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Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and Standardization TMH, Mumbai March 9-11, 2012 Sumeet Gujral, MD Professor, Department of Pathology, Tata Memorial Hospital, Mumbai [email protected] Flow cytometry: An Indian Scenario. History

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Flow cytometry: An Indian Scenario

Multicolor Immunophenotyping: Applications and Standardization

TMH, Mumbai

March 9-11, 2012

Sumeet Gujral, MD

Professor,

Department of Pathology,

Tata Memorial Hospital, Mumbai

[email protected]


Flow cytometry an indian scenario
Flow cytometry: An Indian Scenario

History

The Cytometry Society (TCS)

- Research Arm

- Clinical Cytometry

a. Health care in India

b. Management of HLN (Trained staff, Equipped labs, Cancer Hospitals, Costing)

c. Immunophenotyping

- Indian Data

- First Meeting, 2008 (Indian Guidelines)

- PT program and Standardization

- Training programs

Present meeting

Collaborations

Uniformity in Diagnostics



Mid 80s - Dr. VK Jain’s (NIMHANS), followed by Drs. Ganguly, Pande, Rath, Muthukaruppan, Moudgal, Indranath, Sehgal & Chakraborty.In 90s - Pande & Rath: trg pgms.In 2000 - A Krishan of Univ. of Miami started Indo-US cytometry workshops (12 workshops+).

First fluorescent based FCM developed in

1968 by W Guhde. Pulse cytophotometry

India

Research labs, early 80s

Diagnostic labs,1990s

  • TMC Mumbai

  • AIIMS, New Delhi

  • Hinduja Hospital

  • Pvt Reference Labs

  • Others

  • CD4 counts



The cytometry society tcs 2005
The Cytometry Society (TCS) - 2005

  • 2005 at CCMB

  • 2006 – ICCS meeting in USA, Phil McCoy

  • 2007 – Together, Clinical & Research

  • Self nominations and proposed election…

  • Executive council, President, 2 VPs, 2 Secretaries, various committees.

  • Pande, Amar, Krishnamurthy,..

  • Annual meetings & IndoUS cytometry workshops.

  • Membership and Website: tcs.res.in


7th Indo-US Cytometry Workshop, JNU, New Delhi, 2006


Basic research cytometry
Basic/research cytometry

  • Institute based (government agencies)

  • Last decade – Industry

  • > 1000 cytometers

Total pubmed publications – 126505, first in 1974

Total Indian publications - 1092, first in 1989


Clinical cytometry
Clinical Cytometry

  • Management of HLN in India

  • Cancer Hospitals, Labs, Trained staff, Costing

  • Immunophenotyping

  • - Indian Data

  • - First Meeting, 2008 (Indian Guidelines)

  • - PT program and Standardization

  • - Training programs


Management of leukemia/lymphoma – IndiaCancer HospitalsLabs with Ancillary TechniquesTrained StaffCosting


Dream: Comprehensive diagnostic workup followed by a “protocol based treatment”.

“WHO 2008”

Reality: Protocol based treatment vis-a-vis modified one based on resources available (on individual basis)

No Indian guidelines for most disciplines, opinion/experience based.


Health care in india
Health care in India

  • Hospitals (Government versus Private)

  • Labs with Ancillary techniques

  • Training program

  • Costing


Cancer hospitals 25 n 60 70
Cancer Hospitals (<25) n=60/70

Tertiary Care Cancer Centers: 6-8

Regional Cancer Centers: 15-20

Private/Corporate Hospitals: 35-40

Medical College

Nursing Homes

Management of HLN

Hematolymphoid neoplasm treated at <50 centers

SCT being done at 10-20 centers


Labs with ancillary techniques 15 fcm cytogenetics molecular diagnostics
Labs with Ancillary Techniques <15 FCM, Cytogenetics, Molecular Diagnostics

Tertiary care cancer centers including pvt. hospitals (8-10)

Stand alone private laboratories (3/4)

Regional cancer centers (3/4)

Management of HLN


Trained staff structured training programs
Trained staffStructured training programs

Hematopathologists (DM+fellows+residents): 5+10/year

Management of HLN

  • Medical Oncologists (Ped & Adult): 20-25/year

  • Hematologists: 5/year

  • There are no structured training programs for any of the ancillary techniques (both for pathologists as well as for technologists).


Hematopathology training in india
Hematopathology training in India

Post MD pathologists: 3 year DM, 2 year fellowship and one year residency program.

An occasional center in India train hematopathologists both in lymph nodes and bone marrow.

Management of HLN


Amongst various ancillary techniques flow is better off in

Management of HLN

Amongst various ancillary techniques, flow is better off in..

  • Training programs, conferences/ CMEs

  • Larger pool of young cytometrists

  • Students (DM, Fellows and Residents) get rotation in flow lab (2-5 months, 7-8 centers in India).



Activity Based Costing method is used to calculate per cost test

Indirect costs: Hidden costsalaries, depreciable value,furniture,funds for personnel training and CMEs, ancillary equipments, stationary, electricity and rental chargesMedical insurance, deputation etc

Direct cost: Visible cost1. One time cost of instrumentOutright purchase versus Reagent rental2. Recurring costReagents, antibodies, tubes, fluids, dyes and kits.3. Annual maintenance contract


Direct cost of ipt
Direct cost of IPT test

Indirect cost of IPT

  • Number of SM studies in a year = 1300

  • Per sample indirect cost is Rs 124

  • Indirect cost for SM is 1300 x 124 = Rs 1,61,000


Per sample cost of ipt at tmh 2008 3 color 15 18 markers

Management of HLN test

Per sample cost of IPT at TMH, 20083-color, 15-18 markers

  • Total cost = Direct cost + Indirect Cost

    = 51,96,025 + 1,61,000

    = 53,57,025

  • Per sample cost of SM: 53,57,025 / 1300 = 4120

Costing of one IPT test - Rs. 4120 (USD 100)

Costing of CD34 counts - Rs. 1700 (USD 40)

Gujral, IJPM, 2010


Management of HLN test

Other factorsCost per test decreases as number of samples increase.Cost increases as the number of color/panels increase. Maximum expense is on reagents and consumables, followed by manpower.Cost per test is higher for specialized tests done by a pathologist.

Gujral, IJPM, 2010


Treatment
Treatment test


Population of 1000 million, 6000 children may develop ALL each year

Three tier society (based on socio-economic backgrounds):

Profile I (70%) being extremely poor who cannot afford any treatment

Profile II (25%) from the middle class, and

Profile III (<5%) who can afford to have the best possible treatment

Treatment costs approximately 10% of western costs

Management of HLN

Pediatric Acute Leukemia - India

Government / social organizations fund pediatric cases get treated

Chandy M et al


All patients have a complete work up for diagnosis. each year

Pediatric patients: 70% are treated with a curative intent

(protocol based).

Adult patients: protocol based treatment given to ALL

(70%), AML (70%), CML (100%), CLL (70%), NHL (90%).

Management of HLN

Leukemia/lymphoma - TMH

Gujral, Leukemia 2009


Management of hln at tmh
Management of HLN at TMH each year

Lab tests constitute 2-6% of total cost of management (BMT excluded)


Most labs in India still follow FAB classification systems in diagnosing and sub-typing of hematolymphoid neoplasm.Few centers use WHO 2008 classification system of HLN.

Management of HLN


Immunophenotyping india
Immunophenotyping in diagnosing and sub-typing of hematolymphoid neoplasm. - India


  • 1. Introduction to IPT in diagnosing and sub-typing of hematolymphoid neoplasm.

  • 2. Indian Data

  • 3. First Meeting, 2008 (Indian Guidelines)

  • 4. PT program and Standardization


Flow Cytometry in diagnosing and sub-typing of hematolymphoid neoplasm.

It is the measurement of cellular properties as cells move in a fluid stream (flow), past a stationary set of detectors

Technique of quantitative single cell analysis

It analyses

- physical, and

- chemical properties (immunofluorescence) of cell


Ihc and fcm complementary mandatory for any center doing hln

IHC and FCM – complementary in diagnosing and sub-typing of hematolymphoid neoplasm.Mandatory for any center doing HLN

FCMmulticolor immunophenotypingfluids

Immunohistochemistry mostly single colorbiopsy


>400 labs do CD4 counts (started in mid 80s). in diagnosing and sub-typing of hematolymphoid neoplasm.

>60 labs do leukemia IPT (started in mid 90s).

  • most do 3 colors,

  • few do 4 colors,

  • very few do 6 colors.

    Few do autoimmune workup, PNH studies, CD34

    stem cell counts etc.


2008 WHO classification of in diagnosing and sub-typing of hematolymphoid neoplasm.

Hematolymphoid Neoplasms

WHO classification: still a distant reality

  • Myeloid neoplasms

  • Precursor lymphoid neoplasms

  • Mature B cell neoplasms

  • Mature T- and NK- cell neoplasms

  • Hodgkin lymphoma

  • Immunodeficiency associated LPD

  • Histiocytic and dendritic cell neoplasms


Tmh data
TMH Data in diagnosing and sub-typing of hematolymphoid neoplasm.


Hematopathology lab tmh mumbai
Hematopathology Lab, TMH, Mumbai in diagnosing and sub-typing of hematolymphoid neoplasm.

  • Approx. 50,000 new patients come to TMH/year and 8% of these are hematolymphoid neoplasm.

  • 4000 new cases every year.

Leuk & Lymphoma, 2009 Clinical Cytometry, 2008

IJC, 2010,


Acute leukemia n 2511
Acute Leukemia, n=2511 in diagnosing and sub-typing of hematolymphoid neoplasm.

Common subtypes of AML

AMLM2 (27%),

AMLM5 (15%),

AMLM0 (12%),

AMLM1 (12%),

APML (11%), and

AML t(8;21) (9%)

CMLBC was commonly of myeloid blast crisis

subtype (40 cases)

ALL (58%)

AML (38%)

Common subtypes of ALL vs WestB-cell ALL - 76% (85%)T-cell ALL - 24% (10-15%)


Lymphomas in bm pbs clpds
Lymphomas in BM/PBS in diagnosing and sub-typing of hematolymphoid neoplasm.- CLPDs

B cell Lymphomas

CLL - 68.5%,

FL - 8.5%

MCL - 5.5%

SMZL - 5%)

HCL - 5%

T/NK cell lymphomas

4% (nine cases) of all mature

lymphoid neoplasms.

T-LGL - 4 cases,

T-PLL - 2 (small cell variant),

ATLL – 2

PCGDTCL - 1

IJC, 2010

Leuk Lymphoma, 2009


Hematolymphoid neoplasm one year dmg clinic data
Hematolymphoid Neoplasm - One year DMG/clinic data in diagnosing and sub-typing of hematolymphoid neoplasm.


Pediatric data 2011
Pediatric data, 2011 in diagnosing and sub-typing of hematolymphoid neoplasm.

  • Total number of cases – 1704

  • Solid tumors - 921

  • Hematolymphoid neoplasms - 783

Total number HLN

treated - 665

ALL - 396

AML - 73

NHL - 85

HL - 74

Total number HLN

treated - 665

Newly diagnosed - 587

Previously treated – 65

Second opinion – 7

Investigation only - 5

Newly diagnosed HLN - 665

On protocol – 439 (66%)

Untreated – 85

On other treatment -42

Referred on protocol - 21


March 2005
March 2005 in diagnosing and sub-typing of hematolymphoid neoplasm.


March 2005, Mumbai in diagnosing and sub-typing of hematolymphoid neoplasm.TMH started a ILCP for IPTFive local laboratories joined (sample sent, results, feedback)Quarterly meetings


After 6 cycles of the pt program
After 6 cycles of the PT program in diagnosing and sub-typing of hematolymphoid neoplasm.

Results: Wide variation starting from sample collection, clone and fluorochrome conjugates selection, processing, gating strategies, analysis and reporting format

Planned First Meeting


Focus on in diagnosing and sub-typing of hematolymphoid neoplasm.“Indian Guidelines for Panel selection”Antibody panel selection plays a vital role in obtaining an accurate diagnosis. Lot of diversity in panel selection.Numerous guidelines have addressed antibody panels. Most Guidelines - North America and EuropeOther issues: Sample collection, transport, viability, adequacy of cell yield, storing of samples - recommendations as described elsewhere1,2


Propose guidelines for a minimal antibody panel without compromising on accuracyTo enable uniformity in reporting Educational exercise (evolving technology)PT program

Goals

Avoid ultrashort panels


These documents were circulated, taking opinion from cytometrists, hematopathologists, medical and pediatric oncologists and others

Over next three years (2005-08), consensus Guidelines were formulated based on: - Published Data (Indian and western) - Results of the PT program- Practice Based Questionnaire and- Experience/opinion


“Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry”March 13-15, 2008TMH, Mumbai

First Meeting, 2008


Revised 3 document (consensus) presented Neoplasms by Flow Cytometry”

Presentations: Cytometrists from India, Rest of the Asia, Europe, Australia and America presented their perspective on panel selectionDelegates: 180 delegates including 30 from outside India

Report of proceedings of the national meeting on "Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry". Gujral S, Subramanian PG, Patkar N, Badrinath Y, Kumar A, Tembhare P, Vazifdar A, Khodaiji S, Madkaikar M, Ghosh K, Yargop M, Dasgupta A. Indian J Pathol Microbiol. 2008 Apr-Jun;51(2):161-6


2008 guideline meeting tmh mumbai
2008 Guideline meeting, TMH, Mumbai Neoplasms by Flow Cytometry”


B cell cd10 cd19 t cell cd7 cd5 myeloid cd13 cd33 cd117 other cd34 hla dr cd45

B cell: CD10, CD19 Neoplasms by Flow Cytometry”T cell: CD7, CD5Myeloid: CD13, CD33, CD117Other: CD34, HLA-DR, CD45

Recommended minimal screening panel of Acute Leukemia, (n=10)

Recommended minimal screening panel of CLPD / Mature lymphomas, (n=9)

CD3, CD5,CD19, CD23, CD10, CD20, FMC7, Kappa, Lambda


Review of literature
Review of Literature Neoplasms by Flow Cytometry”


AL - Recommendation by various panels Neoplasms by Flow Cytometry”(n = 10-18)


CLPD - Recommendation by various panels Neoplasms by Flow Cytometry” (n = 7-11)


All lymphoid cells CD45+ (LCA) Neoplasms by Flow Cytometry”

B-cells CD19, CD10, cCD22

T-cells CD3, CD5,cCD3

Myeloid cells CD13, CD33, CD117, anti MPO

Megakaryocytic CD41, CD61

Blasts CD34, Tdt, CD99

Other: HLA-DR, CD23, FMC-7, CD43, CD11c, CD25, CD103, CD38, CD138, CD20, CD79a, Kappa and Lambda light chains, TCR alpha beta, TCR gamma delta, CD4, CD64, CD55, CD59

Recommended markers in a leukemia lab

IJPM. 2008


At same time were published Neoplasms by Flow Cytometry”

2006 Bethesda International Consensus Guidelines.


Bethesda uses a panel of antibodies which are sensitive to pick up cells of a particular lineage.3Most guidelines use a panel of antibodies for diagnosis of AL or CLPDA combination of markers is used for a particular medical indication or symptom (for example lymphadenopathy or blasts in the blood). Wood et al, Clinical Cytometry, 2007


Similarities and differences bethesda versus indian approach
Similarities and Differences pick up cells of a particular lineage.Bethesda versus Indian approach

US – indication based, Indian - morphology (& clinical) based

Both rely on a screening panel - 33 versus 10 antibodies

US - comprehensive panels, more T-cell reagents in screening

Secondary reagents differ

Indian – don’t address maturation pattern, CD45 gating optional

Indian panel includes CD23, FMC7 in primary screen

Leukemia, 2009

Cytometry A, 2009


Acceptability of bethesda consensus guidelines
Acceptability of Bethesda Consensus Guidelines? pick up cells of a particular lineage.

AnemiaPrimary lymphadenopathySplenomegalyStaging of bone marrow in lymphomas


Pancytopenia in india 25 60 is megaloblastic anemia
Pancytopenia in India pick up cells of a particular lineage.25-60% is megaloblastic anemia


Indian guidelines lacunae
Indian Guidelines - Lacunae pick up cells of a particular lineage.

Gujral et al, Cytometry B Clin Cytom. 2008 Aug 25

Gujral et al, Indian J Pathol Microbiol. 2008 Apr-Jun;51(2):161-6.


A beginning
a beginning.. pick up cells of a particular lineage.

Patterns

Lineage associated markers

Gating strategies

Scanty sample size

MRD Studies

Rare tumors are not diagnosed

Increased turn around time

Repeated procedures

Multicolor Immunophenotyping: Applications and Standardization


More colors more issues.. pick up cells of a particular lineage.

How much is enough?..


Selection of fluorochromes and cocktails pick up cells of a particular lineage.Lineage specific markers in one tube

3 color to 10 colors

Third party software

More colors, more issues

15 color

8-10 color

3 to 6 color

or more


Compensation pick up cells of a particular lineage.


Pmt voltage setup using signal noise ratio
PMT Voltage setup using signal/noise ratio pick up cells of a particular lineage.

PE

S/N=481.4

S/N=525.7

S/N=532.2

S/N=513.1


Intracytoplasmic stains pick up cells of a particular lineage.

Normal Peripheral Blood Sample – FSC/SSC

Tube 1- AntiMPO FITC / -PE

Tube 2- -FITC /Cyto CD79aPE

Tube 3- AntiMPO FITC / Cyto CD79aPE

2.5% Formaldehyde Fix for 20 min-wash-0.05% saponin for 10 min


Titration of antibodies
Titration of antibodies pick up cells of a particular lineage.

5 µl

1 µl

2 µl

15 µl

10 µl

20 µl


Spread of cd19 from negative to positive

Contribution of debris to background pick up cells of a particular lineage.

Spread of CD19 from Negative to Positive



Line placing and making quadrants
Line placing and making quadrants background

19/4/8 cells based quadrants

19/4/8 cells based quadrants

Isotype control based quadrants

Isotype control based quadrants


Percp cy5 5 tandem dye split may give false readings
PerCP-Cy5.5 backgroundTandem dye split may give false readings

Tandem fluorochrome split


Major plus of our First Meeting background

FCM got popular

TCS

ILCP / PT program:

Started with 5 local labs in 2005

Presently 16 labs participate

Sponsor – TMH

Delhi ILCP

NARI



3. Second Meeting on Standardization of Reagents for Multicolor ImmunophenotypingMarch 9-11, 2012TMH, Mumbai


Recent spurt in clinical cytometry laboratories, both Institute based as well as stand alone labs.

Many labs have started doing 5-6 color IPT.


To be discussed
To be discussed Institute based as well as stand alone labs.

  • Standardization issues plus a CME on Hematopoietic cells in normal, malignancy and residual disease.

  • Collaborations, multicentric studies.


4. Collaborations Institute based as well as stand alone labs.


ISAC and ICCS in form of holding meetings. Indian cytometrists attend and present in their annual meetings.

IndoUS workshops, an annual event, combined with Annual TCS Meetings.

Panel discussion on day 3


8th indo us cytometry workshop lucknow 2007
8th Indo-US Cytometry Workshop, Lucknow, 2007 cytometrists attend and present in their annual meetings.

9th Indo-US Cytometry Workshop, Bangalore, 2008

10th Indo-US Cytometry Workshop, Bhubaneshwar, 09

11th Indo-US Cytometry Workshop, Bangalore, 2010,

Annual TCS meetings with IndoUS workshops


12th indo us cytometry workshop pu chandigarh 2011
12th Indo-US Cytometry Workshop, PU, Chandigarh - 2011 cytometrists attend and present in their annual meetings.

12th Indo-US Cytometry Workshop, DYP Univ., Pune


Making friends asean cytometry workshops kaula lumpur malayasia singapore turkey thailand
Making friends cytometrists attend and present in their annual meetings.ASEAN Cytometry Workshops, Kaula Lumpur, MalayasiaSingapore, Turkey, Thailand


5. Aiming for Uniformity cytometrists attend and present in their annual meetings.


First meeting - Indian Guidelines on Panel selection cytometrists attend and present in their annual meetings.Second Meeting – Multicolor IPT, Application and StandardizationPT program –16 labsHematopathology FellowshipVarious Training Programs for technologists and pathologists..


FCM Training Programs - TMH cytometrists attend and present in their annual meetings.


TMH Mumbai cytometrists attend and present in their annual meetings.offers various clinical cytometry courses (technologists and pathologists). AIIMS/Vedanta Hospital.BD-NCBS Centre of Excellence, Bangalore offers basic cytometry courses four times a year. TCS offers cytometry programs at various institutions.Centre for Cellular and Molecular Platforms, Bangalore offers basic cytometry courses four times year. Indo-US cytometry workshops at various centers annually.

FCM training programs - India


Conclude we have progressed but still not there
Conclude.. cytometrists attend and present in their annual meetings.We have progressed but still not there


Population: 1.21 Billion (year 2011) cytometrists attend and present in their annual meetings.

289 Medical colleges, 31,548 doctors and 990 pathologists per year.

Only 30 oncologists and 15 hematopathologists per year

Labs with Ancillary Techniques <15

Hematolymphoid neoplasm treated at <50 centers

Treatment costs are 10% of western costs.

Lab tests constitute 2-6% of total cost of management


Hematopathology specialty (flow is a part). cytometrists attend and present in their annual meetings.

Training programs in ancillary techniques for

technologists/pathologists.

Quality assurance program.

Collaboration amongst Indian cytometrists.

Multidisciplinary approach.


Thanks ICCS, faculty and the delegates cytometrists attend and present in their annual meetings.


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