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BRIEF REPORT
Ahead of print publication  

Prevalence of autoantibodies to cellular cytoplasmic and mitotic antigens in routine antinuclear antibody reporting: Implementation of international consensus on antinuclear antibodies patterns guidelines


1 Department of Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Internal Medicine, Division of Clinical Immunology and Rheumatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission22-Jul-2020
Date of Acceptance27-Sep-2020

Correspondence Address:
Ranjana Walker Minz,
Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/injr.injr_198_20

  Abstract 


Background: The international consensus on antinuclear antibody (ANA) patterns (ICAP) guidelines recommends that non-nuclear patterns should be reported as positive for ANA. Therefore, a prospective study was planned to investigate the frequency of cytoplasmic and mitotic ANA patterns in routine reporting in a high-throughput lab in a tertiary referral center in Northern India.
Materials and Methods: Whole blood samples received were centrifuged and serum was separated within 4 hours of collection. Sera were subjected to indirect immunofluorescence (IIF) assay on HEp-2 cells for ANA determination in a dilution of 1:40, per standardized protocol in our laboratory. IIF staining patterns and intensity were evaluated and the results interpreted.
Results: Over a 3-month period, of the 3796 serum samples received for ANA testing, 81 (4.9%) samples were positive for cytoplasmic, 7 (0.4%) for mitotic, and 2 (0.1%) for DNA topoisomerase I (Topo I)-like pattern. The most frequent cytoplasmic fluorescence pattern was dense fine speckled (2%) and the most frequent mitotic pattern was NuMA-like (0.2%). The prevalence of total positive ANA in our study population was found out to be 43.6%.
Conclusions: Implementation of ICAP guidelines into routine ANA reporting helped us in detecting important cytoplasmic antibodies such as antimitochondrial antibodies, anti-ribosomal P protein antibodies, anti-Jo-1antibodies, anti-Topo I antibodies and allowed us to introduce more specific and confirmatory tests for the same. The new format for ANA reporting also encouraged clinician-laboratory crosstalk.

Keywords: Anti-cellular, antinuclear antibodies, cytoplasmic, international consensus on antinuclear antibodies patterns, mitotic



How to cite this URL:
Chhabra S, Kumar Y, Kumar M, Sharma A, Bhardwaj R, Minz RW. Prevalence of autoantibodies to cellular cytoplasmic and mitotic antigens in routine antinuclear antibody reporting: Implementation of international consensus on antinuclear antibodies patterns guidelines. Indian J Rheumatol [Epub ahead of print] [cited 2020 Dec 4]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=299896




  Introduction Top


The indirect immunofluorescence (IIF) assay on HEp-2 cells (IIFA HEp-2) has been recently reinforced as the gold standard for autoantibody screening in systemic autoimmune rheumatic disease (SARD).[1] In the International Consensus on Antinuclear Antibody (ANA) patterns (ICAP) consensus these patterns are divided into three major categories, namely nuclear (“true” ANA), cytoplasmic and mitotic patterns.[2] Therefore, the term anticellular antibodies (ACAs) has been suggested to encompass the ever-expanding spectrum of ACAs and to broaden the definition of classical ANAs to include cytoplasmic and mitotic antigens as well. A new international nomenclature for types of nuclear, cytoplasmic, and mitotic luminescence of ANA in IIF-HEp-2, including 29 variants of anticell (“Anti-cell” [AC]) patterns has been developed.[2]

While “true” ANA patterns have been studied extensively, the prevalence and significance of other cytoplasmic and mitotic patterns, specifically those directed against uncommon antigens are not as well-understood.

Most immunological laboratories report only common ANA patterns, ignoring other less important or rarer ANA patterns as their clinical relevance was previously unknown and more recently with concomitant rise of solid phase assays like immunoblots, this area is an evolving science. Moreover, some ANA patterns are considered to be reportable at a competent level (the simpler, basic patterns) while others are only reportable by an expert (advanced, difficult to recognize patterns) according to the ICAP classification, depending on the clinical relevance and ease of recognition.[2]

Due to the low frequency of the rare ANA patterns, much of our current knowledge regarding their specificity is based on case-series, with their inherent limitations: these datasets tend to overestimate the specificity due to selection and publication bias, for example.[3] Data on less prevalent ANA pattern are sparse, so this prospective cross-sectional study was planned to investigate the frequency of cytoplasmic and mitotic ANA patterns in North Indian population.


  Materials and Methods Top


The study was conducted over a 3-month period from October to December 2019 at the Department of Immunopathology of a tertiary medical center in Northern India. We only included the first sample for each patient to avoid overestimation of a particular pattern and to minimize the risk of bias; a clinical suspicion of SARD makes it more likely that such patients will be retested. All samples were tested by conventional IIF using HEp-2 cells (Innovalite, Cat no. 1053386) in a screening dilution of 1:40, per standardized protocol in our laboratory. Specific profiling for autoantibodies (anti-dsDNA, anti-Ro, anti-La, anti-ribosomalPprotein [RPP], anti-Jo-1, anti-myeloperoxidase antibody, anti-proteinase 3 was performed either using the EUROLINE ANA profile or an autoanalyzer (Phadia) by fluoroenzyme immunoassay. Antimitochondrial antibody profiling was performed by IIF using in-house composite sections and confirmed on ELISA (INNOVA LITE 048521) and liver blot (EURO IMMUNE, D200309AB).


  Results Top


Over a 3-month study period 2, 3796 consecutive samples submitted for ANA determination were studied for the presence of rare ANA patterns [Figure 1]. Of the 3796 sera tested, 1656 (43.6%) were positive for ANA, of which 90 (5.4%) samples showed cytoplasmic and mitotic fluorescence patterns. Eighty-one sera (4.9%) were positive for cytoplasmic fluorescence pattern, 7 (0.4%) showed mitotic and 2 (0.1%) showed DNA topoisomerase I (Topo I)-like pattern. The most frequent cytoplasmic pattern was dense fine speckled (2%), followed by fibrillar filamentous (FF) (0.8%), as shown in [Table 1]. Female outnumbered male patients (3:1). [Table 2] depicts the clinical associations and presence of additional antibodies wherever available. Cytoplasmic dense fine speckled was observed most commonly in systemic lupus erythematosus (SLE) (17 cases) whereas cytoplasmic reticular pattern was noted more commonly in primary biliary cholangitis (PBC).
Figure 1: Indirect immunofluorescence photomicrograph of HEp-2 slide (a) cytoplasmic discrete dots; (b) cytoplasmic fine speckled; (c) rods and rings; (d) cytoplasmic dense fine speckled; (e) mitotic-spindle fibers; (f) Mitotic-NuMA patterns (×400)

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Table 1: Frequency of cytoplasmic indirect immune-fluorescence and/or mitotic indirect immune-fluorescence in unselected routine antinuclear antibodies screening cohorts with groups

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Table 2: Clinical associations and additional antibodies seen in cytoplasmic and mitotic fluorescence patterns

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  Discussion Top


In a panel of 3796 ANA determinations, we found less frequently reported ANA patterns in 5.4% of positive ANA samples. The prevalence of positive ANA in our population was found out to be 43.6%. We noticed a considerably higher positive prevalence since this is a hospital-based study and our laboratory is a tertiary-care referral center with adult and pediatric rheumatology specialties catering to ANA requisitions from all other specialties and super specialties, where all kinds of patients suffering from SARD, viral infections, malignancies, autoimmune liver diseases, and inflammatory bowel disease get tested for ANAs. Chauhan et al. have also observed a positive prevalence of 39.7% at 1:80 dilutions.[4] Betancur et al. have also shown a positive ANA prevalence of 53% in their population-based samples which is almost double the reported positive ANA prevalence in other studies.[5]

Cytoplasmic-dense fine-speckled patterns reveal a dense, fine, granular, cloudy, cytoplasmic fluorescence staining on IIF using Hep-2 cells. This staining pattern is not specific for RPP, as also noted in our study, only 53% of sera showing the corresponding staining pattern were positive for RPP. Anti-ds-DNA antibodies were found in 47% of these sera and 50% cases were already diagnosed known cases of SLE [Table 2].

The next common pattern noted was cytoplasmic FF (AC-16). The importance of this finding is that this pattern is not typically associated with SARD.

The cytoplasmic reticular pattern was noted in 0.5% cases on routine ANA screening. In all these cases, detection of reticular pattern on HEp-2 screening finally led to diagnosis of PBC or PBC/AIH overlap again emphasizing the outstanding potential of HEp-2 screening. These diseases have been shown to be indolent but progressive in North Indian population and can be detected by IIFA HEp-2 and confirmed by ELISA/immunoblot.[6]

Golgi-like pattern is rarely found in routine ANA screening using HEp-2 cells with a reported frequency of 0.05%–0.2%. We also observed this pattern in 0.2% of our cases. If a typical Golgi-like pattern is observed, serological and clinical follow-up examination is indicated in order to detect the potential development of SARD, particularly Sjogren's syndrome (SjS).[7]

In routine screening using HEp-2 cells, a cytoplasmic discrete dot pattern (associated with moderate-to-high anti- Glycine and tryptophan bodies (GWB) titers) is found in approximately 0.4% (0.18% in our study) of samples.[6] If this typical cytoplasmic pattern is observed, a thorough clinical examination should be performed to detect the potential development of SjS or neurological disorders such as ataxia and other sensory/motor neuropathies.[8]

Fine granular or dot cytoplasmic staining was noted in 7 (0.2%) samples only. Auto-antibodies associated with this pattern are primarily anti-Jo-1 antibody that may remain undetected in HEp-2 IIF screening as the antibody titers are often low. Therefore, a negative IIF test using HEp-2 cells does not exclude the possibility of the presence of anti-Jo-1 antibodies.[7] This pattern is typically associated with autoimmune myopathy and interstitial lung disease (ILD). We also noted presence of this pattern in 2 cases of ILD. Thus, Jo-1 may be picked up on IIFA HEp-2 but would require a sensitive immunoblot for detection.

Betancur et al. noted a frequency of 1% for the mitotic patterns in a large cohort of 113,491 ANA determinations[5] which is higher than the prevalence that we observed (0.4%). NuMA-like pattern was the most frequently occurring mitotic pattern in our series with a prevalence of 0.2% as compared to 0.04% in a Chinese study,[9] 0.7% in a European cohort,[3] and 0.4% by Betancur et al.[5] The detection of mitotic patterns is very uncommon. Further, mitotic patterns behave as monospecific antibodies. When present, they are mostly associated with SARDs, mainly SjS and SLE. Clinicians may be aware that in these conditions, anti-NuMA antibodies may be the single serological marker.

The DNA Topo I-like HEp-2 cell IF pattern was noted in two cases only and showed compound fluorescence pattern comprising of a fine-speckled staining of the nucleus, nucleolus and metaphase plate. Both these cases were positive for anti-DNA topo I antibodies on immunoblot. The recognition of the DNA Topo I-like pattern is extremely useful and guides to implementation of specific tests for the identification of anti-Topo I antibodies[10],[11] which characterize systemic sclerosis another common disease in North India.[12]

Rao et al. have reported 1:100 and Ghosh et al. reported 1:80 as the best screening dilution to discriminate healthy individuals from SLE patients.[13],[14] However, we perform routine ANA screening at 1:40 dilution of sera which is already standardized in our laboratory over a period of last 20 years, based on local population data. We screen around 13,567 sera annually with this optimal screening dilution, which is well-accepted by our clinicians.

Another observation was that simultaneous presence of antibodies against 2 cytoplasmic antigens may lead to confounding fluorescence findings, as seen in a case of vasculitis [Table 2], showing cytoplasmic FF pattern along with anti-PR3 antibodies. The co-occurrence of other auto-antibodies should be kept in mind while reporting fluorescence patterns.

The clinical relevance of these patterns remains unclear due to the rarity of their detection, discrepancies between the results reported by different groups (lack of comparability due to differences in study design, methodology, ethnicity of study population), methodological problems or the preliminary nature of the observations.[10] If these uncommon patterns are noted during ANA screening with HEp-2 cells, further serological and clinical examinations should be performed. Collaboration with a research laboratory can be helpful in determining the fine specificity and further evaluation of potential clinical relevance.[6],[9]

To the best of our knowledge, this is the only reported study of uncommon ANA patterns from India. The biggest drawback of this analysis is that the clinical association of these uncommon patterns could not be determined properly due to lack of relevant information in 50% of the patients. Almost all ANA requisitions were from an outpatient department, limiting the traceability of the patients and further evaluation. We also did not have access to all clinical data from the medical records.

The purpose of this paper is to provide an overview with regard to the prevalence of uncommon cytoplasmic and mitotic ANA patterns. As many clinicians are not yet familiar with these patterns, so our main objective is to make clinicians aware of these patterns and to sensitize them for repeated follow-up examinations of these patients to determine the antigen specificity. This information about the types as well as frequency of uncommon patterns will make a commendable contribution in the diagnosis of SARD. Nevertheless, it is important for the clinician to know if their diagnostic laboratory reports on cytoplasmic and mitotic staining in their IIFA HEp-2 results.

Although the newer ICAP nomenclature is scientifically important, the interpretation and clinical relevance of uncommon ANA patterns specifically mitotic patterns remain a challenge. The low prevalence of these patterns adds to the difficulty of establishing close clinical association between fluorescence patterns and clinical syndromes.

Emerging nomenclature guidelines recommend that non-nuclear patterns should be reported as a positive ANA; these efforts will narrow that “seronegative gap” observed in SARD, most commonly in SLE. This is an expanding field of research and a lot remains to be done to define its clinical relevance.

Acknowledgments

We would also like to thank Ms. Rajwant for assistance in carrying out HEp-2 IIF assay and ELISAs for the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Meroni PL, Schur PH. ANA screening: An old test with new recommendations. Ann Rheum Dis 2010;69:1420-2.  Back to cited text no. 1
    
2.
Agmon-Levin N, Damoiseaux J, Kallenberg C, Sack U, Witte T, Herold M, et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis 2014;73:17-23.  Back to cited text no. 2
    
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Vermeersch P, Bossuyt X. Prevalence and clinical significance of rare antinuclear antibody patterns. Autoimmun Rev 2013;12:998-1003.  Back to cited text no. 3
    
4.
Chauhan R, Jain D, Dorwal P, Roy G, Raina V, Nandi SP. The incidence of immune-fluorescence patterns and specific autoantibodies observed in autoimmune patients in a tertiary care centre. Eur Ann Allergy Clin Immunol 2019;51:165-73.  Back to cited text no. 4
    
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Betancur JF, Londoño A, Estrada VE, Puerta SL, Osorno SM, Loaiza A, et al. Uncommon patterns of antinuclear antibodies recognizing mitotic spindle apparatus antigens and clinical associations. Medicine (Baltimore) 2018;97:e11727.  Back to cited text no. 5
    
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Minz RW, Kaur N, Anand S, Aggarwal R, Saikia B, Das A, et al. Complete spectrum of AMA-M2 positive liver disease in north India. Hepatol Int 2012;6:790-5.  Back to cited text no. 6
    
7.
Conrad K, Schößle WR, Hiepe F, Fritzler MJ. Autoantibodies in systemic autoimmune diseases. A diagnostic reference. In: Conrad K, Sack U, eds. Autoantigens, Autoantibodies, Autoimmunity. 3rd ed., Vol. 2. Lengerich, Germany: Pabst Science Publishers; 2015. p. 12-232.  Back to cited text no. 7
    
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Stinton LM, Eystathioy T, Selak S, Chan EK, Fritzler MJ. Autoantibodies to protein transport and messenger RNA pathways: Endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies. Clin Immunol 2004;110:30-44.  Back to cited text no. 8
    
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Xi Q, Wu Y, Li L, Cai B, Zhang J, Yang B, et al. Anti-mitotic spindle apparatus antoantibodies: Prevalence and disease association in Chinese population. J Clin Lab Anal 2016;30:702-8.  Back to cited text no. 9
    
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Dellavance A, Gallindo C, Soares MG, da Silva NP, Mortara RA, Andrade LE. Redefining the Scl-70 indirect immunofluorescence pattern: Autoantibodies to DNA topoisomerase I yield a specific compound immunofluorescence pattern. Rheumatology (Oxford) 2009;48:632-7.  Back to cited text no. 10
    
11.
Damoiseaux J, Andrade LEC, Carballo OG, Conrad K, Francescantonio PLC, Fritzler MJ, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: The International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis 2019;78:879-89.  Back to cited text no. 11
    
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Minz RW, Kumar Y, Saikia B, Anand S, Varma S, Singh S. Use of panel testing for detection of antinuclear antibody in a resource-limited setting: An appraisal. Postgrad Med 2016;128:869-74.  Back to cited text no. 12
    
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Rao JS, Shobha V, Thomas T, Kini U. Standardizing initial dilution titers of antinuclear antibodies for the screening of systemic lupus erythematosus. Indian J Rheumatol 2019;14:211-7.  Back to cited text no. 13
  [Full text]  
14.
Ghosh P, Dwivedi S, Naik S, Agarwal V, Verma A, Aggarwal A, et al. Antinuclear antibodies by indirect immunofluorescence: Optimum screening dilution for diagnosis of systemic lupus erythematosus. Indian J Med Res 2007;126:34-8.  Back to cited text no. 14
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