In the recent past, autoantibody testing for systemic rheumatic diseases has been revolutionized[9]. The integrated approach of ANA patterns and extended panels of anti-ENA antibodies have both reduced the time-lapse in diagnosing the disease and misdiagnosis[10]. Moreover, the Interpretation and reporting of ANA have been standardized to reduce subjectivity[11]. However, testing, reporting, and interpretation of these autoantibodies still pose challenges due to various factors. The testing methodologies and platforms, the expertise of Immunologists and/ or laboratory scientists, and fluorescent microscopes variability, pre-test probability of AID are a few of the variables determining standards of autoantibody reporting[12, 13]. A very important factor is clinicians’ understanding and acceptance of the revised reporting format of these autoantibodies. As they are familiar and more comfortable with the ANA test results and interpretation, introducing the concept of anti-cellular antibodies (ACA) with the importance of anti-cytoplasmic antibodies and anti-mitotic antibodies will require their continuous education[10, 13].
There are certain ANA patterns reported to be specifically associated with anti-ENA. Such as for the nucleolar pattern, anti-ENA usually detected are fibrillarin, PM/Scl, RNA polymerase I, etc. [6, 11, 12, 14]. On the other hand, González DA, et al, have reported a range of nuclear patterns in sera with anti SSA, anti-SSB, and Ro 52 positivity[15]. We correlated anti-ENA antibodies with ANA patterns in our cohort in order to specify follow-up anti-ENA test cost effectively. We found speckled or homogenous ANA patterns were the most common in our anti-ENA positive cohort as previously reported [7]. But interestingly, few anti-ENAs were also seen with patterns like mitotic spindle, mid-body or Numa-like. The autoantibodies detected in these sera were mostly anti-SSA, anti-Ro-52, anti-SSB (table 1). As these anti-ENAs do not correlate with these patterns, it may be that they correlated with negative ANA or cytoplasmic reactivity in these samples. Rodríguez-Orozco, A.R et al[16]. have reported around 3.4% of anti-ENA positivity in patients’ samples negative for ANA. With lower titers used for dilution, they were able to detect cytoplasmic staining in these samples. We have also shown the presence of certain anti-ENAs in ANA negative sera (Fig. 1). Interestingly certain autoantibodies such as anti-ribosomal P proteins (Rib-p-prt) and anti-Jo-1 that are associated with cytoplasmic reactivity, were detected in ANA positive samples of our patients’ cohort. This is also consistent with our earlier report on anti-Rib-P-Prt showing 10.5% positivity in a cohort of ANA positive patients[17]. This shows that we cannot comfortably limit the detection of anti-ENA according to the ANA or cellular pattern observed in our patients.
According to the international consensus on ANA patterns (ICAP), it is advised to change terminology in ANA reporting to ACA on Hep-2 cells. Furthermore, the reports should mention results of nuclear, mitotic and cytoplasmic patterns [18]. In a Korean study, Baek HM et al. mostly found anti-mitochondrial antibodies and anti-Rib-P Prt in patients’ sera showing cytoplasmic staining[19]. We had reported cytoplasmic staining in a very small number of patients. As we have taken out the data form our electronic medical record system, this small number may be due to the inclusion of ANA results reported earlier before our updating of reporting format. A future analysis on a larger sample size with cytoplasmic staining pattern will enable us to look anti-ENA correlation with greater sensitivity and specificity. Also in this small number of samples with cytoplasmic staining, 90% of samples were also positive for ANA.
According to the new algorithm of autoantibody testing for AIDs as per ICAP nomenclature, ANA or ACA should be used as a screening test followed by anti-ENA or anti-ds DNA[11]. In this study we found that ANA was not requested for all patients for whom anti-ENA was done. One possible reason could be ANA was done in some other laboratory but the information was missing in the medical records of these patients. Nevertheless, there is a need to do a local audit and to educate our physicians regarding proper utilization of autoantibody testing for patients with suspicion of AID.
Another important factor is that the correct interpretation of these antibodies needs to be done in accordance with the clinical features. As we already know that most of the features of AIDs are non-specific and overlapping, this makes diagnosis tricky, and initially, patients are labeled for several AIDs until proven otherwise[4, 20]. SLE is the prototype of AID. There is a plethora of autoantibodies that can be found in SLE at varying sensitivity and specificity[21, 22]. Certain autoantibodies such as anti-SSA, anti-SSB, and anti-RNP can be found in more than one AID[23]. In this cohort, we found positivity of almost all anti-ENAs in SLE but limited autoantibodies’ positivity in the rest of the systemic AIDs. Presence of multiple patterns and multiple autoantibodies in some patients may be due to the presence of overlap syndromes. Nevertheless, investigating according to the suggested algorithm[11, 24] may help to narrow down the final diagnosis and to determine targeted therapy and appropriate prognosis accordingly[23, 25].
In our cohort there were 79% patients also had anti-ds-DNA along with anti-ENA. It can be argued that once a diagnosis is made due to the presence of an autoantibody then it is unnecessary to go for extended autoantibody panel of anti-ENA. However, it is a known fact that certain autoantibodies are associated with a more specific symptom. Such as anti-SSA and anti-SSB are associated with congenital heart block and anti-Rib-P-Prt are associated with neuropsychiatric disorders[23]. Also, as more than one AID can co-exist[26], therefore, anti-ENA testing is required even in the presence of a positive anti-ds-DNA antibodies.