flow cytometry: an indian scenario multicolor immunophenotyping: applications and standardization...
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Flow cytometry: An Indian Scenario
Multicolor Immunophenotyping: Applications and StandardizationTMH, MumbaiMarch 9-11, 2012
Sumeet Gujral, MD Professor,Department of Pathology,Tata Memorial Hospital, [email protected]
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Flow cytometry: An Indian Scenario
1. History2. The Cytometry Society (TCS)- Research Arm- Clinical Cytometry
a. 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 programs
3. Present meeting4. Collaborations5. Uniformity in Diagnostics
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1. History
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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+).
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 in1968 by W Guhde. Pulse cytophotometry
India
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2. The Cytometry Society (TCS) of India, 2005
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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
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7th Indo-US Cytometry Workshop, JNU, New Delhi, 2006
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Basic/research cytometry
• Institute based (government agencies)
• Last decade – Industry
• > 1000 cytometers
Total pubmed publications – 126505, first in 1974Total Indian publications - 1092, first in 1989
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Clinical Cytometry
• Management of HLN in IndiaCancer Hospitals, Labs, Trained staff, Costing
• Immunophenotyping - Indian Data- First Meeting, 2008 (Indian Guidelines)- PT program and Standardization- Training programs
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Management of leukemia/lymphoma – India
Cancer HospitalsLabs with Ancillary TechniquesTrained StaffCosting
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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.
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Health care in India
• Hospitals (Government versus Private)
• Labs with Ancillary techniques
• Training program
• Costing
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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 10-20 centers
Management of HLN
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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
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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).
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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
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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
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Costing of Immunophenotyping: Year 2008
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Direct cost: Visible cost
1. One time cost of instrumentOutright purchase versus Reagent rental
2. Recurring costReagents, 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
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Direct 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
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
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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
Gujral, IJPM, 2010
Management of HLN
Costing of one IPT test - Rs. 4120 (USD 100)
Costing of CD34 counts - Rs. 1700 (USD 40)
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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
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Treatment
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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
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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
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Management of HLN at TMH
Neoplasm subtype
Treatmentcost in Indian Rs/ USD
TotalUSD
Diagnostic methodsCost in Indian Rupees / USD
TotalUSD
CT/RT PBSCT others Routine IHC/FCM FISH/PCR Total
AML - Adults 8,00,000 10,00,000 16000 500 8000 15,000 23500 470
ALL - Pediatric 4,00,000 10,00,000 8000 500 8000 15,000 23500 470
Myeloma 10,00,000 500 8000 15,000 23500 470
MDS 10,00,000 500 8000 15,000 23500 470
NHL -others 5,00,000 10,00,000 10000 500 8000 15,000 23500 470
HD 1,50,000 10,00,000 3000 500 3500 ---------- 4000 80
BL 5,00,000 10,00,000 10000 500 8000 15,000 23500 470
T-LL 5,00,000 10,00,000 10000 500 8000 15,000 23500 470
Lab tests constitute 2-6% of to
tal cost of management (B
MT excluded)
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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
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Immunophenotyping - India
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1. Introduction to IPT
2. Indian Data
3. First Meeting, 2008 (Indian Guidelines)
4. PT program and Standardization
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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
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IHC and FCM – complementaryMandatory for any center doing HLN
FCMmulticolor immunophenotypingfluids
Immunohistochemistry mostly single colorbiopsy
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>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.
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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
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TMH Data
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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,
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Acute Leukemia, n=2511
Common subtypes of AMLAMLM2 (27%),AMLM5 (15%), AMLM0 (12%), AMLM1 (12%),APML (11%), and AML 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%)
ALL (58%)AML (38%)
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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
Diagnosis Frequency Adult % Pediatric % Median Age(years)
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Lymphomas in BM/PBS - CLPDs
B cell Lymphomas• CLL - 68.5%, • FL - 8.5% • MCL - 5.5%• SMZL - 5%)• HCL - 5%
Leuk Lymphoma, 2009
T/NK cell lymphomas4% (nine cases) of all maturelymphoid neoplasms. T-LGL - 4 cases, T-PLL - 2 (small cell variant),ATLL – 2PCGDTCL - 1
IJC, 2010
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Hematolymphoid Neoplasm - One year DMG/clinic data
Diagnosis Adultn = 1973
Pediatricn = 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 10 2
JMML - 4
LCH - 13
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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
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March 2005
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March 2005, Mumbai
TMH started a ILCP for IPT
Five local laboratories joined (sample sent, results, feedback)
Quarterly meetings
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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
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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
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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
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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
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“Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry”
March 13-15, 2008TMH, Mumbai
First Meeting, 2008
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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
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
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2008 Guideline meeting, TMH, Mumbai
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B cell: CD10, CD19T cell: CD7, CD5Myeloid: CD13, CD33, CD117Other: 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)
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Review of Literature
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Panels CD5 CD7 CD10 CD19 CD13 CD33 CD117 CD34 HLADR CD45 EXTRAS
US Canadian1997 (12)
Y Y Y Y Y Y Y Y CD2, CD14k,l
ISAC 2000(> 14)
Y Y Y Y Y Y Y T, B, Mye, Eryth, Mega
BCSH 2002 (10)
Y Y Y Y cCD22, CD79,cCD3, aMPO,Tdt, CD2,
Second Latin American2005 (18)
Y Y Y Y Y Y Y Y Y cCD3, aMPO,CD2, CD79a,sIg, k, l, CD15, Tdt
TMHMumbai 2008 (10)
Y Y Y Y Y Y Y Y Y Y
AL - Recommendation by various panels (n = 10-18)
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Panels CD19 CD5 CD23 CD10 FMC7 K L CD3 CD20 EXTRAS
US Canadian1997 (11)
Y Y Y Y Y 3,4,5,7,8,45,
ISAC 2000(8)
Y Y Y Y Y Y Y 45
BCSH 2002 (10)
Y Y Y Y Y Y 2, 22, 79b
Second Latin American2005 (7)
Y Y Y 3, 4, 8, 56
TMHMumbai 2008 (10)
Y Y Y Y Y Y Y Y Y
CLPD - Recommendation by various panels (n = 7-11)
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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
Recommended markers in a leukemia lab
IJPM. 2008
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At same time were published2006 Bethesda International Consensus
Guidelines.
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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
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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, 2009Cytometry A, 2009
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Anemia
Primary lymphadenopathy
Splenomegaly
Staging of bone marrow in lymphomas
Acceptability of Bethesda Consensus Guidelines?
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Pancytopenia in India25-60% is megaloblastic anemia
Pediatric All age groups
Megaloblastic anemia
Aplastic anemia / acute leukemia
Other Megaloblastic anemia
Aplastic anemia / acute leukemia
Others
Gupta et al, Trop Doct. 2008, Varanasi109 cases
7 43/25 32 Khanduri et al, NMJI, 07, Stephens,ND120 cases
71 -- --
Bhatnagar SK , J Trop Pediatr. 2005, LHMC, ND109 cases
28 21/20 30 Khunger et al, IJPM. 2002. Safdarjung, ND200 cases
72 14
Kumar R et al, JAPI. 2001, AHRR, ND166 cases
37 30/49
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Indian Guidelines - Lacunae
Gujral et al, Indian J Pathol Microbiol. 2008 Apr-Jun;51(2):161-6.
Gujral et al, Cytometry B Clin Cytom. 2008 Aug 25
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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
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More colors more issues..How much is enough?..
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8-10 color15 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
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Compensation
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PMT Voltage setup using signal/noise ratio
S/N=532.2 S/N=513.1S/N=525.7S/N=481.4
PE
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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 stainsNormal Peripheral Blood Sample – FSC/SSC
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Titration of antibodies1 µl 2 µl 5 µl
10 µl 15 µl 20 µl
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Spread of CD19 from Negative to Positive
Contribution of debris to background
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Effect of titration of PE-cy7 antibody on Per-CpCy5.5 background
5ul
2.5ul
1.25ul
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Line placing and making quadrants
Isotype control based quadrants
19/4/8 cells based quadrants 19/4/8 cells based quadrants
Isotype control based quadrants
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Tandem fluorochrome split
PerCP-Cy5.5Tandem dye split may give false readings
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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
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Review of the First meeting
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3. Second Meeting on Standardization of Reagents for Multicolor Immunophenotyping
March 9-11, 2012TMH, Mumbai
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• Recent spurt in clinical cytometry laboratories, both Institute based as well as stand alone labs.
• Many labs have started doing 5-6 color IPT.
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To be discussed
• Standardization issues plus a CME on Hematopoietic cells in normal, malignancy and residual disease.
• Collaborations, multicentric studies.
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4. Collaborations
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• 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
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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
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12th Indo-US Cytometry Workshop, PU, Chandigarh - 2011
12th Indo-US Cytometry Workshop, DYP Univ., Pune
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Making friends
ASEAN Cytometry Workshops, Kaula Lumpur, Malayasia
Singapore, Turkey, Thailand
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5. Aiming for Uniformity
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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..
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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
FCM Training Programs - TMH
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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
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Conclude..
We have progressed but still not there
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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
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Hematopathology specialty (flow is a part).
Training programs in ancillary techniques fortechnologists/pathologists.
Quality assurance program.
Collaboration amongst Indian cytometrists.
Multidisciplinary approach.
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Thanks ICCS, faculty and the delegates
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