Basic

Flow Cytometry
Principles






















Presented by: Chrisna Durandt


REVISION STATUS
Version 1.0 (Feb 2008)

COURSE DESCRIPTION
During this course the basic principles of flow cytometry will be discussed and
demonstrated. Commonly used flow cytometry terminology will also be discussed and
explained.

TRADEMARKS
ALTRA, BECKMAN COULTER Logo, COULTER, COULTER CLENZ, CYTO-COMP,
CYTO-TROL, Cytomics, Elite, EPICS, FlowCentre, Flow-Check, Flow-Count,
Flow-Set, Immuno-Brite, ImmunoPrep, Immuno-Trol, IsoFlow,
Multi-Q-Prep, Q-Prep, T-Q-Prep, XL, XL-MCL and Stem-Trol are Trademarks of Beckman
Coulter, Inc.

COURSE DESIGN
Dr. Chrisna Durandt Beckman Coulter South Africa




- i -
BASIC FLOW CYTOMETRY PRINCIPLES
FLOW CYTOMETRY
Flow cytometry is the measurement (meter) of single cells (cyto). Flow cytometry
can be used to simultaneously measure and analyze multiple physical characteristics of
single cells/particles.

Let us look at more DEFINITIONS of flow cytometry:
A method of measuring the number of cells in a sample, the percentage of live cells in a sample, and
certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell
surface. The cells are stained with a light-sensitive dye, placed in a fluid, and passed in a stream
before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts
to the light.
www.dana-farber.org/can/dictionary/
Technique for characterizing or separating particles such as beads or cells, usually on the basis of
their relative fluorescence.
www.combichemistry.com/glossary_f.html

A process in which cell or particle measurements are made while the cells or particles pass,
preferably in single file, through the measuring apparatus in a fluid stream.
www.seagrant.sunysb.edu/BTRI/btriterms.htm

The measurement of cells or cellular properties as they move in a fluid stream past stationary
detectors.
health.enotes.com/nursing-encyclopedia/fetal-cell-screen
Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended
in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical
characteristics of single cells flowing through an optical and/or electronic detection apparatus.
en.wikipedia.org/wiki/Flow cytometry








FLOW CYTOMETRY
A process for measuring the characteristics of
cells or other biological particles as they pass
through a measuring apparatus in a fluid stream.
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BASIC FLOW CYTOMETRY PRINCIPLES

FLUORESCENCE MICROSCOPY & FLOW CYTOMETRY

A flow cytometer can also be described as a high-throughput, automated fluorescence
microscope.

FLUORESCENCE MICROSCOPE FLOW CYTOMETRY



Error!
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SAMPLING AREA
Sample presented on slide.


Produce an image of the cell.

Section of whole tissue can be investigated.

Sample is presented in a liquid stream flowing
through a sensing area.
Does not produce an image of the cell.
Only single cell suspension can be analyzed. To
analyze solid tissues single-cell suspension must
first be prepared.
LIGHT SOURCE
Xenon or mercury-vapor lamp Usually a laser(s), but xenon arc or mercur-vapor
lamps are also used on certain instruments
DETECTION
Human eye
Image might also be projected on computer
screen.
Electronic
OPTICS

DICHROIC mirrors is used on both systems


EMISSION filters are used on both systems

BASIC FLOW CYTOMETRY PRINCIPLES
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ANALYSIS
Objective interpretation. Depending of the
investigators interpretation
Small number of cells analyzed (hundreds)
Slow analysis time 100/sec
Low sensitivity: Only strong protein
expression can be detected
Qualitative results: Cells scored as +/-


Subjective interpretation.

10
3
10
6
cells analyzed. More accurate
High-throughput: 1000 5000 cells/sec
High sensitivity: Weak protein expression can be
clearly identified
Quantitative results: Fluorescence intensity of each
cell individually scored.






















BASIC FLOW CYTOMETRY PRINCIPLES
COMPARISON BETWEEN FLUORESCENCE MICROSCOPY &
FLOW CYTOMETRY RESULTS
As mentioned before the fluorescense intensity of each cell is individually scored. A flow
cytometry plot is usually divided into 1024 channels in which the fluorescence intensity
levels can be displayed. Therefore it can also be explained that 1024 different levels of
fluorescence intensities can be displayed, resulting in excellent sensitivity.

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Increase in fluorescence intensity
FLUORESCENCE
1024 channels
C
O
U
N
T















Each channel represents a specific fluorescence intensity level.
An increase in channel number reflects an increase in fluorescent
intensity.














BASIC FLOW CYTOMETRY PRINCIPLES
Example:
Fibrobrast-like synovial cells (FLS) were transduced with pRET2.ECFP which fluoresces
green. The FLS from patients with rheumatoid arthritis (RA) were transduced with (a) (g)
unconcentrated, (b) (h) 10X concentrated, or (c) (i) 100X concentrated pRET2.EGFP
supernatant. (ac) The percentage of EGFP-positive FLS was determined by flow
cytometry. (gi) Fluorescence microscopy images of cultures following transduction are
shown. These results are representative of data using FLS isolated from 5 RA patients.
Yang et al. Arthritis Res 2002 4:215
Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral
supernatant

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Second peak represents positively stained, green fluorescent cells.
Cells show fluorescent intensity between channel 1 and 100.
Percentages represent % cells that fluoresces green in relation with all cells counted.
First peak represents unstained cells with no or little fluorescence. Therefore low
channel number (0.1 1)

















BASIC FLOW CYTOMETRY PRINCIPLES
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Above-mentioned example also indicates the two main ways flow cytometry results are
reported:
% expression
Mean Fluorescence Intensity (MFI) expressed as mean channel number.

Absolute number of cells (cells/l) can also be obtained using a flow cytometer and will
be discussed later in the course.


WHY IS FLOW CYTOMETRY USED?
Flow cytometry can be used to measure intra- and/or extracellular characteristics of
individual cells. The following information can be obtained for each individual
cell/particle
Size
Complexity / Granularity
percentage of a specific cell population in a sample
absolute number of specific cells/particles in a sample
amount of fluorescence per cell/particle

SAMPLE CRITERIA
The power of flow cytometry is its ability to measure multiple parameters of individual
cells within heterogeneous populations. It is not necessary to purify/isolate cells before
flow cytometry analysis.
The sample should be a single-cell suspension.
The optimal concentration is 2 5 x 10
6
cells/ml and
Most bench-top flow cytometers require at least 0.5 ml of prepared sample for
analysis.
Most flow cytometers are able to measure cells that are between 0.5 m and 40 m
in diameter. Cells, viruses, bacteria, liposomes, etc can be measured using flow
cytometry.




BASIC FLOW CYTOMETRY PRINCIPLES
BASIC PRINCIPLES OF FLOW CYTOMETRY
Flow cytometry uses the principles of light scattering, light excitation, and emission of
fluorochrome molecules to generate specific multi-parameter data from particles. Cells
are hydro-dynamically focused in a sheath of isotonic liquid before intercepting an
optimally focused light source.

HYDRODYNAMIC FOCUSING
In order to make any measurements, you need to have
cells in suspension that flows in single file through the
point of interrogation. This will allow measurements to be
made one cell at a time. The principle of hydrodynamic
focusing is used to achieve this. Sheath fluid flows
through the sensing area known as the flow cell. Within the
flow cell, a slow-moving sample stream is injected into the middle
of the faster moving sheath stream causing the two fluids
(sheath and sample solution), which differ enough in their velocity
and/or density, not to mix. A two-layer stable flow is
formed, with the sheath fluid acting as the wall. By using
the principle of hydrodynamic focusing a much
FOCUSED LASER
smaller sample stream can be created,
down to the micrometer magnitude.
Careful control of the velocity of the two streams allow for accurate control of the width
of the sample stream, aligning the cells to flow in single file past the point of
interrogation.
SHEATH FLUID is a balanced electrolyte solution with very specific characteristics,
including:
Non-fluorescent, allowing improved noise-to-signal ratio
Low background (filtered to 0.2 m)
Bacteriostatic
Fungistatic
Do not alter the characteristics of the sample, i.e pH, protein expression, etc.
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BASIC FLOW CYTOMETRY PRINCIPLES
On most bench-top flow cytometers the operator has the option to select between three
(3) sample flow rates:
Low
Medium
High
The Sample Flow Rate is control by the difference in velocity between the sample and
sheath streams.


SAMPLE PRESSURE =3.72 p.s.i.
FLOW RATE: 10 l/min
Slow analysis time
Increased sensitivity

LOW






SAMPLE PRESSURE =3.92 p.s.i.
FLOW RATE: 30 l/min
MEDIUM





SAMPLE PRESSURE =4.12 p.s.i.
FLOW RATE =60 l/min
Fast acquisition
Decreased sensitivity

HIGH









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BASIC FLOW CYTOMETRY PRINCIPLES
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LIGHT SOURCE
To be able to make measurements there has to be a light source focused on the
interrogation point. On most flow cytometers the light source is a laser.

Laser is an acronym for "Light Amplification by Stimulated Emission of Radiation

A typical laser emits light in a narrow beam and with a well-defined wavelength,
corresponding to a particular color (i.e., monochromatic).

Depending on the flow cytometer, lasers can be inexpensive, air-cooled units or
expensive, water-cooled units. The air-cooled blue argon laser (excitation: 488 nm) is
the most commonly available laser on single laser flow cytometers. On two laser flow
cytometers the second laser is commonly the red helium-neon (HeNe) laser (excitation:
633 nm) or red diode laser (excitation: 635 nm).

Other lasers that are used on flow cytometers are:

UV helium-cadmium (HeCd) laser (325 nm)
UV (HBO) lamp (366 nm) (usually used for DNA work)
violet diode laser (405 nm)
green helium-neon (HeNe) laser (543nm)














BASIC FLOW CYTOMETRY PRINCIPLES
LIGHT SCATTER

As the particles/cells pass the laser, light is scattered in all directions.

Detectors are placed forward of the intersection point as well as perpendicular (90
o
angle
with respect to the laser beam). The detector placed in line with the laser path is known as
the Forward Scatter (FSC) detector. Forward scatter is proportional to cell size; the
bigger the cell, the more light is scattered, the higher the detected signal.

Signal to right of plot.
High channel number.
FSC
FSC
Signal t
Low chann
o left of plot.
el number.
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Laser
Forward Scatter
Detector
Side Scatter Detector
Forward Scatter
Detector






Larger
particle


Smaller
particle

The first detector of a series of detectors placed
perpendicular to the laser path is known as the Side
Scatter (SSC) detector. Side scatter is proportional to
cell complexity/granularity. The more organelles/bits
inside the cytoplasm, the more light scatter, the higher the
detected signal.




BASIC FLOW CYTOMETRY PRINCIPLES
Forward and side scatter may be used to identify cell populations of interest.



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GRANUCLOCYTES:
Increased size, with high
complexity
MONOCYTES:
Intermediate size, with
increased complexity
F
O
R
W
A
R
D

S
C
A
T
T
E
R

SIDE SCATTER
LYMPHOCYTES:
Small , with little
complexity
DEBRIS:
Small in size.
Little complexity
SAMPLE INFORMATION:
Typical, lysed whole blood preparation.
Each dot represents one cellular event.
Although it is possible to analyze platelets
using a flow cytometer, platelets are to
small to be detected using the instrument

The FSC vs SSC plot allows for easy differentiation of small lymphocytes from the larger
monocytes and differentiation of the monocytes from the similarly sized but significantly
more complex neutrophils.







BASIC FLOW CYTOMETRY PRINCIPLES
FLUORESCENCE
Other properties of the cell, such as surface molecules or intracellular constituents, can
also be accurately quantitated if the cellular marker of interest can be labeled with a
fluorescent dye; for example, an antibody conjugated to a fluorescent dye may be used to
bind to specific surface or intracellular receptors. Other dyes have been developed which
bind to particular cellular structures (e.g. DNA, mitochondria) or are sensitive to the local
cellular chemistry (e.g. Ca++concentration, pH, membrane potential, etc.).
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CD4
+
T cell
CD 3
CD 45
CD 4




Depending on the flow cytometer, 3-9 different fluorescent colors may be detected
simultaneously.
Laser
The fluorescent detectors are also placed perpendicular to the laser, after the side scatter
detector. Each detector detects a specific fluorescent wavelength.




Fluorescence detectors
(PMT3, PMT4 etc.)
BASIC FLOW CYTOMETRY PRINCIPLES
The molecules of flourochromes
(fluorescent dyes) are capable to be
excited, via absorption of light
energy, to a higher energy state, also
called an excited state. The process
is known as excitation (). The
energy of the excited state is
unstable and when stimulation of t
fluorescent compound is stopped (by
removing the exciting light source), it quickly adopts a lower-energy excited state, whic
is semi-stable() . Next, the molecules rearrange from the semi-stable excited state ba
to the ground state, and the excess energy is released and emitted as light. This process is
called fluorescence ().
he
h
ck
Fluorescence is always of a lower energy, and hence longer wavelength, than the exciting
light, and this separation in wavelength is known as the Stokes shift. The Stokes shift
enables the exciting and emitted light to be separated by optical filters and therefore the
amount of fluorescence can be quantified.








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BASIC FLOW CYTOMETRY PRINCIPLES
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Knowledge of the excitation wavelength is necessary for the selection of the appropriate
parameters.
Thus if you want to use a certain fluorochrome (fluorescent dye), you need to make sure
it is compatible with the laser in use.
Each fluorochrome (fluorescent dye) has a very specific excitation and emission
wavelength. The excitation wavelength is important for laser selection, while the
emission wavelength is important for filter selection.






The magnitude of the Stokes shift varies between fluorescent molecules, and it is
therefore possible to separate the fluorescence emitted by different molecules excited by
the same light source.
FITC: Fluorescein Isothiocyanate
PE: Phycoerythrin
ECD: Phycoerythrin-Texas Red
PARAMETERS
Signals (information) you want to acquire.

FS (forward scatter)
SS (side scatter)
FL1 (fluorescence 1) usually 525 nm
FL2 (fluorescence 2) usually 575 nm
FL3 (fluorescence 3) usually 625 nm
FL4 (fluorescence 4) usually 675 nm
etc
BASIC FLOW CYTOMETRY PRINCIPLES
NAME EXCITATION
(nm)
APPROPRIATE
LIGHT SOURCE
EMISSION
(nm)
SUITABLE
BAND PASS
FILTER (nm)
Alexa Fluor
*
488 Alexa Fluor
*
488 495 Blue Diode/Argon Laser 520 525
Alexa Fluor
*
647 Alexa Fluor
*
647 650 Red Diode/HeNe Laser 668 675
Alexa Fluor
*
700 Alexa Fluor
*
700 696 Red Diode/HeNe Laser 719 720
AMCA Amino Methylcoumarin Acetic
Acid
354 Hg Arc Lamp or UV
lamp
448 450
APC Allophycocyanin 650 Red Diode/HeNe Laser 660 675
APC-Alexa Fluor
*
700 APC-Alexa Fluor
*
700 650 Red Diode/HeNe Laser 719 720
APC-Alexa Fluor
*
750 APC-Alexa Fluor
*
750 650 Red Diode/HeNe Laser 780 780
CY
*
5 Cyanin 5 580 Red Diode/HeNe Laser 670 675
CY
*
5.5 Cyanin 5.5 630 Red Diode/HeNe Laser 694 700
DAPI 4,6-Diamidino-2-phenylindole 370 Hg Arc Lamp or UV
lamp
455 450
ECD Phycoerythrin-Texas Red
*
480 Blue Diode/Argon Laser 620 620
FITC Fluorescein Isothiocyanate 495 Blue Diode/Argon Laser 520 525
FLMA Fluorescein 5-Maleimide 495 Blue Diode/Argon Laser 520 525
PC5 Phycoerythrin-Cyanin 5 480 Blue Diode/Argon Laser 670 675
PC5.5 Phycoerythrin-Cyanin 5.5 480 Blue Diode/Argon Laser 694 700
COMMONLY USED FLUORESCENT DYES
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BASIC FLOW CYTOMETRY PRINCIPLES
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PC7 Phycoerythrin-Cyanin 7 480 Blue Diode/Argon Laser 767 770
Pacific Blue Pacific Blue 405 Violet/405 nm Diode
Laser
455 450
PE Phycoerythrin 480 Blue Diode/Argon Laser 575 575
PE/CY5 Phycoerythrin-cyanin 5 480 Blue Diode/Argon Laser 670 675
PE/CY5.5 Phycoerythrin-cyanin 5 480 Blue Diode/Argon Laser 694 700
PE/CY7 Phycoerythrin-cyanin 7 480 Blue Diode/Argon Laser 767 770
PI PropidiumIodide 540 Blue Diode/Argon Laser 620 620
RD1 Phycoerythrin 480 Blue Diode/Argon Laser 575 575
Rho 110 Rhodamine 110 496 Blue Diode/Argon Laser 525 525
SPRD SpectralRed
*
(Phycoerythrin-
Cyanin 5)
480 Blue Diode/Argon Laser 670 675
TRITC Tetramethyl Rhodamine
Isothiocyanate
555 Hg Arc Lamp or Green
Laser
580 575
TXRD Texas-Red-x 575 Hg Arc Lamp or Yellow
Laser
620 620
7-AAD 7-Aminoactinomycin D 550 Blue Diode/Argon Laser 660 675
COMMONLY USED FLUORESCENT DYES

BASIC FLOW CYTOMETRY PRINCIPLES
PHOTOMULTIPLIER TUBE
The different wavelenghts (fluorescent colors) are separated by optical filters and directed
to sensors called photomultiplier tubes (PMTs). PMTs are light-sensitive sensors that
convert light energy (photons) into electrical current and generate voltage pulse signals,
by converting photons into electrons through the photoelectric effect. The electrons are
directed towards the electron multiplier, where electrons are multiplied by the process of
secondary emission. The voltage pulses generated rise and fall according to the amount of
light entering the PMTs.
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micro.magnet.fsu.edu
Filter configuration on EPICS XL-MCL
BASIC FLOW CYTOMETRY PRINCIPLES
OPTICAL FILTERS OPTICAL FILTERS
Different types of optical filters are used, each with a very specific function. Different types of optical filters are used, each with a very specific function.
Long pass (LP) filters transmit wavelengths above a cut-on wavelength. For example, a
520 LP filter will allow all light with wavelengths larger than 520 nm trough. All
wavelengths shorter than 520 nm will be blocked out and/or directed to another filter.
Long pass (LP) filters transmit wavelengths above a cut-on wavelength. For example, a
520 LP filter will allow all light with wavelengths larger than 520 nm trough. All
wavelengths shorter than 520 nm will be blocked out and/or directed to another filter.
L Li ig gh ht t S So ou ur rc ce e T Tr ra an ns sm mi it tt te ed d L Li ig gh ht t
> >5 52 20 0 n nm m L Li ig gh ht t
5 52 20 0 n nm m L LP P F Fi il lt te er r

Short pass (SP) filters transmit wavelengths below a cut-off wavelength. For example, a
575 nm SP filter will allow light with wavelengths shorter than 575 nm through. All
wavelengths longer than 575 nm will be blocked out and/or directed to another filter.
Short pass (SP) filters transmit wavelengths below a cut-off wavelength. For example, a
575 nm SP filter will allow light with wavelengths shorter than 575 nm through. All
wavelengths longer than 575 nm will be blocked out and/or directed to another filter.
L Li ig gh ht t S So ou ur rc ce e T Tr ra an ns sm mi it tt te ed d L Li ig gh ht t < < 5 57 75 5 n nm m L Li ig gh ht t
5 57 75 5 n nm m S SP P F Fi il lt te er r

Dichroic filters can be either long pass (LD: long pass dichroic) or short pass (SD: short
pass dichroic) filters. They are placed at a 45
0
angle to the light source, causing the light
that is blocked to be reflected at a 90
0
angle.
Dichroic filters can be either long pass (LD: long pass dichroic) or short pass (SD: short
pass dichroic) filters. They are placed at a 45
0
angle to the light source, causing the light
that is blocked to be reflected at a 90
0
angle.
Filter placed at 45
o

Light Source Transmitted Light


Reflected light


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BASIC FLOW CYTOMETRY PRINCIPLES

Band pass (BP) filters are used to narrow down the amount of light that is transmitted,
allowing only light within a specific wavelength range to pass through the filter. These
filters are usually used to separate the different fluorescence colors according to their
wavelength properties. For example a 525 nm BP filter will only transmit light between
515 nm 535 nm.

T Tr ra an ns sm mi it tt te ed d L Li ig gh ht t L Li ig gh ht t S So ou ur rc ce e
6 62 20 0 - - 6 64 40 0 n nm m

6 63 30 0 n nm m B BP P F Fi il lt te er r


Laser light is very bright and need to be blocked out, to enable emitted fluorescence light
to be detected optimally. A blocking (BK) filter is use for this purpose. The BK filter
completely (99.9%) blocks out light of specified wavelengths, while all other
wavelengths are able to be transmitted. For example, a 457 nm 502 nm BK filter blocks
out wavelengths between 457 nm and 502 nm.












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BASIC FLOW CYTOMETRY PRINCIPLES
HIGH VOLTAGE & GAIN
The voltage pulses generated by the flow cytometer are small and need to be amplified.
The operator is able to manually amplify the pulses by adjusting the
high voltage and/or
gain settings

HIGH VOLTAGE is adjusted to change the sensitivity of the light sensor and is applied
directly to the PMT. This adjustment is used to make small, precise adjustments to the
signal. Increasing the voltage of a specific signal will cause a larger electronic pulse to be
generated, resulting in an increase in channel number.

MFI =500
MFI =0.5
FL3
MFI: Mean Fluorescence Intensity
FL3 voltage
increase i.e from
350 to 450
MFI =600
MFI =0.8 C
o
u
n
t

FL3




C
o
u
n
t





GAIN is the amount of amplification applied to a signal. The signal amplification is done
electronically after the pulses left the PMT. For example, if you increase the gain from
1.0 to 2.0, it will increase the signal by double. Pulses can be amplified 2-, 5-, 10-, 20-,
50- and 75-fold. A single adjustment therefore has a large influence on the signal size and
therefore fluorescent intensity.
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MFI =500
MFI =0.5
FL3
MFI: Mean Fluorescence Intensity
C
o
u
n
t

Adjust gain from
1.0 to 2.0
Signal are doubled
FL3
MFI =5.0
MFI =1000




C
o
u
n
t



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BASIC FLOW CYTOMETRY PRINCIPLES
In flow cytometry, voltages and gains are usually adjusted so that the negative population
within a sample or the isotypic (negative) control falls within the first decade of the plot.









C
O
U
N
T

CD3-FITC
It is very important to note that voltage/gain adjustments have a direct influence on color
compensation settings. Color compensation will covered later in the course. It is
advisable to leave the voltage/gain settings unchanged, while the color compensation is
adjusted.
















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BASIC FLOW CYTOMETRY PRINCIPLES
REGIONS & GATES
Regions and Gates are two of the most common used terminologies in flow cytometry.

REGIONS refer to areas of interest on a plot. A region indicates an area of interest from
which statistics will be generated.

For example:

C is a region indicating the CD4 positive cells. Statistics
for the CD4 positive cells will be generated from a to
b.


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No statistics will be generated for the negative, first peak as
there is no reference points (generated by creating a region)
from where the statistics should be generated.

C
b a
C
O
U
N
T

CD4FITC: FL1

Different types of regions may be created.

The following types of regions may be created on two parameter histograms:
Amorphous (polygonal)







Rectangular



BASIC FLOW CYTOMETRY PRINCIPLES

Quadrant Regions







Only LINEAR regions can be created in single parameter histograms.

C
O
U
N
T

CD3-FITC




















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BASIC FLOW CYTOMETRY PRINCIPLES
Example:


A. A1, A2, A3, A4, B and C are all regions, indicating specific areas
Total CD4
+
Lymphocytes

of interest for which statistics will be generated.

A: Lymphocytes B:
A1: CD4
+
/ CD3
-
Lymphocytes C: Total CD3
+
Lymphocytes
A2: CD4
+
/ CD3
+
Lymphocytes
A3: CD4
-
/ CD3
-
Lymphocytes
A4: CD4
-
/ CD3
+
Lymphocytes
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BASIC FLOW CYTOMETRY PRINCIPLES
A GATE refers to criteria that must be met before an event is included in a histogram.
ion or
ED
A Gate is applied to a specific plot. It is explained in the following diagram.
A region can become a gate in another plot, therefore are the criteria of inclus
exclusion in other plots


UNGAT














A













SIDE SCATTER (SS)
ALL events that passed the laser are
A
Lymphocytes are the area of
interest. Therefore region A
where created around the
lymphocytes.
CD4 FITC: FL1
Refers to criteria of what will be further
n
investigated.
By assigning A to the histogram, it
applies that only what is contained i
region A will be further investigated .
Rest of events will be excluded.
This plot is therefore gated on A
Only lymphocytes is included.


oth CD4 B
-
and CD4
+
events are
present as they are all
lymphocytes
B
B is a region that included all
+
t. CD4 T cells the area of interes
included in the plot.
F
O
R
W
A
R
D

S
C
A
T
T
E
R

(
F
S
)

L
O
G

S
I
D
E

S
C
A
T
T
E
R

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BASIC FLOW CYTOMETRY PRINCIPLES
R
0
20
40
60
80
100
1.00
C
o
u
n
t

Volts
Channels
CHANNEL COUNT
509 0
510 15
511 40
512 100
513 45
514 10
515 0
ESULTS & STATISTICS
meter (histogram) or two-parameters plots. The
he converted pulse for each channel is counted.








tatistics are generated within area indicated.
Results may be presented in single-para
first decade (channel 0 1 (10
0
) usually represents the negative population.














CD3-FITC
C
O
U
N
T

SINGLE-PARAMETER PLOT
A single parameter is used on the x-axis.
CD3 positive
Negative for CD3
The y-axis reflects the number of cells
counted in each channel.

T








S

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BASIC FLOW CYTOMETRY PRINCIPLES
COUNT: Represents all events (cells) counted within a specific area. If we use the
illustration above it can be explained as all the events counted from
channel 509 515.

MODE or PEAK COUNT: Is the channel with the highest number of events. In the
illustration above, it is channel 512.

MEAN: Refers to the Mean Fluorescence Intensity, therefore the average of all the
fluorescent intensity levels within a specific area.

MEDIAN: Refers to the fluorescent intensity level (channel) which allows 50% of the
events at either side.







50% 50%
Mean
50% 50%
Uneven distribution of
events, therefore the
mean and median
differs.
Median
Because the distribution is
symmetric the mean and median,
will be similar.








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BASIC FLOW CYTOMETRY PRINCIPLES


Results may also be presented as a two-parameter plot (dot-plot).


TWO-PARAMETER PLOT


A parameter is plotted on the x- and y-
axis respectively.
Statistics are generated for both the x-
and y-axis.







A1: CD4
+
/ CD3
-
Lymphocytes
A2: CD4
+
/ CD3
+
Lymphocytes
A3: CD4
-
/ CD3
-
Lymphocytes
A4: CD4
-
/ CD3
+
Lymphocytes










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BASIC FLOW CYTOMETRY PRINCIPLES
DISCRIMINATOR
A channel setting for a parameter that lets you ignore events below the setting. This lets
you eliminate signals caused by debris.












Discriminator
Events below discriminator
ignored
Side Scatter
F
o
r
w
a
r
d

S
c
a
t
t
e
r

Please note that a wrong discriminator, may cause partial or total lost of cells of interest.



Part of lymphocyte
population lost
Discriminator










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BASIC FLOW CYTOMETRY PRINCIPLES
COLOR COMPENSATION
The emission curves of the various fluorescent dyes overlap. This is referred to as
Spectral Overlap. The main aim of the process of color compensation is to separate out
the respective emission curves, therefore decreasing the overlap.


























480 500 520 540 560 580 600 620 640 660 680 700 720 740






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BASIC FLOW CYTOMETRY PRINCIPLES
The use of Band Pass filters placed in front of each of the fluorescent detectors assists in
the segregation of the different signals, but is unable to prevent all of the spectral overlap.
















480 500 520 540 560 580 600 620 640 660 680 700 720 740

525
BP
575
BP
625
BP

675
BP


Color compensation must be performed to ensure signals are indicating their true source
and are not as a result of an overlapping fluorescent signal.










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BASIC FLOW CYTOMETRY PRINCIPLES
The overlapping signal (interference signal) may cause

Negative populations to be positive



FALSE POSITIVE
POPULATION due to
incorrect color
compensation
Positive populations to present brighter fluorescence intensity than that is true














FALSE INCREASE IN
FL2 INTENSITY due to
incorrect color
compensation

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BASIC FLOW CYTOMETRY PRINCIPLES
FL1
F
L
2

FL1
F
L
2

UNCOMPENSATED COMPENSATED


















Colour Compensation is an electronic subtraction of the signals originating from the

fluorescence photo multiplier tubes (PMTs) which enables the correction of the
overlapping fluorescent signals.












There are two options:

FL1 FL2

FL2 FL1

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BASIC FLOW CYTOMETRY PRINCIPLES





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L2 is
hat causes FL2 to be more p e?
.
and should be subtracted.













F more positive than it should be.

W ositiv

It only can be FL1 in a FL1 vs FL2 plot

FL1 is therefore the interference signal

Therefore it is FL2 FL1
FL1
F
L
2

Lets compensate the GREEN population
first.

Is it ?
FL1
FL1 FL2
FL2














BASIC FLOW CYTOMETRY PRINCIPLES
The correct adjustment of the colour compensation may be assisted by use of the
atistics, in this example, the Y-mean in quadrants B3 and B4 as shown above. When the
Y-mean is equal in quadrants B3 and B4, then this indicates the correct compensation
ust also be visually
hecked. You can clearly see when a population is under or overcompensated

st
value has been set.



Be careful to depend too much on the numbers. Color compensation m
c





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