Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein.

Objectives: Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods: Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation-mass spectrometry (IP-MS). Results: MND-REF stained 6-20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions: MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

IMP dehydrogenase rod/ring structures in acral melanomas.

Acral lentiginous melanoma (ALM) is a rare subtype of melanoma with aggressive behavior. IMPDH enzyme, involved in de novo GTP biosynthesis, has been reported to assemble into large filamentary structures called rods/rings (RR) or cytoophidium (cellular snakes). RR assembly induces a hyperactive state in IMPDH, usually to supply a high demand for GTP nucleotides, such as in highly proliferative cells. We investigate whether aggressive melanoma tumor cells present IMPDH-based RR structures. Forty-five ALM paraffin-embedded tissue samples and 59 melanocytic nevi were probed with anti-IMPDH2 antibody. Both the rod- and ring-shaped RR could be observed, with higher frequency in ALM. ROC curve analyzing the proportions of RR-positive cells in ALM versus nevi yielded a 0.88 AUC. Using the cutoff of 5.5% RR-positive cells, there was a sensitivity of 80% and specificity of 85% for ALM diagnosis. In ALM, 36 (80%) showed RR frequency above the cutoff, being classified as RR-positive, compared with only 9 (15%) of the nevi (p < .001). Histopathology showed that 71% of the RR-positive specimens presented Breslow thickness > 4.0mm, compared with only 29% in the RR-low/negative (p = .039). We propose that screening for RR structures in biopsy specimens may be a valuable tool helping differentiate ALM from nevi and accessing tumor malignancy.

Future perspective for diagnosis in autoimmune diseases

Human beings have taken successive approaches for the understanding and management of diseases. Initially brewed in supernatural concepts and mystical procedures, a vigorous scientific approach has emerged on the grounds of fundamental disciplines such as anatomy, microbiology, biochemistry, physiology, immunology, pathology, and pharmacology. The resulting integrated knowledge contributed to the current classification of diseases and the way Medicine is carried out today. Despite considerable progress, this approach is rather insufficient when it comes to systemic inflammatory conditions, such as systemic lupus erythematosus, that covers clinical conditions ranging from mild pauci-symptomatic diseases to rapidly fatal conditions. The treatment for such conditions is often insufficient and novel approaches are needed for further progress in these areas of Medicine. A recent breakthrough has been achieved with respect to chronic auto-inflammatory syndromes, in which molecular dissection of underlying gene defects has provided directions for target-oriented therapy. Such approach may be amenable to application in systemic auto-immune diseases with the comprehension that such conditions may be the consequence of interaction of specific environmental stimuli and an array of several and interconnected gene polymorphisms. On the bulk of this transformation, the application of principles of pharmacogenetics may lead the way towards a progressively stronger personalized Medicine.

O homem tem buscado sucessivas abordagens para o entendimento e manejo das doenças. Partindo de conceitos sobrenaturais e procedimentos místicos, uma abordagem científica vigorosa vicejou com base em disciplinas fundamentais como a anatomia, microbiologia, bioquímica, fisiologia, imunologia, patologia e farmacologia. O conhecimento integrado resultante contribuiu para a atual classificação das doenças e a formacom que a Medicina atual é praticada. Apesar deste considerável progresso, esta abordagem é insuficiente quando se trata de condições inflamatórias sistêmicas, como o lúpus eritematoso sistêmico, que abrange condições variando de formas brandas e pauci-sintomáticas até condições rapidamente fatais. O tratamento dessas condições é frequentemente insuficiente e novas abordagens são necessárias para progresso adicional nessas áreas da Medicina. Um avanço recente foi obtido no que tange às síndromes auto-inflamatórias hereditárias, nas quais a dissecção molecular dos defeitos gênicos subjacentes forneceu direcionamento para terapia orientada a alvos moleculares específicos. Esta abordagem é passível de aplicação às doenças auto-imunes sistêmicas com a compreensão de essas condições podem ser conseqüência da interação de estímulos ambientais específicos e uma gama de vários polimorfis mosgênicos interconectados. No escopo dessa transformação, a aplicação dos princípios de farmacogenéticas poderá contribuir para o progressivo desenvolvimento de uma Medicina personalizada vigorosa.

Inflammatory myopathies in childhood: correlation between nailfold capillaroscopy findings and clinical and laboratory data

OBJECTIVE: Nailfold capillaroscopy is an important tool for the diagnosis and follow-up of patients with rheumatic diseases, in particular dermatomyositis and scleroderma. A relationship has been observed in adults between improved capillaroscopic findings and reduced disease activity. Our aim was to correlate disease activity (clinical and laboratory data) and nailfold capillaroscopy findings in 18 patients with inflammatory myopathies. METHODS: This prospective study included 13 juvenile dermatomyositis patients (Bohan and Peter criteria) (mean age of 8.8 years) and five patients with overlap syndrome (mean age of 15.7 years). We evaluated disease activity (skin abnormalities and muscle weakness, muscle enzymes and acute phase reactants) and its correlation with nailfold capillaroscopy findings (dilatation of isolated loops, dropout of surrounding vessels and giant capillary loops). We used a microscope with special light and magnification of 10 to 16X. RESULTS: Eighteen patients underwent a total of 26 capillaroscopic examinations, seven of them on two or more occasions (13 were performed during the active disease phase and 13 during remission). Twelve of the 13 examinations performed during the active phase exhibited scleroderma pattern and 8 of the 13 examinations performed during remission were normal. Therefore, in 20 of the 26 examinations clinical and laboratory data and nailfold capillaroscopy findings correlated (p = 0.01). CONCLUSIONS: Nailfold capillaroscopy is a non-invasive examination that offers satisfactory correlation with disease activity and could be a useful tool for the diagnosis and follow-up of inflammatory myopathies.

The clinical spectrum of antinuclear antibodies associated with the nuclear dense fine speckled immunofluorescence pattern.

OBJECTIVE: Autoantibodies to lens epithelium-derived growth factor (LEDGF) depict a distinctive nuclear dense fine speckled (DFS) pattern in the indirect immunofluorescence antinuclear antibody assay (IIF-ANA). Definition of the clinical spectrum associated with anti-LEDGF antibodies has been evolving over the last decade. We investigated the frequency, clinical spectrum, and immunologic specificity of the DFS pattern in a general clinical laboratory routine. METHODS. All serum samples entered for IIF-ANA determination within a 2 year period were examined for the DFS pattern. Positive samples with consistent clinical information were studied further by IIF with isotype-specific conjugate and immunoblot analysis. RESULTS: Among 13,641 ANA-positive samples, 5081 (37%) presented the DFS pattern. Within a 6 month nested period, there were 650 samples with DFS pattern, and consistent clinical data were available for 81 of these. DFS reactivity was mainly due to IgG. Most samples (86%) presented titer > or = 1/640. Eighty of the 81 DFS samples reacted with a 75 kDa band that comigrated with the band elicited by the standard anti-LEDGF serum. Antibodies that were affinity-purified from the 75 kDa band reproduced the DFS pattern on IIF-ANA. The clinical spectrum associated with DFS reactivity included autoimmune diseases (39%) and an array of nonautoimmune conditions (61%). Among the autoimmune patients, over half presented evidence of autoimmune thyroiditis. CONCLUSION: Anti-LEDGF/p75 antibodies are a common finding among ANA-positive individuals with no evidence of rheumatic autoimmune disease, and should be regarded as a low specificity finding even when in moderate or high titer.

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Especificidades de auto-anticorpos na uveíte anterior aguda segundo a presença do alelo HLA-B27 / Autoantibody specificity in acute anterior uveitis according to the presence of the HLA-B27 allele

Introdução - As uveítes anteriores agudas (UAA) são a foram mais frequente de inflamação do trato uveal e grande proporção dessas está associada à presença do alelo B27 com ou sem espondiloartropatia. Acredita-se que a presença do HLA-27 esteja associada a particularidades clínicas e imunológicas. Objetivos - Estudar pacientes com uveíte anterior aguda HLA-B27 e HLA-B27 quanto à frequência e especificidade de auto-anticorpos órgão-epecíficos e não órgão-específicos. Métodos - Cinquenta e sete pacientes com uveíte anterior aguda foram avaliados por oftalmologista e pelo reumatologista, que realizaram os seguintes testes laboratoriais: immunoblot para anticorpos anti-células HeLa e antiextrato de íris bovina. A pesquisa do HLA-B27 foi realizada por microlinfotoxicidade. A análise estatística foi feita usando-se o teste de Mann-Whitney, teste do qui-quadrado, teste exato de Fisher e o teste de McNemar. Resultados - Dentre so 57 pacientes, 34 possuíam o alelo HLA-B27 (UAA-B27) e 23 não tinham esse alelo (UAA-B27-). Pela técnica de immunoblot com extrato total de células HeLa, no grupo UAA-B27, mas não no grupo UAA-B27, evidenciou-se alta frequência de anticorpos, contra proteínas de diferentes pesos moleculares; as reatividades mais frequentes foram contra proteínas de 46 e 56kDa. Por outro lado, o immunoblot com extrato total de íris evidenciou alta frequência de reatividade nos soros dos dois grupos de pacientes; as reatividades mais frequentes foram as de peso molecular estimado em 35,52 e 54kDa. Conclusão - A UAA, associada ao alelo HLA-B27, apresentou menor expressão de auto-anticorpos não específicos ao tecido uveal. Por outro lado, a expressão de anticorpos antiíris associou-se a UAA, independentemente da presença do alelo HLA-B27

Difosfato de cloroquina em lúpus eritematoso sistêmico: estudo duplo cego e randômico / Randomized double blind trial with chloroquine diphosphate in systemic lupus erythematosus

Objetivo: Avaliar a eficácia do difosfato de cloroquina (DFC) em: a) controlar a atividade do lúpus eritematoso sistêmico (LES), b) diminuir o número de exacerbaçoes da doença e c) diminuir a dose de corticosteróides em pacientes com LES sem manifestaçoes graves. Métodos: Estudo clínico randômico, duplo cego de 37 pacientes com LES (segundo critérios de classificaçao do Colégio Americano de Reumatologia), que estavam em uso de no máximo 0,5mg/kg/d de prednisona. Aleatoriamente, 20 pacientes foram colocados no grupo DFC e receberam 250mg/d de cloroquina e 17 no grupo PL receberam 1cp/d de placebo. Todos fizeram avaliaçao clínica e laboratorial bimestralmente por 6 meses. Resultados: O número de reativaçoes do LES foi significantemente maior nos pacientes do grupo PL quando comparados aos do grupo DFC. Comprometimento cutâneo ocorreu em 58 por cento no grupo PL e em 10 por cento no grupo DFC, acometimento articular foi observado em 41 por cento no grupo PL e em 5 por cento no grupo DFC, enquanto hipocomplementemia ocorreu em 35 por cento e 5 por cento, respectivamente no grupo PL e DFC. Estas diferenças foram estatisticamente significantes. Setenta e cinco por cento dos pacientes do grupo DFC e apenas 23 por cento do grupo PL conseguiram reduzir a dose de prednisona e esta diferença também foi estatisticamente significante. Conclusoes: O uso de DFC preveniu reativaçoes do LES e permitiu a reduçao da dose de prednisona, durante os seis meses de seguimento