Cytometry, Simplifying Cell Selection Task
Since its invention,
flow cytometry has enabled scientists to analyze a variety of cell
types. Today, the applications of this technology are even broader and
Flow cytometry is a technique for counting, examining and sorting
microscopic particles suspended in a stream of fluid. It allows
simultaneous multiparametric analysis of the physical and chemical
characteristics of single cells flowing through an optical and/or
electronic detection apparatus.
Early flow cytometers were generally experimental devices, but recent
technological advances have created a considerable market for the
instrumentation, as well as the reagents used in analysis, such as
fluorescently-labeled antibodies and analysis software.
Modern flow cytometers are capable of analyzing several thousand
particles every second, in real time, and can actively separate and
isolate particles having specified properties.
The new flow cytometers usually have multiple lasers and fluorescence
detectors. Increasing the number of lasers and detectors allow multiple
antibody labeling and can more precisely identify a target population
by their phenotype. Certain instruments can even take digital images of
individual cells and allow analysis of fluorescent signal location
within or on the surface of cells.
Fluorescence-Activated Cell Sorting (FACS) has emerged as a specialized
type of flow cytometry. It provides a method for sorting a
heterogeneous mixture of biological cells into two or more containers
based upon the specific light scattering and fluorescent
characteristics of each cell. It has become 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 acronym FACS is
trademarked and owned by Becton Dickinson.
The first fluorescence-based flow cytometry device (ICP 11) was
developed in the year 1968 by Wolfgang Göhde from the
University of Münster, Germany (patent no. DE1815352) and
first commercialized in 1968-69 by German developer and manufacturer
Partec through Phywe AG in Göttingen. At that time absorption
methods were still widely favored by other scientists over fluorescence
methods. The original name of the flow cytometry technology was pulse
cytophotometry. After 10 years, in 1978, at the conference of the
American engineering foundation in Pensacola, Florida, the name was
changed to flow cytometry, a term which quickly became popular.
Subsequently Bio/Physics Systems Inc. introduced flow cytometry
instrument named Cytofluorograph in 1971. In 1973 Partec
introduced PAS 8000. The first FACS instrument from Becton
Dickinson came in 1974. ICP 22 from Partec/Phywe and Epics from Coulter
were introduced in 1975 and 1977-78 respectively.
The use of flow cytometry has increased considerably during the past
decade. The technology has enabled the rapid measurement and analysis
of multiple characteristics of single cells. Flow cytometric DNA has
been found valuable in determining the biological behavior of various
tumors and predicting clinical outcomes. The technology has
applications in a number of fields, including molecular biology,
pathology, immunology, plant biology and marine biology. In the field
of molecular biology it is especially useful when used with
fluorescence tagged antibodies. It has broad application in medicine
especially in transplantation, hematology, tumor immunology and
chemotherapy, genetics and sperm sorting for sex preselection. In
marine biology, the auto-fluorescent properties of photosynthetic
plankton can be exploited by flow cytometry in order to characterize
abundance and community structure. In protein engineering, flow
cytometry is used in conjunction with yeast display and bacterial
display to identify cell surface-displayed protein variants with
Ram Sharma, managing director, BD, said, “We never imagined
that flow cytometry will become core for monitoring CD4. This is now
the most preferred solution for monitoring HIV/AIDS patients. Almost 90
percent of all CD4 monitoring is done using our flow cytometry.
Although the technology already has a lot of clinical applications in
monitoring cancer, HIV, cord blood banking, and stem cells, it has not
been intensely deployed in the areas of drug discovery, life science
research and basic research. Through partnerships with research
institutes, we can increase the scope of our products in the life
science research applications. Our primary aim lies in enhancing our
scope in drug discovery.”
Compared to other technologies, there are few players operating in the
flow cytometry market space, the important ones being Beckman Coulter,
Guava Technologies, Luminex and Dako which are offering products in
India and globally. Talking about market competition, Rama Sharma of BD
said, “Over a period of time, the technology becomes less
competitive. The real difference is the quality of people who are
training and educating the customers and the support that you provide
to your customers. The manufacturers have to keep a constant check on
the efficiency of the systems and probability of new applications for
The flow cytometry market is composed of instruments, reagents,
devices, and services used across the research and clinical life
sciences areas that span the academic and biomedical, biotechnology,
and pharmaceutical business market sectors. According to a market
research report, the global flow cytometry market in 2008 stood at $1.5
billion and is estimated to grow to $3.7 billion by 2015 with estimated
growth rates of existing product. About 68 percent of the 2008 product
revenue has been from instruments, and reagents make up 32 percent of
the revenue. Cell-based flow cytometry is estimated at $1.3 billion
with a CAGR of 10-15 percent in the coming year. Market leaders Becton
Dickinson and Beckman Coulter account for about 70 percent of the
research and clinical areas of the cell-based flow cytometry market.
Bead-based flow cytometry is estimated at $215 million with a CAGR of
25-30 percent while Luminex and partners comprise approximately 90
percent of the bead-based flow cytometry market.
Researchers expects more fluorescent dyes to become available, with
more color options, and also further technical improvements. These
include dyes with much higher stoke shifts and alternatives for tandem
dyes, which often suffer from inherent problems in multi-laser
applications. Upgradation of some existing dyes are in the pipeline,
which will offer increased brightness for conjugated antibodies. It is
also predicted that new dyes will be offered together with new laser
options from flow cytometer manufacturers, with an emphasis on
accessibility. New solid state lasers in digital instruments are
providing options in bench top machines that were previously restricted
to top-end instruments.
Future Belongs to Cell-based Therapies and Flow Cytometry is the
-Sunit Trivedi, director, BD Biosciences, BD India
How is flow cytometry
making a difference to biotechnology research ?
The life science research in the last few years has moved from genomics
and proteomics to single cell analysis. Flow cytometry’s
power of single cell analysis makes this new research possible. Flow
cytometry posses the ability to sort cells with absolute precision in
terms of purity, recovery and viability. It has the power of measuring
everything in the cell including cell surface antigens, membrane
proteins, intracellular proteins (cytokines), signal transduction
pathways, cell signaling, nuclear proteins, apoptosis, DNA cell cycle,
and ploidy analysis and co-relates all these assays to answer critical
questions in healthcare such as stem cell enumeration, leukemia and
lymphoma phenotyping, and HIV monitoring.
The future belongs to cell-based therapies with flow cytometry as its
integral part. “Cytomics” is the
combination of cytometry technology with other cell biology and imaging
How has flow
cytometry contributed to the development of research ?
Flow cytometry has become the central tool. Almost every question can
be answered with flow cytometry. This technology helps to
answer questions at single cell level, probe what it is and then
understand what it is doing. One can also see the working of a cell and
observe how the cell is being affected by a drug. It enables us to
identify samples with many flurochromes at the same time.
Many colors lead to more answers and more answers lead to more
questions. At the end of the day, researchers are getting to a point
where they can do an analysis on rare samples and understand much more
completely what has happened. If you compare cytometry 20 years ago,
with what researchers can do today, you can’t really say
enough about what the technology can do and where you can go with it.
Researchers can do things that they were never able to do before and
that is really because of the speed of computer processing, the
availability of new solid state lasers with more power, more
control over the lasers and the size of lasers. Now researchers can do
17 colors simultaneously because the processing speed of computers lets
researchers collect the data at that speed. The power, lasers and
flexibility let researchers identify large number of cells, using
different flurochromes and different areas of spectrum that were
Flow cytometers can allow measurements of particles as small
as 200nm and it finds applications in marine biology, microbiology,
environmental biology, and nanotechnology. Its capability is used in
applications such as chlorophyll detection and analysis of sperm cells.
Flow cytometry with spectral analyzer capabilities are used in marine
biology applications and could be used to measure fluoroscence energy
transfer in fluorescent protein pairs. Few of the versions also
features special brackets that easily mount the instrument shipboard.
Quick- exchange fluidics with single use sort assemblies allow
researchers to achieve true aseptic conditions and prevent possible
cross-contamination between samples which may lead to therapeutic cell
sorting and effective cell therapy using stem cells, a much needed
breakthrough required in many incurable diseases.
Development in technology has made it possible to build flow cytometers
on fixed alignment with flow cell which reduce startup time, improve
reproducibility, improve sensitivity and efficiency. But the
most important advantage is ease of use that has upgraded flow
cytometry as a user-friendly sorting device which is so often required
for cellular researchers.
A quick and intuitive alignment procedure has been designed
in the system with pin hole camera to bring near optical alignment
within seconds without using beads. Two way and four way sorting can be
optimized for applications in genomics and proteomics. For
instance, cells can be deflected according to their DNA content to
arrange them by the phase in the cell cycle. While mRNA
hybridization to the stripe of cells of increasing DNA content might
serve to study differential gene expression during the cell cycle.
Software improvements play a major role in flow cytometry and make
researcher’s job easier. Softwares like Cytometer Setup and
Tracking (CS&T) automates flow cytometer setup, adjusts
instrument variability and establishes baseline settings. Chances of
operator error are reduced, and results are more consistent. The setup
features let users define a configuration baseline and run a
performance check. The software will then automatically set up the
cytometer to this established baseline. To match the exact baseline,
the CS&T software automatically adjusts reproducible
performance of the instrument. This ensures the greatest possible
consistency from one day to the next, saves time, and improves
reliability of experiment results. The software also allows creation of
application-specific settings for rapid performance of routine
experiments in a more consistent manner.
Instruments can also be customized to meet customer requirements via
the Special Order Research Products (SORP) programs taking care of
special research needs and configurations.
What are some of the
advances that you foresee?
More dyes will take advantage of larger spectra, moving from visible
color spectrum to far infrared to get more information out of a cell.
How can flow
cytometry technology be further promoted?
Education and training are the key. Organizations should open training
centers and collaborate with leading institutions to
facilitate training of larger group to maximize versatile
utility of flow technology.