Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry.
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.
Lacey
Jean M
JM
Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Minutti
Carla Z
CZ
Magera
Mark J
MJ
Tauscher
Angela L
AL
Casetta
Bruno
B
McCann
Mark
M
Lymp
James
J
Hahn
Si Houn
SH
Rinaldo
Piero
P
Matern
Dietrich
D
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*
D013256:Steroids; C002175:progesterone 17 alpha-hydroperoxide; D000735:Androstenedione; D011374:Progesterone; D006854:Hydrocortisone