Go with the flow – a new algorithm streamlines and improves flow cytometry analysis

Key points:

• A new algorithm developed by researchers at the Babraham Institute provides a fast and effective way to reduce errors in flow cytometry data analysis, overcoming a major restriction on harnessing the full potential of the power of flow cytometry in cell analysis.
• The tool, called AutoSpill, addresses the problem of overlapping signals and autofluoresence, which can be misinterpreted as genuine results.
• Researchers can use the tool, available online and through the software package FlowJo, to easily reduce compensation errors in their flow cytometry data.

Flow cytometry is a key investigative tool used in biomedical research, allowing researchers to identify, separate and study cells according to their characteristics, often working with cell samples containing millions of cells at an analysis pace of a million cells per minute. Cell identification is achieved by labelling cells with fluorescent tags. As with personal gadgets and devices, innovation in molecular biology technologies isn’t standing still. Advances in flow cytometry have allowed scientists to gather data on a growing number of parameters, simultaneously detecting over 30 different tags at a time to allow more sophisticated analyses and much deeper levels of insight. However, while flow cytometry equipment has been updated, the accompanying computational requirements have received less attention, until now. AutoSpill, an algorithm developed by researchers at the Babraham Institute and the VIB Center for Brain Research, brings data processing in line with state-of-the-art machines, simplifying data analysis and increasing accuracy. The new technique is published in Nature Communications today.

Immunology programme senior group leader Prof. Adrian Liston, explained: "Flow cytometry is a foundational technology across many different biomedical research areas, and is a key diagnostic tool in immunology, haematology and oncology. Despite the technical progress over the past decades, the technology has been held back by the mathematical processing of the data. Our new approach reduces error by 100,000-fold, making research and diagnostics more accurate. The collaboration with FlowJo has enabled us to instantly reach 80,000 users. It is very gratifying to see computational biology have a direct and real impact on research and diagnostics."

Using multiple fluorescent signals raises a key issue in flow cytometry called spillover. Spillover occurs because each tag, called a fluorophore, emits light within a range of wavelengths, giving it a unique colour. When multiple fluorophores are used, the signals begin to overlap. To accurately distinguish between two distinct fluorophore signals, researchers must process their data to compensate. Because flow cytometry uses so many different colour tags on each cell, the spillover between colours quickly accumulates, limiting scientists’ power to draw reliable conclusions from their results. The processing of data to remove the spillover between the different colours, known as compensation, is necessary for all flow cytometry experiments. Current methods require many hours of manual work, but AutoSpill reduces the process to minutes.

Dr Rachael Walker, Head of the Institute Flow Cytometry facility, commented: “The new AutoSpill Fluorescence Compensation algorithm is a great tool for quick, simple and accurate compensation. It allows compensation to be accurately calculated on samples where the traditional algorithm is difficult to use. AutoSpill’s integration into the FlowJo post-acquisition software highlights the importance of this new compensation method.”

Another limitation of flow cytometry is autofluoresence, fluorescence produced naturally by cells. The removal of these artefacts by AutoSpill is particularly useful for cancer biologists as tumour cells are high in autofluorescence, which can confuse identification of the type of tumour cell present. By solving these sources of error, AutoSpill can help remove false positives from cell analyses, ensuring more accurate data interpretations.

AutoSpill is available through open source code and a freely-available web service. AutoSpill, and a complementary related tool, AutoSpread, are also available in FlowJo v.10.7. Dr John Quinn, Director of Science and Product Development, FlowJo added: “AutoSpill & AutoSpread have been a revelation for FlowJo users. Compensation has long been one of the most perplexing aspects of cytometry, with the most critical requirement being pristine compensation controls collected for each and every parameter in an experiment. Overall, the combination of these two tools makes compensation both easier and more robust. As an indicator of the popularity of this new approach, the webinar held in conjunction with Nature to introduce AutoSpill / AutoSpread in FlowJo has been viewed over 400 times after the initial live event. We at FlowJo believe the AutoSpill / AutoSpread approach will be the primary means of approaching compensation moving forward.”

Immunology expert Prof Adrian Liston elected Fellow of the Academy of Medical Sciences

Key points:

Postdoc job opportunity in the lab

Happy to say we have a great job opportunity to join our lab! The position is for a bioinformatics or datascience postdoc position, starting in the Babraham Institute. The position is to lead the data analytics of the Eximious Horizon2020 project. An amazing opportunity to unravel the real-world link between environment and immunity, using the largest and most comprehensive datasets to yet be generated. I welcome applications from thoughtful scientists willing to learn the biology and search for the most appropriate computational tools to apply. Time is provided to learn and develop new skills, so consider applying even if you don't perfectly align to the project. Come join us in Cambridge! 

Apply here. 

How to keep your virtual lab meeting from being just another Zoom call

Terrific write up at Cell Mentor on virtual lab meetings. I had the pleasure of discussing our zoom retreat with Claudia Willmes for the article, and could describe all of the fantastic activities designed by our lab social team to keep everyone engaged. The whole article is worth reading, but a highlight is the call-out to Ruben's excellent escape room:

Escape games were already a popular activity for lab outings before 2020, but they have been taken to another level during the pandemic. There are several escape room companies that offer virtual adventures. Liston's group took this a notch further and went for a custom-made game designed by lab member Ruben Vangestel.

"The custom escape Zoom was based in the lab cold room, with a series of puzzles needed to escape," Liston says. "It had the typical ‘communal problem solving' aspect of an escape room, but by using pictures of our lab as the setting and cameos from lab members giving clues, it really created a warm feeling of togetherness. A reminder of the space we used to share, and the common experiences that unify us."

A cynic's guide to getting a faculty position

I gave an academic caeer talk yesterday at the University of Alberta, and on request from the students I am putting the talk online. These are my personal thoughts on how the job selection process works for independent research positions in universities or research institutes, based largely on my experience, the experience of my trainees going through the process and my observations of behind-the-scenes job committee meetings. I am sure that there is enormous variation in experiences, and that systems work differently in different places: hearing the perspective of many people is more valuable than just hearing the perspective on one.

I'd also just note that this is not an endorsement of the system as it exists. There are aspects of the system that I dislike and actively work to change. But I still think it is valuable for job seekers to understand the system, warts and all, rather than believing in an aspiration system that has yet to materalise. I often hear from trainees that they career training is largely directly to non-academic careers, and they rarely hear how the academic pathway works. So, with a little too much honesty, and an expectation of landing in hot water, here is my attempt to open a conversation:

Thesis acknowledgements

It is so lovely to read the words of graduating students in their thesis acknowledgements. I've seen them learn and grow over the years, increase in skill and resiliency, reach depths they didn't know they have. And here they are, just leaving on to their new adventure and they stop to write kind words back to us.

These from (soon to be) Dr Steffie Junius:

Next, I would like to thank my co-promotor Prof. Adrian Liston. While on paper you’re addressed as my ‘co-promotor’, I truly perceived this as rather having two full promotors who both guided me in their own way, complementing each other. I still remember the evening in Boston when I received the email with an offer to start a PhD at your lab. The thrill to be accepted in such an environment of excellent science made me excited to become the best possible immunologist I could be. Throughout this PhD you have guided me with your advice and mentorship. Especially on the dark moments, you always were able to push me in the right direction and to follow through even when I did not know how. As PhD students, we always think the science is the most important part of a PhD, but you made me understand that personal development is just, if not more, important to becoming truly successful. Thank you for your advice and guidance over the years. The lessons you taught me will stay forever with me throughout my career. 

Thank you Steffie, it has been wonderful to be part of your journey. Enjoy the next stage of your career!

Mo Siewcharran Prize to Tenmei

Lovely news that the talented Tenmei won a Mo Siewcharran Prize at the FAB Prizes, discovering new talent and promoting under-represented authors, for her beautiful illustrations in "Battle Robots of the Blood". Gorgeous detail throughout, but my favourite picture is perhaps the evocative eyes staring at an embarrassed Tim. Prize well deserved!

Battle Robots of the Blood reading

Me and Hayden read "Battle Robots of the Blood" together.

Covid-19 vaccines: Discussing how they work, the second dose and timing with Babraham Institute immunologist

Vaccination offers by far the best – perhaps only – route out of the Covid-19 crisis.

And, with two vaccines now being rolled out in the UK and a third available in the spring, progress is being made on delivering this vital protection.

Immunologist Prof Adrian Liston, at the Babraham Institute, tells the Cambridge Independent: “Vaccines all work in a fairly similar way. The key outcome we want are antibodies that bind the infecting virus and either block its entry to cells or flag it for destruction."

read the interview with me here

 

As an aside, back in May 2020 I was interviewed on the prospects of a COVID vaccine. Unlike some other experts, I argued that the first COVID vaccines would likely be designed, tested and approved by the end of 2020, and that the regulators would accept a vaccine efficacy rate as low as 50%. Fortunately my optimistic forecast proved true, with the FDA issuing guidence in June that they would accept efficacy rates of 50% and above, and the first approvals occuring in December 2020. Hopefully my optimism on the dosage change proves equally prophetic!

European “ImmunAID” project for the diagnosis of rare autoinflammatory systemic diseases launched in Belgium

The project wishes to diagnose rare autoinflammatory systemic diseases through the identification of biomarkers

In December 2020 a new project has been launched in the University Hospitals Leuven. The ImmunAID project aims to identify new tools for the diagnosis of systemic auto-inflammatory diseases (SAID). SAID are a complex and evolving group of rare diseases characterised by extensive clinical and biological inflammation. These conditions are caused by a dysregulation of the innate immune system leading to a release of immune cells and mediators provoking fevers, tissue and organ inflammation and damage.

Sometimes it is difficult for the physicians to make a correct diagnosis, since the main symptoms of these diseases (such as fever, rash, joint pain, etc.) are also present in many other conditions. Thus, a patient may have received on average up to 5 inappropriate or ineffective treatments before being properly diagnosed, having a great impact on their health and quality of life. The aim of ImmunAid is to understand the mechanisms that drive the pathology in order to provide better diagnosis and care for patients with these rare but potentially devastating diseases.

An unprecedented body of clinical and biological data in the field of SAID

This new project aims to find new and more effective ways to diagnose SAID. While it is already known that some SAID are due to specific genetic mutations, a large number of SAID can only be detected by a set of clinical signs and symptoms and after other diagnostic possibilities have been excluded. Since SAID are rare conditions, a large group of patients suffering from various SAID is being recruited throughout Europe. As such, the ImmunAID cohort represents a very important tool for researchers defining biological fingerprints, or biomarkers, specific to distinct SAID.

The team expects to find a set of biological features common to all SAID, which will allow to quickly confirm or refute the diagnosis of suspected autoinflammatory syndrome. In addition, for each SAID, a list of characteristic biomarkers and an algorithm will be generated to allow the physician to make an appropriate diagnostic assessment.

In order to achieve the project's objectives, biological samples collected from the patients will be analysed in a European-wide research network by set of state-of-the-art technologies and will generate an unprecedented amount of data (genomics, transcriptomics, proteomics and microbiome). Simultaneously, other analyses will focus on immune cells, molecular mechanisms and specific agents of the immune system (cytokines, etc.). All data generated will be subjected to artificial intelligence and modelling analysis.

Prof. Carine Wouters, paediatric rheumatologist at the University Hospitals Leuven, is highly committed to the success of the project "We are delighted and proud to be able to work with ImmunAID partners as it represents a unique opportunity for the European scientific community to advance research in an important field of rare diseases that can only be tackled at large scale. We will do our best to come up with meaningful results that will improve patients’ diagnosis and medical care.”

Leuven teams are the forefront of the project

The teams of the Leuven University Projects are at the forefront of the project. The activities carried out in the Belgian centre will be two-fold. First, the team from professor Carine Wouters and professor Steven Vanderschueren will be in charge of recruiting patients suffering from monogenic SAID (FMF, CAPS, TRAPS, MKD) or genetically-undiagnosed SAID (Still disease, neutrophilic dermatosis, Schnitzler syndrome, Takayasu arteritis, Kawasaki disease, Behçet disease, chronic osteitis, recurrent pericarditis and chronic systemic inflammation of unknown origin).

Second, professor Wouters, professor Patrick Matthys and professor Paul Proost from the Rega Institute and KU Leuven department for Microbiology, Immunology and Transplantation will be involved in the biochemical and biological analysis of the samples. The team of Carine Wouters and Patrick Matthys will apply their extensive knowledge on Natural Killer cells to identify and characterize their possible altered activity in SAID patients. On the other hand, the team of Paul Proost will study whether modifications of messengers of the immune system (cytokines and chemokines) in patients play a role in regulation of the inflammation processes. The team of professor Stephanie Humblet-Baron and professor Adrian Liston will analyse in-depth the immune cellular compartment of the blood of affected patients in addition to genetic investigation in order to identify new genes responsible for SAID.

These activities are intended to gain insight into the mechanisms triggering the aberrant behaviour of the autoinflammation process. The results will be pooled with other analyses from other European research laboratories to help identify biomarkers of the diseases and possible therapeutic interventions.   

Regarding the ImmunAID project: ImmunAID is a research project (www.immunaid.eu), which aims to identify a set of disease-specific biomarkers to confirm the diagnosis of SAID. ImmunAID is implemented by a large consortium (25 partners in 12 European countries) and has been funded with € 15.8 million by the European Commission. The ImmunAID project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 779295.