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Fluorescence
of
Microscopy
Hemopoietic
with
Acridine
Cells
in Fixed
By
I
HE
sue
of
USE
gained
culture,4
nucleic
acids
viral
has
rihonucleic
the
use
the
blood-forming
of their
been
organs,
recorded.
row.
Variation
quence
tion
of color
plexes
importance
as a cytochemical
In
describes
a
This
report
of fixed
addition,
ervthroid,
acridine
Thin
smears
myeloid.
of peripheral
creases
slides.0
and
the
background
dried
and
three
minutes
The
months.
er1
fixed
with
and
in
absolute
gave
major
bone
methanol
for
difference
three
a
color
in
solution
from a pH of 3.8 to 6.4. Trials
and 7.68.
A pH of 6.4 for the Iiuorochrome
development
follows:
1. Ethyl
2. Ethyl
3. Ethyl
alcohol
alcohol
alcohol
4. Distilled
5. Acetic
1 per
water-2
7. Mcllvane’s
definition
80 per cent-5
70 per cent-S
50 per cent-S
water-S
acid
6. Distilled
and
bufferl.
of
were
cellular
bone
fDisodiuni
hydrogen
Se-
of the
varia-
( AO-RNA
) corn-
cell
fluorescence).
Fixation
slides
hydrogen
ion
concentration
conducted
solution
structure.
was
The
slides
could
by
varying
selected
staining
thick
inn.
(heparin
Carnoy’s
devised
one
One
The
with
the
of the
were
on
avoided
be
inwere
the
pH
air
fhuidl0
for
stored
for
Dart and
of the
Turnbuffer
between
because
of optimal
procedure
was
as
dips
dips
dips
dips
cent-4
dips
minutes
pH
6.4-5
minutes
From
the Department
of Me.diciiw,
Upstate
Medical
Center,
State
Universfrij
York, Syracuse,
N. Y.
This investigation
was supported
by a fellowship,
CF-li,
550, from the National
Institute,
Public
Health
Service.
Submitted
June 30, 1961; accepted
for publication
Sept.
25, 1961.
#{176}ErieScientific
Corp.,
Buffalo,
N. Y.
in distilled
mar-
maturation
made
were
fixation
After
change
fluoro-
and
account
acid
minutes.
AO
0.87
an
aspirate
a modification
being
has
METHODS
altering
results.
was
the
anticoagiilants
without
procedure
during
of
staining,
cells.
marrow
possible,
comparable
blood
give
plasmacytic
AND
and
fluorescence
staining
the
blood
Wherever
will
by
study
reproducible
of the
orange-ribonucleic
MATERIALS
non-fluorescent
simple,
specificity
to the
to supravital
content
paper
its
for
) established
of AO
preparations
the
and
application
RNA
reagent
( DNA
acid
regard
study
the
investigators,
several
by
exfoliative
deoxyribonucleic
with
intracytoplasmic
between
of the
Its
particularly
of
dye
of
The
the
has recently
cytology,’
tis-
a fluorochrorne
niicleases.”
for
is described.
as
disciplines
) and
: A Study
SCHWIER
the
emphasized
( RNA
respective
technic
chrome
in
research.a
been
acid
I\i.
( AO )
orange
I)OPtllarity
and
for
also
acridine
some
Lt:wis
Orange
Preparations
phosphate
19.66
Gm.
anti
citric
acid
6.46
VOL.
19,
Gm.
diluted
to
of
New
Cancer
one
liter
water.
200
BLOOD,
No.
2
(FEBRUARY),
1962
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FLUORESCENCE
MICROSCOPY
8. Acridine
0.01 per
9.
10.
in buffer
buffer,
Mcllvane’s
buffer,
1\iost
buffer
of
the
reduced
use(l
exciting
an
This
minutes
on the
withdrawn
during
the
HBO
was a Jena
of
slide
anti a no.
with absorbent
microscopic
filters
effectively
lumination
to the slide but eliminates
server.
A bright
field condenser
was
a dark
field
condenser
made
with
magnifications
and prints
high
speed
of
the
destained
same
cells
for
Using
were
the
observed
and
five
under
minutes.
Wright’s
vernier
tion of cells was made possible
Pretreatment
with rihonuclease
smears.
The slides were
incubated
by
of
this
of
ii-
the
ohwith
were
exposures
N.
after
and
the
the
slides
had
stag,
the
mechanical
illumination.
with
All film
Y. fter
540X.
water
applied
graduated
tungsten
light
of
replaced
30-second
was
a
mm.
Photomicrographs
stream
stain
tinder
eyes
was
Ot
2.
ultraviolet
the
this
using
a gentle
was
prnhl:lry
a Jeri;s
with
magnifications
of
Laboratories,
Rochester,
coordinates
photographed
long
from
photography.
( EH-135)
removed
anti
filter
purposes;
and
microscope
The
or barrier
screening
Ektachrome
were
methanol
thoroughly.
dried
daylight
coverslips
with
for
examination
Laholux
spectrum
applietl.
nat( rial-
procedure
illuminator.
blue
of the
latter
Leitz
the
secondary
part
used
detailed
as
transmits
this
coverslipt
The
A
lamp
950X,
and 25-secind
exposures
processed
at the Eastman
Kodak
were
examination,
for
1 non-fluorescent
Paper.
examination.
mercury
vapor
BG-] 2, and the
200
4 mm.
combination
minutes
minutes
6.4-4
then
Osram
filter
OG-1.
pH
was
6.4-2
6.4-4
placed
fading
with
pH
pH
was
fluid
201
AO
( 3,6-tetramethyldiaminoacridine)
centf
Mcllvane’s
Fresh
ly
orange0
WITH
Accurate
slides
identifica-
procedure.
w:ts
for
performed
on
one
at
hour
a
of
number
37
C.
in
ribonuclease,j
1.0 mg./ml.,
in glass distilled
water.
Pretreatment
1 mg./ml.,
or streptodornase,#{176} #{176} 1000
units/nil.,
Control
slides were treated
identically
except
for the respective
then carried
through
the staining
procedure.
a
normal
and
solution
of
of slides
was similarly
nucleases.
nuclease,
.thnermal
protease-free
with dleOxyrih()carried
out.1
The slides were
1
RESULTS
Pretreatment
cence
removes
be
by
all green
nucleolus
DNA)
complex.’2
aspirates
The
cytoplasm
With
reticulocyte
Using
and
of
in
the
fluorescent
blood
rubriblast
but
barely
often
Corning
§Eastman
The
Co.,
Kodak
IMann Research
9 Mann Research
Co.,
Corning,
Co., Rochester,
N.
in
and
technic,
the
134
were
normal
fluoreslikewise
2). It can
the
cytoplism
amid
green
red
Philadelphia,
normal
and
series
decreases
fluorescence
stippled
and
(AO-
abnormal
fluoresces
(fig.
which
(fig.
4).
diminution
prorubricyte
yellow
acid
examined.
erythroid
fluorescence
appears
of AO 0.1 per cent in distilled
to each liter of stock stain.
Glass
fluorescence
smears
greatest
the
cytoplasnlic
streptodornase
of cells
(fig.
orange-deoxyribonucleic
the
the
visible
more
46005, Hartman-Leddon
Stock stain
2 ml., is added
of
maturity
red
or
nuclei
complex,
AO
the
and
the
red
acridine
erythrocyte
does not fluoresce.
cence
appears
to occur
between
0C.I.
and
54 peripheral
a fine,
form,
orange
an
increasing
has
orange
of an AO-RNA
demonstrates
marrow
reticular
that
is indicative
fluorescence
intensely.
or yellow
therefore,
prevents
by deoxyribonuclease
fluorescence
from
.
concluded,
and
ribonuclease
( fig. 1 ) Pretreatment
in cytoplasmic
early
3).
The
is sometimes
The
mature
rimbricyte
fluores-
stages.
Pa.
water
is diluted
1:10
with
buffer.
Tween
80,
Louis,
Mo.
Y.
N.
Y.
Laboratories,
New York, N. Y., anti Sigma
Laboratories, New York,
N. Y.
#{176}#{176}Varidase,Lederle Laboratories,
Pearl
River,
New
York.
Chemical
Co.,
St.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
LEWIS
1.
Figs. 1-12.-See
legends,
facing
page.
M.
SCHIF’FES
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
FLUORESCENCE
In the
MICROSCOPY
megaloblastic
is altered
cytoplasmic
The
the
level.
In
fluorescence
increased
In the
over
normal
These
normal
B12 deficiency,
occurs
later
the
cytoplasm
promyelocyte
dominant
cell
and
myelocyte
has
intensely
fluorescence.
There
plasmic
fluorescence
kemia,
vivid
vera,
plasmacytes
orange
in the
multiple
acute
in leukemoid
easily
that maturity
in cytoplasmic
megakaryocytes
fluorescence,
) . Platelets
Lymphocytes
plasmic
of acute
are
leukemia.
bone
to other
in the
The
to
the
marrow
fluorescence
atypical
lymphocytes
a
forms
(fig.
from
of
normal
in cytomyelocytic
leu-
very
by
blood
patients
very
.
cyto.
have
of the
chronic
mononucleosis
in
) It
siginlIcant
usually
appears
with
cell
found
( fig. 8
less
granularity
fluorescence.
intensely
There
their
mature
orange-red
cytoplasm
are
infectious
by
display
faint
pink
The
4).
only
granular
peripheral
however,
of cells
pre-
riucleolar
smears
is the
accompanied
intensity.
forms
the
distinct
forms,
such
as
and more
vivid
have
and
leukemia,
“blast”
is intensely
increase
from
of chronic
in
immature
by their
marrow
slightly
reactions.
is not
found
of intermediate
lymphoblastic
equated
no increase
are
very
( fig. 7 ) . This
series
while
more
distinguished
in the
fluorescence
be
in this
RNA.
the
very
leukemia,
and
recognized
fluorescence
be
With
maturation
in the granuiocvte
to be between
the
myeloblastic
cytoplasm
to be a very
slight
of similar
maturation
and
are
cytoplasmic
Mature
plasmic
( fig. 9
can
In
sequence,
myeloblast
rubriblast.
apparent
appears
marrow
with
such
fluorescence.
Immature
myeloma,
may appear
to be even
brighter
is apparent
decreases
cells
appears
in cells
polycythemia
Mature
stages.
fluorescent
to
of the
more
( hg. 5).
hyperplasia,
appears
fluorescent.
In this respect
it is identical
to the
the fluorescence
decreases
until
it is no longer
( fig. 6 ) . The greatest
decrease
in fluorescence
pattern
have
maturation
erythroid
maturation
series,
preceding
appear
to
maturation
in the
non-megaloblastic
throughout
normal.
myelocytic
the
erythroid
elements
cells of comparable
in fluorescence
rubricyte
cytoplasmic
of vitamin
extent.
than
decrement
late
203
AO
sequence
to a certain
fluorescence
greatest
at
WITH
cvto-
immature
fluorescent
to be
and
little
or
lymphocytic
may
exhibit
Fig.
1.-Normal
marrow.
AO stain
after rihonuclease
pretreatment.
X540
Fig.
2.-Acute
myeloblastic
leukemia.
AO
stain
after
deoxyribonuclease
pretreatment.
The nucleoli
are apparent.
X950
Fig. 3.-Erythroid
cells.
Cvtoplasmic
fluorescence
is red. X950
Fig.
4.-Acute
lymphoblastic
leukemia.
Nucleoli
are visible.
A reticulocyte
is
at the top to the right of center.
X950
Fig.
5.-Megaloblast
completing
telophase.
Note
the small
piece
of chromatin
in the cytoplasm.
X950
Fig.
6.-Myeloid
cells.
Cytoplasmic
fluorescence
(orange-brown)
decreases
with
maturity.
X950
Fig.
7.-Binucleated
plasmacyte
from
normal
marrow.
The
cytoplasm
is intensely
orange.
X950
Fig. 8.-Plasmacytes
of multiple
myeloma.
X950
Fig. 9.-Mature
megakaryocyte.
X540
Fig.
10.-Reticulum
cell.
Nucleoli
are apparent.
X950
Fig. 1 1.-Mitotic
figure
and two rubricytes.
X950
Fig.
12.-LE
cell.
The inclusion
fluoresces
yellow-green.
X950
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2()4
LEWIS
intense
lar
cytoplasmic
fluorescence
in addition
to occasional
well
M.
SCHIFFER
defined
nucleo-
fluorescence.
Monocytes
display
do myelocytes,
as
fluorescence
approximately
and
of
the
Reticulum
It
are
been
function
distinguished
shown
content
of
the
the
parent
that
peared
( such
LE
will
in
served
lines
as
do
complex
throughout
(fig.
7).
myeloma
the
with
fluoresced
yellow.
proportional
the
12)
1.
indicating
the
the
deserves
Thus,
(fig.
5).
The
to fluoresce
series
in
is in
by the
fairly
same
constant
11),
this
immature
a more
(lull
once
plasmacytes
cells
of
the
to
the
a different
dye.
It was
for individual
formed
ob-
in the
as apparent
of the
line,
The
to
how-
AO-RNA
orange-brown
the myeloblast
by their
vivid
of
ap-
dense
was
variation
to
hue.
orange
ap-
of DNA.
is just
orange
fluoresces
more
finding
contrast
complex
and
maturation
distinguished
the
the
This
referring
AO-RNA
often
presence
as
cell
which
complexes
This
most
with
throughout
are easily
apparent
the
3 and
one
was
attention.
metachromasia.
found
( fig. I 1)
Nuclei
AO-RNA
The
. Also
RNA
from
figures
it
a
representing
of
since
the
as
Cytop!asrnic
latter
material.
“metachromasia,”
(figs.
RNA
no fluorescence.
during
the study
fluorescence,
Mitotic
produced
appears
to
of
green,
while
A not unexpected
between
cells
red
granulocytic
is also
(fig.
in
Cytoplasmic
amount
statement,
(fig.
term
series
is evident
Plasmacytes
This
a myelocyte
in table
in nuclear
hue
maturation
the
is
the
plus,
above
cytoplasm
metachromasia
a tendency
in
6). This
passed.
the
increase
green
is distinctly
have
(fig.
been
to
cytopki$mic
and
megaloblastic
ever,
fluorescence
quantitate
to four
plasmacytic
of
series
in the
and
the nucleus
that
cytoplasmic
erythroid
of
iniensity
cytoplasmic
nucleoli.
and
zero
representing
of all cells
observed
cytoplasmic
to
color
distinct
is illustrated
zero
an
fluoresced
referred
interpretation
that
Thorell’s
findings
of the percentage
of ultraviolet
spectrophotometry.13
been
myeloid,
be
and
the
( fig. 10).
dull
of
to
and “open”
often
nuclei
) fluoresced
in
usual
cell
had
variation
erythroid,
from
exception
sole
there
inclusions
The
to
pattern
of variation
in nuclear
be found
during
this investigation.
more
reticular
as plasma
cell
that
intensity
attempt
the “blast”
evaluation
table
are
by means
No consistent
to another,
could
furnished
color
leukemia
very
is often
of fluorescence
graded
was
in the
cytoplasm
included
the
An
intensity
the fluorescence
of
table
is a subjective
in
fluorescent
similar
was
by their
and
that
cells.4
of the
fluorescence
cell
remarkable
is not
has
HNA
same
of monocytic
leukemia.
cells
fluorescence
the
instance
predominant
myelocytic
chronic
in one
stage
has
orange
color
patients
with
multiple
8).
DISCUSSION
It is evident
acids
one
tures
of the
cannot
are
chemical
that
blood-forming
differentiate
invariably
the nucleic
acids
must
be performed
procedures
elements
between
encountered.
of
individual
cytochemically.
for
suffer
the
the
various
It is equally
cell
determination
from
types
at
In 1947,
several
cell
lines,
apparent
various
Thorell
of
the
drawbacks.
nucleic
Notably,
and
population
that
measurement
phases
published
of
mix-
maturation
his
classic
of
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
FLUORESCENCE
MICROSdOPY
WITH
205
AO
Table_1.-Cytoptasmic_Fluorescent
Co!or
and
intensity
of individual
Cells
in
Cytoplaom
(after
Thorell)
RNA
Cytoplasmic
Color
F1uorcent
Intensity
Rubriblast
orange-red
++++
Prorubricyte
red-orange
+++
Rubricyte
red
+
Metarubricyte
Reticulocyte
Erythrocyte
Megaloblast
red
red
trace
none
none
Cell
( pernicious
anemia)
orange-red
5r/(
small
red
3%
Myeloblast
orange
++++
orange-brown
+++
Myelocyte
Metamyelocyte
Band
Segmented
cell
Myeloblast
of acute
myeloblastic
leukemia
orange-brown
orange-brown
++
trace
orange-brown
0 - trace
none
none
Plasmacyte
orange
+++
Plasmacyte
of multiple myeloma
orange
+ + ++
Lymphocyte
orange-red
++
3%
0.5%
-
+
0 ±
++++
-
0.5%
3%
3-5%
orange
5-7%
orange-brown
++
Megakaryocyte
Reticulum
cell
orange-brown
red-orange
++
++
monograph
of the
cytoplasmic
nucleic
by ultraviolet
microspectrophotometry.15
it became
evident
that
RNA
was
during
contained
propose
+++-
of
lymphoblastic
Monocyte
decreased
5’7(
-
orange
acute
leukemia
maturation.
acids
at
its
It was
of the
bone
marrow
as measured
From
this
and
subsequent
maximum
in the “blast”
also
found
that
the
mature
forms
reports
and
plasma
cell
large
amounts
of cytoplasmic
RNA.’4
Although
this paper
does
not
quantitation
of the RNA of blcod
elements
as Thorell
has so elegantly
it does
provides
provide
a basis
RNA
and
for
Recent
studies
have
a function
of the
molecules
available.
molecules
on
available
essentially
the
same
investigation
type
with
of data
(table
1).
microspectrophotometric
This
technic
methods
for
DNA.
complexes
more
0
+
Promyeiocyte
done,
2%
0.5%
++
-
large
Lymphobiast
both
5%
++++
-
of the
shown
number
the
physical
chemistry
that
dye
the
of binding
sites
It is believed
nucleic
binding
acid
sites
that
results
and
of the
molecules
the
acridine
are
on
the
acid
aggregation
in metachromasia.
promotes
stacking,
orange-nucleic
bound
acid
to nucleic
and
the
number
or stacking
RNA
whereas
acids
of the
apparently
DNA
as
of dye
has
dye
has
fewer
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
206
LEWIS
such sites and does
the medium
is also
but
also
the
accounts,
U
not promote
an important
AO-DNA
part,
in
complex
for
the
stacking.’5
variable.’6
The hydrogen
Interestingly,
fluoresces
rnetachromatically
green
and
yellow
colors
M.
SCHIF’FER
ion concentration
of
not only the AO-RNA
at
which
pH
6.4.
This
observed
are
in
the
ucleus.
An
intriguing
AO-RNA
aspect
complexes
different
stage
with
Thlast”
of
differentiation
of
by
more
methods.
classical
the
the
in
For
cells
by
means
the
sensitivity
may
represent
are
system
may
erythroid
to
in
coating
materials
Hemoglobin,
this
can
for
account
changes
the
2. The distribution
strated
cytochemically.
3. The
ration.
RNA
a large
to
pH,
portion
cytoplasmic
also
AND
CONCLUSIONS
a variation
stacking
of the
RNA
formation
Considering
concentration.
in
of
The
cytoplasmic
fluorescence
produced
than
consider
metachromasia
ion
influence
quenches
their
with
the
globulin.
hydrogen
variation
is
study
of fixed
microscopy
of RNA
and
of the
erythroid
content
Mature
It
AO-RNA
plasma
possible
cells
to
complexes
preparations
with
acridine
DNA
contain
large
demonstrate
of the
1. Es presentate
de
un
medulla
meta-
to a certain
extent
cytoplasm
of
and bone
is presented.
marrow
the
erythroid,
per
of blood
orange
the
the
decreases
be
demon-
with
math-
of RNA.
and
between
plasmacvtic
the
cells.
INTERLINGUA
le studio
de
cells
can
metachromasia
myeloid,
pro
cells
quantities
IN
medio
blood
myeloid
cytoplasmic
methodo
ossee
within
and
SUMMARIO
e
rather
possibilities
cell.
1. A technic
for the
means
of fluorescence
4.
in
may
SUMMARY
by
that
the
fluoresce
beyond
the
and
allows
fluorescence
of
concerned
with
immune
intracellular
that
series
It may represent
a difference
in
speculate
example,
for
observation
plasmacytic
apparent
in cells
is reproducible
metachromasia.
and therefore
a difference
or
chromasia.
one
this
repeated
and
a number
erythroid
cell is primarily
that
of the plasma
cell
of
surrounding
the
of cytoplasmic
There
example,
of the maturing
hemoglobin,
and
was
myeloid,
difference
is most
The
color
variation
cause
of the cytoplasmic
chemical
species
of RNA,
capabilities.
and
study
erythroid,
colors.
This
maturation.
of
for
as
this
of the
de
fixate
microscopia
preparatos
de
fluorescentic
sanguine
con
orange
acridinic.
2.
Le
esser
3.
distribution
acido
ribonucleic
cytochimicamente.
Le
de
declina
contento
con
(Ittantitate
4. Il es
de
de
demonstrate
acido
le maturation
ribonucleic.
demonstrar
acridinic
e acido
orange
ribonucleic
de
de acido
possibile
e de
ille
del
cellulas.
acido
cellulas
Matur
metachromasia
ribonucleic
disoxyribonucleic
erythroide
plasmocytos
cytoplasmic
in le cellulas
pote
e
myeloide
contine
inter
grande
le complexos
erythroide,
mveloide,
e
plasmatic.
ADDENDUM
An
elements
article,
with
by
AO
Prof.
Alberto
should
Marmont,
on
be included
among
the
supravital
staining
the references. It can
of
the
be found
formed
in the
blood
Pro-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
FLUORESCENCE
of the
ceedings
II,
MICROSCOPY
pp.
Congress
7th
361-364,
WITH
the
of
207
AO
European
Society
Haematology,
of
London,
1959,
part
1960.
ACKNOWLEDGMENTS
1 he
author
Medical
The
is
grateful
Center,
cost
grant,
of
and
ternity
the
in
and
their
Edward
University
color
part
l)r.
to
State
of
plate
was
by
a leukemia
ladies’
auxiliary.
C.
New
Hughes,
York,
borne
in
research
for
Professor
the
part
by
grant
from
use
of
of
the
N.I.H.
the
the
Obstetrics,
research
Rho
Upstate
equipment.
fluorescence
Pi
fellowship
Phi
supply
pharmaceutical
fra-
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1962 19: 200-207
Fluorescence Microscopy with Acridine Orange: A Study of Hemopoietic
Cells in Fixed Preparations
LEWIS M. SCHIFFER
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