An approach for estimating measurement uncertainty in medical laboratories using data from long-term quality control and external quality assessment schemes

The present study was prompted by the ISO 15189 requirements that medical laboratories should estimate measurement uncertainty (MU)

Genetics in TNF-TNFR pathway: a complex network causing spondyloarthritis and conditioning response to therapy

Background. The seronegative spondyloarthritis (SpA) are a group of chronic inflammatory diseases resulting from a complex interplay among genetic background (mainly represented by HLA-B27) and environmental factors, that leads to the activation of autoinflammation and the dysregulation of the immune-system. In many cases, an early diagnosis and an appropriate monitoring of disease activity can be difficult because of the overlap of clinical features. Furthermore, because of the indices of inflammation, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), are in the normal range in at least half of SpA patients with a clear expression of disease activity, a delay in diagnosis and consequently in treatment in these patients has been documented. This imparts a tremendous symptomatic burden and loss of function in these patients during the productive years of life. For all these reasons, much attention is currently devoted to the identification of biochemical and genetic biomarkers to be used in the diagnosis as well as prognostic factors in evaluating the treatment effectiveness. Among the genetic predisposing factors, a well-known role is that of HLA-B27, which contributes however to only 20–30% of the total heritability, whereas the whole major histocompatibility complex (MHC) accounts for about 40–50% of the genetic risk of developing SpA. This suggested that other genes are involved in pathogenetic mechanism. In fact, in addition to HLA-B27, a number of genetic factors in both, MHC and non-MHC locus, have been claimed to play a role in pathogenesis of SpA. In this context, because of TNF-α is primarily involved in the propagation and perpetuation of inflammation in SpA, the study of TNF-α genetic is of great interest. Several polymorphisms (SNPs) in genes involved in TNF-α signalling, as TNFA, TNFSF15, TNFR1 and TRADD genes, have been identified as associated with SpA, even if results are controversial. Of great interest are also variants in MEFV gene, involved in the pathogenesis of the autoinflammatory disorder Familial Mediterranean Fever (FMF). Recent studies have shown that the SpA, and in particular the ankylosing spondylitis (AS), are very common among patients affected by FMF and that these patients can present with AS as a sole manifestation. The present study, conducted in a cohort of 91 SpA patients and 223 controls, coming from a North-East Italian region, was aimed to identify biohumoral (biochemical and haematological) and genetic factors to support the diagnostic and prognostic (response to therapy) work-up of SpA diseases. In particular, in addition to biochemical and haematological indices, we investigated whether SNPs in the promoter region of TNFA, or SNPs in the autoinflammatory TNFRSF1A and MEFV genes, might concur with HLA-B27 in enhancing the risk of developing SpA disease and/or in predicting the response to anti-TNFα drugs. Methods. The study population comprised 91 patients with a diagnosis of SpA (mean age ± standard deviation: 52.1 ± 12.5 years; 57 males, 34 females) and 223 blood donors (mean age ± standard deviation: 46 ± 11 years; 146 males, 77 females) coming from Veneto Region, a North-East Italian region. Among patients, 36 had a diagnosis of AS and 55 patients of psoriatic arthritis (PsA), which were based on New York and CASPAR criteria respectively. The protocol of this study was approved by the Local Institutional Ethic Committee of University-Hospital of Padua, Italy (Prot.n. 3024P/13), and all participants gave written informed consent before entering the study. Demographic and physiological data, medical and familial history data were collected for each participant. Blood samples were collected and complete blood count, CRP, ESR, uric acid, prealbumin, alanina aminotransferase (ALT) and glucose were evaluated. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. HLA-B27 and TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) were assayed by Real Time-PCR. HLA-CW6 allele presence was analysed by molecular genetic testing using a commercially available CE-IVD microarray. Statistical analysis was performed using STATA software (version 13.1). Results. An higher number of circulating polymorphonuclear cells and higher CRP levels could be detected in SpA patients with respect to controls, and in PsA higher levels of ALT could be observed with respect not only to controls but also to AS. Anyway these indices were not highly elevated and often comprised within the reference intervals. As expected, HLA-B27 was associated with AS (χ2=120.1; p<0.0001). Although a slightly higher frequency of HLA-CW6 carriers was observed among patients with AS (about 6%) or PsA (about 13%) with respect to controls (about 4%), the difference was not statistically significant. Any single studied TNFA SNP was not associated with SpA diagnosis, nor with AS or PsA considered singly. The haplotypes deriving from the pairwise combinations of the five studied SNPs were also statistically inferred. The most frequent haplotypes in controls were selected as references, and only the haplotype -1031C/-308G was significantly associated with AS (p=0.015) exerting in this disease a protective role (Odds Ratio: 0.43; Confidence Interval 95%: 0.22-0.85). Three SNPs were identified in TNFRSF1A gene and among them, only the R92Q (Minor Allele Frequency- MAF=0.034) and the c.625+10A>G (MAF=0.479) were selected for their potential functional implications. Both SNPs were not associated with the presence of SpA (χ2=1.073 and p=0.300 for R92Q; χ2=4.721 and p=0.094 for c.625+10A>G), but c.625+10A>G was associated with the response to anti-TNF therapy, assessed by BASDAI score lower /equal or higher than 4 at 10 months (p=0.031). Twenty-one SNPs were identified in MEFV gene and among them, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF0.05). Conclusions. In conclusion the results of this study indicate the relevant role of TNF-TNFR pathway genetics in the complex network causing SpA and conditioning response to therapy. TNFA was shown to be a predisposing factor for SpA, but mainly for AS, among Italian patients, while genetics of the autoinflammatory gene MEFV appears of no impact in this setting. The haplotype resulting from TNFA-1031C/-308G, potentially associated with lower TNF-α production, exerts a protective role in AS, while the TNFRSF1A c.625+10A>G polymorphism emerged as a potential predictor of response to anti- TNFα therapy

Harmonization of pre-analytical quality indicators

Quality indicators (QIs) measure the extent to which set targets are attained and provide a quantitative basis for achieving improvement in care and, in particular, laboratory services. A body of evidence collected in recent years has demonstrated that most errors fall outside the analytical phase, while the pre- and post-analytical steps have been found to be more vulnerable to the risk of error. However, the current lack of attention to extra‑laboratory factors and related QIs prevent clinical laboratories from effectively improving total quality and reducing errors. Errors in the pre-analytical phase, which account for 50% to 75% of all laboratory errors, have long been included in the ‘identification and sample problems’ category. However, according to the International Standard for medical laboratory accreditation and a patient-centered view, some additional QIs are needed. In particular, there is a need to measure the appropriateness of all test request and request forms, as well as the quality of sample transportation. The QIs model developed by a working group of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) is a valuable starting point for promoting the harmonization of available QIs, but further efforts should be made to achieve a consensus on the road map for harmonization

Verification of examination procedures in clinical laboratory for imprecision, trueness and diagnostic accuracy according to ISO 15189:2012: a pragmatic approach

Background The International Standard ISO 15189 is recognized as a valuable guide in ensuring high quality clinical laboratory services and promoting the harmonization of accreditation programmes in laboratory medicine. Examination procedures must be verified in order to guarantee that their performance characteristics are congruent with the intended scope of the test. The aim of the present study was to propose a practice model for implementing procedures employed for the verification of validated examination procedures already used for at least 2 years in our laboratory, in agreement with the ISO 15189 requirement at the Section 5.5.1.2. Methods In order to identify the operative procedure to be used, approved documents were identified, together with the definition of performance characteristics to be evaluated for the different methods; the examination procedures used in laboratory were analyzed and checked for performance specifications reported by manufacturers. Then, operative flow charts were identified to compare the laboratory performance characteristics with those declared by manufacturers. Results The choice of performance characteristics for verification was based on approved documents used as guidance, and the specific purpose tests undertaken, a consideration being made of: imprecision and trueness for quantitative methods; diagnostic accuracy for qualitative methods; imprecision together with diagnostic accuracy for semi-quantitative methods. Conclusions The described approach, balancing technological possibilities, risks and costs and assuring the compliance of the fundamental component of result accuracy, appears promising as an easily applicable and flexible procedure helping laboratories to comply with the ISO 15189 requirements

Spondyloarthritis: Matrix Metalloproteinasesas Biomarkers of Pathogenesis and Response to Tumor Necrosis Factor (TNF) Inhibitors

The term spondyloarthritis (SpA) is used to describe a group of multifactorial chronic inflammatory diseases characterized by a predisposing genetic background and clinical manifestations typically involving the sacroiliac joint. The absence of pathognomonic clinical and/or laboratory findings generally results in a delay in diagnosis and, consequently, in treatment. In addition, 20-40% of SpA patients are non-responders to tumor necrosis factor (TNF) inhibitor therapies. Given these considerations, it is important to identify biomarkers that can facilitate the diagnosis and assessment of disease activity. As inflammation plays a key role in the pathogenesis of SpA, inflammatory mediators have been investigated as potential biomarkers for diagnosing the disease and predicting response to therapy. Some investigators have focused their attention on the role of matrix metalloproteinases (MMPs), which are known to be markers of synovial inflammation that is generated in the joint in reaction to inflammatory stimuli. Several studies have been carried out to verify if serum MMPs levels could be useful to diagnose SpA, to assess disease severity, and to predict response to TNF inhibitor therapy. The current review focuses on MMPs' role in SpA pathogenesis, diagnosis and therapeutic implications

Performance criteria and quality indicators for the post-analytical phase

Background: Quality indicators (QIs) used as performance measurements are an effective tool in accurately estimating quality, identifying problems that may need to be addressed, and monitoring the processes over time. In Laboratory Medicine, QIs should cover all steps of the testing process, as error studies have confirmed that most errors occur in the pre- and post-analytical phase of testing. Aim of the present study is to provide preliminary results on QIs and related performance criteria in the post-analytical phase. Methods: This work was conducted according to a previously described study design based on the voluntary -participation of clinical laboratories in the project on QIs of the Working Group "Laboratory Errors and Patient Safety" (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Results: Overall, data collected highlighted an improvement or stability in performances over time for all reported indicators thus demonstrating that the use of QIs is-effective in the quality improvement strategy. Moreover, QIs data are an important source for defining the state-of- the-art concerning the error rate in the total testing process. The definition of performance specifications based on the state-of-the-art, as suggested by consensus documents, is a valuable benchmark point in evaluating the performance of each laboratory. Conclusions: Laboratory tests play a relevant role in the monitoring and evaluation of the efficacy of patient outcome thus assisting clinicians in decision-making. Laboratory performance evaluation is therefore crucial to providing patients with safe, effective and efficient care

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

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

Abstract 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

Genetics in TNF-TNFR pathway: a complex network causing spondyloarthritis and conditioning response to therapy

Background. The seronegative spondyloarthritis (SpA) are a group of chronic inflammatory diseases resulting from a complex interplay among genetic background (mainly represented by HLA-B27) and environmental factors, that leads to the activation of autoinflammation and the dysregulation of the immune-system. In many cases, an early diagnosis and an appropriate monitoring of disease activity can be difficult because of the overlap of clinical features. Furthermore, because of the indices of inflammation, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), are in the normal range in at least half of SpA patients with a clear expression of disease activity, a delay in diagnosis and consequently in treatment in these patients has been documented. This imparts a tremendous symptomatic burden and loss of function in these patients during the productive years of life. For all these reasons, much attention is currently devoted to the identification of biochemical and genetic biomarkers to be used in the diagnosis as well as prognostic factors in evaluating the treatment effectiveness. Among the genetic predisposing factors, a well-known role is that of HLA-B27, which contributes however to only 20–30% of the total heritability, whereas the whole major histocompatibility complex (MHC) accounts for about 40–50% of the genetic risk of developing SpA. This suggested that other genes are involved in pathogenetic mechanism. In fact, in addition to HLA-B27, a number of genetic factors in both, MHC and non-MHC locus, have been claimed to play a role in pathogenesis of SpA. In this context, because of TNF-α is primarily involved in the propagation and perpetuation of inflammation in SpA, the study of TNF-α genetic is of great interest. Several polymorphisms (SNPs) in genes involved in TNF-α signalling, as TNFA, TNFSF15, TNFR1 and TRADD genes, have been identified as associated with SpA, even if results are controversial. Of great interest are also variants in MEFV gene, involved in the pathogenesis of the autoinflammatory disorder Familial Mediterranean Fever (FMF). Recent studies have shown that the SpA, and in particular the ankylosing spondylitis (AS), are very common among patients affected by FMF and that these patients can present with AS as a sole manifestation. The present study, conducted in a cohort of 91 SpA patients and 223 controls, coming from a North-East Italian region, was aimed to identify biohumoral (biochemical and haematological) and genetic factors to support the diagnostic and prognostic (response to therapy) work-up of SpA diseases. In particular, in addition to biochemical and haematological indices, we investigated whether SNPs in the promoter region of TNFA, or SNPs in the autoinflammatory TNFRSF1A and MEFV genes, might concur with HLA-B27 in enhancing the risk of developing SpA disease and/or in predicting the response to anti-TNFα drugs. Methods. The study population comprised 91 patients with a diagnosis of SpA (mean age ± standard deviation: 52.1 ± 12.5 years; 57 males, 34 females) and 223 blood donors (mean age ± standard deviation: 46 ± 11 years; 146 males, 77 females) coming from Veneto Region, a North-East Italian region. Among patients, 36 had a diagnosis of AS and 55 patients of psoriatic arthritis (PsA), which were based on New York and CASPAR criteria respectively. The protocol of this study was approved by the Local Institutional Ethic Committee of University-Hospital of Padua, Italy (Prot.n. 3024P/13), and all participants gave written informed consent before entering the study. Demographic and physiological data, medical and familial history data were collected for each participant. Blood samples were collected and complete blood count, CRP, ESR, uric acid, prealbumin, alanina aminotransferase (ALT) and glucose were evaluated. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. HLA-B27 and TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) were assayed by Real Time-PCR. HLA-CW6 allele presence was analysed by molecular genetic testing using a commercially available CE-IVD microarray. Statistical analysis was performed using STATA software (version 13.1). Results. An higher number of circulating polymorphonuclear cells and higher CRP levels could be detected in SpA patients with respect to controls, and in PsA higher levels of ALT could be observed with respect not only to controls but also to AS. Anyway these indices were not highly elevated and often comprised within the reference intervals. As expected, HLA-B27 was associated with AS (χ2=120.1; p<0.0001). Although a slightly higher frequency of HLA-CW6 carriers was observed among patients with AS (about 6%) or PsA (about 13%) with respect to controls (about 4%), the difference was not statistically significant. Any single studied TNFA SNP was not associated with SpA diagnosis, nor with AS or PsA considered singly. The haplotypes deriving from the pairwise combinations of the five studied SNPs were also statistically inferred. The most frequent haplotypes in controls were selected as references, and only the haplotype -1031C/-308G was significantly associated with AS (p=0.015) exerting in this disease a protective role (Odds Ratio: 0.43; Confidence Interval 95%: 0.22-0.85). Three SNPs were identified in TNFRSF1A gene and among them, only the R92Q (Minor Allele Frequency- MAF=0.034) and the c.625+10A>G (MAF=0.479) were selected for their potential functional implications. Both SNPs were not associated with the presence of SpA (χ2=1.073 and p=0.300 for R92Q; χ2=4.721 and p=0.094 for c.625+10A>G), but c.625+10A>G was associated with the response to anti-TNF therapy, assessed by BASDAI score lower /equal or higher than 4 at 10 months (p=0.031). Twenty-one SNPs were identified in MEFV gene and among them, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF0.05). Conclusions. In conclusion the results of this study indicate the relevant role of TNF-TNFR pathway genetics in the complex network causing SpA and conditioning response to therapy. TNFA was shown to be a predisposing factor for SpA, but mainly for AS, among Italian patients, while genetics of the autoinflammatory gene MEFV appears of no impact in this setting. The haplotype resulting from TNFA-1031C/-308G, potentially associated with lower TNF-α production, exerts a protective role in AS, while the TNFRSF1A c.625+10A>G polymorphism emerged as a potential predictor of response to anti- TNFα therapy.Introduzione. Le spondiloartriti sieronegative (SpA) sono un gruppo di malattie infiammatorie croniche risultanti da una complessa interazione tra fattori genetici (tra cui, HLA-B27 è il maggior predisponente) e ambientali. Ed è tale interazione ad indurre l'attivazione di processi autoinfiammatori e la disregolazione del sistema immunitario caratterizzanti la malattia. In molti casi, una diagnosi precoce ed un adeguato monitoraggio dell’ attività di malattia risultano difficili a causa della sovrapposizione delle caratteristiche cliniche tra le diverse forme. Il ritardo nella diagnosi e conseguentemente nel trattamento, è inoltre dovuto al fatto che, gli indici d’infiammazione comunemente utilizzati nella pratica clinica, la velocità di eritrosedimentazione (VES) ed la proteina C-reattiva (PCR), sono nella norma in almeno metà dei pazienti con chiara espressione dell’attività di malattia. Il ritardo nella diagnosi conferisce a questi pazienti un carico sintomatico importante ed una perdita di funzione durante gli anni di vita produttiva. Pertanto, forte attenzione è attualmente rivolta all’identificazione di marcatori biochimici e genetici utili alla diagnosi e di fattori prognostici necessari a valutare l'efficacia del trattamento. Tra i fattori genetici predisponenti, è noto il ruolo di HLA-B27, che contribuisce però solo per il 20-30% all'ereditarietà totale, mentre il complesso maggiore di istocompatibilità (MHC) rappresenta circa il 40-50% del rischio genetico di sviluppare la patologia. Questo dato ha suggerito il probabile coinvolgimento di altri geni nel meccanismo patogenetico. Studi di associazione genetica hanno permesso di identificare un certo numero di altri geni, associati alla patologia, sia nel locus MHC che in altri loci. In questo contesto, di grande interesse è lo studio della genetica di TNF-α, considerato il ruolo di tale citochina nel propagare e perdurare dell'infiammazione. Sebbene numerosi studi abbiano dimostrato l’associazione tra i polimorfismi di geni coinvolti nella via del segnale del TNF-α (es. TNFA, TNFSF15, TNFR1 e TRADD) e la patologia di SpA, i risultati sono discordanti. Di grande interesse sono anche le varianti del MEFV gene, coinvolto nella patogenesi della malattia autoinfiammatoria Febbre Mediterranea Familiare (FMF). Studi recenti hanno, infatti, dimostrato che le SpA, ed in particolare la spondilite anchilosante (AS), sono molto comuni tra i pazienti affetti da FMF e che questi pazienti possono presentarsi con AS come unica manifestazione. Questo studio, condotto su 91 pazienti e 223 controlli, provenienti da una regione italiana del Nord-Est, si propone di identificare fattori bioumorali (biochimici ed ematologici) e genetici al fine di supportare i processi diagnostici e prognostici (risposta alla terapia). In particolare, oltre ai parametri biochimici ed ematologici, è stato valutato se polimorfismi nella regione del promotore del gene TNFA, o dei geni TNFRSF1A e MEFV, possano concorrere con l’allele HLA-B27 all’aumento del rischio di sviluppare la malattia e/o nel predire la risposta agli inibitori del TNF-α. Metodi. La popolazione studiata comprendeva 91 pazienti con diagnosi di SpA (età media ± deviazione standard: 52.1 ± 12.5 anni; 57 maschi, 34 femmine) e 223 donatori di sangue (età media ± deviazione standard: 46 ± 11 anni; 146 maschi, 77 femmine) provenienti dalla Regione Veneto, una regione italiana del Nord-Est. Tra i pazienti, 36 presentavano AS e 55 artrite psoriasica (PsA), con diagnosi formulata sulla base dei criteri rispettivamente di New York e CASPAR. Il protocollo di questo studio è stato approvato dal Comitato Etico Istituzionale locale dell’Università-Azienda Ospedaliera di Padova, Italia (Prot.n. 3024P / 13), e tutti i soggetti arruolati hanno firmato un consenso informato prima di partecipare allo studio. Per ciascun soggetto arruolato, sono stati raccolti i dati demografici e fisiologici, la storia clinica e familiare. Sono stati raccolti poi, campioni di sangue, al fine di valutare l’emocromo e la VES, e di determinare i livelli di PCR, acido urico, prealbumina, alanina aminotransferasi (ALT) e glucosio. L’analisi molecolare dei geni MEFV (esoni 2,3,5 e 10) e TNFRSF1A (esoni 2,3,4 e 6) è avvenuta mediante sequenziamento diretto. La determinazione degli alleli HLA-B27 e dei polimorfismi del gene TNFA (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) è stata condotta mediante PCR in Real-Time. La determinazione degli alleli HLA-CW6 è avvenuta mediante un test genetico molecolare CE-IVD, disponibile in commercio, che adotta la tecnologia microarray. L’analisi statistica è stata effettuata utilizzando il software STATA (versione 13.1). Risultati. Un maggior numero di cellule polimorfonucleate circolanti e livelli di PCR più elevati sono stati rilevati nei pazienti affetti da SpA rispetto ai controlli. Inoltre, i pazienti affetti da PsA hanno mostrato livelli più elevati di ALT, non solo rispetto ai controlli, ma anche rispetto a pazienti affetti da AS. In ogni caso tali indici non erano molto elevati e spesso risultavano compresi entro gli intervalli di riferimento. Come atteso, gli alleli HLA-B27 sono risultati associati all’AS (χ2=120.1; p<0.0001). Sebbene una frequenza leggermente maggiore degli alleli HLA-CW6 sia stata osservata tra i pazienti con AS (circa il 6%) o PsA (circa il 13%) rispetto ai controlli (circa 4%), la differenza non è risultata essere statisticamente significativa. Nessuno dei polimorfismi del gene TNFA è risultato singolarmente associato alla diagnosi SpA, né a quella di AS o PsA, se valutate indipendentemente. Sono stati, poi, statisticamente dedotti gli aplotipi derivanti dalle coppie di combinazioni dei cinque polimorfismi studiati. Gli aplotipi più frequenti nei controlli sono stati selezionati come aplotipi di riferimento, e solo l’aplotipo -1031C/-308G è risultato significativamente associato con l’AS (p=0.015) esercitando in questa malattia un ruolo protettivo (odds ratio: 0.43; intervallo di confidenza al 95%: 0.22- 0.85). Tre polimorfismi sono stati identificati nel gene TNFRSF1A e tra questi, solo i polimorfismi R92Q (Frequenza dell’allele minore- MAF = 0.034) e c.625 + 10A> G (MAF = 0.479) sono stati selezionati a causa del potenziale ruolo funzionale. Entrambi i polimorfismi non sono risultati associati con la diagnosi di SpA (χ2 = 1.073 e p = 0.300 per R92Q; χ2 = 4.721 e p = 0.094 per c.625 + 10A> G). Il polimorfismo c.625 + 10A> G è però, risultato essere associato con la risposta alla terapia con anti-TNF, valutato sulla base di un punteggio BASDAI inferiore / uguale o superiore a 4, a 10 mesi dall’inizio della terapia (p = 0.031). Ventuno polimorfismi sono stati identificati nel gene MEFV e tra questi, 10 noti per il potenziale significato funzionale. Tali varianti alleliche sono risultate estremamente rare nella nostra popolazione (MAF 0.05). Conclusioni. In conclusione, i risultati di questo studio suggeriscono il ruolo rilevante della genetica della via del segnale TNF-TNFR nel complesso sistema che induce la patogenesi di SpA e condiziona la risposta alla terapia. Il gene TNFA, nella popolazione oggetto di studio, si è dimostrato un fattore predisponente per lo sviluppo di SpA, ma soprattutto di AS. Al contrario, la genetica del gene MEFV non sembra mostrare alcun impatto in questo gruppo di malattie. L'aplotipo TNFA-1031C/-308G, potenzialmente associato alla produzione di livelli più bassi di TNF-α, sembra esercitare un ruolo protettivo nella patogenesi di AS, mentre è emerso che il polimorfismo c.625 TNFRSF1A + 10A> G costituisce un potenziale fattore predittivo di risposta alla terapia con anti-TNFα