Anti Nuclear Antibody Testing Using Immunofluorescence

Results:

The aim of this practical was to identify the presence of anti-nuclear antibodies (ANA) in serum samples using immunofluorescence (IFA). The method used for identifying the presence of ANAs was the NOVA LITE Hep-2 ANA Kit. The principle of this method was that it is an indirect immunofluorescence technique, where the samples are incubated with the antigen substrate (Hep2 cells) that is impregnated at the bottom of the wells in the Hep2 slides and any unreacted antibodies are removed by washing. A specific fluorescein labelled conjugate is then added and the wells are then incubated. Any unbound reagent is removed by washing. When the wells are examined with a fluorescence microscope, any positive autoantibody samples will display a specific pattern of apple green fluorescence which  corresponds to parts of the nuclei or cell or where the autoantibody has become bound to.

Fig.1: The NOVA LITE Hep-2 ANA slide used in this practical which contained the antigen substrate. Only six wells were used, wells 1 – 6, as only six samples were used.

The samples used were:

Two patient samples (P1 and P2),

Positive control (ANA titratable pattern), Negative control (IFA system negative control),

Two autoantibody coloured controls: ANA centromere pattern and ANA nucleolar pattern.

The patient samples were diluted 1/80 in phosphate buffered solution (PBS).

The controls used were neat (no dilution required).

An FITC anti-IgG conjugated with fluorescein was used which was added to the wells after the samples were added and then washed to remove unbound sample.

Table 1: The presence or absence of Fluorescence in the Hep2 slide wells

Discussion:

The responsibility of the immune system is to protect the body from infection, however, in a few people healthy cells are repeatedly attacked by the immune system in the body causing an autoimmune disorder. The antinuclear antibody (ANA) term by definition describes a collection of different autoantibodies which bind to various parts of the nuclei of cells such as proteins, DNA and ribonucleoproteins (Cervera et al., 2000).

The detection of ANAs highlights the possible presence of an autoimmune disease and may be diagnosed using a screening test. Even though there may be numerous tests for detecting ANAs, the most commonly used tests for screening are the indirect immunofluorescence (IIF) technique and enzyme-linked immunosorbent assay (ELISA). IIF is one of the most commonly used methods for ANA testing. Generally, Hep-2 cells are the substrate of choice for the detection of antibodies found in the serum of humans. The wells in the microscope slides are impregnated with Hep-2 cells and the wells are incubated with serum. If there is antibodies present they will then bind with the antigens in the Hep-2 cells, as regards to ANAs, these antibodies will then bind to antigens in the cell nucleus. This can be viewed by addition of a fluorescent conjugate (normally FITC) anti-human antibody which attaches to the autoantibodies. The fluorescent conjugate will then fluoresce if a light of a specific wavelength shines on the well, which is able to be visualised under a microscope. Depending on whether the autoantibody is present in the serum and the area of the antigen in the cell nuclei, a specific pattern of fluorescence may be visualised in the cells.

Nowadays, Hep-2 cells are one of the most commonly used substrates for detection of ANAs with immunofluorescence. A native protein array is the Hep-2 cell which has antigens in the hundreds, is an appropriate substrate for detecting ANAs. The detection of ANA in human serum using Hep-2 cells is an important screening tool for connective tissue diseases, systemic lupus erythematosus, scleroderma, primary Sjogren’s syndrome, chronic active hepatitis, hypergammaglobulinemic purpura, juvenile Rheumatoid arthritis and drug induced lupus.

Until about 1975, where Hep-2 cells were first introduced, tissue from animals was used for immunofluorescence as the standard substrate. The animal tissue used began as a laryngeal carcinoma strain, until the cell line got contaminated and was displaced by HeLa cells. Hep-2 cells originate from HeLa cells.

Human epithelial type 2 (Hep-2) cells are used as a antigen substrate in ANA testing due to their large cell size and  also a large nucleus which is important in ANA testing, as nuclear antibodies are being detected. Hep-2 cells have a high rate of mitosis. This enables antibodies to be  detected which are specific for mitosis antigens, for instance centromere antibodies. In addition, Hep-2 cells enable identification of anti-Ro antibodies, as acetone is used in the fixation of cells and while other fixatives might remove the antigen on other cells during washing. They also have better antigen expression allowing recognition of over 30 different nuclear and cytoplasmic patterns that more than 50 different autoantibodies are associated with various autoimmune conditions.

There are numerous nuclear staining patterns observed on Hep-2 cells for instance speckled, homogenous, nucleolar, centromeric, nuclear membranous, pleomorphic and nuclear dot. The homogenous pattern is observed when the interphase chromatin and the condensed chromosomes are stained. This type of pattern is caused by anti-dsDNA antibodies, anti-histone antibodies and  antibodies to components of the nucleosome. Two speckled patterns exist which are coarse and fine. In the fine speckled type of pattern there is fine nuclear staining along with metaphase chromatin which is unstained, caused by anti-La and anti-Ro antibodies. In the coarse staining type of pattern there is coarse granular nuclear staining, associated with anti-Sm and  anti-U1-RNP antibodies. The nucleolar staining type of pattern can be caused by many different antibodies such as anti-PM-Scl, anti-Scl-70, anti-Th/To and anti-Fibrillarin. The staining of the nuclear membrane exhibits a fluorescent ring surrounding the nucleus and are created by anti-p62 and anti-gp210 antibodies. The centromere pattern exhibits numerous nuclear dots in interphase and mitotic cells, which corresponds to the amount of chromosomes in the nucleus. Nuclear dot patterns exhibit around 13 – 25 nuclear dots in interphase cells and are created by anti-sp100 antibodies. Pleomorphic pattern is produced by antibodies specific for the antigen in nucleus of a proliferating cell (Buchner et al., 2014).

In this practical the aim was to identify the presence of ANAs in serum samples from two different patients using indirect immunofluorescence (IFA). Two autoantibodies controls were also used as well as a positive and negative control. Six samples in total were tested and therefore six wells in the Hep-2 slide were only used.

The control results were read first in order to establish that the reagents and method used gave correct results so that the method was then valid and the patient results could then be interpreted. The positive control in well three had fluorescence present and produced a ANA titratable homogenous pattern in the Hep-2 cells. This showed that the positive control worked. The negative control in well four fluorescence was absent. This meant that the negative control worked.

For the autoantibody control in well five fluorescence was present which produced a ANA centromere pattern. According to the expected results, this was the result expected. Therefore the autoantibody control worked for this pattern. When a centromere pattern is produced the antibodies producing this pattern is Cenp A, B and C antibodies. The diseases associated with these antibodies are CREST syndrome, a variant of progressive systemic sclerosis (PSS). CREST is a type of PSS with calcinosis that is prominent, along with Raynaud’s phenomenon, oesophageal dysmotility and reduced engagement of the skin (commonly limited to the face or fingers).

For the autoantibody control in well six fluorescence was present which produced a ANA nucleolar pattern. According to the expected results, this was the result expected. Therefore the autoantibody control worked for this pattern. Antibodies that produce the nucleolar pattern are anti-Scl-70, anti-Pm-Scl, anti-fibrillarin anti- 4-6s RNA and anti-Th/To. The disease associated with these antibodies are if high titres are present are scleroderma and sjogren’s syndrome.

Correct pattern recognition is critical for accurate reporting of ANA results. This is why two positive ANA controls (centromere and nucleolar) were used. From interpreting all the control results, where all four controls gave expected results, the reagents and method used were valid and the patient results could then be read and interpreted.

Two different patient serum samples were tested for ANAs. The patients were called patient one and patient two.

Patient one in well one in the Hep-2 slide result was that fluorescence was absent. Therefore there was no ANAs detected in the patient’s serum.

Patient two in well two result was that fluorescence was present and the pattern on the Hep-2 cells was a ANA coarse speckled pattern. Therefore, there was ANAs detected in the patient’s serum. The subtype of ANA detected that produced the speckled pattern was anti-Sm, anti-RNP, anti-Scl-70, SS-A, SS-B and other antibodies not characterised yet. This type of ANA pattern is diagnostic of autoimmune disorder systemic lupus erythematosus (Sm antibody especially if high titres are present), mixed connective tissue disease (RNP antibody), scleroderma (Scl-70 antibody) or Sjogren’s syndrome-siccca complex (SS-B antibody). Lower titres is suggestive of other connective tissue disorders.

Patient two’s ANA test results is highly suggestive of systemic lupus erythematosus (SLE). This disease also called lupus, is an autoimmune disorder where the person’s immune system attacks the person’s own tissue in various areas of the body. It’s symptoms differ among individuals and can be mild or severe. Typical symptoms are joints which are painful and swollen, chest pain, fever, mouth ulcers, hair loss, fatigue, lymph nodes which are swollen and a red butterfly rash on the face. Episodes of illness are often which are known as flares and episodes of remission where there is very little symptoms.

The exact reason for SLE is still unclear. One SLE manifestation is apoptosis abnormalities. In SLE, the immune system secretes antibodies directed against the host itself, in particular towards proteins found in the nucleus of the cell. SLE is thought to be triggered by factors from the environment which are not known.

The body’s immune system has to balance from being sensitive to fight infections and able to become sensitised to fight the body’s very own proteins (autoimmunity). When the immune system reacts to a foreign trigger, like bacteria, the cells of the immune system that normally are deactivated because of their affinity for the body’s own tissues may be abnormally stimulated via signalling events of antigen presenting cells. Therefore, stimuli can include bacteria, viruses, allergens (IgE plus other hypersensitivity) and may be aggravated via stimulants from the environment like particular drug reactions and ultraviolet light. These triggers start a reaction which can result in destruction of other cells located in the body and exposes their histones, DNA and other proteins, in particular areas of the nucleus of the cell. The body’s B cells which are sensitised now will create antibodies directed at these nuclear associated proteins. These particular antibodies will aggregate together to form complexes of antibodies and proteins which can adhere to surfaces and cause damage to blood vessels in important areas of the body like the kidney glomeruli. These specific attacks from the antibodies are the reason for SLE (Danchenko, Satia and Anthony, 2006).

A chronic inflammatory disease SLE is thought to be a type three hypersensitivity response with the possibility of the involvement of type two. A number of research studies have demonstrated that the quality and quantity of invariant NKT cells have showed that there are marked defects in patient’s with SLE in comparison to a healthy control group (Chen et al., 2018).

The preliminary diagnosis of autoimmune diseases can be confirmed using autoantibodies, to establish prognosis, determine activity of the disease, and to monitor the treatment response and side effects of drugs. Using this fact, autoantibodies have important functions in rheumatological disease management. When carefully used they enable quick diagnosis and relevant treatment. Although, in certain cases instead of assisting the diagnosis, autoantibodies can create more confusion. This is due to some autoantibodies being positive for a lot of autoimmune diseases may also be present in healthy population. False positive test results can result in incorrect treatment  and unnecessary worry for patients. Autoantibody positive test results on their own do not make a final diagnosis. In comparison, a negative autoantibody result on its own doesn’t rule out diagnosis. The test’s success is related closely to specificity and sensitivity.

Rheumatoid factor (RF) is a antibody formed specifically against the Fc portion of the immunoglobulins. However, all the classes of the antibodies contain an Ig structure, the most frequent is the IgM structure. It is thought that RF may play a part in the presentation of antigens and humoral response amplification. Almost 70% of Rheumatoid arthritis patients it is positive for RF and could be indicating a worse prognosis for the patient. High Rheumatoid factor levels can show rheumatoid nodules, aggressive joint disease and accompanied with extra articular involvement. A positive RF result on its own is not enough for diagnosis. In a healthy population about 15% can be positive for RF at low titrations and increases as people get older. In addition, in some other  rheumatologic diseases such as SLE, Sjogren’s syndrome, cryoglobulinemia, silicosis, pulmonary disorders such as interstitial fibrosis and different infectious disorders, RF can be positive. Almost 30% of patients with RA  are seronegative and this can rise to 50% in the early stages of RA. Therefore, a negative RA result cannot rule out diagnosis. Because of results that are contradictory, RF may not be used in monitoring the response of treatment and disease progression.

In most patients with RA, they develop IgG antibodies targeted at citrulline peptides have been found. These type of antibodies are called anti-citrullinated peptide antibodies (ACPAs). Anti-fliagrin, anti-perinuclear factor, anti-keratin antibody, anti-cyclic citrullinated peptide (anti-CCP) and anti-Sa are the main members. Since anti-CCP has a specificity that is higher than RF, it is preferably used for diagnosis of RA. The 1st generation of the anti-CCP test (anti-CCP1) has 96% specificity and 53% sensitivity for RA. The 2nd generation of the anti-CCP test (anti-CCP2) has specificity of 99% and sensitivity of 61.6% for initial RA, 75.2% for late stages of RA and 71.7% for all patients with RA (van Boekel et al., 2002).

There is a change in ANA measurement since lupus erythematous cell was identified to the present day when immunofluorescent techniques are used. Along with the variation in laboratory methods, ANA testing has changed. With an increase in sensitivity, the probability of lupus present with a negative ANA result has reduced, although a ANA positive result in the healthy population has risen. As a result the cut off value has increased from 1/40 to 1/80.

For those with clinical suspicion it is significant if identified at high titrations. Although, at high titres the relationship with the activity of disease and its severity is not possible anymore. So it is not possible to check the progress of disease with ANA values.

An ANA test for testing musculoskeletal disorders and must only be used only if suspicious RD. The sensitivity of the ANA test is 93% for Systemic lupus erythematous and 85% for scleroderma. However, ANA specificity for the same type of diseases are way lower compared with their sensitivity (SLE is 57% and scleroderma is 54%). So a negative ANA result excludes SLE, however its positivity seems not to be important as the specificity is lower. Similarly a negative ANA is useful to rule out scleroderma while a positive result does not confirm diagnosis although it supports.

For SLE that is drug induced and mixed connective tissue disease (MCTD), since their sensitivity is almost 100%, the result of their  ANA test is used to diagnose those two diseases. Diseases with lower ANA sensitivity are secondary Raynaud’s syndrome (64%), polymyositis (61%) and Sjögren’s syndrome (SS) (48%). ANA is useful in diagnosing SS and idiopathic inflammatory myositis despite its lower sensitivity for these diseases (40% and 70%).

Some autoimmune diseases can be diagnosed by specific antibodies. So their specificity is important which is high compared to their sensitivity values which are much lower. Of those antibodies, the most important are:

Anti-dsDNA antibodies which are diagnostic of SLE  with a specificity of 97.4% and a sensitivity of 57.3% sensitivity.

Anti-Sm antibodies are only found in SLE patients (sensitivity: 25%-30% and specificity: high).

Anti-RNP antibodies are found in 30%-60% of SLE patients, however not specific. They have use in diagnosis of MCTD. However, Anti-U1 RNP antibody is usually diagnostic of MCTD.

Anti-histone antibodies are present in 95% of drug induced SLE and 50%-70% with SLE. Most patients who have the antibodies have no symptoms, so a positive sera result doesn’t always mean the disease is present.

Anti-chromatin antibody is found in 50%-90% of patients with SLE.

Anti-La/SSB and anti-Ro/SSA antibodies are present in SLE and SS patients and are diagnostic of SS. These antibodies can be found in patients with SLE that have a negative ANA result.

In the anti-centromere antibody family there are three centromere proteins: CENP A, B, and C. Their target is CENP B. They have been found in patients with limited cutaneous systemic sclerosis and CREST syndrome.The specificity is high of anti-centromere antibodies in CREST syndrome, although their sensitivity is much lower. The development of scleroderma in patients with Raynaud’s syndrome can be estimated using anti-centromere antibodies. However, they are discriminative for excluding CREST.

The anti-Scl-70 antibodies are found in 20%-40% of patients with systemic sclerosis. Although the sensitivity is low, specificity is almost 100%. In the diagnosis of scleroderma it is high as specificity is 98% but the sensitivity is low (28%).

The anti-nucleolar antibody IF pattern is scleroderma specific. These specific antibodies that exhibit this type of pattern are anti-Th/To antibodies, anti-PM/Scl antibodies, anti-RNA polymerase I and III and anti-U3-RNP (Birtane, Yavuz and Taştekin, 2017).

In conclusion to this practical it is possible to diagnose autoimmune disorders by detecting ANAs using immunofluorescence. Hep-2 cells are an ideal antigen substrate due to their large nucleus allowing a variety of nuclear patterns to be produced. The sensitivity and specificity of the different ANAs encountered depends upon their titre along with the particular ANA detected, in diagnosing whether a particular autoimmune disease is present.

References:

• Birtane, M., Yavuz, S. and Taştekin, N. (2017). Laboratory evaluation in rheumatic diseases. World Journal of Methodology, 7(1), p.1.
• Buchner, C., Bryant, C., Eslami, A. and Lakos, G. (2014). Anti-Nuclear Antibody Screening Using HEp-2 Cells. Journal of Visualized Experiments, (88).
• Cervera, R., Font, J., Ramos-Casals, M., García-Carrasco, M., Rosas, J., Morlà, R., Munóz, F., Artigues, A., Pallarés, L. and Ingelmo, M. (2000). Primary Sjoögren’s syndrome in men: clinical and immunological characteristics. Lupus, 9(1), pp.61-64.
• Chen, J., Wu, M., Wang, J. and Li, X. (2018). Immunoregulation of NKT Cells in Systemic Lupus Erythematosus. Hindawi.
• Danchenko, N., Satia, J. and Anthony, M. (2006). Epidemiology of systemic lupus erythematosus: a comparison of worldwide disease burden. Lupus, 15(5), pp.308-318.
• van Boekel, M., Vossenaar, E., van den Hoogen, F. and van Venrooij, W. (2002). Journal search results – Cite This For Me. Arthritis Research, 4(2), p.87.