Joanne V. Mei

Performance Characteristics of a New Less Sensitive HIV-1 Enzyme Immunoassay for Use in Estimating HIV Seroincidence

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.

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.

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.

Development and characterization of dried blood spot materials for the measurement of immunoreactive trypsinogen

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.

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

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.

Effect of specimen storage conditions on newborn dried blood spots used to assess Toxoplasma gondii immunoglobulin M (IgM)

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<0.0001; tyrosine and valine p<0.001). 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.

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 NSQAPs DBS materials allows harmonization of C5OH measurements by newborn screening laboratories worldwide.

A Pilot Study Using Residual Newborn Dried Blood Spots to Assess the Potential Role of Cytomegalovirus and Toxoplasma gondii in the Etiology of Congenital Hydrocephalus

BACKGROUND Congenital hydrocephalus is a condition characterized by accumulation of cerebrospinal fluid in the ventricles of the brain. Prenatal infections are risk factors for some birth defects. This pilot study investigated whether residual dried blood spots (DBS) could be used to assess infections as risk factors for birth defects by examining the associations between prenatal infection with Toxoplasma gondii (T. gondii) or cytomegalovirus (CMV) with congenital hydrocephalus. METHODS Case-infants with hydrocephalus (N=410) were identified among live-born infants using birth defects surveillance systems in California, North Carolina, and Texas. Control-infants without birth defects were randomly selected from the same geographic areas and time periods as case-infants (N=448). We tested residual DBS from case- and control-infants for T. gondii immunoglobulin M and CMV DNA. When possible, we calculated crude odds ratios (cORs) and confidence intervals (CIs). RESULTS Evidence for prenatal T. gondii infection was more common among case-infants (1.2%) than control-infants (0%; p=0.11), and evidence for prenatal CMV infection was higher among case-infants (1.5%) than control-infants (0.7%; cOR: 2.3; 95% CI: 0.48, 13.99). CONCLUSIONS Prenatal infections with T. gondii and CMV occurred more often among infants with congenital hydrocephalus than control-infants, although differences were not statistically significant. This pilot study highlighted some challenges in using DBS to examine associations between certain infections and birth defects, particularly related to reduced sensitivity and specimen storage conditions. Further study with increased numbers of specimens and higher quality specimens should be considered to understand better the contribution of these infections to the occurrence of congenital hydrocephalus.

Standardization and monitoring of laboratory performance and quality assurance by use of the less-sensitive HIV incidence assay: seven years of results.

BackgroundThe Performance Evaluation Program for HIV-1 incidence tests provided quality assurance services to laboratories conducting the serological testing algorithm for recent HIV seroconversion by use of a modified less-sensitive version of the Vironostika HIV-1 MicroElisa System assay. We report on the performance of the assay using proficiency testing and quality control materials tested from 2001 to 2008. MethodsTwo sets of 5 blinded serum panels using common calibration and quality control materials were tested. The mean, standard deviation, and coefficient of variation were calculated. Results were analyzed for misclassifications: false recent HIV infection errors (long-term infection classified as HIV infection less than 1 year), false long-term infection errors (HIV infection less than 1 year classified as long-term infection), and differences in standardized optical density means and variances over time. ResultsThe false recent error rate was 1.26% (n = 2219). The false long-term error rate was 0.25% (n = 1618). No significant trends were observed for misclassification rates by year, and no significant trend in the standardized optical density over 7 years was observed. ConclusionsLaboratories using the less-sensitive Vironostika HIV-1 assay produced consistent results by use of a common calibrator and quality control materials.