K562细胞ssDNA适配子检测方法对慢性粒细胞白血病诊断价值的研究

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查成喜, 邢福军, 张雪燕, 郭鹏, 习静, 韩跃武. K562细胞ssDNA适配子检测方法对慢性粒细胞白血病诊断价值的研究. 检验医学, 2014, 29(8): 835-837[ZHA Chengxi, XING Fujun, ZHANG Xueyan, GUO Peng, XI Jing, HAN Yuewu. Study on the significance of K562 cell ssDNA aptamer detection method for the clinical diagnosis of chronic myeloid leukemia. Labratory Medicine, 2014, 29(8): 835-837] 复制到剪切板

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K562细胞ssDNA适配子检测方法对慢性粒细胞白血病诊断价值的研究

查成喜¹, 邢福军¹, 张雪燕², 郭鹏², 习静², 韩跃武²

1. 甘肃省中医院检验科,甘肃兰州 730050

2. 兰州大学基础医学院生物化学与分子生物学研究所,甘肃兰州 730000

通讯作者:张雪燕,联系电话:0931-8259864。

作者简介:查成喜,男,1986年生,硕士,主管技师,主要研究方向为生物医学诊断技术。

基金:兰州大学中央高校基本科研业务费专项资金资助项目(lzujbky-2010-1E8); 甘肃省自然科学基金资助项目(1107RJZA219)

摘要

目的

评价K562细胞ssDNA适配子检测方法(简称适配子检测法)对慢性粒细胞白血病(CML)的临床诊断价值。

方法

采集18例CML患者、22例其他白血病患者、20名健康体检者(正常对照组)的血液标本各5 mL,分离粒细胞,用建立的适配子检测法检测荧光强度,绘制受试者工作特征(ROC)曲线。

结果

CML组荧光强度为492.26±41.67,与其它白血病组(466.86±33.23)及正常对照组(469.33±37.13)比较,差异无统计学意义(P值分别为0.078、0.243);其它白血病组和正常对照组比较,差异亦无统计学意义(P=0.835)。适配子检测法诊断CML的ROC曲线下面积为0.702,最佳阳性临界值为475.13,灵敏度为83.33%、特异性为54.76%、阳性预测值为44.11%、阴性预测值为88.46%。

结论

适配子检测法对CML的诊断具有较低的准确性,实际应用还有一定的局限性。

关键词: 慢性粒细胞白血病; 核酸适配子; 检测方法; 诊断价值

中图分类号:R446.11 文献标志码:A 文章编号:1673-8640(2014)08-0835-03

Study on the significance of K562 cell ssDNA aptamer detection method for the clinical diagnosis of chronic myeloid leukemia

ZHA Chengxi¹, XING Fujun¹, ZHANG Xueyan², GUO Peng², XI Jing², HAN Yuewu²

1. Department of Clinical Laboratory, Gansu Provincial Hospital of Traditional Chinese Medicine, Gansu Lanzhou 730050, China

2. Institute of Biochemistry and Molecular Biology, School of Basic Medicine, Lanzhou University, Gansu Lanzhou 730000, China

Abstract

Objective

To evaluate the significance of K562 cell ssDNA aptamer detection method for the clinical diagnosis of chronic myeloid leukemia(CML).

Methods

A total of 5 mL blood samples of 18 patients with CML, 22 patients with other leukemia and 20 healthy subjects were collected. The granulocytes were separated, and aptamer detection method was established for the determination of fluorescence intensity. The receiver operating characteristics (ROC) curve was drawn.

Results

The fluorescence intensities were 492.26±41.67 in CML group, 466.86±33.23 in other leukemia group and 469.33±37.13 in healthy control group, and the results showed no statistical significance (P=0.078 andP=0.243). There was no statistical significance between other leukemia group and healthy control group (P=0.835). The area under ROC curve was 0.702, the optimal positive threshold was 475.13, the sensitivity was 83.33%, the specificity was 54.76%, the positive predictive value was 44.11%, and the negative predictive value was 88.46%.

Conclusions

The aptamer detection method has a low accuracy in the clinical diagnosis of CML, and it has some limitations for clinical application.

Keyword: Chronic myeloid leukemia; Aptamer; Detection method; Diagnosis significance

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引言

白血病(leukemia)是起源于骨髓多能造血干细胞的恶性增殖性肿瘤,是十大高发性肿瘤之一。其中慢性粒细胞性白血病(chronic myeloid leukemia,CML)约占各类白血病的20%,占慢性白血病的95%。临床上CML的诊断主要依靠血象、骨髓象、细胞化学染色以及用流式细胞仪检测白血病细胞抗原^{[ 1, 2]}。但是血象检查无特异性;骨髓穿刺具有一定的风险性,且技术要求较高;细胞化学染色结果受影响的因素较多,如试剂、每个实验人员判断标准等,使结果相差很大;流式细胞仪检测价格昂贵,不能作为常规体检的项目对白血病进行人群普查^{[ 3, 4]}。我们利用前期建立的高灵敏度、高特异性的K562细胞ssDNA核酸适配子检测方法(简称适配子检测法)^{[ 5, 6]}检测临床白血病患者血液标本,评价该法在临床CML诊断中的实际应用价值。

材料和方法

一、标本收集及处理

选择2010年6月至2011年10月首次入院,经临床诊断标准^{[ 2]}确诊的CML患者18例(男10例,女8例,平均年龄35岁)、其他白血病患者22例(男10例,女12例,平均年龄37岁),另选取同期健康体检者20名作为正常对照组,男10例,女10例,平均年龄35岁。

二、仪器及试剂

RF-5301PC型荧光分光光度计(日本);Promega链霉亲和素磁珠及磁力架(Promega公司);洗涤缓冲液:4.5 g/L葡萄糖、5 mmol/L MgCl₂·6H₂O溶于含0.1 g/L MgCl₂和0.133 g/L CaCl₂的磷酸盐缓冲液(PBS);结合缓冲液:洗涤缓冲液中加入0.1 mg/mL tRNA、1 mg/mL BSA和0.1%叠氮钠。其它试剂均为分析纯,实验用水均为去离子水。

三、方法

分别采集所有对象静脉血5 mL,分离粒细胞,-20 ℃保存备用。按照构建的检测体系的相关参数^{[ 6]},分别与上述36例血样1×10⁶个中性粒细胞37 ℃黑暗环境孵育,用结合缓冲液洗涤,上磁力架,收集沉淀,加入400 μL结合缓冲液,即适配子-细胞-适配子复合物。用荧光分光光度计测定复合物荧光强度,测定3次,取平均值。

四、统计学方法

计量资料以 ±s表示,应用SPSS 16.0软件进行单因素方差分析和绘制受试者工作特征(ROC)曲线。 P<0.05为差异有统计学意义。

结果

一、各组适配子-细胞-适配子复合物的荧光强度比较

所有数据分布基本符合正态分布,95%分布区间为(463.23,488.71)。CML组适配子-细胞-适配子复合物荧光强度为492.26±41.67,与其它白血病组(466.86±33.23)及正常对照组(469.33±37.13)比较,差异无统计学意义( P值分别为0.078、0.243);其它白血病组与正常对照组之间差异亦无统计学意义( P=0.835)。

二、适配子检测法对CML诊断价值的评价

适配子检测法诊断CML的ROC曲线下面积为0.702,最佳临界值为475.13,见图1。以适配子-细胞-适配子复合物荧光强度≥475.13为阳性,对CML的诊断结果见表1。灵敏度(Sen)为83.33%、特异性(Spe)为54.76%、阳线预测值(pv+)为44.11%、阴性预测值(pv-)为88.46%、诊断效率(DE)为30%、诊断指数(DI)为138.09%、可用度(DA)为0.38、可靠度(DR)为0.86、优势比(RP)为6.05。

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	图1 适配子检测法诊断CML的ROC曲线

表1 临床诊断和适配子检测法的诊断结果比较

讨论

本研究结果显示适配子检测法诊断CML的ROC曲线下面积为0.702;当最佳临界值为475.13时,适配子检测法诊断CML的灵敏度为83.33%、特异性为54.76%;通过ROC曲线结果及与评价指标参考区间^{[ 10]} 的比较,适配子检测法对CML临床诊断具有较低的准确性,实际应用还有一定的局限性,目前还不具备真正的临床应用价值。我们推测造成该法无临床应用价值的原因主要有:(1)收集CML标本数量较少,未能进行大量样本的检测,相对容易产生偏倚;(2)所应用的核酸适配子是在前期试验中以CML K562细胞株为材料,经cell-SELEX筛选得到的,虽然甄别K562细胞具有较高的特异性和亲和力,但是临床CML患者血细胞在形态结构、细胞膜表面物质表达等方面可能具有一定的差异性;(3)收集的血标本放置时间过长,没有及时处理等原因,造成细胞死亡或形态结构改变,引起结合荧光强度降低,本研究中适配子检测法诊断CML的特异性仅为54.76%。

综上所述,适配子检测法目前尚不能应用于临床诊断CML。我们期望通过后续增加样本量、进一步加强与临床合作、重新针对临床细胞筛选核酸适配子等手段将其真正应用于临床诊断中,为临床医师诊断肿瘤提供一种更灵敏、更简便、更经济的检测方法,为社会和广大患者做出积极的贡献。同时,进行有关肿瘤体内外分子成像的研究,为肿瘤诊断提供一种新的分子探针技术。

The authors have declared that no competing interests exist.

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2009

0.0

... 临床上CML的诊断主要依靠血象、骨髓象、细胞化学染色以及用流式细胞仪检测白血病细胞抗原^[1,2] ...

2008

0.0

... 临床上CML的诊断主要依靠血象、骨髓象、细胞化学染色以及用流式细胞仪检测白血病细胞抗原^[1,2] ...

... 材料和方法一、标本收集及处理选择2010年6月至2011年10月首次入院,经临床诊断标准^[2]确诊的CML患者18例(男10例,女8例,平均年龄35岁)、其他白血病患者22例(男10例,女12例,平均年龄37岁),另选取同期健康体检者20名作为正常对照组,男10例,女10例,平均年龄35岁 ...

2010

3.542

0.0

... 流式细胞仪检测价格昂贵,不能作为常规体检的项目对白血病进行人群普查^[3,4] ...

2010

1.681

0.0

. 2010, 91(2):174-179 DOI:10.1007/s12185-010-0529-5

Classification and diagnosis of myeloproliferative neoplasms according to the 2008 World Health Organization criteria

1.Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA 2.Division of Hematology, Department of Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA

The myeloproliferative neoplasms (MPNs) were first recognized by William Dameshek in 1951. The classic MPNs were polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and chronic myelogenous leukemia. They were originally grouped together based on their shared phenotype of myeloproliferation. Since then, important discoveries have been made, identifying a central role of protein tyrosine kinases in the pathogenesis of these disorders. As such, the 2008 WHO diagnostic classification for myeloproliferative neoplasms has incorporated molecular markers with histologic, clinical and laboratory information into the diagnostic algorithms for the MPNs. Important changes include (1) the change of nomenclature of myeloproliferative disorder to myeloproliferative neoplasm emphasizing the clonal nature of these disorders; (2) the classification of mast cell disease as an MPN; (3) the reorganization of the eosinophilic disorders into a molecularly defined category of PDGFRA , PDGFRB and FGFR1 -associated myeloid and lymphoid neoplasms with eosinophilia and chronic eosinophilic leukemia, not otherwise specified; and (4) refinement of the diagnostic criteria for PV, ET and PMF incorporating recently described molecular markers, JAK2 V617F, JAK2 exon 12 mutations and MPL mutations. This review focuses upon the important changes of the 2008 WHO diagnostic criteria for MPNs.

... 流式细胞仪检测价格昂贵,不能作为常规体检的项目对白血病进行人群普查^[3,4] ...

2009

0.0

... 我们利用前期建立的高灵敏度、高特异性的K562细胞ssDNA核酸适配子检测方法(简称适配子检测法)^[5,6]检测临床白血病患者血液标本,评价该法在临床CML诊断中的实际应用价值 ...

2010

0.0

... 按照构建的检测体系的相关参数^[6],分别与上述36例血样1#cod#x000d7 ...

2011

0.0

1998

0.0

1993

2.044

0.0

. 1993, 12(14):1293-1316

Combining independent studies of a diagnostic test into a summary roc curve: Data-analytic approaches and some additional considerations

Lincoln E. Moses 1 ,David Shapiro 1 andBenjamin Littenberg 2

1 Department of Statistics, Stanford University, Stanford, CA 94305–5092, U.S.A. 2 Department of Medicine, Dartmouth Hitchcock Medical Center, Hanover, NH 03756, U.S.A.

> We consider how to combine several independent studies of the same diagnostic test, where each study reports an estimated false positive rate )FPR( and an estimated true positive rate )TPR(. We propose constructing a summary receiver operating characteristic )ROC( curve by the following steps. )i( Convert each FPR to its logistic transform U and each TPR to its logistic transform V after increasing each observed frequency by adding 1/2. )ii( For each study calculate D = V – U , which is the log odds ratio of TPR and FPR, and S = V + U , an implied function of test threshold; then plot each study's point )S i , D i ( . )iii( Fit a robust-resistant regression line to these points )or an equally weighted least-squares regression line(, with V – U as the dependent variable. )iv( Back-transform the line to ROC space. To avoid model-dependent extrapolation from irrelevant regions of ROC space we propose defining a priori a value of FPR so large that the test simply would not be used at that FPR, and a value of TPR so low that the test would not be used at that TPR. Then )a( only data points lying in the thus defined north-west rectangle of the unit square are used in the data analysis, and )b( the estimated summary ROC is depicted only within that subregion of the unit square. We illustrate the methods using simulated and real data sets, and we point to ways of comparing different tests and of taking into account the effects of covariates.

2008

0.0

... 通过ROC曲线结果及与评价指标参考区间^[10] 的比较,适配子检测法对CML临床诊断具有较低的准确性,实际应用还有一定的局限性,目前还不具备真正的临床应用价值 ...