Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin? D Stefanovic´1, M Stefanovic´2, D Laloševic´1,3 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
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Accepted May 30, 2015
Abstract 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 selectively 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 counterstaining 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 stainers. 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 oxidation product, hematein, plus aluminum ions. These stains often are applied in combination with eosin and the dual staining process usually
Correspondence: D. Stefanovic´, 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.
DOI: 10.3109/10520295.2015.1057765
is termed, “hematoxylin and eosin” (H & E) staining. 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 technical 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: 461
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mordant blue 3 and C.I. 43820) is a synthetic sulfonphthalein dye with acid-base indicator properties (Kiernan 2008). ECR can be a red anionic stain when used alone, but forms anionic metal complexes with cations such as iron (Kiernan 1984a). These ironECR (Fe-ECR) mixtures are stable in solution for several years, unlike the metal-hematein complexes of “hematoxylins” (Kiernan 2008). Under appropriate conditions, Fe-ECR solutions could be used for monochromatic selective nuclear or myelin staining, or one-step dichromatic staining that could replace metal-hemateins and H & E, respectively (Kiernan 1984b). Fe-ECR stains nuclei and other basophilic structures blue like aluminum-hematein. Unlike hemalums, however, Fe-ECR stains are resistant to acidic solutions and can be used to stain myelin sheaths selectively like iron-hematein (Page 1965, Kiernan 1984b, 2008). Therefore, Fe-ECR mixtures could provide the benefits of both aluminum and iron hematein in one stain. Other advantages of FeECR include its stability and the reliable supply of the parent dye. Pearse (1957) used his ECR procedure to produce red staining of “enterochromaffin-like” cells, now termed cells of the diffuse neuroendocrine system (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 longer working life than previously published methods, and can be used in automated tissue stainers. We tested the replacement of hemateins with the Fe-ECR stain in several routine histological techniques. 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.
Reagents
Material and methods
Fixative and other solutions
Dyes
Neutral buffered formalin (10% formalin v/v, pH 7.2, 0.02 M) Prepared using distilled water (900 ml), 37% formaldehyde (100 ml), sodium phosphate monobasic dihydrate (1.36 g) and NaOH (0.34 g) (Kiernan 2008).
Alcian blue 8GX and eosin B were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany; 861006 and A5268, respectively). Eriochrome cyanine R and neutral red were obtained from Magnacol Ltd. (Newtown, Wales, UK). Grimelius and van Gieson staining kits, and Papanicolaou EA65 and Papanicolaou OG6 staining solutions were obtained from Bio Optica (Milano, Italy; 04-030802, 04-044822, 05-12017/L and 05-12013/L, respectively). 462
Absolute ethanol, concentrated (37%) hydrochloric acid (HCl), concentrated (70%) nitric acid (HNO3), ferric chloride (FeCl3⋅6H2O), 37% formaldehyde, glacial acetic acid, lithium carbonate (Li2CO3), paraffin oil, sodium hydroxide (NaOH), sodium phosphate monobasic dihydrate (NaH2PO4⋅2H2O) and xylene were obtained from Centrohem (Stara Pazova, Serbia). Canada balsam (Canadabalsam ductil) was obtained from Molar Chemicals KFT (Halásztelek, Hungary), DPX mountant for histology from Sigma-Aldrich Chemie GmbH (44581; Steinheim, Germany) and Histowax special 52⫺54° C from Histolab (Göteborg, Sweden). Distilled water was prepared at Pasteur Institute in Novi Sad, cooled to room temperature, then treated with ion exchange resins. Staining solutions Alcian blue pH 2.5 Dissolve 1 g Alcian blue 8GX in 90 ml 0.02 M acetateacetic 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.
De Castro’s decalcification fluid Dissolve 30 ml concentrated HNO3 in 670 ml distilled water, then add 300 ml absolute ethanol slowly.
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.
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Tissue and cytology samples Fresh samples of pig tissues were obtained from a local slaughterhouse. Samples included aorta, cerebellum, cerebrum, colon, duodenum, epiglottis, ileum, jejunum, kidney, liver, lungs, lymph node, myocardium, esophagus, salivary gland, spinal cord, spleen, sternum, stomach, tongue and trachea. Animal treatment was according to the internal regulations of the vendor. The tissues were fixed for 24 h in neutral buffered 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 regulations of the hospital. These preparations included several cases of routine surgical, endoscopic or gynecologic 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 investigated 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 ethanol, clear with two changes of xylene and mount with DPX or Canada balsam. Selective myelin staining procedure 1. 2. 3. 4.
Bring sections to distilled water. Stain in Fe-ECR solution for 15 ⫺ 20 min. Wash in tap water for 1 min. 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. 2. 3. 4.
Bring sections to distilled water. Stain for 20 ⫺ 30 min in Alcian blue solution. Wash in running tap water for 10 min. Counterstain with Fe-ECR and eosin according to the selective nuclear staining procedure described above. 5. Dehydrate preparations in three changes of ethanol, 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
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3. Rinse in 50% ethanol. 4. Stain with Papanicolaou OG6 staining solution for 1 min. 5. Rinse in 50% ethanol. 6. Stain with Papanicolaou EA65 solution for 2 min. 7. Rinse in 50% ethanol. 8. Dehydrate quickly in absolute ethanol, clear with xylene and mount with Canada balsam. Special stains Grimelius for argyrophilia and van Gieson staining were performed according to the instructions provided with each kit. Nuclei were stained with Fe-ECR (steps 1 ⫺ 5 of the selective nuclear staining procedure described above) before the van Gieson staining was performed. Dyestuff price comparison The relative costs of hematoxylin and ECR have not been investigated previously. To achieve an unbiased price comparison, we searched for prices at online chemical suppliers using the Google search engine. Key words used for the search were “hematoxylin price” and “eriochrome cyanine R price.” Only dye powders were investigated. The price per unit was calculated for each source (Table 1). The search was conducted on February 15, 2015.
Results Fe-ECR selective nuclear staining with eosin counterstaining This staining procedure gave results largely indistinguishable from H & E staining that used a regressive hemalum such as Harris’ or Mayer ’s (Bancroft and Layton 2013) (Figs. 1 ⫺ 6). Cell nuclei, bacteria and other basophilic structures were blue or bluish purple, while acidophilic elements were stained different shades of red, reddish purple and pink.
Table 1. Price comparison of hematoxylin and ECR posted by online vendors. Hematoxylin Price for a pack Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Means
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
$61.65/25 g $134.14/50 g $179.05/25 g $39.89/25 g $79.69/25 g $60.60/25 g $575/100 g $320/25 g $126.44/25 g $252.24/25 g $122.50/25 g $50.55/25 g $14.63/5 g $64.57/10 g $145.79/25 g $101.60/25 g $69/25 g $90/25 g $68.89/10 g
Price/ unit ($/g)
Eriochrome cyanine R Price for a pack
2.47 2.68 $39.10/50 g 7.16 1.60 $95/100 g 3.19 $61.80/25 g 2.42 $25.68/25 g 5.75 $84.40/25 g 12.80 $32.35/10 g 5.06 $379.76/100 g 10.09 4.90 $43/25 g 2.02 $316.80/100 g 2.93 $7.31/5 g 6.46 5.83 $50.91/25 g 4.06 $57.60/25 g 2.76 3.60 $67.50/25 g 6.89 4.88
0.78
0.95 2.47 1.03 3.38 3.23 3.80 1.72 3.17 1.46 2.04 2.30 2.70 2.23
Comparison made February 15, 2015
One difference from routine H & E stains, however, was a characteristic and unusual reddish staining of certain cells in gastrointestinal tract. Most of these cells were in lower two thirds of the intestinal crypts and had rounded or oval nuclei that were displaced toward the luminal part of the cell. The crescent shaped basal cytoplasmic region of these cells (Fig. 7) was filled with granules that were only half the size of specific granules of eosinophils or Paneth cells. A variant type of cell was wedge shaped with a nucleus that was displaced toward the basement membrane and whose triangular luminal region was filled with small stained granules. These wedge shaped cells were located mainly
Fig. 1. Endochondral ossification and bone marrow pig sternum stained with Fe-ECR and eosin.
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Price/ unit ($/g)
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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 granules. The reddish Fe-ECR stained cells had the same distribution and morphology as the argyrophilic cells.
Comparison of dye prices
Selective myelin staining
Discussion
Myelin sheaths were stained aqua blue, while red blood cells were stained deep blue to black. Other structures were colored by the cationic counterstain 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.
Fe-ECR provided selective nuclear counterstaining for Alcian blue (Fig. 9), van Gieson (Fig. 10) and Papanicolaou staining (Fig. 11).
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 control 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. 3. Pig connective tissue with peripheral nerves and blood vessels stained with Fe-ECR and eosin.
Fig. 4. Human placenta with hypovascular villi stained with Fe-ECR and eosin.
Other staining procedures
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.
Eriochrome cyanine R hematoxylin substitute 465
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Fig. 5. Human clear cell renal cell carcinoma of kidney stained with Fe-ECR and eosin.
cell granules. Precise control of the pH of the differentiator 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 cytological smears tested. At the end of the first differentiation 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 “bluing” 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.
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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 various shades of violet, purple and red, and this interferes 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 tissue strainers: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Two xylene baths, 5 min each. Two ethanol baths, 5 min each. Distilled water wash, 3 min. Fe-ECR staining, 5 min. Distilled water wash, 3 min. Acid differentiation (pH 0.95), 30 sec. Two tap water washes, 3 min each. Eosin B staining, 3 min. Three ethanol baths, 3 min each. Two xylene baths, 5 min each. 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 staining 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 resistant 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 hemalums (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 aluminum-ECR mixture gave crimson nuclei; it could not
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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-ethylcarbazole (AEC) or diaminobenzidine (DAB). Further studies are needed to show tinctorial outcomes using ECR mixtures with various cations and their possible application in histology, pathology and histochemistry. We observed a unique staining effect caused by acid differentiation. Fe-ECR stained selectively granules 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 onestep 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 Grimelius technique was used as the “gold standard’ for demonstrating DNES cells. Our comparison of FeECR and Grimelius staining demonstrated that the cells with typical DNES morphology and distribution were stained selectively with both methods. Acid alcohol differentiation produced inconsistent selective red staining by ECR, whereas differentiation in the acid solution described here produced consistent staining. Grimelius (2004) stated that
“most NE [neuroendocrine] cells are visualized by the Grimelius technique; exceptions include cholecystokinin (CCK), insulin, somatostatin and PYY cells.” Whether the latter cells could be stained by Fe-ECR requires further histochemical and immunohistochemical 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 sections stained with the Fe-ECR working solution and differentiated with alkali or iron salts showed different tinctorial properties from those following acid differentiation (Kiernan 2008). Page (1965) obtained selective myelin and erythrocyte staining after prolonged 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 immunohistochemical 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
Fig. 8. Cerebellum of pig stained with Fe-ECR selective staining for myelin and counterstained with neutral red.
Eriochrome cyanine R hematoxylin substitute 467
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Fig. 11. Pap smear stained with Fe-ECR and Papanicolaou technique (OG6 plus EA65).
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 mixtures in general are superior to working hemalum solutions (Hyman and Polding 1961, Page 1965, Llewellyn 1974, Hogg and Simpson 1975, Kiernan
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 hematoxylin substitute for several routine and special stains. It provides both selective nuclear and myelin staining. Nuclear staining with Fe-ECR closely parallels that of regressive aluminum-hemateins plus the resistance to acidic solutions of iron-hemateins. Fe-ECR is applicable to both cytological and histological 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 pathology. It appears to us, however, that H & E is an outmoded method. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
References
Fig. 10. Human kidney chronic pyelonephritis and glomerulosclerosis stained with Fe-ECR and van Gieson method.
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Bancroft JD, Layton C (2013) The hematoxylins and eosin. In: Suvarna SK, Layton C, Bancroft JD, Bancroft’s Theory and Practice of Histological Techniques, 7th ed., Churchill Livingstone, Elsevier Ltd., Philadelphia. pp. 174–178. Chapman DM (1977) Eriochrome cyanin R as a substitute for haematoxylin and eosin. Can. J. Med. Technol. 39: 65–66. Clark G (1979) Staining with chromoxane cyanine R. Stain Technol. 54: 337–344.
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Dapson R, Horobin RW, Kiernan JA (2010) Hematoxylin shortages: their causes and duration, and other dyes that can replace hemalum in routine hematoxylin and eosin staining. Biotech. & Histochem. 85: 55–63. Grimelius L (2004) Silver stains demonstrating neuroendocrine cells. Biotech. & Histochem. 79: 37–44. Hogg RM, Simpson R (1975) An evaluation of solochrome cyanine RS as a nuclear stain similar to haematoxylin. Med. Lab. Technol. 32: 301–306. Hyman JM, Poulding RH (1961) Solochrome cyanin-iron alum for rapid staining of frozen sections. J. Med. Lab. Technol. 18: 107. Kiernan JA (1984a) Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some of its iron complexes. J. Microsc. 134: 13–23. Kiernan JA (1984b) Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes. J. Microsc. 134: 25–39. Kiernan JA (2008) Histological and Histochemical Methods —Theory and Practice, 4th ed. Scion Publishing Ltd., Banbury. pp. 114–116, 131–132, 146–156.
Llewellyn BD (1974) Mordant blue 3: a readily available substitute for hematoxylin in the routine hematoxylin and eosin stain. Stain Technol. 49: 347–349. Llewellyn BD (1978) Improved nuclear staining with mordant blue 3 as a hematoxylin substitute. Stain Technol. 53: 73–77. Page KM (1965) A stain for myelin using solochrome cyanin. J. Med. Lab. Technol. 22: 224–225. Pearse AG (1957) Solochrome dyes in histochemistry with particular reference to nuclear staining. Acta Histochem. 4: 95–101. Pearse AG (1969) The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J. Histochem. Cytochem. 17: 303–313. Young B, O’Dowd G, Woodford P (2014) Wheater ’s Functional Histology–a Text and Colour Atlas, 6th ed. Churchill Livingstone, Elsevier Ltd., Philadelphia. pp. 334–335.
Eriochrome cyanine R hematoxylin substitute 469
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Accepted May 30, 2015
Abstract 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 selectively 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 counterstaining 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 stainers. 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 oxidation product, hematein, plus aluminum ions. These stains often are applied in combination with eosin and the dual staining process usually
Correspondence: D. Stefanovic´, 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.
DOI: 10.3109/10520295.2015.1057765
is termed, “hematoxylin and eosin” (H & E) staining. 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 technical 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: 461
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mordant blue 3 and C.I. 43820) is a synthetic sulfonphthalein dye with acid-base indicator properties (Kiernan 2008). ECR can be a red anionic stain when used alone, but forms anionic metal complexes with cations such as iron (Kiernan 1984a). These ironECR (Fe-ECR) mixtures are stable in solution for several years, unlike the metal-hematein complexes of “hematoxylins” (Kiernan 2008). Under appropriate conditions, Fe-ECR solutions could be used for monochromatic selective nuclear or myelin staining, or one-step dichromatic staining that could replace metal-hemateins and H & E, respectively (Kiernan 1984b). Fe-ECR stains nuclei and other basophilic structures blue like aluminum-hematein. Unlike hemalums, however, Fe-ECR stains are resistant to acidic solutions and can be used to stain myelin sheaths selectively like iron-hematein (Page 1965, Kiernan 1984b, 2008). Therefore, Fe-ECR mixtures could provide the benefits of both aluminum and iron hematein in one stain. Other advantages of FeECR include its stability and the reliable supply of the parent dye. Pearse (1957) used his ECR procedure to produce red staining of “enterochromaffin-like” cells, now termed cells of the diffuse neuroendocrine system (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 longer working life than previously published methods, and can be used in automated tissue stainers. We tested the replacement of hemateins with the Fe-ECR stain in several routine histological techniques. 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.
Reagents
Material and methods
Fixative and other solutions
Dyes
Neutral buffered formalin (10% formalin v/v, pH 7.2, 0.02 M) Prepared using distilled water (900 ml), 37% formaldehyde (100 ml), sodium phosphate monobasic dihydrate (1.36 g) and NaOH (0.34 g) (Kiernan 2008).
Alcian blue 8GX and eosin B were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany; 861006 and A5268, respectively). Eriochrome cyanine R and neutral red were obtained from Magnacol Ltd. (Newtown, Wales, UK). Grimelius and van Gieson staining kits, and Papanicolaou EA65 and Papanicolaou OG6 staining solutions were obtained from Bio Optica (Milano, Italy; 04-030802, 04-044822, 05-12017/L and 05-12013/L, respectively). 462
Absolute ethanol, concentrated (37%) hydrochloric acid (HCl), concentrated (70%) nitric acid (HNO3), ferric chloride (FeCl3⋅6H2O), 37% formaldehyde, glacial acetic acid, lithium carbonate (Li2CO3), paraffin oil, sodium hydroxide (NaOH), sodium phosphate monobasic dihydrate (NaH2PO4⋅2H2O) and xylene were obtained from Centrohem (Stara Pazova, Serbia). Canada balsam (Canadabalsam ductil) was obtained from Molar Chemicals KFT (Halásztelek, Hungary), DPX mountant for histology from Sigma-Aldrich Chemie GmbH (44581; Steinheim, Germany) and Histowax special 52⫺54° C from Histolab (Göteborg, Sweden). Distilled water was prepared at Pasteur Institute in Novi Sad, cooled to room temperature, then treated with ion exchange resins. Staining solutions Alcian blue pH 2.5 Dissolve 1 g Alcian blue 8GX in 90 ml 0.02 M acetateacetic 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.
De Castro’s decalcification fluid Dissolve 30 ml concentrated HNO3 in 670 ml distilled water, then add 300 ml absolute ethanol slowly.
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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.
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Tissue and cytology samples Fresh samples of pig tissues were obtained from a local slaughterhouse. Samples included aorta, cerebellum, cerebrum, colon, duodenum, epiglottis, ileum, jejunum, kidney, liver, lungs, lymph node, myocardium, esophagus, salivary gland, spinal cord, spleen, sternum, stomach, tongue and trachea. Animal treatment was according to the internal regulations of the vendor. The tissues were fixed for 24 h in neutral buffered 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 regulations of the hospital. These preparations included several cases of routine surgical, endoscopic or gynecologic 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 investigated 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 ethanol, clear with two changes of xylene and mount with DPX or Canada balsam. Selective myelin staining procedure 1. 2. 3. 4.
Bring sections to distilled water. Stain in Fe-ECR solution for 15 ⫺ 20 min. Wash in tap water for 1 min. 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. 2. 3. 4.
Bring sections to distilled water. Stain for 20 ⫺ 30 min in Alcian blue solution. Wash in running tap water for 10 min. Counterstain with Fe-ECR and eosin according to the selective nuclear staining procedure described above. 5. Dehydrate preparations in three changes of ethanol, 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
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3. Rinse in 50% ethanol. 4. Stain with Papanicolaou OG6 staining solution for 1 min. 5. Rinse in 50% ethanol. 6. Stain with Papanicolaou EA65 solution for 2 min. 7. Rinse in 50% ethanol. 8. Dehydrate quickly in absolute ethanol, clear with xylene and mount with Canada balsam. Special stains Grimelius for argyrophilia and van Gieson staining were performed according to the instructions provided with each kit. Nuclei were stained with Fe-ECR (steps 1 ⫺ 5 of the selective nuclear staining procedure described above) before the van Gieson staining was performed. Dyestuff price comparison The relative costs of hematoxylin and ECR have not been investigated previously. To achieve an unbiased price comparison, we searched for prices at online chemical suppliers using the Google search engine. Key words used for the search were “hematoxylin price” and “eriochrome cyanine R price.” Only dye powders were investigated. The price per unit was calculated for each source (Table 1). The search was conducted on February 15, 2015.
Results Fe-ECR selective nuclear staining with eosin counterstaining This staining procedure gave results largely indistinguishable from H & E staining that used a regressive hemalum such as Harris’ or Mayer ’s (Bancroft and Layton 2013) (Figs. 1 ⫺ 6). Cell nuclei, bacteria and other basophilic structures were blue or bluish purple, while acidophilic elements were stained different shades of red, reddish purple and pink.
Table 1. Price comparison of hematoxylin and ECR posted by online vendors. Hematoxylin Price for a pack Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Vendor Means
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
$61.65/25 g $134.14/50 g $179.05/25 g $39.89/25 g $79.69/25 g $60.60/25 g $575/100 g $320/25 g $126.44/25 g $252.24/25 g $122.50/25 g $50.55/25 g $14.63/5 g $64.57/10 g $145.79/25 g $101.60/25 g $69/25 g $90/25 g $68.89/10 g
Price/ unit ($/g)
Eriochrome cyanine R Price for a pack
2.47 2.68 $39.10/50 g 7.16 1.60 $95/100 g 3.19 $61.80/25 g 2.42 $25.68/25 g 5.75 $84.40/25 g 12.80 $32.35/10 g 5.06 $379.76/100 g 10.09 4.90 $43/25 g 2.02 $316.80/100 g 2.93 $7.31/5 g 6.46 5.83 $50.91/25 g 4.06 $57.60/25 g 2.76 3.60 $67.50/25 g 6.89 4.88
0.78
0.95 2.47 1.03 3.38 3.23 3.80 1.72 3.17 1.46 2.04 2.30 2.70 2.23
Comparison made February 15, 2015
One difference from routine H & E stains, however, was a characteristic and unusual reddish staining of certain cells in gastrointestinal tract. Most of these cells were in lower two thirds of the intestinal crypts and had rounded or oval nuclei that were displaced toward the luminal part of the cell. The crescent shaped basal cytoplasmic region of these cells (Fig. 7) was filled with granules that were only half the size of specific granules of eosinophils or Paneth cells. A variant type of cell was wedge shaped with a nucleus that was displaced toward the basement membrane and whose triangular luminal region was filled with small stained granules. These wedge shaped cells were located mainly
Fig. 1. Endochondral ossification and bone marrow pig sternum stained with Fe-ECR and eosin.
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Price/ unit ($/g)
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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 granules. The reddish Fe-ECR stained cells had the same distribution and morphology as the argyrophilic cells.
Comparison of dye prices
Selective myelin staining
Discussion
Myelin sheaths were stained aqua blue, while red blood cells were stained deep blue to black. Other structures were colored by the cationic counterstain 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.
Fe-ECR provided selective nuclear counterstaining for Alcian blue (Fig. 9), van Gieson (Fig. 10) and Papanicolaou staining (Fig. 11).
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 control 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. 3. Pig connective tissue with peripheral nerves and blood vessels stained with Fe-ECR and eosin.
Fig. 4. Human placenta with hypovascular villi stained with Fe-ECR and eosin.
Other staining procedures
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.
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Fig. 5. Human clear cell renal cell carcinoma of kidney stained with Fe-ECR and eosin.
cell granules. Precise control of the pH of the differentiator 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 cytological smears tested. At the end of the first differentiation 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 “bluing” 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.
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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 various shades of violet, purple and red, and this interferes 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 tissue strainers: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Two xylene baths, 5 min each. Two ethanol baths, 5 min each. Distilled water wash, 3 min. Fe-ECR staining, 5 min. Distilled water wash, 3 min. Acid differentiation (pH 0.95), 30 sec. Two tap water washes, 3 min each. Eosin B staining, 3 min. Three ethanol baths, 3 min each. Two xylene baths, 5 min each. 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 staining 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 resistant 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 hemalums (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 aluminum-ECR mixture gave crimson nuclei; it could not
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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-ethylcarbazole (AEC) or diaminobenzidine (DAB). Further studies are needed to show tinctorial outcomes using ECR mixtures with various cations and their possible application in histology, pathology and histochemistry. We observed a unique staining effect caused by acid differentiation. Fe-ECR stained selectively granules 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 onestep 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 Grimelius technique was used as the “gold standard’ for demonstrating DNES cells. Our comparison of FeECR and Grimelius staining demonstrated that the cells with typical DNES morphology and distribution were stained selectively with both methods. Acid alcohol differentiation produced inconsistent selective red staining by ECR, whereas differentiation in the acid solution described here produced consistent staining. Grimelius (2004) stated that
“most NE [neuroendocrine] cells are visualized by the Grimelius technique; exceptions include cholecystokinin (CCK), insulin, somatostatin and PYY cells.” Whether the latter cells could be stained by Fe-ECR requires further histochemical and immunohistochemical 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 sections stained with the Fe-ECR working solution and differentiated with alkali or iron salts showed different tinctorial properties from those following acid differentiation (Kiernan 2008). Page (1965) obtained selective myelin and erythrocyte staining after prolonged 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 immunohistochemical 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
Fig. 8. Cerebellum of pig stained with Fe-ECR selective staining for myelin and counterstained with neutral red.
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Fig. 11. Pap smear stained with Fe-ECR and Papanicolaou technique (OG6 plus EA65).
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 mixtures in general are superior to working hemalum solutions (Hyman and Polding 1961, Page 1965, Llewellyn 1974, Hogg and Simpson 1975, Kiernan
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 hematoxylin substitute for several routine and special stains. It provides both selective nuclear and myelin staining. Nuclear staining with Fe-ECR closely parallels that of regressive aluminum-hemateins plus the resistance to acidic solutions of iron-hemateins. Fe-ECR is applicable to both cytological and histological 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 pathology. It appears to us, however, that H & E is an outmoded method. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
References
Fig. 10. Human kidney chronic pyelonephritis and glomerulosclerosis stained with Fe-ECR and van Gieson method.
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