Plasma lipids paediatric reference intervals: Indirect estimation using a large 14-year database.

OBJECTIVES: Establishing direct reference intervals (RIs) for paediatric patients is a very challenging endeavour. Indirect RIs can address this problem, using existing clinical laboratory databases from real-world data research. Compared to the traditional direct method, the indirect approach is highly practical, widely applicable, and low-cost. Considering the relevance of dyslipidemia in the paediatric age, to provide better laboratory services to the local paediatric population, we established population-specific lipid RIs via data mining. METHODS: Our laboratory information system was searched for cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of patients aged less than 18 years, performed from January 2009 until December 2022. RIs were estimated using RefineR algorithm. RESULTS: Values from 215,594 patients were initially collected. After refining data on the basis of specific exclusion criteria that left 17,933 patients, we determined the RIs for each analyte, including corresponding 95% confidence interval (95% CI). Age and sex partitions were required for proper stratification of the heterogenous subpopulations. Age-related variations in TC and TG values were observed mainly in children until 5 years. RIs were defined for children less than 3 years and for those of 3-18 years. In our population, the obtained RIs were comparable with those of the literature, but the upper TG limit in subjects under the age of 3 (2.03 mmol/L with 95% CI: 1.45-2.86) was lower than that previously reported. CONCLUSIONS: Our RIs, necessary for paediatric lipid monitoring, are tailored to the serviced patient population as should be done whenever possible.

Quality indicators in laboratory medicine: state-of-the-art, quality specifications and future strategies.

In the last few decades, quality in laboratory medicine has evolved in concert with the transformation and the changes (technological, scientific and organizational) in this sector. Laboratory professionals have faced great challenges, at times being overwhelmed, yet also involved in this progress. Worldwide, laboratory professionals and scientific societies involved in laboratory medicine have raised awareness concerning the need to identify new quality assurance tools that are effective in reducing the error rate and enhancing patient safety, in addition to Internal Quality Control (IQC) procedures and the participation in the External Quality Assessment Schemes (EQAS). The use of Quality Indicators (QIs), specifically designed for laboratory medicine are effective in assessing and monitoring all critical events occurring in the different phases of Total Testing Process (TTP), in particular, in the extra-analytical phases. The Model of Quality Indicators (MQI), proposed by the Working Group "Laboratory Errors and Patient Safety" (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and validated by experts in consensus conferences, is an important window of opportunity for the medical laboratory to demonstrate the use of an effective quality assurance tool fit for this purpose. Aim of this paper is to provide an update of the state-of-the-art concerning the most used QIs data collected in 2021 and the Quality Specifications (QSs) proposed for their evaluation. Moreover, a strategy for the future is proposed in order to improve the MQI and encourage its use in medical laboratories throughout the world.

New insights into SARS-CoV-2 Lumipulse G salivary antigen testing: accuracy, safety and short TAT enhance surveillance.

OBJECTIVES: The rapid, accurate and safe detection of SARS-CoV-2 is the key to improving surveillance and infection containment. The aim of the present study was to ascertain whether, after heat/chemical inactivation, SARS-CoV-2 N antigen chemiluminescence (CLEIA) assay in saliva remains a valid alternative to molecular testing. METHODS: In 2022, 139 COVID-19 inpatients and 467 healthcare workers were enrolled. In 606 self-collected saliva samples (Salivette), SARS-CoV-2 was detected by molecular (TaqPath rRT-PCR) and chemiluminescent Ag assays (Lumipulse G). The effect of sample pre-treatment (extraction solution-ES or heating) on antigen recovery was verified. RESULTS: Salivary SARS-CoV-2 antigen assay was highly accurate (AUC=0.959, 95% CI: 0.943-0.974), with 90% sensitivity and 92% specificity. Of the 254 antigen positive samples, 29 were false positives. We demonstrated that heterophilic antibodies could be a cause of false positive results. A significant antigen concentration decrease was observed after ES treatment (p=0.0026), with misclassification of 43 samples. Heat had a minimal impact, after treatment the correct classification of cases was maintained. CONCLUSIONS: CLEIA SARS-CoV-2 salivary antigen provides accurate, timely and high-throughput results that remain accurate also after heat inactivation, thus ensuring a safer work environment. This supports the use of salivary antigen detection by CLEIA in surveillance programs.

Salivary proteomic analysis in asymptomatic and symptomatic SARS-CoV-2 infection: Innate immunity, taste perception and FABP5 proteins make the difference.

BACKGROUND AND AIM: SARS-CoV-2 infection spawns from an asymptomatic condition to a fatal disease. Age, comorbidities, and several blood biomarkers are associated with infection outcome. We searched for biomarkers by untargeted and targeted proteomic analysis of saliva, a source of viral particles and host proteins. METHODS: Saliva samples from 19 asymptomatic and 16 symptomatic SARS-CoV-2 infected subjects, and 20 controls were analyzed by LC-MS/MS for untargeted peptidomic (flow through of 10 kDa filter) and proteomic (trypsin digestion of filter retained proteins) profiling. RESULTS: Peptides from 53 salivary proteins were identified. ADF was detected only in controls, while IL1RA only in infected subjects. PRPs, DSC2, FABP5, his-1, IL1RA, PRH1, STATH, SMR3B, ANXA1, MUC7, ACTN4, IGKV1-33 and TGM3 were significantly different between asymptomatic and symptomatic subjects. Retained proteins were 117, being 11 highly different between asymptomatic and symptomatic (fold change ≥2 or ≤-2). After validation by LC-MS/MS-SRM (selected reaction monitoring analysis), the most significant discriminant proteins at PCA were IL1RA, CYSTB, S100A8, S100A9, CA6, and FABP5. CONCLUSIONS: The differentially abundant proteins involved in innate immunity (S100 proteins), taste (CA6 and cystatins), and viral binding to the host (FABP5), appear to be of interest for use as potential biomarkers and drugs targets.

The University of Padua salivary-based SARS-CoV-2 surveillance program minimized viral transmission during the second and third pandemic wave.

BACKGROUND: The active surveillance of students is proposed as an effective strategy to contain SARS-CoV-2 spread and prevent schools' closure. Saliva for molecular testing is as sensitive as naso-pharyngeal swab (NPS), self-collected and well accepted by participants. This prospective study aimed to verify whether the active surveillance of the Padua University employees by molecular testing of self-collected saliva is an effective and affordable strategy for limiting SARS-CoV-2 spread. METHODS: A surveillance program based on self-collection of saliva every 2 weeks (October 2020-June 2021) was conducted. Among 8183 employees of the Padua University, a total of 6284 subjects voluntarily took part in the program. Eight collection points guaranteed the daily distribution and collection of barcoded salivary collection devices, which were delivered to the laboratory by a transport service for molecular testing. Quarantine of positive cases and contact tracing were promptly activated. RESULTS: Among 6284 subjects, 206 individuals were SARS-CoV-2 positive (99 by salivary testing; 107 by NPS performed for contact tracing or symptoms). The cumulative SARS-CoV-2 incidence in this cohort was 3.1%, significantly lower than that of employees not in surveillance (8.0%), in Padua (7.1%) and in the Veneto region (7.2%). Employees with positive saliva results were asymptomatic or had mild symptoms. The levels of serum antibodies after 3 months from the infection were correlated with age and Ct values, being higher in older subjects with greater viral loads. CONCLUSIONS: Salivary-based surveillance with contact tracing effectively allowed to limit SARS-CoV-2 contagion, also in a population with a high incidence.

Hyris bCUBE SARS-CoV-2 rapid molecular saliva testing: a POCT innovation on its way.

OBJECTIVES: The reliable identification of individuals with SARS-CoV-2 infection is the cornerstone for containing viral spread. Rapid molecular point-of-care testing (POCT) of saliva might reduce analysis time, thus increasing the efficacy of contact tracing. In this study, a new POCT RT-PCR assay for the detection of SARS-CoV-2 RNA in saliva was evaluated and compared with an already validated CE-IVD method. METHODS: An evaluation was made of 160 left-over salivary samples (27 frozen, kept at -80 °C and 133 fresh), collected using Salivette (Sarstedt, Germany). Samples were analyzed by TaqPath COVID-19 CE-IVD RT-PCR kit, QuantStudio5 Real-Time (Applied Biosystems, USA) (TaqPath) and bKIT Virus Finder COVID-19 Saliva (Hyris Global Diagnostics, Italy). Performances of three- and fivefold pooling strategies were also evaluated. Blood assay interference in saliva was also tested with Hyris. RESULTS: On using TaqPath, SARS-CoV-2 positivity was detected in 35 samples. Another 10 positive samples were artificially-generated by blind mixing of positive with negative samples. Hyris positive and negative percentages of agreement were 97.6 (95% CI: 87.2-99.9%) and 100 (95% CI: 97.0-100%), respectively. Seventeen positive pools, evaluated for threefold strategy, were all correctly determined by both systems. For the 5-pool strategy, 94.7% (18/19) of samples resulted positive with the Hyris system, and 100% with TaqPath. The presence of 1% of blood (v/v) in saliva did not interfere with the accuracy of Hyris assay. CONCLUSIONS: The sensitivity and specificity of the bKIT Virus Finder COVID-19 Saliva were optimal with respect to TaqPath. In view of the safe and straightforward pre-analytical procedure involved, and the small size of the Hyris bCube, the Hyris system can be used for POCT.

Serum miRNA Profiling for Early PDAC Diagnosis and Prognosis: A Retrospective Study.

BACKGROUND: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. METHODS: Serum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs expression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. RESULTS: 2549 human miRNAs were screened out. At SAM, 76 differentially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large majority (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was associated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signalling pathways. CONCLUSIONS: A series of serum miRNAs was identified as potentially useful for the early diagnosis of PDAC, and for establishing a prognosis.

Salivary SARS-CoV-2 antigen rapid detection: A prospective cohort study.

BACKGROUND AND AIM: SARS-CoV-2 quick testing is relevant for the containment of new pandemic waves. Antigen testing in self-collected saliva might be useful. We compared salivary and naso-pharyngeal swab (NPS) SARS-CoV-2 antigen detection by a rapid chemiluminescent assay (CLEIA) and two different point-of-care (POC) immunochromatographic assays, with results of molecular testing. METHODS: 234 patients were prospectively enrolled. Paired self-collected saliva (Salivette) and NPS were obtained to perform rRT-PCR, chemiluminescent (Lumipulse G) and POC (NPS: Fujirebio and Abbott; saliva: Fujirebio) for SARS-CoV-2 antigen detection. RESULTS: The overall agreement between NPS and saliva rRT-PCR was 78.7%, reaching 91.7% at the first week from symptoms. SARS-CoV-2 CLEIA antigen was highly accurate in distinguishing positive and negative NPS (ROC-AUC = 0.939, 95%CI:0.903-0.977), with 81.6% sensitivity and 93.8% specificity. This assay on saliva reached the optimal value within 7 days from symptoms onset (Sensitivity: 72%; Specificity: 97%). Saliva POC antigen was limited in sensitivity (13%), performing better in NPS (Sensitivity: 48% and 66%; Specificity: 100% and 99% for Espline and Abbott respectively), depending on viral loads. CONCLUSIONS: Self-collected saliva is a valid alternative to NPS for SARS-CoV-2 detection by molecular, but also by CLEIA antigen testing, which is therefore potentially useful for large scale screening.

SARS-CoV-2 identification and IgA antibodies in saliva: One sample two tests approach for diagnosis.

AIM: This study aims to verify whether standardized saliva collection is suitable for SARS-CoV-2 molecular detection and IgA measurement. METHODS: 43 COVID-19 inpatients and 326 screening subjects underwent naso-pharyngeal (NP)-swab and saliva collection (Salivette). Inpatients also underwent repeated blood collections to evaluate inflammation and organs involvement. In all patients and subjects, SARS-CoV-2 (gene E) rRT-PCR was undertaken in saliva and NP-swabs. Salivary IgA and serum IgA, IgG, IgM were measured on inpatients' samples. RESULTS: NP-swabs and saliva were both SARS-CoV-2 positive in 7 (16%) or both negative in 35 (82%) out of 43 patients successfully included in the study. NP-swabs and saliva results did not perfectly match in one patient (saliva positive, NP-swab negative). Positive molecular results were significantly associated with disease duration (p = 0.0049). 326/326 screening subjects were SARS-CoV-2 negative on both NP-swabs and saliva. Among the 27 saliva samples tested for IgA, 18 were IgA positive. Salivary IgA positivity was associated with pneumonia (p = 0.002) and CRP values (p = 0.0183), not with other clinical and molecular data, or with serum immunoglubulins. CONCLUSIONS: A standardized saliva collection can be adopted to detect SARS-CoV-2 infection in alternative to NP-swabs. Preliminary data on salivary IgA support the use of saliva also for patient monitoring.

SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.

Objectives: The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods: Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results: A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions: This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.

Different approaches for estimating measurement uncertainty: An effective tool for improving interpretation of results.

BACKGROUND: The estimation of measurement uncertainty (MU) in clinical laboratories is of crucial importance in improving laboratory testing quality and correctly interpreting results. However, it is difficult for clinical laboratories to reliably estimate MU since current guidelines and standards fail to clearly define and harmonize methods to be used for this purpose. AIMS: To propose a model for MU estimation in relation to test interpretation on the basis of three different scenarios. METHODS: MU estimation was evaluated regard to the inclusion of imprecision and bias components for different test purposes. RESULTS: Three scenarios were identified. The expanded uncertainty values were: 0.56 ng/L and 1.86 ng/L for troponin I at levels 4.95 ng/L and 20.60 ng/L, respectively; 9.4 µmol/L and 27.2 µmol/L for creatinine at levels 121.0 µmol/L and 390.0 µmol/L, respectively. 0.2 mmol/L and 0.3 mmol/L for potassium at levels 4.03 mmol/L and 6.35 mmol/L, respectively; 0.36 mmol/L for glucose at level of around 6 mmol/L. These values represent MU results estimated for the three scenarios, which contemplate test results used for 1a) short term patient monitoring, 1b) longer term patient monitoring, 2) comparison with reference intervals and 3) comparison with a clinical decision point. CONCLUSIONS: Goal-based measurement uncertainty estimation in clinical laboratories provides an opportunity to improve laboratory testing quality and reduce risk in the interpretation of clinical results.

Pre-analytical quality indicators in laboratory medicine: Performance of laboratories participating in the IFCC working group "Laboratory Errors and Patient Safety" project.

Quality indicators (QIs) are key tools for improving the quality of laboratory services, by reducing error rates and safeguarding patient safety. A body of accumulated evidence confirms the relevance of QIs and their impact on the overall quality of laboratory information. The consensus achieved on a list of "harmonized" QIs, along with the system used for data collection and reporting throughout an international benchmarking programme, has enabled achieving realistic performance targets, based on knowledge of the state-of-the-art. Data collected in 2017 and 2018 have been analyzed and performance measures obtained by laboratories participating in the project are summarized in the present article. The laboratory performance measures have been classified into three levels (optimum, desirable or minimum) in agreement with the widely accepted model of analytical quality specifications.

Decision Support and Patient Safety.

In recent years, clinical decision support (CDS) systems have become recognized as increasingly important in assuring patient safety and supporting all phases of the clinical decision-making process. In Laboratory Medicine, CDS systems are usually used to drive test ordering and diagnostic prediction while combining IT components and staff skills. However, educational initiatives, user and provider feedback, and expert consultations should also be considered integral to CDS. The aim of this paper is to provide an overview of some important developments in CDS in supporting the clinical decision-making process and guaranteeing patient safety by reducing medical errors.

The circulating miRNAs as diagnostic and prognostic markers.

A large portion of the human genome transcribes RNA sequences that do not code for any proteins. The first of these sequences was identified in 1993, and the best known noncoding RNAs are microRNA (miRNAs). It is now fully established that miRNAs regulate approximately 30% of the known genes that codify proteins. miRNAs are involved in several biological processes, like cell proliferation, differentiation, apoptosis and metastatization. These RNA products regulate gene expression at the post-transcriptional level, modulating or inhibiting protein expression by interacting with specific sequences of mRNAs. Mature miRNAs can be detected in blood plasma, serum and also in a wide variety of biological fluids. They can be found associated with proteins, lipids as well as enclosed in exosome vesicles. We know that circulating miRNAs (C-miRNAs) can regulate several key cellular processes in tissues different from the production site. C-miRNAs behave as endogenous mediators of RNA translation, and an extraordinary knowledge on their function has been obtained in the last years. They can be secreted in different tissue cells and associated with specific pathological conditions. Significant evidence indicates that the initiation and progression of several pathologies are "highlighted" by the presence of specific C-miRNAs, underlining their potential diagnostic relevance as clinical biomarkers. Here we review the current literature on the possible use of this new class of molecules as clinical biomarkers of diseases.

Validation model of a laboratory-developed method for the ISO15189 accreditation: The example of salivary cortisol determination.

BACKGROUND: The ISO 15189:2012 describes the requirements for quality and competence, which are specific for all sectors of a medical laboratory. Laboratory-developed methods, standard methods used outside their intended scope and validated methods subsequently modified shall be validated. The main objective of validation of an examination procedure (EP) is to demonstrate its fitness-for-purpose. The aim of the present study is to illustrate a model to validate laboratory-developed methods in agreement with the ISO 15189:2012 requirement, through the example of salivary cortisol (sF) measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: The proposed validation model for a laboratory developed method includes 6 steps: 1) analysis of available scientific documentation pertinent to specific EP; 2) evaluation of EP intended use; 3) identification of performance characteristics to evaluate; 4) definition of experimental procedure; 5) identification of acceptability criterion for results evaluation; 6) preparation of a validation plan; 7) production of a validation certificate. RESULTS: sF is an EP sampled during the accreditation audit and resulted in compliance with the Standard. A structured procedure to certify that the achieved performance specifications are appropriate for the purpose of the test is therefore necessary. CONCLUSIONS: A high competence is required for laboratory professionals in order to guarantee that the validated quality specifications have a positive impact on patients' management. The ISO 15189 accreditation is an important tool that allows demonstrating the value of a medical laboratory in the clinical context.

Quality of plasma samples and BD Vacutainer Barricor tubes: Effects of centrifugation.

BACKGROUNDS: The BD Vacutainer® Barricor™ Plasma collection tube (BD Barricor) uses an innovative non-gel separation method. This study compared the plasma residual cell count (PRCC) obtained from BD Barricor and from BD PST II plasma tubes. METHODS: Four BD Barricors and one BD PST II were collected from 40 donors. BD PST II was centrifuged at 1300g/10 min, while the BD Barricors were centrifuged at 1800g/10 min, 4000g/3 min, 4000g/7 min and 4000g/15 min. PRCC was evaluated measuring white blood cells (WBC), red blood cells (RBC) and Platelets (PLT) counts by Siemens ADVIA 2120. Cell-free hemoglobin was quantified by haemolysis index (HI) by Roche Cobas c501. RESULTS: BD PST II Median WBC, RBC and PLT counts were 0.38 (109/L), 0.0291 (1012/L) and 113.5 (109/L), respectively. Considering the BD PST II as reference, PRCC differences were expressed as median bias percentage. WBC showed a significant reduction at all the conditions (p < 0.01), being the reductions: 63.9% (1800g/10 min), 69.9% (4000g/3 min), 75.0% (4000g/7 min) and 82.7% (4000g/15 min). RBC reductions 29.7% (1800g/10 min), 33.8% (4000g/3 min), 39.6% (4000g/7 min) and 66.4 (4000g/15 min) were all significant (p < 0.01). PLT reductions were 1.6% at 1800g/10 min (p = ns), 1.2% at 4000g/3 min (p = ns), 27.1% at 4000g/7 min (p = 0.046) and 46.6% at 4000g/15 min (p = 0.005). BD Barricor centrifuged for 7 and 15 min at 4000g showed an increased haemolysis. CONCLUSIONS: BD Barricors plasma quality improved with increasing the centrifugation times but already at 4000g/3 min, the suggested centrifugation condition, a significant improvement was achieved.

Measurement uncertainty in laboratory reports: A tool for improving the interpretation of test results.

BACKGROUND: Measurement uncertainty (MU) estimation has been introduced by ISO 15189 for the accreditation of clinical laboratories. Although MU reporting is not required, its inclusion in medical reports is of potential assistance to physicians in results interpretation. METHODS: MU reporting was evaluated with respect to different test purposes, namely comparison with reference intervals (RI), patient monitoring or comparison with clinical decision limits. Clinical Biochemistry, Hematology, Coagulation and Clinical Immunology measurands were used as examples. Assuming Gaussian RI distribution, the probability of retesting due to MU was determined by simulations. Significant MU variations were compared against the reference change value (RCV) and clinical decision limits. RESULTS: Three potential scenarios emerged for RI. For 12 measurands, depending on the MU interval, a potential change in results interpretation was found only for Sodium and S-Protein. On considering only the results within RI, simulations confirmed that up to 8.6% of MU intervals encompassed the RI limits, thus potentially leading to retesting. For tests used in patient monitoring, significant MU variations were comparable to those calculated by RCV, with the exception of CEA. For tests results evaluated with respect to clinical decision limits, on including MU, the clinical interpretation may be improved (e.g. for tPSA). CONCLUSION: The findings made in the present study, which considers real MU data and hypothetical results obtained for a series of measurands, support the concept that MU may aid the physician's interpretation thus ensuring reliable clinical decision making.

Genetics in TNF-TNFR pathway: A complex network causing spondyloarthritis and conditioning response to anti-TNFα therapy.

OBJECTIVES: We investigated whether polymorphisms (SNPs) in the promoter region of TNFA, or in the autoinflammatory TNFRSF1A and MEFV genes, concur with HLA-B27 in enhancing the risk of Spondyloarthritis (SpA) and/or in predicting the response to anti-TNFα treatment. METHODS: 373 controls and 137 SpA (82 with Psoriatic Arthritis-PsA and 55 with Ankylosing Spondylitis- AS; 98/137 under TNFα inhibitor therapy) from the Veneto Region (Italy) were studied. TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) and HLA-B27 were assayed by RT-PCR. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. RESULTS: HLA-B27 was associated with AS (χ2 = 120.1; p = 0.000). Only the TNFA -1031T>C was singly associated with SpA, and the haplotype C/G, resulting from -1031T>C/-308G>A combination, was significantly associated with a reduced risk of SpA (OR: 0.67, CI: 0.46-0.97; p = 0.035). Two SNPs were identified in TNFRSF1A, the R92Q (Minor allele frequency-MAF = 0.034) and c.625+10A>G (MAF = 0.479). None of them was associated with SpA (p>0.05). The TNFRSF1A c.625+10 G allele was associated with late response to anti-TNFα therapy (p = 0.031). Twenty-one SNPs were identified in MEFV gene, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF<0.025) except for R202Q (MAF = 0.27). None was associated with SpA diagnosis (p>0.05). CONCLUSION: TNFRSF1A and MEFV gene SNPs are not associated with SpA in the North-East of Italy. AS risk appears to depend not only on HLA-B27, but also on the protective TNFA haplotype -1031C/-308G. The TNFRSF1A c.625+10A>G impacts on the response to anti-TNFα therapy.