title: Multicenter study on in vitro characterization of dendritic cells.
abstract: BACKGROUND
  There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability.
  
  
  METHODS
  CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol.
  
  
  RESULTS
  Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion.
  
  
  CONCLUSIONS
  In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.
authors.affiliation: Institute of Transfusion Medicine and Immunology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Eichler
authors.forename: X D
authors.initials: XD
authors.lastname: Nguyen
authors.forename: D
authors.initials: D
authors.lastname: Roelen
authors.forename: C M
authors.initials: CM
authors.lastname: Celluzzi
authors.forename: D
authors.initials: D
authors.lastname: McKenna
authors.forename: D
authors.initials: D
authors.lastname: Pamphilon
authors.forename: A
authors.initials: A
authors.lastname: Blair
authors.forename: E J
authors.initials: EJ
authors.lastname: Read
authors.forename: T A
authors.initials: TA
authors.lastname: Takahashi
authors.forename: Z M
authors.initials: ZM
authors.lastname: Szczepiorkowski
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D018949:Antigens, CD1 / Q000276:immunology; D051940:B7-2 Antigen / Q000276:immunology; D002452:Cell Count; D002470:Cell Survival / Q000276:immunology; D015925:Cryopreservation / Q000379:methods; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology; D016130:Immunophenotyping / Q000379:methods; D007937:Leukapheresis / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology; D008875:Middle Aged; D010865:Pilot Projects; D011446:Prospective Studies; D017452:Receptors, IgG / Q000276:immunology; D000097802:CD83 Antigen
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018949:Antigens, CD1; D051940:B7-2 Antigen; D006684:HLA-DR Antigens; D007136:Immunoglobulins; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D017452:Receptors, IgG; D000097802:CD83 Antigen; C419393:CD1a antigen