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Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and Standardization

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 s_gujral@hotmail.com. Flow cytometry: An Indian Scenario. History

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Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and Standardization

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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 s_gujral@hotmail.com

  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

  3. 1. History

  4. 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

  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 – 126505, first in 1974 Total Indian publications - 1092, 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 – IndiaCancer HospitalsLabs with Ancillary TechniquesTrained StaffCosting

  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: 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

  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 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).

  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. 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 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

  20. Direct cost of IPT 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. Management of HLN 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

  22. Management of HLN 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

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

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

  26. Management of HLN at TMH 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. 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

  31. IHC and FCM – complementaryMandatory for any center doing HLN FCMmulticolor immunophenotypingfluids Immunohistochemistry mostly single colorbiopsy

  32. >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.

  33. 2008 WHO classification of 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

  34. TMH Data

  35. 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,

  36. 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) ALL (58%) AML (38%) Common subtypes of ALL vs WestB-cell ALL - 76% (85%)T-cell ALL - 24% (10-15%)

  37. Lymphomas in BM/PBS - 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

  38. Hematolymphoid Neoplasm - One year DMG/clinic data

  39. 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

  40. March 2005

  41. March 2005, MumbaiTMH started a ILCP for IPTFive local laboratories joined (sample sent, results, feedback)Quarterly meetings

  42. 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

  43. 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 EuropeOther issues: Sample collection, transport, viability, adequacy of cell yield, storing of samples - recommendations as described elsewhere1,2

  44. 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

  45. 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

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

  47. Revised 3 document (consensus) presented 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

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