1. No category

From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
1966 28: 674-682
Staining of Blood Cells with Periodic Acid/Salicyloyl Hydrazide (PA-SH): A
Fluorescent Method for Demonstrating Glycogen
J. BURNS and P. B. NEAME
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Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
Staining
of
Acid/Salicyloyl
Fluorescent
A
lIE
PERIODIC
the method
as
reaction
of
depends
oxidized
by
densed
J.
with
upon
witim
glycols
intense
blue
salicyloyl
pig
on
tissue
in tissue
Because
and
diagnostic
the periodic
aid
formed
from
standard
in blood
method
was
the
employed
omitte(l.
which
product,
acid
the
of
certain
con-
visible
solutions
for
are
subsequently
by
of
light
salicyloyl
oxidation
sites were
said
He recommended
constituent
its presence
glycogen
with
method
was
accepted
cells.
This
glycols
are
periodic
reactive
light.
reagent
in some
in the differentiation
acid/salicyloyl
hydrazide
PA-SH
of vicinal
acidified
the
The
ultraviolet
is generally
in blood
latter
a magenta-red
of vicinal
to fluoresce
the
use
demonstration
of
and
PAS
was
The
stumdied,
reaction
that
a
of Stoward,’
following
AND
METhODS
an
of
vicinal
was
blood
that
found
and
bone
type
his
to give
abnormal
been
used
as a
to determine
be suitable
if
for
cells.
fluorescent-Schiff
except
procedure
and
has
decided
method
would
marrow
to
cells
we
l)one
applied
and
normal
primitive
of leukemias,
(PA-SH)
MATERIAL
The
REACTION
glycogen
The
conjugated
is a common
because
demonstrating
compared
)
Glycogeii
sections.
glycogen
cells,
the
Periodic
(PA-SH)
NEAME
in glycogen,
now
to
as
B.
dialdehydes.
sections.
exposure
hydrazide
glycols
P.
(PAS
demonstrating
to produce
has
with
Demonstrating
AND
presence,
dialdehydes
in guinea
BURNS
to
reagent
Stoward1
hydrazide
blood
the
acid
Schiff’s
for
ACID-SCHIFF
choice
for
periodic
microscopy.
Cells
Hydrazide
Method
By
T
Blood
nmarrow
solochromne
the
best
smears,
procedure
and
(PA-AF).
black-alunm
The
counterstain
results.
Solutions
Periodic
fronm
Salicyloyl
distilled
a 50
and
temperatumre
the
lost
together
b’
and
htmt the
Sodium
One
was
left
24
water
The
BURNS,
Department
Imours
of
salicyloyl
water
to
about
replaced.
before
solution
prepared
snmears,
on
smears
were
just
thin
glass
then
treated
cent
periodic
3 by
F.I.M.L.T..
F.R.M.S.:
M.D.:
Radcliffe
(Signma)
10-15
in
distilled
was
added
minutes.
adding
5
use.
A brown
remnained
the
deposit
uninmpaired
ml.
of
three
water
to
After
cooling
glacial
acetic
ingredients
appeared
for
Pentacyano-Amino
prior
was
in
several
the
95
nml.
to
acid.
were
solution
of
room
Any
mixed
after
a
weeks.
Ferroate).
A
1 per
cent
soltmtion
to use.
slides,
were
fixed
with
absolute
methyl
alcohol
for
30
as follows:
of Pathology,
Radcliffe
Infirmary,
27, 1965; accepted
for publication
P. B. NEAaE,
of Pathology,
acid
hydrazide
for
Alternatively,
(Tri-$’odium
was
per
(BDH).
boiling
adjusted
of the
From tile Department
First submitted
Dec.
J.
in
was
for
1
solution
granm
warmed
pH
of
stock
evaporation
activity
Air-dried
University.
solution
cent
Amminoprusside
in distilled
minutes.
fresh
per
Hydrazide.
water
water
time
A
Acid.
prepared
Nuffield
Nuffleld
Infirmary,
Research
Lecturer
Oxford,
England.
March
26, 1966.
Technician
in Pathology,
in Pathology,
Oxford
Oxford
University;
Oxford.
674
Bioou,
\TOL.
28,
No.
5
(NovEER).
1966
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
OF
STAINING
BLOOD
a.
Periodic
acid
b.
Washed
in tap
solution
c.
Rinsed
d.
Salicyloyl
e.
Rinsed
in tap
f.
Rinsed
thoroughly
g.
Rinsed
twice
h.
The
barrier
exanmined,
field
(2-4
cleared
to
in
nminutes)
xylol
effect
the
and
of
nmounted
was
for
the
in
a
synthetic
fixed
b.
Old
(4 years)
c.
Fresh
and
alcohol,
the
on
both
bone
and
PA-SH
histochemmmical
Reaction
filter
fluoro
were
snmears
throughout
the
full
nmonocular
(mounted
the
study.
mnethod
substituted,
Marrow
and
the
histochenmical
and
and
its
1 and
studied.
controls
were
as
a UG
and
then
marrow
nmethod
Blood
was
as
procedure,
Various
having
used
binocular
factors
served
reagents
of
full
with
and
and
nmicroscope,
exciter
following
blood
individual
the
PA-SH
applied
Fresh
The
constituents
Wetzlar
The
performed
utilized.
unfixed
the
Leitz
lamp.
autofluorescence
various
conmbinations,
of
and
a
200
was
was
for
with
H.B.
Exanmination
condenser
the
oil,
Osram
Fixed
test
Behavior
under
on
studied
a.
acid
alcohol.
a K 430.
were
mnethod
solution
water
absolute
light
a dark
various
2.
minutes)
in amnminoprusside
were
autofluorescence,
in
(20-40
distilled
Autofluorescence.
order
water
soluton
to
unmounted)
In
distilled
with
filter
and
1.
minutes)
water
imltraviolet
of
vison
675
(Uvinert).
smears
source
PA-SH
(2 minutes)
hydrazide
mountant
the
with
WITH
(10
water
twice
Dehydrated
free
CELLS
on
alone
and
results
were
noted.
Smears.
The
PA-SH
to:
unstained
snmears.
fixed
old
unstained
smears.
May-Grunwald
nmethyl
alcohol
Giemsa
and
periodic
d.
Fresh
and
old
fixed
smears
e.
Fresh
and
old
fixed
snmears,
Blood
and
with
no
(M-G.G.)
stained
snmears
after
destaining
with
acid.
after
digestion
in
were
then
which
salivary
amylase
stained
for
with
an
with
PA-SH.
30
nminutes
improved
at
alum
37
C.
haema-
toxylin.4
3. Appearance
individuals
with
of
a known
known
smears
Comparison
4.
AF methods
were
rinsed
twice
three
changes
were
Staining
blood
and
used.
marrow
Unstained
Blool
smnears
snmears
smears
from
fronm
from
individuals
(1)
above
and
the
same
as controls.
PA-SH,
by
after
and
the
PAS
and
above
three
PA-AF
Procedures.
nmethods
and
absolute
mmmethyl
Smears
compared.
from
The
PAS
and
the
PA-
as follows.
were
with
used
the
stained
Smears
PAS:
treated
disorder
were
of
were
Cells
disorder
haematological
positive
individuals
Marrow
haematological
(a)
fixed
1 per
with
for
cent
30
distilled
of sulphite
(d)
rinse
color
was
in
acid
water;
for
with haematoxylin; (h) rinsed
xylol and mounted
in picolyte.
A magenta-red
nminutes
periodic
for
Schiff’s
observed
minutes;
reagent
2 nminutes
tap
in
10
(C.
each;
(f)
water;
(i)
dehydrated
at
positive
the
alcohol.
(b)
tap
T.
Gurr)
rinsed
sites
The
snmears
water
for
for
in
tap
to
absolute
20-40
water;
when
were
2
(g)
(c)
minutes;
(e)
nuclei
alcohol,
examined
then
minutes;
stained
cleared
by
light
in
micros-
copy.
Acriflavine-Schiff
fluorescence
with
(PA-AF):
obtained
was
the
Leitz
Wetzler
The
at
the
mimethod
sites
nmicroscope
used
of
was
glycogen
using
a BG
that
described
content
12
exciter
by
Culling.5
when
the
smears
filter
and
a K
An
were
530
orange
examined
barrier
filter.
RESULTS
Auto
The
grey
fluorescence
nucleated
white
cells
autofluorescence.
brighter
than
The
in blood
in both
blood
autofluorescence
smears
and
the
and/or
mounting,
it was
slightly
largely
quenched
by
amminoprusside
erythrocytes
fluoresced
the
and
an off-white
nuclei
reduced.
marrow
in
marrow
were
more
The
solution
color.
smears
exhibited
smears
distinct.
autofluorescence
in the
was
usually
After
fixation
was,
PA-SH
a light
however,
technic.
The
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
676
BURNS
Table
h-The
PA-SH
Reaction
tinder
Various
fixed
smears
Fresh
fixed
smears
positivity
salivary
amylase
Fresh
Negative
M-G.G.
stained
destaining
with
01(1
fixed,
imnstained
Old
fixed
smears
smears
acid
after
alcohol
Positive
smears
after
Positive
salivary
anmylase
Old
Reaction
Strong
after
NEAME
Conditions
PA-SH
Condition
Freslm
AND
Variable
M-G.G.
stained
destaining
Smears
fronm
PA-SH
smears
with
acid
fresh
EDTA
after
positivity
after
smears
alcohol
Usually
blood
4 weeks
negative
Positive
of storage
Positive
in the dark
Table
2.-PA-SH
Reaction
on
Normal
Blood
PA-SH
Type
of Blood
Cell
Marrow
Postivity
+++
Diffuse,
often
Eosinoplmil
++
1)iffuse,
mildly
Basophil
+++
Granular
Promyelocyte
+
I)iffuse
\Iyelocyte
+
Diffuse
++
Lymnphocyte
mildly
Difftmse
Gramiular
\lonocyte
0 to +++
l)iffuse. granular
Megakaryocyte
+ to ++++
Diffuse,
++
Plasnma
PA-SH
granular
and
blocks
Granular
to +++
Normoblast
particulate
particulate
0 to +++
Platelet
Cells
Form
Neutrophil
Metanmyelocyte
0
cell
0 to +
Diffuse,
granimlar
Reaction
Positive
sites
exposure
sites
and
Fluorescence
to
exhibited
appeared
form.
well
The
either
nuclei
delineated.
smears
of the
cells
The
under
various
Appearance
conditions
of Blood
2 gives
Table
3 illustrates
of the
positivity
and
the
of the
the
are
of variable
intensity,
cells
and
their
structure
reaction
on
blood
and
in Table
by
in various
normal
blood
in some
pathological
was
0
Cells
with
no
+
Cells
with
a few
or
with
a diffuse,
based
positive
on the
after
was
marrow
1.
Stained
results
Cells
which
The positive
in a granular
to black
with
alum-haematoxylin
by fluorescence
microscopy.
seen
Marrow
grey
PA-SH
shown
smears
findings
the
dark
of
of
recognition
fluorescence
were
results
PA-SH
treated
of cells previously
Staining
Table
a blue
light was reduced
to a white
fluorescence.
as a diffuse
cytoplasmic
fluorescence
or
ultraviolet
the
resulted
PA-SH
and
easy
Reaction
marrow
conditions.
following
in
cells,
The
while
assessment
criteria:
reaction.
scattered
pale
granimles.
blue
with
cytoplasnmic
one
concentric
fluorescence.
ring
of
granules,
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
OF
STAINING
BLOOD
CELLS
Table
WITH
677
PA-SH
3.-PA-SH
Reaction
in Pathological
States
PA-SH
Number
Cases
Disease
Pernicious
Iron
anenmia
deficiency
Acute
crisis
Type
of Blood
Cell
3
Megaloblast
1
Normoblast
anemia
aplastic
of
10
Giant
Reaction
Positivity
Form
0
0 to ++
pro-
++
Diffuse
to +++
Granular
0 to +++
Diffuse,
erythroblast
Erythroleukemia
2
Erytlmroblast
1
Myeloblast
2
Lynmphoblast
0 to ++++
Granular,
1
Monocyte
+ to ++
Diffuse
7
Lymphocyte
0 to +++
Granular
7
Lymphocyte
0 to +++
Granular
granular
Acute
nmyeloblastic
leukemia
Acute
lynmphoblastic
0
leukemia
Acute
block
monocytic
(Schilling
Chronic
leukemia
type)
lymphatic
Infectious
leukemia
mononucleosis
Table
4.-PA-SH
Scores
in Chronic
Lymphatic
Infectious
Mononucleosis
PA-,SH
Score
on
Distribution
Disease
Chronic
Leukemia
100 Consecutive
and
Lymphocytes*
of Positivity
++
Total
Scoret
0
+
+++
1
35
51
13
1
80
2
15
80
5
0
90
lymphatic
leukemial
Cases
3
80
20
0
0
20
4
56
38
6
0
50
5
46
49
4
1
60
6
68
32
0
0
32
7
48
47
5
0
57
93
Infectious
mononucleosis
Cases
1
45
21
30
4
2
39
35
26
0
87
3
39
20
29
12
114
4
31
52
17
0
86
5
6
40
46
36
22
20
22
4
10
88
96
7
41
27
21
11
#{176}Rating according
PA-SH
range
Antimitotic
to
on
20
Quaglino
therapy
in
§ Paul Bunnell
positive
++
Cells
and
normals;
total
all
in
Haylmoe.6
score
11-58.
cases.
all
with
rings
102
cases.
a
of
nmoderate
granules,
concentration
or
a
of
moderately
granules,
intense,
with
diffuse
two
concentric
cytoplasmic
fluo-
rescence.
+++
Cells
with
plasmic
++++
In
Table
mononucleosis
Cells
4
the
are
a
high
showing
a block
findings
shown
concentration
of
granules
or
an
intense
blue
cyto-
fluorescence.
in
or
blocks
in chronic
detail.
The
of
fluorescent
lymphatic
lymphocytes
material.
leukemia
and
infectious
were
rated
according
to
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
BURNS
678
Fig. 1.-NeLmtrophil
Fig.
Q uaglino
comparable
glycogen
and
with
scores
Bergna,
their
2.-Lymphocyte
Mednicoff
cases
of
differences,
above
Figures
showing
1Iayhoe,
those
chrommic
the
the
and
time results
obtained
by them
lymphatic
are
lymphocyte
normal
1-fl show
intense cytoplasmic
fluorescent
found
iim some
of
and Dameslmek,7
however,
perience,
showing
in
leukemia
when
related
in
PAS
using
the
mononucleosis
range.
examples
of some
of the
results
cytoplasm.
leukemia
reaction.
The
the results
of
elevated
scores
to treatment.6
infectious
its
lymphatic
the
our cases
differ
from
who
noted
greatly
apparently
score
chronic
with
NEAME
fluorescence.
stippling
in
AND
obtained.
PAS
In
method.
our
limited
has
been
are
low
Mitus,
in all
These
exwell
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
STAINING
OF
BLOOD
CELLS
PA-SH
WITH
Fig. 3.-Megakaryocyte
679
showing
blocks
of fluorescence.
L
1.
Fig.
4.-Giant
proerythroblast,
from
an
acute
aplastic
crisis,
showing
fluorescent
granules.
of (lie
(70fl1/)ari.S’OH
There
seemed
the
good
the
contrast
technic
PAS
between
of
the
positive
microscope
prove
PA-AF
of
PAS
and
than
to be
reaction.
Imegative
in the
a
more
Methods
time PAS
time PA-Sil
l)etweelm
fluorescence
color
will
and
correlation
i)lue
magenta-red
fluorescent
SH
was
timat
PA-SI!,
ordinary
sensitive
and
PA-SH
methods
reaction
corresponded
The
sites,
light
cytological
however,
tlman
and
the
in
the
Whether
the
PA-
the
reaction
was
microscope.
method
detail
and
it
witlm
clearer
PAS
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
BURNS
680
from
Fig. 5.-Erythroblasts,
p1mg
of one
Fig.
cell and
diffuse
a case
of
cytoplasmic
6.-Lymphoblast,
from
a
erythroleukaemia,
fluorescence
case
of
showing
AND
NEAME
stip-
fluorescent
of another.
acute
leukaemia,
showing
blocks
of
H norescence.
requires
further
investigation
.
Fl uoresceimt
inetimods,
however,
results
wlmen
are
usually
immore
SensitiVe.
Time
PA-AF
Nevertheless,
imietimod
in our
identifying
glycogen
reasonable
results.
gave
opinion
in
blood
It certainly
relatively
time
good
imiethod
PA-AF
cells
could
loses
because
of the
not
comimpare
its
value
vorkimmg
atteimtion
required
with
PA-Sil
time
efficieimtlv.
as a method
for
to obtaimm
method
in
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
STAINING
OF
obtaining
CELLS
consistent
rescent
the
BLOOD
and
cells,
WITH
results,
or in the
nonfluorescent
staiimed
sites.
by the
681
PA-SH
In
PA-AF
striking
contrast
addition
method,
the
tended
obtained
nucleus
between
and
to take
the
fluo-
cytoplasimi
up a yellow
of
staiim.
DIscussIoN
The
method
various
used
ways
by
lmy(lrazide
salicyloyl
length
of
found
time
that
o1)tinmum
is that
method
range
smears.
( P11
given
)
first,
for
the
structure
ated
and
this
technic
at the
the
sites
aimd form
of the
usually
could
no difficulty
and
ph
was
the
best
the
pH
of the
oxidation
the
as
of
i)e easily
encountered
and
additioim,
time
ultimately
on
l)lood
and
periodic
acid
to time
glycols,1
but
subse-
to make
imo differeimce
couimterstain,
used
interfered
In
tried
acid
We
results
vicinal
seemed
black-alum
it
reageimt
lim
exacted.
gave
time
initially
periodic
values.
text
individual
was
We
the
adjusted
of positivity.
cell
Stowarcl.’
of utiliziimg
reagents
procedure
solocimrome
1)ecause
oliTlitte(1
obtained
cence
the
in the
at
(1tlently
dispelmse(1
with
this
th(
l1ltilfl1tt(.
result.
Time
Stoward,’
all
of
We,
by
and
conceimtrations
application
the
described
time solutions
at different
of
marrow
bone
us
of preparnmg
with
addition,
the
without
nucleated
blood
identified.
During
fluores-
time counterstaiii
cell
was
the
the
iii preparing
blue
to
1w
well
deliime-
period
of usiimg
reagents
or
their
iii
utilization.
In centers
prove
where
an ultraviolet
to be a useful
adjunct
lTIa_y
that
only
ai)ly
used
hydrazine-type
for
tiomm of
vicitmal
technic
is also
reagents,
and
imot Schiff-type
dialdehydes
demonstrating
glycols
in
useful
a
microscope
is available,
the
for differentiating
leukemias.
liberated
histochemistry.
method
reagents,
by
the
is little
There
wimeim applied
PA-SH
metlmo(l
Stovard1
felt
could
periodic
doubt
to histological
be
acid
timat
relioxida-
time PA-Sil
sectioims.
SUMMARY
Stowardtm
dialdehydes
pig
tissue
tion,
to
conjugated
formed
from
with
Schiff-type
fiavine
existing
Schiff-type
technic
to
where
The
sections.
demonstrate
compared
aid
PAS
acidified
solutions
the periodic
acid
method
has
periodic
method.
positivity
P.
in
J. Stoward
Ic dialdehydos
with
method
methods
diagnosis
sectioimes
nminor
medullari,
reageimte
Es opinate
fluorescente
de
tissu
e
de
illo
Schiff
que
typo
de
IN
de
porco
pm
demonstrar
ha essite
e con
comparate
mm methodo
Ic methodo
Schiff
that
it will
conditions,
such
with
the
in guinea
minor
and
modificait
has
l)een
a fluorescent
will replace
acrithe
prove
to
as
acute
be
a useful
leukemia,
to occur.
conjugate
solutiones
formate
iii le oxydation
modificatiommes,
and
and
of blood
is known
ha
with
acid-Schiff
reaction
and
It is felt that
the PA-SH
blood
SuMMAIn0
(011
utilized,
cells,
in
fluorescent
in the
been
marrow
glycogen
the
now
of salicyloyl
hydrazide
oxidation
of vicinal
glycols
a
e que
INTEBLINCuA
acidificate
a acido
India.
Iste
de hydrazida
salicyloylic
periodic
de glycoles
vicinal
methodo
glycogeimo
con
typo
esseva
in
cellulas
Ic reaction
a acido
Schiff
a acriflavina
AP-HS
va reimplaciar
illo s’a provar
se tin
Ic
utile
existente
technica
utilisate,
con
sanguinee
periodic
fluorescente.
methodos
de adjuta
e
(‘
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
682
in
BURNS
Ic
de
diagnose
Fmositivit;lte
pro
conditiones
acido
sanguinee,
periodic
incluse
de Schiff
e reagente
leucemia
occurre
AND
NEAME
acute,
in
que
cognoscitemente.
ACKNOWLEDGMENTS
to
We
are grateful
Dr.
S. Callender,
to
Miss
Department
Nimifield
muaterial
was
Dr. A. 11. T.
obtained
Cilliami
Robb-Smith,
Gibbs,
of
Me(licifle
for
this
and
and
the
the
Gibson
in
whose
staff
of
department
the
this
haenmatologv
Laboratories,
work
was
done.
of
the
of
the
laboratories
from
whom
most
from
altmm-haematoxvl
stud.
REFERENCES
1.
Stoward.
J.:
P.
Microscopy.
of
2.
D.
P.
j.,
fluorescence
Thesis,
University
Stoward,
confirmatory
haematoxvlin
Stain
J.:
Studies
III.
The
P.
aldehvdes
5.
L.:
Roy.
for
The
C.
F.
in
D.,
of
Micr.
7.
periodate-
with
salicvlSoc.,
jami.
preparation
of
iron-
Handbook
of
and
Havhoe.
the
F.
and
of
cvtes
in
XV.:
g1cogen
content
lvmphocvtic
1958.
J.: Obdiseases.
Dameshek.
13:748,
B mmt-
acid-Sehiff
in lmphoproliferative
studies
Blood
C.
periodic
J. Path. Bact. 78:521, 1959.
Mitus.
W. J., Bergna,
L. J..
B.,
Histo-
I omu1omm.
p. 500.
on
reaction
fluorescence
demonstration
in
A.:
1963,
Qmmaglino,
in.
1962.
Technk1ues.
terworth,
6.
press).
\l.:
Culling.
37:249,
servations
present
J.
(in
Techn.
pathological
histochemiiistrv.
mucosubstances
hvdrazide.
Formazan
test
in
1964.
oxidised
Krimtsav.
a
204:488.
chemistry.
I 967
Mester.
groups
Nature
the
and
as
al(lehV(le
4.
Phil.
in
Oxford, 1963, p. 272.
Stoward,
reaction
3.
Studies
Mednicoff,
Cytochemuical
of
lvmpho-
proliferations.
I.