Flow cytometry - Tata Memorial Centre
Download
Report
Transcript Flow cytometry - Tata Memorial Centre
Basics of Flow
Cytometry
Prashant Tembhare, MD
Tata Memorial Center, Mumbai
email:[email protected]
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
Page 3
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
Page 7
Page 8
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
Page 9
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.
Page 13
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
Page 19
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
Page 24
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