5th March – 9th March 2018

Multicolour Flow Analysis to the Next Level

Alex has recently been on his travels, this time attending a Multicolour Flow Analysis course at the famous Lorentz Center in Leiden, Holland. This course enabled Alex to gain an important insight into the different methods of analysing complex flow cytometry data.

The course was an intense 5 days for Alex and a steep learning curve, one which he benefitted from immensely as can be seen through the different approaches he is taking to analysing data for his PhD and subsequent publications.

The first day of the course involved talks from: Leo Koenderman (University Medical Center, Utrecht), Kristiaan Wouters (Maastricht University), Antoine Pacheco (Beckman Coulter), Selma van Staveren (University Medical Center, Utrecht), and Boudewijn Lelieveldt (Leiden University).

Prof. Koenderman discussed the many different algorithms available (t-SNE, FlowSOM, SPADE, DAMACY, FLOOD, ECLIPSE) for analysing multicolour flow cytometry data. Dr Wouters then described the importance of immune-phenotyping with flow cytometry and how these algorithms can support this type of data analysis. Antoine Pacheco went on to explain the mechanics of flow cytometry and how data is produced and what affects data outputs. Selma van Staveren provided a talk on the importance of data transformation and considerations to make with compensating data (it was concluded that data is never optimised with or without compensation, and the room was split on when to compensate data (before, during or after). Prof. Lelieveldt then finished the talks by explaining the uses of the t-SNE algorithm for multivariate analysis. The day concluded with ‘hands-on’ data analysis using the t-SNE algorithm.

The second day of the course included talks from Jacqueline Cloos (VU University) & Yvan Saeys (Ghent University).

Dr Cloos opened the day by discussing rare cells and the difficulties within flow cytometry for identifying these cells, particularly in large populations. This talk was followed by Prof. Yvan Saeys who actively works to create algorithms for analysing this kind of data, and explained the role of FlowSOM and SPADE algorithms in relation comparative methods in the analysis of flow cytometry data. The day ended with ‘hands-on’ data analysis using the FlowSOM and SPADE methods with some workshop participants presenting data and comparing the three algorithms covered so far.

The third day consisted of talks from: Nienke Vrisekoop (Utrecht Center for Quantitative Immunology), Oscar van den Brink (TI-COAST), Paul Harrison (Birmingham University) & Jeroen Jansen (Radboud University).

Dr Vrisekoop and Dr van den Brink opened the day by gauging feedback on the various methods covered in the workshop. Dr Harrison followed with a talk around the use of flow cytometry in clinical trials for monitoring the immune system in research conducted at the Institute for Inflammation & Aging in Birmingham. Dr Jansen finished the talks by explaining the complexity of performing analyses using DAMACY, FLOOD and ECLIPSE highlighting the pros and cons of each method. The day finished with an extended ‘hands-on’ session analysing data using the aforementioned algorithms.

Day 4 consisted of one talk by Jolanda Brummelman (Humanitas Research Hospital) who provided an interesting talk on the complexity of analysing multicolour flow cytometry panels, especially when there are 27 parameters being assessed! The rest of the day consisted of further ‘hands-on’ data analysis with all of the algorithms covered in the workshop whilst preparing to report findings back to the group on the final day.

The final day of the workshop consisted of interactive sessions where workshop participants provided feedback on each of the algorithms. The workshop critiqued each algorithm and its suitability for different types of analysis depending on the variables involved and the research question wanting to be addressed. The workshop closed with an afternoon session whereby workshop participants contributed to a consensus document about each of the algorithms for flow cytometry data analysis. The workshop organisers hope to produce a consensus statement as a result of the workshop and intend to hold a follow-up workshop to clarify the consensus further.

Alex benefitted immensely from this course and would like to extent his immense gratitude to the course organisers and participants for the help he received and how welcoming everyone was. In particular, Alex would like to thank Leo Koenderman, Selma van Staveren & Paul Harrison for answering some long-standing queries with regards to his data. Alex has a lot to think about with his data analysis going forwards and has established some great contacts for future research whilst placing Lincoln on the map, yet again!