High Throughput Newborn Screening for Sickle Cell Disease - Application of Two-Tiered Testing with a qPCR-Based Primary screen.

BACKGROUND: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method. METHODS: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation. RESULTS: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences. CONCLUSION: In high-throughput NBS for SCD, qPCR is suitable to focus 2nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

Functional glucocorticoid receptor gene variants do not underlie the high variability of 17-hydroxyprogesterone screening values in healthy newborns.

OBJECTIVE: 17-Hydroxyprogesterone (17-OHP) screening for classical congenital adrenal hyperplasia (CAH) is part of many newborn screening programs worldwide. Cut-off values are relatively high, and screening sensitivity does not reach 100%. Recently, the glucocorticoid receptor (GR) N363S-variant has been linked to relatively low degree of virilization and comparatively lower 17-OHP serum concentrations in clinically diagnosed female CAH patients. We sought to determine whether functional GR gene variants, either increasing (N363S, BclI) or decreasing GR sensitivity (R23K), underlie the variable 17-OHP screening levels in healthy newborns. DESIGN: GR genotypes were compared with 17-OHP screening values in 1000 random samples from routine screening. 17-OHP was measured by conventional immunoassay (TRFIA) and a liquid chromatography-tandem mass spectrometry method (LC-MS/MS), which has been shown to increase screening specificity by steroid profiling and avoiding cross-reactions of the 17-OHP-antibody. RESULTS: There was no significant association of 17-OHP with GR genotypes, even after inclusion of gestational and postnatal age as covariates. However, among LC-MS/MS steroid measurements, we observed some unexpected trends, including lower 11-deoxycortisol concentrations in both 363S- and 23K-carriers. For carriers of the frequent BclI variant, linear regression analysis revealed a significant increase of 4-androstenedione levels with every mutant allele inherited. CONCLUSIONS: Functional GR variants do not underlie the variation of 17-OHP values observed in healthy individuals. However, whether and to which extent genetically determined differences in individual GR sensitivity influence 17-OHP screening levels in conditions of a pathological hypothalamus-pituitary-adrenal gland-axis stimulation and thus may explain false-negative screening results in those affected by CAH remains to be investigated.

EDTA in dried blood spots leads to false results in neonatal endocrinologic screening.

BACKGROUND: Blood samples for neonatal screening for inborn errors of metabolism are collected and shipped on standardized filter paper cards. Occasionally these samples are contaminated with EDTA, which is often used for anticoagulation. EDTA may interfere with newborn screening tests based on lanthanide fluorescence and thus lead to false-negative or false-positive results. METHODS: We used tandem mass spectrometry (MS/MS) to detect EDTA in dried blood spots by use of an extra experiment that was integrated into the standard MS/MS neonatal screening and did not require an additional sample spot, nor extra time or work. We analyzed the influence of different blood sampling procedures on lanthanide fluorescence tests for thyroid-stimulating hormone (TSH) and 17-hydroxyprogesterone (17-OHP). RESULTS: EDTA was increased in 138 of 190 000 newborn screening samples, 27 of which caused false- positive results in the immunoassay for 17-OHP. No false-negative TSH results were found. False-positive results in the 17-OHP test occurred when EDTA concentrations were >2.0 g/L; the TSH test, however, produced false negatives only when EDTA concentrations were >3.0 g/L. Using EDTA-containing devices the procedure of blood collection significantly influenced the concentration of the anticoagulant. CONCLUSION: Addition of EDTA quantification into standard MS/MS tests is a simple and useful method to avoid false-positive or false-negative neonatal screening results in lanthanide fluorescence-based tests.

Newborn screening for hepatorenal tyrosinemia: Tandem mass spectrometric quantification of succinylacetone.

BACKGROUND: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening. METHODS: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile-water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run. RESULTS: Of 61,344 samples, 99.6% had succinylacetone concentrations < or =5 micromol/L. With a cutoff of 10 micromol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 micromol/L, respectively, and the tyrosine concentrations were 54 and 129 micromol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 micromol/L, respectively. CONCLUSIONS: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.

A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands.

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocket A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.