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.

Advantage of the HEp2 cell

Biological materials like the HEp-2 cell line or complete tissue sections present the diagnostically relevant antigens in their natural surroundings; therefore, a variety of autoantibodies can be determined simultaneously and with high sensitivity. Detecting autoantibodies via immunofluorescence on HEp2 cells provides more information than a simple positive or negative signal. Many antibodies show characteristic fluorescence patterns, e.g. homogenous, granular, nucleolar, dotted, cytoplasmic, that allow for differentiation of several groups of autoantibodies (see table 1).

Especially for the determination of antinuclear antibodies (ANA) cultivated HEp2 cells have a higher sensitivity than mammalian tissue sections. The various antibody patterns are easily discernible in HEp2 cell monolayers, but they are difficult to visualize in rat kidney and rat liver tissue sections. [1]

Antibodies against cell-cycle dependent antigens exhibit no immunofluorescence pattern on organ tissue sections. However, they may be of significance in the diagnosis of some autoimmune disorders. Because both the mitotic phase and the metaphase of the cell cycle are identifiable in HEp2 cells, information regarding the patterns of the chromosomes is also available.

The table below gives an overview on the fluorescence patterns produced by autoantibodies directed to different cellular antigens.

Fluorescence patterns of autoantibodies to different nuclear antigens in Hep2 cells

The ideal HEp2 cell, how should it look like?

To confirm a specific positive immunofluorescence result on HEP2 cells, the cell nuclei must be clearly distinguished from the surrounding cytoplasm, and all cells must exhibit a definite fluorescence pattern.

Staining of nuclear pores on HEp2 cells

Describing this pattern includes careful examination of different cellular components.

Are the nuclei glowing homogenously or do they show a granular or nucleolar pattern? The cytoplasm may appear fine or coarse speckled, and antibodies directed to the cytoskeleton may occur. Eventually, the chromosomes may be positive or negative.

Therefore, an ideal HEp2 cell substrate has to be up to a certain standard:

• The cell nuclei have to be large and uniform in size.
• The distribution of the cells should not be too dense, the optimum number of cells varies between 70 and 100 cells in the visible area at a magnification of 400, less than 50 or more than 110 cells is not adequate
• Broad cytoplasms with clearly definable antibody patterns and definite discrimination of the cell nucleus are required.
• A considerable number of cells should be in different phases of the cell cycle.
• A limited number of chromosomes (<10) should be clearly visible. Two to six chromosomes of which one to three are in metaphase is the optimum.
• Minimum background fluorescence desired

To be green or not to be green

Testing for ANA is of special importance for the diagnosis of many rheumatic autoimmune diseases, like systemic lupus erythematosus or Sjoegren’s syndrome. Laboratory tests are helpful in differential diagnosis and for verification of the clinical diagnosis. Indirect immunofluorescence assay of ANA on HEp-2 cells is considered the gold standard for their screening.

Anti-RNP antibodies on HEp2 cells

Due to the large number of different antigens presented by the HEp2 cell substrate, the test is highly sensitive, but for the same reason specificity is limited. The ANA test on HEp2 cells is frequently positive in a considerable number of non-autoimmune subjects. In order to identify features that discriminate positive ANA-HEp-2 tests in autoimmune patients from positive tests in non-autoimmune subjects the authors of a Brazilian study compared the results of ANA determinations in healthy individuals with those of patients suffering from different rheumatic diseases [2].

Mariz et al performed their analysis particularly with regard to the ANA titre and the fluorescence patterns. They found elevated ANA titres in about 13 % of the healthy individuals. In the blood of these people titres generally tended to be low (< 1:80 in 87 % of the control group). In contrast, the majority (90 %) of the patients had high ANA tires (≥ 1:80). For the most part, the non-autoimmune subjects exhibited a fine speckled fluorescence pattern with no staining of the chromosomes. Specific patterns (homogenous, centromere, dividing cells) only appeared in 4 % of the healthy individuals.

Immunofluorescence tests allow for cost-effective and high-quality diagnosis of autoimmune diseases. However, this method still lacks reliable standardisation and is very dependable on the qualification of the observer. Therefore positive findings are subsequently further evaluated with other immunological tests, including ELISAs and immunoblots, in a stepwise diagnostic approach. Enzyme Immunoassays (EIA) and immunoblotting techniques attain good standardisation and comparability. ELISAs are limited to a selection of defined autoantibodies but they allow for quantitative determinations and they can be run in automated systems. [3]

Antibodies to sdDNA and PCNA on HEp2 cells

ANA screening assays by IFA are time consuming, subjective and laborious. Many attempts have been made to find a reliable substitution. A promising study compared the results of 2000 consecutive sera with requests for an ANA in a routine serology laboratory setting tested by an ELISA screening assay with multiple ANA specificities (ANA Detect from ORGENTEC) to those obtained by immunofluorescence. From these results, the authors established an ANA ratio cut-off protocol to guide further action, and assessed its efficacy in a second series of 7000 samples. Their results indicate that the ANA Detect ELISA can be an alternative to IIF for the detection of clinically significant antibodies [4]. However, there are still some antibodies that are exclusively detectable by IIF.

Replacement of standard immunofluorescence methods with bead-based assays for antinuclear antibody (ANA) testing is still under investigation. Bruner et al [5] analysed serum samples from 1540 SLE (systemic lupus erythematous) patients, 1154 unaffected relatives and 906 healthy controls with IIF, immunodiffusion and an automated bead based solid phase assay. The frequencies of ANA in the sera from African American, Hispanic, and European American patients with SLE were 89%, 73%, and 67%, respectively, in the bead-based assay and 94%, 84%, and 86%, respectively, by IIF. The frequencies of ANA detected by the bead-based assay are lower than those detected by IIF, especially in European American patients with SLE. Detecting ANA by IIF on HEp2 cells is superior to the automated assay and still remains the standard technique for the detection of ANA.

Until today, antibody detection on HEp-2 cells is maintaining its central role. In cases with suspected autoimmune disease, the HEp-2 test is the recommended screening method, which allows for cost-effective and high-quality serological diagnosis of various autoimmune diseases. Any positive result is then followed up by a step-wise diagnosis in which, depending on the evaluation of the reactivity with HEp-2 cells, other substrates may be used or other immunological tests like ELISA (enzyme-linked immunosorbent assay) or immunoblot assays are carried out.

References

1.   Hahon,N., Eckert,H.L., and Stewart,J., Evaluation of cellular substrates for antinuclear antibody determinations. J Clin Microbiol 1975. 2: 42-45.  Link to full text article ->

2.   Mariz,H.A., Sato,E.I., Barbosa,S.H., Rodrigues,S.H., Dellavance,A., and Andrade,L.E., Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum 2011. 63: 191-200.  Link to full text article ->

3.   Bayer,P.M., Fabian,B., and Hubl,W., Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics–technique, benefits, limitations and applications. Scand.J Clin.Lab Invest Suppl 2001. 235: 68-76.  Link to abstract in PubMed ->

4.   Sinclair,D., Saas,M., Williams,D., Hart,M., and Goswami,R., Can an ELISA replace immunofluorescence for the detection of anti-nuclear antibodies?–The routine use of anti-nuclear antibody screening ELISAs. Clin.Lab 2007. 53: 183-191.  Link to abstract in PubMed ->

5.   Bruner,B.F., Guthridge,J.M., Lu,R., Vidal,G., Kelly,J.A., Robertson,J.M., Kamen,D.L., Gilkeson,G.S., Neas,B.R., Reichlin,M., Scofield,R.H., Harley,J.B., and James,J.A., Comparison of autoantibody specificities between traditional and bead-based assays in a large, diverse collection of patients with systemic lupus erythematosus and family members. Arthritis Rheum 2012. 64: 3677-3686.  Link to abstract in PubMed ->

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:

In type 1 CGN, antibodies against glomerular basal membrane (GBM) are formed. Type 2 CGN results in immune complexes, and type 3 is identified by the detection of antibodies against cytoplasmic antigens of neutrophil granulocytes (ANCA).

Detection of autoanitbodies against the glomerular basal membrane with an indirect immunoflurescence test on monkey kidney.
© Barbara Fabian, http://www.der-gruene-club.at

Antibodies against GBM and ANCA can also occur together. A variety of studies have thus been carried out to show a relationship between these antibodies and a connection in their formation. Affected patients were also evaluated to identify differences or similarities in their clinical outcomes. Of particular interest was the question of whether antibodies against myeloperoxidase (MPO, pANCA) favour the formation of antibodies against GBM.

Simultaneous occurrence of antibodies against MPO and GBM

In a 2005 study by Rutgers et al., data was evaluated from patients who were entered into a database following diagnosis with CGN as a result of kidney biopsies carried out between 1978 and 2003 (1). Patients with immune complex deposits (type 2 CGN) were excluded at the outset.

There were 127 biopsies with a positive CGN result. In cases where they had not previously been detected, antibodies against GBM and ANCA were confirmed afterward for the study using frozen serum samples taken at the time of biopsy. Only samples positive for MPO and/or GBM antibodies were included, because only one patient gave positive results for both anti-PR3 and GBM antibodies (see Table).  

In the study, three groups of patients that had tested positive for either one or the other of the antibodies (MPO or GBM) or both were compared to each other.

Outcome for patients with GBM and MPO antibodies

Patients from the groups positive for GBM antibodies or both MPO and GBM antibodies generally had a higher level of disease activity. In contrast, the occurrence of chronic symptoms in these two groups was lower than in those patients that only had MPO antibodies.

At the time of diagnosis, only 28 % of the MPO positive patients required dialysis, in contrast to 60 % of the GBM+MPO positive and 69 % of the GBM positive patients.

The patients were followed over an average period of 5.1 years (ranging from 2 days to 22 years). CGN patients who were only positive for GBM had the best chance of surviving the first year (100 %). The survival rate for patients with both antibodies (MPO+GBM) was only 79 % and that of patients with  MPO as the only antibody shrank to 75 %.

In addition to medicinal treatment, patients with MPO-ANCA positive CGN and kidney failure or anti-GBM positive CGN underwent plasma exchange. An isolated description of a case of improvement when both GBM and MPO antibodies were present could not be confirmed by this study.

Do ANCA favour the occurrence of GBM antibodies?

Strikingly, 43 % of the CGN patients positive for GBM antibodies also had MPO-ANCA. One possible explanation for this could be the pathophysiological connections between the formation of MPO and GBM antibodies.

Autoantibodies against myeloperoxidase activate neutrophil granulocytes, initiating degranulation of these cells. This releases reactive enzymes that effect increased proteolysis of the glomerular basement membrane. In addition, MPO activates matrix metalloproteinases, which can cleave Goodpasture antigen, releasing the Goodpasture epitope that is normally embedded in the basement membrane. This makes these normally hidden structures accessible to the immune system, allowing for the formation of GBM antibodies, which lead to the development of Goodpasture syndrome (1). GBM antibodies bind to type IV collagen, which is found in the kidneys and lungs, among other places. Additional triggers include genetic factors, smoking, and reduced immunotolerance (3).

Chronological progression upon appearance of GBM and MPO antibodies

J. Serratrice and his team described the case of a patient with ANCA-associated vasculitis. All criteria for microscopic polyangiitis (MPA) were met. However, within three years, the patient developed the clinical symptoms associated with Goodpasture syndrome and antibodies against GBM became detectable. Earlier serum samples from the patient were tested for GBM antibodies; however, these samples were negative. The GBM antibodies only appeared at the time when the kidneys failed (2). Cross-reactivity of ANCA with the GBM test was ruled out.

This research group also postulated that a possible explanation is that the pANCA could cause damage to the basement membrane, an effect that has previously been observed in animal trials. Antigens normally hidden within the basement membrane are exposed, allowing the immune system to form autoantibodies against these structures. The same conclusion was reached by the research group of S. Clyne, who made similar observations (3).

Because 5 % of all ANCA positive patients also have antibodies against GBM, it is often recommended that a GBM test should be carried out after a positive ANCA result.

Detection of GBM antibodies in the clinical laboratory

The detection of GBM antibodies (IgG) in serum can be carried out by ELISA or immunoblot, as well as indirect immunofluorescence (IIF) on primate kidney. In practice, parallel use of IIF and ELISA or blot test has proven effective for diagnosis and monitoring.

When detecting GBM antibodies by indirect immunofluorescence, the tissue section of primate kidney used as substrate must be treated beforehand (e.g. with glycine) in order to “break up” the glomeruli. This allows the antibodies to bind to the corresponding structures.

References:

1. Rutgers, A. et al. Coexistence of anti-glomerular basement membrane antibodies and myeloperoxidase-ANCAs in crescentic glomerulonephritis, Am. J. Kidney Dis. 46, 253–262, doi:10.1053/j.ajkd.2005.05.003 (2005). Link zum Abstract in PubMed 2. Serratrice, J. et al. Sequential development of perinuclear ANCA-associated vasculitis and anti-glomerular basement membrane glomerulonephritis, Am. J. Kidney Dis. 43, e26-30 (2004). Link to abstract in PubMed

3. Clyne, S., Frederick, C., Arndt, F., Lewis, J. & Fogo, A.B. Concurrent and discrete clinicopathological presentations of Wegener granulomatosis and anti-glomerular basement membrane disease, Am. J. Kidney Dis. 54, 1116–1120, doi:10.1053/j.ajkd.2009.04.026 (2009). Link to fulltext pdf

4. O’Connor, K., Fulcher, D. & Phoon, R.K.S. Development of anti-glomerular basement membrane disease after remission from perinuclear ANCA-associated glomerulonephritis in a patient with HLA susceptibility, Am. J. Kidney Dis. 55, 566–569, doi:10.1053/j.ajkd.2009.07.015 (2010). Link to abstract in PubMed

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.

The picture of celiac disease after two decades of clinical research

Four possible presentations of celiac disease have been recognized:

1) typical, characterized mostly by gastrointestinal signs and symptoms;
2) atypical or extraintestinal, where gastrointestinal signs/symptoms are minimal or absent and a number of other manifestations are present;
3) silent, where the small intestinal mucosa is damaged and celiac disease autoimmunity can be detected by serology, but there are no symptoms;
4) latent, where individuals possess genetic compatibility with celiac disease and may also show positive autoimmune serology, that have a normal mucosa morphology and may or may not be symptomatic.

Determination of serum levels of immunoglobulin A (IgA)  anti-tissue transglutaminase (anti-tTG) is  the first choice in screening for celiac disease, displaying the highest levels of sensitivity (up to 98%) and specificity (around 96%). Anti-endomysium IgA-antibodies (EMA-IgA), on the other hand, have close to 100% specificity and a sensitivity of greater than 90%.

The interplay between gliadin peptides and tTG is responsible for the generation of novel antigenic epitopes, the tTG-generated deamidated gliadin peptides. Such peptides represent much more celiac disease-specific epitopes than native peptides, and deamidated gliadin antibodies (anti-DGP) are promising serological markers for celiac disease. Serology has also been employed in monitoring the response to a gluten-free diet – the very effective but until now also the only possible treatment of celiac disease.

Despite of this large inventory of powerful  tests, serological screening has been utilized primarily to identify those individuals in need of a diagnostic endoscopic biopsy, and  diagnosis of celiac disease has still rested on the demonstration of changes in the histology of the small intestinal mucosa: The classic celiac lesions with histologic changes of villous atrophy, crypt hyperplasia, and increased intraepithelial lymphocytosis. 

Serologic tests may reduce the burden of biopsy 

With a view to bring together the results of two decades of intensive research in a balanced and up to date diagnostic scheme, that helps to avoid unnecessary biopsies and laboratory tests, the ESPGHAN charged a working group consisting of 17 well known experts with the development of new guidelines for the diagnosis of celiac disease. On the basis of a recently published detailed evidence report on antibody testing the working group compiled an updated definition of celiac disease and formulated new guidelines.
Major changes where made concerning the claim for duodenal biopsy: the working group defined subsets of patients for whom biopsies where avoidable. Different diagnostic approaches are recommended for varying groups of patients stratified according to symptoms and clinical presentation.

Definition of celiac disease

Celiac disease (CD) comprises intolerance against dietary gluten present in wheat, rye and barley, and it belongs to the most common food-related life-long disorders in Western countries, but also in India and the Asia-Pacific region, including Australia, Iran, Israel, New Zealand, Syria and Turkey.
Nowadays celiac disease is conceived as an autoimmune-mediated systemic disorder, strongly dependent on HLA-DQ2 and HLA-DQ8 haplotypes, and commonly presenting as enteropathy in genetically susceptible individuals.

The most obvious feature distinguishing celiac disease from other small-intestinal enteropathies is the presence of autoantibodies against tissue transglutaminase (tTG) during a gluten-containing diet. The gluten-derived gliadin peptides and the self antigen, tTG, have an established and well-accepted role in celiac disease pathogenesis. tTG is known to deamidate, and crosslink gluten-derived gliadin peptides, thereby favoring disease progression. Endomysial antibodies (EMA), and antibodies against deamidated forms of gliadin peptides (anti-DGP) complement the repertoire of CD specific antibodies

Identification of patients who should be tested for CD

CD may present with a large variety of nonspecific signs and symptoms. It is important to diagnose CD not only in children with obvious gastrointestinal troubles but also in children with a less clear clinical picture because of the unfavourable  health consequences of untreated celiac disease. Therefore it is recommended to distinguish between symptomatic and asymptomatic patients in two differently defined risk groups. According to the revised guidelines [5]:

Testing for CD should be offered to the following groups

 Group 1 are symptomatic children and adolescents with the otherwise unexplained signs of chronic or intermittent diarrhoea, failure to thrive, weight loss, stunted growth, delayed puberty, amenorrhoea, iron-deficiency anaemia, nausea or vomiting, chronic abdominal pain, cramping or distension, chronic constipation, chronic fatigue, recurrent aphthous stomatitis (mouth ulcers), dermatitis herpetiformis–like rash, fracture with inadequate traumas/osteopenia/osteoporosis, and abnormal liver biochemistry.

 Group 2 are asymptomatic children and adolescents with an increased risk for CD such as type 1 diabetes mellitus (T1DM), Down syndrome, autoimmune thyroid disease, Turner syndrome, Williams syndrome, selective immunoglobulin A (IgA) deficiency, autoimmune liver disease, and first-degree relatives with CD.

All tests should be performed in patients who are consuming a gluten-containing diet to avoid misdiagnosis. If gluten exposure was short or gluten had been withdrawn for a longer period of time a negative result is not reliable.

In addition to classical ELISA-assays, rapid point-of-care tests are an alternative method for initial testing of CD-specific antibodies. However, results of rapid point-of-care tests (either positive or negative) should always be confirmed by a laboratory-based quantitative test.

Diagnostic approach for symptomatic patients

 A test for CD-specific antibodies is the first step. The initial test should be IgA class anti-tTG from a blood sample. If total serum IgA is not known, then this also should be measured. In subjects with either primary or secondary humoral IgA deficiency, at least one additional test measuring IgG class CD-specific antibodies is recommended (IgG anti-tTG, IgG anti-DGP or IgG EMA, or blended kits for both IgA and IgG antibodies).

Testing for antibodies against DGP may be used as an additional test in patients who are negative for other CD-specific antibodies but in whom clinical symptoms raise a strong suspicion of CD, especially if they are younger than two years. Tests for the detection of IgG or IgA antibodies against native gliadin peptides (conventional gliadin antibody test) should not be used for CD diagnosis; however, they may be valuable to document adherence to the gluten-free diet administered by the treating physician.

If IgA class CD antibodies are negative in an IgA-competent symptomatic patient, then it is unlikely that CD is causing the symptom at the given time point. In seronegative cases for anti-tTG, EMA, and anti-DGP but with severe symptoms and a strong clinical suspicion of CD, small intestinal biopsies and HLA-DQ testing are recommended.

When duodenal biopsies,that  have been taken during a routine diagnostic workup for gastrointestinal symptoms, disclose a histological pattern indicative of CD (Marsh 1–3 lesions), then antibody determinations (anti-tTG and, in children younger than two years, anti-DGP) and HLA typing should be performed. In the absence of CD-specific antibodies and/or HLA-DQ2 or HLA-DQ8 heterodimers, other causes of enteropathy (e.g., food allergy, autoimmune enteropathy) must be considered.

See figure 1 for a graphic representation of the diagnostic algorithm for patients with symptoms suggestive for celiac disease:

Figure 1: Diagnostic algorithm for patients with symptoms suggestive for celiac disease

The clinical relevance of a positive anti-tTG or anti-DGP result should be confirmed by histology, unless certain conditions are fulfilled that allow the option of omitting the confirmatory biopsies.

Requirements for diagnosing CD without duodenal biopsy [5]

In children and adolescents with signs or symptoms suggestive of CD and high anti-TTG titers (levels >10 times upper limit of normal), the likelihood for villous atrophy (Marsh 3) is high. In this situation, the paediatric gastroenterologist may discuss with the parents and patient (as appropriate for age) the option of performing further laboratory testing (EMA, HLA) to make the diagnosis of CD without biopsies. Antibody positivity should be verified by EMA from a blood sample drawn at an occasion separate from the initial test to avoid false-positive serology results owing to mislabelling of blood samples or other technical mistakes. If EMA testing confirms specific CD antibody positivity in this second blood sample, then the diagnosis can be made and the child can be started on a gluten free diet. It is advisable to check for HLA types in patients who are diagnosed without having a small intestinal biopsy to reinforce the diagnosis of CD. 

Diagnostic approach for asymptomatic persons at risk for celiac disease

HLA testing should be performed at first, because absence of DQ2 and DQ8 renders CD highly unlikely and no further follow-up with serological tests is needed. If the patient is DQ8 and/or DQ2 positive, anti-tTG IgA test and total IgA determination should be performed, but preferably not before the child is two years old. If antibodies are negative, then repeated testing for CD-specific antibodies is recommended.

In individuals without clinical signs and symptoms, with an increased genetic risk for CD duodenal biopsies with the demonstration of an enteropathy should always be part of the diagnostic algorithm [5].

To avoid unnecessary biopsies in individuals with low CD specific antibody levels (i.e., <3 times upper limit of normal), it is recommended that the more specific test for EMA be performed. If the EMA test is positive, then the child should be referred for duodenal biopsies. If the EMA test is negative, then repeated serological testing on a normal gluten-containing diet in 3 to 6 monthly intervals is recommended.

Figure 2 shows the diagnostic algorithm for asymptomatic persons at increased genetic risk for celiac disease:

Figure 2: Diagnostic algorithm for asymptomatic persons at increased genetic risk for celiac disease

Follow-up

If the diagnosis is definitely made the child can be started on a gluten free diet. The patients should be followed up regularly for symptomatic improvement and normalisation of CD-specific antibody tests. About twelve months after onset of the gluten free diet antibody titers usually decrease below the test‘s cut-off. If there is no clinical response to the gluten free diet in symptomatic patients further small bowel biopsies may be needed.

Gluten challenge is not considered necessary except when there is still doubt about the initial diagnosis. Gluten challenge should be preceded by HLA typing and assessment of mucosal histology and always should be performed under close medical supervision.

Despite the gluten-free diet being so effective, there is a growing demand for alternative treatment options. In the future, new forms of treatment may include the use of gluten-degrading enzymes to be ingested with meals, the development of alternative, gluten-free grains by genetic modification, the use of substrates regulating intestinal permeability to prevent gluten entry across the epithelium, and, finally, the availability of different forms of immunotherapy.

The aim of the ESPGHAN’s new recommendations on antibody testing for the diagnosis of celiac disease was to achieve high diagnostic accuracy while reducing the burden of repeated duodenal biopsies for patients and their families. Now, these new guidelines must stand the test of clinical practice.

Summary:

• The working group defines celiac disease as an immune mediated systemic disorder elicited by gluten and related prolamines in genetically susceptible individuals, characterised by the presence of a variable combination of gluten-dependent clinical manifestations, CD-specific antibodies, HLA-DQ2 and HLA-DQ8 haplotypes, and enteropathy.
• Several classifications of CD have been used, most important with distinctions drawn among classical, atypical, asymptomatic, latent, and potential CD.
• Antibodies against tTG, EMA, and DGP are referred to as disease-specific antibodies and evidence has been accumulating on their diagnostic value. 
•  The leading role of histology for the diagnosis of CD has been challenged; diagnosis depends in equal measure on information from clinical and family data and results from specific antibody testing and HLA typing. In well defined cases serologic testing may replace histology.

References

    1.   Alessio M, Tonutti E, Brusca I, Radice A, Licini L, Sonzogni A, Florena A, Schiaffino E, Marus W, Sulfaro S, Villalta D: Correlation Between IgA Tissue Transglutaminase Antibody Ratio And Histological Finding In Celiac Disease: A Multicentre Study. J Pediatr.Gastroenterol.Nutr. 2011. → Link to abstract in PubMed

   2.   Cummins AG, Roberts-Thomson IC: Prevalence of celiac disease in the Asia-Pacific region. J Gastroenterol.Hepatol. 2009, 24:1347-1351.  → Link to abstract in PubMed

   3.   Giersiepen K, Lelgemann M, Stuhldreher N, Ronfani L, Husby S, Koletzko S, Korponay-Szabo IR: Accuracy of diagnostic antibody tests for coeliac disease in children: summary of an evidence report. J Pediatr.Gastroenterol.Nutr. 2012, 54:229-241. → Link to abstract in PubMed

   4.   Gupta R, Reddy DN, Makharia GK, Sood A, Ramakrishna BS, Yachha SK, Thapa BR, Banerjee R, Anuradha S, Dutta U, Puri AS, Jain AK, Mulder CJ, Kumar A, Boindala S: Indian task force for celiac disease: current status. World J Gastroenterol. 2009, 15:6028-6033. → Link to full text article in pdf format

   5.   Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, Troncone R, Giersiepen K, Branski D, Catassi C, Lelgeman M, Maki M, Ribes-Koninckx C, Ventura A, Zimmer KP: European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr.Gastroenterol.Nutr. 2012, 54:136-160. → Link to full text article in pdf format

   6.   Murray JA: Serodiagnosis of celiac disease. Clin.Lab Med 1997, 17:445-464. → Link to abstract in PubMed

   7.   Tjon J, van Bergen J, Koning F. Celiac disease: how complicated can it get? Immunogenetics 2010, 62:641-651. → Link to full text article in pdf format  

   8.  Walker-Smith JA, Guandalini S,  Schmitz J, Shmerling RH, Visakorpi JK. Revised criteria for diagnosis of coeliac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch.Dis.Child 1990, 65:909-911. → Link to full text article in pdf format

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.

Autoantibodies were first detected many years ago on frozen sections of mouse and rat liver. The samples were generally prepared in the laboratory, and the other required reagents, such as conjugates, were rarely available at the optimal concentrations required for the individual diagnostic test systems. Early autoimmune results were thus found to be highly variable. The use of different substrates for autoantibody detection showed that autoantibodies against nuclear components, such as those against double-stranded DNA, anti-dsDNA antibodies, which exhibited a homogeneous immunofluorescence pattern, were easily detectable on liver sections (Figure 1).

Figure 1: Indirect immunofluorescence test (IFT) on tissue sections: homogeneous immunofluorescence pattern on mouse kidney and stomach – © Barbara Fabian, http://www.der-gruene-club.at

The HEp-2 cell line raises new possibilities

However, not all antinuclear antibodies (ANA) exhibit a typical immunofluorescence pattern on mouse or rat organ tissue sections. For this reason, the additional detection of autoantibodies on human epithelial cells, known as HEp-2 cells, was developed after a few years. The use of this cell line has the advantage that a very broad spectrum of autoantibodies is detectable on HEp-2 cells. Antibodies against cell-cycle dependent antigens exhibit no immunofluorescence pattern on organ tissue sections. However, they are, like proliferating cell nuclear antigen (PCNA), of significance in the diagnosis of systemic lupus erythematosus (SLE), and can be detected on HEp-2 cells in the autoimmune laboratory (Figure 2).

Because both the mitotic phase and metaphase are identifiable in these cells, information regarding the patterns of the chromosomes is also available (Bayer PM, Fabian B, Hübl W: Immunofluorescence Assays (IFA) and Enzyme-Linked Immunosorbent Assays (ELISA) in Autoimmune Disease Diagnostics – Technique, Benefits, Limitations and Applications. – The link leads to the PubMed abstract). This allowed new, previously undetectable antibodies against the HEp-2 cell cytoplasm (cytoplasmic antibodies), such as the anti-SRP antibodies (SRP stands for signal recognition particle) or antibodies against PL7 or PL12 (anti-PL7, anti-PL12), to be detected as well. This led to numerous studies regarding the clinical significance of these new biomarkers. Various diagnostics companies that specialized in the development of test systems for the detection of autoimmune diseases developed tests for these “new” autoantibodies, such as the cytoplasm immunoblot test system.

Figure 2: IFT in autoimmune disease diagnostics: laboratory detection of anti-PCNA antibodies on HEp-2 cells – © Barbara Fabian, http://www.der-gruene-club.at

In order to establish the value of the HEp-2 cell in the diagnosis of antinuclear antibodies, these cells were initially compared to the liver. It was demonstrated that the sensitivity of antinuclear antibodies, ANA, in human cells was better than that in tissue sections. Rat kidney and rat liver demonstrated the least sensitivity in the detection of nuclear antibodies (Hahon N, Eckert HL, Stewart J: Evaluation of Cellular Substrates for Antinuclear Antibody Determinations. – The link leads to a free full-text article in the Journal of Clinical Microbiology).

To this day, antibody detection on HEp-2 cells has retained its central role. When autoimmune disease is suspected, the HEp-2 test is used for screening, which allows for the cost-effective and high-quality serological diagnosis of various autoimmune diseases (Hiemann R et al. Challenges of Automated Screening and Differentiation of Non-Organ-Specific Autoantibodies on HEp-2 Cells. – this link leads to an abstract in Autoimmunity Reviews). The sensitive detection of many clinically relevant autoantibodies is possible on HEp-2 cells. Any positive result is followed up by a step-wise diagnosis in which, depending on the evaluation of the reactivity with HEp-2 cells, other substrates may be used or other immunological tests like ELISA (enzyme-linked immunosorbent assay) or immunoblot assays carried out.

Tissue sections – indispensible for gastroenterological diagnostics

Diagnosis by means of tissue sections remains very important in gastroenterology. In cases of autoimmune hepatitis (AIH) especially, the detection of anti-smooth muscle antibodies, or: ASMA, or antibodies to liver-kidney microsomes, or: anti-LKM antibodies, are important diagnostic tools. Testing on HEp-2 cells alone would not suffice in these cases, because tests for anti-smooth muscle antibodies, the so called ASMA tests, are not always positive, and tests for anti-LKM autoantibodies are not positive. If autoimmune hepatitis is suspected, routine diagnostics call for testing for antibodies on mouse or rat tissues. If required, a positive antibody screening test can be confirmed by enzyme immunoassays (“ELISA tests”) or immunoblot tests (“blot assays”).

Autoantibodies may be detectable years before the diagnosis of an autoimmune disease. For example, the antimitochondrial antibodies (AMA) in cases of primary biliary cirrhosis (PBC), which are also detected by means of mouse or rat tissue (Beleznay Z, Regenass S. Diagnostics of Autoimmune Diseases. – The link leads to an English language abstract in PubMed, the article itself is in German). A consensus report published in 2004 included guidelines and recommendations in this regard (Vergani D et al. Liver Autoimmune Serology: a Consensus Statement from the Committee for Autoimmune Serology of the International Autoimmune Hepatitis Group. – This link leads to a free full-text article in the Journal of Hepatology). The report recommends diagnosis on a section consisting of combined kidney, stomach, and liver tissue (KSL). It also made the first steps toward standardization by establishing the optimal size and histological composition of the tissues. First attempts at analytical standardization, including incubation times, for example, were also covered in this publication.

Indirect immunofluorescence tests (IFT assays) in ANCA diagnostics

Another important area in the diagnosis of autoimmune diseases is the serological diagnosis of systemic autoimmune vasculitis, such as Wegner’s granulomatosis or microscopic polyangiitis. This is achieved through the detection of autoantibodies against cytoplasmic neutrophil granulocytes with the ANCA test (ANCA = anti-nuclear cytoplasmic antibodies). ANCA primarily involves antibodies that display perinuclear (pANCA) or cytoplasmic (cANCA) staining upon exposure to ethanol-fixed granulocytes (Figure 3). In addition to ethanol-fixed granulocytes, formalin- and methanol-fixed granulocytes are also used.

The additional use of formalin-fixed granulocytes allows for the differentiation of the antinuclear antibodies from the ANCA test. Methanol-fixed neutrophil granulocytes are rarely used in routine laboratory analysis because they deliver relatively little information. Diagnostic tests like ELISA and immunoblot assays that are directed toward myeloperoxidase (anti-MPO test systems) and proteinase 3 (anti-PR3 assays) are also available for autoantibodies against neutrophil granulocytes.

A consensus study on the subject of ANCA diagnostics was published in 1999, with an addendum issued in 2003 (Savige J et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). – This link leads to the PubMed abstract. – Savige J. et al. Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies. Quality Control Guidelines, Comments, and Recommendations for Testing in other Autoimmune Diseases. – This link leads to the full-text article in the American Journal of Clinical Pathology.)

The emphasis of these studies is the clinical importance of tests for antibodies against neutrophil granulocytes. However, the goals of these studies also included the standardization of indirect immunofluorescence techniques for the detection of ANCA, including recommendations for the optimal dilution of the serum samples, the conjugate, and the counterstain. A diagnostic scheme for both minimal and optimal antibody detection was established.

Figure 3a: IFT detection of pANCA on ethanol-fixed neutrophil granulocytes, ANCA diagnosis with the aid of indirect immunofluorescence technology – © Barbara Fabian, http://www.der-gruene-club.at

The continuing issue of standardization

Over the years, many publications have discussed the analysis of autoantibodies through indirect immunofluorescence. The emphasis of a 1983 study on the problems with the standardization of immunofluorescence was on the reproducibility of the detection of antinuclear antibodies, known as ANA detection (Beutner EH, Krasny S, Kumar V, Taylor R, Chorzelski TP. Prospects and Problems in the Definition and Standardization of Immunofluorescence. Present Levels of Reproducibility and Disease Specificity of Antinuclear Antibody Tests. – This link leads directly to the article in the Annals of the New York Academy of Sciences). In addition to pointing out the fact that certain autoantibodies such an anti-centromere antibodies react with human cell lines while remaining undetectable on tissue sections or mouse fibroblast cells, this work also covers methodological studies regarding the titre of antibodies.

Figure 3b: IFT detection of pANCA on formalin-fixed neutrophil granulocytes. – © Barbara Fabian, http://www.der-gruene-club.at

In order to minimize variations in titre between different laboratories, the influence of the conjugate was described. At the center of the investigation were the specificity of the concentration, the dilution, and the fluorescein/protein ratio of the conjugate used. The sensitivity of the optical systems used to evaluate the sample slides was also compared. The use of optically standardized sample slides was recommended as a means to control the variation between different systems.

The quality of test products has improved sharply over time. Because diagnostics firms now offer prepared test kits for the serodiagnosis of autoimmune diseases, which contain mutually calibrated “ready-to-use” reagents, the technical aspect of work in the field of autoimmune diagnostics has been significantly simplified.

However, diagnosis by means of indirect immunofluorescence continues to demand a great deal of knowledge and experience, continuing to offer a challenge for laboratory personnel and physicians. This challenge is surely also the reason why most people working in this field exhibit great enthusiasm and intensity in tackling this problem, and why advanced training in this field is of great value.

Please note: All fotographs of immunofluorescence patterns in this article are protected by copyright law, which is ensured by watermarks. If the pictures have attracted your interest, and if you feel the interest to use one or a number of immunofluorescence pictures for a lecture, a presentation, a publication … please feel free to get into contact with Barbara Fabian via the GRÜNER CLUB AUTOIMMUN website.

Author of this article:  Tobias Stolzenberg

References:

Bayer PM, Fabian B, Hübl W. Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics–technique, benefits, limitations and applications. Scand. J. Clin. Lab. Invest. Suppl. 235, 68–76 (2001). – link leads to the abstract in PubMed: http://www.ncbi.nlm.nih.gov/pubmed?term=Bayer%20Fabian%20H%C3%BCbl

Beleznay Z, Regenass S. [Diagnostics of autoimmune diseases]. Ther Umsch 65, 529–537, doi:10.1024/0040-5930.65.9.529 (2008). – link leads to the English-language abstract in PubMed, the article itself is written in German: http://www.ncbi.nlm.nih.gov/pubmed?term=Beleznay%20Regenass

Beutner EH, Krasny S, Kumar V, Taylor R, Chorzelski TP. Prospects and problems in the definition and standardization of immunofluorescence. I. Present levels of reproducibility and disease specificity of antinuclear antibody tests. Ann. N. Y. Acad. Sci. 420, 28–54 (1983). – link leads to the full-text article in the Annals of the New York Academy of Sciences: http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1983.tb22186.x/abstract;jsessionid=1AC6AD5E6E4751591D0CE810B3E1E6D7.d02t02

Hahon N, Eckert HL, Stewart J. Evaluation of cellular substrates for antinuclear antibody determinations. J. Clin. Microbiol. 2, 42–45 (1975). – link to the free full-text article in the Journal of Clinical Microbiology: http://jcm.asm.org/cgi/reprint/2/1/42?view=long&pmid=818105

Hiemann R et al. Challenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells. Autoimmun Rev 9, 17–22, doi:10.1016/j.autrev.2009.02.033 (2009). – link leads to the abstract in Autoimmunity Reviews: http://www.sciencedirect.com/science/article/pii/S1568997209000731

Savige J et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am. J. Clin. Pathol. 111, 507–513 (1999). – link leads to the PubMed abstract: http://www.ncbi.nlm.nih.gov/pubmed/10191771

Savige J. et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am. J. Clin. Pathol. 120, 312–318, doi:10.1309/WAEP-ADW0-K4LP-UHFN (2003). – link leads to the full-text article in the American Journal of Clinical Pathology: http://ajcp.ascpjournals.org/content/120/3/312.long

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).

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.

The investigations of Henrique A. Mariz, Emilia I. Sato, Silvia H. Barbosa, Silvia H. Rodrigues, Alessandra Dellavance, and Luis E. C. Andrade included 918 healthy people with ages ranging from 18 to 66 years. The results of the ANA-HEp-2 immunofluorescence tests (ANA-HEp-2 IIF assays) carried out on serum samples from these healthy participants were compared with those from a control group. The control group consisted of 153 patients who all suffered from an autoimmune rheumatic disease. They included 87 SLE patients (systemic lupus erythematodes), 45 patients with systemic sclerosis, 11 people with confirmed Sjögren’s syndrome, and 10 individuals suffering from idiopathic inflammatory myopathy (autoimmune myopathy).

Indirect immunofluorescence assays on ANA-HEp-2 cells were carried out on serum samples from all of these people. The result was evaluated as positive if the typical fluorescence pattern of indirect immunofluorescence was observed.

The results in brief: Well-defined IIF patterns, and thus a positive ANA-HEp-2 test result, were found for 12.9% of the healthy people. The ANA were observed at low to intermediate titres, and the nuclear fine speckled pattern (NFS) was particularly commonly observed – in nearly half of the ANA-positive healthy individuals (45.8%). The nuclear dense fine speckled pattern (NDFS) was found in nearly one third of the healthy people (33.1%), primarily in conjunction with a high ANA antibody titre. It is noteworthy that none of the healthy patients’ samples demonstrated the nuclear coarse speckled pattern (NCS) or a nuclear homogenous pattern (Ho).

ANA-HEp-2 immunofluorescence pattern: antibodies against CENP-B (centromere) – interphase nuclei: the 46 dots are positive; mitotic cells are positive (400x) – © ORGENTEC Diagnostika, Mainz

In contrast, the serum samples from the autoimmune patients gave positive IIF results at intermediate to high ANA titres. The control group samples were also distinguishable by their completely typical fluorescence patterns. The serum samples from autoimmune patients were the only ones that demonstrated the nuclear coarse speckled (26% of samples), nuclear homogenous (7% of samples), nuclear centromeric (8%), and cytoplasmic dense fine speckled (3%) patterns. At 42%, the nuclear fine speckled pattern was also quite common in this group of patient serum samples; however, the antibodies were present at significantly higher titres than in the sample from healthy individuals. In a follow up after four years, 72.5% of the ANA-positive healthy people still had a positive ANA-HEp-2 test result. As before, they had no symptoms of autoimmune rheumatic disease!

ANA-HEp-2 pattern “is critical in properly diagnosing autoimmune disorders”

In a press release from Wiley-Blackwell (publisher of Arthritis & Rheumatism), Dr. Luis E. C. Andrade drew the following conclusion from his research group’s results: “The ANA-HEp-2 test is positive in a sizeable portion of the general population and our findings established distinguishing characteristics between healthy individuals and patients with autoimmune disease, which is essential to accurately interpret the test results.” In addition, “Our study confirms that the ANA-HEp-2 pattern is critical in properly diagnosing autoimmune disorders and future research should attempt to reproduce the interpretation of test results among different ANA experts and ANA-HEp-2 slide brands.”

[ ... ] the ANA-HEp-2 pattern is critical in properly diagnosing autoimmune disorders and future research should attempt to reproduce the interpretation of test results among different ANA experts and ANA-HEp-2 slide brands.            

IIF pattern on the HEp-2 cells: antibodies against SRP (signal recognition particle) – cytoplasm: diffuse fine speckled positive; mitotic cells: negative (400x) – © ORGENTEC Diagnostika, Mainz

For many studies in autoimmune diagnostics and rheumatism diagnostics, the ANA test for HEp-2 cells is the method of choice; in fact, the ANA HEp-2 test system is often considered the gold standard of diagnosis. A few weeks ago, an Austrian colleague gave me basic instruction in the correct way to carry out indirect immunofluorescence tests and how to evaluate IIF patterns. I was amazed at how easy it is (in principle!) to carry out this technique. I was fascinated by the brilliance of the images I saw under the immunofluorescence microscope – and I was simultaneously humbled intimidated by the tremendous challenge involved in recognizing and evaluating the highly complex immunofluorecence patterns!

The analyst in an ordinary clinical laboratory should be able to recognize the ANA patterns that are clinically relevant and those that are the most probably observed in individuals with no apparent autoimmune disease.

As Dr. Andrade also points out in his article, careful and regular training, as well as a great deal of experience, are a requirement. In any case, he argues for basic knowledge of this highly complex topic: “The analyst in an ordinary clinical laboratory should be able to recognize the ANA patterns that are clinically relevant and those that are the most probably observed in individuals with no apparent autoimmune disease.” – I highly recommend you read the original text, it is worth it!

Re©ognise ©opyright law!!! — Please note that all immunofluorescence pictures used in this blog post are copyrighted material! All these IIF pattern fotos are taken and kindly left by Barbara Fabian, Austria. If you’re interested in using the pictures, please ask for permission: pr@orgentec.com 

References:
Mariz HA, Sato EI, Barbosa SH, Rodrigues SH, Dellavance A, Andrade LE. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum. 2011 Jan;63(1):191-200. – doi: 10.1002/art.30084.

Author of this article:  Tobias Stolzenberg

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.

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. 

The classification and diagnostic criteria for SLE are not covered in this article; neither are the pathogenesis and treatment of systemic lupus erythematosus (SLE). I do recommend two outstanding reviews for those of you who wish to learn more about the subject of lupus and lupus diagnostics:  

• Manson JJ, and Rahman A. Systemic Lupus Erythematosus. Orphanet J Rare Dis. 2006 Mar 27;1:6. Review. – doi:10.1186/1750-1172-1-6 – open source, free full text
• Kumar Y, Bhatia A, Minz RW. Antinuclear Antibodies and Their Detection Methods in Diagnosis of Connective Tissue Diseases: a Journey Revisited. Diagn Pathol. 2009; 4: 1. – doi: 10.1186/1746-1596-4-1. – free full text  

Both of these articles can be downloaded free of charge through these links (as of April 21, 2010). Among the other literature references at the end of this post you will also find a number of open-source and free articles to download. – I hope you find what you are looking for and enjoy a stimulating read!  

Antinuclear antibodies (ANA): characteristic of inflammatory rheumatic diseases   

Antinuclear antibodies (ANA) are considered characteristic of inflammatory rheumatic diseases. ANA are antibodies that bind to components of the cell nucleus. These antigens may be localized in the chromatin, nucleolus, nucleoplasm, nuclear matrix, or the nuclear membrane.  

Common signs and symptoms of systemic lupus erythematosus.

Important target antigens of these nuclear antibodies are double-stranded deoxyribonucleic acid (dsDNA), DNA-associated proteins like histones or centromere proteins, and enzymes relevant to cell biology (splicing enzyme, RNA polymerases, DNA topoisomerase I).  

In a narrower sense, ANA are the autoantibodies that induce nuclear staining in indirect immunofluorescence tests on tissue sections (for example rat livers) or human tumour cell monolayers (such as HEp-2 cells). Today, there is a broad consensus that at least some of these autoantibodies are clearly involved in the pathogenesis of SLE.  

Elevated values for ANA are detected in the blood of nine out of ten SLE patients. The high diagnostic sensitivity of over 90% for ANA detection makes the ANA test the most sensitive confirmatory test when systemic lupus erythematosus is suspected.  

However, the high diagnostic sensitivity of ANA detection contrasts with its low specificity in the diagnosis of lupus. In other words, antinuclear antibodies (ANA) are also detected in patients with nearly all other autoimmune diseases, such as systemic scleroderma or polymyositis, as well as relatively often in patients with tumours. Antinuclear antibodies are often detected in some cases of chronic infection, and some healthy patients also occasionally test positive for ANA.  

Various test methods are used for ANA detection in patient serum, including indirect immunofluorescence microscopy (indirect immunofluorescence technique, IFT; also indirect immunofluorescence assay, IFA) or ELISA technology (enzyme-linked immunosorbant assay). Within the next few weeks, I plan to describe the individual test methods in more detail in another post.  

Expert opinion states that a positive ANA result should be followed up with assays to detect the individual specific ANAs, depending on the diagnostic question and the immunofluorescence pattern obtained by IFT (IFA). All such patients should be tested for anti-ENA, antibodies against extractable nuclear antigens.  

Significant for the diagnosis of lupus are anti-Sm, anti-U1RNP (also anti-U1-RNP or anti-RNP), anti-SS-A/Ro, and anti-SS-B/La. The various ENA are associated with different manifestations of the lupus disease. For example, anti-Sm are specific to systemic lupus erythematosus (SLE), and are particularly found in cases of lupus with kidney involvement. On the other hand, anti-Ro antibodies are indicative of secondary Sjögren’s syndrome.     

Unlike undifferentiated ANA detection, individual detection of antibodies against double-stranded DNA (anti-dsDNA antibodies) and the differentiated detection of anti-nucleosome antibodies are highly specific to systemic lupus erythematosus (SLE).   

Like anti-Sm antibodies, the anti-dsDNA antibodies indicate involvement of the kidneys in SLE. In addition, the anti-dsDNA titres correlate with disease activity, which makes the anti-dsDNA antibodies a marker for SLE as well as an aide for monitoring the disease. Typically, a flare up of the disease is accompanied by rising anti-dsDNA titres, as well as an increased erythrocyte sedimentation rate (ESR), lowered complement protein values, and reduced lymphocyte count.   

Usually, the C-reactive protein value (CRP) does not increase with rising disease activity. When this does occur, it could be due to arthritis or serositis. – Warning: an increased CRP value in SLE patients may indicate infection!   

The following are “individual portraits” of the autoantibodies whose detection is helpful for the diagnosis and monitoring of SLE:    

Anti-dsDNA antibodies: markers for SLE  

Autoantibodies against double-stranded DNA (anti-dsDNA) are detected in over 30% of lupus patients. Anti-dsDNA antibodies are considered markers for SLE. This autoantibody is rare in healthy individuals, occurring in just 1% of the population, which makes it useful for confirmation

Immunfluorescence test with Crithidia luciliae: anti-dsDNA antibodies are considered markers for SLE.

of suspected systemic lupus erythematosus. Anti-dsDNA antibodies are thus a component of the SLE classification criteria of the ACR (American College of Rheumatology).    

Literature reports indicate that a positive anti-dsDNA result is indicative of a grave progression of the disease, specifically development of lupus nephritis (kidney lupus). When the disease is active and involves the kidneys, various studies indicate that high titres of anti-dsDNA antibodies are usually detectable.    

Within this clinical framework, experts believe that anti-dsDNA antibodies are very valuable for monitoring disease. Increasing levels of antibodies can thus be used to predict oncoming flare-ups. – Warning: The absence of anti-dsDNA antibodies does not rule out SLE!    

To my knowledge, there are basically three tests in use for the detection of anti-dsDNA antibodies at present: the ELISA (enzyme-linked immunosorbant assay), the immunofluorescence test or immunofluoreszence assay (IFT resp. IFA) with Crithidia luciliae cells(crithidia luciliae immunofluorescence technique, CLIFT), and the Farr assay with radiolabeled double-stranded DNA.    

Anti-Sm antibodies  

Antibodies against the Sm antigen (anti-Smith antibodies, anti-Sm antibodies), a ribonucleoprotein from the cell nucleus, are almost exclusively found in patients with lupus. The diagnostic specificity is high at 99%.    

In contrast, anti-Sm antibodies are rarely found in patients who suffer from other rheumatic diseases; they occur in less than 1% of healthy individuals. Anti-Sm antibodies thus have a marker function in lupus diagnostics, and are an ACR criterion for the diagnosis of SLE (ACR = American College of Rheumatology).    

The diagnostic sensitivity of anti-Sm antibodies, on the other hand, is slight: only about 20% of people with lupus have anti-Sm antibodies. I find it noteworthy that the occurrence of this biomarker can vary significantly within individual ethnic groups. I have thus found that the incidence of anti-Sm antibodies in SLE patients of Asian or African descent lies between 30 and 40%, whereas for lupus patients of Caucasian ancestry it is significantly lower, at 10 to 15%.    

As to the question of whether the appearance of anti-Sm antibodies correlates with disease activity, and flare-ups in particular, I have found contradictory claims. This still appears to be a bit unclear. Claims of correlation with specific manifestations of the disease are also partially contradictory, although severe organ manifestations, such as involvement of the central nervous system or kidneys, seem to go hand-in-hand with the appearance of anti-Sm antibodies.    

In contrast to the anti-dsDNA antibodies, the anti-Sm antibodies do not correlate with the degree of kidney involvement in lupus nephritis. On this point the literature is in agreement.    

Anti-U1RNP antibodies (anti-RNP)  

In general, anti-U1RNP antibodies (also known as anti-RNP antibodies) are found together with anti-Sm antibodies in the serum of SLE patients. The incidence of anti-U1RNP antibodies in people with lupus is 25%, while less than 1% of healthy individuals have these antibodies.     

IFT with HEp-2 cells: anti-RNP antibodies show a spreckled pattern.

Unlike anti-dsDNA and anti-Sm antibodies, anti-U1RNP antibodies are not specific for lupus. They also occur in other rheumatic diseases, including rheumatoid arthritis (RA), systemic sclerosis, Sjögren’s syndrome, and polymyositis.    

It has been demonstrated that anti-U1RNP antibodies are detectable in patients with scleroderma, including Raynaud’s phenomenon. There is also a connection to other diseases, such as Jaccoud’s arthropathy.    

The titre of anti-U1RNP in an individual may vary. However, I have found no literature reference indicating a correlation between the level of anti-RNP antibodies and disease activity. There thus seems to be no relationship.    

Anti-Ro/SS-A and Anti-La/SS-B antibodies

Anti-Ro/SS-A and anti-La/SS-B antibodies are mainly found in people with systemic lupus (30 to 40%) or primary Sjögren’s syndrome (for Sjögren’s syndrome, anti-Ro/SS-A are diagnostic markers and an ACR classification criterion).    

Anti-Ro and anti-La antibodies can also be detected in a number of other rheumatic diseases, such as systemic sclerosis, rheumatoid arthritis (RA), and polymyositis. According to the literature, these autoantibodies are also found in over 15% of healthy individuals.    

Anti-Ro and anti-La are thus not specific for diagnosing lupus. However, as has been described a number of times, they are connected to a number of specific symptoms typical of lupus, such as extreme sensitivity to sunlight, or subacute cutaneous lupus erythematosus (SCLE). They are also linked to a deficiency in certain complement factors.    

In addition, the children of mothers whose serum contains anti-Ro and anti-La antibodies have an increased risk of developing neonatal lupus erythematosus (NLE), a disease that can lead to congenital heart block (CHB). I found it recommended that SLE patients wishing to have children should always be tested for anti-Ro/SS-A and anti-La/SS-B antibodies.    

Anti-histone antibodies (AHA)

Antibodies against histone, known as anti-histone or AHA, are detectable in patients with a variety of diseases, primarily rheumatic conditions.    

Among specialists in this field, there is consensus that they are not in themselves sufficiently specific for the diagnosis of lupus. High titres of anti-histone antibodies are almost exclusively detectable in patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL). This means that in the absence of SLE marker antibodies, a high-titre of anti-histone antibodies can be considered characteristic of drug-induced lupus.    

That’s about it for autoantibody detection in SLE diagnosis. If you would like to know more about the individual antibody tests or individual antibodies and my literature references do not suffice, please refer to our list of literature references about ANA at the ORGENTEC website, which I will update in the next few days.    

Earlier in this post I touched briefly on the laboratory tests for the detection of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). I would thus like to briefly mention the detection of the complement proteins and the significance these laboratory tests may have for confirming a lupus diagnosis.    

If the overall level of complement proteins is low, an infection may be indicated. If the clinical picture otherwise indicates it, a low level of complement proteins is also supportive of a lupus diagnosis, according to rheumatologists. If the values of complement proteins C3 and C4 are low and the ANA result is positive, an active phase of disease may be indicated, possibly with kidney involvement.    

Author of this article:  Tobias Stolzenberg

Literature:

Bardin N, Ragot C, Sanmarco M. Clinical evaluation of a new quantitative enzyme-linked immunosorbent assay for detection of double-stranded DNA autoantibodies. Ann N Y Acad Sci. 2007 Aug;1109:511-8 – doi 10.1196/annals.1398.057 – to the abstract    

Bizzaro N, Tozzoli R, Shoenfeld Y. Are we at a stage to predict autoimmune rheumatic diseases? Review. Arthritis Rheum. 2007 Jun;56(6):1736-44. – doi 10.1002/art.22708 – free full text    

Habash-Bseiso DE, Steven HY, Glurich I, Goldberg JW. Serologic testing in connective tissue diseases. Clin Med Res. 2005;3:190–193. – free full text    

Isenberg DA, Manson JJ, Ehrenstein MR, Rahman A. Fifty years of anti-ds DNA antibodies: are we approaching journey’s end? Rheumatology (Oxford). 2007 Jul;46(7):1052-6. Review. – doi:10.1093/rheumatology/kem112 – free full text    

Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagn Pathol. 2009; 4: 1. – doi: 10.1186/1746-1596-4-1. – free full text    

Manson JJ, Ma A, Rogers P, Mason LJ, Berden JH, van der Vlag J, D’Cruz DP, Isenberg DA, Rahman A. Relationship between anti-dsDNA, anti-nucleosome and anti-alpha-actinin antibodies and markers of renal disease in patients with lupus nephritis: a prospective longitudinal study. Arthritis Res Ther. 2009 Oct 14;11(5):R154. – free full text    

Manson JJ, Rahman A. Systemic lupus erythematosus. Orphanet J Rare Dis. 2006 Mar 27;1:6. Review. – doi:10.1186/1750-1172-1-6 – open source, free full text    

pictures are from: Wikimedia Commons (File:Lupus facial rash.jpg, File:Symptoms of SLE.png) and from ORGENTEC Diagnostika (IFT patterns)