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Steroid Profiling by Tandem Mass Spectrometry Improves the Positive Predictive Value of Newborn Screening for Congenital Adrenal Hyperplasia

Departments of Laboratory Medicine and Pathology (C.Z.M., J.M.L., M.J.M., S.H.H., P.R., D.M.), Pediatric and Adolescent Medicine (S.H.H., D.Z., P.R., D.M.), and Biostatistics (J.F.L.), Mayo Clinic College of Medicine, Rochester, Minnesota 55905; Department of Pediatrics (C.Z.M.), John Stroger Jr. Hospital of Cook County, Chicago, Illinois 60612; Minnesota Department of Health (M.M.), Minneapolis, Minnesota 55440; University Children’s Hospital (A.S.), 69120 Heidelberg, Germany; Department of Biochemistry (D.C., C.D.), Hospital Debrousse, Lyon, 69322 France; and Pediatrix Screening (D.H.C.), Bridgeville, Pennsylvania 15017

Address all correspondence and requests for reprints to: Dr. Dietrich Matern, Biochemical Genetics Laboratory-Hilton 330, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota 55905. E-mail: matern{at}mayo.edu.

	Abstract

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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.

	Introduction

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CONGENITAL ADRENAL HYPERPLASIA (CAH) is a family of inborn errors of steroid biosynthesis. More than 90% of cases result from a defect in 21-hydroxylation blocking cortisol production and causing accumulation of 17-hydroxyprogesterone and androstenedione (1). Clinical manifestations include life-threatening salt-wasting crises in the newborn period and incorrect gender assignment of virilized females, outcomes that make early diagnosis by newborn screening highly desirable. In 1977, Pang et al. (2) described a RIA technique for the determination of 17-hydroxyprogesterone in blood collected on filter paper. This method was later applied to a pilot study in Alaska that demonstrated the clinical utility of newborn screening for CAH (3). In the United States, newborns in 36 states are currently screened for CAH (http://genes-r-us.uthscsa.edu/resources/newborn/screenstatus.htm); additional regional, national, and pilot programs have been implemented in several countries worldwide.

Despite the proven clinical benefits of early diagnosis, the limited implementation of CAH screening stems from the significant resources required for the follow-up of false-positive results (4). These events have been related to different causes, most commonly a physiologically delayed expression of the enzyme 11-ß-hydroxylase in premature babies that causes transiently elevated concentrations of 17-hydroxyprogesterone (5). Other determining factors causing false-positive results are elevations of 17-hydroxyprogesterone levels due to illness, poor kidney function, and stress (6). Immunoassays for 17-hydroxyprogesterone are also hampered by cross-reactivity of antibodies with other steroids, particularly 17-hydroxy pregnenolone, which tends to be very high in newborns (7). This is related to the observation that 3-ß-hydroxysteroid dehydrogenase activity is uniformly low among newborns, perhaps due to inhibition by maternal estrogens (8). Cross-reactivity of antibodies against 17-hydroxyprogesterone has been reported to be as high as 8% for different steroids and for several medications (9, 10), greatly reducing discrimination between normal neonates and those with CAH. The introduction of more specific antibodies and of preanalytical processing such as solvent extraction have not sufficiently enhanced the screening efficiency of existing immunoassays (11). Similarly, cutoff values for 17-hydroxyprogesterone adjusted for birth weight, gestational age, and stress factors have not significantly improved the positive predictive value of this test, which remains the lowest of all screening programs (6, 12).

To improve the current situation, it is necessary to develop new methods with enhanced specificity that are applicable to population screening. Tandem mass spectrometry (MS/MS) is rapidly being incorporated in newborn screening laboratories worldwide because of the ability to perform simultaneous analyses of metabolic markers in a multicomponent and cost-efficient manner (13, 14), minimizing the potential interference of chemically similar but unrelated substances. The determination of 17-hydroxyprogesterone in plasma and blood spots using MS/MS has been recently described (15, 16, 17). To improve the specificity of newborn screening for CAH, we recently developed a steroid-profiling assay for the simultaneous analysis of 17-hydroxyprogesterone, androstenedione, and cortisol (18). We report here a retrospective, blinded study of 1222 original newborn-screening cards using this MS/MS method.

	Subjects and Methods

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Experimental subjects

Six screening programs in the Unites States, Germany, France, and Korea were asked to supply a blinded selection of anonymized blood spots inclusive of a high number of cases that had an abnormal screening by either fluorescent immunoassay or ELISA that were later found to represent false-positive results. Data provided at submission included only birth weight and age at sample collection; the concentration of 17-hydroxyprogesterone and the final clinical outcome were made available after completion of the retrospective analysis (Table 1). A total of 1222 blood spots were analyzed. Thirty-one samples were from patients diagnosed with CAH subsequent to an abnormal newborn screening result, and 190 samples were from cases that initially had abnormal results but were classified as false positives after clinical and laboratory evaluation. The remaining 1001 blood spots were from newborns with negative screening results (Table 1). This study was approved by each of the participating centers’ institutional review board.

Standards of 17-hydroxyprogesterone, androstenedione, and cortisol were purchased from Sigma-Aldrich (St. Louis, MO), and d₈-17-hydroxyprogesterone was purchased from CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada). Dried blood spots for calibration and quality control monitoring were obtained from the Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention (Atlanta, GA).

Sample preparation and analysis

The analyses were performed as described by Lacey et al. (18). In short, a 3/16-in. disc is punched from each blood spot and placed in a screw-capped glass tube. As an eluant, 0.5 ml of internal standard solution (d₈-17-hydroxyprogesterone in water) is added. Three milliliters of diethyl ether are added, and the aqueous phase is extracted by agitation. After a repeat extraction step after transfer of the diethyl ether phase to a new vial, the extract is dried under nitrogen and then dissolved in 50 µl of methanol and water (50:50, vol/vol) mobile phase and loaded in the autosampler. The four steroids are chromatographically separated by a reverse-phase (Symmetry C18, 50 x 2.1 mm; Waters, Milford, MA) liquid chromatography column using a gradient system of two solvents (water and methanol) before MS/MS analysis using an API 3000 instrument (Applied Biosystems/MDS Sciex, Toronto, Ontario, Canada). Data acquisition was optimized in positive ion mode to detect by selected reaction monitoring the transitions m/z 331.2 (precursor ion) to m/z 97.0 (product ion), m/z 339.2 to m/z 100.0, m/z 287.2 to m/z 97.0, and m/z 363.3 to m/z 121.2 for 17-hydroxyprogesterone, d₈-17-hydroxyprogesterone, androstenedione, and cortisol, respectively. The retention times of these compounds range from 2 to 4 min (Fig. 1), but a 12-min total run is required to elute an unrelated, water-soluble compound released by the filter paper.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Steroid profiling by MS/MS in blood spots. A, Normal control; B, false-positive result by fluorescent immunoassay; C, false-negative result by MS/MS due to early initiation of cortisol treatment in a patient clinically diagnosed before newborn screening (see text for details); D, confirmed case with CAH. Peak identification as follows: 1, cortisol; 2, androstenedione; 3, d8-17-hydroxyprogesterone (internal standard); 4, 17-hydroxyprogesterone. Conversion factor: 1 ng/ml = 3 nmol/liter.

To assess the characteristics of the method, we selected various cutoff values for 17-hydroxyprogesterone and for the ratio between the sum of 17-hydroxyprogesterone plus androstenedione divided by the value calculated in the same manner for cortisol (18). For each cutoff value, sensitivity and specificity were examined. Exact 95% binomial confidence intervals were computed for each of these characteristics (19). We considered a test positive only if both parameters were above the respective cutoff value. Because our population study was enriched with respect to both true-positive and false-positive subjects, the positive predictive value of this test was calculated using correction factors to predict how many false positives we would have encountered in a random population large enough, according to published data (20), to include 31 true positives (Table 2).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

View larger version (31K): [in this window] [in a new window]   FIG. 2. Comparison of 17-hydroxyprogesterone (17-OHP) levels by immunoassays and liquid chromatography-MS/MS measured in the false-positive group of the population study. A, Birth weight less than 1500 g (n = 66); B, birth weight between 1500 and 2500 g (n = 62); C, birth weight more than 2500 g (n = 62); D, confirmed cases with CAH (n = 31). Please note the different scale used in panel D.

To further improve the specificity of this method, we calculated the ratio between the sum of 17-hydroxyprogesterone plus androstenedione divided by cortisol. A cutoff value for this ratio was set at 3.75 (100% sensitivity, 88–100% confidence interval; 98% specificity, 97–99% confidence interval) for all birth-weight groups. Using this ratio, only 21 of the remaining 65 false positives with 17-hydroxyprogesterone greater than 12.5 ng/ml (38 nmol/liter) had an abnormal result that would have required follow-up and additional investigations. A subject who was reported to have CAH was born prematurely (birth weight, 790 g) and had a 17-hydroxyprogesterone value of 110.6 ng/ml (335 nmol/liter) by immunoassay and 53.5 ng/ml (162 nmol/liter) by MS/MS. However, the calculated ratio was merely 2.62 due to a prominent cortisol peak, a finding not consistent with a block of the biosynthetic pathway. Unfortunately, no follow-up information was available to the testing site, and we have been unable to conclusively establish whether these findings represent a false-negative outcome. The prominent cortisol peak implies that this case represents a false-positive result unrecognized by the original testing site. Alternatively, this result could be explained by early initiation of hormone replacement therapy in an affected newborn as observed by the authors in the following case brought to our attention independent of this study. This patient was born with ambiguous genitalia and gastroschisis. CAH was considered a diagnosis, and hydrocortisone treatment was initiated before surgical repair of the abdominal wall defect on the first day of life. The newborn screening sample was collected on the second day of life and revealed a 17-hydroxyprogesterone of 380.5 ng/ml (1153 nmol/liter) by immunoassay. Testing by our method also yielded an elevated 17-hydroxyprogesterone level [25.3 ng/ml (77 nmol/liter)], but the result was deemed normal because of a very low peak area ratio of 17-hydroxyprogesterone plus androstenedione divided by cortisol (0.18) (Fig. 1C). The possibility of medication-induced false-negative results must therefore be considered under special circumstances, recipients of screening results must be educated, and all results must be interpreted in clinical context.

Table 3 summarizes the outcome of the retrospective screening by MS/MS based on the serial application of the two parameters. Using these criteria, 169 (89%) of the 190 false-positive cases were reported as negative; eight of the 21 remaining false-positive cases had a birth weight less than 1500 g (Table 3). For comparison, 66 of the 104 cases with a birth weight less than 1500 g had an abnormal screening by immunoassay (Table 1). With the exception of two cases described above, testing of all other patients with CAH yielded distinctively abnormal results. The clinical phenotype was verified in 19 of the 31 cases. Although four confirmed patients with a simple virilizing phenotype showed only moderately elevated levels of 17-hydroxyprogesterone [median, 23.6 ng/ml (72 nmol/liter)], levels of this steroid intermediate were higher than 50 ng/ml (152 nmol/liter) in 13 of 15 patients with the severe salt-wasting phenotype [median, 127 ng/ml (385 nmol/liter)].

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Testing for CAH is not included in all newborn-screening programs because the immunoassays traditionally used for the determination of 17-hydroxyprogesterone in blood spots are associated with a high false-positive rate. Variable cutoff values based on birth weight have been applied to these screening tests to reduce the frequency of false-positive results caused by endogenous metabolites, encountered particularly in premature newborns, that cross-react with 17-hydroxyprogesterone antibodies (12). When 17-hydroxyprogesterone was determined by MS/MS, the cutoff value was lower than by immunoassay. Nevertheless, in this retrospective study of 1220 blood spots collected between the second and fifth day of life in four countries, the application of steroid profiling by MS/MS to the newborn screening for CAH yielded an 89% reduction of false-positive results. This is achieved through the added effect of two factors. One is the high analytical specificity inherent in MS/MS-based methods. Fifty-four percent (104 of 190) of the false-positive results could be eliminated because compounds unintentionally measured by the immunoassay due to cross-reactivity with the 17-hydroxyprogesterone antibodies did not interfere with the analysis by MS/MS. The second factor is rooted in the ability to simultaneously analyze several compounds within a metabolic pathway by MS/MS. Our assay not only determines 17-hydroxyprogesterone as a direct substrate for 21-hydroxylase but also a downstream product of this enzyme’s reaction, cortisol, and another steroid, androstenedione, which accumulates secondarily in CAH. The ratio of the sum of the two analytes before the enzymatic step catalyzed by 21-hydroxylase to the end product of cortisol biosynthesis can therefore be calculated. The rationale for the use of this ratio lies in the fact that newborns under stress (i.e. due to prolonged delivery or an infection) will have elevated cortisol levels with secondary accumulation of 17-hydroxyprogesterone. Measuring only the concentration of 17-hydroxyprogesterone will yield elevated results in these cases, which are not related to CAH and do not require follow-up investigations. Consideration of the additional steroids allows better discrimination between elevated 17-hydroxyprogesterone levels due to CAH and those due to stress reactions. This is documented by the further reduction of false-positive results to 89% (169 of 190) in our comparative investigation.

Our estimate of the positive predictive value (4.7%) of CAH screening by steroid profiling is significant in light of a recent review of the magnitude of false-positive results in newborn screening programs in the United States (20). This review concluded that among five disorders studied (biotinidase deficiency, CAH, congenital hypothyroidism, classic galactosemia, and phenylketonuria), CAH screening had the lowest positive predictive value (111 true-positive cases among 20,647 abnormal screening results over a period of 2 yr, or 0.53%; compared with 6.36% for biotinidase deficiency, 1.84% for congenital hypothyroidism, 0.56% for classic galactosemia, and 2.9% for phenylketonuria). According to this estimate, 200 unaffected newborns require clinical and laboratory follow-up for every true case of CAH. This places a significant burden on health care resources and causes physicians to become desensitized and reluctant to initiate follow-up, especially when an abnormal screening result is attributed to a low-birth-weight newborn. The unnecessary stress and anxiety caused to so many parents who live in states offering this screening is compounded by the bitter recrimination voiced by the families of affected cases who were not screened at birth and suffered catastrophic consequences.

In recent years, the potential application of MS/MS to expand newborn-screening programs has started an intense debate between public health programs and the general public, with special attention given to this issue by mass and even scientific media (21). Despite considerable pressure, concerns about a drastically different analytical platform and the degree of complexity of result interpretation have contributed to the slow implementation of MS/MS by public health screening laboratories (22).

Under these circumstances, steroid profiling by MS/MS may offer two different avenues to universal newborn screening for CAH: first, public health and private laboratories with adequate expertise and available resources could implement it as a primary screening tool. Although the current run time limits testing volume to no more than 100 analyses per instrument per day (25,000 samples per year), instrument capacity could be doubled with relative ease by adding valve-switching technology between two parallel injection systems (23).

The total direct and indirect cost of the procedure is $26 per sample, which is considerably higher than the analytical cost for immunoassay methods in the six testing sites that contributed to this study (range, $2.00–5.20 per test). However, we predict that the increased cost by MS/MS would be offset by the reduction of additional expenses triggered by false-positive results. From January 2000 to December 2002, for example, 66 of 6213 babies born at Mayo Clinic Rochester had an abnormal screening reported by the state laboratory. None was affected, and the cost of follow-up, including only medical evaluation and laboratory tests, was over $55,000 ($848 per patient). Comparing the cost of screening by immunoassay plus follow-up expenses to the cost of screening 25,000 newborns by MS/MS, the 89% reduction of false positives resulting from conversion to MS/MS would greatly reduce the cost of repeat analysis of unsatisfactory samples (defined as those collected at less than 24 h of age), other follow-up costs (personnel time and effort to track down cases), and parental factors (loss of productivity). Another advantage of using MS/MS is avoidance of parental anxiety engendered by the false-positive results inherent in the use of conventional immunoassays. As was recently shown, false-positive newborn screening results can have lasting negative effects on families by causing increased stress and dysfunctional parent-child relationships (24).

Alternatively, MS/MS could be used as a second-tier test to follow up abnormal results by immunoassay rather than using it in primary screening. Although on-site availability of the test would be desirable, the original blood spot could be sent to a regional center for MS/MS analysis, eliminating the need to recall the patient to draw additional specimens. The overall delay in the process is less than 24 h in our laboratory, and the additional MS/MS analysis would not preclude immediate referral of a newborn with a significantly abnormal immunoassay result to the care of an endocrinologist.

In recent years, second-tier testing for CAH using molecular genetic methods has been proposed to confirm a diagnosis on the DNA level. However, this approach is not comprehensive because CAH is a genetically heterogenous disorder with less than 95% of mutant alleles being carriers of the 10 most common CYP21 mutations (25, 26). Molecular studies would therefore increase specificity but not sensitivity of CAH screening in light of the fact that not all mutations can be reliably detected in a screening setting (6).

We hope our assay and its proposed use as a first- or second-tier screening test could lead public health programs and their advisory committees to more readily embrace the addition of CAH to the panel of disorders screened at birth in their state. This action would be consistent with the ongoing effort by federal agencies and professional organizations to define a uniform panel of disorders (27). With no exceptions, there is a unanimous consensus that CAH should be included without further delays in all public health and private newborn-screening programs.

	Acknowledgments

 
We thank April S. Studinski, Dr. Roy F. House, Mary J. Schibursky (Mayo Clinic), and Bethany A. Sgroi (Pediatrix Screening) for collecting clinical information, Charles H. Darby and Dr. Terry M. Therneau (Division of Biostatistics, Mayo Clinic) for data analysis and statistical advice, respectively, and Dr. Julian H. Barth (Leeds Teaching Hospitals NHS Trust, UK) for critical review of the manuscript.

	Footnotes

 
Abbreviations: CAH, Congenital adrenal hyperplasia; MS/MS, tandem mass spectrometry.

Received December 30, 2003.

Accepted April 1, 2004.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Minutti, C. Z. Articles by Matern, D. Search for Related Content PubMed PubMed Citation Articles by Minutti, C. Z. Articles by Matern, D. Pubmed/NCBI databases OMIM Compound via MeSH Substance via MeSH Hazardous Substances DB 17ALPHA-HYDROXYPROGESTERONE HYDROCORTISONE Genetics Home Reference Medline Plus Health Information Newborn Screening