Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers
Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers
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DOI：
10.1034/j.1399-3003.2000.16b22.x
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发表时间：
2000-08-01
影响因子：
24.3
通讯作者：
Jörres, RA
中科院分区：
文献类型：
--
作者：
Loppow, D;Böttcher, M;Jörres, RA
在分析前，必须用二硫苏糖醇（DTT）进行预处理，以溶解诱导痰样品中的粘液。然而，DTT可能会影响细胞表面标志物，这是必要的淋巴细胞亚型。因此，本研究的目的是：评价DTT对适当的表面标志物组的影响。使用外周血白细胞是因为这些细胞与痰细胞不同，可以在没有DTT处理的情况下获得。根据标准痰程序，将来自健康供体的外周血与DTT或磷酸盐缓冲盐水（PBS）孵育，洗涤并与荧光染料标记的抗体孵育。红细胞裂解后，使用校准的流式细胞仪进行分析。白细胞群通过其光散射特性鉴定。DTT处理后，CD 16阳性粒细胞的荧光强度显著增加; CD 2阳性淋巴细胞、CD 45阳性淋巴细胞和CD 14阳性单核细胞的荧光强度降低（p <0.001）。这些变化发生在所有样品中。DTT对CD 3-、CD 4-、CD 8-、CD 19-、CD 56-和组织相容性白细胞DR抗原阳性淋巴细胞的荧光强度无明显影响。然而，有统计学意义（p < 0.001），虽然很小，白细胞的百分比的变化，目前的数据表明，虽然二硫苏糖醇作为痰液分析中使用的影响外周血白细胞的一些表面标志物，关于淋巴细胞表面标志物的样品之间的可比性是保留。因此，这表明，用二硫苏糖醇处理痰液样本不会使淋巴细胞的免疫细胞化学分析无效。
Pretreatment with dithiothreitol (DTT) is necessary to dissolve mucus in samples of induced sputum prior to analysis. However, DTT may affect cell surface markers which are essential for lymphocyte subtyping. Therefore, the aim of this study was: to evaluate the effect of DTT on an appropriate panel of surface markers. Peripheral blood leukocytes were used because these cells, in contrast to sputum cells, could be obtained without DTT treatment.Peripheral blood from healthy donors was incubated with either DTT according to standard sputum procedures or phosphate-buffered saline (PBS), washed and incubated with fluorochrome-labelled antibodies. After lysis of erythrocytes, analysis was performed using a calibrated flow cytometer. Leukocyte populations were identified by their Light scattering properties. For analysis, fluorescence intensity was compared between DTT- and PBS-treated samples.After treatment with DTT, fluorescence intensity was significantly increased in CD16-positive granulocytes; it was reduced in CD2-positive lymphocytes, CD45-positive lymphocytes and CD14-positive monocytes (p less than or equal to 0.001). These changes occurred in all samples. The fluorescence intensity of CD3-, CD4-, CD8-, CD19-, CD56- and histocompatibility leukocyte antigen DR-positive lymphocytes was not altered by DTT. However, there were statistically significant (p < 0.001), although small, changes in the percentages of leukocytes.The present data demonstrate that, although dithiothreitol as used in sputum analysis affects some surface markers of peripheral blood leukocytes, comparability between samples concerning lymphocyte surface markers is preserved. Therefore, it is suggested that treatment of sputum samples with dithiothreitol does not invalidate the immunocytochemical analysis of lymphocytes.