Basics of Flow
Cytometry
Prashant Tembhare, MD
Tata Memorial Center, Mumbai
email:docprt@gmail.com
What is Flow Cytometry?
Cyto = cells
Metry = Measurement
Flow = in a flow or a stream
Page  2
Flow cytometry
Flow cytometer is an instrument that
- illuminates cells as they flow in front of a light source
- detects and correlates the signals from illumination.
Unique Ability – rapid analysis of thousands of cells
Analyze 500-5000 cells/second
- simultaneous illustration of multiple antigens
Two major principles 1. Measurement of physical properties
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2. Measurement of antigenic properties
Page  4
Principles of flow cytometry
1. Measurement of physical properties i.e. size and complexity
(granularity).
Right Angle
Light Detector
c
LASER
BEAM
Page  5
Forward
Light
Detector
Principles of flow cytometry
2. Measurement of
ANTIGENIC properties of cell surface and inside the cell
with the help of antibodies labeled with different fluorochromes.
c
LASER
BEAM
Page  6
Instrument Components
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
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Fluidics
 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
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Fluidics
↓D by 10-40 = ↑V by 100-1600 fold
Page  10
HYDRODYNAMIC FOCUSING
Page  11
2. OPTICS
(a) LASER (argon)
(b) Dichroic Filters and Mirrors
(b) Photodiode
(d) PMT (photo multiplier tubes )
Page  12
What is Fluorescence ?
 = 488 nm
O
HO
 = 520 nm
C
Incident
Light Energy
CO2H
Fluorescein
Molecule
Antibody
Emitted Fluorescent
Light Energy
 The fluorochrome absorbs energy from the laser.
•
The fluorochrome releases the absorbed energy by:
vibration and heat dissipation.
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emission of photons of a longer wavelength.
Mechanism of fluorochrome
Page  14
Excitation & Emission
Page  15
Fluorescence
Emitted fluorescence intensity is proportional to
binding sites
FITC
FITC
FITC
Number of Events
FITC
FITC
Page  16
FITC
FITC
0
FITC
Log scale of Fluorescent Intensity
FITC
FITC
Emission Spectra
100%
PE
APC
PerCP
Normalized Intensity
FITC
0%
400
Page  17
500
600
Wavelength (nm)
700
800
Emission Spectra
Cascade Blue FITC Alexa 430
PE
PI APC PerCP PerCP-Cy5.5 PE-Cy7
Normalized Intensity
100%
0%
400
Page  18
500
600
Wavelength (nm)
700
800
Fluorescent Light Filteration
Control
Absorption
No blue/green light
red filter
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Filters
Page  20
Dichroic Filters
 Can be a long pass or short pass filter
 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.
Detector 1
Detector 2
Page  21
Dichroic
Filter
PMT – Photomultiplier Tubes
Page  22
Coulter optical system - Elite
PMT4
PMT2
PMT3
PMT1
555 - 595
575 BP
525 BP
488 BP
655 - 695
L
L
D
5
2
5
6
5
0
L
D
0
9
4
APC
 The Elite optical system uses 5
side window PMTs and a
number of filter slots into which
any filter can be inserted
PMT6
TM
PMT7
Purdue Cytometry Labs
Page  23
D
488 BK
632 BP
675 BP
PMT5
BD optical system – Canto-II
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Page  25
Optical Design
Page  26
Electronics





Page  27
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
Laser
Voltage
Electronics:
Triggering on a voltage pulse
Laser
Voltage
Time
Laser
Page  28
Voltage
Time
Time
Optical to Digital
PMT
Voltage
Signal
Out
Log amplification of signals
2 Options for SSC and fluorescence channels
Photon
In
Analog to
Digital
Converter
Linear amplification of signals
Voltage In
PMT
Power Supply
Levels 0–1000V
adjusted by slider control
on computer
Page  29
compensation
circuit
Gain levels from 0–9.99
adjusted by slider control
on computer
Amplifier output voltage
ranging between 10mV to 10V
Optical to Digital
Page  30
Data Analysis by Software
Display
Plots
Create
Gates
Display
Statistics
Analyze
Statistics
Plot Types:
Gate Types:
Statistics Types:
Results:
Histogram
Polygon
# of Events
% positive for
Dot
Ellipse
% of Gated
particular markers:
Contour
Histogram
% of Total
Density
Quadrant
Page  31
-viable cells
-immunophenotype
mean
mean fluorescence intensity
geometric mean
DNA content
standard deviation
absolute counts
Sample processing
 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
 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:
Page  32
VIMP – Needs experience and knowledge
Common Clinical Applications
 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
Page  33  Cell Function analysis
Analysis Approach
Page  34
Management of Leukemia
 Accurate diagnosis and classification
 Knowledge of prognostic factors
 Monitoring response
 Diagnosis of early relapse at other sites
like CNS
Page  35
ALL
naïve
B-lymphocytes
AUL
Lymphoid
progenitor
Plasma
cells
T-lymphocytes
Mixed Lineage Leukemia
AML
Hematopoietic
stem cell
Myeloid
progenitor
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Page  36
FCM in diagnosis and classification
 Identification of blasts
 Enumeration of blasts
 Assignment of blast lineage

Identification of abnormal blasts

Subclassification
Page  37
Identification of blasts
Low side light scatter
Weak CD45 expression

Markers of immaturity
such as CD34 and TdT
10
196608
SSC-A


84.98%
3
cyTdT-FITC

4
10 0.00%
262144
2
131072
10
65536
1
10
Lack markers of maturation
00
B lymphoblasts – surface light chains
10
CD34 PerCP
T lymphoblasts – Surface CD3
15.02%
5
10 0 10 1 10
10
210
3
4
10
10 CD4510PerCP10
10
CD45-ECD
Myeloblasts - CD11b, CD15, CD16.
kappa/lambda
0.00%
10
10
10
2
3
4
4
3
2
1
0
10 0
10
10
1
10
2
10
CD45 FITC
Page  38
3
10
4
Enumeration of Blasts
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
Page  39
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,
Page  40
- Other on PB subset CD2, CD5, CD7
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
Page  41
AML M0
Page  42
AML M2
t(8;21)(q22;q22) RUNX1-RUNX1T1
Page  43
AML M5a
Page  44
AML Monocytic differentiation (M5b)
Page  45
AML M6
Page  46
AML M7
Page  47
B - ALL
Page  48
T - ALL
Page  49
Borowitz M, Bene M, Harris N and Matutes E, (2008) Acute leukaemias of ambiguous lineage.,
Page Health
50
World
Organization Classification of Tumours IARC Press, Lyon, pp. 150–155.
Page  51
EG Weir and MJ Borowitz. Leukemia (2007) 21, 2264–2270.
Page  52
Bi-lineal Leukemia
Antigens
Early
(St-1)
Intermediate
(st 2 & 3)
Mature
B 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
-
-/+
+
Page  53
ALL in various cluster patterns
Page  54
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
Page  55
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
Page  56
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
Page  57
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
Page  58
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
Page  59
Page  60
CLL
MANTLE CELL LYMPHOMA
Page  61
Page  62
HAIRY CELL LEUKEMIA
Page  63
ATLL
PERIPHERAL T CELL LYMPHOMA - NOS
Page  64
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
Page  65
Multiple Myeloma
0.15%
8c icPC 38v450+ 19+45+
5
1.20%
5 10.83%
10
10
CD56 PC7
CD19 APC
32.66%
4
10
3
CD19 neg PCs
10
4
10
3
10
9.63%
2
10
2
10
78.34%
67.02%
2
10
3
4
2
10
5
10
10
CD45 V500
10
3
4
10
10
CD19 APC
5
10
0.18%
Br CD81+
3
10
2
10
10
3
10
Dim CD81+
2
Page  66
3
4
5
10
10
10
CD19 PerCP Cy55
4
3
10
2
10
2
10
63.90%
3
4
10
10
8c icKappa PE
12.39%
20.49%
10
2
10
3.05%
10
5
10
4
CD19 APC
4
10
20.32%
5 12.73%
10
CD19 APC
5
10
5
10
63.35%
3.76%
2
10
3
4
5
10
10
10
8c icLambda FITC
Page  67
THANK YOU!
Page  68