A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies

Abstract

Large population-based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 participants in the Health and Retirement Study (HRS) are described. The extensive quality control involved in a large-scale immunophenotyping epidemiological study is also outlined. The existing literature on the effect of cryopreservation on various immune cell subsets including T, B, NK cells, monocytes, and dendritic cells is provided. © 2018 by John Wiley & Sons, Inc.

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

“…Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40). Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40).…”

“…Manual gating can be a laborious and time-consuming task when dealing with large data sets and may be a source of errors, as the gating strategy and order are critical to resolve the interplay between various markers. Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40). If no other options are feasible and manual gating is to be performed, it should be centralized to one location of a multisite study to help minimize variability.…”

“…Another potential solution is to integrate automation to the sample processing workflow. Instruments such as liquid handlers offer protocols for automated processing which have been shown to perform as efficiently as the goldstandard Ficoll density gradient method, as measured by overall yield, viability, recovery, white blood cell subpopulation distribution, and gene expression (16)(17)(18)(19).…”

Validation of a hybrid approach to standardize immunophenotyping analysis in large population studies: The Health and Retirement Study

“…At the 2016 HRS survey wave, venous blood samples were collected from 9,934 HRS participants in 7,227 households. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained from a CPT TM tube (BD Biosciences, San Jose, CA) as described previously 18 . The cryopreserved PBMCs were used for immunophenotyping in this study.…”

“…flow cytometry panels and detection of immune subsets. Two panels consisting of 17 fluorochrome-color cytometry panels targeting 24 cell populations were evaluated and have been described in detail previously 18 . The immune cell subsets were identified using minor modifications to the standardized protocol published by the Human Immunology Project 19 to identify different subsets of effector cytotoxic T cells by including CD95 antibody.…”