The utility of alizarin red s staining in calcium pyrophosphate dihydrate crystal deposition disease

• Published in: Journal of rheumatology 2003-05, Vol.30 (5), p.1032-1035
• Main Authors: YAMAKAWA, Koji, IWASAKI, Hiroshi, MASUDA, Ikuko, OHJIMI, Yuko, HONDA, Itsuo, SAEKI, Kazuhiko, JINGFAN ZHANG, SHONO, Eisuke, NAITO, Masatoshi, KIKUCHI, Masahiro
• Format: Article
• Language: English
• Subjects: Aged; Anthraquinones; Biological and medical sciences; Calcium Pyrophosphate - analysis; Chondrocalcinosis - pathology; Citric Acid; Coloring Agents; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Investigative techniques, diagnostic techniques (general aspects); Male; Medical sciences; Osteoarticular system. Muscles; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Staining and Labeling - methods
• ISSN: 0315-162X; 1499-2752

OBJECTIVE: To determine the most suitable staining method for preservation and detection of calcium pyrophosphate dihydrate (CPPD) crystals in histological sections of patients with CPPD crystal deposition disease. METHODS: Paraffin sections of CPPD crystal-bearing tissues of 31 patients were stained with hematoxylin and eosin (H&E) and Alizarin red S (ARS). For H&E, the sections were treated with Mayer's hematoxylin (pH 2.3) for 5 min and with eosin alcohol (pH 4.1) for 1 min. For ARS, 1% ARS dissolved in distilled water was adjusted to pH 6.4 by adding 0.1% ammonia solution drop by drop while stirring. As controls, unstained sections were soaked in 1% citric acid monohydrate solution (CAMS, pH 2.3) for 5 or 10 min. The histological preparations were examined under a compensated polarized light using a first-order red compensator. We counted the number of weakly positive birefringent CPPD crystals in 3 high power fields (HPF, 0.272 mm2). RESULTS: CPPD crystals were seen clearly in most specimens stained with ARS, but were markedly reduced in tissue sections stained with H&E or CAMS. The number of CPPD crystals detected in sections stained by ARS (1723 +/- 683 per 3 HPF, mean +/- standard deviation) was significantly higher compared with H&E, CAMS (5 min), and CAMS (10 min) (401 +/- 374, 1022 +/- 616, and 494 +/- 636 per 3 HPF, respectively; p < 0.001, each). CONCLUSION: Standard H&E staining reduces the number of visible CPPD crystals, probably due to the strong acidity of both hematoxylin and eosin solutions, whereas the ARS stain seems to preserve a large number of CPPD crystals. The utility of ARS staining may improve the identification of CPPD crystals and contribute to a correct diagnosis of CPPD crystal deposition.