Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells

Charuta Agashe, David Chiang, Alexander Grishin, Madhan Masilamani, Stacie M. Jones, Robert A Wood, Scott H. Sicherer, A. Wesley Burks, Donald Y.M. Leung, Peter Dawson, Hugh A. Sampson, M. Cecilia Berin

Research output: Contribution to journalArticle

Abstract

In centralized immune monitoring for a multi-center allergen immunotherapy trial, we observed frequent loss of CD4+ T cell integrity following staining of cultured PBMCs with our regulatory T cell flow cytometry panel. Samples were marked by a loss of total cellular events, altered scatter properties, and reduced CD3+ CD4+ events. This occurred only in samples that were stained with Foxp3 and were therefore treated with Foxp3 fixation-permeabilization buffer. We identified granulocyte contamination in samples associated with a loss of integrity, and went on to test the impact of granulocyte depletion on day-old blood samples. Granulocyte depletion prevented loss of cell integrity and CD3+ CD4+ events, and reduced variability in detection of Foxp3+ cells. Addition of purified neutrophils back to PBMCs altered scatter properties and detection of CD4+ T cells. Implementation of a granulocyte depletion step in our standard operating protocols has reduced assay failure due to loss of sample integrity from 31% to 0%. Routine incorporation of a granulocyte depletion step during PBMC isolation is recommended prior to downstream immune monitoring in blood with next-day processing.

Original languageEnglish (US)
JournalJournal of Immunological Methods
DOIs
StateAccepted/In press - 2016

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Agashe, C., Chiang, D., Grishin, A., Masilamani, M., Jones, S. M., Wood, R. A., Sicherer, S. H., Burks, A. W., Leung, D. Y. M., Dawson, P., Sampson, H. A., & Berin, M. C. (Accepted/In press). Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. Journal of Immunological Methods. https://doi.org/10.1016/j.jim.2017.06.004