Utility of measuring very long-chain fatty-acyl carnitines in dried blood spots for newborn screening of X-linked Adrenoleukodystrophy

Abstract 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.

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Newborn Screening for Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase and Mitochondrial Trifunctional Protein Deficiencies Using Acylcarnitines Measurement in Dried Blood Spots—A Systematic Review of Test Accuracy

Frontiers in Pediatrics ◽

10.3389/fped.2021.606194 ◽

2021 ◽

Vol 9 ◽

Author(s):

Chris Stinton ◽

Hannah Fraser ◽

Julia Geppert ◽

Rebecca Johnson ◽

Martin Connock ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Dried Blood Spots ◽

Test Accuracy ◽

Tandem Mass ◽

Blood Spots ◽

Hydroxyacyl Coa Dehydrogenase ◽

Sensitivity Specificity ◽

Long Chain

Background: Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies are rare autosomal recessive fatty acid β-oxidation disorders. Their clinical presentations are variable, and premature death is common. They are included in newborn blood spot screening programs in many countries around the world. The current process of screening, through the measurement of acylcarnitines (a metabolic by-product) in dried blood spots with tandem mass spectrometry, is subject to uncertainty regarding test accuracy.Methods: We conducted a systematic review of literature published up to 19th June 2018. We included studies that investigated newborn screening for LCHAD or MTP deficiencies by tandem mass spectrometry of acylcarnitines in dried blood spots. The reference standards were urine organic acids, blood acylcarnitine profiles, enzyme analysis in cultured fibroblasts or lymphocytes, mutation analysis, or at least 10-year follow-up. The outcomes of interest were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Assessment of titles, abstracts, and full-text papers and quality appraisal were carried out independently by two reviewers. One reviewer extracted study data. This was checked by a second reviewer.Results: Ten studies provided data on test accuracy. LCHAD or MTP deficiencies were identified in 23 babies. No cases of LCHAD/MTP deficiencies were identified in four studies. PPV ranged from 0% (zero true positives and 28 false positives from 276,565 babies screened) to 100% (13 true positives and zero false positives from 2,037,824 babies screened). Sensitivity, specificity, and NPV could not be calculated as there was no systematic follow-up of babies who screened negative.Conclusions: Test accuracy estimates of screening for LCHAD and MTP deficiencies with tandem mass spectrometry measurement of acylcarnitines in dried blood were variable in terms of PPVs. Screening methods (including markers and thresholds) varied between studies, and sensitivity, specificity, and NPVs are unknown.

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Newborn screening for Pompe disease with dried blood spots in a Japanese region: a pilot study

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2013.12.183 ◽

2014 ◽

Vol 111 (2) ◽

pp. S79

Author(s):

Ken Momosaki ◽

Shirou Matsumoto ◽

Kimitoshi Nakamura ◽

Hiroshi Mitsubuchi ◽

Toshika Okumiya ◽

...

Keyword(s):

Pilot Study ◽

Newborn Screening ◽

Pompe Disease ◽

Dried Blood Spots ◽

Blood Spots

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Development of a newborn screening tool for mucopolysaccharidosis type I based on bivariate normal limits: Using glycosaminoglycan and alpha‐L‐iduronidase determinations on dried blood spots to predict symptoms

JIMD Reports ◽

10.1002/jmd2.12093 ◽

2020 ◽

Vol 52 (1) ◽

pp. 35-42 ◽

Cited By ~ 3

Author(s):

Thomas J. Langan ◽

Kabir Jalal ◽

Amy L. Barczykowski ◽

Randy L. Carter ◽

Molly Stapleton ◽

...

Keyword(s):

Newborn Screening ◽

Screening Tool ◽

Dried Blood Spots ◽

Type I ◽

Mucopolysaccharidosis Type ◽

Mucopolysaccharidosis Type I ◽

Bivariate Normal ◽

Blood Spots ◽

Normal Limits

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Incorporating the measurement of EDTA in dried blood spots (DBS) by tandem mass spectrometry in routine newborn screening

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2014.07.058 ◽

2014 ◽

Vol 47 (15) ◽

pp. 144

Author(s):

Lawrence J. Fisher ◽

Osama Y. Al-Dirbashi ◽

Svetlana Ogrel ◽

Nathan McIntosh ◽

Michael T. Geraghty ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Newborn Screening ◽

Dried Blood Spots ◽

Tandem Mass ◽

Blood Spots

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Toward newborn screening of metachromatic leukodystrophy: results from analysis of over 27,000 newborn dried blood spots

Genetics in Medicine ◽

10.1038/s41436-020-01017-5 ◽

2020 ◽

Author(s):

Xinying Hong ◽

Jessica Daiker ◽

Martin Sadilek ◽

Nicole Ruiz-Schultz ◽

Arun Babu Kumar ◽

...

Keyword(s):

Newborn Screening ◽

Metachromatic Leukodystrophy ◽

Dried Blood Spots ◽

Blood Spots

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Contamination of dried blood spots – an underestimated risk in newborn screening

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-0270 ◽

2018 ◽

Vol 56 (2) ◽

pp. 278-284 ◽

Cited By ~ 3

Author(s):

Theresa Winter ◽

Anja Lange ◽

Anke Hannemann ◽

Matthias Nauck ◽

Cornelia Müller

Keyword(s):

Newborn Screening ◽

Infant Formula ◽

False Positive ◽

Dried Blood Spots ◽

Substantial Effect ◽

Blood Collection ◽

Inborn Errors ◽

Blood Spots ◽

Filter Papers ◽

The Impact

Abstract Background: Newborn screening (NBS) is an established screening procedure in many countries worldwide, aiming at the early detection of inborn errors of metabolism. For decades, dried blood spots have been the standard specimen for NBS. The procedure of blood collection is well described and standardized and includes many critical pre-analytical steps. We examined the impact of contamination of some anticipated common substances on NBS results obtained from dry spot samples. This possible pre-analytical source of uncertainty has been poorly examined in the past. Methods: Capillary blood was obtained from 15 adult volunteers and applied to 10 screening filter papers per volunteer. Nine filter papers were contaminated without visible trace. The contaminants were baby diaper rash cream, baby wet wipes, disinfectant, liquid infant formula, liquid infant formula hypoallergenic (HA), ultrasonic gel, breast milk, feces, and urine. The differences between control and contaminated samples were evaluated for 45 NBS quantities. We estimated if the contaminations might lead to false-positive NBS results. Results: Eight of nine investigated contaminants significantly altered NBS analyte concentrations and potentially caused false-positive screening outcomes. A contamination with feces was most influential, affecting 24 of 45 tested analytes followed by liquid infant formula (HA) and urine, affecting 19 and 13 of 45 analytes, respectively. Conclusions: A contamination of filter paper samples can have a substantial effect on the NBS results. Our results underline the importance of good pre-analytical training to make the staff aware of the threat and ensure reliable screening results.

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