Li N, Halldén G, Hjemdahl P
Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.
Eur J Haematol. 2000 Jul;65(1):57-65. doi: 10.1034/j.1600-0609.2000.90198.x.
A flow cytometric assay for measurements of leukocyte CD11b expression in whole blood has been developed and evaluated. The method is based on triggering of the flow cytometer by a fluorescent pan leukocyte marker, RPE-CD45. This enabled flow cytometric analysis in whole blood, and avoidance of in vitro artefacts related to cell purification and hemolysis. Our methodological evaluation suggested the following routine procedure: sampling with sodium citrate as the anticoagulant, sample incubation at 22 degrees C, and mild sample fixation with 0.5% formaldehyde saline. The latter provided good sample stability during 24 h. Moreover, the assay provided good assay reproducibility, low labelling antibody consumption, and minimal sample manipulation (< 30 min) and acquisition time demands. The assay seems to reflect the CD11b expression of circulating leukocytes, and is also suitable for studies of agonist stimulated CD11b expression in leukocyte subpopulations in vitro. When full CD11b responsiveness to agonist stimulation is desired, samples should be incubated at 37 degrees C, but this also elevated CD11b expression in unstimulated samples. The present whole-blood technique is thus suitable for analyses of CD11b expression for both research and clinical routine laboratory use. The assay can easily be modified for measurements of other leukocyte antigens by use of other specific fluorescent antibodies.
已开发并评估了一种用于测量全血中白细胞CD11b表达的流式细胞术检测方法。该方法基于用荧光全白细胞标志物RPE-CD45触发流式细胞仪。这使得能够对全血进行流式细胞术分析，并避免了与细胞纯化和溶血相关的体外假象。我们的方法学评估建议采用以下常规程序：以柠檬酸钠作为抗凝剂进行采样，在22℃下孵育样品，并用0.5%甲醛盐水进行轻度样品固定。后者在24小时内提供了良好的样品稳定性。此外，该检测方法具有良好的检测重现性、低标记抗体消耗，以及最少的样品处理（<30分钟）和采集时间要求。该检测方法似乎反映了循环白细胞的CD11b表达，也适用于体外研究激动剂刺激的白细胞亚群中CD11b表达。当需要对激动剂刺激有完全的CD11b反应性时，样品应在37℃下孵育，但这也会提高未刺激样品中的CD11b表达。因此，目前的全血技术适用于研究和临床常规实验室对CD11b表达的分析。通过使用其他特异性荧光抗体，该检测方法可以很容易地进行修改以测量其他白细胞抗原。
