FLOW CYTOMETRY; BASIC PRINCIPLES

AND APPLICATIONS IN HAEMATOLOGY

A SEMINAR PRESENTATION
BY
ADEBAYO GANIYAT OLAITAN
INTERN, MEDICAL LABORATORY SCIENTIST
HAEMATOLOGY DEPARTMENT
UNIVERSITY OF ILORIN TEACHING HOSPITAL, ILORIN

SUPERVISED BY: MRS OLUTUNDE

1
JUNE, 2022
 OUTLINE
• INTRODUCTION
• PRINCIPLE OF FLOW CYTOMETRY
• TYPES OF FLOW CYTOMETRY
• COMPONENTS OF A FLOW CYTOMETER
• DATA ANALYSIS
• QUALITY CONTROL
• APPLICATIONS
• LIMITATIONS
• CONCLUSION
• REFERENCES
2
 INTRODUCTION
• Flow cytometry is a technique used to simultaneously detect and measure physical and
chemical characteristics of a population of cells or particles as they flow in a fluid stream
through a beam of light.

• Each cells or particles is analysed for visible light scatter and relative fluorescence intensity.

• Visible light scatter is measured in two different directions, the forward direction (Forward
Scatter or FSC) which can indicate the relative size of the cell.

(McKinnon, 2018) 3
 INTRODUCTION CONT'D
• And at 90° (Side Scatter or SSC) which indicates the internal complexity or
granularity of the cell.

• Most commonly analysed samples include;

Bone marrow aspirate
Blood
Lymph node suspension
Body cavities and
Solid Tissue

(Adan et al., 2017) 4

 INTRODUCTION CONT'D

Dot plot showing the light scattered profile of lysed whole blood (Bajgelman, 2019).
5
 PRINCIPLE OF FLOW CYTOMETRY
• The basic principle of flow cytometry involves the passage of cells in single file in front
of a highly focused extremely bright beam of monochromatic light from a laser.

• These cells components are fluorescently labelled and are excited by the laser to emit
light at varying wavelengths.

• Several detectors are then carefully placed around the stream at the point where the fluid
passes through the light beam to measure the light scatter and fluorescent intensity

(Chelkhar and Panda, 2020) 6

PRINCIPLE OF FLOW CYTOMETRY (CONT’D)

The underlying working principle of a flow cytometer

(Adan et al., 2017) 7
DIAGRAM OF A FLOW CYTOMETER

Bioaster, 2018
8
 TYPES OF FLOW CYTOMETRY
• There are two different types of flow cytometry named the non-sorting and
sorting type

• Non-sorting type can perform light scattering and fluorescence emission while
the sorting type has the ability to sort particles as well.

(Adan et al., 2017)

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 COMPONENTS OF A FLOW CYTOMETER
• A flow cytometer is made up of three components

Fluidics
Optics
Electronics

(Kussick, 2018)
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 FLUIDICS SYSTEM
• The purpose of fluidics is to transport particles in a fluid stream to the laser beam for
interrogation.

• The design of the flow chamber allows the sample core to be focused in the centre of the sheath
fluid where the laser beam then interacts with the particles.

• Focusing is achieved by injecting the sample suspension into the centre of a sheath liquid
stream. The flow of the sheath fluid moves the particles and restricts them to the centre of the
sample core.
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 OPTICS SYSTEM
• The optical system of the cytometer consists of excitation optics and collection optics. The
former are used to shape and focus the laser beam to the flow of the sample.

• The collection optics collect light emitted after the particle interacts with the laser beam
and divert the specified wavelengths of the collected light to designated optical detectors.

• After the laser light passes through a cell or particle, the rays emitted and fluorescence
signals are detected by the photomultiplier tubes, or photodiode which will collects the
signals.
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(Kussick, 2018)
 ELECTRONICS SYSTEM
• The electronic system converts the signals from the detectors into digital signals that can be
read by a computer and a software.

• Once the light signals strike one side of the PMT or the photodiode, they are converted into a
relative number of electrons that are multiplied to create a more significant electrical current.

• The electrical current moves to the amplifier and is converted to a voltage pulse. The Analog-to-
Digital Converter (ADC) then converts the pulse to a digital number.

(Anupama, 2022)
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Schematic overview of a flow cytometer (Anupama, 2022) 14
 DATA ANALYSIS CONT’D
•The information obtained after the passage of samples

in a flow cytometer may be stored in the form of;

monoparametric histograms wherein fluorescence

intensity versus the number of cells is counted

Dot plot which is a two parameter graphical

representation of a cell population

(Virgo and Gibbs, 2012) 15

 DATA ANALYSIS
• The major principles of data analysis are to selectively quantify the cells of
interest and to find out more about those cells. This method is called ‘‘gating’’
in flow cytometry

• A gate is a numerical or graphical boundary that can be used to define the

characteristics of cells to include for further analysis.

(Jahan-Tigh et al., 2012)

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 QUALITY CONTROL
• To assure that a flow cytometer provides consistent results, optical and
fluorescent standards are run daily by using Commercially-available calibration
beads and software

• Normal controls must be tested periodically to ensure accurate sample

processing and proper performance of the flow cytometer

(Henel and Schmitz, 2017) 17

 APPLICATIONS OF FLOW CYTOMETRY

• Diagnosis of hematological malignancies through technique called immunophenotyping in

which cells can be studied for expression of several surface molecules

• Quantification and analysis of reticulocyte and platelet

• Detection of Minimal Residual Disease

• It can also be employed in the detection of HLA alloantibodies

(Chelkhar and Panda, 2020) 18

 APPLICATIONS OF FLOW CYTOMETRY CONT’D

• The different stages of cell death, apoptosis, and necrosis can be detected based on

the differences in the morphological and biochemical changes.

• Sensitive detection and accurate quantitation of Fetal Hemoglobin

• Measuring Residual White Cells in Leukocyte-Reduced Blood

• Immunologic monitoring of HIV infected patients

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APPLICATION OF FLOW CYTOMETRY CONT’D

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 LIMITATIONS
• The laser can only analyse one cell at a time so cells must always be in
suspension to be analysed.

• Cells must be viable before it can be analysed.

• Data analysis can become very complicated and relies almost exclusively on
gating by a human expert

(Virgo and Gibbs, 2012) 21

 CONCLUSION
Flow cytometry been a powerful techniques for correlating multiple
characteristics on a single cell, has emerged to be the supreme diagnostic tool for
disease surveillance. Due to its great potential, the use of flow cytometry has been
expanded to diverse fields of various health areas and is routinely used in clinical
diagnostics, biotechnology and applied research.

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 REFERENCES
Adan, A., Alizada, G., Kiraz, Y., Baran, Y. and Nalbant, A. (2017). Flow cytometry: basic
principles and applications. Critical reviews in biotechnology, 37(2);163-176.

Anupama, S. (2022). Flow Cytometry-Definition, Principle, Parts, Steps, Types, Uses

Bajgelman, M. (2019). Principles and applications of flow cytometry. In Data Processing

Handbook for Complex Biological Data Sources, 119-124.

Bioaster (2018). Flow cytometer. https://www.bioaster.org/bioasteracquiredthenew-bd-

facsymphony- flow- cytometer. Accessed June 2022 23
 REFERENCES
Chelkar, M. and Panda, S. (2020). Flow cytometry: Principle and applications.

Henel, G. and Schmitz, J. (2017). Basic theory and clinical applications of flow
cytometry. Laboratory Medicine, 38(7);428-436.

Jahan-Tigh, R., Ryan, C., Obermoser, G. and Schwarzenberger, K. (2012). Flow cytometry. The
Journal of investigative dermatology, 132(10);1.

Keohane, E., Otto, C. and Walenga, J. (2019). Rodak's Hematology-E-Book: Clinical

Principles and Applications. Elsevier Health Sciences. 24
 REFERENCES
Kussick, S. (2018). Flow cytometric principles in hematopathology. In Hematopathology,
686-711.

McKinnon, K. (2018). Flow cytometry: an overview. Current protocols in

immunology, 120(1);5-1.

Virgo, P. and Gibbs, G. (2012). Flow cytometry in clinical pathology. Annals of clinical

biochemistry, 49(1);17-28.

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YOU

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