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In France, sickle cell disease newborn screening (SCD NBS) has been targeted to at-risk regions since 1984, but generalization to the whole population will be implemented from November 2024. Although tandem mass spectrometry (MS/MS) is already used for the NBS of several inherited metabolic diseases, its application for SCD NBS has not been widely adopted worldwide. The aim of this study was to evaluate a dedicated MS/MS kit (Targeted MS/MS Hemo, ZenTech, LaCAR Company, Liege, Belgium) for SCD NBS and to compare the results obtained with those from an NBS reference center using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and cation-exchange high-performance liquid chromatography (CE-HPLC, Variant NBS, Biorad Laboratories, Inc., Hercules, CA, USA) as confirmatory method. The MS/MS Hemo kit was used according to the manufacturer's instructions and performed on a Waters Xevo TQ-D (Waters Corporation, USA). The software provided by the manufacturer was used for the calculation and analysis of peptide signal ratios. Among the 1333 samples, the results of 1324 samples were consistent with the HPLC and/or MALDI-TOF results (1263 FA, 50 FAS, 7 FAC, 1 FAO-Arab, and 3 FS). All the discordant results (one FAS on MS/MS vs. FA in CE-HPLC, one FA on MS/MS vs. FAS in CE-HPLC, seven FS on MS/MS vs. FAS in CE-HPLC) were corrected after modifying the peptide signal ratios thresholds, allowing the MS/MS Hemo kit to achieve near-100% sensitivity and specificity for SCD NBS. In conclusion, the MS/MS Hemo kit appears to be an effective method for SCD NBS, particularly for laboratories already equipped with MS/MS technology. However, these results should be confirmed in a larger cohort including a greater number of positive samples for SCD.

Previous research has shown that a MALDI-MS technique can be used to screen for sickle cell disease (SCD), and that a system combining automated sample preparation, MALDI-MS analysis and classification software is a relevant approach for first-line, high-throughput SCD screening. In order to achieve a high-throughput "plug and play" approach while detecting "non-standard" profiles that might prompt the misclassification of a sample, we have incorporated various sets of alerts into the decision support software. These included "biological alert" indicators of a newborn's clinical status (e. g., detecting samples with no or low HbA), and "technical alerts" indicators for the most common non-standard profiles, i.e., those which might otherwise lead to sample misclassification. We evaluated these alerts by applying them to two datasets (produced by different laboratories). Despite the random generation of abnormal spectra by one-off technical faults or due to the nature and quality of the samples, the use of alerts fully secured the process of automatic sample classification. Firstly, cases of β-thalassemia were detected. Secondly, after a visual check on the tagged profiles and reanalysis of the corresponding biological samples, all the samples were correctly reclassified without prompting further alerts. All of the samples for which the results were not tagged were well classified (i.e., sensitivity and specificity = 1). The alerts were mainly designed for detecting false-negative classifications; all the FAS samples misclassified by the software as FA (a false negative) were marked with an alert. The implementation of alerts in the NeoScreening ® Laboratory Information Management System's decision support software opens up perspectives for the safe, reliable, automated classification of samples, with a visual check solely on abnormal results or samples. It should now be possible to evaluate the combination of the NeoSickle ® analytical solution and the NeoScreening ® Laboratory Information Management System in a real-life, prospective study of first-line SCD screening., Competing Interests: Conflicts of InterestThe authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2019 by the authors.)

The reference methods used for sickle cell disease (SCD) screening usually include two analytical steps: a first tier for differentiating haemoglobin S (HbS) heterozygotes, HbS homozygotes and β-thalassemia from other samples, and a confirmatory second tier. Here, we evaluated a first-tier approach based on a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform with automated sample processing, a laboratory information management system and NeoSickle ® software for automatic data interpretation. A total of 6701 samples (with high proportions of phenotypes homozygous (FS) or heterozygous (FAS) for the inherited genes for sickle haemoglobin and samples from premature newborns) were screened. The NeoSickle ® software correctly classified 98.8% of the samples. This specific blood sample collection was enriched in qualified difficult samples (premature newborns, FAS samples, late and very late samples, etc.). In this study, the sensitivity of FS sample detection was found to be 100% on the Lille MS facility and 99% on the Dijon MS facility, and the specificity of FS sample detection was found to be 100% on both MS facilities. The MALDI-MS platform appears to be a robust solution for first-tier use to detect the HbS variant: it is reproducible and sensitive, it has the power to analyze 600-1000 samples per day and it can reduce the unit cost of testing thanks to maximal automation, minimal intervention by the medical team and good overall practicability. The MALDI-MS approach meets today's criteria for the large-scale, cost-effective screening of newborns, children and adults., Competing Interests: Conflicts of InterestPatrick Ducoroy was an employee of the University of Burgundy at the time of the research described here. He then founded the company Biomaneo, and currently serves as its CEO., (© 2019 by the authors.)

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes. Recently, we diagnosed T2 deficiency in two patients (GK45 and GK47) by the absence of potassium ion-activated acetoacetyl-CoA thiolase activity, whereas these patients were previously misinterpreted as normal by a coupled assay with tiglyl-CoA as a substrate. This method has been widely used for the enzymatic diagnosis of the T2 deficiency in the United States and Europe. We hypothesized that some residual T2 activity showed normal results in the assay. To prove this hypothesis, we analyzed these two patients together with three typical T2-deficient patients (GK46, GK49, and GK50) at the DNA level. Expression analysis of mutant cDNAs clearly showed that GK45 and GK47 had "mild" mutations (A132G, D339-V340insD) that retained some residual T2 activity, at least one of two mutant alleles, whereas the other three patients had null mutations (c.52-53insC, G152A, H397D, and IVS8+1g>t) in either allele. These results raise the possibility that T2-deficient patients with mild mutations have been misinterpreted as normal by the coupled assay with tiglyl-CoA.