Screening Of High-risk Children And Patients From Neonatal Intensive Care Units For Inborn Errors Of Metabolism Using Quid Chromatography-tandem Mass Spectrometry (LC-MS/MS)

Screening of high-risk children and patients from neonatal intensive care units for inborn errors of metabolism using quid chromatography-tandem mass spectrometry (LC-MS/MS)Background Tandem mass spectrometry now allows newborn screening for more than 30 inborn errors of metabolism. We have initiated screening for inborn errors of metabolism in north China by analyzing acylcarnitines and amino acids in a dried blood filter-paper samples using LC-MS/MS.Objective1. To investigate the effects of LC-MS/MS in the screening of inborn errors of metabolism in normal newborn and the patients admitted to neonatal intensive care units, the incidence of IEM in the population of patients admitted to NICU, to investigate the effects of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the diagnosis of inherited metabolic diseases in high risk children.2. To investigate hypoglycaemia as an indicator for screening high risk patients with inborn errors of metabolism (IEM) in neonatal intensive care unit (NICU).Methods1. The dry blood filter papers were tested by LC-MS/MS among 724 patients from a maternal and child health hospital'neonatal intensive care unit,11240 normal newborns who taken part in newborn screening from our laboratory,2760 children suspected to be with inherited metabolic diseases from more than sixty hospitals in north China.Before testing the filter papers should be punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCL. Amino acids and acylcarnitines were quantitated against dried-blood spot calibrators. The results were transferred to a central data base, where each result was checked against predefined algorithms and reference ranges, including reference ranges for ratios and for second samples, when requested. The positive results were further confirmed by gas chromatography-mass spectrometry (GC-MS) and other biochemical genetic tests, including enzyme and molecular analyses (DNA mutation analysis). Clinical follow-up of all positive patients in a median observation period was 11 months2.160 patients from neonatal intensive care unit were enrolled. Blood glucose was measured by the glucometer on site, the abnormal neonatal were then tested by Roche Modular chemistry. The dry blood on filter papers, collected from 160 patients, was tested by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect 35 inborn errors of metabolism. The specimen was extracted out of the dry blood on filter paper, derivatized before injected into LC-MS/MS. Clinical follow-up of all the patients at least in an interval 6 months. The mean observation period was 7.2 months per child.Results1.16 of the 11240 newborns (0.14%) were diagnosed as with 11 kinds of disorders:5 with MMA,2 with hyperphenylalanaemia,1 with MCAD,1 with MADD,1 with PA, 1with IVA, 1with GA 1,1 with MUSD,1 with tyosinaemia,1 with homocystinuria,1 with argininaemia; and the collective estimated prevalence amounted to 1 in 700 with a sensitivity of 100%, a specificity of 99.48%, a false positive rate of 0.52%, a positive predictive value of 21.33%, and no false negative appear.8 of the 724 patients(1.1%) from NICU were diagnosed as with 5 kinds of disorders:3 with MMA, 1 with MADD,1 with PA,2 with tyosinaemia,1 with MUSD. The difference of the diagnosed rate between two groups is significant (x2=26.86, p<0.005). The patients from NICU is 7.76 (95% CI:3.58,16.84) times more likely to suffer from 35 inborn errors of metabolism than normal newborns.249 of the 2760 children (9%)were diagnosed with twenty-one kinds of disorders: 41 of the 249 (16.5%) were diagnosed as fatty acid disorders; 71 of the 249 (28.5%) were diagnosed as amino acid diseases; 137 of the 249 (55%) were diagnosed as organic acidemias.48 of the 249 patients (19.3%) were neonates, including 11 (22.9%) with fatty acid disorders,15 (31.3%) with amino acid diseases,22 (45.8%) with organic acidemias.201 of the 249 patients aged> 28days, including 30 (14.9%) with fatty acid disorders,56 (27.9%) with amino acid diseases,115 (57.2%) with organic acidemias.2. Based on the ROC curve, the optimal cut-off value of hypoglycaemia as an indicator for testing for IEM was projected to be 2.8mmol/Lwhich yielded a sensitivity of 71.4%and a specificity of 76.5%. Using the blood glucose cut off level of 2.8mmol/L, the patients were divided into two groups:hypoglycaemia group (48 cases) and the control group (112 cases).The average blood glucose level of the hypoglycaemia group and the control were (2.05±0.45)mmol/L and (4.17±1.30)mmol/L, respectively. A total of 5(10.4%) out of 48 patients in the hypoglycaemia group were positive in the testing for IEM including four with methylmalonic acidemia and one with tyrosinemia, while only 2(1.8%)out of the 112 patients in the control group were positive including one with methylmalonic acidemia and one with maple syrup urine disease.. The difference of the positive rate in the testing for IEM between the hypoglycaemia group and the control group was significant (x2=4.10, p<0.05); the relative risk (RR) was 5.83 (95% CI:1.06-32.12).Conclusion1. This technology of LC-MS/MS is a fast, accurate and effective method for amino acid and acylcarnitine profiles analysis, more than thirty IEM could be detected in a single test. The incidence of IEM in the population of patients admitted to NICU was 1.1%, indicates an underestimation of the incidence of metabolic disorders prior to implementing LC-MS/MS screening in NICU. This study provides important pilot screening results and incidence data with regard to the north china population.2. The risk of patients with hypoglycaemia suffering from IEM was significant higher than the control group based on cut-off value of 2.8mmol/L. We recommend that hypoglycaemia is considered to be used as an indicator for screening for IEM in NICU.