慢性髓细胞性白血病BCR::ABL1基因p210转录本实验室自建荧光定量PCR检测方法的性能确认  

Analytic performance validation of laboratory-developed real-time quantitative PCR (RQ-PCR) test for BCR::ABL1 p210 transcript in chronic myeloid leukemia

作　　者：韩聪 宋鸽 马娇 徐世才 孙琦 王慧君 肖志坚 姚瑶 Han Cong;Song Ge;Ma Jiao;Xu Shicai;Sun Qi;Wang Huijun;Xiao Zhijian;Yao Yao(National Clinical Research Center for Blood Diseases,State Key Laboratory of Experimental Hematology,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所),实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,细胞生态海河实验室,血液病理诊断中心,天津300020

出　　处：《中华检验医学杂志》2023年第10期1040-1047,共8页Chinese Journal of Laboratory Medicine

基　　金：中国医学科学院医学与健康科技创新工程项目(2022-I2M-1-022);实验血液学国家重点实验室转化项目(Z22-06, Z21-09)。

摘　　要：目的建立BCR::ABL1 p210转录本荧光定量PCR(RQ-PCR)实验室自建检测方法和分析性能确认。方法本研究采用欧洲抗癌计划(EAC)公布的BCR::ABL1 p210转录本RQ-PCR检测的引物和探针序列建立了BCR::ABL1 p210转录本RQ-PCR实验室自建检测(LDT)方法, 通过WHO国际参考品获得转换系数(CF), 并使用K562和HL60细胞系混合物构建两个不同水平的实验室内部质控品。回顾性分析慢性髓细胞性白细胞(CML)患者阳性标本, 参考CLSI MM01-A3等指南评价实验室自建BCR::ABL1 p210 RQ-PCR 方法的精密度、正确度、线性范围、分析灵敏度和分析特异性等参数。采用Bland-Altman法对LDT方法与美国FDA批准的BCR::ABL1 p210转录本定量检测体外诊断(IVD)试剂盒检测结果进行一致性分析。结果本中心通过WHO国际参考品校准获得的BCR::ABL1国际标准值(BCR::ABL1IS)的CF为0.535。本研究建立的LDT方法在4个分子学反应(MR)水平(0.5、1.5、2.5、3.5)的BCR::ABL1IS 的重复性变异系数(CV)分别为7.44%、5.33%、9.12%、18.06%, 室内总不精密度CV分别为7.99%、5.49%、10.95%、17.99%。2020至2022年本实验室所有美国病理学家协会(CAP)能力验证(PT)样本的MR值均在CAP允许的变异范围内, MR结果与CAP-PT MR平均值强相关(r=0.999, P<0.01), 所有差值均在一致性界限范围内。本研究建立的LDT方法与Qiagen IVD试剂盒检测结果相关性高(r=0.997, P<0.01)。e13a2转录本的BCR::ABL1IS线性范围为0.001%~7.454%, 最大CV值为64.09%。e14a2转录本的BCR::ABL1IS线性范围为0.002%~12.398%, 最大CV值为43.37%。e13a2转录本和e14a2转录本的最低检测限(LoD)分别为MR5.0(0.001% IS)和MR4.8(0.002% IS);e13a2和e14a2转录本定量检测限(LoQ)均为MR4.7(0.002% IS)。该方法分析特异性为100%, 与BCR::ABL1 p190转录本检测无交叉反应性。结论本实验室自建的BCR::ABL1 p210转录本RQ-PCR定量检测体系分析性能良好, 可满足CML患者BCR::ABL1融合基因的临床检测需求。Objective To establish and validate analytic performance of laboratory-developed real-time quantitative polymerase chain reaction(RQ-PCR)test(LDT)for BCR::ABL1 p210 transcript.Methods Using the primes and probes released by the Europe Against Cancer Program(EAC),we have established BCR::ABL1 p210 RQ-PCR test.The laboratory-specific conversion factor(CF)was determined by the WHO 1st International Genetic Reference Panel,and a two-level internal control is developed using a mixture of K562 and HL60 cell lines was created to ensure traceability.Analytical performance,including analytical accuracy,analytical precision,linearity range,analytic sensitivity and specificity of RQ-PCR LDT test for BCR::ABL1 p210 transcript were validated according to CLIS guidelines.Furthermore,a comparison was made with an FDA-cleared RQ-PCR in vitro diagnostics(IVD)kit by Bland-Altman analysis.Results The laboratory specific conversion factor(CF)for LDT RQ-PCR was determined to be 0.535 based on WHO 1st International Genetic Reference Panel,which can be used to convert to the BCR::ABL international scale(BCR::ABL1IS)reliably.The repeatability of BCR::ABL1IS results at 4 different molecular response(MR0.5,MR1.5,MR2.5,MR3.5)levels are 7.44%,5.33%,9.12%and 18.06%,respectively,with total precision of 7.99%,5.49%,10.95%and 17.99%.The previous CAP proficiency test(PT)results from our laboratory were within the acceptable range of variation.MR results of our laboratory and MR mean value of all CAP-PT laboratory is highly correlated(r=0.999,P<0.01),and consistent according to Bland-Altman analysis.Furthermore,the LDT method in our laboratory has a high correlation with the test results of FDA-cleared Qiagen IVD kit(r=0.997,P<0.01).BCR::ABL1IS results of BCR::ABL1 e13a2 transcript showed linearity within the range of 0.001%-7.454%,with a maximum coefficient of variation(%CV)64.09%.The linearity range of e14a2 transcript BCR::ABL1IS was 0.002%-12.398%,with a maximum%CV of 43.37%.The test has a limit of detection(LoD)of MR5.0(0.001%IS)for e13a2 an

关 键 词：聚合酶链反应 BCR::ABL1 实验室自建检测 性能确认 

分 类 号：R733.72[医药卫生—肿瘤]