title: Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function.
abstract: We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.
authors.affiliation: Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, United States. mckennakc@upmc.edu
authors.forename: Kyle C
authors.initials: KC
authors.lastname: McKenna
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Beatty
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Vicetti Miguel
authors.forename: Richard A
authors.initials: RA
authors.lastname: Bilonick
mesh_terms: D001781:Blood Component Removal / Q000379:methods*; D039481:CD11b Antigen; D017252:CD3 Complex / Q000276:immunology*; D049109:Cell Proliferation; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000276:immunology*; D006098:Granulocytes / Q000166:cytology / Q000276:immunology*; D006801:Humans; D016256:Lewis X Antigen; D008297:Male; D011309:Preservation, Biological; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013997:Time Factors
chemical_list: D039481:CD11b Antigen; D017252:CD3 Complex; C509295:ITGAM protein, human; D016256:Lewis X Antigen