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

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Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
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Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
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Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




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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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Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
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Professor and Head
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Saraswati Dental College
Lucknow
On Sep 2018




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Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




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Best regards,
C.S. Ramesh Babu,
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Muzaffarnagar Medical College,
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On Aug 2018




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Dr. Arundhathi. S
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.
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
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
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.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
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)
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

Important Notice

Original article / research
Year : 2008 | Month : August | Volume : 2 | Issue : 4 | Page : 963 - 968

qRT-PCR Compliments Immunohistochemistry In Archival Breast Cancer Samples

PRABHU J S *, WANI S *, KORLIMARLA A, PAYAL K., VICTOR J M , ANAND A, SAHOO R .

Triesta Sciences, India Pvt. Ltd Institute Of Population Health And Clinical Research Building St John’s Medical College CampusOpp. Bda Complex,Koramngala,Bangalore-560034 India

Correspondence Address :
Dr.Prabhu J S Triesta Sciences, India Pvt. Ltd Institute Of Population Health And Clinical Research Building St John’s Medical College CampusOpp. Bda Complex,Koramngala,Bangalore-560034
India

Abstract

Background: Molecular characterization of tumour tissue in limited archived formalin fixed, paraffin embedded (FFPE) sample is always challenging. Better molecular characterization will not only help in accurate diagnosis but also in prognosis and guides therapeutic decisions.
Study Design: In the present study we tested the prognostic markers of breast cancer, estrogen receptor (ER) and progesterone receptor (PgR) by immunohistochemistry and quantitative real time polymerase chain reaction (qRT-PCR) on archived FFPE samples.
Results: High concordance was observed between the two methods for ER and PgR.
Conclusion: We conclude qRT-PCR may be used as an alternative method for the study of prognostic factors in archived tissues. Moreover, qRT-PCR can be a high throughput method for evaluation of markers with limited archived tissue.

Keywords

Formalin fixed paraffin embedded (FFPE), hormone receptors, breast cancer, IHC, qRT- PCR

Introduction
Archived formalin fixed, paraffin embedded blocks have become valuable for validation of biomarkers in retrospective observational studies as they represent by far, the most abundant supply of solid tissue specimens associated with clinical records.

Until recent past, hormone receptor status in breast cancer was assessed in fresh tissues using ligand-binding assays (1). Currently immunohistochemistry on formalin fixed paraffin embedded (FFPE) tissue has become the method of choice for determining the biomarkers like hormone receptors which are important regulators of growth and differentiation in the normal mammary gland and are of considerable diagnostic/prognostic value in breast cancer (2). However it has inherent disadvantages like, intra and inter-observer variation between different labs, dependence on fixation conditions and lack of calibration (2). In molecular biology, reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process. Polymerase chain reaction itself is the process used to amplify specific parts of a DNA molecule, via the temperature-mediated enzyme DNA polymerase.

In the recent past great advances have been made in the development of other sensitive techniques like quantitative reverse transcriptase polymerase chain reaction (qRT- PCR) for studying gene expression patterns using FFPE blocks (3) in retrospective observational studies. These techniques have the advantage of being robust, quantitative, and accurate and are less amenable to inter observer variation due to automated calibration when compared to subjective assessment in immunohistochemistry (IHC).

To evaluate the feasibility of using FFPE as a substrate for molecular analysis, we explored the utilization of qRT-PCR as a method for determining the hormone receptor status like oestrogen receptor (ER) and progesterone receptor (PgR) in archival breast cancer specimens. Here we report the concordance between conventional techniques like immunohistochemistry and qRT- PCR for hormone receptors like ER and PgR in archival breast cancer specimens.

Material and Methods

Tissue Specimens
We studied formalin fixed paraffin embedded blocks of 53 patients who had undergone surgery for primary breast cancer. These blocks were obtained from various hospital laboratories after obtaining the approval from the ethics committee of respective hospitals. All 53 patients had undergone surgery between the periods 1994 to 2004. Of these, 46 were diagnosed as Invasive ductal carcinoma, five were invasive lobular carcinoma, and two were papillary carcinomas. Histological analysis was performed on Haematoxylin and Eosin (H&E)-stained slides of the blocks to confirm the diagnosis. One block containing the representative tumour and having more than 50 % of area showing tumour cells was selected for the study in each case.

Immunohistochemistry
5-micron sections were cut from these blocks on Poly-L-Lysine coated slides. The slides were kept in an incubator overnight at 56ÂşC. Sections were deparaffinised through two changes of Xylene, rehydrated through graded alcohols to distilled water. After blocking endogenous peroxidase activity with 3 % hydrogen peroxide in methanol, sections subjected to antigen retrieval by heating the slides in a pressure cooker in 1 mM ethylene diamine tetra acetic acid (EDTA) buffer, pH 8. Mouse monoclonal anti-ER- α primary antibodies (DAKO M7047) and anti-PgR mouse monoclonal antibody (DAKO M3569) were applied for 60 min at suggested dilutions. DAKO Envision kit was used for application of secondary antibody. All incubations were performed at room temperature. Sections were developed with diaminobenzidine followed by light counter stain with haematoxylin. Each test batch was run with a known positive and negative control.

Immunostaining Analysis
The stained sections were independently examined by two pathologists and were categorized as positive (antigen present) when more than 10 % of the cells showed brown nuclear staining. Intensity of staining was graded from 1 to 3 according to suggested guidelines.

RNA extraction
Total RNA was extracted from two 20-micron sections taken from each patient’s tumour block. After deparaffinization by heat, sections were subjected to overnight digestion using proteinase K (Qiagen #19133). Total RNA was then extracted using a modified TriReagent protocol (Ambion # 9738). Quantitation of RNA was done using the Ribogreen flourimetry system (Molecular probes, Turner Biosystems). On and average each patient sample yielded 5-6 µg of total RNA. 1µg of total RNA was then reverse transcribed using the ABI high capacity cDNA archive kit (ABI # 4322171) as per manufacturer’s protocol.

Gene Expression Analysis
Expression levels of two-test genes oestrogen receptor and progesterone receptor were determined along with a panel of 6 reference genes (HPRT1, B2M, RPL13A, β-actin, RPLP0 and GAPDH). Ready to use primer-probe reagents for all genes tested were obtained from Applied Biosystem. The reference genes normalize for any variations that may be introduced through varied sample processing (considering they are from different laboratories) and handling methods which in turn lead to varied levels of RNA preservation in the FFPE blocks. Using 10 ng cDNA per reaction qRT-PCR was done in triplicate using TaqMan chemistry on the ABI 7000 sequence detection system. Universal human reference RNA (Stratagene, USA, Cat No# 740000) was also reverse transcribed and 0.5 ng of this was used as a positive control for all genes. Total reaction volume was 20 µl. Pre-incubation and initial denaturation of the template cDNA was performed at 95˚ for 10 min, followed by amplification for 45 cycles with 95˚ for 15 sec and 60˚ for 1 min. The qRT-PCR yielded CT values for the test genes, which were in turn normalized relative to the mean CT value of the six reference genes. Expression levels of the test genes were calculated relative to the Universal human reference RNA.

Gene expression calculations were done using standard and established methods and values greater than or equal to 4 normalized units (Log 2) were considered indicative of over-expression of the gene. Concordance between data of immunohistochemistry and gene expression data was calculated by over all percentage and using Cohen’s Kappa statistics (17).

Results

qRT-PCR versus conventional IHC in the detection of the hormone receptors ER and PgR
Parallel analysis of qRT-PCR versus IHC was carried out on 53 breast cancer samples.
Concordance between the two methods was compared using Cohen’s kappa statistical method (17) [Table/Fig 1a and (Table/Fig 2).

Thirty-six samples were ER positive and 14 were ER negative by both IHC and qRT-PCR. The percentage agreement between the two methods for ER is 94.4 % (Table/Fig 1). In three samples, there was discrepancy between IHC and qRT-PCR (5.6 %). Of these three samples, one sample was negative by IHC but showed over expression by qRT-PCR (1.8 %). Two samples were positive by IHC but did not show over expression by qRT-PCR (3.7 %).

Of the 53 samples tested, 26 were positive and 18 were negative for PgR by both methods. The percentage agreement between the two methods was high, 83.1 % (Table/Fig 2). In nine samples, the results of IHC and qRT-PCR for PgR were discordant (16.9%). Of these 9 samples, 7 samples were negative by IHC but showed over expression by qRT-PCR (13.2 %) and 2 samples were positive by IHC but showed no over expression by qRT-PCR (3.7 %).

Discussion

Archived formalin fixed paraffin embedded blocks serve as valuable material in observational studies. Although great advances have been made in the development of molecular techniques using FFPE blocks (4), sensitivity of these techniques are highly dependant on the preservation of molecules like protein, RNA and DNA in the archived material. Several factors are known to affect the preservation of these molecules like duration of fixation, type and the amount of fixative used and duration for which the archived material is stored (5),(6) making FFPE a difficult substrate for molecular analysis. Moreover, many studies have also reported chemical modifications induced by formalin such as random base damage in DNA (6) and extensive fragmentation of the RNA (5). Despite such reports, successful attempts have been made to use mRNA from FFPE tissues for gene expression assays (3),(7).
Though enzyme immunoassays were frequently used in the past (8), immunohistochemistry is considered the gold standard (10) for the evaluation of hormone receptors on FFPE blocks of breast cancer. It is a technique used by most pathology laboratories, as it is simple, relatively inexpensive and does not need highly trained personnel/equipment. It also has the advantage of topoanatomical localization of the antigen in question. Though this technique is best suited for surgical pathology practice, the results are semi quantitative, and limited by subjective interpretation. Therefore, reproducibility and standardization are critical factors in the assay (10). In addition, many studies have reported a loss of immunostaining intensity, in stored paraffin sections (11), (12) giving false negative results by IHC. We have also observed this loss of immunogenicity in archived FFPE blocks in house.

Our results demonstrate that the material extracted from archived FFPE tissues can be used for quantitative RT-PCR, which has the advantage of reproducibility, sensitivity, and quantitation over a dynamic range and hence could be used for confirmation/complementing of IHC results in observational studies. Moreover, qRT-PCR is a high throughput (4), (9) method for mRNA quantitation of many genes using limited archived tissue. Our results of high level of concordance between the two techniques for hormone receptors are in agreement with that seen in other studies (13), (14),(15),(16).

Differences in intracellular stability and biological variations in preservation of different mRNA species may explain variation in concordance levels between oestrogen receptor and progesterone receptor.

The discrepancies seen in few cases between the two techniques could be explained as follows. Expression of oestrogen receptor in adjacent normal breast tissue could have contributed to high level of expression in qRT-PCR whereas IHC showed negative results. In cases where IHC showed the presence of antigen but qRT-PCR did not show, over expression, is probably due to loss of the tumour tissue in the block after repeated trimming.

Conclusion

In conclusion, qRT-PCR analysis from FFPE (archived breast tumours) is feasible and results correlate with immunohistochemistry. However, a combined IHC and quantitative RT-PCR approach for determining ER and PgR in archived breast cancer may be an effective and efficient strategy. RNA based techniques may be sufficient for high through put analysis of many markers when tissue is a limiting factor.

Abbreviations
B2M - Beta 2 micro globulin
cDNA - Complimentary deoxyribonucleic acid
EDTA - Ethylene Diamine Tetra acetic acid
ER - Oestrogen receptor
FFPE - Formalin fixed paraffin embedded
GAPDH - Glyceraldehyde 3 phosphate dehydrogenase; µg - microgram
H&E - Haematoxylin and Eosin
HPRT1 - hypoxanthine-guanine phosphoribosyltransferase
IHC - Immunohistochemistry
mRNA - messenger Ribonucleic acid
PgR - Progesterone receptor
qRT-PCR - quantitative reverse trascriptase polymerase chain reaction
RPL13a - Ribosomal protein L13a
RPLP0 - Ribosomal protein Large 0s

References

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Hirotaka I, Yukashi I, Tatsuya K, Hiroko Y, Hiroji I, Tatsuya T, Yasuo H, Shunzo K. Simultaneous quantitative analyses of c-erbb-2 protein, epidermal growth factor receptor, cathepsin d, and hormone receptors in breast cancer. Cancer Detect Prev 1997; 21(1):29-35.
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Pawlowski V, RĂ©villion F, Hornez L, Peyrat JP. A real-time one-step reverse transcriptase - polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer. Cancer Detect Prev 2000; 24(3):212-23.
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Bertheau P, Cazals-Hatem D, Meignin V, de Roquancourt A, Verola O. Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 1998; 51: 370-74.
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Jacobs TW, Prioleau JE, Stillman IE, Schniit SJ. Loss of tumour marker immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst 1996; 88(15) 1054-59
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