Chiara Bonaguri

A comparative study on the reliability of an automated system for the evaluation of cell-based indirect immunofluorescence

BACKGROUND Automated interpretations systems for anti-nuclear antibody (ANA), anti-double stranded DNA antibody (dsDNAab), and anti-neutrophil cytoplasmic antibody (ANCA) assessment by indirect immunofluorescence (IIF) have been recently introduced. The aim of this study was to compare the diagnostic performance of the automated IIF reading system AKLIDES with both traditional visual interpretation of IIF by laboratory experts and confirmatory tests. METHODS Visual and automated autoantibody interpretations of IIF findings using AKLIDES pattern recognition algorithms were performed for ANA on HEp-2 cells (n=182), dsDNAab on Crithidia luciliae (n=44) and ANCA on human neutrophils (n=46). All serum samples tested by IIF for ANCA and dsDNAab were also assessed with the corresponding enzyme-linked immunosorbent assays (ELISAs). Out of the 182 sera tested for ANA by IIF, 116 were also assessed for antibodies to extractable nuclear antigens (ENA) by ELISA and dot immunoassay (DIA). RESULTS ANA testing showed an excellent agreement between visual and AKLIDES reading (98.9%). The overall agreement of dsDNAab testing on C. luciliae substrate slides was 91.0%, whereas ANCA showed a concordance of 89.1%. There was a remarkable agreement of AKLIDES findings for dsDNAab with confirmatory tests. CONCLUSION Visual and automated interpretations of IIF findings for ANA, ANCA, and dsDNAab demonstrated a good agreement when assessing patients with suspected autoimmune diseases. Automated interpretation systems such AKLIDES may improve laboratory efficiency and support standardization of IIF in clinical laboratories.

Current state of diagnostic technologies in the autoimmunology laboratory

Abstract The methods for detecting and measuring autoantibodies have evolved markedly in recent years, encompassing three generations of analytical technologies. Many different immunoassay methods have been developed and used for research and laboratory practice purposes, from the early conventional (or monoplex) analytical methods able to detect single autoantibodies to the more recent multiplex platforms that can quantify tens of molecules. Although it has been in use for over 50 years, indirect immunofluorescence remains the standard method for research on many types of autoantibodies, due to its characteristics of diagnostic sensitivity and also to recent technological innovations which permit it a greater level of automation and standardization. The recent multiplex immunometric methods, with varying levels of automation, present characteristics of higher diagnostic accuracy, but are not yet widely diffused in autoimmunology laboratories due to the limited number of autoantibodies that are detectable, and due to the high cost of reagents and systems. Technological advancement in autoimmunology continues to evolve rapidly, and in the coming years new proteomic techniques will be able to radically change the approach to diagnostics and possibly also clinical treatment of autoimmune diseases. The scope of this review is to update the state of the art of technologies and methods for the measurement of autoantibodies, with special reference to innovations in indirect immunofluorescence and in multiple proteomic methods.

Italian multicentre study for application of a diagnostic algorithm in autoantibody testing for autoimmune rheumatic disease: conclusive results.

AIM The presence of specific auto-antibodies in serum (i.e., antinuclear antibodies or ANA, anti-extractable nuclear antigens or anti-ENA, and anti-double stranded DNA or anti-dsDNA ) is one of the major criteria in the diagnostics of Autoimmune Rheumatic Disease. As such, the request for these tests has grown exponentially in laboratory practice. The aim of this study is to describe the implementation of a joint laboratory-clinics guideline for reducing clinically inappropriate requests for autoantibody testing in a broad geographic area (Parma, Modena, Piacenza, Reggio-Emilia) for the diagnosis of Autoimmune Rheumatic Disease. METHODS This study, supported by a Regional grant for innovative research projects started in January 2008, is an observational research aimed at comparing the number of ANA, anti-dsDNA and anti-ENA testing as well as the percentage of positive test results before and after implementation of the diagnostic algorithm in hospitalized patients. A multidisciplinary team consisting of clinical immunologist and laboratory scientists was established, with the aim of collecting and analysing diagnostic criteria, clinical needs, laboratory report formats, analytical procedures, as well as the number of tests performed. The laboratory results and the clinical protocol were both validated by data emerging from the clinical follow-up studies. RESULTS A joint guideline for auto-antibody testing, placing ANA test at the first level, has been developed and implemented since January 2009. The results for the period January-June 2009 (12,738 tests) were compared with those of the same period in 2008 (13,067 tests). A significant reduction in the number of anti-dsDNA (-26%) and anti-ENA (-15%) was observed. The percentage of second-level tests positivity after implementation of the diagnostic protocol had also consistently increased for both ENA (13% vs 17%) and dsDNA (9% vs 11%). DISCUSSION The development and implementation of algorithms for the diagnostics of Autoimmune Rheumatic Disease in hospitalized patients was associated with a reduction in the number of second-level tests, but also with an increased diagnostic specificity. This outcome attests that close collaboration and audit between clinicians, laboratory specialists and healthcare services is effective to develop efficient diagnostic algorithms for both hospitalized patients and outpatients.

Anti-68 kDa antibodies in autoimmune sensorineural hearing loss: are these autoantibodies really a diagnostic tool?

Objectives: Autoimmune sensorineural hearing loss (ASNHL) is a relatively rare disorder which can lead to total deafness. At present, no specific laboratory test with adequate sensitivity and specificity is available to confirm the clinical suspicion of ASNHL. The aim of this study was to identify if evaluation of anti-hsp70 antibodies is an accurate diagnostic tool in patients affected by ASNHL. Study design: Prospective study. Methods: During 4-year (2001–2005), all patients with SNHL who were referred to the Eye, Ear, Nose and Throat Department of Parma University, Italy, underwent specific tests to determine the autoimmune origin of the disease. Patients with a consistent suspicion of ASNHL underwent the routine serologic tests and a test for determination of anti-hsp70 antibodies. The same patients were divided into three groups: (1) idiopathic ASNHL; (2) ASNHL associated with ocular inflammation, i.e. Cogans Syndrome; (3) ASNHL associated with a systemic autoimmune disease (SAD). The control group included: (1) healthy subjects; and (2) patients affected by SAD, without any ocular or audiovestibular disease. Results: 88 subjects (67 patients, defined as “study group”, and 21 controls) were evaluated. Anti-hsp70 antibodies were isolated in 52% of the study group patients, and in 4% of the control group (χ2 = 13.009, p < 0.01). In the idiopathic ASNHL patients, 59.5% were found positive for anti-hsp70 antibodies. About 50% of patients affected by CS and 37.5% of patients affected by SAD with SNHL were found positive. In the control group, anti-hsp70 antibodies were found in 8.3% of healthy subjects and in none of the patients with SAD and no hearing loss. Conclusions: The present study confirms the value of the anti-hsp70 test in the serological diagnosis of autoimmune hearing loss. It is still the only available diagnostic marker that identifies an autoimmune origin of hearing loss.

An Italian Multicenter Study for Application of a Diagnostic Algorithm in Autoantibody Testing

The presence in the serum of specific autoantibodies, such as antinuclear antibodies (ANA), anti‐double‐stranded DNA (anti‐dsDNA), and antiextractable nuclear antigens (anti‐ENA), is one of the diagnostic criteria for autoimmune rheumatic disease, and the requests for these tests in the last few years have grown remarkably. A guideline for reducing clinically inappropriate requests in autoantibody testing (ANA, anti‐dsDNA, anti‐ENA) has been applied in the Parma Hospital since 2007. The results for the period January–December 2007 were compared to those of the previous period January–December 2006, and a significant reduction in the number of anti‐dsDNA (23.9%) and anti‐ENA (20.7%) was found. The aim of this study was to assess the applicability of a similar guideline in a wide area (Parma, Modena, Piacenza, Reggio‐Emilia) with reference to the diagnosis of autoimmune rheumatic disease. This project, supported by a regional grant for innovative research projects, was started in January 2008 and consists of three different steps: (1) a study group of clinicians and laboratory physicians to evaluate the diagnostic criteria, the analytical procedures, and the number of tests performed in different hospitals; (2) developing common guidelines for autoantibody testing that takes into account the different clinical needs with the aim of improving efficiency and clinical effectiveness of diagnosis and monitoring; and (3) assessing compliance with the guidelines in the different hospitals that are evaluating the second‐level test (anti‐dsDNA, anti‐ENA) decrease. We think that the validation of guidelines for the laboratory diagnosis of autoimmune rheumatic disease can represent a tool for improving patients’ outcomes and economic efficiency.

Effects of acute exercise and xanthine oxidase inhibition on novel cardiovascular biomarkers

Several sports have been associated with a postexercise increase of cardiac, liver, and skeletal muscle biomarkers of injury. Exhaustive or acute physical exercise causes an increased generation of reactive oxygen species, resulting in cellular injury. Thus, exercise and training may trigger pathophysiological changes in serum concentrations of a variety of biomarkers. In this study, we aimed to evaluate the variation of novel biomarkers of stress and cardiovascular disease such as copeptin, midregional part of proadrenomedullin (MR-proADM), growth differentiation factor 15 (GDF15), soluble vascular endothelial growth factor receptor, and placental growth factor along with uric acid before and after acute high-intensity exercise and allopurinol administration. We also assessed whether allopurinol administration may affect the circulating levels of these biomarkers by inhibition of XO activity. This is a double-blind, placebo-controlled study in which 12 professional football players were divided into 2 experimental groups. An oral dose of 300 mg of allopurinol was administered to one group of six participants 4 hours before a match of the Spanish Football League, whereas the other 6 participants received placebo (cellulose). Venous blood samples were obtained before the match (baseline) and twelve hours afterwards (post-match). Serum MR-proADM levels increased significantly in the placebo group, whereas serum GDF15 levels increased significantly in both the placebo and allopurinol group after the match. No differences in the other parameters tested were found after the match in any experimental group. The trend toward postexercise increase of serum MR-proADM and GDF15 levels shows that the metabolism of these proteins is clearly imbalanced after exercise, which thereby represents a potential source of biological variability in their clinical assessment.

The serum concentrations of leptin and MCP-1 independently predict low back pain duration

Abstract Background: Low back pain (LBP) is a very frequent condition, affecting most people at some point throughout their life. This cross-sectional study was aimed to investigate a selected panel of cytokines and inflammatory biomarkers in patients with or without LBP. Methods: The study population consisted of 104 patients diagnosed with LBP (52 non-persistent and 52 persistent) and 52 healthy subjects with no LBP. Blood samples were collected for assessment of adiponectin, leptin, monocyte chemoattractant protein-1 (MCP-1) and C reactive protein (CRP). The duration of LBP was categorized as “no pain”, “non-persistent LBP” and “persistent LBP”. Results: Higher values of CRP and lower concentrations of both leptin and MCP-1 were found in LBP patients compared to controls, whereas adiponectin did not differ among groups. MCP-1 was also lower in patients with non-persistent than in those with persistent LBP. Age, leptin (relative risk, 11.8; 95% CI, 3.9–35.8) and MCP-1 (relative risk, 2.7; 95% CI, 1.7–4.4) were independently associated with presence and duration of LBP. The combination of age, leptin and MCP-1 predicted 61% of the risk of LBP duration. The area under the curve of MCP-1 for distinguishing persistent from non-persistent LBP was 0.65 (95% CI, 0.54–0.76). Conclusions: Then results of our study suggest that leptin and MCP-1 may be promising biomarkers for diagnosis of acute LBP and its risk to become chronic.

DNA injury is acutely enhanced in response to increasing bulks of aerobic physical exercise

The aim of this study was to evaluate DNA damage in response to increasing bulks of aerobic physical exercise. Fifteen adult and trained athletes performed four sequential trials with increasing running distance (5-, 10-, 21- and 42-km) in different periods of the year. The γ-H2AX foci parameters were analyzed before and 3h after the end of each trial. The values of all γ-H2AX foci parameters were enhanced after the end of each trial, with values gradually increasing from the 5- to the 42-km trial. Interestingly, a minor increase of γ-H2AX foci was still evident after 5- to 10-km running, but a much higher increase occurred when the running distance exceeded 21km. The generation of DNA injury was then magnified by running up to 42-km. The increase of each γ-H2AX foci parameter was then found to be associated with both running distance and average intensity. In multivariate linear regression analysis, the running distance was significantly associated with average intensity and post-run variation in the percentage of cells with γ-H2AX foci. We can hence conclude that aerobic exercise may generate an acute DNA damage in trained athletes, which is highly dependent upon running distance and average intensity.

Mobile phone radiofrequency exposure has no effect on DNA double strand breaks (DSB) in human lymphocytes

BACKGROUND The use of mobile phones has been associated with an increased risk of developing certain type of cancer, especially in long term users. Therefore, this study was aimed to investigate the potential genotoxic effect of mobile phone radiofrequency exposure on human peripheral blood mononuclear cells in vitro. METHODS The study population consisted in 14 healthy volunteers. After collection of two whole blood samples, the former was placed in a plastic rack, 1 cm from the chassis of a commercial mobile phone (900 MHz carrier frequency), which was activated by a 30-min call. The second blood sample was instead maintained far from mobile phones or other RF sources. The influence of mobile phone RF on DNA integrity was assessed by analyzing γ-H2AX foci in lymphocytes using immunofluorescence staining kit on AKLIDES. RESULTS No measure of γ-H2AX foci was significantly influenced by mobile phone RF exposure, nor mobile phone exposure was associated with significant risk of genetic damages in vitro (odds ratio comprised between 0.27 and 1.00). CONCLUSIONS The results of this experimental study demonstrate that exposure of human lymphocytes to a conventional 900 MHz RF emitted by a commercial mobile phone for 30 min does not significantly impact DNA integrity.

Inusuale pattern immunofluoroscopico su cellule HEp-2

RiassuntoPremesseLa ricerca degli anticorpi anti-nucleo (ANA) in immunofluorescenza indiretta (IFI) su cellule HEp-2 rappresenta una tappa fondamentale nella diagnostica delle patologie autoimmuni. Riportiamo il caso di 5 pazienti, che all’IFI per la ricerca di ANA eseguita con kit Nova Lite™ HEp-2 (Inova Diagnostics, San Diego, CA), presentavano un pattern citoplasmatico particolare.MetodiI sieri analizzati appartengono a cinque pazienti (età compresa tra 45 e i 69 anni). I sieri sono stati testati per la ricerca degli anticorpi anti-antigeni nucleari estraibili con metodo ELISA (ENA: anti-Sm, RNP, SSA/Ro, SSB/La, Scl-70 e anti-Jo-1) (ENA Screen, Inova Diagnostics) per la ricerca di ANA su HEp-2 di altri produttori (MBL International, Nagoya, Giappone e Alphadia, Waver, Belgio), per la ricerca di AMA, ASMA, APCA, LKM e Lc-1 in IFI su triplo tessuto di roditore (Inova Diagnostics) e per la ricerca di anticorpi anti-Sm, RNP, SSA/Ro, SSB/La, Scl-70, Jo-1, PM/Scl, Ku, Cenp-A/B, PCNA, LKM-1, Lc-1, SLA, F-actina e AMA-M2 con metodica Dot Blot (Alphadia).RisultatiQuattro pazienti presentavano un pattern nucleare speckled a titolo 1:80, mentre il quinto risultava negativo (<1:80), ma tutti presentavano un pattern citoplasmatico, con la presenza di una struttura rotondeggiante relativamente grande, presente in circa un terzo delle cellule. La ricerca di tutte le altre specificità anticorpali ricercate in ELISA, in IFI e in DotBlot ha dato esito negativo.ConclusioniLe informazioni cliniche sui 5 pazienti sono state ottenute dai rispettivi medici curanti; due pazienti non avevano una diagnosi definitiva di malattia, uno risultava affetto da ulcera duodenale e uno da gastrite cronica, entrambi in trattamento con inibitori della pompa protonica. Il quinto paziente era affetto da ipotiroidismo ed era trattato con terapia sostitutiva. Interessante appare l’osservazione di un tale pattern citoplasmatico non ascrivibile a una struttura citoplasmatica facilmente identificabile. Sono ovviamente necessari ulteriori studi per verificare se tale pattern abbia significatività clinica.SummaryBackgroundThe detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) on HEp-2 cells is a fundamental step in the diagnosis of autoimmune diseases. We report the case of five patients that, on IFA for detection ANA (Nova Lite™ HEp-2, Inova Diagnostics), presented an unusual cytoplasmatic pattern.MethodsSerum samples belonging to 5 patients (3 women and 2 men, age 45–69 years). All the samples were analysed with ELISA for detection of antiextractable nuclear antigens (ENA) (anti-Sm, RNP, SSA/Ro, SSB/La, Scl-701 and anti-Jo-1) (ENA Screen, Inova Diagnostics). ANA detection was performed also with IFA using HEp-2 cells from different manufacturers (MBL and Alphadia). Furthermore we performed the detection of anti-mitochondrial antibodies, antismooth muscle antibodies, anti-gastric parietal cell antibodies, anti-liver/kidney microsomal and anti-liver cytosol antibodies on IFA on rat tissue (Inova Diagnostics), and the detection of anti-Sm, RNP, SSA/Ro, SSB/La, Scl-70, Jo-1, PM/Scl, Ku, Cenp-A/B, PCNA, LKM-1, Lc-1, SLA, F-actin and AMA-M2 with Dot Blot (Alphadia).ResultsFour patients showed a nuclear speckled pattern at 1:80 dilution, while the fifth patient was negative (<1:80), but all of them showed a cytoplasmic pattern with a round-shaped body of relatively large dimension in about one third of the cells. Detection of all other autoantibodies assayed was negative.ConclusionsWe contacted the clinicians of these five patients to obtaine clinical information. Two patients had no definite clinical diagnosis; one was affected by chronic gastritis and one was affected by duodenal ulcer; both the latter patients were in therapy with protonic pump inhibitors. The fifth patient suffered of hypothyroidism and was treated with substitutive therapy.