A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans.

Michael P Gustafson,Patrick B Johnston,Dennis A Gastineau,Tobias Peikert,Virginia P Van Keulen,Mary L Maas,Allan B Dietz,Yi Lin,Derya Unutmaz

doi:10.1371/journal.pone.0121546

Michael P Gustafson, Patrick B Johnston + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0121546

Copy DOI

Journal: PloS one	Publication Date: Mar 23, 2015
Citations: 103	License type: CC BY 4.0

Affiliation: Mayo Clinic

Abstract

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.

Highlights

Myeloid cells are a diverse group of cells that serve critical roles in the regulation of innate and adaptive immunity
We have shown that there is considerable variability in how myeloid derived suppressor cells (MDSCs) are measured and reported and that reporting MDSC percentages without additional context of the larger “parent” or “grandparent” population can lead to erroneous conclusions[13]
We have since developed eight 10-color flow cytometric protocols that enable the absolute quantification of all major leukocyte populations (Table 1)

Summary

Introduction

Myeloid cells are a diverse group of cells that serve critical roles in the regulation of innate and adaptive immunity. Myeloid cells traffic to sites of injury, internalize and present foreign objects and pathogens, and secret pro- and anti- inflammatory cytokines. Common myeloid progenitors originate from hematopoietic stem cell progenitors and give rise to granulocytes and monocytes. Monocytes can further differentiate into macrophages and dendritic cells. In addition to regulating normal immune physiology, myeloid cells participate in regulating both positive and negative responses to tumor formation [1,2,3].

Objectives

Methods

Results

Discussion

Conclusion