Jean M. Lacey

Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD).

BACKGROUND Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. METHODS Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. RESULTS Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. CONCLUSIONS The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

Comparison of multiple steroid concentrations in serum and dried blood spots throughout the day of patients with congenital adrenal hyperplasia

Background/Aim: Periodic measurement of plasma concentrations of cortisol precursors on a clinic visit may be of limited value in patients with congenital adrenal hyperplasia because it does not reflect a patient’s circadian patterns of adrenal steroid secretion. Steroid profiling in dried blood spots (DBS) may allow for more frequent and sensitive monitoring. Methods: We compared the agreement between 17α-hydroxyprogesterone (17-OHP) and androstenedione (D4A) levels determined from DBS samples and concurrently collected serum samples. Blood was drawn from 9 congenital adrenal hyperplasia patients every 4 h over a 24-hour period. Serum and DBS steroid levels were measured by liquid chromatography tandem mass spectrometry. Results: DBS determinations of 17-OHP overestimated corresponding serum levels (mean difference 1.67 ng/ml), and underestimated D4A serum levels (mean difference 0.84 ng/ml). However, the DBS assay yielded excellent agreement (97%) with serum 17-OHP, but did considerably poorer for D4A (31%). Conclusions: Our results indicate an excellent agreement between DBS and serum 17-OHP measurements to identify the peaks and troughs associated with an individual’s circadian pattern. Larger-scale studies are required to evaluate the utility of DBS for home monitoring and to determine if more frequent monitoring leads to improved clinical outcomes.

Precision newborn screening for lysosomal disorders

PurposeThe implementation of newborn screening for lysosomal disorders has uncovered overall poor specificity, psychosocial harm experienced by caregivers, and costly follow-up testing of false-positive cases. We report an informatics solution proven to minimize these issues.MethodsThe Kentucky Department for Public Health outsourced testing for mucopolysaccharidosis type I (MPS I) and Pompe disease, conditions recently added to the recommended uniform screening panel, plus Krabbe disease, which was added by legislative mandate. A total of 55,161 specimens were collected from infants born over 1 year starting from February 2016. Testing by tandem mass spectrometry was integrated with multivariate pattern recognition software (Collaborative Laboratory Integrated Reports), which is freely available to newborn screening programs for selection of cases for which a biochemical second-tier test is needed.ResultsOf five presumptive positive cases, one was affected with infantile Krabbe disease, two with Pompe disease, and one with MPS I. The remaining case was a heterozygote for the latter condition. The false-positive rate was 0.0018% and the positive predictive value was 80%.ConclusionPostanalytical interpretive tools can drastically reduce false-positive outcomes, with preliminary evidence of no greater risk of false-negative events, still to be verified by long-term surveillance.

Detection of Hypo‐N‐Glycosylation Using Mass Spectrometry of Transferrin

Many congenital disorders of glycosylation (CDG) can be diagnosed by observing the extent of glycosylation of the abundant serum glycoprotein transferrin (Trf). Trf is an N‐glycosylated protein with two asparagine glycation sites. CDG types I are those genetic defects which occur prior to transfer of the complex oligosaccharide to the acceptor asparagine in the cotranslated polypeptide chain. CDG Ia constitutes by far the most frequent form of CDG and is the result of mutations in the phosphomannomutase gene. CDG Ia and the Ib subtype (Phosphomannoisomerase deficiency) result in low cellular mannose‐1‐phosphate levels, a required precursor for oligosaccharide assembly in the endoplasmic reticulum. The deficiency in oligosaccharides with branched mannose structures is thereafter expressed by the appearance of glycoproteins with unoccupied N‐glycosylation sites (hypoglycosylation). Currently, there have been at least 11 Type I defects, type Ia being by far the most frequently occurring. Most, if not all type I defects result in unoccupied N‐glycation sites. Hypoglycosylated Trf, also known as carbohydrate‐deficient Trf (CDT), can be detected using mass spectrometry (MS) to measure the masses of the serum Trf. The methods for sample preparation using affinity chromatography and MS analysis are described in this unit. Curr. Protoc. Hum. Genet. 54:17.4.1‐17.4.9

Potential of inner cell mass outgrowth and amino acid turnover as markers of quality in the in vitro fertilization laboratory

OBJECTIVE To compare sensitivity of inner cell mass (ICM) outgrowth assay and analysis of culture media amino acid turnover with the sensitivity of the human sperm motility assay (HSMA) and murine embryo assay (MEA) for detection of formaldehyde toxicity. DESIGN Prospective in vitro study. SETTING University hospital-based infertility center. ANIMAL(S) Murine embryos. INTERVENTION(S) The HSMA, MEA, and ICM outgrowth assays were performed with media containing 0-64-μM concentrations of formaldehyde. These assays were compared with dynamics of amino acid turnover in culture media. MAIN OUTCOME MEASURE(S) The lowest concentration of formaldehyde in culture media detected by each quality control assay. RESULT(S) Sperm forward progression, but not motility, detected formaldehyde at a concentration of 32 μM. Sperm motility index identified formaldehyde toxicity at 64 μM, whereas blastocyst rates in the MEA were affected at 32 μM formaldehyde. Evaluation of ICM using outgrowth and grade detected 16 μM formaldehyde. Leucine turnover in culture media detected 64 μM formaldehyde in the amino acid assay. CONCLUSION(S) Inner cell mass outgrowth is a more sensitive bioassay than MEA and HSMA for the detection of formaldehyde in culture media. Amino acid metabolism may also provide a sensitive quality control measure for detection of formaldehyde.