ABSTRACT Skin biopsy with investigation of small-diameter nerve fibers in human epidermis and dermis has been proven to be a useful method for confirming small-fiber neuropathy. In medical practice, small-fiber neuropathy is increasingly recognized as a leading cause of neuropathic pain. It is a prevalent complaint in medical offices, brought by patients often as a “painful burning sensation”. The prevalence of neuropathic pain is high in small-fiber neuropathies of different etiologies, especially in the elderly; 7% of population in this age group present peripheral neuropathy. Pain and paresthesia are symptoms which might cause disability and impair quality of life of patients. The early detection of small-fiber neuropathy can contribute to reducing unhealthy lifestyles, associated to higher incidence of the disease.

Abstract Objective To evaluate the frequencies of iNKT cells and their subsets in patients with deep endometriosis. Methods A case-control study was conducted between 2013 and 2015, with 73 patients distributed into two groups: 47 women with a histological diagnosis of endometriosis and 26 controls. Peripheral blood, endometriosis lesions, and healthy peritoneal samples were collected on the day of surgery to determine the frequencies of iNKT cells and subtypes via flow cytometry analysis. Results The authors observed a lower number of iNKT (p= 0.01) and Double-Negative (DN) iNKT cells (p= 0.02) in the blood of patients with endometriosis than in the control group. The number of DN iNKT IL-17+ cells in the secretory phase was lower in the endometriosis group (p= 0.049). There was an increase in the secretion of IL-17 by CD4+ iNKT cells in the blood of patients with endometriosis and severe dysmenorrhea (p= 0.038), and severe acyclic pelvic pain (p= 0.048). Patients with severe dysmenorrhea also had a decreased number of CD4+ CCR7+ cells (p= 0.022). Conclusion The decreased number of total iNKT and DN iNKT cells in patients with endometriosis suggests that iNKT cells play a role in the pathogenesis of endometriosis and can be used to develop new diagnostic and therapeutic agents.

Abstract Background: The VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report. Mainbody: Sixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP. Conclusion: The VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.

RESUMO Objetivo Avaliar o desempenho das metodologias de ensaio imunoenzimático e imunofluorescência indireta para a detecção de anticorpos anticitoplasma de neutrófilos em um contexto de laboratório clínico de rotina. Métodos Foram testadas 227 amostras pelas metodologias de imunofluorescência indireta e ensaio imunoenzimático com especificidades para anticorpos antiproteinase-3 e antimieloperoxidase. As proporções de amostras positivas foram comparadas por hipóteses de McNemar, e a concordância foi descrita pelo coeficiente Kappa de Cohen. Resultados A concordância dos testes foi 96,5%, e o coeficiente Kappa obtido foi 0,70 (IC95%: 0,50-0,90; p<0,001). Utilizando a imunofluorescência indireta como padrão-ouro, a sensibilidade do ensaio imunoenzimático foi de 0,62 e a especificidade, 0,99, com acurácia diagnóstica em 96% dos casos. Algumas amostras apresentaram resultados negativos por ensaio imunoenzimático e positivos por imunofluorescência. Isso ocorreu em amostras com vários padrões de fluorescência, mas particularmente nos casos com padrões atípicos. Duas amostras com positividade antiproteinase 3 foram consideradas negativas por imunofluorescência. Conclusão Os métodos de ensaio imunoenzimático tiveram alta especificidade, mas sensibilidade inferior. A realização da imunofluorescência indireta aumenta a sensibilidade diagnóstica, ao mesmo tempo que a pesquisa de antiproteinase 3 por ensaio imunoenzimático também pode agregar poder diagnóstico.

ABSTRACT Objective To evaluate the performance of enzyme-linked immunosorbent assay and indirect immunofluorescence methods for the detection of antineutrophil cytoplasmic antibodies in a routine clinical laboratory setting. Methods A total of 227 samples were tested by indirect immunofluorescence and enzyme-linked immunosorbent assay with antigen specificity for antiproteinase 3 and antimyeloperoxidase. The proportions of positive samples were compared by McNemar hypotheses and agreement was described by Cohen’s Kappa coefficient. Results The agreement of the tests was 96.5%, and the Kappa coefficient obtained was 0.70 (95%CI: 0.50-0.90; p<0.001). Considering indirect immunofluorescence as the gold standard, the sensitivity of the enzyme-linked immunosorbent assay was 0.62 and the specificity was 0.99, with diagnostic accuracy in 96% of cases. Some samples were negative in enzyme-linked immunosorbent assay and positive in indirect immunofluorescence. This situation occurred in all immunofluorescence patterns, but particularly in atypical patterns. Two samples with antiproteinase 3 positivity were considered negative in indirect immunofluorescence. Conclusion The enzyme-linked immunosorbent assay had high specificity but lower sensitivity. The performance of indirect immunofluorescence increases diagnostic sensitivity, while the search for antiproteinase 3 by enzyme-linked immunosorbent assay may also add diagnostic power.

RESUMO Objetivo: Avaliar o desempenho da imunofluorescência indireta no diagnóstico sorológico de dengue em uma população com alta prevalência de arboviroses. Métodos: Duzentas amostras de soro de pacientes com suspeita clínica de dengue foram testadas por ensaio imunoenzimático e imunofluorescência indireta mosaico BIOCHIP®. Foram calculados especificidade, sensibilidade e coeficiente Kappa. Nas amostras discordantes, realizou-se reação em cadeia da polimerase como método confirmatório. Resultados: Das 200 amostras, 20% foram positivas e 80% negativas para IgM antivírus da dengue no ensaio imunoenzimático. Das 40 positivas, 25% foram negativas na imunofluorescência indireta. Destas dez negativas, apenas 20% eram também negativas na reação em cadeia da polimerase. Das 160 negativas no ensaio imunoenzimático, 5% foram positivas na imunofluorescência indireta. Por fim, dentre as nove discordantes, 33% tiveram vírus da dengue detectado na reação em cadeia da polimerase. O coeficiente Kappa foi 0,70 (0,57-0,82). Sensibilidade e especificidade por imunofluorescência indireta foram, respectivamente, 75% e 94%. Para IgG antivírus da dengue, de 200 amostras, 15,5% foram positivas e 84,5% negativas no ensaio imunoenzimático. Das 31 positivas, 12,9% foram negativas na imunofluorescência indireta. Destas quatro discordantes, 25% apresentaram vírus da dengue não detectado na reação em cadeia da polimerase. Das 169 negativas, 8% foram positivas na imunofluorescência indireta. Destas, 64% foram positivas também na reação em cadeia da polimerase. O coeficiente Kappa foi 0,695 (0,56-0,83). Sensibilidade e a especificidade por imunofluorescência indireta foram, respectivamente, 87,1% e 91,7%. Conclusão: Ensaio imunoenzimático seria suficiente para diagnóstico sorológico de infecção aguda, não justificando a incorporação da imunofluorescência indireta. Substituir ensaio imunoenzimático pela imunofluorescência indireta poderia comprometer a sensibilidade para IgM. Contudo, a imunofluorescência indireta auxilia diferenciar três arboviroses simultaneamente, sendo vantajoso em epidemias concomitantes.

ABSTRACT Objective: To evaluate the performance of indirect immunofluorescence for serological diagnosis of dengue virus in a population with high prevalence of arboviruses. Methods: Two-hundred serum samples from patients with clinical suspicion of dengue fever were tested by immunoenzymatic and indirect immunofluorescence assay BIOCHIP® mosaic. Specificity, sensitivity and Kappa coefficient were calculated. Discordant samples were tested by polymerase chain reaction for confirmation. Results: Of the 200 samples, 20% were positive and 80% negative for anti-dengue virus IgM antibodies in the immunoenzymatic test. Of the 40 positives, 25% were negative in indirect immunofluorescence. Of these ten discordant results, only 20% were also negative in the polymerase chain reaction (PCR). Of the 160 negatives in the immunoenzymatic test, 5% were positive in indirect immunofluorescence. Of these nine discordant results, 33% were positive in the PCR. The Kappa coefficient was 0.7 (0.572-0.829). Sensitivity and specificity of indirect immunofluorescence were respectively 75% and 94%. For anti-dengue virus IgG antibodies, of the 200 samples, 15.5% were positive and 84.5% were negative in the immunoenzymatic test. Of the 31 positives, 12.9% were negative in indirect immunofluorescence. Of these four discordant results, 25% were negative in the PCR. Of the 169 negatives, 8% were positive in indirect immunofluorescence. Of these 14 discordant results, 64% were also positive in the PCR. The Kappa coefficient was 0.695 (0.563-0.83). Sensitivity and specificity of indirect immunofluorescence were 87.1% and 91.7%, respectively. Conclusion: For diagnosis of acute infection, the immunoenzymatic test is enough, and the use of additional methods is not warranted. Replacing the immunoenzymatic test by indirect immunofluorescence would compromise the sensitivity for IgM. However, indirect immunofluorescence can distinguish three arboviruses simultaneously, an advantage during concomitant epidemics.

RESUMO: Introdução: A glicemia de jejum é um teste usado para o monitoramento do diabetes mellitus, bem como para seu rastreamento e diagnóstico. O objetivo do estudo foi analisar resultados de glicemia de jejum de pacientes da rede pública e sua correlação com hemoglobina glicada e lipídios. Métodos: Estudo transversal, com 77.581 pacientes, atendidos em 2014. Resultados: A maioria é do sexo feminino (65%), com idade entre 18 e 115 anos (53 ± 15,5 anos). A concordância entre glicemia de jejum e hemoglobina glicada foi moderada (Kappa = 0,416), entretanto foi substancial para níveis compatíveis com diabetes (Kappa = 0,689) e pobre para pré-diabetes (Kappa = 0,188). Glicemia de jejum ≥ 100 mg/dL foi encontrada em 41,1% dos pacientes e hemoglobina glicada ≥ 5,7% em 61,5%. As alterações lipídicas são mais frequentes nos indivíduos com alterações na glicemia. Dos 14.241 indivíduos com glicemia de jejum ≥ 126 mg/dL, a microalbuminúria foi pesquisada em apenas 883 (6,2%) indivíduos, com resultado alterado em 201 (22,8%). Conclusões: Nos indivíduos que realizaram mais de uma dosagem de glicemia de jejum, a maioria permaneceu com exames alterados, principalmente os que apresentavam valores compatíveis com o diagnóstico de diabetes, sugerindo que não conseguem um controle adequado. A baixa frequência de pesquisa de microalbuminúria em indivíduos com glicemia de jejum sugestiva de diabetes reflete a pequena preocupação com o rastreio de suas complicações crônicas. A elevada frequência de dislipidemia nesses indivíduos evidencia ser uma população de elevado risco cardiovascular.

ABSTRACT: Introduction: Fasting glucose is a test used for monitoring diabetes mellitus, as well as its screening and diagnosis. The objective of this study was to evaluate fasting glucose results and their correlation with glycated hemoglobin and lipids. Methods: Cross-sectional study, involving 77,581 patients, attended in 2014. Results: The majority of the patients are women (65%). The age of the patients ranged from 18 to 115 years (mean of 53 ± 15.5). The agreement between fasting glucose and glycated hemoglobin was moderate (kappa = 0.416); however, it was substantial for the levels used for the diagnosis of diabetes (kappa = 0.689) and poor for pre-diabetes (kappa = 0.188). Fasting glucose ≥ 100 mg/dL was observed in 41.1% of the patients and 61.5% present glycated hemoglobin ≥ 5.7%. Lipid abnormalities are likeliest in patients with elevated fasting glucose. From those 14,241 individuals that had fasting glucose ≥ 126 mg/dL, the microalbuminuria test was performed in only 883 (6.2%) patients, with abnormal results in 201 (22.8%). Conclusions: The high frequency of fasting glucose with abnormal results may reflect the high proportion of exams performed by individuals with diagnosis of diabetes, to evaluate their glycemic control. The low frequency of requests for microalbuminuria tests in those with probable diagnosis of diabetes reflects the little attention paid for the screening of chronic complications of diabetes. It calls attention the high frequency of dyslipidemia in those individuals, highlighting the fact that this is a population with high cardiovascular risk.

RESUMO: Introdução: A glicemia de jejum é um teste usado para o monitoramento do diabetes mellitus, bem como para seu rastreamento e diagnóstico. O objetivo do estudo foi analisar resultados de glicemia de jejum de pacientes da rede pública e sua correlação com hemoglobina glicada e lipídios. Métodos: Estudo transversal, com 77.581 pacientes, atendidos em 2014. Resultados: A maioria é do sexo feminino (65%), com idade entre 18 e 115 anos (53 ± 15,5 anos). A concordância entre glicemia de jejum e hemoglobina glicada foi moderada (Kappa = 0,416), entretanto foi substancial para níveis compatíveis com diabetes (Kappa = 0,689) e pobre para pré-diabetes (Kappa = 0,188). Glicemia de jejum ≥ 100 mg/dL foi encontrada em 41,1% dos pacientes e hemoglobina glicada ≥ 5,7% em 61,5%. As alterações lipídicas são mais frequentes nos indivíduos com alterações na glicemia. Dos 14.241 indivíduos com glicemia de jejum ≥ 126 mg/dL, a microalbuminúria foi pesquisada em apenas 883 (6,2%) indivíduos, com resultado alterado em 201 (22,8%). Conclusões: Nos indivíduos que realizaram mais de uma dosagem de glicemia de jejum, a maioria permaneceu com exames alterados, principalmente os que apresentavam valores compatíveis com o diagnóstico de diabetes, sugerindo que não conseguem um controle adequado. A baixa frequência de pesquisa de microalbuminúria em indivíduos com glicemia de jejum sugestiva de diabetes reflete a pequena preocupação com o rastreio de suas complicações crônicas. A elevada frequência de dislipidemia nesses indivíduos evidencia ser uma população de elevado risco cardiovascular.

ABSTRACT: Introduction: Fasting glucose is a test used for monitoring diabetes mellitus, as well as its screening and diagnosis. The objective of this study was to evaluate fasting glucose results and their correlation with glycated hemoglobin and lipids. Methods: Cross-sectional study, involving 77,581 patients, attended in 2014. Results: The majority of the patients are women (65%). The age of the patients ranged from 18 to 115 years (mean of 53 ± 15.5). The agreement between fasting glucose and glycated hemoglobin was moderate (kappa = 0.416); however, it was substantial for the levels used for the diagnosis of diabetes (kappa = 0.689) and poor for pre-diabetes (kappa = 0.188). Fasting glucose ≥ 100 mg/dL was observed in 41.1% of the patients and 61.5% present glycated hemoglobin ≥ 5.7%. Lipid abnormalities are likeliest in patients with elevated fasting glucose. From those 14,241 individuals that had fasting glucose ≥ 126 mg/dL, the microalbuminuria test was performed in only 883 (6.2%) patients, with abnormal results in 201 (22.8%). Conclusions: The high frequency of fasting glucose with abnormal results may reflect the high proportion of exams performed by individuals with diagnosis of diabetes, to evaluate their glycemic control. The low frequency of requests for microalbuminuria tests in those with probable diagnosis of diabetes reflects the little attention paid for the screening of chronic complications of diabetes. It calls attention the high frequency of dyslipidemia in those individuals, highlighting the fact that this is a population with high cardiovascular risk.

Abstract Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

ABSTRACT Objective We sought to investigate the impact of self-reported fasting duration times on the lipid profile results and its impact on the cardiovascular risk stratification and metabolic syndrome diagnosis. Subjects and methods We analyzed data from all consecutive individuals evaluated in a comprehensive health examination at the Hospital Israelita Albert Einstein from January to December 2015. We divided these patients in three groups, according to the fasting duration recalled (< 8h, 8-12h and > 12h). We calculated the global cardiovascular risk and diagnosed metabolic syndrome according to the current criteria and estimated their change according to fasting duration. Results A total of 12,196 (42.3 ± 9.2 years-old, 30.2% females) patients were evaluated. The distribution of cardiovascular risk was not different among groups defined by fasting duration in both men and women (p = 0.547 for women and p = 0.329 for men). Similarly, the prevalence of metabolic syndrome was not influenced by the fasting duration (p = 0.431 for women and p = 0.166 for men). Conclusion Self-reported fasting duration had no significant impact on the lipid profile results, including triglyceride levels. Consequently, no changes on the cardiovascular risk stratification using the Framingham risk score nor changes on the prevalence of metabolic syndrome were noted.

RESUMO A análise do líquido cefalorraquidiano tem sido empregada para avaliação diagnóstica da esclerose múltipla e a exclusão dos diagnósticos diferenciais. Os achados clássicos refletem a natureza inflamatória da doença, incluindo discreta pleocitose, leve hiperproteinorraquia, aumento da síntese intratecal de imunoglobulina G e, mais tipicamente, a presença de bandas oligoclonais. Nos últimos anos, surgiram novos biomarcadores para esclerose múltipla, e esta busca por marcadores reflete a necessidade de melhor avaliar a atividade e a progressão da doença, bem como a eficácia terapêutica. Uma avaliação mais refinada da atividade da doença e da resposta aos medicamentos pode contribuir para melhores decisões terapêuticas, particularmente no que se refere à troca de medicação. Isto é muito importante nos dias de hoje, quando surgem novas opções medicamentosas. Neste artigo de revisão, avaliamos criticamente a literatura atual referente aos novos marcadores liquóricos na esclerose múltipla. A mensuração destes marcadores, como a quimiocina CXCL13, fetuína A e, principalmente, o neurofilamento de cadeia leve, demonstrou resultados promissores na avaliação da doença, provendo informações que, em conjunto com dados clínicos e de neuroimagem, podem contribuir para melhores decisões terapêuticas.

ABSTRACT The cerebrospinal fluid analysis has been employed for supporting multiple sclerosis diagnosis and ruling out the differential diagnoses. The most classical findings reflect the inflammatory nature of the disease, including mild pleocytosis, mild protein increase, intrathecal synthesis of immunoglobulin G, and, most typically, the presence of oligoclonal bands. In recent years, new biomarkers have emerged in the context of multiple sclerosis. The search for new biomarkers reflect the need of a better evaluation of disease activity, disease progression, and treatment efficiency. A more refined evaluation of disease and therapy status can contribute to better therapeutic choices, particularly in escalation of therapies. This is very relevant taking into account the availability of a greater number of drugs for multiple sclerosis treatment in recent years. In this review, we critically evaluate the current literature regarding the most important cerebrospinal fluid biomarkers in multiple sclerosis. The determination of biomarkers levels, such as chemokine ligand 13, fetuin A, and mainly light neurofilament has shown promising results in the evaluation of this disease, providing information that along with clinical and neuroimaging data may contribute to better therapeutic decisions.

RESUMO O uso de anticoagulantes e de agentes antiplaquetários tem aumentado nos últimos anos. A realização de punção lombar diagnóstica em pacientes utilizando tais medicamentos representa um desafio, em função dos riscos de complicações hemorrágicas ou trombóticas, estas últimas em pacientes que interrompem o tratamento para a realização do procedimento. Não há ainda estudos controlados nem diretrizes específicas nesta área. Neste artigo de revisão, algumas recomendações são feitas, levando-se em conta aspectos farmacológicos destas medicações, o risco de complicações trombóticas de acordo com a doença de base, e a urgência na coleta do líquor. A avaliação cuidadosa destas informações e uma monitorização neurológica rigorosa visando a detecção e o tratamento precoce de complicações podem reduzir o risco de sequelas neurológicas decorrentes de hemorragia. Uma decisão individualizada e uma efetiva comunicação entre o médico assistente e o responsável pela realização da punção lombar é essencial para minimizar potenciais riscos.

ABSTRACT The use of anticoagulants and antiplatelet agents has largely increased. Diagnostic lumbar puncture in patients taking these drugs represents a challenge considering the opposing risks of bleeding and thrombotic complications. To date there are no controlled trials, specific guidelines, nor clear recommendations in this area. In the present review we make some recommendations about lumbar puncture in patients using these drugs. Our recommendations take into consideration the pharmacology of these drugs, the thrombotic risk according to the underlying disease, and the urgency in cerebrospinal fluid analysis. Evaluating such information and a rigorous monitoring of neurological symptoms after lumbar puncture are crucial to minimize the risk of hemorrhage associated neurological deficits. An individualized patient decision-making and an effective communication between the assistant physician and the responsible for conducting the lumbar puncture are essential to minimize potential risks.

Medicina personalizada é o uso de biomarcadores, em sua maioria marcadores moleculares, para a detecção de traços genéticos específicos, a fim de orientar diversas abordagens para a prevenção e o tratamento de diferentes doenças. A identificação de vários genes relacionados a doenças hereditárias, oncológicas e infecciosas permite a detecção de polimorfismos genéticos que estão envolvidos em diferentes evoluções clínicas dessas doenças, bem como com variações na resposta ao tratamento. Atualmente, já é possível detectar esses polimorfismos utilizando diversas metodologias: a detecção de polimorfismos de nucleotídeo único pela reação de polimerização em cadeia; a detecção de microarranjos de ácidos nucleicos; e o sequenciamento de ácidos nucleicos com sequenciadores de DNA automatizados usando métodos derivados de sequenciamento Sanger ou de nova geração. Os ensaios de medicina personalizada são dirigidos para detectar variações genéticas que alteram interações de fármacos com alvos ou vias metabólicas de fármacos (anabólicas e catabólicas), podendo ser utilizados para a seleção de formulações farmacêuticas e para detectar diferentes imunogenicidades da droga. As aplicações de medicina personalizada já foram descritas em várias áreas da Medicina e permitem que abordagens de tratamento específicas sejam aplicadas para cada paciente e para cada doença, de acordo com os resultados dos ensaios utilizados. A aplicação de um protocolo desse tipo exige uma relação intensa entre o laboratório e o corpo clínico. Para sua execução, é necessária uma equipe coordenada, composta por investigadores de pesquisa básica e médicos altamente especializados em suas áreas, apoiada por um time bastante especializado de analistas clínicos treinados em testes de biologia molecular.

Personalized medicine is the use of biomarkers, most of them molecular markers, for detection of specific genetic traits to guide various approaches for preventing and treating different conditions. The identification of several genes related to heredity, oncology and infectious diseases lead to the detection of genetic polymorphisms that are involved not only in different clinical progression of these diseases but also in variations in treatment response. Currently, it is possible to detect these polymorphisms using several methodologies: detection of single nucleotide polymorphisms using polymerase chain reaction methods; nucleic acid microarray detection; and nucleic acid sequencing with automatized DNA sequencers using Sanger-derived methods and new generation sequencing. Personalized medicine assays are directed towards detecting genetic variations that alter interactions of drugs with targets or the metabolic pathways of drugs (upstream and downstream) and can be utilized for the selection of drug formulations and detect different immunogenicities of the drug. Personalized medicine applications have already been described in different areas of Medicine and allow specific treatment approaches to be applied to each patient and pathology according to the results of these assays. The application of such a protocol demands an increasing interaction between the clinical laboratory and the clinical staff. For its implementation, a coordinated team composed of basic researchers and physicians highly specialized in their areas supported by a highly specialized team of clinical analysts particularly trained in molecular biology assays is necessary.

Objetivo Testar e validar um método molecular multiplex para detecção de infecções sanguíneas, além de comparar os resultados com os obtidos pela hemocultura convencional.Métodos Os testes de hemocultura e o LightCycler® SeptiFast foram realizados em 114 pacientes consecutivos com evidência clínica de sepse.Resultados Mais amostras positivas (23; 20,2%) foram detectadas pelo LightCycler® SeptiFast do que pela hemocultura (17; 14,9%), mostrando concordância de 86,8%. Os resultados discordantes foram de quatro pacientes positivos apenas para hemocultura, dez positivos apenas para LightCycler® SeptiFast e um com patógenos diferentes encontrados em cada método. Infecções por micro-organismos não reconhecidos pelo LightCycler® SeptiFast e detectados apelas pela hemocultura confirmam a necessidade da realização dos dois métodos. O tempo médio para os resultados da hemocultura foi de 5 dias para amostras negativas e de 3,5 dias para as positivas. Os resultados pelo LightCycler® SeptiFast foram obtidos em menos de 8 horas.Conclusão O LightCycler® SeptiFast mostrou ser um teste de grande potencial para ser realizado simultaneamente à hemocultura para diagnóstico de sepse em doentes graves, permitindo um diagnóstico mais rápido de infecções por bactérias e fungos e, dessa forma, auxiliando a redução do tempo de hospitalização e racionalização do uso de antibióticos. Eventualmente, o LightCycler® SeptiFast pode detectar inclusive infecções por micro-organismos dificilmente detectáveis via hemocultura, especialmente aquelas causadas por Candida nãoalbicans.

Objective To test and validate a multiplex real-time polymerase chain reaction method for bloodstream infections, as well as to compare the results with conventional blood culture.Methods A total of 114 consecutive patients with clinical evidence of sepsis were submitted to blood culture and LightCycler™ SeptiFast tests.Results More positive specimens (23; 20.2%) were detected using the LightCycler™ SeptiFast than the blood culture (17; 14.9%), with an agreement of 86.8%. Discordant results were seen in four patients positive only to blood culture, ten positive only to LightCycler™ SeptiFast and one to different pathogens found by each test. Infections with microorganisms detected only using blood culture reassured the need to perform both tests. The mean time to results for blood culture was 5 days for negative and 3.5 days for positive results. LightCycler™ SeptiFast results were achieved in less than 8 hours.Conclusion LightCycler™ SeptiFast showed a high potential as a test to be carried out concomitantly with blood culture for sepsis diagnosis in severely ill patients. This test allowed a faster diagnosis of bacterial and fungal infections that helped to reduce hospital stay and to control the use of antibiotics. LightCycler™ SeptiFast can also eventually detect microorganism and infections that are hardly detected by blood culture, especiallyCandidanon-albicans infections.

Objetivo: O IV Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) realizado em Vitória (ES), no dia 18 de setembro de 2012, objetivou discutir estratégias e recomendações relacionadas ao procedimento técnico, à padronização e à interpretação dos resultados da pesquisa de autoanticorpos em células HEp-2. Métodos: Participaram do evento 23 pesquisadores e especialistas de Universidades e laboratórios brasileiros. Foram abordados diferentes tópicos, discutidos amplamente a fim de se estabelecer recomendações específicas. Resultados e conclusão: O IV Consenso integrou à árvore de decisão o padrão citoplasmático em Anéis e Bastões, o padrão nuclear pontilhado Quasi-homogêneo (QH) e o padrão misto CENP-F. Discutiu-se ainda a necessidade de atenção para a classificação do padrão misto relacionado à presença de anticorpos anti-DNA topoisomerase I (Scl70), compreendendo os componentes nuclear pontilhado fino, nucleolar homogêneo, NOR na placa metafásica e citoplasmático pontilhado fino. Foram sugeridas diretrizes para o controle de qualidade do teste, diluição de triagem e diluição de esgotamento, e foi emitido alerta quanto à necessidade de atenção em relação à heterogeneidade de substratos disponíveis no mercado e a utilização de metodologias automatizadas para detecção de autoanticorpos.

Objective: The Fourth Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) was held in Vitória, Espírito Santo, and aimed to discuss strategies and recommendations about the technique, standardization, interpretation and quality control of the indirect immunofluorescence reaction on HEp-2 cells. Methods: Twenty three ANA experts from university centers and private laboratories in different areas from Brazil discussed and agreed upon recommendations for the fourth edition of the Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells. Results and conclusion: The 4th ANA Consensus included three novel patterns into the existing algorithm (cytoplasmic Rods and Rings, nuclear Quasi-homogeneous, and CENP-F). Emphasis was given to the need of attention in describing the peculiar mixed pattern elicited by anti-DNA topoisomerase I (Scl-70) autoantibodies, comprising nuclear fine specked, nucleolar homogeneous pattern, NOR staining in metaphase plates, and cytoplasmic fine speckled patterns. The group also emphasized the need for continuous quality control in indirect immunofluorescence assays, the establishment of screening dilutions, as well as conjugate titration. An alert was made regarding the heterogeneity of commercial kits in defining patterns and the use of solid phase methodologies to determine the presence of autoantibodies.

INTRODUÇÃO: A imunofluorescência indireta (IFI) utilizando células HEp-2 como substrato antigênico é o teste padrão-ouro para a pesquisa de autoanticorpos contra antígenos celulares. Contudo, as condições de cultivo, fixação e permeabilização celular interferem diretamente na preservação e na distribuição espacial dos antígenos. Portanto, pode-se presumir que distintas condições no preparo das células possam interferir no reconhecimento dos padrões de imunofluorescência associados aos diversos autoanticorpos. OBJETIVO: Avaliar um painel de amostras de soro representativo de padrões nuclear, nucleolar, citoplasmático, de aparelho mitótico e de placa cromossômica em substratos de células HEp-2 de diferentes fornecedores. MATERIAIS E MÉTODOS: Sete observadores blindados e independentes de três centros de referência avaliaram 17 amostras que apresentavam diferentes padrões nucleares, nucleolares, citoplasmáticos e associados ao aparelho mitótico em lâminas com células HEp-2 de oito procedências. As lâminas foram codificadas para manter a confidencialidade das marcas, bem como dos centros participantes. RESULTADOS: Os 17 padrões de imunofluorescência em células HEp-2 foram reconhecidos na maioria dos substratos. No entanto, alguns substratos mostraram déficit na apresentação de alguns padrões (nuclear pontilhado grosso/U1-ribonucleoprotein associado a anticorpos contra o RNP (U1 ribonucleoproteína), sugestivo da presença de anticorpos anti-CENP-F (proteína centromérica F), sugestivo de anticorpos contra antígenos de célula em proliferação (proliferating cell nuclear antigen [PCNA]), citoplasmático pontilhado fino associado a anticorpos anti-Jo-1 (histidil sintetase), anti-NuMA-1 (nuclear mitotic apparatus protein 1) e anti-NuMA-2 (nuclear mitotic apparatus protein 2). CONCLUSÃO: Em que pese a boa qualidade geral dos substratos avaliados, existe divergência nos resultados obtidos entre os diferentes substratos comerciais. As variações observadas podem ser devidas aos lotes avaliados, portanto não se pode generalizar para as respectivas marcas. Recomenda-se que cada novo lote ou marca de lâmina sejam testados com diferentes soros referência representativos dos diversos padrões.

INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP), centromeric protein F (CENP-F), proliferating cell nuclear antigen (PCNA), cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase), nuclear mitotic apparatus protein 1 (NuMA-1) and nuclear mitotic apparatus protein 2 (NuMA-2). CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.