A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials
Fabien Pitoiset, Michèle Barbié, Guillaume Monneret, Cécile Braudeau, Pierre Pochard, Isabelle Pellegrin, Jacques Trauet, Myriam Labalette, David Klatzmann, Michelle Rosenzwajg, Fabien Pitoiset, Michèle Barbié, Guillaume Monneret, Cécile Braudeau, Pierre Pochard, Isabelle Pellegrin, Jacques Trauet, Myriam Labalette, David Klatzmann, Michelle Rosenzwajg
Abstract
Background: Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4+ CD25+ CD127low FoxP3+ cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3.
Method: A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms.
Results: The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands.
Conclusions: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.
Keywords: flow cytometry; regulatory T cells; standardization.
© 2018 International Clinical Cytometry Society.
Source: PubMed