Flow Cytometry Buying Guide

Flow Cytometry Buying Guide image
1. Introduction
     1.1 What is Flow Cytometry?
     1.2 Clinical Applications of Flow Cytometry
     1.3 Research Applications of Flow Cytometry
2. Flow Cytometry Technology
     2.1 The Flow Cytometer
           2.1.1 Fluidics System
           2.1.2 Optics System
           2.1.3 Electronics System
     2.2 Data analysis
     2.3 Application Specific Technology
           2.3.1 Cell Sorting
           2.3.2 Imaging Flow Cytometry
3. Key Considerations for Choosing a Flow Cytometer
     3.1 Technological Considerations
     3.2 General Considerations
     3.3 Software Considerations
     3.4 Considerations Summary
4. Fluorochrome Selection
5. Future of Flow Cytometry
6. Summary

Buying a new flow cytometer is a huge investment, and with the variety and diversity of flow cytometers on the market, it is often difficult to ensure you purchase the instrument that best fits your needs.

In addition to providing information on the technology and applications of flow cytometry, this guide highlights key considerations for purchasing new instrumentation and reagents in order to help you make the right decision when choosing new flow cytometry equipment.


Introduction

1.1 What is Flow Cytometry?

Flow cytometry is a powerful technique that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Flow cytometry, which can be abbreviated to FCM, is widely used for analyzing expression of cell surface and intracellular molecules, characterizing and defining different cell types in heterogeneous cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence (Figure 2, Section 2.1).

Although cells (mammalian, plant, algae, yeast, bacteria) are usually analyzed, other particles, such as nuclei, chromosomes or small beads, can also be studied using flow cytometry. Some organisms, such as marine algae, are inherently fluorescent but, in general, the fluorescence arises from different labels. Fluorescent chemicals may be used to label cell components, such as DNA, directly; others are attached to antibodies against a wide variety of cellular proteins.

Over the past 30 years flow cytometry has become an indispensible tool for many scientific researchers and clinicians. It has applications in a number of fields including cell biology, drug discovery, cancer research, neuroscience, stem cell research, pathology, immunology, hematology, plant biology, food science and marine biology. As the technology continues to evolve, it remains a relevant and sophisticated platform for the development of new applications in the clinical laboratory, as well as in basic and clinical research.

1.2 Clinical Applications of Flow Cytometry

Flow cytometry has become an important component in the diagnosis and monitoring of patients with a diverse array of diseases. While the basic principals underlying flow cytometry remain relatively unchanged, the technology and array of available reagents have continued to evolve, thus expanding the list of applications in laboratory medicine.

One of the most common applications performed on the cytometer is immunophenotyping. This technique identifies and quantifies populations of cells in a heterogeneous sample - usually blood, bone marrow or lymph. All normal cells express a variety of cell surface markers, dependent on the specific cell type and degree of maturation. However, abnormal growth may interfere with the natural expression of markers resulting in overexpression of some and under-representation of others. Flow cytometry can be used to immunophenotype cells and thereby distinguish between healthy and diseased cells. It is therefore unsurprising that today immunophenotyping is one of the major clinical applications of flow cytometry, and is used to assist the diagnosis of myelomas, lymphomas and leukemias.

Flow cytometry is important for the detection of minimal residual disease (MRD). MRD is defined as disease beyond the limit of morphological detection using conventional microscopy. Flow cytometric methods can detect far lower levels of disease, which can be important for monitoring the effectiveness of clinical treatments and the clinical management of diseases such as leukemia. In leukemia residual tumor cells are detected using immunofluorescence of surface markers.

In a clinical laboratory flow cytometry often plays an import role in solid organ transplant procedures. For example, flow cytometry can be used to crossmatch a recipient's serum with donor lymphocytes to detect antibodies that could interfere with engraftment. After the organ transplant, analysis of the peripheral blood lymphocytes may help to indicate early rejection and bone marrow toxicity during immunosuppressive therapies, and to help in the differentiation of infections from transplant rejection. A variety of cell surface markers and activation antigens can be used depending on the clinical condition and the organ transplanted.

Other current clinical applications of flow cytometry include detection of autoantibodies, diagnosis of immunodeficiency disorders, monitoring of HIV infection, measuring the efficacy of cancer chemotherapy, quantifying fetal maternal hemorrhage, stem cell and reticulocyte enumeration, platelet counting and quality control. With the continual development of new kits and technology, the number of applications for the clinical laboratory will continue to rise.

1.3 Research Applications of Flow Cytometry

The ability to simultaneously measure multiple parameters on a cell by cell basis is probably the most powerful aspect of analytical flow cytometry. This allows research scientists to use flow cytometry for a wide range of applications, with many institutes having a dedicated flow cytometry core. Research applications of flow cytometry include:

Cell Cycle Analysis - One of the most common applications of flow cytometry is measurement of the DNA content of cells. In this way different treatments can be assessed for their effect on cell cycle distribution, for example, treatment with a drug or transfection with a gene of interest. The technique can easily be applied to both mammalian and yeast cells, although due to their much smaller size and DNA content, yeasts show less defined phases of the cell cycle. There are many different fluorochromes that can be used to determine DNA content, each having differing excitation and emission wavelengths. Many manufacturers offer a wide range of fluorescent dyes to allow accurate cell cycle analysis in either live or fixed cell populations.

Protein Expression Analysis - Flow cytometry can accurately quantitate reporter gene expression (for example, Green Fluorescent Protein, GFP) in each cell in a population being transfected. Co-transfection of a reporter plasmid and a reference plasmid can be very variable in normal human cells, making interpretation difficult in reporter gene assays. However, using flow cytometry, reporter and reference plasmid expression can both be quantitated at the single-cell level, even in cases of low transfection efficiency. In addition, heterogeneity in reporter expression and transient effects in gene expression can be examined.

Apoptosis - Apoptosis, or programmed cell death, is a normal part of the life cycle of eukaryotic cells. Cells die for a variety of reasons: through necrosis, brought on by external physical and chemical changes to the cell or through apoptosis, a process in which cells initiate a "suicide" program through internally controlled factors. These two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells. Apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple points, providing many opportunities to evaluate the activity of the proteins involved. The initiator and effector caspases are particularly good targets for detecting apoptosis in cells and their detection by flow cytometry have become widespread.

Cell Proliferation Assays - Cell proliferation assays are widely used in cell biology to measure cellular metabolic activity in response to stimuli such as growth factors, cytokines and other media components. The flow cytometer can measure proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). When the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide, half of the original dye is passed on to each daughter cell. By measuring the reduction of the fluorescence signal, researchers can calculate cellular activation and proliferation. Successful cell proliferation analysis by dye dilution requires sensitive instrumentation and an extremely bright dye to accurately distinguish fluorescently labeled cells from autofluorescence after several cell divisions.

Intracellular Calcium Flux - Cells interact with each other and their environment through signal transduction pathways. When these pathways are activated, membrane-bound calcium ion channels pump calcium into the cell and rapidly increase the intracellular calcium concentration. The cytometer can monitor the flux of calcium into the cell and measure the extent to which cells respond to the stimuli. The most common method is to use the UV-excited dye Indo-1, which fluoresces at a different wavelength when it is bound to calcium than when it is not. By measuring fluorescence at two different wavelengths and by ratioing the two signals, a flux can be seen.

Cell Sorting - If a cell or particle can be specifically identified by its physical or chemical characteristics, it can be separated using a cell sorter. A cell sorting cytometer interrogates and characterizes each cell as it passes through the laser. The sorter then uses sophisticated electronics and fluidics to identify and separate the cells of interest from the fluidic stream into a collection vessel.

Flow Cytometry Technology

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. A flow cytometer is made up of three main systems (Figure 2). The fluidics system transports particles in a sample stream to the interrogation point and takes away the waste (Figure 1). The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. The electronics system converts the light signals from the detectors into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect particles.

2.1.1 The Fluidics System

The fluidics system within the flow cytometer transports particles in a fluid stream to the laser beam for interrogation (Figure 1). When a sample in solution is injected into a flow cytometer, the particles are randomly distributed in three-dimensional space. For accurate data collection, it is important that the particles or cells are passed through the laser beam one at a time. Most flow cytometers accomplish this by injecting the sample stream into a stream of faster flowing sheath fluid within the flow chamber. The flow of the sheath fluid accelerates the particles and restricts them to the center of the sample core such that a single stream of particles is created. This process is known as hydrodynamic focusing (Figure 1). Operators can establish the flow characteristics of the fluid using the mathematical equation, Reynolds Number (Re).

Hyrdodynamic focussing

Figure 1. Hydrodynamic focusing within the fluidics system of the flow cytometer

2.1.2 The Optics System

After the process of hydrodynamic focusing, each particle passes through one or more beams of light (Figure 2). Light scattering or fluorescence emission (if the particle is labeled with a fluorochrome) provides information about the particle’s properties. The laser and the arc lamp are the most popular light sources in modern flow cytometry.

Lasers produce a single wavelength of light (a laser line) at one or more discreet frequencies (coherent light). Arc lamps tend to be less expensive than lasers and exploit the color emissions of an ignited gas within a sealed tube. However, this produces unstable incoherent light of a mixture of wavelengths, which needs subsequent optical filtering.

Light scattering occurs when a particle deflects incident laser light. Light scattered in the forward direction, typically up to 20° offset from the laser beams axis, is collected by the forward scatter channel (FSC) (Figure 2). The magnitude of forward scatter is roughly proportional to the size of the cell, and this data can be used to quantify that parameter. Light scattered at larger angles is known as side scatter. The side scatter channel (SSC) provides information on cell granularity or internal structural complexity.

Fluorescent measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules such as DNA and cytokines (Figure 2). The argon ion laser is most commonly used in flow cytometry. This is because the 488 nm light that it emits excites more than one fluorochrome.

2.1.3 The Electronics System

Once a cell or particle passes through the laser light, emitted side scatter and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a silicon photodiode collects the forward scatter signals (Figure 2). The number of detectors will vary according to the instrument and its manufacturer. All of the signals are routed to their photodetectors via a system of mirrors and optical filters. Optical filters control the specificity of detection by blocking certain wavelengths, and transmitting others. The three main types are long pass (>500nm wavelength), short pass (<560nm wavelength) and band pass (615 – 645nm band width). Beam splitters, such as dichroic mirrors, are devices that direct light of different wavelengths in different directions.

The photodetectors convert the light signals into electronic signals. The electronic currents are first processed by a series of linear and log amplifiers to convert them into a voltage pulse. The voltage pulse is then assigned a digital value by an Analog-to-Digital Converter (ADC), which in turn allows for events to be plotted on a graphical scale. Log amplification is normally used for fluorescence studies because it expands weak signals and compresses strong signals, resulting in a distribution that is easy to display on a histogram. Linear scaling is preferable where there is not such a broad range of signals e.g. in DNA analysis.

The measurement from each detector is referred to as a ‘parameter’ e.g. forward scatter, side scatter or fluorescence. The data acquired in each parameter are known as the ‘events’ and refer to the number of cells displaying the physical feature or marker of interest.

An electronic threshold can be used to limit the number of events that the flow cytometer acquires. A threshold is set on one parameter. When a threshold value is defined, only signals with intensity greater than or equal to the threshold channel value will be processed and sent to the computer. For example, when running immunophenotyping samples, the threshold can be set on FSC to eliminate events such as debris that are smaller than the threshold channel number. Other parameters can be used to set the threshold depending on the application.

A second threshold parameter is available on some benchtop analyzers equipped with the second-laser option. If two threshold parameters are chosen, then the particle must meet the values of both thresholds to be processed as an event.

Schematic

Figure 2. Schematic overview of a typical flow cytometer setup

2.2 Data Analysis

Flow cytometry can be applied in a high-throughput fashion to process thousands of samples per day. Recent advances in flow cytometry instrumentation and reagent development permits fine cell analysis up to 20 parameters. As a result, this technology generates very complex data sets that demand highly efficient tools for analysis. Data analysis can be a significant challenge for researchers because each data set is a multi-parametric description of millions of individual cells.

In flow cytometry, once light signals have been converted to electronic pulses and then converted to channel numbers by the ADC, the data must be stored by the computer system. Once the data is collected, there is no need to stay connected to the flow cytometer. For this reason, analysis is most often performed on a separate computer. This may be an important consideration, especially in core facilities where usage of these machines is in high demand.

Flow cytometric data is stored according to a standard format, the flow cytometry standard (FCS) format, developed by the Society for Analytical Cytology. According to the FCS standard, a data storage file includes a description of the sample acquired, the instrument on which the data was collected, the data set, and the results of data analysis.

The data generated by flow cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even three-dimensional plots. An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating. A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis.

Manual gating is often considered to be a time consuming and highly subjective process. Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Specific gating protocols exist for different applications, thus the choice of software is an important consideration in flow cytometry data analysis.

As the level of sophistication of flow cytometry experimentation has increased, so too have the methods by which data analysis can be performed beyond routine fluorescence intensity. Experiments that involve changes in fluorescence intensity over time, such as calcium flux studies, can be performed, displayed and observed in greater detail by using the time parameter combined with fluorescence ratio. Other arithmetic functions can also be applied to extend the capabilities of the flow cytometry technique.

Flow cytometric analysis, although capable of measurements with high precision, is prone to the introduction of systematic errors. Sources of measurement degradation can include poor sample preparation and staining, nonspecific and non-homogeneous staining, fluorochrome emission variations with pH and temperature, optical and electronic noise, laser noise and fluidic instability. Any degradation in the accuracy of measurements can be detected rapidly if standard particles are used routinely to monitor and log instrument performance.

2.3 Application Specific Technology

Depending on your application you may require your instrument to perform additional functions to that of a conventional standard flow cytometer. For example you may require your system to be capable of electrostatic sorting and/or imaging.

2.3.1 Cell Sorting

One factor to consider when choosing a flow cytometer is whether you would like it to include a cell sorting function. In most applications, after a particle exits the laser beam, it is sent to waste. Some flow cytometers are equipped with a sorting feature, which enables the user to capture and collect cells of interest for further analysis.

Many flow cytometers can now be upgraded to perform this function known as fluorescence-activated cell sorting (FACS™), or electrostatic cell sorting. The light scattering and fluorescent characteristics of each cell are compared to criteria programmed into the chosen instrument. If there is a match the fluid stream is charged as it exits the nozzle of the fluidics system. When the droplets pass through an electrostatic field they are deflected in different directions based on their charge. The speed of flow sorting depends on several factors including particle size and the rate of droplet formation. A nozzle producing 30,000 – 100,000 droplets per second is ideal for accurate sorting.

2.3.2 Imaging Flow Cytometry

Another facet of flexibility is determining whether your flow cytometry requirements include imaging. Imaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. Imaging enables researchers not only to obtain fluorescence data from millions of cells but also to visualize and quantitate the location of that fluorescence within the cell.

Imaging flow cytometry can be used to study cell function morphology, internalization, cell signaling, co-localization, cell death, stem cell differentiation and cell-cell interactions to name but a few. The technique is also a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids such as blood and urine. Many flow cytometers are now designed to be small, compact and portable enabling them to be used outside the traditional lab setting. Portable flow cytometers can be used globally for the diagnosis and monitoring of diseases such as HIV/AIDS, leukemia, tuberculosis and malaria.

Key Considerations for Choosing a Flow Cytometer

When buying a flow cytometer, like with any major piece of equipment, in addition to considering your budget there are several important considerations that should be taken into account. This section of the guide will outline the main technological, general and software considerations that need to be carefully thought about before going ahead with a purchase.

3.1 Technological Considerations

When purchasing a flow cytometer you will firstly need to consider the number of parameters you wish to measure. The number of parameters you can measure simultaneously in one assay will depend largely on how many lasers and detectors the flow cytometer is equipped with. The wavelengths/colors of the laser will dictate the excitation frequencies of the fluorochromes that you can use. The possible number of fluorochrome colors will also be determined by the number of detectors you use at the respective emission wavelengths. In addition, detectors for scattered light will provide estimates of the dimensions of the cells or particles.

Another important consideration when choosing a flow cytometer is resolution and data quality. Modern flow cytometers can analyze several thousand particles or cells every second. It is important to ensure that when you purchase a flow cytometer designed for quick data acquisition times that the resolution and quality of the data is not compromised by these rapid sampling rates. Pre-enrichment of target cell populations can aid in acquiring higher-quality data.

Some flow cytometers are now fitted with a magnetic pre-enrichment column, which is particularly useful when studying rare cells. If you are working with rare or precious samples, or trying to isolate a cell population of low abundance from a large mixture, you may wish to consider looking for a flow cytometry capable of addressing these challenges.

Buying Guide Tip: Conserve your precious samples - One way to protect rare or precious samples is to use an entirely closed flow cytometry system. Some manufacturers offer solutions that protect your cells from environmental influences and maintain sterile working conditions.

Another important technological consideration is sample handling. To ensure that sample and, therefore, data integrity is maintained, it is important that the damaging external effects of light exposure, temperature variation and mechanical agitation are minimized to the sample under investigation. Consideration should also be given to the suitable selection of sheath fluid so that cells can be analyzed and/or sorted with minimum disturbance.

Sample contamination can occur when the fluidic lines and other surfaces that come in contact with sample fluid are not cleaned sufficiently or provide a location for carry-over of sample fluid and particles. This issue can be minimized by following appropriate daily cleaning practices and by adhering to instrument replacement and maintenance schedules.

In addition to considering your sample type, you should also consider your sample volume. Most flow cytometers can utilize sample volumes ranging from several microliters to hundreds of microliters. If you have a small volume of precious cells you may wish to consider using a microfluidics-based flow cytometry chip. The development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. Attempts providing such groundbreaking capabilities have already been made and microfluidic chip-based cytometry is slowly entering a commercial stage. It is worth considering whether such technology may be relevant for your applications.

When buying a flow cytometer an extremely important factor to consider is throughput. The number of lasers you use will affect how many ways you can sort your cells, for example, into 2, 4, or 6 sorted populations. The more sorted groups you have, the more you need to think about where they will be placed in your sample vessels. Typically, flow cytometers sort cells into sample tubes or microplates of up to 384 wells. While it is possible to run single samples on most flow cytometers, the ability to use microplates makes higher throughput possible.

Buying Guide Tip: Determine how much automation you require - Alongside throughput you will also need to decide how much automation you want in your system. Many variations and degrees of automation are available, from automated liquid handling systems that carry out particular sampling and dispensing jobs to fully automated robotic systems that execute all the functions of the work flow, including moving plates and samples around the flow cytometer.

Automated introduction of samples to the flow cytometer allows for walk-away operation to be performed on large numbers of samples using a variety of formats from single test tubes to multi-well plates. Single tubes are often contained within rectangular grid arrangements or carousel formats to match laboratory equipment used for sample preparation.

A large number of samples can be analyzed using multi-well plate feeders that automatically feed samples to the instrument for periods of a day or more. A number of other routine instrument functions are being automated as well. These include self calibration and self check utilities, start-up and shutdown procedures, biosafety monitoring and intervention, fluorescence compensation and sort monitoring and control.

Reduced operator involvement removes human errors, and frees up additional human time for higher-level activities such as planning future experiments and data interpretation. It is therefore strongly recommended you think about the degree of automation you require.

3.2 General Considerations

When buying a flow cytometer there are several general considerations that should not be overlooked. Perhaps the first question you should ask yourself is “who will be using the instrument and for what applications will it be used for?” This will immediately help you clarify the sort of system you are looking for. For example, if you decide you require a flow cytometer that will be used by many researchers from a variety of laboratories it is likely a highly flexible instrument will be preferred. Once you have considered both your application needs and the different technologies that are available, the chances are you will have narrowed down the choice to just a few flow cytometers.

Buying Guide Tip: Ask for a demo - Before going ahead and purchasing a flow cytometer it is highly recommended that you try it out. Most manufacturers will let you demo the instrument for a trial period of time and it is important to use this time wisely to ensure the instrument is capable of fulfilling all of your requirements.

You should consider the extent of the technical support and training that is available from the manufacturer at the point on purchase. It is also recommended that you look into the company’s policy on after sales service. While everything is operating well, this may be the last thing on your mind, but in the event that after sales support is required, it is important to know how will this be achieved.

You will need to carefully evaluate how much of your laboratory space can be dedicated to your new instrument. The good news is that flow cytometer manufacturers appreciate laboratory space is extremely valuable and consequently are trending towards smaller, more compact systems. Benchtop friendly systems that do not compromise on function are becoming more readily available. Personal flow cytometers, which are commonplace on the market today, tend to have an extremely compact footprint and are often portable for field work.

The issue of biosafety is attracting increasing attention in the field of flow cytometry. Risks are similar to those faced in other biological laboratories dealing with hazardous solvents, infectious agents and blood products. The particular challenges with flow cytometry are the high pressures applied to samples and the aerosols that can be associated with sorting.

Buying Guide Tip: Investigate biosafety accessories - Though standard laboratory safety procedures address biosafety issues in many cases, instrument manufacturers also offer special biosafety accessories, such as custom biosafety cabinets, sort shields and aerosol evacuation pumps. You should consider the sample type you are working with and take the necessary precautions to ensure the safety of yourself and colleagues.

When purchasing flow cytometry technology it is important to establish just how flexible you need the system to be. By measuring multiple parameters on individual cells, flow cytometry enriches your research. Most researchers seek a system that can grow with the laboratories needs, for example, with regard to changes in throughput and application. In order for a system to grow with your changing requirements you will need to investigate the potential for upgradability. For example you may which to increase the number of lasers and detectors to enable multiple labeling and facilitate more targeted population studies. Will you be able to expand your instrument or perhaps trade it for a more flexible model?

Another extremely important factor that you will need to consider when buying a flow cytometer is its compatibility. In order to reliably assess the suitability of your potential new flow cytometer you may wish to investigate its compatibility with automated or robotic lab systems, with LIMS or other data storage systems, as well as with reagents and consumables.

When approaching flow cytometer vendors you should have a thorough idea of your current and future needs. Contact multiple vendors and have a list of questions prepared. Explore all of the available options and where possible request a demo before purchasing.

3.3. Software Considerations

Early flow cytometers used an oscilloscope for simple pulse height display and analysis. Today, most instruments use software for instrument control, data acquisition, analysis and display. Many instruments provide software control for many or all instrument hardware functions including fluidics, lasers, electronics and sorting. Further, operations such as start-up and shutdown sequences, cleaning cycles, calibration cycles and self-test functions can be monitored and regulated by software, so that instrument operation can be simplified and optimized for the user.

When purchasing a flow cytometer it is imperative to consider the ease-of-use of the system. This is in part determined by the system’s software. You should look for an instrument with a software package that enables you to get up and running in a short period of time, but make sure the software has expanded capabilities so you can perform more complex analyses in the future.

It is important to investigate how regularly the analysis software is updated, and whether you will be able to receive automatic upgrades. You should consider whether the software will integrate with hardware you already have in the laboratory. In addition you should determine how the software will present the data to you and establish whether this meets your needs. Depending on who will be using the instrument, you may wish to consider whether the software is available in multiple languages?

Buying Guide Tip: Speak with the manufacturer - In order to thoroughly evaluate the software you will need to know your exact requirements for data analysis, so that when you speak with manufacturers you can ask questions such as “will I be able to merge multiple files for better analysis, how does the software compare different data sets and will I be able to see plots in 3D?”

3.4 Considerations Summary

Buying Guide Tip: Create a matrix - Once you have a list of all of your wants and needs, collated various marketing materials and spoken with multiple vendors, put all your information together in a tabular format and request a demo from the top two or three contenders. Following this step and further negotiation with the manufacturer you should be ready to go ahead and purchase your new flow cytometer (Table 1).

Instrument

# Lasers (Total # Available)

Total Detectors

Field Upgradable

Automated Sample Handling

Total Events Per File

Optical Upgrades Available

A

1-3 (3)

3-8

No

Yes

100,000

No

B

2-4 (7)

4-12

Yes

Yes

5,000,000

Yes

C

2-6 (12)

4-18

Yes

Yes

10,000,000

Yes

D

2-4 (4)

4-10

No

No

2,000,000

No

E

2-5 (8)

4-14

Yes

Yes

10,000,000

Yes

F

1-3 (4)

3-6

No

No

100,000

No

Table 1. Fictitious instrument comparison chart, with the three contenders highlighted in green

Fluorochrome Selection

As has been highlighted throughout this guide, the most powerful aspect of flow cytometry is the fast and simultaneous multiparametric analysis of proprieties of single cells. Sophisticated flow cytometers capable of analyzing signals of up to 18 fluorochromes at the same time are available today. It is becoming routine to do 5 to 7 color experiments without any major difficulties. Advancements in flow cytometer instrumentation have spurred the development of new fluorochromes, also known as fluorophores, and antibody conjugates that take advantage of these capabilities. Choosing the optimal combination of fluorochromes for your particular antibody specificities remains a complex process. There are a few basic rules that you should consider when choosing a fluorochrome or a combination of fluorochromes:

Buying Guide Tip: Know your instrument configuration - Reagent selection starts with your knowing your instrument configuration. The type and number of lasers and detectors dictate whether the optical system can excite a given fluorochrome and properly detect a given combination of fluorochromes. The design of the optical system also impacts the efficiency with which particular dyes are detected, as do the instrument settings, including PMT voltages. The choice of optical filters that are used with each detector greatly influences the effective brightness of one fluorochrome versus another. Filter selection is a give-and-take process: using a wider band pass filter can increase the ability to detect a given fluorochrome, but may also increase the amount of spillover background contributed into that detector from other neighboring fluorochromes.

Buying Guide Tip: Maximize the usage of your lasers - As discussed previously many cytometers include several lasers. Try to choose a color in each excitation range before considering a second color for the same laser. This considerably reduces the amount of compensation you need to perform between the different colors.

Buying Guide Tip: Choose the brightest fluorochromes that can be used on your instrument - When choosing a fluorochrome or combination of fluorochromes it is advisable to use the brightest fluorochrome for the least well expressed protein/antigen and the dimmest fluorochrome for the most well expressed protein/antigen. It is also recommended that you choose the brightest fluorochromes that can be used on your instrument.

Buying Guide Tip: Choose fluorochromes with emissions having the least spectral overlap - Every fluorochrome has a wide fluorescence emission spectrum that extends beyond the narrow window of light allowed by an optical filter for a specific fluorochrome. It is therefore important to select fluorochromes with emissions that have the least spectral overlap. There are many spectra viewers available on the web that can help you check whether two or more fluorochromes can be combined together.

Buying Guide Tip: Use tandem dyes with caution - Tandem dyes are being used more and more frequently in flow cytometry. They consist of two conjugated dyes that are covalently linked and in close proximity (30-50 nm). When one of the fluorochromes is excited, the energy is transferred to the nearby fluorochrome, which in turn produces fluorescence emission. The process is called FRET (fluorescence resonance energy transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increase the number of colors that can be analyzed from a single laser wavelength. Tandem dyes are however prone to uncoupling and are sensitive to photobleaching and extended incubation in fixation buffers. Always follow the manufacturer’s storage and manipulation guidelines and use a single-stained control for any tandem dye in order to verify that the tandem has not uncoupled.

The difficulty in choosing the best suited fluorochromes for any given application means we often stick with fluorochrome combinations that have been historically used in our labs rather than the ones that are best suited for the given application. While panel design can be a complex process, speaking with other scientists and manufacturers should enable you to better optimize the results of your flow cytometry experiments.

The Future of Flow Cytometry

From its development in the 1960s, flow cytometry has evolved into a technique that is now used routinely in the clinic, as well as in the research setting. The largest users are immunologists and hematologists who use it for cell sorting and analysis. Increasing requirements for processing larger numbers of patient samples has led to the development of automated tube loading carousels and, later, multi-well plate-based sampling systems.

While there are numerous technologies and methods available to study cell populations, flow cytometers are distinguished by their capacity to collect and process large amounts of data expeditiously. As new genes are discovered in the post-genomics age, understanding their biological significance within cells, in the context of other genes and gene products, is essential. By having a system to detect, view and analyze the expression patterns of multiple proteins involved in complex biological processes such as apoptosis, oncogenesis or cell division, investigators are able to better understand the possible combinatorial roles specific proteins play in these processes. The genetic activity can then be correlated to morphologic and phenotypic changes in the cells. In the future, the advantages of flow cytometry, including data storage and analysis, should allow this science to retain its prominence in the continued study and understanding of complex cellular processes.

Recently a new platform has been developed that couples flow cytometry with mass spectrometry. This technology, known as mass cytometry, offers a new approach in which antibodies are tagged with isotopically pure rare earth elements, rather than fluorophores, allowing simultaneous measurement of greater than 40 parameters while circumventing the issue of spectral overlap. Hundreds of cells per second are passed through argon plasma, ionizing the multi-atom metal tags, which are then analyzed by a time-of-flight mass spectrometer. By exploiting the resolution, sensitivity and dynamic range of mass spectrometry on a time-scale that allows the measurement of 1000 individual cells per second, this configuration offers a new approach to high-content cytometric analysis. Mass cytometry is very much in its infancy, but has the potential to become an important tool for drug discovery and clinical applications in the future.

Initially the introduction of flow cytometry to the clinical laboratory was limited by the cost of instrumentation and the need for well trained operators. However, improvements in the ease of operation, the software and the cost of flow cytometers, have seen these instruments become valuable tools in the clinical laboratory. Furthermore, another key characteristic of flow cytometry is its versatility: the same instrument can be used to perform direct diagnosis, antimicrobial susceptibility testing and serum antibody detection, for example. Today’s flow cytometers allow the analysis of several cell functions in a rapid and multiparametric fashion, and also enable both qualitative and quantitative analysis. With this in mind, it is likely that in the future the applications of flow cytometry in the clinical laboratory will continue to expand.

One major limitation in conventional flow cytometry is that hydrodynamic focusing is normally required to align single cells in a laminar flow, which prevents this technology from being used for quantitative in vivo detection. However, the nascent field of in vivo flow cytometry (IVFC) has begun to bear fruit in applications such as monitoring circulating cancer cells (CTCs). IVFC has the capability to measure the dynamics of fluorescently labeled CTCs continuously and non-invasively. Studies to date have shown that IVFC has higher specificity and sensitivity than conventional flow cytometry methods. IVFC have been used to investigate several cancers including leukemia, prostate cancer, breast cancer, hepatocellular carcinoma and melanoma, among others. In the future IVFC may be an important tool for elucidating mechanisms that drive metastasis and may be used to monitor the efficacy of cancer therapy. With advances in the technique, IVFC could potentially be used clinically in both cancer diagnosis and treatment.

The high speed quantitative high content analysis of cells also makes flow cytometry an attractive technology for drug discovery applications and it is currently used at many stages of this process. Recently it is also finding a niche in drug screening laboratories sharing bench space with other high throughput technologies. Flow cytometry is becoming an ideal tool, particularly in an environment where primary cell-based assays are increasingly being deployed to monitor drug responses. The potential of flow cytometry as a drug screening platform was first realized upon the introduction of plate-based sampling on flow cytometers. The introduction of faster sampling technologies, and the use of peripheral automation and liquid handling, has transformed flow cytometry into a powerful drug screening tool. Ongoing development efforts in the flow cytometry industry are aimed at automation and laboratory integration. Input/output robotics, pushbutton operation and automated sample preparation will increase throughput rates and make the technology more accessible to a wider user base, as new fluorescent dyes and creative screening approaches expand applications into the proteomic arena. High throughput flow cytometry is still at ‘the lower end’ of the capacity scales in a typical drug screening laboratory where one to two million compounds can be profiled in just a few weeks, but further improvements are currently in progress, such as 1536 well sampling. Other important, platform independent, factors such as assay costs and cell supply will influence whether flow cytometry becomes a routine tool for drug screening laboratories.

Flow cytometry is increasingly recognized as an invaluable technology in biomarker research. Owing to its multiparametric nature it can provide highly detailed information on any single cell in a heterogeneous population. Its versatility means it can be conducted in both the preclinical and clinical setting, generating biomarker data that can drive decisions pertaining to dose selection in clinical trials, treatment options for cancer sufferers and even suitability of patients to receive transplants. Most tissue types can be utilized by the flow cytometrist, allowing the technology to be applied to many fields of research, yet consensus still needs to be reached on standardization, regulation and validation of multiparametric flow cytometry assays. In parallel, continual innovation in analysis software to manage the huge datasets that can be generated is also needed. Nevertheless, the flexibility of flow cytometry means that it remains at the forefront of both routine and exploratory biomarker studies.

Flow cytometry does, without a doubt, have a colorful future for cell analysis. As discussed in this section, there are several hurdles that must be overcome before this application will become more widely used. First, we need flow cytometers that are easier to validate and have consistent implementations of hardware (such as lasers and filter combinations). In addition, better software systems are needed to guide the user through what is still a fairly complex setup and optimization procedure. Second, manufacturers must devise a new model for distributing reagents so that laboratories can try a large variety of different fluorescently labeled monoclonal antibodies at minimal cost, before committing to purchase larger amounts of optimized panels. This necessitates a huge inventory requirement for manufacturers, as the number of reagent-fluorochrome combinations is an order of magnitude greater than what is needed today. Finally, we desperately need new tools to aid in the exploration of these complex data sets, as well as new techniques for the presentation of the data. Journals will need to accommodate the requirement for the presentation of far more complex graphics and analyses; the inclusion of such information as online supporting documentation will become ubiquitous.

Summary

Flow cytometry is a powerful technique that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Over the past 30 years flow cytometry has become an indispensible tool for many scientific researchers and clinicians. It has applications in a number of fields ranging from life science and drug discovery to clinical diagnostics and marine biology. As the technology continues to evolve, it remains a relevant and sophisticated platform for the development of new applications in the clinical laboratory, as well as in basic and clinical research.

Whether you are a first time buyer of flow cytometry equipment, or an experienced user, there are a number of factors to consider if you are choosing a new platform. It is also essential that you thoroughly examine your current and your future application needs.

Visit the SelectScience product directory for an overview of the latest flow cytometry technology from leading manufacturers and read user reviews. Keep up to date with the latest cytometry methods by visiting the SelectScience application note and video libraries.

Download a PDF version of the flow cytometry buying guide.

Editor's picks

Kerry Parker

Editor-in-Chief

Guava easyCyte 8HT Base System (EMD Millipore)

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4 out of 5

"It’s very easy to use and I cannot imagine working without it. It’s such a perfect system. It’s worth every dollar. There is nothing better."
Raffaella Spina, Case Western Reserve University


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CyFlow® SL (Partec)

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4 out of 5

"Very easy to operate and gives consistent results. It’s low cost and can handle a good amount of samples. Operator friendly interface."
Wei-Hsuan Yu, National Taiwan University


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Luminex 200 System (Luminex)

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4 out of 5

"Luminex 200 flow cytometry system is a good choice for HLA typing. Easy, fast and good HLA-SSO typing results which are compatible with SSP detection.”
Turker Toktay, Acibadem Labcell




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BD FACSCalibur Flow Cytometer (BD Biosciences)

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4 out of 5

"This equipment is very easy to use, software is very friendly. The results are very easy to understand. I like it to use and I am happy to recommend to my colleagues."
Pamela Leal, University de la Frontera

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CyAn™ ADP Analyzer (Beckman Coulter)

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3 out of 5

"It is easy to use and useful for both graduate students and undergraduates. I definitely recommend this machine."
Lucas Horn, University of Maryland, Baltimore


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BD Accuri™ C6 Flow Cytometer (BD Biosciences)

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4 out of 5

"Amazing benchtop flow cytometer. Easy to use, quick and very user friendly personal flow cytometer for various cell types. The four lasers cover almost all the most commonly used fluorophores."
Pranav Gupta, Morehouse School of Medicine

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