1. 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

2. 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 2 2

3. 1. History

4. India First fluorescent based FCM developed in
1968 by W Guhde. Pulse cytophotometry
India
Research labs, early 80s
Diagnostic labs,1990s
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 A Krishan of Univ. of Miami started Indo-US cytometry workshops (12 workshops+).
TMC Mumbai
AIIMS, New Delhi
Hinduja Hospital
Pvt Reference Labs
Others
CD4 counts

5. 2. The Cytometry Society (TCS) of India, 2005

6. 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

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

8. Basic/research cytometry
Institute based (government agencies)
Last decade – Industry
> 1000 cytometers
Total pubmed publications – , first in 1974
Total Indian publications , first in 1989

9. Clinical Cytometry Management of HLN in India Immunophenotyping
Cancer Hospitals, Labs, Trained staff, Costing
Immunophenotyping
- Indian Data
- First Meeting, 2008 (Indian Guidelines)
- PT program and Standardization
- Training programs

10. Management of leukemia/lymphoma – India Cancer Hospitals Labs with Ancillary Techniques Trained Staff Costing

11. No Indian guidelines for most disciplines, opinion/experience based.
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. 11

12. Health care in India Hospitals (Government versus Private)
Labs with Ancillary techniques
Training program
Costing

13. Cancer Hospitals (<25) n=60/70
Management of HLN
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 centers

14. 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)

15. Trained staff Structured training programs
Management of HLN
Trained staff Structured training programs
Medical Oncologists (Ped & Adult): 20-25/year
Hematologists: 5/year
Hematopathologists (DM+fellows+residents): 5+10/year
There are no structured training programs for any of the ancillary techniques (both for pathologists as well as for technologists).

16. Hematopathology training in India
Management of HLN
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.

17. 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).

18. Costing of Immunophenotyping: Year 2008

19. Activity Based Costing method is used to calculate per cost test
Direct cost: Visible cost 1. One time cost of instrument Outright purchase versus Reagent rental 2. Recurring cost Reagents, 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

20. Per annum cost of Immunophenotyping
Direct cost of IPT
Indirect 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
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

21. Per sample cost of IPT at TMH, 2008 3-color, 15-18 markers
Management of HLN
Per sample cost of IPT at TMH, color, markers
Total cost = Direct cost + Indirect Cost
= 51,96, ,61,000
= 53,57,025
Per sample cost of SM: 53,57,025 / 1300 = 4120
Costing of one IPT test - Rs (USD 100)
Costing of CD34 counts - Rs (USD 40)
Gujral, IJPM, 2010

22. Management of HLN
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

23. Treatment

24. Pediatric Acute Leukemia - India
Management of HLN
Pediatric Acute Leukemia - India
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
Government / social organizations fund pediatric cases get treated
Chandy M et al 24

25. Leukemia/lymphoma - TMH
Management of HLN
Leukemia/lymphoma - TMH
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%).
Gujral, Leukemia 2009 25

26. Management of HLN at TMH
Neoplasm subtype
Treatment
cost in Indian Rs/ USD
Total
USD
Diagnostic methods
Cost in Indian Rupees / USD
CT/RT
PBSCT
others
Routine
IHC/FCM
FISH/PCR
AML - Adults
8,00,000
10,00,000
16000
500
8000
15,000
23500
470
ALL - Pediatric
4,00,000
Myeloma
MDS
NHL -others
5,00,000
10000 HD
1,50,000
3000
3500
4000 80 BL
T-LL
Lab tests constitute 2-6% of total cost of management (BMT excluded) 26

27. Management of HLN
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. 27 27

28. Immunophenotyping - India

29. 1. Introduction to IPT
2. Indian Data
3. First Meeting, 2008 (Indian Guidelines)
4. PT program and Standardization

30. Flow Cytometry
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 30 30 30

31. 31

32. 32 32 32

33. IHC and FCM – complementary Mandatory for any center doing HLN
FCM multicolor immunophenotyping fluids
Immunohistochemistry mostly single color biopsy 33 33

34. >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, CD34
stem cell counts etc.

35. Hematolymphoid Neoplasms
2008 WHO classification of
Hematolymphoid Neoplasms
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
WHO classification: still a distant reality 35 35 35

36. TMH Data

37. 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, 2008
IJC, 2010,

38. Acute Leukemia, n=2511 ALL (58%) AML (38%) 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)
Common subtypes of ALL vs West B-cell ALL - 76% (85%) T-cell ALL - 24% (10-15%) 38

39. Diagnosis Frequency Adult % Pediatric % Median Age (years)
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 39

40. Lymphomas in BM/PBS - CLPDs
B cell Lymphomas
CLL %,
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 40

41. Hematolymphoid Neoplasm - One year DMG/clinic data
Diagnosis
Adult
n = 1973
Pediatric
n = 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 2
JMML - 4
LCH 13

42. 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

43. March 2005

44. March 2005, Mumbai TMH started a ILCP for IPT Five local laboratories joined (sample sent, results, feedback) Quarterly meetings 44

45. 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 45

46. 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 46

47. Avoid ultrashort panels
Goals
Propose guidelines for a minimal antibody panel without compromising on accuracy To enable uniformity in reporting Educational exercise (evolving technology) PT program
Avoid ultrashort panels 47 47

48. Over next three years ( ), consensus Guidelines were formulated based on: - Published Data (Indian and western) - Results of the PT program - Practice Based Questionnaire and - Experience/opinion
These documents were circulated, taking opinion from cytometrists, hematopathologists, medical and pediatric oncologists and others 48

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

50. Revised 3 document (consensus) presented
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
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 Apr-Jun;51(2):161-6 50

51. 2008 Guideline meeting, TMH, Mumbai

52. Recommended minimal screening panel of Acute Leukemia, (n=10)
Recommended minimal screening panel of CLPD / Mature lymphomas, (n=9)
B cell: CD10, CD19 T cell: CD7, CD5 Myeloid: CD13, CD33, CD117 Other: CD34, HLA-DR, CD45
CD3, CD5, CD19, CD23, CD10, CD20, FMC7, Kappa, Lambda 52

53. Review of Literature 53

54. AL - Recommendation by various panels (n = 10-18)
CD5
CD7
CD10
CD19
CD13
CD33
CD117
CD34
HLADR
CD45
EXTRAS
US Canadian
1997 (12) Y
CD2, CD14
k,l
ISAC 2000
(> 14)
T, B,
Mye, Eryth, Mega
BCSH
2002 (10)
cCD22, CD79,
cCD3, aMPO,
Tdt, CD2,
Second Latin American
2005 (18)
CD2, CD79a,
sIg, k, l,
CD15, Tdt
TMH
Mumbai 2008 (10) 54

55. CLPD - Recommendation by various panels (n = 7-11)
CD19
CD5
CD23
CD10
FMC7 K L
CD3
CD20
EXTRAS
US Canadian
1997 (11) Y
3,4,5,7,8,45,
ISAC 2000
(8) 45
BCSH
2002 (10)
2, 22, 79b
Second Latin American
2005 (7)
3, 4, 8, 56
TMH
Mumbai 2008 (10) 55

56. Recommended markers in a leukemia lab
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
IJPM. 2008 56 56 56

57. At same time were published
2006 Bethesda International Consensus Guidelines. 57

58. 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 58

59. 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, 2009
Cytometry A, 2009 59

60. Acceptability of Bethesda Consensus Guidelines?
Anemia Primary lymphadenopathy Splenomegaly Staging of bone marrow in lymphomas 60

61. Pancytopenia in India 25-60% is megaloblastic anemia
Pediatric
All age groups
Megaloblastic anemia
Aplastic anemia / acute leukemia
Other
Others
Gupta et al, Trop Doct. 2008, Varanasi
109 cases 7
43/25 32
Khanduri et al, NMJI, 07, Stephens,ND
120 cases 71 --
Bhatnagar SK , J Trop Pediatr. 2005, LHMC, ND 28
21/20 30
Khunger et al, IJPM Safdarjung, ND
200 cases 72 14
Kumar R et al, JAPI. 2001, AHRR, ND
166 cases 37
30/49

62. Indian Guidelines - Lacunae
Gujral et al, Cytometry B Clin Cytom Aug 25
Gujral et al, Indian J Pathol Microbiol Apr-Jun;51(2):161-6. 62

63. 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 63

64. More colors more issues..
How much is enough?..

65. More colors, more issues
Selection of fluorochromes and cocktails 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 65

66. Compensation

67. PMT Voltage setup using signal/noise ratioPE
S/N=532.2
S/N=513.1
S/N=525.7
S/N=481.4 67

68. Intracytoplasmic stains 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

69. Titration of antibodies
5 µl
1 µl
2 µl
15 µl
10 µl
20 µl

70. Spread of CD19 from Negative to Positive
Contribution of debris to background
Spread of CD19 from Negative to Positive

71. Contribution of debris to background

72. Effect of titration of PE-cy7 antibody on Per-CpCy5.5 background
5ul
Effect of titration of PE-cy7 antibody on Per-CpCy5.5 background
2.5ul
1.25ul

73. Line placing and making quadrants
19/4/8 cells based quadrants
19/4/8 cells based quadrants
Isotype control based quadrants
Isotype control based quadrants

74. Tandem dye split in a case of AML

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

76. Tandem fluorochrome split

77. Major plus of our First Meeting
FCM got popular
TCS
ILCP / PT program:
Started with 5 local labs in 2005
Presently 16 labs participate
Sponsor – TMH
Delhi ILCP
NARI 77

78. Second ILCP Group (AIIMS, Delhi)
B.L. Kapur Memorial Hospital
Dr. RML Hospital
Indraprastha Apollo Hospital
AIIMS, IRCH
Dr. Lal’s Path Labs
Vimta Labs Ltd.
Quest Diagnostics India Pvt. Ltd.
Cryobanks International India
OncQuest (Dabur)
BD Biosciences

79. Review of the First meeting

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

81. Recent spurt in clinical cytometry laboratories, both Institute based as well as stand alone labs.
Many labs have started doing 5-6 color IPT.

82. To be discussed
Standardization issues plus a CME on Hematopoietic cells in normal, malignancy and residual disease.
Collaborations, multicentric studies.

83. 4. Collaborations

84. ISAC and ICCS in form of holding meetings
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

85. 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

86. 12th Indo-US Cytometry Workshop, PU, Chandigarh - 2011
12th Indo-US Cytometry Workshop, DYP Univ., Pune

87. Making friends ASEAN Cytometry Workshops, Kaula Lumpur, Malayasia Singapore, Turkey, Thailand

89. 5. Aiming for Uniformity 89 89

90. 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.. 90

91. FCM Training Programs - TMH
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

92. FCM training programs - India
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.

93. Conclude.. We have progressed but still not there

94. 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

95. Hematopathology specialty (flow is a part).
Training programs in ancillary techniques for
technologists/pathologists.
Quality assurance program.
Collaboration amongst Indian cytometrists.
Multidisciplinary approach.

96. 96

98. Thanks ICCS, faculty and the delegates98 98