An introduction to flow cytomatry

1. MOHAMED NIJAS V
SEMESTER 7
I.S.P.

2. INTRODUCTION
• A flow cytometer is an optical diagnostic device which is used in
research and clinical laboratories for disease profiling by measuring
the physical and/or chemical characteristics of cells.
• also suitable for rapid and sensitive screening of potential sources
of deliberate contamination, an increasing source of concern of
bioterrorism.
• also emerges as a powerful technique for agriculture research and
livestock development.

3. WHAT IS CYTOMETRY???
• The term cytometry refers to the measurement of physical and/or
chemical characteristics of cells or, in general, of any biological
assemblies.
• In flow cytometry, such measurements are made while the cells, the
biological assemblies, or microbeads (as calibration standards) flow in
suspension, preferably in a single file, one by one, past a sensing
point.
• The sensing is conducted by using an optical technique where the
beam from a light source interacting with each individual cell, a bio
assembly, or a microbead produces scattering or fluorescence.
• The optical response is used to determine cellular features and
organelles, providing counts and ability to distinguish different types
of cells in a heterogeneous population

4. A LITTLE MORE…
• Another name used for flow cytometry and thus applied
interchangeably is fluorescence-activated cell sorting (FACS), which
emphasizes the utilization of fluorescence detection and the ability
of the instrument to sort cells that meet specific measured criteria.
• In a regular microscope with point detection (such as in a confocal
microscope),the light (laser) beam moves (scanned) to detect and
image cells, but the cells are moving (flowing in a single file) in a
flow cytometer.

5. THE COMPONENTS OF A FLOW
CYTOMETER
• Light Source. A flow cytometer may use a single excitation wavelength or a
number of excitation wavelengths from different laser sources.
• Flow Cell. The flow cell is designed to hydrodynamically focus the sample
• Illumination Optics. Optical elements between the laser and the sample are
referred to as illumination optics and are used to shape and focus the laser
beam.
• Collection Optics. The collection optics consists of a train of optical ports to
separate and collect various optical responses produced by illumination of each
flowing cell.
• Detection and Electronics. In most flow cytometers, the photodetectors used for
flourescence detection are photomultiplier tubes.
• Cell Sorter. The two types of cell sorting devices used in flow cytometry are (a)
electrostatic sorting, (b) mechanical sorting

6. LIGHT SOURCE
• A flow cytometer may use a single excitation wavelength or a
number of excitation wavelengths from different laser sources.
• The laser beams can be coaxial or separated so that one or more
interrogation point occurs.
• The critical requirements for a laser in flow cytometry are power
stability, high-quality beam characteristics, and low-level high-
frequency noise.
• Currently used ones are compact solid-state lasers.
• Another new prospect is the use of near IR lasers (700–800nm) to
excite IR dyes, whereby the problem due to autofluorescence
interference is considerably reduced.

7. FLOW CELL
• The flow cell is designed to hydrodynamically focus the
sample stream.
• A cell suspension is introduced through a core inlet which
has an inner diameter of 20mm and is surrounded by a
larger (~200mm) stream of flowing saline (sheath liquid).
• Therefore,in this arrangement,a core stream of the cell
suspension is injected into the center of the sheath stream.
• During the flow,the sheath fluid produces hydrodynamic
focusing of coaxial flow of the core fluid, whereby the two
streams maintain their relative positions and do not mix
significantly and move at the velocity of the sheath fluid.

8. • The hydrodynamic focusing produces the flow of cells in a
single file (one cell at a time).
• The core and sheath streams are driven by syringe pumps
or by sources of pressure that deliver a known volume of
sample per unit time with minimum pulsation.
• From the sample flow rate, one can easily derive the cell
count per unit volume.

9. ILLUMINATION OPTICS
• Optical elements between the laser and the sample are referred to as
illumination optics and are used to shape and focus the laser beam.
• The beam shaping optics most frequently used in current flow cytometers
utilize a pair of anomorphic prisms or two crossed cylindrical lenses that
provide an elliptical spot of 10– 20mm in dimension parallel to the
direction of cell flow and 60mm in dimension perpendicular to the flow
dimensions.
• .The elliptical beam provides a wider illumination field across the width of
the flow
• At the same time, the smaller (20mm) dimension of the elliptical beam
parallel to the flow direction allows cells to pass in and out of the light
illumination quickly and avoids simultaneous

10. COLLECTION OPTICS
• The collection optics consists of a train of optical ports to separate
and collect various optical responses produced by illumination of
each flowing cell.
• These optical responses are (i) forward scattering count (FSC), (ii)
side scattering count (SSC),and (iii) various fluorescence signals at
different wavelengths collected at 90° to the laser beam.
• The number of optical responses detected (hence the number of
photodetectors used) define what is known as the number of
measured parameters in a flow cytometer.

11. DETECTION AND ELECTRONICS.
• In most flow cytometers, the photodetectors used for flourescence detection are
photomultiplier tubes.
• An electrical preamplifier following the photodetector is required because a dc offset
voltage that establishes zero baseline is used to account for steady-state stream
fluorescence.
• This stream fluorescence is the result of fluorochromes remaining in solution.
• Subsequent amplifiers can be either logarithmic or linear. The logarithmic amplifier allows
one to process signals over a wide range of intensities, while a linear amplifier restricts
sensitive measurements to signals in a small linear range.
• The logarithmic amplifiers also have an “offset”control to select an intensity range to be
analyzed without changing the amplification.

12. CELL SORTER
• Most commercial flow cytometers are not equipped with a cell
sorter capability.
• In the electrostatic sorting device, the cells of a specific type,
after passing the interrogation point, are charged and
electrostatically deflected to a collection point.
The electrostatic sorting method can be
operated at rates up to 50,000 cells per second. The necessary
time delay to trigger the vibrating nozzle and the charging collar
can be determined from the flow rate of the cells.
• The second method utilizes a mechanical gate that swings back
and forth to direct a particular type of cell into a desired
pathway. While this method is considered to be more gentle on
cells ,it has only a maximum rate of 300 sorted cells per
second.

13. BASICS OF FLOW CYTOMETRY
Fluorescence
labelling of biological
particles
Hydrodynamic
focusing to produce
single file flow of
these biological
particles
Light (laser)
illumination of
individual biological
particles
Sorting of biological
cells
Multiparameter
detection of optical
response
Optical response
generated by
interaction between
laser beam and and
biological particles
Data acquisition and
processing

14. APPLICATIONS
 HIV monitoring
 Leukaemia or lymphoma
immunophenotyping
 Organ transplant monitoring
 DNA analysis for tumour ploidy and SPF
 Primary and secondary immunodeficiency
 Hematopoietic reconstitution
 Paroxysmal nocturnal haemoglobinuria
 Multiplexing immunoassays
 Multiparameter immunophenotyping
 Measurement of intracellular cytokines
 Signal transduction pathways
 Cell cycle analysis
 Measuring cellular function
 Multiplexing oligonucleotide assays
 Measuring gene expression
 In situ hybridization
 Drug discovery
Clinical Applications
Research Applications
Molecular Flow Cytometry

15. IMMUNOPHENOTYPING
• Identification of cells using fluorochrome conjugated antibodies
as probes for proteins (antigens) expressed by cells.
• The reasons for using the term immunophenotyping are twofold
(I) it relates to the activities of immunological
species, namely, antibodies
(Ii) it is primarily used to identify lymphoid and hematopoietic
cells, which are constituents of immune
systems.
• So it deals with classification of normal or abnormal white blood
cells according to their multiparameter surface antigen
characteristics, which can then be used as a profile for a specific
disease or malignancy.
• Hiv immunophenotyping is another common clinical application
of flow cytometry.

16. FUTURE DIRECTIONS
• Flow cytometry is a rapidly expanding field worldwide where an
enormous increase in its capability can be expected over the
coming years.
• There has been renewed interest in flow cytometry from the point
of view of research where a major impetus is derived from its
applications to genomics and proteomics.
• Recent advances in solid-state lasers, microfluidics, microarray
technology, micro-optics, and miniaturized detectors provide
challenging technological opportunities for developing small and
compact flow cytometers with enhanced capabilities to
simultaneously monitor many more parameters than currently
possible.
• It is refuelling the expectation that perhaps a flow cytometer-on-
a-chip is not such a distant dream.

17. REFERENCES
• Introduction To Biophotonics : P.N. Prasad
• Castro, A., Fairdield, F. R., and Shera, E. B., Fluorescence Detection and
Size Measurement of Single DNA-Molecules, Anal. Chem. 65, 849–852
(1993).
• Givan,A. L.,Flow Cytometry:First Principles,2nd edition,Wiley-Liss,New
York,2001.