, Melissa Yssel, MB ChB, FC Path(SA) Chem
139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.
Background: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17␣-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-isotopelabeled 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
Congenital adrenal hyperplasia (CAH) is primarily caused by 21-hydroxylase deficiency and leads to an accumulation of 17-hydroxyprogesterone and reduced cortisol levels. Newborn screening for CAH is traditionally based on measuring 17-hydroxyprogesterone by different immunoassays. Despite attempts to adjust cutoff levels for birth weight, gestational age, and stress factors, the positive predictive value for CAH screening remains less than 1%. To improve this situation, we developed a method using liquid chromatography-tandem mass spectrometry to measure 17-hydroxyprogesterone, androstenedione, and cortisol simultaneously in blood spots. A total of 1222 leftover blood spots from six different screening programs using different immunoassays (fluorescent immunoassay and ELISA) were reanalyzed in a blinded fashion by liquid chromatography-tandem mass spectrometry. Thirty-one samples were from babies with CAH, 190 had yielded false-positive results by immunoassay, and the remaining 1001 samples were from babies with normal screening results. Steroid profiling allowed for an elimination of 169 (89%) of the false-positive results and for an improvement of the positive predictive value from the reported 0.5 to 4.7%. Although this method is not suitable for mass screening due to the length of the analysis (12 min), it can be used as a second-tier test of blood spots with positive results for CAH by the conventional methods. This would prevent unnecessary blood draws, medical evaluations, and stress to families.
This cohort study assesses the sensitivity of dried blood spots polymerase chain reaction for newborn screening for congenital cytomegalovirus infection using saliva as the reference standard for screening, followed by collection of a urine sample for confirmation of congenital infection
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