Surprising causes of C5-carnitine false positive results in newborn screening.

During an 18-month period, we noticed an alarming increase of newborn screening false positivity rate in identifying isovaleric acidemia. In 50 of 50 newborns presenting elevated C5-carnitine, we confirmed the presence of pivaloylcarnitine. Exogenous pivalate administration had been previously identified as the causal agent of this concern. No pivalic-ester prodrug is commercially available in Belgium, but pivalic derivates are also used in the cosmetic industry as emollient under the term "neopentanoate". We have identified neopentanoate-esters in a nipple-fissure unguent that was provided to young mothers. Ceasing distribution of this product hugely reduced the C5-carnitine false positivity rate.

Enhanced interpretation of newborn screening results without analyte cutoff values.

PURPOSE: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. METHODS: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. RESULTS: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. CONCLUSION: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%.

Newborn screening for sickle cell disease using tandem mass spectrometry.

BACKGROUND: Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening. METHODS: We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and beta-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens. RESULTS: Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was <0.03%, and the limit of detection was fixed at 1% of Hb S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E, and beta-globin production defects), the comparative study did not yield any discrepant results between the 2 techniques. CONCLUSIONS: MS/MS is a reliable method for hemoglobinopathy neonatal screening.

Neonatal screening for sickle cell disease in Central Africa: a study of 1825 newborns with a new enzyme-linked immunosorbent assay test.

OBJECTIVES: To evaluate the feasibility of systematic neonatal screening for sickle cell disease in the region of Great Lakes in Central Africa using a new approach with limited costs. METHODS: Between July 2004 and July 2006, 1825 newborn dried blood samples were collected onto filter papers in four maternity units from Burundi, Rwanda and the East of the Democratic Republic of Congo. We tested for the presence of haemoglobin C and S in the eluted blood by an enzyme-linked immunosorbent assay (ELISA) test using a monoclonal antibody. All ELISA-positive samples (multiple of the median (MoM) > or = 1.5) were confirmed by a simple molecular test. The statistica software version 7.1 was used to create graphics and to fix the MoM cut-off, and the chi(2) of Pearson was used to compare the genotype incidences between countries. RESULTS: Of the 1825 samples screened, 97 (5.32%) were positive. Of these, 60 (3.28%) samples were heterozygous for Hb S, and four (0.22%) for Hb C; two (0.11%) newborns were Hb SS homozygotes. CONCLUSIONS: The lower cost and the high specificity of ELISA test are appropriate for developing countries, and such systematic screening for sickle cell anaemia is therefore feasible.

Screening for sickle cell disease on dried blood: a new approach evaluated on 27,000 Belgian newborns.

SETTING: Early diagnosis of sickle cell disease decreases morbidity. However, cost-effective screening programmes are not yet available. METHODS: We explored the feasibility of systematic screening performed on dried blood harvested from five-day-old newborns. RESULTS: A total of 27,010 samples were collected in Belgian maternity units between June 2003 and February 2005, and the presence of haemoglobin (Hb) C or S in the eluted blood was examined by an enzyme-linked immunosorbent assay (ELISA) test performed with a monoclonal antibody detecting both mutated forms. As this antibody slightly cross-reacts with Hb A, better specificity is achieved if the test is performed not later than day 5. Among the 27,010 samples, 132 (0.49%) were positive. Molecular biology tests performed on dried blood from positive samples showed that 106 of these babies were heterozygotes for the Hb S mutation and three were heterozygotes for the Hb C mutation, while three newborns were SS homozygotes (0.011%). Seventeen samples (0.063%) were false-positives as we could not detect any mutation. CONCLUSIONS: We have developed a new immunological approach in the field of haemoglobinopathy neonatal screening. This ELISA test is cheap (0.2 euro/test or 1800 euro/detected SS homozygote) and could be centralized. Its cost-effectiveness in the whole Belgian population is comparable with that of screening for phenylketonuria or congenital adrenal hyperplasia. Further improvements should obviously be achieved in order to better discriminate heterozygotes and homozygotes, but the accessibility and the low cost of the test are relevant arguments for the screening extension in a wide range of countries, especially in Central Africa.

[Quantification of physiological aminoacids using aTRAQ(®) kit: evaluation and implementation of new markers]. / Quantification des acides aminés physiologiques par le kit aTRAQ(®) : évaluation et implémentation de nouveaux paramètres.

Nowadays, physiological amino acids profiling is based primarily on ion exchange chromatography (IEC) coupled to a post-column derivatization with ninhydrin and UV detection at two wavelengths. Unfortunately, this technique suffers various drawbacks such as long analysis time, high sample volume and specific costs related to the maintenance of a dedicated equipment. These reasons have led us to consider a technology switch to a mass spectrometry method. We tested the kit aTRAQ amino acids analysis for physiological samples (AB Sciex), offering a selective quantification of more than 40 amino acids, and have implemented the acquisition of various original markers to the initial method. The accuracy profiles established for each amino acid show that the results are very reliable. The linearity is assured between 1 and 1.000 µmol/L for most analytes. Result comparison with IEC method showed good agreement. Reference ranges are similar to those defined for the IEC method and patients with inborn errors of metabolism were readily identified. The aTRAQ method offers a valid alternative to IEC method with several advantages: reduced sample volume, decreased run time and increased specificity. However, the procedure requires a thorough review of all chromatographic peaks, process that considerably lengthens the overall time of the procedure. Finally, financial and practical considerations of both techniques have to be counterbalanced before initiating any methodological transition.

Quantification of hypoglycin A in serum using aTRAQ(®) assay.

BACKGROUND: Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. METHODS: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. RESULTS: Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50µmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. CONCLUSIONS: The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.

Evaluation of physiological amino acids profiling by tandem mass spectrometry.

BACKGROUND: Nowadays, the most conventional method to quantify physiological amino acids consists in ion exchange chromatography (IEC) followed by post-column ninhydrin derivatization and UV detection at two wavelengths. Unfortunately, the technique presents some drawbacks such as long run time, large sample volume, and specific costs associated to the maintenance of a dedicated instrument. Therefore, we aimed to switch towards a mass spectrometry approach. METHODS: We have tested the aTRAQ kit for Amino Acid Analysis of Physiological Fluids (AB Sciex), affording the selective quantification of about 40 amino acids, and present here the results of our assessments. RESULTS: Outlined accuracy profiles for each amino acid demonstrated very reliable data. A good linearity was observed from 1 to 1,000 µmol/L. RESULTS comparison with IEC showed a right concordance. Reference intervals established were very similar to those obtained by IEC and patients suffering from inborn error of metabolism have been readily identified. CONCLUSIONS: The present approach offers a valid alternative to the IEC method, with several advantages: lower sample volume, run time reduction, and improved specificity. However, the aTRAQ method requires minute data reviewing, expending the overall time of procedure. Finally, financial and practical considerations of both techniques have to be counterbalanced before engaging any transition.

Neonatal thyroid-stimulating hormone concentrations in Belgium: a useful indicator for detecting mild iodine deficiency?

It has been proposed that neonatal thyroid-stimulating hormone (TSH) concentrations are a good indicator of iodine deficiency in the population. A frequency of neonatal TSH concentrations above 5 mU/L below 3% has been proposed as the threshold indicating iodine sufficiency. The objective of the present study was to evaluate feasibility and usefulness of nation-wide neonatal TSH concentration screening results to assess iodine status in Belgium. All newborns born in Belgium during the period 2009-2011 (n = 377713) were included in the study, except those suffering from congenital hypothyroidism and premature neonates. The frequency of neonatal TSH concentrations above 5 mU/L from 2009 to 2011 in Belgium fluctuated between 2.6 and 3.3% in the centres using the same TSH assay. There was a significant inverse association between neonatal TSH level and birth weight. The longer the duration between birth and screening, the lower the TSH level. Neonatal TSH levels were significantly lower in winter than in spring or autumn and significantly lower in spring and summer than in autumn while significantly higher in spring compared to summer. In conclusion, despite that pregnant women in Belgium are mildly iodine deficient, the frequency of neonatal TSH concentrations above 5 mU/L was very low, suggesting that the neonatal TSH threshold proposed for detecting iodine deficiency needs to be re-evaluated. Although neonatal TSH is useful to detect severe iodine deficiency, it should not be recommended presently for the evaluation of iodine status in mildly iodine deficient regions.

3-years experience review of neonatal screening for hemoglobin disorders using tandem mass spectrometry.

BACKGROUND: Neonatal screening programs for sickle cell disease are common in North America and in some European countries. Isoelectric Focusing or High Performance Liquid Chromatography is the main technique used for hemoglobin variant detection. METHODS: Since tandem mass spectrometry is being used for screening of inherited metabolic disorders and allows protein identification, we had developed an application to identify the most relevant hemoglobin mutations with this technology. RESULTS: This approach had been previously validated and has been routinely applied in our laboratory for the last three years. We report here our experience with this new method in the field, applied to our East-Belgian population. CONCLUSIONS: To conclude, mass spectrometry provides an efficient alternative approach for laboratories performing neonatal screening of hemoglobin disorders.

Analytical validation based on total error measurement and cut-off interpretation of a neonatal screening TSH-immunoassay.

To prevent the severe developmental and physical morbidities associated with congenital hypothyroidism, we developed a home-made Enzyme-Linked Immunosorbent Assay (ELISA) method to quantify Thyroid Stimulating Hormone (TSH) levels on newborn dried blood spots. In order to agree with actual clinical laboratory quality referential (ISO 15189), we desired to update our analytical validation protocol. For this purpose, an approach using accuracy profiles based on tolerance intervals for the total error measurement was for first time applied to an immunological assay. According to acceptance limits fixed at +/-30%, the method was found accurate over a concentration range from 17.48 to 250 mIU/L. Based on 99.5 percentile of a 16,459 newborn population, cut-off was fixed at 20.1 mIU/L and validated against normal and pathologic neonatal populations. Additionally, uncertainty regions around this value were obtained applying four different approaches. Finally, we demonstrated here our in-house immunological technique fulfils criterions of a neonatal screening policy.

Molecular analysis in two siblings African patients with severe form of Hunter Syndrome: identification of a novel (p.Y54X) nonsense mutation.

Hunter syndrome (or Mucopolysaccharidosis type II, MPS II) is an X-linked recessive disorder due to the deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in the accumulation of heparan and dermatan sulfates in the lysosomes. The heterogeneity of clinical phenotypes, ranging from mild-to-severe forms, is a result of different mutations in the IDS gene. We report here, a novel nonsense mutation (p.Y54X) in two siblings MPS II African patients affected with a severe form of the disease. We postulated that the p.Y54X mutation which causes a loss of the IDS region highly conserved among sulfatase enzymes, could be predicted as a severe disease-causing mutation for Hunter syndrome.