Blood Spot Punch Research Articles

Epigenomic newborn screening for conditions with intellectual disability and autistic features in Australian newborns.

This study describes a protocol to assess a novel workflow called Epi-Genomic Newborn Screening (EpiGNs) on 100,000 infants from the state of Victoria, Australia. The workflow uses a first-tier screening approach called methylation-specific quantitative melt analysis (MS-QMA), followed by second and third tier testing including targeted methylation and copy number variation analyzes with droplet digital PCR, EpiTYPER system and low-coverage whole genome sequencing. EpiGNs utilizes only two 3.2mm newborn blood spot punches to screen for genetic conditions, including fragile X syndrome, Prader-Willi syndrome, Angelman syndrome, Dup15q syndrome and sex chromosome aneuploidies. The program aims to: identify clinically actionable methylation screening thresholds for the first-tier screen and estimate prevalence for the conditions screened.

B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device

Abstract Background Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample. Methods According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively. Results The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² > 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² > 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges. Conclusion The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.

Tandem Mass Spectrometry Enzyme Assays for Multiplex Detection of 10-Mucopolysaccharidoses in Dried Blood Spots and Fibroblasts.

The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders, as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultrarare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.

A highly multiplexed biochemical assay for analytes in dried blood spots: application to newborn screening and diagnosis of lysosomal storage disorders and other inborn errors of metabolism

PurposeTo develop a multiplexed assay for the newborn screening of lysosomal storage disorders and additional inborn errors in a flexible, comprehensive, and affordable manner to keep up with the expansion of the newborn screening panel. MethodsUltraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was chosen as the detection platform for its superiority compared to traditional flow-injection MS/MS. ResultsA high-throughput, 18-plex UPLC-MS/MS assay was developed for screening purposes with a sample turnaround time of 2.7minutes. The assay was consolidated such that only four dried blood spot punches were required, and it displayed good precision and reproducibility. ConclusionWe report a highly multiplexed UPLC-MS/MS assay that is appropriate for the newborn screening of 15 lysosomal storage diseases and 3 additional inborn errors. It can be further expanded to include additional conditions for which presymptomatic diagnosis may facilitate optimum treatment outcome.

New Advances in Amino Acid Profiling in Biological Samples by Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI

To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan. More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype. Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency. The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.

Demonstration of a digital microfluidic platform for the high throughput analysis of 12 discrete fluorimetric enzyme assays using a single newborn dried blood spot punch

A biomarker is an indicator or molecular signature that is observed in normal bodily biological processes or disease conditions. Biomarkers exist in various forms, such as proteins, peptides, antibodies, cells, metabolites, hormones, lipids, and enzymes. Developing sensitive methods for the detection of diseases at an earlier stage via these structures is essential in terms of better treatment options. Therefore, researchers have recently become interested in designing nanosensors by integrating nano-scale materials with optical, mechanical, electrical, and magnetic sensors. The use of nanostructures, such as nanotubes, nanofibers, quantum dots, and nanowires, contributes to the development of cheaper, faster, and smaller sensors, which increase sensitivity as well as enhance the detection limit and response time. This chapter examines the contributions of nanotechnology in detecting and determining biomarkers and the significance of this process in the diagnosis and treatment of diseases. Moreover, the methods of biomarker detection are analyzed via different studies as presented in the literature.

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

Aims: Archived newborn bloodspots are valuable sample collections for genetic and epigenetic disease research. However, they have often been stored for long periods of time, under less than ideal circumstances, and nucleic acid yields can be low, particularly when samples become limiting. We wished to determine whether the quantity and quality of genomic DNA (gDNA) isolated from a single, surgical bloodspot punch (2 mm dia.) was adequate for accurate and reliable DNA methylome profiling on the Illumina HumanMethylation450 array.Methods: A total of 25–750 ng of archived bloodspot or Jurkat cell gDNA were bisulfite converted and analyzed on the array without any additional DNA amplification steps. Methylation profiles were assessed for call rate, call confidence (detection p-value), and reproducibility.Results: Using 25 ng gDNA from either Jurkat cells or dried bloodspots, array-wide call rates (∼99.9%) and detection p-values (99.9% with p < 5 × 10−6) were excellent. There was good agreement between methylation profiles generated from 25 ng gDNA and those generated from 750 ng (ρ > 0.98), although a fraction of CpG sites (2–8% depending on experiment) exhibited quantitative differences. Genome-wide methylation levels were strikingly reproducible from 25 ng DNA in both replicate and interindividual samples (ρ > 0.98).Conclusions: Twenty-five nanograms of gDNA, isolated from a single, surgical punch (2 mm dia.) of an archived newborn bloodspot, generate a genome-wide methylation profile on the Illumina HumanMethylation450 array that is robust, reproducible, and suitable for differential methylation studies.

Successful newborn screening for Gaucher disease using fluorometric assay in China.

Gaucher disease (GD) is an inherited metabolic disorder that involves accumulation of glycolipid glucocerebroside in monocyte–macrophage cells, which can result in multiple organ damage. Enzyme replacement and substrate reduction therapies have improved the potential for early diagnosis and treatment. Determining the true incidence of this rare disease is critical for relevant policy establishment. Newborn screening allows for early diagnosis and an comparatively accurate incidence of GD. A fluorometric method to detect acid β-glucocerebrosidase (GBA) activity on a dried blood spot punch was developed. Validity and feasibility of the fluorometric method was demonstrated by examining 116 healthy controls, 19 confirmed GD patients and 19 obligate carriers. GBA activity was measured on dried blood spots of 80 855 newborns. Samples from positively screened newborns were reanalyzed by a leukocyte GBA activity test and GBA gene analysis. Plasma glucosylsphingosine level was determined as a biomarker of the pathophysiology of GD. GD patients were distinguished from healthy controls and obligate carriers using the fluorometric method. Mean GBA activity in newborn screening specimens was 145.69±44.76 μmol l−1 h−1 (n=80 844). Three children had low GBA activity, of which one child had low GBA activity on the second dried blood spot specimen. Leukocyte, genetic and biomarker analysis confirmed the diagnosis and indicated that this child was in the early stages of GD. In conclusion, the incidence of GD in Shanghai of China is approximately 1 in 80 855. Screening for GD by fluorometric analysis of GBA activity is an efficient and feasible technology in newborns.

Simultaneous quantitation of hexacosanoyl lysophosphatidylcholine, amino acids, acylcarnitines, and succinylacetone during FIA–ESI–MS/MS analysis of dried blood spot extracts for newborn screening

ObjectivesThe goal of this study was to include the quantitation of hexacosanoyl lysophosphatidylcholine, a biomarker for X-linked adrenoleukodystrophy and other peroxisomal disorders, in the routine extraction and analysis procedure used to quantitate amino acids, acylcarnitines, and succinylacetone during newborn screening. Criteria for the method included use of a single punch from a dried blood spot, one simple extraction of the punch, no high-performance liquid chromatography, and utilizing tandem mass spectrometry to quantitate the analytes. Design and methodsDried blood spot punches were extracted with a methanolic solution of stable-isotope labeled internal standards, formic acid, and hydrazine, followed by flow injection analysis–electrospray ionization–tandem mass spectrometry. ResultsQuantitation of amino acids, acylcarnitines, and hexacosanoyl lysophosphatidylcholine using this combined method was similar to results obtained using two separate methods. ConclusionsA single dried blood spot punch extracted by a rapid (45min), simple procedure can be analyzed with high throughput (2min per sample) to quantitate amino acids, acylcarnitines, succinylacetone, and hexacosanoyl lysophosphatidylcholine.

Reliable analysis of phenylalanine and tyrosine in a minimal volume of blood

ObjectivesPhenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to a defect in phenylalanine hydroxylase (PAH). Treatment principle is to reduce phenylalanine concentration sufficiently to prevent neuropathological effects. Dietary management is performed and the effect of treatment is monitored by regular analysis of phenylalanine and tyrosine. The aim of the study was to develop a rapid method to routinely measure both metabolites in minimal bloodspot volume (1.5mm Ø, corresponding with a volume of 1.3μL blood). MethodWhole blood was spiked with phenylalanine and tyrosine at 24 different concentrations. Dried blood spots (DBS) were prepared, after which punches of 1.5mm Ø and 6mm Ø (corresponding with a volume of 12.4μL) were taken. Additionally, punches of both sizes were prepared from DBS of PKU-patients (n=77). All samples were analyzed by tandem mass-spectrometry and results between both punches were compared. ResultsA good correlation between concentrations of phenylalanine and tyrosine in 1.5 and 6mm punches was found (r2=0.9917 and r2=0.9892, respectively). Analysis of phenylalanine and tyrosine in punches of PKU-patients (n=77) showed similar results and fitted within the procentual range of the between run variation. ConclusionWe developed an accurate and rapid method to analyze phenylalanine and tyrosine concentrations in a 1.5mm Ø bloodspot punch with an estimated whole blood volume of 1.3μL. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30–60min time saving in sample preparation.

Dried Blood Spot Punches for Confirmation of Suspected γ-Hydroxybutyric Acid Intoxications: Validation of an Optimized GC–MS Procedure

γ-hydroxybutyric acid (GHB), notorious as a club- and date-rape drug, was quantified in dried blood spots (DBS) by punching out a disc, followed by 'on-spot' derivatization and analysis by GC-MS. A homogenous distribution in DBS was demonstrated and accurate results were obtained when analyzing a disc punched out from a 20-35 µl spot, regardless the hematocrit of the blood sample. Validation based on US FDA and European Medicines Agency guidelines was performed, with a calibration range covering 2-100 µg/ml. A sensitive GC-MS method for GHB analysis in DBS was successfully optimized and validated. The successful analysis of DBS collected from GHB abusers suggests the routine applicability of the DBS sampling technique for GHB analysis in toxicological cases.

A Tandem Mass Spectrometry Triplex Assay for the Detection of Fabry, Pompe, and Mucopolysaccharidosis-I (Hurler)

We sought to develop a tandem mass spectrometry assay in which the enzymatic activities of 3 lysosomal enzymes (α-glucosidase, α-galactosidase A, and α-l-iduronidase) could be quantified in dried blood spots by using a single assay buffer. A 3-mm dried blood spot punch was incubated in a single assay buffer with 3 different substrates and internal standards. The sample was processed by a simple liquid-liquid extraction by using ethyl acetate. The extract was dried down and resuspended in solvent for injection into the tandem mass spectrometer. Products and internal standards were monitored by multiple reaction monitoring. Assay for the 3 lysosomal enzymes was successfully achieved with acceptable statistics. The assay can be performed by using a minimal quantity of disposable supplies and equipment. The entire procedure fits into a 48-h cycle including data analysis. Data from 5990 anonymous newborn dried blood spots showed an approximate bell-shaped distribution of enzymatic activities (mean values of 19.0, 11.5, and 3.5 μmol · h(-1) · (L blood)(-1) for α-glucosidase, α-galactosidase A, and α-l-iduronidase, respectively. Blank values obtained in the absence of blood were 0.13, 0.24, and 0.45 μmol · h(-1) · (L blood)(-1), respectively). By assaying 3 enzymes at once, problematic samples are spotted for reanalysis if enzyme activity values are low for all enzymes (for example, if insufficient blood is present in the assay). This method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high-throughput newborn screening laboratory for the analysis of Pompe, Fabry, and mucopolysaccharidosis-I (Hurler) diseases.

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using 2H 8-Val, 2H 3-Leu, 2H 3-Met and 2H 5-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.

High-Throughput Multiplexed T-Cell–Receptor Excision Circle Quantitative PCR Assay with Internal Controls for Detection of Severe Combined Immunodeficiency in Population-Based Newborn Screening

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Detection of HIV-1 in alternative specimen types using the APTIMA ® HIV-1 RNA Qualitative Assay

Peripheral blood mononuclear cells (PBMCs), saliva, seminal plasma, and dried blood spots were evaluated as specimen types for the APTIMA HIV-1 RNA Qualitative Assay (APTIMA HIV-1 Assay), which employs a target capture step to recover HIV-1-specific sequences from complex specimen types. Analytical sensitivity studies were carried out using samples that were either diluted or eluted with a buffered detergent and spiked with different concentrations of HIV-1 ranging from 1 to 10,000 copies/mL. PBMC samples spiked with HIV-1 had comparable analytical sensitivity to HIV-1 spiked plasma with a 95% limit of detection of 13.1 and 17.2 copies/mL, respectively. Analytical sensitivity in seminal plasma specimens diluted 1:5 and saliva diluted 1:2 was comparable to HIV-1 spiked dilution buffer alone. Whole blood and dried blood spot specimens spiked with HIV-1 had equivalent reactivity at 250 copies/spot (5000 copies/mL). However, the 95% limit of detection values were significantly different (293.7 copies/mL for whole blood and 2384 copies/mL for dried blood spot specimens). No significant effect on analytical sensitivity was observed when one HIV-1 positive dried blood spot punch was pooled with up to 9 HIV-1 negative dried blood spot punches. Together, these studies demonstrate that the APTIMA HIV-1 RNA Qualitative Assay can be used to process a diverse array of specimen types with minimal impact on analytical sensitivity for most specimen types.

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)

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. 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. 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. 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.