, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.

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. Conclusions: 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%

General mitochondrial trifunctional protein (TFP) deficiency leads to a wide clinical spectrum of disease ranging from severe neonatal/infantile cardiomyopathy and early death to mild chronic progressive sensorimotor poly-neuropathy with episodic rhabdomyolysis. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency resulting from the common Glu510Gln mutation usually gives rise to a moderately severe phenotype with multiorgan involvement with high morbidity and mortality. However, isolated LCHAD deficiency can also be consistent with long-term survival in patients identified and treated from an early age. We present biochemical, clinical and mutation data in 9 patients spanning the full spectrum of disease. Fibroblast acylcarnitine profiling shows good correlation with clinical phenotype using the ratio C18(OH)/(C14(OH)+C12(OH)). This ratio shows a gradation of values, from high in four patients with severe neonatal disease (2.5+/-0.8), to low in two neuromyopathic patients (0.35, 0.2). Fibroblast fatty acid oxidation flux assays also show correlation with the patient phenotype, when expressed either as percentage residual activity with palmitate or as a ratio of percentage activity of myristate/oleate (M/O ratio). Fibroblasts from four patients with severe neonatal disease gave an M/O ratio of 4.0+/-0.6 compared to 1.97 and 1.62 in two neuromyopathic patients. Specific enzyme assay of LCHAD and long-chain 3-ketothiolase activity in patient cells shows lack of correlation with phenotype. These results show that measurements in intact cells, which allow all determinative and modifying cellular factors to be present, better reflect patient phenotype. Mutation analysis reveals a number of alpha- and beta-subunit mutations. Peripheral sensorimotor polyneuropathy, often as the initial major presenting feature but usually later accompanied by episodic rhabdomyolysis, is a manifestation of mild TFP protein deficiency. The mild clinical presentation and relative difficulty in diagnosis suggest that this form of TFP is probably underdiagnosed.

BackgroundMedium chain acyl-CoA dehydrogenase deficiency (MCADD) is a rare, life-threatening condition. Early diagnosis by screening asymptomatic newborns may improve outcome, but the benefit to newborns identified with variants not encountered clinically is uncertain.ObjectiveTo estimate, overall and by ethnic group: screen-positive prevalence and predictive value (PPV); MCADD prevalence; proportion MCADD variants detected of predicted definite or uncertain clinical importance.SettingAll births in areas of high ethnic minority prevalence in England.MethodsProspective multicentre pilot screening service; testing at age five to eight days; standardized screening, diagnostic and management protocols; independent expert review of screen-positive cases to assign MCADD diagnosis and predicted clinical importance (definite or uncertain).ResultsApproximately 1.5 million babies (79% white; 10% Asian) were screened. MCADD was confirmed in 147 of 190 babies with a positive screening result (screen-positive prevalence: 1.20 per 10,000; MCADD prevalence: 0.94 per 10,000; PPV 77% [95% CI 71–83]), comprising 103 (70%) with MCADD variants of definite clinical importance (95 white [95%]; 2 Asian [2%]) and 44 (30%) with variants of uncertain clinical importance (29 white [67%]; 12 Asian [28%]).ConclusionOne baby in every 10,000 born in England is diagnosed with MCADD by newborn screening; around 60 babies each year. While the majority of MCADD variants detected are predicted to be of definite clinical importance, this varies according to ethnic group, with variants of uncertain importance most commonly found in Asian babies. These findings provide support for MCADD screening but highlight the need to take account of the ethnic diversity of the population tested at implementation.

Objectives This paper reports early screening results from the newborn sickle cell disease screening programme recently implemented in England. Setting England. Screening is offered at 5-8 days of age as part of the existing bloodspot test and offered to all babies irrespective of ethnicity. Methods The laboratory methods recommended are high performance liquid chromatography (HPLC) and iso-electric focusing (IEF). 15 Two methods of analysis must be applied to all screen positive results. The conditions screened for are:- Sickle cell anaemia (Hb SS), Hb SC disease, Hb S/β-thalassaemia, Hb S/DPunjab, Hb S/OArab, Hb S/HPFH. Carriers identified for the common haemoglobin variants are reported to parents and follow-up counselling is offered. A bespoke laboratory quality assurance programme has been established which has defined standards of satifactory performance. Results Provisional figures from the first seven months of screening (up to March 2004) 108,255 infants were screened gave a screen positive rate of 1:900 for these high prevalence areas and a carrier rate of 2.7%. Figures for 2004-2005 show about 250 significant screen positive results for sickle cell disorders and about 6,500 carriers were identified. The birth prevalence for screen positive results from 2004-05 is 1:1500. We estimate that when there is countrywide data, the national birth prevalence will be about 1:2000-1:2,500. Conclusion The results from the national newborn sickle cell screening programme in England - show that the sickle cell disorders are as common as cystic fibrosis (CF) in England, although the distribution of cases is concentrated in London and other urban areas. The findings and approach to implementation adopted in England may be of interest to other Western European countries with increasing rates of sickle cell disease who are considering such programmes and also to other developed countries.