Can Antinuclear Antibodies (ANA) be Monoclonal?

Background Nuclear staining by immunofluorescence in a kidney biopsy is often seen in patients with positive antinuclear antibodies (ANA) in the serum. These ANA are usually polyclonal, but herein we report 9 cases with an unusual finding of monoclonal nuclear staining by immunofluorescence on kidney biopsy. Case Presentation. Nine cases with predominant stain for kappa or lambda light chain were identified by searching the renal pathology laboratory database for the past 10 years. All cases had positive stain for only kappa (six cases) or lambda (three cases) light chain in the nuclei. Eight out of nine cases had positive nuclear IgG stain, and one case had positive nuclear IgA stain. Among cases with positive nuclear IgG staining, six cases were positive for IgG1 subclass, one case was positive for IgG2 subclass, and one case was positive for IgG3 subclass. All patients with positive IgG nuclear stain, who had testing for ANA, had positive ANA. Patients with positive IgG1 subclass did not have monoclonal protein in the serum or urine, but the patient with positive IgG2 subclass and lambda light chain stain in the nuclei had IgG lambda monoclonal gammopathy. Conclusions We identified a new unique pattern of nuclear stain by immunofluorescence in kidney biopsies that suggests the presence of monoclonal ANA. Workup for underlying monoclonal gammopathy is warranted in such patients.


Introduction
Antinuclear antibodies (ANA) are autoantibodies that are often present in patients with autoimmune diseases, such as systemic lupus erythematous (SLE), Sjogren's syndrome, scleroderma, mixed connective tissue disease, polymyositis, and dermatomyositis. e first method to identify ANA is considered one of the milestones in the history of clinical immunology over the last 60 years [1]. ANA is a screening test to detect autoimmune antibodies, and, if positive, other tests to detect specific markers such as anti-dsDNA antibodies or antiextractable nuclear antigens (anti-ENA) antibodies (such as SS-A, SS-B, Sm, Sm/ RNP, Jo-1, and Scl-70) are performed [1]. e traditional methods for detecting ANA are indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). Renal pathologists use immunofluorescence to detect deposition of different immunoglobulins and other proteins in the kidney. If in a kidney biopsy there is a positive nuclear stain for an immunoglobulin, usually IgG, by immunofluorescence, this indicates the presence of ANA in the patient. In addition to positive IgG, positive nuclear stain is usually seen for both kappa and lambda light chains [2]. Herein, we report nine cases of positive nuclear stain by immunofluorescence in kidney biopsies that show the presence of monoclonal ANA. positive nuclear staining for only kappa or lambda light chain were selected for studies. Direct immunofluorescence with antibodies to IgG subclasses was performed in cases with positive nuclear staining for IgG (eight cases). Clinical history and laboratory data were analyzed for the presence of a monoclonal protein in the serum and urine.
Demographic and laboratory data for patients are present in Table 1. ere were 6 females and 3 males, and all patients were Caucasian. e mean age was 58 ± 24 years (range 22-84 years). ree out of 9 patients had acute kidney injury at the time of the kidney biopsy. ree patients had nephrotic range proteinuria and two had severe hematuria. Seven patients had positive ANA in the serum, one case had negative ANA in the serum, and one case did not have ANA data available. Monoclonal gammopathy workup was performed in 5 of the 9 patients. Two of these 5 patients(cases # 1 and 7) did not have monoclonal protein in the serum and/or urine. ree patients had abnormalities detected on protein electrophoresis or immunofixation (Table 1). Bone marrow biopsies were performed in two out of these three patients and both were negative for multiple myeloma.
Immunofluorescence findings in kidney biopsies are present in Table 2. ere was positive nuclear stain for IgG in all of the cases except case #6, where IgA nuclear staining was seen (Figures 1(a)-1(c)). ere was predominant staining for one of the light chains in all 9 cases, with 6 (66.7%) cases having predominance of kappa light chain and 3 (33.3%) cases with predominant lambda light chain nuclear stain. Among the 8 cases with positive IgG nuclear stain, 6 had dominant IgG1 subclass stain, one case (#4) had dominant IgG2 subclass (Figures 2(d)-2(g)), and one case (#8) had dominant IgG3 subclass stain ( Table 2).
Main pathologic findings in the kidney biopsies are present in Table 3. Four patients had immune complexmediated glomerulonephritis (three cases of lupus nephritis and one with predominant C3-containing immune complex deposits), and five patients did not have immune complex depositions in the glomeruli.

Discussion and Conclusions
To the best of our knowledge, this is the first report describing the monoclonal pattern of staining of tissue ANA in kidney biopsies. ANA are an important diagnostic marker in the variety of autoimmune diseases that have predictive and prognostic values [3]. ANA are a heterogeneous group that include several different antibodies against specific components of the nucleus and nuclear envelope [1]. We identified 9 cases where there was strong predominance for kappa or lambda light chain stain in the nuclei by immunofluorescence. Nuclear stain in a kidney biopsy indicates presence of ANA in the patient. Indeed, 7 out of 9 patients in this study had positive ANA, and only one patient had negative ANA testing, but this patient had positive staining for IgA, not IgG, in the nuclei. is patient underwent testing by using the Euroimmun immunofluorescence assay (IFA) for ANA screening. is methodology uses goat anti-human IgG-conjugated antibodies and does not recognize IgA [4]. erefore, this patient may still have ANA in the serum which are not detectable by this testing method.
In 8 patients with positive IgG nuclear stain, 6 cases had predominant stain for IgG1 subclass. While IgG1 is being the predominant subclass in the circulation, these data should be interpreted with caution. However, the predominance of IgG1 should also not discount these data entirely. As it was illustrated in the case series that describe proliferative glomerulonephritis with monoclonal IgG deposits, 9 out of 32 patients also had predominance of IgG1 subclass, and in these patients, there was evidence of IgG1-containing immune complex deposits on a renal biopsy, indicating that this IgG1 was pathogenic [5]. Interestingly, in our cases and in those described by Nasr et al., monoclonal protein in the serum or urine usually was Intensity of the staining was quantitated by a semiquantitative scale, where 0 represents absent; 1+ represents mild; 2+ represents moderate, and 3+ represents prominent staining. BL, baseline; SCr, serum creatinine; ANA, antinuclear antibody; SPEP/UPEP, serum and urine protein electrophoresis.
not found in patients who had predominance of the IgG1 subclass [5]. Case #4 showed specificity for IgG2 subclass and lambda light chain in the nuclei, and this patient had monoclonal IgG lambda in the serum. is is notable because this is the only patient with the monoclonal paraprotein in the serum that corresponds to the kidney biopsy immunofluorescence findings.
Case #4 makes a compelling argument that monoclonal ANA stain can be caused by a paraprotein in a subset of patients. Based on these results, another subset of patients may have a detectable paraprotein that does not correspond to the ANA stain on immunofluorescence. e differential diagnosis includes monoclonal gammopathy with very low levels of monoclonal protein that is below the current methodology detection limits [6]. Dreher in 1977 described that in patients with rheumatoid arthritis, the majority of ANA are IgG ANA (43%), followed by IgM ANA (20%) with as little as 2% of IgA ANA [7]. Our data also indicate that this trend largely holds for monoclonal ANA as well. In our study, the majority are IgG ANA (8 out of 9 cases, 89%) with one case (11%) of IgA ANA. Several of our patients had diagnosis of SLE. In patients with lupus, it is not uncommon to see nuclear ANA stain by immunofluorescence, though it is nearly always polyclonal. e significance of the monoclonal ANA stain in these patients is unclear. SLE and other lupus-like inflammatory conditions [8,9] are associated with an increased malignancy risk, especially lymphomas. Although this monoclonal nuclear stain may not indicate a paraprotein, work up for neoplastic processes may be prudent in these patients event though polyclonal nuclear stain is relatively common.
In summary, we identified the unusual pattern of stain in the nuclei that suggests the presence of a monoclonal ANA. Even though only one patient had the corresponding monoclonal protein in the serum, several patients showed different monoclonal paraprotein on Data Availability e data and pathology images used to support the findings of this study are available from the corresponding author upon request that will not violate HIPA rules about privacy of patient information.

Consent
e consent for research and publications is a part of the kidney biopsy consent.
ere are only entirely anonymized images (pathology slides) from which the individual cannot be identified.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Case Reports in Nephrology 5