Measuring properties of cell (sample) as they flow in a fluid suspension across an illuminated light path.

1. FLOW CYTOMETRY (FCM)
Amer Ali Khaleel
M.SC in Medical Immunology
Hawler Medical University

2.  Measuring properties of cell (sample) as they flow
in a fluid suspension across an illuminated light
path.
FLOW CYTOMETRY

3. 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.

4. FLOW CYTOMETRY
• The properties measured include a particle’s
relative size, relative granularity or internal
complexity (all these without stain), and
relative fluorescence intensity.

5. SAMPLES
Most commonly analyzed materials are:
 Blood.
 Bone marrow aspirate.
 lymph node suspensions.

7. Permeabilized (ethanol) is used for enter stain inside the sample

8.  Most important step is optimization for any
procedure.
 Everything dye or fluorescent should be in dark
place.
 Contain 8 controls.
 1-laser 2-filter 3-mirror 4- detectors

9. PRINCIPLE OF FLOW CYTOMETRY
• Flow cytometer is composed of three main components:
1-The Flow system (fluidics) brain of flow cytometry
Cells in suspension are brought in single file past.
2-The Optical system (light sensing)
A focused laser which scatter light and emit fluorescence that
is filtered and collected.
3-The Electronic system (signal processing)
Emitted light is converted to digitized values that are stored
in a file for analysis.

10. 1-FLUIDICS
The fluidic system consists of a Flow
cell (Quartz Chamber):
Central core - through which the
sample is injected.
Outer sheath - contains faster flowing
fluid i.e. Sheath fluid, enclosing the
central core.

11. 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.'

12. WHAT HAPPEN IF TWO CELLS PASS
TOGETHER.
 1- data not appear accurate on plot.
 2- error in the result.
 3- your procedure in not correct.
 4- your obstruct the system.

13.  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

14. 2-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.

15. • When a light intersects a laser beam at the so
called interrogation point two events occur:
a)Light scattering. = size
b)Emission of light. = fluorescence

16.  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.
A) LIGHT SCATTER

17. 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).

18. 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,…).
Side scatter detector
Measuring cell granularity

19. Figure: Light scattering.
FSC is proportional to size while SSC is proportional to cell
granularity or internal complexity.
More granules = more scatter

20.  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.
B) EMISSION OF LIGHT (FLUORESCENCE)

21. OPTICS : 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.

22. TYPES OF FILTERS
1. Long Pass: Transmit all wavelengths greater than
specified wavelength.
2. Short Pass: Transmits all wavelengths less than
specified wavelength.
3. Band Pass: Transmits a specific band of wavelengths.
4. Dichroic: Long pass or short pass filters.

23. OPTICS : DETECTOR
• The photodetector
convert the photons to
electrical impulses.
• Two common types of
detectors used in flow
cytometry:
• Photodiode.
• Photomultiplier tube
(PMT)

24. OPTICS : DETECTOR
1-Photodiode
used for strong signals, forward scatter detector.
2-Photomultiplier tube (PMT)
• Used for side scatter and fluorescent signals.
• More sensitive than photodiode but can be destroyed by
exposure to too much light.

25. 3-ELECTRONICS
• The electronic system converts photons to photoelectrons, It
measures the amplitude of the photoelectrons pulse and
coverts it into voltage pulse.
• The process involves signal shaping, amplification, and
conversion from analog to digital format.
• The voltage pulse is assigned a digital value by the Analog-
to-Digital Converter (ADC).
• The voltage pulse is transferred to the computer and the light
signal is then displayed in an appropriate position on the plot.

26. DATA ANALYSIS- PLOT TYPES
Contour Plot Density Plot
Greyscale Density Dot Plot
Histogram
both

27. DATAANALYSIS - 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.

28. GATING
 Isolate populations of interest.
 Gating an area will make your analysis more specific.
 Can remove dead cells and debris.
 Cannot discriminate between cells with the same
scattering properties.

29. 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.

30. 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).

31. COMPENSATION
 In cytometry, compensation is a mathematical
correction of a signal overlap between the
channels of the emission spectra of
different fluorochromes.

32. APPLICATIONS
Flow cytometry is used to perform several procedures
including:
1. Cell sorting.
2. Cell counting.
3. Cell cycle analysis.
4. Studies on chromosome.
5. It can used for leukocyte characterization.

33. WHAT IS UNIQUE IN FLOWCYTOMETRY
1. Multiparametric.
2. High statistical power.
3. Information at a single cell level.
4. Detection of rare cell populations.
5. Rapid analysis of large number of cells.
6. Allows physical isolation of cells of interest.

34. LIMITATIONS
1. Aggregates or debris can give false results.
2. Expensive and needs highly-trained technicians.
3. Can not tell the intracellular location and distribution of
proteins.
4. Pre-treatment of the cells for fluorescent staining is time-
consuming • samples such as tissue or cells in culture
have to be treated to separate cells.

35. 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

37. SUMMARY

38. THANK YOU