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Flow cytometry: An Indian Scenario

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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:

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 ratio
PE 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 delegates
98 98


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