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

2017 10

Journal Article

Agashe, C.; Chiang, D.; Grishin, A.; Masilamani, M.; Jones, S.M.; Wood, R.A.; Sicherer, S.H.; Burks, W.; Leung, D.Y.M.; Dawson, P.; Sampson, H.A.; Berin, C.

J Immunol Methods





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 CD3CD4 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 CD3CD4 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.

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