flow cytometry is a laser- or impedance-based, biophysical technology

employed in cell counting, cell sorting, biomarker detection and protein

engineering, by suspending cells in a stream of fluid and passing them through
an electronic detection apparatus. A flow cytometer allows
simultaneous multiparametric analysis of the physical
and chemical characteristics of up to thousands of particles per second.

Flow cytometry is routinely used in the diagnosis of health disorders,

especially blood cancers, but has many other applications in basic research,
clinical practice and clinical trials. A common variation involves linking the
analytical capability of the flow cytometer to a sorting device, to physically
separate and thereby purify particles of interest based on their optical
properties. Such a process is called cell sorting, and the instrument is
commonly termed a "cell sorter"

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 at similar rates having specified optical properties. A flow
cytometer is similar to a microscope, except that, instead of producing an
image of the cell, flow cytometry offers high-throughput, large-scale,
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.
The flow cell has a liquid stream (sheath fluid), which carries and aligns the
cells so that they pass single file through the light beam for sensing. The
measuring system commonly use measurement of impedance (or conductivity)
and optical systems - lamps (mercury, xenon);

The process of collecting data from samples using the flow cytometer is termed
'acquisition'. Acquisition is mediated by a computer physically connected to the
flow cytometer, and the software which handles the digital interface with the
cytometer. The software is capable of adjusting parameters (e.g., voltage,
compensation) for the sample being tested, and also assists in displaying initial
sample information while acquiring sample data to ensure that parameters are
set correctly. Early flow cytometers were, in general, experimental devices, but
technological advances have enabled widespread applications for use in a
variety of both clinical and research purposes. Due to these developments, a
considerable market for instrumentation, analysis software, as well as the
reagents used in acquisition such as fluorescently labeled antibodies has
developed.

Modern instruments usually have multiple lasers and fluorescence detectors.

The current record for a commercial instrument is ten lasers[6] and 30
fluorescence detectors.[7]Increasing the number of lasers and detectors allows
for multiple antibody labeling, and can more precisely identify a target
population by their phenotypic markers. Certain instruments can even take
digital images of individual cells, allowing for the analysis of fluorescent signal
location within or on the surface of cells.

Fluorescence-activated cell sorting[edit]

Fluorescence-activated cell sorting (FACS)

Fluorescence-activated cell sorting (FACS) is a specialized type of flow

cytometry. It provides a method for sorting a heterogeneous mixture of
biological cells into two or more containers, one cell at a time, based upon the
specific light scatteringand fluorescent characteristics of each cell.[26] It is a
useful scientific instrument as it provides fast, objective and quantitative
recording of fluorescent signals from individual cells as well as physical
separation of cells of particular interest. The technique was expanded by Len
Herzenberg, who was responsible for coining the term FACS.[27] Herzenberg
won the Kyoto Prize in 2006 for his seminal work in flow cytometry.

The cell suspension is entrained in the center of a narrow, rapidly flowing

stream of liquid. The flow is arranged so that there is a large separation
between cells relative to their diameter. A vibrating mechanism causes the
stream of cells to break into individual droplets. The system is adjusted so that
there is a low probability of more than one cell per droplet. Just before the
stream breaks into droplets, the flow passes through a fluorescence measuring
station where the fluorescent character of each cell of interest is measured. An
electrical charging ring is placed just at the point where the stream breaks into
droplets. A charge is placed on the ring based immediately prior to
fluorescence intensity being measured, and the opposite charge is trapped on
the droplet as it breaks from the stream. The charged droplets then fall
through an electrostatic deflection system that diverts droplets into containers
based upon their charge. In some systems, the charge is applied directly to the
stream, and the droplet breaking off retains charge of the same sign as the
stream. The stream is then returned to neutral after the droplet breaks off.

The acronym FACS is trademarked and owned by Becton, Dickinson and

Company.[28]

Labels[edit]

Use of flow cytometry to measure copy number variation of a specific DNA sequence
(Flow-FISH)
Fluorescent labels[edit]
Main article: Fluorophore

A wide range of fluorophores can be used as labels in flow

cytometry.[10] Fluorophores, or simply "fluors", are typically attached to an
antibody that recognizes a target feature on or in the cell; they may also be
attached to a chemical entity with affinity for the cell membrane or another
cellular structure. Each fluorophore has a characteristic
peak excitation and emission wavelength, and the emission spectra often
overlap. Consequently, the combination of labels which can be used depends on
the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and
on the detectors available.[29] The maximum number of distinguishable
fluorescent labels is thought to be 17 or 18, and this level of complexity
necessitates laborious optimization to limit artifacts, as well as
complex deconvolution algorithms to separate overlapping spectra.[30] Flow
cytometry uses fluorescence as a quantitative tool; the utmost sensitivity of
flow cytometry is unmatched by other fluorescent detection platforms such
as confocal microscopy. Absolute fluorescence sensitivity is generally lower
in confocal microscopy because out-of-focus signals are rejected by the
confocal optical system and because the image is built up serially from
individual measurements at every location across the cell, reducing the amount
of time available to collect signal.[31]

Quantum dots[edit]

Quantum dots are sometimes used in place of traditional fluorophores because

of their narrower emission peaks.

Isotope labeling[edit]
Main article: Mass cytometry

Mass cytometry overcomes the fluorescent labeling limit by

utilizing lanthanide isotopes attached to antibodies. This method could
theoretically allow the use of 40 to 60 distinguishable labels and has been
demonstrated for 30 labels.[30] Mass cytometry is fundamentally different
from flow cytometry: cells are introduced into a plasma, ionized, and
associated isotopes are quantified via time-of-flight mass spectrometry.
Although this method permits the use of a large number of labels, it currently
has lower throughput capacity than flow cytometry. It also destroys the
analysed cells, precluding their recovery by sorting.[30]

Cytometric bead array[edit]

In addition to the ability to label and identify individual cells via fluorescent
antibodies, cellular products such as cytokines, proteins, and other factors may
be measured as well. Similar to ELISA sandwich assays, cytometric bead array
(CBA) assays use multiple bead populations typically differentiated by size and
different levels of fluorescence intensity to distinguish multiple analytes in a
single assay. The amount of the analyte captured is detected via a biotinylated
antibody against a secondary epitope of the protein, followed by a
streptavidin-R-phycoerythrin treatment. The fluorescent intensity of
R-phycoerythrin on the beads is quantified on a flow cytometer equipped with
a 488 nm excitation source. Concentrations of a protein of interest in the
samples can be obtained by comparing the fluorescent signals to those of a
standard curve generated from a serial dilution of a known concentration of
the analyte. Commonly also referred to as cytokine bead array (CBA).

Impedance flow cytometry[edit]

Impedance-based single cell analysis systems are commonly known as Coulter
counters. They represent a well-established method for counting and sizing
virtually any kind of cells and particles. The label-free technology has recently
been enhanced by a "lab-on-a-chip" based approach and by applying high
frequency alternating current (AC) in the radio frequency range (from
100 kHz to 30 MHz) instead of a static direct current (DC) or low frequency
AC field.[32] This patented technology allows a highly accurate cell analysis and
provides additional information like membrane capacitance and viability. The
relatively small size and robustness allow battery powered on-site use in the
field.

Measurable parameters[edit]
 Apoptosis (quantification, measurement of DNA degradation,
mitochondrial membrane potential, permeability changes, caspase activity)
 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

 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
 Glutathione
 Intracellular antigens (various cytokines, secondary mediators, etc.)

 Membrane fluidity
 Monitoring electropermeabilization of cells

 Nuclear antigens
 Oxidative burst
 pH, intracellular ionized calcium, magnesium, membrane potential
 Protein expression and localization
 Protein modifications, phospho-proteins

 Scattering of light can be used to measure volume (by forward scatter)

and morphological complexity (by side scatter) of cells or other particles,
even those that are non-fluorescent. These are conventionally abbreviated
as FSC and SSC respectively.
 Total DNA content (cell cycle analysis,
cell kinetics, proliferation, ploidy, aneuploidy, endoreduplication, etc.)
 Total RNA content

 Transgenic products in vivo, particularly the green fluorescent

protein or related fluorescent proteins

 Various combinations (DNA/surface antigens, etc.)

Applications
The technology has applications in a number of fields, including molecular
biology, pathology, immunology, virusology,[33] plant biology and marine
biology.[34] It has broad application in medicine especially in transplantation,
hematology, tumor immunology and chemotherapy, prenatal diagnosis,
genetics and sperm sorting for sex preselection. Flow cytometry is widely
applied to detect sperm cells abnormality associated with DNA
fragmentation[35] in male fertility assays.[36] Also, it is extensively used in
research for the detection of DNA damage,[37][38] caspase cleavage
and apoptosis.[39] In neuroscience, co-expression of cell surface and
intracellular antigens can also be analyzed.[40] In marine biology, the
autofluorescent properties of photosynthetic plankton can be exploited by flow
cytometry in order to characterise abundance and community structure. In
microbiology, it can be used to screen and sort transposon mutant libraries
constructed with a GFP-encoding transposon (TnMHA).[41] In protein
engineering, flow cytometry is used in conjunction with yeast
display and bacterial display to identify cell surface-displayed protein variants
with desired properties.

ANNEXIN A5 AFFINITY ASSAY

In molecular biology, an annexin A5 affinity assay is a test to quantify the

number of cells undergoing apoptosis. The assay uses the protein annexin
A5 to tag apoptotic and dead cells, and the numbers are then counted using
either flow cytometry or a fluorescence microscope.[1]

The annexin a5 protein binds to apoptotic cells in a calcium-dependent

manner using phosphatidylserine-containing membrane surfaces that are
usually present only on the inner leaflet of the membrane.

Background[edit]
Apoptosis is a form of programmed cell death that is used by the body to
remove unwanted, damaged, or senescent cells from tissues. Removal of
apoptotic cells is carried out via phagocytosis by white blood cells such as
macrophages and dendritic cells. Phagocytic white blood cells recognize
apoptotic cells by their exposure of negatively charged phospholipids
(phosphatidylserine) on the cell surface.
In normal cells, the negative phospholipids reside on the inner side of the
cellular membrane while the outer surface of the membrane is occupied by
uncharged phospholipids. After a cell has entered apoptosis, the negatively
charged phospholipids are transported to the outer cell surface by a
hypothetical protein known as scramblase. Phagocytic white blood cells express
a receptor that can bind to and detect the negatively charged phospholipids on
the apoptotic cell surfaces. After detection the apoptotic cells are removed.

Detection of cell death with annexin A5[edit]

Healthy individual apoptotic cells are rapidly removed by phagocytes. However,
in pathological processes, the removal of apoptotic cells may be delayed or even
absent. Dying cells in tissue can be detected with annexin A5. Labeling of
annexin A5 with fluorescent or radioactive molecules makes it possible to
detect binding of labeled annexin A5 to the cell surface of apoptotic cells. After
binding to the phospholipid surface, annexin A5 assembles into a trimeric
cluster. This trimer consists of three annexin A5 molecules that are bound to
each other via non-covalent protein-protein interactions. The formation of
annexin A5 trimers results in the formation of a two-dimensional crystal
lattice on the phospholipid membrane. This clustering of annexin A5 on the
membrane greatly increases the intensity of annexin A5 when labeled with a
fluorescent or radioactive probe. Two-dimensional crystal formation is believed
to cause internalization of annexin A5 through a novel process of endocytosis if
it occurs on cells that are in the early phase of executing cell
death.[2]Internalization amplifies additionally the intensity of the annexin A5
stained cell.

Annexin A5 has been used to successively detect apoptotic cells in vitro and in
vivo.[1][3] Pathological processes in which apoptosis occurs include
inflammation, ischemia damage of the heart caused by myocardial infarction,
apoptotic white blood cells and smooth muscle cells present in atherosclerotic
plaques of blood vessels, transplanted organs in the donor patient that are
rejected by the immune system or tumour cells that are exposed to cytostatic
drugs during chemotherapy.
The non-invasive detection of diseased tissue with, for example, radioactively
labeled annexin A5 is the goal of a recently developed line of research known as
Molecular Imaging.

Molecular Imaging of cell death using radioactive annexin A5 can become of

clinical significance to diagnose vulnerability of atherosclerotic plaques
(unstable atherosclerosis),[4]heart failure,[5] transplant rejection,[6] and to
monitor efficacy of anti-cancer therapy.