Preservation of calcium pyrophosphate dihydrate crystals: effect of Mayer's haematoxylin staining period

Objective-To clarify the deleterious effects of Mayer's haematoxylin staini ng procedure which result in a decrease in, or complete loss of, the number of calcium pyrophosphate dihydrate (CPPD) crystals, and to determine the p roper staining period for preserving the crystals in a histological paraffi n section of articular tissues. Methods-Paraffin sections of CPPD crystal-bearing articular tissues of six patients were stained with Mayer's haematoxylin for 3, 8, or 15 minutes, an d subsequently with eosin for one minute. The specimens were examined with an Olympus BHS polarised light microscope. The pH of Mayer's haematoxylin s olution was measured with a TOA pH meter. Results-Positive birefringent CPPD crystals were seen clearly in all specim ens stained with Mayer's haematoxylin for three minutes. The specimens stai ned for eight minutes showed a reduced number of crystals. No crystals were seen in the specimens stained for 15 minutes. Ordinary light microscopy sh owed no notable differences in the stainability of nucleus, cell membrane, and their surrounding tissues among specimens when stained with Mayer's hae matoxylin for either 3, 8, or 15 minutes. The pH of Mayer's haematoxylin so lution was 2.31. Conclusions-To find CPPD crystals in the paraffin sections of articular tis sues, the staining period with Mayer's haematoxylin should be limited to th ree minutes. The longer the staining period, the greater the reduction in t he number of crystals owing to the strong acidity of the haematoxylin solut ion. A staining period of 15 minutes causes a complete loss of CPPD crystal s.