1. Context

Like other technologies, flow cytometry has been evolving to enable researchers and scientists to get more for less: In this case to get more cell information from less material, which is key in investigating precious clinical samples for example.

Until about 2015, traditional flow cytometers were able to measure as many as 20 or 25 different parameters simultaneously. 

The advent of spectral flow cytometry over the past 5 years has been a breakthrough in the evolution of flow cytometry: Instruments such as the Cytek Auroras or the Sony IDs now allow for the phenotyping of more than 40 or 50 cell parameters simultaneously. 

2. What is spectral flow cytometry?

Spectral flow cytometers are relatively similar to conventional ones. The fluidics systems are essentially very similar but spectral cytometers have a more elaborate optical setup. 

Rather than having a specific bandpass optical filter for the detection of every fluorochrome in or around its peak region, spectral flow cytometers optically capture the full emission of every fluorochrome using a large set of detectors across a wide wavelength range (from 300nm to 900nm for example). The full fluorescent spectrum of each fluorochrome is thus collected, recorded and recognized as a spectral signature that can be differentiated from others. 

In other words, full spectrum cytometers exploit the inherent emission pattern of each fluorochrome to generate a unique spectral signature that can be unmixed from other ones using unmixing algorithms. 

Using traditional cytometers, it was never recommended to design panels including fluorchromes such as PerCP and PerCP-efluor710 simultaneously for example because of high spillover and because of the resulting difficulty in distinguishing one signal from the other. The optical setup and the spectral unmixing algorithms of spectral cytometers are now perfectly able to differentiate both signals.

Although this leap allows for considerably more flexibility in fluorochromes selection, it is still extremely important to determine the appropriate combination of colors, especially when designing large panels. 

As for traditional flow cytometry panel design, the first step is to ask biological questions and determine the cell targets of interest that would help address them.

What are the cells populations and subpopulations of interest? What cell markers do they express/coexpress?

Such questions are important: Material and data for both human and murine models that could be helpful include BD’s handbook of CD markers and the OMIPs (Optimized Multicolor Immunofluorescence Panels). 

Once cell markers are set, the steps below should guide you toward the design or optimization of a spectral flow cytometry panel. They can be performed instantly using EasyPanel, our automated and intelligent panel designer. 

3. How to design spectral flow cytometry panels?

3.1. Minimizing Similarity Between Fluorochrome Pairs and Minimizing Panel Complexity Index

We developed a proprietary Python script that returns the combination of fluorochromes (of a given size, as requested by the user) with:

• the lowest panel complexity score
• the lowest total similarity score (i.e., the lowest sum of similarity indices in the similarity matrix corresponding to the panel).

The similarity index characterizes any given pair of fluorochromes and measures how unique are their spectrum signatures; A similarity index of 0 means that the signatures have no commonality between them, whereas a similarity index of 1 indicates that the two signatures are identical. A panel of N fluorochromes, we will have (N x N)/2 similarity indices.

Fluorochrome combinations containing similarity scores higher than 0.9 between any 2 fluorochromes are rejected.

The complexity index considers eigenvalues of the reference matrix for a combination of fluorochromes. It is defined by the “Condition Number” of the reference matrix used for spectral unmixing.

Such work which could take countless hours can now be performed instantly using EasyPanel. 

For example, the 40-color Cytek Aurora panel designed in OMIP-069 probably took days of laborious trial and error work (manually combining/mixing and matching different fluorochrome combinations) to reach a complexity index of more than 53.

A significantly in silico-superior panel was designed using EasyPanel almost instantly with a complexity score of about 30 and with almost all products (fluorochrome-conjugated antibodies) readily commercially available.

3.2. Matching Fluorochromes to Antigens

We also developed a proprietary script that matches fluorochromes returned in the optimized combination to antigens added by users as per the considerations below, ordered by higher to lower priority:

1. Antigens marked as co-expressed on the same cell population (cell population 1, 2 or 3) are matched to fluorochromes in “Fluorochromes Coexpressed group 1, 2 or 3”. In each of the 3 fluorochrome groups are 5 fluorochromes with minimal total spillover score (or minimal total similarity score or minimal total spillover spread score). 
2. Antigens not marked as co-expressed on the same cell population are matched to fluorochromes in the group “Remaining_FCs” (which are fluorochromes in none of the “Fluorochromes Coexpressed Group 1, 2 nor 3”) or to fluorochromes not yet matched (even if they are in the group “Fluorochromes Coexpressed Group 1”)
3. Antigens marked as “Low Expression Level” or “Unspecified Expression Level” are matched to the group of fluorochromes with the highest brightness scores (or stain index). Antigens marked as “High Expression Level” are matched to the group of fluorochromes with the lowest brightness/stain index.
4. Antigens marked as “Dump Channel” are matched to the Fluorochrome with the lowest brightness/stain index.
5. At this stage, further to all the above-mentioned matching criteria, specific matching of one antigen to one fluorochrome is not done yet, i.e., each antigen is matched to a group of fluorochromes, not a specific one.

Fluorochromes of the optimized fluorochromes combination are queried (vs each of the user-entered antigens and user-specified Species reactivity) in the commercial database to quantify and rank them in terms of “commercial availability”. The commercial availability score of each fluorochrome is the number of different antigens (in the corresponding matching group) for which the fluorochrome commercially exists.

Fluorochromes with lowest commercial availability score are first matched to “eligible” antigens.

About EasyPanel

EasyPanel is an Automated and Intelligent tool for flow cytometry panel design (traditional and spectral cytometers). It is licensed by leading biotech, pharma and academic flow core facilities to design optimized panels of up to 48 colors in seconds or minutes.
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