干血片法中孕期唐氏综合征产前筛查的多中心研究
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摘要
一、目的
对干血片法应用于中孕期产前筛查进行多中心的研究,探讨干血片法中孕期产前筛查的可行性,对其方法学进行初步评价,比较干血片法及血清法检测的相关性,并获取基于本研究人群干血片法各筛查指标在不同孕周的中位数值和计算中位数-孕周(天数)拟合曲线,确定中孕期干血片法唐氏筛查的风险切割值,初步建立干血片法中孕期DS筛查技术。
二、材料与方法
1.收集2011年4月至2011年9月间在云南省第一人民医院、陕西省妇幼保健院、四川大学华西医学中心第二医院、湖南省妇幼保健院、北京市海淀区妇幼保健院接受产前筛查的15-20+6周孕妇血清及干血片标本,并记录相关临床信息。
2.对干血片法AFP/free-β-HCG检测的精密度及灵敏度,及打孔位置的选择进行评价。
3.对收集的全部标本进行血清法中孕期三联筛查及干血片法AFP、Free β-HCG的检测,获得干血片法AFP、Free β-HCG的中位数-孕周(天数)拟合曲线,并计算风险。
4.随访妊娠结局。
5.分别比较干血片法与血清法AFP、Free β-HCG测量值、中位数值及中位数倍数(MoM)值的相关性,比较的血片法二联筛查与血清法三联筛查的一致性、筛查效率及诊断价值。获得干血片法二联筛查的风险切割值。
三、结果
1.本研究干血片法精密度检测变异系数(CV)控制在15%以内。AFP、free-βHCG检测底限分别为0.27u/ml,0.57ng/ml；功能灵敏度分别为1.2u/ml、5.9ng/ml。
2.共10252例标本纳入研究,三个干血片检测中心检测值一致性良好,年龄及体重的分布存在差异。干血片法AFP、Free β-HCG的MoM值分布为中位数和众数均为接近1的单峰曲线。
3.随访妊娠结局7685例,随访率为75%,各标本采集中心随访率不一致。发现胎儿先天异常共64例,其中21三体10例,18三体1例,开放性神经管畸形2例。
4.干血片法与血清法AFP、Free β-HCG的测量值、中位数值及MoM值成明显直线相关。DS筛查风险切割值设定为1/270时,干血片法二联筛查与血清法三联筛查结果比较,Kappa系数为0.511,筛查结果一致性中等。干血片法二联筛查检出率60%,假阳性率4.96%；血清法三联筛查检出率80%,假阳性率8.26%；各标本检测中心筛查效率不同。血清法三联筛查ROC曲线下面积(0.927)大于干血片法二联筛查(0.813),干血片法筛查的诊断价值略逊于血清法。
四、结论
干血片法中孕期DS产前筛查的方法是可行的。但是干血片法产前筛查系统与血清法筛查系统还存在一定的差异,干血片法产前筛查系统在各地的应用也存在差异,需建立各地自己的中位数-孕周(天数)拟合曲线,重新计算适合的cut-off值。
Objective
This is a multicenter research to evaluate the feasibility and the methodology of dried bloods spots (DBS) as a method for Down syndrome (DS) screening. To compare the correlation of the two prenatal screening programs--DBS and serum. And to set up the curve of median-weeks, and to obtain the cutoff value, and to establish second-trimester screening for Down syndrome in dried blood spots.
Materials and Methods
1. The serum and DBS samples of gravidas were collected from the obstetric clinics of yunnan province first people's hospital, maternity and child care centers of shaanxi province, sichuan university huaxi medical center hospital, the maternity and child care centers of hunan province, maternity and child care centers of haidian district Beijing city from April2011to Sept2011. The gestational week of all the gravidas was between15to20+6weeks, and record the relevant clinical information simultaneously.
2. To discuss the detection precision and sensitivity of DBS, and the selection of drilling position.
3. AFP, free β-HCG concentrations were measured for all the serum and DBS samples to set up the the curve of median-weeks.
4. Follow-up the pregnancy outcome.
5. Correlation of all parameters between DBS and serum were compared. The detection rates of DBS and serum prenatal screening program were compared, and their correlation was evaluated. To get the cutoff value of DBS DS screening.
Results
1. In this study, the precision detection of DBS control within15%, The functional sensitivity of AFP and free-β-HCG in DBS were1.2u/ml,5.9ng/ml, and the low limit of detection of AFP and free-β-HCG in DBS were0.27u/ml,0.57ng/ml.
2. A total of10252cases were collected, the measurement values of three certer were accordance, the distribution of age and weight in three certer were different. The MoM value of free β-HCG and AFP distribution in DBS were described as unimodal curves.
3.7685cases were followed up, follow-up rate was75%, the rate of follow-up in specimen collection centers were inconsistency. A total of64Fetal congenital anomaly was found, DS10cases,18-trisom1cases, NTD2cases.
4. When DS screening cutoff value was seted to1/270, the performance of DBS screening program was moderate consistent with that of the serum screening program, Kappa coefficient was0.511. The detection rate of DBS DS screening was60%, false-positive rate was4.96%; and for serum DS screening, the detection rate was80%, false-positive rate was8.26%. The screening efficiency of each specimen test center were different. AUC of serum method (0.927) were larger than that of DBS method (0.813), the diagnostic value of the DBS was inferior than the serum method.
Conclusions
DS prenatal screening in DBS is feasible. But it excisted differences between DBS screening system and serum system. The application of DBS screening system in various regions was different, still need to establish their own median-weeks (days) curve, to calculation suitable cut-off value.
对干血片法应用于中孕期产前筛查进行多中心的研究,探讨干血片法中孕期产前筛查的可行性,对其方法学进行初步评价,比较干血片法及血清法检测的相关性,并获取基于本研究人群干血片法各筛查指标在不同孕周的中位数值和计算中位数-孕周(天数)拟合曲线,确定中孕期干血片法唐氏筛查的风险切割值,初步建立干血片法中孕期DS筛查技术。
二、材料与方法
1.收集2011年4月至2011年9月间在云南省第一人民医院、陕西省妇幼保健院、四川大学华西医学中心第二医院、湖南省妇幼保健院、北京市海淀区妇幼保健院接受产前筛查的15-20+6周孕妇血清及干血片标本,并记录相关临床信息。
2.对干血片法AFP/free-β-HCG检测的精密度及灵敏度,及打孔位置的选择进行评价。
3.对收集的全部标本进行血清法中孕期三联筛查及干血片法AFP、Free β-HCG的检测,获得干血片法AFP、Free β-HCG的中位数-孕周(天数)拟合曲线,并计算风险。
4.随访妊娠结局。
5.分别比较干血片法与血清法AFP、Free β-HCG测量值、中位数值及中位数倍数(MoM)值的相关性,比较的血片法二联筛查与血清法三联筛查的一致性、筛查效率及诊断价值。获得干血片法二联筛查的风险切割值。
三、结果
1.本研究干血片法精密度检测变异系数(CV)控制在15%以内。AFP、free-βHCG检测底限分别为0.27u/ml,0.57ng/ml；功能灵敏度分别为1.2u/ml、5.9ng/ml。
2.共10252例标本纳入研究,三个干血片检测中心检测值一致性良好,年龄及体重的分布存在差异。干血片法AFP、Free β-HCG的MoM值分布为中位数和众数均为接近1的单峰曲线。
3.随访妊娠结局7685例,随访率为75%,各标本采集中心随访率不一致。发现胎儿先天异常共64例,其中21三体10例,18三体1例,开放性神经管畸形2例。
4.干血片法与血清法AFP、Free β-HCG的测量值、中位数值及MoM值成明显直线相关。DS筛查风险切割值设定为1/270时,干血片法二联筛查与血清法三联筛查结果比较,Kappa系数为0.511,筛查结果一致性中等。干血片法二联筛查检出率60%,假阳性率4.96%；血清法三联筛查检出率80%,假阳性率8.26%；各标本检测中心筛查效率不同。血清法三联筛查ROC曲线下面积(0.927)大于干血片法二联筛查(0.813),干血片法筛查的诊断价值略逊于血清法。
四、结论
干血片法中孕期DS产前筛查的方法是可行的。但是干血片法产前筛查系统与血清法筛查系统还存在一定的差异,干血片法产前筛查系统在各地的应用也存在差异,需建立各地自己的中位数-孕周(天数)拟合曲线,重新计算适合的cut-off值。
Objective
This is a multicenter research to evaluate the feasibility and the methodology of dried bloods spots (DBS) as a method for Down syndrome (DS) screening. To compare the correlation of the two prenatal screening programs--DBS and serum. And to set up the curve of median-weeks, and to obtain the cutoff value, and to establish second-trimester screening for Down syndrome in dried blood spots.
Materials and Methods
1. The serum and DBS samples of gravidas were collected from the obstetric clinics of yunnan province first people's hospital, maternity and child care centers of shaanxi province, sichuan university huaxi medical center hospital, the maternity and child care centers of hunan province, maternity and child care centers of haidian district Beijing city from April2011to Sept2011. The gestational week of all the gravidas was between15to20+6weeks, and record the relevant clinical information simultaneously.
2. To discuss the detection precision and sensitivity of DBS, and the selection of drilling position.
3. AFP, free β-HCG concentrations were measured for all the serum and DBS samples to set up the the curve of median-weeks.
4. Follow-up the pregnancy outcome.
5. Correlation of all parameters between DBS and serum were compared. The detection rates of DBS and serum prenatal screening program were compared, and their correlation was evaluated. To get the cutoff value of DBS DS screening.
Results
1. In this study, the precision detection of DBS control within15%, The functional sensitivity of AFP and free-β-HCG in DBS were1.2u/ml,5.9ng/ml, and the low limit of detection of AFP and free-β-HCG in DBS were0.27u/ml,0.57ng/ml.
2. A total of10252cases were collected, the measurement values of three certer were accordance, the distribution of age and weight in three certer were different. The MoM value of free β-HCG and AFP distribution in DBS were described as unimodal curves.
3.7685cases were followed up, follow-up rate was75%, the rate of follow-up in specimen collection centers were inconsistency. A total of64Fetal congenital anomaly was found, DS10cases,18-trisom1cases, NTD2cases.
4. When DS screening cutoff value was seted to1/270, the performance of DBS screening program was moderate consistent with that of the serum screening program, Kappa coefficient was0.511. The detection rate of DBS DS screening was60%, false-positive rate was4.96%; and for serum DS screening, the detection rate was80%, false-positive rate was8.26%. The screening efficiency of each specimen test center were different. AUC of serum method (0.927) were larger than that of DBS method (0.813), the diagnostic value of the DBS was inferior than the serum method.
Conclusions
DS prenatal screening in DBS is feasible. But it excisted differences between DBS screening system and serum system. The application of DBS screening system in various regions was different, still need to establish their own median-weeks (days) curve, to calculation suitable cut-off value.
引文
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1. Sherman SL, Freeman SB, Allen EG, Lamb NE. Risk factors for nondisjunction of trisomy 21. Cytogenet Genome Res.2005; 111(3-4):273-80.
2. Rodriguez-Hernandez ML, Montoya E. Fifty years of evolution of the term Down's syndrome. Lancet.2011; 378(9789):402.
3. Kubas C. Noninvasive means of identifying fetuses with possible Down syndrome:a review. J Perinat Neonatal Nurs.1999; 13(2):27-46.
4. Irving CA, Chaudhari MP. Cardiovascular abnormalities in Down's syndrome: spectrum, management and survival over 22 years. Arch Dis Child.2012; 97(4):326-30.
5. Buitenkamp TD, Pieters R, Gallimore NE, van der Veer A, Meijerink JP, Beverloo HB, et al. Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations. Leukemia.2012.
6. Livingston G, Strydom A. Improving Alzheimer's disease outcomes in Down's syndrome. Lancet.2012; 379(9815):498-500.
7. Hook EB. Rates of chromosome abnormalities at different maternal ages. Obstet Gynecol.1981; 58(3):282-5.
8. Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities. Am J Obstet Gynecol.1984; 148(7):886-94.
9. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L, Mackinson AM. First and second trimester antenatal screening for Down's syndrome:the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen.2003; 10(2):56-104.
10. Miao ZY, Liu X, Shi TK, Ge JM, Xu Y. [First trimester and second-trimester integrated screening for Down's syndrome]. Zhonghua Yi Xue Za Zhi.2011; 91(3): 185-8.
11. Grati FR, Barlocco A, Grimi B, Milani S, Frascoli G, Di Meco AM, et al. Chromosome abnormalities investigated by non-invasive prenatal testing account for approximately 50% of fetal unbalances associated with relevant clinical phenotypes. Am J Med Genet A.2010; 152A(6):1434-42.
12. Mizejewski GJ, Bellisario R, Beblowski DW, Carter TP. Commercial radioimmunoassay kit for measurement of alpha-fetoprotein adapted for use with dried blood specimens from newborns. Clin Chem.1982; 28(5):1207-10.
13. ACOG. Education Bulletin Maternal serum screening Number 228. September1996. Committee on Educational Bulletins of the American College of Obstericians and Gynecologists [J].Int J Gynaecol Obstet,1996,55(3):299-308.
14. Yu DY, Fu P, Zhang ZH, Wang F, Han MY, Ren HY, et al. [Evaluation of Down's syndrome screening methods using maternal serum biochemistry in the second trimester pregnancy]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.2011; 28(3):332-5.
15. Liu F, Liang H, Jiang X, Zhang Y, Xue L, Yang C, et al. Second trimester prenatal screening for Down's syndrome in Mainland Chinese subjects using double-marker analysis of alpha-fetoprotein and beta-human chorionic gonadotropin combined with measurement of nuchal fold thickness. Ann Acad Med Singapore.2011; 40(7):315-4.
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17. Benn P, Cuckle H, Pergament E. Non-invasive prenatal diagnosis for Down syndrome:the paradigm will shift, but slowly. Ultrasound Obstet Gynecol.2012; 39(2): 127-30.
18. Guthrie R, Susi A. A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics.1963; 32:338-43.
19. Duquette D, Rafferty AP, Fussman C, Gehring J, Meyer S, Bach J. Public support for the use of newborn screening dried blood spots in health research. Public Health Genomics.2011; 14(3):143-52.
20.区小冰,佟丽贞,张力等.干血纸片法用于葡萄糖-6磷酸脱氢酶基因突变型的检测。.中华儿科杂志.2000；7：22.
21.赵晏,伍欣星,干纸血直接用于DNA扩增。临床检验杂志,2001,1-55.
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26. Verloes A, Schoos R, Herens C, Vintens A, Koulischer L. A prenatal trisomy 21 screening program using alpha-fetoprotein, human chorionic gonadotropin, and free estriol assays on maternal dried blood. Am J Obstet Gynecol.1995; 172(1 Pt 1):167-74.
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32.刘娜.血片法中孕期唐氏综合症筛查的初步研究。北京协和医院,中国医学科学院北京协和医学院;2009.
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34. Vieira Neto E, Carvalho EC, Fonseca A. Adaptation of alpha-fetoprotein and intact human chorionic gonadotropin fluoroimmunometric assays to dried blood spots. Clin Chim Acta.2005; 360(1-2):151-9.
35.周玉侠.滤纸干血片唐氏综合征产前筛查AFP和β-hCG检测的影响因素.医学检验与临床.2010；21(5).
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47.周玉侠.滤纸干血片唐氏综合征产前筛查AFP和β-hCG检测的影响因素.医学检验与临床.2010；21(5).
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2. Rodriguez-Hernandez ML, Montoya E. Fifty years of evolution of the term Down's syndrome. Lancet.2011; 378(9789):402.
3. Kubas C. Noninvasive means of identifying fetuses with possible Down syndrome:a review. J Perinat Neonatal Nurs.1999; 13(2):27-46.
4. Irving CA, Chaudhari MP. Cardiovascular abnormalities in Down's syndrome: spectrum, management and survival over 22 years. Arch Dis Child.2012; 97(4):326-30.
5. Buitenkamp TD, Pieters R, Gallimore NE, van der Veer A, Meijerink JP, Beverloo HB, et al. Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations. Leukemia.2012.
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8. Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities. Am J Obstet Gynecol.1984; 148(7):886-94.
9. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty L, Mackinson AM. First and second trimester antenatal screening for Down's syndrome:the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen.2003; 10(2):56-104.
10. Miao ZY, Liu X, Shi TK, Ge JM, Xu Y. [First trimester and second-trimester integrated screening for Down's syndrome]. Zhonghua Yi Xue Za Zhi.2011; 91(3): 185-8.
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14. Yu DY, Fu P, Zhang ZH, Wang F, Han MY, Ren HY, et al. [Evaluation of Down's syndrome screening methods using maternal serum biochemistry in the second trimester pregnancy]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.2011; 28(3):332-5.
15. Liu F, Liang H, Jiang X, Zhang Y, Xue L, Yang C, et al. Second trimester prenatal screening for Down's syndrome in Mainland Chinese subjects using double-marker analysis of alpha-fetoprotein and beta-human chorionic gonadotropin combined with measurement of nuchal fold thickness. Ann Acad Med Singapore.2011; 40(7):315-4.
16. Midtrimester amniocentesis for prenatal diagnosis. Safety and accuracy. JAMA. 1976; 236(13):1471-6.
17. Benn P, Cuckle H, Pergament E. Non-invasive prenatal diagnosis for Down syndrome:the paradigm will shift, but slowly. Ultrasound Obstet Gynecol.2012; 39(2): 127-30.
18. Guthrie R, Susi A. A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics.1963; 32:338-43.
19. Duquette D, Rafferty AP, Fussman C, Gehring J, Meyer S, Bach J. Public support for the use of newborn screening dried blood spots in health research. Public Health Genomics.2011; 14(3):143-52.
20.区小冰,佟丽贞,张力等.干血纸片法用于葡萄糖-6磷酸脱氢酶基因突变型的检测。.中华儿科杂志.2000；7：22.
21.赵晏,伍欣星,干纸血直接用于DNA扩增。临床检验杂志,2001,1-55.
22. Karthipan SN, George E, Jameela S, Lim WF, Teh LK, Lee TY, et al. An assessment of three noncommercial DNA extraction methods from dried blood spots for beta-thalassaemia mutation identification. Int J Lab Hematol.2011; 33(5):540-4.
23. UNAIDS/WH01 Working Group on Global HIV/AIDS/STI Survei1 lance. Guidelines for Using HIV testing Technologies in Suryeillance 2001 [Z]. ISBN 9173—92—063—71.
24. Huang S, Erickson B, Mak WB, Salituro J, Abravaya K. A novel RealTime HIV-1 Qualitative assay for the detection of HIV-1 nucleic acids in dried blood spots and plasma. J Virol Methods.2011; 178(1-2):216-24.
25. Wong PY, Mee AV, Doran TA. Studies of an alpha-fetoprotein assay using dry blood-spot samples to be used for the detection of fetal neural tube defects. Clin Biochem. 1982; 15(3):170-2.
26. Verloes A, Schoos R, Herens C, Vintens A, Koulischer L. A prenatal trisomy 21 screening program using alpha-fetoprotein, human chorionic gonadotropin, and free estriol assays on maternal dried blood. Am J Obstet Gynecol.1995; 172(1 Pt 1):167-74.
27. Resnick L, Veren K, Salahuddin SZ, Tondreau S, Markham PD. Stability and inactivation of HTLV-Ⅲ/LAV under clinical and laboratory environments. JAMA.1986; 255(14):1887-91.
28. Bond WW, Favero MS, Petersen NJ, Gravelle CR, Ebert JW, Maynard JE. Survival of hepatitis B virus after drying and storage for one week. Lancet.1981; 1(8219):550-1.
29. Tsao D, Hsiao KJ, Wu JC, Chou CK, Lee SD. Two-site enzyme immunoassay for alpha-fetoprotein in dried-blood samples collected on filter paper. Clin Chem.1986; 32(11):2079-82.
30. Lambert-Messerlian GM, Eklund EE, Malone FD, Palomaki GE, Canick JA, D'Alton ME. Stability of first-and second-trimester serum markers after storage and shipment. Prenat Diagn.2006; 26(1):17-21.
31. Rashed MS, Ozand PT, Bucknall MP, Little D. Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry. Pediatr Res.1995; 38(3):324-31.
32.刘娜.血片法中孕期唐氏综合症筛查的初步研究。北京协和医院,中国医学科学院北京协和医学院;2009.
33. Cowans NJ, Stamatopoulou A, Liitti P, Suonpaa M, Spencer K. The stability of free-beta human chorionic gonadotrophin and pregnancy-associated plasma protein-A in first trimester dried blood spots. Prenat Diagn.2011; 31(3):293-8.
34. Vieira Neto E, Carvalho EC, Fonseca A. Adaptation of alpha-fetoprotein and intact human chorionic gonadotropin fluoroimmunometric assays to dried blood spots. Clin Chim Acta.2005; 360(1-2):151-9.
35.周玉侠.滤纸干血片唐氏综合征产前筛查AFP和β-hCG检测的影响因素.医学检验与临床.2010；21(5).
36. Mei JV,Alexander JR,Adam BW,et al.Use of filter paper for the collection and analysis of human whole blood specimens[J].J Nutr.2001; 131 (5):1631S-6S.
37.周玉侠.红细胞压积对干血片法Down综合征产前筛查结果的影响.山东医药.2010；50(43).
38. Macri JN, Anderson RW, Krantz DA, Larsen JW, Buchanan PD. Prenatal maternal dried blood screening with alpha-fetoprotein and free beta-human chorionic gonadotropin for open neural tube defect and Down syndrome. Am J Obstet Gynecol. 1996; 174(2):566-72.
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