W. Harry Hannon

Development and Evaluation of Quality Control Dried Blood Spot Materials in Newborn Screening for Lysosomal Storage Disorders

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 (micromol/L/h). RESULTS Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%-14.3% for galactocerebroside alpha-galactosidase, 6.8%-24.6% for acid alpha-galactosidase A, 7.36%-22.1% for acid sphingomyelinase, 6.2%-26.2% for acid alpha-glucocerebrosidase, and 7.0%-24.8% for lysosomal acid alpha-glucosidase (n = 9). In addition, DBS stored at -20 degrees and 4 degrees C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37 degrees and 45 degrees 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.

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.

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. .

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

BACKGROUND The analysis of amino acids (AA) and acylcarnitines (AC) by tandem mass spectrometry (MS/MS) is performed in newborn screening laboratories worldwide. While butyl esterification assays are routine, it is possible to detect AAs and ACs as their native free acids (underivatized). The Centers for Disease Control and Preventions 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.

Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters

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.

Improving and Assuring Newborn Screening Laboratory Quality Worldwide: 30-Year Experience at the Centers for Disease Control and Prevention

Newborn screening is the largest population-based genetic screening effort in the United States. The detection of treatable, inherited congenital disorders is a major public health responsibility. The Centers for Disease Control and Preventions (CDCs) 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 CDCs 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 Programs milestones and services to the newborn screening community.

Performance properties of filter paper devices for whole blood collection

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

Teratogen update: iodine deficiency, a community teratogen.

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.

Maternal and Neonatal Vitamin B12 Deficiency Detected through Expanded Newborn Screening—United States, 2003–2007

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

Evaluation of four reagents for delipidation of serum

Four reagents, Aerosil 380, Freon 113, Dextran sulfate 500-S, and a mixed organic solvent were tested for their abilities to produce optically clear, pooled human serum. Aerosil-380, a silicon dioxide, removed 95% of serum cholesterol and triglycerides, and 80% of the free fatty acids. A mixed organic solvent (n-butanol:diisopropyl ether) was equally effective, but also removed nearly all endogenous alkaline phosphatase and lactate dehydrogenase. Freon-113 and Dextran sulfate 500-S removed about half of the serum cholesterol and triglycerides. The serum content of several non-lipid components was unaffected by Aerosil-380, Freon-113, and Dextran sulfate treatments; however, the mixed organic solvent removed 69% of the endogenous calcium. Light scattering data revealed that treatment with all reagents except the mixed organic solvent resulted in optically-clear serum products.

Quantification of malonylcarnitine in dried blood spots by use of MS/MS varies by stable isotope internal standard composition.

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 ((2)H(3)-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.