Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric whole blood immunophenotyping

Abstract

Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.

A comprehensive assessment of immunophenotyping performed in cryopreserved peripheral whole blood

“…To our knowledge, these findings are the first to describe the performance of immunophenotyping in cryopreserved blood, and are very similar to that reported in fresh blood (29). We found that while the correlation of estimates obtained on different days was consistently high (>0.78), coefficient of variation (CV) estimates (inter-or intra-assay) were slightly higher for pDCs, basophils and NKT cells.…”

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

“…Over the past three decades, flow cytometry has been widely applied for immune‐phenotyping in clinical and research settings 1, 2, 29, 30, 47, 65, 66, 67, 68, 69, 70. A 17‐multiparamentric assay has been developed in the past for analyzing in details PB mature cells 68.…”

Computational flow cytometry: helping to make sense of high-dimensional immunology data

“…A reference dataset is constructed and new samples, analysed with the same panels, can be mapped onto this reference to classify a patient. Also in larger community-wide efforts, such as the Human Immunology Project Consortium (HIPC) 25 or the Milieu Interieur project 26 , the design of standardized panels is of key importance. To facilitate sharing and dissemination of optimized panels, the cytometry community has recently introduced a new series of protocols to improve reproducibility, referred to as optimized multicolour immunofluorescence panels (OMIPs) 27 .…”