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


CONCLUSIONS
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.
Department of Pathology, Fukuoka University School of Medicine, Fukuoka, Japan. 6789@d3.dion.ne.jp
Koji
K
Yamakawa
Hiroshi
H
Iwasaki
Ikuko
I
Masuda
Yuko
Y
Ohjimi
Itsuo
I
Honda
Kazuhiko
K
Saeki
Jingfan
J
Zhang
Eisuke
E
Shono
Masatoshi
M
Naito
Masahiro
M
Kikuchi
D000368:Aged; D000880:Anthraquinones; D002131:Calcium Pyrophosphate / Q000032:analysis; D002805:Chondrocalcinosis / Q000473:pathology*; D019343:Citric Acid; D004396:Coloring Agents; D004801:Eosine Yellowish-(YS); D005260:Female; D006416:Hematoxylin; D006801:Humans; D008297:Male; D013194:Staining and Labeling / Q000379:methods
D000880:Anthraquinones; D004396:Coloring Agents; D019343:Citric Acid; C004468:Alizarin Red S; D004801:Eosine Yellowish-(YS); D002131:Calcium Pyrophosphate; D006416:Hematoxylin