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 ->

For the launch of our Liver-9-Line immunoblot test (to our press release “Liver Disease Diagnostics by Immunoblot” of May 16, 2011), I dug through a pile of literature on the topic of autoantibody-based diagnosis of autoimmune diseases of the liver. In the last week I picked it all up again and worked through it systematically.

The interior surface of the liver. A reproduction of a lithograph plate from Gray’s Anatomy.

The reason for my renewed interest is that we brought four more ELISA tests for liver diagnostics to the market two weeks ago. They are the Anti-LKM-1, Anti-SLA, Anti-gp210, and Anti-Sp100 tests, all designed for fully automated autoimmune diagnosis with our Alegria system. All four test systems assist the formulation of a diagnosis when autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are suspected, or for differential diagnosis when another disorder of the liver is assumed.

Autoimmune diseases of the liver

The requirements for successful treatment of liver disease naturally include a precise diagnosis. As a result, unexplained elevated liver enzymes generally lead the treating physician on a complicated search for the source of the abnormally elevated liver values. In addition to anamnesis and clinical findings, laboratory tests and the correct interpretation of the laboratory results play a critical role in this process.

If the doctor is able to rule out alcohol abuse, drug-induced hepatitis, and viral hepatitis (for example hepatitis B, C, or D), and if nothing points to hemochromatosis, alpha-1-antitrypsin deficiency, or Wilson’s disease (also called: hepatolenticular degeneration), the most likely suspects remaining are autoimmune liver diseases.

In cases of autoimmune liver disease, the body loses its ability to tolerate its own hepatocellular or cholangiocellular tissues in the liver. The causes of these diseases remain a mystery.

When it comes to autoimmune diseases of the liver, there are essentially three conditions to differentiate:

1. autoimmune hepatitis (AIH), of which there are two variants, type 1 AIH and type 2 AIH.
2. primary biliary cirrhosis (PBC)
3. primary sclerosing cholangitis (PSC)

The individual clinical presentations of these diseases may overlap, which is what makes the appraisal and interpretation of laboratory values, as well as the formation of a diagnosis, so difficult for these autoimmune diseases. Overlap syndromes are particularly common between autoimmune hepatitis and PBC; overlap between the autoimmune liver diseases and viral hepatitis C is also not uncommon. (Here you will find a background article on autoimmune liver disease diagnostics.)

Autoimmune hepatitis (AIH)

Type 1 autoimmune hepatitis (type 1 AIH) is considered “classic” autoimmune hepatitis. This disorder mostly occurs in young women and is typically characterized by the presence of antinuclear antibodies (ANA) and/or anti-smooth muscle antibodies (ASMA, or: SMA), with F-actin generally acting as the target antigen. Autoantibodies against the soluble liver antigen/liver pancreas antigen (anti-SLA/LP antibodies) are often also detected. Depending on the study, this is the case in up to 25% of affected patients.

In the past, this form of “SLA-positive AIH” was considered to be its own variant of autoimmune hepatitis and was formerly known as “type 3 autoimmune hepatitis” (“type 3 AIH”). Because this anti-SLA positive form of the disease is clinically and therapeutically no different from type 1 autoimmune hepatitis, the differentiation between type 1 and type 3 AIH was abandoned. Clearly different from type 1 AIH, type 2 AIH is primarily characterized by the presence of anti-LKM-1 and anti-LC-1 antibodies.

Figure 1 – A simplified scheme: serological differentiation of type 1 autoimmune hepatitis and type 2 autoimmune hepatitis.

The autoantibodies typically detected in cases of autoimmune hepatitis are: antinuclear antibodies (ANA), anti-smooth muscle antibodies (ASMA), LKM-1 antibodies, LC-1 antibodies, and anti-SLA/LP antibodies.

Primary biliary cirrhosis (PBC)

Primary biliary cirrhosis (PBC) is a chronic, progressive disease involving destruction of the small and intermediate bile ducts. It runs in families and is more common in women over 40 years of age. The diagnostic marker for PBC is detection of AMA, in particular AMA-M2. This test is complemented by the detection of anti-Sp100 and anti-gp210 autoantibodies.

Figure 2 – Simplified scheme: serodiagnosing primary biliary cirrhosis (PBC).

The autoantibodies typical of primary biliary cirrhosis (PBC) are: antimitochondrial antibodies (in particular AMA of subtype M2) and the antinuclear antibodies directed against the antigens sp100 and gp210.

Sclerosing cholangitis

Sclerosing cholangitis presents in at least two variants: the first is classical primary sclerosing cholangitis (PSC), which mostly affects adult men and for which the only (and nonspecific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (“xANCA pattern”), the second is a childhood disease called autoimmune sclerosing cholangitis (ASC) with serological characteristics resembling those of a type 1 autoimmune hepatitis.

Antibody diagnostics for cases of …

… autoimmune hepatitis (AIH) …

After ASMA, antinuclear antibodies (ANA) are the most frequently detected autoantibodies in patients with type 1 AIH. Although ANA have no specificity for type 1 AIH, they are considered to be marker antibodies for this disease. In a large proportion (up to 60%) of patients with type 1 AIH, both ANA and ASMA can be detected. Up to 15% of patients with type 1 AIH have only ANA; about a third (30-35%) of patients have exclusively ASMA.

Depending on the test, antibodies against SLA/LP (SLA/LP = soluble liver antigen / liver pancreas) are found in up to 25% of serum samples from patients with type 1 AIH. These SLA-positive patients were previously considered to have a variant of the disease called “type 3 autoimmune hepatitis”; today they are included with the type 1 autoimmune hepatitis patients.

… and of primary biliary cirrhosis (PBC) …

Serologically, an elevated level of IgM is typical of the symptoms of primary biliary cirrhosis (PBC). In over 90% of affected individuals, antimitochondrial antibodies (AMA) are also detected, especially those of the AMA-M2 subtype. Especially for PBC patients whose serum test negative for AMA, complementary detection of autoantibodies against Sp100 and gp210, as well as the detection of anti-centromere antibodies, are of particular diagnostic importance.

… and of primary sclerosing cholangitis

In case of primary sclerosing cholangitis (PSC), no particular antibodies are known to offer useful information for the diagnosis of this autoimmune disease. However, in up to 90% of PSC patients, atypical pANCA (“xANCA”) are detected, a result that does provide the physician with an important indicator for making a diagnosis. Occasionally, antinuclear antibodies (ANA) and/or anti-smooth muscle antibodies (ASMA) may be detected in low titres. – Please note: antimitochondrial antibodies are always absent in cases of PSC!

The individual autoantibodies “in close-up”:

Antinuclear antibodies (ANA)

After ASMA, ANA are the most commonly detected autoantibodies in cases of type 1 AIH. Although ANA have no specificity for type 1 AIH, they are considered a marker antibody for this disease. For the majority of type 1 AIH patients (60%), ANA and ASMA are both detectable. Exclusively ANA (without ASMA) are found in 10-15% of patients with type 1 AIH; exclusively ASMA (without ANA) are found in 30-35% of patients with type 1 AIH. – Notice! In 20-40% of cases, ANA and ASMA are also quite frequently found in cases of type II chronic hepatitis C infection!

Figure 3 – Serodiagnosis and laboratory testing for autoimmune liver diseases. Please contact me for references.

Anti-smooth muscle antibodies (ASMA)

At high titres, anti-smooth muscle antibodies (abbreviated: ASMA) are considered to be marker antibodies and a recognized diagnostic criterion for type 1 autoimmune hepatitis. The relevant autoantigen is F-actin. Autoantibodies against F-actin can be separately detected with our Liver-9-Line immunoblot test.

The diagnostic sensitivity of ASMA detection is 80%, which means that a negative ASMA result does not completely exclude autoimmune hepatitis. ASMA are very often associated with antinuclear antibodies (ANA). The ASMA titre has limited correlation to the activity of the disease.

In addition, low ASMA titres may also be found in cases of various viral infections, such as infectious mononucleosis, Epstein-Barr virus (EBV), or chronic hepatitis C. ASMA are also occasionally detected in patients with rheumatic diseases, primary biliary cirrhosis (PBC), or neoplastic disease.

SLA/LP antibodies

Antibodies against the SLA/LP autoantigen (the abbreviation stands for soluble liver antigen / liver pancreas antigen) are often detected in conjunction with ASMA or ANA (about 30% of anti-SLA positive serum samples also contain ASMA and/or ANA).

In patients who are assumed to have type 1 AIH, but test negative for ASMA or ANA (!), the individual detection of SLA/LP antibodies is particularly important for the formation of a diagnosis. In addition, SLA antibodies are indicative of a more severe progression of autoimmune disease. In addition to this, antibodies against the SLA/LP antigen are not detected in cases of viral hepatitis!

LKM-1 antibodies

Antibodies to liver kidney microsomal type 1 (anti-LKM-1) are markers for type 2 autoimmune hepatitis. The detection of anti-LKM-1 antibodies also allows for the differentiation of type 2 AIH from type 1 AIH.

In young patients, LKM-1 antibodies have a very high diagnostic sensitivity (70-93%). However, LKM-1 antibodies are also quite often found in patients infected with the hepatitis C virus (6-10%).

LC-1-Antibodies

Anti-LC-1 antibodies (LC-1 stands for liver cytosol type 1) are detectable in nearly half of young patients with type 2 autoimmune hepatitis. Their appearance partially overlaps with the anti-LKM-1 autoantibody; about half of the patients who are anti-LKM-1 positive also have the LC-1 antibodies as a second marker of type 2 AIH. Each of these antibodies may also come up alone.

Antibodies against LC-1 are largely specific for type 2 AIH. They correlate better with disease activity than do anti-LKM-1 antibodies.

Antimitochondrial antibodies (AMA)

Antimitochondrial antibodies, AMA, act as marker antibodies for primary biliary cirrhosis (PBC). They are detected in 90-95% of cases and are primarily directed against the M2-antigen (AMA-M2). AMA have a predictive value for PBC.

However, AMA-M2 antibodies are also detected in one out of every five SLE patients (17-23%) and nearly one out of five patients with Sjögren’s syndrome (22%). They are also regularly found in patients with scleroderma (8-18%) and rheumatoid arthritis (10%). Presumably, these patients have a greater risk of developing primary biliary cirrhosis in addition to their existing autoimmune disease.

Caution! – AMA detected by immunofluorescence (IFT) are not all associated with PBC. It is thus recommended that a positive immunofluorescence test be followed up with a separate test for AMA-M2 antibodies!

AMA detection: an AMA pattern in an immunofluorescence test is not always associated with PBC! A positive IFA should always be followed up with a separate AMA-M2 test. – Picture: antibodies against mitochondria (AMA-M2?), rat liver-kidney-stomach triple tissue by ORGENTEC Diagnostika. – © Barbara Fabian MSc, http://www.der-gruene-club.at

Sp100 antibodies

Antibodies to Sp100, directed against the multiple nuclear dots, a soluble nuclear protein of 100 kDa, have 95% specificity for primary biliary cirrhosis. These antibodies are detectable in up to 25% of patients with PBC.

Relatively often, in 48% of cases, Sp100 antibodies are detected among the PBC patients who are AMA-negative. Anti-Sp100 antibodies are only very rarely detected in patients with autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PBC). Occasionally, these autoantibodies turn up in patients with rheumatic diseases (systemic lupus erythematosus, SLE: up to 10%, progressive systemic scleroderma, PSS: 5%, rheumatoid arthritis, RA: 3%, Sjögren’s syndrome, SS: 2%).

gp210 antibodies

With a specificity of nearly 100% (!), anti-gp210 antibodies are highly specific to primary biliary cirrhosis (PBC). They are regularly found in samples from AMA-negative patients with clinically observed PBC (in 21-47% of cases, depending on test), which underlines the significance of anti-gp210 detection for the diagnosis of PBC.

They are very rarely found in patients with autoimmune hepatitis, rheumatoid arthritis, polymyositis/dermatomyositis, or Sjögren’s syndrome. Antibodies against the gp210 antigen are associated with extrahepatic manifestations, such as arthritis. They are a prognostic marker and are indicative of an unfavourable outcome of PBC.

Suspicion of PSC: ANCA detection can help

Whereas type 1 autoimmune hepatitis, type 2 autoimmune hepatitis, and primary biliary cirrhosis (PBC) are characterized by typical autoantibody test results, no disease-specific autoantibodies are known for primary sclerosing cholangitis (PSC). However, pANCA and the “atypical pANCA pattern”, xANCA, are detected in up to 90% of cases.

In fact, ANCA detection for the diagnosis of primary sclerosing cholangitis is not specific. However, in some cases, an ANCA test may be helpful in the formation of a diagnosis when PSC is suspected.

Author of this article:  Tobias Stolzenberg

References:

1. Bogdanos DP, Invernizzi P, Mackay IR, Vergani D. Autoimmune liver serology: Current diagnostic and clinical challenges. World J Gastroenterol 2008 June 7; 14(21): 3374-3387 – DOI: 10.3748/wjg.14.3374. ==>  free full text article
2. Bogdanos DP, Liaskos C, Pares A, Norman G, Rigopoulou EI, Caballeria L, Dalekos GN, Rodes J, Vergani D. Anti-gp210 antibody mirrors disease severity in primary biliary cirrhosis. Hepatology 45 (6):1583-1584, 2007.==> free full text article
3. Crosignani A, Battezzati PM, Invernizzi P, Selmi C, Prina E, Podda M. Clinical features and management of primary biliary cirrhosis. World J Gastroenterol 2008 June 7; 14(21): 3313-3327 – DOI:10.3748/wjg.14.3368. ==> free full text article
4. Granito A, Muratori L, Muratori P, Pappas G, Guidi M, Cassani F, Volta U, Ferri A, Lenzi M, Bianchi FB. Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis. J Clin.Pathol. 59 (3):280-284, 2006. ==>  free full text article
5. Muratori P, Granito A, Pappas G, Pendino GM, Quarneti C, Cicola R, Menichella R, Ferri S, Cassani F, Bianchi FB, Lenzi M, and Muratori L. The serological profile of the autoimmune hepatitis/primary biliary cirrhosis overlap syndrome Am J Gastroenterol. 104 (6):1420-1425, 2009. ==> free full text article
6. Rigopoulou EI, Mytilinaiou M, Romanidou O, Liaskos C, and Dalekos GN. Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis. J Autoimmune.Dis 4:2, 2007. ==> free full text article
7. Rust C, Beuers U. Overlap syndromes among autoimmune liver diseases. World J Gastroenterol 2008 June 7; 14(21): 3368-3373 – DOI:10.3748/wjg.14.3368. ==> free full text article

You will find more references on the ORGENTEC website >> Literature Service

Pictures:
The interior surface of the liver: reproduction from Gray’s Anatomy taken from the Wikimedia Commons Project

AMA detection: IFA picture by Barbara Fabian, MSc, Der GRÜNE CLUB AUTOIMMUN, http://www.der-gruene-club.at

The schemes are own work.

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

Autoantibodies against dsDNA are diagnostic markers for Systemic Lupus Erythematosus.

Autoantibodies are specific and sensitive biomarkers for autoimmune diseases and indispensible diagnostic tools. They may also be involved in pathogenic processes underlying the disease and will potentially occur in sera of apparently healthy people long before onset of the first symptoms.

C. Eriksson, S. Raantapaa-Dalquist and their colleagues from Umeå University in Sweden have focused on this preclinical phase in the development of Systemic Lupus Erythematosus (SLE).

Catharina Eriksson, Heidi Kokkonen, Martin Johansson, Goran Hallmans, Goran Wadell, and Solbritt Rantapaa-Dahlqvist.
Autoantibodies predate the onset of Systemic Lupus Erythematosus in northern Sweden.
Arthritis Research & Therapy 2011, 13 (1):R30.

The Swedish scientists performed a comprehensive study of the autoantibody profiles before disease onset and after diagnosis in 35 patients who had donated blood before the first disease symptoms occurred and in 152 age- and sex-matched healthy subjects from northern Sweden. They investigated the production of autoantibodies that bind to major autoantigens associated with the disease, e.g. anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP, anti-Scl70, anti-Jo-1, anti-dsDNA, anti-centromere protein B, anti-histone and antinuclear antigens. These antibodies were then correlated to the first recorded symptom of the disease.

 In 63% of the 35 pre-symptomatic individuals autoantibodies could be detected before onset of disease symptoms by a median of 4.2 years. Ten of these patients expressed one autoantibody, 12 had two or more autoantibodies. The sensitivity was highest for ANA at 45.7 % with a specificity of 95 %, followed by anti-dsDNA and anti-Ro/SSA antibodies both with a sensitivity of 20 % but with specificities of 98.7 % and 97.4 %, respectively. The sensitivities for the other autoantibodies varied between 14.3 % and 2.9 % at 98 % to 100 % specificity. In sera of unaffected controls antibody titers ranged between 6.7 % and 0 %.

The first antibody seen in the preclinical phase was anti-Ro/SSA; anti-RNP and anti-histone also appeared very early. Anti-centromere B and anti-Sm were detected on the verge of disease onset. In general, the number of patients expressing autoantibodies as well as the number of autoantibodies increased while approaching the date of disease manifestation.

Patients presenting with serositis as the first symptom had more autoantibodies and a shorter period between the positive blood sample and disease onset, indicating that severe symptoms at disease onset may be related to faster disease development.

The limited number of patients with pre-symptomatic blood tests certainly is a drawback of the present study. However, the results support the role of these autoantibodies in the first steps of the pathogenic process leading to the full-blown clinical picture of SLE. Analysis of autoantibody profiles in the preclinical phase of disease development may therefore contribute to improved diagnosis and clinical management of patients with SLE.

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)