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

Abstract

Arthropathy induced by monosodium salt of uric acid [C 5 H 4 N 4 O 3 ] (MSU) (gout), by calcium pyrophosphate dihydrate [Ca 2 P 2 O 7 .2H 2 O] (CPPD) crystals (chondrocalcinosis, pseudogout, pyrophosphate arthropathy) and arthropathy induced by hydroxyapatite [Ca 5 (PO 4) 3 (OH)] (HA) crystals (apatite rheumatism, hydroxyapatite arthritis, calcifying tenosynovitis, Milwaukee syndrome, calcific tendinitis, calcific periarthritis) are regarded as distinct clinical entities. The solubility of MSU, CPPD and HA crystals in conventional fixatives (aqueous formaldehyde solution), in alcohol, acetone, and xylene or in solutions of dyes vary. The crystals in tissues may dissolve during fixation in aqueous formaldehyde solution, embedding in paraffin or during staining. Only those minerals or crystals can be detected microscopically with stains or histochemical reactions which remain in tissue sections after fixation, paraffin embedding or staining. The probability of identification of crystals is much higher in unstained sections viewed under polarized light than in traditionally stained ones. The aim of this study was to compare the "non-staining" technique according to Bély and Apáthy (2013) with worldwide accepted stains and histochemical methods: hematoxylin-eosin (H-E) staining, Gömöri's methenamine silver method, Schultz staining, Alizarin red S staining, and von Kossa's reaction in tissue samples of patients with clinically diagnosed gout, chondrocalcinosis and apatite rheumatism in order to demonstrate the effectivity of crystal detections by these standard methods in comparison with the non-staining technique. Patients and methods: One hundred and five (105) tissue samples of 47 patients with clinically diagnosed gout, 25 tissue samples of 16 patients with clinically diagnosed chondrocalcinosis, and 19 tissue samples of 4 patients with clinically diagnosed apatite rheumatism were studied. The tissue blocks were fixed in an 8% aqueous solution of formaldehyde [CH 2 (OH) 2 ] at pH 7.6 for > 24 hours at room temperature (20 °C) and embedded in paraffin. Serial tissue sections (5 microns thin) were cut. Using Bély and Apáthy's "non-staining" technique, the fixation of tissue blocks, and embedding were the same as with the standard stainings and reactions. Unstained tissue sections were deparaffinized, mounted and cover slipped with Canada balsam. The standard and unstained sections were examined with a professional polarizing light microscope (Olympus BX51). Results and conclusions: Bély and Apáthy's non-staining technic was more effective: MSU was demonstrated in 83 (79.05% of 105) tissue samples of 37 (78.72% of 47) patients; CPPD in 15 (60.00% of 25) tissue samples of 10 (62.50% of 16) patients. HA crystals were detected exclusively with this method: in all tissue samples (in 19 of 19; 100.0%) of all patients (in 4 of 4; 100.0%), none with the traditional stains and reactions. The non-staining technique is a simple and very effective method to demonstrate crystal deposits in tissue samples. Handbooks of histologic...

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

“…In unstained sections, using a professional polarizing microscope with high, at least 100-Watt brightness, the clusters (1–5 µm) of HA crystals were visible, and were birefringent under polarized light (×100) [ 13 , 14 ]. The clusters formed aggregates of 100 µm or larger, which were visible with an objective of ×40 with non-staining techniques [ 13 , 14 ].…”

“…The birefringence of HA prisms was weakly positive (δ = 0.007) compared to the stronger positive birefringence of CPPD crystals (δ = 0.017); the large and strongly birefringent CPPD crystals dominated deceptively the microscopic fields, in cases of coexisting HA and CPPD crystals at 60-Watt illumination, or using a Red I compensator (the Red I compensator notably reduces the intensity of illumination) [ 13 , 14 ].…”

“…Serial sections were examined without staining [1,[12][13][14][15][16], with HE staining [18], as well as with special stains recommended in the literature, and were examined with the light microscope and under polarized light, respectively.…”

“…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].…”

“…In contrast to the classical staining methods the unstained technique is much more effective in the detection of crystals and crystalline structures of various metabolic disorders [1,[12][13][14][15][16].…”