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.

A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry.

Early diagnosis of severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD) improves health outcomes by providing a specific treatment before the onset of symptoms. A high-throughput nucleic acid-based method in newborn screening (NBS) has been shown to be fast and cost-effective in the early detection of these diseases. Screening for SCD has been included in Germany's NBS Program since Fall 2021 and typically requires high-throughput NBS laboratories to adopt analytical platforms that are demanding in terms of instrumentation and personnel. Thus, we developed a combined approach applying a multiplexed quantitative real-time PCR (qPCR) assay for simultaneous SCID, SMA, and 1st-tier SCD screening, followed by a tandem mass spectrometry (MS/MS) assay for 2nd-tier SCD screening. DNA is extracted from a 3.2-mm dried blood spot from which we simultaneously quantify T-cell receptor excision circles for SCID screening, identify the homozygous SMN1 exon 7 deletion for SMA screening, and determine the integrity of the DNA extraction through the quantification of a housekeeping gene. In our two-tier SCD screening strategy, our multiplex qPCR identifies samples carrying the HBB: c.20A>T allele that is coding for sickle cell hemoglobin (HbS). Subsequently, the 2nd tier MS/MS assay is used to distinguish heterozygous HbS/A carriers from samples of patients with homozygous or compound heterozygous SCD. Between July 2021 and March 2022, 96,015 samples were screened by applying the newly implemented assay. The screening revealed two positive SCID cases, while 14 newborns with SMA were detected. Concurrently, the qPCR assay registered HbS in 431 samples which were submitted to 2nd-tier SCD screening, resulting in 17 HbS/S, five HbS/C, and two HbS/ß thalassemia patients. The results of our quadruplex qPCR assay demonstrate a cost-effective and fast approach for a combined screening of three diseases that benefit from nucleic-acid based methods in high-throughput NBS laboratories.

Identification of potential interferents of methylmalonic acid: A previously unrecognized pitfall in clinical diagnostics and newborn screening.

OBJECTIVES: Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. METHODS: Techniques included quadrupole time-of-flight high-resolution mass spectrometry (QTOF HR-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). RESULTS: The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-hydroxyisovalerate, 3-hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. CONCLUSIONS: The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B12 deficiency, ketosis and lactic acidosis.

Closing the gap - inclusion of ultrashort-chain perfluoroalkyl carboxylic acids in the total oxidizable precursor (TOP) assay protocol.

An improved protocol of the total oxidizable precursor (TOP) assay was developed for precursors to C2-C14 perfluoroalkyl carboxylic acids (PFCAs) and C4-C8 and C10 perfluoroalkyl sulfonic acids (PFSAs). The proposed protocol was tested and validated for contaminated soil samples. The perfluoroalkyl acids (PFAAs) present in the soil extract solutions after oxidation with persulfate were separated from the inorganic salts by vacuum-assisted drying of the digestion solution followed by solid-liquid extraction of the PFAAs with acetonitrile from the dry residue. Ion chromatography (for C2-C4 PFCAs) and reversed phase liquid chromatography (for all other PFASs), both coupled to tandem mass spectrometry, were used for quantification. High procedural recoveries of PFAAs between 68% and 123% with RSDs between 0.2% and 25% (n = 3) were achieved. The method was validated using selected polyfluoroalkyl phosphoric acid esters (PAPs) and bis-[2-(N-ethyl perfluorooctane-1-sulfonamido)ethyl] phosphoric acid ester (diSAmPAP) as model precursors in pure solutions and in the presence of soil matrix. The oxidation led to characteristic and reproducible PFCA patterns (in the case of PAPs) or PFOA (in the case of diSAmPAP) with total reaction yields between 92 ± 4% and 123 ± 13% (n = 3). The impact of the soil matrix on transformation yields was negligible. In a soil core from a PFAS-polluted agricultural site, precursors were concentrated in the upper 40 cm with long-chain precursors being prevalent. After oxidative digestion, the total molar PFAA-concentrations increased by factors of 1.6 to 5.0. More than 40 cm below ground precursors of TFAA, PFPrA and PFBA accounted for ∼50% of the reaction products, underlining the importance of their inclusion in mass balances based on the TOP assay.

Robust trace analysis of polar (C2-C8) perfluorinated carboxylic acids by liquid chromatography-tandem mass spectrometry: method development and application to surface water, groundwater and drinking water.

A simple and robust analytical method for the determination of perfluorinated carboxylic acids (PFCAs) with C2 to C8 chains, based on solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed, validated and applied to tap water, groundwater and surface water. Two stationary phases for LC (Obelisc N and Kinetex C18) and two materials with weak anion-exchange properties for SPE (Strata X-AW and Oasis WAX) were evaluated. Robust separation and retention was achieved with the reversed phase column and an acidic eluent. Quantitative extraction recoveries were generally achieved for PFCAs with C > 3, but extraction efficiencies were different for the two shortest chained analytes: 36 to 114% of perfluoropropanoate (PFPrA) and 14 to 99% of trifluoroacetate (TFA) were recovered with Strata X-AW, while 93 to 103% of PFPrA and 40 to 103% of TFA were recovered with Oasis WAX. The sample pH was identified as a key parameter in the extraction process. One-step elution-filtration was introduced in the workflow, in order to remove sorbent particles and minimise sample preparation steps. Validation resulted in limits of quantification for all PFCAs between 0.6 and 26 ng/L. Precision was between 0.7 and 15% and mean recoveries ranged from 83 to 107%. In groundwater samples from sites impacted by per- and polyfluoroalkyl substances (PFASs), PFCA concentrations ranged from 0.056 to 2.2 µg/L. TFA and perfluorooctanoate were the predominant analytes. TFA, however, revealed a more ubiquitous occurrence and was found in concentrations between 0.045 and 17 µg/L in drinking water, groundwater and surface water, which were not impacted by PFASs.

Small, mobile, persistent: Trifluoroacetate in the water cycle - Overlooked sources, pathways, and consequences for drinking water supply.

Elevated concentrations of trifluoroacetate (TFA) of more than 100 µg/L in a major German river led to the occurrence of more than 20 µg/L TFA in bank filtration based tap waters. Several spatially resolved monitoring programs were conducted and discharges from an industrial company were identified as the point source of TFA contamination. Treatment options for TFA removal were investigated at full-scale waterworks and in laboratory batch tests. Commonly applied techniques like ozonation or granulated activated carbon filtration are inappropriate for TFA removal, whereas TFA was partly removed by ion exchange and completely retained by reverse osmosis. Further investigations identified wastewater treatment plants (WWTPs) as additional TFA dischargers into the aquatic environment. TFA was neither removed by biological wastewater treatment, nor by a retention soil filter used for the treatment of combined sewer overflows. WWTP influents can even bear a TFA formation potential, when appropriate CF3-containing precursors are present. Biological degradation and ozonation batch experiments with chemicals of different classes (flurtamone, fluopyram, tembotrione, flufenacet, fluoxetine, sitagliptine and 4:2 fluorotelomer sulfonate) proved that there are yet overlooked sources and pathways of TFA, which need to be addressed in the future.