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Untargeted metabolomics profiling and hemoglobin normalization for archived newborn dried blood spots from a refrigerated biorepository.

ABSTRACT: Archived dried blood spots (DBS) following newborn screening are an attractive resource for interrogating early-life biology using untargeted metabolomics. Therefore, they have the potential to substantially aid etiological studies, particularly for rare and low-frequency childhood diseases and disorders. However, metabolite quantification in DBS is hindered by variation sources not present in serum and plasma samples such as the hematocrit effect and unknown initial blood volumes. Hemoglobin (Hb) is an appropriate correlate for hematocrit in experimentally-generated DBS punches. However, since many biorepositories worldwide archive DBS at 4-5 °C, there is a need to validate the utility of Hb for DBS archived under refrigeration. We evaluated two simple spectroscopic methods for measuring Hb in DBS stored at 4 +/- 2 °C for up to 21 years, obtained from the newborn screening program at the Karolinska University Hospital, Sweden. Spearman correlation analysis and Akaike Information Criterion model selection found that measurement of a Hb sodium lauryl sulfate complex at 540 nm better described nuisance variation than Hb measured at 404 nm, or using age of spot alone. This is the first study to profile metabolites and to propose a normalization factor for metabolite measurements from DBS archived for decades at 4 °C.

SUBMITTER: Yu M

PROVIDER: S-EPMC7899729 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Untargeted metabolomics profiling and hemoglobin normalization for archived newborn dried blood spots from a refrigerated biorepository.

Yu Miao M Dolios Georgia G Yong-Gonzalez Vladimir V Björkqvist Olle O Colicino Elena E Halfvarson Jonas J Petrick Lauren L

Journal of pharmaceutical and biomedical analysis 20200823

Archived dried blood spots (DBS) following newborn screening are an attractive resource for interrogating early-life biology using untargeted metabolomics. Therefore, they have the potential to substantially aid etiological studies, particularly for rare and low-frequency childhood diseases and disorders. However, metabolite quantification in DBS is hindered by variation sources not present in serum and plasma samples such as the hematocrit effect and unknown initial blood volumes. Hemoglobin (H ...[more]

PMID: 32896810

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Project description:Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited mitochondrial metabolic disease of fatty acid β-oxidation, especially in newborns. MCADD is clinically diagnosed using Newborn Bloodspot Screening (NBS) and genetic testing. Still, these methods have limitations, such as false negatives or positives in NBS and the variants of uncertain significance in genetic testing. Thus, complementary diagnostic approaches for MCADD are needed. Recently, untargeted metabolomics has been proposed as a diagnostic approach for inherited metabolic diseases (IMDs) due to its ability to detect a wide range of metabolic alterations. We performed an untargeted metabolic profiling of dried blood spots (DBS) from MCADD newborns (n = 14) and healthy controls (n = 14) to discover potential metabolic biomarkers/pathways associated with MCADD. Extracted metabolites from DBS samples were analyzed using UPLC-QToF-MS for untargeted metabolomics analyses. Multivariate and univariate analyses were used to analyze the metabolomics data, and pathway and biomarker analyses were also performed on the significantly identified endogenous metabolites. The MCADD newborns had 1034 significantly dysregulated metabolites compared to healthy newborns (moderated t-test, no correction, p-value ≤ 0.05, FC 1.5). A total of 23 endogenous metabolites were up-regulated, while 84 endogenous metabolites were down-regulated. Pathway analyses showed phenylalanine, tyrosine, and tryptophan biosynthesis as the most affected pathways. Potential metabolic biomarkers for MCADD were PGP (a21:0/PG/F1alpha) and glutathione, with an area under the curve (AUC) of 0.949 and 0.898, respectively. PGP (a21:0/PG/F1alpha) was the first oxidized lipid in the top 15 biomarker list affected by MCADD. Additionally, glutathione was chosen to indicate oxidative stress events that could happen during fatty acid oxidation defects. Our findings suggest that MCADD newborns may have oxidative stress events as signs of the disease. However, further validations of these biomarkers are needed in future studies to ensure their accuracy and reliability as complementary markers with established MCADD markers for clinical diagnosis.

Project description:IntroductionHeel pricks are performed on newborns for diagnostic screenings of various pre-symptomatic metabolic and genetic diseases. Excess blood is spotted on Guthrie cards and archived by many states in biobanks for follow-up diagnoses and public health research. However, storage environment may vary across biobanks and across time within biobanks. With increased applications of DNA extracted from spots for genetic studies, identifying factors associated with genotyping success is critical to maximize DNA quality for future studies.MethodWe evaluated 399 blood spots, which were part of a genome-wide association study of childhood leukemia risk in children with Down syndrome, archived at the Michigan Neonatal Biobank between 1992 and 2008. High quality DNA was defined as having post-quality control call rate ≥ 99.0% based on the Illumina GenomeStudio 2.0 GenCall algorithm after processing the samples on the Illumina Infinium Global Screening Array. Bivariate analyses and multivariable logistic regression models were applied to evaluate effects of storage environment and storage duration on DNA genotyping quality.ResultsBoth storage environment and duration were associated with sample genotyping call rates (p-values < 0.001). Sample call rates were associated with storage duration independent of storage environment (p-trend = 0.006 for DBS archived in an uncontrolled environment and p-trend = 0.002 in a controlled environment). However, 95% of the total sample had high genotyping quality with a call rate ≥ 95.0%, a standard threshold for acceptable sample quality in many genetic studies.ConclusionBlood spot DNA quality was lower in samples archived in uncontrolled storage environments and for samples archived for longer durations. Still, regardless of storage environment or duration, neonatal biobanks including the Michigan Neonatal Biobanks can provide access to large collections of spots with DNA quality acceptable for most genotyping studies.

Dataset Information

Untargeted metabolomics profiling and hemoglobin normalization for archived newborn dried blood spots from a refrigerated biorepository.

Publications

Untargeted metabolomics profiling and hemoglobin normalization for archived newborn dried blood spots from a refrigerated biorepository.

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