by Dr James McCracken

Most of us will have some sort of knowledge or experience of flow cytometers, even if it’s just at the level of knowing that they are large instruments that have their own lab space and a dedicated team of people to run them and that they usually seem to be used for analysing white blood cells. This article explains how they can now be used in many other areas of study and that advances in technology mean that they are becoming items that individual lab groups can think about owning and running.

Flow cytometry is one of the most powerful tools in a researcher’s toolkit. By identifying cells based on certain molecular characteristics, flow cytometry can be used to isolate information about specific cell types from organs or bodily fluids that contain a variety of cell types that are otherwise difficult to separate. Flow cytometry can be performed at speeds of thousands of cells per second allowing substantial amounts of data to be collected in a reasonable amount of time. This allows for cells to be characterized at an individual level, and even sorted into tubes or plates in pure populations for further testing, culture or other uses.

Before being loaded into a flow cytometer, cells are labelled with fluorescent probes such as covalently tagged antibodies to mark cells that have specific proteins on their surface. Cells in suspension enter through the flow cytometer fluidics arriving at a focused laser or lasers. These lasers excite the fluorescent tags, and then emitted light, as well as laser light scattered by the cell itself, is measured with the cytometer’s electronics. Based on the specificity of the probes, different cell types can be identified and collected from a heterogenous sample in a process known as fluorescence-activated cell sorting. These measurements of fluorescence levels in individual cells gives the scientist discrete information about the relative level of a protein on the cell as well as allowing for separation of cells of interest based on combinations of protein markers. Flow cytometry data can be used for identifying cell populations in human samples or animal models, and is also used in diagnostic tests for a wide variety of clinical applications including HIV monitoring, cancer diagnosis and organ transplantation. One of the biggest advantages of flow cytometry is the flexibility of applications that are possible. It has become popular in many areas of laboratory science because it can be moulded to fit the needs of almost any research project involving single cells. Sorting is also used for generating single-cell clones of induced pluripotent stem cells (iPSCs), a rapidly growing field of inquiry.

The miniature future of cytometers

In recent years, there have been several improvements in flow cytometers across the industry that have improved workflows through automation and innovation. This has improved efficiency, and the accompanying software is becoming better at troubleshooting errors as well as visualizing data. As a whole, flow cytometers have become more sensitive over time; this, however, brings a need for additional attention to sample preparation, as greater instrument sensitivity makes it more likely that small debris particles will be detected as a result of scatter. There have been two key areas of improvement in flow cytometer design. First, the advances in software guided tools have increased usability, making set-up easier and experimental design faster. Second, with the reduction in size of the devices, they have become more compact and easier to maintain. These changes can help drive the future of flow cytometry towards more accessible solutions for more labs.

Finding the basis of neurological diseases

Partnering with the life sciences community is paramount to what we do. Our partnership with Dr Christopher Bare (who has nearly 30 years’ experience in the biosciences industry) over the years has yielded valuable feedback for us to make enhancements. His history of leadership experience in academia with graduate students using flow cytometry and cell sorting in research in a variety of systems shows that there is more to flow cytometry than characterizing immune cell populations in blood samples.

The flow cytometry result of whole blood sample that analyses in forward and side scatter parameter and separated to granulocyte, monocytes and lymphocytes (Shutterstock.com)

“Nearly half of our flow cytometry work was used for neuroscience research,” Bare explained. “Some researchers were sorting cells from human brains to study neurological afflictions such as schizophrenia and addiction. These studies were looking at the biological and biochemical correlations of these cognitive disorders. By isolating and purifying the brain cells and cell nuclei from this complex organ, scientists are able to explore further using downstream tools such as epigenetics or sequencing and compare to similar cells from unafflicted brains. Many neurodegenerative diseases are predicted to be genetically driven, so they were interested in isolating purified populations of nuclei from different brains to identify the cellular correlates of these diseases” [1].

Tracking lung cancer risk after exposure to fallout from 9/11

Bare also worked with oncology researchers on a unique project tracking a cohort of people who were exposed to debris on the September 11, 2001, terrorist attacks in New York [2].

“During the attack, several toxic chemicals like asbestos were released into the air, exposing first responders and civilians in the surrounding area,” Bare recalled. “Researchers could use cell sorting to isolate individual cells for DNA sequencing. This allowed them to track the progression through life of the people who were exposed to these chemicals during 9/11 and see if those exposed now have a predisposition or additional genetic variation related to lung cancer.”

Immunology research

“Other projects include the classic immunology approach of sorting out immune cells in the blood,” Bare said. “Researchers looked at populations of dendritic cells, or infectious disease researchers would use flow cytometry to sort the lymphocytes in the blood. And lastly there are other researchers looking at more ‘exotic’ cell types that aren’t as often used in flow cytometry like epithelial cells, cartilage, and cardiomyocytes.”

Looking to the future

The global flow cytometry market is expected to grow from 6.3 billion USD in 2021 to 17.3 billion USD in 2030 [3]. This growth is reflected in the growing research that’s anticipated in not only the examples mentioned in this article, but through disease research including HIV-AIDS, biological drug development, and more.

As an instrument provider, we continue seeking to lead the way with cutting-edge technology in flow cytometry, from providing superior sensitivity and resolution, to making it all fit in the smallest possible benchtop packaging. We also strive to make it as intuitive and userfriendly as possible, enabling laboratory staff to get to work right after installation. As great as any technology is, the major challenge is adopting and accepting new workflows. When a new automation solution is installed, many lab managers and staff will spend unnecessary time watching the instrument to ensure it is accurately performing the tasks they previously had to gruellingly and slowly do by hand. For veteran cell sorter operators, learning to trust the software tools and allowing more unaided access to sorting can allow increased output from the labs they serve.

“Academic scientists exist in a sort of paradox. They crave innovation but fear change,” Bare said. “Reluctance to adopt successive generations of a platform can slow research to a glacial pace. Cytometry is no exception but with the miniaturization and ease of use of new cytometers, I’d expect to see a paradigm shift. It will be easier for individual labs to operate them, and use might shift away from centralized shared labs and into individual labs. This could also carry over to clinical devices, which would allow basic cytometry to be done at point of care as technology for rapid diagnostics.” With such large growth expected in the coming years, more labs will be under pressure to accomplish more in less time, and the benefits of automation will come to the forefront, releasing laboratory staff to focus on other critical work and not the workflow. After all, that’s what fuels the passion in the first place.

“It is personally gratifying to see how this research can shape future treatments,” Bare said. “I can’t wait to see what happens next.”

References

1. Wei J, Lambert TY, Valada A et al. Single nucleus transcriptomics reveals pervasive glial activation in opioid overdose cases. bioRxiv [Preprint] 2023;2023.03.07.531400. doi: 10.1101/2023.03.07.531400.
2. Wang L, Xu Y, Zhang L et al. World Trade Center dust exposure promotes cancer in PTEN-deficient mouse prostates. Cancer Res Commun 2022;2(6):518–532. doi: 10.1158/2767-9764.crc-21-0111.
3. Flow cytometry market (by product: instruments, reagents & consumables, accessories, software, services; by technology: cell-based, bead-based; by application: research, industrial, clinical; by end-use: commercial organizations, academic institutes, hospitals, clinical testing labs) – global industry analysis, size, share, growth, trends, regional outlook, and forecast 2022-2030. Precedence Research 2022; Report code: 2315  (https://www.precedenceresearch.com/flow-cytometry-market).

The author

James McCracken PhD
Beckman Coulter Life Sciences, Indianapolis, IN, USA

Email: jmccracken@beckman.com