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Use of eriochrome cyanine R in routine histology

and histopathology: is it time to say goodbye to
hematoxylin?
a b ac
D Stefanović , M Stefanović & D Lalošević
a
Department of Histology and Embryology, Medical Faculty, University of Novi Sad, Novi
Sad, Serbia
b
Department of Pathological Anatomy, General Hospital of Leskovac, Leskovac, Serbia
c
Pasteur Institute-National Reference Laboratory for Rabies, Novi Sad, Serbia
Published online: 14 Aug 2015.

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To cite this article: D Stefanović, M Stefanović & D Lalošević (2015) Use of eriochrome cyanine R in routine histology
and histopathology: is it time to say goodbye to hematoxylin?, Biotechnic & Histochemistry, 90:6, 461-469, DOI:
10.3109/10520295.2015.1057765

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 Use of eriochrome cyanine R in routine histology
and histopathology: is it time to say goodbye to
hematoxylin?
D Stefanović1, M Stefanović2, D Lalošević1,3
1Department of Histology and Embryology, Medical Faculty, University of Novi Sad, Novi Sad, 2Department of

Pathological Anatomy, General Hospital of Leskovac, Leskovac, and 3Pasteur Institute-National Reference Laboratory
for Rabies, Novi Sad, Serbia

Accepted May 30, 2015

Abstract
Downloaded by [Dušan Stefanovi] at 08:51 22 August 2015

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such
as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining
depending on the differentiator used. Selective nuclear staining was obtained by differentiation
in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH
difference facilitated control of differentiation. When used with an eosin B counterstain, results
were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear
staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as
well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selec-
tively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an
H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear coun-
terstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques.
With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible
with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used
for both cytological and histological samples, and was suitable for use in automated tissue stain-
ers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear
counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too
acidic.

Key words: APUD, DNES, diffuse neuroendocrine system, eosin, eriochrome, hematoxylin,
mordant blue 3, myelin staining, nuclear staining

Hematoxylin (natural black 1, C.I. 75290) has been
used, in combination with various metal ions, for
biological staining since the 19th century. The most
widely used stains, commonly termed hemalums
or “hematoxylins,” contain the hematoxylin oxi-
dation product, hematein, plus aluminum ions.
These stains often are applied in combination
with eosin and the dual staining process usually
Correspondence: D. Stefanović, Department of Histology and
Embryology, Medical Faculty, University of Novi Sad,
Hajduk Veljkova 3, Novi Sad, 21000 Serbia. E-mail: dulef90@
gmail.com
© 2015 The Biological Stain Commission
Biotechnic & Histochemistry 2015, 90(6): 461–469.
is termed, “hematoxylin and eosin” (H & E) stain-
ing. Although not perfect, H & E has become the
standard overview stain for animal histology and
routine diagnostic histopathology. Consequently,
any hematoxylin substitute must match closely
the tinctorial features of H & E. Owing to the tech-
nical and commercial complexities of hematoxylin
production and distribution, repeated shortages
of hematoxylin have occurred, most recently in
2008. Consequently, appropriate substitutes have
been sought; eriochrome cyanine R (ECR) and
celestine blue are among the preferred candidates
(Dapson et al. 2010).
ECR, also known as chromoxane cyanine R or
solochrome cyanine R (Colour Index identifiers:

DOI: 10.3109/10520295.2015.1057765 461

 mordant blue 3 and C.I. 43820) is a synthetic sulfon- Reagents
phthalein dye with acid-base indicator properties
Absolute ethanol, concentrated (37%) hydrochloric
(Kiernan 2008). ECR can be a red anionic stain when
acid (HCl), concentrated (70%) nitric acid (HNO3),
used alone, but forms anionic metal complexes with
ferric chloride (FeCl3⋅6H2O), 37% formaldehyde, gla-
cations such as iron (Kiernan 1984a). These iron-
cial acetic acid, lithium carbonate (Li2CO3), paraffin
ECR (Fe-ECR) mixtures are stable in solution for
oil, sodium hydroxide (NaOH), sodium phosphate
several years, unlike the metal-hematein complexes
monobasic dihydrate (NaH2PO4⋅2H2O) and xylene
of “hematoxylins” (Kiernan 2008). Under appropri-
were obtained from Centrohem (Stara Pazova,
ate conditions, Fe-ECR solutions could be used for
Serbia). Canada balsam (Canadabalsam ductil)
monochromatic selective nuclear or myelin staining,
was obtained from Molar Chemicals KFT (Halász-
or one-step dichromatic staining that could replace
telek, Hungary), DPX mountant for histology from
metal-hemateins and H & E, respectively (Kiernan
Sigma-Aldrich Chemie GmbH (44581; Steinheim,
1984b). Fe-ECR stains nuclei and other basophilic
Germany) and Histowax special 52⫺54° C from His-
structures blue like aluminum-hematein. Unlike
tolab (Göteborg, Sweden). Distilled water was pre-
hemalums, however, Fe-ECR stains are resistant
pared at Pasteur Institute in Novi Sad, cooled to room
to acidic solutions and can be used to stain myelin
temperature, then treated with ion exchange resins.
sheaths selectively like iron-hematein (Page 1965,
Kiernan 1984b, 2008). Therefore, Fe-ECR mixtures
Staining solutions
Downloaded by [Dušan Stefanovi] at 08:51 22 August 2015

could provide the benefits of both aluminum and

iron hematein in one stain. Other advantages of Fe-
ECR include its stability and the reliable supply of
the parent dye.
Pearse (1957) used his ECR procedure to pro-
duce red staining of “enterochromaffin-like” cells,
now termed cells of the diffuse neuroendocrine sys-
tem (DNES), but he gave no details. No one appears
to have commented on this observation, but Pearse
had a particular interest in these cells, which he and
his co-workers called amine precursor uptake and
decarboxylase (APUD) cells (Pearse 1969).
We present here a selective Fe-ECR nuclear
staining technique that is standardized, has a lon-
ger working life than previously published meth-
ods, and can be used in automated tissue stainers.
We tested the replacement of hemateins with the
Fe-ECR stain in several routine histological tech-
niques. We also compared the prices of hematoxylin
and ECR dyes from several vendors to provide an
estimate of the cost-effectiveness of ECR. Finally, we
describe the selective staining of DNES cells using
Fe-ECR.
Alcian blue pH 2.5
Dissolve 1 g Alcian blue 8GX in 90 ml 0.02 M acetate-
acetic acid buffer, pH 2.5, made from distilled water,
sodium hydroxide and glacial acetic acid. Filter the
solution and add distilled water to 100 ml.
Eosin B
Dissolve 0.25 g eosin B in a solution of 40 ml water,
40 ml ethanol and 10 ml glacial acetic acid. Filter the
solution and add ethanol to 100 ml.
Fe-ECR
Acidify 450 ml distilled water with HCl until the pH
is 1.5. Add 1 g ECR powder and 1.12 g FeCl3⋅6H2O
to the solution, mix, filter and add water to 500 ml.
Adjust the pH of the final solution to 1.5 if necessary.
Neutral red
Dissolve 0.5 g neutral red in a solution of 90 ml
distilled water and 1 ml glacial acetic acid. Filter the
resulting solution and add water to 100 ml.

Fixative and other solutions

Material and methods

Dyes Neutral buffered formalin (10% formalin v/v, pH 7.2,

0.02 M)
Alcian blue 8GX and eosin B were obtained from Sig-
Prepared using distilled water (900 ml), 37% form-
ma-Aldrich Chemie GmbH (Steinheim, Germany;
aldehyde (100 ml), sodium phosphate monobasic
861006 and A5268, respectively). Eriochrome cya-
dihydrate (1.36 g) and NaOH (0.34 g) (Kiernan
nine R and neutral red were obtained from Magna-
2008).
col Ltd. (Newtown, Wales, UK). Grimelius and van
Gieson staining kits, and Papanicolaou EA65 and
Papanicolaou OG6 staining solutions were obtained De Castro’s decalcification fluid
from Bio Optica (Milano, Italy; 04-030802, 04-044822, Dissolve 30 ml concentrated HNO3 in 670 ml distilled
05-12017/L and 05-12013/L, respectively). water, then add 300 ml absolute ethanol slowly.

462 Biotechnic & Histochemistry 2015, 90(6): 461–469

 Ethanol solutions (90 and 50% v/v)
Prepare from distilled water and absolute ethanol.
HCl differentiation solution
Dilute concentrated HCl with distilled water until
the pH is 0.95.
Li2CO3 differentiation solution
Add 1 g Li2CO3 to 100 ml distilled water.
Tissue and cytology samples
Fresh samples of pig tissues were obtained from a
local slaughterhouse. Samples included aorta, cer-
ebellum, cerebrum, colon, duodenum, epiglottis,
ileum, jejunum, kidney, liver, lungs, lymph node,
Downloaded by [Dušan Stefanovi] at 08:51 22 August 2015
myocardium, esophagus, salivary gland, spinal
cord, spleen, sternum, stomach, tongue and trachea.
Animal treatment was according to the internal reg-
ulations of the vendor.
The tissues were fixed for 24 h in neutral buff-
ered formalin. Bony tissues were decalcified in de
Castro’s fluid prior to further processing. After
fixation, tissue samples were processed manually
using an ethanol, xylene, paraffin oil, paraffin wax
sequence and embedded in paraffin. Sections were
cut at 4 ⫺ 6 μm using a Leica RM2125 RTS rotary
microtome (Leica Biosystems, Nussloch, Germany).
After mounting on slides, sections were heated for
90 min at 54 ⫺ 56° C in a ventilated histological oven
(SVF100; Bio Optica, Milano, Italy), cooled to room
temperature and washed twice for 5 min each in
both xylene and absolute ethanol.
Paraffin blocks of human tissues and monolayer
cell smears prepared for cytology were obtained from
the Department of Pathology of the General Hospital
in Leskovac, Serbia. Human material was obtained in
accordance with internal ethical guidelines and regu-
lations of the hospital. These preparations included
several cases of routine surgical, endoscopic or gyne-
cologic material (cervical Pap smears) that had been
sent for histopathological or cytological analysis.
Tissue samples were processed and cut as described
above, and monolayer cell smears were fixed with
90% (v/v) ethanol prior to staining.
Pig tissues were used to demonstrate tinctorial
features of Fe-ECR on various normal histological
samples. Human samples were used to demonstrate
features of Fe-ECR on pathologically altered tissues.
Pap smears were used to demonstrate features of
Fe-ECR on cytological samples. Thus, we investi-
gated Fe-ECR for use in cytology, histology and
histopathology.
Staining procedures
Selective nuclear staining procedure
1. Bring sections to distilled water.
2. Stain in Fe-ECR solution for 3 ⫺ 5 min.
3. Wash in distilled water until excess dye is
removed.
4. Immerse in HCl differentiation solution until
preparations turn bright red or light pink, which
usually requires up to 30 sec.
5. Wash in two changes of tap water (the pH should
be 7.0–8.0; if not, add sodium bicarbonate or
other alkali salt to the wash fluid), 1 min each
with agitation. Inspect the slides to determine
whether steps 2–5 need adjustment.
6. Counterstain with the eosin solution for 3 min,
or according to the relevant procedure if another
stain is used.
7. Dehydrate preparations in three changes of eth-
anol, clear with two changes of xylene and mount
with DPX or Canada balsam.
Selective myelin staining procedure
1. Bring sections to distilled water.
2. Stain in Fe-ECR solution for 15 ⫺ 20 min.
3. Wash in tap water for 1 min.
4. Immerse in Li2CO3 differentiation solution for
30 ⫺ 60 sec, with periodic washing in tap water
and checks by microscopy, until selective myelin
staining is achieved.
5. Counterstain in neutral red staining solution for
5 min.
6. Rinse in distilled water, dehydrate quickly in
ethanol, clear with xylene and mount in Canada
balsam or DPX.
Alcian blue staining procedure
1. Bring sections to distilled water.
2. Stain for 20 ⫺ 30 min in Alcian blue solution.
3. Wash in running tap water for 10 min.
4. Counterstain with Fe-ECR and eosin according
to the selective nuclear staining procedure
described above.
5. Dehydrate preparations in three changes of eth-
anol, clear with two changes of xylene and mount
with DPX or Canada balsam.
Papanicolaou staining procedure for cell smears
1. Fix with 90% ethanol
2. Stain nuclei using steps 2 ⫺ 5 of the selective
nuclear staining procedure described above.
Eriochrome cyanine R hematoxylin substitute 463
 3. Rinse in 50% ethanol. Table 1. Price comparison of hematoxylin and ECR
4. Stain with Papanicolaou OG6 staining solution posted by online vendors.
for 1 min. Eriochrome
5. Rinse in 50% ethanol. Hematoxylin cyanine R
6. Stain with Papanicolaou EA65 solution for
2 min. Price/ Price/
7. Rinse in 50% ethanol. Price for a unit Price for a unit
8. Dehydrate quickly in absolute ethanol, clear pack ($/g) pack ($/g)
with xylene and mount with Canada balsam. Vendor 1 $61.65/25 g 2.47
Vendor 2 $134.14/50 g 2.68 $39.10/50 g 0.78
Vendor 3 $179.05/25 g 7.16
Special stains Vendor 4 $39.89/25 g 1.60
Grimelius for argyrophilia and van Gieson stain- Vendor 5 $95/100 g 0.95
ing were performed according to the instructions Vendor 6 $79.69/25 g 3.19 $61.80/25 g 2.47
provided with each kit. Nuclei were stained with Vendor 7 $60.60/25 g 2.42 $25.68/25 g 1.03
Vendor 8 $575/100 g 5.75 $84.40/25 g 3.38
Fe-ECR (steps 1 ⫺ 5 of the selective nuclear staining
Vendor 9 $320/25 g 12.80 $32.35/10 g 3.23
procedure described above) before the van Gieson Vendor 10 $126.44/25 g 5.06 $379.76/100 g 3.80
staining was performed. Vendor 11 $252.24/25 g 10.09
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Vendor 12 $122.50/25 g 4.90 $43/25 g 1.72

Vendor 13 $50.55/25 g 2.02 $316.80/100 g 3.17
Dyestuff price comparison Vendor 14 $14.63/5 g 2.93 $7.31/5 g 1.46
The relative costs of hematoxylin and ECR have not Vendor 15 $64.57/10 g 6.46
been investigated previously. To achieve an unbi- Vendor 16 $145.79/25 g 5.83 $50.91/25 g 2.04
ased price comparison, we searched for prices at Vendor 17 $101.60/25 g 4.06 $57.60/25 g 2.30
Vendor 18 $69/25 g 2.76
online chemical suppliers using the Google search
Vendor 19 $90/25 g 3.60 $67.50/25 g 2.70
engine. Key words used for the search were “hema- Vendor 20 $68.89/10 g 6.89
toxylin price” and “eriochrome cyanine R price.” Means 4.88 2.23
Only dye powders were investigated. The price per
unit was calculated for each source (Table 1). The Comparison made February 15, 2015
search was conducted on February 15, 2015.
One difference from routine H & E stains, how-
Results ever, was a characteristic and unusual reddish stain-
ing of certain cells in gastrointestinal tract. Most of
these cells were in lower two thirds of the intestinal
Fe-ECR selective nuclear staining with eosin
crypts and had rounded or oval nuclei that were
counterstaining
displaced toward the luminal part of the cell. The
This staining procedure gave results largely indis- crescent shaped basal cytoplasmic region of these
tinguishable from H & E staining that used a cells (Fig. 7) was filled with granules that were
regressive hemalum such as Harris’ or Mayer ’s only half the size of specific granules of eosinophils
(Bancroft and Layton 2013) (Figs. 1 ⫺ 6). Cell nuclei, or Paneth cells. A variant type of cell was wedge
bacteria and other basophilic structures were shaped with a nucleus that was displaced toward
blue or bluish purple, while acidophilic elements the basement membrane and whose triangular
were stained different shades of red, reddish luminal region was filled with small stained gran-
purple and pink. ules. These wedge shaped cells were located mainly

Fig. 1. Endochondral ossification and bone marrow pig sternum stained with Fe-ECR and eosin.

464 Biotechnic & Histochemistry 2015, 90(6): 461–469

 Fig. 2. Pig lung tissue stained with Fe-ECR and eosin.
in the luminal halves of the intestinal crypts. The
morphological features of these cells corresponded
to cells of the DNES (formerly known as APUD)
cells (Young et al. 2014). To check this, we used
the Grimelius procedure for staining argyrophilic
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granules. The reddish Fe-ECR stained cells had the
same distribution and morphology as the argyro-
philic cells.
Selective myelin staining
Myelin sheaths were stained aqua blue, while red
blood cells were stained deep blue to black. Other
structures were colored by the cationic counter-
stain in various shades of pink and reddish purple
(Fig. 8). At high magnification, the stained myelin
sheaths appeared as hollow tubules of varying
diameter; the axons were unstained.
Other staining procedures
Fe-ECR provided selective nuclear counterstaining
for Alcian blue (Fig. 9), van Gieson (Fig. 10) and
Papanicolaou staining (Fig. 11).
Fig. 3. Pig connective tissue with peripheral nerves and
blood vessels stained with Fe-ECR and eosin.
Comparison of dye prices
Price per pack and per unit of both hematoxylin and
ECR are given in Table 1. The lowest price per unit
for hematoxylin was $1.60/g, the mean price was
$4.88/g and highest price was $12.80/g. The least
expensive ECR was $0.78/g, the mean price was
$2.23/g, and the highest price was $3.80/g.
Discussion
The selective nuclear staining method using Fe-ECR
proposed here is based on techniques similar to
those of Chapman (1977) and Kiernan (1984b). The
working stain solution that we used, however, had a
relatively high Fe:ECR ratio and had been carefully
acidified with HCl. Differentiation was done in two
steps: acid wash and tap water wash.
The use of the HCl solution provided more con-
trol over differentiation than the acidified alcoholic
solution that often is recommended (Llewellyn
1974, Hogg and Simpson 1975, Chapman 1977). The
use of HCl increased technical convenience and was
critical for achieving specific red staining of DNES
Fig. 4. Human placenta with hypovascular villi stained
with Fe-ECR and eosin.
Eriochrome cyanine R hematoxylin substitute 465

Fig. 5. Human clear cell renal cell carcinoma of kidney
stained with Fe-ECR and eosin.
Downloaded by [Dušan Stefanovi] at 08:51 22 August 2015
cell granules. Precise control of the pH of the differ-
entiator proved to be the most important factor for
successful differentiation. At pH ⬍ 0.90, prolonged
acid washing tended to remove too much dye from
tissue sections, while at pH ⬎ 1.00, differentiation
tended to be incomplete and results were erratic.
When the pH of differentiation solution was set at
0.95, however, tissue sections could be left in the
acid differentiation bath for as long as 5 min with no
reduction in the intensity of the stain. We found that
a 30 sec wash in the pH 0.95 acid bath was sufficient
for differentiation of all tissue sections and cytologi-
cal smears tested. At the end of the first differentia-
tion step, sections turned bright red or pink.
The second step in the differentiation process
was a wash in tap water; this step is similar to “blu-
ing” of hemalum stained sections. The pH of tap
water should be between 7.0 and 8.0. The objective
of this step is to remove stain from all acidophilic
Fig. 6. Human intestinal metaplasia stained with Fe-ECR
and eosin.
466 Biotechnic & Histochemistry 2015, 90(6): 461–469
components of tissues and to retain blue color only
in nuclei and other basophilic structures, although
red blood cells often retained the deep red color
conferred by Fe-ECR. If the pH of tap water is too
low, some tissue components become stained vari-
ous shades of violet, purple and red, and this inter-
feres with subsequent counterstaining, e.g., eosin; if
the pH is too high, nuclear staining tends to fade.
Prolonged washing in tap water at appropriate
pH did not change the intensity of the expected
staining.
Based on our investigation, we propose the
following ECR plus eosin staining scheme as a
replacement for H & E for use with automated tis-
sue strainers:
1. Two xylene baths, 5 min each.
2. Two ethanol baths, 5 min each.
3. Distilled water wash, 3 min.
4. Fe-ECR staining, 5 min.
5. Distilled water wash, 3 min.
6. Acid differentiation (pH 0.95), 30 sec.
7. Two tap water washes, 3 min each.
8. Eosin B staining, 3 min.
9. Three ethanol baths, 3 min each.
10. Two xylene baths, 5 min each.
11. Mounting.
Other investigators also have suggested that
selective nuclear staining with Fe-ECR plus eosin
could replace H & E for histology and pathology
(Llewellyn 1965, 1974, Chapman 1977, Clark 1979,
Kiernan 1984b). In our hands, Alcian blue stain-
ing followed by Fe-ECR plus eosin gave excellent
results (Fig. 9). Unlike hemalums, Fe-ECR nuclear
staining was resistant to acidic solutions, which
made it a suitable nuclear counterstain for the
van Gieson technique (Fig. 10). An earlier Fe-ECR
method has been tested in various trichrome and
other histochemical methods by Hogg and Simpson
(1975). These investigators also obtained acid resis-
tant selective nuclear staining that was compatible
with most histochemical staining methods.
Llewellyn first dismissed (Llewellyn 1965),
then later recommended (Llewellyn 1974) Fe-ECR
nuclear staining for cytological preparations before
Papanicolaou staining. We found that Fe-ECR was
an excellent substitute for Harris’ and Gill’s hemal-
ums (Fig. 11). Bacteria were clearly stained, but ECR
could not match Romanowsky-Giemsa stains for
this particular application.
The use of ECR as a nuclear counterstain for
immunohistochemistry is problematic. Fe-ECR
mixtures are probably too acidic to be used for this
purpose. Chapman (1977) reported that an alumi-
num-ECR mixture gave crimson nuclei; it could not
 Fig. 7. Duodenum of pig with DNES cells. A) Stained with Fe-ECR and eosin. B) Stained by the Grimelius technique.
be used after chromogens such as 3-amino-9-ethyl-
carbazole (AEC) or diaminobenzidine (DAB). Fur-
ther studies are needed to show tinctorial outcomes
using ECR mixtures with various cations and their
Downloaded by [Dušan Stefanovi] at 08:51 22 August 2015
possible application in histology, pathology and his-
tochemistry.
We observed a unique staining effect caused by
acid differentiation. Fe-ECR stained selectively gran-
ules of what appeared to be DNES cells. Only Pearse
(1957) mentioned this phenomenon; he reported
in passing the specific red staining of granules of
mammalian “enterochromaffin-like” cells by a one-
step dichromatic staining method that resembled H
& E. He provided no further information despite
the fact that he was among the originators of the
APUD/DNES concept (Pearse 1969).
We stained serial sections with either Fe-ECR
plus eosin or the Grimelius method. The Grime-
lius technique was used as the “gold standard’ for
demonstrating DNES cells. Our comparison of Fe-
ECR and Grimelius staining demonstrated that the
cells with typical DNES morphology and distribu-
tion were stained selectively with both methods.
Acid alcohol differentiation produced inconsistent
selective red staining by ECR, whereas differentia-
tion in the acid solution described here produced
consistent staining. Grimelius (2004) stated that
Fig. 8. Cerebellum of pig stained with Fe-ECR selective staining for myelin and counterstained with neutral red.
“most NE [neuroendocrine] cells are visualized by
the Grimelius technique; exceptions include chole-
cystokinin (CCK), insulin, somatostatin and PYY
cells.” Whether the latter cells could be stained by
Fe-ECR requires further histochemical and immu-
nohistochemical investigation. In any case, at least
some types of DNES cells are stained by Fe-ECR
under the conditions described above.
It has been known for many years that tissue sec-
tions stained with the Fe-ECR working solution and
differentiated with alkali or iron salts showed differ-
ent tinctorial properties from those following acid
differentiation (Kiernan 2008). Page (1965) obtained
selective myelin and erythrocyte staining after pro-
longed differentiation in 10% iron alum. Later, Clark
(1979) used aqueous NH4OH for differentiation for
selective myelin staining, which reduced staining
time from an hour to a couple of minutes. In our
hands, 1% aqueous Li2CO3 provided effective and
rapid differentiation for selective myelin staining
(Fig. 8). The selective myelin staining should be
compared to Luxol fast blue and to immunohis-
tochemical staining for myelin basic protein (MBP)
in terms of complexity, cost and potential diagnostic
application in neuropathology.
Finally, we compared the cost effectiveness of
ECR and hematoxylin stains. It has been shown by
Eriochrome cyanine R hematoxylin substitute 467

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Fig. 9. Human colon, stained with Alcian blue, pH 2.5,
Fe-ECR and eosin.
others that the shelf-life and stability of Fe-ECR mix-
tures in general are superior to working hemalum
solutions (Hyman and Polding 1961, Page 1965,
Llewellyn 1974, Hogg and Simpson 1975, Kiernan
Fig. 10. Human kidney chronic pyelonephritis and
glomerulosclerosis stained with Fe-ECR and van Gieson
method.
468 Biotechnic & Histochemistry 2015, 90(6): 461–469
Fig. 11. Pap smear stained with Fe-ECR and Papanicolaou
technique (OG6 plus EA65).
1984b, 2008). A complete comparison would require
researching the effectiveness of different lots of each
dye and an accounting of the non-dye reagent costs.
Table 1 clearly indicates, however, that ECR has a
significant cost advantage over hematoxylin.
We have shown that Fe-ECR is a reliable hema-
toxylin substitute for several routine and special
stains. It provides both selective nuclear and myelin
staining. Nuclear staining with Fe-ECR closely par-
allels that of regressive aluminum-hemateins plus
the resistance to acidic solutions of iron-hemateins.
Fe-ECR is applicable to both cytological and histo-
logical samples. Another potentially useful feature
of Fe-ECR is its selective staining of DNES cells.
Although prices of ECR lots generally are lower
than those of hematoxylin, a full cost-effectiveness
study is required to determine the economic aspects
of applying ECR in routine histology and pathol-
ogy. It appears to us, however, that H & E is an
outmoded method.
Declaration of interest: The authors report no con-
flict of interest. The authors alone are responsible
for the content and writing of the paper.
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