Newborn Screening for Congenital Adrenal Hyperplasia: Additional Steroid Profile using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Our liquid chromatography-tandem mass spectrometry procedure as a second-tier test can be used to reduce false-positive results of standard 21-CAH screening. The short total run time of 6 min allows for immediate reanalysis of all immunoassay results above the cutoff.

Dried blood spot sampling in combination with LC‐MS/MS for quantitative analysis of small molecules

“…However, for small biomarker molecules, it might be diffi cult because these molecules are endogenous with varying concentrations. A common approach to overcoming this is to mix the washed blood cells with artifi cial serum or plasma to make artifi cial 'whole blood' , as demonstrated in several applications for the simultaneous analysis of multiendogenous compounds (Higashi et al, 2008;Lacey et al, 2004;Janzen et al, 2007Janzen et al, , 2008Minutti et al, 2004). Listed below is the detailed procedure used by Higashi et al for the determination of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone in dried blood spots from low birth weight infants: whole blood (10 mL) was collected from a healthy volunteer into a heparinized tube and centrifuged at 1500g at room temperature for 15 min to separate plasma and blood red cells.…”

“…Listed in Table 1 are some representative applications in the primary and/or confi rmatory newborn screening for the diagnosis of some inherited metabolic disorders, including congenital adrenal hyperplasia (CAH), hyperhomocysteinemia, tyrosinemia type 1, methylmalonic acidemia and homocytinuria, etc. (Janzen et al, 2007;Lacey et al, 2004;la Marca et al, 2008c;McCann et al, 2003;Oglesbee et al, 2008;Zoppa et al, 2006). There are many situations in newborn screening where absolute concentration of a single biomarker molecule may not be diagnostic; instead, accurate quantifi cation of multiple biomarker molecules using LC-MS/MS, followed by comparison of response ratios of one species to the other(s), is more powerful (Carpenter and Wiley, 2002).…”

“…By implementing this approach with a lower cutoff concentration value of 10.2 ng/mL of 17-OHP and 2.50 of the above MS response ratio, the overall specifi city of newborn screening for CAH using LC-MS/MS was signifi cantly improved with false positive rate decreased approximately 10 times Matern et al, 2007). The method was later modifi ed by shortening the analysis cycle time from 12 to 6 min and including 11-deoxycortisol, 21-deoxycortisol, instead of androstenedione to the response ratio comparison , respectively, to cortisol] for the confi rmatory test, from which high concentration levels of 21-deoxycortisol were only found in children with CAH (Janzen et al, 2007). It is possible that accurate measurement of some other steroids might contribute to the diagnosis of CAH in newborn screening, thus, developing and validating a more comprehensive DBS-LC-MS/MS assay is necessary as shown in a recent report (Janzen et al, in 2008).…”

“…Furthermore, the possible in-source fragmentation in the case that the target analyte is a derivative of another intact biomarker molecule and natural isotope contribution might add another challenge to MS/MS (infusion or fl ow injection) only assays (Garg and Dasouki, 2006;Piraud et al, 2003). Consequently, LC-MS/MS has been increasingly explored in the confi rmatory analysis (second-tier test) of those 'diffi cult' biomarker molecules, resulting in a reduced false-positive rate (Janzen et al, 2007;Lacey et al, 2004;la Marca et al, 2007 and2008a;Matern et al, 2007;Minutti et al, 2004).…”

Functional glucocorticoid receptor gene variants do not underlie the high variability of 17-hydroxyprogesterone screening values in healthy newborns

“…LC-MS/MS steroid profile included quantification of 17-OHP, 21-deoxycortisol (21F), cortisol (F), 4-androstenedione (4A), and 11-deoxycortisol (S) as well as calculation of the 21FC17-OHP/F ratio, which has been shown to further improve screening sensitivity and specificity. A detailed description of the assay procedure and performance is given elsewhere (10). A numbered scrap of each screening card together with anonymous steroid measurement data and information on birth weight, gestational age, and postnatal age at the time samples had been taken were then sent to the University Children's Hospital Bonn for genotyping and statistical analyses.…”