Made available in DSpace on 2016-01-26T12:51:44Z (GMT). No. of bitstreams: 1 ricardoacayabadetoledo_dissert.pdf: 765730 bytes, checksum: bac7d4d4322f0a4d4a32d9be65a91344 (MD5) Previous issue date: 2013-03-20 / Introduction: Spondyloarthritis is a group of diseases with clinical, laboratory and image similar. Analysis of clinical manifestations of spondyloarthritis in patients with and without the HLA-B*27 were performed, but the results revealed great heterogeneity. Objectives: The aim of this study was to evaluate the clinical manifestations of spondyloarthritis according to the classification criteria of the European Study Group Spondyloarthropathy (ESSG) in the presence and absence of the HLA-B*27. Patients and methods: From a total of 156 patients with clinical suspicion referred for the investigation of gene HLA-B*27, 73 were diagnosed with spondyloarthritis according to the criteria of ESSG. The HLA-B*27 were identified using commercial kits (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Clinical data were collected from medical records of patients. The results were compared using the chi-square or Fisher exact test. The values of Odds Ratio (OR) and confidence interval of 95% were also calculated. The p-value equal to or less than 0.05 was considered significant. Results: Of the 73 selected patients, 53 (72.6%) were male and 20 (27.4%) were female. The mean age was 49.4 and did not differ between genders (p = 0337). The spondyloarthritis found among the 73 patients were: ankylosing spondylitis (n = 47; 64.4%), psoriatic spondyloarthritis (n = 9; 12.3%), undifferentiated spondyloarthritis (n = 9; 12.3%), spondyloarthritis enteropathica (n = 6; 8.2%) and reactive arthritis (n = 2; 2.7%). The average age of onset was equal to 39.1 (± 11.7) and did not differ between genders (p = 0.9057). Of the total, 35 (47.9%) patients were HLA-B*27 positive and 38 (52.1%) were negative. This gene was positively associated with ankylosing spondylitis (OR: 5.37, 95% CI: 1813-15905, p = 0.003) and negatively with spondyloarthritis enteropathica (OR: 0.07, 95% CI: 0003-1301, p = 0.025). The sacroiliitis was associated with the presence of the gene (OR: 10 552, 95% CI: 1260-88256, p = 0.014) and intestinal injury absence (OR: 0.195, 95% CI: 0038-0978, p = 0.048). Conclusions: The HLA-B * 27 was associated with ankylosing spondylitis, enteropathic but not to spondyloarthritis. The radiological signs of sacroiliitis prevailed in patients positive for HLA-B*27, while intestinal involvement was associated with the absence of this gene. especially in cases of dystrophic scoliosis. In both cases, studies with larger samples are needed to assess whether these trends are evident. / Introdução: Espondiloartrite é um grupo de doenças com características clínicas, laboratoriais e imagenológicas semelhantes. Análises das manifestações clínicas das espondiloartrites em pacientes com e sem o gene HLA-B*27 foram realizadas, mas os resultados revelaram grande heterogeneidade. Objetivos: O objetivo deste trabalho foi avaliar as manifestações clínicas das espondiloartrites utilizadas nos critérios de classificação do European Spondyloarthropathy Study Group (ESSG) na presença e na ausência do gene HLA-B*27. Casuística e método: De um total de 156 pacientes com suspeita clínica, encaminhados para investigação do gene HLA-B*27, 73 tiveram diagnóstico de espondiloartrites confirmado, de acordo com os critérios do ESSG. O gene HLA-B*27 foi identificado com o uso de kits comerciais (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Os dados clínicos foram colhidos dos prontuários médicos dos pacientes. Os resultados foram comparados com o uso dos testes Qui-quadrado ou o teste exato de Fisher. Os valores de Odds Ratio (OR) e intervalo de confiança a 95% também foram calculados. O valor p igual ou menor que 0,05 foi considerado significante. Resultados: Dos 73 pacientes selecionados, 53 (72,6%) eram do sexo masculino e 20 (27,4%), femininos. A média de idade foi igual a 49,4 e não diferiu entre os sexos (p=0.337). As espondiloartrites encontradas entre os 73 pacientes foram: espondilite anquilosante (n=47; 64,4%), espondiloartrite psoriásica (n=9; 12,3%), espondiloartrite indiferenciada (n=9; 12,3%), espondiloartrite enteropática (n=6; 8,2%) e artrite reativa (n=2; 2,7%). A média de idade de início dos sintomas foi igual a 39,1 (±11.7) e não diferiu entre os sexos (p=0,9057). Do total, 35 (47.9%) pacientes eram HLA-B*27 positivo e 38 (52.1%), negativos. Este gene foi associado positivamente à espondilite anquilosante (OR: 5.37; IC 95%: 1.813-15.905; p=0.003) e negativamente à espondiloartrite enteropática (OR: 0.07; IC 95%: 0.003-1.301; p=0.025). A sacroiliíte se associou à presença do gene (OR: 10.552; IC 95%: 1.260-88.256; p=0.014) e o comprometimento intestinal à ausência (OR: 0.195; IC 95%: 0.038-0.978; p=0.048). Conclusões: O gene HLA-B*27 foi associado à espondilite anquilosante, mas não à espondiloartrite enteropática. Os sinais radiológicos de sacroiliíte prevaleceram nos pacientes positivos para o gene HLA-B*27, enquanto o comprometimento intestinal foi associado à ausência deste gene.

Background: There is an unacceptable delay in diagnosis of axial Spondyloarthritis (axSpA) especially in its early stages among patients with inflammatory bowel disease (IBD). Objective: to develop a sensible and reliable questionnaire to identify undetected axSpA among IBD patients. Methods: Candidate items for the questionnaire were selected on 3 domains (IBD, inflammatory back symptoms, and extra-axial features). Sensibility of the Toronto axSpA Questionnaire (TASQ) was assessed leading to drafting 18 items. Test-retest reliability study was conducted among 77 patients with established IBD and axSpA and kappa agreement coefficients were calculated for items. Results: The TASQ was developed using multiple steps of sensibility assessment resulting in 16 items. Kappa coefficients ranged from 0.81 to 1.00 for all items indicating almost perfect agreement. Conclusion: TASQ is a newly developed, sensible and reliable questionnaire that should facilitate identification and referral of IBD patients to rheumatologists and should avoid delay in diagnosis of axSpA.

Background: There is an unacceptable delay in diagnosis of axial Spondyloarthritis (axSpA) especially in its early stages among patients with inflammatory bowel disease (IBD). Objective: to develop a sensible and reliable questionnaire to identify undetected axSpA among IBD patients. Methods: Candidate items for the questionnaire were selected on 3 domains (IBD, inflammatory back symptoms, and extra-axial features). Sensibility of the Toronto axSpA Questionnaire (TASQ) was assessed leading to drafting 18 items. Test-retest reliability study was conducted among 77 patients with established IBD and axSpA and kappa agreement coefficients were calculated for items. Results: The TASQ was developed using multiple steps of sensibility assessment resulting in 16 items. Kappa coefficients ranged from 0.81 to 1.00 for all items indicating almost perfect agreement. Conclusion: TASQ is a newly developed, sensible and reliable questionnaire that should facilitate identification and referral of IBD patients to rheumatologists and should avoid delay in diagnosis of axSpA.

La spondylarthrite ankylosante (SA), la forme la plus commune des spondylarthrites (SpA), est fortement associée à la molécule du CMH de classe I HLA-B27 mais le rôle de cet antigène d'histocompatibilité dans le développement de ces pathologies reste encore inexpliqué. L’étude des rats transgéniques HLA-B27, développant une pathologie inflammatoire spontanée ressemblant aux SpA, a permis de confirmer l’implication directe du HLA-B27 et de corréler l’apparition des symptômes avec une forte expression de cette molécule. De plus, il a été montré que l’HLA-B27 présentait une propension particulière au mauvais repliement et à la formation d’oligomères de chaînes lourdes. L’objectif de mon travail de thèse était de déterminer si le trafic et/ou la formation d’oligomères du HLA-B27 étaient corrélés à sa surexpression. Pour cela, notre équipe a développé des protéines de fusion (HLA-BYFP et HLA-BRLuc) ainsi que la technique BRET afin d’étudier les interactions HLA-B/HLA-B. Au moyen de ce système expérimental, nous avons montré la formation de vésicules intracellulaires riches en protéines HLA-B mal repliées lorsqu’elles étaient fortement exprimées, qu'il s'agisse d'allèles associés à la SA (HLA-B*2702, -05, et -07) ou non (HLA-B*2706, et -09, HLA-B*0702). Cependant, ce phénomène était significativement plus prononcé pour les sous-types associés à la SA. Dans les conditions de forte expression, nous avons également observé que les sous-types associés à la SA formaient des oligomères qui se comportent différemment de ceux formés par la protéine HLA-B7. Ce phénomène ne semble pas être dû au déclenchement de la réponse cellulaire « Unfolded Protein Response » (UPR) et n’est pas abrogé par l’inhibition du protéasome. / Ankylosing spondylitis (AS), the most common form of spondyloarthritis (SpA), is strongly associated with the MHC class I HLA-B27 molecule. Although this association has been largely studied, mechanisms of pathology remain unclear. Development of a spontaneous inflammatory disease resembling human SpA in HLA-B27 transgenic rats confirmed the direct involvement of HLA-B27 and allowed to associate disease development with high expression levels of this molecule. Moreover, the HLA-B27 protein has an enhanced propensity to misfold and form aberrant disulfide linked heavy chain oligomers in the endoplasmic reticulum and at the cell surface. The goal of my thesis work was to determine if the HLA-B27 traffic and/or its ability to form oligomers are involved in this requirement of overexpression. For that, our team has developed fusion proteins ((HLA-BYFP and HLA-BRLuc) and the BRET technique to study, in vitro, the HLA-B/HLA-B interactions. Using this experimental system, we have shown the formation of intracellular vesicles, in which misfolded/unfolded HLA-B proteins accumulated when they were highly expressed, for both AS-associated alleles (HLA-B*2702, -05, et -07) or not (HLA-B*2706, et -09, HLA-B*0702). This phenomenon is strongly pronounced for AS-associated subtypes. For high-level expression, we also observed that the AS-associated subtypes form oligomers that behave differently from those formed by the HLA-B7 control protein. This phenomenon doesn’t appear to be due to unfolded protein response (UPR) triggering and is not abrogated by proteasome inhibition.

Introduction: Axial spondyloarthritis (ax-SpA) is an inflammatory rheumatic disease. It is a unique model of bone remodeling disorders because, although one of the main diagnostic parameters is the rate of bone formation, inflammation present in patients' bodies increases the risk of pathological bone resorption, which can lead to osteoporosis. The processes of pathological resorption in ax-SpA have not been fully investigated, both in the disease as such and in the individual forms of the disease, i.e. non-radiographic (nr-axSpA), radiographic axial spondyloarthritis (r-axSpA) and ankylosing spondylitis (AS). This work deals with the influence of inflammatory serum of patients on the process of osteoclast differentiation from peripheral precursors of patients and healthy donors. Material and methods: Monocytes separated from the peripheral blood of either axSpA patients or healthy donors were stimulated for 14 days in vitro with serum from patients and in parallel with serum of age and sex of the corresponding healthy donors. Osteoclasts were evaluated as multinucleated, TRAP positive cells. Their numbers were statistically processed. Results: The inflammatory serum environment of patients with axSpA stimulated the osteoclastogenesis of axSpA monocytes significantly more (P <0,05) than the...

BACKGROUND: Distinguishing patients with axial spondyloarthritis (axial SpA) from patients with other causes of chronic back pain remains a challenge. The lack of reliable biomarkers contributes to the diagnostic delay in axial SpA. Recently, macrophage migration inhibitory factor (MIF) has been proposed as a candidate diagnostic and prognostic biomarker. MIF is a proinflammatory cytokine that was shown to be upregulated in several autoimmune diseases, including axial SpA. The putative role of CD8+ T cells in the disease process suggests further that serum markers of cytotoxicity might have value as serological biomarkers in axial SpA, and that subpopulations of cytotoxic lymphocytes might deserve attention as candidate cellular biomarkers. OBJECTIVE: The goal of this study was to compare serum levels of MIF and other candidate serum proteins in patients with axial SpA and controls, and to develop a flow cytometry panel to analyze cytotoxic lymphocyte cell subpopulations in these cohorts, including KIR+CD8+ T cells, Granzyme B+ CD8+ T cells, MAIT cells, and InEx cells. METHODS: Study subjects were recruited from the Brigham and Women’s Hospital Orthopedic and Arthritis Center. Four cohorts were compared: healthy controls (HC), patients with chronic low back pain (cLBP), axial SpA patients not on a biologic (axSpA/-), and axial SpA patients treated with a TNF inhibitor (axSpA/TNFi). Study subjects were matched for age, sex, and race, when possible. Serum was evaluated using the LEGENDplex Human CD8/NK panel (BioLegend) for thirteen markers including IL-17A, IL-6, TNF, granzyme B, and perforin. CRP and MIF were evaluated by DuoSet ELISA (R&D Systems). A high-dimensional flow cytometry panel was designed to evaluate 14 cell populations of interest. RESULTS: The severity of back pain in the cLBP controls and axSpA/- patients was comparable (BASDAI Q2 mean 5.0 +/- 1.9 vs. 5.0 +/- 3.0). axSpA/- patients had higher back pain, BASDAI and ASDAS scores than axSpA/TNFi patients consistent with higher disease activity in the biologic naïve group. Serum CRP values were significantly higher in axSpA/- patients compared with HC, cLBP controls, and axSpA/TNFi patients (P= 0.01, P=0.0029, P=0.004 respectively). Serum MIF levels were not statistically different between all four groups (P= 0.8069). Additionally, there were no statistically significant differences between the groups for any of the markers included in the LEGENDplex Human CD8/NK panel. A 32-color staining panel was developed to evaluate cytotoxic cell populations. CONCLUSION: In contrast to a previous study, we did not find differences in serum MIF levels between axial SpA patients and controls. Of the evaluated serum biomarkers, only CRP values correlated with active axial SpA. We have developed a promising flow cytometry panel that will help analyze subpopulations of cytotoxic cells. This ultimately could shed light on a candidate cellular biomarker. Our results underscore the need for more research into diagnostic biomarkers in axial SpA.

Introdução: A Espondiloartrite axial (EpA-ax) é uma doença inflamatória e crônica do grupo das Espondiloartrites (EpA). Ela acomete o sistema músculo esquelético ocasionando inflamação das articulações axiais (especialmente da sacroilíaca). Quando essas manifestações são evidenciadas por exames de imagem, temos a caracterização da Espondilite Anquilosante (EA). Do contrário, chamamos de EpA-axial não radiográfica (EpA-ax-nr). A Espondilite Anquilosante (EA) se caracteriza pelo envolvimento inflamatório de articulações do esqueleto axial e apendicular. Seu diagnóstico é baseado em achados clínicos e radiológicos, além da elevação de marcadores inflamatórios e presença do HLA-B27. Várias são as metodologias que se propõem a identificar o HLA-B27 e a Polymerase Chain Reaction (PCR) é tida como referência. Sua ampla utilização esbarra, no entanto, em questões que levam em conta o custo, oferta do exame e o tempo até a obtenção do resultado. Neste contexto, a Citometria de Fluxo (CF) surge como uma alternativa capaz de ajudar o médico na identificação deste marcador genético que pode ser importante para o diagnóstico e prognóstico dessa doença. Objetivo: Avaliar a correlação entre as técnicas laboratoriais mais utilizadas na prática clínica (CF e PCR), comparando sensibilidade e especificidade para detecção do HLA-B27 em pacientes com diagnóstico estabelecido de EpA-ax. Métodos: Estudo transversal, comparativo, com pacientes maiores de 18 anos de idade selecionados por conveniência, coletados ao longo de 2015, com diagnóstico estabelecido de EpA-ax, segundo os do grupo internacional ASAS (working group - Assessment of SpondyloArthritis Intenational Society). Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. O teste Exato de Fisher foi utilizado para comparar as variáveis categóricas, o teste t de Student, para variáveis quantitativas com distribuição simétrica de amostras independentes. E as variáveis com distribuição assimétrica foram comparadas pelo teste de Mann-Whitney. Resultados: O coeficiente Kappa obtido para a determinação da concordância entre os testes foi de 0,454. Foram incluídos no estudo 62 pacientes, desses, sessenta preencheram os critérios para diagnóstico de EA e 2 para EpA-ax não radiográfica (EpA-ax-nr), 64,5% dos pacientes eram do sexo masculino, 88,7% se autodeclararam brancos, a idade média (± desvio padrão) foi de 54,5±12 anos e o tempo mediano (percentis 25 e 75) de diagnóstico de 14 (9 e 24) anos. Dentre as características clínicas apresentadas pela população estudada houve diferença estatisticamente significativa para a artrite periférica, sendo mais frequente no grupo HLA-B27 negativo que no grupo HLA-B27 positivo (P=0,032). Na análise de correlação, 90,3% apresentaram tipagem HLA-B27 positiva por CF e 79,0% pela técnica de PCR. Tendo o PCR como padrão ouro, a CF apresentou uma sensibilidade de 98,0%, especificidade de 38,5% e uma acurácia de 85,5%. Conclusão: Apesar da baixa especificidade apresentada pela CF, nosso estudo demonstrou que a CF tem alta sensibilidade e boa acurácia o que a torna uma boa alternativa para ser utilizada como um teste de triagem na busca da caracterização da doença. Apesar da moderada concordância com a técnica de referência, a CF poderia ajudar o médico a excluir resultados falsamente negativos, racionalizando assim a investigação laboratorial para o diagnóstico da EA. / Introduction: Axial spondyloarthritis (SpA-ax) is an inflammatory and chronic disease of the Spondyloarthritis (SpA) group. It affects the skeletal muscle system causing inflammation of the axial joints (especially of the sacroiliac). When these manifestations are evidenced by imaging tests, a characterization of Ankylosing Spondylitis (AS). Otherwise, we call the non-radiographic SpA-axial (SpA-ax-nr). AS marked by the inflammatory involvement of the axial and appendicular skeletal joints. The diagnosis is based on clinical and radiological findings, as well as the on the elevation of inflammatory markers and presence of HLA-B27. There are several methodologies to identify the HLA-B27 gene and a Polymerase Chain Reaction (PCR) is considered the reference method. However, its use on a large scale does not progress because it takes into account the cost, offer of the exam and the running time to obtain the result. In this context, Flow Cytometry (FC) emerges as an alternative method, helping with the physician in the determination of this genetic marker, important for the diagnosis and prognosis of disease. Objective: To evaluate the correlation between FC and PCR, comparing sensitivity and specificity for detection of HLA-B27 in patients with established diagnosis of SpA-ax. Methods: A cross sectional study including 62 patients recruited during 2015 month was conducted in Hospital de Clínicas de Porto Alegre, an university public hospital. The sample, recruited by convenience in the SpA clinic, was composed of patients ≥ 18 years old, fulfilling the Assessment of Spondyloarthritis (ASAS) criteria SpA-ax. All participants underwent HLA-B27 typing through FC and PCR. The kappa statistic was used to calculate the concordance between the FC and PCR. Taking PCR as the gold standard, sensitivity and specificity of FC to detect HLA-B27 were calculated. Results: The Kappa coefficient obtained to determine the agreement between the tests was 0.454. Sixty two patients were included in the study, sixty met the criteria for diagnosis of AS and two for SpA-ax-nr, 64.5% of the patients were male, 88.7% were self-declared mean age (± standard deviation) was 54.5 ± 12 years and median time (25th and 75th percentiles) for diagnosis was 14 (9 and 24) years. Among the clinical characteristics presented by the population studied, there was a statistically significant difference for peripheral arthritis, wich was more frequent in the HLA-B27 negative group than in the HLA-B27 positive group (P = 0.032). In the correlation analysis, 90.3% presented HLA-B27 positive typing by FC and 79.0% by PCR technique. When PCR was considered the gold standard, CF had a sensitivity of 98.0%, specificity of 38.5% and an accuracy of 85.5%. Conclusion: Despite the low specificity presented by FC, our study demonstrated that FC has high sensitivity and good accuracy, which makes it a good alternative to be used as a screening test in the search for the characterization of the disease. Despite the moderate agreement with the reference technique, FC could help the physician to exclude falsely negative results, thus rationalizing laboratory investigation for the diagnosis of AS.

Introdução: A Espondiloartrite axial (EpA-ax) é uma doença inflamatória e crônica do grupo das Espondiloartrites (EpA). Ela acomete o sistema músculo esquelético ocasionando inflamação das articulações axiais (especialmente da sacroilíaca). Quando essas manifestações são evidenciadas por exames de imagem, temos a caracterização da Espondilite Anquilosante (EA). Do contrário, chamamos de EpA-axial não radiográfica (EpA-ax-nr). A Espondilite Anquilosante (EA) se caracteriza pelo envolvimento inflamatório de articulações do esqueleto axial e apendicular. Seu diagnóstico é baseado em achados clínicos e radiológicos, além da elevação de marcadores inflamatórios e presença do HLA-B27. Várias são as metodologias que se propõem a identificar o HLA-B27 e a Polymerase Chain Reaction (PCR) é tida como referência. Sua ampla utilização esbarra, no entanto, em questões que levam em conta o custo, oferta do exame e o tempo até a obtenção do resultado. Neste contexto, a Citometria de Fluxo (CF) surge como uma alternativa capaz de ajudar o médico na identificação deste marcador genético que pode ser importante para o diagnóstico e prognóstico dessa doença. Objetivo: Avaliar a correlação entre as técnicas laboratoriais mais utilizadas na prática clínica (CF e PCR), comparando sensibilidade e especificidade para detecção do HLA-B27 em pacientes com diagnóstico estabelecido de EpA-ax. Métodos: Estudo transversal, comparativo, com pacientes maiores de 18 anos de idade selecionados por conveniência, coletados ao longo de 2015, com diagnóstico estabelecido de EpA-ax, segundo os do grupo internacional ASAS (working group - Assessment of SpondyloArthritis Intenational Society). Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. O teste Exato de Fisher foi utilizado para comparar as variáveis categóricas, o teste t de Student, para variáveis quantitativas com distribuição simétrica de amostras independentes. E as variáveis com distribuição assimétrica foram comparadas pelo teste de Mann-Whitney. Resultados: O coeficiente Kappa obtido para a determinação da concordância entre os testes foi de 0,454. Foram incluídos no estudo 62 pacientes, desses, sessenta preencheram os critérios para diagnóstico de EA e 2 para EpA-ax não radiográfica (EpA-ax-nr), 64,5% dos pacientes eram do sexo masculino, 88,7% se autodeclararam brancos, a idade média (± desvio padrão) foi de 54,5±12 anos e o tempo mediano (percentis 25 e 75) de diagnóstico de 14 (9 e 24) anos. Dentre as características clínicas apresentadas pela população estudada houve diferença estatisticamente significativa para a artrite periférica, sendo mais frequente no grupo HLA-B27 negativo que no grupo HLA-B27 positivo (P=0,032). Na análise de correlação, 90,3% apresentaram tipagem HLA-B27 positiva por CF e 79,0% pela técnica de PCR. Tendo o PCR como padrão ouro, a CF apresentou uma sensibilidade de 98,0%, especificidade de 38,5% e uma acurácia de 85,5%. Conclusão: Apesar da baixa especificidade apresentada pela CF, nosso estudo demonstrou que a CF tem alta sensibilidade e boa acurácia o que a torna uma boa alternativa para ser utilizada como um teste de triagem na busca da caracterização da doença. Apesar da moderada concordância com a técnica de referência, a CF poderia ajudar o médico a excluir resultados falsamente negativos, racionalizando assim a investigação laboratorial para o diagnóstico da EA. / Introduction: Axial spondyloarthritis (SpA-ax) is an inflammatory and chronic disease of the Spondyloarthritis (SpA) group. It affects the skeletal muscle system causing inflammation of the axial joints (especially of the sacroiliac). When these manifestations are evidenced by imaging tests, a characterization of Ankylosing Spondylitis (AS). Otherwise, we call the non-radiographic SpA-axial (SpA-ax-nr). AS marked by the inflammatory involvement of the axial and appendicular skeletal joints. The diagnosis is based on clinical and radiological findings, as well as the on the elevation of inflammatory markers and presence of HLA-B27. There are several methodologies to identify the HLA-B27 gene and a Polymerase Chain Reaction (PCR) is considered the reference method. However, its use on a large scale does not progress because it takes into account the cost, offer of the exam and the running time to obtain the result. In this context, Flow Cytometry (FC) emerges as an alternative method, helping with the physician in the determination of this genetic marker, important for the diagnosis and prognosis of disease. Objective: To evaluate the correlation between FC and PCR, comparing sensitivity and specificity for detection of HLA-B27 in patients with established diagnosis of SpA-ax. Methods: A cross sectional study including 62 patients recruited during 2015 month was conducted in Hospital de Clínicas de Porto Alegre, an university public hospital. The sample, recruited by convenience in the SpA clinic, was composed of patients ≥ 18 years old, fulfilling the Assessment of Spondyloarthritis (ASAS) criteria SpA-ax. All participants underwent HLA-B27 typing through FC and PCR. The kappa statistic was used to calculate the concordance between the FC and PCR. Taking PCR as the gold standard, sensitivity and specificity of FC to detect HLA-B27 were calculated. Results: The Kappa coefficient obtained to determine the agreement between the tests was 0.454. Sixty two patients were included in the study, sixty met the criteria for diagnosis of AS and two for SpA-ax-nr, 64.5% of the patients were male, 88.7% were self-declared mean age (± standard deviation) was 54.5 ± 12 years and median time (25th and 75th percentiles) for diagnosis was 14 (9 and 24) years. Among the clinical characteristics presented by the population studied, there was a statistically significant difference for peripheral arthritis, wich was more frequent in the HLA-B27 negative group than in the HLA-B27 positive group (P = 0.032). In the correlation analysis, 90.3% presented HLA-B27 positive typing by FC and 79.0% by PCR technique. When PCR was considered the gold standard, CF had a sensitivity of 98.0%, specificity of 38.5% and an accuracy of 85.5%. Conclusion: Despite the low specificity presented by FC, our study demonstrated that FC has high sensitivity and good accuracy, which makes it a good alternative to be used as a screening test in the search for the characterization of the disease. Despite the moderate agreement with the reference technique, FC could help the physician to exclude falsely negative results, thus rationalizing laboratory investigation for the diagnosis of AS.

Introdução: A Espondiloartrite axial (EpA-ax) é uma doença inflamatória e crônica do grupo das Espondiloartrites (EpA). Ela acomete o sistema músculo esquelético ocasionando inflamação das articulações axiais (especialmente da sacroilíaca). Quando essas manifestações são evidenciadas por exames de imagem, temos a caracterização da Espondilite Anquilosante (EA). Do contrário, chamamos de EpA-axial não radiográfica (EpA-ax-nr). A Espondilite Anquilosante (EA) se caracteriza pelo envolvimento inflamatório de articulações do esqueleto axial e apendicular. Seu diagnóstico é baseado em achados clínicos e radiológicos, além da elevação de marcadores inflamatórios e presença do HLA-B27. Várias são as metodologias que se propõem a identificar o HLA-B27 e a Polymerase Chain Reaction (PCR) é tida como referência. Sua ampla utilização esbarra, no entanto, em questões que levam em conta o custo, oferta do exame e o tempo até a obtenção do resultado. Neste contexto, a Citometria de Fluxo (CF) surge como uma alternativa capaz de ajudar o médico na identificação deste marcador genético que pode ser importante para o diagnóstico e prognóstico dessa doença. Objetivo: Avaliar a correlação entre as técnicas laboratoriais mais utilizadas na prática clínica (CF e PCR), comparando sensibilidade e especificidade para detecção do HLA-B27 em pacientes com diagnóstico estabelecido de EpA-ax. Métodos: Estudo transversal, comparativo, com pacientes maiores de 18 anos de idade selecionados por conveniência, coletados ao longo de 2015, com diagnóstico estabelecido de EpA-ax, segundo os do grupo internacional ASAS (working group - Assessment of SpondyloArthritis Intenational Society). Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. O teste Exato de Fisher foi utilizado para comparar as variáveis categóricas, o teste t de Student, para variáveis quantitativas com distribuição simétrica de amostras independentes. E as variáveis com distribuição assimétrica foram comparadas pelo teste de Mann-Whitney. Resultados: O coeficiente Kappa obtido para a determinação da concordância entre os testes foi de 0,454. Foram incluídos no estudo 62 pacientes, desses, sessenta preencheram os critérios para diagnóstico de EA e 2 para EpA-ax não radiográfica (EpA-ax-nr), 64,5% dos pacientes eram do sexo masculino, 88,7% se autodeclararam brancos, a idade média (± desvio padrão) foi de 54,5±12 anos e o tempo mediano (percentis 25 e 75) de diagnóstico de 14 (9 e 24) anos. Dentre as características clínicas apresentadas pela população estudada houve diferença estatisticamente significativa para a artrite periférica, sendo mais frequente no grupo HLA-B27 negativo que no grupo HLA-B27 positivo (P=0,032). Na análise de correlação, 90,3% apresentaram tipagem HLA-B27 positiva por CF e 79,0% pela técnica de PCR. Tendo o PCR como padrão ouro, a CF apresentou uma sensibilidade de 98,0%, especificidade de 38,5% e uma acurácia de 85,5%. Conclusão: Apesar da baixa especificidade apresentada pela CF, nosso estudo demonstrou que a CF tem alta sensibilidade e boa acurácia o que a torna uma boa alternativa para ser utilizada como um teste de triagem na busca da caracterização da doença. Apesar da moderada concordância com a técnica de referência, a CF poderia ajudar o médico a excluir resultados falsamente negativos, racionalizando assim a investigação laboratorial para o diagnóstico da EA. / Introduction: Axial spondyloarthritis (SpA-ax) is an inflammatory and chronic disease of the Spondyloarthritis (SpA) group. It affects the skeletal muscle system causing inflammation of the axial joints (especially of the sacroiliac). When these manifestations are evidenced by imaging tests, a characterization of Ankylosing Spondylitis (AS). Otherwise, we call the non-radiographic SpA-axial (SpA-ax-nr). AS marked by the inflammatory involvement of the axial and appendicular skeletal joints. The diagnosis is based on clinical and radiological findings, as well as the on the elevation of inflammatory markers and presence of HLA-B27. There are several methodologies to identify the HLA-B27 gene and a Polymerase Chain Reaction (PCR) is considered the reference method. However, its use on a large scale does not progress because it takes into account the cost, offer of the exam and the running time to obtain the result. In this context, Flow Cytometry (FC) emerges as an alternative method, helping with the physician in the determination of this genetic marker, important for the diagnosis and prognosis of disease. Objective: To evaluate the correlation between FC and PCR, comparing sensitivity and specificity for detection of HLA-B27 in patients with established diagnosis of SpA-ax. Methods: A cross sectional study including 62 patients recruited during 2015 month was conducted in Hospital de Clínicas de Porto Alegre, an university public hospital. The sample, recruited by convenience in the SpA clinic, was composed of patients ≥ 18 years old, fulfilling the Assessment of Spondyloarthritis (ASAS) criteria SpA-ax. All participants underwent HLA-B27 typing through FC and PCR. The kappa statistic was used to calculate the concordance between the FC and PCR. Taking PCR as the gold standard, sensitivity and specificity of FC to detect HLA-B27 were calculated. Results: The Kappa coefficient obtained to determine the agreement between the tests was 0.454. Sixty two patients were included in the study, sixty met the criteria for diagnosis of AS and two for SpA-ax-nr, 64.5% of the patients were male, 88.7% were self-declared mean age (± standard deviation) was 54.5 ± 12 years and median time (25th and 75th percentiles) for diagnosis was 14 (9 and 24) years. Among the clinical characteristics presented by the population studied, there was a statistically significant difference for peripheral arthritis, wich was more frequent in the HLA-B27 negative group than in the HLA-B27 positive group (P = 0.032). In the correlation analysis, 90.3% presented HLA-B27 positive typing by FC and 79.0% by PCR technique. When PCR was considered the gold standard, CF had a sensitivity of 98.0%, specificity of 38.5% and an accuracy of 85.5%. Conclusion: Despite the low specificity presented by FC, our study demonstrated that FC has high sensitivity and good accuracy, which makes it a good alternative to be used as a screening test in the search for the characterization of the disease. Despite the moderate agreement with the reference technique, FC could help the physician to exclude falsely negative results, thus rationalizing laboratory investigation for the diagnosis of AS.

Background: Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting primarily the spine and its adjacent structures. The disease is characterized not only by destructive joint changes but also by excessive osteoproduction, which can lead to gradual ankylosis of the spine and thus significantly reduce the mobility and quality of life. The pathogenesis of the disease is not yet fully understood, but a strong genetic background is suggested, along with dysregulation of tissue metabolism resulting from an imbalance of pro- and anti-inflammatory immune mechanisms. We are still lacking biomarker with sufficient sensitivity and specificity which could help to identify early diagnosis, to monitor subchondral damage, and to differentiate rapidly progressing patients. The aim of this work was to determine the levels of potential biomarkers of connective tissue metabolism, fat metabolism and new promising biomarkers for both disease subtypes, their relationship to disease activity and progressive structural changes. Results: We have shown increased serum/plasma levels of connective tissue metabolism biomarkers (especially matrix metalloproteinase mediated metabolites), which were able to differentiate patients with early and late forms of axSpA from healthy individuals (HC), were...