串联质谱法和荧光分析法检测滤纸干血片苯丙氨酸的比较

doi:10.3969/j.issn.1673-8640.2016.09.018

摘要/Abstract

摘要：

目的比较荧光分析法和串联质谱法检测滤纸干血片上苯丙氨酸（Phe）水平的分布和差异,为实验室开展新生儿高苯丙氨酸血症（HPA）的筛查提供方法学依据。方法选择进行遗传代谢病筛查的滤纸干血片样本62 510例。采用荧光分析法和串联质谱法同步检测新生儿滤纸干血片的Phe浓度,同时还用串联质谱法检测酪氨酸（Tyr）浓度。采用配对Wilcoxon检验和等级相关分析评价2种方法间的差异和相关性,采用Bland-Altman一致性分析评价2种方法间的一致性。结果荧光分析法和串联质谱法均在62 510例新生儿滤纸干血片样本中检出3例HPA,敏感性均为100%。荧光分析法的阳性预期值为33.3%,串联质谱法的阳性预期值为18.8%,若联合Phe/Tyr比值,串联质谱法的阳性预期值可达100%。正常新生儿Phe浓度呈偏态分布,荧光分析法和串联质谱法检测结果<60 μmol/L者分别占96.31%和95.08%,≥120 μmol/L者仅占0.01%和0.03%。荧光分析法的测定值先低于串联质谱法,至97%位点（Phe≈63 μmol/L）处基本持平,然后逐渐高于串联质谱法,2种方法的测定值呈正相关（r=0.43,P<0.01）。以不同浓度的Phe测定值作Bland-Altman分析,Phe浓度越高,2种方法的偏移越小,Phe>120 μmol/L的19个样本点全部落在95%的一致性限内,2种方法有很好的一致性,检测结果有可替代性。结论荧光分析法和串联质谱法检测Phe在低浓度时有差异,高浓度时一致性好。2种方法对于HPA的临床判断无影响,均能用于新生儿筛查。串联质谱法可同时检测Phe和Tyr浓度,若联合Phe/Tyr比值,检出HPA的阳性预期值很高。

关键词: 苯丙氨酸, 串联质谱法, 荧光分析法, 滤纸干血片样本

Abstract:

Objective To compare the distribution and differences of phenylalanine（Phe） concentration on dry blood spot by fluorescence assay and tandem mass spectrometry,and to provide a methodological reference for newborn hyperphenylalaninemia（HPA）screening program. Methods A total of 62 510 dry blood spot samples were collected for metabolic disease screening,and the Phe concentration was determined by fluorescence assay and tandem mass spectrometry. Tandem mass spectrometry was used to determine tyrosine（Tyr） concentration. The differences and correlation between the 2 methods were analyzed by paired Wilcoxon test and rank correlation analysis,and the consistency was compared by Bland-Altman analysis. Results Both fluorescence assay and tandem mass spectrometry determined 3 cases of HPA in 62 510 dry blood spot samples,and the sensitivities were 100%. The positive predictive value of fluorescence assay was 33.3%. The positive predictive value of tandem mass spectrometry was 18.8%,and it can be up to 100% combined with Phe/Tyr ratio. The distribution of Phe concentrations of normal newborn samples was skewed,and the results of fluorescence assay and tandem mass spectrometry as <60 μmol/L accounted for 96.31% and 95.08%,respectively,and the results as ≥120 μmol/Laccounted for only 0.01% and 0.03%. The concentration by fluorescence assay was lower than that by tandem mass spectrometry. To the 97% percentile （Phe≈63 μmol/L）,the concentration went to flat,then gradually was higher than that by tandem mass spectrometry. The 2 methods were related positively （r=0.43,P<0.01）. For Bland-Altman analysis,the higher the concentration of Phe was,the smaller the bias of the 2 methods was. When Phe was >120 μmol/L,19 sample points all fell within the 95% limit of consistency,the 2 methods were in good consistency,and the results can be replaceable. Conclusions There are differences by fluorescence assay and tandem mass spectrometry to determine Phe with low concentrations,while the 2 methods are in good consistency for that with high concentrations. The 2 methods had no effect on clinical judgment for HPA,and they can be used in newborn screening. Tandem mass spectrometry can simultaneously determine Phe and Tyr concentrations,and the positive predictive value for HPA can be up to 100% combined with Phe/Tyr ratio.

Key words: Phenylalanine, Tandem mass spectrometry, Fluorescence assay, Comparative study, Dry blood spot

中图分类号:

R446.1

田国力, 王燕敏, 许洪平, 郭静, 周卓, 姚静. 串联质谱法和荧光分析法检测滤纸干血片苯丙氨酸的比较[J]. 检验医学, 2016, 31(9): 814-819.

TIAN Guoli, WANG Yanmin, XU Hongping, GUO Jing, ZHOU Zhuo, YAO Jing. Comparative study of determining phenylalanine on dry blood spot by tandem mass spectrometry and fluorescence assay[J]. Laboratory Medicine, 2016, 31(9): 814-819.

图/表 7

参考文献 13

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病例	性别	荧光分析法 Phe（μmol/L）	串联质谱法			诊断
病例	性别	荧光分析法 Phe（μmol/L）	Phe（μmol/L）	Tyr（μmol/L）	Phe/Tyr比值	诊断
1	女	1 282.2	1 131.86	43.59	25.97	PKU
2	男	219.6	261.46	29.61	8.86	PKU
3	女	286.8	244.70	60.52	3.57	轻度HPA

病例	性别	荧光分析法 Phe（μmol/L）	串联质谱法			诊断
病例	性别	荧光分析法 Phe（μmol/L）	Phe（μmol/L）	Tyr（μmol/L）	Phe/Tyr比值	诊断
1	女	1 282.2	1 131.86	43.59	25.97	PKU
2	男	219.6	261.46	29.61	8.86	PKU
3	女	286.8	244.70	60.52	3.57	轻度HPA

Phe（μmol/L）	荧光分析法			串联质谱法
Phe（μmol/L）	例数	构成比（%）	累计频率（%）	例数	构成比（%）	累计频率（%）
<60	60 202	96.31	96.31	59 435	95.08	95.08
60~<120	2 300	3.68	99.99	3 059	4.89	99.97
≥120	8	0.01	100.00	15	0.03	100.00

Phe（μmol/L）	荧光分析法			串联质谱法
Phe（μmol/L）	例数	构成比（%）	累计频率（%）	例数	构成比（%）	累计频率（%）
<60	60 202	96.31	96.31	59 435	95.08	95.08
60~<120	2 300	3.68	99.99	3 059	4.89	99.97
≥120	8	0.01	100.00	15	0.03	100.00

方法	例数	x	s	Z值	P值	百分位点
方法	例数	x	s	Z值	P值	5%	25%	50%	75%	95%	96%	97%	98%	99%
荧光分析法	62 507	21.8	18.9	32.67	<0.01	0.60	5.40	19.20	33.00	56.40	59.4	63.0	68.40	78.00
串联质谱法	62 507	42.2	9.9	13.24	<0.01	28.41	35.21	40.95	47.72	59.88	61.47	63.48	66.44	71.59