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Lot-to-lot stability of antibody reagents for flow cytometry

S. Böttcher, VHJ. van der Velden, N. Villamor, M. Ritgen, J. Flores-Montero, HM. Escobar, T. Kalina, M. Brüggemann, G. Grigore, M. Martin-Ayuso, Q. Lecrevisse, CE. Pedreira, JJM. van Dongen, A. Orfao,

. 2019 ; 475 (-) : 112294. [pub] 20170330

Language English Country Netherlands

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc20023416

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.

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