流式细胞术检测急性髓系白血病微小残留病的现状与进展

doi:10.3969/j.issn.1004-583X.2021.10.005

摘要/Abstract

摘要：

流式细胞术(FCM)检测微小残留病(MRD)具有很高的灵敏度和覆盖率,已经成为急性髓系白血病(AML)重要的疗效监测、预后评估和指导治疗方法选择的生物标记。然而,FCM检测AML患者MRD仍有许多问题亟待解决。本文介绍了FCM检测AML MRD的现状以及存在的问题,提出相应的解决方案与注意事项,并展望了新技术带来的新希望。

关键词: 白血病,髓样,急性, 流式细胞术, 肿瘤,残余, 分子靶向治疗

Abstract:

Minimal residual disease(MRD) of acute myeloid leukemia (AML) detected by flow cytometry(FCM) has high sensitivity and coverage, which has been an important biomarker for efficacy evaluation, outcome prediction, and therapy selection. However, a number of questions remain uncertain. This paper introduced the status and problems of FCM detection in AML MRD, discussed the corresponding solutions and precautions with the new hope by the progress of FCM.

Key words: leukaemia,myeloid acute, flow cytometry, minimal residual disease, neoplasm,residual, molecular targeted therapy

中图分类号:

R733.72

王卉, 陈曼. 流式细胞术检测急性髓系白血病微小残留病的现状与进展[J]. 临床荟萃, 2021, 36(10): 889-895.

Wang Hui, Chen Man. Flow cytometry in minimal residual disease detection of acute myeloid leukemia: current status and progress[J]. Clinical Focus, 2021, 36(10): 889-895.

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链接本文: https://huicui.hebmu.edu.cn/CN/10.3969/j.issn.1004-583X.2021.10.005

https://huicui.hebmu.edu.cn/CN/Y2021/V36/I10/889

图/表 6

参考文献 29

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抗原性质	标志	发生率
原始细胞/骨架标志	CD45 CD34 CD117 HLA-DR和(或)CD33
伴系表达标志	CD4	≥50%
	CD7^▼ CD9^#	≥30%~<50%
	CD56^△ CD25^*	≥10%~<30%
	CD19^△ CD2^▲ CD5 CD10	<10%
过早获得的成熟阶段标志	CD18^▽	≥50%
	CD15^▼ CD65 CD36 CD24	≥30%~<50%
	CD11b CD64^▽ CD11c	≥10%~<30%
	CD14^▼↓	<10%
表达强度改变标志	CD81	≥50%
(包括丢失、获得)	CD96^△↑ CD123^#↑ CD371(CLL1)^△↑ CD200^△ CD366(tim3)^△↑	≥30%~<50%
	CD38 CD33^▼↑ CD13^#↑▼↑ CD34^#↓ CD117^#↑ HLA-DR^#↓▼↑ CD184(CXCR4)	≥10%~<30%
其他可选标志	CD244 CD52^△↑ CD97 CD54^▲↑ CD170 CD300f GPR56 CD11a^★↓	≥50%
	CD59^△↓ CD44 CD300a/c^△↓ CX3CR1 CD86 CD32 CD1c CD26 CD66c CD273(PDL2)	≥30%~<50%
	CD47 CD99 CD68^▽↑ CD93^▲↑	≥10%~<30%
肿瘤干细胞标志	CD9 CD69 CD93 CD371 IL-1RAP^* CD114 CD135 CD123	≥50%
	CD25^# CD26^# CD96 CD116 CD332	≥30%~<50%
	CD115 CD221	≥10%~<30%

抗原性质	标志	发生率
原始细胞/骨架标志	CD45 CD34 CD117 HLA-DR和(或)CD33
伴系表达标志	CD4	≥50%
	CD7^▼ CD9^#	≥30%~<50%
	CD56^△ CD25^*	≥10%~<30%
	CD19^△ CD2^▲ CD5 CD10	<10%
过早获得的成熟阶段标志	CD18^▽	≥50%
	CD15^▼ CD65 CD36 CD24	≥30%~<50%
	CD11b CD64^▽ CD11c	≥10%~<30%
	CD14^▼↓	<10%
表达强度改变标志	CD81	≥50%
(包括丢失、获得)	CD96^△↑ CD123^#↑ CD371(CLL1)^△↑ CD200^△ CD366(tim3)^△↑	≥30%~<50%
	CD38 CD33^▼↑ CD13^#↑▼↑ CD34^#↓ CD117^#↑ HLA-DR^#↓▼↑ CD184(CXCR4)	≥10%~<30%
其他可选标志	CD244 CD52^△↑ CD97 CD54^▲↑ CD170 CD300f GPR56 CD11a^★↓	≥50%
	CD59^△↓ CD44 CD300a/c^△↓ CX3CR1 CD86 CD32 CD1c CD26 CD66c CD273(PDL2)	≥30%~<50%
	CD47 CD99 CD68^▽↑ CD93^▲↑	≥10%~<30%
肿瘤干细胞标志	CD9 CD69 CD93 CD371 IL-1RAP^* CD114 CD135 CD123	≥50%
	CD25^# CD26^# CD96 CD116 CD332	≥30%~<50%
	CD115 CD221	≥10%~<30%

荧光素方案	标本 (管)	FITC	PE	PerCP- Cy5.5或 PE-Cy5	PE-CY7	APC	APC-H7 或其他	BV421或深蓝或 V450	V500或深黄	BV605	APC R700 或APC- Alexa Fluor700	PE-TR	A594
中国免疫学会流式细胞学组^[9]	全	CD38	X^*	CD33	CD34	CD13	HLA-DR	CD117	CD45	X^*	X^*
华盛顿大学^[12]	1	CD15	CD33	CD117	CD13	CD34	CD45	HLA-DR			CD71	CD19	CD38
	2	CD64	CD123	CD14	CD13	CD34	CD45	HLA-DR			CD16	CD4	CD38
	3	CD56	CD7	CD5	CD33	CD34	CD45						CD38
MD Anderson^[13]	1	CD7	CD33	CD19	CD34	CD13		CD38	CD45
	2	HLA-DR	CD117	CD4	CD34	CD123	CD19 eF780	CD38	CD45
	3	HLA-DR	CD36	CD56	CD34	CD64	CD19 eF780	CD14	CD45
	4	CD5	CD2	CD22	CD34	CD38	CD19 eF780	CD15	CD45
荷兰瑞士协作组^[14]	白血病干细胞	CD45RA	CD371^△	CD123	CD34	CD38	CD44	CD33	CD45
	1	CD7	CD56	CD34	CD117	CD33	HLA-DR	CD13	CD45
	2	CD15	CD22	CD34	CD117	CD19	HLA-DR	CD13	CD45
	3	CD36	CD14	CD34	CD117	CD11b	HLA-DR	CD13	CD45
	4^#	CD2	CD133	CD34	CD117	CD33	HLA-DR	CD13	CD45
陆道培医院1^▲	1	CD7	CD117	CD34	CD13	CD33	HLA-DR	CD11b	CD45	CD56
	2	CD15	CD64	CD34	CD117	CD14	HLA-DR	CD19	CD45
陆道培医院2^▽	1	CD33	CD96	CD34	CD13	CD117	HLA-DR	CD11b	CD45
	2	CD7	CD117	CD34	CD19	CD56	CD14	CD38	CD45

荧光素方案	标本 (管)	FITC	PE	PerCP- Cy5.5或 PE-Cy5	PE-CY7	APC	APC-H7 或其他	BV421或深蓝或 V450	V500或深黄	BV605	APC R700 或APC- Alexa Fluor700	PE-TR	A594
中国免疫学会流式细胞学组^[9]	全	CD38	X^*	CD33	CD34	CD13	HLA-DR	CD117	CD45	X^*	X^*
华盛顿大学^[12]	1	CD15	CD33	CD117	CD13	CD34	CD45	HLA-DR			CD71	CD19	CD38
	2	CD64	CD123	CD14	CD13	CD34	CD45	HLA-DR			CD16	CD4	CD38
	3	CD56	CD7	CD5	CD33	CD34	CD45						CD38
MD Anderson^[13]	1	CD7	CD33	CD19	CD34	CD13		CD38	CD45
	2	HLA-DR	CD117	CD4	CD34	CD123	CD19 eF780	CD38	CD45
	3	HLA-DR	CD36	CD56	CD34	CD64	CD19 eF780	CD14	CD45
	4	CD5	CD2	CD22	CD34	CD38	CD19 eF780	CD15	CD45
荷兰瑞士协作组^[14]	白血病干细胞	CD45RA	CD371^△	CD123	CD34	CD38	CD44	CD33	CD45
	1	CD7	CD56	CD34	CD117	CD33	HLA-DR	CD13	CD45
	2	CD15	CD22	CD34	CD117	CD19	HLA-DR	CD13	CD45
	3	CD36	CD14	CD34	CD117	CD11b	HLA-DR	CD13	CD45
	4^#	CD2	CD133	CD34	CD117	CD33	HLA-DR	CD13	CD45
陆道培医院1^▲	1	CD7	CD117	CD34	CD13	CD33	HLA-DR	CD11b	CD45	CD56
	2	CD15	CD64	CD34	CD117	CD14	HLA-DR	CD19	CD45
陆道培医院2^▽	1	CD33	CD96	CD34	CD13	CD117	HLA-DR	CD11b	CD45
	2	CD7	CD117	CD34	CD19	CD56	CD14	CD38	CD45

特点	传统流式细胞术	二代流式细胞术	全光谱流式细胞术	质谱流式细胞术
原理	多采用光电倍增管	同传统流式	使用光电倍增管或者雪崩二极管,多个探测器检测一个荧光素形成连续光谱	使用各种金属元素作为标签;使用质谱技术作为检测手段
激光	1~3	3激光	3~5激光	使用质谱,非激光
荧光素(色)	4~13	≥8	理论上38~184通道,目前抗体限制检测24~40色	理论可以检测130多种蛋白,目前受标志物限制,检测50多个蛋白或者RNA
获取细胞数	10⁵~10⁶	5×10⁶~2×10⁷	同传统流式	几百万细胞中进行单细胞分析
灵敏度	10^-3~10^-4	10^-5~10^-6	同传统流式	速度限制(获取1000个/s),目前灵敏度有限
操作	多数先标记后溶血	建议增加样本量,先溶血后标记	多数同传统流式	金属标签标记
方案	个体化	标准化	个体化	个体化
分析方法	差别大,多数人工	相对固定,人工+半自动	人工	人工
软件要求	多使用Diva、Kaluza等二维参数分析软件,极少使用高维软件进行分析。	可以使用Diva、Kaluza等常规软件,建议使用Infinicyt配套软件。除了常规平面图,使用多维自动群聚分析图进行多参数群聚分析。	除了Spectroflo二维软件,一般会使用Flowjo、Cytobank等多维软件及插件进行多维和群聚分析。	一般会使用Flowjo、Cytobank等多维软件及插件进行多维和群聚分析。
优点	临床适用性最高:操作简单,速度快,分析简单,相互补偿干扰小,数据容量小,一般电脑满足需求。	灵敏度最高:本质是对传统高端流式进行操作和分析的规范化。操作简单,标本量少的实验室一般电脑可以满足需求。	性价比最高:可以实现多参数检测,做好单阳管质控,可以减除自发荧光。	技术高度最高:可以同时进行上百个独立通道检测, 且通道间无干扰,无需补偿。除了免疫表型分析,还能检测胞内信号通路等深入研究。
缺点	检测参数少,灵敏度较低。	耗时长,数据占用空间大,对电脑容量和内存要求高。	需要找到合适的单阳管做解析,数据分析复杂,对软件和电脑要求高。	获取速度慢,成本高,分析复杂,需要高端电脑和软件。