title: Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry.
abstract: 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 microg/L. At a cutoff for 17-OHP of 12.5 microg/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.
  
  
  CONCLUSIONS
  Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Jean M
authors.initials: JM
authors.lastname: Lacey
authors.forename: Carla Z
authors.initials: CZ
authors.lastname: Minutti
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Magera
authors.forename: Angela L
authors.initials: AL
authors.lastname: Tauscher
authors.forename: Bruno
authors.initials: B
authors.lastname: Casetta
authors.forename: Mark
authors.initials: M
authors.lastname: McCann
authors.forename: James
authors.initials: J
authors.lastname: Lymp
authors.forename: Si Houn
authors.initials: SH
authors.lastname: Hahn
authors.forename: Piero
authors.initials: P
authors.lastname: Rinaldo
authors.forename: Dietrich
authors.initials: D
authors.lastname: Matern
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000097:blood / Q000175:diagnosis*; D000735:Androstenedione / Q000097:blood; D002853:Chromatography, Liquid; D005189:False Positive Reactions; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007118:Immunoassay; D007231:Infant, Newborn; D008403:Mass Screening / Q000379:methods*; D011374:Progesterone / Q000031:analogs & derivatives* / Q000097:blood; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013256:Steroids / Q000097:blood*
chemical_list: D013256:Steroids; C002175:progesterone 17 alpha-hydroperoxide; D000735:Androstenedione; D011374:Progesterone; D006854:Hydrocortisone