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The efficacy of the improved zirconyl hematoxylin in the diagnosis of Barrett’…


Biology Articles » Methods & Techniques » Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett’s esophagus » Discussion

Discussion
- Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett’s esophagus

The original zirconyl hematoxylin used the oxidant to hematoxylin ratio recommended by Lillie [10, 23] which left most of the hematoxylin intact for later oxidation by atmospheric oxygen during the working life of the solution. It worked well on the acidic mucins of rodent salivary glands [21] and the abnormal acidic mucin of prostatic cancer [22]. It was later observed that the staining of goblet cells by the original zirconyl hematoxylin improved markedly with the age of the solution as atmospheric oxygen converted more hematoxylin to hematein. Thus, the formula for zirconyl hematoxylin was improved by quintupling the amount of oxidant (iodate) and slightly decreasing the amount of anti-oxidant (glycerol). This is a little over the oxidant to hematoxylin ratio recommended by Harris [24]. It matches the optimal oxidant to hematoxylin ratio found by Palmer and Lillie [25] for their best samples of hematoxylin. Hematoxylin is now more uniform in quality than it was then [14]. The improved zirconyl hematoxylin stains goblet cells strongly, even when fresh, and it has a working life of one year.

The improved zirconyl hematoxylin stain is a useful alternative to alcian blue for the diagnosis of Barrett’s intestinal metaplasia of the esophagus. Zirconyl hematoxylin staining takes 20 min less time than alcian blue staining. Although alcian blue now costs 4 times as much, the difference in cost remains trivial. The real advantage of zirconyl hematoxylin is the ready availability of the ingredients. A minor advantage is that zirconyl hematoxylin can be easily removed by 30 min immersion in 1% HCl in 70% ethanol so that unique tissue sections can be restained with another stain. The removal of alcian blue requires 2 hr in 2.5 % trifluoroacetic acid in dichloromethane, which also removes some of the mucins [26].

Zirconyl hematoxylin poses no special hazards. Zirconium salts are often used in antiperspirants [27]. Hematoxylin, extracted from sappanwood in India and Malaya or from logwood in Central America, is a folk remedy for dysentery [28]. The alum lake of hematoxylin is sometimes used to stain surgical sutures [29]. Hematein (“ripened hematoxylin”) has been administered orally to hypercholesterolemic rabbits [30] and intraperitoneally to hyperlipidemic mice [31] to prevent atherosclerosis.

Lillie [32] warmly recommended methylene green as a nuclear counterstain for the periodic acid-Schiff procedure but gave no details. The procedure used here was arrived at by trial and error. Boric acid was added to the staining solution because Pease [33] suggested that borates improved staining by basic aniline dyes. Boric acid was substituted for sodium borate in order to make the stain specific for nuclei. Methylene green maximizes the color contrast between the nuclei and the goblet cell mucin. If one wished to maximize the contrast in optical density rather than color, a lighter counterstain, such as kernechtrot and tropaeolin O could be used [14, 26].

The value of screening patients with gastroesophageal reflux disease who do not have bleeding, pain, or difficulty in swallowing is debatable [7, 34]. The risk of Barrett’s esophagus progressing to adenocarcinoma is only 0.005 per patient year [34]. However, multiplying that risk by a conservative estimate of the patient’s remaining years gives a 10% risk of progression to adenocarcinoma [35]. Although the cost of screening patients with gastroesophageal reflux disease for Barrett’s esophagus and maintaining surveillance of patients with Barrett’s for adenocarcinoma is high (~$38,000 per cancer detected), it is substantially lower than the cost of mammographic screening for breast cancer (~$55,000 per cancer detected) [36].


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