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Published on: December 16, 2022
Intracellular Flow Cytometry Improvements in Clinical Studies
Julie Demaret1,2, Morgane Gossez1,2, Fabienne Venet1,2
1Immunology Laboratory, Hospices Civils de Lyon, E. Herriot Hospital, Pavillon E, 5 Place d'Arsonval, Lyon, Cedex 03, 69437, France.
Insights
Novel intracellular flow cytometry protocols simplify clinical use, reducing time and improving staining for research and personalized medicine. These methods enhance feasibility for multicenter studies and patient stratification.
Area of Science:
- Immunology and Cell Biology
- Clinical Diagnostics
Background:
- Intracellular flow cytometry is vital but faces challenges in routine clinical use and multicenter studies.
- Current methods are time-consuming, cause cell loss, and lead to nonspecific binding or reduced fluorescence, hindering standardization.
Purpose of the Study:
- To present novel, clinic-ready intracellular flow cytometry protocols.
- To overcome limitations of existing methods for improved standardization and feasibility in clinical settings.
Main Methods:
- Development of one-step, whole-blood, lyse-no-wash-no-centrifuge protocols.
- Utilization of improved staining quality and lyophilized reagents in ready-to-use tubes.
- Application in immunomonitoring of septic patients, evaluating myeloperoxidase, lactoferrin, FOXP3, and STAT5 phosphorylation.
Main Results:
- Protocols offer shorter time-to-results and improved staining quality.
- One-step procedures minimize cell loss and nonspecific binding.
- Demonstrated feasibility in evaluating specific biomarkers in neutrophils and lymphocytes.
Conclusions:
- Novel protocols enhance standardization and feasibility of intracellular flow cytometry in clinical studies.
- These advancements support drug efficacy monitoring and patient stratification for personalized medicine.
- The methods are applicable to immunomonitoring in conditions like sepsis.
Abstract:
Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.
