,
which
the
Furthermore,
platelets
of the
from
under
normoblasts
antisera
normoblasts
difference
of the
receptors
on such
donor
conspicuous
control
on
specific
of the
that
pronormoblasts,
the
us
paper.
available
erythrocytic
small
in
B
the
reveal
normoblasts
are similar
to
)
with
group
erythrocytes.
of
by
blood,
myeloid
of
surface
mature
cells
and
corroborated
peripheral
cells
the
numbers
as free
A1
groups
corresponding
orthrochromatic
method
(
Although
the
the
ABO
is no
on
to
on
normohiasts
The
presented
there
receptors
same
data
described,
of isoantigen
present
blood
The
ability
the
normoblasts.
the
63
NORMOBLASTS
to type
0
is obtained.
velopment.
ON
it is possible
A1,
corresponds
bone
ANTIGENS
be
the
give
the
more
demonstrated
presence
of
a false
a
negative
reaction.
The
models
portant
described
information
antigens
on
Coombs’
mixed
the
“pure”
detection
by
paper
studies.
suspensions
of
and
currently
leukocytes
the
antibody
been
with
imof iso-
precursors
population
in immune
also
us
demonstration
their
minor
fluorescent
have
the
and
the
normoblasts
normoblasts
providing
include
and
coating
methods
on
are
These
method
of antibodies
antigens
two
this
agglutination
agglutination
group
in
in other
by
the
technic,
hemolytic
technics.
and
anemias
Other
demonstrated
by
blood
the
latter
technics.14
The
direct
However,
cells
agglutination
with
in the
will
elements
be
shown
that
problem
maturation
are
table
2).
not
to the
known
and
to be
of isoantigens
normoblasts
pearance
of isoantigens
of
lacking
ABO
suggest
cell
useful
of the
a small
that
methods.
of
white
is mainly
due
on myeloid
similar
numbers
and
four
number
this
of isoantigens
normoblasts
on
system
that
on the
believe
of
erythrocytes
to
cells.
myeloid
showing
leukocytes.
of appearance
of cells
of the
dividing
We
find
publication,”
relationship
specialization
most
to
presence
in agglutinates
of the
and
common
in a subsequent
present
are
The
and
simplest
it is
(see
trapping”
antigens
ence
is the
method
agglutinates
“mechanical
As
this
of antigens
is interesting.
as early
maturation
Our
as the
on
findings
cells
and
of the
pronormoblasts
is independent
of
the
pres-
and
on
the
ap-
cell.
SUMMARY
This
of the
article
describes
presence
of
the
specific
application
isoantigens
of
on
different
normoblasts.
four
models
to the study
The
findings
demon-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
64
YUNIS
strate
be
the
A, B and
shown
ment
appear
H receptors
on
on the
surface
to be present
and on mitotic
early
in cell
normoblasts.
formation
human
Iste
communication
studio
del
statationes
blastos
human.
etiam
de
de
demonstra
superficie
possibile
del
mitotic.
appare
maturation
le
stadios
Iste
constatationes
in
le
suggest
of cell
that cell
maturation.
isoantigens
quatro
differente
super
omne
precocemente
can
of develop-
receptores
demonstrar
in
receptors
stages
de
isoantigenos
de
YUNIS
INTERLLNGUA
application
presentia
normoblastos
de normoblastos
cellula
le
specific
le
Il es
de
IN
describe
presentia
These
in all
These
findings
independently
and
SuxIMARI0
al
normoblasts.
of normoblasts
AND
A,
B,
e
presentia
del
modellos
normoblastos.
Le
H
de
super
iste
receptores
disveloppamento
suggere
formation
del
con-
normoal
de
que
istos
e
le isoantigenos
cellulas
e non
depende
cellular.
ACKNOWLEDGMENT
Tile
Both
authors
she and
material.
parts
are
Dr.
The
grateful
Victor
technical
of the
work
to Dr.
Gilbertsen
Dorothy
Sundberg
for her advice
gave
valuable
help
in obtaining
of Miss
Mary
Jane Kiun and Miss
with thanks.
assistance
is acknowledged
and encouragement.
the bone
marrow
Patricia
Hanauer
in
REFERENCES
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erythrocyte-platelet
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STUDY
13.
OF
BLOOD
Cohen,
F.,
M. M.:
antigens
by the
Blood
GROUP
Zuelzer,
ANTIGENS
W.
W.,
ON
and
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of
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Laboratory,
of
Instructor
and
Director
Department
of
Laboratory
Minnesota,
School
of
Medicine,
Clinical
Medicine,
Minneapolis,
Minn.
Edmond
Department
Yunis,
M.D.,
Instructor
of Laboratory
School
of
Medicine,
and
Medicine,
Director
University
Minneapolis,
of Blood
Bank,
of Minnesota,
Minn.
on
normo-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1963 22: 53-65
Cell Antigens and Cell Specialization: I. A Study of Blood Group Antigens
on Normoblasts
JORGE J. YUNIS and EDMOND YUNIS
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