held on 6 -7 January 2012 on ‘Biotechnology in Diagnostics’vpmthane.org/BNB/Seminar/Biotechnology conf proceeding.pdf · held on 6th-7th January 2012 on ‘Biotechnology in Diagnostics

I

Proceedings of National Conference

held on 6th -7th January 2012 on

‘Biotechnology in Diagnostics’

Organized by

Department of Biotechnology and Microbiology,Vidya Prasarak Mandal’s

B. N. Bandodkar College of Science,NAAC reaccredited ‘A’ grade

Best College Award, University of Mumbai

Edited By

Mrs. Jayashree PawarAssist. Prof. Dept of Biotechnology and Microbiology,

B. N. Bandodkar College of Science, Thane

®

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Please Note:The Authors of the papers are aloneresponsible for technical content of thepapers and references cited therein

Published by :Department of Biotechnology and MicrobiologyVPM’s B.N. Bandodkar College of Science“Jnanadweepa”, Chendani, Bunder Road,Thane (W) 400 601. MaharashtraTel. : 2533 6507E-mail: [email protected]

Printed atPerfect Prints22, Jyoti Industrial Estate,Nooribaba Darga Road, Thane 400 601.Tel. : 2534 1291 / 2541 3546Email : [email protected]

Advisory Panel:Dr. Siddhan Subramanium

Dr. Sudhakar Agarkar

Dr. P. M. Kelkar

Dr. V. V. Bedekar

Dr. Meghana Joshi

Dr. Kalpita Mulye

Convenor:Dr. (Mrs.) M. K. Pejevar

Program Committee

Teaching Staff Non-teaching StaffDr. (Mrs.) M. K. Pejevar Ms. Priyaka Manore

Dr. Vijaya Srinivasan Mrs. Vasudha Shetye

Dr. Meghana Joshi Mr. Sachin Adivarekar

Dr. Kalpita Mulye Mr. Sunil Babar

Mrs. Jayashree Pawar Mr. Jitendra Khairnar

Mrs. Nilambai Daripkar

Ms. Rajitha Radhakrishnan

Ms. Sonal Mathias

Dr. Ashwini Pande

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From the Convenors deskFrom the Convenors deskFrom the Convenors deskFrom the Convenors deskFrom the Convenors desk

Biotechnology and Microbiology departments are comparatively younger departments of our college.In comparison with other colleges of University of Mumbai we were late to enter in the field. But withthe inception of this department, we started with many innovative methods for students’ interaction.

We run a movie club in which short movies are shown to the students which help in understandingthe subject better. Students also get acquainted with the working environment of laboratories.

Students are given small research projects, due to which the students get acquainted with the researchmethodologies and get training in the laboratory techniques.

We run a journal club where the students have to go through the research journals including journalslike Lancet and have to discuss the research papers published in the journals. This not only gives a deepinsight, but also introduces the research aptitude in students.

With these practices we also thought of conducting the National Conference on a very importanttopic ‘Biotechnology in Diagnostics’. Keeping in mind the field of human health which is dependent onnew Biotechnological tools, the conference will be helpful for the students to get a chance of interactionwith the experts in the field. This will help students to choose their path in the research career if theywish to.

I am very happy as a convener of the conference and principal of the college to hand over theseproceedings in your hands.

Dr. (Mrs.) M. K. PejavarPrincipal,


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Messege From The Department In-ChargeMessege From The Department In-ChargeMessege From The Department In-ChargeMessege From The Department In-ChargeMessege From The Department In-Charge

Our Biotechnology and Microbiology departments are fairly new and I am glad to be a part of it.The chairman of Vidya Prasarak Mandal and the Principal encouraged me to put my ideas into practicesuch as journal club, movie club, guest seminars and I hope the students will benefit by it in the comingyears.

This is the first national-level conference organized by our department and we hope to engage inmany more such initiatives that will bring together academicians, scientists, students and industryprofessionals at our VPM campus.

The diagnostic market in India is one of the most profitable markets in the world. The market willcontinue to grow in the coming years with increasing disease prevalence and patient awareness. Wehave players innovating in diagnostics ranging from point of care diagnostics to basic research supportingdiagnostics.

I hope this conference exposes the students to the field of diagnostic research and creates enoughinterest for them to pursue it to later on commercialize diagnostic technology.

Dr. Meghana JoshiIn- Charge,

Department of Biotechnology and Microbiology.B. N. Bandodkar College of Science, Thane

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Editor’s NoteEditor’s NoteEditor’s NoteEditor’s NoteEditor’s Note

This publication contains the Conference Proceedings of the two-day conference on ‘Biotechnologyin Diagnostics’ held at B. N. Bandodkar college of Science, Thane, on January 6-7, 2012.

On behalf of VPM’s B. N. Bandodkar College of Science, I take this opportunity to welcome all theHonourable speakers and participants for this conference on ‘Biotechnology in Diagnostics’

Over the years, B. N. Bandodkar college of Science, Thane, has been known to pioneer efforts toprovide innovative programs in newer domains of Biotechnology. This conference is another effort toinculcate research aptitude in the students.

The theme of Conference is ‘Biotechnology in Diagnostics’. Many eminent personalities from allover India, working on different aspects of diagnostics from Academia, Industry and Research Institutions,will be presenting recent breakthroughs in diagnostics. The conference aims to open a dialogue amongstthese eminent personalities and the participants.

One of the academic objective of the conference is to give the students, an insight of the researchfield. Two preparatory workshops have been conducted as a part of the conference on 24th Septemberand 26th November 2011 at B. N. Bandodkar College of Science, Thane. We have tried to impart thebasic technical information about the subject to the students, which will widen their understanding oftoday’s conference theme.

This conference also includes poster presentations by undergraduate and post graduate students.We aim to give them a platform to interact with the scientific community and prepare them for thecompetitive world which they will be facing in near future. We got an overwhelming response fromstudents for poster presentations.

I request all of you to actively participate in this valuable discussion. I am sure that at the end of thesecond day we will have ample vital information as output of this conference for academic, industrialand research reference.

I am grateful to Vidya Prasarak Mandal, Our Principal Dr. M. K. Pejavar and our Department in-charge, Dr. Meghana Joshi for giving me this opportunity to work on the conference proceedings. Last,but not the least, I would like to thank my Departmental collegues, and the non- teaching staff for theirimmense support.

Mrs. Jayashree Pawar Assistant Prof.,

Department of Biotechnology and Microbiology.B. N. Bandodkar College of Science, Thane

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VII

Table of Contents

Section I Inaugural SessionOpening Address : Molecular medicine in the “Omics” era Dr. B. B. Chattoo ..................... 3

Key-note Address: Monoclonal Antibodies in CancerDiagnosis and Therapy Dr. S. V. Chiplunkar ................. 4

Section II Invited lecturesCopy Number Variations as new tools formolecular diagnostics Dr. Anu Ghosh .......................... 7

Nuclear Magnetic Resonance Study for Drug andBiological Molecules Dr. Ashes Ganguly ................... 8

Study of Cardiac Markers as Independent Risk Factorsfor Early Detection of Coronary Heart Disease Dr. Rekha Bhagwat .................. 9

Expressions” matter: RNAs in diagnosis andmolecular medicine Dr. Bhakti Pathak ................... 10

Application of Bioinformatics in Medical Diagnostics Dr. Suruchi Jamkhedkar ......... 11

Biosensors and biochips: advances in biological andmedical diagnostics Dr. Vandana Singh ................. 12

Pharmacogenomics: Towards Personalized Medicine Dr. Supriya Bhalerao ............. 14

Metabolomics as a tool for Diagnostics Dr. Ajit Datar ......................... 15

Section III Concluding SessionEmerging Strategies for Effective Cancer TreatmentBased on Cell Electroporation Technology Dr. K. P. Mishra .................... 19

Section IV Poster presentations

Section V Pre-conference Workshops

Conventional Diagnostic Tests Ms. Rajitha Radhakrishnsn .... 33

Immunological Diagnostics Mrs. Nilambari Daripkar ....... 33

Modern Immunodiagnostic technique Dr. Kalpita Mulye .................. 34

Molecular Diagnosics- Polymerase Chain Reaction Mrs. Jayashree Pawar ........... 34

Techniques in Animal Cell Culture Dr. Sharmila Raikar ............... 35

Molecular Diagnosics Ms. Sonal Mathias ................. 35

Diagnosis of Diabetes Dr. Sangeeta Bhagat .............. 36

VIII

National Conference ''Biotechnology in Diagnostics'', January 06-07, 2012.

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SECTION I

INAUGURAL SESSION


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Molecular medicine in the “Omics” era

Hon’ble Dr. Bharat B. Chattoo

Founder Vice Chancellor of the Shri Mata Vaishno Devi University, Jammu.Director, Genome Research Centre

Department of Microbiology and Biotechnology CentreM. S. University of Baroda, Vadodara – 390002 (Gujarat), India

Recent advances in genomics have made itpossible to analyse various aspects of genome structureand dynamics in much greater depth. It is now possibleto simultaneously monitor the expression of a largenumber of genes and to study the interaction amongvarious proteins that are critical to many cellularprocesses, using various tools involving judicious use ofgenetic systems and advances in analytical methods.Information now available from various genomesequencing projects is leading towards an increasinglyimproved understanding of the genome architecture ofvarious organisms and is providing new insights intothe evolutionary relationships among various species.The documentation of genetic polymorphisms in humansis leading towards a better understanding of howdifferent genetic differences might influencepredisposition to disease and response to various drugs.This opens up new avenues for molecular diagnostics

and identification of novel targets for drug developmenton the one hand and towards development ofpersonalized medicine on the other. Some of these toolsare also making it possible to study the vast majority ofmicroorganisms that have not been possible so far tocultivate in culture. This is providing new insights forthe study of the microbiome and its implications forhuman health. One of the major challenges that theavailability of huge amounts of data from various“Omics” projects poses is to develop innovativemethods of analysis to handle large data sets and todesign experiments so that the biological significanceof various processes can be studied experimentally.Understanding of how the instructions from the genomeand epigenome are modulated in various cellularprocesses within a cell also promises to be an excitingarea of future work.


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Monoclonal Antibodies in Cancer Diagnosis and Therapy

Prof. S. V. Chiplunkar

Principle investigation, Advanced centre for treatment, research, and education in cancer,Tata Memorial Centre, Kharghar, Navi Mumbai - 410210 India

The potential for using antibodies as ‘magic bullets’against cancer has been alluring investigators sinceKohler and Milstein described the making of monoclonalantibodies. These are invaluable tools in research,medicine, diagnosis and treatment of diseases. Themonoclonal antibody market has experienced explosivegrowth since the process for creating monoclonalantibodies was introduced. More than fifteen therapeuticmonoclonal antibodies have been approved, several ofwhich have reached blockbuster status. Numerousdiagnostic monoclonal antibodies have also beenapproved, making this one of the fastest growing fieldsin biotechnology. Today, the industry is working asfuriously as ever to perfect the design and productionof monoclonal antibodies for therapeutic, diagnostic, andother purposes. The recent clinical and commercialsuccess of anticancer antibodies such as rituximab andtrastuzumab has created great interest in antibody-baseddiagnostic and therapeutics for hematopoietic malignantneoplasms and solid tumors. Radioimaging also knownas radioimmunoscintigraphy uses radionuclide labeledantibody or antibody fragments to diagnose tumors incancer patients in an antigen dependant manner. Severalmonoclonal antibody based ELISA kits are available inthe market for diagnosis of colon, breast, and prostatecancers. Immunophenotyping of leukemias andlymphomas using monoclonal antibodies are now inroutine practice for diagnosis and prognosis of thedisease.

In cancer therapy, the purpose of antibodyadministration is to induce the direct or indirectdestruction of cancer cells, either by specificallytargeting the tumor or the vasculature that nourishesthe tumor. Traditionally monoclonal antibodies have beenproduced as native molecules in murine hybridoma lines.The development of genetic engineering has beencentral to the clinical use of antibodies. This technologyhas allowed the conversion of existing mouse monoclonalantibodies into mouse-human chimerized antibody andhumanized reagents where only the antibodycomplementarity determining regions (CDR) are ofmurine origin. More recently, the production of fullyhuman monoclonal antibody has been made using phagedisplay technology or transgenic mice. The ‘humanized’antibodies have facilitated improvement in thetherapeutic potential of monoclonal antibodies as theseare less immunogenic, more effective, and do not evokea human anti-mouse antibody response(HAMA).Therapeutic antibodies have become a majorstrategy in clinical oncology owing to their ability to bindspecifically to primary and metastatic cancer cells withhigh affinity and create antitumor effects bycomplement-mediated cytolysis and antibody-dependent,cell-mediated cytotoxicity (naked antibodies) or by thefocused delivery of radiation or cellular toxins(conjugated antibodies).


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SECTION II

INVITED TALKS


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Copy Number Variations as new tools for molecular diagnostics

Dr. Anu Ghosh

Low Level Radiation Studies Section, Radiation Biology & Health Sciences Division,Bio-Medical Group, Bhabha Atomic Research Centre, Mumbai – 400085

Email: [email protected]

Copy Number Variations (CNVs) are nowrecognized to be a highly prevalent form of geneticvariation in humans. These can be microscopic orsubmicroscopic and result when the number of copiesof a particular DNA segment shows difference betweenindividuals. CNVs may involve either copy number gains(insertions/ duplications) or losses (deletions/ nullgenotypes). De novo locus specific mutation rates forCNVs are estimated to be in the range of 1.7 x 10-6 to1.0 x 10-4 per locus per generation which is 100-1000times higher than nucleotide substitution rates. CNVsmay represent benign polymorphic variations or conveyclinical phenotypes either by disrupting genes or byaltering gene dosage. Inherited and de-novo CNVshave increasingly being linked not only to sporadic birthdefects, sporadic and mendelian diseases but also tocomplex human traits such as susceptibility to Alzheimer,Parkinson’s, mental retardation, cleft lip/palate, autism,schizophrenia, HIV infection and anatomicaldeformaties. Germline CNVs have been implicated

with increased cancer risks including neuroblastoma,breast and prostrate cancers. Numerous genomeanalysis platforms have been used for the identificationof CNVs including single nucleotide polymorphism(SNP) genotyping platforms, array comparative genomehybridization (aCGH) and next generation sequencing(NGS) platforms. Over 41% of all CNVs identifiedoverlap with known genes, emphasising the importantrole played by them in modulating expression of genes.CNVs have thus, opened up new analytical avenuesfor clinical cases which had previously eluded diagnosis.However, pathogenicity of a given CNV can be difficultto establish and the connection between genomicobservation and clinical implication is not always clear.The real challenge for laboratories and clinicianstherefore, lies in using this knowledge of CNVs notonly for prognosis but also in formulating apharmacological strategy for prevention and treatmentof genomic disorders.


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Nuclear Magnetic Resonance Study for Drug and Biological Molecules

Dr. Ashes Ganguly

Cryogen Instruments India Pvt. Ltd.Thane – 400602, MH. India

(Website: www.cryogen.co.in)

Nuclear Magnetic Resonance (NMR) techniqueis widely used for structural elucidation for drug andbiological molecules. For the molecular weight of upto>2000, the medium field NMR (1H/13C) are used upto400MHz (9.4 T) or so. But the molecular weight morethan 2000 i.e even upto 10-15 Kilodaltons, the high fieldNMR (13C/15N/31P) like from 500MHz (11.7 T) to900MHz (21.2 T) are generally used, now a days.

In general, the biological molecules are beingcomplex as the sizes are bigger, so, the interpretation

of structural elucidation are also bit tedious. In thesecases high field NMR with special techniques and cryoprobes are being used based on the nature, size andcomplexity of molecules.

In the coming days, the work in above directionwill be continued with new sequences of biologicalrelevance using a variety of NMR techniques,computational methodologies with high field NMR.


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Independent Risk Factors for Early Detection of Coronary Heart Disease

Dr. Rekha Bhagwat

Associate Professor, Department Of Biochemistry,Padamashree Dr. D. Y. Patil Medical College, Nerul

In Indian population highest numbers of patientsare suffering from Coronary heart disease (CHD). Itis projected to be 40 million Indians may suffer due toCoronary heart disease by the year 2020. Present studyrevealed that conventional markers for CHD areinsufficient to predict the risk. The measurement of hs-CRP and Lp-PLA

2 level & clinical indication may

predict as a diagnostic marker for early detection ofcoronary heart disease. The present study was carriedat Padamashree Dr. D. Y. Patil Hospital & ResearchCentre, Navi Mumbai. Fasting blood samples werecollected from male and female patients attending

Cardiology & Diabetic Clinics. The study was designedbetween the different groups as diabetic, diabetic withhypertension and Myocardial Infarction & normalhealthy individuals between the ages 25-60 years. Itwas concluded that hs-CRP and Lp-PLA

2 level

increased significantly in diabetic, diabetic withhypertension and MI, though their lipid levels werewithin normal range.

Key words: hs-CRP- Lp-PLA2

– diabetes,diabetes with hypertension - lipid profile- myocardialinfarction


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Expressions” matter:RNAs in diagnosis and molecular medicine

Dr. Bhakti Pathak

Scientist C, Structural Biology, NIRRH, Mumbai.

Apart from acting as an intermediate moleculelinking the genome and the proteome, Ribonucleic acid(RNA) is also performing an important task ofcontrolling the genome and the proteome. Only a smallfraction of the RNA species found in eukaryotic cellshas protein-coding function- the traditional role for RNA.During the past decade, a multitude of RNAs arisingfrom the huge non-coding part of the genome wasdiscovered to exert regulatory functions of fundamentalimportance to maintain normal cell function.Understanding this regulatory role of RNA in geneexpression is gaining importance and is being exploitedin the field of molecular medicine.

At the same time RNAs as messengers of geneticinformation for protein synthesis, are being utilized forthe diagnostic purposes in cases where detection of

the key protein is not possible. Specific examples withreference to prostate cancer diagnosis would bediscussed in detail. From the point of regulation of geneexpression, discovery of RNAi technology has not onlyprovided a tool for fundamental loss-of-function studiesbut it has also opened up new possibilities in the field ofmolecular medicine. The rational design of RNAsequences that can specifically block the expression ofselected genes responsible for various diseases, includingcancer, viral infection and genetic disease has receivedmajor attention in recent years. Multiple challenges, suchas optimization of selectivity, stability, delivery, and long-term safety, have to be addressed in order for ‘RNAdrugs’ to become successful therapeutic agents.Progress made in this field will be reviewed with someexamples.


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Application of Bioinformatics in Medical Diagnostics

Dr. Suruchi Jamkhedkar

B-55/535, MIG Colony, Bandra (E), Mumbai 400051, IndiaEmail: [email protected]

Bioinformatics is an interdisciplinary field, whichencompasses branches like biology, computer science,engineering, information technology, statistics andmathematics. It is science of managing and analyzingvast biological data using advanced computationaltechniques.With the advent of Bioinformatics the basicconcept of Central Dogma has changed from traditional:Gene to mRNA to Protein to newer version of Genometo Transcriptome to Proteome with direct applicationto bench research work.

In 2001, an NIH working group standardized thedefinition of a biomarker as “a characteristic that isobjectively measured and evaluated as an indicator ofnormal biological processes, pathogenic processes, orpharmacologic responses to a therapeutic intervention”.In the post- genome era, efforts are focused onbiomarker discovery and the early diagnosis of a diseasethrough the application of various omics technologies

like transcriptomics, proteomics, metabonomics (geneticand metabolic network)- on body fluids and tissuesamples. For biomarker development, bioinformaticstools are required to extract the diagnostic or prognosticinformation from the complex data like cell expressionprofile, microarray and high- through output sequencingdata. Thus, medical applications of Bioinformatics wouldbe in:

(a) understanding genetic diseases like cysticfibrosis, sickle cell anemia (SNPs), cancer,cardiovascular diseases

(b) gene therapy

(c) pharmacogenetics and pharmacogenomics topersonalize drugs for better bioavailability, todesign new and better drugs, to develop betterdrug delivery system.


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Biosensors and biochips: advances in biological and medical diagnostics

Dr. Vandana Singh

Faculty, Department of Biochemistry, Govt. Holkar Science College, Indore (M.P.).

The development of biochips is a major thrust ofthe rapidly growing biotechnology industry, whichencompasses a very diverse range of research effortsincluding genomics, proteomics, and pharmaceuticalsetc. The merging of semiconductor industry andbiotechnology has enabled biotechnologists to beginpacking their traditionally bulky sensing tools ofbiotechnology into smaller and smaller spaces, onto so-called biochips. Easy sample preparation, standardoperating protocols and a portable biochip reader makethe system suitable for use in the various fields.Advances in these areas are giving scientists newmethods for unraveling the complex biochemicalprocesses occurring inside cells, with the larger goal ofunderstanding and treating human diseases.

A biosensor can be generally defined as a devicethat consists of a biological recognition system, oftencalled a bioreceptor, and a transducer. In general, abiochip consists of an array of individual biosensors thatcan be individually monitored and generally are usedfor the analysis of multiple analytes. The interaction ofthe analyte with the bioreceptor is designed to producean effect, such as an electrical signal. Biosensors andbiochips can be classified either by their bioreceptor ortheir transducer type. A bioreceptor is a biologicalsystem (e.g., cells, tissue, or whole organisms) that utilizea biochemical mechanism for recognition. The samplingcomponent of a biosensor contains a bio- sensitive layer.The layer can either contain bioreceptors or be madeof bioreceptors covalently attached to the transducer.The most common forms of bioreceptors used in

biosensing are based on 1) antibody/antigen interactions,2) nucleic acid interactions, 3) enzymatic interactions,4) cellular interactions and 5) interactions usingbiomimetic materials (i.e., synthetic bioreceptors).Fortransducer classification, conventional techniquesinclude: 1) optical measurements 2) electrochemical and3) mass-sensitive measurements.

Biosensors have seen a wide variety ofapplications primarily in two major areas, environmentalsensing and biological monitoring. Biosensors arepowerful tools aimed at providing selective identificationof toxic chemical compounds at ultra trace levels inindustrial products, chemical substances, environmentalsamples (e.g., air, soil, and water) or biological systems(e.g., bacteria, virus, or tissue components) forbiomedical diagnosis.

Cancer Diagnosis :-Biochips can detect cancersbefore symptoms develop.Laser-induced fluorescence(LIF) is used for in vivo cancer diagnosis (LIF).

Biochip Array Technology is used for BloodAnalysis as well as in detection of blood Glucose level.

The biodetectors in the SIMBAS (Self-poweredIntegrated Micro fluidic Blood Analysis System)chipprovided a readout of the biotin levels in 10 minutes.The biochip system can identify infectious diseasestrains in less than 15 minutes when testing proteinarrays and in less than two hours when testing nucleicacid arrays.


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Diagnosis of ASPV (Apple stem pitting virus):- Adirect method of detecting plant viruses using anaptamer based biochip. ASPV is a worldwide virus thathas been associated with complex growth disorders infruits.

Biochip technology is a powerful tool for studyingthe specificity and pathogenesis of autoantibodyresponses in autoimmune diseases.

Routine analytical measurement of folic acid, biotin,vitamin B12 and pantothenic acid as an alternative tomicrobiological assay.

Determination of drug residues in food, such asantibiotics and growth promoters. Drug discovery andevaluation of biological activity of new compounds.

The biochip offer a chance to determine the“signatures” of weaponizable biological agents, mostnotably anthrax.

Biochips technology are most attractive in thearea of human health. Biochips enable researchers toquickly screen large numbers of biological analytes fora variety of purposes, from disease diagnosis todetection of bioterrorism agents.


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Pharmacogenomics: Towards Personalized Medicine

Dr. Supriya Bhalerao, Renuka Munshi, Falguni Panchal

Dept. of Clinical Pharmacology, TNMC & BYL Nair Ch. Hospital

Technology and resources promoted by the HumanGenome Project have profound impacts on biomedicalresearch. As a major outcome of this research, a newbranch of clinical medicine, Pharmacogenomics, acombination of functional genomics and molecularpharmacology, has been emerged recently.Pharmacogenomics is defined as the study of the effectof variation in multiple genes that influence drugmetabolism and response.

There are often large differences amongindividuals in the way they respond to medications, bothin terms of efficacy and safety. Although there can bevarious potential causes for this variability in drug effectsviz. nature and severity of the disease being treated,individual’s age and race, organ function, druginteraction and concomitant illnesses etc., geneticvariation in drug metabolizing enzymes is one of themajor causes of inter-individual variation of drugeffects.

Genetic variations i.e. genetic polymorphisms ofdrug-metabolizing enzymes give rise to distinctsubgroups in the population that differs in their ability toperform certain drug biotransformation reactions.Polymorphisms are generated by mutations in the genescoding these enzymes, which lead to decreased,increased, or absent enzyme expression or activity bymultiple molecular mechanisms.

Since Pharmacogenomics aims at discoveringassociation between specific genotypes and phenotype(drug metabolism), it helps physicians to select a rightdrug and right dose for an individual patient. Thisultimately facilitates effective management of thepatient’s disease condition without producing sideeffects (or personalized therapy). The present paperdiscusses the concept of Pharmacogenomics in detailwith the examples of the work carried out at theMolecular Biology lab, Dept. of Clinical Pharmacology,BYL Nair Hospital.


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Metabolomics as a tool for Diagnostics

Dr. Ajit Datar

Shimadzu Analytical (India) Pvt. Ltd.

In the field of Biology, the functional analyses at“Omics” levels have been growing very rapidly fromthe beginning of 21st century. These include Genomics,Transcriptomics, Proteomics, Metabolomics, andLipidomics etc. For disease diagnosis, Metabolomicsoffers unique advantages over other ‘Omics”techniques. Hence Metabolic Fingerprinting hasbecome one of the very important tools. In diagnosticsthere are several techniques used for MetaboliteFingerprinting. Most commonly used analyticaltechniques are based on Spectroscopy orChromatography or Hyphenation of these techniques.These include Infra-Red (IR) Spectroscopy, Ramanmicro spectroscopy, Nuclear Magnetic Resonance(NMR) Spectroscopy, Mass Spectroscopy (MS) andChromatographic Techniques like Gas Chromatography(GC), High Performance Liquid Chromatography(HPLC) and Hyphenated Techniques like GC-MS orLC-MS. Tandem MS is also used in most of theapplications, e.g. GC-MS/MS or LC-MS/MS. The MSmethods are used predominantly because MS offersexquisite sensitivity and the great selectivity inidentifying many hundreds of metabolites in a singlerun. Tandem MS has proven itself in the Diagnosis ofNeonatal Screening for Inherited Disorders ofIntermediary metabolism, in the field of identificationof biomarkers from Type 2 diabetes. There are several

examples in literature for the disease diagnostics usingMS, e.g. cancer, coronary heart disease, neurologicaldisease, many infectious diseases and so on.

It is essential then to understand the Tandem MStechnique with GC or LC as front end for Metabolitefingerprinting. The power of Chromatography toseparate the complex mixture of metabolites from thecomplex matrices such as blood plasma, urine, tissuesetc. and the ability of MS for its accurate massmeasurement are essential for understanding thestructure and pathways of metabolite formation and thushelp in

treatment of disease. Current development in thefield of Analytical Biochemistry (or Bio-analyticalSciences) allows simultaneous measurements ofmultiple analytes in biological matrix. Despite the currentadvances in this field, the goal of analyzing all themetabolites in the metabolome is still a challenge. Itrequires continued development of libraries of standards,instrument platforms with higher and higher sensitivitiesand mass accuracies by MS and integration of MS andNMR methods. Not to forget is the development incomputational methods for analysis of complexmetabolomic datasets and their integration with equallycomplex other “Omics” and database available as thesetechniques grow.


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17

SECTION III

CONCLUDING SESSION


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Emerging Strategies for Effective Cancer Treatment Based on CellElectroporation Technology

Hon’ble Vice Chancellor Dr. K. P. MishraNehru Gram Bharati University, Allahabad 211002

Ex Radiation Biology and Health Sciences Division, Bhabha Atomic Research Center, MumbaiEmail: [email protected], [email protected]

Living cells have tight regulation of entering andoutgoing molecules through sophisticated barrierprovided by cellular plasma membrane. Failure of drugaction on cells and, therefore, its ineffectiveness ontarget disease cells/tissues poses challenge to medicalscience in clinic. Cancer arises due to transformationof normal cell to cancer cell which is known to escapethe built-in control mechanisms of membranepermeability, protein transformation and gene regulationand expression affected by various causative factors.Cancer kills millions of patients each year the worldover. There is a critical need for safe, effective, andaffordable alternative treatment modalities, especiallyfor inoperable, recurring and chemo-resistant cancers,that do not respond well to current treatment modalities.A physical technique based on exposure of cells to shorthigh voltage electrical pulse, discovered a few yearsago, allows impermeant drugs, enzymes and otherdiagnostic and therapeutic agents enter the target cellsopening many new opportunities in medicine andbiotechnology. This method of electric pulse-mediatedpermeabilization of cell membrane known as

electroporation permits enhanced drug deliverytechnique has provided one of the novel approach toeffectively treat cancer patients which seems especiallysuitable for low and middle-income countries, wheredue to lack of infrastructure and resources, manycancers are diagnosed at late stages and this safe,effective, economical, out-patient-based and quicktechnique is a boon to these patients for palliative andother care with enhanced quality of life. This talk isdesigned to

present basic aspects of cell electroporation whichhas important implications to cell biotechnology and givesexamples of clinical applications of electroporation. Anattempt is made to discuss novel and recent clinicalapplications of electrochemotherapy for drug deliveryin treating various types of cancers and other diseases.The talk points out possibilities of developing noveltechnologies with potential applications in medicine,biotechnology, agriculture and food science with the useof novel and effective diagnostic and therapeutic tools.


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21

SECTION IV

POSTER ABSTRACTS


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Biomedical ImagingSwapnil Vichare, Amit Adsule, BhooshanMankame, B. N. Bandodkar College of Science, Thane

Biomedical imaging is the technique and processused to create images of the human body or parts forclinical purposes. Medical imaging constitutes a sub-discipline of biomedical engineering and of medicalphysics.

Medical imaging is often perceived to designatethe set of techniques that non-invasively produce imagesof the internal aspect of the body. The term non-invasiveis a term based on the fact that medical imagingmodalities do not penetrate the skin physically. But onthe electromagnetic and radiation level, they are quiteinvasive. There are several techniques under medical

imaging like radiography, ultrasound, MagneticResonance Imaging (MRI), tomography etc.

Medical imaging has become a major tool in clinicaltrials since it enables rapid diagnosis with visualizationand quantitative assessment. A typical clinical trial goesthrough multiple phases and can take up to severalyears. An imaging technique is used as an indicator ofpharmacological response to a therapy, obtaining quickresults. Imaging is able to reveal change that is indicativeof the progression of therapy that may be missed outby, traditional approaches. Thus Biomedical Imagingplays an important role in the medical fields.

Microarray Analysis as a Diagnostic ToolSneha Tawde, Humera Bhattiwala, Manali Patel

B.N. Bandodkar College of Science, Thane

The advent of microarray technology has a majorimpact on the molecular classification and understandingof human diseases. It has revolutionized the study ofgene expression and gene regulation.This technologypromises to monitor the whole genome on a single chipso that researchers can have a better picture ofinteractions among thousands of genes simultaneously.There are 2 types of microarray techniques:- 1) DNAmicroarray and 2) Protein microarray.

In this technique, mRNA is isolated from a givensample; then when cDNA synthesis is initiated the firststrand of the cDNA is labeled with the tag to form a

pool of target sequences. The next step is to hybridizethe labeled cDNA to nucleic acid affixed in amicroarray. The process of arraying the cDNA isaccomplished using robotics. In case of proteinmicroarray, instead of DNA externally synthesised,purified proteins are attached

to the solid surface like glass slide or nano wells.Taken together, DNA or protein microarray hasfacilitated the high throughput genomic and proteomicstudies and diagnosis of human cancer and otherdiseases.

Gas Chromatography- Mass Specrometry (GC-MS) in DiagnosticsAnuja Kumbhar, Priyanka Pawar, B. N. Bandodkar College of Science, Thane

Gas chromatography-mass spectrometry (GC-MS)is a hybrid technique which couples the powerfulseparation potential of gas chromatography with specificcharacterization ability of mass spectroscopy. Gaschromatography-mass spectrometry (GC-MS) is animportant technique for the identification and detectionof bacteria and bacterial constituents. Certain classesof structural components, or secreted metabolites, areuseful as chemical markers for different groups oforganisms. The GC works on the principle that a mixturewill separate into individual substances when heated.

The heated gases are carried through a column withan inert gas (such as helium). As the separatedsubstances emerge from the column opening, they flowinto the MS. Mass spectrometry identifies compoundsby the mass of the analyte molecule. The GCMS candetect chemicals in amounts as small as 0.1 to100 ng.A gas chromatography–mass spectrometry (GC–MS)method with minimum sample preparation is describedfor early diagnosis of tuberculosis(Mycobacteriumtuberculosis).The technique is also used in detectionof Urine d-Arabinitol/l-Arabinitol Ratio in Diagnosis of


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Invasive Candidiasis(Candida spp) in NewbornInfants. It is used for rapid diagnosis of infectiousDiarrhea caused by Clostridium difficile& diagnosisof bacterial meningitis by the detection of a fatty acidmarker in CSF. GC-MS is the most widely used

application in metabolite research & disease diagnosis.It enables the identification of small molecules as aminoacids, fatty acids, organic acids in biofluid as blood andurine providing diagnostic information in inheriteddiseases, including numerous metabolic disorders.

Molecular Markers in DiagnosticsNikita Diwan, Pooja Kadhane, Rashmi Joshi, B. N. Bandodkar College of Science, Thane

Recent revolutionary progress in human genomicsis reshaping our approach to therapy and diagnosis. Notwo human individuals have exactly the same genomeand this has led to development of DNA typing. DNAtyping relies on DNA analysis using molecular markerssuch as RFLPs, RAPD, AFLP, SNPs, and SSLPs.

Restriction fragment length polymorphism (RFLP)generated when natural mutations at site of restrictionenzymes as well as insertion or deletion between thesites differentiate individuals. Random amplifiedpolymorphic DNA (RAPD) markers are generated byPCR amplification of genomic DNA segments using asingle, short primer under low annealing temperature.Amplified fragment length polymorphism (AFLP) hasbeen widely used in genome mapping and gene tagging.This versatile method is able to detect the presence of

restriction fragments in almost any DNA, regardlessof its complexity.This approach can be used for indirectdetection of a disease causing mutation or simplydistinguishes DNA fragments of different sizes fromthe same region or also can be used as markers withina family who is likely to carry a disease – causingmutation and who is not . The identification of suchmarkers has shown some important consequences i.e.it may offer a diagnostic procedure for detecting someof the human diseases that are genetically wellcharacterized but ill defined in molecular terms cannotbe easily diagnosed . This is a clear indication that asmany molecular techniques have made their expectedtransitions into the clinical arena. Molecular diagnosticsis becoming an integral part of clinical practice.

Prognostic MarkersHemant Shirvalkar, Satish Mirgal, Ruchi Khadtale, B.N. Bandodkar College of Science, Thane

Prognostic markers (biomarkers) arecharacteristics that help to identify or categorise peoplewith different risks of specific future outcomes. Theymay be simple clinical measures such as body massindex, but are more often pathological, biochemical,molecular or genetic measures or attributes. Identifyingthose who are or who are not at risk can facilitateintervention choice, and aid patient counselling.

Prognostic markers help to stratify patients fortreatment by identifying patients with different risks ofoutcome (e.g. recurrence of disease), and are importanttools in the management of cancer and many otherdiseases. Systematic review and meta-analyticalapproaches to identifying the most valuable prognosticmarkers are needed because (sometimes conflicting)evidence relating to markers is often published acrossa number of studies.

In oncology, prognostic markers are clinicalmeasures used to help elicit an individual patient’s riskof recurrence of disease after primary treatment.Evidence based results regarding prognostic markersare therefore very important to both clinicians andpatients. Cervical cancer remains the leading cause ofcancer deaths in women. The researchers of UnitedStates National Cancer Institute(NCI), The GynecologicCancer Intergroup found out that para-aortic and pelviclymph node involvement is clearly one of the mostimportant prognostic markers, as well as one of themost important factors to influence treatment decisions.Additional adverse prognostic markers includeperitoneal spread as well as presence of supraclavicularnodes in those with more advanced diseases.TejashriPatil.


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Diagnosis of HIVPriyanka Bhalerao, Anuja Bharati, B. N. Bandodkar College of Science, Thane

Human Immunodeficiency Virus (HIV) wasunknown until the early 1980s but since that time it hasinfected millions of people worldwide. HIV is mostcommonly diagnosed by testing the blood or saliva forthe antibodies to the virus. However, different types ofHIV diagnostic techniques have been used over a periodof time which include antibody test:-ELISA (rapid test)which detects HIV antibodies & the results areconfirmed by Western Blot which is one of the oldestbut most accurate confirmatory tests. But it takes timefor a person’s body to develop these antibodies, antigentests:- protein P24 most commonly provokes an antibodyresponse for HIV & are now most often used as acomponent of fourth generation tests. Some of the mostmodern HIV tests combine P24 antigen tests withstandard antibody tests, PCR test which can detect thegenetic material of HIV within 2-3 weeks, HIV homesampling & HIV home testing which is done using homesampling kits & home testing kits. Several types of testshelp the doctor determine what stage of the disease aperson has :-CD4 count, viral load, drug reistance. Alongwith them come the test for complications for TB,

Hepatitis, Toxoplasmosis, STD, Liver or kidney damage,Urinary tract infection.

The period between HIV infection & theproduction of antibodies is known as ‘’WindowPeriod’’.During this time,an antibody test may give afalse negative results which means the test will benegative even though a person is infected with HIV. Toavoid false negtive result, antibody test recommended3 months after potential exposure to HIV infection.

Diagnostic techniques provide prognosticinformation in patients with estblished disease. Thecriterion (reference) standard test definatively decideseither presence or absence of a disease. However, thesetest routinely come with drawbacks such as they areusually expensive, less widely available, more invasive& riskier.

This presentation provides an overview of diffrentHIV diagnostic tests describing how they work & theadvantages & limitaion of various tests. It also includeswhat molecular marker these tests used to pinpoint HIVinfection

Enzyme Based DiagnosticsRucha Kulkarni, Mansi Lad, Darshana Sakpal, B.N. Bandodkar College of Science, Thane

The major importance of enzymes in diagnosticsis that they are used to determine the specific levels ofenzymes in blood, urine, body fluids and tissues. Theyare also helpful in determining the concentration ofsubstances and metabolites. There is a lot of progressin diagnosticsusing enzymes, as they are comparativelyeasy to use and are now commercially available. Highlysophisticated equipments and assays are available forenzyme diagnoses that are easy to assay and automate.They are often preferred over older tests as thedifferences between the normal specimen and thedisease specimen are clearly demonstrated anddiagnostically significant. The enzymes utilized aresufficiently stable and allow storage for limited butreasonable time. Available diagnostic tests candemonstrate plasma-specific enzymes that areassociated with normal functioning of plasma such asblood coagulation components, complement activationcomponents, and lipoprotein metabolism. Newer tests

can also determine non-plasma specific enzymes suchas those resulting from abnormal cellular metabolismthat have no actual function in plasma where coenzymesor substrates are lacking they are present in plasma asa result of other body problems such as liver diseaseheart attacks, muscle disease, disease of pancreas orcancer. Examples include phosphatases, transaminases,lipases and amylases. There are certain serum enzymeswhich are released in blood only when the cells to whichthey are confined are damaged. The presence of theseenzymes in blood indicates the site of tissue injury. Someof the examples of such enzymes areAldolase, Creatinephosphokinase, Lipase. There are many other exampleslikeTransaminases like Glutamic-oxaloacetic acid(GOT), Glutamic-pyruvic transaminase lacticdehydrogenase etc. The common enzymes for clinicaldiagnosis are Acid phosphatase, alanineaminotransferase, cholinesterase lactate dehydrogenaseetc. Enzymes are also used for the prognosis of adisease.


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Differential Staining Of BloodDr. Kalpita Mulye, Anjali Tidke, Priyanka Barmukh, B.N. Bandodkar college of Science

Hematology encompasses the study of bloodcomponents and coagulation. It includes, analysis of theconcentration, structure &functions of the cells & repairtheir precursors in the bone marrow, analysis of chemicalconstituents of plasma or serum intimately, intimately,linked with blood cell structure & functions, Study offunctions of the platelets & proteins involved in bloodcoagulation.

Importance of Differential Staining :-. Differentialcount is useful to identify changes in the distribution ofWBCs which may be related to specific types ofdisorders. It also gives idea regarding the severity ofthe disease & the degree of response of the body.

Principle :- Giemsa stain contains methylene blue& eosin. These basic and acidic dyes includes multiple

colors applied to cells. Methanol acts as a fixative andalso as a solvent. The basic component of WBCs i.e.cytoplasm is stained by acidic dye & they are describedas eosinophilic. The acidic component i.e. nucleus withnucleic acid take blue colour by basic dye & they arecalled as basophilic. The neutral component of the cellsare stained by both dyes.

Screening of blood smears :- 25 blood smears weretaken & screened for White Blood Cells count withGiemsa staining. Different abnormalities detected arelisted below :-Toxic Granulation which may be due tosevere bacterial infection & also in some hereditarydisorders. Hypersegmentation which may be due toinherited disorder also seen in macrocytic anemias.

Cholescintigraphy (HIDA Scan)Shrikant Sonawane, Uttara Patil, Noopur Patil, B. N. Bandodkar College of Science, Thane

HIDA( Hepato Iminodiacetic Acid) Scan is animaging procedure that helps track the production andflow of bile from liver to small intestine. Bile, fluidproduced by liver that helps digestive system breakdownfats into the food we eat. HIDA scan, one of the nuclearmedicine scan, uses a radioactive chemical or tracerthat helps highlight certain organs like liver, gallbladder,bile ducts and small intestine on the scan.

HIDA scans are ordered for patients who aresuspected of having an obstruction in biliary tract, mostcommonly those who are thought to have a stoneblocking the cystic duct leading out of the gallbladder.Such a scenario is consistent with acute cholecystitis,which often requires surgical removal of gallbladder.In cholecystitis, HIDA will appear in the bile ducts, butwill not enter the cystic ducts or gallbladder- a finding

that indicates obstruction. If HIDA enters bile ductsbut does not enter the small intestine, then an obstructionin bile duct is suspected.

The patient receives an intravenous injection ofradioactive material i.e. technetium 99m iminodiaceticacid derivative, taken up by the liver and excreted intothe biliary tract. HIDA imaging is done by a nuclearscanner, which takes pictures of patient’s biliary tractover the course of about an hour or two. The imagesare then examined by a radiologist, who interprets theresults.

HIDA scan for acute cholecyctitis has a sensitivityof 97%, specificity of 94%. It is generally a very safetest and well tolerated by most patients.

Polymerase Chain ReactionPranali Chowre, Tejal Niphade, Sudha Shukla, Vaibhavi Ghag,


Polymerase chain reaction is a method forproducing many copies of a specific DNA sequencefrom a DNA mixture without having to clone thesequence in host organism. The three main steps

involved in the PCR techniques are repeateddenaturation, annealing and extension cycle.

PCR is mainly used for amplifying small (generallyless than 10 kb) region of DNA. It is much more


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sensitive and quicker method than cloning.

PCR is mainly used for mapping DNA sequence,DNA fingerprinting, DNA sequencing, forensics, diseasediagnosis, sex determination of embryos, tissue typing.PCR is a key point in the process of DNAsequencing.PCR is routinely used tool in thelaboratories of molecular geneticists.

PCR technology has become an essential researchand diagnostic tool for improving human health andquality of life.PCR based diagnostics tests are availablefor detecting and quantifying several hazardouspathogens. In diagnosis, it is used to detect the bacterialpathogens or viral pathogens such as HIV and HepatitisB. it is also widely used in the genetic disease diagnosis.

Fluorescence in situ HybridizationRuchita more, Priyanka Arekar, Chaitali Chachad, B. N. Bandodkar College of Science, Thane

Fluorescence in-situ hybridization (FISH) is acytogenetic technique developed by biomedicalresearchers in the early 1980s that is used todetect and localize the presence or absence ofspecific DNA sequence on chromosomes. FISH isa process which ‘paints’ chromosomes or parts ofchromosomes with fluorescent molecules.Fluorescence in-situ hybridization involves the useof probes (short sequence of single-stranded DNA)which are labeled with fluorescent tags to hybridizeor bind to only those parts of the chromosomewith which they show a high degree of sequencecomplementarity within the intact cell.

Fluorescence microscopy can be used to findout where the fluorescent probe is bound to thechromosomes. FISH is a technique which helps in

identifying chromosomal abnormalities. FISH alsoaids in gene mapping, toxicological studies, analysisof chromosome structural aberrations, and ploidydetermination. It is also used to identify the presenceand location of a region of DNA or RNA withinmorphologically preserved chromosome preparations,fixed cells or tissue sections. A variety of proceduresare available to cytogeneticists, who use them todiagnose many types of chromosomal abnormalitiesin patients.

The methodology is being continuously improvedso far however, microscopic analysis by FISH hasnot been automated sufficiently which would bedesirable in many investigations. Accuratequantification still remains a challenging task andstudy needs careful controls.

Cancer DiagnosticsSonam Patil, Nayana Choudhari, Pooja Mandluskar, B. N. Bandodkar College of Science

Cancer is a group of diseases in which abnormalbody cells in one part of the body start to grow out ofcontrol. Oncology is the study of cancer and tumors.

Diagnostic procedures for cancer may includeimaging, laboratory tests (including tests for tumormarkers), tumor biopsy, endoscopic examination,surgery, or genetic testing.

Blood test: A complete blood count (CBC) tomeasures the size, number, and maturity of the differentblood cells. A variety of blood tests are used to checkthe levels of substances in the blood

Urinalysis: Urinalysis breaks down the componentsof urine to check for the presence of drugs, blood,protein, and other substances.

Tumor markers: Tumor markers are substanceseither released by cancer cells into the blood or urine

or substances created by the body in response to cancercells. Some of useful tumor markers are prostatic acidphosphatase (PAP), CA 125, carcinoembryonicantigen(CEA), alpha-fetoprotein (AFP), human chorionicgonadotropin (HCG), CA 19-9, CA15-3, CA 27-29.

Imaging: Imaging is the process of producingvaluable pictures of body structures and organs. It isused to detect tumors and other abnormalities, todetermine the extent of disease, and to evaluate theeffectiveness of treatment .There are three types ofimaging used for diagnosing cancer: transmissionimaging, reflection imaging, and emission imaging.

Transmission Imaging: X-ray, computedtomography scan (CT scan or CAT scan),bone scan,lymphangiogram (LAG), mammogram, ReflectionImaging: ultrasound Emission imaging, MagneticResonance Imaging (MRI)


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Haematological DiagnosticsSwati Kolte, Poonam Shukla, B. N. Bandodkar College of Science, Thane

Haematology encompasses the study of bloodcomponents and coagulation. It includes, the analysisof the concentration, structure and functions of the cellsand their precursors in the bone marrow, analysis ofchemical constituents of plasma or serum intimatelylinked with blood cell structure and functions, study offunctions of the platelets and proteins involved in bloodcoagulation. Changes in one or more of thecharacteristics mentioned above may producehaematological disease or manifestations.

Some of the Haematological disorders arehaemoglobinopathy, and haemolytic anemias.Haemoglobinopathy is a kind of genetic defect thatresults in abnormal structure of one of the globin chainsof the haemoglobin molecule. Haemoglobinopathies areinherited single-gene disorders; in most cases, they areinherited as autosomal co-dominant traits. Commonhaemoglobinopathies include sickle-cell disease.

Methods of detecting Haemoglobinopathy arealkaline and acid gel electrophoresis, Isoelectric focusing(IEF), High Pressure Liquid Chromatography (HPLC)and Capillary electrophoresis (CE)

Haemolytic anaemia is a form of anaemia due tohaemolysis, the abnormal breakdown of red blood cells(RBCs), either in the blood vessels (intravascularhaemolysis) or elsewhere in the human body(extravascular). The general classification ofhaemolytic anaemia is either inherited or acquired.Treatment depends on the cause and nature of thebreakdown. Methods of detecting haemolytic anaemiaare absolute reticulocyte count, red blood cell count(RBC), haemoglobin, and haematocrit (HCT), Serumhaptoglobin levels, Serum indirect bilirubin levels andSerum LDH determination.

Use of monoclonal antibody-based biosensors in diagnostics testsSandhya Dumbre, K. B. P. College, Vashi

Biosensors are analytical tools of bio-materialsamples to gain an understanding of their bio-composition, structure and functions by converting abiological response into an electrical signal. Theanalytical devices composed of a biological recognitionelement directly interfaced to a signal transducer whichtogether relates the concentration of an analyte (or agroup of related analytes) to a measurableresponse.Biosensor provides similar sensitivity asprovided by other conventional detection instruments& techniques.

Some biosensors reached even detection limitssimilar to the PCR techniques. Typical time ofimmunodetection is 15 min, but some devices likeresonant mirror, quartz crustal microbalance are ableto provide signal within 5 min. A new generation ofbiosensors discussed in this presentation uses antibodyand DNA probes, like Antibody-basedfluoroimmunosensors (FISs), Biochips,NANOSENSORS, etc. Monoclonal antibodies are

proving to be very useful as diagnostic, imaging, &therapeutic reagents in clinical medicine. Among themany monoclonal antibody diagnostic reagents nowavailable are products for detecting pregnancy,diagnosing numerous pathogenic microorganisms,matching histocompatibility antigens, & detectingantigens shed by certain tumors. Thus, by combiningthe properties of monoclonal antibodies & principlebehind the biosensors, various biosensors can bedeveloped which would be useful in diagnosing diseases/infections whose cure is still a mystery.Due to theirsmall size & low cost, biosensors are convenient notonly for laboratory routine but also for mobilelaboratories & portable systems in the field. In the future,one can expect development of new biosensors givingreliable detection results with amount of individual agentsunder their infectious doses, with multi-channelarrangement for several agents, & with furtherminiaturized designs


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Karyotype AnalysisPrathamesh Chaughule, K. B. P. College, Vashi

Organisms may be identified by using the size andshape of their genetic material at a particularpoint inthe cell division cycle, termed metaphase. At this pointDNA condenses to form anumber of very distinctchromosome structures. Various morphologicalcharacteristics ofchromosomes may be identified at thisstage including the centromere and the telomere.Thedisplay of the 46 human chromosomes at mitosis iscalled the human karyotype. If parts ofchromosomesare lost or are switched between chromosomes, thesechanges can be detectedby changes in the bandingpatterns or by changes in the pattern of chromosomepainting.Using chromosome banding alone, a disturbinglyhigh number of chromosomal aberrationscannot becharacterized comprehensively. Thus, a specialapproach is applied, called spectralkaryotyping, that isbased on the simultaneous hybridization of 24combinatorially labeledhuman chromosome painting

probes. Spectral karyotyping was developed for thedetection ofnon-homologous structural and numericalchromosomal aberrations on the single metaphaselevelhybridizing a 24-color whole chromosome probe mix.Each homologue chromosome isdisplayed in a differentcolor, based on the use of computer-generated false-color chromosomeimages and karyotyping. Spectralkaryotyping combines Fourier transform spectroscopy,CCDimaging, and optical microscopy to measuresimultaneously at all points in the sampleemissionspectra in the visible and near-infrared spectralrange. This technique is widely used tostudyconstitutional chromosome abnormalities isparticularly useful for identifying de novo balancedandunbalanced translocations. Spectral karyotyping is alsoused in multiparameter analysis ofcytologicalpreparations to routine diagnostic preparations.

Diagnostic Techniques for Human Diseases caused by Fungal Pathogens:A Brief Review

Moses Kolet, Amit Bendre, Sohil Pawar, B. N. Bandodkar College of Science, Thane

Fungi constitutes a large and diversified group ofheterotrophic organisams comprising parasites as wellas saprophytes. Their occurance can be described asboth overt and covert. Of approximately 100,000 fungiknown to mankind, less than 500 have been implicatedin various cases of mycosis affecting man and animals.Some of these are capable of affecting exposedindividual while others are opportunistic pathogenes.The infections themselves vary in degree of seriousness;some apperently ignorable while others may prove fetal.

The present work deals with an overview ofepidemiology of the diseases of fungal origin, theirincidence and different types of fungal infectionsaffecting human beings. A brief review of classical andmodern diagnostic techniques inclusive of latestserological techniques and application of Polymerasechain reaction for diagnosis of fungal infection isundertaken.


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SECTION V

PRE CONFERENCE WORKSHOPS

ABSTRACTS


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Conventional Diagnostic tests

Rajitha RadhakrishnanLecturer, Department of Microbiology and Biotechnology, B. N. Bandodkar College of Science, Thane

Diseases are on rise all over the world. The abilityto control and manage diseases is largely dependenton the ability to detect the etiological agents in the clinicalmicrobiology laboratory. Hence diagnostic plays veryimportant role in disease management.

Though the last few years of the twentieth centuryhave seen an explosion of molecular biology techniquesin diagnostics, the underlying principle of many suchtechniques still lies in conventional diagnostics methods.Conventional Diagnostic technique includes techniqueslike microscopy, culturing of sample, antigen or antibodydetection, immunoserology as well as Enzymatic test.Conventional identification of microbial pathogens usingmicroscopy relies on phenotypic characteristics of theorganisms, morphology, bacterial metaboliccharacteristics, fungal structures, parasitic morphologyand viral cytopathic effect .These primarily includesGram staining, differential staining using Giemsa stains,HE stains, ZNCF, wet mounts of sample as in KOH/fungal wet mount. Culturing of sample includescollection of Sample, followed by macroscopic and

microscopic observation of sample, isolation on suitableselective media, and based on colonies obtained furtherdetection of etiological agent is done using variousbiochemicals. A variety of test systems have beendeveloped to identify the antigens of infectious diseasesas well as antibodies secreted as part of protectivemechanisms of body. Test systems used in immuneserology have classically included methods of detectingantigen-antibody reactions which range fromcomplement fixation to immunoassay methods. Similarlymany diseases like sickle cell anemia, phenylketonuria(PKU) and Tay-Sachs disease are detected by checkingabnormal enzymatic levels in blood or tissues.

In many developing countries even now manydiseases are detected using conventional methods asthey are simple, affordable and accurate. Althoughconventional diagnostic methods are dependable andreliable too, most approaches still have certain limitations.Thereby for improved sensitivity and specificity onegoes for advanced molecular techniques.

Immunological Diagnostics

Nilambari DaripkarLecturer, Department of Microbiology and Biotechnology, B. N. Bandodkar College of Science, Thane

E-mail: [email protected]

Immunity is a biological term that describes astate of having sufficient biological defenses to avoidinfection, disease, or other unwanted biological invasion.The human immune system is a truly amazingconstellation of responses to attacks from outside thebody. It has many facets, a number of which can changeto optimize the response to these unwanted intrusions.The system is remarkably effective, most of the time.An immune system is a system of biological structuresand processes within an organism that protects againstdisease. In order to function properly, an immune systemmust detect a wide variety of agents, from viruses toparasitic worms, and distinguish them from theorganism’s own healthy tissue.

An antigen is any substance that elicits an immuneresponse, from a virus to a sliver. Detection of particular

antibodies raised against these antigens is a verycommon form of medical diagnostics. For example, inbiochemical assays for disease diagnosis, a titer ofantibodies directed against Epstein-Barr virus or Lymedisease is estimated from the blood. If those antibodiesare not present, either the person is not infected, or theinfection occurred a very long time ago. Practically,several immunodiagnostic methods based on detectionof complex antigen-antibody are used to diagnoseinfectious diseases, for example ELISA,Immunofluorescence,Westernblot, Immunodiffusion,Immunoelectrophoresis and Magnetic immunoassay.

Example, Antibodies raised against HumanChorionic Gonadotropin is used in Pregnancydetection tests.


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Modern Immuno-Diagnostic Techniques

Kalpita MulyeAssistant professor, Department of Biotechnology and Microbiology

B. N. Bandodkar College of Science, thane

Modern methods of immunodiagnostics are basedon the much familiar phenomenon of Antigen – Antibodyinteraction viz. Specificity, Affinity and avidity,Reversibility and Cross reactivity.

High Sensitivity, Precision, Rapidity, Reproducibilityand sophisticated Automation render modernimmunodiagnostic techniques their much deservingpopularity.

The milestone discovery “ELISA” revolutionizeddiagnostics by attaining detection sensitivity up to0.0001–0.01 micrograms of antibody from the sample.The technique is based on the use of specific enzymetagged antibodies which will bring about the conversionof substrate to chromogenic product that can beanalyzed by spectrophotometer.

The similar technique which uses the radioactivelabel instead of an enzyme label is Radio Immuno Assay(RIA). Principle of RIA involves competitive binding

of radio-labeled antigen and unlabeled antigen to a high-affinity antibody.

Variation to the conventional ELISA, which is usedfor detection of specific biomolecules e.g. Cytokinesin situ is ELISPOT. Most of the cytokine moleculessecreted by a particular cell react with nearby well-bound antibodies and is in wide use to track the cytokinelevels as one of the prognostic markers for diseaseslike HIV/AIDS.

Automated analysis and separation of cells ispossible now with help of flow cytometry, which makethe use of fluorescent monoclonal antibodies developedfor specific cell surface markers.

Western blotting, Immuno-electron microscopy,Immuno fluorescence, Immuno-histochemistry (IHC)are few more important techniques based on use ofspecific antibodies tagged with different moieties forvaried diagnostic goals.

Molecular Diagnostics - Polymerase Chain Reaction

Jayashree PawarAssistant Professor, B. N. Bandodkar College of Science, Thane

Polymerase chain reaction, PCR, the quick, easymethod for generating unlimited copies of any fragmentof DNA, is one of those scientific developments thatactually deserves time-worn superlatives like“revolutionary” and “breakthrough.” PCR hastransformed the diagnostic methodology utterly. PCRtakes analysis of tiny amounts of genetic material—even damaged genetic material—to a new level ofprecision and reliability.

The central scientific fact that makes PCR souseful is this: the genetic material of each living organismpossesses sequences of its nucleotide building blocksthat are uniquely and specifically present only in its ownspecies. These unique variations make it possible totrace genetic material back to its origin, identifying withprecision at least what species of organism it came from,and often which particular member of that species.

PCR requires a template molecule—the DNA orRNA one wants to copy—and two primer moleculesto get the copying process started. Primer designing isan important aspect of polymerase chain Reaction. Thereaction also requires dNTPs, a thermostable DNApolymerase, e. g., Taq DNA polymerase and bufferfor the enzyme with correct magnesium ionconcentration.

There are three basic steps in PCR. Denaturationof the template DNA molecules, primer annealing, andextention of annealed primers by polymerase. The resultis two new helices in place of the first, each composedof one of the original strands plus its newly assembledcomplementary strand. The three steps are repeated25-30 times to get millions of copies of the templateDNA flanked by the primers. The reaction is perfomedin a ‘Themal Cycler’. Use of capillry systems instead


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of tubes and forced turbulant air systems havedramatically lowered the time required for a round ofPCR.

Various modifiactions of the basic PCR are carriedout for special applications e. g., Reverse TranscriptasePCR is used to amplify RNA templates. Nested PCRis used to assure specificity of the reaction as twoprimer pairs in two rounds of PCR cycling are used.

Medical research and clinical medicine areprofiting from PCR mainly in two areas: detection ofinfectious disease organisms, and detection of variationsand mutations in genes, especially human genes. Themethod is especially useful for searching out diseaseorganisms that are difficult or impossible to culture, suchas many kinds of bacteria, fungi, and viruses, becauseit can generate analyzable quantities of the organism’sgenetic material for identification

Techniques in Animal Cell Culture

Dr. Sharmila RaikarCo-ordinator, M. Sc. Neutraceuticals, G. N. Khalsa College, Matunga, Mumbai.

Animal tissue culture (ATC) deals with removalof cells, tissues, or organs (organ culture) from an animaland their growth in vitro. ATC finds many applicationsin cell and molecular biology. Cell cultures provide agood model system for studying basic cell biology andbiochemistry, interactions between disease-causingagents and cells, drug pharmacokinetics andpharmacodynamics, ageing and nutritional studies.Mechanisms of carcinogenesis can be studied and novelanti-cancer drugs can be screened using cell lines. Someother applications include vaccine production, proteinengineering, stem cell therapy, gene therapy, drugscreening and development etc. In vitro conditions foranimal cell growth are more complex in terms ofmedium (serum requirement) and environmental factorssuch as temperature, carbon-dioxide requirements etc.Normal cell lines display certain characteristics suchas anchorage dependence (certain exceptions are bloodcells that grow as suspension cultures) and contact

inhibition. Transformed cell lines display no contactinhibition and have lower serum requirement than normalcells.

Reliable cell quantitation methods must beemployed in order to ensure accuracy andreproducibility of any assay. Methods for animal cellquantitation include cell counting (hemocytometer, cellcounters, FACS), proliferation assays (thymidine uptakeassays), metabolic assays such MTT reduction assay,colorimetric assays (using dyes such as Crystal Violet,or fluorescence DNA binding assays using dyes suchas Propidium iodide, Hoechst dyes etc.). Cytotoxicityis an important parameter for studying the effect ofenvironmental factors (e.g. drugs) on viability andsurvival of cells. Cytotoxicity may studied as a functionof change in cell viability (uptake/exclusion of vitaldyes), cell survival (by testing plating efficiency),metabolic ability (MTT reduction) etc.

Biotechnology in Diagnosis of Genetic Disorders

Sonal MathiasLecturer, Dept. of Biotechnology, B. N. Bandodkar College of Science, Thane

A genetic disorder is an illness caused byabnormalities in genes or chromosomes, especially acondition that is present from before birth. Most geneticdisorders are quite rare and affect one person in everyseveral thousand or millions. A genetic disorder may ormay not be a heritable disorder. Some genetic disordersare passed down from the parents’ genes, but othersare always or almost always caused by new mutationsor changes to the DNA. The four different types ofgenetic disorders are (1) single-gene, (2) multifactorial,

(3) chromosomal, and (4) mitochondrial. Gene testingis carried out only after informed consent of theindividuals. Samples used are blood, bone marrow,amniotic fluid, skin , semen and hair. If the disorder ischromosomal in nature then KARYOTYPING isadvised.Genetic tests are used for several reasons,including: carrier screening, which involves identifyingunaffected individuals who carry one copy of a genefor a disease that requires two copies for the disease tobe expressed, preimplantation genetic diagnosis, prenatal


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diagnostic testing (amniocentesis &CVS ),new-bornscreening, presymptomatic testing for predicting adult-onset, presymptomatic testing for estimating the risk ofdeveloping adult-onset cancers and Alzheimer’s disease,confirmational diagnosis of a symptomatic individual.Diagnostic test are of two types: Non-invasivetechniques: Fetal echocardiography, Fetal visualizationUltrasound, Screening for neural tube defects (NTDs)- Measuring maternal serum alpha-fetoprotein(MSAFP),Screening for fetal Down syndromeMeasuring MSAFP, measuring maternal serum beta-human chorionic gonadotropin (HCG) and Invasivetechniques :Fetal tissue sampling Amniocentesis,

Chorionic villus sampling (CVS),Percutaneous umbilicalblood sampling (PUBS),Percutaneous skin biopsy,Preimplantation biopsy of blastocysts obtained by invitro fertilization, Cytogenetic investigations, Detectionof chromosomal aberrations, Fluorescent in situhybridization, Restriction fragment lengthpolymorphisms (RFLPs),Single nucleotidepolymorphisms (SNPs) .Many of the risks associatedwith genetic testing involve the emotional, social, orfinancial consequences of the test results. Majorlimitation is the lack of treatment strategies for manygenetic disorders once they are diagnosed.

Diagnosis of Diabetes

Dr. Sangeeta BhagatHead, Dept. of Biotechnology, Modern college, Shivajinagar, Pune

Diabetes is defined as a state in which homeostasisof carbohydrate and lipid metabolism is improperlyregulated by insulin. This results in elevated fasting andpostprandial glucose levels. If this imbalance does notreturn to normalcy and continues for protracted periodof time, it leads to hyperglycaemia that in due courseturns into a syndrome called “Diabetes mellitus”. Thereare two main categories of this disease. Type onediabetes mellitus called as insulin dependent diabetesmellitus (IDDM) and type two the non-insulin dependentdiabetes mellitus (NIDDM).Diabetic hyperglycaemiacauses variety of pathological changes in small vessel,arteries and peripheral nerves. Symptoms of diabetesare Polydipsia, polyuria and polyphagia.

Diabetes is diagnosed using different methods, bymeasuring plasma glucose level, by oral glucose

tolerance test (OGTT) and glycosylated haemoglobin.If fasting BGL is >100 but <126 mg/ml condition iscalled impaired fasting Glucose (IFG). This condition iscalled prediabetes. Oral glucose Tolerance Testmeasures blood glucose after a person fasts at least 8hours and 2 hours after the person drinks 75 gm. of aglucose-containing beverage. If BSL after a drink isaround 200 mg/dl, patient is diabetic. Glycosylated Hbis best for diabetes follow up than to diagnose thediabetes good indicator of blood glucose control. 8% orgreater indicate BSL is too high.

Research area of diabetes are Oxidative stress,Measurement of AGEs, Measurement of free radicals,Use of natural dietary antioxidants and herbal medicinein prevention of diabetic complications.

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held on 6 -7 January 2012 on ‘Biotechnology in Diagnostics’vpmthane.org/BNB/Seminar/Biotechnology conf proceeding.pdf · held on 6th-7th January 2012 on ‘Biotechnology in Diagnostics