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J Clin Pathol 1984;37:1187-1194

Technical methods
Single incubation double esterase Material and methods
cytochemical reaction using a MATERIAL
single coupling reagent Buffered formalin/acetone (20 mg Na2HPO4, 100
mg KH2PO4, 30 ml distilled water, 45 ml acetone,
25 ml concentrated formalin)
DM SWIRSKY Department of Haematological 50 ml 0-1 mol/I phosphate buffer pH 8-0
Medicine, University Clinical School, Cambridge 2 5 mg naphthol AS-D chloroacetate (Sigma no
CB2 2QL N-0758) in 1 ml acetone
4 mg (4,ul) a-naphthyl butyrate (Sigma no N-8000)
in 1 ml acetone
80 mg fast blue BB salt (Gurr/BDH no 34177).
Since 1953, whem Gomoril reported on the use of
chloroacyl esters as histochemical substrates, ester- METHOD
ase cytochemistry has come to play an important 1 Fix air dried smears .n buffered formalin/acetone
part in the identification and classification of the for 30 s.
acute myeloid leukaemias.23 The substrate 2 Wash briefly in distilled water and air dry.
specificities of normal granulocytes (naphthol AS-D 3 Mix the fast blue BB vigorously with the buffer.
-chloroacetate) and normal monocytes (a-naphthyl 4 Add 1 ml of acetone to the naphthol AS-D
butyrate)4 now form the basis of identifying poorly chloroacetate, agitate until dissolved, and mix with
differentiated myeloid leukaemia cells as committed the buffer/fast blue BB.
to the granulocytic or monocytic lineages. Methods 5 Add the a-naphthyl butyrate to 1 ml of acetone,
for combining two esterase stains on a single slide35 agitate until dispersed, and mix with the buffer/fast
have enabled the simultaneous visualisation of blue BB/naphthol AS-D chloroacetate.
leukaemic cells exhibiting different substrate prefer- 6 Pour substrate solution into a Coplin jar in which
ences. All methods previously described require the the fixed slides have been placed and allow to incu-
blood or marrow smear to be stained twice, using bate at room temperature for 15-30 min.
separate coupling reagents with each substrate-for 7 Wash in distilled water.
example, fast blue BB with naphthol AS-D 8 Counterstain with aqueous haematoxylin for 2-5
chloroacetate and fast garnet GBC with a-naphthyl min.
butyrate. 9 Rinse in distilled water, air dry, and coverslip
Fast blue BB is an excellent coupling reagent using aqueous mounting medium.
when used with either naphthol AS-D chloroacetate Steps 4 and 5 should be carried out as rapidly As
or a-naphthyl butyrate as substrates. When naph- possible. Step 7 should be carried out by running
thol AS-D chloroacetate is the substrate, the reac- distilled water into the Coplin jar until the substrate
tion product is bright blue; the reaction occurs pre- solution (deep violet) has been cleared. The dark
dominantly in polymorphonuclear neutrophils and brown reaction product with a-naphthyl butyrate is
their precursors and only minimally in monocytes. soluble in organic solvents.
In sharp contrast, when a-naphthyl butyrate is the
substrate the reaction product is dark brown; the Results
reaction occurs predominantly in monocytes and
their precursors and as dot like positivity in a vari- Granulocytes and their normal and leukaemic pre-
able number of T lymphocytes.6 This difference has cursors stain bright blue. Monocytes and their nor-
allowed the development of a single incubation mal and leukaemic precursors stain dark brown. A
medium containing naphthol AS-D chloroacetate, proportion of normal T lymphocytes show localised
a-naphthyl butyrate, and fast blue BB, which pro- dark brown positivity, as do a variable proportion of
vides the same information with similar sensitivity to cells in T cell malignancies. The typical appearances
the traditional sequential staining methods used in in normal peripheral blood and bone marrow and in
performing "double esterase" stains. a variety of cases of acute myeloid leukaemia are
Accepted for publication 27 June 1984 shown in Figs. 1-6.
1188 Technical methods

Fig. 1 Normal peripheral

blood buffy coat. Three
neutrophils show blue
Fig.~~ ~ ~ ~ ~ ~~~~~~~~~~~~~clooctt ositivity.
Three monocytes show dark
'4~~~~~~~~~~~~ butyrate positivity.
One lymphocyte shows
localised dark brown
butyrate positivity, the other
is negative.
Fig.2 Normal bone
marrow. Six neutrophils
and granulocyte precursors
AMIN ~~~~~~~~~~~~~~~~~show
blue chloroacetate
One monocyte
shows dark brown butyrate
positivity. One lymphocyte
and five normoblasts are
Fig. 3 Acute myeloblastic
leukaemia (M2) marrow
~~~~smear.Eight cells
show blue
~~~~~~~~~Three cells and a
e e.
ly1mphocyte are negative.
Technical methods 1189

Fig. 4 Acute
promyelocytic leukaemia
(M3) buffy coat. Nine cells
show localised or heavy
overall blue chloroacetate
positivity. Multiple Auer
rods are present in two cells.
The remainder show fine
scattered blue chloroacetate
Fig. 5 Acute
myelomonocytic leukaemia
(M4) buffy coat. Ten blast
cells show dark brown
butyrate positivity. One
blast cell shows blue
chloroacetate positivity only
and one shows a mixture of
chloroacetate and butyrate
positivity. The remaining
cells are negative or show a
fine scatter of blue
chloroacetate granules.
Fig. 6 Acute monoblastic
leukaemia (MS) marrow
smear. Twelve blast cells
and promonocytes show
dark brown butyrate
positivity of varying
intensity. One normoblast
and the remaining
leucocytes are negative.
1190 Technical methods
Phosphate buffer at pH 8*0 gave the sharpest 2 Rozenszajn L, Leibovich M, Shoham D, Epstein J. The esterase
staining reactions, although there was little differ- activity in megaloblasts, leukaemic and normal haemopoietic
ence at pH 7-0 or pH 7-5. As the buffer pH was cells. Br J Haematol 1968; 14:605-19.
3Hayhoe FGJ, Quaglino D. Haematological cytochemistry. Edin-
increased above pH 8-0 staining with both substrates burgh: Churchill Livingstone, 1980.
became progressively weaker, especially above pH 4Li CY, Lam KW, Yam LT. Esterases in human leucocytes. J
9.0. Below pH 7-0 staining with a-naphthyl butyrate Histochem Cytochem 1973;21:1-12.
Yam LT, Li CY, Crosby WH. Cytochemical identification of
became weaker, and below pH 5*0 staining with monocytes and granulocytes. Am J Clin Pathol 1971;55:283-
naphthol AS-D chloroacetate began to disappear. 90.
6 Armitage RJ, Linch DC, Worman CP, Cawley JC. The morphol-
This work was supported by a Medical Research ogy and cytochemistry of human T-cell subpopulations defined
by monoclonal antibodies and Fc receptors. Br J Haematol
Council project grant. I thank Professor FGJ 1983;51:605-13.
Hayhoe for valuable advice.
References Requests for reprints to: Dr DM Swirsky, Department of
Gomori G. Chloroacyl esters as histochemical substrates. J His- Haematological Medicine, University Clinical School, Hills
tochem Cytochem 1953;1:469-70. Road, Cambridge CB2 2QL, England.

Simple technique to identify identification of the two compounds on the same

haemosiderin in
immunoperoxidase stained Material and methods
sections Sections were cut from biopsy specimens of surgi-
cally removed pituitary gland which had been
embedded in paraffin and fixed in formalin. Ten
CAROLE D NICKOLS Department of Morbid 3 ,tm serial sections were prepared and dried in a
Anatomy. The London Hospital, Whitechapel, Lon- 37C incubator for 12 h.
don El IBB The immunoperoxidase staining method used was
an adaptation of the method of Kovacs et al.' The
Identifying iron compounds in immunoperoxidase method differed from the original technique by
stained sections usually presents little or no problem using antihuman prolactin antiserum (Mercia
to the trained eye. The distinction between peroxi- Brocades) at a dilution of 1/1000 (diluent 0-15 M
dase positive staining and haemosiderin becomes phosphate buffered saline, pH 7.2) at 4C for 1 h on
important when one is in doubt as to which com- material obtained from surgical biopsies. Before
pound is giving the "brown positive" result. staining, the natural endogenous peroxidase activity
Recent biopsies have presented this problem. was blocked using the modified technique that
Although naturally occurring endogenous peroxi- Slocombe et a12 used in the demonstration of blood
dase activity has been blocked in the sections there group antigens.
has been confusion between the brown staining of After treatment with 1% osmium, the last stage of
haemosiderin and the positive brown staining of the the original technique before mounting the section,
3-3' diaminobenzidine tetrahydrochloride, es- the sections were gently washed in running tap water
pecially in weakly positive sections. for 5 min. They were then washed several times in
The staining of serial sections with haematoxylin distilled water. The original technique of Perls3-
and eosin and by Perls' Prussian blue is routinely heated potassium ferrocyanide and 1 % hydrochloric
performed when this problem arises. But unless a acid-was then applied to show the haemosiderin in
comparator microscope is used to check the identity the section.
of the stained cell, the problem remains. Counter- The Perls' Prussian blue and the uncounterstained
staining the peroxidase stained slide with Perls' peroxidase serial slides were compared with the
Prussian blue technique, however, permits the peroxidase section counterstained with Perls' Prus-
sian blue using a Zeiss comparison bridge mounted
on two Zeiss Standard 18 microscopes. (Carl Zeiss
Accepted for publication 14 May 1984 (Oberkochen) Limited)