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Metabolic Diseases and Crystal Induced Arthropathies Technic of Non-Staining Histologic Sections

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Metabolic Diseases and Crystal Induced Arthropathies Technic of Non-Staining Histologic Sections ISSN: 2643-4091 Bély and Apáthy. Clin Arch Bone Joint Dis 2018, 1:007 Volume 1 | Issue 2 Open Access Clinical Archives of Bone and Joint Diseases RESEARCH ARTICLE Metabolic Diseases and Crystal Induced Arthropathies Technic of Non-Staining Histologic Sections - A Comparative Study of Stan- dard Stains and Histochemical Reactions Miklós Bély1 and Ágnes Apáthy2 1 Check for Department of Pathology, Hospital of the Order of the Brothers of Saint John of God in Budapest, Hungary updates 2Department of Rheumatology, St. Margaret Clinic, Budapest, Hungary *Corresponding author: Miklós Bély, M.D., Ph.D., D.Sc. Acad. Sci. Hung, Department of Pathology, Hospital of the Order of the Brothers of Saint John of God, H- 1027 Budapest, Frankel L. 17-19, Hungary, Tel: +361-438-8491; 063-0219-4142 Abstract tissue blocks were fixed in an 8% aqueous solution of form- aldehyde [CH2(OH)2] at pH 7.6 for > 24 hours at room tem- Arthropathy induced by monosodium salt of uric acid perature (20 °C) and embedded in paraffin. Serial tissue [C H N O ] (MSU) (gout), by calcium pyrophosphate dihy- 5 4 4 3 sections (5 microns thin) were cut. Using Bély and Apáthy’s drate [Ca P O .2H O] (CPPD) crystals (chondrocalcinosis, 2 2 7 2 “non-staining” technique, the fixation of tissue blocks, and pseudogout, pyrophosphate arthropathy) and arthropathy embedding were the same as with the standard stainings induced by hydroxyapatite [Ca (PO ) (OH)] (HA) crystals 5 4 3 and reactions. Unstained tissue sections were deparaffin- (apatite rheumatism, hydroxyapatite arthritis, calcifying te- ized, mounted and cover slipped with Canada balsam. The nosynovitis, Milwaukee syndrome, calcific tendinitis, calcific standard and unstained sections were examined with a pro- periarthritis) are regarded as distinct clinical entities. The fessional polarizing light microscope (Olympus BX51). solubility of MSU, CPPD and HA crystals in conventional fixatives (aqueous formaldehyde solution), in alcohol, ace- Results and conclusions: Bély and Apáthy’s non-stain- tone, and xylene or in solutions of dyes vary. The crystals ing technic was more effective: MSU was demonstrated in in tissues may dissolve during fixation in aqueous formal- 83 (79.05% of 105) tissue samples of 37 (78.72% of 47) dehyde solution, embedding in paraffin or during staining. patients; CPPD in 15 (60.00% of 25) tissue samples of 10 Only those minerals or crystals can be detected micro- (62.50% of 16) patients. HA crystals were detected exclu- scopically with stains or histochemical reactions which re- sively with this method: in all tissue samples (in 19 of 19; main in tissue sections after fixation, paraffin embedding or 100.0%) of all patients (in 4 of 4; 100.0%), none with the staining. The probability of identification of crystals is much traditional stains and reactions. The non-staining technique higher in unstained sections viewed under polarized light is a simple and very effective method to demonstrate crystal than in traditionally stained ones. The aim of this study was deposits in tissue samples. Handbooks of histologic meth- to compare the “non-staining” technique according to Bély ods and histochemistry do not mention this simple tech- and Apáthy (2013) with worldwide accepted stains and nique. In case of clinically or histologically suspected meta- histochemical methods: hematoxylin-eosin (H-E) staining, bolic or crystal-induced diseases it is advisable to analyze Gömöri’s methenamine silver method, Schultz staining, unstained tissue sections as well, supplemented with the Alizarin red S staining, and von Kossa’s reaction in tissue traditional hematoxylin-eosin stained ones. This approach samples of patients with clinically diagnosed gout, chondro- may also be useful in other crystal deposition induced dis- calcinosis and apatite rheumatism in order to demonstrate eases or identification of foreign bodies and refractile arte- the effectivity of crystal detections by these standard meth- facts. ods in comparison with the non-staining technique. Keywords Patients and methods: One hundred and five (105) tis- Non-staining technique, Tradicional stainings and histo- sue samples of 47 patients with clinically diagnosed gout, chemical methods, Gout, Chondrocalcinosis, Apatite rheu- 25 tissue samples of 16 patients with clinically diagnosed matism chondrocalcinosis, and 19 tissue samples of 4 patients with clinically diagnosed apatite rheumatism were studied. The Citation: Bély M, Apáthy A (2018) Metabolic Diseases and Crystal Induced Arthropathies Technic of Non-Staining Histologic Sections - A Comparative Study of Standard Stains and Histochemical Reactions. Clin Arch Bone Joint Dis 1:007 Accepted: August 02, 2018: Published: August 04, 2018 Copyright: © 2018 Bély M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Bély and Apáthy. Clin Arch Bone Joint Dis 2018, 1:007 • Page 1 of 15 • ISSN: 2643-4091 out foreign bodies and/or refractile artefacts) may exist Abbreviations simultaneously in synovial fluid, synovium or adjacent HE: Hematoxylin Eosin; MSU: Monosodium Urate- [C H- 5 bone and cartilage. Most common crystals and foreign N O ]; CPPD: Calcium Pyrophosphate Dehydrate- [Ca- 4 4 3 bodies are summarized in Table 1 [1-3]. 2P2O7.2H2O]; HA: Calcium Hydroxyapatite- [Ca5(PO4)3(OH)] Arthropathy induced by monosodium salt of uric acid [C H N O ] (MSU) (gout), by calcium pyrophosphate di- Introduction 5 4 4 3 hydrate [Ca2P2O7.2H2O] (CPPD) crystals (chondrocalci- Metabolic diseases and crystal induced arthrop- nosis, pseudogout, pyrophosphate arthropathy) and athies are characterized by deposits of crystal and/ arthropathy induced by hydroxyapatite [Ca5(PO4)3(OH)] or non-crystalline (amorphous) calcium phosphates in (HA) crystals (apatite rheumatism, hydroxyapatite ar- synovial membranes (synovium) and periarticular soft thritis, calcifying tenosynovitis, Milwaukee syndrome, tissues. Bone and cartilage may also be involved, and calcific tendinitis, calcific periarthritis) are regarded as crystals may be present in the synovial fluid as well. distinct clinical entities [4-14]. Foreign bodies and refractile artefacts may vary the his- The solubility of MSU, CPPD and HA crystals in con- topathological findings. Different crystals (with or with- ventional fixatives (aqueous formaldehyde solution), Table 1: Characteristics of most common crystals and foreign bodies in synovial fluid or synovium*. Breaking direction Breaking Crystal [Chemical formule] Shape Size (Elongation) intensity Monosodium urate (MSU)-[C H N O ]- Submicroscopic -40 μm 5 4 4 3 negative intensive needle,rod, sperule Figure 2 5-25 μm Calcium pyrophosphate dihydrate positive strong rod, rhomboid < 40 µm (CPPD) [Ca2P2O7.2H2O]- Figure 3 individual crystals: submicroscopic [1] Calcium hydroxyapatite - HA rod, clusters: shiny 50-500 nm [2] [Ca5(PO4)3(OH)], [Ca10(PO4)6OH2] or positive weak ** coins clusters of crystals: 1-5 [Ca5(PO4)3OH.2H2O] - Figure 4 μm [2] 1.9-15.6 μm [3] Octocalcium phosphate- ND ND ND ND [Ca8H2(PO4)6.5H2O] Tricalcium phosphate-[Ca (PO ) ] 3 4 2 ND ND ND ND (whitelockite) Dicalcium phosphate dehydrate ND ND ND ND [Ca2(PO4).2H2O] Calcium hydrogen phosphate dihydrate- [CaH(PO4).2H2O] (orthophosphate, positive moderate rod 1-2 μm brushite) Calcium oxalate [CaC2O4.H2O] positive variable tetrahedron, rod 1-2 μm rhomboidal, notched, negative and/or Cholesterol- [C H O]- Figure 5 variable needle-shaped cloven 5-40 μm [3] 27 46 positive -separate or clusters Maltese cross, Liquid lipid crystals- Figure 5 variable spherules 0.5-30 μm [3] positive Lithium heparin positive weak polymorphous 2-5 μm mostly Corticosteroid variable polymorphous 1-40 μm strong Maltese cross, Talc strong ovoid 1-40 μm negative Glass fiber positive strong orbed-oval variable Surgical sutures negative strong clusters variable Methylmetacrylate intensive fragments variable Metallosis (swarf) none none spotty deposits variable Maltese cross, Hemosiderin crystals weak groups 5-40 μm positive Osmium none none spotty deposits variable Ochronosis (artifact) colored refractile positive variable fragments variable collagen fragments Remark to Table 1: *Modified from Gatter and Schumacher [3] and from Gardner and McClure [4]. ** ND- no data; the terminology of hydroxyapatite is not uniform in the pertinent literature; the formula of HA is: [Ca5(PO4)3(OH)] or [Ca10(PO4)6OH2]. Bély and Apáthy. Clin Arch Bone Joint Dis 2018, 1:007 • Page 2 of 15 • ISSN: 2643-4091 Table 2: Sex, average age (range) of patients with gout, chondrocalcinosis and apatite rheumatism. Clinical diagnosis Number of patients Average age in years at biopsy Range (in years) Gout 47 52.59 85-36 Female 6 57.67 62-51 Male 41 52.17 85-36 Chondrocalcinosis 16 61.86 81-39 Female 15 61.00 81-39 Male 1 73.00 - Apatite rheumatism 4 71.33 79-66 Female 3 74.00 79-69 Male 1 66.00 - in alcohol, acetone, and xylene or in solutions of dyes as with the standard stainings and reactions. Unstained vary. The crystals in tissues may dissolve during fixation tissue sections were deparaffinized, mounted and cover in aqueous formaldehyde solution, embedding in par- slipped with Canada balsam. affin or during staining [15,16]. Only those minerals or Serial sections (5 micron thin) were stained: in case crystals can be detected microscopically by staining or of gout with H-E [20], according to Gömöri [18,20], and histochemical reactions which
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