Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels

Łukasz Sędek, Juan Flores-Montero, Alita van der Sluijs, Jan Kulis, Jeroen Te Marvelde, Jan Philippé, Sebastian Böttcher, Marieke Bitter, Joana Caetano, Vincent H.J. van der Velden, Edwin Sonneveld, Chiara Buracchi, Ana Helena Santos, Margarida Lima, Tomasz Szczepański, Jacques J.M. van Dongen, Alberto Orfao*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Scopus)
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Objective interpretation of FC results may still be hampered by limited technical stan-dardization. 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.

Original languageEnglish
Article number473
Issue number3
Publication statusPublished - 18 Jan 2022

Bibliographical note

Funding Information:
Funding: 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).

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© 2022 by the authors. Licensee MDPI, Basel, Switzerland.


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