The gold-standard diagnostic test for peroxisomal disorders (PDs) is plasma concentration analysis of very long-chain fatty acids (VLCFAs). However, this method’s time-consuming nature and ...limitations in cases which present normal VLCFA levels necessitates alternative approaches. The analysis of C26:0-lysophosphatydylcholine (C26:0-LPC) in dried blood spot samples by tandem-mass spectrometry (MS/MS) has successfully been implemented in certain newborn screening programs to diagnose X-linked adrenoleukodystrophy (ALD). However, the diagnostic potential of very long-chain LPCs concentrations in plasma remains poorly understood. This study sought to evaluate the diagnostic performance of C26:0-LPC and other very long-chain LPCs, comparing them to VLCFA analysis in plasma. The study, which included 330 individuals affected by a peroxisomal β-oxidation deficiency and 407 control individuals, revealed that C26:0- and C24:0-LPC concentrations demonstrated the highest diagnostic accuracy (98.8% and 98.4%, respectively), outperforming VLCFA when C26:0/C22:0 and C24:0/C22:0 ratios were combined (98.1%). Combining C24:0- and C26:0-LPC gave the highest sensitivity (99.7%), with ALD females exhibiting notably higher sensitivity compared with the VLCFA ratio combination (98.7% vs. 93.5%, respectively). In contrast, C22:0-LPC exhibited suboptimal performance, primarily due to its low sensitivity (75%), but we identified a potential use to help distinguish between ALD and Zellweger spectrum disorders. In summary, MS/MS analysis of plasma C24:0- and C26:0-LPC concentrations represents a rapid and straightforward approach to diagnose PDs, demonstrating superior diagnostic accuracy, particularly in ALD females, compared with conventional VLCFA biomarkers. We strongly recommend integrating very-long chain LPC plasma analysis in the diagnostic evaluation of individuals suspected of having a PD.
The exposure to a fetal inflammatory response (FIR) is often followed by a postnatal systemic inflammation, increasing the risk of perinatal brain injury. We profiled the transcriptome and proteome ...in neonatal dried blood spots from extremely preterm newborns (< 28 weeks of gestation), affected and unaffected by FIR. The observed molecular changes confirm the activation of the innate immune system in FIR‐affected neonates and reveal an impairment of their adaptive immunity.
The fetal inflammatory response (FIR) increases the risk of perinatal brain injury, particularly in extremely low gestational age newborns (ELGANs, < 28 weeks of gestation). One of the mechanisms contributing to such a risk is a postnatal intermittent or sustained systemic inflammation (ISSI) following FIR. The link between prenatal and postnatal systemic inflammation is supported by the presence of well‐established inflammatory biomarkers in the umbilical cord and peripheral blood. However, the extent of molecular changes contributing to this association is unknown. Using RNA sequencing and mass spectrometry proteomics, we profiled the transcriptome and proteome of archived neonatal dried blood spot (DBS) specimens from 21 ELGANs. Comparing FIR‐affected and unaffected ELGANs, we identified 782 gene and 27 protein expression changes of 50% magnitude or more, and an experiment‐wide significance level below 5% false discovery rate. These expression changes confirm the robust postnatal activation of the innate immune system in FIR‐affected ELGANs and reveal for the first time an impairment of their adaptive immunity. In turn, the altered pathways provide clues about the molecular mechanisms triggering ISSI after FIR, and the onset of perinatal brain injury.
Databases
EGAS00001003635 (EGA); PXD011626 (PRIDE).
Alteration of vitamin B.sub.12 metabolism can be genetic or acquired, and can result in anemia, failure to thrive, developmental regression and even irreversible neurologic damage. Therefore, early ...diagnosis and intervention is critical. Most of the neonatal cases with acquired vitamin B.sub.12 deficiency have been detected by clinical symptoms and only few of them trough NBS programs. We aim to assess the usefulness of the second-tier test: methylmalonic acid (MMA), methylcitric acid (MCA) and homocysteine (Hcys) in our newborn screening program and explore the implications on the detection of cobalamin (vitamin B.sub.12) related disorders, both genetic and acquired conditions. A screening strategy using the usual primary markers followed by the analysis of MMA, MCA and Hcys as second tier-test in the first dried blood spot (DBS) was developed and evaluated. During the period 2015-2018 a total of 258,637 newborns were screened resulting in 130 newborns with acquired vitamin B.sub.12 deficiency (incidence 1:1989), 19 with genetic disorders (incidence 1:13,613) and 13 were false positive. No false negatives were notified. Concerning the second-tier test, the percentage of cases with MMA above the cut-off levels, both for genetic and acquired conditions was very similar (58% and 60%, respectively). Interestingly, the percentage of cases with increased levels of Hcys was higher in acquired conditions than in genetic disorders (87% and 47%, respectively). In contrast, MCA was high only in 5% of the acquired conditions versus in 53% of the genetic disorders, and it was always very high in all patients with propionic acidemia. When screening for methylmalonic acidemia and homocystinuria, differential diagnosis with acquired vitamin B.sub.12 deficiency should be done. The results of our strategy support the inclusion of this acquired condition in the NBS programs, as it is easily detectable and allows the adoption of corrective measures to avoid the consequences of its deficiency.
Acylcarnitine and amino acid analyses of dried blood spot (DBS) samples using tandem mass spectrometry in newborn screening (NBS) programmes can generate false positive (FP) results. Therefore, ...implementation of second-tier tests (2TTs) using DBS samples has become increasingly important to avoid FPs. The most widely used 2TT metabolites include methylmalonic acid, 3-hydroxypropionic acid, methylcitric acid, and homocysteine.
We simultaneously measured 46 underivatised metabolites, including organic acids, acylglycine and acylcarnitine isomers, homocysteine, and orotic acid, in DBS samples using tandem mass spectrometry. To validate this method, we analysed samples from 147 healthy newborns, 160 patients with genetic disorders diagnosed via NBS, 20 patients with acquired vitamin B12 deficiency, 10 newborns receiving antibiotic treatment, and nine external quality control samples.
The validation study revealed that 31 metabolites showed good analytical performance. Furthermore, this method detected key metabolites for all diseases associated with increased levels of the following acylcarnitines: C3, C4, C5, C4DC/C5OH, and C5DC. The sensitivity of this method to detect all diseases was 100 %, and the specificity was 74-99 %, except for glutaric aciduria type 1. This method can also be used to diagnose mitochondrial fatty acid β-oxidation disorders (FAODs) and urea cycle defects (UCDs).
We have described a 2TT panel of 31 metabolites in DBS samples based on an easy and rapid method without derivatisation. Its implementation allowed us to distinguish between different organic acidurias, some FAODs, and UCDs. This new strategy has increased the efficiency of our NBS programme by reducing FP and false negative results, second sample requests, and the time required for diagnosis.
Alteration of vitamin B
metabolism can be genetic or acquired, and can result in anemia, failure to thrive, developmental regression and even irreversible neurologic damage. Therefore, early ...diagnosis and intervention is critical. Most of the neonatal cases with acquired vitamin B
deficiency have been detected by clinical symptoms and only few of them trough NBS programs. We aim to assess the usefulness of the second-tier test: methylmalonic acid (MMA), methylcitric acid (MCA) and homocysteine (Hcys) in our newborn screening program and explore the implications on the detection of cobalamin (vitamin B
) related disorders, both genetic and acquired conditions.
A screening strategy using the usual primary markers followed by the analysis of MMA, MCA and Hcys as second tier-test in the first dried blood spot (DBS) was developed and evaluated.
During the period 2015-2018 a total of 258,637 newborns were screened resulting in 130 newborns with acquired vitamin B
deficiency (incidence 1:1989), 19 with genetic disorders (incidence 1:13,613) and 13 were false positive. No false negatives were notified. Concerning the second-tier test, the percentage of cases with MMA above the cut-off levels, both for genetic and acquired conditions was very similar (58% and 60%, respectively). Interestingly, the percentage of cases with increased levels of Hcys was higher in acquired conditions than in genetic disorders (87% and 47%, respectively). In contrast, MCA was high only in 5% of the acquired conditions versus in 53% of the genetic disorders, and it was always very high in all patients with propionic acidemia.
When screening for methylmalonic acidemia and homocystinuria, differential diagnosis with acquired vitamin B
deficiency should be done. The results of our strategy support the inclusion of this acquired condition in the NBS programs, as it is easily detectable and allows the adoption of corrective measures to avoid the consequences of its deficiency.
Newborn Screening for SCID: Experience in Spain (Catalonia) Argudo-Ramírez, Ana; Martín-Nalda, Andrea; González de Aledo-Castillo, Jose Manuel ...
International journal of neonatal screening,
07/2021, Letnik:
7, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Newborn screening (NBS) for severe combined immunodeficiency (SCID) started in Catalonia in January-2017, being the first Spanish and European region to universally include this testing. In Spain, a ...pilot study with 5000 samples was carried out in Seville in 2014; also, a research project with about 35,000 newborns will be carried out in 2021–2022 in the NBS laboratory of Eastern Andalusia. At present, the inclusion of SCID is being evaluated in Spain. The results obtained in the first three and a half years of experience in Catalonia are presented here. All babies born between January-2017 and June-2020 were screened through TREC-quantification in DBS with the Enlite Neonatal TREC-kit from PerkinElmer. A total of 222,857 newborns were screened, of which 48 tested positive. During the study period, three patients were diagnosed with SCID: an incidence of 1 in 74,187 newborns; 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns who also benefited from the NBS program. The results obtained provide further evidence of the benefits of early diagnosis and curative treatment to justify the inclusion of this disease in NBS programs. A national NBS program is needed, also to define the exact SCID incidence in Spain.
Liquid chromatography coupled with tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants has been widely adopted in clinical chemistry laboratories. However, UPLC is ...replacing classical LC techniques, providing higher resolution and speed. We developed and validated an UPLC–MS/MS method for the simultaneous measurement of cyclosporine A, everolimus, sirolimus and tacrolimus concentrations in human blood. Following extraction with a zinc sulfate solution and acetonitrile, the chromatographic separation was achieved using an Acquity® UPLC® BEH™ (2.1 × 30 mm id, 1.7 µm) reverse-phase C₁₈ column, with a water/methanol linear gradient containing 2 mM ammonium acetate with 0.1 % formic acid at a 0.5 mL min⁻¹ flow rate. All immunosuppressants were detected by ESI mass spectrometry in positive ion multiple reaction monitoring mode using mass-to-charge transitions of 1219.8 → 1202.6/1184.4, 975.5 → 908.3/891.6, 931.5 → 864.3/883.3, 821.4 → 768.2/719.9 for cyclosporine A, everolimus, sirolimus and tacrolimus, respectively. Coefficients of variation and relative bias were less than 5.8 and 9.7 % for cyclosporine A, 8.7 and 6.4 % for everolimus, 8.5 and 7.2 % for sirolimus and 6.7 and 4.7 % for tacrolimus. Limits of quantification were 15.4 µg L⁻¹ for cyclosporine A, 1.42 µg L⁻¹ for everolimus, 1.58 µg L⁻¹ for sirolimus and 0.65 µg L⁻¹ for tacrolimus. Mean recoveries were greater than 77.6 % for all immunosuppressants. Evaluation of the matrix effect showed ion suppression for all the immunosuppressants, except for cyclosporine A, which suffered ion enhancement. No carry-over was observed. The validated method appears to be well adapted for therapeutic drug monitoring of multiple immunosuppressants in daily clinical practice.
The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure ...of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p < 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p < 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p < 0.00001) compared to samples collected during TKI treatment: r2 = 0.76; p < 0.00001 for SQI and VAF and r2 = 0.75; p < 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.
Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas
EGFR Mutation Test v2 (cobas
EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced ...non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the
gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of
testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis.
A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naïve patients analyzed by the cobas
EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively.
The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r
=0.70) and did not correlate with cfDNA concentration (r
=0.17, P=0.28) or disease stage (stage III patients SQI =9.1±3.1 and stage IV patients SQI =11.5±4.8, P=0.41). We found differences in SQI values according to the type of
mutation (Ex19Del mutations, SQI =13.6; p.L858R, SQI =8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86
. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naïve advanced NSCLC patients.
The study indicates that cfDNA analysis in plasma for
testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.
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