Flow cytometry
INTRODUCTION
What is Flow Cytometry?
Flow cytometry is a laser-based analytical technique used to:
o Identify
o Count
o Analyze
o Sort cells or particles
suspended in a fluid as they pass one by one through a laser beam.
o Why is it called “Flow” cytometry?
Because cells flow in a narrow stream inside the
instrument and are analyzed individually.
o Simple definition :
Flow cytometry is a technique that analyzes physical
and chemical characteristics of cells as they flow in
suspension through a laser beam.
Principle of flow cytometry
The principle of flow cytometry is based on the passage of cells in a
single-file stream through a focused beam of laser light.
Core Principle:
Cells are:
➢ Suspended in fluid
➢ Passed single-file through a laser
➢ Light interaction is measured
➢ Data is converted into digital signals
Cells are first suspended in a liquid medium and introduced into
the flow cytometer
As each cell passes through the laser beam, the light interacts with
the cell and produces scattering and fluorescence signals
When laser hits the cell:
Three things happen:
• Forward Scatter (FSC) → cell size
• Side Scatter (SSC) → cell granularity/complexity
• Fluorescence emission → presence of specific markers
These signals are collected by detectors and converted into
electrical impulses.
The electrical signals are processed by a computer and displayed
as graphs, allowing the identification and characterization of
different cell populations.
Example:
• Lymphocyte → small FSC, low SSC
• Neutrophil → high SSC (granules)
FLUIDICS SYSTEM & HYDRODYNAMIC
FOCUSING
The principle of flow cytometry is based on the passage of cells
in a single-file stream through a focused beam of laser light.
The fluidics system is responsible for transporting cells through
the flow cytometer. It ensures that cells pass through the laser
beam one at a time to avoid overlapping signals.
Hydrodynamic focusing is a key concept in flow cytometry. In
this process, the sample fluid containing cells is injected into a
faster-flowing sheath fluid. The sheath fluid surrounds the
sample fluid and narrows it into a thin central stream.
This controlled flow ensures accurate detection and measurement
of each individual cell
OPTICAL SYSTEM, LIGHT SCATTER &
FLUORESCENCE
The optical system of a flow cytometer consists of
lasers, lenses, optical filters, and detectors. The laser
serves as the light source that illuminates the cells.
When laser light strikes a cell, forward scatter occurs due to
light diffracted in the same direction as the laser beam.
Forward scatter provides information about cell size.
Side scatter occurs due to light scattered at a right angle and
reflects the internal complexity or granularity of the cell.
Fluorescence occurs when fluorescent dyes attached to
antibodies are excited by the laser and emit light of a longer
wavelength. This emitted light is detected and used to
identify specific cellular components
FLUORESCENT ANTIBODIES &
IMMUNOPHENOTYPING
Flow cytometry uses monoclonal antibodies tagged with
fluorescent dyes to detect specific antigens on the surface
or inside cells. These antigens are commonly known as
cluster of differentiation, or CD markers.
Each monoclonal antibody binds specifically to a particular
antigen, allowing precise identification of cell types. For
example, CD4 markers identify helper T lymphocytes,
while CD8 markers identify cytotoxic T lymphocytes.
This process of identifying cells based on antigen
expression is called immunophenotyping and is
widely used in the diagnosis of hematological
malignancies.
DATA ANALYSIS, GATING & CELL SORTING
Flow cytometry uses monoclonal antibodies tagged with
fluorescent dyes .
The data generated by flow cytometry is displayed using
histograms and dot plots. Histograms represent a single
parameter such as fluorescence intensity, while dot plots
represent two parameters simultaneously.
Gating is a critical step in data analysis. It involves selecting
specific cell populations based on their characteristics while
excluding debris and unwanted cells.
Some flow cytometers are capable of fluorescence-activated cell
sorting, or FACS. In this process, cells are physically separated
into different containers based on their fluorescence
characteristics.
CLINICAL APPLICATIONS
It is essential in the diagnosis and classification of leukemias
and lymphomas. It is also used for monitoring immune
status in HIV patients by measuring CD4 T-cell counts.
In addition, flow cytometry is used in transplant medicine,
autoimmune disease evaluation, and research studies
involving apoptosis and cell cycle analysis .
ADVANTAGES & LIMITATIONS
Advantages
• Rapid analysis (thousands of cells/sec)
• High sensitivity
• Multiparametric analysis
• Quantitative & qualitative
Limitations:
• Expensive equipment
• Requires skilled personnel
• Cells must be in suspension
• Limited tissue architecture information