Improving Newborn Screening for CAH Using Tandem Mass Spectrometry

Winter 2003 CARES Foundation, Inc.

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Improving Newborn Screening for CAH Using Tandem Mass Spectrometry


Following the development of an immunoassay to determine the concentration of 17-hydroxy progesterone (17-OHP) in blood spots in 1977, newborn screening for CAH was introduced into several newborn screening programs worldwide. However, in the USA babies are currently screened for this disorder in only 34 states. The reason for this limited penetrance is not a disagreement about CAH being an important disorder that should be identified and treated as early in life as possible, but the fact that the immunoassays available are associated with a high number of false positive results. In other words too many babies not affected with CAH have an abnormal screening test. This is particularly true for premature neonates because their blood often contains compounds that the available screening tests cannot differentiate from 17-OHP. However, every abnormal result has significant consequences because they must be followed up to ensure that no affected baby is mistakenly left behind. This requires collecting additional blood specimens to measure 17-OHP and other steroids. Minimizing the number of these false positive results is important because there would be fewer families experiencing the emotional stress of an abnormal screening result and of the inevitable consequences (consultation with endocrinologist, laboratory testing, unnecessary care). To achieve the goal of improving newborn screening for CAH, we have developed an assay using tandem mass spectrometry (MS/MS). This technology has gained a prominent position among analytical methods due to its versatility, unmatched sensitivity, and specificity. Since the 1990s, MS/MS is becoming an integral part of newborn screening programs because of its ability to detect more then 30 disorders in a single blood spot and a single analysis. CAH is not one of these disorders, but it is possible to specifically detect 17-OHP utilizing this technology. Furthermore, using MS/MS one is not limited to a single analyte but other steroids can be identified simultaneously. As indicated in the figure, CAH deficiency not only causes the elevation of 17-OHP and other steroids (i.e., androstenedione), but also results in relatively low cortisol concentrations. Using this multi-analyte approach, the specificity of newborn screening for CAH can be improved significantly. In a retrospective study we determined 17-OHP, androstenedione, and cortisol in more than 700 blood spots that had previously been tested by immunoassay. Using MS/MS 95% of the initially labeled false positives were eliminated. The analytical time required for this MS/MS method is currently not compatible with its use as a primary screening test of hundreds of samples per day. However, it can still significantly reduce the false positive rate of the conventional immunoassays as a second tier test which means that newborn blood spots that yield elevated 17-OHP concentrations are re-analyzed using MS/MS to determine the final screening result. This approach can also be taken by molecular genetic testing for the most common mutations in the CAH gene (CYP21) as described by Dr. Naylor in the previous CARES newsletter (CARES Foundation 2002;1(3):10). However, genetic testing has limitations because patients carrying less common mutations would remain unidentified. Biochemical testing using MS/MS on the other hand is typically abnormal in a patient with CAH independent of the frequency or type of the gene defect involved. The MS/MS assay described here is currently being validated and is anticipated to become available by the spring of 2003. ------------------------------ Figure: Pathway of steroid synthesis. 21-Hydroxylase deficiency accounts for approximately 95% of disorders affecting this pathway and causes classic Congenital Adrenal Hyperplasia (CAH).



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