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A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies
Sabine Ivison, … , Ryan R. Brinkman, Megan K. Levings
Sabine Ivison, … , Ryan R. Brinkman, Megan K. Levings
Published December 6, 2018
Citation Information: JCI Insight. 2018;3(23):e121867. https://doi.org/10.1172/jci.insight.121867.
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Technical Advance Immunology Transplantation

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies

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Abstract

The analysis and validation of flow cytometry–based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman’s correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.

Authors

Sabine Ivison, Mehrnoush Malek, Rosa V. Garcia, Raewyn Broady, Anne Halpin, Manon Richaud, Rollin F. Brant, Szu-I Wang, Mathieu Goupil, Qingdong Guan, Peter Ashton, Jason Warren, Amr Rajab, Simon Urschel, Deepali Kumar, Mathias Streitz, Birgit Sawitzki, Stephan Schlickeiser, Janetta J. Bijl, Donna A. Wall, Jean-Sebastien Delisle, Lori J. West, Ryan R. Brinkman, Megan K. Levings

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Figure 6

Detecting differences between 2 cohorts using unmanipulated blood versus cryopreserved PBMCs.

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Detecting differences between 2 cohorts using unmanipulated blood versus...
Populations from all panels that significantly differed between healthy controls and patients 100 ± 20 days after HSCT in either (A) whole blood or (B) cryopreserved PBMCs. Note: Post-HSCT, but not healthy control, PBMCs were incubated 24 hours before cryopreservation. Proportions of parent gates for each population are shown as box-and-whisker plots; midline is the median, box is the interquartile range, and whiskers show minimum and maximum values. Means are indicated by a thick black band. See Supplemental Table 1 for a list of parent gates. Only samples that were evaluable in both unmanipulated blood and PBMCs were compared; n = 9 healthy and n = 8 post-HSCT subjects (except n = 4 for B cell–derived populations). *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t test with Holm-Šidák corrections.

Copyright © 2021 American Society for Clinical Investigation
ISSN 2379-3708

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