Automated Spectral Panel Design
Advances in both flow cytometry reagents and instrumentation, including the advent of spectral flow cytometry (Cytek Aurora, Sony ID7000 flow cytometers…), allowed researchers to perform larger and more complex multicolor experiments. This has been a significant progress in the field as it enabled the more accurate investigation and definition of cell populations while requiring smaller sample volumes.
However, such advances and increased panel complexity created a new need: Automated and Intelligent tools for flow cytometry panel design. In fact, one of the biggest challenges in multiparameter flow cytometry is selecting the right combination of fluorochromes and judiciously matching them to antigens so that the need for compensation is minimized while the quality and accuracy of the data are not compromised.
Although there are some excellent online tools for guiding multicolor flow cytometry panel design, none of them is able to automatically suggest concrete optimized panels based on users inputs and requirements, as of April 1st 2022. Current tools such as FluoroFinder consist of commercial catalogues showing users which fluorochrome-conjugated antibodies are commercially available for each channel/filter, but it is ultimately up to users to design the panels themselves and to make the selections. Although these tools also feature spectrum viewers and automated notification systems informing users whenever their fluorochrome selections are highly suboptimal, they still require significant time and experience from users to complete panel design. Moreover, generated panels are by no means optimized and their quality is not guaranteed in silico. In other words, panels quality are not quantified nor ranked, which leaves room for much better selections of fluorochrome combinations and their matching to antigens.
To address such unmet need, EasyPanel was developed as an automated and intelligent flow cytometry panel designer.
Spectral Panel Design Considerations
- Minimizing Similarity Between Fluorochromes and Panel Complexity Indices
Spectral Signature (normalized emission) of the different Fluorochromes in each of the channels of spectral cytometers (including the Cytek Auroras and the Sony ID7000s)
Similarity Matrix between all Fluorochromes for each spectral cytometer.
A similarity score is an angle between 2 vectors in multi-dimensional space. Each Vector represents the spectral signature of a given fluorochrome in the different channels, the number of channels being the number of dimensions of the vector.
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; 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.
- Matching Fluorochromes to Antigens
Fluorochromes brightness and stain index scores as shared by vendors.
Commercial Database of fluorochrome-conjugated antibodies used for flow cytometry
Alias for antigen names. This helps with matching fluorochrome-antigen suggestions in the commercial database
We 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:
- 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 similarity score.
- 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”)
- 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.
- Antigens marked as “Dump Channel” are matched to the Fluorochrome with the lowest brightness/stain index.
- 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.
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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