Optimization of Laboratory Diagnostics of Primary Biliary Cholangitis: When Solid-Phase Assays and Immunofluorescence Combine.

The laboratory diagnostics of primary biliary cholangitis (PBC) have substantially improved, thanks to innovative analytical opportunities, such as enzyme-linked immunosorbent assays (ELISA) and multiple immunodot liver profile tests, based on recombinant or purified antigens. This study aimed to identify the best diagnostic test combination to optimize PBC diagnosis. Between January 2014 and March 2017, 164 PBC patients were recruited at the hospitals of Parma, Modena, Reggio-Emilia, and Piacenza. Antinuclear antibodies (ANA) and anti-mitochondrial antibodies (AMA) were assayed by indirect immunofluorescence (IIF), ELISA, and immunodot assays (PBC Screen, MIT3, M2, gp210, and sp100). AMA-IIF resulted in 89.6% positive cases. Using multiple immunodot liver profiles, AMA-M2 sensitivity was 94.5%, while anti-gp210 and anti-sp100 antibodies were positive in 16.5% and 17.7% of patients, respectively. PBC screening yielded positive results in 94.5% of cases; MIT3, sp100, and gp210 were detected by individual ELISA test in 89.0%, 17.1%, and 18.9% of patients, respectively. The association of PBC screening with IIF-AMA improved the diagnostic sensitivity from 89.6% to 98.2% (p < 0.01). When multiple immunodot liver profile testing was integrated with AMA-IIF, the diagnostic sensitivity increased from 89.1% to 98.8% (p < 0.01). The combination of IIF with solid-phase methods significantly improved diagnostic efficacy in PBC patients.

The Clinical Significance of Procalcitonin Elevation in Patients over 75 Years Old Admitted for COVID-19 Pneumonia.

AIM: To investigate the clinical significance of procalcitonin (PCT) elevation on hospital admission for coronavirus disease-19 (COVID-19) and its association with mortality in oldest old patients (age > 75 years). METHODS: The clinical records of 1074 patients with chest high-resolution computed-tomography (HRCT) positive for interstitial pneumonia and symptoms compatible for COVID-19, hospitalized in medical wards during the first pandemic wave in a single academic center in Northern Italy, were retrospectively analyzed. All patients had serum PCT testing performed within six hours from admission. Information on COVID-19-related symptoms, comorbidities, drugs, autonomy in daily activities, respiratory exchanges, other routine lab tests, and outcomes were collected. Clinical characteristics were compared across different admission PCT levels and ages. The association of admission PCT with mortality was tested separately in participants aged > 75 and ≤75 years old by stepwise multivariate Cox regression model with forward selection. RESULTS: With increasing classes of PCT levels (<0.05, 0.05-0.49, 0.5-1.99, and ≥2 ng/ml), there was a significant trend (P < 0.0001) towards older age, male gender, wider extension of lung involvement on HRCT, worse respiratory exchanges, and several other laboratory abnormalities. Each incremental PCT class was associated with increased risk of hospital death at multivariate models in subjects older than 75 (hazard ratio for PCT ≥ 2 vs. <0.05 ng/ml: 30.629, 95% confidence interval 4.176-224.645, P = 0.001), but not in subjects aged 75 or younger. CONCLUSIONS: In patients admitted for COVID-19, PCT elevation was associated with several clinical, radiological, and laboratory characteristics of disease severity. However, PCT elevation was strongly associated with hospital mortality only in oldest old subjects (age > 75).

A robust machine learning framework to identify signatures for frailty: a nested case-control study in four aging European cohorts.

Phenotype-specific omic expression patterns in people with frailty could provide invaluable insight into the underlying multi-systemic pathological processes and targets for intervention. Classical approaches to frailty have not considered the potential for different frailty phenotypes. We characterized associations between frailty (with/without disability) and sets of omic factors (genomic, proteomic, and metabolomic) plus markers measured in routine geriatric care. This study was a prevalent case control using stored biospecimens (urine, whole blood, cells, plasma, and serum) from 1522 individuals (identified as robust (R), pre-frail (P), or frail (F)] from the Toledo Study of Healthy Aging (R=178/P=184/F=109), 3 City Bordeaux (111/269/100), Aging Multidisciplinary Investigation (157/79/54) and InCHIANTI (106/98/77) cohorts. The analysis included over 35,000 omic and routine laboratory variables from robust and frail or pre-frail (with/without disability) individuals using a machine learning framework. We identified three protective biomarkers, vitamin D3 (OR: 0.81 [95% CI: 0.68-0.98]), lutein zeaxanthin (OR: 0.82 [95% CI: 0.70-0.97]), and miRNA125b-5p (OR: 0.73, [95% CI: 0.56-0.97]) and one risk biomarker, cardiac troponin T (OR: 1.25 [95% CI: 1.23-1.27]). Excluding individuals with a disability, one protective biomarker was identified, miR125b-5p (OR: 0.85, [95% CI: 0.81-0.88]). Three risks of frailty biomarkers were detected: pro-BNP (OR: 1.47 [95% CI: 1.27-1.7]), cardiac troponin T (OR: 1.29 [95% CI: 1.21-1.38]), and sRAGE (OR: 1.26 [95% CI: 1.01-1.57]). Three key frailty biomarkers demonstrated a statistical association with frailty (oxidative stress, vitamin D, and cardiovascular system) with relationship patterns differing depending on the presence or absence of a disability.

Genetic susceptibilty and celiac disease: what role do HLA haplotypes play?

Celiac disease is a chronic immune-mediated enteropathy triggered by exposure to dietary gluten in genetically predisposed individuals. Many genes involved in the pathogenesis have been identified and a crucial role is known to be played by the Human Leukocyte Antigen (HLA) system. The main determinants for genetic susceptibility are HLA-DQA1 and HLA-DQB1 genes encoding for HLA-DQ2 and HLA-DQ8 molecules, carried by almost all patients affected. However, since HLA-DQ2 and HLA-DQ8 heterodimers explain almost 40% of the disease heritability, HLA typing should not be applied in diagnosis, but exclusively to clarify uncertain diagnoses, considering its negative predictive value.

Non-invasive tests for the diagnosis of helicobacter pylori: state of the art.

Usually, non-invasive tests are the first methods for diagnosing Helicobacter pylori (HP) infection. Among these, serological test, stool antigen research and urea breath test are the most used. Antibodies anti-HP are not recommended in low prevalence population, moreover they cannot reveal an ongoing infection, but they only prove a contact with the bacterium. Also, they can persist for a long time after the eradication of the infection, therefore, they should not be used to verify the success of eradication therapy. Stool antigen research and Urea Breath Test (UBT) are useful both in diagnosis and during follow-up after eradication treatment. The stool antigen test is cheaper than Urea breath test with similar sensitivity and specificity.  Non-invasive tests are not able to diagnose the associated complications to HP infection.

The New Test for Monitoring Infliximab Therapy: From Laboratory to Clinical Practice.

BACKGROUND: Biological agents for anti-tumor necrosis factor-α therapy have revolutionized treatments for autoimmune diseases; however, approximately 20% of rheumatology and 40% of gastroenterology patients do not respond to the therapy, or they show reduced drug efficacy because of anti-drug antibody (ADA) formation. OBJECTIVES: To evaluate laboratory tools for individual monitoring of infliximab therapy and the relationship between ADA and infliximab serum levels, ADA and clinical response, and ADA and autoantibodies. METHODS: Our study comprised patients treated with infliximab and affected by selected rheumatology and gastroenterology diseases. Sera were analyzed for infliximab, total-anti-drug antibodies (Total-ADA), and free-anti-drug antibodies (Free-ADA) serum levels and for the detection of specific autoantibodies. RESULTS: We analyzed 73 patients. Total-ADA were detected in 26 rheumatology and 21 gastroenterology patients. Serum infliximab levels were significantly lower in Total-ADA positive patients (P = 0.01 for rheumatology group, P = 0.02 for gastroenterology group). A lack of response was observed in 7 rheumatology and 15 gastroenterology samples. Total-ADA serum levels were statistically significantly higher in patients with treatment failure in both groups (P = 0.01 and P = 0.001, respectively). There was no significant association between the presence of Total-ADA and other autoantibodies. Free-ADA were detected in only 27 rheumatology patients. Results showed a significant correlation with clinical outcome (P = 0.006). CONCLUSIONS: The correlation with clinical response suggests that the presence of ADA could interfere with efficacy of therapy. The tests for monitoring therapy may be an important tool to assist clinicians in early detection and prevention of therapy failure.

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.

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

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.

Cogan's syndrome: anti-Hsp70 antibodies are a serological marker in the typical form.

BACKGROUND: Cogan's syndrome (CS) is a rare autoimmune vasculitis characterized by ocular inflammation and sensorineural hearing loss. CS is divided into a "typical" form with non-syphilitic interstitial keratitis and audiovestibular symptoms, and an "atypical" form with ocular involvement affecting structures other than the cornea. Anti-Hsp70 antibodies were found at variable levels in patients presenting with various forms of autoimmune sensorineural hearing loss (ASNHL). OBJECTIVES: To assess the correlation between anti-Hsp70 antibodies and specific ASNHL subgroups. METHODS: We divided 112 subjects into four groups: 14 subjects with typical CS, 24 with atypical CS, 55 with ASNHL, and 19 control subjects (healthy subjects and patients with systemic autoimmune diseases but no sensorineural hearing or audiovestibular alterations). Patients were tested for serological autoimmunity markers including anti-Hsp70. RESULTS: Positivity of the anti-Hsp70 antibody test was highest in the typical CS group (92.9%) and lowest in the control group (5.2%). The test was positive in 52.7% of patients in the ASNHL group and 16.6% in the atypical CS group. The paired comparison analysis between groups showed that sensitivity of anti-Hsp70 in the typical CS group was significantly higher, as compared to the other three study groups. CONCLUSIONS: Anti-Hsp70 antibodies can be considered a serological marker of "typical" CS. "Atypical" CS is conceivably a sort of "melting pot" of different forms of autoimmune diseases still characterized by ocular inflammation and sensorineural hearing loss but whose antigenic characteristics need to be further defined.

Autoantibody induction and adipokine levels in patients with psoriasis treated with infliximab.

This study was aimed to analyse the prevalence of antinuclear antibodies in patients with psoriasis after treatment with infliximab and correlates the development of antibodies with both response to treatment and adipokines levels. Serum levels of ANA, anti-dsDNA, anti-histone, anti-nucleosome and anti-ENA antibodies at baseline after 2 and 12 months of treatment with infliximab were measured in 27 patients with psoriasis, as well as in 27 matched controls. Serum C-reactive protein (CRP), chemerin, visfatin and resistin were also assessed. The prevalence of ANA increased from 22 to 37% and 63% (p < 0.01) during treatment with infliximab, with a gradual progressive increase both in ANA titre and in percentage of ANA pattern. The prevalence of other antibodies also increased from 7 to 30% and 48% (p < 0.01) for anti-ds-DNA and from 7 to 26% and 37% for anti-nucleosome antibodies (p < 0.05), whereas the prevalence of anti-histone and anti-ENA antibodies was unchanged throughout the study period. Basal chemerin, resistin and CRP levels were higher in patients than in controls, and their levels progressively normalized during treatment (p < 0.01). Conversely, visfatin levels gradually increased (p < 0.01). ANA+ patients tended to show a faster decrease in PASI score, CRP and chemerin levels after 2 months, but the PASI score did not differ between ANA+ and ANA- patients at 12 months. A higher increase of visfatin was also found in ANA+ patients at 2 and 12 months. The antinuclear antibody response induced by infliximab was restricted to ANA, anti-dsDNA and anti-nucleosome antibodies. Patients who developed ANA positivity showed a faster clinical, inflammatory and immunological response to infliximab therapy.

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.

Effects of acute exercise and allopurinol administration on soluble urokinase plasminogen activator receptor (suPAR).

BACKGROUND: Although physical exercise acutely increases the most widely used inflammatory biomarkers, there is no information on its effect on soluble urokinase plasminogen activating receptor (suPAR), a circulating biomarker increasingly used for the assessment of systemic inflammation. METHODS: suPAR was assessed with the quantitative suPARnostic Standard ELISA Assay (Virogates, Birkerød, Denmark) in 12 professional football players before and after a football match. The athletes were divided into two experimental groups. An oral dose of 300 mg of allopurinol was administered to one group of six participants four hours before a match; the other six participants received placebo. RESULTS: Serum suPAR concentration did not vary significantly after the match in both the placebo and allopurinol group. No significant differences were observed between placebo and allopurinol groups at baseline and after the game. CONCLUSIONS: At variance with other consolidated inflammatory biomarkers, suPAR is not influenced by either physical exercise or administration of xanthine oxidase inhibitors.

Current state of diagnostic technologies in the autoimmunology laboratory.

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.

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.

Overcoming a "probable" diagnosis in antimitochondrial antibody negative primary biliary cirrhosis: study of 100 sera and review of the literature.

Serum anti-mitochondrial antibodies (AMA) are the serological hallmark of primary biliary cirrhosis (PBC), yet up to 15% of PBC sera are AMA negative at routine indirect immunofluorescence (IIF) while being referred to as "probable" cases. The diagnostic role of PBC-specific antinuclear antibodies (ANA) remains to be determined. We will report herein data on the accuracy of new laboratory tools for AMA and PBC-specific ANA in a large series of PBC sera that were AMA-negative at IIF. We will also provide a discussion of the history and current status of AMA detection methods. We included IIF AMA-negative PBC sera (n=100) and sera from patients with other chronic liver diseases (n=104) that had been independently tested for IIF AMA and ANA; sera were blindly tested with an ELISA PBC screening test including two ANA (gp210, sp100) and a triple (pMIT3) AMA recombinant antigens. Among IIF AMA-negative sera, 43/100 (43%) manifested reactivity using the PBC screening test. The same test was positive for 6/104 (5.8%) control sera. IIF AMA-negative/PBC screen-positive sera reacted against pMIT3 (11/43), gp210 (8/43), Sp100 (17/43), both pMIT3 and gp210 (1/43), or both pMIT3 and Sp100 (6/43). Concordance rates between the ANA pattern on HEp-2 cells and specific Sp100 and gp210 ELISA results in AMA-negative subjects were 92% for nuclear dots and Sp100 and 99% for nuclear rim and gp210. Our data confirm the hypothesis that a substantial part of IIF AMA-negative (formerly coined "probable") PBC cases manifest disease-specific autoantibodies when tested using newly available tools and thus overcome the previously suggested diagnostic classification. As suggested by the recent literature, we are convinced that the proportion of AMA-negative PBC cases will be significantly minimized by the use of new laboratory methods and recombinant antigens.

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.

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.