不同保存方式对流式细胞术检测阵发性睡眠性血红蛋白尿症室间质量评价样本稳定性的影响

Effect of different preservation methods on stability of samples in external quality assessment of paroxysmal nocturnal hemoglobinuria by flow cytometry

目的分析不同保存方式对流式细胞术检测阵发性睡眠性血红蛋白尿症(PNH)室间质量评价(EQA)样本稳定性的影响,为建立PNH流式细胞术检测的EQA体系奠定基础。方法收集9例PNH阳性新鲜乙二胺四乙酸抗凝血液样本。将每例样本混匀后分成4份,其中3份分别使用国产细胞保存液(A组)、进口细胞保存液(B组)、枸橼酸-枸橼酸钠-葡萄糖(ACD)细胞保存液(C组)进行处理;1份作为对照,不添加细胞保存液(D组)。将处理后的样本充分混匀,每组再各分为5份,分别于2~6℃环境中保存1、3、5、7、14 d时,采用美国临床实验室标准化协会(CLSI)H52-A2文件推荐的规范化方法进行流式细胞检测,以确定不同保存方式的样本稳定性。结果与2~6℃环境保存1 d比较,A组、B组、D组保存3、5、7、14 d红细胞PNH克隆比例均呈现下降趋势(P<0.05),C组不同保存时间红细胞PNH克隆比例差异无统计学意义(P>0.05)。B组、C组白细胞PNH克隆比例(粒细胞和单核细胞)较稳定;A组、D组保存7、14 d与保存1 d比较,差异有统计学意义(P<0.05);A组、D组保存3、5 d,B组、C组保存3、5、7、14 d与保存1 d比较,差异均无统计学意义(P>0.05)。结论在PNH克隆检测中,无论红细胞PNH克隆还是白细胞PNH克隆,用ACD保存样本,均可以满足14 d内EQA样本稳定的要求,能最大限度确保样本的稳定性。

Objective To investigate the effect of different preservation methods on the stability of samples in external quality assessment(EQA)of paroxysmal nocturnal hemoglobinuria(PNH)by flow cytometry,and to lay the foundation for establishing an EQA system for PNH flow cytometry determination.Methods Totally,9 PNH positive fresh ethylenediaminetetracetic acid anticoagulated samples were collected.For each sample,the sample was mixed and then classified into 4 parts.The 3 parts of them were treated with 3 different cell preservation solutions,domestic company's cell preservation solution(Group A),foreign company's cell preservation solution(Group B)and acid-citrate-dextrose(ACD)cell preservation solution(Group C).The remaining 1 part was used as control without adding cell preservation solution(Group D).After the samples were treated,they were thoroughly mixed and divided into 5 parts and placed at 2-6℃.The sample stability under different preservation methods for 1,3,5,7 and 14 d was determined by standard flow cytometry recommended by the Clinical and Laboratory Standards Institute(CLSI)H52-A2.Results Compared with that at 2-6℃for 1 d,the red blood cell PNH clone ratio exhibited a decreasing trend in Group A,B and D at 3,5,7 and 14 d(P<0.05),while Group C showed no statistical significance(P>0.05).The white blood cell PNH clone ratio remained stable in Group B and C.There was statistical significance between 7,14 d and 1 d for Group A and D(P<0.05),and there was no statistical significance for Group A and D at 3 and 5 d as well as for Group B and C at 3,5,7 and 14 d compared to 1 d(P>0.05).Conclusions In PNH determination,whether it is red blood cell PNH clone or white blood cell PNH clone,the samples with ACD preservation can meet the requirement of EQA sample stability within 14 d,ensuring the sample stability to the maximum extent possible.