Quantification des acides amines physiologiques par le kit aTRAQ : evaluation et implementation de nouveaux parametres.

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 mumol/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

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 (HbS, HbC, and HbE) 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/HbA 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 HbS. According to the MS/MS settings (detection of HbS, HbC, HbE, 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

A Prenatal Trisomy 21 Screening Program Using Alpha-Fetoprotein, Human Chorionic Gonadotropin, and Free Estriol Assays on Maternal Dried Blood

OBJECTIVE: The feasibility of large-scale Down syndrome maternal screening with dried blood samples and nonradioactive methods was examined. STUDY DESIGN: A prospective observation study was performed on a nonselected population of 11,241 pregnant women sampled between January 1991 and September 1992, between 14 and 24 weeks' gestation (ultrasonographic scanning available for 91.6%), through a multicenter collaborative network. Enzyme-linked immunosorbent assays for alpha-fetoprotein, human chorionic gonadotropin, and free estriol were performed on dried blood samples. Risk determination was made with an in-house software implementing the multivariate gaussian log likelihood method. RESULTS: A total of 10,450 samples were eligible for the study. Mean age at term was 27.9 years. A total of 6.84% of the patients were > or = 35 years old with a prior risk of trisomy 21 > 1:350. The general positive rate of our sample was 8.15%. After calculation 31.7% with prior risk > 1:350 were still in the high-risk group; 6.36% of the low-risk group were found to be at high risk for Down syndrome. Fifteen trisomic pregnancies were observed, of which 11 had a calculated risk higher than the selected cutoff value (1:350). The overall detection rate was 73%, specificity was 92%, and positive predictive power was 1.2%. CONCLUSION: Our pilot study has shown performances within the range of conventional serum screening programs. Dried blood assays are a handy alternative to serum assays. Blot paper cards represent a simple method of sampling, well fitted for large population screening. Combined with nonradioactive methods, this method appears to be both low cost and effective. The current work apparently is the first large-scale Down screening program performed with dried blood

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