Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells.
J Immunol Methods
; 449: 23-27, 2017 10.
Article
en En
| MEDLINE
| ID: mdl-28629732
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Leucocitos Mononucleares
/
Linfocitos T CD4-Positivos
/
Linfocitos T Reguladores
/
Granulocitos
Tipo de estudio:
Clinical_trials
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Immunol Methods
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos