Preservation of calcium pyrophosphate dihydrate crystals: eVect of Mayer’s haematoxylin staining period

Objective—To clarify the deleterious effects of Mayer’s haematoxylin staining procedure which result in a decrease in, or complete loss of, the number of calcium pyrophosphate dihydrate (CPPD) crystals, and to determine the proper staining period for preserving the crystals in a histological paraYn section of articular tissues. Methods—ParaYn sections of CPPD crystal-bearing articular tissues of six patients were stained with Mayer’s haematoxylin for 3, 8, or 15 minutes, and subsequently with eosin for one minute. The specimens were examined with an Olympus BHS polarised light microscope. The pH of Mayer’s haematoxylin solution was measured with a TOA pH meter. Results—Positive birefringent CPPD crystals were seen clearly in all specimens stained with Mayer’s haematoxylin for three minutes. The specimens stained for eight minutes showed a reduced number of crystals. No crystals were seen in the specimens stained for 15 minutes. Ordinary light microscopy showed no notable diVerences in the stainability of nucleus, cell membrane, and their surrounding tissues among specimens when stained with Mayer’s haematoxylin for either 3, 8, or 15 minutes. The pH of Mayer’s haematoxylin solution was 2.31. Conclusions—To find CPPD crystals in the paraYn sections of articular tissues, the staining period with Mayer’s haematoxylin should be limited to three minutes. The longer the staining period, the greater the reduction in the number of crystals owing to the strong acidity of the haematoxylin solution. A staining period of 15 minutes causes a complete loss of CPPD crystals. (Ann Rheum Dis 2001;60:80–82) Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is diagnosed by confirming both the radiological evidence of articular chondrocalcinosis and the presence of weakly positive birefringent monoclinic or triclinic crystals. When characteristic crystals are not seen in a synovial fluid, a histological examination of crystals in articular tissues becomes crucial. As a routine procedure, histological specimens are treated with haematoxylin and eosin (H&E). The staining periods of haematoxylin—namely, from five minutes to 15 minutes 3 or more, are often determined by the investigator’s personal preference. Histological specimens of articular tissues bearing CPPD crystals, treated with H&E, occasionally show no light microscopic evidence of crystals. 5 This false negative result seems to be caused by decalcification of calciumcontaining CPPD crystals through the strong acidity of the haematoxylin solution. This study was carried out to clarify the eVects of Mayer’s haematoxylin staining procedure causing a loss of CPPD crystals and to determine the proper staining period for preserving the crystals in histological specimens.