Flow Cytometry Principles and applications

Basic Hematopathology Course

J une 12-13, 2010
TMH
Dr Sumeet Gujral, MD
Associate Professor
Department of Pathology
Tata Memorial Hospital, Mumbai
s_gujral@hotmail.com
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
Flow cytometry
Immunohistochemistry
Immunofluorescence
Monoclonal antibodies
Sensitize mouse to antigen
Harvest spleen B cells
Fuse with myeloma cells
Select hybridoma clones for antibody production
Label antibody with fluorochrome dye / color
FCM and IHC: complementary
FCM
multicolor immunophenotyping
fluids
Immunohistochemistry
mostly single color
biopsy
flow + cyto + metry
The first impedance-based flow cytometry
device, using the coulter principle was issued in
1953 (Wallace A Coulter).
The first fluorescence-based flow cytometry
device (ICP 11) was developed in 1968 by
Wolfgang Ghde, University of Munster
Present
- 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
It requires a suspension of single cells or
other particles, with minimum clumps and
debris.
To analyze solid tissues, a single-cell suspension must
first be prepared
No information on tissue architecture
Shortcomings of a FCM
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
randomphase 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
PMT
PMT
PMT
LASER
Sample in a hydrodynamically focused stream
Detectors
Amplification and
computer
Properties of FCM
Physical properties
Hydrodynamic
focussing
Forward scatter Side scatter
Size Granularity
SS detector - Granularity
SS detecter Granularity
FS
size
FS
size
Laser
Laser
Size and granularity
Data shown either as a
- single parameter histograms, or
- two parameter correlated plots
Data may be shown as
Linear scale The scale on which the output is directly
proportional to the input.
Logarithmic scale The scale on which the values
increase logarithmically
Data
Single parameter (Histogram), with dye (PI),
tissue showing DNA content, linear scale
DNA
Count
256
384
256
128
128
384
G
0
G
1
G
2
M
S phase
Two Parameter (Dot Plots), without dye,
lysedperipheral blood, logarithmic scale
Dot plot Density plot Contour plot
Contours as a percentage of the
maximum event number
Contours as a percentage
of the total number of events
Scatter pattern lysed peripheral blood
Forward
Side
Properties of FCM
Chemical properties
Flow cytometry measures fluorescence per cell or
particle
Spectrophotometry measures the percent absorption
and transmission of specific wavelengths of light for a
bulk volume of sample
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 malignancies
3. Monitoring Minimal Residual Disease
4. Determining CD34 counts
5. Reticulocyte Counts
6. Diagnosis of PNH
7. DNA analysis of S-phase fraction
8. Platelet counts
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
Certain issues with FCM
Garbage in garbage out
Background, non specific staining
Antigen expression:
RBCs, WBCs, Platelets, Others
CD3
Cyto CD3
Tdt
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
Leukemia/lymphoma
immunophenotyping
Lysis of red cells
Add reagents
Forward scatter Side scatter
CD45 Side scatter
CD19 Side scatter
Gating (cells of interest)
FSC vs SSC: Dot plot / Scatter plot
Forward
Side
Cells of interest
Forward
Side
Normal peripheral blood
Leukemic peripheral blood
Scatter pattern showing a
single dense cluster
Scatter pattern peripheral blood
Normal peripheral blood
Peripheral blood full of tumor cells
Different patterns on
FS versus SS
Problem when the tumor cells are scanty
1. FSC vs SSC
Peripheral blood - 92% tumor cells
2. CD45 gating
Helps differentiate blasts from lymphocytes
Problem when the tumor cells
are scanty
Cells of interest are few
CD45 gating
CD45 strongest in lymphocytes
CD45 weakest in blasts
CD45 gating means CD45 in each tube
Tracking marker
CD45
Myeloblasts
2. CD45 vs SSC
Peripheral blood - 12% blasts
Guess
1. FSC vs SSC
3. CD19 vs SSC
2. CD3 vs SSC
T and B cells
3. CD19 vs SSC
4. Reverse gating
ISHAGE
5. Sequential gating
CD19+, CD5- B cell lymphoma
6. Multiple gates (6 color IPT)
CD3 - FITC
CD19 - PE
CD13 PerCP
CD45 TR
Multicolor immunophenotyping
Brent Wood et al
Multicolor FCM - issues
Expression of myeloid antigens
CD117APC
CD11bPECy7
CD15FITC
CD45APC Cy7
CD13PE
CD16PECy5
CD3 FITC
Diagnostic Hematopathology
Morphology
H&E stain - Biopsy
Giemsa stain Aspirate
Cytochemistry (MPO, NSE)
Immunohistochemistry, Flow cytometry
Cytogenetics, FISH
Molecular methods
Centers doing management of
hematolymphoid malignancies
IHC/FCM - Must
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
Common CD markers Leukemia lab
CDs: Large number of antibodies are available
Most of the leukocyte surface antigens are lineage
associated, and not specific to a single lineage or stage
of cellular maturation
Lineage specific markers
Blasts, Maturation patterns
Clonality
Minimal panels Guidelines
10 antibodies plus controls for AL
9 antibodies plus controls for CLPD
IJ PM, 2008
B-cell maturation (Brent wood et al)
B-cell maturation (Brent wood et al)
Case
56 year old male with lymphocytosis
Provisional diagnosis: Lymphocytosis
Advise: ?
Provisional diagnosis: Lymphocytosis
Advise:
FCM immunophenotyping Lymphoma panel
Lymph node biopsy
CD45, CD20, CD19, CD5, CD23, CD22wk, CD20,
Kappa LC, CD200
Impression: B-cell lymphoma, CLL
Another case
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
Patterns of antigen expression - Granulocytic maturation
CD45 vs SSC
Normal MDS
CD13 CD16
CD11c CD16
MDS and Hematogones
Pattern analysis
Lymphocytosis
CD10+, CD19+, HLADR+
CALLA,
BL
FL,
DLBCL
Hematogones
Lymph node FNAC and FCM
Lymph node Biopsy and FCM
Primary follicles
IgM+IgD+
Secondary follicles, IgD-
Mantle zone
IgM+IgD+
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
CD
41/61
RBC
Single Platform Immunoplatelet method
Single Platform Bead Assay
Absolute platelet count =
Gated Plt events X Bead count
Gated bead events (fixed value)
Sehgal K, Cytomerty B, Clinical Cytometry, 2010
Lymphocyte subset analysis
CD4/CD8 Counts
Lymphocyte subset
analysis
Normal peripheral blood Lymphocyte gate
For CD4 counts, add CD3
CD4 PE
CD8
FITC
Normal peripheral blood Lymphocyte gate
For CD4 counts, add CD3
PNH studies
Normal Neutrophils
Abnormal Neutrophils
Reticulocyte counts
Minimal residual disease
DNA Ploidy
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 controls
are 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
Minimal residual disease in ALL
(eg., MRD Lite)
Three color Immunophenotyping
5 year old boy with fever 1 month
6 year old girl,
Diagnosis
MPO negative NSE negative
Diagnosis
Immunophenotyping
3 Color FCM
FSC vs SSC
Blasts only
Diagnosis
CALLA- ALL
FL, BL, DLBCL,
Hematogones
MCP 841 protocol
Day 18 BM
Morphology
MRD Lite by flow
Day 18 post induction
MRD lite
Day 18 post induction, bone marrow is
done
No hematogones
CD19, HLADR, CD34, syto 13
Quality control
Instrument setup, compensation, titration/validation, Isotype
(background staining), FMO (spillover)
EQAS/Proficiency Testing
Cell viability
Clinical history, morphology
Panels: adequate, combinations, weak fluorochromes
Signatory
Final report (Positive/negative, intensity, CD45 gating or
FSC/SSC, how many cells gated and studied)
Single/double platform, CVs
strong
dim
crossroads
do not give
percentages
For Referring Oncologist/Pathologist
Discuss with cytometrist, history and choice of panels
Indications and Transportation
Stem cells, CSF, Lymph nodes
Reading the report
Indian data on hematolymphoid
malignancies and guidelines for IPT
Panel selection
ICMR Taskforce
EQAS / PT
Cytometry B Clin Cytometry, 2008
IJ PM, 2009
Leukemia Lymphoma 2009
Leukemia 2009
IJ C, 2010
Final comprehensive report
Morphology
Cytochemistry
Flow cytometry / IHC
Cytogenetics
Molecular diagnostics