Last data update: Jun 24, 2024. (Total: 47078 publications since 2009)
Records 1-24 (of 24 Records) |
Query Trace: Hannon WH [original query] |
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Technological journey from colorimetric to tandem mass spectrometric measurements in the diagnostic investigation for phenylketonuria
Chace DH , Hannon WH . J Inborn Errors Metab Screen 2016 4 Phenylalanine analysis for phenylketonuria (PKU) detection in newborn screening (NBS) was chosen as the model system to describe how advancements in laboratory technology improved laboratory performance. These advancements have made NBS programs better and have improved the health outcomes of the affected newborn through improvements in accurate early detection over the past 50 years. The most current state-of-the-art technology, tandem mass spectrometry (MS/MS), has proven that it is now the choice in almost all modern NBS facilities because it is a versatile instrument that continues to grow in its application not just for amino acid and acylcarnitine detection but for other metabolites and disorders such as lysosomal storage diseases and second-tier detection of some screen-positive results. The use of MS/MS will continue to expand, even with the anticipated introduction and expansion of molecular screening methods into NBS programs. Regarding technological advancements, the future of NBS will include even newer technologies and approaches that will enhance the detection and treatment of newborns affected by PKU and other inborn errors of metabolism. |
Single newborn screen or routine second screening for primary congenital hypothyroidism
Shapira SK , Hinton CF , Held PK , Jones E , Hannon WH , Ojodu J . Mol Genet Metab 2015 116 (3) 125-32 Routine second screening of most newborns at 8-14days of life for a panel of newborn conditions occurs in 12 U.S. states, while newborns in the other states typically undergo only a single routine newborn screen. The study objective was to evaluate screening consequences for primary congenital hypothyroidism (CH) in one- and two-screen states according to laboratory practices and medical or biochemical characteristics of screen-positive cases. Individual-level medical and biochemical data were retrospectively collected and analyzed for 2251 primary CH cases in one-screen (CA, WI) and two-screen (AL, DE, MD, OR, TX) states. Aggregate data were collected and analyzed for medical and biochemical characteristics of all screened newborns in the states. Among the states evaluated in this study, the detection rate of primary CH was higher in the one-screen states. In the two-screen states, 11.5% of cases were detected on the second screen. In multivariate analyses, only race/ethnicity was a significant predictor of cases identified on the first versus second screen, which likely reflects a physiologic difference in primary CH presentation. Newborn screening programs must heed the potential for newborns with CH not being detected by a single screen, particularly newborns of certain races/ethnicities. If the two-screen states converted to a single screen using their current algorithms, newborns currently identified on the routine second screen would presumably not be detected, resulting in probable delayed diagnosis and treatment. However, based on the one-screen state experiences, with appropriate modifications in screening method and algorithm, the two-screen states might convert to single screen operation for CH without loss in performance. |
Congenital adrenal hyperplasia cases identified by newborn screening in one- and two-screen states.
Held PK , Shapira SK , Hinton CF , Jones E , Hannon WH , Ojodu J . Mol Genet Metab 2015 116 (3) 133-8 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.
Taylor JL , Lee FK , Yazdanpanah GK , Staropoli JF , Liu M , Carulli JP , Sun C , Dobrowolski SF , Hannon WH , Vogt RF . Clin Chem 2014 61 (2) 412-9 BACKGROUND: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of the motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn bloodspot 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. |
Performance of succinylacetone assays and their associated proficiency testing outcomes
Adam BW , Hall EM , Meredith NK , Lim TH , Haynes CA , De Jesus VR , Hannon WH . Clin Biochem 2012 45 (18) 1658-63 BACKGROUND: Succinylacetone (SUAC) is the primary metabolic marker for hepatorenal tyrosinemia. MATERIALS AND METHODS: We used results reported for dried-blood-spot proficiency testing (PT) specimens and hepatorenal tyrosinemia patients' newborn screening (NBS) samples to demonstrate analytic biases in SUAC recoveries and differences in presumptive clinical classifications. RESULTS: SUAC recoveries from non-kit and NeoBase kit tandem mass spectrometry methods were markedly different. Kit users that set high cutoff values submitted discordant clinical assessments of "within normal limits" for PT specimens enriched with 10-15mcmol SUAC/L in blood. SUAC levels in tyrosinemia patients' NBS samples analyzed by NeoBase kit were lower than those in samples analyzed by non-kit methods. CONCLUSIONS: From 2009 to 2011, analytic biases in SUAC recoveries were consistent. Discordant clinical assessments of PT specimens were associated with high cutoff values for NeoBase kit results. Method-related differences in SUAC concentrations of tyrosinemia patients' samples were consistent with those of PT specimens. |
CFTR mutation analysis and haplotype associations in CF patients.
Cordovado SK , Hendrix M , Greene CN , Mochal S , Earley MC , Farrell PM , Kharrazi M , Hannon WH , Mueller PW . Mol Genet Metab 2012 105 (2) 249-54 Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies. |
The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States
Adam BW , Hall EM , Sternberg M , Lim TH , Flores SR , O'Brien S , Simms D , Li LX , De Jesus VR , Hannon WH . Clin Biochem 2011 44 1445-50 OBJECTIVE: We aimed to measure separately the contributions of heat and humidity to changes in levels of 34 markers of inborn disorders in dried-blood-spot (DBS) samples. DESIGN AND METHODS: We stored paired sets of DBSs at 37 degrees C for predetermined intervals in low-humidity and high-humidity environments. Marker levels of all samples in each complete sample set were measured in a single analytic run. RESULTS: During the 30+/-5day studies, galactose-1-phosphate uridyltransferase and biotinidase lost almost 65% of initial activities in low-humidity storage; most of the degradation in 27 other markers was attributable to adverse effects of high-humidity storage; seven markers in DBSs stored at high humidity lost more than 90% of initial levels by the end of the study and 4 of the 7 lost more than 50% of initial levels within the first week of storage. CONCLUSIONS: Minimizing both humidity and temperature in DBS transportation and storage environments is essential to maintaining sample integrity. |
Detection of TPN contamination of dried blood spots used in newborn and metabolic screening and its impact on quantitative measurement of amino acids
Chace DH , De Jesus VR , Lim TH , Hannon WH , Clark RH , Spitzer AR . Clin Chim Acta 2011 412 1385-90 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 4g/kg/day of Premasol(R) 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 7.6mmol/l at 1:80v/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.
Earley MC , Laxova A , Farrell PM , Driscoll-Dunn R , Cordovado S , Mogayzel PJ Jr , Konstan MW , Hannon WH . Clin Chim Acta 2011 412 1376-81 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. |
The preparation and storage of dried-blood spot quality control materials for lysosomal storage disease screening tests
Adam BW , Orsini JJ Jr , Martin M , Hall EM , Zobel SD , Caggana M , Hannon WH . Clin Biochem 2011 44 704-10 OBJECTIVE: We aimed to prepare dried-blood spot (DBS) quality control (QC) materials for lysosomal storage disease (LSD) screening tests and to determine optimum blood and DBS storage conditions. METHODS: We compared enzyme activities of five LSD markers in adult blood, umbilical-cord blood, and leukocyte-reduced blood. We measured activities in liquid blood and DBSs after predetermined intervals at controlled temperatures and humidities. RESULTS: Lysosomal-enzyme activity levels in umbilical-cord blood mimicked those in newborn screening samples. Lysosomal-enzyme activities in leukocyte-reduced blood were lower than in LSD-positive patient samples. Enzyme activities were stable in refrigerated liquid blood for 32days and in frozen DBSs stored at low humidity for a year. Activity losses from DBSs after 34days at 37+/-1 degrees C were 35-66% in low humidity and 61-100% in high humidity. CONCLUSIONS: Umbilical-cord blood is the preferred matrix for LSD-normal DBS QC materials. Leukocyte-reduced blood is lysosomal enzyme-deficient. Failure to control humidity during DBS storage results in loss of lysosomal-enzyme activities. |
Proficiency testing outcomes of 3-hydroxyisovalerylcarnitine measurements by tandem mass spectrometry in newborn screening
Lim TH , De Jesus VR , Meredith NK , Sternberg MR , Chace DH , Mei JV , Hannon WH . Clin Chim Acta 2010 412 631-5 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 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 2.67mumol/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. |
Impact of second-tier testing on the effectiveness of newborn screening
Chace DH , Hannon WH . Clin Chem 2010 56 (11) 1653-5 The goal of newborn screening (NBS)3 for inherited disorders of metabolism is the early detection and confirmation of disease, thus enabling early medical intervention, treatment, and improved outcomes (1). Important characteristics of a screening method include analytical specificity and sensitivity, coupled with rapid, high throughput and timely reporting of abnormal results. Routine primary screening methods are designed to identify as many abnormal infants as possible, with diagnostic sensitivity favored over specificity for disorder detection. This approach not only increases the numbers of false-positive test results, thus adding to the cost of operating NBS programs, but also places unnecessarily increased stress, anxiety, and possibly parent–child dysfunction on families (2). As the number of disorders in the NBS test panels grows, however, so does the overall number of false-positive results, which has increased severalfold per true case (3). One solution to this problem is to use improved methods or to couple primary screening methods with second-tier tests that improve selectivity. | The use of tandem mass spectrometry (MS/MS) for detecting phenylketonuria is an example of an NBS method that improves detection as a primary screen while also being more selective than older, classic NBS methods such as fluorometry. In one study, MS/MS analysis of newborn spot samples of dried blood collected ≤24 h after birth was compared with fluorometric analysis of the same samples. Because of this early time of collection, the decision level for an increased phenylalanine concentration was lowered by the public health laboratory using fluorometry to ensure that no infants with phenylketonuria were missed. MS/MS analysis of the identical samples demonstrated that disease detection could be sustained while improving selectivity (4). The ability to measure multiple analytes in the same analysis enabled the calculation of the phenylalanine/tyrosine molar ratio, which reduced false-positive rates a 100-fold. This screen for phenylketonuria was the first instance of a new paradigm in NBS, in which both current screens could be improved and new screens could be added for other disorders, such as fatty acid oxidation defects and organic acidemias (5). The ability of MS/MS to improve efficacy without the need for collecting a second sample reduces the false-positive rate. |
Effect of specimen storage conditions on newborn dried blood spots used to assess Toxoplasma gondii Immunoglobulin M (IgM)
Mei JV , Li L , Rasmussen SA , Collier S , Frias JL , Honein MA , Shaw GM , Lorey F , Meyer R , Chaing S , Canfield MA , Jones J , Hannon WH . Clin Chim Acta 2010 412 455-9 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, citrulline p<0.0001; tyrosine, 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. |
Tandem mass spectrometric identification of dextrose markers in dried-blood spots from infants receiving total parenteral nutrition
Chace DH , De Jesus VR , Lim TH , Hannon WH , Spitzer AR . Clin Chim Acta 2010 411 1806-16 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. |
Potential loss of methionine following extended storage of newborn screening samples prepared for tandem mass spectrometry analysis
Chace DH , Luo Z , De Jesus VR , Haynes CA , Hannon WH . Clin Chim Acta 2010 411 1284-6 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. |
Proficiency testing of human leukocyte antigen-DR and human leukocyte antigen-DQ genetic risk assessment for type 1 diabetes using dried blood spots
Dantonio P , Meredith-Molloy N , Hagopian WA , She JX , Akolkar B , Cordovado SK , Hendrix M , Henderson LO , Hannon WH , Vogt RF . J Diabetes Sci Technol 2010 4 (4) 929-941 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
De Jesus VR , Simms DA , Schiffer J , Kennedy M , Mei JV , Hannon WH . Clin Chim Acta 2010 411 1684-7 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-alpha-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, cortisol, reflected small differences in their analytical methods. Average quantitative values for 17-OHP increased from 81% to 107% recovery over the 3.5-y 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. |
Maternal and neonatal vitamin B12 deficiency detected through expanded newborn screening--United States, 2003-2007
Hinton CF , Ojodu JA , Fernhoff PM , Rasmussen SA , Scanlon KS , Hannon WH . J Pediatr 2010 157 (1) 162-3 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)
Therrell BL Jr , Schwartz M , Southard C , Williams D , Hannon WH , Mann MY . Semin Perinatol 2010 34 (2) 105-20 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
De Jesus VR , Mei JV , Bell CJ , Hannon WH . Semin Perinatol 2010 34 (2) 125-33 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
De Jesus VR , Chace DH , Lim TH , Mei JV , Hannon WH . Clin Chim Acta 2010 411 684-9 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
Adam BW , Lim TH , Hall EM , Hannon WH . Clin Chem 2009 55 (12) 2207-13 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 ≥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. |
National Academy of Clinical Biochemistry laboratory medicine practice guidelines: follow-up testing for metabolic disease identified by expanded newborn screening using tandem mass spectrometry; executive summary
Dietzen DJ , Rinaldo P , Whitley RJ , Rhead WJ , Hannon WH , Garg UC , Lo SF , Bennett MJ . Clin Chem 2009 55 (9) 1615-26 BACKGROUND: Almost all newborns in the US are screened at birth for multiple inborn errors of metabolism using tandem mass spectrometry. Screening tests are designed to be sufficiently sensitive so that cases are not missed. The NACB recognized a need for standard guidelines for laboratory confirmation of a positive newborn screen such that all babies would benefit from equal and optimal follow-up by confirmatory testing. METHODS: A committee was formed to review available data pertaining to confirmatory testing. The committee evaluated previously published guidelines, published methodological and clinical studies, clinical case reports, and expert opinion to support optimal confirmatory testing. Grading was based on guidelines adopted from criteria derived from the US Preventive Services Task Force and on the strength of recommendations and the quality of the evidence. Three primary methods of analyte measurement were evaluated for confirmatory testing including measurement of amino acids, organic acids, and carnitine esters. The committee graded the evidence for diagnostic utility of each test for the screened conditions. RESULTS: Ample data and experience were available to make strong recommendations for the practice of analyzing amino acids, organic acids, and acylcarnitines. Likewise, strong recommendations were made for the follow-up test menu for many disorders, particularly those with highest prevalence. Fewer data exist to determine the impact of newborn screening on patient outcomes in all but a few disorders. The guidelines also provide an assessment of developing technology that will fuel a refinement of current practice and ultimate expansion of the diseases detectable by tandem mass spectrometry. CONCLUSIONS: Guidelines are provided for optimal follow-up testing for positive newborn screens using tandem mass spectrometry. The committee regards these tests as reliable and currently optimal for follow-up testing. . |
Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters
Chace DH , Lim T , Hansen CR , De Jesus VR , Hannon WH . Clin Chim Acta 2009 407 6-9 BACKGROUND: The utilization of succinylacetone (SUAC) as the primary metabolic marker for tyrosinemia Type I is now well known, thus new methods have been developed to analyze SUAC as a first tier test in newborn screening. One approach is to prepare a SUAC hydrazine derivative from the dried blood spots (DBS) previously utilized in the extraction of acylcarnitine (AC) and amino acids (AA). The final derivatized products of SUAC, AA and AC are combined in a single tandem mass spectrometric (MS/MS) analysis. However, butyl esterification techniques may result in contamination of underivatized acylcarnitines by as much as 20%. We have developed a simple wash step to improve the combined analysis of SUAC, AA and AC in DBS by MS/MS. METHODS: AA and AC were extracted with methanol containing labeled internal standard from 3.2mm punches taken from the DBS specimen. The previously extracted blood spot that remains after removal of the methanol extraction solvent was used in the preparation of SUAC with and without additional washing of the blood spot. The butyl ester eluates of AA and AC, and SUAC hydrazine derivatives were recombined and measured by MS/MS. RESULTS: Three additional methanol wash steps of the remaining DBS punches prior to SUAC derivatization reduced the presence of underivatized acylcarnitines, resulting in a 4-fold reduction of underivatized palmitoylcarnitine. Palmitoylcarnitine butyl ester is detected at m/z 456 while the underivatized species is detected at m/z 400, which is also the mass of dodecanoylcarnitine butyl ester. The linearity of the SUAC assay was unchanged by the additional wash steps. For butyl esterification methods, the preferred analytic procedure, the presence of AC can compromise the results of a newborn screen for the actual concentrations of acylcarnitines. It is essential to remove any underivatized acylcarnitines prior to SUAC analysis. CONCLUSION: The additional methanol wash steps did not alter SUAC assay results but did remove underivatized acylcarnitines which could result in the incorrect quantification of acylcarnitines. |
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