Presentation on theme: "Flow cytometry: An Indian Scenario"— Presentation transcript:
1 Flow cytometry: An Indian Scenario Multicolor Immunophenotyping: Applications and StandardizationTMH, MumbaiMarch 9-11, 2012Sumeet Gujral, MDProfessor,Department of Pathology,Tata Memorial Hospital, Mumbai
2 Flow cytometry: An Indian Scenario HistoryThe Cytometry Society (TCS)- Research Arm- Clinical Cytometrya. 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 programsPresent meetingCollaborationsUniformity in Diagnostics22
4 India First fluorescent based FCM developed in 1968 by W Guhde. Pulse cytophotometryIndiaResearch labs, early 80sDiagnostic labs,1990sMid 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 MumbaiAIIMS, New DelhiHinduja HospitalPvt Reference LabsOthersCD4 counts
6 The Cytometry Society (TCS) - 2005 2005 at CCMB2006 – ICCS meeting in USA, Phil McCoy2007 – Together, Clinical & ResearchSelf 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 cytometersTotal pubmed publications – , first in 1974Total Indian publications , first in 1989
9 Clinical Cytometry Management of HLN in India Immunophenotyping Cancer Hospitals, Labs, Trained staff, CostingImmunophenotyping- 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 techniquesTraining programCosting
13 Cancer Hospitals (<25) n=60/70 Management of HLNCancer Hospitals (<25) n=60/70Tertiary Care Cancer Centers: 6-8Regional Cancer Centers: 15-20Private/Corporate Hospitals: 35-40Medical CollegeNursing HomesHematolymphoid neoplasm treated at <50 centersSCT being done at centers
14 Management of HLNLabs with Ancillary Techniques <15 FCM, Cytogenetics, Molecular DiagnosticsTertiary 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 HLNTrained staff Structured training programsMedical Oncologists (Ped & Adult): 20-25/yearHematologists: 5/yearHematopathologists (DM+fellows+residents): 5+10/yearThere 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 HLNHematopathology training in IndiaPost 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 HLNAmongst various ancillary techniques, flow is better off in..Training programs, conferences/ CMEsLarger pool of young cytometristsStudents (DM, Fellows and Residents) get rotation in flow lab (2-5 months, 7-8 centers in India).
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 contractIndirect 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 IPTIndirect cost of IPTCost CentersTotal CostMonoclonal Antibodies41,25,000Equipment4,77,313Electricity1,26,256Reagents1,57,137Quality Control88,435Spares and maintenance1,34,687Consumables87,197Per annum cost of Immunophenotyping 51,96,025Number of SM studies in a year = 1300Per sample indirect cost is Rs 124Indirect 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 HLNPer sample cost of IPT at TMH, color, markersTotal cost = Direct cost + Indirect Cost= 51,96, ,61,000= 53,57,025Per sample cost of SM: 53,57,025 / 1300 = 4120Costing of one IPT test - Rs (USD 100)Costing of CD34 counts - Rs (USD 40)Gujral, IJPM, 2010
22 Management of HLNOther 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
24 Pediatric Acute Leukemia - India Management of HLNPediatric Acute Leukemia - IndiaPopulation of 1000 million, 6000 children may develop ALL each yearThree tier society (based on socio-economic backgrounds):Profile I (70%) being extremely poor who cannot afford any treatmentProfile II (25%) from the middle class, andProfile III (<5%) who can afford to have the best possible treatmentTreatment costs approximately 10% of western costsGovernment / social organizations fund pediatric cases get treatedChandy M et al24
25 Leukemia/lymphoma - TMH Management of HLNLeukemia/lymphoma - TMHAll 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 200925
26 Management of HLN at TMH Neoplasm subtypeTreatmentcost in Indian Rs/ USDTotalUSDDiagnostic methodsCost in Indian Rupees / USDCT/RTPBSCTothersRoutineIHC/FCMFISH/PCRAML - Adults8,00,00010,00,00016000500800015,00023500470ALL - Pediatric4,00,000MyelomaMDSNHL -others5,00,00010000HD1,50,00030003500400080BLT-LLLab tests constitute 2-6% of total cost of management (BMT excluded)26
27 Management of HLNMost 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.2727
29 1. Introduction to IPT2. Indian Data3. First Meeting, 2008 (Indian Guidelines)4. PT program and Standardization
30 Flow CytometryIt is the measurement of cellular properties as cells move in a fluid stream (flow), past a stationary set of detectorsTechnique of quantitative single cell analysisIt analyses- physical, and- chemical properties (immunofluorescence) of cell303030
33 IHC and FCM – complementary Mandatory for any center doing HLN FCM multicolor immunophenotyping fluidsImmunohistochemistry mostly single color biopsy3333
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, CD34stem cell counts etc.
35 Hematolymphoid Neoplasms 2008 WHO classification ofHematolymphoid NeoplasmsMyeloid neoplasmsPrecursor lymphoid neoplasmsMature B cell neoplasmsMature T- and NK- cell neoplasmsHodgkin lymphomaImmunodeficiency associated LPDHistiocytic and dendritic cell neoplasmsWHO classification: still a distant reality353535
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, 2008IJC, 2010,
38 Acute Leukemia, n=2511 ALL (58%) AML (38%) Common subtypes of AML AMLM2 (27%),AMLM5 (15%),AMLM0 (12%),AMLM1 (12%),APML (11%), andAML 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%)38
39 Diagnosis Frequency Adult % Pediatric % Median Age (years) B-Cell ALL1120 (44.4%)29.362.310T-cell ALL351 (13.8%)12.415.615AML964 (38.3%)53.220.731BAL28 (1.1%)1.81.119CMLBC45 (1.8%)3.00.335AUL2TAL139
44 March 2005, Mumbai TMH started a ILCP for IPT Five local laboratories joined (sample sent, results, feedback) Quarterly meetings44
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 formatPlanned First Meeting45
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 elsewhere1,246
47 Avoid ultrashort panels GoalsPropose guidelines for a minimal antibody panel without compromising on accuracy To enable uniformity in reporting Educational exercise (evolving technology) PT programAvoid ultrashort panels4747
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/opinionThese documents were circulated, taking opinion from cytometrists, hematopathologists, medical and pediatric oncologists and others48
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 IndiaReport 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):161-650
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, 200758
59 Similarities and Differences Bethesda versus Indian approach US – indication based, Indian - morphology (& clinical) basedBoth rely on a screening panel - 33 versus 10 antibodiesUS - comprehensive panels, more T-cell reagents in screeningSecondary reagents differIndian – don’t address maturation pattern, CD45 gating optionalIndian panel includes CD23, FMC7 in primary screenLeukemia, 2009Cytometry A, 200959
60 Acceptability of Bethesda Consensus Guidelines? Anemia Primary lymphadenopathy Splenomegaly Staging of bone marrow in lymphomas60
61 Pancytopenia in India 25-60% is megaloblastic anemia PediatricAll age groupsMegaloblastic anemiaAplastic anemia / acute leukemiaOtherOthersGupta et al, Trop Doct. 2008, Varanasi109 cases743/2532Khanduri et al, NMJI, 07, Stephens,ND120 cases71--Bhatnagar SK , J Trop Pediatr. 2005, LHMC, ND2821/2030Khunger et al, IJPM Safdarjung, ND200 cases7214Kumar R et al, JAPI. 2001, AHRR, ND166 cases3730/49
62 Indian Guidelines - Lacunae Gujral et al, Cytometry B Clin Cytom Aug 25Gujral et al, Indian J Pathol Microbiol Apr-Jun;51(2):161-6.62
63 a beginning.. Patterns Lineage associated markers Gating strategies Scanty sample sizeMRD StudiesRare tumors are not diagnosedIncreased turn around timeRepeated proceduresMulticolor Immunophenotyping: Applications and Standardization63
64 More colors more issues.. How much is enough?..
65 More colors, more issues Selection of fluorochromes and cocktails Lineage specific markers in one tube3 color to 10 colorsThird party softwareMore colors, more issues15 color8-10 color3 to 6 coloror more65
77 Major plus of our First Meeting FCM got popularTCSILCP / PT program:Started with 5 local labs in 2005Presently 16 labs participateSponsor – TMHDelhi ILCPNARI77
78 Second ILCP Group (AIIMS, Delhi) B.L. Kapur Memorial HospitalDr. RML HospitalIndraprastha Apollo HospitalAIIMS, IRCHDr. Lal’s Path LabsVimta Labs Ltd.Quest Diagnostics India Pvt. Ltd.Cryobanks International IndiaOncQuest (Dabur)BD Biosciences
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
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 CoursesPathologistsTechnologists5 Days Advance CBC Course5 Days Advance Clinical Cytometry CourseOne month ObservershipSix month Training programOne year Residency programTwo year Hematopathology FellowshipTwo 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 yearLabs with Ancillary Techniques <15Hematolymphoid neoplasm treated at <50 centersTreatment 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 fortechnologists/pathologists.Quality assurance program.Collaboration amongst Indian cytometrists.Multidisciplinary approach.