http://www.bloodjournal.org/content/bloodjournal/31/3/381.full.pdf?sso-checked%3Dtrue

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Immunofluorescent
with
Studies
Antiserum
to
of
a
Human
Murine
(Rauseher
By
ANGELIKI
IN
1963,
FINK
cence,
showed
reactive
with
Rauscher,
and
In
cells
studies
with
serum
against
reacted
rabbit
L.
using
the
in muring
of
studies
that
the
LEE
technic
leukemia
Further
with
ROSNER,
some
murine
by
Fink,
leukemic
using
plasma
blood
( Rauscher)
virus
reacted
specifically
with
leukemia.2’3
The present
tissue
against
Malmgren,
leukemic
prepared
murine
immunofluorescontains
antigen
human
humans
with
leukemic
Rauscher
Virus
antisera
leukemic
of patients
human
Fium
STANLEY
demonstrated
also
addition,
like particles
from
significant
number
AND
antibody.
Karon2
marrow
antibody.
BRENNER
anti-viral
and
IoANNmss,
MALMGREN,1
lymphoid
tissue
specific
Orr
bone
AND
that
Leukemic
Cells
Strain)
KATRAHOURA
MARTIN
Leukemic
containing
virus-
leukocytes
of a
report
describes
fluorescein-tagged
monkey
anti-
leukemia.
MATERIALS
METhODS
AND
Antiserum
Antimurine
leukemia
serum prepared
against
the Rauscher
u#{176}was made by immunizing rhesus
monkeys
against
Balb/c
mouse
plasma
containing
Rauscher
virus.
The monkey
antiserum
was absorbed
with Balb/c
mouse
erythrocytes
and plasma
until no further
reaction was obtained
with either
of these.
Fluorescein
isothiocyanate
conjugation
of the antiserum
was
effected’
antiserum
was
was
lyophilized
restored
and
to solution
of phosphate
Materials
then
purified
Rhodamine
by
stored
at
by
addition
buffered
the
saline
labeled
Sephadex
4 C.
(pH
and
Prior
bovine
DEAE
to
use,
albumin
cellulose
each
of one
miiiiliter
smears
were
was
added.
filtration
ampoule
of
of distilled
and
the
lyophilized
water
The
monkey
final
product
material
and
four
was
milliliters
7.1).
Tested
Peripheral
and
and
then
chronic
whose
bone
bone
marrow
blood
and
leukemia
marrows
bone
marrow
seen
during
were
examinations
the
being
examined
were
available
study
prepared
period
diagnostically.
for
study.
from
and
In
In
all
from
three
31
54
some
patients,
with
patients
nonleukemic
cases,
two
imprints
acute
patients
to
four
of
serial
surgical
From the Division
of Hematology,
Department
of Medicine,
Maimonides
Medical
Centet,
Brooklyn,
N. 1.
First submitted
April 21, 1967; accepted
for publication
Sept. 25, 1967.
ANGELIXI
KATRAHOURA
IOANNIDES,
M.D. : Fellow
in Clinical
Pathology,
Maimonides
Medical
Center,
Brooklyn,
N. Y. FRED R05NER,
M.D.:
Assistant
Director,
Division
of Hematology,
Department
of Medicine,
Maimonides
Medical
Center,
Brooklyn,
N. Y. MAinniN
BRENNER,
B.A.:
Third
Year Student,
State
University
of New
York,
College
of Medicine,
Buffalo,
N. Y. STANLEY
L. LEE,
M.D.:
Director,
Division
of Hematology,
Maimonides
Medical
Center,
Brooklyn,
N. Y.
#{176}Kindly supplied
BLOOD,
VOL.
31,
by
No.
Dr.
Mary
3 (MA.acu),
Alexander
1968
Fink
of the
National
Cancer
Institute.
381
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382
IOANNIDES
or autopsy
specimens
were used only when
of
spleen,
liven,
the peripheral
kidney
white
and
blood
bone
marrow
cell count
were
exceeded
utilized.
AL.
ET
Blood
smears
SO,000/mm3.
Technic
The
smears
in methyl
alcohol for 15-20
minutes
and
found
to be preferable
to acetone
for storing
for prolonged
periods
of time
( weeks to months).
After
being
brought
to room
temfor 30 minutes,
the smears
were refixed
in acetone
for 10 minutes.
Then 0.05-0.1
cc.
stored
were
air dried,
at 4 C. until
smears
used.
fixed
Methyl
immediately
alcohol
was
perature
of reconstituted
fluorescent
Rauscher
antiserum
glass tray to prevent
evaporation
and allowed
hour. The excess
phosphate
buffered
After
air
the Leitz
drying
for
of the
graded
of
for
were
off
20
minutes,
not
the
made
weak
and
the
smears
were
at 20 C. for
the
slides
smears
were
light source,
as results
were
cytoplasmic
moderate,
added,
were
45
covered
with
minutes
to one
washed
a
thoroughly
in
minutes.
( HB200
apple-green
as strong,
poured
microscope
smears
intensity
was
30-40
fluorescence
mounts
The
antiserum
was
saline (pH 7.1)
was
to incubate
recorded
(and,
or none.
examined
under
ultraviolet
light
2 and 4 mm. UC1 filters).
Permanent
when
In most
immediately
present,
after
nuclear)
instances
the
in
examination.
fluorescence
predominant
was
fluorescence
but
its presence
did not
localization
of the fluorescent
stain
smears
were
identified
by code
number
only and were examined
and interpreted
independently
by two observers.
Random
duplicate
smears
were tested
with fluorescein
labelled
anti human
globulin
and in no instance was more than weak non specific fluorescent
detected.
cytoplasmic.
follow
Nuclear
fluorescence
any consistent
pattern.
has been
ignored
in the
was
For this
analysis
occasionally
noted,
reaosn
the cytologic
of these
data.
All
RESULTS
Acute
Lymphoblastic
Leukemia
Twenty-four
kemia
sets
were
seven
in all
examined.
of these
instances
patient
at
a time
differences
in
from
when
count
Bone
and
case
Nine
examined.
sets
Rauscher
blood
fluoresced
strongly
strongly.
on three
and
Acute
with
not
failed
the
cells
were
from
was observed
tested
on one
1000,000/mm.3
marrow
during
and
smears
of four patients
subsequent
relapse.
No
observed
during
five
the
cases
cells
of chronic
demonstrated
of
these
patients,
to fluoresce
at a time
peripheral
occasions.
blood
of
in
remission
three
than
lymphocytic
strong
cells
when
of
during
these.
relapse.
to fluoresce
with
leukemia
fluorescence
from
cells
blood
cells
of
In one patient
failed
smears
examined.
from
Rauscher
In
of
intensely
of cells
blood
exceeded
leu-
available
the
from
buffy
the
bone
another
patient
the cells
from
Rauscher
were
with
coat
of
marrow
fluoresced
both
bone
antiserum.
Leukemia
available
all.
one
The
separate
sets
were
at
staining
more
In
peripheral
Twenty-seven
was
white
lymphoblastic
were
Leukemia
Myelogenous
leukemia
row
of
stained
antiserum.
marrow
his
smears
from
four
patients,
peripheral
of acute
aspirations
blood
Lymphocytic
of
cases
marrow
entirely
of lymphoblasts.
during
complete
remission4
Chronic
In
eleven
bone
Moderate
or strong
fluorescence
for the cells of a single
peripheral
intensity
one
from
Multiple
patients.
except
consisted
almost
were
examined
Cells
of smears
Of
from
the
eleven.
antiserum;
three
cases
twelve
In five
in the
cells
from
fifteen
patients
of the
remaining
bone
patients
with
studied
patients
six
marrow
the
the
acute
myelogenous
during
life,
cells
reacted
cells
reacted
demonstrating
bone
mar-
strongly
weakly
complete
or
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IMMUNOFLUORESCENT
Table
OF
STUDIES
1.-Immunofluorescence
and Other
LEUKEMIC
CELLS
383
of Nucleated
Cells from Patients
with
with Antibody
Prepared
Against
Disorders
Rauscher
Murine
Leukemia
Leukemia
Virus
Iminunofloureecence
Number
of
Specimens
Relapse
6
13
6
12
1
1
Remission
9
11
9
11
0
0
Diagnosis
(Acute
)
(Lymphatic)
(Leukemia)
Total
(Myelogenous)
24
11
23
15
23
7
8
Remission
) Total
(Leukemia
Positive
( Strong or Moderate)
Patients
Specimens
11
) Relapse
(Acute
Results
Number
of
Patients
Negative
( Weak
Negative)
Patients
Specimens
1
1#{176}
10
15t
4
4
1
1
3
3
15
27
7
9
11
18t
9
4
6*
2
3t
5
5
Chronic
Lymphatic
Leukemia
5
Plasma
Cell
Myeloma
7
7
2
2
Lymphoma
3
5
1
1
3
4
Carcinomatosis
7
7
3
3
4
4
6
6
5
5
1
1
2
2
2
2
0
0
2
2
2
2
0
0
28
29
4
4
24
25
Anemias
Megaloblastic
Idiopathic
Thrombocytopenic
Purpura
Leukemoid
Reaction
Anemias
of
Diverse
Origins
#{176}Onespecimen
of peripheral
blood.
tlncludes
two samples
of peripheral
lncludes
three
samples
of peripheral
remission
reacted
of relapse.
became
weak
made
no
However,
the
Rauscher
antiserum.
strongly.
but liver
other
case
Chronic
one
one
one
case did
strongly.
patient.
post-mortem
table
( Case
instance,
cells
Rauscher
obtained
antiserum
In three
patients
and their
cells
9
imprints
not. On
)
cells
from
at the
time
during
relapse
imprints
tested
were
against
splenic
imprints
from this patient
also stained
the other
hand,
cells in splenic
not react
while
cells in liver
Cells
in imprints
from
all
imprints
organs
from
tested
the same
from
an-
reacted
weakly
Leukemia
patient
was
studied.
Cells
from
his
bone
marrow
antiserum.
Myeloma
Seven
loma
with
to fluoresce.
Rauscher
Multiple
corresponding
Cells in lymph
node
and kidney
cells did
Granulocytic
Only
with
failed
in
at the
In
from one
fluoresced
than
reaction
remission
spleen
fluoresced
intensely,
imprints
patient
differently
a strong
during
from
blood.
blood.
were
fluorescence
sets
of bone
examined.
was
marrow
smears
No reactions
observed
in
the
from
occurred
cells
of
seven
patients
with
multiple
in the cells of four patients.
one case,
the reaction
was
myeWeak
weak
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384
IOANNIDES
to moderate
in the
moderate
intensity
their
was
cells
of another
of staining.
noted
Miscellaneous
with
case,
In
the
nonspecific
and
the
latter
cells
three
of another
case
weak
fluorescence
cases,
anti-human
AL.
showed
of
globulin.
Conditions
Bone
marrow
smears
Weak
fluorescence
studied.
bone
cells
El’
marrows
of
from
seven
patients
of cells
the
was
with
iron
detected
other
six
patients
from
five
patients
deficiency
in one
failed
anemia
were
Cells
in the
instance.
to
react
with
Rauscher
antiserum.
Bone
marrow
smears
studied.
In four
cells. Cells from
instances,
moderate
bone marrow
smears
Five
sets
of
lymphoma
were
smears
studied.
did not fluoresce.
weak
to moderate
another
case
Cells
secondary
from
tients
idiopathic
three
out
of
are
stained
four
indicated
carcinoma
smears
case
did
were
in the
patients
of these
with
cases
in one case
reacted
not react
and
cells
two patients
respectively
with
and
purpura
fluoresced
were
moderately
weakly
or
patients
failed
to fluoresce
on bone
marrow
marrow
in one
thrombocytopenic
three
reacted
bone
Cells
anemia
were
observed
to fluoresce.
aspirates
from
three
node
imprints
in two
smears
from
and infection
metastatic
from
cells
megaloblastic
with
from
weakly.
bone
marrow
to hemorrhage
with
smears
the
bone
marrow
Cells
in lymph
Cells
from
intensity.
fluoresced
with
patients
of
with
to strong
reactions
in one case failed
or
not
at
with
when
smears
1.
bone
marrow
Cells
but
cirrhosis
from
and
from
four
Seven
bone
in the four
bone
marrow
from
Rauscher
patients
reactions
other
pa-
strongly.
Cells
strongly
all.
treated
with
in additional
in Table
studied.
leukemoid
from
two
marrow
other
patients
smears
from
patients
with
antiserum.
Results
with
miscellaneous
uremia
obtained
conditions
DIscussIoN
Cells
from
from
consecutive
reactivity
nic of
patients
in whom
bone
Since
selection
of
lymphoblastic
leukemia
marrow
leukemia
reacted
of only half
demonstrated.
all the work
leukemia
from
liver,
chronic
aspirates
in
were
this
latter
comparisons
The bone
and
strongly
and
and
kidney
of four
with
available
group
tissues
the
failed
been
other
imprints
were
tested
between
marrow
out
of five
Rauscher
the
similarly
studied.
and non-leukemic
all patients
with
patients
with
chronic
In the
techcondi-
( although
unavoidable
leukemic
cells of
antiserum.
for
bone
paacute
lymphatic
marrow
cells
could
such
reactions
he
of the fact that
in nearly
agents,
the type
of leu-
lymphocytic.
than
peripheral
to
spleen
were
was
patients
with
granulocytic
leukemia
These
findings
are interesting
in view
on transmission
of leukemia
by filterable
has
and
leukemias
the
involved
Human
acute
( Rauscher)
vrius
antibody
employing
Patients
with
various
non-hematological
patients
without
bias ), statistical
tients
were
not attempted.
Cells
with
to murine
leukemia
immunofluorescence.
lions
kemia
aspirates
fluoresce
bone
blood
marrow
from
with
aspirates
a
Rauscher
patient
produced
erratic
with
acute
antiserum
at
results.
lymphoblastic
a
time
when
From www.bloodjournal.org by guest on July 8, 2017. For personal use only.
IMMUNOFLUORESCENT
the
cells
of the
findings
bone
occurred
prints
marrow
in
obtained
acute
node
in
another
spleen
one
patient
leukemia
lymph
and
same
patient
with
( spleen
and
not
fluoresced
chronic
various
from
in one
case;
show
strongly.
lymphocytic
organs
liver
in another
did
385
CELLS
from
spleen
case
LEUKEMIC
of the
post-mortem
myelogenous
row,
OF
STUDIES
Im-
patients
with
kidney,
kidney,
consistent
leukemia.
three
case;
liver,
Similar
bone
fluorescence
bone
mar-
marrow
and
with
Rauscher
antiserum.
Since
bone
dividuals
marrow
tested
certainly
nic
cence
Recent
not
in bone
two
or
represent
situation
to find
The present
an antigen
experimental
with one of the
Bone
marrow
cells
harboring
results
causative
from
strongly
four
with
be
due
the
out
of such
antigen
individuals-that
Rauscher
specific
to erythropoietic
suggestion
is the
scher,
fact
that
picture
very
immunofluorescent
Orr
and
Karon2’3
one
of the
of five
marked
may
hematological
the strongest
for
nonleukemic
for leukemia.
patients
requires
indicate
human
viruses
at least
in some
clinical
disease
for
leukemia,
and
patients
with
It has
erythroid
it would
yet
free
Rauscher
in
the
produces
in bone
marrow
this
marrows
an antibody
to
viral.
In support
in mice
cells
anemias
that
bone
to human
erythroleukemia.
reactions
reported
by Fink,
occurred
be
shares
virus
suggested6
activity
virus
not
of leukemia.
megaloblastic
been
antiserum
may contain
cells but not necessarily
similar
intech-
That
fluoresexplanation.
that
present
antiserum.
finding
this
hold
of
immunofluorescent
would
fit this model
if Rauscher
agents
of human
leukemia.
Rauscher
to
test
simultaneously
should
persons
third
the
in animals
be
must
surprising
fluoresced
leukemias
viruses
one
antiserum,
a diagnostic
of nonleukemic
cells
of viral
more
approximately
Rauscher
marrow
If this
develop.
from
with
investigations
cases
to
does
occurs
cells
reacted
a clinical
an
of
and
Furthermore,
Malmgren,
Rau-
of patients
with
eryth-
roleukemia.
The
present
marrow
bone
and
marrow
tients
studied.
activity
A viral
gators79
Certain
There
etiology
for human
since
the earliest
Ellerman
by
although
were
a recent
a variant
tissues
extended
shown
prepared
a viral
this
thesis,
correlation
however.
between
leukemia
has
description
and
Bang
in 1908.10
Gross
in 195111
and this
report
by
between
against
etiology
describes
the
and
same
human
marine
rats
the
and
( Rauscher)
leukemia
virus
also be caused
is etiologically
and,
subsequently,
some
viral
virus.
patients
These
virus.2”
suggest
by viruses.9
related
to a
that
lympho-
monkeys.’2
detected
Rauscher
the
pa-
was
first
confirmed.8
of Burkitt’s
into
to
the
many
investiof avian
leu-
leukemia
subsequently
with
leukemia
regard
in all
postulated
by
viral
etiology
leukemia,
from
bone
of erythroid
with
but
transmission
tissue
and
amount
only
anemias
Malmgren,1
investigators
Differential
the
A murine
work
was
infected
leukemic
of human
been
of the
lymphoblastic
Fink
of mice
not
cattle
leukemias
may
that human
leukemia
of human
immunofluorescence,
By
confirm
any
the degree
of immunofluorescence,
cells
of patients
with
megaloblastic
probably
leukemic
late
not
forms
of dog, cat and
is as yet no direct
proof
virus
was
do
to show
ever
kemia
by
demonstrated
ma,
studies
failed
counts
antigenic
antigen
These
in
studies
cross
reactivity
and
antiserum
authors
postu-
similarities
From www.bloodjournal.org by guest on July 8, 2017. For personal use only.
386
IOANNIDES
occur
among
The present
viral
strains
of leukemia
studies
confirm
etiology
of human
equally
strong
blastic
tested
leukemia
by serial
body
does
state
whether
marrow
that
indicate
with
Rauscher,
( type
unspecified
“leukemia
virus”
antibody
marrow
cells
findings
reactivity
the
stage
with
Malmgren
leukemia
present
findings
a possible
to indicate
with
acute
that
lympho-
in remission
or in relapse
Karon,
Rauscher,
Malmgren
in other
leukemias
with the anti-Rauscher
(other
virus
as
than
anti-
of the disease.”
These
preliminary
findings.
On the other
hand,
our
and
in
whereas
for
seem
of patients
such
patients
are
aspirations.
Fink,
agreement
in
The
“preliminary
that the
correlate
are
Karon,
in bone
occur
bone
not
servations
leukemia.
reactions
and On3
also
erythroleukemia)
virus infecting
various
species.
the work
of others3
in the search
AL.
ET
ohFink,
own
Orr3 found
that 79 percent
of cases
of acute
relapse
fluoresced
strongly
with
antihuman
this occurred
in only 25 percent
of patients
)
in remission.
SUMMARY
Cells
from
from
patients
hematological
( Rauscher)
cells
bone
marrow
with
leukemia
disorders
virus
antibody
of all eleven
five
patients
aspirates
and
were
tested
using
the
patients
with
chronic
lymphatic
with
and
other
acute
liver,
kidney
for
reactivity
technique
of
with
murine
immunofluorescence.
lymphoblastic
leukemia
leukemia
reacted
15 patients
with
myelogenous
leukemia
and
the
47
with
miscellaneous
disorders
( Table
SUMMARIO
IN
Cellulas
ab aspiratos
de medulla
ossee
con leucemia
e con altere
non-leucemic
testate
pro reactivitate
con anticorpore
utilisation
del techncia
immunofluorescentic.
acute
leucemia
lymphatic
circa
lymphoblastic
reageva
un
poteva
four
In
approximately
1
)
non-
leukemia
The
and
strongly.
imprints
and
only
out
of
six
of
one-third
strong
was
of
immuno-
demonstrated.
fluorescence
con
spleen
hematological
the
patients
and
nonleukemic
tertio
esser
e
fortemente.
de
47
In
de
solo
patientes
con
INTERLINGUA
e impriniitas
de hepate,
ren, e splen
ab
disordines
hematologic
e non-hematologic
anti virus
de leucemia
munin
(Rauscher)
Le cellulas
de omne
le dece-un
patientes
esseva
quatro
leucemia
sex
del
del
cinque
15 patientes
miscellanee
disordines
patientes
con
con
leucemia
un
forte
chronic
myelogene
con
le
patientes
e in
inimunofluorescentia
demonstrate.
ACKNOWLEDGMENTS
The
authors
Institute
by
for
Mrs.
are very
her
Frances
much
and
advice
indebted
to Dr. Mary Alexander
Fink
encouragement.
Secretarial
assistance
of the National
Cancer
was kindly
contributed
Rose.
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1968 31: 381-387
Immunofluorescent Studies of Human Leukemic Cells with Antiserum to a
Murine Leukemic Virus (Rauscher Strain)
ANGELIKI KATRAHOURA IOANNIDES, FRED ROSNER, MARTIN BRENNER and STANLEY L. LEE
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