From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Relative
Labeling
Platelets
By
in Human
J.
HARMON
Relative
labeling
cells
by
by means
51-chromium
of a sucrose
separation
of
were
heavily
labeled
R
confirmed
than
to
small
and
of
leukocyte
been
on
but
types
found
terpretation
were
relatively
differences
in
portant
in evaluating
is utilized
for
of
the
study
Erythrocytes
and
lighfly
labeling
labeled.
are
im-
data
51-chromium
( 51Cr)
it was
and
not
been
has
been
organ
used
obtained
as
leukocyte
when
a cell
label
kinetics.
widely
for
this
largely
distribution
and
distribution
used
as
a label
of intravascular
as a leukocyte
label
again
concerned
quantitative
in normal
data
obtained
of
granulocytes.
for the determination
was first utilized
have
the
PERRY
SEYMOUR
lymphocytes
studies
kinetics
reported
cell
more
platelets
51Cr
patients,4
These
56
and
by
CHROMIUM
erythrocytes
and
organ
sequestration.
AND
platelets
These
and
be
and
by 51Chromium
ROSEN
blood
lymphocytes
ADIOACTIVE
in leukemic
J.
was
determined
gradient
density
found
Erythrocytes
Blood
PETER
peripheral
Large
monocytes
EynE,
and
technic
radioautography.
of Leukocytes,
purpose
with
very
of the
the
little
of
survival
in 1955
until
very
determination
information
label
among
has
the
various
blood.
This information
is important
for valid
inwhen
this label
is employed
to study
leukocyte
kinetics.
In
this
investigation,
erythrocytes,
and
gradient
the
separation
autography,
relative
by
platelets
technic.
a
labeling
51Cr
The
potentially
is
data
useful
of
normal
determined
obtained
technic
human
using
were
with
a
leukocytes,
sucrose
density
confirmed
51Cr
recently
by
radio-
by
described
Ronai.7
MATERIALS
Fifty
ml.
of normal
The
leukocyte-rich
with
and
an equal
0.45 per
was
removed
blood
cells
then
incubated
In
five
From
Institutes
Submitted
Supported
human
whole
AND
blood
were
METhoDs
collected
in a vial
containing
10
cc.
ACD-A.
obtained
by sedimentation
at 1 g. for 15-30
minutes
amount
of a solution
containing
three per cent dextran,
2.5 per cent dextrose
cent
sodium
chloride.
The supernatant
containing
leukocytes
and platelets
and centrifuged
at 2000 RPM for five minutes,
and the contaminating
red
fraction
were
subjected
with
experiments
to
200
pCi.
hypotonic
of
a known
the Human
of Health,
October
by
was
Tumor
Bethesda,
27,
1969;
lysis.
Na.51CrO4
aliquot
Cell
Md.
for
of
Biology
publication
The
for
red
45
blood
Branch,
February
final
leukocyte-platelet
minutes
cells
at
was
National
room
added
Cancer
concentrate
was
temperature.
prior
to
incubation
Institute,
National
2, 1970.
NIH.
J. EYIIE,
M.D.:
Clinical
Associate,
Human
Tumor
Cell
Biology
Branch,
NaCancer
Institute,
NIH,
Bethesda,
Md.;
PETER
J. ROSEN,
M.D.:
Clinical
Associate,
Tumor
Cell
Biology
Branch,
National
Cancer
lnstitute,
NiH,
Bethesda,
Md.;
SEvIoun
PERRY,
M.D.:
Associate
Scientific
Director
for Clinical
Trials,
Chemotherapy,
National
Cancer
Institute,
NIH,
Bethesda,
Md.
HARMON
tional
Human
250
BLOOD,
VOL.
36, No. 2 (AuGusT),
1970
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
LABELING
OF
Table
LEUKOYTES,
1.-Relative
Leukocytes,
Labeling
by
AND
Table
2.-Leukocyte
and
Range
Cell
of
Per
Cent
#{176}
Mean
Granulocyte
29-56
(9)
Monocyte
212-346
(4)
Monocyte
Erythrocyte
2.9-6.0
(4)
Platelet
1.2-1.7
(5)
*
Number
with
51Cr
of
determinations
in
based
on
Per Cent
Based
*
Grain
Distribution
Type
Cranulocyte
Small
lymphocyte
Large
lymphocyte
Relative
Labeling
Lymphocyte
100
Mean
Count
51Chromium
Type
251
PLATELETS
of
Erythrocytes
Platelets
Cell
ERYTHROYTES
on
Grain
Count
*
5.0
11.1
21.8
2.8.8
200
cells
of each
type.
parenthe-
ses.
to
contained
determine
At the
conclusion
25
of
in
ml.
filtered.
After
allowed
to
Evans.8
A
was
then
details
this
procedure
was
in 0.9
by
per
not
of
a
toluenet
was
then
counts
a
direct
after
saline
per
settling
cent
to
devised
during
of
specific
measurement
At
the
Peterson
Hanks
for
two
the
then
suspension
by
in
of 51Cr
separation.
and
the
sucrose
sediment
suspended
sucrose
counts,
chamber
per
obtained
and
cent
differential
allowed
stained
and
the
AR
and
dissolved
EC
Quenching
counts
an
solution
hours.
The
sedimentation
radioactivities
conclusion
by
phase
of
an
of
the
sedi-
settling
fashion.
reagent.f
Liquifluor-
counted
in
a
standard
Red
blood
experiments,
standard
external
microscopy.
in the
four
the
was
by
red
in
of NCS
chromium
counter;
In
film
in 1 ml.
of
cell
stain.
stripping
monitored
done
electronic
Wright’s
10
decay
was
were
in
with
Kodak
concentrated
determined
the
cells
by
and
using
added
Platelet
were
the
and
prepared
spectrometer.
significant.
and
0.3-1.75
with
0.05
thus
platelets.
50 ml. were collected
in 0.2 ml. of 10 per cent bovine
albumin
ml. of each fraction
were used for cell counts.
Slides were made
of
were
of
determined
cytocentrifuge*
cells
cell
and
twice
elsewhere.#{176} Elution
before
Ten
were
bottom
and
as
washed
for
suspension
cells
containing
containing
reported
elements
NaCl.
the
chamber
significant
fractions
scintillation
by
the
are
cell
remaining
not
in
cent
means
into
final
red
were
solution
removed
gradient
The
leukocytes,
cells
salt
was
rapidly
cellular
radioautographs
The
flow
procedure
various
mentation,
aliquot
labeling.
of
the
balanced
continuous
generated
of
numbers
of incubation
Basic
a small
and
erythrocyte
equal
Hanks
was
the
relative
approximately
and
cells
white
were
liquid
and
was
blood
cell
also
counted
method.
RESULTS
At
the
conclusion
discrete
bands
( The system’s
described
in
peniments
aliquots
results
counting
of
INCS
in
Table
erythrocytes.
is consistently
Co.,
blood
England
Packard
Instrument
Co.,
possible
IlCotilter
Electronics,
Inc.,
Corp.,
Corp.,
LaGrange,
Hialeah,
Fla.
Des
most
variation
several
reasons
at high
Plaines,
Boston,
Ill.
to
Ill.
Mass.
or
more
obtain
but
were
for
dilutions,
and
in several
ex-
obtained.
by liquid
was
is
fractions
eosinophils
pure,
monocytes
Pa.
Nuclear
three
and leukocytes.
of the blood
( including
is less
51Cn as determined
counting
Sewickley,
Inc.,
cent
The
were
cell
Nuclear-Chicago
New
per
with
There
of red
reagent,
It
1.
chamber,
of platelets,
erythrocytes
the cellular
elements
60-80
labeling
shown
Scientific
jLiquifluor,
)
containing
inaccuracy
*5haI(lon
consisting
in separating
lymphocytes
or granulocytes
The monocyte
fraction
of relative
are
labeling
sedimentation
elsewhere.9
),
basophils
basic
detail
95 per cent
respectively.
containing
The
of
were
visible
capability
scintillation
observed
this including
and
in
a small
the
the
but
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
EYRE,
252
1.-Representa51-chromium
radiograph
of leukocyte
suspension
demonstrating heavier
labeling
of
large
lymphocytes
and
monocytes.
ROSEN
AND PERRY
Fig.
tive
I
I___
variable
overlap
was
established
3-6
per
cent
Dresch
labeling
that
of
lymphocytes
confidence
compared
to the
lymphocytes
studies
separation
of
methods
In
the
granulocyte
in
Because
the
monocyte
column.11’12
data
in Table
of
were
NIean
labeled
are
grain
counts
a representative
of the large
lymphocyte
revealed
that the large
most-heavily
cell
much
types
The small
lymphocyte,
while
more-heavily
much-less-heavily
labeled
than the large
it
order
of
data
of
higher
it was
found
granulocytes.
labeling
retained
In
following
technics.
since
along
it
with
the
the results
of previous
investigators,
made
in order
to validate
the data
with
2 and
than
gnanulocyte
lymphocyte
cells
counting.
listed
the heavier
labeling
The radioautographs
the
large
conflicted
to the
labeling
was estimated
fiber
or glass
wool
column
overestimate
separated
scintillation
are
heavily
the
On
contradistinction
more
nylon
an
and
the
of the
liquid
to
present
nadioautographs
leukocytes
by
lead
Nevertheless,
labeled
Scott,t
who
reported
in the present
study
granulocyte
lymphocytes
fraction.
enythrocyte
labeled
relative
would
that
is known
were
erythrocyte
the
lymphocyte.
consistently
previous
These
from
the
that
and
Najea&#{176} and
McMillan
and
of granulocytes
than
lymphocytes,
these
the
in
with
for
the
various
classes
radioautograph
and monocyte
lymphocytes
and
had
labeled
lymphocyte.
similar
than
of
illustrating
is shown
and the
mean
the
in Fig.
monocytes
grain
1.
counts.
granulocyte,
was
DIscussIoN
The significantly
granulocytes
with
kinetic
bers
data,
of these
especially
cells
of lymphocytes
such
as
the
higher
51Cr
as,
with
one
labeling
of lymphocytes
is of importance
in the
when
for
51Cr
outlined
the
example,
differential
in the
combined
here,
with
should
count
and monocytes
interpretation
compared
of leukocyte
reveals
appreciable
neutropenic
patient.
The
improved
methods
make
reliable
of
intravascular
num-
high
cell
labeling
separation,
lymphocyte
to
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
LABELING
OF
kinetic
LEUKOCYTES,
studies
possible
could
only
other
clinical
situations
finding
of low,
The
be
confirms
leukocytes
for
patients.
in patients
with
although
the
significant.
kinetic
of
studies.
levels
the
The
RonaiT
recently
demonstrated
radioautography
is possible
resulting
from
4.5
keV.
quality
electron
which
of the
films
data
Analysis
lymphocytes
and
large
not
as
be of further
to
of
of
the
3H
here,
10-20
procedure.
of the
Auger
and
It
from
quantitative
as liquid
also
the
served
other
to
in elucidating
the
of 51Cr
with
soft
beta
an
distinguish
The
stripping
time
heavy
the
labeled
energy
emitters.
the
counting,
relative
but
disintegrations
electrons
lightly
scintillation
of
erythro-
High-resolution
confirmed
relatively
and
one granulocyte.
generally
appreciated,
cent
to
radioautographs
monocytes.
in
preparations
processed
in the usual
manner
with
of the short
halflife
of 51Cr developing
lymphocyte
value
as
not
of this
rise
that
or
of erythrocytes
pure
presented
70 per
give
is similar
short.
While
because
of information
leukemia
of labeling
relatively
feasibility
capture
radioautognaphs
and because
is excellent,
relatively
labeled
the
type
lymphocytic
counts.
obtaining
From
this
chronic
lymphocyte
necessity
253
Previously,
with
high
I’LATELETS
AND
would
contribute
the same
radioactivity
technic
of 51Cr
radioautography
is
cytes
of
in normal
obtained
platelets
of
ERYTHROCYTES
more-heavily
small
lymphocyte.
radioautography
labeling
is
labeling
of leukemic
may
cells
by
Cr.
ACKNOWLEDGMENT
We
wish
to
acknowledge
with
appreciation
the
technical
assistance
of
Miss
Ada
Brooks.
REFERENCES
1. Gray,
tagging
with
S.
of
J.,
red
2.
Sterling,
cells
radioactive
29:1604,
and
and
K.:
plasma
J. Clin.
chromium.
The
Invest.
Isotopes
F.
J.
F. :
for
the
C.,
as
survival
in
Emerson,
The
chromium-51
agent
use
an
C.
of
P.,
radioactive
erythrocyte
determination
vivo.
J. Clin.
8.
and
red
cell
32:1260,
Invest.
20:
3.
Aster,
R.
H.,
1964.
4. McCall,
Eisentraut,
at
of
NI.
S.,
NI.,
43:843,
9. Brubaker,
survival.
Duvall,
D.
Lanz,
H.:
leukocytes
with
and
measurement
J. Lab.
Clin.
A.,
The
radioof
Med.
the
45:717,
C. P.,
and
Perry,
S. : The
in
the
study
of
51-chromium
kinetics
in
chronic
myelocytic
labeling
nique
and
with
results
normal
J.
leukemia.
51-chromium.
in
use
leukocyte
Lab.
Clin.
Med.
71:614,
1968.
6. McMillan,
R., and Scott, J. L.:
cyte
of
and
Evans,
marrow
cells
gravity.
Nature
L.
H.,
and
Leuko-
subjects.
erythrocytes,
purified
262,
TechBlood
H.:
sedi-
214:824,
and
and
relative
W.
H.:
monocytes,
lym-
platelets
from
tagging
( DFP).
J.
with
Lab.
Clin.
Med.
73:1036,
1969.
10. Dresch,
C., and Najean,
Y. : Study
of
the
kinetics
of polymorphonuclear
leukocytes
after
in vitro
labeling
with
radiochromium.
( Translated
from
the French)
Nouv.
Rev.
Franc.
Hemat.
7:27,
1966.
11. Oppenheim,
J. J., Leventhal,
B., and
Hersh,
E. M. : The transformation
of column
specific
I.
W.
by
Evans,
granulocytes,
blood
human
1955.
5.
unit
A.,
1967.
phocytes,
: Platelet
Sutherland,
and
leukemic
chromium
in vivo
J. H.
Invest.
Jandl,
J. Clin.
bone
diisopropylfluorophosphate
A.
tagging
active
and
in man.
1968.
E.
of
Separation
sequestration
471,
Peterson,
mentation
tagging
of
P. : High
resolution
autoradiog51Cr.
Int.
J. AppI.
Radiat.
Separation
1953.
of
1968.
Ronai,
rahpy
with
1950.
Ebaugh,
Ross,
32:738,
7.
proteins
with
stimuli.
nonspecific
and
J. Immunol.
101:
1968.
12.
To
lymphocytes
antigenic
be
Seeger,
published.
R.,
and
Oppenheim,
J.
J.:
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1970 36: 250-253
Relative Labeling of Leukocytes, Erythrocytes and Platelets in Human
Blood by 51Chromium
HARMON J. EYRE, PETER J. ROSEN and SEYMOUR PERRY
Updated information and services can be found at:
http://www.bloodjournal.org/content/36/2/250.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.