Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels

Sedek, Lukasz; Flores-Montero, Juan; Sluijs-Gelling, Alita J. van der; Kulis, Jan; Marvelde, Jeroen G. te; Philippé, J.; Böttcher, Sebastian; Bitter, Marieke; Caetano, J.; Velden, Vincent H. J. van der; Sonneveld, Edwin; Buracchi, Chiara; Santos, Ana Helena; Lima, Margarida; Szczepanski, Tomasz; Dongen, J. J. M. van; Orfao, Alberto

Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on d...

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Autores:	Sedek, Lukasz, Flores-Montero, Juan, Sluijs-Gelling, Alita J. van der, Kulis, Jan, Marvelde, Jeroen G. te, Philippé, J., Böttcher, Sebastian, Bitter, Marieke, Caetano, J., Velden, Vincent H. J. van der, Sonneveld, Edwin, Buracchi, Chiara, Santos, Ana Helena, Lima, Margarida, Szczepanski, Tomasz, Dongen, J. J. M. van, Orfao, Alberto
Tipo de recurso:	artículo
Estado:	Versión publicada
Fecha de publicación:	2022
País:	España
Institución:	Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:	DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:	oai:digital.csic.es:10261/282910
Acceso en línea:	http://hdl.handle.net/10261/282910
Access Level:	acceso abierto
Palabra clave:	Flow cytometry Standardization Immunophenotyping Protocol Anticoagulant Sample storage Leukemia Lymphoma Multiple myeloma

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network_acronym_str	ES
network_name_str	España
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dc.title.none.fl_str_mv	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
title	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
spellingShingle	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels Sedek, Lukasz Flow cytometry Standardization Immunophenotyping Protocol Anticoagulant Sample storage Leukemia Lymphoma Multiple myeloma
title_short	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
title_full	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
title_fullStr	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
title_full_unstemmed	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
title_sort	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels
dc.creator.none.fl_str_mv	Sedek, Lukasz Flores-Montero, Juan Sluijs-Gelling, Alita J. van der Kulis, Jan Marvelde, Jeroen G. te Philippé, J. Böttcher, Sebastian Bitter, Marieke Caetano, J. Velden, Vincent H. J. van der Sonneveld, Edwin Buracchi, Chiara Santos, Ana Helena Lima, Margarida Szczepanski, Tomasz Dongen, J. J. M. van Orfao, Alberto
author	Sedek, Lukasz
author_facet	Sedek, Lukasz Flores-Montero, Juan Sluijs-Gelling, Alita J. van der Kulis, Jan Marvelde, Jeroen G. te Philippé, J. Böttcher, Sebastian Bitter, Marieke Caetano, J. Velden, Vincent H. J. van der Sonneveld, Edwin Buracchi, Chiara Santos, Ana Helena Lima, Margarida Szczepanski, Tomasz Dongen, J. J. M. van Orfao, Alberto
author_role	author
author2	Flores-Montero, Juan Sluijs-Gelling, Alita J. van der Kulis, Jan Marvelde, Jeroen G. te Philippé, J. Böttcher, Sebastian Bitter, Marieke Caetano, J. Velden, Vincent H. J. van der Sonneveld, Edwin Buracchi, Chiara Santos, Ana Helena Lima, Margarida Szczepanski, Tomasz Dongen, J. J. M. van Orfao, Alberto
author2_role	author author author author author author author author author author author author author author author author
dc.contributor.none.fl_str_mv	European Commission European Hematology Association Polish National Agency for Academic Exchange Silesian University of Technology Instituto de Salud Carlos III Ministerio de Economía y Competitividad (España) Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv	Flow cytometry Standardization Immunophenotyping Protocol Anticoagulant Sample storage Leukemia Lymphoma Multiple myeloma
topic	Flow cytometry Standardization Immunophenotyping Protocol Anticoagulant Sample storage Leukemia Lymphoma Multiple myeloma
description	Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.
publishDate	2022
dc.date.none.fl_str_mv	2022 2022 2022 2022
dc.type.none.fl_str_mv	info:eu-repo/semantics/article http://purl.org/coar/resource_type/c_6501 Publisher's version info:eu-repo/semantics/publishedVersion
format	article
status_str	publishedVersion
dc.identifier.none.fl_str_mv	http://hdl.handle.net/10261/282910
url	http://hdl.handle.net/10261/282910
dc.language.none.fl_str_mv	Inglés
language_invalid_str_mv	Inglés
dc.relation.none.fl_str_mv	Sedek, Lukasz; Flores-Montero, Juan; Sluijs-Gelling, Alita J. van der; Kulis, Jan; Marvelde, Jeroen G. te; Philippé, J.; Böttcher, Sebastian; Bitter, Marieke; Caetano, J.; Velden, Vincent H. J. van der; Sonneveld, Edwin; Buracchi, Chiara; Santos, Ana Helena; Lima, Margarida; Szczepanski, Tomasz; Dongen, J. J. M. van; Orfao, Alberto; 2022; Supplementary files of the article Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels [Dataset]; Multidisciplinary Digital Publishing Institute; http://dx.doi.org/10.3390/cancers14030473 http://dx.doi.org/10.3390/cancers14030473 Sí
dc.rights.none.fl_str_mv	info:eu-repo/semantics/openAccess
eu_rights_str_mv	openAccess
dc.format.none.fl_str_mv	application/pdf application/pdf
dc.publisher.none.fl_str_mv	Multidisciplinary Digital Publishing Institute
publisher.none.fl_str_mv	Multidisciplinary Digital Publishing Institute
dc.source.none.fl_str_mv	reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str	Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str	DIGITAL.CSIC. Repositorio Institucional del CSIC
collection	DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling	Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panelsSedek, LukaszFlores-Montero, JuanSluijs-Gelling, Alita J. van derKulis, JanMarvelde, Jeroen G. tePhilippé, J.Böttcher, SebastianBitter, MariekeCaetano, J.Velden, Vincent H. J. van derSonneveld, EdwinBuracchi, ChiaraSantos, Ana HelenaLima, MargaridaSzczepanski, TomaszDongen, J. J. M. vanOrfao, AlbertoFlow cytometryStandardizationImmunophenotypingProtocolAnticoagulantSample storageLeukemiaLymphomaMultiple myelomaObjective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.This research was funded by the EuroFlow Consortium which received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA); the grant of the Polish National Center for Research and Development (no. STRATEGMED3/304586/5/NCBR/2017 Person ALL); and internal grant of the Medical University of Silesia (no. PCN-1-050/K/0/K); the grant of CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER (no. CB16/12/00400).Multidisciplinary Digital Publishing InstituteEuropean CommissionEuropean Hematology AssociationPolish National Agency for Academic ExchangeSilesian University of TechnologyInstituto de Salud Carlos IIIMinisterio de Economía y Competitividad (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2022202220222022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10261/282910reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSedek, Lukasz; Flores-Montero, Juan; Sluijs-Gelling, Alita J. van der; Kulis, Jan; Marvelde, Jeroen G. te; Philippé, J.; Böttcher, Sebastian; Bitter, Marieke; Caetano, J.; Velden, Vincent H. J. van der; Sonneveld, Edwin; Buracchi, Chiara; Santos, Ana Helena; Lima, Margarida; Szczepanski, Tomasz; Dongen, J. J. M. van; Orfao, Alberto; 2022; Supplementary files of the article Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels [Dataset]; Multidisciplinary Digital Publishing Institute; http://dx.doi.org/10.3390/cancers14030473http://dx.doi.org/10.3390/cancers14030473Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2829102026-05-22T06:33:51Z
score	15,811543

Impact of pre-analytical and analytical variables associated with sample preparation on flow cytometric stainings obtained with EuroFlow panels

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