AB0842 A Simple Method for the Microscopic Identification of Calcium Pyrophosphate Dihydrate and Hydroxyapatite Deposits in Metabolic and Crystal Induced Diseases

Abstract

Background Arthropathy induced by calcium pyrophosphate dihydrate [Ca2P2O7.2H2O] (CPPD) crystals (chondrocalcinosis, pseudogout, pyrophosphate arthropathy) and arthropathy induced by hydroxyapatite [Ca5(PO4)3(OH)] (HA) crystals (apatite rheumatism) are separate entities. CPPD and HA crystals may exist together in association with more or less abundant amorphous calcium phosphate, or carbonate deposits. CPPD has a rhomboid shape, they range in size is from submicroscopic to 40μm. HA crystals are small, 50-500 nm, rod-shaped and are arranged typically in 1-5 μm spheroid microaggregates. Under polarized light both types of crystals show positive birefringence, but the intensity of birefringence of HA is much weaker. Objectives The aim of this study was to introduce a simple and sensitive method for identification of CPPD and HA crystals. Methods At the National Institute of Rheumatology and at the Hospital of the Order of the Brothers of Saint John of God 101,855 surgical specimens were examined histologically between 1985 and 2010, among them 10 (0.01%) with histological diagnosis of chondrocalcinosis and 3 (0.003%) with clinically diagnosed apatite rheumatism (Milwaukee syndrome). Seventeen (17) tissue samples of 10 patients with CPPD and 13 tissue samples of 3 patients with HA deposits were studied in serial sections stained with haematoxylin-eosin and in unstained sections viewed under polarized light. Results In formaldehyde fixed and haematoxylin-eosin stained sections, viewed under polarized light, CPPD crystals were detected in 8 of 17 tissue samples (47.1%), whereas in unstained sections CPPD crystals were present in 16 of them (94.1%). In formaldehyde fixed and haematoxylin-eosin stained sections, viewed under polarized light, HA crystals were detected in 1 of 13 tissue samples (7.69%), whereas in unstained sections HA crystals were present in 11 of them (84.61%). In unstained sections the dominant CPPD crystal deposition was accompanied with less pronounced amounts of HA in unstained sections of 2 of 10 patients (HA crystals were not detectable in haematoxylin-eosin stained sections viewed under polarized light). HA were associated sporadically with CPPD crystals in unstained sections of 1 of 3 patients of Milwaukee syndrome (scattered CPPD crystals remained visible in haematoxylin-eosin stained sections). Conclusions In formalin fixed, paraffin embedded and serially sectioned unstained sections the CPPD and HA crystals may be identified by polarizations optical methods as positively birefringent crystals of different size, shape and arrangement. The combined deposition of CPPD and HA crystals characterizes the complexity of metabolic disorders and arthropathies. The solubility of CPPD and HA crystals are different in fixatives (formaldehyde water solution), in acetone, and in solutions of dyes. The solubility and the weak birefringence of HA crystals may lead to the missed diagnosis of apatite rheumatism, whereas the presence of the less soluble CPPD crystals may lead to the misinterpretation of...

Crystal induced arthropathies—a comparative study of 40 patients with apatite rheumatism, chondrocalcinosis and primary synovial chondromatosis

“… 1. Tissue blocks of surgically removed specimens are fixed in 8% neutral buffered formalin (at pH 7.6 for >24 h at 20°C room temperature) [ 1 , 12 – 16 ]. 2.…”

“…Some authors believe that HA crystals are identical with the amorphous mineral deposits stained with Alizarin red S [6-10], although Alizarin red S staining is not specific for HA crystals [11]. Moreover, calcium and phosphorus in crystalline structure do not stain with Alizarin red S or with the von Kossa reaction [12][13][14][15][16].…”

Metabolic Diseases and Crystal Induced Arthropathies Technic of Non-Staining Histologic Sections - A Comparative Study of Standard Stains and Histochemical Reactions

“…In sections stained with H-E: MSU crystals were present in 24 and hydroxyapatite arthritis (in case of standard stains and reaction in comparison with Bély and Apáthy's "non-staining" technique) is summarized in Table 3.…”

“…In case of suspected metabolic or crystal induced disorders, we suggest analyzing the tissue samples with unstained tissue sections as well, supplemented with traditional stainings and reactions. Crystals remain detectable in unstained sections viewed under polarized light in the great majority of cases which appear negative with H-E staining [22][23][24][32][33][34].…”

“…Only those minerals or crystals can be detected microscopically by staining or histochemical reactions which remain in tissue sections after fixation, paraffin embedding and staining [17][18][19][20][21]. The probability of identification of crystals is much higher in unstained sections viewed under polarized light than in traditionally stained ones [22][23][24][25]26].…”