Rinaldo, Piero

The expansion of the recommend uniform screening panel to include more than 50 primary and secondary target conditions has resulted in a substantial increase of false positive results. As an alternative to subjective manipulation of cutoff values and overutilization of molecular testing, here we describe the performance outcome of an algorithm for disorders of methionine, cobalamin, and propionate metabolism that includes: (1) first tier screening inclusive of the broadest available spectrum of markers measured by tandem mass spectrometry; (2) integration of all results into a score of likelihood of disease for each target condition calculated by post-analytical interpretive tools created byCollaborative Laboratory Integrated Reports (CLIR), a multivariate pattern recognition software; and (3) further evaluation of abnormal scores by a second tier test measuring homocysteine, methylmalonic acid, and methylcitric acid. This approach can consistently reduce false positive rates to a <0.01% level, which is the threshold of precision newborn screening. We postulate that broader adoption of this algorithm could lead to substantial savings in health care expenditures. More importantly, it could prevent the stress and anxiety experienced by many families when faced with an abnormal newborn screening result that is later resolved as a false positive outcome.

AbstractAnalyses of 19 amino acids, 38 acylcarnitines, and 3 creatine analogues (https://clir.mayo.edu) were implemented to test the hypothesis that succinic semialdehyde dehydrogenase deficiency (SSADHD) could be identified in dried bloodspots (DBS) using currently available newborn screening methodology. The study population included 17 post‐newborn SSADHD DBS (age range 0.8‐38 years; median, 8.2 years; 10 M; controls, 129‐353 age‐matched individuals, mixed gender) and 10 newborn SSADHD DBS (including first and second screens from 3 of 7 patients). Low (informative) markers in post‐newborn DBS included C2‐ and C4‐OH carnitines, ornithine, histidine and creatine, with no gender differences. For newborn DBS, informative markers included C2‐, C3‐, C4‐ and C4‐OH carnitines, creatine and ornithine. Of these, only creatine demonstrated a significant change with age, revealing an approximate 4‐fold decrease. We conclude that quantitation of short‐chain acylcarnitines, creatine, and ornithine provides a newborn DBS profile with potential as a first tier screening tool for early detection of SSADHD. This first tier evaluation can be readily verified using a previously described second tier liquid chromatography‐tandem mass spectrometry method for γ‐hydroxybutyric acid in the same DBS. More extensive evaluation of this first/second tier screening approach is needed in a larger population.

Enzyme-based newborn screening for Mucopolysaccharidosis type I (MPS I) has a high false-positive rate due to the prevalence of pseudodeficiency alleles, often resulting in unnecessary and costly follow up. The glycosaminoglycans (GAGs), dermatan sulfate (DS) and heparan sulfate (HS) are both substrates for α-l-iduronidase (IDUA). These GAGs are elevated in patients with MPS I and have been shown to be promising biomarkers for both primary and second-tier testing. Since February 2016, we have measured DS and HS in 1213 specimens submitted on infants at risk for MPS I based on newborn screening. Molecular correlation was available for 157 of the tested cases. Samples from infants with MPS I confirmed by IDUA molecular analysis all had significantly elevated levels of DS and HS compared to those with confirmed pseudodeficiency and/or heterozygosity. Analysis of our testing population and correlation with molecular results identified few discrepant outcomes and uncovered no evidence of false-negative cases. We have demonstrated that blood spot GAGs analysis accurately discriminates between patients with confirmed MPS I and false-positive cases due to pseudodeficiency or heterozygosity and increases the specificity of newborn screening for MPS I.

Abstract Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B12 deficiency. Methods: We added 1 nmol of the internal standard MMA-d3 to 500 μL of plasma or 100 μL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 °C for 15 min. For separation, we used a Supelcosil LC-18, 33 × 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d3, respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. Results: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 μmol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030x − 0.032 (Sy|x = 1.03 μmol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3.8–8.5% and 1.3–3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 μmol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 μmol/L), 96.0% (0.60 μmol/L), and 94.8% (2.02 μmol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100–150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. Conclusions: This method is well suited for large-scale MMA testing (≥100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.

Abstract Background: Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C6 to C16. Methods: We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay. Results: The assay was linear from 0.2 to 50 μmol/L for all six 3-OH-FAs. CVs were 5–15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in μmol/L were as follows: 3-OH-C6, 0.8 (0.3–2.2); 3-OH-C8, 0.4 (0.2–1.0); 3-OH-C10, 0.3 (0.2–0.6); 3-OH-C12, 0.3 (0.2–0.6); 3-OH-C14, 0.2 (0.0–0.4); and 3-OH-C16, 0.2 (0.0–0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C14 and 3-OH-C16 concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C14 to 3-OH-C16. Conclusion: Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.

Abstract Background: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. Methods: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 μL of either plasma or urine with homocystine-d8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d4 were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d4 was 1.5 min in a 2.5-min analysis. Results: Daily calibrations between 2.5 and 60 μmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 μmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x − 1.377 (r = 0.975; Sy|x =1.595 μmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; Sy|x =1.146 μmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 μmol/L. Mean recovery of tHcy was 94.2% (20 μmol/L) and 97.8% (50 μmol/L). Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.

AbstractBackground and aimsCell‐based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids.MethodsPorcine hepatocytes were isolated by a three‐step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106/mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine‐tryptophan‐ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N‐acetyl‐L‐cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage.ResultsIn this study, we observed that cold‐induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48‐ and 72‐hour time points (P < 0.05).ConclusionsThe beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid‐based bioartificial liver.

Abstract BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were &lt;7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.