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"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."

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Dr. Arundhathi. S
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Sanjay Gandhi institute of trauma and orthopedics,
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.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
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.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".

Dr. Mamta Gupta
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Aug 2018

Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
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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Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."

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)
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.
On April 2011

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
On Jan 2020

Important Notice

Original article / research
Year : 2022 | Month : November | Volume : 16 | Issue : 11 | Page : EC05 - EC08 Full Version

Immunofluorescence on Formalin Fixed Paraffin Embedded Renal Tissue Sections: A Retrospective Study

Published: November 1, 2022 | DOI:
Renuka Malipatel, Gnanapriya Vellaisamy, Pritilata Rout

1. Assistant Professor, Department of Pathology, St. John’s Medical College, Bengaluru, Karnataka, India. 2. Assistant Professor, Department of Pathology, St. John’s Medical College, Bengaluru, Karnataka, India. 3. Professor, Department of Pathology, St. John’s Medical College, Bengaluru, Karnataka, India.

Correspondence Address :
Dr. Renuka Malipatel,
Assistant Professor, Department of Pathology, St. John’s Medical College, Sarjapur Road, Bengaluru, Karnataka, India.


Introduction: Direct Immunofluorescence (DIF)/Routine Immunofluorescence (R-IF) on frozen sections is vital in the work-up of renal diseases. Sometimes, the unfixed sample may not be available for DIF or the sample may be inadequate. Paraffin Immunofluorescence (P-IF) can be used as a salvage technique in these situations. R-IF is more sensitive than P-IF in detecting Immunoglobulins (Ig) and complements. P-IF detects characteristic immunoglobulins and complements in the majority of glomerular diseases.

Aim: To evaluate the sensitivity of the P-IF in comparison to the gold standard R-IF in renal biopsies with proliferative and non proliferative glomerular diseases.

Materials and Methods: The present study was a retrospective study done on 52 selected cases, at St. John’s Medical College, Bangalore, Karnataka, India, data collected from January to December 2016. Based on the clinical differential diagnoses, light microscopy and R-IF findings; selected panels of immunostains (IgG, IgA, IgM, C3, C1q) were done. Proteinase K was used for enzymatic digestion. Immunofluorescence intensity was scored by two pathologists independently. Any specific pattern of staining of atleast 1+ intensity was considered as positive on P-IF. Sensitivity, specificity and confidence intervals were estimated for P-IF.

Results: P-IF was done on a total of 52 selected cases. In this study P-IF showed 100% sensitivity for diagnosis of lupus nephritis, infection related glomerulonephritis, Henoch-Schönlein Purpura (HSP) nephritis and 78% for IgA nephropathy. Overall sensitivity in the diagnoses of common glomerular diseases studied was 90% (95% CI=78.97-96.80, p-value=0.025). It was less sensitive for detecting C3.

Conclusion: This retrospective study demonstrated that, P-IF has a good sensitivity for diagnosing common glomerulopathies like IgA nephropathy and lupus nephritis. P-IF is a good adjunct to R-IF testing with 100% specificity.


Biopsy, Complement, Glomerular diseases, Immunoglobulins

Direct immunofluorescence (DIF)/routine immunofluorescence (R-IF) on frozen sections is vital in the aetiological work-up of renal diseases (1),(2). Routinely one core of renal biopsy is sent separately for R-IF in Michel’s transport media/normal saline. Sometimes the unfixed sample may not be available for DIF or the sample may be inadequate due to sampling of medullary tissue. P-IF performed on formalin fixed paraffin embedded tissue submitted for light microscopy can be used as a salvage technique in these situations. Formalin fixation causes cross linking of globular proteins and preserves the secondary structure of proteins in tissues (3). It is difficult to detect immunoglobulins and complements in formalin fixed paraffin embedded tissue sections. Antigen retrieval helps antibodies to bind to unmasked antigens for detection (4). P-IF with antigen retrieval by enzyme treatment was described long ago (5),(6),(7),(8),(9). However, only in the recent past, it is put into use as an adjunct to R-IF on fresh frozen tissue (10). In P-IF procedure enzymes like trypsin, pronase, pepsin and Proteinase K are used in enzymatic digestion and followed by direct or indirect method of IF. Heat treatment can also be used for the P-IF (11). R-IF is more sensitive than P-IF in detecting immunoglobulins and complements. P-IF detects characteristic immunoglobulins and complements in the majority of glomerular diseases, such as, IgA nephropathy, lupus nephritis, and infection related glomerulonephritis and membranous nephropathy (7),(8),(9). The concordance rate between R-IF and P-IF reported in the literature varies from 83-100% (8),(9),(10),(12),(13),(14). The present study was undertaken to evaluate the sensitivity and specificity of the P-IF technique in the diagnosis of common proliferative as well as non proliferative glomerular diseases.

Material and Methods

The present study was a retrospective study done in the Department of Pathology, St. John’s Medical College, Bangalore, Karnataka, India, data collected from January to December 2016. P-IF was performed on selected cases from 2018-2021. Analysis of the data done from January to April 2022. This study was approved by the Institutional Ethics Committee (IEC), St. John’s Medical College and Hospital, Bangalore (IEC Study Ref. No.174/2017).

Inclusion criteria: Renal biopsies from cases of all age groups with adequate cortical tissue were included in the study.

Exclusion criteria:

1. Renal biopsies with inadequate cortical tissue/diffuse glomerulosclerosis.

2. Paraffin block not available.

Sample size calculation: To estimate sensitivity of 80% with 15% relative precision and 95% Confidence Interval (CI), sample size required was 42, but final sample was taken as 52. P-IF procedure was validated, by doing it on two cases of lupus nephritis with full house positivity.

P-IF Procedure

1. 3 to 4 μ sections were cut from the formalin fixed paraffin embedded tissue block. Sections were taken on poly -L-Lysine (PLL) coated slides. Each slide was labelled appropriately.

2. Deparaffinisation was done in a slide incubator for 1 hour and later kept in xylene for 10 minutes.

3. Slides were immersed for 30 minutes in Tris EDTA buffer (pH=9) at room temperature.

4. Proteinase K (ready to use from Dako) was used for enzymatic digestion. Slides were kept in a moisture chamber on a level surface. PAP (Peroxidase Anti-Peroxidase/buffer) pen was used to circle the tissue. One or two drops of Proteinase K solution pipetted to completely cover the sections. Incubated for 45 minutes.

5. Enzymatic digestion was stopped by transferring the slides to Tris EDTA buffer at 4°C and left for 40 minutes.

6. Slides were rinsed in PBS buffer for 10 minutes.

7. Fluorescein isothiocyanate (FITC) conjugated polyclonal rabbit antibodies (Dako) were applied and incubated for two hours in a moist chamber in the dark. (All antibodies were used in dilution of 1:30).

8. Rinsed in PBS buffer and mounted in glycerol.

9. Slides were examined in fluorescence microscope and representative images were captured.

Based on the clinical differential diagnoses, light microscopy and R-IF findings; selected panel of immunostains (IgG, IgA, IgM, C3 and C1q) were performed on P-IF.

Following parameters were assessed on P-IF: The immunofluorescence intensity of P-IF was evaluated and scored on a semi-quantitative scale of 0-3+ (0-absent, 1+-mild, 2+- moderate,3+- strong), by two pathologists independently and were blinded to R-IF findings. Intensity and location of the immune deposits on P-IF were compared with the corresponding R-IF. Any specific pattern of staining of atleast 1+ intensity was considered as positive on P-IF. Trace positivity was taken as negative. P-IF results were categorised as positive (1+ and above) or negative (score 0) for analysis. R-IF is the gold standard test for detecting immunoglobulins and complements. The Concordance rate between P-IF and R-IF was estimated.

Statistical Analysis

Sensitivity, specificity, 95% confidence intervals were estimated for P-IF. McNemar’s test was used to calculate the p-value. Categorical variables were expressed as numbers and percentages. Sensitivity, specificity and confidence intervals were estimated for each immunostain (IgG, IgA, IgM, C3 and C1q) on P-IF irrespective of the glomerular disease.


The P-IF was performed on 52 selected cases. Concordance rate of P-IF with R-IF in various glomerular diseases was calculated (Table/Fig 1). Among the common glomerulopathies studied by P-IF, 100% sensitivity was observed for lupus nephritis, infection related glomerulonephritis and HSP nephritis. No false positive staining was observed in cases of diabetic nephropathy, minimal change disease and focal segmental glomerulosclerosis. For IgA nephropathy and membranoproliferative glomerulonephritis, sensitivity of 78% and 75%, respectively was noted.

Sensitivity and specificity of each immunostains on P-IF in comparison to gold standard R-IF is depicted in (Table/Fig 2). Of the immunostains performed none showed non specific/false positive staining. IgG, IgA, IgM and C1q showed better sensitivity than C3.

[Table/Fig-(3),(4),(5) shows P-IF and R-IF of Lupus nephritis, IgA nephropathy, membranous nephropathy and focal segmental glomerulosclerosis.


The DIF testing on frozen tissues is essential in the aetiological work-up of renal biopsies (1),(2). R-IF is essential in the diagnosis of glomerular diseases and it requires an unfixed sample in Michel’s transport media/normal saline. In certain situations, separate unfixed sample may not be available or the sample may be inadequate due to lack of glomeruli. In such instances, P-IF can be performed on paraffin sections as a salvage technique. Many studies have been performed P-IF on renal tissue sections using different enzymes like trypsin, pronase and proteinase K for enzymatic digestion. In the present study, proteinase K was used and the P-IF method described by Singh G et al., was followed (13). P-IF staining intensity and pattern matched with R-IF, in most of the cases. The concordance was good when the immune complex deposits were bright and abundant (3+) on R-IF. P-IF, a salvage technique has fair sensitivity in the diagnoses of common glomerulopathies. Studies have shown that the R-IF and P-IF results were in agreement for immunoglobulins, characteristic of a particular glomerular disease (Table/Fig 6) (6),(8),(9),(10),(12),(13),(14),(15). In the present study, 100% sensitivity was observed for diagnosis of lupus nephritis (Table/Fig 3), infection related glomerulonephritis, Henoch-Schönlein purpura nephritis and 78% for IgA nephropathy (Table/Fig 4). Nasr SH et al., have shown slightly lower sensitivity of P-IF in membranous nephropathy and anti-GBM disease. They also observed reduced sensitivity of P-IF for C3 immunostain (10). In this study, sensitivity of 67% for membranous nephropathy (Table/Fig 5) and 55% for C3 immunostain, was observed.

In cases of diabetic nephropathy, minimal change disease and focal segmental glomerulosclerosis no non specific staining was observed. They were clearly negative. Linear IgG staining and segmental sclerosis (Table/Fig 5) were well demonstrated on P-IF in diabetic nephropathy and focal segmental glomerulosclerosis cases, respectively. Singh G et al., observed that P-IF interpretation was possible in 87% (214/246) (13). On P-IF granular staining, pattern of immunoglobulins and complements were less evident and appeared smudgy (16). Similar staining pattern was observed in the present study. Apart from its use as a salvage technique, Messias NC et al., have shown the unmasking effect of P-IF on immune complex deposits in C3 dominant glomerulonephritis, which precludes unnecessary labeling of these cases as C3 glomerulopathies (17). Nasr SH et al., demonstrated the usefulness of P-IF in dysproteinaemia associated with renal diseases. P-IF is more sensitive than R-IF in light chain Fanconi syndrome (10). Membranous-like Glomerulopathy with Masked IgG kappa Deposits (MGMID) and Membranoproliferative Glomerulonephritis (MPGN) with masked monotypic Ig deposits require P-IF for diagnosis (18),(19),(20). Recent studies have applied P-IF for light chain detection in amyloidosis and dysproteinaemia in extrarenal locations, as it is more sensitive than immunohistochemistry (21),(22),(23).

As a salvage technique, P-IF exhibits fairly good sensitivity in diagnosing immune-complex mediated glomerular diseases, especially when the immune deposits are abundant. Future studies on larger samples, encompassing various glomerular diseases with weak to abundant immune deposits, are necessary.


Since, renal biopsies with adequate cortical tissue on paraffin sections were retrospectively selected for the study; there could have been a selection bias, as glomerular diseases with better defined and evolved morphological patterns might have been favored. Chances of false negative results are higher in early glomerular diseases. Secondly, limited number of common glomerular diseases were included in the study. Also, kappa and lambda light chain immunostains were not performed.


In the present study, sensitivity of 90% (95% CI=78.97-96.80, p-value=0.025) was observed for P-IF in diagnosis of various common glomerular diseases and false positive results were none. To conclude, P-IF is a good salvage technique, with 100% specificity.


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Schneider W. Stellenwert der Immunfluoreszenz in der Diagnostik von Nierenerkrankungen [Value of immunofluorescence in the diagnosis of kidney diseases]. Z Gesamte Inn Med. 1983;38(4):115-20. German. PMID: 6344465. (Abstract).
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Choi YJ, Reiner L. Immunofluorescence of renal lesions in paraffin-embedded and fresh- frozen sections. Am J Clin Pathol. 1980;73(1):116-19. Doi: 10.1093/ ajcp/73.1.116. PMID: 6986075. [crossref] [PubMed]
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DOI and Others

DOI: 10.7860/JCDR/2022/58418.17003

Date of Submission: Jun 14, 2022
Date of Peer Review: Jul 28, 2022
Date of Acceptance: Oct 05, 2022
Date of Publishing: Nov 01, 2022

Author declaration:
• Financial or Other Competing Interests: This study was funded by Rajiv Gandhi University of Health Sciences- Advanced research project (Grant number 17M011).
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA

• Plagiarism X-checker: Jun 27, 2022
• Manual Googling: Sep 19, 2022
• iThenticate Software: Oct 01, 2022 (5%)

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