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Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and Standardization TMH, Mumbai March 9-11, 2012 Sumeet Gujral, MD Professor,

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Presentation on theme: "Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and Standardization TMH, Mumbai March 9-11, 2012 Sumeet Gujral, MD Professor,"— Presentation transcript:

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 1.History 2.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 3.Present meeting 4.Collaborations 5.Uniformity in Diagnostics

3 1. History

4 Mid 80s - Dr. VK Jains (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+). 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 in 1968 by W Guhde. Pulse cytophotometry India

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

6 The Cytometry Society (TCS) 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 7 th 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 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 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.

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

13 Cancer Hospitals (<25) n=60/70 Tertiary Care Cancer Centers: 6-8 Regional Cancer Centers: Private/Corporate Hospitals: Medical College Nursing Homes Hematolymphoid neoplasm treated at <50 centers SCT being done at centers Management of HLN

14 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

15 Trained staff Structured 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).

16 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

17 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

18 Costing of Immunophenotyping: Year 2008

19 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 Activity Based Costing method is used to calculate per cost test

20 Direct cost of IPT Cost CentersTotal Cost Monoclonal Antibodies41,25,000 Equipment4,77,313 Electricity1,26,256 Reagents1,57,137 Quality Control88,435 Spares and maintenance1,34,687 Consumables87,197 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

21 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 Gujral, IJPM, 2010 Management of HLN Costing of one IPT test - Rs (USD 100) Costing of CD34 counts - Rs (USD 40)

22 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

23 Treatment

24 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

25 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

26 Management of HLN at TMH Neoplasm subtype Treatment cost in Indian Rs/ USD Total USD Diagnostic methods Cost in Indian Rupees / USD Total USD CT/RT PBSCT othersRoutineIHC/FCMFISH/PCRTotal AML - Adults8,00,000 10,00, , ALL - Pediatric4,00,000 10,00, , Myeloma 10,00, , MDS 10,00, , NHL -others5,00,000 10,00, , HD1,50,000 10,00, BL5,00,000 10,00, , T-LL5,00,000 10,00, , Lab tests constitute 2-6% of total cost of management (BMT excluded)

27 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

28 Immunophenotyping - India

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

30 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



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

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 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

36 TMH Data

37 Hematopathology Lab, TMH, Mumbai Approx. 50,000 new patients come to TMH/year and 8% of these are hematolymphoid neoplasm new cases every year. Leuk & Lymphoma, 2009 Clinical Cytometry, 2008 IJC, 2010,

38 Acute Leukemia, n=2511 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%) ALL (58%) AML (38%)

39 B-Cell ALL1120 (44.4%) T-cell ALL351 (13.8%) AML964 (38.3%) BAL28 (1.1%) CMLBC45 (1.8%) AUL2 TAL1 DiagnosisFrequencyAdult %Pediatric %Median Age (years)

40 Lymphomas in BM/PBS - CLPDs B cell Lymphomas CLL %, FL - 8.5% MCL - 5.5% SMZL - 5%) HCL - 5% Leuk Lymphoma, 2009 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

41 Hematolymphoid Neoplasm - One year DMG/clinic data Diagnosis Adult n = 1973 Pediatric n = 772 AML16581 ALL (46%) Multiple Myeloma101 Acute Promyelocytic Leukemia2310 Chronic Myeloid Leukemia28619 CLPDs60 NHL551 (27%)89 HD17975 Acute Leukemia - others1935 Others108 MDS102 JMML-4 LCH-13

42 Pediatric data, 2011 Total number of cases – 1704 Solid tumors Hematolymphoid neoplasms Total number HLN treated ALL AML - 73 NHL - 85 HL - 74 Total number HLN treated Newly diagnosed Previously treated – 65 Second opinion – 7 Investigation only - 5 Newly diagnosed HLN 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

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

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 elsewhere 1,2

47 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

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

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

50 Report of proceedings of the national meeting on "Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry".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 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

51 2008 Guideline meeting, TMH, Mumbai

52 B cell:CD10, CD19 T cell: CD7, CD5 Myeloid: CD13, CD33, CD117 Other: 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)

53 Review of Literature

54 Panels CD5CD7CD10CD19CD13CD33CD117CD34HLADRCD45EXTRAS US Canadian 1997 (12) YYYYYYYYCD2, CD14 k,l ISAC 2000 (> 14) YYYYYYYT, B, Mye, Eryth, Mega BCSH 2002 (10) YYYYcCD22, CD79, cCD3, aMPO, Tdt, CD2, Second Latin American 2005 (18) YYYYYYYYYcCD3, aMPO, CD2, CD79a, sIg, k, l, CD15, Tdt TMH Mumbai 2008 (10) YYYYYYYYYY AL - Recommendation by various panels (n = 10-18)

55 Panels CD19CD5CD23CD10FMC7KLCD3CD20EXTRAS US Canadian 1997 (11) YYYYY3,4,5,7,8,45, ISAC 2000 (8) YYYYYYY45 BCSH 2002 (10) YYYYYY2, 22, 79b Second Latin American 2005 (7) YYY3, 4, 8, 56 TMH Mumbai 2008 (10) YYYYYYYYY CLPD - Recommendation by various panels (n = 7-11)

56 All lymphoid cellsCD45+ (LCA) B-cellsCD19, CD10, cCD22 T-cells CD3, CD5, cCD3 Myeloid cellsCD13, CD33, CD117, anti MPO MegakaryocyticCD41, 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

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

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

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 – dont address maturation pattern, CD45 gating optional Indian panel includes CD23, FMC7 in primary screen Leukemia, 2009 Cytometry A, 2009

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

61 Pancytopenia in India 25-60% is megaloblastic anemia PediatricAll age groups Megaloblastic anemia Aplastic anemia / acute leukemia Other Megaloblastic anemia Aplastic anemia / acute leukemia Others Gupta et al, Trop Doct. 2008, Varanasi 109 cases 743/2532Khanduri et al, NMJI, 07, Stephens,ND 120 cases 71-- Bhatnagar SK, J Trop Pediatr. 2005, LHMC, ND 109 cases 2821/2030Khunger et al, IJPM Safdarjung, ND 200 cases 7214 Kumar R et al, JAPI. 2001, AHRR, ND 166 cases 3730/49

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

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

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

65 8-10 color 15 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

66 Compensation

67 PMT Voltage setup using signal/noise ratio S/N=532.2S/N=513.1S/N=525.7S/N=481.4 PE

68 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 stains Normal Peripheral Blood Sample – FSC/SSC

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

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


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

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

74 Tandem dye split in a case of AML

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

76 Tandem fluorochrome split

77 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

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

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. 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, 20079th Indo-US Cytometry Workshop, Bangalore, th 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 th Indo-US Cytometry Workshop, DYP Univ., Pune

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


89 5. Aiming for Uniformity

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..

91 CoursesPathologistsTechnologists 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

92 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

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.



98 Thanks ICCS, faculty and the delegates


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