Flow cytometry has many of the technology attributes (eg multi-parameter
analysis of cell suspensions) needed when attempting to profile a phenotypic
drug response. However, for flow cytometry to be considered the phenotypic
drug screening platform of choice it needs to interface with the real screening
world, ie be able to support higher throughput and to acquire low sample
volumes from high density microplates.

Flow cytometry is recognised for its ability to perform high-speed quantitative analysis of suspended cells and other particles and for the level of multiplexing (multi-parameter analysis) that can be achieved (1).

This has led to the technique being extensively used for rare cell analysis and cell sorting (2). Flow cytometry has also many of the technology attributes needed to support phenotypic screening, which is currently experiencing something of a rebirth in drug discovery labs. Flow cytometry suitability for phenotypic screens include its ability to assay physiologically relevant cell models in suspension, including human primary cells and cell mixtures; its aptitude to screen multiple cell functions and cell health endpoints simultaneously providing a high content alternative to imaging; and its capacity to monitor multiple secreted proteins (including chemokines and cytokines) simultaneously enabling better biomarker profiling.

However, for flow cytometry to be considered the phenotypic screening platform of choice it needs to interface with the real screening world, ie have the capacity to process with rapidity 100,000s of samples which are most often assayed and presented for analysis in low volume microplates. Many flow cytometry systems have been adapted in recent years to accept sample acquisition enabled direct from microplate wells or have been coupled with an auto-sampler option accepting microplates. However, the majority of these options are limited to processing an individual discrete sample every few minutes, owing to a combination of data processing delays and the mechanics concerned with running one sample into the next1.

It was not until the development of IntelliCyt’s unique aspiration and sample feed method of sequentially transferring assay components in solution to the flow cytometer detector in a continuous, air gap delimited stream, that dramatic reductions in sample processing time and sample dead volume were achieved. IntelliCyt’s HyperCyt autosampler, which can interface with most flow cytometer systems or its fully integrated iQue Screener system, is now able to process an entire 96-well plate in 3-4 minutes using as little as 1-2μl of sample per well. With the possibility of using these platforms and other approaches to high throughput flow for the fast and efficient screening of suspension cells, phenotypic drug discovery, antibody screening, immunophenotyping and biomarker research are now more readily accomplished.

In December 2014, HTStec undertook a market survey on high throughput flow cytometry (HT flow)1 mainly among research labs in pharma, biotech and academia. The main objectives of this survey were to understand the current implementation of HT flow assays, where and how and for what it was being used today and user’s expectations for the future. In this article highlights from this market survey are reported and the findings are discussed together with some of latest vendor products and developments supporting flow cytometry and phenotypic screening.

How HT flow assays are achieved to date
Survey respondents have achieved HT flow analysis mostly to date by using a flow cytometer with sample acquisition enabled direct from microplate wells. This was followed by adding an auto-sampler option to the flow cytometer, using an IntelliCyt iQue Screener system, and then interfaced IntelliCyt’s HyperCyt with one flow cytometer (Figure 1).

The typical throughput realised to date by survey respondents’ current approach to HT flow was a median of 500-1,000 samples or microplate wells assayed by flow per 24-hour day. It is clear from the results presented only a very small minority (6%) of survey respondents have currently achieved what might be considered high throughput (>50,000 samples per day) (Figure 2).

Where do HT flow assays fit
The majority (83%) of survey respondents believed that implementing HT flow will mainly complement existing screening technologies. Only 17% believed HT flow would displace existing technologies (Figure 3).

Survey respondents rated they were most interested in applying HT flow assays to other membrane receptors (eg cytokines, transporters, adhesion). This was followed by phenotypic (non-target based) screens; protein-protein interactions; and then GPCRs. Very limited interest was shown for applying HT flow assays to phosphodiesterases (PDEs) (Figure 4).

The majority (53%) of survey respondents selected antibody screening as one of the key application areas most suited to an HT flow approach. This was followed by immunophenotyping (42% selecting); and then phenotypic drug discovery or immunoassays (both 38% selecting). A HT flow approach was considered least suited to FISH, clinical diagnosis and hypersensitivity (Figure 5).

Survey respondents ranked ability to analyse multiple cell populations within each microplate well as the greatest benefit (advantage) of HT flow versus other techniques. It was followed in ranking by ability to work with suspension cells/blood cells; level of multiplexing achieved (both cell and bead-based readouts); throughput of cells per second; and then novel approach to phenotypic screening. Ranked least advantage of HT flow versus other techniques was resolution of free and bound signals for cellular and molecular targets (Figure 6).

Cell markers of interest regularly targeted by survey respondents’ HT flow assays were mainly located on the cell surface (88% of assays), followed by intracellular (77% of assays) and secreted proteins (49% of assays) (Figure 7).

The type of end points of most interest to survey respondents were a combination of those that report cell function(s) and cell health (61% responding), followed by endpoints that report only cell function(s) (22% responding) and then endpoints that report only cell health (only 6% of respondents) (Figure 8).

Future requirements for HT flow assays
Survey respondents reported their median future requirements for HT flow assays as follows: the time required to aspirate all 96 samples from a 96-well microplate and detect up to 10,000 object events per second using a flow cytometer was 3min; the preferred sample presentation/ handling format was the 96-well plate; the target sample (aspirate) volume wanted was 5-10uL; with 5-7 parameters of information wanted to be acquired (multiplexed) simultaneously from the same sample.

HT flow assays vendor perception
The vendor of HT flow assays that first comes to the mind of survey respondents was IntelliCyt followed by BD Biosciences and then to a lesser extent Beckman Coulter, Fluidigm and Bio-Rad. All other vendors had minimal survey respondent recognition with respect to HT flow assays (Figure 9).

Current thinking on HT flow
Survey respondent’s level of agreement with statements about the status of HT flow assays was most positive (showed greatest agreement) with ‘HT flow is the ideal high content platform for phenotypic screening’. This was very closely followed by agreement with the statement ‘HT flow has made a significant impact on phenotypic screening’. Survey respondents were most negative (showed greatest disagreement) with the statement ‘HT flow is nothing more than a prescreen for more sophisticated imaging-based analyses’. Opinion on all other statements was broadly neutral (neither agree nor disagree strongly) (Figure 10).

Possible limitations of HT flow
Survey respondents rated investment in additional expensive instrumentation as the main limitation of HT flow assays. This was followed by not suited to cells grown in complex matrices, with feeder cells, model organisms etc, and then only indirectly suited to 3D cultures (after spheroid/microtissue dissociation). Rated least limiting was no references images, still need to undertake confirmatory image-based analysis. However, since the responses to most factors rated fell between 2.50 and 3.00 we have to conclude they are only moderately limiting the uptake and future adoption of HT flow assays (Figure 11).

Flow vendor updates
The following snapshots were provided by vendors about their latest products and developments supporting flow cytometry, HT flow and phenotypic screening:

The ACEA Biosciences (www.aceabio.com) NovoCyte™ flow cytometer is a new high-performance benchtop flow cytometer designed for everyone. It is an affordable instrument that is capable of detecting up to 15 parameters with enhanced sensitivity and resolution. Its light collection optics design allows fluorescent light excited by spatially-separated laser beams to be collected and detected with the same sets of detectors. The shared PMT (Photo Multiplier Tubes) configuration of NovoCyte maximises its optical bench design while providing multiplexed cell analysis capabilities. The NovoExpress acquisition and analysis software enables the automation of various fluidic functions including declogging and getting rid of bubbles with a push of a button. Novocyte is customisable for single, two, or three laser configurations, exchangeable filters, multiple sampling options and flexible analysis formats. NovoCyte offers versatility in automated sample acquisition with its fully integrated NovoSampler™ sample loader. Users can acquire samples using single or multiple tubes, as well as 24- and 96-well plates for increased sample processing throughput. The NovoSampler is a walk-away system. Users can programme sample acquisition protocols such as sample injection port rinse/wash following each acquisition or after multiple tube acquisitions minimising carryover issues. Pre-programmed sample loader mixing protocol ensures cell suspension during acquisition. The typical plate processing speed for 96- well plate acquisition is less than an hour. The flow operators’ quality of life is improved with the unique system maintenance features of NovoCyte. Fluid levels are monitored with indicators for appropriate required actions. Start-up and shutdown protocols are automated, eliminating operators’ idle and tedious bleach and water cleaning routine. Other fluidic features include the fast acquisition rate of 35,000 events per second, volumetric absolute counts acquisition through syringe pump sample aspiration. The NovoCyte flow cytometry is ideal for the core as well individual lab facilities (Figure 12).

BD Biosciences (www.bdbiosciences.com) continues to deliver innovative solutions in both system capability and fluorochromes that help make flow cytometry-based assays more capable and accessible. The latest in its line of high-performance flow cytometers is the BD LSRFortessa™ X-20 cell analyser. Like its predecessor, it can be configured with up to five lasers to detect up to 20 parameters simultaneously. Integration of the newest laser and light technologies greatly increases the sensitivity, allowing resolution of populations that might otherwise be obscured. The optional BD™ High Throughput Sampler (HTS) allows sample loading from 96- or 384-well microtiter plates for screening assays. In high-throughput mode using a two-second acquisition time it takes less than 15 minutes per 96-well plate. The BD FACSVerse™ system supports analysis of up to 10 parameters. Designed to address the growing need for both reproducibility of data and ease-of-use, it delivers users a seamless workflow. The ability to save assay parameters makes it possible to reproduce results on different BD FACSVerse systems. The optional BD FACS™ Universal Loader provides walkaway operation with samples loaded in either microtiter plates or tube racks. The capability of these platforms for multiparameter analysis is supported by a whole range of new fluorochromes. Perhaps the most significant advancement in reagent technology in a decade, the new BD Horizon Brilliant™ polymer dyes offer unprecedented brightness, allowing improved resolution of cell populations with dim markers. Now available for multiple wavelengths off the blue, violet and ultraviolet lasers, the consistent spillover values of BD Horizon Brilliant™ dyes also make them easier to combine into multicolour panels (Figure 13).

The Beckman Coulter (www.CytoFLEXFlow.com) CytoFLEX research flow cytometer is the first of its kind to utilise technologies from the telecommunications industry to advance signal detection and processing for exceptional fluorescence sensitivity in cytometry applications. The proprietary Wavelength Division Multiplexing (WDM) and Fiber Array Photo Diodes (FAPD)s showcase two examples of powerful technologies from the fields of fibre optic communication and computing, now applied to flow cytometry creating a new performance standard of red channel and violet channel fluorescence sensitivity. The FAPD provides lownoise detection with high quantum efficiency and minimum light loss ensuring high signal to noise ratio and optical resolution especially with small particle measurements and dim fluorescence detection. Additionally, the bench-top design of the CytoFLEX flow cytometer offers built-in system flexibility for highly multiplexed flow cytometry applications. The unprecedented 21 configuration options accommodate growth for simple to complex assays on the same platform with a newlylaunched internal Plate Loader option for higher throughput applications. CytoFLEX Plate Loader option can analyse a 96-well plate in 32 minutes with walk-away convenience. With the Plate Loader’s front loading design, the possibility of robot-loading solutions is easy. Beckman will next investigate the needs of high throughput labs, including requirements for hardware and software, to provide a more comprehensive solution. There are several exciting research applications where the CytoFLEX technology is keenly positioned such as for the measurement of nanoparticles and extracellular vesicles as well as rare event detection where sensitivity and resolution are critical. The compactsize CytoFLEX has superior sensitivity and resolution for robust performance throughout all configurations (Figure 14).

EMD Millipore (www.emdmillipore.com) has a strong heritage and culture of innovation that has led to the most unique offering of flow cytometric analysers available. The Amnis® imaging flow cytometers are the first and only cell analysers to combine the best of flow cytometry and microscopic imaging, yielding novel insights into cell biology and powerfully expanding the range of cell analysis applications. The Amnis® brand imaging flow cytometers provide high sensitivity, multicolour images of every cell to visualise fluorescence at the cell membrane, within the cytoplasm, or in the nucleus. Effortlessly identify cell doublets and distinguish debris to improve gating and analysis at 20X magnification with the Amnis® brand FlowSight® compact, high-sensitivity, multi-colour flow cytometer. Or use the Amnis® brand ImageStream®X Mark II Imaging Flow Cytometer which combines up to 60X magnification of up to 5,000 cells per second with the speed, sensitivity and phenotyping abilities of a high sensitivity flow cytometer. The Guava easyCyte™ benchtop flow cytometers are based on novel patented microcapillary fluidics that consume less sample, generate less waste and are easier to use and maintain. The Guava easyCyte™ flow cytometry systems are now more flexible and sensitive than ever before with up to three lasers and 12 parameters and detection of particles as small as 0.2μm. The 96-well auto sampler on the Guava easyCyte™ systems enables walk away high throughput capabilities to enable more efficient discovery (Figure 15).

Fluidigm (www.fluidigm.com) is the only company to offer mass cytometry to meet researchers’ comprehensive phenotyping and functional profiling needs. Mass cytometry utilises heavy metal tags attached to specific antibodies that can be discreetly quantified using high resolution mass spectrometry. At the moment, flow cytometers can process around a dozen fluorescent dyes at once before the spectra of some colours overlap with the others, making the crests extremely difficult to discern. In place of fluorescent dyes, Fluidigm’s mass cytometry uses metal tags to label the proteins. The mass cytometer uses high-temperature plasma to split the cells into their component atoms. These atoms, which now include the metal labels, then get fed into a mass spectrometer to measure the mass and abundance of each metal, and thus each matched protein. There are therefore no problems with signal overlap as there are in fluorescent dyes. Fluorescence-based flow cytometry is faster. It can analyse up to 50,000 cells per second. Mass cytometry manages 1,000 cells per second. The CyTOF® mass cytometry technology provides deep measurement across a broad array of cell types in a single tube. As a discovery tool, mass cytometry delivers a systemlevel perspective that lower-dimensional panels cannot match. The CyTOF 2 simultaneously quantifies protein expression on and within cells. These attributes provide researchers with an unparalleled ability to generate high resolution phenotypic and functional profiles of cells from normal and diseased states. Fluidigm is working on several enhancements to the CyTOF platform. Sample barcoding will allow researchers to multiplex up to 20 samples, delivering improved data consistency and increased throughput. Fluidigm is also developing imaging mass cytometry in which the metal tags are being used to create images of the individual cells in situ. Images produced by this technique will illuminate how cells respond to local conditions within a tumour, such as low-oxygen environments. Imaging mass cytometry could be applied beyond cancer, to examine (for example) how neurons are distributed in the brain or spinal cord (Figure 16).

IntelliCyt’s (www.intellicyt.com) iQue® Screener is the first truly high throughput flow cytometry platform for screening cells and beads in microplates. By integrating patented instrumentation, software and reagent kits that are optimised for phenotypic screening, antibody discovery and immunology, IntelliCyt has created an automated, benchtop workhorse that can process and analyse microplates in minutes (96 wells in as little as five minutes) while delivering rich, high content data from a powerful flow cytometry engine. Recent innovations in probe design enable microvolume sampling (1μl) from miniaturised assay volumes as low as 6μl in 384- or 1536-well plates creating attractive cost savings for reagents and sparing of scarce biological/clinical samples. Two exciting examples illustrate the novel synergy of iQue capabilities: 1) immunotherapy, with a mixed cell and bead-based assay allowing simultaneous cellular and molecular measurements to be profiled for optimal response, as might be needed for checkpoint inhibition assays; and 2) oncolytic polypharmacy supporting new drug and repurposing evaluation in mixtures, with multi-pathway targeting of tumour cell populations requiring high-dimensional optimisation of mixtures while still evaluating the heterogeneity of individual cellular response. ForeCyt® data analysis and visualisation software rapidly transforms the large, information rich data sets acquired into actionable results. A screening centric, interactive, assay development, data analysis and result visualisation environment that makes creating highly multiplexed assays, including measuring cells and beads together, intuitive to learn and a pleasure to use. Users can easily assess the data on a per-cell, per-well or per-plate level, shortening the time to discovery (Figure 17).

Stratedigm (www.stratedigm.com) manufactures configurable, scalable and upgradeable flow cytometry solutions that fit every lab and every budget. Users can configure their S1000EXi cytometer for up to four lasers and 22 parameters. Even though it has the smallest footprint in its class, Stratedigm flow cytometers maintain industry- leading sensitivity and resolution. A dedicated microparticle mode powered by Stratedigm’s patent pending laser trimming technology enables analysis and accurate counting of a wide range of particle sizes from 0.2μm to 7μm in the same sample. Complementing any S1000EXi or SE520EXi cytometer, the Stratedigm automation suite provides automated reagent mixing, digital temperature control, sterilisation and 100% automated runs of up to 320 plates using the A700 High Throughput Hotel and A600 High Throughput Auto Sampler (Figure 18).

The Thermo Fisher Scientific (www.lifetechnologies. com/attune) offering in flow cytometry includes the Attune® NxT Acoustic Focusing Cytometer and the Molecular Probes® line of reagents from Invitrogen™. The Attune NxT is a modular flow cytometer with choices of one laser/six detection channels to four lasers/16 detection channels. It also features the novel acoustic focusing technology which uses acoustic waves and hydrodynamic focusing to focus cells within the sample input stream and allows for sample input rates of up to 1mL/min (10 times faster than traditional cytometers) and 35,000 events/sec. Acoustic focusing enables these fast sample input speeds with minimal loss in fluorescence sensitivity and minimal variation (resulting in tight co-efficient of variations (CVs) at any sample input rate). For the high throughput market, an Attune NxT autosampler can be added which enables 96 and 384-well plates as well as deep well plates. The autosampler is designed for minimal clogging using a 200μM flow cell along with hydrodynamic and acoustic focusing. It is also designed with high flexibility: customers can choose to run plates quickly or thoroughly by simply choosing the number of mixes and washes within the software. In high throughput mode (no washes) a processing time <45 minutes for 96-well plate is achieved. Molecular Probes® fluorescence reagents are among the most peer-referenced in all of life science research. Trusted product brands including Alexa Fluor®, SYTOX® and Click-iT® empower scientists working in cell biology, flow cytometry, protein biology, and high throughput screening to make the discoveries that advance our understanding today and catalyse the research goals of tomorrow (Figure 19).

While preparing this article we contacted nearly all the vendors of flow cytometry equipment and asked them to specifically comment on how their instrumentation addressed HT flow cytometry assays and/or phenotypic screening. Several vendors declined to participate in the article citing that either their product range is not targeted at the high throughput market or it was too early for them to talk about flow products that addressed these market needs. Of those 11 vendors that did respond it is evident that few are engaging with true HT flow or most feel this is adequately covered by an optional add-on autosampler. Clearly the capacity of some autosamplers and associated automation does provide a reasonable degree of unattended walkaway processing which is adequate for many user’s needs. The inclusion of various automated tweaks to sample acquisition protocols, such as washing or mixing protocols further extend the usefulness of the automated experience. However, from a purely drug screening perspective, most of these set-ups only offer low to medium throughput capability and could only be used to investigate relatively small scale screens.

A theme that is evident from many vendors is their focus on the number of detection channels (parameters) which can be simultaneously measured with their instruments, such a capability is of increasing value when attempting to profile a phenotypic response. Multi-parameter assays are significantly enhanced by the availability of the new fluorochromes offered by some vendors, including those whose superior brightness allows for improved resolution of cell populations with dim markers.

Of those novel or unique features incorporated into some flow cytometers the following are worthy of note from a screening perspective: acoustic focusing enables faster sample input speeds with minimal loss in fluorescence sensitivity and minimal variation (Thermo Fisher Scientific); the use of technologies from the telecommunications industry to advance signal detection and processing for exceptional fluorescence sensitivity in cytometry applications (Beckman Coulter); internal front loading plate holder design opens up the possibility of robot-loading and integrated automated solutions (Beckman Coulter); continuous, air gap delimited stream offering big reductions in sample processing time and innovations in probe design enable microvolume sampling (1μl) from miniaturised assay volumes (IntelliCyt); and laser trimming technology enabling analysis and accurate counting of a wider range of particle sizes in the same sample (Stratedigm). Surprisingly only one vendor (IntelliCyt) chose to comment specifically on the suitability of its data analysis and software package for HT flow, this is telling as better computational analysis tools are still required to handle the quantity and complexity of the data obtained when attempting phenotypic screens using HT flow1.

Several vendors (Fluidigm and Amnis) have reported on new variants or alternatives to flow cytometry with the potential to provide researchers with a greater ability to generate high resolution phenotypic and functional profiles of cells from normal and diseased states and to gain novel insights into cell biology and powerfully expanding the range of cell analysis applications. Unfortunately neither of these unique developments currently supports high throughout analysis. In conclusion, if your main application is true HT flow sample acquisition and analysis at a level that might be useable for primary phenotypic drug screening, your choices remain rather limited today. The IntelliCyt iQue is the fastest platform and currently most screening-friendly (eg supports high density microplates and low volume sample utilisation). Other platforms have slower sample acquisition rates but within workstation set-ups with autosamplers, robotic plate movers and plate hotels could provide significant unattended sample processing. These may offer cost-effective solutions for those unable to invest in faster instrumentation. Overall the prospects for HT flow assays being adopted into drug screening laboratories remains challenging.

Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation; assay methodologies and reagent offerings) to drug discovery and the life sciences. Since its formation 11 years ago, HTStec has published more than 110 market reports on enabling technologies and Dr Comley has authored 53 review articles in Drug Discovery World. Please contact info@htstec.com for more information about HTStec reports.