Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry
Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry
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DOI：
10.1373/clinchem.2003.027193
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发表时间：
2004-03-01
影响因子：
9.3
通讯作者：
Matern, D
中科院分区：
文献类型：
--
作者：
Lacey, JM;Minutti, CZ;Matern, D
背景资料：新生儿先天性肾上腺增生症（CAH）筛查包括测量17 α-羟孕酮（17-OHP），通常通过免疫测定法。由于这种检测的特点是假阳性率高，我们开发了一种类固醇分析方法，使用液相色谱-串联质谱法采用高效液相色谱-串联质谱法（LC-MS/MS）同时测定血斑中的17-OHP、雄烯二酮和皮质醇。用含氘标记内标物d（8）-17-OHP的水溶液从4.8 mm（3/16英寸）干血斑上洗脱全血。将17-OHP、雄烯二酮和皮质醇萃取至乙醚中，随后蒸发，并将残留物溶于LC移动的相中。将该浸提液进样至配备有电喷雾辅助的LC-MS/MS中。在选择反应监测模式下，通过使用稳定同位素标记的内标物的峰面积对类固醇进行定量。结果：17-OHP在2、30和50 μ g/L浓度下的批内和批间变异系数分别为7.2-20%和3.9- 18%。当17-OHP的临界值为12.5 μ g/L，17-OHP和雄烯二酮的峰面积之和除以皮质醇的峰面积的临界值为3.75时，101份假阳性样本中有86份在参考值范围内LC-MS/MS，而常规筛查获得的742份正常结果和14份真阳性结果被正确分类。结论：血斑中的类固醇分析可以识别CAH常规新生儿筛查获得的假阳性结果。(C)2004年美国临床化学协会。
Background: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots.Methods: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening.Results: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 mug/L. At a cutoff for 17-OHP of 12.5 mug/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified.Conclusion: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH. (C) 2004 American Association for Clinical Chemistry.