The histochemical and immunohistochemical differential diagnosis of amyloidosis in surgical pathology in a referral center is presented. Different forms of amyloidosis are considered e.g. systemic generalized amyloidosis: secondary (AA), primary (AL), senile, hemodialysis-associated, hereditary and organ (tissue)-limited (localized) amyloidosis: cerebral, dystrophic (age-related, so-called "senile"), endocrine-related, localized to tumours, focal (concentrated secretion), and isolated plasma cell (solitary plasmacytoma, B-cell) dyscrasia related amyloidosis. The amyloid deposits were identified and characterized histochemically by Congo red staining after performate pre-treatment at 20 degrees C for 1, 3, 5, 10, 15, 20 or 25 sec, and with oxidation induced proteolysis by trypsin digestion at 20 degrees C for 5, 10, or 30 sec, 1, 2, 3, 4, 5, 6 or 10 min and covered with gum-arabic according to Romhányi, and confirmed by streptavidin-biotin-complex/horseradish peroxidase immunohistochemical reactions. The "sensitivity" or "resistance" to pre-treatment of amyloid deposits depends on the type of amyloid, and the length of pre-treatment. Secondary (AA) amyloid is sensitive to KMnO4 oxidation, followed by trypsin digestion (for 1 min), and its green birefringence under polarized light disappears, while primary (AL) (for 1-5 min), senile (for 1-10 min), and most forms of organ (tissue)-limited (localized) amyloid (for 1-10 min) are resistant. Performate pre-treatment is followed by pronounced congophilia. Secondary (AA) is sensitive to performate pre-treatment (for 1 sec), while primary (AL) amyloid (for 1-20 sec), senile (for 1-25 sec), and most forms of organ (tissue)-limited (localized, isolated) amyloid deposits (for 1-25 sec) are resistant, and are constantly positively birefringent. Early identification and differentiation of amyloid deposits is important for the prognosis and for the choice of therapy. The authors conclude that the presented classical histochemical methods are useful as first line screens for the histological identification of amyloidosis.

BackgroundApatite rheumatism (AR), chondrocalcinosis (Ch-C) and primary synovialis chondromatosis (prSynCh) are calcium hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) crystal induced progressive metabolic diseases, accompanied by more or less amorphous calcium phosphate [CaPO4], calcium carbonate [CaCO3]) deposits, chondroid and/or bone formation [1 – 5].ObjectivesTheaimof this study was.(1)to assess the mineralization, chondroid and/or bone formation ofAR,Ch-CandprSynChwith conventional histologic stains and histochemical reactions,(2)to identifyHA[Ca5(PO4)3(OH)] andCPPD[Ca2P2O7.2H2O] crystal deposits in conventionally fixed and stained tissue sections in comparison with unstained sections (Bély and Apáthy 2013) [6].MethodsTenjoints (6 knees, 1 hip, 3 shoulders) of5patients withAR,16joints (8 knees, 4 hips, 1 shoulder, 1 elbow, 2 wrists) of16patients withCh-C, and21joints (14 knees, 5 hips, 2 elbows) of20patients withprSynChwere studied histologically.The amount of amorphous calcium phosphate and/or calcium carbonate deposits, furthermore chondroid and/or bone formation were evaluated by a semiobjective score system in conventionally stained tissue sections (0- no mineral deposits, chondroid and/or bone formation,1– minimal,2– moderate, and3– abundant).Standard stained and unstained tissue sections were examined with the light microscope and under polarized light, respectively.ResultsSummary of Patients’ Demographics.Table 1.Clinical diagnosisTotal n of patients (n=41)Mean age in years at surgery ± SDRange of age (In years)Apatite rheumatism (n=5)5 of 4174.80±6.9166 – 82Female477.00±5.6069 – 82Male166.0066.00Chondrocalcinosis (n=16)16 of 4163.67±21.1739 – 81Female1462.00±21.9339 – 81Male274.00±1.4173 – 74Primary synovial chondromatosis – (n=20)20 of 4150.20±12.5130 – 76Female1353.15±10.4940 – 74Male744.71±14.9030 – 76The amount of amorphous calcium phosphate and/or calcium carbonate deposits was different in all patient groups; the average value of scores in tissue samples of patients with clinical diagnosis ofARwas (1.80), withCh-C(2.60), and withprSynCh(0.556).Chondroid formation in synovial membranes was minimal in tissue samples of patients with the clinical diagnosis ofAR(0.058) orCh-C(0.050); osteoid or bone formation was not detected (0.0).Massive chondroid formation (with or without osteoid or bone formation) was characteristic forprSynCh(2.1351).In tissue sections stained withHE,HAcrystals were found only in3(4.054%), andCPPDin15(20.27%) of 74 tissue sections of28patients with the clinical diagnosis ofAR,Ch-C, or withprSynCh.Using Bély and Apáthy’s non-staining technique (2013), different amounts ofHAandCPPDcrystals were demonstrated in all28patients (100%) with the clinical diagnosis ofAR,Ch-CandprSynCh.In unstained sectionsHAcrystals were found in49, (66.216%), andCPPDin44(59.459%) of 74 tissue samples.The unstained sections were more efficient to demonstrateHAorCPPDcrystals than conventionally stained ones.ConclusionOur study indicates thatAR,Ch-CandprSynChare crystal induced maladies and belong to the same group of metabolic diseases.All of these diseases start withHAandCPPDcrystal deposition which provokes an inflammatory reaction.The inflammatory reaction is inhibited or moderated by calcium carbonate [CaCO3] and/or calcium phosphate [CaPO4] deposition in case ofARorCh, and is inhibited or moderated by chondroid and/or osteoid formation in case ofprSynCh.In our viewprSynChis a defect variant of metabolic disorders, characterized by diminished calcium phosphate and/or carbonate production.References[1]Bély M, Apáthy A:Clinical Archives of Bone and Joint Diseases,2018; 1.2DOI: 10.23937/cabjd-2017/1710007[2]Bély M, Apáthy Á:Orvosi Hetilap,2013; 154(23): 908-913. [Hungarian]Acknowledgements:NIL.Disclosure of InterestsNone Declared.

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...

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...

The aim of this study was to determine: the prevalence, and histological characteristics of vasculitis in the pancreas, and to follow the formal pathogenesis of multifocal pancreatitis due to arteritis and/or arteriolitis (multifocal vasculogenic pancreatitis). A randomized autopsy population of 161 in-patients with rheumatoid arthritis (RA) was studied. Systemic vasculitis (SV) complicated RA in 36 (22.36%) of 161 cases; tissue samples of pancreas were available for histologic evaluation in 28 patients. Pancreatitis and vasculitis were characterized histologically and immunohistochemically. Vasculogenic, multifocal pancreatitis was not recognized clinically. Vasculitis of the pancreatic arterioles and small arteries (branches of splenic artery, upper and lower gastroduodenal arteries) can lead to local ischaemia and to regressive changes in the pancreas. This vasculogenic process is more or less widespread and multifocal, depending on the number of involved vessels and is followed by reactive inflammation, depending on the stages of the pathological process. Because of the recurrent nature of vasculitis with time these regressive changes accumulate within the pancreas and may contribute to an unexpected circulatory failure and sudden death of the patient. Vasculogenic microinfarcts in the pancreas may be clinically characterized by unexplained recurrent abdominal symptoms and spontaneous remissions which insidiously may lead to metabolic failure resistant to therapy.