J Clin Pathol: first published as 10.1136/jcp.42.8.881 on 1 August 1989. Downloaded from J Clin Pathol 1989;42:881-884 Orcein-alcian blue staining: a new technique for demonstrating acid mucins in gastrointestinal epithelium R SINGH, A W P GORTON Department ofHistopathology, Manor Hospital, Walsall, West Midlands SUMMARY Orcein-alcian blue staining, a new method for the simultaneous demonstration of sulphated and sialomucins in gastrointestinal epithelium was compared with the standard high iron diamine-alcian blue technique. Sections were oxidised with potassium permanagate and decolourised in oxalic acid. They were stained with orcein for four hours, differentiated for a few seconds in acid alcohol, and then counterstained with alcian blue for halfto one minute. There was a good correlation of results between the two methods. Orcein-alcian blue is a safer, cheaper, and quicker method than high iron diamine-alcian blue which can be safely introduced into routine laboratories for the study of acid mucins in the gastrointestinal diseases. Orcein, a naturally occurring vegetable dye, has been Table used for staining elastic fibres for many years. It is also used for the demonstration of hepatitis B antigen and Histological diagnosis No ofcases copper-associated proteins in chronic liver diseases.'2 copyright. Recently, the orcein technique has been studied for Normal mucosa (stomach, ileum, appendix, large bowel) 25 Intestinal metaplasia of stomach 5 showing the presence of sulphated mucin in gastroin- Adenomas (stomach, appendix, large bowel) 6 testinal epithelium.34 It has also been reported to be Adenocarcinomas (stomach, appendix, ileum, large bowel) II selectively positive for mucin-producing adenocarcin- Total 47 omas of the lower gastrointestinal tract; therefore, adenocarcinomas in sites other than the colon and rectum are negative for orcein staining.5 We report our BDH Poole, Dorset) in 100 ml 70% alcohol and then http://jcp.bmj.com/ findings of combining a modified orcein staining adding 1-0 ml concentrated hydrochloric acid. Sec- method with alcian blue to show the presence of tions were washed well in tap water and differentiated sulphated and sialomucins in normal, non-neoplastic, for a few seconds in acid-alcohol. Sections were again and neoplastic gastrointestinal epithelium. washed in water and stained in 1% alcian blue in 3% acetic acid (pH 2-5) for one minute. This was followed Material and methods by dehydration, clearing and mounting. The details of the 47 cases studied are shown in the CONTROL STUDY on September 28, 2021 by guest. Protected table. The resected surgical specimens were fixed in To establish that orcein stained sulphomucins in the 10% formol-saline and routinely processed for paraf- goblet cells and not sulphur-containing amino acids, fin wax embedding. Serial sections, 5 um thick, were control sections of keratinised skin, normal stomach, stained with haematoxylin and eosin, orcein, orcein- colon and rectum were subjected to a two stage alcian blue, high iron-diamine and high iron-diamine- blocking technique before orcein-alcian blue staining, alcian blue (modifications of Spicer's method).6 using sodium dithionite and mercuric chloride which Shikata's orcein method was used with a slight effectively block any S-S and S-H groups which might modification.' The sections were taken to water, then be present. The presence of such sulphur-containing oxidised in a solution of 0-25% potassium perman- amino acids can then be determined by comparison agate in 0-25% concentrated sulphuric acid for one with untreated, stained duplicate sections (HC Cook, minute. Sections were decolourised in 2% oxalic acid personal communication). and rinsed in water. They were then stained in orcein solution for four hours. This staining solution was Results prepared by dissolving 1-0 g natural orcein (Gurr, Accepted for publication 2 February 1989 Throughout the entire gastrointestinal tract, the 881 J Clin Pathol: first published as 10.1136/jcp.42.8.881 on 1 August 1989. Downloaded from 882 Singh, Gorton * .4 140-4.- A"iR.ci --i "I5I 'o* 4Wi: 'p 4A X~ ;, ' ; I- _ -. copyright. Fig 1 Section showing goblet cells ofstomach intestinal Fig 2 Section ofterminal ileum showing ocassional goblet metaplasia containing sulphated mucin (brown) and cells in the villi and crypts containing sulphomucin (brown). sialomucin (blue). (Orcein-alcian blue.) (Orcein-alcian blue.) goblet cells containing sulphated mucins stained were similar to that obtained with high iron diamine- http://jcp.bmj.com/ brown with orcein and sialomucins stained blue with alcian blue. alcian blue. With the exception of the elastic fibres, all The six benign and 11 malignant tumours stained other cells and tissues showed a faint brown staining with orcein-alcian blue and high iron diamine-alcian that was regarded as negative in this study. To blue had identical results. The benign tumours showed compare the staining results of orcein-alcian blue and a production of mixed mucins with predominance of high iron diamine-alcian blue identical areas were either sulphated or sialomucins. The malignant studied in serial sections. tumours varied from well differentiated mucin-secret- on September 28, 2021 by guest. Protected Surface foveolar epithelial cells of the stomach did ing to poorly differentiated with a corresponding not stain. Apical granules of the parietal cells of decrease in mucin content. The secretory product in all stomach showed a faint brown staining with orcein- the adenocarcinomas was a mixture of mucins with alcian blue. There was comparable negative staining of predominance of either sulphated or sialomucin. The cells with high iron diamine-alcian blue. The goblet poorly differentiated adenocarcinomas showed scanty cells in gastric intestinal metaplasia-complete and mucin in occasional goblet cells and in the lumen of incomplete type-however, stained for both sulphated gland-like structures. In mucoid carcinomas with and sialomucins (fig 1). High iron diamine-alcian blue extracellular mucin "lakes", however, there was a also showed similar results. In the terminal ileum, predominance of intra- and extracellular sialomucin close to the ileocaecal valve, traces of sulphomucins and little sulphomucin production. The sections were seen in occasional goblet cells (fig 2). In the left treated with dithionite stained with orcein-alcian blue colon sulphomucins were seen in the goblet cells in the showed undiminished staining in the goblet cells. The lower half of the crypt and sialomucins in the upper specificity and the intensity ofstaining with orcein was crypt (figs 3a and b); a reverse pattern was observed in similar to that seen in untreated sections stained with the right colon and the appendix. The above results orcein-alcian blue. J Clin Pathol: first published as 10.1136/jcp.42.8.881 on 1 August 1989. Downloaded from Orcein-alcian blue staining 883 F m f !aC .. ! 0 it' I ' copyright. Fig 3 Normal mucosa ofleft colon showingpredominance ofsulphatedmucin (brown) in lower crypt and sialomucin (blue) in the upper third ofcrypt: (a) orcein-alcian blue; (b) high iron diamine-alcian blue. Discussion this method, however, has yet to be investigated in other routine laboratories, and the lengthy staining Most research work carried out to study mucin time (more than 24 hours) and the relative complexity http://jcp.bmj.com/ secretion ofgastrointestinal epithelium has been based may deter laboratories from using it routinely. on only one technique-that is, the high iron diamine- Sipponen studied the distribution of cells positive alcian blue method.6 This stains sulphated mucins for orcein in the gastrointestinal tract and compared black and sialomucins blue. This technique has two the results with high iron diamine and AB 1-0 and 2-5.3 fundamental disadvantages, however: (i) The diamine With or without prior oxidation orcein, high iron reagents (N,N-dimethyl-m-phenylene-diamine and N, diamine and AB 1-0 gave similar results: it was N dimethyl-p-phenylenediamine) are carcinogenic concluded that, like high iron diamine or AB 1-0, the on September 28, 2021 by guest. Protected and hence require careful handling; (ii) it requires at orcein reactions stain sulphate groups in epithelial least an 18 hour staining period. The other drawbacks mucosubstances and that sulphonic acid residues, ofthis method are that some ofthe steps are crucial for resulting from the oxidation of sulphur groups (S-S or correct results. These include the commercial source of S-H) in the protein core of mucus glycoproteins were the reagents, the water rinse, pH of the solutions, responsible for the orcein, high iron diamine and concentration of the ferric chloride solution, and the alican blue reactions. The application ofthe dithionite use of freshly prepared diamine salts. Above all, the blocking technique, however, which gave an unchan- variations in staining method have also been reported ged orcein staining effect, meant that we were able to as giving different results.'4 show that orcein was stainipg sulphated mucin only in Recently, the KOH-AB1 0-PAPS method9 has been the material studied in spite of the oxidation step. used for detecting sulphated and sialomucins in the In this study we found that the intensity of orcein gastrointesinal epithelium and the results were com- staining was stronger with oxidation than without. pared with high iron diamine-alcian blue. It is claimed This can be explained by the fact that potassium that this technique is more sensitive in detecting cells permanganate oxidation enhances polyanionic reac- containing both types of mucins. Reproducibility of tions and also that orcein has a natural selective J Clin Pathol: first published as 10.1136/jcp.42.8.881 on 1 August 1989. Downloaded from 884 Singh, Gorton affinity for the sulphate moiety which is intensified only four hours for staining. It can therefore be after oxidation. Natural orcein also gave more stable routinely used as an aid to the diagnosis of pre- results than artificial orcein and that it could be malignant and malignant diseases of the gastrointes- combined with alican-blue.