gujral fcm 2
DESCRIPTION
TMH proceedings 2010-2011,pdfTRANSCRIPT
Flow Cytometry – Principles and Applications
Second Basic Hematopathology CourseTMHJune 11-12, 2011
Dr Sumeet Gujral, MDAssociate ProfessorDepartment of PathologyTata Memorial Hospital, [email protected]
Role of a Hematopathologist
• Establishing Diagnosis
• Prognostication (sub-classification)
• Prediction
• Evaluation of treatment effectiveness (MRD)
Leukemia/Lymphoma - Diagnosis
• Clinical: History and Examination
• Routine investigations
• Morphology (smear, biopsy)
• Ancillary techniques
Ancillary techniques
• Immunohistochemistry, • Flow Cytometry, • Cytogenetics, • Molecular Diagnostics
• Diagnostic• Prognostic• Predictive
100bpladder
1 2 3 4 5 6
RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) detects diagnostic translocation markers like BCR/ABL in CML - BCR/ABL: b3a2 437bp
Flow Cytometry
Localization of antigens or proteins in cells using labeled antibodies through antigen-antibody interactions,
Reaction visualized by a marker (fluorescent dye, enzyme, colloidal gold etc)
Immunophenotyping
Complementary
FCMmulticolor immunophenotypingfluids
Immunohistochemistry mostly single colorbiopsy
Our practice
Flow cytometry – Liquid and Solid
Immunohistochemistry – Solid and Liquid
flow + cyto + metry
• 1953 - The first impedance-based flow cytometry device, using the coulter principle (Wallace A Coulter)
• 1968 - The first fluorescence-based flow cytometry device (ICP 11) by Wolfgang Göhde, University of Munster
It is the measurement of cellular properties as cells move in a fluid stream (flow), past a stationary set of detectors (thousand events per second)
Technique of quantitative single cell analysis
Principle
It analyses - physical, and - chemical properties (immunofluorescence) of cell
Components of a Flow Cytometer
• Fluidics: a flow cell with sheath fluid (hydrodynamic focussing)
• Optics: LASERS, single wavelength,
coherent light (however incoherent light is of random phase varying with time and position)
• a detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer
• an amplification system – linear or
logarithmic
• a computer for analysis of the signals- Single or multiple Lasers- Sorter
Types
- Single Laser or Multiple Lasers(1 laser three color, 4 lasers 18 fluorescence detectors)
- Sorter (so as to purify populations of interest )
- Laser scanning cytometers
Advantages of a FCM
• Study of cells, chromosomes and particles (analysis, counting and sorting)
• Thousand of particles per second
• Multiparametric analysis at a single cell level
• Pattern studies
• Sorting
Research Applications• Autofluorescent Proteins • Antigen or Ligand Density • Apoptosis • Enzyme activity • DNA, RNA content and changes in the cell cycle • Membrane Potential • Cytokine receptors and it's synthesis • Drug uptake and efflux • Phagocytosis • Viability • Changes in Intracellular pH • Changes in Intracellular calcium • Changes in Intracellular glutathione • Changes in Oxidative Burst
Diagnostic Applications
1. Monitoring AIDS patients 2. Immunophenotyping: Diagnosis, subtyping and
prognostication of hematolymphoid malignancies3. Monitoring Minimal Residual Disease 4. Determining CD34 counts5. Reticulocyte Counts 6. Diagnosis of PNH 7. DNA analysis of S-phase fraction8. Platelet counts
It requires a suspension of single cells No information on tissue architecture
Expensive, maintenance
Shortcomings of a FCM
ImmunophenotypingSampleProcessAcquireAnalysisFinal report
Flow cycle
Referring physician Sample collection/transportation
Preparation of cells of interest Add antibodies tagged with fluorochromes
Acquire (flow) the cells LASER
Amplified signals Digitized
Analyze Report Referring physician
Antigen expression:RBCs, WBCs, Platelets, Others
CD3
Cyto CD3
Tdt
Lysis of red cells
Add reagents
Process
Intracellular staining
For Intracellular staining, cells are first fixed in suspension and then permeabilised before adding the fluorochrome
This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact
Commercial kits/In-house
Cells are fixed – permeabilised –add antibody (FL)
CD3
Cyto CD3
Tdt
Results
Physical Properties Scatter pattern, FSC vs SSC
Forward - Size
Side - Granularity
Note: no RBCs
Chemical propertiesImmunofluoresence
How do we select cells of interest?Gating
Scatter pattern, FSC vs SSC
Forward - Size
Side - Granularity
Cells of interest: FSC vs SSC
Forward - Size
Side - Granularity
FSC vs SSC CD45 Gating CD19 Gating
Cells of interest may be scanty
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CD3 - PerCP
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FSC vs SSC
Study B-cellsStudy T-cells
T and B cells
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GatingSmall cell Lymphoma
What is CD45 gating?
How is CD45 gating better than SSC/FSC gating in Acute Leukemia?
Importance of CD45 gating when blasts are few
Guess
CD45 gating means CD45 in each tube
CD45
Myeloid tube T cell tube B cell tube MDS tube Other tube
CD45 CD45 CD45CD45
Generally 3-6 tubes,How to select antibody combinations?
CD3 - FITCCD19 - PE CD13 – PerCPCD45 – TR
Four color means a cocktail of four antibodies
CD3 - FITCCD4 - PE CD8 – PerCPCD45 – TR
or of same lineage..
of different lineages..
Expression of myeloid antigens – 6 colors
CD117APC
CD11bPECy7
CD15FITC
CD45APC Cy7
CD13PECD16PECy5
CD13 FITC
Indian Guidelines, 2008
Most labs still do 3-4 colors (single Laser)
10 antibodies plus controls for Acute Leukemia
9 antibodies plus controls for CLPD
Published minimal panels may be INADEQUATE
Recommendations
IJPM, 2008
All lymphoid cells CD45+ (LCA)
B-cells CD19, CD10, CD20, CD23, FMC7, k/l LC, cCD22
T-cells CD3, CD4, CD5, CD7, CD8, cCD3
Myeloid cells CD13, CD33, CD117, anti MPO
Megakaryocytic CD41, CD61
Blasts CD34, Tdt, CD99, HLADR
Others: CD1a, CD2, CD11c, CD15, CD16, CD25, CD38, CD43, CD56,CD55, CD59 CD64, CD79a, CD103, CD138, TCR alpha/beta, TCRgamma/delta, IgM, IgD
Cytometry B Clin Cytom. 2009 May;76(3):199-205
6 color and more
CD23APC
CD5PECy7
Lambda FITC
CD19APC H7
Kappa PECD20PerCpCy5.5
Lambda FITC
Single color 6 color, all B cell lineage
Normal 6 color pattern with T and B cell markers
Multiple gates
Normal 6 color pattern with T and B cell markers
Colors unstoppable…
• Euroflow - 8 colors
• Brentwood et al - 10 colors
• Roederer et al - 15 colors
• Most labs do 3-4 color IPT
In Immunophenotyping, FCM helps in detecting following:
- presence or absence of an antigen- intensity- blasts- Clonality: LCR, V-beta repertoire- scanty cells, Maturation patterns
CD3 antibody by FCM detects the fully assembled TCR-CD3 complex, which is present on the surface of T cells only.
In contrast, the CD3 IHC stain usually detects only the epsilon component of CD3, therefore cannot distinguish between T & NK cells.
Diagnosis of Leukemia/lymphoma
• Morphology H&E stain - BiopsyGiemsa stain – Aspirate
• Cytochemistry (MPO, NSE)
• Immunohistochemistry, Flow cytometry
• Cytogenetics, FISH
• Molecular methods
Case
55 year old male
Bilateral cervical and axillary lymph nodes
Normal Complete Blood Counts
Provisional diagnosis: LymphomaAdvise: Biopsy diagnosisFNAC and flow
3 color panel
3 color panel
CD45, CD20, CD19, CD5, CD23, CD22wk, CD20, Kappa LC, CD200
Impression: B-cell lymphoma, SLL
Prognostic markers: CD38, ZAP70
3 color panel
CD19 gating
Same patient of CLL, however, CD19 gating was done
4 color panel
B-CLPD – 6 color panel
Debris
B-CLPD – 6 color panel
CD45, CD20, CD19, CD5, CD23, CD22wk, CD20, Kappa
LC, CD200
Impression: B-cell lymphoma, SLL
Subtyping of Small B cell lymphomas
Patterns recognition
• MDS• Hematogones
Normal BM Maturation patterns of myeloid / monocytic cells
NeutrophilsCD13+, CD16+
NeutrophilsCD11b+, CD117-
B-cell maturation
Cyto muheavy chain
IgM+, IgD+CD5+
IgM
IgG IgA
CD10, BCL6+
Pax5 (My+)
CD79a (T+)
IgG,A,M.D.EIgMIgM
BCR
T-cell maturation
Prothymocyte Subcapsular Th Cortical Th Medullary Th PTC
CD7
Tdt
CD1a
CD2/CD5
CD3
CD4/CD8
surface
DPT CD4+
CD8+
cytoplasmic
Myelodysplastic syndrome
MDS Scatter pattern and maturation pattern
• FSC versus SSC• Myeloid cells: CD11c, CD13, CD15, CD16• Monocytic: CD33, CD56, CD14• Erythroid: CD235, CD71• Megakaryocytic: CD41, CD61, Morphology
Normal MDS
Patterns of antigen expression - Granulocytic maturation
FSC vs SSC
Patterns of antigen expression - Granulocytic maturation
CD45 vs SSC
Normal MDSCD13 CD16CD11c CD16
Normally Maturing Granulocytes
Sickle/Concave pattern Wave pattern
Granulocytic maturation in MDS (CD13 vs CD16)
NORMAL MDS
Normal MDS
Granulocytic maturation in MDS (CD11C vs CD16)
NORMAL MDS
Normal MDS
Hematogones
Hematogones
medium sized lymphoid cells,scant cytoplasm, round to irregular nuclei, dense homogeneous chromatin no - very small nucleoli
Bone marrow from a 3-year-old boy with megakaryocytic thrombocytopenia. Hematogones bear close resemblance to the neoplastic lymphoblasts
Numerous lymphoblasts are present in this bone marrow smear from a 5-year-old girl with precursor B-ALL
Hematogones B-cell ALL
Another case
CD19+, CD10+, FMC7+, CD79b+, CD38+, LLC+ CD3-, CD5-, CD23-, KLC -
Diagnosis: High grade B cell lymphoma
Pleural fluid, lymphoid rich effusion
CD10+, CD19+, HLADR+
B cell ALL, Burkitt’s Lymphoma
CD10+, CD19+, HLADR+
FL, DLBCL
B cell ALL, Burkitt’s Lymphoma
CD10+, CD19+, HLADR+
FL, DLBCL
Hematogones(Tdt+/-)
B cell ALL, Burkitt’s Lymphoma
Lymph node FNAC and FCM
Lymph node Biopsy and FCM
Primary folliclesIgM+ IgD+
Secondary follicles, IgD-
Mantle zoneIgM+ IgD+
B cell clonalitySmall B cell lymphomas
CD20+
Brent Wood et al
?? Role of FCM
Follicular lymphoma , Grade 1 Follicular hyperplasia
FL (Grade 3) Reactive
Bcl2 might not help
flow flow flow
Light chain restriction
CD20 and lambda
Quantitation by FCM
Platelet counts
Various method
International Flow Reference Method
RBC/Platelet Ratio Method (Dual Platform Method)
Absolute Platelet Count=
RBCevents X RBC count Platelet events (Automated Cell Analyzer)
ISLH Task Force, Am J Clin Pathol 115, 460-464.(2001)
CD41/61
RBC
Platelets
Single Platform Immunoplatelet method
Single Platform Bead Assay
Absolute platelet count =
Gated Plt events X Bead count Gated bead events (fixed value)
PlateletsBead
s
Sehgal K, Cytomerty B, Clinical Cytometry, 2010
Lymphocyte subset analysis
CD4/CD8 Counts
Lymphocyte subset analysis
Normal peripheral blood
CD4 PE
CD8FITC
PNH studies (WBCs and RBCs)
It is a rare clonal stem cell disorder of hematopoietic cells that is associated with intravascular hemolysis, venous thrombosis, and cytopenias related to bone marrow aplasia.
Somatic mutations of PIGA gene
Loss of GPI (CD55, CD59)
FLAER test: In vitro binding of a fluorescent-labeled modified toxin that is dependent on the presence of GPI anchors.
Normal Neutrophils
Abnormal Neutrophils
CD34 stem cells enumeration
ISHAGE protocol
Dual Platform - Lyse wash method
Performed in duplicate
Acquire at least 100 CD34+ events for an intra assay C.V of 10%
Four parameters are used– FSC– SSC – Intensity of CD34 staining– Intensity of CD45 staining
All initial ungated events must be acquired and isotype controlsare not required
Then analyzed in a sequential manner (BOOLEAN gating)as per the ISHAGE protocol
Isotype controls are not required with ISHAGE gating system
CD34 % =G4/G1 x100
Absolute CD34=CD34% X WBC 100
N=CD34+ events in R4
D=CD45+ events in R1
ISHAGE Single Platform
Subtract 7AAD + cells from R1
R7=bead events
Lyse no wash processing
Reverse pipetting is essential
Reticulocyte counts
DNA Ploidy
FCM studies cell surface antigens (phenotype), DNA content (ploidy) (DNA index) and DNA flow cytometric proliferation analysis (S-phase fraction or %S-phase).
Minimal residual disease in ALL
(eg., MRD Lite)
5-year-old boy presented with fever - 1 month
On examination: Pallor, Hepatosplenomegaly
Complete Blood CountsHb - 6gm%TLC – 44,000/cmmPlatelets – 1.25lakh/cmm
Peripheral Blood Smear Examination
DiagnosisMPO, NSE negative
MPO negative NSE negative
Diagnosis
3 Color FCM
FSC vs SSC
Blasts only
CD19, CD10, HLADR positive blasts
CD34 negative
Diagnosis: B-cell ALL
Morphological differentials CD19, CD10, HLADR positive blasts
1. B-cell ALL Tdt, CD34
2. Burkitt’s Lymphoma Kappa, Lambda
LymphocytosisCD10+, CD19+, HLADR+, Tdt+
B cell ALL Hematogones
Diagnosis: Tdt + B-cell ALL
Prognostication: Cytogenetic studies
Chemotherapy: MCP 841 protocol
Evaluation of Treatment: Day 18 BM
- Morphology
- MRD Lite by flow
Day 18 post induction
MRD lite
• Hypothesis: Day 18 post induction, no hematogones in BM
• CD19, HLADR, CD34, syto 13
MRD Tube
FSC-AF
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MRD Tube
FSC-A
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Gate 2
MRD Tube
CD10 PE-A
CD
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Gate3
MRD Tube
KAPPA/LAMBDA FITC-A
CD
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P-A
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10410
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Gate 4MRD Tube
CD34 PerCP-A
CD
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CD10 PE-A
CD
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MRD Tube
KAPPA/LAMBDA FITC-A
CD
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CD10 PE-W
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CD19 APC-A
SS
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Gate 5
FCS Filename Gate # of Events % of Gated Cells
% of All Cells
1,3a,100_MRD TUBE.fcs
None 104464 100.0 44.75
1,3a,100_MRD TUBE.fcs
Gate 1 104464 100.0 44.75
1,3a,100_MRD TUBE.fcs
Gate 2 58041 55.56 24.86
1,3a,100_MRD TUBE.fcs
Gate3 1157 1.11 0.5
1,3a,100_MRD TUBE.fcs
Gate 4 1126 1.08 0.48
1,3a,100_MRD TUBE.fcs
Gate 5 6481 6.2 2.78
1,3a,100_MRD TUBE.fcs
Gate6 12102 11.58 5.18
1 in 100 tumor cell dilution – MRD Tube
MRD = 1.43%
Quality control
• Instrument quality controlExperiment setup includes:
- PMT Voltage setup
- Compensation setup and
- Titration of Antibodies
• Reagent/Antibody quality control
• Process quality control
• Tandem dyes
• Trained staff, CMEs
For Referring Oncologist/Pathologist
Indications of FCM, short cuts
Transportation/viability of sample
Multicolor analysis and reading the report
QC - Final comprehensive report
• Morphology• Flow cytometry / IHC• Cytogenetics• Molecular Diagnostics