title: Validation of Immunomonitoring Methods for Application in Clinical Studies: The HLA-Peptide Multimer Staining Assay.
abstract: Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA-peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen-specific T cell responses after immunotherapy.
  
  
  
  Parameters of the assay, specificity, precision, linearity, sensitivity, and robustness were assessed systematically. Experiments were designed to address specifically each parameter and are detailed.
  
  
  
  Nonspecific multimer staining was below the acceptance limit of 0.02% multimer(+)CD8(+)cells. The assay showed acceptable precision in all dimensions it was repeated (CV < 10%) and also demonstrated a linear detection (R2> 0.99) of antigen specific cells.
  
  
  
  We succeeded in validating the HLA-multimer staining assay in a systematic manner. Additionally, we propose a technical framework and recommendations that can be applied for validating other T cell assessment methods. © 2016 International Clinical Cytometry Society.
authors.affiliation: Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: P Anoop
authors.initials: PA
authors.lastname: Chandran
authors.affiliation: Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: Karoline
authors.initials: K
authors.lastname: Laske
authors.affiliation: Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, United Kingdom.
authors.forename: Angelica
authors.initials: A
authors.lastname: Cazaly
authors.affiliation: Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Rusch
authors.affiliation: Center for Clinical Transfusion Medicine GmbH, University Hospital, Tuebingen.
authors.forename: Barbara
authors.initials: B
authors.lastname: Schmid-Horch
authors.affiliation: Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Rammensee
authors.affiliation: Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, United Kingdom.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ottensmeier
authors.affiliation: Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
authors.forename: Cécile
authors.initials: C
authors.lastname: Gouttefangeas
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D006680:HLA Antigens / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007167:Immunotherapy / Q000379:methods; D010455:Peptides / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D006680:HLA Antigens; D010455:Peptides
keywords: HLA-multimer; assay validation; biomarker; immunotherapy; mmunomonitoring