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.

2nd Department of Medicine University Hospital of Schleswig Holstein Campus Kiel Kiel Germany

Cancer Research Center and Department of Medicine University of Salamanca Salamanca Spain

Cytognos SL Salamanca Spain

Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam The Netherlands

Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University Prague Czech Republic

Dept of Immunohematology and Blood Transfusion Leiden The Netherlands

Hospital Clínic de Barcelona Barcelona Spain

Rostock University Medical Center Division of Internal Medicine Medical Clinic 3 Hematology Oncology and Palliative Medicine Special Hematology Laboratory Rostock Germany

Rostock University Medical Center Division of Internal Medicine Medical Clinic 3 Hematology Oncology and Palliative Medicine Special Hematology Laboratory Rostock Germany; 2nd Department of Medicine University Hospital of Schleswig Holstein Campus Kiel Kiel Germany

Systems and Computing Department Rio de Janeiro Brazil

References provided by Crossref.org

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

EuroFlow-Based Flowcytometric Diagnostic Screening and Classification of Primary Immunodeficiencies of the Lymphoid System