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Flow cytometer manufacturers provide technical specification sheets (tech specs or spec sheets) that describe the instruments&#; key performance characteristics, and these documents can contain a wealth of information for those interested in purchasing a flow cytometer. Comparing various tech specs, however, can be challenging because their values may have been calculated in different ways, despite sharing the same terminology.
The purpose of the spec sheet is to help you identify design attributes of the flow cytometer, such as performance, size, environment, and software, to determine if the instrument is a good fit for your research.
Technical specifications can be used as a basis for comparison, helping you assess the value of different instruments for the price. The spec sheet is also a guide to the performance that the manufacturer will warrant. For this reason, you should have a good understanding of the stated values and how they pertain to your intended use of the instrument. When using the spec sheet as a comparison guide across platforms, be inquisitive. There are many performance values that appear comparable across instruments but in reality are quite different. A specification is derived from a specific test or calculation, but these tests are not standardized across instrument developers and may be misleading in a side-by-side comparison.
Comparing various tech specs can be challenging because their values may have been calculated in different ways, despite sharing the same terminology. This white paper provides helpful hints about how to decipher the variations.
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Sections within a technical specification sheet vary from one manufacturer to another, adding additional variability. Commonly published categories of information are described in Figures 1&#;8 using the spec sheet for the Invitrogen Attune NxT Flow Cytometer as an example. Key specifications that require special attention during flow cytometer evaluations are discussed, including helpful hints about how to decipher the variations.
Figure 1. Fluidics. The fluidics system of a flow cytometer transports the sample from the sample tube to the flow cell. Once through the flow cell (and past the laser and detector), the sample is transported to waste. The fluidics section in a spec sheet provides information about the sample, volume, flow rates, flow cell, and fluidic capacity.
Figure 2. Optics. As an analysis platform, flow cytometry relies on interrogation of individual cells by laser light and the collection of the resulting fluorescence and scatter. The optics system handles illumination and light collection within the instrument. The optics section in the sheet provides overviews of optical specifications such as laser type and power, laser profile, alignment, and photomultiplier tubes (PMTs).
The power specification is most frequently defined as the laser&#;s output as published by the laser manufacturer (Figure 2). However, the power specification stated does not necessarily correspond with the power that is actually delivered at the point of interrogation, because light losses in the optical components between the laser and the flow cell can be large. This light loss varies from system to system. High light loss means that some of the laser&#;s power is not being fully utilized in the experiment; reduced light loss means higher laser intensity on the flow cell, leading to greater excitement of the fluorophores and greater sensitivity. Because of this variability, a higher number for laser power indicated on the spec sheet does not mean that the instrument is more sensitive than another.
Be cautious in how much you rely on this specification when comparing instruments. Most manufacturers don&#;t report the actual power that is reaching the flow cell, but this is where the comparison should be made.
This specification, which is not reported by every manufacturer, refers to the number of pinholes that determine if the fluorescence signal generated by each laser is separated at the detectors (also called PMTs). You should know if the system&#;s lasers are spatially separated by internal pinholes or whether the system is collinear. Pinholes allow for maximal excitation of fluorophores and minimal crosstalk between the laser lines. Several flow cytometer manufacturers utilize collinear lasers. When lasers are aligned in a collinear fashion through a single pinhole, there is a collection of signal from more than one laser in the same optical path. This configuration means that the response of several dyes excited by different lasers is measured by the same detector, which can affect compensation values and lead to difficulty in analysis. Other risks in this design include susceptibility to alignment issues (increased coincidence rate if the beams are not exactly collinear) or reduced sensitivity for dim labels. An alternate design is a spatially separated system (Figure 2). This configuration has several benefits, including resistance to alignment problems, more choices for laser colors, and improved compensation for multicolor panels.
Be sure you have a clear understanding of the type and quantity of lasers that are assigned to each pinhole, and ask if the system under consideration uses spatially separated or collinear lasers.
Figure 3. Performance. Of the typical specifications published widely among manufacturers, the performance section of a spec sheet contains several common features, such as MESF calculations, data acquisition rate, and parameters for forward scatter (FSC) and side scatter (SSC).
The event rate is the physical count of the cells or particles as they pass through the instrument&#;s interrogation point. Two basic yet quite different methods are used to determine what the value is, even though this value is generally cited in the same way (i.e., events/sec). The two methods for calculating event rate are:
It&#;s important to understand how the manufacturer arrived at this value in the spec sheet.
The first method is theoretical and disregards the rate of coincidence and other instrument design features such as the system fluidics. Thus, while the electronics may be able to process events at 10,000&#;100,000 events/sec, this event rate may correspond to a coincidence rate well above the generally accepted 10% rate limit according to Poisson distribution. This way of presenting the specification for event rate has become more popular in recent years due to the advent of faster (though not necessarily higher-quality) electronic circuitry; however, event rates calculated this way don&#;t take coincidence into consideration.
The second method of determining an instrument&#;s event rate is based on a 10% level of coincidence, which is more relevant to researchers (Figure 3). Lower coincidence rates indicate higher data integrity. Therefore, using this method best represents acceptable coincidence at an actual event rate that can be used to generate high-quality data. Users can be more confident that the data will be within acceptable coincidence rate levels at a given event rate when manufacturers report their specification for event rate using this method.
When deciding between instruments, be sure that you know how the manufacturer arrived at the specification for event rate. If this value was calculated using the first methodology (speed of electronics only), you should carefully examine the coincidence rates when running your samples.
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Carryover refers to the amount of an original sample that carries over and contaminates the next sample, resulting in data inaccuracies (Figure 3). The percent carryover specification is usually measured by acquiring a fixed volume of sample, followed by acquiring a fixed volume of a particle-free, buffer-only solution such as phosphate-buffered saline (PBS). Events representing the contaminating cells are then identified in the buffer only solution. Many manufacturers run a specific cell line or set of beads to determine this carryover value under defined conditions.
It&#;s important to find out what these defined conditions are and what sample was used to determine a carryover value. For example, the number of washes or the size or type of particles or cells used for the specification test may be vastly different from what a researcher would use. Ask the manufacturer how they arrived at their stated carryover value. Knowing how different manufacturers calculate this value can help you make a direct comparison of carryover just by referring to the specification.
Figure 4. Software. The software section of a spec sheet enables researchers to identify key capabilities and features built into the system. This section also indicates the extent of functionality, user-definable features, maintenance features, and user account administration. Software features vary among manufacturers and some systems have unique, exclusive options.
Figure 5. Quality and regulatory. Quality and regulatory specifications indicate manufacturing integrity, warranty, field engineering procedures, and ISO credentials.
Figure 6. Data management. This section is key for successful computer installation and setup. Specifications within this section detail RAM, hard drives, and FCS format.
This specification is often overlooked, yet can be important to the lifetime value of your instrument because optical alignment and fluidics are highly coupled to these values (Figure 7).
Determine if the lab remains at relatively constant temperature and if the instrument will be used in a variety of places. Instrument performance is tested and warranted only within the specified temperature range, so be sure to consider the conditions of operation.
Figure 7. Installation requirements. A typical requirements section denotes information about the environmental impact of the instrument, operating conditions, and footprint.
Consider whether the instrument will fit in the desired space or area available in a fume hood. Be sure to include the space requirements for accessories such as an autosampler and find out if any space will be necessary for the fluidics (Figure 7). An external fluidics system is common for many flow cytometers and can add substantially to the overall space needed for the instrument. If the instrument will be moved periodically, think about how difficult this might be. Be aware that not all benchtop instruments have the same space requirements or portability.
When a demo is being performed, request to see the full system with all components set up so that you&#;re able to directly compare the space needed for each system.
Plate-speed values are typically defined as the time required to complete analysis of a 96- or 384-well plate (Figure 8). There are two aspects to this definition: one is the sample volume, the other is the sample-processing rate. Be aware that the time value reported in this specification may represent a sacrifice in data quality. Also, be sure to get clarity on what volume of sample was analyzed and what sample processing rate was used to determine the value in this specification. Some manufacturers choose to show a specification figure that minimizes plate times by collecting low-volume samples. However, in practice this could require samples to be highly concentrated, resulting in data quality issues like higher coincidence and abort rates. Conversely, if samples aren&#;t concentrated enough, a low volume may lead to a lack of enough events to be statistically significant.
The sample-processing rate (the rate at which the sample is being introduced to the fluidics system) can also affect the data quality. In flow cytometers that rely solely on hydrodynamic focusing, the sample is spread across a wider core stream as the flow rate increases. Higher sample rates produce greater variability, less precise measurements, and compromised data quality. With instruments that utilize acoustics-assisted hydrodynamic focusing, the cells remain tightly aligned in the center of the stream regardless of the sample rate, resulting in less signal variation and improved data quality. Therefore, if you choose to increase the sample input rate in order to lower plate times, you should use a system that offers acoustic focusing to avoid loss of data quality.
Another variable in calculating the plate analysis speed is whether the probe is rinsed between samples. Probes that are not rinsed introduce the potential for higher carryover&#;a tradeoff that should be considered before making a decision to run experiments at the given specification for plate analysis time.
Make sure you understand the tradeoff in data quality that may be incurred to achieve the times the manufacturer represents with this specification.
Figure 8. Sampler specifications. A sampling device is an accessory to the flow cytometer. Specifications of this device include acquisition time, carryover, and mixing method. Additional information regarding compatible plate types and fluidics options may also be included.
Always inquire about the tests associated with pertinent specification values, so you can be confident that you&#;re making accurate comparisons between the features of the instruments under consideration.
Figure 2. Cerebral Organoids Generated with the STEMdiff&#; Cerebral Organoid Kit Are Transcriptionally Similar to Those from Published Protocols
RNA-seq data was extracted from a publication (C Luo et al. Cell Rep, ) that generated cerebral organoids (open blue circles) and compared this transcriptional profile to that of cerebral organoids generated with the STEMdiff&#; Cerebral Organoid Kit (filled blue circles). Principal component analysis was performed on these data. The cerebral organoids from the STEMdiff&#; Cerebral Organoid Kit cluster together, and cluster with the previously published cerebral organoids. The first principal component accounts for the majority of variance seen (PC1; 80%) and distinguishes the cerebral organoid samples from the hPSCs (green circles). The second principal component accounts for only 9% of the variation, and highlights the modest expression differences between cultured organoids and primary embryonic fetal brain samples (19 post-conceptional weeks, brown circles).
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