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

作者：Łukasz Sędek, Juan Flores‐Montero, Alita van der Sluijs, Jan Kulis, Jeroen te Marvelde, Jan Philippé, Sebastian Böttcher, M. 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 Órfão · 发表于：Cancers · 年份：2022 · DOI：10.3390/cancers14030473 · 被引用次数：12 · 研究领域：Hematopoietic Stem Cell Transplantation、Acute Myeloid Leukemia Research、Acute Lymphoblastic Leukemia research

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...