We correlated ANA-specific antibodies with ANA patterns in our cohort in order to specify follow-up autoantibody test cost effectively. We found speckled or homogenous ANA patterns were the most common in our ANA-specific antibodies-positive cohort as previously reported [7]. But interestingly, few ANA-specific antibodies 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. As these ANA-specific antibodies do not correlate with these patterns, it may be that they correlated with negative ANA or cytoplasmic reactivity in these samples.
In the recent past, autoantibody testing for systemic rheumatic diseases has been revolutionized[9]. The integrated approach of ANA patterns and extended panels of ANA-specific 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, fluorescent microscopes variability, and pre-test probability of SAID 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 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 ANA-specific-autoantibodies. Such as for the nucleolar pattern, ANA-specific antibodies 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]. Rodríguez-Orozco, A.R et al[16]. have reported around 3.4% of ANA-specific antibodies 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 ANA-specific-autoantibodies in ANA negative sera.
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 ANA-specific antibodies 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 with ANA-specific antibodies will enable us to look at ANA-specific antibodies correlation with greater confidence and significance. 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 SAID as per ICAP nomenclature, ANA or ACA should be used as a screening test followed by ANA-specific antibodies or anti-ds DNA[11]. In this study we found that ANA was not requested for all patients for whom ANA-specific antibodies 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 SAID.
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 SAID are non-specific and overlapping, this makes diagnosis tricky, and initially, patients are labeled for several SAID until proven otherwise[4, 20]. SLE is the prototype of SAID. 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 SAID[23]. In this cohort, we found positivity of almost all ANA-specific-autoantibodies in SLE but limited autoantibodies’ positivity in the rest of the systemic SAID. 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 62%% patients also had anti-ds-DNA along with ANA-specific-autoantibodies. 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 ANA-specific-autoantibodies. 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 SAID can co-exist[26], therefore, ANA-specific antibodies testing is required even in the presence of a positive anti-ds-DNA antibodies.