title: Standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring--Development of an algorithm.
abstract: Flow cytometry, as a powerful tool for immunomonitoring and quality control of peripheral blood mononuclear cells (PBMCs), is routinely used in clinical studies. However, flow cytometry based assays for cryopreserved peripheral blood mononuclear cells (cPBMCs) constitute a challenge. Down-regulation of surface and intracellular markers, as well as impairment of cell function might result from cryopreservation. Furthermore, protocols for resting cPBMCs are available but diverse. Therefore, we performed a standardization of the resting process concerning resting position, cell concentration, resting period and material of cell culture tubes as well as culture media. We further investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cPBMCs. Polychromatic flow cytometry staining, peptide-MHC class I restricted tetramer staining and intracellular cytokine staining as major methods were used. Our results revealed that a horizontal position, a cell concentration of 2 to 5 × 10(6) cells/ml and an overnight resting phase is beneficial to eliminate dead or dying cells in cPBMCs with a mean cell loss of 14% overall cell populations. In addition, the quality and quantity of regulatory T cells and antigen specific T cells recovered upon resting. For multifunctional T cells a decrease of activation threshold in the way of a twofold mean fluorescence intensity (MFI) and increase of degranulation marker CD107a, co-stimulatory marker CD28, adhesion molecule CD62L as well as the ability to secrete antiviral cytokines like interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) comparable to fresh PBMCs were observed. However, based upon our data resting is not helpful for the flow cytometric analyses of myeloid-derived suppressor cells (MDSCs) and large/intermediate size lymphocytes which rather decreased/vanished ex vivo. Therefore, we developed an algorithm to indicate for which cell population and for which type of analyses the resting process is useful or not.
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Lei
authors: L
authors: Wang
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Angela
authors: A
authors: Hückelhoven
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Jian
authors: J
authors: Hong
authors: Department of Hematology, Zhongda Hospital, Southeast University, Nanjing, China.
authors: Nan
authors: N
authors: Jin
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Jiju
authors: J
authors: Mani
authors: Department of Hematology, Zhongda Hospital, Southeast University, Nanjing, China.
authors: Bao-an
authors: BA
authors: Chen
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Michael
authors: M
authors: Schmitt
authors: Department of Internal Medicine V, University Clinic Heidelberg, University of Heidelberg, Germany.
authors: Anita
authors: A
authors: Schmitt
mesh_terms: D000465:Algorithms; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D015925:Cryopreservation / Q000592:standards*; D005434:Flow Cytometry / Q000592:standards*; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology; D019041:L-Selectin / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000276:immunology; D061251:Primary Cell Culture; D013048:Specimen Handling / Q000592:standards*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology
chemical_list: D018106:CD28 Antigens; D015395:Histocompatibility Antigens Class I; D007376:Interleukin-2; D052118:Lysosomal-Associated Membrane Protein 1; D014409:Tumor Necrosis Factor-alpha; D019041:L-Selectin; D007371:Interferon-gamma
keywords: T lymphocyte function; flow cytometry; immunomonitoring; myeloid-derived suppressor cells; regulatory T cells; resting peripheral blood mononuclear cells