Going with the flow

Transplantation of cells, tissues and organs restores essential functions, saving the lives of patients with a wide range of conditions, from haematological disorders and immunodeficiencies to organ failure.

In 2022 alone, more than 157,000 organ transplantations were conducted globally, and more than 45,000 haematopoietic cell transplantations occurred in Europe alone, highlighting the critical role of these medical interventions to improving the quality of life of countless individuals.

Current cell therapies – such as CAR-T treatments – are predominantly autologous, with allogeneic approaches (when cells are sourced from donors) increasingly being explored.

In order to advance these innovative therapeutic approaches, the crucial role of the donors’ and recipients’ immune status must be appreciated, and the characteristics and specificity of the associated immune responses must be clarified.

With this in mind, the discovery of immunological biomarkers and their precise assessment at the cellular level is becoming paramount to revealing biological mechanisms and identifying therapeutic targets, as well as anticipating the risk of transplant rejection, ‘living drug’ failure and other potential complications, which will ultimately increase the success rate of therapies.

Through advanced techniques such as single-cell analysis or multi-omics, clinicians and researchers can gain valuable insights into an individual’s immunological profile, and apply effective identification and exploitation of biomarkers in therapeutic decisions.

A core technology in this comprehensive cellular interrogation is flow cytometry – a tool capable of multi-parameter analysis of thousands of cells per second, that enables the identification of specific cell populations involved in immune responses. In addition, it provides insights on cell-to-cell interactions.

By analysing cells labelled with fluorescent markers targeted to specific antigens, flow cytometry provides critical data on cell surface markers, intracellular proteins and the functional responses of cells to various external stimuli, including alloreactivity vs donor cells and antigens.

The reliance on flow cytometry in the development and study of biomarkers highlights its crucial role, emphasising the need to design integrated and automated workflows to enhance and streamline the decision-making process in clinical research settings.

Until recently, manually setting up multiple panels for immunological profiling through flow cytometry could be time-consuming, labour-intensive, and prone to human error and data variability, which taken together has created a significant bottleneck to the workflow, and limited sample analysis throughput.

Streamlining, standardising and simplifying flow cytometry are crucial steps to guaranteeing the robustness and reliability of experimental results, and as a consequence, repeatability and consistency across both clinical and translational research.

Introducing automation into the workflows is one way to streamline current processes.

Flow cytometers integrate automation at various stages, from sample preparation through to data tracking to data analysis, thereby providing end-to-end solutions with automatised operations that are flexible for specific experimental needs.

Dr James Hutchinson, a transplant immunologist from the Department of Surgery at the University Hospital Regensburg, when highlighting the importance of automation in revolutionising flow cytometry, stated:

“My laboratory uses a flow cytometer for research use. Using a sample-barcoding strategy, we greatly reduce technical variability, reduce handling and data-acquisition time, and reduce reagent costs.”

Standardisation is also a pivotal aspect in research applications, primarily because variability in sample processing, analysis and interpretation can lead to discrepancies in data, thus hindering the ability to draw reliable conclusions and conduct comparative studies.

For example, the quality of data obtained from samples can be compromised by temperature fluctuations including during transport of reagents, variability in reagent composition, or human error when mixing reagents.

“The dried-down reagents are part of the solution because they’re very consistent. What we found was that the variability in our measurements in various ways was greatly reduced using DURAClone compared to the same antibody panels made up of liquid reagents,” says Dr Hutchinson.

The use of dry reagents, which are pre-formulated and pre-aliquoted in antibody panels, can minimise the variability introduced by the manual preparation of liquid reagents, which are more prone to human error and variability between operators and laboratories.

Dried-down reagents are also more stable and have a longer shelf life, which helps to produce comparable results in different studies carried out over a duration of time, enhancing standardisation in research studies.

Unlike liquid reagents, they don’t require cold chain logistics, which also helps support global standardisation efforts, including regions with limited infrastructure.

After streamlining and standardising flow cytometry, there is simplifying, especially in the context of the vast amount of data generated through this tool.

Multi-omics technologies leveraging combinations of genomics, epigenetics, transcriptomics, proteomics and secretomics, allow for the examination of multiple biological layers within single cells, producing large volumes of information.

The vast amount of flow cytometry data comes with challenges, starting with storage and analysis of high-dimensional data, which can easily encompass 20 fluorescent parameters.

In this regard, the extensive array of channels now available offers the possibility of barcoding, whereby samples labelled with a unique fluorescent tag or barcode can be analysed in combination.

This enhances the capacity to conduct large-scale high-throughput experiments, as demonstrated by Dr Hutchinson’s transplantation research studies immunoprofiling samples from patients undergoing cell therapy.

The technological complexities of analysing multiparametric data sets can also effectively be addressed through digitalisation, the integration of software solutions that further automate workflows and limit the subjectivity that comes with operators and manual gating.

Integrating automation, standardisation and digitalisation in flow cytometry is streamlining and simplifying the technology, helping to advance research studies investigating the immune mechanisms associated with cellular and organ transplantation.

As we enhance the sophistication of these technologies, it is crucial to keep in mind the ultimate goal – the identification of biomarkers that may be used for more accurate outcomes.

By pinpointing relevant parameters, we can improve accuracy and accelerate research, leading to critical developments faster.

Michael Kapinsky, Senior Manager Product Management, Beckman Coulter Life Sciences.
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