Department of Laboratory Medicine, Green Cross Laboratories, Yongin, Korea.
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Seoul, Korea.
Ann Lab Med. 2019 May;39(3):263-270. doi: 10.3343/alm.2019.39.3.263.
Screening for congenital adrenal hyperplasia (CAH) using immunoassays for 17α-hydroxyprogesterone generates many false-positive results. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of nine steroid hormones in dried blood spot (DBS) samples, and established reference intervals for these hormones.
We examined our method for linearity, precision, accuracy, extraction recovery, and matrix effects and determined the reference intervals of cortisol, 17α-hydroxyproges-terone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, 11-deoxycorticosterone, testosterone, and progesterone in 1,146 DBS samples (from 272 preterm and 874 full-term neonates). Immunoassay and LC-MS/MS methods were compared for 17α-hydroxyprogesterone. Fourteen additional samples were tested to validate the clinical applicability of the LC-MS/MS method.
The linearity range was 2.8-828.0 nmol/L for cortisol and 0.9-40.0 nmol/L for the other steroids (R²>0.99). Intra-day and inter-day precision CVs were 2.52-12.26% and 3.53-17.12%, respectively. Accuracy was 80.81-99.94%, and extraction recovery and matrix effects were 88.0-125.4% and 61.7-74.2%, respectively. There was a negative bias, with higher values measured by immunoassay compared with LC-MS/MS (=0.8104, <0.0001). The LC-MS/MS method was successfully applied to the analysis of nine steroids in DBS for screening and diagnosis of CAH using the 14 additional samples.
Our method enables highly sensitive and specific assessment of nine steroids from DBS and is a promising tool for clinical analysis of CAH.
使用免疫分析法检测 17α-羟孕酮对先天性肾上腺皮质增生症（CAH）进行筛查会产生许多假阳性结果。我们开发并验证了一种用于同时定量测定干血斑（DBS）样本中九种类固醇激素的液相色谱-串联质谱（LC-MS/MS）检测方法，并建立了这些激素的参考区间。
我们考察了本方法的线性、精密度、准确度、提取回收率和基质效应，并在 1146 份 DBS 样本（来自 272 份早产儿和 874 份足月儿）中测定了皮质醇、17α-羟孕酮、11-脱氧皮质醇、21-脱氧皮质醇、雄烯二酮、皮质酮、11-脱氧皮质酮、睾酮和孕酮的参考区间。比较了免疫分析法和 LC-MS/MS 方法对 17α-羟孕酮的检测结果。另外还测试了 14 份样本，以验证 LC-MS/MS 方法的临床适用性。
皮质醇的线性范围为 2.8-828.0 nmol/L，其他类固醇的线性范围为 0.9-40.0 nmol/L（R²>0.99）。日内和日间精密度 CV 分别为 2.52-12.26%和 3.53-17.12%。准确度为 80.81-99.94%，提取回收率和基质效应分别为 88.0-125.4%和 61.7-74.2%。与 LC-MS/MS 方法相比，免疫分析法的检测结果偏高，存在负偏倚（=0.8104，<0.0001）。该 LC-MS/MS 方法成功应用于另外 14 份样本中 DBS 中九种类固醇的分析，可用于 CAH 的筛查和诊断。
本方法能够对 DBS 中的九种类固醇进行高灵敏度和高特异性的评估，是 CAH 临床分析的一种有前途的工具。
