It still works okay in most cases, it is just that the small errors start piling up when you are making 250+ calculations on a high parameter dataset.ĪutoSpill is actually fairly simple at heart: Traditional compensation defines a positive and a negative population, finds the slope and uses that as the spillover matrix. For decades we've been building more-and-more expensive machines, and haven't updated the basic maths that the machines run on! How does AutoSpill work? It is a huge surprise to me, but with the enormous effort over decades to add extra lasers and new flurophores onto machines, the mathematics behind compensation hasn't been updated since 1993, where it was designed for 3 colour flow on computers with 100,000-fold less capacity. AutoSpill is designed to run through the same operations that a skilled flow user does, just faster. But always remember - the compensation can still only be as good as the quality of the single colour controls! If you want to run 28 colour flow cytometry on a 28 colour machine, you can spend hours-upon-hours compensating your data by hand, or 2 minutes with AutoSpill. (if you are using the script, you can reduce the error further - we stop the improvement at this point because it is functionally perfect). This means the large over/under-compensation effects can be completely removed from your data. What does a "perfect" spillover matrix mean? An error reduction of 100,000-fold on average, to the point where error is practically zero. AutoSpill creates a perfect spillover matrix. In high dimensional flow cytometry traditional compensation errors create artefacts. Here are a few examples of the error reduction you can get with AutoSpill: If you program your flow cytometry analysis in R, we have also released the AutoSpill full code, so you can add this to your bioinformatics pipeline. Dr Carly Whyte made this easy two minute tutorial: So how does AutoSpill work? If you just want to compensate your data, simply upload your single colour controls to and then copy the spillover matrix to your flow cytometry program of choice. It is thanks to AutoSpill that we can push machines to their max colours, and actually get good quality 40+ parameter flow cytometry data. AutoSpill is a beautiful example of what maths can add to #immunology, led by the talented Dr Carlos Roca. AutoSpill is a novel method for applying compensation to flow cytometry data, which reduces the error by ~100,000-fold. I am really thrilled to release AutoSpill onto BioRxiv.
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