Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Nijmegen Medical Center, Geert Grooteplein 8, 6525 GA Nijmegen, The Netherlands.
J Immunol Methods. 2011 Sep 30;372(1-2):42-51. doi: 10.1016/j.jim.2011.06.025. Epub 2011 Jul 14.
Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO.
Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO.
We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings.
多色免疫表型分析可改善白细胞亚群及其恶性对应物的特征描述。然而，缺乏各种荧光标记的单克隆抗体（MoAb）阻碍了多色面板的形成。CD45 似乎是这些细胞免疫表型分析的重要 MoAb。与侧向散射相对照，CD45 提供免疫细胞分化，并能够识别各种正常和恶性白细胞亚群。CD45 通常被广泛使用，并与各种荧光染料标记，因此作为较少可用的荧光染料（如紫激光染料）的缀合物被纳入多色面板。然而，这些染料（例如太平洋橙/PO）通常荧光强度较低，可能太弱以致无法区分群体。新型有机染料 Krome Orange（KO，发射波长为 528nm）似乎是一种更强烈的紫激光染料，可作为 PO 的替代品。
在不同的细胞来源中测试 FITC、PE、ECD、PE-Cy5、PE-Cy7、PO 和 KO 标记的 CD45 强度。通过 CD45-KO 对各种谱系标志物进行反向门控，以鉴定亚群。组成了一个用于确定小细胞样本异常的十色 MoAb 面板以测试 CD45-KO 的特异性。
我们在来自不同来源的各种固定和未固定细胞中表明，KO 是一种具有明显更高量子产率的合适荧光染料，甚至比其他紫激光染料（例如太平洋蓝）更亮。CD45-KO/SS 使我们能够区分和描述各种正常和恶性白细胞亚群。通过使用十色 MoAb 面板筛选异常，我们表明 CD45-KO 可在少量细胞中提供可靠的免疫表型分析，从而提高十色染色的质量。
