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Modified Triple Stain (Carbol Fuchsin/Alcian Blue/Hematoxylin-Eosin) for the Identification of Helicobacter pylori.

To the Editor.--We recently described an inexpensive stain that is easy to apply on the autostainer for the simultaneous visualization of Helicobacter pylori, gastritis, intestinal metaplasia, and other pathologies.[1] After adopting the procedure in our laboratory and applying it to many biopsy and surgical specimens, we have been able to shorten the procedure. The major change was to eliminate the 30-minute incubation with hydrogen peroxide at the beginning of the procedure. Instead, we have found that hydrogen peroxide can be added to the fresh carbol fuchsin stock to produce a 0.25% (vol/vol) solution (eg, 1.25 mL of hydrogen peroxide to 500 mL of carbol fuchsin stock). This stock can be used for 1 to 2 months. As with other special stains, the quality of the stain is checked with a control slide (preferably from the gastric corpus). If the bacteria do not stain satisfactorily dark, the slides are placed in carbol fuchsin for 2 minutes. If the background is too high, the slides are placed in carbol fuchsin for only 30 seconds to 1 minute. On average, the time needed in a single batch of carbol fuchsin will be 2 minutes for the first week, then 30 seconds for the second and third weeks, then 1 minute for the fourth and fifth weeks. As the solution ages, exposed slides will become murky, and a flesh carbol fuchsin batch should be used. Thick sections ([is greater than] 4 [micro]m) should be avoided to prevent the oxyntic mucosa from becoming darker than desired. Using this stain, H pylori has a red surface, the mucus is light blue, and the gastric biopsy tissue has the regular hematoxylin-eosin (H&E) staining with areas of intestinal metaplasia highlighted (Figure).


Revised Procedure

The stain is performed in our laboratory with a Sakura autostainer.

1. Deparaffinize and rehydrate to distilled water.

2. Place in carbol fuchsin (with 0.25% hydrogen peroxide) for 30 seconds to 2 minutes, as described above.

3. Rinse thoroughly in tap water for 2 minutes. Increase the wash time if the tissue is too dark.

4. Place in 1% Alcian blue in 3% acetic acid, pH 2.5, for 1 minute.

5. Rinse with tap water for 5 minutes.

6. Stain with Gill's hematoxylin for 7 minutes.

7. Rinse with tap water for 5 minutes.

8. Rinse with 0.25% ammonia water for 30 seconds.

9. Stain with eosin for 30 seconds.

10. Rinse with tap water.

11. Dehydrate to absolute alcohol, clear in xylene, and coverslip.

It has become increasingly recognized that evaluating sections stained only with H&E has a reduced accuracy for the detection of H pylori, and special stains have been recommended.24 Factors influencing the choice of a special stain include the time and expense of processing. The described triple stain uses commonly available reagents and can be performed on an autostainer,[1] which has its advantage in general laboratories.

HALA M. T. EL-ZIMAITY, MD Gastrointestinal Mucosa Pathology Laboratory, Departments of Medicine and Pathology

Veterans Affairs Medical Center and Baylor College of Medicine

Houston, TX 77030

[1.] El-Zimaity HMT, Ota H, Scott S, Killen DE, Graham DY. A new triple stain for Helicobacter pylori suitable for the autostainer. Arch Pathol Lab Med. 1998;122:732-736.

[2.] Deltenre M, Glupczynski Y, De Prez C, et al. The reliability of urease tests, histology and culture in the diagnosis of Campylobacter priori infection. Scand J Gastroenterol Suppl. 1989;160:19-24.

[3.] Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161-1181.

[4.] Kolts BE, Joseph B, Achem SR, Bianchi T, Monteiro C. Helicobacter pylori detection: a quality and cost analysis. Am J Gastroenterol. 1993;88:650-655.
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Publication:Archives of Pathology & Laboratory Medicine
Date:Oct 1, 2000
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