Performance of succinylacetone assays and their associated proficiency testing outcomes.

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 10-15 µmol 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.

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.

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°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 ± 5 day 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.

The preparation and storage of dried-blood spot quality control materials for lysosomal storage disease screening tests.

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 32 days and in frozen DBSs stored at low humidity for a year. Activity losses from DBSs after 34 days at 37±1°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.

First ISNS Reference Preparation for Neonatal Screening for thyrotropin, phenylalanine and 17alpha-hydroxyprogesterone in blood spots.

BACKGROUND: Neonatal screening for congenital disorders like phenylketonuria (PKU), congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) is generally performed in dried blood spots on filter paper. The analytes of interest for testing for PKU, CH and CAH are phenylalanine, thyrotropin (TSH) and 17alpha-hydroxyprogesterone (17OHP), respectively. The International Society for Neonatal Screening (ISNS) decided to prepare a combined reference preparation for the three analytes on filter paper Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545. This 'First ISNS Reference Preparation for Neonatal Screening for TSH, phenylalanine and 17OHP in blood spots' (1st ISNS-RPNS) has been prepared by the RIVM (Bilthoven). METHOD: The number of filter paper cards prepared, each with two sets of six blood spot calibrators, was 480, 42 and 69 for Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545, respectively. The volume of blood dispensed was 50 microl. The range of concentrations for TSH was 1-121 mIU/L blood, for phenylalanine 65-865 micromol/L blood and for 17OHP 2.2-302 nmol/L blood. RESULTS: The linearity of the blood spot calibrators and the homogeneity of the batch (only tested for Schleicher & Schuell) were good. The differences between the three filter papers were small: i.e. the potency of the ISNS-RPNS on Whatman and Toyo Roshi in terms of Schleicher & Schuell was between 0.98 and 1.09 for the three analytes. CONCLUSION: The 1st ISNS-RPNS for TSH, phenylalanine and 17OHP can be said to be suitable as formal reference preparation and as a source for (re)calibrating kit calibrators.

Measures of maternal tobacco exposure and infant birth weight at term.

This study was undertaken to determine the relation between self-reported number of cigarettes smoked per day and urine cotinine concentration during pregnancy and to examine the relations between these two measures of tobacco exposure and birth weight. Data were obtained from the Smoking Cessation in Pregnancy project, conducted between 1987 and 1991. Cigarette smoking information and urine cotinine concentration were collected for 3,395 self-reported smokers who were receiving prenatal care at public clinics in three US states (Colorado, Maryland, and Missouri) and who delivered term infants. General linear models were used to quantify urine cotinine variability explained by the number of cigarettes smoked per day and to generate mean adjusted birth weights for women with different levels of tobacco exposure. Self-reported number of cigarettes smoked per day explained only 13.9% of the variability in urine cotinine concentration. Birth weight declined as tobacco exposure increased; however, the relation was not linear. The sharpest declines in birth weight occurred at low levels of exposure. Furthermore, urine cotinine concentration did not explain more variability in birth weight than did number of cigarettes smoked. These findings should be considered by researchers studying the effects of smoking reduction on birth outcomes.

Use of filter paper for the collection and analysis of human whole blood specimens.

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.

Fluorescence intensity calibration for immunophenotyping by flow cytometry.

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.

Oral zidovudine during labor to prevent perinatal HIV transmission, Bangkok: tolerance and zidovudine concentration in cord blood. Bangkok Collaborative Perinatal HIV Transmission Study Group.

OBJECTIVES: To evaluate tolerance for the oral administration of zidovudine (ZDV) during labor and measure the resulting ZDV concentrations in umbilical cord blood. DESIGN: A cross-sectional study of women in a placebo-controlled trial of short-course ZDV (twice a day from 36 weeks' gestation until labor and every 3 h during labor) to prevent perinatal HIV transmission in Bangkok. METHODS: Umbilical cord blood was collected. Sixty control specimens and specimens from 372 women (182 in the ZDV group, 190 in the placebo group) were tested for ZDV by radioimmunoassay (lower detection limit < 1 ng/ml). RESULTS: All women in the ZDV group took one or more labor dose, 170 (93%) took their last dose within 3 h of delivery, and only five (3%) experienced nausea or vomiting, a proportion similar to the placebo group. The median concentration of ZDV in the cord blood in the ZDV group was 252 ng/ml (range, < 1-1133 ng/ml); 31 (17%) specimens were less than 130 ng/ml (0.5 microM), the concentration thought to be active against HIV in vitro. Median concentrations were 189 ng/ml in specimens from women taking one or two labor doses, 290 ng/ml in those taking three or four doses, and 293 ng/ml in those taking more than four doses (P < 0.01). The ZDV concentration was not associated with time since the last dose, body weight, or perinatal transmission. CONCLUSION: Oral intrapartum ZDV was feasible and well tolerated. Most ZDV concentrations in the cord blood after oral dosing during labor were at therapeutic concentrations but were lower than those reported after continuous intravenous administration. Although concentrations were not associated with perinatal transmission, these data do not exclude the possibility that intrapartum and neonatal chemoprophylaxis is effective.

Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays.

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 degrees C, or -20 degrees 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.

Current activities at the Centers for Disease Control and Prevention's National Diabetes Laboratory.

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.

Medium chain acyl-CoA dehydrogenase deficiency human genome epidemiology review.

Medium chain acyl-CoA dehydrogenase (MCAD) is a tetrameric flavoprotein essential for the beta-oxidation of medium chain fatty acids. MCAD deficiency (MCADD) is an inherited error of fatty acid metabolism. The gene for MCAD is located on chromosome one (1p31). One variant of the MCAD gene, G985A, a point mutation causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD protein, has been found in 90% of the alleles in MCADD patients identified retrospectively. There is a high frequency of MCADD among people of Northern European descent, which is believed to be due to a founder effect. MCADD is inherited in an autosomal recessive manner. Of patients clinically diagnosed with MCADD, 81% who have been identified retrospectively are homozygous for K304E, and 18% are compound heterozygotes for K304E. Clinical data on the probability of clinical disease indicates that MCADD patients are at risk for the following outcomes: hypoglycemia, vomiting, lethargy, encephalopathy, respiratory arrest, hepatomegaly, seizures, apnea, cardiac arrest, coma, and sudden and unexpected death. Long-term outcomes include developmental and behavioral disability, chronic muscle weakness, failure to thrive, cerebral palsy, and attention deficit disorder (ADD). Differences in clinical disease specific to allelic variants have not been documented. Factors that may increase risk for disease onset or modify disease severity are age when the first episode occurred, fasting, and presence of infection. Acute attacks must be treated immediately with appropriate intravenous doses of glucose. For those diagnosed, long-term management of the disease includes preventing stress caused by fasting and maintaining a high-carbohydrate, reduced-fat diet, and carnitine supplementation. Hospitalization costs attributable to morbidity and mortality from MCADD are unknown; MCADD is not a diagnosis in the International Classification of Disease, 10th Revision (ICD-10) codebook. Furthermore, the penetrance of the MCAD genotypes is unknown; there appears to be a substantial number of asymptomatic MCADD individuals and some uncertainty regarding which individuals will manifest symptoms and which individuals will remain asymptomatic. Several technologies are available to detect MCADD. Diagnostic technologies include DNA-based tests for K304E mutations using the polymerase chain reaction (PCR), and the detection of abnormal metabolites in urine. Screening technologies include tandem mass spectrometry (MS/MS), which detects abnormal metabolites mostly in blood. State programs are beginning to offer screening in newborns for MCADD using MS/MS. In addition, a private company currently offers voluntary supplemental newborn screening for MCADD to birthing centers.

Quality assurance and standardization: summary of the satellite meeting, Turku, Finland, 11-12 June 1999.

The Quality Assurance (QA) and Standardization satellite meeting addressed five major issues in newborn screening: (i) Pre- and post-analytical phases are important in the overall quality of screening, and quality assurance programs should include these aspects. (ii) It is possible to run a single screening program using laboratories on more than one site, as is done in Europe, California and Cuba. Special quality assurance procedures are necessary for success. (iii) It is possible to achieve improved analytical quality and international comparison of results by use of common reference materials such as the amino acid materials developed in Europe and the US. It will be important to use tested paper with defined performance characteristics. (iv) It is appropriate for newborn screening programs to be accredited, but it will also be important to develop criteria for pre- and post-analytic phases of screening. (v) A new QA program is being developed for Latin America. The Australasian QA program now includes amino acids and acylcarnitines in a form suitable for tandem mass spectrometry blind QA. The European Society for Paediatric Endocrinology has developed guidelines for CAH screening.

Terminology and nomenclature for standardization in quantitative fluorescence cytometry.

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.

Iodine nutrition in the United States. Trends and public health implications: iodine excretion data from National Health and Nutrition Examination Surveys I and III (1971-1974 and 1988-1994)

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.

Radioimmunoassay for monitoring zidovudine in dried blood spot specimens.

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 micrograms/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.

Stability of urine creatinine.

The measurement of urinary creatinine is important in many situations, but perhaps most important when measuring the urinary content of substances other than creatinine. Because urine flow changes unpredictably during the day, but total creatinine output is generally constant, many investigators normalize their results to creatinine content (i.e. mg chromium/mg creatinine). Because the widespread use of creatinine levels make the reliability of their measurement important, we decided to test this by studying the effects of storage time and temperature on urine specimens obtained from 10 healthy adults. Our results showed that only prolonged storage time at high temperatures (30 days, 55 degrees C) could cause significant decreases in urine creatinine levels. When stored for 2 days at 55 degrees C the decrease in urine creatinine levels was < 3%. We conclude that in all but extreme cases urine creatinine is virtually unaffected by storage time and temperature.