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This question lies at the heart of a study, currently in preparation for publication, led by University of Melbourne’s Dr Laura Cook, Senior Research Officer at the Doherty Institute. Dr Cook and her team are on a mission to unravel the hidden dynamics of the immune system in critically ill patients in Intensive Care Units (ICU), with the help of state-of-the-art technology – flow cytometry.

In the evolving landscape of medical cell-based diagnostics, one tool stands out for its precision and versatility — flow cytometry. Flow cytometry is one of the original single-cell analysis platforms, a powerful tool for the study of cell biology.

Used in various fields of biomedical research, especially in immunology and oncology, this technique enables scientists to examine multiple cell characteristics simultaneously, including cell types and numbers, levels of protein expression, viability status (live, dying or dead cells) and the levels of cell proliferation (which occurs during immune responses).

Dr Cook uses flow cytometry to investigate the complex behaviour of immune cells (her specialty area being CD4+ ‘helper’ T cells, particularly regulatory T cells) in infectious and autoimmune diseases. 

“The basic principle of flow cytometry involves passing cells, one at a time, in a fluid stream through a laser beam,” said Dr Cook.

“As each cell passes through the laser, it scatters light and emits fluorescent signals based on the markers or molecules it contains, including fluorescent markers added by the researcher that bind to proteins of interest in the cells. The emitted fluorescence is then recorded and analysed to provide information about each cell.”

In her study on the early immune changes in ICU patients, Dr Cook and colleagues in the Department of Critical Care at the University of Melbourne used flow cytometry to assess innate immune cells’ ability to combat invading pathogens and activate other immune cells.

One of the challenges with analysing early responses of the innate immune system is the need to examine patients’ blood samples within just a few hours of collection, to ensure the cells of interest remain functional. Thanks to their collaboration with the Royal Melbourne Hospital’s ICU nearby, the team managed to process samples within 30 minutes of collection.

“By combining flow cytometry-based immune cell and plasma analysis with patient clinical data, we were able to detect early changes in the immune system during critical illness that distinguished trauma and severe COVID-19 patients” she said.

“This is significant as this approach could help identify biomarkers related to illness outcomes, revealing new targets for initial treatment intervention to prevent immune-related complications.”

In 2024, the MEGA-SCORES research group, of which Dr Cook is part, will commence a Phase 1 clinical trial to test a new treatment for sepsis patients in ICU, with the help of the flow cytometry-based blood assays.

In parallel, Dr Cook is working on the development of predictive models to improve treatment and patient care, with the help of the technology.

“By integrating data from flow cytometry, plasma protein levels, patient symptoms and other clinical analyses, we aim to validate computational and mathematical algorithms to guide treatment approaches, bringing innovative technology to the patient’s bedside,” she said.

Researchers are increasingly turning to flow cytometry-based Activation Induced Marker (AIM) assays to study protective adaptive immunity (the body's ability to remember and defend against specific pathogens after an initial encounter). This method is a valuable tool for gaining deep insights into memory T cells derived from prior pathogen exposures or vaccinations and understanding how they initiate rapid and effective responses upon encountering these pathogens again.

In a review published in Immunology and Cell Biology, Dr Cook and her co-authors outline the diverse applications of AIM assays in both research and clinical studies, including their role in studying immunity against SARS-CoV-2 and in broadening our understanding of immune responses following infection or vaccination.

“I am now working with the latest technology, called spectral flow cytometry, which allows us to measure more proteins in each sample, compared to conventional flow cytometry," said Dr Cook.

“As an immunologist, I started using flow cytometry as an undergraduate student and have witnessed the rapid evolution and uptake of this technology across various areas of research. From examining early immune responses in ICU patients to unlocking the secrets of memory T cells, this technology has remarkable versatility to help us study the immune system and design treatment approaches to improve patient outcomes.”

At the Doherty Institute, scientists have access to the latest equipment and expert technical advice for designing and executing flow cytometry experiments at the Melbourne Cytometry Platform, located within the Institute, conveniently close to the researchers’ labs.

Content of this article was presented by Dr Laura Cook at the Oz Single Cell conference in Melbourne, October 2023, where she was invited to talk about her work with flow cytometry and how it can be used to ensure that cellular DNA/RNA analyses incorporate a measure of the cells’ functional abilities.