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

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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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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.
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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.
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Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

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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 : January | Volume : 17 | Issue : 1 | Page : EC21 - EC24 Full Version

Morphological Characteristics, Immunophenotyping and Cytogenetics in Acute Myeloid Leukaemia Patients at a Tertiary Care Centre, Gujarat, India


Published: January 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/59299.17423
Rahul Ranka, Beena Brahmbhatt

1. Senior Resident, Department of Oncopathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India. 2. Assistant Professor, Department of Oncopathology, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India.

Correspondence Address :
Beena Brahmbhatt,
100 Shivganesh Bunglows Zydus Hospital Road SG Highway,
Thaltej-380059, Ahmedabad, Gujarat, India.
E-mail: beena.brahmbhatt@gcriindia.org

Abstract

Introduction: Acute Myeloid Leukaemia (AML) is a group of disorders characterized by a spectrum of clinical, morphological, immunophenotypic and associated chromosomal abnormalities. The identification of cytogenetic abnormalities at diagnosis is important for the evaluation of the response to therapy and the identification of an early re-emergence of disease.

Aim: To examine the morphological characteristics of AML and emphasize the role of immunophenotyping and cytogenetics in subtyping AML.

Materials and Methods: The cross-sectional study was a prospective study that took place from June 2018 to November 2021 at a tertiary-care cancer center, Department of Oncopathology in Gujarat, India. Following the inclusion and exclusion criteria, a total of 232 patients were diagnosed, with 21 being eliminated owing to the unavailability of samples for cytogenetic testing. Diagnosis of AML was based on morphology of Bone Marrow (BM) aspirates and flow cytometric immunophenotyping. Chromosomal analysis was performed on BM and peripheral blood by using standard cytogenetic technique.

Results: There were 115 (54.50%) males and 96 (45.49%)females with age group between 1-72 years. The most common subtype was diagnosed as AML M1. Flow cytometry was done on 178 (84.36%) bone marrow and 33 (15.63%) peripheral blood samples. The CD33, CD13, MPO and CD117 were expressed in the vast majority of AML patient, aberrant expression of CD7and CD19 was seen. Total case 121 (57.34%) had normal karyotypes (the majority of cases), 86 (40.75%) cases had anomalous karyotypes consistent with t (8;21), t (15;17), and Inv16, and 4 (1.89%) cases were non informative.

Conclusion: The study concluded that flow cytometry and cytogenetics should be performed routinely in all cases of AML. A multimodal diagnostic approach combining cytomorphology, multiparametric flow cytometry accompanied by cytogenetic is needed to arrive at definitive diagnosis of AML.

Keywords

Flow cytometry, Fluorescent in situ hybridisation, Karyotype

In terms of clinical characteristics, morphology, immunophenotype, and molecular genetics, AML is a heterogeneous disease (1). Based on morphological characteristics, blast proportion, blast maturation, and cytochemistry, the FAB (French-American-British) classification method divides AML into eight subtypes (1). Several more subgroups defined by recurring chromosomal abnormalities have been identified as a result of the development of immunophenotyping, cytogenetics, and molecular genetics (2),(3). As a result, AML is divided into six major groups by the World Health Organization (WHO) (4).

The diagnosis and classification of acute leukaemia begins with morphology, which may be complemented by cytochemistry. Multiparameter flow cytometry is the preferred method for immunophenotypic analysis in AML because it can analyse a large number of cells in a short amount of time while simultaneously recording information about several antigens for each individual cell. At the time of diagnosis, cytogenetic and molecular genetic tests are required not only to identify specific genetically defined entities, but also to provide a baseline against which follow-up studies can be read to assess disease progression and prognosis (5),(6),(7).

Research efforts in the last decade have expanded the pathophysiologic molecular subsets of AML, through identification of prognostic, predictive and targetable molecular abnormalities (8),(9).

This research examines the morphological characteristics of AML and emphasises the role of immunophenotyping and cytogenetics in subtyping AML and provide prognostic importance for illness prediction.

Material and Methods

The current study was a cross-sectional study that took place from June 2018 to November 2021 at a tertiary-care cancer center, Department of Oncopathology in Gujarat, India.

Inclusion and Exclusion criteria: All adult and paediatric patients having a complete blood count, peripheral smear, bone marrow aspirate showing any blast cell population, as well as samples for immunophenotyping and cytogenetics, met the inclusion criteria. Patients without sufficient samples or data were excluded. A total of 232 patients were diagnosed, with 21 being eliminated owing to the unavailability of samples for cytogenetic testing.

All of the specimens were acquired using conventional procedures. The Complete Blood Count (CBC) was performed using the BC 6800 Plus Mindray cell counter (7 parts) and the LH 750 Beckman coulter (five parts). Peripheral Smears (PS) and bone marrow aspirate smears were stained with the Wright stain.

The revised 2017 edition of the World Health Organization’s haematopoietic and lymphoid tissues was used to report AML diagnosis and subtyping (4).

Study Procedure

Total 178 bone marrow and 33 peripheral blood samples were collected in a Potassium Ethylenediamine Tetraacetic Acid (K2EDTA) vial for immunophenotyping. Total 100 μL of whole blood and/or bone marrow samples were obtained for the immunophenotyping assay and treated with the appropriate panel of AML antibodies for 10 minutes at room temperature (CD45, CD117, CD33, CD13, CD15, HLADR, CD14, CD34, and others). Following incubation, the cells were treated with an erythrocyte lysing solution (1:10) and incubated at room temperature for 10 minutes. The White Blood Cells (WBCs) were pelleted and washed twice with 2 mL of sheath fluid after centrifugation at 1000 rpm for five minutes.

Before being acquired, the final pellet was resuspended in a 500 μL Phosphate Buffered Saline (PBS) solution. On a Fluorescence Activated single Cell Sorting (FACS) Canto II eight-color flow cytometer with BD Diva software, data was collected and analysed (6),(7). Blasts were subtyped based on marker expression and side-scattered versus CD45 plots.

Traditional cytogenetic and Fluorescent In Situ Hybridization (FISH) techniques were used to analyse chromosomal data from pre treatment BM. For traditional cytogenetics, at least 20 metaphases were examined, and 200 cells in interphase were examined using FISH. The worldwide system of cytogenetic or cytogenetic nomenclature was used to report the diagnoses {International System for Human Cytogenomic Nomenclature (ISCN)} (7).

Statistical Analysis

Descriptive statistics were used to analyse the data and the data was represented as number and percentages.

Results

Adults accounted for 169 (80.09%) cases and children accounted for 42 (19.90%) cases, with the age range ranging from 1 to 72 years (median 37 years).There were 115 (54.50%) males and 96 (45.49%) females among the 211 cases. The M:F ratio of 1.2:1 showed a male preponderance.

Haematological findings: The average Haemoglobin (Hb) level was 7.5 gm/dL, accounting for 86% of cases and normal Hb levels accounting for the remaining instances. A total leucocyte count of more than 11,000/cumm was found in 60% of the patients, whereas leucopoenia (4000/cumm) was found in 31%. Seventy percent of the people had moderate thrombocytopenia (platelet counts greater than 20,000×103), while the rest had normal platelet counts.

Most common type was AML M1 accounting for 61 (28.90%) of the cases and AML M2 contributing to 56 (26.54%).

Flow cytometry: It was done on 178 (84.36%) bone marrow and 33 (15.63%) peripheral blood samples. The following antigens were the most commonly expressed

• Myeloid markers CD33, CD13, MPO, and CD117 are expressed in the vast majority of AML patients, with frequencies of 80.5% (170), 90.04% (190), 78.19% (165), and 81.99% (173), cases respectively. A significant proportion of AML M5 cases, 39.39% (13) were reported as MPO negative.
• Human Leukocyte Antigen (HLA)-DR and CD34 were expressed at a rate of 151 (71.56%) and 181 (85.78%), respectively, in stem/progenitor cells. Five of the 34 AML M3 cases expressed CD34, while one expressed HLA-DR.
• Aberrant expression of CD7 was the most commonly expressed lymphoid marker 48 (22.74%) in AML patients, followed by CD19 15 (7.1%) (Table/Fig 1).

Cytogenetics: In 211 cases of AML, karyotype tests revealed that 121 cases (57.34%) had a normal karyotype, 86 cases (40.75%) had an aberrant karyotype, and four cases (1.89%) were non informative. The AML M2, AML M3, and AML M4 were reported to have karyotype aberrations consistent with t (8;21), t (15;17), and Inv16, respectively. The relationship between morphology (FAB subtype) and immunophenotyping and cytogenetics is summarized in (Table/Fig 2).

All the findings of three methods ie blood smear, flowcytometry and FISH technique is depicted in (Table/Fig 3).

Discussion

The 211 cases of AML were diagnosed by cytomorphology, flow cytometry, and cytogenetics in our analysis included 19% of childhood cases and 81% of adult cases, which differed somewhat from Bashrat M et al., (10) findings (27.3, 72%, respectively). In contrast, paediatric cases were 24% and 76% in research by Ghosh S et al., (11). AML M1 (28.90%) was the most common subtype in this study, which is similar to Faleh AA et al., (12) study (AML M1=86%). In contrast, the M2 subtype predominated in a study by Bashrat M et al., (10) and Ghosh S et al., (11). (47.2% and 34.3%, respectively).

The use of flow cytometry in the diagnosis and subclassification of AML is essential. Flow cytometric immunophenotyping offers the advantage of great sensitivity and efficiency when using a multiparametric method.

Each FAB subtype has distinct immunophenotypes, including AML M3, which can be distinguished from other AML FAB subtypes. Five out of 34 cases of AML M3 in our investigation exhibited CD34, which is similar to the findings of Faleh AA et al., (12) and Zheng J et al., (13) (1/4,3/31, respectively). The AML-M4 and other the AML subtypes have varying levels of CD14 and CD15 expression. Strong CD64 expression distinguished AML M5 from M0 to M4 subtypes. However, dim or moderate CD64 expression did not distinguish M0 to M4 subtypes from M5 (5). However, in the study by Dunphy CH (14), any CD64 expression associated with strong CD15 expression distinguished AML M4 or M5 from other AML subtypes, which was a similar finding in our study.

The presence of CD41, a marker for megakaryocytic lineage, in AML-M7 (5) was consistent with Faleh AA et al., findings (12). In our study, CD7, a T cell antigen known to have aberrant expression, was the most commonly expressed 48 (22.74%), followed by CD19 15 (7.10%). The same pattern was observed in studies conducted by Faleh AA et al., (12) (10%, 8%), Zheng J et al., (13) (14%, 10%), and Bashrat M et al., (10) (26.4%), 1.2%).

Although morphological evaluation of BM aspiration and biopsy is still important for AML diagnosis, the presence or absence of specific cytogenetic abnormalities and acquired genetic mutations is clearly a cornerstone in predicting prognosis (favorable, intermediate, and unfavorable risk groups) and treatment (15),(16).

Chromosomal abnormalities were found in 39.6%of AML cases in the present study. Balanced translocation was found in proportions comparable to other large series and was found to be correlated with FAB subtypes. The t (15;17) was observed in 13.27% of the cases in this study, whereas it was observed in 7% of the cases in Faleh AA et al., (12), 14.3% of Cheng’s Y et al., (17), and 11% of Enjeti’s AK et al., (18) studies.The benefit of cytogenetic analysis is that it has the fundamental potential to detect structural or numerical anomalies, as well as unique and uncharacterized abnormalities. Total 10% of AML patients have chromosomal abnormalities. The relevance of cytogenetic abnormalities and multilineage dysplasia in leukaemia subtyping was also highlighted in a recent WHO classification (19),(20),(21).

In AML M3, our study found variant abnormalities such as {t (15;17) and t (15;11)} (0.5%) as well as additional chromosomal abnormalities such as [t (15;17), trisomy 8] (1%) and [t (15;17), trisomy 21] (0.5%). Faleh AA et al., (12) and Cheng Y (17) found an incidence of t (8.21%) in their AML patients (8.0% and 8.3%, respectively), which was confirmed in our study (7%). In comparison to Ahmed’s study, we found a higher incidence of AML with Inv 16 (8%) in our study (1%). The comparision of Karyotypic pattern in AML patients in the present study with various previous study has been shown in (Table/Fig 4) (12),(17),(18),(22),(23),(24).

Limitation(s)

Limitation of study was the fact that molecular testing could not be performed due to non availability at the institute.

Conclusion

Flow cytometry and cytogenetics should be performed routinely in all cases of AML. Morphology combined with cytochemistry are used to reliably diagnose acute myeloid leukaemia. Flow cytochemistry is a very effective method for diagnosing and subclassifying AML efficiently and precisely.Cytogenetic analyses aid in the biological classification of AML and should be performed routinely in all cases of AML as required for risk stratification (prognostic index).

References

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Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposals for the classification of the acute leukaemias. French-American-British co-operative group. Br J Haematol. 1976;33:451-58. [crossref] [PubMed]
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Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative group. Ann Intern Med. 1985;103:620-25. [crossref] [PubMed]
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Steven H, Elias C, Nancy LH,WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. Acute Myeloid leukaemia.ed 2017:10-17.
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Kottler M.Cytological technique,preconception and the counting of the human chromosomes.Bull.Hist.Med. 1974;48:455-02.
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Liqaa M, Comparison between Morphological Aided Cytochemical Stains(Sudan Black B, PAS) and Flowcytometry in Diagnosis and Classification of Acute Leukaemia. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2016;7.
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Knapp W, strobalh,Majdic O, Flow cytometric analysis of cell-surface and intracellular Antigens in leukemiadiagnosis.cytometery. 1994;18:187-98. [crossref] [PubMed]
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DOI and Others

DOI: 10.7860/JCDR/2023/59299.17423

Date of Submission: Jul 26, 2022
Date of Peer Review: Sep 03, 2022
Date of Acceptance: Nov 22, 2022
Date of Publishing: Jan 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? No
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Jul 31, 2022
• Manual Googling: Nov 03, 2022
• iThenticate Software: Nov 21, 2022 (8%)

ETYMOLOGY: Author Origin

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