Barbara W. Adam

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.

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.

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.

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.

Congenital Hypothyroid Screening Programs and the Sensitive Thyrotropin Assay: Strategies for the Surveillance of Iodine Deficiency Disorders

Iodine deficiency disorders (IDD) are among the most important global public health problems with approximately one billion people at risk worldwide(1). Iodine deficiency is not restricted to developing countries and persists on the European Continent(1). The successful elimination of this nutritional deficiency, which may be associated with a wide range of neurologic, developmental, and intellectual impairments, requires an effective monitoring and surveillance program.

Preliminary Proficiency Testing Results for Succinylacetone in Dried Blood Spots for Newborn Screening for Tyrosinemia Type I

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

Stabilities of hemoglobins A and S in dried blood spots stored under controlled conditions

OBJECTIVE We aimed to measure separately the contributions of heat and humidity to changes in levels of hemoglobins A and S 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. Hemoglobin A and S levels of all samples in each complete set were measured in a single high performance liquid chromatography run. RESULTS During the one-month storage intervals, both hemoglobin species lost about 35% of initial levels in low-humidity storage and almost all of initial levels in high-humidity storage. CONCLUSIONS Minimizing both humidity and temperature in the transportation and storage environments of DBS samples is essential to maintaining the integrity of the hemoglobin tetramer molecules.