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Routine second screening of most newborns at 8–14 days 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 2,251 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.

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.

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.

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.

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.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.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.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.

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.

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 p 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.

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.

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.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.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.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.

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.

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.

Background: Lysosomal storage disorders (LSDs) comprise more than 40 genetic diseases that result in the accumulation of products that would normally be degraded by lysosomal enzymes. A tandem mass spectrometry (MS/MS)-based method is available for newborn screening for 5 LSDs, and many laboratories are initiating pilot studies to evaluate the incorporation of this method into their screening panels. We developed and evaluated dried blood spot (DBS) QC materials for LSDs and used the MS/MS method to investigate their suitability for LSD QC monitoring. Methods: We incubated 3.2-mm punches from DBS controls for 20–24 h with assay cocktails containing substrate and internal standard. Using MS/MS, we quantified the resulting product and internal standard. Samples were run in triplicate for 3 consecutive days, and results were reported as product-to-internal standard ratios and enzyme activity units (μmol/L/h). Results: Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%–14.3% for galactocerebroside α-galactosidase, 6.8%–24.6% for acid α-galactosidase A, 7.36%–22.1% for acid sphingomyelinase, 6.2%–26.2% for acid α-glucocerebrosidase, and 7.0%–24.8% for lysosomal acid α-glucosidase (n = 9). In addition, DBS stored at −20° and 4 °C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37° and 45 °C had lower activity values over the 187-day evaluation time. Conclusions: Suitable QC materials for newborn screening of LSDs were developed for laboratories performing DBS LSD screening. Good material linearity was observed, with goodness-of-fit values of 0.953 and higher. The QC materials may be used by screening laboratories that perform LSD analysis by MS and/or more conventional fluorescence-based screening methods.

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.

Investigators have reported that certain metabolic disorders (fatty acid oxidation (FAO) disorders and organic acidemias) contribute to unexpected early childhood deaths. We estimated the contribution of these metabolic disorders to a population-based sample of unexpected early childhood deaths. The study population included children less than 3 years of age who died during 1996-2001 and whose deaths were investigated by the Virginia Office of the Chief Medical Examiner (ME). Dried post-mortem blood on filter paper was sent to a reference laboratory for metabolic screening by tandem mass spectrometry. When molecular DNA analysis was available to identify known gene mutations, positive screens were confirmed. If molecular DNA analysis for a suspected disorder was not available, tandem mass spectrometry was performed on newborn blood spots when available. If DNA analysis was not available and newborn blood spots could not be obtained, an independent expert biochemical geneticist confirmed the post-mortem interpretation. We obtained screening results for 793 (88%) of 904 children examined. Eight children had a positive screen for FAO disorders or organic acidemias. One child would not have benefited from identification in the newborn period. However, seven children's outcomes might have been improved had they been identified during the newborn period and effectively treated. Post-mortem metabolic screening may identify a cause of death for about 1% of children who die unexpectedly before 3 years of age, allowing for identification and treatment of affected siblings. Identifying and treating affected children during the newborn period may offer an opportunity to reduce early childhood mortality.

Less sensitive (LS) HIV-1 enzyme immunoassays (EIAs) have significantly improved the quantity and quality of HIV surveillance data. The first LS-HIV-1 EIA, the Abbott 3A11-LS, provided reliable incidence data, but the assay required specialized equipment, and the lack of available reagents made testing difficult. This study evaluated the use of an alternate assay, a modified version of the Vironostika HIV-1 EIA (Vironostika-LS), to be used for LS testing. The Vironostika-LS has similar performance characteristics to the Abbott 3A11-LS with additional advantages. This 96-well formatted assay is commonly found in public health laboratories for routine HIV-1 testing and can be used with both serum and dried blood spot specimens. The estimated mean time from seroconversion (defined using a standardized optical density cutoff of 1.0) with the Vironostika-LS was 170 days (95% CI, 145-200 days). When the Vironostika-LS was applied to a matched serum set previously tested with the Abbott 3A11-LS, the Vironostika-LS accurately identified 97% of specimens with recent or long-standing HIV infection. The paper also reports Vironostika-LS quality control guidelines and the results from 3 rounds of proficiency testing.

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications.

The Centers for Disease Control and Prevention and its partners have been operating the Newborn Screening Quality Assurance Program for >20 y. The program helps participating laboratories to evaluate and improve the quality of their newborn-screening testing efforts by providing quality control dried blood spot materials and proficiency-testing materials for the external evaluation of screening programs. The Newborn Screening Quality Assurance Program provides an independent evaluation of filter papers approved by the Food and Drug Administration for the collection of blood for clinical tests. These activities have created a mechanism for the validation of the filter paper blood collection device and the standardization of materials and methods for the analysis of dried blood spots.

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 …

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 μl) 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.

In 1997, the Centers for Disease Control and Prevention established the National Diabetes Laboratory in order to help prevent and treat type 1 diabetes. This state-of-the-art laboratory collaborates with research scientists and key national and international organizations throughout the world to identify and study risk factors for type 1 diabetes by developing measurements for glycosylated proteins, developing and evaluating technology for measuring genetic risk factors for the disease, and working to standardize autoantibody measurements. Developing improved technologies for diagnosing and managing diabetes and developing reference materials for properly calibrating and standardizing blood glucose meters are also critical aspects of the laboratory's work. In addition, the laboratory provides quality storage for valuable collections of biologics and other materials and facilitates sharing of specimens, associated epidemiologic data, and test results. Working with our partners in diabetes research, we are improving the diagnosis, treatment, and prevention of type 1 diabetes.

Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage. Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs. Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested. Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.

Iodine deficiency in a population causes increased prevalence of goiter and, more importantly, may increase the risk for intellectual deficiency in that population. The National Health and Nutrition Examination Surveys [NHANES I (1971-1974) and (NHANES III (1988-1994)] measured urinary iodine (UI) concentrations. UI concentrations are an indicator of the adequacy of iodine intake for a population. The median UI concentrations in iodine-sufficient populations should be greater than 10 microg/dL, and no more than 20% of the population should have UI concentrations less than 5 microg/dL. Median UI concentrations from both NHANES I and NHANES III indicate adequate iodine intake for the overall U.S. population, but the median concentration decreased more than 50% between 1971-1974 (32.0+/-0.6 microg/dL) and 1988-1994 (14.5+/-0.3 microg/dL). Low UI concentrations (5 microg/dL) were found in 11.7% of the 1988-1994 population, a 4.5-fold increase over the proportion in the 1971-1974 population. The percentage of people excreting low concentrations of iodine (UI,5 microg/dL) increased in all age groups. In pregnant women, 6.7%, and in women of child-bearing age, 14.9% had UI concentrations below 5 microg/dL. The findings in 1988-1994, although not indicative of iodine deficiency in the overall U.S. population, define a trend that must be monitored.

Terminology in any field is a complex mix of established conventions, accepted usages, disputed terms, and occasional misnomers. The terminology that has evolved for quantitative fluorescence cytometry (QFCM) is especially multifarious, in part because QFCM encompasses a range from subjective visual assessments to objective photon counts. Thus, while descriptive terms such as “dim” and “bright” are still quite useful, quantitative terms such as “binding capacity” should be used with collective understanding of their exact meanings. This article reviews current usage and proposes definitions that, with refinement from suppliers and users of QFCM technology, can provide the required clarity. Cytometry 33:97–105, 1998. Published 1998 Wiley-Liss, Inc.1

We modified and evaluated a RIA for serum and used the modified RIA to measure zidovudine in dried blood spot specimens (DBSs) routinely collected for newborn screening and tested anonymously for maternally acquired HIV antibodies in the national HIV Seroprevalence Survey Among Childbearing Women. DBS calibration and quality-control materials were used to adapt the serum assay to the DBS matrix. The assay had a limit of detection of 24 μg/L serum and was used to measure zidovudine from both whole DBSs and the eluate remaining after HIV antibody screening. We initiated a pilot study to investigate the assay’s performance and assess its potential to determine the implementation of the US Public Health Service recommendations that HIV-infected pregnant women and newborns receive zidovudine treatment to reduce the risk of perinatal HIV transmission.

We describe a sensitive and specific method for measuring cotinine in serum by HPLC coupled to an atmospheric pressure chemical ionization tandem mass spectrometer. This method can analyze 100 samples/day on a routine basis, and its limit of detection of 50 ng/L makes it applicable to the analysis of samples from nonsmokers potentially exposed to environmental tobacco smoke. Analytical accuracy has been demonstrated from the analysis of NIST cotinine standards and from comparative analyses by both the current method and gas chromatography/high-resolution mass spectrometry. Precision has been examined through the repetitive analysis of a series of bench and blind QC materials. This method has been applied to the analysis of cotinine in serum samples collected as part of the Third National Health and Nutrition Examination Survey (NHANES III).

A collaborative March of Dimes study was designed to examine the utility of dried blood spot (DBS) materials routinely collected from newborns as a source for monitoring cocaine exposure and to assess the prevalence of cocaine use among childbearing women in Georgia. We used a modified urinary radioimmunoassay (RIA) to anonymously detect the cocaine metabolite benzoylecgonine (BE) in DBSs. Extensive efforts were undertaken to assure absolute nonlinkage of BE data to any individual. The positive results found by RIA were confirmed by a mass spectrometry (MS) method specifically developed to detect BE in DBSs. BE was measured in 23,141 DBSs collected during 2 months of routine newborn screening in Georgia. A good correlation was observed for RIA results versus MS results (r2= 0.97). The estimated minimal statewide BE prevalence was 4.8 per 1000 childbearing women. We demonstrated that immunoassay testing for cocaine without confirmatory testing can yield falsely elevated prevalence rates. When proper confirmatory testing is done, DBSs are a valuable source for population-based monitoring of substance abuse among childbearing women.

In the last decade, iodine deficiency disorders (IDD) have become recognized as the most common preventable cause of mental retardation worldwide. Iodine deficiency interferes severely with prenatal and postnatal growth and neurologic development of individuals. It has condemned tens of millions of children to cretinism—characterized by mental and growth retardation, rigid spastic motor disorders, deaf mutism, and severe hypothyroidism—and hundreds of millions of children to milder degrees of mental and physical impairments. Accompanying effects include increased rates of congenital anomalies, fetal wastage, infant mortality, goiter, and hypothyroidism in children and adults. Endemic cretinism has been classically divided into two types: a chronic neurological disorder and a condition with myxedema and severe hypothyroidism. Recent studies have shown considerable overlap in the findings of these two types, which are now thought to have a common etiology differing primarily by the timing and severity of the pre- and postnatal deficiency of iodine and maternal thyroxine. The severity of iodine deficiency and hypothyroidism in the mother during early and midgestation is related to the severity of the neural damage in the fetus. The most logical intervention for a community or population is the introduction of iodine prophylaxis. Although salt iodization is technically straightforward, community leadership is essential to any effort. The mobilization of global efforts through WHO, UNICEF, Program Against Micronutrient Malnutrition (PAMM), and International Council for Control of Iodine Deficiency Disorders (ICCIDD) since 1990 has led to goals to eliminate new cases of IDD by the year 2000. Most countries at great risk for IDD have met or are close to meeting the middecade goal of having iodized salt for 90% of households. This provisional success indicates that the goal of eliminating new IDD cases by the year 2000 may be achieved.

These guidelines provide scientific information for policy development by state health departments considering appropriate use of newborn screening specimens after screening tests are finished. Information was collected, debated, and formulated into a policy statement by the Newborn Screening Committee of the Council of Regional Networks for Genetic Services (CORN), a federally funded national consortium of representatives from 10 regional genetics networks. Newborn screening programs vary widely in approaches and policies concerning residual dried blood spot samples (DBS) collected for newborn screening. Recognition of the epidemiological utility of DBS samples for HIV seroprevalence surveys and a growing interest in DBSs for DNA analysis has intensified consideration of issues regarding retention, storage, and use of residual DBS samples. Potentially these samples provide a genetic material “bank” for all newborns nationwide. Their value as a resource for other uses has already been recognized by scientists, administrators, and judicial officials. Programs should promulgate rules for retention and use of residual newborn screening DBS samples based on scientifically valid information. Banking of newborn samples as sources of genetic material should be considered in light of potential benefit or harm to society.

A preliminary survey has been made of quality assurance programs in newborn screening. These have been summarized according to the contact information for the program, the distribution characteristics of the program, the reporting characteristics of the program and the analytes covered.

used as urinary screening tests followed by confirmation by GC/MS. Immunoassay tests have been used primarily on urine specimens because of the ease of specimen collection and the relative abundance of the metabolite in urine following cocaine use. Levels of BE in blood are lower than those found in urine in concurrent samples, but they remain in the detectable range of immunoassays. The predictable half-life of BE in blood for extended periods has not been calculated, and the relationship between levels in blood and urine has not been correlated. Moreover, metabolism of BE in the blood during pregnancy and its half-life in the blood of newborns have not been investigated. Previously, investigators have described several techniques for the extraction of BE from blood using organic solvent extraction and subsequent assay in an aqueous or methanolic medium (5-7). Here, the authors describe the use of a commercial urinary BE immunoassay procedure with standards, calibrators, and quality control materials (QC) made up in aqueous eluates from blood spots or BE spiked into whole blood, spotted onto filter paper, and then eluted in aqueous solution. Postmortem blood samples were quantitated as blood spots for BE, and quantitation was confirmed by GC/MS analysis. The correlation of blood and urine levels of BE was measured on samples from pregnant women. Blood samples were collected from mothers; cord and blood spots from neonates were measured for BE. Finally, the quantitative immunoassay was used to measure BE levels in more than 500 anonymous residual blood spots obtained routinely from infants born in three states, at hospitals where high prevalence of cocaine use among childbearing women is suspected.

The use of tandem mass spectrometry (MS-MS) in the analysis of filter paper blood spots from newborns for acylcarnitines and amino acids has expanded significantly in recent years (1)(2). With estimates of 1 million specimens analyzed by MS-MS per year throughout the world, the demand is acute for assay standardization and harmonization. Programs exist at the CDC for amino acid standardization and quality assurance pertaining to newborn screening (3)(4). This program is being extended to include acylcarnitines, and the data in this report stem from that extension. Five metabolites are key in the diagnosis of several disorders of fat and organic acid metabolism. Preliminary results demonstrated excellent linearity for each of the five acylcarnitines added to blood. However, an extremely unusual result was observed for octanoylcarnitine: the recovery of octanoylcarnitine was significantly lower than that of other acylcarnitines. This observation is supported by Turner and Dalton (5), who report a 40% loss of octanoylcarnitine after addition to whole blood and plasma. We investigated the cause of this loss of octanoylcarnitine so that this serious error could be prevented or accounted for. The results of this study demonstrate the importance of assay standardization and the validation required in clinical screening that goes well beyond this particular quality-assurance/quality-control program for acylcarnitines. We obtained isotope-labeled internal standards (l-2H3-propionylcarnitine, l-2H3-butyrylcarnitine, l-2H3-octanoylcarnitine, l-2H9-myristoylcarnitine, and l-2H3-palmitoylcarnitine) from Cambridge Isotope Laboratories. Unlabeled standards (d,l-octanoylcarnitine, d,l-myristoylcarnitine, and l-palmitoylcarnitine) were obtained from Sigma, and l-propionylcarnitine, l-butyrylcarnitine, and l-octanoylcarnitine were obtained from Life Sciences Resources. The unlabeled standards were used to prepare a series of blood specimens at the CDC using procedures described previously (3)(4) with the following …

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.

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 13 C 6 -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 13 C 6 -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.

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.

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.

Dried blood spots (DBSs) are used to screen newborns for phenylketonuria and other aminoacidopathies. The calibrators for this testing are usually DBSs with values for Phe. Two DBS reference materials have been prepared, the European Working Standard for Phe (EWS-Phe-01) (1) and the amino acid reference material (AARM) from the CDC (2). The two reference materials are not interchangeable because they differ in blood hematocrit, blood-spot size, and filter paper, each of which (3)(4)(5)(6) affects analyte recovery. We measured quantitatively the effects of these differences on analyte recovery from DBSs and used results from our measurements to predict expected Phe recoveries from tandem analyses of the two sets of materials. In EWS-Phe-01 (1)(7), human blood with a 50.5% hematocrit and intact red cells was divided into five portions for enrichment with 0, 20, 40, 80, and 120 mg Phe/L blood (0, 120, 240, 480, and 720 μmol/L blood). The liquid added during enrichment (7) was sufficient to reduce the hematocrit to 50.1%. The Phe-enriched blood portions were dispensed in 35-μL aliquots (7) onto Schleicher & Schuell (S&S) Grade 2992 (lot no. 121576) filter paper (1). The AARM was prepared (2) by dividing human blood with a 57% hematocrit and intact red cells into six portions for enrichment with pure amino acids to cover the usual analytic ranges of Phe, Tyr, Leu, Met, and Val. The Phe enrichments were 0, 40, 80, 120, 160, and 200 mg Phe/L blood (0, 240, 480, 720, 960, and 1200 μmol/L blood). The liquid added during enrichment was sufficient to reduce the hematocrit to 53%. The whole-blood pools were dispensed in 100-μL portions onto S&S Grade 903 (lot no. W941) filter paper with dashed-line 13-mm printed circles (2). To examine the effect of blood hematocrit …

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. .

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 3.2 mm 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.

Lysosomal storage disorders (LSDs) embody a collection of >40 unique genetic diseases that cause the accumulation of macromolecular substrates normally degraded by lysosomal (and in some cases, nonlysosomal) enzymes involved in lysosomal metabolism (1). Although individual LSDs are rare, their combined incidence has been estimated at 1 per 7700 live births (1). Recently, 2 reports that describe the use of a tandem mass spectrometry-based multiplex assay for newborn screening for LSDs have been published in Clinical Chemistry (2)(3). A third report describes the availability of QC dried blood spot materials for monitoring the quality of the multiplex assay for Krabbe (galactocerebrosidase), Gaucher (acid β-glucocerebrosidase), Niemann-Pick types A and B (acid sphingomyelinase), Pompe (acid α-glucosidase), and Fabry (acid α-galactosidase) disorders (4). The multiplex assay has been shown to be robust and is currently in use or under evaluation in many screening and diagnostic laboratories for newborns around the world. Current protocols call for the use of acetonitrile as a major component of the mobile phase. Acetonitrile is a widely used solvent in newborn-screening laboratories for LSD and other mass spectrometry-based assays (e.g., for amino acids and acylcarnitines). Several chemical suppliers have …

To determine the precision of cellular fluorescence intensity (FI) measurements derived from labeled microbead standards, FI results were compared from 43 different flow cytometers in 34 laboratories. All laboratories analyzed prepared aliquots of fluoresceinated calf thymocyte nuclei (Fluorotrol), human lymphocytes stained with fluoresceinated anti-CD4 antibody, and fluoresceinated microbeads used as both internal and external standards. Measurements were conducted by most laboratories on the third and fourth days after sample preparation. Results for percent of events within the gates and the histograms returned by participants indicated that the samples had remained stable and that gated populations had been properly identified. All standard curves showed strong linearity, and the pooled results from all standards produced a best-fit curve that was in close agreement with the assigned values. Nonetheless, results for cellular FI were highly variable, with CVs of 20-34%. Agreement within lab/instrument was much better, with CVs ranging from 3.0 to 9.9%. The overall variability was not obviously attributable to differences in the types of cytometer, nor could it be explained by attributes of the standard curves or any other single variable examined. However, the application of a corrective factor based on FI results for Fluorotrol allowed a two-fold improvement in the precision of FI measurements on CD4-stained lymphocytes, with an overall CV of 11%. Uncharacterized differences in the operating conditions of flow cytometers can influence cellular FI measurements, but consistent results can be obtained if a stained cellular calibrator is analyzed in addition to the proper microbead standards.

Background The utilization of MS/MS for the analysis of amino acids and acylcarnitines from dried blood spots (DBS) is routine in many newborn screening (NBS) laboratories. Recently, malonylcarnitine (C3DC) was shown to be elevated in the DBS of affected infants with malonic acidemia. Quantitative features were unknown, so that its measurement was an approximation. Synthesis of malonylcarnitine enabled both a study in the analytical characteristics of C3DC and a survey of its measurement in NBS laboratories. Methods Malonylcarnitine was enriched in blood and spotted onto filter paper cards. The DBS were sent to several laboratories for analysis, and the results were returned to the Centers for Disease Control and Prevention (CDC) for evaluation. Reports included a description of the MS/MS method utilized. Results A pilot proficiency survey shows a bimodal distribution of data from 98 laboratories. Analysis of proficiency data reveals the use of different stable isotope internal standards for quantification. Analysis of standard, labeled or unlabelled (2H3-octanoylcarnitine (C8), glutarylcarnitine (C5DC) and malonylcarnitine (C3DC) revealed significantly different ion detection values. Quantification in laboratories is based on the ratio of the metabolite in question to a reference stable isotope standard. Conclusions Quantification of metabolites depends upon the reference isotope standard utilized. Quantification requires describing the standards used for estimation of concentration (a pseudo-concentration) and a notation that includes a reference to the isotope standard used. This descriptive method will enable harmonization of data in screening laboratories.

Newborn screening has existed as a state-based public health service since the early 1960s. Every state and most territorial jurisdictions have comprehensive newborn screening programs in place, but in the United States a national newborn screening policy does not exist. This results in different administrative infrastructures, screening requirements, laboratory and follow-up services, medical management approaches, and related activities across the country. Federal initiatives and support have contributed to limited evaluations of various aspects of individual newborn screening programs at the national level, but funding is an issue. The national evaluation strategies have taken various forms, all with the intent of improving the screening system through review of actions taken and suggestions for future improvements. While participation in the national evaluation effort for newborn screening laboratory practices includes all US programs, and this has aided in improving quality and harmonizing protocols, other national evaluation activities have been only moderately successful. National data reporting of quality indicators for various program elements must be comprehensive and timely, and the elements must be universally accepted in order to meet the evaluation and improvement needs of the national newborn screening system. A comprehensive real time national evaluation activity will likely require additional resources and enforcement incentives. Limited federal actions through grant incentives and selected reporting requirements provide a possible means of stimulating programs to participate in national harmonization efforts.

Certain alleles among the genes that code for the human leukocyte antigens (HLA) confer susceptibility or resistance to the development of autoimmunity that causes type 1 diabetes (T1D). A number of ongoing diabetes research studies analyze dried blood spots (DBS) from newborn infants for HLA-D alleles to identify higher-risk children as early as possible. A commercially available assay to detect such alleles has recently become available using a dissociation- enhanced lanthanide fluorescence system found in many newborn screening laboratories.We adapted the system for use with DBS and improved the sample set-up for greater efficiency. We also developed an independent system for data analysis based on a spreadsheet program. These modifications were applied to HLA-DQB1 gene locus (DQB) analysis of 117 newborn DBS, and the results we obtained were compared with independent reference values.Our assay modifications and independent data analysis improved sample throughput and result tabulation. DQB results from the modified assay were consistent with the reference values in all but one sample, which showed a partial match.The modifications described here make this commercially available assay more suitable for high-throughput applications such as newborn screening. Our results show that this system allows highly accurate detection of DQB alleles that influence T1D risk.

Objectives: In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. Methods: DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. Results: IRT was stable in the filter paper matrix when stored for one year at either −20°C or 4°C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. Conclusions: IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.

The Maternal and Child Health Bureau commissioned the American College of Medical Genetics to outline a process of standardization of outcomes and guidelines for state newborn screening programs and to define responsibilities for collecting and evaluating outcome data, including a recommended uniform panel of conditions to include in state newborn screening programs. The expert panel identified 29 conditions for which screening should be mandated. An additional 25 conditions were identified because they are part of the differential diagnosis of a condition in the core panel, they are clinically significant and revealed with screening technology but lack an efficacious treatment, or they represent incidental findings for which there is potential clinical significance. The process of identification is described, and recommendations are provided.

Objectives To provide current data for policy discussions and to assess future needs among newborn screening programs regarding the storage and use of residual dried blood spots (DBS) in the United States. Study design An electronic questionnaire was administered to U.S. state health department laboratory directors in 2003. Results Responses were received from 49 of the 50 states. Approximately half of them stored residual DBS for more than 6 months, 57% did not have a written policy that determines how residual DBS can or cannot be used, and 16% informed parents that DBS might be retained. Residual DBS were used by 74% of respondents for evaluation of newborn screening tests, by 52% for clinical or forensic testing, and by 28% for epidemiologic studies. Use of DBS was reported more frequently by states with extended storage. When asked if they might participate in an anonymous multistate epidemiologic study by contributing unlinked DBS, 41% responded affirmatively. Conclusions More states have used residual DBS for evaluating newborn screening tests than for epidemiologic studies. There is potential interest among states in using unlinked DBS for multistate studies and a need for written policies addressing all uses of residual DBS.

The Centers for Disease Control and Prevention (CDC) has a long history of involvement in quality assurance (QA) activities for support of newborn screening laboratories. Since 1978, CDC's Newborn Screening Quality Assurance Program (NSQAP), has distributed dried-blood spot (DBS) materials for external QA and has maintained related projects to serve newborn screening laboratories. The first DBS materials were distributed for congenital hypothyroidism screening in 1978 and by 2001, NSQAP had expanded to over 30 disorders and performance monitoring for all filter paper production lots from approved commercial sources. In 2001, there were 250 active NSQAP participants, 167 laboratories from 45 countries and 83 laboratories in the United States. Of these laboratories, 31 are from the Asia Pacific Region representing nine countries primarily for two disorders. In 1999, US laboratories had more errors for Performance Evaluation (PE) specimens than other laboratories; but in 2000, US laboratories had fewer errors. International laboratories reported 0.3% false-negative PE clinical assessments for congenital hypothyroidism and 0.5% for phenylketonuria (0.5%) in 2000. Paperless PE data-reporting operation using an Internet website has recently been implemented.

Primary immunodeficiency (PI) diseases are a group of primarily single-gene disorders of the immune system. Approximately 100 separate PI diseases have been described, but20 probably account for90% of cases. Although diverse, PI diseases share the common feature of susceptibility to infection and result in substantial morbidity and shortened life spans. Most important, prompt diagnosis and treatment can now lead to life-saving treatment and result in marked improvements in the quality and length of life for persons with PI diseases. In November 2001, a workshop was convened by CDC in Atlanta, Georgia, to discuss ways to improve health outcomes among persons with PI disease. A multidisciplinary panel of persons knowledgeable in PI diseases and public health met to identify and discuss public health strategies that can be applied to PI diseases and possibly for other genetic disorders. A systematic assessment based on the established public health framework was applied to the growing group of PI diseases, whose diverse genetic mutations span multiple components of the immune system but all lead to increased incidence and severity of infections. During the meeting, specialists in clinical immunology, public health, genetics, pediatrics, health communication, and ethics from state and federal agencies, academic centers, professional organizations, and advocacy foundations discussed the four components of the public health framework as they relate to PI diseases. These four components include 1) public health assessment (application of traditional public health methods to assess the occurrence and impact of PI diseases on communities); 2) population-based interventions (development, implementation, and evaluation of screening tests administered to newborns and clinical algorithms for early recognition of symptomatic persons to facilitate the earliest possible diagnosis and treatment for PI diseases); 3) evaluation of screening and diagnostic tools (to ensure their quality and appropriateness for identification of patients with PI diseases); and 4) communication (communication with and information dissemination to health-care providers and the public to facilitate prompt and appropriate diagnosis and intervention). The working group's deliberations focused on challenges and opportunities, priority research questions, and recommendations for future action for these four components. These recommendations, developed by workshop participants, will be useful to medical and public health professionals who are evaluating methods to increase recognition of PI diseases and other genetic disorders.