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

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Dr Mohan Z Mani

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Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



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
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"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."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
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I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



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.
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
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
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 : June | Volume : 17 | Issue : 6 | Page : EC01 - EC05 Full Version

Chimerism Revisited: Perspective of an Immunopathologist


Published: June 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/58099.17971
Devika Gupta, Lavan Singh, Kamlesh Kumar Singh, Tathagata Chatterjee

1. Professor, Department of Laboratory Science and Molecular Medicine, Army Hospital (Research and Referral), New Delhi, India. 2. Professor, Department of Laboratory Science and Molecular Medicine, Army Hospital (Research and Referral), New Delhi, India. 3. Associate Professor, Department of Laboratory Science and Molecular Medicine, Army Head Quarters and Army Hospital (Research and Referral), New Delhi, India. 4. Professor and Head, Department of Laboratory Science and Molecular Medicine, Army Hospital (Research and Referral), New Delhi, India.

Correspondence Address :
Dr. Devika Gupta,
Professor, Department of Laboratory Science and Molecular Medicine, Army Hospital (Research and Referral), New Delhi, India.
E-mail: devikalives5h@gmail.com

Abstract

Introduction: Chimerism analysis is an important diagnostic tool not only for assessing the risk of relapse after allo-Haematopoietic Stem Cell Transplant (HSCT) in patients with malignant diseases, it is also the predominant method for monitoring post-transplant engraftment status in both haematological malignancies and non malignant haematological disorders.

Aim: To show the chimerism data observed in post-HSCT cases of the hospital over last four years and to emphasise on timely and close monitoring of these patients in the laboratory.

Materials and Methods: The present study was a retrospective study in which 40 HSCT cases comprising of both haematological malignancies and non malignant haematological disorders were monitored in the molecular laboratory of Army Hospital (Research and Referral), at regular intervals by Short Tandem Repeats-Polymerase Chain Reaction (STR-PCR) for quantification of donor chimerism. The pretransplant workup included Human Leukocyte Antigens (HLA) typing of all recipients and donors, serum Panel Reactive Antibody (PRA) testing and Single Antigen Bead (SAB) assays for detecting donor specific antibodies in all haploidentical transplants.

Results: Male patients formed majority, with only nine female cases. After complete HLA matching and preconditioning, 35 patients underwent matched related stem cell transplant, two were Matched Unrelated Donor (MUD) and three haploidentical transplants. Complete donor chimerism at D+90 was reported in 19 patients (73%) of haematologic malignancies with two cases of relapse and five showing evidence of Graft Versus Host Disease (GVHD). Amongst non malignant disorders 10 patients (71.4%) showed complete donor chimerism at D+90 with two cases of GVHD.

Conclusion: Post-HSCT, progressive chimerism monitoring is an essential molecular test that predicts engraftment status of the patient by verifying the dynamic relationship between recipient and donor cells.

Keywords

Allogeneic haematopoietic stem cell transplantation, Graft versus host disease, Haploidentical transplant, Minimal residual disease, Short tandem repeat

Allogenic HSCT is a potentially curable treatment option for patients suffering from both haematologic malignancies and various non malignant haematologic conditions like aplastic anaemia, haemoglobinopathies, storage disorders and primary immunodeficiency diseases etc., (1),(2). Allogenic HSCT is performed after chemo/radio ablation of the recipient marrow followed by infusion of donor stem cells that facilitates sustained donor cell engraftment. Post-HSCT, what is desirable is a state of engraftment where donor cells completely replace the recipient’s haematopoietic lineage. This type of donor immune reconstitution is synonymous to tolerance (3). Though efforts to develop tolerance inducing strategies and agents are gaining rapid recognition, it still remains a distant possibility. Allogenic HSCT is associated with complications related to use of preconditioning regimen, HLA match, type of graft (T cell deplete or replete), underlying molecular aberrations of the disease etc., therefore, a need for frequent monitoring of transplant recipient for development of sinister events like graft failure, relapse or GVHD. Various molecular techniques are used to predict re-emergence of disease clone or recipient haematopoiesis. It is here that the role of immuno/haematopathologist becomes crucial. For all patients of allogenic HSCT, a surveillance protocol for engraftment evaluation post-HSCT is followed using chimerism studies. Chimerism testing helps predict graft rejection or recurrence of disease. Chimerism testing is routinely done between 30 and 100 days after transplantation. Post-HSCT, the recovery of haematopoiesis in recipient depends on ability of donor stem cells to generate progenitor cells and repopulate the marrow niche. This dynamic donor-recipient mix is named chimera which is measured qualitatively and quantitatively in post-transplant patients (4),(5),(6).

Regular monitoring of chimerism status provides an early indication of incipient relapse of underlying malignancy. Increasing mixed chimerism warrants initiation of immunotherapy with Donor Lymphocyte Infusion (DLI) and reduction of Immunosuppression (IS). The clinical importance of chimerism has increased further with introduction of Reduced Intensity Conditioning (RIC), as more patients show mixed chimerism (7),(8).

Chimerism analysis also serves as a surrogate marker for Minimal Residual Disease (MRD) detection especially in acute leukaemia’s without suitable molecular alterations. It remains the only most important tool in predicting early disease relapse or graft failure in non malignant disorders. The authors report here, the spectrum of chimerism data observed at our centre with emphasis on following analgorithmic approach in the post-HSCT period which may serve to monitor and predict disease status.

Material and Methods

The present study was a retrospective study in which 40 patients were analysed who underwent HSCT at Army Hospital (Research and Referral) over four years i.e. January 2015 to June 2019 and analysed over next six months. Patients and donors gave written informed consent and study was approved by Hospital Ethical Committee. The study consisted of 40 patients of haematological malignancies and non malignant haematological conditions who underwent HSCT. They were monitored at regular intervals for disease relapse, graft failure or GVHD, postallogenic HSCT by chimerism. Recipients included those suffering with haematological malignancies and non malignant disorders for which a bone marrow transplant was done.

Inclusion criteria: All patients with atleast six months of initial clinical and laboratory follow-up were included in the study.

Exclusion criteria: Cases that were not on regular follow-up and non compliant patients were excluded from the study.

Study Procedure

The HLA typing was done by Sequence Specific Primer (SSP) method in the laboratory for all patients (9). For haploidentical match transplants the pretransplant immunological monitoring also included performing screening of serum Panel Reactive Antibodies (PRA) in the recipient for Class-I and Class-II HLA antigens by flow cytometry and further using luminex platform for SAB assay for detecting donor specific antibodies. Blood and bone marrow samples were taken at various time periods after transplantation and tested for chimerism using Amp FLSTR Identifiler kit (Applied Biosystems). STR-PCR chimerism for engraftment purpose was performed every 30 days for first six months, then at three monthly intervals for two years post-transplant and six monthly thereafter. This time interval protocol was strictly adhered especially, in cases of haematologic malignancies.

Conditioning: Intravenous infusion of cyclophosphamide, Fludarabine, busulphan, Antithymocyte Globulin (ATG) and melphalan were used as chemotherapeutic agents in different combinations and dosages. ATG was given primarily to patients with mismatched donors and to MUDs. Post-transplant IS consisted of cyclosporine in combination with short course of methotrexate, mycophenolate mofetil. In cases of haematological non malignant conditions who undergo RIC, the immunosuppressive agents were continued for one year as against malignant cases where the drugs were tapered over a period of three months. So as part of conditioning regime, the more cytotoxic drugs were replaced by less toxic agents that enabled donor engraftment. In absence of GVHD, post-transplant IS was tapered from three months onwards. Patients who had an episode of GVHD thereafter were treated with prednisolone and resumption of full dose IS, if applicable. Patients with evidence of impending relapse detected by increasing mixed recipient chimerism were initiated on chemotherapeutic drugs along with DLI.

Chimerism testing:

a) Isolation of genomic DNA: DNA was isolated from peripheral blood or bone marrow sample of recipient and donor before the transplant using a Qiagen kit (Qiagen, CA). The column exchange DNA preparation method ensured high quality DNA devoid of PCR inhibitors. DNA quantification was performed using spectrophotometry. Post-transplant, DNA was extracted from recipient at frequent intervals to determine chimerism status.

b) Short Tandem Repeat (STR-PCR): The method is based on discrimination of donor and recipient alleles using a set of 15 STR-PCR exhibiting a high degree of size polymorphism. PCR was performed using AmpFLSTRIdentifiler kit (Applied Biosystems, Foster city, CA) according to the manufacturer instructions. All STR markers were amplified in a multiplex PCR reaction (Thermocycler ABI). One μL of PCR product was added to 09 μL deionised formamide/gene scan-500 (LIZ) as per manufacturers protocol, denatured at 95° for two minutes and placed on ice for atleast one minute before electrokinetic injection on the ABI3100 OR 3130 capillary electrophoresis instrument.

c) Quantification of donor chimerism: After electrophoresis, the fragments were analysed using Gene mapper software. Post-transplant chimerism value was calculated using average of atleast three informative STR markers [10,11]. The chimerism values were estimated from observed peak areas of the informative markers (12).

Statistical Analysis

Categorical variables were summarised as percentages. Data in tables were presented as median values and range (minimum-maximum) or as absolute numbers.

Results

The patient demographic profile is detailed in (Table/Fig 1). The median age was 18.5 years (range 02-47). Sixteen (40%) were sex mismatch HSCT was performed at this centre. Thirty-one patients were male (77.5%%) and nine were female (22.5%).Twenty six cases were of haematologic malignancies (65%) and 14 of non malignant conditions (35%) who underwent stem cell transplant.

HLA matching: Complete HLA match at HLA-A, B DR locus occurred in 35 (87.5%) patients for whom matched related donor transplant was performed. Two cases were of HLA MUDs (5%) and three cases (7.5%) of haploidentical transplant for whom the donor was from within the family. The two successful MUD cases, was a three-year-old male child suffering from severe combined immunodeficiency disorder and other a case of Acute Myeloid Leukaemia (AML). Two haploidentical transplant cases included were diagnosed patients of WiskottAldrich syndrome and third a case of AML. The AML haplotransplant patient relapsed after three months whereas the two patients with underlying primary immunodeficiency disorder suffered episodes of GVHD.

Chimerism status/Prevalance of chimerism: The follow-up chimerism status for all cases was atleast initial six months. The median Day+30 whole blood chimerism level was 99.6% (range 87-100%) and 96.75% (87-100%) for haematologic malignancies and non malignant conditions, respectively, whereas the D+90 values were 74.8% (range 88-99.2%) and 99% (range 81-99%) (Table/Fig 2). Complete donor chimerism at D+90 was reported in 19 patients (73%) of haematologic malignancies with two cases of relapse and five showing evidence of GVHD. Amongst non malignant disease category, 10 patients (71.4%) showed complete donor chimerism at D+90 with two cases affected with GVHD. Follow-up and correlation with MRD levels was done only in two haematological malignancy cases with disease relapse.

Highlighted, here is the longitudinal follow-up of chimerism pattern as observed and its clinical implication in two pertinent cases from the study cohort. As is seen in (Table/Fig 3) the graph shows increasing mixed recipient chimerism values in a patient of AML who despite chemotherapeutic therapy, subsequently relapsed. Correlation with MRD was suggested ongoing relapse though his bone marrow aspirate was in morphological remission. Patient was treated with azacytidine, chemotherapeutic agent, to which he did not respond.

The second case was of haemoglobinopathy (Thalassaemia) (Table/Fig 3)a who throughout longitudinal follow-up on chimerism maintained a state of stable mixed status with periods of complete donor chimerism. There was no evidence of increasing recipient lineage cells and patient remained symptom as well as transfusion free.

Discussion

Allogenic stem cell transplantation is currently a well-established procedure for treatment of various malignant and non malignant haematologic diseases. It not only helps in restoring normal haemopoiesis but is also the preferred therapeutic modality for malignant disorders due to graft versus leukaemia effect where donor T cells show immune reactivity against the host leukaemic cell epitopes (13). Chimerism analysis by STR-PCR method helps to determine the genetic origin of haematopoietic cells in patient circulation, post-transplant (14),(15). It is one of the most significant tools for monitoring donor engraftment. In this study, authors evaluated the engraftment status of 40 cases with diverse haematological disorders (malignant and non malignant). The chimeric status helped predict relapse in a case of AML showing no overt clinical evidence. Gambacorta V et al., in their study have shown that reappearance of host specific haematopoietic chimerism has been associated with relapse (16). If performed at frequent intervals in post-transplant period it helps to predict disease relapse, level of engraftment and information on GVHD/GVT effects (17). This may prevent catastrophic event by timely therapeutic intervention either by reduction of IS, use of immunotherapy (DLI) or use of chemotherapeutic agents like azacitidine (18). In malignant disorders, the chief concern is to maintain a balance between graft failure or disease relapse and GVHD by close laboratory monitoring. In non malignant disorders, timely monitoring helps to prevent graft failure by optimisation of IS therapy. In the 14 cases of non malignant diseases, authors observed that a stable mixed chimerism status was adequate to control the underlying disease. This was contrary to haematological malignancies where complete donor derived haematopoiesis is desirable for cure (19). Persistence of host haematopoietic lineage cells in malignancies is definitively suggestive of relapse or impending relapse. However, in non malignant diseases where GVL has no significance the main purpose is to lower risk of GVHD. Seven patients with 100% donor chimerism were found to have GVHD. Hence, a stable mixed chimerism (where both donor and host cells co-exist) is an accepted norm, provided the dynamics of chimerism status is monitored serially (20). If an increase in ratio of recipient haematopoiesis (as depicted by increasing mixed chimerism) is observed there is need for therapeutic intervention. Also, it is important to note that a stable mixed chimerism in non malignant disorders is sufficient to revert the clinical manifestations of the primary disease, as was observed in present study. Arwen S et al., in their study have demonstrated that stable long-term MC did not negatively affect patient well-being and long-term outcome (21). Few of present study Thalassaemia and primary immunodeficiency disorder patients post-transplant remained in good health suggestive of immunologically synergistic environment between donor and recipient cells.

Chimerism needs to be measured longitudinally at frequent intervals to accurately predict disease status. One isolated value has no significance as authors are required to see the increase or decrease graph of chimerism levels (22). Kristt D et al., in their review have focused on longitudinal and sequential chimerism evaluation for predicting the kinetics of each patient’s chimeric state (23). Single absolute values of percentage chimerism are not useful except for a few rare cases. In present cohort too, the longitudinal monitoring in different diseases (AML and thalassaemia) has helped to create a quantitative record of patient’s chimeric status and timely therapeutic intervention helped to prevent relapse.

As of today, various techniques are available for chimerism testing but the most robust is amplification of highly polymorphic STR-PCR informative markers. Chimerism testing has a sensitivity of only 1% when performed on bone marrow or whole blood and hence, should be supplemented with more sensitive assays for MRD detection in haematologic malignancies. The two most sensitive methods being used are multiparameter flow cytometry to detect leukaemia associated aberrant immunophenotypes and real time quantitative Polymerase Chain Reaction (qPCR) for fusion gene transcripts or clonal immunoglobulin or T cell receptor (Ig/TCR) gene rearrangements (24). These MRD assays have a sensitivity of atleast 1 to 2 log greater than chimerism by STR-PCR method (25),(26).

Also, MRD techniques detect residual disease or re-emerging leukaemia clones based on known aberrant surface or genetic markers. Chimerism on the other hand picks up recipient haematopoiesis. Despite, the known fact that patients with complete donor chimerism have shown MRD positivity, it remains one of the imperative tools to monitor donor engraftment especially in non malignant immune-haematologic conditions and in malignant diseases with no known underlying genetic aberrations (27). Hence, chimerism analysis is complementary to MRD measurement for relapse prediction after transplant in malignant conditions and serves as a surrogate in cases with extramedullary disease or leukaemia/Myelodysplastic syndrome with no known molecular markers (28),(29),(30).

In the present study, authors have proposed a diagnostic algorithm for monitoring of post-transplant engraftment by chimerism assay in a transplant and immunogenetics laboratory (Table/Fig 4). The algorithm is interpretation of chimerism levels reported in HSCT patients and a step-wise logical correlation with clinical parameters and MRD assays for predicting disease outcome. The sensitivity of the above technique can be increased substantially to 0.1% by using lineage specific leucocyte subsets for testing. These cell subsets may be enriched for cells that may contain MRD. This especially applies to high risk cases when other suitable disease markers are not available for monitoring MRD (31). By employing variant-allele-specific quantitative PCR-based approaches to detect small DNA insertions or deletion sensitivity can be further increased to a level of 0.01-0.001% (32).

Limitation(s)

Present study included a heterogenous group of haematological disorders, however the limitation was small sample size and short follow-up time of six months of the cases.

Conclusion

Chimerism analysis is an indispensable technique for routine post-HSCT monitoring of donor engraftment. It is especially valuable in setting of RIC as well as patients with no underlying MRD molecular marker. At times, regular time bound chimerism analysis along with clinical assessment may serve the purpose of predicting disease relapse earlier than MRD. Each laboratory should formulate standard operating procedures and guidelines for judicious use of the assay and increasing the test robustness and result reliability. Nonetheless, communication between clinicians, haematologists and immunopathologist accompanied by a methodical step-wise laboratory diagnostic approach can facilitate early relapse diagnosis and timely therapeutic intervention.

Acknowledgement

The authors thank the study subjects, for providing samples and supporting this work; the participating clinical staff, for their dedication to this research; A special thanks to Mr. B. Saravanan and Mr. Vineeth Kumar S. (Department of Laboratory Medicine and Molecular Sciences), Army Hospital Research and Referral, for their help in processing of samples for chimerism assay by STR-PCR method.

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DOI and Others

DOI: 10.7860/JCDR/2023/58099.17971

Date of Submission: May 31, 2022
Date of Peer Review: Jul 22, 2022
Date of Acceptance: Jan 23, 2023
Date of Publishing: Jun 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Jun 20, 2022
• Manual Googling: Dec 07, 2022
• iThenticate Software: Dec 10, 2022 (7%)

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