scholarly journals Combined Newborn Screening for Succinylacetone, Amino Acids, and Acylcarnitines in Dried Blood Spots

2008 ◽  
Vol 54 (4) ◽  
pp. 657-664 ◽  
Author(s):  
Coleman Turgeon ◽  
Mark J Magera ◽  
Pierre Allard ◽  
Silvia Tortorelli ◽  
Dimitar Gavrilov ◽  
...  
Keyword(s):  
Amino Acids ◽  
Newborn Screening ◽  
False Negative ◽  
Internal Standard ◽  
Early Death ◽  
Cost Effective ◽  
Dried Blood Spots ◽  
Type I ◽  
Blood Spots ◽  
Negative Results

Abstract Background: Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). Methods: We extracted 3/16-inch DBS punches with 300 μL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 μL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155–137) and 13C5-SUAC (m/z 160–142). Analysis time was 1.2 min. Results: SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13–81 μmol/L), with Tyr concentrations ranging from 65 to 293 μmol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 μmol/L. Conclusion: The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

scholarly journals 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)

2008 ◽  
Vol 54 (3) ◽  
pp. 542-549 ◽  
Author(s):  
Devin Oglesbee ◽  
Karen A Sanders ◽  
Jean M Lacey ◽  
Mark J Magera ◽  
Bruno Casetta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Newborn Screening ◽  
Dried Blood Spots ◽  
Branched Chain Amino Acids ◽  
Maple Syrup ◽  
Blood Spots ◽  
Urine Disease ◽  
Second Tier ◽  
Branched Chain ◽  
Positive Results

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.

scholarly journals Preliminary Proficiency Testing Results for Succinylacetone in Dried Blood Spots for Newborn Screening for Tyrosinemia Type I

2009 ◽  
Vol 55 (12) ◽  
pp. 2207-2213 ◽  
Author(s):  
Barbara W Adam ◽  
Timothy H Lim ◽  
Elizabeth M Hall ◽  
W Harry Hannon
Keyword(s):  
Newborn Screening ◽  
Proficiency Testing ◽  
False Negative ◽  
Dried Blood Spots ◽  
Screening Tests ◽  
Type I ◽  
Primary Metabolite ◽  
Screening Practices ◽  
Tyrosinemia Type I ◽  
Stable Performance

Abstract Background: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I—an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. Methods: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. Results: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries ≥100% used DBS matrix calibrators. Conclusions: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.

scholarly journals Validation of Accuracy-based Amino Acid Reference Materials in Dried-Blood Spots by Tandem Mass Spectrometry for Newborn Screening Assays

1999 ◽  
Vol 45 (8) ◽  
pp. 1269-1277 ◽  
Author(s):  
Donald H Chace ◽  
Barbara W Adam ◽  
S Jay Smith ◽  
J Richard Alexander ◽  
Steven L Hillman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Amino Acid ◽  
Tandem Mass Spectrometry ◽  
Newborn Screening ◽  
Dried Blood Spots ◽  
Tandem Mass ◽  
Blood Spots ◽  
Amino Acid Contents ◽  
Amino Acid Concentrations

Abstract Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage. Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs. Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested. Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.

scholarly journals Evaluation of the Ultrasensitive Human Immunodeficiency Virus Type 1 (HIV-1) p24 Antigen Assay Performed on Dried Blood Spots for Diagnosis of HIV-1 Infection in Infants

2007 ◽  
Vol 15 (2) ◽  
pp. 388-391 ◽  
Author(s):  
Janet C. Patton ◽  
Ashraf H. Coovadia ◽  
Tammy M. Meyers ◽  
Gayle G. Sherman
Keyword(s):  
False Negative ◽  
Dried Blood Spots ◽  
Blood Spots ◽  
Negative Results ◽  
Laboratory Services ◽  
False Negative Results ◽  
Antigen Assay ◽  
Immunodeficiency Virus ◽  
P24 Antigen ◽  
Hiv 1

ABSTRACT The diagnostic accuracy of the modified p24 antigen assay performed on pediatric dried blood spots was evaluated. Samples analyzed within 6 weeks of collection yielded no false-positive results (specificity, 100%) and few false-negative results (sensitivity, 96.5% to 98.3%). Laboratory services with limited resources should assess this option for routine infant diagnosis.

scholarly journals Newborn Screening for Hepatorenal Tyrosinemia: Tandem Mass Spectrometric Quantification of Succinylacetone

2006 ◽  
Vol 52 (3) ◽  
pp. 482-487 ◽  
Author(s):  
Johannes Sander ◽  
Nils Janzen ◽  
Michael Peter ◽  
Stefanie Sander ◽  
Ulrike Steuerwald ◽  
...  
Keyword(s):  
Newborn Screening ◽  
False Positive ◽  
False Negative ◽  
Internal Standard ◽  
Mass Spectrometric ◽  
Absolute Methanol ◽  
Tandem Mass ◽  
Screening Programs ◽  
Blood Spots ◽  
Tandem Mass Spectrometric

Abstract Background: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening. Methods: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile–water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run. Results: Of 61 344 samples, 99.6% had succinylacetone concentrations ≤5 μmol/L. With a cutoff of 10 μmol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 μmol/L, respectively, and the tyrosine concentrations were 54 and 129 μmol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 μmol/L, respectively. Conclusions: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.

scholarly journals Effect of Dried Blood Spot Quality on Newborn Screening Analyte Concentrations and Recommendations for Minimum Acceptance Criteria for Sample Analysis

2016 ◽  
Vol 62 (3) ◽  
pp. 466-475 ◽  
Author(s):  
Roanna S George ◽  
Stuart J Moat
Keyword(s):  
Newborn Screening ◽  
Filter Paper ◽  
Statistical Significance ◽  
False Negative ◽  
Dried Blood Spots ◽  
Sample Analysis ◽  
Acceptance Criteria ◽  
Blood Spots ◽  
Spot Quality ◽  
Blood Spot

Abstract BACKGROUND The analysis of dried blood spots has been used routinely for newborn screening since the early 1970s, and the number of disorders screened has expanded substantially in recent years. However, there is a lack of evidence regarding minimum blood spot quality acceptance criteria for sample analysis. METHODS Blood pools were spiked with phenylalanine, tyrosine, leucine, methionine, octanoylcarnitine, decanoylcarnitine, isovalerylcarnitine, glutarylcarnitine, thyroid-stimulating hormone, and immunoreactive trypsinogen to concentrations at the analytical cutoffs used in UK screening protocols. We evaluated the effect of sample volume applied to the card (10, 20, 50, 75, and 100 μL), punch location (central vs peripheral), and sample quality (double layering, applying blood to both sides of the filter paper, multispotting, applying insufficient sample, and compressing the sample after application). RESULTS Compression of blood spots produced significantly lower results (14%–44%) for all analytes measured (P < 0.001). Smaller blood spots produced significantly lower results (15%–24% for 10-μL vs 50-μL sample size) for all analytes at all concentrations measured (P < 0.001). Results obtained from peripheral punches were higher than those from a central punch, although this did not reach statistical significance for all analytes. Insufficient and multispotted samples demonstrated heterogeneous results. CONCLUSIONS All blood spots containing ≤20 μL (blood spot diameter <8 mm), those in which blood has not fully penetrated the filter paper, and all samples with evidence of compression should be rejected, since there is a risk of producing false-negative results.

scholarly journals EDTA in Dried Blood Spots Leads to False Results in Neonatal Endocrinologic Screening

2008 ◽  
Vol 54 (3) ◽  
pp. 602-605 ◽  
Author(s):  
Ute Holtkamp ◽  
Jeanette Klein ◽  
Johannes Sander ◽  
Michael Peter ◽  
Nils Janzen ◽  
...  
Keyword(s):  
Newborn Screening ◽  
False Positive ◽  
Neonatal Screening ◽  
False Negative ◽  
Dried Blood Spots ◽  
Screening Tests ◽  
Blood Collection ◽  
Blood Spots ◽  
Sampling Procedures ◽  
Positive Results

Abstract Background: Blood samples for neonatal screening for inborn errors of metabolism are collected and shipped on standardized filter paper cards. Occasionally these samples are contaminated with EDTA, which is often used for anticoagulation. EDTA may interfere with newborn screening tests based on lanthanide fluorescence and thus lead to false-negative or false-positive results. Methods: We used tandem mass spectrometry (MS/MS) to detect EDTA in dried blood spots by use of an extra experiment that was integrated into the standard MS/MS neonatal screening and did not require an additional sample spot, nor extra time or work. We analyzed the influence of different blood sampling procedures on lanthanide fluorescence tests for thyroid-stimulating hormone (TSH) and 17-hydroxyprogesterone (17-OHP). Results: EDTA was increased in 138 of 190 000 newborn screening samples, 27 of which caused false- positive results in the immunoassay for 17-OHP. No false-negative TSH results were found. False-positive results in the 17-OHP test occurred when EDTA concentrations were >2.0 g/L; the TSH test, however, produced false negatives only when EDTA concentrations were >3.0 g/L. Using EDTA-containing devices the procedure of blood collection significantly influenced the concentration of the anticoagulant. Conclusion: Addition of EDTA quantification into standard MS/MS tests is a simple and useful method to avoid false-positive or false-negative neonatal screening results in lanthanide fluorescence–based tests.

scholarly journals Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots: Application to Screening Newborns for Mucopolysaccharidosis I

2008 ◽  
Vol 54 (12) ◽  
pp. 2067-2070 ◽  
Author(s):  
Sophie Blanchard ◽  
Martin Sadilek ◽  
C Ronald Scott ◽  
Frantisek Turecek ◽  
Michael H Gelb
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Newborn Screening ◽  
Lysosomal Enzymes ◽  
Internal Standard ◽  
Dried Blood Spots ◽  
Simple Liquid ◽  
Tandem Mass ◽  
Mucopolysaccharidosis I ◽  
Blood Spots

Abstract Background: Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, α-L-iduronidase. Methods: We synthesized a new α-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liquid-liquid extraction step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concentration of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision. Results: When the assay was tested on dried blood spots, the α-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the α-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands. Conclusions: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this laboratory and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.

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