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Immunocytochemical
Distinction
Between
Primary
and Secondary
Granule
Formation
Developing
Human
Neutrophils:
Correlations
With
Romanowsky
Stains
By Katherine
B. Pryzwansky,
John
P. Gregory
K. Spitznagel,
and
UMAN
POLYMORPHONUCLEAR
distinct
round,
large,
cathepsin
been
dase
G,
and
Front
the
many
Department
of
Submitted
June
29,
Supported
by
Grants
HL07149).
North
Carolina,
Health
Address
University
1979
secondary
denote
Blood,
accepted
from
the
Hill
by
and
(VB
161,
Group
K.B.P.,
the
of North
25.
of the
the
UNC
supported
by
are
cell in
have
stains
for peroxithere
are no stains
Department
of
Carolina,
Medicine
Chapel
and
Hill,
the
NC.
1978.
Society
Development
VC 795),
two
granules
color of the
and lysozyme
Histochemical
granules,
but
University
Cancer
and
(specific)*
Immunology,
September
American
Research
Studies
reprint
by Grune
terms
only
the
53,
requests:
North
Chapel
& Stratton,
Inc.
frequently
used
those
granules
No.
Katherine
Carolina,
are
to denote
Vol.
Secondary
for the characteristic
PMN
only lactoferrin
Disease,
have
(IN-l5R.
IN-I5QR),
Administration
Rothrock
School
NIH
(E 40-I).
Research
of Public
Institute
Fund
Health.
of
NIH
the
(AI02430.
University
in Hematology,
fPartial
General
of
and
fulfillment
Medical
Science
the
of a
Grant
138-15.1
for
of
TMThese
1978;
Energy
dissertation
5-T0l-GMOJ
Bacteriology
Infectious
(PMN)
Primary
(azurophilic)
granules
are
myeloperoxidase,
elastase,
lysozyme,
hydrolases.25
responsible
In human
and
Chapel
Occupational
doctoral
granules.
contain
associated
with these
particles.
routinely
as markers
for primary
of Hematology
AI13464.
acid
lucent,’
and
preparations.
definitively
are used
Division
NEUTROPHILS
types
of cytoplasmic
electron
dense,’
and
smaller,
electron
Wright-stained
Rausch,
C. Herion
Romanowsky
agents. Normal human marrow
cells
were
stained
with
May-GrUnwaldGlemsa and photographed.
The slides were
decolorized
in buffered
glycerine
and saline
for 24 hr and then stained with fluorescelnand rhodamine-conjugated
monospecific
antisera to human granulocyte
myeloperoxldase, cathepsin
G, elastase,
lysozyme,
and
lactoferrin.
The same cells were then located
and examined
for conjugate
binding. Double
stains
with
fluoresceinand rhodaminelabeled
antisera
offered
the additional
advantage
of simultaneous
Identification
of
primary
and secondary
granules.
This approach
confirmed
the partItion
of primary
and secondary
granule proteins
and related
their
appearance
to the maturation
of developing neutrophils
In normal
human
bone
marrow.
Electron-microscopic
studies with peroxidase cytochemistry
have shown that primary
(azurophilic)
granules
in human neutrophils
are synthesized
in promyelocytes,
while
secondary
(specific)
granules
are formed In
myelocytes.
However,
these
studies
were
limited by the lack of specific
markers
for
secondary
granules
and by the Inability
to
make direct comparisons
of electron-microscopic
morphology
with the light-microscopic appearance
of the same cell. Thus
secondary
granules cannot be Identified
reliably and the relevance
of these findings to
the light-microscopic
InterpretatIon
of clinIcal bone marrow specimens
cannot be evaluated.
To circumvent
these
problems,
we
developed
a method to permit lmmunofluorescent
demonstration
of primary
and secondary granule markers in cells stained with
H
John
in
granules
of the
2 (February),
NC.
Dept.
of
Bacteriology
and
Immunology.
275/4.
0006-497//79/5302-0002$0l.0O/0
interchangeably,
defined
appropriate
1979
B. Pryzwansky,
Hill,
but
by specific
color
protein
in
this
markers,
in Romanowsky-stained
paper
and
we
shall
azurophilic
use
primary
and
specific
and
to
cells.
1 79
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1 80PRVZWANSKY
specific
ET
for
logic
lactoferrin;
appearance
secondary
and
granules
myeloperoxidase
Electron-microscopic
have
been
identified
by their
AL.
morpho-
negativity.’
studies”6’7
have
shown
that
primary
granules
are
formed
in
promyelocytes,
nately,
these
while secondary
granules
are synthesized
in myelocytes.
Unfortustudies were limited
by the lack of a marker
specific
for secondary
granules
by the
and
light-microscopic
inability
to relate
the
of the
same
appearance
Thus the identification
of secondary
results
to the clinical
interpretation
has not been determined.
Mason
found
that
lactoferrin
was
electron-microscopic
cell
a
triple-staining
simultaneous
granules
and
present
in metamyelocytes,
technique.
approach
is specific
within
a single cell.9
correlate
cell.
stains
elastase,
and permits
In addition,
immunofluorescent
With
this
marrow,
granules
approach
appearance
of the
Preparation
of labeled
specificity
PMN.
between
in human
granules
neutrophil
of cellular
correlated
antisera.
resultant
mature
were
marrow
identified
with
for secondary
granules.
This
components
agents
appearance
development
the
to
of the same
in normal
human
bone
maturation
at which primary
and secondary
these
findings
with the light-microscopic
cells.
G, elastase,
or fluorescein.
of the
granulopoiesis
light-microscopic
MATERIALS
oxidase,
cathepsin
with
rhodamine
and
the relationship
using lactoferrin
as a marker
or cathepsin
G for primary
with
we traced
same
agents.
the simultaneous
localization
of two
the cells were stained
with Romanowsky
results
identified
the stage
are formed,
and
to the
Romanowsky
bands,
thus
Cytoplasmic
immunofluorescent
myeloperoxidase,
with
granules
is unreliable,
and the relevance
of the
of conventionally
stained
marrow
specimens
et al.,8 using an immunoperoxidase
technique,
However,
they did not identify
primary
granules;
two granule
types was not determined.
To circumvent
these difficulties,
we studied
with
morphology
stained
AND
High-titer
METHODS
monospecific
antisera
to human
lysozyme,
and lactoferrin,
raised
in goats
The
purity
of the antigen
preparations
antisera,
and
the
procedures
used
granulocyte
myeloper-
and rabbits,
were conjugated
used
for immunization,
the
to label
antisera
have
been
previously
described.’0
Preparation
patients
those
samples
through
found
with
pH
(20
mg
disodium
[40%
Gnunwald-Giemsa
immersion
without
layered
mounted
enhance
The
Leitz
excitation
7.4.
The
stains
the
and
mg
the
for different
protein
fluorescein,
conjugates,
Some
were
slides
were
at
was
30
They
in PBS
performed
with
also
counterstained
Onthoplan
microscope
30
25 ml
with
(ASA
50)
May-
under
oil
recorded.
xylene.
The
at 4#{176}C
for
antisera
separately
The
decolonized
with
on
formalin
water,
two
washed
deposited
stained
film
7.4
saline
and
dilutions.
mm,
forced
ml distilled
with
pH
were
in buffered
were
were
removed
washed
4#{176}C
for
I mm
professional
in appropriate
incubated
for
coordinates
oil
and
together
7.2.
Ektachrome
immersion
markers
mixed
pH
micrometer
30 mm
fixed
Only
particles
solution
from
surgery.
in phosphate-buffered
in the same
and
was aspirated
cardiac
Marrow
(BSA)
phosphate,
acetone)
substage
for
dried
monopotassium
ml
during
included.
albumin
to l06/ml
air
Kodak
The
(9:1)
suspended
45
with
were
serum
were
at sternotomy
for
0.01%
30
were
conjugated
slides
mm
methyl
cells
24 hr.
were
in PBS,
green
and
in PBS
to
detail.
were
with
100
and
glycerol:PBS
diluted
nuclear
slides
slides
photographed
in glycerol:PBS.
Ploempak
The
Fishenl
a covenslip,
and
with
were
Bone marrow
staining.
and
of disease
cells
staining.
rhodamine
histochemical
or lymphoma
in 5% bovine
phosphate,
Immunocytochemical
with
and
of evidence
washed
v/v.
and
decolonized
Double
free
and
by cytocentnifugation.
formaldehyde
with
cells
for anemia
to be
needle
5% EDTA
slides
acetone
marrow
evaluation
a 25-gauge
(PBS)
glass
of bone
undergoing
examined
2.1
edge
and
filter
with
filter
K480
a Leitz
system
H for
to minimize
fluorescein
quenching.
using
excitation
Photographs
incident-light
and
filter
were
excitation
system
taken
N for
with
the
with
the
rhodamine
Leitz
NPL
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NEUTROPHIL
GRANULOPOIESIS
Fluotar
100/1.32
objective
push-processed
to ASA
Controls.
were
To
(4)
for
check
prior
automatic
conjugates
for
and
the
camera
triple
the
using
staining
Kodak
method
overnight
Each
cells.
experiment
globulins
stained
Ektachrome
200
film
determined
with
the
were
The
antisera
conjugates
reaction
g for
with
cells
the
same
preincubated
with
each
were
absorbed
mixture
10 mm,
was
and
the
and
with
myeloperoxi-
incubated
for 30 mm
supernatant
lysozyme
at
was
tested
also
absorbed
were
for
antigens.
the
that
from
cells.
antiserum.
lactoferrin,
respective
by staining
and
the
The
1500
obtained
were
decolorized
wavelengths.
of target
cells
conjugated
to myeloperoxidase,
their
controlled
(above)
reagent,
at
antisera,
the
rhodamine
for staining
same
lactofernin.
centrifuged
against
was
as in (2)
and
the
the
and
immunoglobulins
tested
of
with
autofluorescence,
fluorescein
antisera,
and
of each
lysozyme,
chromatography
the
specificity
were
at 4#{176}C,
and
of target
of
produced
at the
conjugated
monospecificity
G, elastase,
stain
excited
binding
were
and
To determine
by affinity
when
immunologic
antiserum
37#{176}C,held
May-Grtlnwald-Giemsa
nonspecific
evaluate
cathepsin
staining
the
fluorescence
to immunization
unconjugated
dase,
of the
if
observed
To
animals
(3)
Orthomat-W
experiments:
( I ) To determine
(2)
and
800.
Specificity
following
cells
181
had
slides
not
with
been
conjugated
stained
preimmunization
with
immuno-
May-Grtlnwald-Giemsa.
RESULTS
General
comments.
nofluorescence
stains,
stained
the cells with
ing,
The
In initial
experiments
we stained
with
the
May-Grtinwald-Giemsa
because
the May-Grtinwald-Giemsa
PBS wash during
the fluorescent
this effect
occurred
GrUnwald-Giemsa).
In subsequent
Romanowsky
glycerin
and
stained
and
regardless
experiments,
of
we
stain
leukocytes
did
not
cellular
fixatives
dehyde
investigated
vapor,
and
staining
with
Fluorescent
shrinkage
and
(absolute
heat)
were
or
eliminated
failed
to reveal
cytoplasmic
detail.
responsible
for the poor results,
and
stain
this
produces
(Wright,
problem
Giemsa,
by
Slight
residual
clefts.
red
blocked
the
autofluo-
stains,
by antisera
affinity
chromatography
or in liquid
phase
and by preincubation
with unconjugated
antisera.
Fluorescent
staining
was not blocked
with
cell
However,
methanol,
ethanol,
1% paraformaldehyde,
either
no better
or resulted
in diffuse
the fluorescent
conjugates.
staining
of cells was completely
May-
with buffered
were not visibly
specific
immunofluorescence.
fixative
for Romanowsky
paranuclear
or
staining
dyes were then extracted
At completion,
the cells
autofluoresce.
rescence
did not interfere
with subsequent
Formalin
acetone
is not the optimal
causes
stain
was
fixation
agent
first. The methanolic
prolonged
washing
in PBS.
the
combining
Romanowsky
and immuimmunofluorescent
reagent
first and
second.
The results
were disappoint-
since
all
it
other
formalcytoplasmic
absorbed
by
of the target
by incubation
cells
of
the antisera
with other
granule
proteins.
The conjugated
preimmunization
immunoglobulins
did not stain
the cells.
The
May-Grunwald-Giemsa
stain
did not
diminish
or alter
fluorescent
binding.
Neutrophils
were
intensely
stained
with
these
reagents
(Figs.
IA-IC).
The
conjugates
were bound
in a granular
pattern,
and no nuclear
or membrane
binding
was observed.
When
the cells were stained
simultaneously
for two primary
granule
markers,
the conjugates
appeared
to be bound
by the same
granules.
However,
combinations
of a primary
granule
marker
and lactoferrin
produced
dissimilar
reaction
patterns,
and the two markers
appeared
to be present
in separate
granules.
This
distinction
was most
easily
demonstrated
in ruptured
cells
in which
the
individual
granules
were
more
dispersed
(Figs.
ID
and
lE).
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
182
PRYZWANSKY
Fig.
1.
See
legend
facing
page.
ET
AL.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
NEUTROPHIL
GRANULOPOIESIS
183
Triple
staining
ofbone
marrow
cells.
Myeloid
cells, monocytes,
and reticulum
cells were stained
with one or more
of these
reagents.
Monocytes
were strongly
positive
for lysozyme
and weakly
positive
for myeloperoxidase
but were negative
for
elastase,
cathepsin
G, and lactoferrin.
Reticulum
cells were occasionally
positive
for lysozyme
but negative
for all other
markers.
macrophages
was strongly
autofluorescent,
as were
marrow
elements
did not stain
(Figs.
IA-IC).
Promyelocytes
were
demonstrated.
(Figs
I D and
azurophilic
They
I E).
granule
earliest
myeloid
cells
in which
were positive
for myeloperoxidase,
The immunofluorescent
intensity
granules
primary
the
observed
marker
in the
proteins
not as intense
as myeloperoxidase
distinct
granule.
Lactoferrin
was
were
identified
or cathepsin
not present
distinguished
the
in pigmented
granules.
Other
markers
elastase,
appeared
could
be
and cathepsin
to correlate
May-GrUnwald-Giemsa
were
stain.
G
with
Occasionally,
immunocytochemically
azurophilic
granules
were seen in the Romanowsky
faint and difficult
to photograph
due to fluorescent
long exposure
times required.
Lysozyme
was also
Myelocytes
The
pigment
eosinophilic
before
stains,
but the reactions
were
quenching
occurring
during
the
present
in promyelocytes
but was
G and may
in promyelocytes
have
in May-Grunwald-Giemsa
been
(Figs.
stains
present
in a
I F-I H).
by the
charac-
teristic
“dawn
of neutrophilia”;
identification
of lactoferrin
was the immunofluorescent
counterpart
of this tinctorial
change
(Figs.
11-1 K). In early
myelocytes
with predominantly
basophilic
cytoplasm,
lactoferrin
fluorescence
was localized
a small
perinuclear
cytoplasm
even
the cytoplasm
increase
remained
and
region,
but
in most
cells
it was
distributed
though
the Romanowsky
stain showed
was “neutrophilic.”
Both
lactoferrin
as myelocytes
matured,
the same or decreased
Mature
cells.
did not differ
but myeloperoxidase,
in intensity
(Figs.
Metamyelocytes,
from circulating
bands,
PMN.
and
that
and
the
only a small portion
lysozyme
appeared
cathepsin
1L-IN).
PMN
to
throughout
were
G,
positive
and
of
to
elastase
for all markers
DISCUSSION
This
report
demonstrates
protein markers
same cells. The
antigens
Fig.
Bone
(B),
and
1.
the
specificity
Simultaneous
marrow
and
reacted
rhodamine-labeled
conjugates
demonstrates
idase
rhodamine-labeled
(D),
In identical
Giemsa
(F),
Lactoferrln
is not
specific
granule
Giemsa
(L),
decreased
(F-H)
In the
cell.
fluorescein-labeled
more
Intense
or remained
and
more
same.
staIns
(I-K)
(1-N)
mature
and
Similar
results
stained
myelocytes
(top
were
and
human
bone
marrow.
(A-C)
marrow
elements
green.
Rupture
conjoint
binding
stained
(M)
with
bottom),
with
rhodamine-labeled
In more
one
mature
showing
band
while
lactoferrin.
of
the
of both
May-Gr#{252}nwaldcells
the
stained
an
the
antllactoferrin
(H).
In this
field.
(I), fluorescein-
May-Gr#{252}nwald-Glemsa
(K)
by
antlmyeloperox-
the
reactive
with
were
antlmyeloperoxldase
other
rhodamine-labeled
seen
study
methyl
and
and
of the
original
with
antllactoferrin
G (N),
in this
fluorescein-labeled
myelocyte
specific
staining
of the
emphasizes
and
Is strongly
Myelocyte
cells
and
(G),
but
Three
of
with
counterstained
(P)
used
of normal
staining
granules
rhodamlne-labeled
antlcathepsin
In the
the
(E)
of the
promyelocyte
and
antisera
stained
of
Romanowsky
on the purity
(A), fluorescein-Iabeled
Negative
E) Promyelocyte
Promyelocyte
In all cells.
G (J),
marker
The
antlmyeloperoxldase
In the
Is present
anti-cathepsin
(C).
(0,
dispersion
granules.
present
labeled
identification
and Romanowsky
antlelastase
allowed
antisera.
May-Gr#{252}nwald-Giemsa
antilactoferrin
fluorescein-labeled
Myeloperoxidase
appears
with
specifIcIty.
membrane
markers
immunocytochemical
of the
immunofluorescent
neutrophil
cytoplasmic
that
can be successfully combined
with
success
of the method
is dependent
appearance
with
of
May-Gr#{252}nwald-
antilysozyme
(N).
azurophillc
granule
Lysozyme
marker
a
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1 84
PRYZWANSKY
highly
reactive
and
monospecific.
antigens,
and
conjugate
observed.
This
is not
weaker
than
The
patterns
specificity
and
due
results
confirm
which
contain
azurophilic
lactoferrin.
and
with
Thus
secondary
are
and
are
granules
and
data
and
antecedent
immunologic
are
synthesized
that
for the
preparations
can
yield
at
promyelocytes,
primary
reliably
valuable
are
Maymeth-
granule
cathepsin
G and that myelocytes
always
positive
for the secondary
Romanowsky-stained
secondary
the
Fc
reaction
demonstrate
the
and normoblasts
granules
positive
not
is much
the
biochemical
we showed
always
was
to demonstrate
that
Also,
subsequent
In addition,
elastase,
granules
used
AL.
respective
lgG
granule
markers
of lymphocytes
consistent
and
granules,
markers
myeloperoxidase,
ing specific
(neutrophilic)
primary
primary
maturation.
their
receptors
demonstration
and secondary
lack of staining
are
to
to unaggregated
usually
the
of reagent
specificity.
did not interfere
with
that
of cellular
bound
Fc
due
IgG
and
of monocytes
stages
tightly
binding
experiments
staining
distinct
marker
since
were distinct
for primary
of each antiserum.
The
the limited
were
membrane-associated
to aggregated
blocking
another
demonstration
GrUnwald-Giemsa
stains
ods.
The
by
surprising,
binding
receptors.”
They
binding
ET
containgranule
demonstrate
information
concerning
these particles.
However,
which
Whether
these
apparatus6
cannot
primary
azurophilic
granule
granules
proteins
were
or in mature
be
considered
granule
markers
were
not
present
granules
determined.
were
seen
that
in the
were
Nevertheless,
myeloblasts
proteins.
The
present
with
the
endoplasmic
not stained
it is clear
in conventionally
results
also establish
in some
stained
that
immature
cells
May-Grunwald-Giemsa
reticulum
with
that
the
and
Golgi
Romanowsky
some
cells
preparations
most
primary
in
stain.
that
agent
would
contain
granule
be
primary
proteins
appear
simultaneously
in promyelocytes.
Lysozyme
is unique
because
it is present
in both classes
of granules.23
Spitznagel
et al.3 showed
that
primary
granules
can be separated
into “slow”
and “fast”
sedimenting
bands
by careful
cell fractionation
techniques
and that myeloperoxidase predominates
in the slow fraction
while lysozyme
is a major
constituent
of the
fast band.
Accordingly,
strongly
reactive
for
it was interesting
myeloperoxidase,
to find
contained
that some early
minimal
amounts
This suggests
that slow granules
are synthesized
first.
sensitivity
of the antisera
could also explain
the finding,
required
to resolve
this issue.
The
finding
that
the primary
granule
markers
lactoferrin
undoubtably
entirely
and
lysozyme
became
due to continued
consistent
with
synthesis
a previous
more
intense
of secondary
report
by
in
However,
differences
and further
studies
became
less
developing
granules
Bainton
promyelocytes,
of lysozyme.
et
intense
while
myelocytes
in these
al.6
in the
will be
cells,
The
demonstrate
that lactoferrin
is an excellent
marker
for secondary
granules
provide
an index of neutrophil
maturation.
In other
studies
we have
shown
that
the dual-labeled
fluorescent
technique
is useful
in studying
abnormal
blood
cells.9”2
The integration
fluorescent
technique
with conventional
Romanowsky
stains
now adds
dimension
to cellular
identification
and is a powerful
technique
anticipated
valuable
data in other
studies.
is
a result
results
and
also
can
antibody
of this
a further
to yield
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
NEUTROPHIL
185
GRANULOPOIESIS
ACKNOWLEDGMENT
We
thank
assistance,
Dr.
and
ieffnies
ian
Palmer
Hinsch
for
and
valuable
David
advice
Lonnquest
and
(E.
encouragement,
Leitz,
Inc.)
Manva
for expert
Dowdy
technical
for
technical
advice.
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1979 53: 179-185
Immunocytochemical distinction between primary and secondary granule
formation in developing human neutrophils: correlations with Romanowsky
stains
KB Pryzwansky, PG Rausch, JK Spitznagel and JC Herion
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