Stewart Judith C, Villasmil Michelle L, Frampton Mark W
Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
Cytometry A. 2007 Jun;71(6):379-85. doi: 10.1002/cyto.a.20392.
Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied.
Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison.
Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils.
The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.
血液白细胞的免疫表型分析通常在流式细胞术分析前用多聚甲醛固定。然而，细胞类型和标志物特异性对固定后荧光强度稳定性的影响尚未得到充分研究。
使用一组异硫氰酸荧光素标记的表面标志物抗体对人全血进行染色。在染色后0、2、4、6、24、48和96小时，通过流式细胞术对未固定和固定的样本进行分析。将荧光测量值转换为等效可溶性荧光染料分子进行比较。
固定在48小时时导致前向和侧向散射显著降低，这需要进行门控调整以实现细胞群体的分辨率。固定样本中的自发荧光逐渐增加（单核细胞在96小时时增加了九倍）。在校正自发荧光后，标志物相关荧光的可变降低变得明显。96小时时降低的幅度因细胞类型和标志物而异，从单核细胞上CD32的5%到中性粒细胞上CD16的39%。
白细胞染色和固定后荧光强度的变化因细胞类型和表面标志物而异。应针对每种使用的细胞类型和标志物确定荧光稳定性，并考虑固定对细胞自发荧光的混杂影响。
