Impact of Granulocyte Contamination on PBMC Integrity of Shipped Blood Samples: Implications for Multi-Center Studies Monitoring Regulatory T Cells

Publication Type
Journal Article
Year of Publication
Agashe, Charuta; Chiang, David; Grishin, Alexander; Masilamani, Madhan; Jones, Stacie M; Wood, Robert A; Sicherer, Scott H; Burks, A Wesley; Leung, Donald Y M; Dawson, Peter; Sampson, Hugh A; Berin, M Cecilia
J Immunol Methods
Start Page
Date Published
Blood Cells; CD4-Positive T-Lymphocytes; Cell Survival; Flow cytometry; Forkhead Transcription Factors; Granulocytes; Humans; Leukocytes, Mononuclear; Multicenter Studies as Topic; Specimen Handling; Staining and Labeling; T-Lymphocytes, Regulatory

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.