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Basics of Flow Cytometry
Prashant Tembhare
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Flow Cytometry is the automated measurement of Physical, Chemical and Biological properties of individual cells (Cytometry) or particles flowing in a single stream (Flow) in a fluidic system.
What is Flow Cytometry?
Cyto = cells
Metry = measurement
Flow = in a flow or a stream
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Flow cytometer is an instrument that
- illuminates cells as they flow in front of a light source &
- detects and correlates the signals from the illumination.
Unique Ability – rapid analysis of thousands of cells
cells flow at a velocity of 5–50 m/s
Analyze 500-5000 cells/second
- simultaneous illustration of multiple antigens
Two major principles 1. Measurement of physical properties
2. Measurement of antigenic properties
Flow cytometry
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Principles of flow cytometry
Right Angle Light Detector
Forward Light
Detector
LASER BEAM
1. Measurement of physical properties i.e. size and complexity (granularity).
c
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2. Measurement of ANTIGENIC properties of cell surface and inside the cell
with the help of antibodies labeled with different fluorochromes.
Principles of flow cytometry
LASER BEAM
c
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1. Fluidics: Specimen, Sheath fluid, flow chamber.
2. Optics: Light source(s), mirrors, filters, detectors, spectral separation
3. Electronics: Controls pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control
4. Data Analysis: SOFTWARE - Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation
Instrument Components
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Crosland-Taylor - Hydrodynamic focussing = coaxial flow
→ a narrow stream of cells flowing in a core within a wider sheath stream
• Provides a highly controlled fluid stream.
• Provides exact location of a cell in three dimensions
• Maintains sample handling compartment (Flow Cell)
• Forced under pressure through a conical nozzle assembly geometrically designed to produce a laminar flow
• This fluid is SHEATH FLUID - Isotonic fluid
Fluidics
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Fluidics
↓D by 10-40 = ↑V by 100-1600 fold
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HYDRODYNAMIC FOCUSING
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(a) LASER (argon)
(b) Dichroic Filters and Mirrors
(b) Photodiode
(d) PMT (photo multiplier tubes )
OPTICS
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· The fluorochrome absorbs energy from the laser.
• The fluorochrome releases the absorbed energy by:
-vibration and heat dissipation.
-emission of photons of a longer wavelength.
= 488 nm
Emitted Fluorescent Light EnergyAntibody
IncidentLight Energy Fluorescein
Molecule
= 520 nmHO
CO2H
O
C
What is Fluorescence ?
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Mechanism of fluorochrome
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FITC FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Emitted fluorescence intensity is proportional to binding sites
FITC
Log scale of Fluorescent Intensity
Num
ber
of E
vent
s
Fluorescence
0
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Emission Spectra
APC PerCP
Wavelength (nm)400 500 600 700
100%
0%
Nor
mal
ized
Int
ensi
ty
FITC PE
800
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Emission Spectra
APC PerCPPI
Wavelength (nm)400 500 600 700
100%
0%
Cascade Blue
Nor
mal
ized
Int
ensi
ty
FITC PEAlexa 430 PerCP-Cy5.5
800
PE-Cy7
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Absorption
Control
No blue/green light red filter
Fluorescent Light absorption
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Can be a long pass or short pass filter or band pass
Filter is placed at a 45º angle to the incident light
Part of the light is reflected at 90º to the incident light, and part of the light is transmitted and continues on.
DichroicFilter
Detector 1
Detector 2
Dichroic Filters
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Coulter optical system - Elite
The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted
555 - 595
PMT4
APC 655 - 695
PMT6
PMT7
49
0
DL
488
BK
05
5
DL
62
5
DL
675
BP
488 BP525 BP575 BP
632
BP
TM
PMT3 PMT2 PMT1
PMT5
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Optical Design
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
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·Compute pulse height
·Perform calculations for pulse area and pulse
width
·Calculate ratios
·Convert analog signals to proportional digital
signals
·Interface with the computer for data transfer
Electronics
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Electronics:Triggering on a voltage pulse
Laser
Laser
Laser
Time
Volt
age
Time
Volt
age
Time
Volt
age
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Voltage In
PMTPower Supply
Levels 0–1000Vadjusted by slider control on computer
PhotonIn
VoltageSignal Out Analog to
Digital Converter
Log amplification of signalsPMT
Linear amplification of signals
Gain levels from 0–9.99adjusted by slider control on computer
2 Options for SSC and fluorescence channels
Amplifier output voltage ranging between 10mV to 10V
compensationcircuit
Optical to Digital
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Plot Types: Gate Types: Statistics Types: Results:Histogram Polygon # of Events % positive for
Dot Ellipse % of Gated particular markers:
Contour Histogram % of Total -viable cells
Density Quadrant -immunophenotype
mean mean fluorescence intensity
geometric mean DNA content
standard deviation absolute counts
Display Plots
Create Gates
Display Statistics
Analyze Statistics
Data Analysis by Software
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Single cell suspension: all specimens with cells in suspension
PB, BMA, CSF, PF, BAL
Solid tissue» Fine needle aspirations» Tissue suspensions - slicing, mincing and teasing = Filtering
Sample stabilization: Anticoagulant - EDTA or Heparin – Transport at RT
Enrichment of cells: For leucocytes - RBC Lysis - NH4CL or
- Density gradient centrifugation – Ficoll medium
Antibody staining: Separate cells-wash-incubate with Ab-F in dark Acquisition: Acquire the stained cells at earliest or
Fixed and store in refrigerator Data Analysis: VIMP – Needs experience and knowledge
Sample processing
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CLINICAL APPLICATIONS OF FLOW CYTOMETRY
Enumeration of lymphocyte subsets (CD4/CD8) Immunophenotyping of hematologic malignancies Minimal Residual Disease (MRD) Myelodysplatic Syndrome (MDS) HLA B27 typing PNH diagnosis (CD55-/CD59-) DNA/RNA analysis & Cell cycle studies Reticulocyte analysis Hemotopoietic stem cell (CD34+)analysis Platelet analysis Antigen quantitation e.g. CD20, CD22, CD33 etc
Other uncommon Microbiology Determination of drug resistance to chemotherapy Cell Function analysis
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Analysis Approach
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Accurate diagnosis and classification
Knowledge of prognostic factors
Monitoring response
Diagnosis of early relapse at other sites like CNS
FCM in management of Acute Leukemia
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Hematopoieticstem cell
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Myeloidprogenitor
Lymphoidprogenitor
B-lymphocytes
T-lymphocytes
Plasmacells
naïve
ALL
AML
AUL
Mixed Lineage Leukemia
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Identification of blasts
Enumeration of blasts
Assignment of blast lineage
Identification of abnormal blasts
Subclassification
FCM in diagnosis and classification
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Identification of blasts
Low side light scatter
Weak CD45 expression
Markers of immaturity
such as CD34 and TdT
Lack markers of maturation
Myeloblasts - CD11b, CD15, CD16.
B lymphoblasts – surface light chains
kappa/lambda
T lymphoblasts – Surface CD3
CD45 PerCP
SS
C-A
102
103
104
105
0
65536
131072
196608
262144
CD45-ECD
cyT
dT
-FIT
C
100
101
102
103
10410
0
101
102
103
104
0.00% 84.98%
0.00% 15.02%
CD45 FITC
CD
34
Pe
rCP
100
101
102
103
10410
0
101
102
103
104
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Flow cytometric count lower than manual count
Dilution with peripheral blood
Some blasts lack expression of CD34 and CD117
CD45 expression may very
Flow cytometric count higher than manual count
Loss of NRBCS during red cell lysis.
Ficoll Hypaque separation
Blast identifications may be difficult due to poor
preservation or may be disrupted during smear
preparation
Enumeration of Blasts
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Immunophenotypic markers Markers of Immaturity – TdT, CD34
Lineage Specific markers
Myeloid - cMPO
B cell - cCD22/cCD79a
T cell - cCD3
Lineage Associated markers
Myeloid - Common - CD13, CD33, CD117
- Other - CD11b, CD15
Monocytic - CD13, CD33, CD64, CD68, CD117, CD11b, CD14, CD4, cLysozyme
Erythroid - CD36, CD71, CD105, CD235a (Glycophorin A), Hb
Megakaryocytic - CD36, CD41, CD42, CD61 andCD62
B cell - CD19, CD22, CD20, cCD79a, CD10, cIgM, sIg
T cell - Common - CD1a, CD2, CD5, CD7, CD10
- Other - CD4, CD8, CD3,
NK cell - CD16, CD56, CD57, CD94, KIR
PDC - CD123, CD4, CD56, CD68, CD33, CD43, BDCA,
- Other on PB subset CD2, CD5, CD7
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Lineage Infidelity markers
(Leukemia associated immunophenotype; LAIP)
Lymphoid markers in AML - CD7, CD56, CD2, CD5 and CD19.
Myeloid markers in ALL – CD13, CD33, CD117, CD15
Other Markers useful for MRD detection
Associated with AML – CD38, CD45, CD68, HLADR
Associated with ALL – CD9, CD24, CD25, CD52, CD58, CD81, CD123
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AML M0
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AML M2
t(8;21)(q22;q22) RUNX1-RUNX1T1
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AML M5a
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AML Monocytic differentiation (M5b)
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AML M6
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AML M7
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B - ALL
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T - ALL
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Biphenotypic or mixed lineage leukemia
Borowitz M, Bene M, Harris N and Matutes E, (2008) Acute leukaemias of ambiguous lineage., World Health Organization Classification of Tumours IARC Press, Lyon, pp. 150–155.
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Bi-lineal Leukemia
EG Weir and MJ Borowitz. Leukemia (2007) 21, 2264–2270.
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Hematogones
Antigens Early(St-1)
Intermediate(st 2 & 3)
MatureB cells
TdT + - -
CD34 + - -
CD10 bright dim -
CD19 dim intermediate bright
CD22 dim dim intermediate
CD20 - (-/+) weak intermediate
CD38 bright bright variable
CD45 dim intermediate bright
CD58 dim dim dim
CD81 bright bright intermediate
Cyt IgM - + +
K/L - -/+ +
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ALL in various cluster patterns
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Role of flow cytometry in CLPD & MM
Diagnosis
Staging of lymphoma – Bone marrow involvement or body
fluids
Prognostication eg Zap 70 in CLL
Minimal residual disease
Diagnosis of relapse
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Analysis Approach
I. Isolation of cells using lineage specific markers
like CD19 for B cells and CD3 for T cells
II. detection of abnormal immunophenotype
III. Clonality evaluation eg kappa or lambda
IV. Note size of cells – FSC
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Antibody panels- B CLPD
Mature B cells CD19, CD20, CD22, cyto79a, CD79b
Mature T cells CD2, CD3, CD4, CD5, CD7, CD8, TCR αβ/γδ
NK cells CD2, cytoCD3, CD7, vCD8, CD16, CD56, vCD57, CD94, CD158 (KIRs)
Plasma cells CD138, bCD38, CD19, cyto79a, cyto-Kappa, cyto-Lambda
Clonality markers– B cells - sKappa, sLambda,
– PCs - cyto-Kappa, cyto-Lambda
– T cells – TCR V beta repertoire
Other important Markers
CD45, CD38, HLADR, Granzyme, Perforin, TIA
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Disease oriented
B CLPD – CLL – CD19,CD5, CD23, d-n CD20, d-n CD22, d-n FMC7, CD43,
CD81, CD200
– HCL – CD11c, CD25, CD103, CD123
– FCL/DLBCL – CD10
– MCL – CD5 & CCD
MM – CD19, CD20, CD27, CD45, CD56, CD81, CD117
T CLPD– ATLL/CTCL – CD25, CD26, CD27
– AILT – CD10
– ALCL – CD30
– EATCL – CD103
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Approach to immunophenotyping CLPD
Identification of lineage: expression of lineage specific markers.
B cell lineage- CD 19 or CD20 (CD20 may be lost after treatment with rituximab).
Immunoglobulin Light chain restriction
T cell lineage- CD7, CD3, CD2, CD5 (many markers may be lost in null cell phenotype)
TCR V beta repertoire restricted usage
NK cell – CD7, cytoCD3, CD2, CD16, CD56, CD57
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Page 55 CLL
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Page 56MANTLE CELL LYMPHOMA
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Page 58HAIRY CELL LEUKEMIA
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PERIPHERAL T CELL LYMPHOMA - NOS
ATLL
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Immunophenotype of plasma cells
Normal plasma cells
Specific markers- CD138, CD38 (strong)
B cell lineage – weak CD19, strong CD27
Moderate expression of CD45
Neoplastic plasma cells
Aberrant expression- CD20, bCD56, CD28, CD117, CD200
Loss of CD19, CD27, CD45, CD81
Surface/Cytoplasmic light chain restriction
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CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105 10.83%1.20%
78.34%
9.63%
CD19 PerCP Cy55
CD
81 F
ITC
102 103 104 105
102
103
104
105Br CD81+
Dim CD81+
8c icKappa PE
CD
19 A
PC
102 103 104 105
102
103
104
105 12.73% 20.32%
3.05% 63.90%
CD45 V500
CD
19 A
PC
102 103 104 105
102
103
104
105
32.66%
0.15%
67.02%
0.18%
8c icPC 38v450+ 19+45+
CD19 neg PCs
8c icLambda FITC
CD
19 A
PC
102 103 104 105
102
103
104
105
3.76%63.35%
12.39%20.49%
Multiple Myeloma
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Immunophenotyping in Myelodysplastic Syndrome
Normal Granulocytic Maturation
Granulocytic dysplasia in MDS
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Normal Monocytic Maturation
Monocytic dysplasia in MDS
Immunophenotyping in Myelodysplastic Syndrome
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Paraxysmal Nocturnal Hemoglobinuria (PNH)
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THANK YOU!