Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
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Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
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Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

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Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
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Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2023 | Month : January | Volume : 17 | Issue : 1 | Page : EC10 - EC14 Full Version

Rare Antinuclear Antibody Patterns: Relevance in Routine Laboratory Reporting


Published: January 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/60084.17321
Vidya Bhakta

1. Specialist, Pathologist, Department of Laboratory Medicine, Dr Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia.

Correspondence Address :
Dr. Vidya Bhakta,
Medical Laboratory, Dr Sulaiman Al Habib Medical Group,
Olaya Medical Complex, P.O. Box 91877; Riyadh 11643, Saudi Arabia.
E-mail: vidya.bhakta@rediffmail.com

Abstract

Introduction: Uncommon patterns on Human Epithelial 2 (Hep 2) substrate during Antinuclear Antibody (ANA) screening by Indirect Immunofluorescence (IIF) microscopy are not routinely reported by many laboratories since their clinical relevance is not well established.

Aim: To elucidate rare ANA patterns on Hep 2 and their possible association with clinical presentation.

Materials and Methods: A retrospective study was conducted on ANA reports in the duration January 2021 to March 2022 at the Department of Laboratory Medicine, Dr. Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia, to recognise rare ANA patterns. Gold standard method of IIF on Hep 2 was used for screening. Statistical evaluation was done to obtain frequencies of various ANA patterns. Those with frequency of less than 1% were classified as rare patterns.

Results: Overall, 4207 consecutive ANA reports were evaluated out of which 1388 were positive and 210 (4.99%) demonstrated rare ANA patterns including nuclear, cytoplasmic and mitotic subtypes. Most commonly encountered among the rare ANA patterns was intercellular bridge (AC 27) with frequency of 0.78% (n=33). Systemic Lupus Erythematosus (SLE) (10/210) was the most often observed clinical association with rare cytoplasmic and mitotic patterns at titer ≥1:160.

Conclusion: Uncommon ANA patterns may be useful in initial work-up of autoimmune illness hence, should be routinely reported. Further studies to enlighten the significance of these patterns, analogous antibodies could be of diagnostic relevance in autoimmune and other diseases.

Keywords

Autoimmunity, Fluorescence microscopy, Spindle apparatus

Antibodies against various cellular antigens, better known as ANA have been a significant screening tool in approaching patients suspected of having autoimmune illness (1). ANA is specifically important in the evaluation and follow-up of patients with systemic autoimmune rheumatic diseases such as SLE. ANA positivity is one of the criteria set by the American College of Rheumatology (ACR) in order to classify patients with SLE (2). Furthermore, positive ANA may not always hint to Systemic Autoimmune Rheumatic Diseases (SARD) as low titres may be found in some healthy individuals (3). The IIF has long been considered as the gold standard screening method to detect ANA (4) which uses the Hep 2 substrate derived from human laryngeal carcinoma. Capturing monolayer cells in different phases of mitosis and allows detection of antibodies to a broad array of nuclear antigens, and those located in the cytoplasm or associated with mitotic apparatus can also be detected by this assay (5). Based on fluorescence patterns observed, the technique enables identification of antibodies against cell antigens favourably expressed in specific mitotic phases like spindle fibre related antigen and others of unknown significance (6). Hence, it still stays as the method of choice for initial assessment of ANA even with the emergence of commercial assays for antibodies against specific cellular antigens (7).

International Consensus on ANA Patterns (ICAP) describes 29 distinct Anti Cellular (AC) patterns named as AC 1 to AC 29 and categorised under nuclear, cytoplasmic and mitotic subtypes with defined competence level for identifying each of them. AC 0 represents as negative for any specific fluorescence for cellular autoantibodies (8),(9),(10). Common patterns have undergone extensive research and shown to have specific disease association whereas relevance of rare patterns is still unknown due to confined studies. Rare patterns have been defined as those with prevalence of less than 1% (11). Most patterns so far described as rare in various studies (11),(12),(13),(14) are classified under ‘expert level’ category in the ICAP classification (15) and so could be the reason that some laboratories do not report them in addition to lack of certainty about their significance.

The present study was conducted in a tertiary care hospital laboratory with high turnover of patient samples for ANA screening by IIF. The study intends to analyse the range of rare ANA patterns, estimate their frequency and possible clinical relevance.

Material and Methods

Data Collection

Retrospective observational study was conducted at the Department of Laboratory Medicine, Dr. Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia. Data for duration between 1st January 2021 to 31st March 2022 was collected retrospectively and subsequently analysed in the period 1st April 2022 to 31st July 2022. Total 4.207 ANA reports were reviewed and their respective patterns and titers were noted. Rare ANA patterns were based on frequency of less than 1% (11). Clinical diagnoses associated with rare patterns as mentioned in medical record were noted and data was anonymously saved. No human or animal experiments were conducted in the study and all protocols were in accordance with the Helsinki Declaration of 1975.

Inclusion criteria: All ANA reports of consecutive patients referred for ANA screening by IIF in study duration were reviewed in the study.

Exclusion criteria: In case of multiple ANA requests for a patient, only the first sample was considered for study purpose and subsequent were excluded from the study.

Slide preparation and microscopy: ANA testing was performed using Euroimmun, Germany kits. Mosaic biochips containing two substrates Hep 2 cells and primate liver in each reaction well were incubated with diluted patient samples. Fluorescein labeled secondary antibodies bound to patient’s antibodies, if present were made visible with a fluorescence microscope at 40X magnification.

Serum samples were screened at 1:80 dilution, titers were reported by serial dilutions 1:160, 1:320, 1:640. ANA reaction at 1:80 was considered as borderline and titers 1:160 and above as positive (16). For study purpose samples showing immunofluorescence at titer 1:80 and above were considered positive and rest negative. ANA patterns were reported in accordance with the ICAP nomenclature guidelines (10). Samples with more than one pattern also called mixed patterns were reported in the order of nuclear, cytoplasmic and then mitotic pattern (17). Positive, negative controls were performed with each batch of testing. The laboratory is enrolled with an external proficiency program for ANA titer and pattern identification by the College of American Pathologists. The diagnoses at lower titer of 1:160 and higher titer of ≥1:320 were considered possibly relevant to rare patterns (16). Preliminary or final diagnosis of patients reported with rare patterns were reviewed from health records and noted under two titer categories, 1:160 and ≥1:320.

STATISTICAL ANALYSIS

Descriptive statistics were used to deduce frequency (%).

Results

As depicted in (Table/Fig 1) out of 4.207 samples analysed for ANA by IIF method during the study period, 33% (n=1388) were positive. Among the positive cases, 210 (4.99%) exhibited rare ANA patterns, (Table/Fig 2) lays out the frequencies of different ANA patterns observed in the study. Rare patterns were delineated based on their observed frequencies viz., less than 1%. Overall, 24 different ANA patterns from subcategories of nuclear (n=822. 19.53%), cytoplasmic (n=78. 1.85%) and mitotic (n=80. 1.90%) were identified while mixed patterns constituted 9.70% (n=408). The most common ANA pattern was fine speckled (AC 4) with prevalence of 3.14% (n=132) whereas the rarest pattern was cytoplasmic fibrillar filamentous (AC 16) with prevalence of 0.02 % (n=1).

Total 14 ANA patterns fulfilled the criterion of being rare, that is frequency of <1%. The rare patterns were from all three categories: nuclear, cytoplasmic and mitotic. The most commonly occuring rare ANA pattern was Intercellular bridge-AC 27 with frequency of 0.78% (n=33) (Table/Fig 1).

As reflected in the (Table/Fig 3) the most frequently encountered autoimmune disorder in patients with rare ANA patterns was SLE, Other associations were of Rheumatoid Arthritis (RA), Autoimmune Hepatitis (AIH), Primary Biliary Cholangitis (PBC) and Mixed Connective Tissue Disease (MCTD). Higher titers of ≥1:320 were more frequently related to these diagnoses.

The SLE could be linked to cytoplasmic and mitotic patterns and so did RA that exhibited cytoplasmic (AC 22, 23) and mitotic patterns (AC 27). Autoimmune liver disease namely, PBC and AIH were possible diagnoses related to patterns AC 6, 12, 15, 21 while MCTD was linked to AC 27.

(Table/Fig 4) shows immunofluorescence microscopic images of rare nuclear- AC 6, AC 12 (Table/Fig 4)a,b and cytoplasmic ANA patterns- AC 15, 16, 18, 19, 20, 21, 22, 23 (Table/Fig 4)c,d,e,f,g,h,i,j on Hep 2 substrate, as observed in the study. (Table/Fig 5) shows the images of rare mitotic ANA patterns-AC 24, 25, 26, 27 (Table/Fig 5)a,b,c,d as noted in the study.

Discussion

The retrospective analysis of ANA reports in the study duration showed 4.99% rare patterns which was comparable to another similar study that reported it to be 6.39% (14). Present study included rare patterns among all categories- nuclear, cytoplasmic, mitotic and demonstrated possible association with autoimmune diseases both at low and high titers.

Nuclear pattern: Multiple Nuclear dots (AC 6) and punctate nuclear envelope (AC 12) were the rare types among nuclear patterns. AC 6 pattern has been mainly associated with PBC however, out of total cases reported with this pattern, only one was diagnosed with PBC. The pattern was also noted in some patients with pneumonia, upper respiratory tract infection and conjunctivitis. Infectious agents are considered possible triggers of autoimmunity (18) and still remain a topic of interest to various scientists. Nuclear envelope pattern is classified further as Smooth (AC 11) and punctate (AC 12). In this study, 20 patients had AC 12 pattern out of which 2 patients were diagnosed with autoimmune liver disease. A follow-up testing for antiglycoprotein-210 antibodies is however recommended, other target antigens for this pattern include p62 nucleoporin, lamin B receptor (19).

Cytoplasmic pattern: All the cytoplasmic patterns observed in the study fulfilled the criterion of being a rare pattern and most frequent among all was Reticular/AMA like (AC 21). AC-21 has been associated with PBC and antibodies are directed against the E2 subunit of PDH complex (19). In present study, two cases with this pattern had PBC while eight other cases were suspected of liver disease. Fibrillar Linear (AC 15) is commonly found in patients with AIH 1 and F actin being the main target antigen of Anti Smooth Muscle Antibodies (ASMA) associated with this pattern. Only one case had an established diagnosis of AIH. Both fibrillar filamentous (AC 16) and Discrete dots (AC 18) are not typically associated with autoimmune disorders and present study too did not reveal any relationship. Similar to this study Nanda R et al., also did not report association of AC 16 with SARD (14).

There is a fine distinction between dense fine speckled (AC 19) and fine speckled (AC 20) that may depend on detection of anti Jo1 antibodies primarily linked to AC 20, although not specific (19). The AC 19 may be found in SLE as in present study, two cases showed this pattern. The antigens recognised are Ribosomal P phosphoproteins, included under Extractable Nuclear Antigen (ENA) profile, which were not done in the observed two cases however, dsDNA antibodies were positive. Tomic´ Sremec N et al., reported several antibodies associated with AC 19 pattern including antids DNA, antiTRIM21, antihistones and antiribosomes and all may be present in SLE (11). AC 20 may be found in patients with antisynthetase syndrome, Interstitial Lung Disease (ILD), polyarthritis (19). In present study, one patient with myositis showed AC 20, antiJo1 antibodies associated with this pattern, directed to histidyl t RNA synthetase, were also positive. Another patient exhibiting AC 20 was diagnosed as ILD however autoimmune aetiology was not further confirmed. Patients with RA and Bullous Pemphigoid showed fluorescence for Polar/Golgi Like pattern (AC 22) at high titre. First described in a patient with lymphoma and Sjogren syndrome, the pattern cannot be indicative of a specific autoimmune disease in current clinical practice (20) however, one study mentioned that the pattern may be an early sign of future autoimmune disease (14). Rods and rings pattern (AC 23) has been described in patients on ribavirin/interferon treatment for Hepatitis C virus infection, the target antigen being inosine-5’-mono- phosphate dehydrogenase 2 (19),(21). Though, none of the patients in this study had Hepatitis C infection two were suspected of RA, similar to a recent case series report where the pattern has been described in various other diseases including autoimmune (22).

Mitotic pattern: These patterns mark the antigens associated with mitotic apparatus. The antibodies directed against various antigenic markers of mitotic apparatus have not been identified well but the clinical association of corresponding ANA patterns reported in previous literature makes them crucial markers of SARD (23). The intercellular bridge or Midbody pattern (AC 27) was found to be the commonest among all rare patterns in current study. It is the residual part of dividing daughter cells just before separation and has been associated with systemic Sclerosis and malignancies (14),(21). Nanda R et al., have reported a strong association with autoimmune aetiology (14), similar to the present study where patients with SLE, RA and MCTD exhibited this pattern. Spindle Fiber (AC 25) was the next commonly encountered mitotic pattern. Two of the SLE patients exhibited this pattern which corroborates with another study where the pattern has demonstrated association with SLE (14).

NuMA-like pattern (AC 26) is found in SARD. NuMA represents several proteins involved in the nuclear reconstruction after mitosis and spindle microtubule organisation (24). Out of total, three patients with AC 26 manifested as SLE. In a recent study, NuMA was found to be significant in patients with cardiovascular disease and autoantibodies probably pointed to coexisting autoimmune disease or an underlying autoimmune process leading to heart disease (24). Centrosome pattern (AC 24) is characterised by fluorescence of 1 or 2 centrioles at the opposing poles and is mostly described in non specific infections. The present study did not find any possible association with SARD although Vermeersch P et al., have reported AC 24 in Raynaud’s (21) and another study found it in patients with small vessel vasculitis (14).

Limitation(s)

The major limitation was the retrospective nature which was the reason for incomplete diagnoses in some cases and hence some rare ANA patterns could not be related to any disease. Also, there were slight variations in ANA pattern-disease associations compared to some previously published studies which could be attributed to ethnic diversity. A single centre study results in lack of representation of a varied population and hence could have led to a bias.

Conclusion

With increasing worldwide prevalence of autoimmune diseases, it seems pertinent to implement effective screening for early diagnosis as timely intervention could bring better outcomes in these patients and ANA by immunofluorescence, being the gold standard, could be an effective tool. Rare ANA patterns, especially cytoplasmic and mitotic patterns, which are not routinely reported, could also be of significance in diagnosis of autoimmune diseases as evinced in present study and hence should be identified and reported in routine laboratory practice. Though, it is essential to investigate further through prospective studies to support the existing finding.

References

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Pisetsky DS, Bossuyt X, Meroni PL. ANA as an entry criterion for the classification of SLE. Autoimmun Rev. 2019;18(12):102400. [crossref] [PubMed]
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DOI and Others

DOI: 10.7860/JCDR/2023/60084.17321

Date of Submission: Sep 17, 2022
Date of Peer Review: Nov 10, 2022
Date of Acceptance: Nov 25, 2022
Date of Publishing: Jan 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? No
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Sep 19 2022
• Manual Googling: Nov 17, 2022
• iThenticate Software: Nov 24, 2022 (4%)

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