即将举行的网络研讨会:实现无需离心的流式细胞术样品制备工作流程的标准化
在过去,平均荧光强度(MFI)或相对荧光强度(RFI)被用来表征细胞各种成分的表达,如某些抗原或受体。由于流式细胞仪的条件随时间而变化,使用的仪器类型因实验室而异,细胞抗原或受体的表达如MFI或RFI没有可比性,虽然流式细胞仪具有较高的荧光敏感性,但不可能准确地应用这些信息。
近年来,定量流式细胞术逐渐发展起来,用于流式细胞术对某些细胞成分的准确定量分析。定量分析主要有两个原则:
①抗体微球定量法:特殊包装上的微球是已知数量的绵羊抗小鼠IgG分子,然后将包装上的不同数量的分子微球混合,形成含有不同数量分子的绵羊抗大鼠IgG混合微球,将微球与被测标本在相同条件下与荧光素标记的单克隆抗体(McAb)反应后在流式细胞仪上测定荧光强度,根据微球包裹绵羊抗小鼠IgG分子数和对应的对数荧光强度计算回归方程。然后将待测样品中阳性细胞的对数荧光强度代入方程,得到每个细胞上抗原分子的平均数量(抗原结合位点数)。
②荧光素分子定量微球法:将荧光素分子直接包覆在一种特殊的微球上,然后将包覆不同分子数的微球混合,形成含有不同分子数荧光素的混合微球。在仪器中设置细胞,在相同条件下测定被测细胞的微球和荧光强度,根据微球对荧光素分子包上的荧光数量和荧光强度并取对数对相应的回归方程进行计算,然后将被测细胞的荧光强度取对数代入方程,根据用于样品测定的单克隆抗体(McAb)的荧光素结合分子的比例,计算每个细胞上的平均抗原分子数。单细胞抗原或受体定量是流式细胞术的一个重要进展,它为细胞生物学、分子生物学、生物化学和免疫学提供了更准确的方法。
从单色到多色荧光分析
流式细胞术从间接免疫荧光染色、单色或双色直接荧光染色迅速发展到三色、四色甚至五色或六色荧光分析,使细胞亚群的鉴定和分选和细胞功能的评价更加准确。淋巴细胞亚群和白血病免疫表型分析应采用至少三种颜色的荧光分析更可靠。
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