Congenital adrenal hyperplasia cases identified by newborn screening in one- and two-screen states.

There is no clear consensus among state newborn screening programs on whether routine second screening of newborns identifies clinically relevant cases of congenital adrenal hyperplasia. This retrospective study evaluated laboratory practices, along with biochemical and medical characteristics of congenital adrenal hyperplasia (CAH) cases (1) detected on the first newborn screen in one-screen compared to two-screen states, and (2) detected on the first versus the second screen in the two-screen states, to determine the effectiveness of a second screen. A total of 374 confirmed cases of CAH from 2 one-screen states and 5 two-screen states were included in this study. Demographic data and diagnostic information on each reported case were collected and analyzed. Additionally, laboratory data, including screening methodologies and algorithms, were evaluated. The one-screen states reported 99 cases of CAH out of 1,740,586 (1 in 17,500) newborns screened: 88 (89%) identified on the first screen and 5 (5%) identified on the targeted second screen. The two-screen states reported 275 cases of CAH out of 2,629,627 (1 in 9500) newborns screened: 165 (60%) identified on the first screen and 99 (36%) identified on the second screen. Using a multivariate model, the only significant predictor of whether a case was identified on the first or the second screen in the two-screen states was the type of CAH. Compared with classical salt-wasting CAH, classical simple virilizing and non-classical CAH cases were less likely to be detected on the first versus the second screen. The routine second newborn screen is important for identifying children with CAH, particularly simple virilizing and non-classical forms, which might otherwise not be captured through a single screen.

Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

BACKGROUND: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. METHOD: An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. RESULTS: DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. CONCLUSIONS: SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: challenges in newborn dried blood spot screening.

On May 23-24, 2011, a workshop entitled "Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: Technical Issues and Challenges" was held in Annapolis, Maryland. The two-day workshop was co-hosted by the National Newborn Screening and Genetics Resource Center, Austin, Texas, and the Association of Public Health Laboratories, Silver Spring, Maryland, in collaboration with the Health Resources and Services Administration and the Centers for Disease Control and Prevention. Participants included nearly 40 representatives from U.S. state public health and commercial laboratories performing newborn dried blood spot screening tests for cystic fibrosis (CF), the federal government, academic research institutions, and commercial vendors of products used in newborn screening. Representatives from selected European CF newborn screening programs were also present. The workshop focused on identifying key IRT testing issues and mechanisms for achieving their resolution and laboratory harmonization in order to reduce, or eliminate completely, the late identified CF cases following a negative newborn screen. Informative findings are reported, their impacts on improving IRT screening are described, and their implications are discussed.

Committee report: Considerations and recommendations for national guidance regarding the retention and use of residual dried blood spot specimens after newborn screening.

Newborn screening programs are state based with variable policies. Guidance regarding the retention, storage, and use of portions of newborn screening dried blood spots that remain after screening (residual specimens) was first published in 1996. Since then, newborn screening programs have paid increased attention to specimen storage and usage issues. Standard residual specimen uses include quality assurance and program evaluation, treatment efficacy, test refinement, and result verification. In all cases, privacy and security are primary concerns. In general, two distinct state practices regarding the storage and use of residual newborn screening specimens exist: (1) short-term storage (<3 years), primarily for standard program uses and (2) long-term storage (>18 years), for standard program uses and possible important public health research uses. Recently, there have been concerns in some consumer communities regarding both the potential uses of residual specimens and patient (newborn and family) privacy. To assist in policy improvements that can protect the individual's privacy and allow for important public health uses of residual newborn screening specimens, the Secretary of Health and Human Services' Advisory Committee on Heritable Disorders in Newborns and Children has developed recommendations (with requested action by the Secretary where applicable). This report presents the Committee's recommendations and reviews the pertinent associated issues.

Detection of TPN contamination of dried blood spots used in newborn and metabolic screening and its impact on quantitative measurement of amino acids.

BACKGROUND: Markers derived from dextrose (d-glucose) are observed in the MS/MS-based acylcarnitine profiles from dried-blood spots of some premature infants receiving intravenous nutrition. The presence of these markers at m/z 325, 399 and 473 are thought to arise from contamination of blood by total parenteral nutrition (TPN) solutions during specimen collection from premature infants. These solutions contain high concentrations of amino acids and as a result, false-positive screening results for amino acid disorders may occur. This study investigates quantitative parameters of dextrose and amino acids in blood samples enriched with different TPN solutions. METHODS: Whole blood collected in heparin was enriched with three different TPN solutions containing 5, 10 or 12.5% dextrose and amino acids that were originally prepared for delivery of 2.5, 3 or 4 g/kg/day of Premasol® then spotted onto filter paper cards. Acylcarnitine and amino acid profiles using MS/MS were obtained. Ion ratios of dextrose relative to specific acylcarnitine stable isotope internal standards and amino acid concentrations were obtained. RESULTS: The ion ratios for each of the dextrose markers at m/z 325, 399 and 473 exhibit linearity with the concentration of the dextrose component of TPN added to blood. The lowest detectable dextrose concentration added to blood was 7.6 mmol/l at 1:80 v/v TPN in blood. Furthermore, the concentrations of amino acids were linear with the concentration of the amino acid component of TPN added to blood. At the lowest detectable concentrations of dextrose marker, the amino acid concentrations were at or above the values considered abnormal in newborn screening laboratories. The molar ratios of amino acids approached the relative quantity of amino acid in the TPN solution with increasing enrichments in blood. CONCLUSIONS: Detection of the combinations of dextrose markers, very high elevations of amino acids and unusual molar ratios can be used to reject a specimen as improperly collected rather than declaring it a false positive and hence reduce false positive rates. This process enhances efficiency, reduces parental anxiety, and improves positive predictive values.

Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.

BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

Proficiency testing outcomes of 3-hydroxyisovalerylcarnitine measurements by tandem mass spectrometry in newborn screening.

BACKGROUND: The use of tandem mass spectrometry (MS/MS) for the analysis of amino acids and acylcarnitines from dried-blood spots (DBS) has become routine practice in newborn screening laboratories. The Newborn Screening Quality Assurance Program (NSQAP) added 3-hydroxyisovalerylcarnitine (C5OH) into its routine quality control and proficiency testing (PT) DBS materials for MS/MS to assure the quality of C5OH screening. We report the results from NSQAP evaluations for C5OH-enriched DBS, and summarize participant screening practices based on their analytical methods. METHODS: NSQAP prepared C5OH-enriched DBS materials for its participants. Laboratories reported quantitative and qualitative results. Bias plots of quantitative results were constructed using reported data and the results were sorted by an analytical method. RESULTS: NSQAP participants reported PT specimen 3964 as outside of normal limits for C5OH. The mean C5OH value for derivatized and non-derivatized methods was 2.80 and 2.67 µmol/l, respectively. Reported data from other specimens showed a similar trend in derivatized vs. non-derivatized assay results. Differences in C5OH quantitative values were observed among laboratories using different internal standards. CONCLUSIONS: C5OH MS/MS measurements in DBS assays varied by method and the choice of internal standards. The use of NSQAP's DBS materials allows harmonization of C5OH measurements by newborn screening laboratories worldwide.

Effect of specimen storage conditions on newborn dried blood spots used to assess Toxoplasma gondii immunoglobulin M (IgM).

BACKGROUND: Newborn screening programs store-under varying conditions-residual dried blood spots (DBS). Residual DBS were used to investigate the contribution of congenital infection with Toxoplasma gondii to the etiology of hydrocephalus and as a key step, we assessed the effect of storage conditions on the stability of newborn screening biomarkers. METHODS: Infants with hydrocephalus (410 cases) were identified using population-based birth defects surveillance systems in California, North Carolina, and Texas. Infants without birth defects (448 controls) were randomly selected from the same geographic areas and time periods. California stores DBS with controlled temperature, while North Carolina and Texas store DBS under ambient conditions. After removal of personal identifiers, DBS were tested for Toxo-specific immunoglobulin-M (Toxo-IgM). Because of poor elution of DBS stored in ambient conditions, additional biomarkers were tested on a specimen subset. RESULTS: Among 858 DBS tested, Toxo-IgM was found in 3 cases and no controls from California (N=515) and in no specimens from North Carolina or Texas (N=343). Among the 98 specimens tested for selected biomarkers, statistically significant differences were found for California vs. combined North Carolina and Texas DBS (thyroid stimulating hormone, phenylalanine, methionine, leucine and citrulline p<0.0001; tyrosine and valine p<0.001). CONCLUSIONS: Storage conditions for residual DBS had an effect on the ability to extract, recover, and accurately measure Toxo-IgM and other biomarkers from the filter paper matrix.

Performance properties of filter paper devices for whole blood collection.

BACKGROUND: The Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention assesses the adherence to established performance standards of manufactured lots of whole blood filter paper collection devices that are registered by the US FDA. We examined 26 newborn screening analytes measured from blood applied to filter papers from two FDA-cleared sources, Whatman(®) Grade 903 and Ahlstrom Grade 226. The dried blood spots contained analytes at both single levels and dose-response series. RESULTS: We observed overlap at one standard deviation for each analyte, with no more than 4-5% difference between the papers. CONCLUSION: The data demonstrated similarities of analyte recovery between the papers, indicating comparability of the devices for newborn screening and other applications.

Tandem mass spectrometric identification of dextrose markers in dried-blood spots from infants receiving total parenteral nutrition.

BACKGROUND: The false positive rate for the newborn screening of disorders of amino acid metabolism for premature infants is higher than full term infants. This may be due to very low birth weight infants receiving high concentrations of amino acids from total parenteral nutrition (TPN) administration and/or immature metabolism. An investigation of the possible influence of TPN on screening of premature infants resulted in the detection of three unusual peaks in the tandem mass spectrometry (MS/MS) acylcarnitine profile. These markers were closely correlated with the detection of very high multiple amino acid increases in the profiles of newborns administered with TPN and who were ultimately found to be normal and free of inherited metabolic disorders. METHODS: TPN solutions contain a concentrated mixture of amino acids and dextrose and other nutrients in saline. Due to its high concentration and suggestion of a carbohydrate, it was hypothesized that dextrose (D-glucose) was the contaminant and source of the markers detected. Dextrose, stable isotope-labeled 13C6-dextrose and various TPN solutions were analyzed directly or after enrichment in whole blood by multiple MS/MS acquisition modes including MS-only, product and precursor ion and neutral loss scans. RESULTS: Analysis of dried-blood spots (DBS) prepared from whole blood spiked with TPN solutions containing 12.5% dextrose and amino acid formulations designed to deliver 2.5 gm/kg/day of an amino acid mixture had moderate increases of all 3 dextrose markers detected at m/z 325, 399 and 473 as compared to controls. MS-only scans, product and precursor ion scans of dextrose and 13C6-dextrose in positive ion mode confirmed that these 3 peaks are derived from dextrose. Mass spectral analysis of labeled and unlabeled dextrose suggested that these peaks were dimers derived from dextrose. CONCLUSION: The identification of dextrose markers in DBS indicates that high concentrations of dextrose were present in blood and the likely source was contamination by TPN solutions most likely occurring during a sample collection process.

Proficiency testing of human leukocyte antigen-DR and human leukocyte antigen-DQ genetic risk assessment for type 1 diabetes using dried blood spots.

BACKGROUND: The plurality of genetic risk for developing type 1 diabetes mellitus (T1DM) lies within the genes that code for the human leukocyte antigens (HLAs). Many T1DM studies use HLA genetic risk assessment to identify higher risk individuals, and they often conduct these tests on dried blood spots (DBSs) like those used for newborn bloodspot screening. One such study is The Environmental Determinants of Diabetes in the Young (TEDDY), a long-term prospective study of environmental risk factors. To provide quality assurance for T1DM studies that employ HLA genetic risk assessment, the Centers for Disease Control and Prevention (CDC) conducts both a voluntary quarterly proficiency testing (VQPT) program available to any laboratory and a mandatory annual proficiency testing (PT) challenge for TEDDY laboratories. METHODS: Whole blood and DBS samples with a wide range of validated HLA-DR and HLA-DQ genotypes were sent to the participating laboratories. Results were evaluated on the basis of both the reported haplotypes and the HLA genetic risk assessment. RESULTS: Of the reported results from 24 panels sent out over six years in the VQPT, 94.7% (857/905) were correctly identified with respect to the relevant HLA-DR or HLA-DQ alleles, and 96.4% (241/250) were correctly categorized for risk assessment. Significant improvement was seen over the duration of this program, usually reaching 100% correct categorization during the last three years. Of 1154 reported results in four TEDDY PT challenges, 1153 (99.9%) were correctly identified for TEDDY eligibility. CONCLUSIONS: The different analytical methods used by T1DM research centers all provided accurate (>99%) results for genetic risk assessment. The two CDC PT programs documented the validity of the various approaches to screening and contributed to overall quality assurance.

Pilot proficiency testing study for second tier congenital adrenal hyperplasia newborn screening.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is caused by inherited defects in steroid biosynthesis. The Newborn Screening Quality Assurance Program (NSQAP) initiated a pilot, dried-blood spot (DBS)-based proficiency testing program designed to investigate materials and laboratory performance for second tier CAH screening by tandem mass spectrometry (MS/MS). METHODS: The ratio of 17-α-hydroxyprogesterone (17-OHP), androstenedione (4-AD) and cortisol is used as an indicator of CAH in laboratory protocols for second tier analysis of DBS specimens. DBS prepared by NSQAP contained a range of steroid concentrations resulting in different clinical ratios. Laboratories received blind-coded DBS specimens and reported results to NSQAP for evaluation. RESULTS: Quantitative values reported by participants for 17-OHP, 4-AD, and cortisol, reflected small differences in their analytical methods. Average quantitative values for 17-OHP increased from 81% to 107% recovery over the 3.5-year period; cortisol recoveries increased from 61.9% to 89.5%; and 4-AD recoveries decreased from 184% to 68%. CONCLUSIONS: Laboratory participation in the CAH second tier proficiency testing program has resulted in improved analyte recoveries and enhanced sample preparation methodologies. NSQAP services for the second tier CAH analysis in DBS demonstrate the need for surveillance to ensure harmonization and continuous improvements, and to achieve sustained high-performance of newborn screening laboratories worldwide.

Potential loss of methionine following extended storage of newborn screening samples prepared for tandem mass spectrometry analysis.

BACKGROUND: Methionine (Met) is a key metabolite used in the newborn screening of homocystinuria by tandem mass spectrometry (MS/MS). Recently, a loss of ion counts in both Met and its deuterium-labeled internal standard ((2)H(3)-Met) was observed by the CDC's Newborn Screening Quality Assurance Program laboratory. We report on the stability of labeled and unlabeled Met solutions and their storage in two types of 96 well microtiter plates to illustrate the potential loss of Met following storage of samples prior to MS/MS analysis. METHODS: Neat labeled and unlabeled Met standards were prepared and added (25, 50 and 100 microl) to two different types of microtiter plates, dried under nitrogen and stored for up to 168 h. All samples were reconstituted in mobile phase and analyzed as free acids for simplification of the study. RESULTS AND CONCLUSIONS: Met appears to interact significantly with polystyrene microtiter plates and to a much lesser extent with polypropylene microtiter plates. Furthermore, the loss is greatest for lower concentrations of methionine. While this loss of Met signal may be unimportant due to a presumption of equal loss of (2)H(3)-Met, a significant decline in ion signals will cause greater error in the calculation of concentration. These results suggest that polypropylene may be a better choice for Met analysis. Furthermore, storing prepared samples prior to analysis may impact the quality of the MS/MS analysis for Met and potentially other metabolites. Plates used by newborn screening laboratories should be evaluated periodically if the signal intensity for Met is reduced.

Maternal and neonatal vitamin B12 deficiency detected through expanded newborn screening--United States, 2003-2007.

The incidence of neonatal vitamin B12 (cobalamin) deficiency because of maternal deficiency was determined by surveying state newborn screening programs. Thirty-two infants with nutritional vitamin B12 deficiency were identified (0.88/100,000 newborns). Pregnant women should be assessed for their risk of inadequate intake/malabsorption of vitamin B12.

Newborn Screening System Performance Evaluation Assessment Scheme (PEAS).

Newborn screening (NBS) reaches approximately all of the 4 million newborns in the United States each year and has been effective in significantly reducing the morbidity and mortality that results from certain congenital conditions. The comprehensive NBS system can be divided into preanalytic (education and screening), analytic (laboratory testing), and postanalytic (reporting, short-term follow-up/tracking, diagnosis, treatment/management, ancillary services, and outcome evaluation) activities. To monitor and improve the screening system, there has been increasing emphasis on evaluation models. Federal sponsorship of a model performance evaluation and assessment scheme (PEAS) has resulted in a comprehensive listing of quality indicators for system self-assessment. We review the PEAS evolution process in an effort to illustrate the necessary infrastructure considerations in a well-functioning NBS system. Readers are encouraged to identify their role in the system and to interact appropriately at the local level. The comprehensive PEAS indicator list is provided as an Appendix.

Improving and assuring newborn screening laboratory quality worldwide: 30-year experience at the Centers for Disease Control and Prevention.

Newborn screening is the largest population-based genetic screening effort in the United States. The detection of treatable, inherited congenital disorders is a major public health responsibility. The Centers for Disease Control and Prevention's (CDC's) Newborn Screening Quality Assurance Program helps newborn screening laboratories ensure that testing accurately detects these disorders, does not delay diagnosis, minimizes false-positive reports, and sustains high-quality performance. For over 30 years, the CDC's Newborn Screening Quality Assurance Program has performed this essential public health service, ensuring the quality and accuracy of screening tests for more than 4 million infants born each year in the United States and millions more worldwide. The Program has grown from 1 disorder in 1978 for 31 participants to more than 50 disorders for 459 participants in 2009. This report reviews the Program's milestones and services to the newborn screening community.

Comparison of amino acids and acylcarnitines assay methods used in newborn screening assays by tandem mass spectrometry.

BACKGROUND: The analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Prevention's Newborn Screening Quality Assurance Program provides dried blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs. METHODS: DBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols. RESULT: Minor differences (<15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters. CONCLUSIONS: The use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not.

Preliminary proficiency testing results for succinylacetone in dried blood spots for newborn screening for tyrosinemia type I.

BACKGROUND: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I--an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. METHODS: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. RESULTS: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries > or =100% used DBS matrix calibrators. CONCLUSIONS: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.