3. Basic mechanism
Biological sample
Label it with a fluorescent marker
Cells move in a linear stream through a focused light
source (laser beam)
Fluorescent molecule gets activated and emits light
that is filtered and detected by sensitive light detectors
(usually a photomultiplier tube)
Conversion of analog fluorescent signals to digital
signals
4. Flow Cytometry
This method allows the quantitative and
qualitative analysis of several properties of cell
populations from virtually any type of fresh
unfixed tissue or body fluid.
The properties measured include a particle’s
related size, relative granularity or internal
complexity, and relative fluorescence intensity
Most commonly analyzed materials are:
blood,
bone marrow aspirate and
lymph node suspensions.
5. Principle of Flow Cytometry
Flow cytometer is composed of three main
components:
The Flow system (fluidics)
Cells in suspension are brought in single file past
The Optical system (light sensing)
a focused laser which scatter light and emit
fluorescence that is filtered and collected
The Electronic system (signal processing)
emitted light is converted to digitized values that
are stored in a file for analysis
6. The Flow System
One of the fundamentals of flow cytometry is the ability
to measure the properties of individual particles, which
is managed by the fluidics system.
When a sample is injected into a flow cytometer, it is
ordered into a stream of single particles.
The fluidic system consists of a FLOW CELL (Quartz
Chamber):
Central channel/ core - through which the sample is
injected.
Outer sheath - contains faster flowing fluid k/a
Sheath fluid (0.9% Saline / PBS) , enclosing the
7. Hydrodynamic Focusing
Once the sample is injected
into a stream of sheath fluid
within the flow chamber, they
are forced into the center of
the stream forming a single
file by the PRINCIPLE OF
HYDRODYNAMIC
FOCUSING.
'Only one cell or particle can
pass through the laser beam
at a given moment.'
8. • The sample pressure is always higher than the
sheath fluid pressure, ensuring a high flow rate
allowing more cells to enter the stream at a given
moment.
• High Flow Rate - Immunophenotyping analysis of
cells
• Low Flow Rate - DNA Analysis
Sheath
Tank
Waste
Tank
Line Pressure
Vacuum
Sample
Pressure
(Variable)
Sheath
Pressure
(Constant)
Sample
Tube
9. OPTICS
After the cell delivery system, the need is to excite the
cells using a light source.
The light source used in a flow cytometer:
Laser (more commonly)
Arc lamp
Why Lasers are more common?
They are highly coherent and uniform. They can be easily
focused on a very small area (like a sample stream).
They are monochromatic, emitting single wavelengths of light.
ARGON Lasers - 488nm wavelength (blue to blue
green)
10. When a light intersects a laser beam at the so called
'interogation point' two events occur:
a) light scattering
b) emission of light (fluorescence )
Fluorescence is light emitted during decay of excited
electron to its basal state.
11. OPTICS
a) LIGHT SCATTER
When light from a laser interrogates a cell, that cell
scatters light in all directions.
The scattered light can travel from the interrogation point
down a path to a detector.
12. OPTICS - FORWARD SCATTER
(FSC)
• Light that is scattered in the forward direction
(along the same axis the laser is traveling) is
detected in the Forward Scatter Channel.
• The intensity of this signal has been attributed to
cell size, refractive index (membrane
permeability).
13. OPTICS - SIDE SCATTER
(SSC)
Laser light that is scattered at 90 degrees to the axis of
the laser path is detected in the Side Scatter Channel.
The intensity of this signal is proportional to the amount
of cytosolic structure in the cell (eg. granules, cell
inclusions, etc.) Side scatter detector
Measuring cell granularity
16. The light scattered in the forward direction is
proportional to the square of the radius of a sphere, and
so to the size of the cell or particle.
The cells are labelled with fluorochrome-linked
antibodies or stained with fluorescent membrane,
cytoplasmic or nuclear dye.
17. Commonly used Fluorochromes
FLUOROCHROMES EMISSION
MAXIMUM
Fluorescein Isothiocynate (FITC) 530nm
Phycoerythrin (PE) 576nm
Peridin-chlorophyll alpha complex
(PerCP)
680nm
Allophycocyanin (APC) 660nm
Texas red 620nm
ECD( PE - Texas Red Tandem) 615nm
PC5 (PE - cyanin 5 dye tandem) 667nm
18. Optics
B) EMISSION OF FLUORESCENT LIGHT
(FLUORESCENCE)
As the fluorescent molecule present in or on the particle
is interrogated by the laser light, it will absorb energy
from the laser light and release the absorbed energy at
longer wave length.
Emitted photons pass through the collection lens and are
split and steered down specific channels with the use of
filters.
Emitted fluorescence intensity is proportional to the
19. Optics- Filters
Different wavelengths of light are scattered
simultaneously from a cell
Need to split the light into its specific wavelengths in
order to measure and quantify them independently.
This is done with filters.
The system of filters ensures that each photodetector
receives light bands of various wavelengths.
Optical filters are designed such that they absorb or
reflect some wavelengths of light, while transmitting
others.
Types of filters
1. Long Pass 2. Short Pass
3. Band Pass 4. Dichroic
20. Optics- Long Pass Filters
Transmit all wavelengths greater than specified
wavelength
Example: 500LP will transmit all wavelengths greater
than 500nm
400nm 500nm 600nm 700nm
Transmittance
Original from Cytomation Training Manual
21. Optics- Short Pass Filter
Transmits all wavelengths less than specified
wavelength
Example: 600SP will transmit all wavelengths less
than 600nm.
400nm 500nm 600nm 700nm
Transmittance
Original from Cytomation Training Manual
22. Optics- Band Pass Filter
Transmits a specific band of wavelengths
Example: 550/20BP Filter will transmit wavelengths
of light between 540nm and 560nm (550/20 = 550+/-
10, not 550+/-20)
400nm 500nm 600nm 700nm
Transmittance
Original from Cytomation Training Manual
23. Optics- Dichroic Filters
Long pass or short pass filters
Placed at a 45º angle of incidence
Part of the light is reflected at 90º , and part of the light is
transmitted and continues.
Dichroic Filter
Detector 1
Detector 2
24. OPTICS - DETECTORS
The photodetectors convert the photons to electrical
impulses.
Two common types of detectors used in flow cytometry:
Photodiode
used for strong signals, when saturation is a potential
problem (eg, forward scatter detector).
Photomultiplier tube (PMT)
more sensitive than photodiode but can be destroyed
by exposure to too much light.
used for side scatter and fluorescent signols.
25. ELECTRONICS
The electronic subsystem converts photons to
photoelectrons.
Measures amplitude, area and width of photoelectron
pulse.
It amplifies pulse either linearly or logarithmically and
then digitalizing the amplified pulse.
27. Data Analysis- Plot Types
There are several plot choices:
Single Color Histogram
Fluorescence intensity (FI) versus the number of cells
counted.
Two Color Dot Plot
FI of parameter 1 versus FI of Parameter 2
Two Color Contour Plot
Concentric rings form around populations. The more
dense the population, the closer the rings are to each
other
Two Color Density Plot
Areas of higher density will have a different color than
29. DATA ANALYSIS - GATING
Gating is in essence electronic window that sets
upper and lower limits on the type and amount
of material that passes through.
Selection of only a certain population of cells
for analysis on a plot.
Allows the ability to look at parameters specific
to only that subset.
30. Interpretation of Graphs
An important tool for evaluating data is the dot
plot.
The instrument detects each cell as a point on
an X-Y graph. This form of data presentation
looks at two parameters of the sample at the
same time.
31. Three common modes for dot plots
are:
Forward scatter (FSC) vs. side scatter (SSC)
To look at the distribution of cells based upon size &
granularity
Single color vs. side scatter
To visualize the expression of the fluorescence of the
cells
Two-color fluorescence plot.
To differentiate between those cells that express only one
of the particular fluorescent markers, those that express
neither, and those that express both.
used to discriminate dead cells from the live ones that
are expressing the desired fluorescence.
32. When to say an antigen is positive
or negative?
A sample that has some
cells single positives for
CD8 along the x-axis
(green arrow)
some single positives for
CD4 along the y-axis (red
arrow).
Upper right quadrant of the
plot - cells positive for both
fluorescent markers
(purple arrow).
Lower left quadrant - cells
negative for both markers
(orange arrow).
33. How to differentiate dim & bright
expression of an antigen?
Dim : cells are
present more towards
the origin(0) on x(red)
- y axis (pink)
Bright : cells are
present away from
the origin(0) on
x(green) & y(yellow)
axis.
DIM
BRIGHT
Y-axis
CD4
X-axis
CD8
34. WHAT IS UNIQUE IN
FLOWCYTOMETRY
MULTIPARAMETRIC
RAPID ANALYSIS OF LARGE NUMBER OF
CELLS
INFORMATION AT A SINGLE CELL LEVEL
DETECTION OF RARE CELL POPULATIONS
ALLOWS PHYSICAL ISOLATION OF CELLS
OF INTEREST
43. How to differentiate between
Normal and Neoplastic B
cells
1) Imunoglobulin light
chain class restriction.
2) Aberrant antigen
expression.
MONOCLONALITY CD 13, CD 33, CD 5 ON B
CELLS
44. Normal, polyclonal B-cells are a
mixture of kappa-B-cells and
lambda-B-cells.
A B-cell carries either kappa- or
lambda-light chain on its surface. And
normal polyclonal B-cells are a mixture
of kappa-B-cells and lambda B-cells as
can be seen in the left-hand figure.
Monoclonal mature B-cells are either
kappa or lambda.
If a malignant B-cell clone proliferates
this will result in a B-cell population
consisting of either only kappa- or only
lambda-B-cells. The latter case (i.e.
lambda-monoclonal B-cells) is
symbolized in the left-hand figure.
45. Expression of CD5
The arrow in the right panel
points to the abnormal,
strong expression of CD5 by
B-cells. CD5 expression as
strong as this can usually
only be found on T-cells.
Normal B-cells show no or
only a weak expression of
CD5 (left-hand panel)
Weak expression of CD20
The B-cells in the right panel
show only a weak
expression of CD20 (arrow).
For comparison: normal
CD20 expression in the left-
hand panel.
47. Chronic lymphocytic leukemia
Typical phenotype: CD20 (d), CD22 (d), sIg
(d), CD23+
FMC-7-
Characteristic morphology
Testing for the prognostic markers CD38 and
ZAP-70 can be considered
48.
49. Mantle cell lymphoma
Variable phenotype not typical for CLL;
often CD20 (i), sIg (i), CD23-, FMC-7 +
IHC : Cyclin-D1
FISH : t(11;14)/CCND1 rearrangement
50.
51. Hairy cell lekaemia
Typical pheotype: CD20 (b), CD22 (b), CD11c
(b),
CD25+, CD103+, sIg (i)
Confirm characteristic morphology of a hairy
cell and TRAP +
A small subset of HCL are CD10+ but are
morphologically similar to CD10- HCL.
52.
53. Follicular lymphoma
Usually bcl-2, CD43.
Some follicular growth.
t(14;18)/BCL-2 rearrangement.
D/D
1. DLBCL : diffuse growth pattern against the
nodular growth pattern in FL
2. BL : morphologial (vacuoles), High S phase
fraction.
54.
55. 80/F
c/o cervical adenopathy
On CBC : an absolute
lymphocytosis ≥5 ×
109/L;
PBF is flooded with
small mature
lymphocytes with
condensed chromatin
and scant cytoplasm
along with numerous
smudge cells.
CASE
57. Not that simple
A certain immunophenotype may be typical but
is by no means obligatory.
The significance of one marker depends on
the expression of other markers.
The strength of antigen expression is
important.
58. Flow cytometric approach to the diagnosis
and classification of T- cell lymphoid
neoplasms.
T CELL LYMPHOMA
59. Finding abnormal T/ NK cells.
1) Search for
monoclonal T cells
2) T cells with aberrant
antigen expression.
CD4/CD8 Ratio
Loss of CD3
Overexpression of CD5
60. Normally, the CD4/CD8-T-
Cell ratio in peripheral
blood is about 2:1.
In a T-lymphocytic leukemia
this ratio can shift
dramatically. Unfortunately,
this ratio may also be altered
by many non-malignant
diseases. eg viral infections.
Therefore, only extreme
alterations of this ratio can
be regarded as a sign for T-
lymphocytic malignancy.
61. CD4/CD8 coexpression
In the right-hand dot-plot you can
see cells that express both the CD4
and the CD8-antigen (arrow) which
is highly irregular. In addition both
antigens are expressed weakly
(compared to normal T-cells). Left-
hand panel shows a normal
situation.
Loss of CD3
Overexpression of CD5
In the right-hand dot-plot you can
see T-cells which overexpress CD5
while they lack CD3 (arrow). Only a
few normal T-cells are present.
(blue oval). Left-hand panel shows
a normal situation.
62. PROBLEMS IN DIAGNOSIS OF T-
CLPD
Relatively low incidence.
5-25% of all lympoid neoplasms.
Clinico-biological heterogeneity.
Lack of distinctive genetic markers.
68. Cell cycle analysis
The percentage of the cells in each region is
analyzed. In normal tissues –
95% cells - G0/G1 phase
2.5% cells - S phase
2.5% cells - G2/M phase
In neoplasm, percentage of cells in S and G2/M
phase increases which is expressed as S phase
fraction or the proliferation index
69. S Phase, synthesis phase.
It is the part of cell cycle in which DNA is
replicated occurring between G1phase and G2
phase.
70. S phase has strong correlation with
grading.
DNA ploidy has no correlation with
grading.
ADVANTAGES OVER IHC
73. STEPS
Finding the blast population
Defining the immunophenotype
Diagnosis
74. A malignant blast population
may be detected because of
Increase of
immature cells
Abnormal marker
expression of
immature cells
CD38 / CD45 AGAINST
SSC
CD19, 7 on non lymphoid
cell
75. Finding immature
cells using CD45-
CD34 dot-plots
The arrows points at the
blast populations, which
is very conspicuous in
case AL 1 (upper right)
and AL 3 (lower right).
In case AL 2 (lower left),
the difference between
the normal picture is
more subtle and the
blasts may be missed
because in this case the
blasts are CD34
negative.
77. Finding immature
cells using CD45-
Side Scatter dot-plots
The three cases of
acute leukemia: The
arrows point to the
blast populations which
are clearly visible in all
three cases.
Even the blasts of case
AL 2 can easily be
spotted.
79. Example of an abnormal antigen
expression on myeloid blasts
Compare the normal blasts (upper
dot-plot, blue oval) with those of an
acute myeloid leukemia (lower dot-
plot, red oval)): the malignant
blasts abnormally express CD15
and they show an increased
expression of CD34.
Note: CD34-negative cells have
been removed for reason of clarity.
80. DIAGNOSIS
WHICH ONES TO IMMUNOPHENOTYPE?
1. Equivocal morphology
2. Cytochemistry is noncontributory
3. Specific subtypes
LEUKAEMIA VS NON LEUKAEMIA
1. Overlapping morphology. Eg: hematogones, viral
infections.
2. Partially treated acute leukaemia
81. PROGNOSTIFICATION
CYTOGENIC AND MOLECULAR
ABNORMALITIES
• Association with specific cytogenic
abnormalities
• DNA ploidy
RESIDUAL DISEASE MONITORING
82. CLASSIFICATION
Acute leukaemia is classified on the basis of
immunological markers into
B lineage ALL
T lineage ALL
Acute myeloid leukaemia
Acute leukaemia of ambiguous lineage
83. How to define the lineage of
leukaemia
THE FLOW CYTOMETRIC EVALUATION OF HEMATOPOIETIC
NEOPLASIA Brent L. Wood, Michael J. Borowitz. Henry’s, 22nd edition,
Chapter 34
85. How to diff ALL from NHL
CD 34
TdT
Bcl2
CD99
NHL cases with spillover demonstrate bright
CD45 expression while it is moderate in B
ALL.
86. Subtypes of ALL
Flow cytometric immunophenotyping does not
provide a suitable surrogate tool for detection
of these subtypes of ALL.
Subtype
ALL
HLA-
DR
TdT CD 10 CD 19 SmIg CyCD79
a
Pro- B ALL +/- + - + - +
Common
ALL
+ + + + - +
Pre B ALL + - - + - +
Mature B
ALL
- - - + +K/L +
103. CASE
29yrs/ F
O/E : Fever, Pallor, Gum
hyperplasia,
Hepatosplenomegaly.
CBC : Hb-5.2 g%, Plt-
19,000/cu.mm
PBF : shows blasts and dual
differentiation to
granulocytes and
monocytoid cells (large cells,
abundant pale blue
cytoplasm, lobulated or
indented nucleus with
variable nucleoli).
DLC
Blasts40 P8 L10 Monocytoid41
E1
104. red - dim CD45and low
side scatter.
Positive for - CD13,
cyMPO, CD34, HLA-
DR, CD33, CD11c
Negative for - CD10,
CD19, CD3, CD79a.
Blue - bright CD45 &
moderate side scatter.
Positive for - CD14,
CD11c, CD13.
Negative for - CD34,
cyMPO, CD3, CD10,
CD19
ACUTE MYELOMONOCYTIC LEUKEM
105. Hematogones
Physiologic precursors of maturing B-cells.
Confused with neoplastic immature lymphoid
cells of B lymphoblastic leukemia/ lymphoma
or B-ALL.
Increased in:
Autoimmune or congenital cytopenias
Solid organ tumors e.g. neuroblastoma
AIDS
NHL
Post-chemotherapy and after BMT
Copper deficiency
106. Morphologically, hematogones resemble
lymphoblasts.
Hematogones can be differentiated from
lymphoblasts by
Unique Immunophenotypic pattern :
CD34 < TdT < CD20 < PAX5
Variable CD10 & CD20
"J shaped trail pattern" : on CD10/20 Dot plot
112. At diagnosis the tumour burden is
approximately 10^12 leukaemic cells.
Induction chemothereapy achieves a 3 log cell
kill bringing it down to 10^9 leukaemic cells.
Light microscopy of BMA can detect leukaemia
only when there are more than 5 blasts/ 100
nucleated cells. Anything less than that is
termed remission.
Introduction
113. Introduction
What is Minimal residual disease or MRD?
It is that submicroscopic disease that cannot be
detected by conventional light microscopic
examination of the BMA.
It could be as high as 1 billion leukaemic cells.
It can be performed by two techniques: FCM &
PCR.
114. Used mainly in -
1. Acute leukaemia for guiding thereapy as well
as prognostic purposes.
2. Patients with low grade B cell malignancies
undergoing high dose chemotherapy.
3. Post stem cell transplant and
immunothereapy.
4. Lymphoma spillover.
115.
116. REFERENCES
THE FLOW CYTOMETRIC EVALUATION OF
HEMATOPOIETIC NEOPLASIA Brent L. Wood, Michael
J. Borowitz. Henry’s, 22nd edition, Chapter 34
ATLAS AND TEXT OF HEMATOLOGY. Dr Tejinder
singh
Manual: 6th Advanced TCS Flowcytometry workshop on
hematological malignancies.
Flow Cytometry in Hematopathology. A visual approach
to data analysis and interpretation. Doyen, Lawrence
and Raul.
Flow Cytometric Analysis of Leukemia and Lymphoma -
The Basics Univ.Doz.Dr.med. Wolfgang Hübl
