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浙江大学学报(医学版)  2020, Vol. 49 Issue (5): 565-573    DOI: 10.3785/j.issn.1008-9292.2020.10.03
专题报道     
递送及储存条件对新生儿干血斑标本中氨基酸和肉碱浓度的影响
胡凌微1(),胡真真1,杨建滨1,张玉2,施叶珍2,祝莎莎3,杨茹莱1,黄新文1,*()
1. 浙江大学医学院附属儿童医院遗传与代谢科 国家儿童健康与疾病临床医学研究中心 国家儿童区域医疗中心, 浙江 杭州 310052
2. 浙江博圣生物技术股份有限公司, 浙江 杭州 310012
3. 台州市妇女儿童医院儿科, 浙江 台州 318000
Effects of delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots
HU Lingwei1(),HU Zhenzhen1,YANG Jianbin1,ZHANG Yu2,SHI Yezhen2,ZHU Shasha3,YANG Rulai1,HUANG Xinwen1,*()
1. Department of Genetics and Metabolism, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, National Regional Medical Center for Children, Hangzhou 310052, China
2. Zhejiang Biosan Biochemical Technologies Co., Ltd., Hangzhou 310012, China
3. Department of Pediatrics, Taizhou Maternal and Child Health Hospital, Taizhou 318000, Zhejiang Province, China
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摘要:

目的: 探讨不同递送和储存条件对新生儿干血斑标本中氨基酸和肉碱浓度的影响,为提高串联质谱筛查新生儿遗传代谢病的准确性及可靠性提供依据。方法: 根据不同递送和储存条件将1 254 616名在浙江省新生儿疾病筛查中心进行遗传代谢病筛查新生儿的干血斑标本分为3组:室温递送储存(室温组,n=338 467)、冷链递送+低温储存(冷链+低温组,n=480 021)、冷链递送+低温低湿储存(冷链+低温低湿组,n=436 128)。应用串联质谱技术检测干血斑标本中氨基酸和肉碱浓度,SPSS 24.0软件进行数据分析,分析递送和储存过程中温度和湿度对氨基酸和肉碱浓度稳定性的影响。结果: 三组样本氨基酸和肉碱浓度均呈偏态分布,氨基酸和肉碱浓度组间差异均有统计学意义(均P < 0.01)。酪氨酸浓度中位数冷链+低温组与冷链+低温低湿组相近,室温组较冷链+低温组和冷链+低温低湿组分别降低18%和16%;室温组的蛋氨酸浓度中位数与冷链+低温组相近,分别较冷链+低温低湿组降低15%和11%;3组精氨酸浓度中位数均有差异,室温组和冷链+低温组分别较冷链+低温低湿组降低12%和25%。室温组的游离肉碱浓度中位数与冷链+低温组相近,较冷链+低温低湿组增高12%。冷链+低温组的乙酰肉碱、丙酰肉碱、丙二酰肉碱+3-羟基-丁酰肉碱、戊二酰肉碱+3-羟基-己酰肉碱、十六碳酰肉碱等浓度中位数与冷链+低温低湿组相近,室温组较冷链+低温组和冷链+低温低湿组降低21%~64%。其他氨基酸和酰基肉碱浓度组间差异较小。除瓜氨酸、甲基丙二酰肉碱+3-羟基-异戊酰肉碱、戊酰基肉碱外,室温组其他氨基酸和肉碱浓度月中位数变异系数均高于冷链+低温组和冷链+低温低湿组。结论: 使用冷链递送和低温低湿储存可以有效延缓干血斑标本中部分氨基酸和肉碱的降解速度,提高串联质谱检测结果的准确性和可靠性,从而保证新生儿遗传代谢病筛查质量。

关键词: 干血斑检测冷链运输储存温度湿度氨基酸类肉碱遗传代谢病筛查    
Abstract:

Objective: To explore effects of different delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots (DBS), so as to provide evidence for improving accurate and reliable detection by tandem mass spectrometry. Methods: A total of 1 254 616 newborn DBS samples in Newborn Screening Center of Zhejiang Province were delivered and stored at room temperature (group A, n=338 467), delivered by cold-chain logistics system and stored at low temperature (group B, n=480 021), or delivered by cold-chain logistics system and stored at low temperature and low humidity (group C, n= 436 128), respectively. The concentrations of amino acids and carnitines in DBS were detected by tandem mass spectrometry. Data analysis was performed by SPSS 24.0 to explore the influence of temperature and humidity on the concentrations of amino acids and carnitines. Results: The concentrations of amino acids and carnitines in the three groups were skewed, and the differences in amino acid and carnitine concentrations among groups were statistically significant (all P < 0.01). The median concentration of tyrosine was lower in group A than those in group B and group C by 18%and 16%respectively, while there was no significant difference between the last two groups. The median concentrations of methionine were lower in group A and group B than that in group C by 15%and 11%, respectively. The median concentrations of arginine were lower in group A and group B than that in group C by 12%and 25%, respectively. The median concentration of free carnitine (C0) was higher in group A than that in group C by 12%, while there was no significant difference between group A and group B. The median concentrations of acetylcarnitine (C2), propionyl carnitine (C3), C3DC+C4OH, C5DC+C6OH and hexadecanoyl carnitine (C16) were lower in group A than those in group B and group C by 21%-64%. The concentrations of other amino acids and acylcarnitines differed little among three groups. The monthly median coefficients of variation of other amino acids and carnitines in group A were higher than those in group B and group C except for citrulline, C4DC+C5OH and isovalerylcarnitine (C5). Conclusion: Cold-chain logistics system and storage in low temperature and low humidity can effectively reduce degradation of some amino acids and carnitines in DBS, improve the accuracy and reliability of detection, and thus ensures the quality of screening for neonatal metabolic diseases.

Key words: Dried blood spot testing    Cold-chain transportation    Storage    Temperature    Humidity    Amino acids    Carnitine    Genetic and metabolic diseases screening
收稿日期: 2020-05-12 出版日期: 2020-11-19
:  R446.1  
基金资助: 国家重点研发计划(2018YFC1002204);浙江省公益技术研究计划(LGC19B050013)
通讯作者: 黄新文     E-mail: hulw@zju.edu.cn;6305022@zju.edu.cn
作者简介: 胡凌微(1991-), 女, 学士, 技师, 主要从事儿童遗传代谢病筛查工作; E-mail:hulw@zju.edu.cn; https://orcid.org/0000-0001-8442-0579
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引用本文:

胡凌微,胡真真,杨建滨,张玉,施叶珍,祝莎莎,杨茹莱,黄新文. 递送及储存条件对新生儿干血斑标本中氨基酸和肉碱浓度的影响[J]. 浙江大学学报(医学版), 2020, 49(5): 565-573.

HU Lingwei,HU Zhenzhen,YANG Jianbin,ZHANG Yu,SHI Yezhen,ZHU Shasha,YANG Rulai,HUANG Xinwen. Effects of delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots. J Zhejiang Univ (Med Sci), 2020, 49(5): 565-573.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.10.03        http://www.zjujournals.com/med/CN/Y2020/V49/I5/565

组别 n 采血时间 递送条件 储存条件
室温组 338 467 2014年3月—2015年2月 室温 室温
冷链+低温组 480 021 2018年3月—2019年2月 冷链(温度4~8 ℃,湿度25%~65%) 低温(4~8 ℃储存)
冷链+低温低湿组 436 128 2019年3月—2020年2月 冷链(温度4~8 ℃,湿度25%~65%) 低温低湿(温度4 ℃,湿度30%)
表 1  三组新生儿干血斑标本递送和储存条件
组别 丙氨酸 精氨酸 瓜氨酸 甘氨酸 亮氨酸 蛋氨酸 鸟氨酸 苯丙氨酸 脯氨酸 酪氨酸 缬氨酸
中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%)
与室温组比较,*P < 0.01;与冷链+低温组比较,#P < 0.01.X0.5%:0.5%分位数;X99.5%:99.5%分位数.
室温组 337.48(153.51, 765.43) 9.06 9.95(1.66, 47.29) 40.84 14.72(7.06, 37.87) 2.91 501.82(194.86, 1252.35) 7.78 160.35(80.16, 318.75) 8.13 18.49(6.63, 46.48) 26.27 125.68(50.37, 394.95) 10.42 53.22(28.55, 102.78) 7.38 209.04(111.40, 435.93) 3.68 92.93(38.25, 279.11) 7.12 147.50(70.26, 308.93) 10.15
冷链+低温组 316.93(161.62, 681.14)* 3.36 8.48(1.16, 43.83)* 37.45 14.17(7.09, 34.35)* 4.63 531.04(278.70, 1187.46)* 3.03 166.28(92.51, 311.10)* 2.38 19.38(8.99, 41.71)* 16.85 122.79(54.53, 346.45)* 2.72 57.38(34.37, 102.35)* 3.92 204.20(114.45, 420.13)* 3.28 113.36(47.31, 341.59)* 2.93 145.14(80.59, 271.66)* 3.23
冷链+低温低湿组 341.77(159.76, 650.53)*# 4.01 11.36(1.37, 47.31)*# 28.29 14.47(7.11, 33.74)*# 4.77 541.75(259.40, 1189.72)*# 2.92 165.50(92.00, 313.55)*# 1.82 21.87(9.31, 42.46)*# 11.31 130.70(57.59, 354.70)*# 3.10 54.96(32.65, 98.47)*# 2.71 211.17(111.24, 411.68)*# 2.82 110.24(44.64, 329.13)*# 4.43 157.61(78.38, 275.11)*# 2.69
表 2  不同递送和储存条件下1 254 616名新生儿干血斑标本中各种氨基酸浓度
图 1  不同递送和储存条件下1 254 616名新生儿干血斑标本中各种氨基酸浓度月中位数变化趋势图
组别 游离肉碱 乙酰肉碱 丙酰肉碱 丁酰肉碱 丙二酰肉碱+3-羟基丁酰肉碱 戊酰肉碱 己酰肉碱 辛酰肉碱 C4DC+C5OH 癸酰肉碱 十二碳酰肉碱 十四碳酰肉碱 戊二酰肉碱+3-羟基己酰肉碱 十四碳烯酰肉碱 十六碳酰肉碱 十八碳酰肉碱
中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%) 中位数(X0.5%, X99.5%, μmol/L) 变异系数(%)
与室温组比较,*P < 0.01;与冷链+低温组比较,#P < 0.01.X0.5%:0.5%分位数;X99.5%:99.5%分位数.C4DC+C5OH:甲基丙二酰肉碱+3-羟基异戊酰肉碱.
室温组 23.59(10.38, 58.17) 8.25 14.51(2.98, 40.01) 19.36 1.33(0.42, 4.00) 9.71 0.20(0.10, 0.47) 5.82 0.09(0.03, 0.36) 16.54 0.11(0.05, 0.37) 3.85 0.04(0.02, 0.18) 30.76 0.06(0.02, 0.24) 33.89 0.18(0.08, 0.40) 2.44 0.07(0.03, 0.27) 6.43 0.07(0.03, 0.30) 10.16 0.16(0.06, 0.38) 4.25 0.04(0.01, 0.22) 50.33 0.05(0.03, 0.26) 16.76 2.46(0.53, 6.02) 6.69 0.79(0.26, 1.90) 7.22
冷链+低温组 23.39(10.49, 55.54)* 5.35 19.14(6.74, 45.39)* 7.13 1.69(0.55, 4.83)* 5.25 0.22(0.11, 0.54)* 4.97 0.12(0.04, 0.41)* 5.37 0.11(0.05, 0.37)* 6.34 0.04(0.02, 0.11)* 10.19 0.06(0.02, 0.18)* 8.32 0.21(0.10, 0.45)* 4.22 0.08(0.03, 0.28)* 4.76 0.08(0.02, 0.34)* 5.25 0.19(0.07, 0.45)* 0.00 0.11(0.05, 0.24)* 4.66 0.06(0.03, 0.30)* 0.00 3.25(0.72, 7.46)* 1.53 0.89(0.30, 1.98)* 1.80
冷链+低温低湿组 20.91(9.79, 49.25)*# 5.63 19.70(7.34, 46.64)*# 4.39 1.75(0.58, 4.99)*# 3.34 0.22(0.11, 0.54)*# 4.38 0.13(0.04, 0.44)*# 6.42 0.11(0.05, 0.36)*# 5.56 0.04(0.02, 0.11)*# 0.00 0.07(0.03, 0.21)*# 12.09 0.21(0.10, 0.46)*# 5.17 0.08(0.03, 0.29)*# 6.13 0.08(0.02, 0.35)*# 6.50 0.18(0.06, 0.44)*# 4.31 0.10(0.04, 0.21)*# 6.38 0.08(0.03, 0.31)*# 5.48 3.16(0.66, 7.34)*# 4.07 0.86(0.27, 1.93)*# 3.11
表 3  不同递送和储存条件下1 254 616名新生儿干血斑标本中各种肉碱浓度
图 2  不同递送和储存条件下1 254 616名新生儿干血斑标本中各种肉碱浓度月中位数变化趋势图
1 LIM M D . Dried blood spots for global health diagnostics and surveillance:opportunities and challenges[J]. Am J Trop Med Hyg, 2018, 99 (2): 256- 265
doi: 10.4269/ajtmh.17-0889
2 RYCKMAN K K , BERBERICH S L , SHCHELOCHKOV O A et al. Clinical and environmental influences on metabolic biomarkers collected for newborn screening[J]. Clin Biochem, 2013, 46 (1-2): 133- 138
doi: 10.1016/j.clinbiochem.2012.09.013
3 MEI J V , LI L , RASMUSSEN S A et al. Effect of specimen storage conditions on newborn dried blood spots used to assess Toxoplasma gondii immunoglobulin M (IgM)[J]. Clin Chim Acta, 2011, 412 (5-6): 455- 459
doi: 10.1016/j.cca.2010.11.028
4 THERRELL B L , HANNON W H , PASS K A et al. Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis:statement of the Council of Regional Networks for Genetic Services[J]. Biochem Mol Med, 1996, 57 (2): 116- 124
doi: 10.1006/bmme.1996.0017
5 中华人民共和国卫生部.新生儿疾病筛查技术规范(2010年版)[A/OL].(2010-11-10)[2020-06-11].http://www.nhc.gov.cn/cmsresources/mohfybjysqwss/cmsrsdocument/doc10798.doc.
Ministry of Health of the People's Republic of China. Technical guide of newborn screening in China(2010)[A/OL].(2010-11-10)[2020-06-11].http://www.nhc.gov.cn/cmsresources/mohfybjysqwss/cmsrsdocument/doc10798.doc. (in Chinese)
6 ADAM B W , HALL E M , STERNBERG M et al. The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States[J]. Clin Biochem, 2011, 44 (17-18): 1445- 1450
doi: 10.1016/j.clinbiochem.2011.09.010
7 GOLBAHAR J , ALTAYAB D D , CARREON E . Short-term stability of amino acids and acylcarnitines in the dried blood spots used to screen newborns for metabolic disorders[J]. J Med Screen, 2014, 21 (1): 5- 9
doi: 10.1177/0969141314525367
8 尚世强, 杨建滨, 王治国 . 新生儿遗传代谢病串联质谱筛查存在的问题及展望[J]. 中华检验医学杂志, 2016, 39 (4): 237- 239
SHANG Shiqiang , YANG Jianbin , WANG Zhiguo . Current problems and prospect on tandem mass spectrometry based newborn inherited metabolic diseases screening[J]. Chinese Journal of Laboratory Medicine, 2016, 39 (4): 237- 239
doi: 10.3760/cma.j.issn.1009-9158.2016.04.001
9 LEVY H L , SIMMONS J R , MACCREADY R A . Stability of amino acids and galactose in the newborn screening filter paper blood specimen[J]. J Pediatr, 1985, 107 (5): 757- 760
doi: 10.1016/s0022-3476(85)80411-x
10 CHACE D H , ADAM B W , SMITH S J et al. Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays[J]. Clin Chem, 1999, 45 (8): 1269- 1277
doi: 10.1093/clinchem/45.8.1269
11 STRNADOVá K A , HOLUB M , MVHL A et al. Long-term stability of amino acids and acylcarnitines in dried blood spots[J]. Clin Chem, 2007, 53 (4): 717- 722
doi: 10.1373/clinchem.2006.076679
12 FINGERHUT R , ENSENAUER R , R?SCHINGER W et al. Stability of acylcarnitines and free carnitine in dried blood samples:implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency[J]. Anal Chem, 2009, 81 (9): 3571- 3575
doi: 10.1021/ac8022235
13 JOHNSON D W , TRINH M U . Stability of malonylcarnitine and glutarylcarnitine in stored blood spots[J]. J Inherit Metab Dis, 2004, 27 (6): 789- 790
doi: 10.1023/b:boli.0000045830.82698.90
14 HAN J , HIGGINS R , LIM M D et al. Short-term stabilities of 21 amino acids in dried blood spots[J]. Clin Chem, 2018, 64 (2): 400- 402
doi: 10.1373/clinchem.2017.278457
15 PRENTICE P , TURNER C , WONG M C et al. Stability of metabolites in dried blood spots stored at different temperatures over a 2-year period[J]. Bioanalysis, 2013, 5 (12): 1507- 1514
doi: 10.4155/bio.13.121
16 SANTER R , FINGERHUT R , L?SSKER U et al. Tandem mass spectrometric determination of malonylcarnitine:diagnosis and neonatal screening of malonyl-CoA decarboxylase deficiency[J]. Clin Chem, 2003, 49 (4): 660- 662
doi: 10.1373/49.4.660
17 李惠中, 周伟, 王传霞 et al. 串联质谱筛查新生儿肉碱吸收障碍的临床研究[J]. 中华实用儿科临床杂志, 2019, 34 (14): 1053- 1059
LI Huizhong , ZHOU Wei , WANG Chuanxia et al. Clinical study on screening carnitine absorption deficiency in neonates by tandem mass spectrometry[J]. Journal of Applied Clinical Pediatrics, 2019, 34 (14): 1053- 1059
doi: 10.3760/cma.j.issn.2095-428X.2019.14.004
18 郭元芳, 李改杰, 田丽萍 et al. 串联质谱筛查新生儿遗传代谢疾病中氨基酸浓度波动性及其影响因素分析[J]. 中国妇幼保健, 2018, 33 (24): 5886- 5889
GUO Yuanfang , LI Gaijie , TIAN Liping et al. Tandem mass spectrometry screening of fluctuation of amino acid concentration in neonatal genetic metabolic diseases and analysis of influencing factors[J]. Maternal and Child Health Care of China, 2018, 33 (24): 5886- 5889
doi: 10.7620/zgfybj.j.issn.1001-4411.2018.24.74
[1] 黄新文 等. 浙江省新生儿氨基酸代谢疾病筛查及随访分析[J]. 浙江大学学报(医学版), 2017, 46(3): 233-239.
[2] 郝爱平. 人类pygo1基因的生物信息学分析[J]. 浙江大学学报(医学版), 2014, 43(4): 453-457.
[3] 章向群,吴凌华,陈方军,张伊,陈璐. 高效液相色谱-质谱法测定伏格列波糖片中伏格列波糖含量[J]. 浙江大学学报(医学版), 2014, 43(2): 141-144.
[4] . 小鼠体温对体积描记箱内压力变化的影响[J]. 浙江大学学报(医学版), 2011, 40(3): 315-320.
[5] 徐卫华. 小鼠呼出与吸入气体容量差值的测定[J]. 浙江大学学报(医学版), 2010, 39(3): 322-325.
[6] 应跃斌;孙红颖;丁丁;李丹曦;薛乔;陈枢青. 金黄色葡萄球菌肠毒素C2中缺口的形成及影响因素的研究[J]. 浙江大学学报(医学版), 2009, 38(5): 505-510.
[7] 谢志强. 温度敏感型瞬时受体电位通道与皮肤神经源炎症和瘙痒[J]. 浙江大学学报(医学版), 2009, 38(4): 409-414.
[8] 张恒义;施弘毅;王恩禹. 环境温度对单胺类神经递质微透析探针回收率的影响[J]. 浙江大学学报(医学版), 2009, 38(3): 271-275.
[9] 方雪玲,章云涛,方强,卢阳珍,傅素珍. 静脉营养对重症脓毒症患者血氨基酸谱及免疫的影响[J]. 浙江大学学报(医学版), 2007, 36(3): 298-302.
[10] 张科平;张恒义;郑筱祥. 微透析探针在体透析时间对氨基酸回收率的影响[J]. 浙江大学学报(医学版), 2006, 35(6): 642-647.
[11] 王林波, 张苏展, 丁克峰, 郑树. 大肠癌区域动静脉血氨基酸的研究[J]. 浙江大学学报(医学版), 1997, 26(3): 114-117.
[12] 江米足, 许蓓, 叶瑞云. 小儿腹泻时血清氨基酸浓度分析[J]. 浙江大学学报(医学版), 1997, 26(2): 71-73.
[13] 郑树森, 胡清凌, 孙义国, 林建灿, 李兰娟, 傅素贞. 阻塞性黄疸患者手术前后血清氨基酸谱变化分析[J]. 浙江大学学报(医学版), 1992, 21(5): 211-213.
[14] 胡永洲, 吴益均, 何建平. 固-液相转移催化烃化合成α-氨基酸[J]. 浙江大学学报(医学版), 1992, 21(2): 54-57.
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