Sędek Łukasz, Flores-Montero Juan, van der Sluijs Alita, Kulis Jan, Te Marvelde Jeroen, Philippé Jan, Böttcher Sebastian, Bitter Marieke, Caetano Joana, van der Velden Vincent H J, Sonneveld Edwin, Buracchi Chiara, Santos Ana Helena, Lima Margarida, Szczepański Tomasz, van Dongen Jacques J M, Orfao Alberto
Department of Microbiology and Immunology, Medical University of Silesia in Katowice (SUM), 41-808 Zabrze, Poland.
Cancer Research Center (IBMCC, USAL-CSIC), Department of Medicine and Cytometry Service (NUCLEUS), University of Salamanca (USAL), 37007 Salamanca, Spain.
Cancers (Basel). 2022 Jan 18;14(3):473. doi: 10.3390/cancers14030473.
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.
流式细胞术（FC）结果的客观解读可能仍会受到技术标准化有限的阻碍。欧洲流式细胞术联盟开展了一系列实验，以确定不同变量对来自健康供体和患有不同血液系统恶性肿瘤患者样本中不同细胞群体上标记物的相对分布和中位荧光强度（MFI）的影响。评估了不同抗凝剂的使用；样本采集、制备和获取之间的时间间隔；洗涤缓冲液的pH值；以及仅使用细胞表面膜（SM）与细胞表面加胞浆内（SM+CY）染色方案的情况。我们的结果表明，在乙二胺四乙酸（EDTA）抗凝与肝素抗凝的样本中，只有单核细胞的占比更高。应用SM或SM+CY方案仅在特定抗体组合下，中性粒细胞和碎片的百分比会产生细微差异。反过来，当使用浆细胞疾病检测板时，样本在室温下储存24小时会导致碎片和细胞双联体的百分比更高。此外，染色细胞在室温下储存24小时对成熟B细胞和T细胞上CD19和CD45的MFI水平有选择性损害（但对白血病原始细胞、克隆性B细胞和浆细胞、中性粒细胞及自然杀伤细胞无影响）。所得结果表明，所评估的变量可能需要根据样本和细胞类型以及所比较的特定标记物进行调整；然而，为储存时间、染色至获取的延迟以及洗涤缓冲液的pH值确定平衡良好的界限，对于本文所述的大多数应用和情况将是一个有效的建议。
