Autoantibody detection plays an important role in the clinical management of patients with connective tissue disease. Detection of Anti-nuclear antibody (ANA) is a common screening test ordered during initial workup. Though ANA testing is simple, different laboratories vary in their test methodology and reporting format, and that could be confusing. In this review, a general introduction on the common diagnostic assays employed in ANA test, along with issues related to the interpretation would be discussed. In addition, an update on anti-DSF70 antibodies and its associated clinical significance will be discussed.
2 ANA assays methodology
Traditionally, ANA was detected by indirect immunofluorescence (IIFA). It was first described in 1950 by Coons and Kaplan. The method consists of initial screening, followed by serial dilutions of positive sera and visual determination of ANA staining patterns on HEp-2 slides. IIFA detects antibody directing against the reagent cell nucleus forming distinct fluorescence patterns. Different ANA patterns on HEp-2 cells are associated with different autoantibodies. This technique is well established. It is the most sensitive method for ANA detection and it essentially detects all autoantibodies against the cellular constituents, which varies in clinical significance. However, it is labor intensive and the result interpretation could be subjective.
With an increasing test load, nowadays, there is a trend for service laboratories to move from the traditional laborious method to quantitative automated high volume solid phase platforms, such as bead-based multiplex immunoassays, enzyme linked immunosorbent assay (ELISA), chemiluminescence immunoassays, and so on. Among these, ELISA is a common choice, and is gaining popularity over IIFA. There are two main types of ANA ELISA, namely a generic assay, which detects ANA of broad specificity, and the antigen specific assay, which detects ANA reacting with specific antigens such as, dsDNA, Ro, La, Scl-70, Sm, RNP/Sm, and so on. The source of antigens used in ANA ELISA can be very different. These antigens could be extracted from tissues such as calf and rabbit thymus, or purified recombinant antigens. The source of antigen used in coating the ELISA plate has significant implication on the test sensitivity and specificity, and hence the performance of the ELISA.
With increasing numbers of available methods, issues and problems related to the variable assays’ sensitivity, specificity and clinical interpretations arise. Our existing knowledge on the disease association of autoantibodies are based on the historical methods. Nevertheless, the performance of these newer platforms is different from the traditional assay. For example, ANA is known to be a sensitive test for SLE, with the traditional IIFA method, it is expected that over 95% of SLE patients will be tested positive. However, solid phase assays based on limited number of antigens mixture do not have comparable sensitivity. There has been reports and concerns on cases of lupus patients being delayed in diagnosis because of false negative ANA results by non-IIFA methods. In 2007, the American College of Rheumatology (ACR) created an ANA Task Force in view of these issues.[2,3] After reviewing relevant literature, the task force has concluded that solid phase immunoassays are not substitute for IIFA for the detection of ANA, and that IIFA remains the gold standard in ANA testing.
3 Inconsistencies and limitations of IIFA
While IIF is the preferred method for ANA screening as recommended by the ACR and the European autoimmunity standardization initiative (EASI), it entails substantial technical expertise and the assays’ performance is highly dependent on the laboratory expertise and experience. In reality, inconsistency of IIFA among different laboratories is not uncommon. There are several reasons to account for this. Firstly, slides from different vendors vary in sensitivity, especially in anti-SSA/Ro detection. This may be due to the fixatives used, which may cause variability in cell and nuclear morphology as well as effect on antigen preservation. Other factors leading to variability include growth time of the HEp-2 culture, pH of the reagents, and so on. Moreover, reading and interpreting the slides are observer dependent, which is skill dependent and subjective.
Variation in the reporting format among different laboratories is another source of inconsistencies. Laboratories vary in terms of screening dilution they perform their ANA tests with. The common screening dilution varies from 1/40 to 1/160 in different laboratories. And all samples with results lower than screening dilution will be reported as negative. Hence, the assay sensitivity and specificity varies depending on the screening dilution performed. For example, a patient will be reported as ANA positive with a titer 1/80 in one laboratory but being ANA negative in another laboratory which starts at a higher screening dilution (e.g., 1/160). Moreover, there is disagreement on what constitutes a positive ANA. Some laboratories consider ANA positive only in the presence of nuclear staining, whereas cytoplasmic staining without nuclear staining as negative, while these may be considered ANA positive in other laboratories. These inconsistencies could only be minimized by the standardization of ANA testing and reporting among different laboratories, though this is not an easy task.
4 Issues related to ANA results interpretation
Though there are a number of limitations of the ANA test, careful review and interpretation of the test does provide useful information for patients’ workup. Although ANA by IIFA is a sensitive screening test for the workup of connective tissue disease, it is not specific. A positive ANA at 1:80 dilution has been reported in up to 13.3% of healthy subjects in the literature.[6,7,8,9,10,11] In general, the ANA titers and patterns do provide some additional information on the significance of a positive test result.
It is generally believed that individuals without autoimmune disease would present a lower titer compared to those with autoimmune disease.[12,13] In one study, it was reported that up to 31.7% of healthy controls have positive ANA at 1:40, 13.3% at 1:80 and 5% at 1:160. Therefore, high-titer results in general are clinically more significant than low titer results. Nevertheless, the titer of ANA has no bearings on the disease activity.
Other than titer, ANA pattern also provides useful information to discriminate between patients with autoimmune rheumatic conditions from those who do not. IIFA pattern reflects the topographic distribution of the targeted autoantigens and bear association with certain autoantibodies specificities.[14,15,16,17,18,19,20] In one study on ANA patterns in healthy subjects versus patients with ANA related autoimmune rheumatic disease conditions, nuclear fine speckled (45.8%) and nuclear dense fine speckled (DFS) (33.1%) were the two most commonly encountered patterns in healthy subjects. In contrast, fine speckled (42%) and coarse speckled (26.1%) were the two most common patterns in patients. It’s interesting that certain ANA patterns are exclusively found in the patients’ ANA test results, including coarse speckled, nuclear homogeneous and centromere patterns. Moreover, none of these patients had positive ANA with DFS, which is relatively prevalent among the healthy subjects. Though ANA titers were in general low to moderate in the healthy subjects, in this study, it was found that high titer at or above 1:640 was not infrequent among the DFS positive samples (61.5%). Hence, identifying DFS pattern is important.
5 Update on Anti-DSF70 antibodies
In recent years, DFS pattern is one of the hot topics in ANA. This pattern is included in the list of “competent level” patterns by the International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) (http://www.anapatterns.org/). External quality assurance program by some professional bodies also include this pattern as part of the ANA competency assessment. DFS pattern is associated with antibodies to a 75 kDa protein. The target antigen was also termed lens epithelium-derived growth factor (LEDGF). The pattern is characterized by uniformly distributed granular staining throughout the interphase nucleus and dense fine speckled staining localized on metaphase chromosomes plate (Figure 1). The DFS 70 antibody was first described in 1990s during surveys of ANA in patients with interstitial cystitis and chronic fatigue syndrome.[19,21] Subsequently, it was also reported in a variety of other conditions, including atopic dermatitis, prostate cancer, autoimmune thyroiditis and even healthy subjects.[19,20,22,23]
Indeed, anti-DFS70 is not infrequent in ANA positive healthy subjects. Watanabe et al.  reported in their study on healthy hospital workers that 11% of their cohort were positive for anti-DSF70 (54% of all ANA positive cases). Mahler et al.  also reported a prevalence of 8.9% of anti-DFS70 in a cohort of 124 healthy subject samples. Though anti-DFS70 are relatively common findings among ANA positive individuals with no evidence of autoimmune rheumatic disease conditions, it is not common (2–4%) among patients with ANA related autoimmune rheumatic condition, despite a high prevalence of positive ANA in these subjects. From the reported literature, among those infrequent occurrence of anti-DSF 70 antibodies in the context of ANA related autoimmune rheumatic disease, majority of these cases also had at least one other related autoantibodies, such as anti-ENA, anti-dsDNA, and so on.[12,24,25,26,27] Hence, patients with ANA related autoimmune rheumatic disease like lupus, producing anti-DSF 70 antibodies as the only serum ANA IIFA pattern, without the presence of other autoimmune markers are rare, that these findings help to exclude the diagnosis.
Lately, there is an interesting study on the cost effectiveness showing potential cost saving and decreased outpatient clinic visits by including anti-DSF70 in the ANA workup algorithm. In this study, they evaluate 181 patient samples taken from their Autoimmune Serum Collection, along with their follow-up medical records and history. These patients were suspected of having ANA associated autoimmune rheumatic disease and were positive for ANA but negative for anti-ENA. The oldest available serum for each patient was used for the study. The follow-up time of these cases was up to 10 years (mean 4.75 years). The final diagnoses in this cohort included SLE (n = 44), Sjogren’s syndrome (n = 23), systemic sclerosis (n = 18) and non-ANA related autoimmune rheumatic disease (n = 96). The cost effectiveness of routine practice versus new algorithms including anti-DSF70 were compared. In the conventional algorithm, repetition of entire antibody panel and specialist clinic referrals were the usual norm. In the new algorithm, patients who were ANA positive but negative for anti-ds DNA and anti-ENA were considered as potential non-autoimmune rheumatic disease cases if their anti-DSF70 were positive, and inconclusive case that required annual follow-up if their anti-DSF70 were negative. In this cohort, none of those patients, who were assigned as potential non-autoimmune rheumatic disease under the new algorithm, developed ANA associated autoimmune rheumatic disease during the follow-up period of the study. With the new algorithm, the visits for outpatient clinic decreased by 70% compared to the conventional practice. The finding in this study is encouraging, though prospective study in this area is still awaited.
Accurately identifying anti-DFS70 is important. Nevertheless, identifying the DFS IIFA pattern in HEp-2 screening is not an easy task.[12,24,29,30,31] The pattern could be mixed up with other mixed ANA patterns, and antibodies other than anti-DFS 70 may also be presented as DFS pattern seen in IIFA. Discriminating among these various ANA patterns is challenging. In addition, HEp-2 slides from different commercial sources gave inconsistent results, probably related to different fixation methods or cell growth conditions. Therefore, the detection of anti-DFS70 should not be relied exclusively on IIFA pattern, but supported by analyte specific immunoassays, such as immunoblot, immunoprecipitation, ELISA and chemiluminescence.[24,32,33,34]
ANA is a commonly employed first line assay in a number of settings, in particular for the workup for patients with suspected autoimmune rheumatic disease conditions. With the changes in the field of autoimmune diagnostics and lack of standardization in practice by different laboratories, the interpretation of results could be complicated. Better understanding of the assay interpretation facilitates patient management, while minimizing unnecessary investigations and anxiety.
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