Flow cytometry

Meroj A. Jasem

ALI Saad ALbadri Eman Abbas

 Introduction
Flow (noun) = the motion characteristics of fluids.
Cytometry (noun) = is a general name for a group of
biological methods used to measure various parameters
of cells.
Cytometry depend on the basic laws of
physics, including those of fluidics, optics,
and electronics
 Flow cytometry
is a system for sensing individual cells in a
physiologic saline solution as they move in a
focused liquid stream through a fixed laser
beam scattering light and emitting fluorescence
that is measured and converted into digitized
data.
• Principle work of Flow cytometry:
The sample is focused to ideally flow one
cell at a time through a laser beam and the
light scattered is characteristic to the cells
and their components. Cells are often
labeled with fluorescent markers so that light
is first absorbed and then emitted in a band
of wavelengths. Tens of thousands of cells
can be quickly examined and the data
gathered are processed by a computer
 Jv

The first Wallace H. Coulter in 1953 . impedance-based flow

cytometry device, using the Coulter principle(is an instrument for
counting and sizing particles suspended in electrolytes.)
Principle :
A typical Coulter counter has one or more microchannels that separate two
chambers containing electrolyte solutions. As fluid containing particles or cells is
drawn through each microchannel, each particle causes a brief change to the
electrical resistance of the liquid. The counter detects these changes in electrical
resistance
It is used for cells, bacteria, prokaryotic cells and virus particles
 Mack Fulwyler was the inventor of the forerunner to
today's flow cytometers - particularly the cell
sorter. Fulwyler developed this in 1965.

 The first fluorescence-based flow cytometry device (ICP 11)

was developed in 1968 by Wolfgang Göhde from
the University of Münster, filed for patent on 18 December
1968[.

 flow cytometry instruments were developed, including the

Cytofluorograph (1971) from Bio/Physics Systems.

 he first FACS (fluorescence-activated cell sorting) instrument

from Becton Dickinson (1974).
Name of the technology •
The original name of the fluorescence-based
flow cytometry technology was "pulse cytopho-
tometry", based on the first patent application
on fluorescence-based flow cytometry.
Modren Flow cytometers:
Modern flow cytometers are able to analyze many
thousand particles per second, in "real time," and, if
configured as cell sorters, can actively separate and
isolate particles with specified optical properties at
similar rates.
A flow cytometer is similar to a microscope, except
that, instead of producing an image of the cell
flow cytometry offers high-throughput
, automated quantification of specified optical
parameters on a cell-by-cell basis. To analyze
solid tissues, a single-cell suspension must first be
prepared.
 A flow cytometer has five main components:
 a flow cell,
a measuring system,
a detector,
 an amplification system,
and a computer for analysis of the signals
Schematic diagram of a flow cytometer
Measurable parameters

 Apoptosis (quantification, measurement of DNA degradation

, mitochondrial membrane potential, permeability changes, )

 Cell adherence (for instance, pathogen-host cell adherence)

 Cell pigments such as chlorophyll or phycoerythrin

 Cell surface antigens (Cluster of differentiation (CD) markers)

 Cell viability

 Circulating tumor cells: isolation and purification

 Characterising multidrug resistance (MDR) in cancer cells

 Chromosome analysis and sorting (library construction, chromosome

paint)

 DNA copy number variation (by Flow-FISH or BACs-on-Beads technology)

 Enzymatic activity

 Intracellular antigens (various cytokines, secondary mediators,

etc.)

 Nuclear antigens

 total DNA content

 Total RNA content

 What are the advantages / disadvantages?
ADVANTAGE
 User friendly
 Possible standardization
 Simple office technique
 Rapid diagnosis( high speed analyses )
 Flexibility of the data acquisition
 Statistical information immediately available
 Ability to reanalyze with new gate gives us new Information from old
acquisitions
 Internal control .
 Measure single cell and a large number of cell .
 Simultaneous analysis multiple parameters
 Identifies small population
 Portable equipment .
 study heterogeneous populations of cells.
Disadvantage :
 Limited sensitivity
 Need for liquid cell suspension .
 Requires 1 .00000 events acquired by tubes , which often
limits its use in CSF.
 Must carefully choose combination of fluorochrome
conjugate
 Very expensive .
 Requires management by a highly trained specialist and on-
Going maintained by service engineers .

 Complex instrument are porn to problem with the

microfluidics system (blockage ) and also required warm , cleaning.
 Great potential for error in compensation (proper control required )
 It also takes off any debris or dead cells when providing the final data
Principle of Flow cytometry

Asst.Prof.
Meroj A. Jasem
Ph.D. Student
Immunological techniques Course
• There are four general components of a flow cytometer:
• Fluidics
• Lasers
• Optics Detectors
• Detectors
• Electronics
• Understanding how a flow cytometer operates is critical to the design
and execution of flow cytometry experiments.
 Instrument overview

Fluidics
Lasers
Optics
Detectors
Electronics
Fluidics
Lasers
Optics
Detectors
Electronics
Fluidics
Lasers
Optics
Detectors
Electronics
Fluidics
Lasers
Optics
Detectors
Electronics
Fluidics
Lasers
Optics
Detectors
Electronics
Fluidics
Lasers
Optics
Detectors
Electronics
 Core

Sheath

Laminar flow occurs when a fluid flows in

parallel layers, with no disruption between
the layers
Interrogation point
Data presentation
 TUNEL assay
• Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) is a method for detecting DNA fragmentation by
labeling the 3′- hydroxyl termini in the double-strand DNA breaks
generated during apoptosis
• TUNEL is a method for detecting apoptotic DNA fragmentation,
widely used to identify and quantify apoptotic cells, or to detect
excessive DNA breakage in individual cells.[3] The assay relies on the
use of terminal deoxynucleotidyl transferase (TdT),
an enzymethat catalyzes attachment of deoxynucleotides, tagged
with a fluorochrome or another marker, to 3'-hydroxyl termini of DNA
double strand breaks.
• The fluorescein-labeled DNA can then be analyzed by flow cytometry
with Ex/Em 488/520 nm. Propidium iodide is also included in the kit
as a counterstain with Ex/Em 488/623nm.
DNA strand breaks labeling with Br-dUTP utilizing
exogenous terminal deoxynucleotidyl transferase
(TdT)
The methodology is
based on the use of
exogenous terminal
deoxynucleotidyl
transferase (TdT) to
label 3′-OH ends of the
DSBs with
fluorochromes, defined
as the TUNEL assay. This
chapter describes the
variant based on DSBs
labeling using 5-
Bromo-2′-deoxyuridine-
5′-triphosphate
(BrdUTP) as a TdT
substrate.
Thank Thee
 Applications
of Flow cytometry
by:
Eman Abbas Muhsin
Ph.D student ⁄ Microbiology
Applications in clinical laboratories:
DNA content analysis:
Leukocyte analysis:
Platelet analysis:
Apoptosis and chemotherapy:

*A major cause of failure to many of the chemotherapeutic

agents is “multiple drug resistance”
*DNA replication and repair, or other mechanisms such as
cell viability and apoptosis are involved to improve the
diagnosis and biological therapy for a wide variety of
human diseases, including cancer and autoimmune disease.
Applications in Microbiology:

Modern flow cytometry allows single- or multiple- microbe detection

(bacteria, fungi, parasites and viruses) easy, reliable, and fast on the
basis of their cytometric parameters or by means of certain
flourochromes used either alone or bound to specific antibodies.
Future expectations ↗↗↗↗

Lower
Fine
samples cost

Bioinformatics and Single cell level

laser will be applied multiparameters
Thank
You
Questions

• What are the benefits and disadvantages of flow cytometry?

• What are the uses of flow cytometry?
• One of the clinical applications of flowcytometry is ..... , ........ , or/
and .........
• Explain the role of flow cytometry in microbiology .
• What is the principle of flow cytometry in measuring cell
components?
• What is the dynamic fluidics?
• What is the difference between forward and side scatter?