Department of Laboratory Medicine, Donauspital, 1220 Vienna, Austria.
Cytometry A. 2013 Mar;83(3):316-23. doi: 10.1002/cyto.a.22245. Epub 2013 Jan 11.
Identification and quantification of lymphocyte subsets is based on the expression of specific cell surface antigens. As only a minority of these structures is lineage-restricted gating strategies were established, which should permit to include a maximum of lymphocytes, to reach a high purity within the gate and to avoid specific loss of subsets. Two problems remain: First, the incomplete removal of nonlymphoid cells when CD14 is used to exclude them from the lymphocyte gate. Second, the lack of a restricted marker to identify NK cells that are usually defined as CD3(-) /CD16/56(+) lymphocytes, though contaminating monocyte subsets share the expression of CD16, respectively, CD56. This study demonstrates the advantage of CD33 over CD14 at the creation of a pure lymphocyte gate, because CD33 enables the exclusion of all monocyte subpopulations as well as basophils and granulocytes. Independent of the applied NK cell marker mean purity was significantly higher, when CD33 was used (P < 0.001). For the identification of NK cells, CD7 was compared with CD16/56 and with single stained CD56. CD7 and CD16/56 exhibited as equivalent in various CD33 settings (P ≥ 0.173), whereas the mean proportion of CD56(+) NK cells was significantly lower (P ≤ 0.008). Use of CD14 entailed a significantly higher amount of CD3(-) /CD16/56(+) cells than of CD3(-) /CD7(+) cells (P = 0.008) because of the remaining CD14(-) /CD16(+) monocytes. As CD7 is restricted to T cells and NK cells in peripheral blood, misclassification of contaminating monocytes is avoided and CD7(+) NK cells can be identified by lack of CD3. Applying this new selection of mAbs, we reached a mean purity of ≥99.50% within the revised lymphocyte gate. As gates can be set very broadly, high inclusivity and high purity are not mutually exclusive. We propose the adoption of CD7 and CD33 for the quantification of lymphocyte subsets.
淋巴细胞亚群的鉴定和定量是基于特定细胞表面抗原的表达。由于只有少数这些结构是谱系受限的，因此建立了门控策略，这应该允许包含最多的淋巴细胞，在门内达到高纯度，并避免亚群的特异性丢失。仍然存在两个问题：第一，当使用 CD14 将其排除在淋巴细胞门之外时，不能完全去除非淋巴细胞。第二，缺乏用于识别 NK 细胞的受限标志物，NK 细胞通常被定义为 CD3(-)/CD16/56(+)淋巴细胞，尽管污染的单核细胞亚群分别表达 CD16 和 CD56。这项研究表明，与 CD14 相比，CD33 在创建纯淋巴细胞门时具有优势，因为 CD33 可以排除所有单核细胞亚群以及嗜碱性粒细胞和粒细胞。独立于应用的 NK 细胞标志物，当使用 CD33 时，平均纯度显著更高（P < 0.001）。为了鉴定 NK 细胞，将 CD7 与 CD16/56 以及单独染色的 CD56 进行了比较。在各种 CD33 设置中，CD7 和 CD16/56 表现出等效性（P≥0.173），而 CD56(+) NK 细胞的平均比例显著更低（P≤0.008）。由于剩余的 CD14(-)/CD16(+)单核细胞，使用 CD14 会导致明显更多的 CD3(-)/CD16/56(+)细胞，而不是 CD3(-)/CD7(+)细胞（P=0.008）。由于 CD7 在周围血液中仅限于 T 细胞和 NK 细胞，因此避免了对污染单核细胞的错误分类，并且可以通过缺乏 CD3 来识别 CD7(+)NK 细胞。应用这种新的单克隆抗体选择，我们在修订后的淋巴细胞门内达到了≥99.50%的平均纯度。由于门可以设置得非常宽，高包容性和高纯度并不相互排斥。我们建议采用 CD7 和 CD33 来定量淋巴细胞亚群。
