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Systemic lupus erythematosus is a multi-systemic autoimmune disease that was first described in 1941, by Klemperer and colleagues (Gonzalez-Buitrago and Gonzalez, 2006). It is a disease that can attack almost any organ or system in the body, where imbalances in self tolerance create an abnormal immune response to self proteins resulting in autoimmunity (Male et al, 2006). SLE is a disease that has a strong correlation to defects in apoptosis; however no specific cause of the disease is known (Arbuckle et al, 2003). The prevalence of the disease is worldwide; however it commonly affects people of African descent, particularly in Europe and Northern America (Kumar et al, 2009). Environmental triggers are known to contribute to the disease manifestation; although genetic links have also shown association with all HLA classes (I, II, III) on chromosome 6. Other transcription factors such as IRF5, STAT and proteins such as PTPN22 have also been seen to contribute to the manifestation (Male et al, 2006).
SLE is particularly common between the ages of 15-50, where patients present with positive antinuclear antibodies (ANA). ANA are a group of heterogenous antibodies that are capable of binding to components of the nucleus, resulting in damage of DNA. The initial screening method for patients with AIDs such as SLE is via the ANA test. 80-90% of patients with SLE present with a positive ANA (Bonilla et al, 2007), however other AID such as Sjögren's syndrome, Rheumatoid arthritis, Autoimmune hepatitis, Scleroderma and Polymyositis & Dermatomyositis, also see positive results. Antigen specific assays such as extractable nuclear antigen (ENA) and double stranded DNA (dsDNA) must then be performed to confirm a diagnosis, as approximately 70% of patients with SLE have antibodies to dsDNA (Rahman & Isenberg, 2008). Positive results can be seen within the aging population as the immune system begins to deteriorate. Nilsson et al, (2006) supports this and found that positive ANA results were found particularly in elderly patients over 85 years. 90% of patients with SLE are women, suggesting a hormonal link (Rahman et al, 2008). Hormonal imbalances are seen in women with SLE, thus it becomes difficult to maintain immune tolerance. Increased oestrogen levels result in increased antibody production and Th2 response, whilst decreased levels of androgens depress the response resulting in an abnormal immune response (Danchenko et al, 2006).
1.2 The clinical significance of ANA testing
The diagnosis of SLE is dependent on a variety of factors including clinical details, family history, age, race, sex, medication and infection (Stinton & Fritzler, 2007). The classical symptom for SLE is a butterfly-shaped rash which is commonly seen on the face (Figure 1.1).
Figure 1.1: Classical symptom of SLE
In 1982 the American College of Rheumatology (ACR) described a set criterion (Table 1) (updated in 1997), for the diagnosis of SLE aiding clinicians to correctly diagnose patients. Four points of the criteria must be met, for a definite diagnosis of SLE. The criterion for SLE includes symptoms, immunological and haematological tests. Points 10 and 11 are of particular importance, as they are confirmatory of SLE. A study by Arbuckle et al, (2003) examined the onset of SLE in 130 patients and found that 115 patients had positive indirect immunofluorescence (IIF) ANA, before diagnosis.
1. Malar Rash
A butterfly rash usually seen on the face
2. Discoid rash
red, scaly patches on skin that cause scarring
Skin rash as a result of unusual reaction to sunlight
4. Oral ulcers
Oral or nasopharyngeal ulceration
5. Nonerosive Arthritis
tenderness or swelling of joints
6. Pleuritis or Pericarditis
Pleuritis inflammation of the pleura, the lining of the pleural cavity surrounding the lungs
Pericarditis - small amount of fluid builds up between the two layers of the pericardium.
7. Renal Disorder
Cellular casts--may be red cell, hemoglobin, granular, tubular, or mixed
8. Neurologic Disorder
9. Hematologic Disorder
Hemolytic anemia--with reticulocytosis
Lyphopenia--< 1,500/ mm3
10. Immunologic Disorder
Anti-DNA: antibody to native DNA in abnormal titer
Anti-Sm: presence of antibody to Sm nuclear antigen
Positive finding of antiphospholipid antibodies on:
11. Positive Antinuclear Antibody
An abnormal antinuclear antibody by immunofluorescence
Table 1: Criterion for the diagnosis of SLE
(Obtained from American College of Rheumatology)
Once a positive ANA test has been performed there is no reason to repeat the test, however if clinicians have a strong suspicion of an evolving connective tissue disease (CTD) negative ANAs should be re-requested (Blerk et al, 2008). Other immunological tests such as complement components (C3 and C4), C-reactive protein, anti-phospholipid antibodies and anti-histone can also be tested to investigate SLE; however these may not always aid all patients (Egner, 2000).
1.3 History of ANA testing and how the diagnosis of SLE evolved
The ANA test has been around for over 40 years and is the most widely performed autoantibody test, worldwide. The test is commonly performed within Immunology laboratories and has evolved very little over the years. ANA's originated from lupus erythrocytosms, also known as the 'LE' cell phenomenon. LE cells were discovered in 1948 by Hargrave, who saw that patients with SLE have polymorphonuclear leukocytes, which had phagocytosed nuclei, within the bone marrow (Hepburn, 2001). Following the discovery, Lee et al, (1957) showed that the LE cells were formed by gamma proteins in leukocytes which were thought to be antibody. Fluorescent labels were also introduced in 1957, to show homogenous patterns on human tissue (Hughes et al, 2008). By 1961 rat sections substrates were introduced, enabling patterns such as homogenous, speckled and nucleolar to be seen in patients with rheumatic diseases. The use of rat substrates brought about a new discovery, which saw that washing cells in saline, caused alterations to cells within slides, thus altering patterns seen, thus the precursor of the ENA screen was introduced. By the 1970-80's Human epithelioma type 2 cells: CCL-23 (HEp-2) substrates were widespread and National quality assurance schemes began to establish.
1.4 Techniques implemented in laboratories for ANA detection
There are many techniques available for the testing of ANAs; these can be seen in the UK National External Quality Assessment Service (UKNEQAS) report found in Appendix 1.
1.4.1 Indirect immunoflourescent (IIF)-ANA
Indirect immunoflourescent (IIF) is a general screening technique performed to identify patients with autoantibodies. It enables scientist to link autoantibody patterns present within a patient sera, to help diagnose and monitor their progress during treatment.
ANA testing using IIF was developed by George Friou in 1957, where initially substrates such as chicken erythrocytes were used (Kumar et al, 2009). ANA substrates were traditionally prepared in-house using rodent tissue where thin layers of tissue were sliced using a cryostat. However as demand for the screening of autoantibodies increased (Figure 1.2), preparing slides was no longer feasible, as it was time consuming and laboratories could no longer manage rodent houses as they required expert attention.
Figure 1.2: Shows the cumulative increase in ANA screens since 1957 (Unit -thousands)
(Obtained Hughes et al, 2008)
Commercial companies then began to produce ready to use tissues substrates, offering a greater sensitivity. However as many commercial substrates are now available, variability between kits, manufactures, substrate, conjugate and the degree of cellularity (good monolayer of cells and a number of mitotic spindles), make it difficult to standardise methods of detection and reporting.
In order to produce accurate results, substrates must be present in the correct phase of the cell cycle (Figure 1.3). Identification of IIF-ANA patterns is dependant on the true state of chromosome. Most autoantibodies are directed against antigens expressed during interphase. Interphase is divided into 3 stages: G1, S and G2, where cytoplasmic organelles and fibres structure are most visible and the nucleoli appear well differentiated. A mix of mitotic and non mitotic forms of cells are needed in the metaphase stage as it is influential in interpreting IIF-ANA patterns, especially centromeres and homogenous patterns (Sacks et al, 2009).
Figure 1.3: Shows the stages of the cell cycle
(Obtained Hughes et al, 2008)
The HEp-2 substrate is commonly used in ANA detection and was introduced commercially in 1975 (Kavanaugh et al, 2000). HEp-2 provided a greater sensitivity for the testing of SLE as they were composed of human laryngeal squamous cell carcinoma, allowing the recognition of over 30 nuclear and cytoplasmic antigens (Gonzalez-Buitrego & Gonzalez, 2006). HEp-2 substrate contains various organelles (Figure 1.4) allowing uniform distribution of cells, showing large nucleolus, meaning no interference of the intercellular matrix is seen (Gonzalez et al, 2002).
Figure 1.4: Shows the different organelles that can be seen on HEp-2/ HEp-2000 slides
(Obtained Hughes et al, 2008)
The introduction of the HEp-2 substrate was a big step forward in identifying patients with the ribonucleoprotein complex (anti-Ro). The anti-Ro antigen is particularly significant in patients with SLE as it offers a poor prognosis. However this antigen is seen to overlap between different autoimmune diseases such as Sjögren's syndrome, thus the detection of the antigen must be precise. The Ro (SS-A) antibody is seen to target protein antigens associated with small RNA molecules known as hY-RNAs11, 12 and are of unknown function (Cozzani et al, 2008). HEp-2 cells were seen to destroy the Ro antigens during fixation, so commercial companies began to devise ways around this. To overcome this problem, HEp-2 cells were genetically modified to produce extra Ro antigen and this substrate was known as HEp-2000. HEp-2000 substrate is uniquely produced by ImmunoConcepts (Sacramento CA, USA). The slides have 10-25% mitotic human epithelia and offer a greater sensitivity (Table 2) in the diagnosis of SLE. They have aided in reducing the number of ANA negative SLE patients; however detection of Ro is dependent on the stability of actin, as it can denature easily.
Table 2: Shows IIF-ANA to have greatest sensitivity (Cozzani et al, 2008)
Although HEp-2000 substrates were seen to be more beneficial in detection of Ro antigen, they limit the identification of the different epitopes of the Ro antigen. At present HEp-2000 substrate can only identify the 60kDA Ro antigen; but since the 52kDA Ro antigen also exists, patients with this epitope are missed. A study by Cozzani and colleagues (2008) looked at 5,949 people over a 5 year period. All participants were photosensitive and 2,315 of these had connective tissue disease (CTD) such as SLE. The study found that the anti-Ro was easy to identify on HEp-2000 slides with a sensitivity of 81% according to the Altman test, of accuracy. However a study by Bossuyt and Luyckx (2005) compared IIF to EIA and saw that patients with anti-Ro antibodies were missed using HEp-2000 slides, as the undetected patients contained the Ro 52 antibody; although they reported a sensitivity of 82.9%. One patient in this study was negative for IIF-ANA, but was shown to have a positive Ro antigen by EIA. A study by Dahle et al, (2004), looked at HEp-2 and compared three ANA methods; Enzyme immunoassay (EIA), double radial immunodiffusion (DRID) and IIF. 3,079 patients were examined and overlapping results between IIF and DRID were seen and 60% of IIF-ANA gave a positive homogenous pattern. However results for EIA showed that positive IIF results appeared negative by EIA.
In 2006 the LGI performed a study looking at 18,320 samples, requesting ANA tests by IIF. The study found that 1 in 5 patients, identified as negative or weak positive by IIF, showed positive for anti-Ro via EIA. This proved that Hep2000 cells can't detect the different epitope of Ro, thus concludes that antigen-specific testing is required following the ANA test. This agrees with Morozzi et al, (2000), who suggest that a combination of 2 or more methods are required for the detection of the anti-Ro antibody in patients. This study looked at 64 people with connective tissue disorders and tested them by IIF, EIA and DRID. Results showed that 54 people were positive by at least one method and the specificity of each technique was good, whilst sensitivity varied. Sensitivity for IIF-ANA via HEp-2000 was 89%, EIA (Ro60) was 89%, EIA (Ro52) was 67% and DRID presented with a sensitivity of 76%. Although the NEQAS report shows that DRID is no longer used within laboratories, results from this study suggest that EIA has the ability to detect the different epitopes, preventing misreading of the anti-Ro antigen. Thus to ensure that all SLE patients are identified antigen-specific tests such as extractable nuclear antigen (ENA) should be used to detect the various epitopes (Cozzani et al, 2008).
Conjugates play a significant role in the determination of IIF and EIA results. Fluorescein-conjugated antibodies produced from goat, sheep or rabbit are commonly used. These are usually bought from commercial companies, which produce pre-diluted conjugate, raised against mouse or human, which aims to achieve optimal sensitivity and reactivity. Immunoglobulin fraction can be also be used; however fluorescein conjugates such as fluorescein isothiocyanate (FITC) are preferred as they produce less background staining. A fluorescein/protein (FP) molar ratio is employed, with in-house diluted conjugates. The ratio varies between kits, however a 1:3 dilution with phosphate buffered saline (PBS) is usually used (Egner, 2000). At LGI the conjugate used for detection of ANAs is IgG, as it allows accurate diagnosis and monitoring of diseases such as SLE. IgM-ANA can also be employed, although this indicates milder or non-specific diseases, whilst IgA-ANA gives little information so aren't used. Due to the use of fluorescence conjugate, slides fade overtime, thus it is particularly important to determine results as soon as possible as photographs are not taken. As IIF varies daily due to slides and condition of the microscope, it would be appropriate to carry out daily checkerboards to see which working dilution is best for the conjugate, improving consistency; however this is no longer feasible in high-throughput laboratories.
When reporting ANA three factors require evaluation: the pattern observed; substrate used and the titre of the positive test. Experienced scientist can interpret ANA slides and distinguish titre levels; however this takes years of experience. The screening dilution is important in patients presenting with positive results, as it helps determine an individual's severity of disease and can prove beneficial to clinicians. Serial dilutions at 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 can be performed, where the titre value is the one at which positive sample becomes negative. 5% of a healthy population can present with a positive low ANA titre, with no disease activity and are commonly women aged over 60 (Shmerling, 2003). Peterson et al, (2009) found that beside patients with SLE patients, other diseases also present with positive ANA titres. 1:20 healthy people presented with a positive ANA and the number of positives increased to 1:3, with a dilution of 1:40. To reduce the number of false positives, titres are commonly performed at 1:80. At LGI titres were performed on all positive samples and pregnant women, regardless of whether they are positive or negative. Pregnant women are closely monitored as a precaution as IgG antibodies cross the placenta, thus anti-Ro/La antigen is capable of causing fetal heart block (Rahman & Isenberg, 2008). Patients who presented with symptoms for SLE were also titrated; however lots of weak positive results were seen as a dilution of 1:40 was employed. As workload increased titrations became laborious and impractical, thus performing titres routinely was abolished and titres are now only performed upon request.
Cut-offs exist, however these are modified around the local population, to give a better sensitivity (Stinton & Fritzler, 2007). Shmerling, (2003) has suggested that ANA titres can correlate with disease activity, but as positive samples undergo antigen specific testing via EIA, titres should be abolished, unless there are specifically requested by the clinicians to monitor changes to disease.
Wieser et al, (2001) found that there was a lack of correlation between the clinical features of patients and laboratory results obtained. The study looked at 3 cases with varying antibody titres and established algorithms seen in Figure 1.5. Similarly Hanley et al, (2009) suggested algorithms help in diagnostics (Appendix 2). As a small number of cases were analyses, it appears that there is not sufficient evidence to develop an algorithm; however both the studies have been adapted in Europe as they were seen to prevent patients with detectable antibodies being missed and to avoid the unnecessary testing and time of laboratory staff.
Figure 1.5: Algorithms for the diagnosis of SLE, (Wieser et al, 2001)
Slide processors are available to prepare IIF slides. They first appeared in the late 1990s and include platforms such as ASP1200 and AFT from Binding Site (Figure 1.6). These slide processors ensure that all samples are prepared quickly, reliably and accurately, avoiding cross reactivity in sample preparation.
Figure 1.6: The ASP1200 automated substrate slide processor used
in the Immunology Department at LGI.
Slide processors perform IIF via indirect antibody reactions as seen in Figure 1.7. Patient serum is incubated with a substrate, followed by washing to remove any unbound protein. A second antibody, FITC is added and this reacts with immunoglobulins which have combined with the substrate. Another washing stage is performed and slides are ready to be mounted and interpreted manually, however this causes subjectiveness.
Figure 1.7: Shows IIF on human tissue (Hughes et al, 2008)
IIF-ANA result interpretation is dependent on the operator's setup of the microscope, type and number of hours the bulb (mercury) has been used, type of objective lens, filters and most importantly magnification. At the LGI the Leica DMRB mercury microscope is employed and allows cells to magnify at X200, X400 and X500. Positive results fluoresce an apple-green colour (Table 3), whilst negative samples have little fluorescence. Two independent observers interpret the slides to prevent reading errors and any conflicting results are followed by an anti-ENA and anti-DNA screen.
Automated commercial slide readers are now available to allow interpretation of ANAs. Images are automatically scanned and stored within computer systems, where positive and negative ANA results are determined by the amount of flourenscene emitted. The operator can then scan through positive ANAs, identifying their patterns. This aims to improve the subjectiveness seen between scientists and aims to improve accuracy; however these are not robust so not widely used.
The advantage of IIF-ANA is that it is easy, inexpensive, available from a wide range of commercial companies, sensitive, reliable and has reduced cross reactivity and background fluorescence. The disadvantages of IIF-ANA are that it is laborious and requires a high degree of technical expertise. Within most Immunology laboratories the ANA test is not linked to the pathology computer systems, so tests cannot be picked up via an interface. This can be problematic as wrong samples can be analysed and reported. The use of barcode readers can overcome this problem.
Dull cells seen
Homogenous Pattern is the most common pattern seen in 60% of Systemic Lupus Erythematosus (SLE) patients. However it can be seen in drug induced lupus, Rheumatoid Arthritis.
Positive patients are then further evaluated against: Anti-dsDNA, Anti-Smith
Speckled Pattern can exist as coarse expressing is Sm, U1-RNP antigen or fine expressing Ro or La.
Sm positive is seen in 4-40% of SLE patients, whilst RNP is seen in high titres in patients with Mixed Connective Tissue Disease (MCTD). Patients with Scleroderma and Sjogren's Syndrome also present with positive results.
Centromere pattern is seen in 57-82% of patients with CREST syndrome and Raynauds.
The suspected antigen is CENP A, CENP B, CENP C.
Nucleolar Pattern seen in patients with Scleroderma. There are multiple nuclear antigens, such as fibrilliarin. Positive patients are then further tested against Scl-70 (Anti-Topoisomerase I).
Table 3: Shows the various ANA patterns seen by IIF on the HEp-2000 substrate
(Produced by Nisha Lad, 2010)
As different laboratories use different substrates and conjugates, IIF-ANA lacks standardisation worldwide (Bonilla, 2009). A study by Blerk et al, (2008) showed that if laboratories employed the same cells, substrate and conjugate they were able to report the same staining patterns. Over 157 laboratories across Belgium participated and each looked at 9 different samples. Looking at the results it is clear that after considering the variable factors, participants that employed the same HEp-2 slide substrates (Medica, USA) and method of detection were able to produce consistant results, suggesting standardization can be achieved.
Although IIF-ANA is subjective, replacement with EIA or bead technology is suggested to increase sensitivity. Bonilla et al (2007) performed a study in the USA suggesting that IIF had a sensitivity of 90.6%, whilst bead technology had a sensitivity of 41.9% and the specificity of IIF was lower at 76%; however for bead technology was 87%. Having tested 385 patients a conclusion was made saying IIF was a better technique for diagnosis of patients with SLE. Olaussen and Rekvig (1999) also produced similar results, where two commercial IIF assays and two commercial ELISA kits consisting of a range of antigens, significant in the diagnosis of SLE were used. The study showed correlation between IIF and ELISA, where sensitivity for IIF was 88%, whilst that for ELISA was 86%. Specificity however varied with 67% for IIF and 60% for ELISA. Another study by Gonzalez et al, (2002), analysed 709 samples comparing IIF and EIA for the diagnosis of ANA. Results showed good reproducibility in both assays, but found that the antibodies which produced a homogenous and speckled IIF patterns were best detected via EIA. On the other hand a study by Nifli et al, (2006) compared routine technology in a selection of Clinical Immunology laboratories and analyzed 11088 samples, using IIF and ELISA at the University Hospital of Heraklion in Greece. Results showed a highly significant correlation for ANA performed by ELISA; however it suggested that as IIF had a low sensitivity of 58%, this could be replaced by multiplex technology, allowing multiple antigen measurement. Looking at these studies closely it appears that although there were similarities between technologies, different kits and manufacturers were used, producing variable results.
1.4.2 Antigen-specific assays for the detection of ANA
Many different patterns can be seen by IIF-ANA, however to determine autoantibody specificity further antigen-specific assays are needed. Antibodies against Sm, native dsDNA and chromatin are used in the diagnosis of patients with SLE (Hanley et al, 2009). Currently ANAs are categorised into two main groups; ANA to DNA and histones (dsDNA) and ANA to extractable nuclear antigens (ENA). Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA) are now available for antigen specific testing, providing a new horizon for SLE testing, as they are able to identify individual antigens. ELISA/EIA is the most commonly performed technique, implemented in laboratories today. In the past, ELISA plates were assembled in-house, however as a successful assay requires careful assembly of the different layers, this soon became difficult to achieve, thus commercial ELISA kits were developed in the 1980s to overcome assay failure and to overcome the subjectiveness of IIF-ANA.
The ELISA assay can be performed either manually or via automated technologies. 96 well plates coated with the same antigens are commonly used, however Phadia produce an EIA platform, whereby pens containing singles wells with individual antigens can be used, allowing multiple antigen recognition and analysis. Both ELISA/EIA operate via immunometric methods of detection for anti-ENAs and anti-DNAs. The principle (Figure 1.8) of this technique is via microplates which are coated with purified antigens of interest. Patient serum is incubated in the wells and unbound antibody is then washed away, followed by the addition of a conjugate such as alkaline phosphotase (AP) or horseradish peroxidase (HRP). Another wash stage is performed and colorimetric results develop, which are proportional to the initial concentration of antibody in the patient's sample. Results are dependant on kit standards, which produce a calibration curve and then the optical density of the wells is taken to give a quantitative result (Branda et al, 2009).
Figure 1.8: Shows the principle of ELISA
(Obtained Piercenet Proteins, URL: http://www.piercenet.com/media/ELISAFormats575x214.jpg)
ELISA are a versatile assay, where the amplification of the signal, increases the overall sensitivity of the assay, as it uses an antibody which are specific to the type of antigen/protein being measured. Studies suggest that ELISA is a sensitive assay, however lacks specificity so false positives results are detected (Castro and Gourley, 2009). The advantage of ELISA is that it can be performed both manually and via automation. Analysers can also be linked to the pathology computer systems, preventing transcription errors in result interpretation. However disadvantages for ELISA are that purified antigens need to be prepared via HPLC, meaning assays are not cost effective and can be time-consuming. As microtitre plates are now purchased with one antigen, there is a limited dynamic range of detection; however EIA pens now overcome this problem. To produce successful assays, instrumental conditions need to be carefully considered. Washing errors, contamination of substrate or inadequate incubation times may produce little signal amplification resulting in false negative results (Castro and Gourley, 2010).
Anti-dsDNA were first described in 1957, by Ceppelini and colleagues. Anti-dsDNA are found in patients with SLE and are mainly found in the form of nucleosomes. Nucleosomes are fragments of chromatin that cells release during apoptosis. dsDNA antibodies bind to the nucleosome to form complexes which settle in the glomeruli, resulting in glomerulonephritis and increasing the risk of lupus nephritis flare, thus detection is crucial as it helps to determine the therapy required for treatment. ï¡-actinin (100kDA) is a microfilament skeletal muscle protein, which aids in maintaining the function of podocytes in the kidney. This protein is not specific for SLE, although it can act as a marker for renal involvement (Raheman et al, 2008).
The dsDNA assay can be performed via (Figure 1.9); IIF with Crithidia luciliae substrate (CLIF), Farr assay also known as radioimmunoassay (RIA), however the most commonly used technique is EIA/ELISA as described in 1.4.2.
Figure 1.9: Shows the different technologies used for dsDNA testing (Data obtained from UKNEQAS).
The Farr assay is regarded as the gold standard technique for the detection of dsDNA (Launey et al, 2010). It uses cultured cells labelled with thymidine and idocythidine, which act as radioactive DNA. In the assay bound and free DNA is separated by precipitating immuglobulins and ammonium sulphate. Although this method is good, it misses low avidity anti-DNA antibodies due to a nitrocellular filter, which allows the passage of free DNA and however double stranded DNA (dsDNA) cannot be filtered. Thus the radioactivity is said to be proportional to serum anti-DNA (Isenberg & Smeenk, 2002). The Farr assay can detect high affinity antibodies, with relatively high specificity; however it requires precision in pipetting as there must be sufficient labelled DNA to bind to samples in order to reach an endpoint.
Although the use of radiolabels within the Farr assay provides highly reproducible results, it becomes very costly, dangerous and difficult to dispose of the radioactive isotopes. Other limitations with this assay are that it only detects IgG and cannot determine any other immunoglobulin isotopes (IgA/IgM), thus patients presenting with dsDNA antibodies to IgA/IgM can be missed (Egner 2000). UK NEQAS shows that the Farr assay is still being used (Figure 1.9), as it is a more accurate confirmatory test that can be used in the diagnosis of SLE. The accuracy of the Farr assay can be seen in many studies. A study by Launey and colleagues (2010) compared the Farr radioimmunoassay to three commercial enzyme immuoassays and CLIF staining. The study looked at 99 patients with SLE and found that the Farr assay was the best assay, offering greater sensitivity and specificity of 95%, than the three other ELIA and CLIF assays. Derksen et al, (2002) also showed similar results. He compared the Farr assay with the Varelisa EIA assay and found that the Farr assay was superior to the EIA assay as it presented with a specificity of 95% and a sensitivity of 72%, whilst in EIA specificity corresponded to sensitivities at 44%.
Many laboratories also perform follow-up DNA tests by EIA, using CLIF to determine the avidity of anti-dsDNA antibodies. However CLIF can also be used alongside IIF to measure anti-DNA (IIF-DNA) and this does not requiring any specialist equipment, other than a fluorescence microscope. The CLIF assay allows detection of high affinity antibodies through titrations, however this requires precise pipetting. CLIF detects antibodies to kinetoplast of organisms, which consists of circular dsDNA and allows both IgG-anti-dsDNA and IgM-anti-dsDNA to be tested (Gonzalez-Buiterego & Gonzalez, 2006). The test is highly reproducible and is particularly suitable for a limited number of samples. Although the assay offers the highest specificity for ANA testing, it has a relatively low diagnostic sensitivity for SLE.
Due to the degree of accuracy of the Farr assay, it is undoubtedly the best assay for the detection of dsDNA and so has been approved by the World Health Organisation (WHO) and operates under the WHO80-IRP standard. However due to the risk of handling radioactive substance and the cost of the assay; this is not routinely used within Immunology.
Positive IIF-ANA are typically followed up by extractable nuclear antigens (ENA). ENAs were discovered in 1966 by Smith and colleagues, offering a greater specificity, to allow a more accurate disease diagnosis, in correlation to the initial IIF-ANA screen. Originally ENA's referred to proteins found in a saline extract of cell nuclei, however since then the components have been identified and these consist of cytoplasmic molecules. A whole spectrum of approximately 100 antigens can be screened; however most have no clinical significance. In order to cover the majority of inflammatory autoimmune diseases 6 clinically significant antigens (Table 4); Ro, La, Sm, RNP, Scl-70 and Jo1 are used within most laboratories across the UK.
SLE, Sjogrens syndrome, neonatal lupus, neonatal heartblock
SLE, Sjogrens syndrome, neonatal lupus
SLE, Mixed connective tissue diseases
Progressive systemic sclerosis
Table 4: ENA screen and disease significance
It can be seen that SLE is associated with many of the antigens in the screen.
Although ENAs are commonly performed via EIA (Figure 1.10), other methods such as qualitative gel precipitation assays, passive haemagglutination, immunoblotting, counter current immunoelectrophoresis (CIE) and antigen microarray can also be used (Kumar et al, 2009).
Figure 1.10: Shows the different technologies used for ENA testing
(Data obtained from UKNEQAS).
Sceening of ENAs is expensive in comparison to IIF-ANA as it allows specific antigen detection, offering a greater sensitivity as approximately 90% of positive IIF-ANA produce negative results via EIA (Dahle et al, 2004). Gel precipitation assays such as double immunodiffusion (DID) and counter current immunoelectrophoresis (CIE) are still being used within laboratories; however these were discovered over 5 decades ago. CIE uses an electric current to accelerate the migration of antibody and antigen through a buffered gel diffusion medium, where the antibody in the medium is less negatively charged and will migrate in an opposite or 'counter' direction toward the cathode. If the antigen and antibody are specific for each other, they form immunocomplexes resulting in a distinct precipitin line (Bossuyt & Luyckx, 2005). The sensitivity and the specificity of precipitation assays vary depending on the matrix used and this is dependent on the commercial kit employed by laboratories. The assay is able to screen out 70-80% of negative results (Kumar et al, 2009), however it can be seen from Figure 1.10, that CIE is now not widely used within the UK. As these assays do not require purified antigen, they prove to be inexpensive and require minimum equipment, thus is not labour intense. However it can present with drawbacks, as incubation times are prolonged and experienced staff are required to read the gels.
A variety of blotting techniques are still in use for the determination of ENAs. Western blots were first introduced in the 1980s; these used molecular weight markers to separate nuclear and cytoplasmic antigens. Western blots are sensitive for anti-ENA, however specificity is poor as the structure of epitopes can be destroyed, or appear to show non-linear bands making interpretation inaccurate. Dot blot and line blots are similar to western blot; however these are performed on nitrocellulose, where purified antigens are blotted on pre-located spots or strips, giving qualitative results. Although blotting is inexpensive it presents with drawbacks as RNP antigens can combine with Sm antigens. The ACR suggests that autoantibodies to ENA should not be used in isolation and it is recommended that a definite diagnosis of SLE, patients must present with a positive ANA or dsDNA.
1.4.3 Current technologies for the detection of ANA
Modern, innovated techniques are now available for the testing of ANAs. Multiplexing immunoassays (MIA) are the most recently introduced technologies within Immunology laboratories, however descriptions of MIA; have been found in literature as far back as 1977 (Elshal & McCoy, 2006). MIA can be used for multiple antigen detection. This technique is built around fluorescent dyed microspheres (beads) which are coated with individual synthetic nuclear antigens. A cocktail of antigens can be used and incubated with patient serum in a single tube. If ANA are present they bind antigen on the beads. Washing then takes place to remove any unbound antibody and a fluorochrome conjugate usually an anti-human IgG antibody is then added (Bonilla, 2009; Tozzoli, 2007). Flow cytometry is then used to detect the fluorescent signals produced by the beads. Two lasers are used; one laser excites the molecular tags so fluorescence intensity can be reported and the second laser excites the microsphere to identify the antigen specificity of the antibody (Castro & Gourley, 2009). The amount of fluorescence that is emitted is proportional to the amount of analyte captured in the immunoassay, allowing quantification of each analyte in the sample (Bonilla, 2009).
MIA-ANA is seen to be more efficient and technically less challenging than IIF-ANA. They aim to remove the subjectiveness and decrease the number of false positives seen by ELISA. As MIA allows multiple antigen analysis, flexibility is created and improved turnaround times are seen. Multiple antigens analysis also minimises sample volumes, thus patients who are not easy to bleed can also be tested for multiple antigens. This method is cost effective and provides quantitive results (Elshal &McCoy, 2006). MIA beads have the potential to become lodged within equipment resulting in technical problems within instrumentation, resulting in increased downtime (Elshal & McCoy, 2006).
Various studies have been performed to understand whether MIA is suitable for the testing of ANAs. A study performed by The College of American Pathologist's Proficiency Test (CAP PT), in 2006 suggests that MIA is a less sensitive assay to IIF-ANA and ELISA. The study showed higher false negative results, especially in detecting the nucleolar antigen. However Elshal and McCoy, (2006) found that ELISA was accepted as a gold standard technique, however good correlation to multiplex bead assays was seen, but a poor concurrence of qualitative values were achieved. A current study performed by Hanley et al, (2010) determined the ability of MIA to measure multiple autoantibodies and also monitor disease activity in patients with SLE. The study looked at 192, predominantly (87%) female, of Caucasian origin with mean disease duration of 8.8 years. The autoantibodies were measured using the Bioplex 2200 system and 3 commercial kits; Bio-Rad, INOVA diagnostics and Mikrogen were used, each consisting of multiple antigens such as dsDNA, chromatin, Ro60, Ro52, La, Sm, RNP, centromere and Jo-1. The overall study indicating that laboratories can produce similar results when using different MIA kits, however there were slight variations in sensitivity for some autoantibodies.
Although flow cytometry is incorporated within MIA, it can also be used on its own to diagnose patients with SLE. Flow cytometry is fully automated and cost effective, however as it operates via hydrodynamic focusing it produces single results for each analysis. This increases sensitivity, however results cannot be confirmed as a single reading are taken (Castro & Gourley, 2009).
At present, the use of Microarrays is not widely used however it can be used to detect ENA specific antigens. They allow simultaneous analysis of thousands of molecular parameters (Gonzalez-Buitrogo & Gonzalez, 2006). Chip synthesis strategies which contact printing or inkjet are used on planar and non-planar microarrays to identify different subsets of autoimmune disease (Robinson et al, 2002). Planar arrays are incorporated into microspots (within slides), microplates or nitrocellulose membrane. Whilst non-planar arrays use microparticles or bar-coded microbeads and are recognised by laser nephelometry or fluorimetry in the flow cytometer (Tozzoli, 2007). Microarrays are a semi-automated technique as washing is performed manually. Patient sera is added to the chips, followed by addition of horseradish peroxidise conjugated, secondary antibody and chemiluminescent substrate. Light signals are then captured by a camera based chip reader; which quantifies the results, based upon a calibration curve. Microarrays offer the ability to undergo serial dilution of various antigens on a single chip, giving an accurate titre level (Gonzalez-Buitrogo and Gonzalez, 2006). Microarrays offer consistent performance, cost effectiveness and precise measurement of antibody levels, producing a quantitative and highly sensitive and specific result (Plebani et al, 2009). Microarrays are still evolving as it is a recent introduced technique. The technique is highly costly, time consuming, requiring skilled staff and is currently not widely available.
1.5 Other factors that influenced ANA reporting
1.5.1 Quality assurance schemes in ANA testing
As patients are dependent on laboratory results for treatment, it is important to address quality control (QC). All laboratories need to participate in internal and external quality assurance schemes, in order to obtain precise and accurate results. The World Health Organisation (WHO) suggests in-house controls must be comparable to their reference sera (International Reference Preparation 66/233) (Kavanaugh et al, 2000). Internal QCs are in place to show precision and reproducibility and measure equipment, operator performance, lot variation and kit stability and can aid in troubleshooting. Internal QC's are produced from a pool of positive ANA samples and are aliquoted and frozen, until they are required. The disadvantage of in-house produced controls is that they are not 'CE' marked, thus do not comply with the essential requirements of the relevant European health, safety and environmental protection legislation. Manufacturer controls are also available and are run alongside internal QC's; however these are costly. In-house positive or negative controls are easily produced high throughput laboratories such as the LGI, thus prove inexpensive. Clinical Pathology Accreditation (CPA) UK, requires all laboratories to participate in external quality assurance programmes. United Kingdom National External Quality Assessment Service (UKNEQAS) is an external quality (EQA) scheme used in ANA screening to help assess the laboratories performance. EQA aims to highlight problematic areas such as lot variations and instrumental errors that need to be re-assessed to improve the services provided by laboratories.
1.5.2 The future of pathology testing and its impact on patients with SLE
Pathology is now evolving and UK health departments are currently developing proposals to redesign and redefine the careers of healthcare scientists. Modernising scientific careers (MSC) aims to provide a framework to scientist enabling them to undertake further education and training in order to progress through healthcare. This new plan means that staff may soon be trained in multi-discipline laboratories and the implications of this mean that Immunological tests such as ANA, for the screening of SLE could soon be employed by other departments such as Biochemistry or Haematology, thus will no longer be known as a specialised test.
ANA testing has evolved from LE cells substrate to new commercial multi-block substrates. IIF microscopy offers the possibilities to screen a broad range of antigens, including nucleosomal or chromatin-associated, nucleoplasmic and nucleor antigens. It has a high negative predictive value, where most patients screened have a negative result, thus don't have SLE and the positive predictive value is low as most symptomatic patients with a positive result will have the disease. Currently there is no standardisation between assays for the diagnosis of SLE. The American Pathologist survey, 2006 suggests that differences in reporting ANA patterns exists due to commercial kit variations, microscope magnification, differences in calibration and variations in reference ranges which are affected by the local population (Egner, 2000; Peterson et al, 2009).
After considering the screening techniques on offer for ANA testing I believe that anti-dsDNA assays need to be reviewed as they are crucial in the diagnosis and monitoring of patients with SLE. Laboratory investigation of SLE should always be carried out in order to benefit patients, ensuring a precise and accurate result is obtained. Assays are continuously being evaluated within laboratories to improve diagnosis, sensitivity and specificity, reducing labour time and cost.
Multiplex bead is becoming the future of ANA testing, as multiple antigens can be analysed in a single well (Dahle et al, 2004; Bonilla, 2009). Other technologies such flow cytometry and microchips are still evolving and may soon also be employed by more laboratories across the world, thus the diagnosis of SLE continues to develop.
I believe that IIF-ANA should still remain the gold standard technique and dsDNA and ENA screens should be performed alongside ANAs to determine the diagnosis of SLE, until the criteria for SLE is reviewed. As the clinical knowledge of SLE increases, more clinicians are requesting ANA results and improvements in service, turnaround times are required to meet demands. Technologies need to be kept up to date to optimise staff levels and as MSC takes effect soon more laboratories will explore new technologies.
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