title: Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection.
abstract: Umbilical cord blood has been used for a wide variety of immunologic investigations including assessments of developmental perturbations by antenatal exposures. Recent advances in multiparameter flow cytometry have allowed finer characterization of lymphocyte phenotype and function, revealing important differences between the fetal and adult immune systems. The degree of variability between human subjects confounds the ability to draw firm conclusions. Artifacts resulting from processing techniques exacerbate this variability. The unpredictable nature of deliveries, especially of premature infants, makes it difficult to control variables such as timing of umbilical cord mononuclear cell (UCMC) isolation and method of collection. Additionally, in multicenter studies dependent on central processing, delays are inevitable. However, little available literature describes systematic testing of the degree to which processing variations affect UCMC phenotype and function. Using multiparameter flow cytometry, we tested the effect of collection technique and length of time prior to UCMC isolation on T cell phenotype and function, with the goal of creating a standardized operating procedure for a multicenter investigation. The study also provides a benchmark data set including extensive surface and functional phenotyping of umbilical cord T cells. UCMC isolation delay of up to 24 h produced similar T cell phenotype and function as tested by in vitro SEB stimulation. There were few statistically significant differences between time points based on data medians. We conclude that, for the purpose of immunologic investigations, a 24-h time delay from sample collection to mononuclear cell isolation does not introduce a significant degree of variation in T cell phenotype and function when adhering to strict standard operating procedures.
authors.affiliation: Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, USA. Kristin_Scheible@urmc.rochester.edu
authors.forename: Kristin
authors.initials: K
authors.lastname: Scheible
authors.forename: Shelley
authors.initials: S
authors.lastname: Secor-Socha
authors.forename: Terry
authors.initials: T
authors.lastname: Wightman
authors.forename: Hongyue
authors.initials: H
authors.lastname: Wang
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Mariani
authors.forename: David J
authors.initials: DJ
authors.lastname: Topham
authors.forename: Gloria
authors.initials: G
authors.lastname: Pryhuber
authors.forename: Sally
authors.initials: S
authors.lastname: Quataert
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D001800:Blood Specimen Collection / Q000379:methods / Q000592:standards*; D018106:CD28 Antigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D016923:Cell Death; D016207:Cytokines / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry; D006493:Heparin; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D010641:Phenotype; D015203:Reproducibility of Results; D013997:Time Factors; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000276:immunology
chemical_list: D018106:CD28 Antigens; D016207:Cytokines; D004768:Enterotoxins; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; C031818:enterotoxin B, staphylococcal; D006493:Heparin