{"title":"液相色谱串联质谱法测定干血斑类固醇谱:在新生儿先天性肾上腺增生筛查中的应用","authors":"Xia Zhan, Lianshu Han, Wenjuan Qiu, Xuefan Gu, Jun Guo, Siyu Chang, Yu Wang, Huiwen Zhang","doi":"10.1016/j.steroids.2022.109056","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Newborn screening for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA) generates a large number of false-positive results. The present study aimed to improve the sensitivity of the CAH neonatal screening by including second-tier steroid profiling in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS).</p></div><div><h3>Methods</h3><p>We developed and validated a LC-MS/MS method for simultaneous determination of six steroids in DBS, including androstenedione, testosterone, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, and cortisol. Two 5-mm blood spots were eluted by internal standard working solution. We analyzed 1170 DBS samples from neonates to determine gestational age-specific reference intervals. In order to test the specificity of the second-tier method, we analyzed 707 cards with a positive screening by DELFIA.</p></div><div><h3>Results</h3><p>Values of intra- and inter-day precision coefficients of variance and accuracy were 2.0%–13.3% and 85.8%–114.5%, respectively. Recovery ranged from 85.0% to 106.9%. The lower limit of quantification was 0.5 ng/mL for 21-deoxycortisol, 0.25 ng/mL for 17-hydroxyprogesterone and cortisol, and 0.1 ng/mL for testosterone, androstenedione, and 11-deoxycortisol. In addition, the linearity range was 0.25–50 ng/mL (R<sup>2</sup> > 0.99). According to the 17-hydroxyprogesterone levels and ratios of (androstenedione + 17-hydroxyprogesterone)/cortisol in the 707 positive screening samples, 77 neonates should receive recall visit. The number of false-positive results reduced by 89.1%. Totally, 18 newborns were diagnosed with 21-hydroxylase deficiency, one with P450 oxidoreductase deficiency and one with 11β-hydroxylase deficiency. With two-tier screening, the positive predictive value increased to 26.0%.</p></div><div><h3>Conclusions</h3><p>The second-tier steroid profiling by LC-MS/MS reduced the false-positive rate and improved the positive predictive value of CAH screening. We suggest applying this steroid profiling assay as a second-tier test for CAH screening in China.</p></div>","PeriodicalId":21997,"journal":{"name":"Steroids","volume":"185 ","pages":"Article 109056"},"PeriodicalIF":2.3000,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"4","resultStr":"{\"title\":\"Steroid profile in dried blood spots by liquid chromatography tandem mass spectrometry: Application to newborn screening for congenital adrenal hyperplasia in China\",\"authors\":\"Xia Zhan, Lianshu Han, Wenjuan Qiu, Xuefan Gu, Jun Guo, Siyu Chang, Yu Wang, Huiwen Zhang\",\"doi\":\"10.1016/j.steroids.2022.109056\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Newborn screening for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA) generates a large number of false-positive results. The present study aimed to improve the sensitivity of the CAH neonatal screening by including second-tier steroid profiling in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS).</p></div><div><h3>Methods</h3><p>We developed and validated a LC-MS/MS method for simultaneous determination of six steroids in DBS, including androstenedione, testosterone, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, and cortisol. Two 5-mm blood spots were eluted by internal standard working solution. We analyzed 1170 DBS samples from neonates to determine gestational age-specific reference intervals. In order to test the specificity of the second-tier method, we analyzed 707 cards with a positive screening by DELFIA.</p></div><div><h3>Results</h3><p>Values of intra- and inter-day precision coefficients of variance and accuracy were 2.0%–13.3% and 85.8%–114.5%, respectively. Recovery ranged from 85.0% to 106.9%. The lower limit of quantification was 0.5 ng/mL for 21-deoxycortisol, 0.25 ng/mL for 17-hydroxyprogesterone and cortisol, and 0.1 ng/mL for testosterone, androstenedione, and 11-deoxycortisol. In addition, the linearity range was 0.25–50 ng/mL (R<sup>2</sup> > 0.99). According to the 17-hydroxyprogesterone levels and ratios of (androstenedione + 17-hydroxyprogesterone)/cortisol in the 707 positive screening samples, 77 neonates should receive recall visit. The number of false-positive results reduced by 89.1%. Totally, 18 newborns were diagnosed with 21-hydroxylase deficiency, one with P450 oxidoreductase deficiency and one with 11β-hydroxylase deficiency. With two-tier screening, the positive predictive value increased to 26.0%.</p></div><div><h3>Conclusions</h3><p>The second-tier steroid profiling by LC-MS/MS reduced the false-positive rate and improved the positive predictive value of CAH screening. We suggest applying this steroid profiling assay as a second-tier test for CAH screening in China.</p></div>\",\"PeriodicalId\":21997,\"journal\":{\"name\":\"Steroids\",\"volume\":\"185 \",\"pages\":\"Article 109056\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2022-09-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"4\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Steroids\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0039128X22000940\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Steroids","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0039128X22000940","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Steroid profile in dried blood spots by liquid chromatography tandem mass spectrometry: Application to newborn screening for congenital adrenal hyperplasia in China
Background
Newborn screening for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA) generates a large number of false-positive results. The present study aimed to improve the sensitivity of the CAH neonatal screening by including second-tier steroid profiling in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS).
Methods
We developed and validated a LC-MS/MS method for simultaneous determination of six steroids in DBS, including androstenedione, testosterone, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, and cortisol. Two 5-mm blood spots were eluted by internal standard working solution. We analyzed 1170 DBS samples from neonates to determine gestational age-specific reference intervals. In order to test the specificity of the second-tier method, we analyzed 707 cards with a positive screening by DELFIA.
Results
Values of intra- and inter-day precision coefficients of variance and accuracy were 2.0%–13.3% and 85.8%–114.5%, respectively. Recovery ranged from 85.0% to 106.9%. The lower limit of quantification was 0.5 ng/mL for 21-deoxycortisol, 0.25 ng/mL for 17-hydroxyprogesterone and cortisol, and 0.1 ng/mL for testosterone, androstenedione, and 11-deoxycortisol. In addition, the linearity range was 0.25–50 ng/mL (R2 > 0.99). According to the 17-hydroxyprogesterone levels and ratios of (androstenedione + 17-hydroxyprogesterone)/cortisol in the 707 positive screening samples, 77 neonates should receive recall visit. The number of false-positive results reduced by 89.1%. Totally, 18 newborns were diagnosed with 21-hydroxylase deficiency, one with P450 oxidoreductase deficiency and one with 11β-hydroxylase deficiency. With two-tier screening, the positive predictive value increased to 26.0%.
Conclusions
The second-tier steroid profiling by LC-MS/MS reduced the false-positive rate and improved the positive predictive value of CAH screening. We suggest applying this steroid profiling assay as a second-tier test for CAH screening in China.
期刊介绍：
STEROIDS is an international research journal devoted to studies on all chemical and biological aspects of steroidal moieties. The journal focuses on both experimental and theoretical studies on the biology, chemistry, biosynthesis, metabolism, molecular biology, physiology and pharmacology of steroids and other molecules that target or regulate steroid receptors. Manuscripts presenting clinical research related to steroids, steroid drug development, comparative endocrinology of steroid hormones, investigations on the mechanism of steroid action and steroid chemistry are all appropriate for submission for peer review. STEROIDS publishes both original research and timely reviews. For details concerning the preparation of manuscripts see Instructions to Authors, which is published in each issue of the journal.
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