Flow cytometry: An Indian Scenario

Multicolor Immunophenotyping: Applications and StandardizationTMH, MumbaiMarch 9-11, 2012

Sumeet Gujral, MD Professor,Department of Pathology,Tata Memorial Hospital, Mumbais_gujral@hotmail.com

Flow cytometry: An Indian Scenario

1. History2. The Cytometry Society (TCS)- Research Arm- Clinical Cytometry

a. Health care in Indiab. 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

3. Present meeting4. Collaborations5. Uniformity in Diagnostics

1. History

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+).

Research labs, early 80s• TMC Mumbai • AIIMS, New Delhi• Hinduja Hospital• Pvt Reference Labs• Others

CD4 counts

Diagnostic labs,1990s

First fluorescent based FCM developed in1968 by W Guhde. Pulse cytophotometry

India

2. The Cytometry Society (TCS) of India, 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

• Institute based (government agencies)

• Last decade – Industry

• > 1000 cytometers

Total pubmed publications – 126505, first in 1974Total Indian publications - 1092, first in 1989

Clinical Cytometry

• Management of HLN in IndiaCancer Hospitals, Labs, Trained staff, Costing

• Immunophenotyping - Indian Data- First Meeting, 2008 (Indian Guidelines)- PT program and Standardization- Training programs

Management of leukemia/lymphoma – India

Cancer 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

• Hospitals (Government versus Private)

• Labs with Ancillary techniques

• Training program

• Costing

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

Hematolymphoid neoplasm treated at <50 centers

SCT being done at 10-20 centers

Management of HLN

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 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

• 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..

• 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).

Management of HLN

Costing of Immunophenotyping: Year 2008

Direct cost: Visible cost

1. One time cost of instrumentOutright purchase versus Reagent rental

2. Recurring costReagents, antibodies, tubes, fluids, dyes and kits.

3. Annual maintenance contract

Indirect costs: Hidden cost

salaries, depreciable value,furniture,funds for personnel training and CMEs, ancillary equipments, stationary, electricity and rental charges

Medical insurance, deputation etc

Activity Based Costing method is used to calculate per cost test

Direct cost of IPT

Cost Centers Total Cost

Monoclonal Antibodies 41,25,000

Equipment 4,77,313

Electricity 1,26,256

Reagents 1,57,137

Quality Control 88,435

Spares and maintenance 1,34,687

Consumables 87,197

Per annum cost of Immunophenotyping 

51,96,025

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, 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

Gujral, IJPM, 2010

Management of HLN

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

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

Other factors

Cost 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

Management of HLN

Treatment

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

Chandy M et al

Pediatric Acute Leukemia - India

Management of HLN

Government / social organizations fund pediatric

cases get treated

All patients have a complete work up for diagnosis.

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%).

Leukemia/lymphoma - TMH

Management of HLN

Gujral, Leukemia 2009

Management of HLN at TMH

Neoplasm subtype

Treatmentcost in Indian Rs/ USD

TotalUSD

Diagnostic methodsCost in Indian Rupees / USD

TotalUSD

CT/RT PBSCT others Routine IHC/FCM FISH/PCR Total

AML - Adults 8,00,000 10,00,000 16000 500 8000 15,000 23500 470

ALL - Pediatric 4,00,000 10,00,000 8000 500 8000 15,000 23500 470

Myeloma 10,00,000 500 8000 15,000 23500 470

MDS 10,00,000 500 8000 15,000 23500 470

NHL -others 5,00,000 10,00,000 10000 500 8000 15,000 23500 470

HD 1,50,000 10,00,000 3000 500 3500 ---------- 4000 80

BL 5,00,000 10,00,000 10000 500 8000 15,000 23500 470

T-LL 5,00,000 10,00,000 10000 500 8000 15,000 23500 470

Lab tests constitute 2-6% of to

tal cost of management (B

MT 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

1. Introduction to IPT

2. Indian Data

3. First Meeting, 2008 (Indian Guidelines)

4. PT program and Standardization

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

Flow Cytometry

It analyses - physical, and - chemical properties (immunofluorescence) of cell

IHC and FCM – complementaryMandatory for any center doing HLN

FCMmulticolor immunophenotypingfluids

Immunohistochemistry mostly single colorbiopsy

>400 labs do CD4 counts (started in mid 80s).

>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, CD34stem cell counts etc.

A. Myeloid neoplasms

B. Precursor lymphoid neoplasms

C. Mature B cell neoplasms

D. Mature T- and NK- cell neoplasms

E. Hodgkin lymphoma

F. Immunodeficiency associated LPD

G. Histiocytic and dendritic cell neoplasms

2008 WHO classification of Hematolymphoid Neoplasms

WHO classification: still a distant reality

TMH Data

Hematopathology Lab, TMH, Mumbai

• 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, 2008IJC, 2010,

Acute Leukemia, n=2511

Common subtypes of AMLAMLM2 (27%),AMLM5 (15%), AMLM0 (12%), AMLM1 (12%),APML (11%), and AML t(8;21) (9%)

CMLBC was commonly of myeloid blast crisissubtype (40 cases)

Common subtypes of ALL vs West

B-cell ALL - 76% (85%)

T-cell ALL - 24% (10-15%)

ALL (58%)AML (38%)

B-Cell ALL 1120 (44.4%) 29.3 62.3 10

T-cell ALL 351 (13.8%) 12.4 15.6 15

AML 964 (38.3%) 53.2 20.7 31

BAL 28 (1.1%) 1.8 1.1 19

CMLBC 45 (1.8%) 3.0 0.3 35

AUL 2

TAL 1

Diagnosis Frequency Adult % Pediatric % Median Age(years)

Lymphomas in BM/PBS - CLPDs

B cell Lymphomas• CLL - 68.5%, • FL - 8.5% • MCL - 5.5%• SMZL - 5%)• HCL - 5%

Leuk Lymphoma, 2009

T/NK cell lymphomas4% (nine cases) of all maturelymphoid neoplasms. T-LGL - 4 cases, T-PLL - 2 (small cell variant),ATLL – 2PCGDTCL - 1

IJC, 2010

Hematolymphoid Neoplasm - One year DMG/clinic data

Diagnosis Adultn = 1973

Pediatricn = 772

AML 165 81

ALL 297 355 (46%)

Multiple Myeloma 101

Acute Promyelocytic Leukemia 23 10

Chronic Myeloid Leukemia 286 19

CLPDs 60

NHL 551 (27%) 89

HD 179 75

Acute Leukemia - others 193 5

Others 108

MDS 10 2

JMML - 4

LCH - 13

Pediatric data, 2011

• 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, Mumbai

TMH started a ILCP for IPT

Five local laboratories joined (sample sent, results, feedback)

Quarterly meetings

After 6 cycles of the PT program

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

Planned First Meeting

Focus on “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 Europe

Other 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 accuracy

To 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

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

Presentations: Cytometrists from India, Rest of the Asia, Europe, Australia and America presented their perspective on panel selection

Delegates: 180 delegates including 30 from outside India

Revised 3 document (consensus) presented

2008 Guideline meeting, TMH, Mumbai

B cell: CD10, CD19T cell: CD7, CD5Myeloid: CD13, CD33, CD117Other: CD34, HLA-DR, CD45

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

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

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

Review of Literature

Panels CD5 CD7 CD10 CD19 CD13 CD33 CD117 CD34 HLADR CD45 EXTRAS

US Canadian1997 (12)

Y Y Y Y Y Y Y Y CD2, CD14k,l

ISAC 2000(> 14)

Y Y Y Y Y Y Y T, B, Mye, Eryth, Mega

BCSH 2002 (10)

Y Y Y Y cCD22, CD79,cCD3, aMPO,Tdt, CD2,

Second Latin American2005 (18)

Y Y Y Y Y Y Y Y Y cCD3, aMPO,CD2, CD79a,sIg, k, l, CD15, Tdt

TMHMumbai 2008 (10)

Y Y Y Y Y Y Y Y Y Y

AL - Recommendation by various panels (n = 10-18)

Panels CD19 CD5 CD23 CD10 FMC7 K L CD3 CD20 EXTRAS

US Canadian1997 (11)

Y Y Y Y Y 3,4,5,7,8,45,

ISAC 2000(8)

Y Y Y Y Y Y Y 45

BCSH 2002 (10)

Y Y Y Y Y Y 2, 22, 79b

Second Latin American2005 (7)

Y Y Y 3, 4, 8, 56

TMHMumbai 2008 (10)

Y Y Y Y Y Y Y Y Y

CLPD - Recommendation by various panels (n = 7-11)

All lymphoid cells CD45+ (LCA)

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 published2006 Bethesda International Consensus

Guidelines.

Bethesda uses a panel of antibodies which are sensitive to pick up cells of a particular lineage.3

Most guidelines use a panel of antibodies for diagnosis of AL or CLPD

A 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

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, 2009Cytometry A, 2009

Anemia

Primary lymphadenopathy

Splenomegaly

Staging of bone marrow in lymphomas

Acceptability of Bethesda Consensus Guidelines?

Pancytopenia in India25-60% is megaloblastic anemia

Pediatric All age groups

Megaloblastic anemia

Aplastic anemia / acute leukemia

Other Megaloblastic anemia

Aplastic anemia / acute leukemia

Others

Gupta et al, Trop Doct. 2008, Varanasi109 cases

7 43/25 32 Khanduri et al, NMJI, 07, Stephens,ND120 cases

71 -- --

Bhatnagar SK , J Trop Pediatr. 2005, LHMC, ND109 cases

28 21/20 30 Khunger et al, IJPM. 2002. Safdarjung, ND200 cases

72 14

Kumar R et al, JAPI. 2001, AHRR, ND166 cases

37 30/49

Indian Guidelines - Lacunae

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

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

a beginning..

• 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..How much is enough?..

8-10 color15 color

3 to 6 color or more

Selection of fluorochromes and cocktails

Lineage specific markers in one tube

3 color to 10 colors

Third party software

More colors, more issues

Compensation

PMT Voltage setup using signal/noise ratio

S/N=532.2 S/N=513.1S/N=525.7S/N=481.4

PE

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

Intracytoplasmic stainsNormal Peripheral Blood Sample – FSC/SSC

Titration of antibodies1 µl 2 µl 5 µl

10 µl 15 µl 20 µl

Spread of CD19 from Negative to Positive

Contribution of debris to background

Effect of titration of PE-cy7 antibody on Per-CpCy5.5 background

5ul

2.5ul

1.25ul

Line placing and making quadrants

Isotype control based quadrants

19/4/8 cells based quadrants 19/4/8 cells based quadrants

Isotype control based quadrants

Tandem fluorochrome split

PerCP-Cy5.5Tandem dye split may give false readings

FCM got popular

TCS

ILCP / PT program:Started with 5 local labs in 2005

Presently 16 labs participate

Sponsor – TMH

Delhi ILCP

NARI

Major plus of our First Meeting

Review of the First meeting

3. Second Meeting on Standardization of Reagents for Multicolor Immunophenotyping

March 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

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

• Collaborations, multicentric studies.

4. Collaborations

• 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 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, DYP Univ., Pune

Making friends

ASEAN Cytometry Workshops, Kaula Lumpur, Malayasia

Singapore, Turkey, Thailand

5. Aiming for Uniformity

First meeting - Indian Guidelines on Panel selection

Second Meeting – Multicolor IPT, Application and Standardization

PT program –16 labs

Hematopathology Fellowship

Various Training Programs for technologists and pathologists..

Courses Pathologists Technologists

5 Days Advance CBC Course

5 Days Advance Clinical Cytometry Course

One month Observership

Six month Training program

One year Residency program

Two year Hematopathology Fellowship

Two day Basic Hematopathology Course for PG

FCM Training Programs - TMH

TMH Mumbai 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

Population: 1.21 Billion (year 2011)

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).

Training programs in ancillary techniques fortechnologists/pathologists.

Quality assurance program.

Collaboration amongst Indian cytometrists.

Multidisciplinary approach.

Thanks ICCS, faculty and the delegates