Antinuclear antibodies (ANA) represent a family of autoantibodies targeting ubiquitous cellular constituents and are a hallmark of systemic inflammatory autoimmune rheumatic diseases named connective ...tissue diseases (CTD). The gold standard method for ANA determination is indirect immunofluorescence (IIF) on the human laryngeal epidermoid carcinoma cell line type 2 substrate (HEp-2), but with increasing demand for ANA testing, novel methods eased for automation emerged, which allows testing by staff less experienced in this specific field of laboratory diagnostic. In 2016 The working group (WG) for laboratory diagnostics of autoimmune diseases as part of the Committee for the Scientific Professional Development of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM) published the data of a survey regarding general practice in laboratory diagnostics of autoimmune diseases in Croatia. Results indicated high diversity in the performance of autoantibody testing as well as reporting of the results and indicated the need of creating recommendations for the assessment of ANA that would help harmonize diagnostics of systemic autoimmune rheumatic diseases in Croatia. This document encompasses twenty-seven recommendations for ANA testing created concerning indications for ANA testing, preanalytical, analytical, and postanalytical issues, including rational algorithm and quality control assurance. These recommendations are based on the relevant international recommendations and guidelines for the assessment of ANA testing and relevant literature search and should help to harmonize the approach in ANA testing and clarify differences in interpretation of the results obtained using different methods of determination.
Osteoarthritis (OA) is a chronic joint disease caused by both mechanical damage and metabolic factors, which intertwine in their pathogenetic pathways. We hypothesise that the oxidised cholesterol ...derivative, 7-ketocholesterol (7-KCh), represents a danger signal in the synovia of patients with OA and promotes low-grade inflammation. The study would aim to elucidate the possible immune mechanisms initiated by 7-KCh that contribute to oxidative stress and inflammation in the synovia and synovial CD68+ cells, which are mostly macrophages. The polarisation of synovial CD68+ cells, their tissue distribution in relation to T and natural killer (NK) cells, and the influence of 7-KCh on the phenotype and intracellular cytokine and chemokine production in suspension is worth analysing. We envisage that this research would contribute to a better understanding of the biology of CD68+ macrophages influenced by 7-KCh and encourage the development of therapeutic approaches based on directing macrophage polarisation.
We aimed to evaluate the diagnostic accuracy of the proinflammatory monocyte chemotactic protein-1 (MCP-1) in the diagnosis of asymptomatic diastolic dysfunction (DD) in patients with psoriatic ...arthritis (PsA). The disease activity in psoriatic arthritis (DAPSA) was determined using clinical and laboratory parameters, and echocardiography was performed to estimate DD. Serum MCP-1 concentrations were elevated in PsA patients with DD diagnosed with ultrasound (median (25th percentile, 75th percentile): 366.6 pg/mL (283, 407.1 pg/mL) vs. 277.5 pg/mL (223.5, 319.1 pg/mL) in controls; P<0.0017). PsA patients with serum MCP-1 concentration higher than the cut-off value of 347.6 pg/mL had a 7.74-fold higher chance of developing DD than PsA patients with lower serum MCP-1 concentrations (controls), with a specificity of 86.36% and sensitivity of 55%, as verified using ultrasound. The group with MCP-1 concentrations above the cut-off value also showed a higher late peak diastolic mitral inflow velocity, A-wave value (P=0.000005), E/E′ ratio (P=0.00005), and a lower E/A ratio (P=0.000002), peak systolic left atrial reservoir strain, SA value (P=0.0066), early peak diastolic displacement of the mitral septal annulus, E′ wave value (P=0.003), than controls. Systolic blood pressure (P=0.01), LDL cholesterol concentration (P=0.012), glucose concentration (P=0.011), and DAPSA (P=0.0000) increased in the PsA group with higher MCP-1 concentrations, although there were no differences in comorbidities and therapy between the groups compared. Thus, the serum MCP-1 concentration was a significant and independent prognostic indicator for asymptomatic DD in PsA patients (area under the curve=0.730, P=0.001). The DAPSA score in PsA patients might indicate the need for echocardiography and adjustment of anti-inflammatory treatment in terms of DD prevention.
Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low-grade inflammation. Increased levels of T helper 1 ...pro-inflammatory cytokines in the serum of OA patients may support granulysin (GNLY) mediated cytotoxicity, which in-turn may contribute to the pathogenesis of OA. In the present study, GNLY expression and cytotoxic/apoptotic mechanisms mediated by GNLY in the peripheral blood of OA patients were assessed. A total of 40 non-obese women (median age of 64 years old) with knee OA, and 40 controls (median age 62 years old) were enrolled in the study. GNLY, IFN-γ and IL-4 expression levels were investigated in peripheral blood lymphocytes (PBLs) using flow cytometry, immunocytochemistry and/or confocal microscopy. Natural killer (NK) GNLY-mediated apoptosis through NK effectors against K-562 targets was analyzed using the PKH-26 18-h cytotoxicity assay. Serum GNLY levels were assessed using ELISA. The percentage of GNLY
PBLs was higher in the OA patients than that in the controls due to the increase in the proportions of GNLY
cells in the natural killer (NK), T and natural killer T (NKT) subsets. GNLY localization inside exocytotic lysosomal-associated membrane protein-1
granules was ~40% in both groups. However, the intensity of GNLY labeling in PBLs was higher in OA patients than in the controls, and it was supported by the increased expression of IFN-γ relative to IL-4 in NK and T cells from OA patients. The serum GNLY concentration was <0.3 ng/ml in both groups. RC8 anti-GNLY mAb by itself was unable to significantly alter early apoptosis, whereas RC8 anti-GNLY mAb combined with anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients, whilst not exerting a notable effect in the controls. Anti-perforin mAb by itself did not affect apoptosis significantly. These results suggest that in women with knee OA, GNLY expression in the PBL subsets and GNLY-mediated early apoptosis of K-562 targets are increased compared with the controls and accompanied by intracellular dominance of IFN-γ over IL-4 in NK cells.
With the trend of increasing incidence of autoimmune diseases, laboratories are faced with exponential growth of the requests for tests relating the diagnosis of these diseases. Unfortunately, the ...lack of laboratory personnel experienced in this specific discipline of laboratory diagnostic, as well as an unawareness of a method limitation often results in confusion for clinicians. The aim was to gain insight into number and type of Croatian laboratories that perform humoral diagnostics with the final goal to improve and harmonize laboratory diagnostics of autoimmune diseases in Croatia.
In order to get insight into current laboratory practice two questionnaires, consisting of 42 questions in total, were created. Surveys were conducted using SurveyMonkey application and were sent to 88 medical biochemistry laboratories in Croatia for the first survey. Out of 33 laboratories that declared to perform diagnostic from the scope, 19 were selected for the second survey based on the tests they pleaded to perform. The survey comprised questions regarding autoantibody hallmarks of systemic autoimmune diseases while regarding organ-specific autoimmune diseases was limited to diseases of liver, gastrointestinal and nervous system.
Response rate was high with 80 / 88 (91%) laboratories which answered the first questionnaire, and 19 / 19 (1.0) for the second questionnaire. Obtained results of surveys indicate high heterogeneity in the performance of autoantibody testing among laboratories in Croatia.
Results indicate the need of creating recommendations and algorithms in order to harmonize the approach to laboratory diagnostics of autoimmune diseases in Croatia.
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