21st INDO-US Flow Cytometry Workshop

“Clinical Applications of Flow Cytometry”

Jointly organized by

Trust for Education and Training in Cytometry (TETC)

Live Education Task Force -International Society for
Advancement of Cytometry (LETF-ISAC)
Department of Pathology, Ramaiah Medical College (RMC)

PROGRAM BOOK

Venue
Ramaiah Medical College,
Bengaluru, INDIA

January 29th- 30th, 2020

1
We Thank All Our Supporters & Sponsors

2
21st INDO-US Flow Cytometry Workshop
“Clinical Applications of Flow Cytometry”

Jointly organized by

Trust for Education and Training in Cytometry (TETC)

Live Education Task Force -International Society for
Advancement of Cytometry (LETF-ISAC)
Department of Pathology, Ramaiah Medical College (RMC)

PROGRAM BOOK

Venue
Ramaiah Medical College,
Bengaluru, INDIA

January 29th- 30th, 2020

3
 Ramaiah Medical College
Bengaluru, INDIA

WELCOMES ALL
FACULTIES, DELEGATES, SUPPORTERS
AND SPONSORS

4
INDEX

S. No. CONTENTS PAGE NUMBER

1. ORGANIZING COMMITTEE 6

2. PROGRAM 7-9

3 ORGANIZERS 10-13

4. MESSAGES 14-22

5. FACULTY 23-31

6. ABSTRACTS 32-38

7. WORKSHOP SLIDES 39-97

8. WORKSHOP PROTOCOLS 98-113

9. LIST OF PARTICIPANTS 114-122

10. ADVERTISEMENTS 123- 131

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 Organizing Committee

Organizing Chairpersons
Prof. Awtar Krishan, USA
Prof. Pushpati N Razdan, INDIA

Organizing Co-Chairpersons
Dr. Prasanna Shetty B
Dr. H. Krishnamurthy

Convener
Dr. Rekha Gour

Organizing Secretaries
Dr. Kalpana Kumari MK
Dr. Hemant Agrawal

Coordinators
Dr. Nandakishore Alva
Dr. Vivek Tanavade

Members
Dr. Vijaya V Mysorekar Dr. Mangalagouri S R Dr. H M Sudha
Dr. Aarathi R Rau Dr. Sulata M Kamath Dr. Usha M
Dr. Clement Wilfred D Dr. Sridhar H Dr. Rashmi K

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 Program

7
DAY 1 (29th January, 2020)

TIMING PROGRAM FACULTY

8.00-08:30 BREAKFAST
8:30-9:00 Registration

Basics of Flow Cytometry Dr. H. Krishnamurthy

9:00-9:45

Welcome and Inauguration (RMC, TETC and LETF-ISAC)

09:45-10:15

10:15 - 10:30 TEA BREAK

10:30 - 11:10 Quality Control and Trouble Shooting Dr. Zosia Maciorowski

RED- High Speed, High Resolution Mr. Badri N.Narayan-

11:10 - 11:30
Flow Cytometry in Diagnostics ThermoFisher Scientific

Multicolor Clinical Flow Cytometry Mr. Swapnil Walke-BD Life

11:30-11:50
with BD FACSLyricTM Sciences-Biosciences

Polychromatic Flow Cytometry and

11:50 - 12:30 Dr. Paul Edward Hutchinson
Panel Designing

The use of multiparametric Flow

12:30 - 13:10 Cytometry in Myelodysplastic Dr. Brent Wood
Syndrome Syndrome

13:10 – 14:00
LUNCH BREAK

Lab 1: Instrumental startup and Dr. Hemant Agrawal and

demonstration of instrument setup.
14:00 – 15:00 Dr. Rekha Gour
DNA Estimation and Cell Cycle
Analysis Dr. H Krishnamurthy

Lab 2: Multicolor Flow Cytometry for Dr. Paul Edward Hutchinson Dr.
15:00-16:15
TBNK, CD4/CD8 analysis Zosia Maciorowski

16:15 – 16:30 TEA BREAK

16:30-17:15 Leukemia/Lymphoma Phenotyping Dr. Sumeet Gujral

17:15-18:00 Minimal Residual Disease (MRD) Dr. Brent Wood

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DAY 2 (30th January, 2020)
TIMING PROGRAM SPEAKER

9:00-9:45 The use of multiparametric Flow Dr. Brent Wood

Cytometry in Multiple Myeloma

9:45-11:15 Lab 3: Hematopoietic Stem Cell Dr. Vivek Tanavde

Enumeration

11:15 - 11:30 TEA BREAK

11:30-11:45 DxFLEX- Flexible For Your Lab, Ms Pooja Dalvi

Now and in the Future
Beckman Coulter Life
Sciences

11:45 - 12:30 Flow cytometry in PNH and Primary Dr. Ananthvikas Jayaram
immunodeficiency disease

12:30-13:15 Flow Data Analysis and Presentation Dr. Hemant Agrawal

13:15 - 14:00 LUNCH

14:00-16:00 Lab 4: Leukemia/Lymphoma Dr. Brent Wood

Phenotyping
Dr. Monica Singh

16:00-17:00 Case Studies Dr. Brent Wood

Dr. Monica Singh

17:00-17:15 Quiz for Dr. Awtar Krishan Award Dr. Rekha Gour

Mission and Objectives of TETC Dr. Hemant Agrawal

17:15 -17:30 Award, Valedictory and feedback All Delegates, Faculties and
Organizing committee
members

17:30 High Tea

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 Organizers

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 Ramaiah Medical College

The Ramaiah Medical College (RMC) was established in 1979 by Gokula Education
Foundation (Medical), a charitable trust with the vision of providing quality medical
education to all. The College is affiliated to the Rajiv Gandhi University of Health Sciences
(RGUHS), Karnataka and is recognized by the Medical Council of India (MCI). To meet the
need of training the next generation of doctors, the Ramaiah Medical College Hospital
(RMCH) was set up on the campus in 1984. The Hospital accommodates 1,331 beds and
offers all super specialties. The College is well-equipped with the latest infrastructure,
technology-enabled classrooms and advanced laboratories, and continually focuses on
teaching, clinical practice, and research. The College offers degrees in MBBS, MD and MS in
all specialties, DM and MCH in the super specialties, PG Diploma and Ph.D. courses as well
as Bachelors (BPT), Masters (MPT) and Ph.D. in Physiotherapy. At RMC, we strive to
nurture a well-rounded professional. Here, our focus is on building doctors with a strong,
comprehensive foundation of medical knowledge. Our students le arn through rigorous
academics and a balanced focus on extra-curricular activities. They learn to practice in
well-equipped OTs as well as in the rural and urban field practice areas. They learn to
make decisions independently and when to collaborate with a fellow student of nursing,
physiotherapy or pharmacy. At RMC, we support students to i nitiate, organise and
participate in co-curricula r activities. Because we believe a Medic al Leader needs skill
that go beyond treatment and cure.

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The Indo-US flow cytometry workshops in India were started by Prof. Awtar
Krishan in collaboration with Dr. Ranbir Sobti and Dr. Arvinder Singh in 2002.
These workshops were started with a vision of bringing experts from India, USA
and abroad to the same platform, where their expertise can be harnessed by the
participants to understand the basics and advanced concepts in flow cytometry and
to apply this insight to their clinical or non-clinical research.

Looking at the increasing demand and requirement of these educational programs,

Indo-US Workshops committee decided to form and register a formal non-profit
Trust, which can carry on these educational activities all over in collaboration with
interested organizations. We are happy to inform that this above mentioned trust
has been registered as “Trust for Education and Training in Cytometry (TETC)” in
2018, which is officially organizing 21st Indo-US Cytometry Workshops in
collaboration with LETF-ISAC at 4 major Institutes of India at

CSIR-NIO, Goa, 27th-28th January

Ramaiah Medical College, Bangalore 29th-30th January
Panjab University, Chandigarh 2nd-5th February
Eternal University, Baru Sahib 6th-7th February 2020

TETC works under the leadership of Prof. Awtar Krishan, Patron, INDO-US
Flow Cytometry Workshops, India.

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 International Society for Advancement of Cytometry (ISAC)

w ww.isac-net.org

The International Society for Advancement of Cytometry (formerly International Society for
Analytical Cytology) is a scientific and educational organization that leads the way in
development of cytometry, transfer of new methodologies, and exchange of cutting-edge
scientific and technical information in quantitative cell sciences.

ISAC is the professional organization for scientists utilizing multidisciplinary, advanced

technology for the measurement of cells and cell processes. ISAC members range from new
graduates, and graduate students, to senior and emeritus members who have distinguished track
records of scientific achievement. ISAC members cover a very broad range of technological
expertise, thus increasing the probability of identifying collaborators for multidisciplinary
projects, both academic and commercially oriented. Also, The Society has a strong track record
of mentoring over the 25 years of its existence.

Amongst the principal benefits to you and your organization other than joining and participating
in ISAC events, is access to Cytometry Part A, the Society's official journal. Current Protocols in
Cytometry is also available to ISAC members at a special rate.

Every year ISAC holds the annual CYTO meeting and this year’s CYTO
(https://cytoconference.org/) will be held in Philadephia, USA from June 19th -23th 2020.

We have some of the world's top developers of technology within our organization. Additionally,
we sponsor specialty technology development meetings, science-based meetings and interactive
workshops on a variety of topics making ISAC is a premier organization available for
networking in today's highly technological world. Looking forward to having you on board!!

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 Messages

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 MESSAGE FROM TETC

We welcome you to the 21st Indo-US Flow Cytometry Workshop hosted by Ramaiah Medical College,
Bengaluru.

As we have been involved in the Indo-US flow cytometry workshops and conferences in India from very
beginning, we have seen the continuous development of this state-of-the-art technology in our country.
This revolutionary initiative towards imparting the knowledge of flow cytometry in India was taken by
Prof. Awtar Krishan in collaboration with Prof. Ranbir Sobti and Dr. Arvinder Singh in 2002, which gave
the birth to the “Indo-US Cytometry Workshops”.These workshops were started with a vision of bringing
experts from India, USA and abroad to same platform, where their expertise can be harnessed by the
participants to understand the basics and advanced concepts in flow cytometry and to apply this insight to
their clinical or non-clinicalresearch.

To realize the vision of the founders of the “Indo-US Cytometry workshops” for a good flow cytometry
education in India, we have been continuously supported by faculties (National and International), ISAC,
Host Institutes/Universities, Government and Non-Government sponsors. Recently, we have started
supporting the workshops outside India too. In the recent past, workshops in Nepal and Sri Lanka were
successfully organized by Indo-US Workshop committee. We also took an initiative to propagate the
knowledge of flow cytometry to the students of colleges and state funded Universities. Looking at the
increasing demand and requirement of these educational programs, committee decided to form and
register a formal non-profit Trust, which can carry on these educational activities all over in collaboration
with interested organizations. We are happy to inform that this above-mentioned trust has been registered
as “Trust for Education and Training in Cytometry (TETC)”, which is officially organizing 21st Indo-US
Cytometry Workshops.

We would like to take this opportunity to thank all our enthusiastic participants, our elite faculty and our
sponsors for making this event happen. Also, we would like to thank the organizers for their foresight and
efforts in making these workshops a great platform for learning.

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 9650 Rockville Pike Bethesda, Maryland 20814-3998 USA

PHONE301-634-7435 EMAILisac@isac-net.org

FAX 301-634-7429 WEBwww.isac-net.org

21st Indo-US Flow Cytometry Workshop

Ramaiah Medical College
Bengaluru, India
29 – 30th January, 2020
th

Message
On behalf of the Organizing Committee for the Indo-US Workshops and the Live Education
Task Force of the International Society for Advancement of Cytometry, I welcome you to our
21st Flow cytometry workshops.

The Cytometry Workshops (www.cytometryworkshops.com) were started in 2002 with the

express purpose of interfacing visiting experts in flow Cytometry for teaching applications of
flow cytometry in bio-medical research. We have used the Indian model to conduct flow
cytometry workshops in various cities of Egypt, India and China and in Kuala Lumpur,
Singapore, Jakarta, Bangkok, Istanbul, Ankara, Antalya, Riyadh, Glasgow, Prague, Warsaw and
Miami.

We are thankful to the local organizing committee and the organizing committee of the Indo-US
workshops (Drs. Rekha Gour, Hemant Agrawal, H. Krishnamurthy, and Vivek Tanavde) to have
organized an excellent program of lectures and wet labs using latest protocols and instruments.

We are glad to inform you that a non-profit educational trust for teaching cytometry named Trust
for Education and Training for Cytometry (TETC) has been set up in India for continuation of
these workshops.

We are thankful to our vendors for continuous support of our workshops with seed funds,
instruments and technical staff.

I hope you enjoy positive interactions with the faculty and continue your association with the
Indo-US cytometry workshops in future.

Yours truly,

Awtar Krishan

Emeritus Professor
Pathology Department
University of Miami School of
Medicine Miami, Fl. USA.
Co-Chair, Live Education Task Force, International Society for Advancement of Cytometry

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 Message from Organizing Co–Chairperson

Flow cytometry is an important noninvasive upcoming diagnostic tool which is being used
widely.

However not all centers are equipped and familiar with this.

This workshop will definitely help all the delegates in acquiring the skills and knowledge
necessary.

I extend a warm welcome to all the delegates to the 21st Indo – US Flow Cytometry Workshop
jointly organized by Department of Pathology, Ramaiah Medical College, TETC and LETF-
ISAC

Prof. Pushpati N. Razdan

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 MESSAGE

Dr. Medha Y. Rao

Principal and Dean

Ramaiah Medical College

I am happy to note that the 21st Indo – US Flow Cytometry Workshop “Clinical Applications of Flow
Cytometry “is being held on 29th– 30th January 2020 at Ramaiah Medical College, Bengaluru.

The workshop is designed with relevant and interesting topics and I am sure the takeaway for all the
delegates is going to be greatly helpful in day to day practice.

The healthcare professionals will be enormously benefitted as the information acquired will go long way
in designing effective care plans for good outcomes.

I congratulate the organizing committee of this conference for their huge efforts to organize this
conference and wish them success.

I extend a warm welcome to all the delegates to Ramaiah Medical College and wish them a rich
experience on the campus.

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 Message from Co–Chairperson

I cordially welcome all delegates and faculty to this

academic extravaganza in the field of flow cytometry. As
the organizing co-chairperson of this 21st Indo-US flow
cytometry workshop jointly organized by Live Education
Task Force, International Society for Advancement of
Cytometry (LETF-ISAC) , Trust for Education and Training
in Cytometry (TETC)and Ramaiah Medical College (RMC) it gives me immense
pleasure and joy to host this workshop in our premier Medical institute of
excellence. My heartfelt gratitude to our ever-encouraging management and my
colleagues in the department who have left no stones unturned in organizing the
workshop. I am sure the delegates attending this workshop will benefit from the
rich experience of the esteemed faculty and pave the way for further advancement
in laboratory medicine.

Welcome you all……

Warm Regards

Dr Prasanna Shetty

Prof and HOD of Pathology

Ramaiah Medical College, Bengaluru

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 Message from Co-founder-TETC &
Workshop Convenor

It is my great honor to be the convener for the 21st Indo-US Flow

Cytometry Workshops which is happening at 4 sites in India. Ramaiah
Medical College is one of the sites, the other three workshops are being
organized CSIR-NIO, Goa, 27th-28th Jan; Panjab University,
Chandigarh 2nd-5th Feb and at Eternal University, Baru Sahib 6th-7th
Feb 2020.

In last few years, flow cytometry has rapidly changed both in its
dimension and direction. With the advancement of the technology, it’s
very important to get updated with the current knowledge available in the field. This workshop
will provide an excellent platform for all the participants to learn the technology and its
applications directly from the experts.

As I have been involved in the Indo-US flow cytometry workshops and conferences in India
from more than a decade, I have seen the continuous development of this state-of-the-art
technology in our country. The revolutionary initiative towards imparting the knowledge of flow
cytometry to the Indian scientists was take by Dr. Awtar Krishan in collaboration with Dr.
Ranbir Sobti and Dr. Arvinder Singh in 2002, which gave the birth to the “Indo-US flow
cytometry workshops”. These workshops were started with a vision of bringing experts from
India, USA or abroad to a same platform, where their expertise can be utilized by the
participants to understand the basics and advanced concepts in flow cytometry and to apply this
insight to their clinical or non-clinical research interests.

To take forward the vision of the founders of the “Indo-US Cytometry workshops”, we have
registered a non-profit Trust known as Trust for Education and Training in Cytometry (TETC)
and started an initiative to propagate the knowledge of flow cytometry to the students of colleges,
research institutes, and universities in India. On the same line, this current workshop at Ramaiah
Medical College is our earnest move in this direction.

I would like to take this opportunity to thank all our enthusiastic participants, our elite faculty
and our sponsors for making this event happen. Also, I like to thank the Ramaiah Medical
College and whole Team at Ramaiah and TETC for their foresight and earnest efforts in making
this workshop a great platform for learning.

Dr. Rekha Gour

Workshop Convenor
Co-founder and Managing Trustee-TETC

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 Message from Organizing Secretary

It gives me immense pleasure to welcome all the delegates to

this 21st INDO-US Flow Cytometry Workshop “Clinical
Applications of Flow Cytometry,” jointly organized by
Department of Pathology, Ramaiah Medical College, TETC
and LETF-ISAC.

The use of flow cytometry as a diagnostic tool for leukemia

and lymphoma is most important.

I am glad that eminent personalities, national and international

from the field of flow cytometry will share their experiences
which will benefit one and all. I hope this workshop will be a platform for postgraduates, post
doctoral students and pathologists to have meaningful interactions with the speakers.

The organizing team has worked tirelessly to make this conference a memorable event.

I take this opportunity to invite you all to attend the workshop in our esteemed institution.

I wish this workshop a grand success.

Dr. Kalpana Kumari MK

Current designation:

ASSOCIATE PROFESSOR,

Dept of pathology,Ramaiah Medical College.

In charge pathologist at Clinical hematology and bone marrow transplantation Unit

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 Message from Organizing Secretary

It is my great pleasure to be the part of the 21st INDO-US Flow

Cytometry Workshop at Ramaiah Medical College, Bangalore
from 29th-30th Jan 2020, which is jointly organized by TETC,
Ramaiah Medical College and LETF-ISAC.

Flow cytometry is a rapidly growing state of the art technique,

and to harness the real power of this complex and rapidly
evolving technique, formal organized training plays a major role.
Correct formal training is not only important but also critical to
avoid poor experimental design, inaccurate data analysis, wrong
presentations and incorrect conclusions.

Keeping this in mind, INDO-US Flow Cytometry meetings has been started, which promotes the
flow cytometry education in India and neighboring countries. The aim of these workshops is to
promote and facilitate the formal training for the scientists who are using or intending to use
flow cytometry in their research. In last few years, the number of parameters (colors)
simultaneously used in flow cytometry experiments has increased, which has added even more
complexity to the researchers and made formal training of paramount importance. The goal is to
provide correct insight about the core concepts of flow cytometry, which will help researchers to
generate accurate and meaningful results that can further be utilized in research or clinics.

I would like to take this opportunity to thank all our participants, our elite faculty and our
sponsors for making this event happen. Also, I like to thank the Ramaiah Medical College and
whole Team at Ramaiah and TETC for their hard work and earnest efforts in making this
workshop a great platform for learning.

Dr. Hemant Agrawal

Founder Director-Flowcytometry Solutions

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 Workshop
Faculty

23
 Know Your Faculty

Name Affiliation Email

Dr. Brent Wood University of Washington, USA woodbl@uw.edu
Live Education
Task Force of the International
Society for Advancement of
Dr. Zosia Maciorowski Cytometry (ISAC) zosiamaciorowski@gmail.com

Dr. Paul Edward National University of

Hutchinson Singapore lsipeh@nus.edu.sg

National Centre for Biological

Dr. H Krishnamurthy Sciences, Bengaluru, India krishna@ncbs.res.in

Dr. Sumeet Gujral Tata Memorial Centre, Mumbai. flowtmh@gmail.com

Trust for Education and Training
in Cytometry (TETC),
Dr. Rekha Gour India rekhagour@gmail.com
Flowcytometry Solutions (P)
Dr. Hemant Agrawal Ltd, India flowsols@gmail.com

Dr. Vivek Tanavade Ahmedabad University vivek.tanavde@ahduni.edu.in

Anand Diagnostic Laboratory and
Dr. Ananthvikas Neuberg Anand
Jayaram ReferenceLaboratory, Bangalore. ananthvikas@neuberganand.com

Dr. Monica Singh Pathkind Diagnostics, India doctor.monica21@gmail.com

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Dr. Brent Wood
Brent Wood obtained his MD and PhD from Loma Linda
University followed by a residency in Anatomic and Clinical
Pathology at the University of Washington in Seattle. After a
fellowship in Hematopathology at the University of Washington,
Dr. Wood accepted a faculty position in the Department of
Laboratory Medicine at the University of Washington where he
is currently Professor and Director of the Hematopathology and
Seattle Cancer Care Alliance Laboratories. His responsibilities
include clinical service work, translational research, and trainee
education. Flow cytometry is an area of particular interest. He is responsible for implementing
the first use of 9 and 10 color flow cytometry in the clinical laboratory and exploiting its
potential for the identification of minimal residual disease in acute lymphoid leukemia and
acute myeloid leukemia. His laboratory serves as one of two national reference laboratories for
the identification of minimal residual disease in childhood acute lymphoblastic leukemia for
the Children’s Oncology Group, is involved in similar protocols with the Southwest Oncology
Group, and provides contract testing for a number of biotechnology and pharmaceutical
companies. Dr. Wood lectures both nationally and internationally on clinical applications of
flow cytometry and is Past President of the International Clinical Cytometry Society.

Dr. Zosia Maciorowski

Zosia Maciorowski received her B.Sc. in Microbiology from McGill
University in Montreal and M.A. in Biology from Wayne State
University in Detroit. She has worked in many labs and countries over
the years on a variety of different subjects, from the early days of tissue
culture to small animal surgery, monoclonal antibody production and
early immunological and molecular biology techniques. In the 80’s she
specialized in solid tumor preparation for multicolor and cell cycle
analysis. For 28 years she was responsible for the Flow Cytometry Core Facility at the Curie
Institute in Paris, France, from which she retired last year. Zosia is Co-Chair of the Live
Education Task Force of the International Society for Advancement of Cytometry (ISAC)
which has organized international flow cytometry workshops around the world.

25
Dr Paul Edward Hutchinson
Paul Hutchinson began working in flow cytometry in 1983 at the
Peter MacCallum Cancer Institute in Melbourne while completing
his Applied Physics degree at RMIT. In this lab, flow cytometry
was used for clinical and research work, including the diagnosis
of AIDS patients at the beginning of the HIV epidemic and early
research on the isolation of haemopoietic stem cells. In 1986 he
moved to Prince Henry’s Hospital to be in charge of the Cell
Sorter in the Department of Nephrology. During his time in this lab he completed his Master of
Science degree (Monash University) on macrophage function. In 1992 Paul joined the Clinical
Immunology department at Monash Medical Centre and was in charge of the core flow
cytometry facility, which had 3 Mo-Flo cell sorters along with one analyser. The lab was used
for clinical tests and research, and it was here that he did his PhD (Monash University) on the
use of flow cytometry to quantify the level of immune function in renal transplant recipients. In
February 2008 he was appointed the head of the Unidade de Citometria de Fluxo at the Instituo
de Medicina Molecular in Lisbon, Portugal. This flow cytometry lab serviced more than 250
researchers, and had a 11 color FACSAria cell sorter, and three analysers. Since July 2009 Paul
has been in charge of the core flow cytometry facility at the Immunology Programme of the
National University of Singapore which has 4 analysers (5 laser X-20, 4 laser Fortessa, 4 laser
Attune, and 3 laser CyAN), plus a Mo-Flo XDP cell sorter, Sony Sy3200 sorter, and a 5 laser
20 parameter BD FACSFusion cell sorter. Besides running this busy lab, Paul has pursued his
research interests in using flow cytometry to develop diagnostic tests for tuberculosis and
investigating new technologies for doing single cell measurements.

Dr. H. Krishnamurthy
Dr. H. Krishnamurthy obtained his PhD from the Manipal
Academy of Higher Education, Manipal, India (1997) in Radiation
Biology. From 1997 to 2001, he underwent postdoctoraltraining at the
Institute for Reproductive Medicine,Muenster, Germany: Institute for
Clinical Research, Montreal, Canada and University of Iowa, Iowa,
USA. Later, he moved to Herbert Irving Comprehensive Cancer
Center, Columbia University, New York, USA as Associate Research Scientist and Managing
Director of the flow cytometry facility. At present, he is working as Scientific Officer ‘G’ and
In‐ charge of Central Imaging and Flow Cytometry at the National Centre for Biological
Sciences, Tata Institute of Fundamental Research, Bangalore, India.

26
His research interest is hormonal regulation of spermatogenesis and trafficking and signaling
of gonadotropin receptors. He is a member of live education task force of the International
Society for Advancement of Cytometry (ISAC). He has hosted the 9th and 11th Indo‐US
Cytometry workshops in Bangalore and participated in other workshops organized by the Asia
Task Force of ISAC.

Dr. Rekha R. Gour

Convenor, 21stIndo-US Cytometry Workshops, 2020
Co-founder and Managing Trustee, Trust for Education and Training in Cytometry (TETC)
Advisory Member-Biotechnology Society of Nepal (BSN), Nepal
rekhagour@gmail.com; 09284999738

Dr. Rekha R. Gour, graduated with a major in chemistry from

Mumbai University in 1991 and got her Diploma in Medical
Laboratory Technology. She was associated with the Flow
Cytometry Facility at the Cancer Research Institute, ACTREC, Tata
Memorial Centre, Mumbai, INDIA for 25 years. As Scientific
Assistant at ACTREC, she was actively involved in Flow Cytometry
training of scientists and students from ACTREC and other Institutes.
Rekha has been actively associated with the Indo-US Cytometry workshops since the first
workshop in Chandigarh in 2002. She has been an invited faculty and member of the
Organizing Committee for most of the INDO-US Cytometry workshops. With her experience
and expertise in Flow Cytometry she is able to assist organizers, involved in Indo-US Flow
Cytometry workshops in India. She was an Executive member of The Cytometry Society
(TCS-India) & has played a key role in expanding the TCS strength by registering new
members and raising fund.
Understanding the lack of adequate Flow Cytometry training in remote corners of India, Rekha
has assisted in Organizing Indo-US and TCS workshops in CCHRC (Silchar) and IIT-G
(Guwahati), Assam. Her idea of teaching Flow Cytometry to young students has led her to start
Flow Cytometry workshops in Mumbai Colleges, which has trained many college students and
faculties. She is member of the Organizing Committee for conduct of Indo-US cytometry
Workshops in India and a Founder and Managing Trustee for Trust for Education and Training
in Cytometry (TETC). She is playing a key role by assisting and connecting the flow cytometry
community to organize and conduct Flow cytometry workshops in India, Nepal and Sri Lanka.
Rekha opted for Voluntary Retirement, but her commitment and support for Flow Cytometry
continues and in a great way.

27
Dr. Hemant Agrawal
flowsols@gmail.com
Director—Flowcytometry Solutions Pvt. Ltd.
Organizing Secetray-21st Indo-US Cytometry Workshops, 2020
Member—Trust for Education and Training in Cytometry (TETC)
Advisory Member-Biotechnology Society of Nepal (BSN), Nepal
Application Support Consultant—FCS Express Software
(DENOVO Software, USA)
flowsols@gmail.com; +91-7665130114

Hemant Agrawal is an immunologist with a passion for flow cytometry. Hemant obtained PhD
in Immunology from the University Hospital Essen, Germany (2006). He joined Oklahoma
Medical Research Foundation, Oklahoma City, USA as an Associate Research Scientist in the
Immunology and Arthritis Program (2006-2009). There he performed extensive studies on
dendritic cells and macrophages using multicolor flow cytometry. He subsequently joined the
Rheumatology Department at Northwestern Memorial Hospital, Chicago from 2009 to 2011 as
a Research Associate Scientist and continued studies in autoimmunity using flow cytometry.
From 2011 to 2013, he worked as an Application and Product Manager with FlowJo, TreeStar
Inc, USA for the Indian subcontinent and Middle East. His knowledge of flow cytometry is
highly regarded, which led him to be served as consultant for companies like Bio-Rad,
TTPLabtech and Zydus Cadila. Hemant has co-authored many peer-reviewed scientific
publications and has been invited as speakers at various national and international meetings &
conferences. He is also an organizing committee member of the prestigious Indo-US
Cytometry Workshops registered as Trust for Education and Training in Cytometry (TETC),
which conducts annual cytometry meetings in India and neighboring countries in collaboration
with International Society of Advancement of Cytometry (ISAC). He is experienced in flow
cytometry experimental design, data acquisition, data analysis and presentation of
immunophenotyping, intracellular staining, cell proliferation, cell cycle, apoptosis, cytometric
bead array etc. At present, he is based in Jaipur and running a flow cytometry consultancy
company, “Flowcytometry Solutions (P) Ltd”, which imparts training and consultancy in the
field of flow cytometry in India and neighboring countries. His organization has conducted
over 70 flow cytometry workshops all over India and neighboring countries in last 5 years,
where participants got the training to design, acquire and analyze the flow cytometry
experiments. He is also a consultant to De Novo Software (FCS Express software), Venture
Centre (CSIR-Bioincubator) and Quantum Technology Group, USA. Recently, he has been
appointed as an advisory member of Biotechnology Society of Nepal (BSN) to support and
promote flow cytometry and biotechnology education in Nepal.

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Dr. Sumeet Gujral, MD

Sumeet Gujral, Professor of Pathology at the Tata Memorial

Hospital, Mumbai received his MBBS from PGIMS, Rohtak, and his
MD in Pathology from AIIMS, New Delhi. He is an oncopathologist
with a special interest in hematopathology, immunohistochemistry
and flow cytometry. He is a founder-Member and First Secretary
(clinical) of the The Cytometry Society. His laboratory has published
extensively on Indian data on hematolymphoid neoplasms. His
laboratory conducts proficiency testing program for hematolymphoid
neoplasms, and advanced clinical cytometry and hematology training programs for students
from India and other Asian countries.

Dr. Vivek Tanavde

Dr. Vivek Tanavde obtained his Ph.D from the Cancer Research
Institute, Mumbai (1999) in Applied Biology. From 1999 to 2002 he
was a post doctoral fellow at the Sidney Kimmel Cancer Centre,
Johns Hopkins University working on expansion of hematopoietic
stem cells. He returned to India in 2002 and was heading the
Hematopoietic Stem Cell Lab at Reliance Life Sciences, Mumbai.
His laboratory also provided diagnostic services using flow
cytometry, mainly in the areas of CD4/CD8 enumeration & Single platform CD34 enumeration.
In 2006, he joined the Bioinformatics Institute, Singapore as a Research Scientist in the
Genome & Gene Expression Data Analysis Division, where he was a Principal Investigator
until Oct 2016. Most of his work has focused on developing protocols for expansion and
targeted differentiation of hematopoietic and mesenchymal stromal cells. His current research
focuses on the use of bioinformatics and systems biology tools to understand mesenchymal
stromal cell biology and differentiation. He recently joined Ahmedabad University as an
Associate Professor at the School of Arts and Sciences.

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Dr. Ananthvikas J

Dr. Ananthvikas J obtained MD in Pathology from Christian

Medical College, Vellore in 2017. He obtained further training in
Molecular Hematology from ACTREC, Tata Memorial Hospital,
Mumbai in 2018. He is currently a pathologist at Anand
Diagnostic Laboratory and Neuberg Anand Reference Laboratory,
Bangalore.
His primary area of interest is hematopathology and is involved in
the reporting of lymph node biopsies, bone marrows and flow cytometry assays in his
laboratory.
He is passionate about development and reporting of flow cytometry assays and has been
responsible for the standardization and development of assays including diagnostic
Leukemia/Lymphoma assays, B-ALL Minimal residual disease, Naïve T cell subset
enumeration and diagnostic assays for Primary immunodeficiency disorders. He has
standardized a low cost screening panel for Primary immunodeficiency disorders at Neuberg
Anand Reference Laboratory, Bangalore, which has helped in screening of close to 250
children with suspected primary immunodeficiencies over the last year.
He has also worked extensively on cell counter parameters and scattergrams and maximizing
their utility in adding value to patient care. His team have identified several specific scatterplot
patterns that aid in diagnosis, ranging from viral illnesses including dengue and infectious
mononucleosis to malignancies including acute leukemias and lymphoproliferative disorders.
This work has been presented at various state and national level conferences.

Dr. Monica Singh

MBBS, DNB Pathology, Ccy (ICCE)

Hematopathologist

Cofounder. Cellnomics Ltd.

Consultant Hematopathologist-Pathkind Diagnostics, India.

Manipal Hospitals, India. Strand Lifesciences, India.

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Trained in Hematopathology from Tata Memorial Hospital, Mumbai.

Trained in Minimal residual disease detection by Flow Cytometry at NUH,

Singapore and Seattle Cancer Care Alliance, Univ. of Washington, USA.

Also trained in Primary Immune Deficiency Disorder detection by Flow

Cytometry at Cincinnati Children’s Hospital, Cincinnati, USA.

Trained in Molecular diagnosis of leukaemias from Univ. of Washington, USA.

Area of Interest: Primary Immunodeficiency Disorders

Minimal residual disease detection by flow cytometry.

Post transplant immune monitoring

Areas of Expertise in Flow Cytometry

Planning and implementing multicolor assays on 10 color flow cytometer

especially comprising:

 Flow cytometric analysis of minimal residual disease (MRD) for B ALL, T

ALL, AML, CLL and Multiple Myeloma.
 Flow cytometric analysis for diagnosis of Acute Leukaemias, Multiple
Myeloma, Chronic Lymphoproliferative Disorders, Myeloproliferative
Neoplasms and Myelodysplastic Syndromes.
 Flow cytometric analysis of Primary Immunodeficiency Disorders.
 Flow cytometric assays for post-hematopoietic transplant immune
monitoring and T regulatory cell monitoring.

31
 Abstracts

32
 Basics of Flow Cytometry
H. Krishnamurthy
National Centre for Biological Sciences
Tata Institute of Fundamental Research
Bangalore

To drive an automobile one does not need to know how to design an alternator but should
understand if it’s operating correctly and optimally. Likewise with a flow cytometer or cell sorter; to
properly operate a cytometer and interpret data, one must understand its basic components and
their operation in order to obtain valid and optimized data. The goal of this lecture is to familiarize
those new to the field of flow cytometry with its basic components and their correct usage. The key
components including fluidics, lasers, optics, electronic detectors, analog to digital converters and
pulse processors will be described in sufficient detail to give the new operator/user a basic
understanding.

Leukemic Cell Phenotype and MRD Monitoring.

Dr. Brent Wood
Although morphology with cytochemistry can diagnose many cases of acute leukemia, there remain
a significant minority of cases that cannot be definitively classified by these methods. Such cases
require immunophenotyping by flow cytometry (FCM). Similarly, chronic lymphoproliferative
disorders (CLPD) need flow cytometric analysis for further characterization. Subclassification of
acute leukaemia and CLPD is important, as the treatment and prognosis of subtypes is different. In
addition, the monitoring of response after therapy is increasingly recognized as one of the single
more important prognostic factors in acute leukemia. Consequently, FCM has become a standard
and routine practice for the evaluation of lymphomas and leukemia in most diagnostic laboratories
and commonly used for detecting residual disease after therapy. In the lecture and lab, the
principles of immunophenotyping for the diagnosis, classification and monitoring of hematopoietic
neoplasms will be presented and illustrated with clinical case material.

Leukemia Phenotype Analysis

Dr. Sumeet Gujral
Morphology with cytochemistry (myeloperoxidase and non specific esterase) can diagnose
most cases of acute leukemia. However, there remains a significant minority of cases that
cannot be definitively diagnosed by these methods. Such cases require immunophenotyping
by flow cytometry (FCM). Similarly chronic lymphoproliferative disorders (CLPD) need
flow analysis for further characterization. Sub typing of acute leukemias and CLPDs is
important, as treatment and prognosis of different types is different. FCM has become a
standard and a routine practice for the evaluation of lymphomas and leukemia in most
diagnostic laboratories and in detecting minimal residual disease. FCM constitutes an
extremely important tool in the diagnosis of PNH and in lymphocyte subset analysis.

33
 Polychromatic flow and Panel design for
Immunophenotyping of B, T and NK cells

Dr. Paul Edward Hutchinson

Lymphocytes are vital cells of the immune system. They consist of T-, B-, and Natural Killer (NK) –
cells; who in turn can be subdivided into further subsets each with their own particular functions in
host defense. These different subsets of lymphocytes can be identified and enumerated by
measuring the expression of specific proteins using fluorescently tagged antibodies and flow
cytometry, in a technique known as Polychromatic Flow. In this talk I will describe the principals of
doing this, along with examples and tips for the design of antibody panels to measure the different
lymphocyte subsets

CD34 Enumeration
Dr. Vivek Tanavde
CD34 enumeration is a useful tool to enumerate the numbers of hematopoietic stem cells in bone
marrow, cord blood or mobilized peripheral blood grafts used for transplantation. This lab will
introduce the general principles involved in rare cell analysis, their application to CD34 enumeration
and discuss the ISHAGE protocol which enumerates the numbers of viable CD34+ cells using single
platform flow cytometry (1). Critical issues in this assay as outlined by Sutherland & Keeney (2) will
also be discussed.

Flow Data Analysis and Presentation

Dr. Hemant Agrawal
Accurate data analysis and display is not only important but also critical to adequately identify or
functionally characterize the multiple populations by flow cytometry. In this module, we will cover
analysis of single or multicolor flow cytometry data and guidelines for the correct data analysis and
presentation. Also, there will be demonstration and discussion of different flow data files (FCS files)
using offline data analysis software. Participants will be provided with the knowledge to understand
potential technical issues with the flow data analysis and presentation.

34
Flow cytometry in Paroxysmal Nocturnal hemoglobinuria
Dr. Ananthvikas Jayaram
Paroxysmal Nocturnal Hemoglobinuria is an acquired clonal hematopoietic stem cell disorder
characterized by an intravascular hemolytic anemia. This disease is characterized by the inability of
cells to produce glycosylphosphatidylinositol (GPI)-anchored proteins on the surface, which include
decay accelerating factor (CD55) and Membrane inhibitor of reactive lysis (CD59), leading to
complement mediated hemolysis and hemoglobinuria. The genetic basis of this disease involves
mutation in the PIG-A gene which is located on the short arm of the X chromosome.

Historically, the Ham acidified serum lysis test has been used in the diagnosis of PNH. Ham, between
1937 and 1939, demonstrated that bicarbonate diminished hemolysis in PNH and that an acidifying
agent, ammonium chloride, increased PNH hemolysis, thereby implicating acidosis as a cause of
hemolysis. The only other disease associated with a positive HAM test was Congenital
dyserythropoieticanemia type II (HEMPAS). During the 1960s Dacie and Rosse, using the
complement lysis sensitivity test, identified phenotypic heterogeneity, or mosaicism, among PNH
RBCs, and later identified 3 PNH RBC phenotypes: I, II, and III, that exhibit normal, moderate, and
severe complement sensitivity, respectively. In the 1970s and 1980s, it was demonstrated that C3
binding on PNH cells was increased due to lack of decay accelerating factor which is an inhibitor of
C3 convertase. Subsequently, it was also demonstrated that MIRL was deficient on the surface of
PNH RBCs.

Flow cytometry has recently replaced Ham test as a definitive test for PNH. Early flow cytometry
assays for PNH relied on detection of CD55 and CD59. Typically, RBCs were identified on FSC and SSC
and assessed for expression of CD55 and CD59. Expression of these markers was used to enumerate
the type III (complete GPI-antigen deficiency) and type II (partial GPI-antigen deficiency) PNH clones.
This approach, while it led to an improved detection of PNH, was neither accurate nor sensitive
below the 1-4% clone size. Detection of small clones that are often associated with Aplastic Anemia
or MDS was often a limitation. As per the latest consensus guidelines, CD235a is recommended to
gate the erythrocyte population as opposed to using only FSC and SSC.

With regard to WBC testing, a wide array of monoclonal antibodies have become available to detect
GPI anchored proteins (See table below).

Examples of GPI linked proteins:

Leucocyte antigens CD14, CD24, CD48, CD52, CD55, CD59, CD73, CD87, ART1, ART2, Sca-2,
Qa-2, Ly-6

Neural cell antigens NCAM, F3/F11, TAG-1, BIG-1, CNFTFR-alpha, ceruloplasmin,

Cerebroglycan

Receptors/ Adhesion Folate binding, protein, NCAM, F3/F11, TAG-1, BIG-1, CNFTFR-alpha,

35
 molecules Glypicans, Cerebroglycan, CD14, CD87, LFA-3

At least two different GPI protein deficiencies within two different cell lines from granulocytes,
monocytes or erythrocytes need to be shown with flow cytometry for diagnosis. Granulocytes and
monocytes are the preferred lineages for routine testing of PNH clone. It is advised to test for RBCs
in at least those cases which show a PNH clone on WBC analysis. Routine analysis of RBC alone is no
more recommended.

The utilization of FLAER (Fluorescent Aerolysin) has also improved the sensitivity of these assays as
this binds directly to the GPI anchor. This improved sensitivity has made it possible to detect PNH
clones of as small as 0.01%. For gating of neutrophils and monocytes, it is recommended to use
lineage specific markers (CD15 for neutrophils, and CD64 for monocytes), and gating based solely on
light scatter with/without CD45 is not considered optimal. A combination of FLAER and CD24 is often
used for Neutrophils, and FLAER and CD14 for monocytes. CD157 is another GPI linked marker that
can be used for both neutrophils and monocytes.

Gating strategies involve isolation of singlets followed by viability gating (FSC-SSC). Neutrophils and
monocytes are further gated using lineage specific markers as described. The gated neutrophils and
monocytes are then analysed further for the expression of GPI linked proteins - CD24 (neutrophils)
and CD14 (monocytes). Based on the limit of quantification in each lab, as many as 5,00,000 events
or more may be acquired to achieve a desired sensitivity of 0.01%.

References: PMID: 22499484; 29236353; 10539884; 14584753; 18557633

Flow cytometry in Primary immunodeficiency disorders (PID)

Dr. Ananthvikas Jayaram
PIDs are a group of inherited disorders that affect the development and /or function of the immune
system. These are rare disorders that have a prevalence of approximately 1 per 10,000 births. This
incidence however is increased in populations with a higher degree of consanguinity. These diseases
usually result in recurrent or persistent severe infections, autoimmunity, autoinflammation or
malignancies.

While most children with persistent infections may have a normal underlying immune system, it is
important to recognize a child with an underlying PID so that further investigations can be
performed. Prompt and early diagnosis not only helps initiate appropriate treatment, but also helps
further genetic counselling for the family.

The 2017 classification of the International union of Immunological Societies identifies close to 354
inborn errors of immunity, further classified as those affecting cellular and humoral immunity, with
syndromic features, antibody deficiencies, diseases of immune dysregulation, phagocyte defects,
defects of innate immunity, autoinflammatory disorders, complement deficiencies and phenocopies.

Flow cytometry is one of the tools in the diagnostic armamentarium for Primary immunodeficiency
disorders. The Euroflow consortium has recently described a PID orientation tube which is a fairly
comprehensive screening panel for lymphoid PIDs.

36
In the following passages, a few of the more common applications of flow cytometry shall be
discussed.

Lymphocyte subset analysis

Flow cytometric analysis for lymphocyte subsets including marker of B cells (CD19), T cells (CD3) and
NK cells (CD16+56) is often a first line investigation, especially in cases of severe combined
immunodeficiency. Results of lymphocyte subset analysis can also aid in further classification of SCID
(Eg. T-B-NK- SCID often associated with ADA and PNP gene defects, T-B-NK+ SCID often associated
with RAG1, RAG2, DCLRE1C, LIG4 and NHEJ1 gene defects, etc).

Further, flow cytometry for Naïve T cells and Memory T cells subsets in CD4 and CD8 positive T cells
adds value in diagnosis of cases of SCID, as Naïve T cells are often markedly decreased in cases of
SCID.

Functional assays for Phagocyte defects

Chronic granulomatous disease results from dysfunctional NADPH oxidase activity leading to
defective oxidative burst in neutrophils. Patients with CGD have reduced or absent NADPH oxidase
activity leading to reduced or no conversion of DHR into fluorescent rhodamine. Neutrophil
oxidative index is markedly decreased in patients with CGD.

Neutrophils are stimulated by a stimulant, commonly Phorbol 12-Myristate 13-Acetate in the

presence of DHR123 dye. The release of oxidases converts DHR123 to Rhodamine which is highly
green fluorescent when excited by a 488nm Laser. Neutrophil oxidative index is calculated as the
ratio of mean peak channel fluorescence in the PMA stimulated cells over the PMA unstimulated
cells. Appropriate healthy controls are included with every test sample.

Pre analytical factors heavily influence the outcome of the DHR flow cytometry assay. Sample
transport and storage lead to a progressive decline in the neutrophil oxidative burst in the samples,
and hence testing of a normal control sample sent along with the test, ie. a transport control, is
essential for comparison of the resultant DHR flow cytometry findings.

Rare cases of Myeloperoxidase deficiency may give a false decreased oxidative burst by
Dihydrorhodamine, but a preserved NBT dye reduction. The reason for this is that both NADPH
oxidase and myeloperoxidase enzyme systems play a role in the DHR test, which the NBT test relies
predominantly on the NADPH oxidase system.

CD18, CD11a, CD11b and CD11c in Leucocyte Adhesion Defect type 1

Leucocyte Adhesion deficiency is a rare autosomal recessive disorder with increased susceptibility to
infections resulting from defects in neutrophil adhesion. These defects result in poor neutrophil
chemotaxis, marked neutrophilia and the inability to form pus. Of the 3 types of Leucocyte Adhesion
Defeciency, LAD-1 is the more common, caused by a defect in the integrin Beta 2 (ITGB2) gene,
which codes for the beta 2 integrin subunit, also known as CD18. Upon binding with one of a number
of alpha chains, CD18 is capable of forming multiple heterodimers, which play significant roles in
cellular adhesion and cell surface signaling, as well as important roles in immune responses. The
known binding partners of CD18 are CD11a, CD11b,CD11c and CD11d.

37
Suspected cases of LAD-1 often present with marked neutrophilic leucocytosis with a history of
delayed detachment of umbilical stump at birth. Reduced or absent expression of CD18 or CD11 in
neutrophils in LAD1 can be detected by flow cytometry.

Double negative T cell estimation for Autoimmune lymphoproliferative syndrome.

ALPS is characterized by lymphoproliferation, multilineage cytopenias, and an increased risk of B cell

lymphoma. Defective apoptosis of developing lymphocytes in thymus results in an increase of
double-negative T cells (CD3+TCRαβ+CD4–CD8–). Percentage of DNTs >1.5% of all lymphocytes or
2.5% of CD3+ T lymphocytes is considered abnormal.

References: PMID: 30886612, 3594570, 31244832, 31572360, 29226301

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PANEL DESIGNING FOR LEUKEMIA AND LYMPHOMAS

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Flow Cytometry Data Analysis & Presentation

Dr. Hemant Agarwal

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Workshop
Protocols

98
 Cell Cycle Analysis Using Hypotonic Propidium Iodide Solution
Dr. Awtar Krishan

Crissman and Steinkamp (1973) published a paper on use of propidium iodide for flow cytometric
analysis of DNA in cells fixed with ethanol and digested with RNase. Subsequently, Krishan (1975)
reported that propidium iodide solutions made in hypotonic sodium citrate could lyse the cells and
directly stain the cells for DNA content and cell cycle analysis. The method described below is
rapid, uses small amount of material and has been extensively quoted and used for flow
cytometric analysis of DNA aneuploidy and cell cycle distribution.

Reagents:

Propidium iodide – Sigma P-4170

Trisodium citrate dihydride – Sigma S-4641
Nonidet P-40 (NP-40) – Sigma N-6507
NP-40 equivalent – Sigma I-3021 Igepal CA-630
Tween 20 – Sigma P-1379
RNase A – Sigma R-4875

Solution for live cells (100ml hypotonic solution)

100ml DDW
100 mg Sod. Citrate
4 mg RNase
5 mg PI
30 ul NP40/IGEPAL/Tween-20
Solution should be stored in a dark amber bottle or glass bottle al-foil wrapped in a
refrigerator (4-8 °C) and will keep for years.
Protocol:

Buffy coats from Ficol-Hypaque treated peripheral blood or bone marrow

1. Add approximately 106 cells/ml directly to the hypotonic propidium iodide stain and
vortex vigorously.
Suspension cell cultures
1. Centrifuge the cells to collect a cell pellet.
2. Remove all medium by inverting and holding the test tube over a paper towel to drain.
3. Add hypotonic propidium iodide stain and vortex vigorously.
Monolayer cultures
1. Remove all medium and rinse the flasks or Petri dishes with 1x phosphate buffered
saline (PBS).
2. Add the hypotonic propidium iodide citrate stain to the monolayer and scrape with a
rubber policeman, shake vigorously and use a fine tipped pipette to dislodge any
cytoplasmic remnants from the lysed cells.
3. Centrifuge and suspend the nuclear pellet in fresh hypotonic propidium iodide citrate
stain for analysis. Samples can be stored for up to 24 hrs in a refrigerator before flow
analysis. The isolated nuclei will need more centrifugal force than cells and we
normally use approximately 100 RCF.

Comments: Do not use trypsin to remove the monolayer as it may interfere with dye
binding.

99
 Basic Multicolor Flow Cytometry Wet Lab

Dr. Zosia Maciorowski

SURFACE STAINING PROTOCOLS for cells and beads

CELLS (peripheral blood cells)

3 ml EDTA anti-coagulated freshly collected human peripheral blood.

Antibodies and Buffers:

Reagent Other details

In this wet lab we will use:
Anti CD3 antibodies conjugated to
CD3 FITC, CD3 PE, CD3 PeCY5, PerCP-CY5.5, CD3 Pe-CY7,
different fluorochromes
CD3 APC, CD3 Alexa 700, CD3 BV421, CD3 BB515

Lysing solution (1x) Manufacturer’s protocol

PBS or stain buffer (PBS with 1%
BSA)

STAIN:
 Add 100 ul of whole blood (about 1 million cells) to 12x15 round bottom
tube
 Add antibody to the appropriate concentration as determined by
titration or as recommended by manufacturer.
 Incubate in dark at room temperature (RT) for 10-15 minutes

LYSE:
 Add 2 ml lysing (1x) solution.
 Vortex
 Incubate in dark at RT for 10-12 minutes (no less and no more!).
 Centrifuge cells at 300 g for 5 minutes.
 Discard supernatant and break the pellet by gently flicking the
bottom of tube.
WASH:
 Add 2ml of PBS
 Vortex.
 Centrifuge at 300g for 5 minutes
 Discard the supernatant and break the pellet.
 Resuspend cells in 0.5 ml of Stain Buffer or PBS.

100
BEADS

Use Compensation beads appropriate to your antibody;

 some compensation beads are generic and will bind any species of
antibody
 others are species-specific and will bind mouse, rat, or hamster
antibodies
 many kinds available from different vendors

STAIN:

 Add 1 drop of positive and 1 drop of negative compensation beads

to a 12 × 15–mm round-bottom tube.
 Add 100 μl of PBS or staining buffer
 Add antibody to the appropriate concentration as determined by
titration or as recommended by manufacturer.
 Incubate in dark at room temperature (RT) for 10-15 minutes

WASH:
 Add 2ml of PBS
 Vortex.
 Centrifuge at 300g for 5 minutes
 Discard the supernatant and break the pellet.
 Resuspend in 0.5 ml of Stain Buffer or PBS.

101
 Exercise 1: STAIN INDEX

In this exercise we will calculate stain index (Bigos, 2007), which is used to quantify the
effective brightness of a fluorochrome/antibody on a cytometer.

Stain Index = median positive – median negative

2 x rSD negative

The stain index uses the separation of medians of the positive and the negative populations,
normalized to the width (robust Standard Deviation: rSD), of the negative population.

Stain Index is fluorochrome and cytometer specific: it is affected by the intrinsic fluorochrome
brightness, antigen density, antibody affinity, and importantly here, by cytometer
characteristics and detector voltage or gain.

SAMPLES:

Prepare cells or compensation beads stained with CD3 FITC antibody as in Staining Protocols

DATA ACQUISITION AND ANALYSIS

1. Ensure cytometer is functioning correctly by running daily quality-control procedure.

2. Create a new experiment

3. Create dot plots and histograms as follows:

a. Create Forward scatter vs side scatter dot plot

b. Set fsc ssc gate on lymphocytes or beads

c. Create histogram in log scale for FITC parameter, gated on fsc ssc

d. Set appropriate detector gain (PMT Voltage) (see Exercise 2) and threshold

e. Record data on 5000 beads/cells.

f. Create gates on negative and positive populations.

g. Create statistics view to show median fluorescent intensity and rSD (robust
standard deviation) of positives and negatives

4. Calculate stain index according to the formula:

Stain Index = Median positive − median negative

2 × rSD negative

102
 Exercise 2: VOLTRATION

DETERMINATION OF BEST GAIN OR PMT VOLTAGE SETTINGS

In this exercise we will determine the optimum gain setting or photomultiplier tube (PMT)
voltage for one parameter(FITC in this case), in order to maximize the sensitivity of the FITC
detector. Beads or cells stained with FITC coupled antibody will be run at a range of gain or
voltage settings, and the Stain Index calculated for each setting. This would generally be done
for all parameters on a cytometer to determine the best settings for general use.

Hint: If doing this on all parameters, it saves time to create a generic template with a tube for
each gain setting, with the same gain on all the parameters. For example, tube ‘gain 400’ would
have a setting of 400 V on all parameters. This also gives a good idea of how much spillover you
are seeing in the other detectors at equivalent gain settings.

SAMPLE:

Prepare cells or compensation beads stained with CD3 FITC antibody as in “Staining Protocols”

DATA ACQUISITION AND ANALYSIS (as in Exercise 1)

1. Ensure cytometer is functioning correctly by running daily quality-control procedure.

2. Create a new experiment

3. Create dot plots and histograms as follows:

a. Create Forward scatter vs side scatter dot plot

b. Set fsc ssc gate on lymphocytes or beads

c. Create histogram in log scale for FITC parameter, gated on fsc ssc

4. Set FITC detector gain at low setting, eg 350 volts

a. Record data on 5000 beads/cells.

5. Increase FITC detector gain by 50 volts, eg to 400 volts

a. Record data on 5000 beads/cells

6. Continue creating tubes in this way and record data at 50 volt increments for the entire
voltage range

7. Analysis: (as in Exercise 1)

a. Create gates on negative and positive populations.

103
 b. Create statistics view to show median fluorescent intensity and rSD (robust
standard deviation).

8. Calculate stain index according to the formula:

Stain Index = Median positive − median negative

2 × rSD negative

9. Plot stain index versus gain or PMT voltage setting. The optimal gain is the lowest gain
that gives a maximal stain index, where it reaches a plateau.

If the positive population goes off the top of the scale before a maximum stain
index plateau, for example at a medium gain setting, then your sample is too
bright to test the full range of gain settings. Restain the beads/cells with either
less antibody or diminish staining by adding unlabeled antibody.

Positive Negative rSD

Voltage/gain 2 x rSD Stain Index
median median negative
25

50

100

200

300

400

500

800

1000
1500

2000

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 Exercise 3: Fluorochrome Brightness Comparison

This exercise uses calculation of the Stain Index to compare different fluorochromes coupled to
the same antibody. The differences seen depend on the intrinsic fluorochrome brightness and
the individual cytometer characteristics. Spillover can also be visually evaluated. This
information will aid in choosing fluorochromes to detect antigens, bright fluorochromes are
usually used for low density antigens and vice versa.

SAMPLES:

Prepare cells or compensation beads stained with CD3 antibody coupled to different
fluorochromes (see table below), as in Staining Protocols

DATA ACQUISITION AND ANALYSIS

1. Ensure cytometer is functioning correctly by running daily quality-control procedure.

2. Create a new experiment

3. Create dot plots and histograms as follows:

a. Create Forward scatter vs side scatter dot plot

b. Set fsc ssc gate on lymphocytes or beads

c. Create histograms in log scale for all parameters, gated on fsc ssc. Set these up in
order, laser by laser, so that all parameters for each laser are lined up in rows.
This makes it easy to see where the fluorochromes spillover into other detectors.

d. Set appropriate detector gains (PMT Voltage) (see Exercise 2) and threshold

e. Record data on 5000 beads/cells for each sample.`

f. Create gates on negative and positive populations for each parameter.

g. Create statistics view to show median fluorescent intensity and rSD (robust
standard deviation) of positives and negatives

4. Calculate stain index according to the formula:

Stain Index = Median positive − median negative

2 × rSD negative

105
 Positive Negative rSD
CD3 2 x rSD Stain Index
median median negative
Fluorochrome
FITC

PE

BB515

PE-Cy5

PerCP-Cy5.5

PE-Cy7

APC

AL700

BV421

Which fluorochromes are the brightest?

Which show the most spillover?

Which would be most appropriate for a high density antigen?

Which for a low density antigen?

106
 Exercise 4 : Manual Compensation

This exercise will show how to perform manual compensation for a simple 2 color experiment.
Compensation is the procedure by which the level of fluorochrome spillover into other channels
is calculated using single color controls and then corrected for, so that only the fluorochrome of
interest is measured in each detector.

Keep in mind that this is an exercise to understand the compensation process, and it is
preferable to use the automatic compensation calculation available in almost all software.
Automated software calculationis more accurate, as all colors are compensated simultaneously,
not sequentially as in the manual procedures.

In part B we will look at the effect that changing the gain or voltage has on the calculated
compensation.

SAMPLES:

Prepare cells or compensation beads stained with CD3 FITC, or CD19 PE, or double stained with
both, as per Staining Protocols.

DATA ACQUISITION AND ANALYSIS

1. Ensure cytometer is functioning correctly by running daily quality-control procedure.

2. Create a new experiment

3. Create dot plots and histograms as follows:

a. Create Forward scatter vs side scatter dot plot

b. Set fsc ssc gate on lymphocytes or beads

c. Create dot plot in log scale for FITC vs PE, gated on fsc ssc.

d. Set appropriate detector gain (PMT Voltage) (see Exercise 2) and threshold

e. Record data on 5000 beads/cells.

f. Create gates on negative and positive populations.

g. Create statistics view to show median fluorescent intensity of positive and

negative populations.

A Compensation calculation:

h. Using the FITC single color: adjust the compensation PE- x%FITC so that on the
PE axis (i.e. in the PE channel), the median of the FITC positive is the same as the
FITC negative. This correctly compensated sample now shows the single color
FITC shows a positive population only in the FITC channel: all cells are negative
in the PE channel.

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 i. Vice versa, on the PE single color, adjust the compensation FITC-x%PE so that on
the FITC axis (i.e. in the FITC channel), the median of the PE positive population
is the same as the PE negative.

j. These are the compensation values (PE- x%FITCand FITC-x%PE)that should be

applied to the double stained cells.

B The effect of voltage change on compensation

1. Rerun the same FITC single color using the compensation value calculated above,
but this time, increase the FITC PMT voltage by 50 volts. What happens to the
medians? Is the compensation still valid?
2. Rerun the same FITC single color using the compensation value calculated above,
but this time, decrease the PMT voltage by 50 volts. What happens to the medians?
Is the compensation still correct?
3. Repeat this test with the PE single color using the compensation value calculated
above, but increase or decrease the PMT voltage. What happens to the medians? Is
the compensation still valid?

The lesson here is that compensation is dependent on the voltage or gain setting. If you
change your voltage or gain setting for your samples, you must recalculate the
compensation values using your single colors.

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Supplementary Protocol

ANTIBODY TITRATION

Each antibody should be titrated before use to determineoptimal antibody concentration to

maximize discrimination of positive and negative populations. A serial dilution of the antibody
is prepared, the cells are added, incubated and washed and the stain index at each dilution is
calculated.This protocol is an 8-point doubling dilution series starting at 2× the manufacturer’s
recommendation; more points can be added or the dilution factors changed if needed.

Staining Buffer: PBS/1% BSA

SAMPLES:

Cells are prepared and treated exactly the same as the experimental sample (fixation,
permeabilization, FC block etc.) at 1–5 × 106 cell/ml, andshould contain 2 populations: 1
positive for the antigen and 1 negative for the antigen.

ANTIBODY SERIAL DILUTIONS: Dilute the antibody as per the Table below

 Add 50 μl of staining buffer to each tube

 Add 50 μl of antibody at 4× the manufacturer’s recommendation to the first tube
 Mix well and transfer 50 μl to the next tube according to the Table
 Discard the excess 50 μl from tube 7
 All tubes should contain 50 μl of antibody except tube 8 which is an unstained
control.
 Add cells: Add 100 μl of the cell suspension to all tubes and mix well.
 Incubate 30 min at room temperature in the dark, or the recommended
conditions for your antibody.
 Wash: Add 2 ml staining buffer, centrifuge 5 min at 300 × g, 4°C, remove the
supernatant
 Vortex the pellet, and resuspend in 200 μl staining buffer

DATA ACQUISITION AND ANALYSIS

1. Create a new experiment

2. Create dot plots and histograms as follows:

a. Create Forward scatter vs side scatter dot plot

b. Create gate on cells

c. Create histogram in log scale for antibody/fluorochrome used, gated on fsc ssc.

d. Set appropriate detector gain (PMT Voltage) (see Exercise 2) and threshold

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 e. Create gates on negative and positive populations.

f. Create statistics view to show median fluorescent intensity and rSD (robust
standard deviation).

g. Record data on 5000 cells for each antibody dilution

3. Calculate stain index according to the formula:

Stain Index = Median positive − median negative

2 × rSD negative

4. Plot stain index against antibody dilution/concentration. Choose the lowest antibody
concentration that gives the highest stain index.

Stain Transfer from Temporary Transfer to Final Ab Antibody Stain

Tube Buffer previous tube volume next tube volume concentration Index

1 50 ul 50 ul Antibody 100 ul 50 ul 50 ul

2 50 ul 50 ul 100 ul 50 ul 50 ul

3 50 ul 50 ul 100 ul 50 ul 50 ul

4 50 ul 50 ul 100 ul 50 ul 50 ul

5 50 ul 50 ul 100 ul 50 ul 50 ul

6 50 ul 50 ul 100 ul 50 ul 50 ul

7 50 ul 50 ul 100 ul discard 50 ul 50 ul

8 50 ul 0 ul (no antibody) 50 ul NA 50 ul

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 Sample preparation for surface staining by Stain Lyse-Wash
Method

Dr. Monica Singh

Objective:
To perform staining for surface markers

Sample:
2 ml EDTA/Heparin anti-coagulated peripheral blood/ bone marrowaspirate

Reagents:
1. Antibodies: As per the different panels provided in the lab
2. BD FACSLyse solution (10x) (349202)
a. Prepare 1x working solution by diluting 1:10 in distilled water

Protocol:

Adjust cell count to 20000/ul for MRD and 5-1000/ul for Non MRD

1) Label appropriate number of 12x75mm FACS tubes as per the panel.

2) Take 100 ul of sample (about 1 million cells) in each tube.
3) Add 20 ul (or titrated volume) of Antibody/ antibody cocktail. Vortex (Vx).
4) Incubate in dark at RT for 20 minutes
5) Add 2 ml BD FACSLyse (1x) .Vx
6) Incubate in dark at RT for 12 minutes (critical)
7) Spin cells at 1500 rpm for 5 minutes
8) Aspirate/Discard supernatant. Break the pellet
9) Wash cells once/twice with sheath
a. Add 2ml of sheath, Vx, spin at 1500 rpm for 5 minutes, discard
supernatant
10) Resuspend cells in 0.5 ml of sheath or PBS with 2% paraformaldehyde
11) Acquire 1 million viable cells ( based on FSC SSC gate) for MRD and 1.5 lakh cells
( based on FSC SSC gate) for non MRD samples on flow cytometer.

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 Sample preparation for simultaneous surface and intracellular
staining

Dr. Monica Singh

Objective:
1) To perform staining of intracellular markers
Sample:
2 ml EDTA/Heparin anti-coagulated Hu peripheral blood/ bone marrow aspirate

Reagents:
1) Antibodies: as per panel
2) BD FACSLyse solution (10x) (349202)
a. Prepare working solution by diluting 1:10 in distilled water
3) BD Perm II permeabilization buffer (10x) (340973)
a. Prepare working solution by diluting 1:10 in distilled water

Protocol:
Adjust cell count to 5000-10000/ul

1) Take 100 ul of sampleinlabelled BD FACS tubes

2) Add pre-titrated volume of surface antibodies to the tubes.
3) Vortex (Vx). Incubate for 20 minutes at RT, dark.
4) Add 1 ml FACSLyse (1x). Vortex .Incubate in dark at RT for 12 minutes (critical)
5) Spin cells at 1500rpm for 5 minutes. Aspirate/Discard supernatant. Break the pellet
6) Add 0.5 ml of Perm2 (1x, Cat 340973) Vortex and incubate in dark for 10 minutes
7) Wash cells once with sheath - Add 1ml of sheath, Vortex. Spin at 1500 rpm for 5 minutes
and discard supernatant
8) Add pre-titrated volume of intracellular antibodies. Vortex.
a) Incubate in dark at RT for 15 minutes
9) Wash cells once/twice with sheath - Add 1 ml of sheath, Vortex. Spin at 1500rpm for 5
minutes. Discard supernatant
10) Resuspend cells in 0.5 ml of sheath or PBS with 1% formaldehyde (methanol free)
11) Acquire 1.5 lakh viable cells ( based on FSC SSC plot) on a flow cytometer.

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 Sample preparation method for Surface Kappa Lambda
staining using BD PharmLyse (Lyse-wash-Stain-wash)

Dr. Monica Singh

Objective:
To perform staining for surface immunoglobulins (kappa/lambda) on lymphocytes

Sample:
2 ml EDTA/Heparin anti-coagulated peripheral blood/ Bone Marrow Aspirate

Reagents:
l) Monoclonal Antibodies:

Lambda FITC (BD 644059 clone TB28-2)

Kappa PE (BD 642925 clone 1-155-2)

Surface Antibodies as per panel

2) BD PharmLyse solution (10x) (cat no)

a. Prepare working solution by diluting 1:10 in distilled water
Protocol:

Adjust cell count to 20000/ul for MRD and 5000-1000/ul for Non MRD

1) Take 100 ul of sample (about 1 million cells) in a 12x75mm tube

2) Wash the sample up front 2 times with Sheath fluid. Aspirate off each wash
fluid with a transfer pipette (do not dump).
2) Add 2 ml of BD Pharm Lyse (1x solution) to the tube. Vortex. Incubate for 12
minutes at RT in dark. Spin at 1500 rpm for 5 minutes and discard the
supernatant.
3) Wash cellstwice with 2 ml of sheath fluid and resuspend in 100ul of sheath
fluid. (The residual drop after decanting the supernatant)
4) Add pre-titrated volume of antibodies. Vortex.
5) Incubate in dark at RT for 15 minutes
6) Wash cells once with 2 ml sheath at 300g for 5 minutes
7) Resuspend cells in 0.5 ml of sheath fluid or PBS with 1 % formaldehyde
(Methanol free)
8) Acquire 1 million events cells on flow cytometer.

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 List of
Participants

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 NAME EMAIL ID COLLEGE/ORGANIZATION

Abdul Razak Rilwan abdulrazaklurwan59@gmail.com Mewar University, Chittorgarh

Dr. Adarsh Sanikop adarsh.sanikop@gmail.com KLE Hospital, Belagavi

Dr. Alice Gifty John alicegifty2004@yahoo.com

Aron J Aronjeeva007@gmail.com Technical Supervisor, Manipal Hospitals

Bangalore.

Dr. Anand Kalia DRANANDKALIA@gmail.com Fellowship, Bharati Vidyapeeth Hospital, Pune

Dr. Ananthvikas ananthvikas@neuberganand.com Neuberg Anand Reference laboratory

Jayaram

Dr. Anitha saturn424@gmail.com St. Martha's, Bangalore

Ramakrishna

Dr. Aparna Narasimha sonrichie14@gmail.com East Point College of Medical Sciences

Dr. Arathi C. A ca.arathi@yahoo.com PES medical college and hospital

Dr. Archana M drarchanamanohar@gmail.com Kidwai Memorial Institute of Oncology

Dr. Arundhathi S arundhathi19@yahoo.co.in Asso.Professor AIIMS, (Mangalagiri)

Dr. Bala Bhasker P.M balag4g@gmail.com Yashoda hospital(consultant)

Dr. Balachandra Bhat kumtabalu@gmail.com Bangalore Baptist Hospital

Dr. Chandana N chandz.laks@gmail.com Adichunchangiri Institute of Medical Sciences

Dr. Chethan Manohar chethanm15@gmail.com Pathologist, Anand Diagnostic Lab

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Dr. Dattaraj Shivaji docrajkarade@gmail.com HCG Cancer hospital
Karade
Dr. D. Deepa drdeepamudha4@gmail.com M S Ramaiah Medical College and Hospital

Dr. Deepak S Jois deepaksjois@gmail.com Anand Diagnostic Laboratory

Dr. Deepak M B deepakmb.skt@gmail.com Asso. consultant. Narayana hrudayalaya

Dr. Deepika V deepikavunnam2287@gmail.com Fellow in Oncopathology, Sri Shankara Cancer

Hospital and Research Centre

Dr. Deepthi P Kambi deepthi.kambi@sakraworldhospital.com Consultant, Sakra Hospital, Bangalore

Dr. Dhanalakshmi B dhanalakshmi81@gmail.com Adichunchangiri Medical College

Gowtham Kumar G gowthamkumar.net@gmail.com Sri Ramachandra Insitute of Higher education

and research

Gulista Bano gulistabano@iisc.ac.in Student, Indian Institute of Science, Bangalore

Dr. Hafsa Shabeer hafsa.9186@gmail.com KIMS Bangalore

Hamenth Kumar P hamenth@cmcvellore.ac.in Christian Medical College

Dr. Hema N Anadure drheman.anadure@gmail.com HCG(Junior consultant)

Dr. A. N. Hemalatha drhmlthan@yahoo.com Private consultant

Dr. Hemavathi N hemavathinarayan123@gmail.com MVJ Medical College & Research Hospital

Ibrahim Haruna dawandoluda@gmail.com NIMS University India

Ibrahim Bala Baba ibrahimbala548@gmail.com Mewar University

Jasmine. J jasminesheeba@gmail.com Section Technical Coordinator, SRL

Dr. Jeena Johns drjeenas@gmail.com —

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 Dr. Joan Maria Vynettad@gmail.com Gokula Metropolis Clinical Laboratories
Vynetta Devadoss

Kamini Khatak mskamini7@gmail.com Anna University, Chennai

Dr. Kavitha K P kpkavi@gmail.com Aster MIMS Hospital, Kozhikode, Kerala

Dr. Kavitha R kavi_kavir@yahoo.co.in Sriramachandra Institute of Medical Sciences

Thangaraj

Dr. Kavya J greeny.239@gmail.com Bowring & Lady Curzon Medical College &
Research Institute

Dr. Kavya Shri H B kavyashrihb22@gmail.com KIMS, Bangalore

Dr. Komal komaldc.3636@gmail.com Consultant Pathologist, Cytecare Cancer

Dheerendra Hospital.
Chippalkatti

Dr. Kunal Sharma drkunalsharma@yahoo.com SRL diagnostics(consultant)

Maheshwarappa M mahidsm@gmail.com Columbia Asia Hospital(technologist)

Dr. Madhu A.B Madhu.aramane@gmail.com KMC, hubli

Dr. D. S. Madhumathi madhuds123@gmail.com HCG(consultant)

Dr. Majitha Mohamed majithalungal@gmail.com Korambayil Hospital & Diagnostic Centre,

Manjeri

Dr. Mayur M Nigalye drmayurnigalye@gmail.com

Metropolis Healthcare limited, Mumbai
Dr. Mohit Agrawal mohit.medico@gmail.com Kidwai Memorial Institute

Dr. Monika Singh monika04apr@gmail.com Junior Resident, BJGMC,PUNE.

Muhammad Lawan 12kolomi@gmail.com Mewar University, Chittorgarh

Kolomi
Muhammad Maidala muhammadmaidala@gmail.com Integral University

Dr. Namrata N R nrnamrata@yahoo.com Kidwai Cancer Centre

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 Neha Guleria neha.gul5@gmail.com Senior Research Fellow, ICAR-IVRI

Nasir Abubakar nasirgamji2014@gmail.com Mewar University, Chittorgarh

Dr. Nethra R dr.nethra05@gmail.com Kidwai Memorial Institute of

Oncology,Bangalore

Dr. Nidha Gaffoor nidhanabil@gmail.com Consultant pathologist, SRL

Dr. Nitin Agarwal nuts_medico@rediffmail.com Sparsh Hospital(Consultant)

Nura Murtala nuramurtala2000@gmail.com NIMS University, India

Dr. Pomilla Singh pomillasingh@gmail.com BVP Medical College, Pune

Dr. Prabha M prabhamg2gmail.com Ramaiah Institute of Technology

Dr. Pratibha Singh varunspratibha@gmail.com Apollo Speciality Hospital, Chennai

Dr. M. Pranathi pranathi.mahankali58@gmail.com Dr. Pinnamaneni Siddhartha Institute of

Medical Sciences & Research Foundation

Dr. M. V. R. K. ramya.mynampati@gmail.com PSIMS&RF

Prashanthi

Dr. Preethi M S msluvs99@gmail.com PSG Institute of Medical Sciences and

Research

Dr. K. R. Priyadarshini pdraj89@gmail.com Tutor, PSG Institute of medical science and

research, tamil nadu

Poonam Rani poonamrani@iisc.ac.in Student, Indian Institute of Science ,

Bangalore

Dr. Raghav Kapoor Raghavkapoor2008@gmail.com JNMC(PG)

Dr. P Raghavendra prkaranth@gmail.com Eurofins Central Lab

Karanth

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 Dr. Rahul Bhagat rahulbhagat1234@gmail.com Scientist, Sri Shankara Cancer Hospital and
Research Centre

Dr. Rajan Shah rajan.shah@bpkihs.edu B.P Koirala institute of health sciences,

Nepal.

Rajneshwari.J rajneeshwarij@gmail.com Technical Supervisor,Manipal Hospitals

Bangalore.

Raseed I Munawalli raseed09@gmail.com Medgenome Labs Limited

Rekha Nambudiry rekha.nambudiry@thermofisher.com Thermofisher Scientific

Dr. Renuka P drrenuka@chanrediagnostic.com Chan Re Diagnostic centre

Dr. Reshma Anegundi rashmi.raghavendra703@gmail.com PES medical college and hospital

Dr. Sandeep Sanprof707@gmail.com Columbia Asia Hospital(consultant)

Sandeep Kumar S psriramsandeep@gmail.com Kidwai Cancer Institute

Dr. Sandhya Rohit phadke.sandhya@gmail.com Consultant Pathologist

Phadke
Dr. Sarah Khan drsarahkhan17@gmail.com Aster Labs

Sathya P sathyapandu92@gmail.com

Dr. Savithri M.C savithrimc@yahoo.co.in Professor, Amala Medical College

Dr. Seema seema0319@yahoo.com Narayana Multispeciality Hospital

Sivasankaran

R. Seenuvasan seenuvasan77088@gmail.com JIPMER

R. Sundharamurthy r.sundhar26@gmail.com JIPMER

Dr. Shalini K S shaliniyajman@yahoo.co.in Junior Consultant, Narayana hrudayalaya

Dr. Shilpa T Patil shilpapatil2590@gmail.com Vinayaga Mission’s Medical College,

Karaikal

Dr. Shireesha Mallik shireeshamallik@gmail.com JJM Medical College

119
 Dr. Shivani Joshi dr.joshishivani@gmail.com Dr. Patel Metropolis Lab Nashik

Dr. Shubham dr.shubhamvarshney@gmail.com JNMC(MD Pathology)

Varshney
Dr. Spoorthy M spoom1992@gmail.com

Dr. Sridevi H B drsri.20@gmail.com Kasturba Medical College, Mangalore

S. Sushya dolly_96@yahoo.com JIPMER

Dr. Subhan Ali R subhanalir@gmail.com Kidwai Memorial Institute of Oncology

Dr. Sunilkumar K. B. drsunilkb@gmail.com JJM Medical College

Dr. Sunitha S sunithakiran84@gmail.com DM Resident GCRI,Ahmedabad, Gujarat

Dr. Suresh H. surbh2001@gmail.com Principal Scientist, ICAR-IVRI, Bengaluru

Basagoudanavar
Tammanna Bhajantri tammanna212@gmail.com MGR medical university (Phd student)

Dr. Uma D B druma.bakkappa@asterhospital.com Aster CMI Hospital

Usman Salahuddeen alsaada2018@gmail.com Mewar University, Chittorgarh

Abdullah

Dr. K. S. Vaisakhi kasivaisakhi@gmail.com Star Hospital

Dr. Vaishali susrisha04@gmail.com Sumukh Prayogalaya, Hubli

Choukimath

Dr. Vanesa John T treesa1980@yahoo.co.in Assistant Professor, Amala Medical College

Dr. Veda. P vedalallu@yahoo.com Senior Specialist, ESIC Hospital,rajajinagar

Dr. Venugopal A dravenugopal2008@gmail.com Clearmedi Radiant Hospital, Mysuru

Dr. Vidyadhar vrrmmc_doc@yahoo.co.in Kannur Medical College, Anjarakandy

N.K.Rama Rao

120
 Dr. Vignesh C vikkyajj@gmail.com Vinayaga Mission’s Medical College,
Karaikal

Zainal Abidina Garba garbazainalabideen@gmail.com Mewar University, Chittorgarh

Dr. Prasanna Shetty B drbadilaprasanna@rediffmail.com M S Ramaiah medical college

Dr. Vijaya V vijayamysorekar@yahoo.com M S Ramaiah medical college

Mysorekar

Dr. Mangalagouri S R mangalagouri22@yahoo.com M S Ramaiah medical college

Dr. H M Sudha sudharaviprakash98@gmail.com M S Ramaiah medical college

Dr. Aarathi R Rau aarathirau@gmail.com M S Ramaiah medical college

Dr. Nandakishore dr.nkalva@yahoo.com M S Ramaiah medical college

Alva

Dr. Sulata M Kamath drsmkamath@gmail.com M S Ramaiah medical college

Dr. Clement Wilfred D clement.wilfred@yahoo.com M S Ramaiah medical college

Dr. Kalpana Kumari M kalpank@gmail.com M S Ramaiah medical college

K

Dr. Sridhar H drsridhar24@rediffmail.com M S Ramaiah medical college

Dr. Rashmi K rashmikrishnappa@yahoo.co.in M S Ramaiah medical college

Dr. Usha M usharavihitha@gmail.com M S Ramaiah medical college

Dr. Ganraj Bhat. S gansi.bhat@gmail.com M S Ramaiah medical college

Dr. Lavish Tayal tayal.lavish@gmail.com M S Ramaiah medical college

Dr. Prathibha Patil patilprati1708@gmail.com M S Ramaiah medical college

Dr. Shalini R rj.shalini@gmail.com M S Ramaiah medical college

Dr. Mansi Khamesra mansikhamesra12@gmail.com M S Ramaiah medical college

Dr. Anisha Hari anishari.16@gmail.com M S Ramaiah medical college

Dr. Shivani Gupta drshivani5.3@gmail.com M S Ramaiah medical college

Dr. Poornima R poorni0298@gmail.com M S Ramaiah medical college

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Dr. Anusha. C anushac2192@gmail.com M S Ramaiah medical college

Dr. Likitha S R likithasram@gmail.com M S Ramaiah medical college

Dr. Greeshma Prasad greeshprasad48@gmail.com M S Ramaiah medical college

Dr. Sravanthi sravanthi.ss@gmail.com M S Ramaiah medical college

Sunkarnaneni

Dr. Shruti shru113_90@yahoo.co.in M S Ramaiah medical college

chidambaram Iyer

Dr. Gowri R gowri.raghunath@gmail.com M S Ramaiah medical college

Dr. Shah Sagar Ajay sagarshah9999@gmail.com M S Ramaiah medical college

Dr. Sana Fathima najsan10@gmail.com M S Ramaiah medical college

Dr. Disha Rai disharai1493@gmail.com M S Ramaiah medical college

Dr. Pragya Mishra doc.pragya08@gmail.com M S Ramaiah medical college

Dr. Chinki Anupam c.anupam5@gmail.com M S Ramaiah medical college

Dr. Safeena Taranum safeena.taranum123@gmail.com M S Ramaiah medical college

Dr. Abhinav Ashish mail2meabhinav@gmail.com M S Ramaiah medical college

Dr. Atira Mirza atiramirza25@gmail.com M S Ramaiah medical college

Dr. Pranjali V C pranj92@gmail.com M S Ramaiah medical college

Dr Prachi saxena dr.saxena.prachi@gmail.com M S Ramaiah medical college

Sachin BMTU, Ramaiah Memorial Hospital

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 Building Cytometry Community

TETC works under the leadership of

Prof. Awtar Krishan
PATRON, INDO-US Flow Cytometry Workshops, India.

Team TETC
(Drs. Rekha Gour, Arvinder Singh, P.N. Razdan, Hemant Agrawal)

CONTACT US
indiatetc@gmail.com; +91-9284999738; +91-7665130114
FOLLOW US AT
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