ORGENTEC Autoimmunity Blog

Covering Autoimmune Diseases

IFT / IFA

tag for articles about immunofluorescence technology / immunofluorescence assays for detecting autoantibodies and for diagnosing autoimmune diseases

Portrait of the HEp2 cell, the pet of immunofluorescence professionals

HEp2 cells -- centromere B

Anti-Centromere B on HEp2 cells

HEp2 cells are held dear in autoimmune diagnostics. They are invaluable for people engaged in analysing autoantibodies, as E. coli is for molecular biologists or mice for toxicologists.

In spite of a wide range of other suitable methods and technologies, determination of autoantibodies with indirect immuno-fluorescence assays (IFA) on human epithelioma (HEp2) cells still contributes significantly to the diagnosis of autoimmune diseases. The widely recognised advantages of this method are high sensitivity and a broad spectrum of antibodies that can be analysed simultaneously. In addition to mere detection of antibodies a characteristic fluorescence pattern and staining of metaphase and cytoplasmic cells offer supplementary information.

When an autoimmune disease is suspected, the HEp-2 test usually is the first line test. Any positive result is then followed up by a step-wise diagnostic approach, including other immunological tests like ELISA (enzyme-linked immunosorbent assay) for single antibody specificities or immunoblot tests.
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Immunofluorescence Tests in Crescentic Glomerulonephritis

The web community GRÜNER CLUB AUTOIMMUN is a voluntary association of scientists, laboratory specialists, medical doctors, students and immunofluorescence enthusiasts from Austria. In their internet blog these experienced IFT professionals  discuss questions, ideas and concepts of immunofluorescence tests in autoimmune disease diagnostics.

DER GRÜNE CLUB AUTOIMMUN

In a recent posting my Austrian colleague Barbara Fabian, community manager of GRÜNER CLUB AUTOIMMUN, refered to the relationship  between the formation of  autoantibodies against glomerular basal membrane (GBM) and ANCA (antibodies against cytoplasmic antigens of neutrophil granulocytes). The article was originally written in German language, but we had it translated  for the not German speaking readers of the Autoimmunity Blog.

Autoantibodies against Glomerular Basal Membrane and Myeloperoxidase in Crescentic Glomerulonephritis

by Barbara Fabian, Vienna

Crescentic Glomerulonephritis (CGN) is an autoimmune disease of the kidney that leads to vasculitis of the capillaries in the glomeruli. The appearance of characteristic autoantibodies or antibody complexes is indicative of CGN and allows for the differentiation of three groups:

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New Guidelines for the Diagnosis of Celiac Disease

Celiac disease diagnostics revised 

Anti-endomysial antibodies on monkey esophagus

Anti-endomysial antibodies on monkey esophagus

The diagnostic criteria for celiac disease (CD) have remained unchanged for more than 20 years, after the 1990 revision of the guidelines originally formulated in 1969.  During this period the disease has been intensively studied and scientific findings have unveiled the genetic background of celiac disease, linked to the human leukocyte antigen (HLA)-DQ2 and HLA-DQ8 haplotypes. The key autoantigen tissue transglutaminase (tTG) has been identified and reliable laboratory tests for disease specific autoantibodies now contribute to diagnostics and complement the methodological repertoire of clinical observations and histologic findings in duodenal biopsy samples. Finally, the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) has now published New Guidelines for the Diagnosis of Celiac Disease.

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A short History of Indirect Immunofluorescence Technology

The GRÜNER CLUB AUTOIMMUN blog featured a fine post about the history of indirect immunofluorescence. In that article my Austrian colleague Barbara Fabian, community manager of GRÜNER CLUB AUTOIMMUN, described in great detail how indirect immunofluorescence technology, or: IFT, and also referred to as IIF assay,  has become an indispensible tool of autoimmune disease diagnostics over the last two decades, and how IFT has become a standard laboratory technique used in serological autoimmune diagnostics.

Without further ado I have translated Barbara’s post in order to make you this text, and especially the interesting images, available. – Here it is:

The Development of Indirect Immunofluorescence Technology (IFT)

by Barbara Fabian, MSc, Community Manager of GRÜNER CLUB AUTOIMMUN

Over the last 20 years, the detection of autoantibodies has developed into an indispensible component of autoimmune diagnostics. Along with serological and clinical data, autoimmune status has become an important building block in the formation of diagnoses. (more…)

Rheumatology Diagnostics: Indirect Immunofluorescence Tests (IIF assays) on HEp-2 Cells

Immunofluorescence patterns help eliminate “false positives” in diagnosing autoimmune rheumatic diseases

The detection of anti-nuclear antibodies, the ANA test, is a clear (laboratory-) diagnostic indicator of rheumatic autoimmune disease. One of the standard laboratory tests for the detection of these antinuclear antibodies is IIF, the indirect immunofluorescence assay, on human HEp-2 cells (ANA-HEp-2 test).

This pictures shows anti-RNP pattern on HEp-2 cells after ANA-HEp-2 indirect immunofluorescence assay

ANA-HEp-2 indirect immunofluorescence test (IIF): antibodies against RNP (ribonucleoproteins) – interphase nucleoli: coarse granular positive, nucleoli neglected; mitotic cells: negative (400x) – © ORGENTEC Diagnostika, Mainz

However, for up to 13% of healthy individuals, indirect immunofluorescence may detect anti-nuclear antibodies. Most of these healthy people will not develop an autoimmune disease – despite the positive ANA test. It is thus a challenge for the physician to differentiate these healthy, false-positive patients from those ANA-positive patients who already have an inflammatory rheumatic disease or who truly have an increased risk of developing such an autoimmune disease.

 

Several very specific IIF patterns

In a large study, Brazilian IIF experts have now worked out the fundamental differences between the ANA-HEp-2 test results on serum samples from healthy individuals and the immunofluorescence patterns from serum samples of patients with rheumatic disease; they have described various IIF patterns that can be used to differentiate between the two patient groups (Mariz et al. 2011). This study was published a few weeks ago in the January issue of Arthritis & Rheumatism, the journal of the American College of Rheumatology (ACR). In their article, the scientists from the Universidade Federal de São Paulo, Brazil, explain in detail that there are several very specific immunofluorescence patterns in the ANA-HEp-2 assay with which the autoimmune rheumatic diseases (ARD) are truly associated. (more…)

Blood Tests for the Diagnosis of Lupus

Blood Tests for the Diagnosis of Lupus

Welcome to our Autoimmunity Blog! The subject of this post is blood tests for the diagnosis of lupus.  

Lupus facial rash in a typical wolf-like distribution.

Lupus facial rash in a typical wolf-like distribution.

The emphasis of this article is on the detection of autoantibodies relevant to the diagnosis of SLE. Specifically, this includes detection of ANA (antinuclear antibodies) by immunofluorescence and individual tests for various ANA, including anti-dsDNA, anti-Sm, anti-U1RNP (also anti-U1-RNP or anti-RNP), and anti-histone, as well as anti-SS-A/Ro and anti-SS-B/La.  

Tests for ANA are also highly useful in differential diagnostics, especially when diseases with symptoms resembling SLE must be distinguished from lupus itself, for example fibromyalgia, infections like tuberculosis and HIV/AIDS, or certain malignant tumours, particularly lymphoma and leukaemia.  (more…)

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