Pénélope Bourgoin1, Jack Hayman2, Thomas Rimmelé3, Fabienne Venet2, Fabrice Malergue4, Guillaume Monneret2. 1. Department of Research and Development, Beckman Coulter-Immunotech, Marseille, France. 2. Hospices Civils de Lyon, Edouard Herriot Hospital, Immunology Laboratory, Lyon, France. 3. Hospices Civils de Lyon, Edouard Herriot Hospital, Surgical Intensive Care Unit, Lyon, France. 4. Department of Research and Development, Beckman Coulter-Immunotech, Marseille, France. Electronic address: fmalergue@beckman.com.
Abstract
BACKGROUND: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression. METHODS: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR. RESULTS: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 μL to 5 μL, lysis from 1 mL to 500 μL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed. CONCLUSIONS: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications.
BACKGROUND: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression. METHODS: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR. RESULTS: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 μL to 5 μL, lysis from 1 mL to 500 μL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed. CONCLUSIONS: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications.
Authors: Pénélope Bourgoin; Thomas Soliveres; Alexandra Barbaresi; Anderson Loundou; Inès Ait Belkacem; Isabelle Arnoux; Denis Bernot; Marie Loosveld; Pierre-Emmanuel Morange; Pierre Michelet; Fabrice Malergue; Thibaut Markarian Journal: Cytometry A Date: 2021-02-08 Impact factor: 4.714