title: Lot-to-lot stability of antibody reagents for flow cytometry.
abstract: The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers. These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry.
authors.affiliation: Rostock University Medical Center, Division of Internal Medicine, Medical Clinic III, Hematology, Oncology and Palliative Medicine, Special Hematology Laboratory, Rostock, Germany; Second Department of Medicine, University Hospital of Schleswig Holstein (UKSH), Campus Kiel, Kiel, Germany. Electronic address: Sebastian.Boettcher@med.uni-rostock.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Böttcher
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Vincent H J
authors.initials: VHJ
authors.lastname: van der Velden
authors.affiliation: Hospital Clínic de Barcelona, Barcelona, Spain.
authors.forename: Neus
authors.initials: N
authors.lastname: Villamor
authors.affiliation: Second Department of Medicine, University Hospital of Schleswig Holstein (UKSH), Campus Kiel, Kiel, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Ritgen
authors.affiliation: Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Flores-Montero
authors.affiliation: Rostock University Medical Center, Division of Internal Medicine, Medical Clinic III, Hematology, Oncology and Palliative Medicine, Special Hematology Laboratory, Rostock, Germany.
authors.forename: Hugo Murua
authors.initials: HM
authors.lastname: Escobar
authors.affiliation: Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University (DPH/O), Prague, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Kalina
authors.affiliation: Second Department of Medicine, University Hospital of Schleswig Holstein (UKSH), Campus Kiel, Kiel, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Brüggemann
authors.affiliation: Cytognos SL, Salamanca, Spain.
authors.forename: Georgiana
authors.initials: G
authors.lastname: Grigore
authors.affiliation: Cytognos SL, Salamanca, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Martin-Ayuso
authors.affiliation: Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Lecrevisse
authors.affiliation: Systems and Computing Department (PESC), COPPE, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Pedreira
authors.affiliation: Dept. of Immunohematology and Blood Transfusion (IHB), Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Jacques J M
authors.initials: JJM
authors.lastname: van Dongen
authors.affiliation: Cancer Research Center (IBMCC-CSIC/USAL-IBSAL), Cytometry Service (NUCLEUS) and Department of Medicine, University of Salamanca, Salamanca, Spain. Electronic address: orfao@usal.es.
authors.forename: Alberto
authors.initials: A
authors.lastname: Orfao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards*; D005456:Fluorescent Dyes / Q000592:standards; D051379:Mice; D055550:Protein Stability; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes
keywords: Flow cytometry; Fluorescence intensity; Fluorochrome-to-antibody ratio; Variability