Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry.
BACKGROUND
Neonatal screening programs for congenital adrenal hyperplasia (21-CAH) using an immunoassay for 17alpha-hydroxyprogesterone (17-OHP) generate a high rate of positive results attributable to physiological reasons and to cross-reactions with steroids other than 17alpha-OHP, especially in preterm neonates and in critically ill newborns.


METHODS
To increase the specificity of the screening process, we applied a liquid chromatography-tandem mass spectrometry method quantifying 17alpha-OHP, 11-deoxycortisol, 21-deoxycortisol, cortisol, and androstenedione. The steroids were eluted in aqueous solution containing d8-17alpha-OHP and d2-cortisol and quantified in multiple reaction mode.


RESULTS
Detection limit was below 1 nmol/liter, and recovery ranged from 64% (androstenedione) to 83% (cortisol). Linearity was proven within a range of 5-100 nmol/liter (cortisol, 12.5-200 nmol/liter), and total run time was 6 min. Retrospective analysis of 6151 blood samples and 50 blood samples from newborns with clinically confirmed 21-CAH, as well as prospective analysis of 1609 samples of a total of 242,500 testing positive in our routine 17-OHP immunoassay, allowed clear distinction of affected and nonaffected newborns. High levels of 21-deoxycortisol were only found in children with 21-hydroxylase deficiency. Calculating the ratio of 17alpha-OHP to 21-deoxycortisol divided by cortisol further increased the sensitivity of the method.


CONCLUSIONS
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.
Janzen
N
N
Screening-Labor Hannover, D-30952 Ronnenberg, Germany. n.janzen@metabscreen.de
Peter
M
M
Sander
S
S
Steuerwald
U
U
Terhardt
M
M
Holtkamp
U
U
Sander
J
J
D019326:17-alpha-Hydroxyprogesterone / Q000032:analysis / Q000097:blood*; D000312:Adrenal Hyperplasia, Congenital / Q000097:blood* / Q000175:diagnosis*; D000735:Androstenedione / Q000032:analysis / Q000097:blood; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D003350:Cortodoxone / Q000032:analysis / Q000097:blood*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis / Q000097:blood; D007231:Infant, Newborn; D007234:Infant, Premature; D008297:Male; D015997:Neonatal Screening / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D011446:Prospective Studies; D015203:Reproducibility of Results; D012189:Retrospective Studies; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards
D000735:Androstenedione; C003556:21-deoxycortisol; D019326:17-alpha-Hydroxyprogesterone; D003350:Cortodoxone; D006854:Hydrocortisone