Newborn Screening Quality Assurance Program Research Articles

Multi-Laboratory Evaluation of Prototype Dried Blood Spot Quality Control Materials for Creatine Kinase-MM Newborn Screening Assays

Pilot studies to detect newborns with Duchenne Muscular Dystrophy (DMD) by newborn bloodspot screening (NBS) have been conducted under the New York State Newborn Screening Program (NYS) and are currently in progress as part of the Early Check Program at Research Triangle Institute (RTI) International. The Newborn Screening Quality Assurance Program (NSQAP) at the U.S. Centers for Disease Control and Prevention (CDC) produced a set of seven prototype dried blood spot (DBS) reference materials spiked with varying levels of creatine kinase MM isoform (CK-MM). These DBS were evaluated over a 3-week period by CDC, NYS, and RTI, all using the same CK-MM isoform-specific fluoroimmunoassay. Results from each laboratory were highly correlated with the relative proportion of CK-MM added to each of the six spiked pools. Based on reference ranges established by NYS and RTI for their pilot studies, these contrived DBS collectively spanned the CK-MM ranges found in typical newborns and the elevated ranges associated with DMD. This set allows quality assessment over the wide range of fluctuating CK-MM levels in typical and DMD-affected newborns.

Comprehensive Evaluation of the NeoBase 2 Non-derivatized MSMS Assay and Exploration of Analytes With Significantly Different Concentrations Between Term and Preterm Neonates.

Despite the popularity of the NeoBase 2 Non-derivatized MSMS assay (PerkinElmer, Turku, Finland), there are no reports of its comprehensive evaluation, including the ability to distinguish transient tyrosinemia of the newborn (TTN) from tyrosinemia type 1 (TYR 1) using succinylacetone (SUAC). No newborn screening (NBS) cutoffs for preterm neonates in the Korean population have been suggested. We evaluated the NeoBase 2 assay and identified analytes requiring different cutoffs in preterm neonates. Residual NBS dried blood spot samples and proficiency testing (PT) materials of the Newborn Screening Quality Assurance Program and the Korean Association of External Quality Assessment Service were used. Precision, accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), linearity, recovery, carryover, and performance of SUAC were evaluated. Cutoffs were determined, and analytes requiring different cutoffs in preterm neonates were investigated. Mean CVs for within-run and between-day precision were within 15%. Accuracy analysis indicated high agreement with in-house derivatized assay results and results of other PT participants. All analytes demonstrated acceptable LOD, LLOQ, and linearity. Recoveries were acceptable, except for SUAC. Carryover was negligible. Cutoffs were established for all analytes; Tyr, adenosine, and C20:0-lysophosphatidylcholine required different cutoffs in preterm neonates. Differential diagnosis of TYR 1 and TTN was successful with simultaneous Tyr and SUAC measurement. The NeoBase 2 assay demonstrated satisfactory performance. The additional analytes provide a wider diagnostic coverage, and the simultaneous measurement of Tyr and SUAC is efficient in excluding TYR 1. The new cutoffs for preterm neonates may decrease false-positive rates, without compromising diagnostic sensitivity.

Arginine, as a Key Indicator for Real-Time Stability Monitoring of Quality Control in the Newborn Screening Test Using Dried Blood Spot

Dried blood spots (DBS) have advantages such as minimizing blood collection volume and the distress to neonate. DBS have been used for tandem mass spectrometry (MS/MS)-based newborn screening tests (NST) of amino acid (AA) and acylcarnitine. The Newborn Screening Quality Assurance Program (NSQAP) have been provided quality control (QC) materials for MS/MS, as DBS cards. The NSQAP is generally provided within 14 months of the shelf life and the recommended storage condition is at −10 °C to −30 °C. Previously, several accelerated degradation studies had been performed to determine the transportation stability and short-term stability of AAs and acylcarnitines in DBS. However, the experimental condition is markedly different to the storage condition. We performed long-term monitoring for the real-time stability of seven AAs and 14 acylcarnitines from three levels of 2012 NSQAP QC materials across a time period of 788 days. Arginine suddenly yielded a catastrophic degeneration pattern, which started around D300. When comparing this with previous accelerated degradation studies, methionine, tyrosine, citrulline, and acetylcarnitine did not show a remarkable measurand drift for the real-time stability, except for arginine. Our study showed that arginine would require intensive QC monitoring in routine practice, and should be used for the assessment of the stability in long-term storage of DBS samples for biobanking.

Harmonizing Newborn Screening Laboratory Proficiency Test Results Using the CDC NSQAP Reference Materials.

Newborn screening (NBS) laboratories cannot accurately compare mass spectrometry-derived results and cutoff values due to differences in testing methodologies. The objective of this study was to assess harmonization of laboratory proficiency test (PT) results using quality control (QC) data. Newborn Screening Quality Assurance Program (NSQAP) QC and PT data reported from 302 laboratories in 2019 were used to compare results among laboratories. QC materials were provided as dried blood spot cards which included a base pool and the base pool enriched with specific concentrations of metabolites in a linear range. QC data reported by laboratories were regressed on QC data reported by the Centers for Disease Control and Prevention (CDC), and laboratory’s regression parameters were used to harmonize their PT result. In general, harmonization tended to reduce overall variation in PT data across laboratories. The metabolites glutarylcarnitine (C5DC), tyrosine, and phenylalanine were displayed to highlight inter- and intra-method variability in NBS results. Several limitations were identified using retrospective data for harmonization, and future studies will address these limitations to further assess feasibility of using NSQAP QC data to harmonize PT data. Harmonizing NBS data using common QC materials appears promising to aid result comparison between laboratories.

Newborn Screening Quality Assurance Program for CFTR Mutation Detection and Gene Sequencing to Identify Cystic Fibrosis.

All newborn screening laboratories in the United States and many worldwide screen for cystic fibrosis. Most laboratories use a second-tier genotyping assay to identify a panel of mutations in the CF transmembrane regulator (CFTR) gene. Centers for Disease Control and Prevention’s Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing CFTR mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment. Recently, CFTR mutation detection has increased in complexity with expanded genotyping panels and gene sequencing. To accommodate the growing quality assurance needs, the repository samples were characterized with several multiplex genotyping methods, Sanger sequencing, and 3 next-generation sequencing assays using a high-throughput, low-concentration DNA extraction method. The samples performed well in all of the assays, providing newborn screening laboratories with a resource for complex CFTR mutation detection and next-generation sequencing as they transition to new methods.

The Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention: Thirty-five Year Experience Assuring Newborn Screening Laboratory Quality.

Newborn screening is the largest genetic testing effort in the United States and is considered one of the ten great public health achievements during the first 10 years of the 21st century. For over 35 years, the Newborn Screening Quality Assurance Program (NSQAP) at the US Centers for Disease Control and Prevention has helped NBS laboratories ensure that their testing does not delay diagnosis, minimizes false-positive reports, and sustains high-quality testing performance. It is a multi-component program that provides comprehensive quality assurance services for dried blood spot testing. The NSQAP, the Biochemical Mass Spectrometry Laboratory (BMSL), the Molecular Quality Improvement Program (MQIP) and the Newborn Screening Translation Research Initiative (NSTRI), aid screening laboratories achieve technical proficiency and maintain confidence in their performance while processing large volumes of specimens daily. The accuracy of screening tests could be the difference between life and death for many babies; in other instances, identifying newborns with a disorder means that they can be treated and thus avoid life-long disability or severe cognitive impairment. Thousands of newborns and their families have benefited from reliable and accurate testing that has been accomplished by a network of screening laboratories and the NSQAP, BMSL, MQIP and NSTRI.

Influence of Hematocrit and Total-Spot Volume on Performance Characteristics of Dried Blood Spots for Newborn Screening.

Dried blood spots (DBS) have been used in newborn screening (NBS) tests for over 50 years. The Newborn Screening Quality Assurance Program (NSQAP) at the Centers for Disease Control and Prevention (CDC) conducted studies to assess the individual impacts of hematocrit and total-spot volume on characteristics of DBS samples. Per-punch serum volumes decreased 27%, RBC volumes more than doubled, absorption times increased over 300%, and spot diameters decreased marginally between the hematocrits of 40% to 65%. Per-punch serum and RBC volumes decreased logarithmically with lowering total-spot volumes. Patient hematocrit is an uncontrollable variable and inevitably affects the resulting punch from a DBS sample. It may be possible, though, to identify samples that fall outside of an acceptable range by noting certain physical characteristics of the DBS.

Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening

BackgroundCDC'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. MethodsAdult 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. ResultsThe 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. ConclusionsResults 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.

Proficiency testing outcomes of 3-hydroxyisovalerylcarnitine measurements by tandem mass spectrometry in newborn screening

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.

Performance Properties of Filter Paper Devices for Whole Blood Collection

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. We observed overlap at one standard deviation for each analyte, with no more than 4-5% difference between the papers. The data demonstrated similarities of analyte recovery between the papers, indicating comparability of the devices for newborn screening and other applications.

Pilot proficiency testing study for second tier congenital adrenal hyperplasia newborn screening

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). 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. 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%. 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.

Haplotype analysis of the CFTR gene from the CDC Newborn Screening Quality Assurance Program's proficiency testing collection

Haplotype analysis of the CFTR gene from the CDC Newborn Screening Quality Assurance Program's proficiency testing collection

Potential loss of methionine following extended storage of newborn screening samples prepared for tandem mass spectrometry analysis

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 ( 2H 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 2H 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.

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 Prevention's Newborn Screening Quality Assurance Program provides dried blood spot (DBS) quality control (QC) and proficiency testing (PT) programs for numerous MS/MS analytes. We describe empirical differences between derivatization and non-derivatization techniques for selected AAs and ACs. Methods DBS materials were prepared at levels near, above and below mean domestic laboratory cut-offs, and distributed to program participants for MS/MS analysis. Laboratories reported quantitative and qualitative results. QC DBS materials were assayed in-house following established protocols. Result Minor differences (< 15%) between quantitative values resulting from butyl esters and free acid techniques were observed for the majority of the analytes. Mass spectrometric response from underivatized dicarboxylic acid acylcarnitines was less intense than their butyl esters. Conclusions The use of underivatized techniques may also result in the inability to differentiate isobaric acylcarnitines. Laboratories should establish their own protocols by focusing on the decisions that identify test results requiring additional follow-up testing versus those that do not.

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.