Harmonization of Light Scatter and Fluorescence Flow Cytometry Profiles Obtained After Staining Peripheral Blood Leucocytes for Cell Surface-Only Versus Intracellular Antigens with the Fix & Perm™ Reagent
Harmonization of Light Scatter and Fluorescence Flow Cytometry Profiles Obtained After Staining Peripheral Blood Leucocytes for Cell Surface-Only Versus Intracellular Antigens with the Fix & Perm™ Reagent
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DOI：
10.1002/cyto.b.20486
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发表时间：
2010-01-01
影响因子：
3.4
通讯作者：
Orfao, Alberto
中科院分区：
文献类型：
--
作者：
da Costa, Elaine Sobral;Peres, Rodrigo Tosta;Orfao, Alberto
用Fix & PerM（TM）试剂染色细胞内标记物与白细胞散射特性的变化有关，限制了流式细胞术（FCM）数据的自动分析。在这里，我们研究了这些变量显着贡献的光散射，自体荧光，和bcl 2染色特性的外周血（PB）白细胞的变化后，固定和Perm（TM）。我们的主要目的是评估一种新的数学方法，用于自动协调FCM数据，这些数据来自对应于用仅细胞表面与Fix & Perm细胞内染色技术处理的样品等分试样的样品。总体而言，既没有使用抗凝剂，也没有使用样本储存15分钟）对PB白细胞的FCM特性影响最小。相反，细胞/蛋白浓度和固定液/样品（体积/体积）比的变化对某些白细胞群体的光散射特征有明显影响。因此，较低的细胞/蛋白质浓度与较低的散射值相关，特别是对于中性粒细胞。这种变化可以通过使用更高的固定剂与样品体积比来部分纠正。尽管用仅细胞表面染色程序与细胞内染色程序处理的相同样品的等分试样之间检测到可变变化，但本文提出并评价的用于两种方法中共同参数的自动协调的新数学方法可以校正来自经历常规固定/透化程序的细胞的白细胞的FCM谱，并使它们与对应于用仅细胞表面染色技术处理的相同样品的等分试样的那些不可区分。
Staining for intracellular markers with the Fix & PerM (TM) reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & Perm (TM). Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for 15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques.