A day in the life of a PhD candidate

Early detection of sepsis using reactive oxygen species

In this series, the HTRIC PhD candidates will give a little insight in their life during their PhD. Britt is one of our PhD candidates and currently 6 months into her HTRIC PhD. Within this blog she will update you about the latest developments within her research and share her thoughts.

To start, a little bit of background: My PhD project is a multi-disciplinary project involving three research groups within the University of Groningen and the UMCG (department of Molecular Immunology and the department of Biomedical Engineering) and the emergency department at the UMCG (including Acutelines, a biobank for acute medicine). The research aims to develop a fast and sensitive method to detect sepsis by measuring radical production in immune cells. In this blog post, I’ll share some general information and updates on my project.

I’ll start with a brief introduction on sepsis. Sepsis is a life-threatening dysregulated host response to infection, leading to organ dysfunction. The global burden is astonishingly high: 20% of all deaths worldwide are due to sepsis. Every year, more people in the Netherlands die of sepsis than of any type of cancer, myocardial infarction or traffic accidents. Early recognition of sepsis is essential for timely initiation of adequate care and lower morbidity and mortality. However, sepsis recognition in the early phase and prediction of the clinical course are difficult as signs and symptoms may be nonspecific and vary among individuals. At this point, the golden standard in recognition is based on a scoring system assessing the performance of multiple organ systems. And the specificity of this scoring system? An “astonishing” 47%, meaning that you might as well flip a coin to diagnose a patient.

Next to this scoring system, inflammatory markers that can be measured in the blood are used in sepsis diagnosis. The production of radicals (reactive oxygen species [ROS] to be specific) by immune cells is one of the earliest responses in inflammation, and precedes the production of these inflammatory markers by multiple hours. In sepsis, the function of immune cells is disturbed leading to increased ROS production, eventually resulting in organ damage. So why not look at this early response molecule to diagnose sepsis? Well, ROS are challenging to detect due to their high reactivity and low abundance. Fortunately, this challenge has recently been overcome by the development of nanoscale MRI, which allows fast measurement of ROS using fluorescent defects in Nano diamonds. The goal of my project is therefore to develop a fast and sensitive diagnostic test for the early detection of sepsis using this nanoscale MRI method.

In the past six months, I’ve been busy reading a lot of literature and gathering my first results in the lab. Moreover, I developed an optimized method for the isolation of immune cells that are mainly responsible for ROS production, the monocytes and neutrophils. This is important because I need to be able to isolate these immune cells from blood without them getting activated in the process. Next to that, I also had a first try in using the nanoscale MRI method to detect free radical production in freshly isolated neutrophils. It was the first time ever that we tried to do this, and I’m happy to share with you that it worked! I was able to do a “baseline” measurement of these neutrophils, after which I stimulated them with a microbial stimulus to start producing ROS. Over time (20 minutes to be precise), a clear increase in ROS production was measured every 2 minutes, eventually resulting in the orchestrated death of the neutrophils. This is also known as NETosis, but I will not go into detail about that for now. In the attached picture (special thanks to Alina Sigaeva) you can see the outline of a single neutrophil (purple) with the characteristic lobular nucleus (darker areas in the middle), and a diamond (bright yellow/orange spot).

Overall, I’m excited about the potential implications of my research and the positive impact it could have on sepsis diagnosis. I’m looking forward to continuing my work in the upcoming years and exploring the mechanisms behind ROS in sepsis.

Britt Coenen