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24. Development of an enhanced screening strategy for classical galactosaemia in the Republic of Ireland

Roche G., McGarrity J., O’Grady L., Flynn A., Lim K., Bennett D., Mayne P.

Children’s University Hospital, National Newborn Screening Laboratory, Dublin, Ireland

Screening for Classical Galactosaemia has been part of the Irish National Newborn Screening Programme since 1972. The method is based on Guthrie’s Bacterial Inhibition assay (BIA) for free galactose in combination with a qualitative Beutler assay for Galactose-1-phosphate uridyl transferase. The overall incidence is 1 in 19,500 births, comprising an incidence of 1 in 32,000 in the general population and 1 in 480 in Irish Travellers – an identifiable cultural and nomadic sub-group within the Irish community. This project was designed to select and implement an enhanced screening strategy by assessing methods for measuring Total Galactose (TGAL) and Galactose-1-phosphate uridyl transferase (GALT) and to establish cut-offs to ensure no increase in the false positive rate. Ethical approval was granted. Two commercially available methods for each of TGAL and GALT and an in-house semi quantitative fluorometric Beutler assay were evaluated using 1,000 randomly selected, anonymised blood spots from routine newborn screening samples. DNA analysis has been performed on 600 samples for Q188R (allele frequency 89% in the general population; 100% in Irish Travellers) and N314D (incidence of Duarte variant 1 in 650 in the general population). There was poor correlation between the Bio-Rad and PerkinElmer GALT assays with just one sample (heterozygous for Q188R) having an enzyme activity below the 99% cut-off for both methods. GALT assay performance characteristics were spread across a range with total variation (% CV) of 7.7% (Bio-Rad) and 25.3% (PerkinElmer). With a 99% cut-off for the Bio-Rad TGAL assay of 500 µmol/L no sample had a GALT activity below the 99th percentile by any method. No compound heterozygote for Q188R and N314D has been identified. Based on the analytical performance, assay characteristics, experimental design criteria and cost, Bio-Rad TGAL with subsequent in-house semi quantitative Beutler assay was selected as the screening strategy of choice.

25. Newborn screening for tyrosinemia I, simultaneous versus separate quantitation of succinylacetone

Sander J.¹, Sander S.¹, Janzen N.², Peter M.¹, Dragoey J.¹

¹Screening-Labor Hannover, Laboratory, Hannover, Germany

²Medical School Hannover, Pediatrics, Hannover, Germany

Measurement of succinylacetone (SA) in blood spots of metabolic screening programs is an effective means of presymptomatic diagnosis of Tyrosinemia typ I (Tyr I). Since SA is found in blood mainly covalently linked to proteins it has to be liberated by hydrazine during extraction. Separate extraction together with separate measurement (method 1) doubling the number of analytic runs may be considered to be too costly with respect to the low prevalence of Tyr I in most screening populations. Separate extraction and pooling of extracts (method 2) will reduce expenses. This is also true for addition of hydrazine to the extraction solution for simultaneous extraction of amino acids (AS), acylcarnitines (AC) and SA (method 3). Prior to routine application of one of the more cost effective methods, however, it has to be demonstrated for method 2 that it is of sufficient sensitivity and for method 3 that it does not unfavourably affect quality of the standard screening parameters. Measuring SA after pooling of 8 extracts increased the level of discrimination of a positive case from 8 µmol/L SA (cut off for single determinations) to 12 µmol/L SA in 1 of 8 samples. SA concentrations in positive cases were in the range of 23 to >250 µmol/L. Adding hydrazine to the extraction solution caused reduction of MS/MS signals for AC and AS as well as for the relevant internal standards up to 20%. Quantitative results for metabolites, however, did not differ significantly provided the concentration of hydrazine did not exceed 10 µmol/L in the extraction solution. We conclude that both the pooling of hydrazine containing extracts as well as the simultaneous extraction of SA, AC and AS in the presence of hydrazine can be used for effective screening for Tyr I.

26. Quantitation of Amino Acids in Dried Blood Spots by iTRAQ^® Reagent Derivatization Reaction and LC/MS/MS Analysis

Sasaki T. A.¹, Yang S.², Han J.², Kwon H.³, Kim S.³, Krol J.⁴, Daniels S.⁴, Leonard S.⁴

¹Applied Biosystems, Life Technologies, Foster City, United States

²Green Cross, Reference Lab., Yongin, South Korea

³Applied Biosystems, Life Technologies, Seoul, South Korea

⁴Applied Biosystems, Life Technologies, Framingham, United States

Introduction: Dried blood spots (DBS) are the standardized sampling method for the screening of inborn errors of amino acid metabolism. Recent publications report quantitation of amino acids (AAs) in physiological fluids such as plasma, urine and cerebrospinal fluid (CSF) by LC/MS/MS using iTRAQ^® Reagents. This method allows measurement of individual isobaric AAs and provides quantitative information on almost twice the number of AAs than the conventional screening method. However, this method has not been applied to dried blood spots. This study investigates an extraction protocol to quantify 45 physiological Aas from DBS using the iTRAQ labeling method.

Method: Dried blood spots on filter paper and plasma samples were prepared from the same whole blood sample. A 3/16 inch punch from the dried blood filter paper was extracted with trichloracetic acid for 10 minutes. The extracted spot samples and 40 µL of plasma were prepared using the iTRAQ derivatization protocol and analyzed by LC/MS/MS. Total run time was 25 minutes. A labeled internal standard for each analyte was used for quantitation.

Preliminary data: Thirty sets of dried blood spot and plasma samples were analyzed and data compared. Several different extraction methods were investigated. Modifications to extraction solvent, solvent modifiers, and extraction time were explored. Derivatization efficiency for whole blood as a function of incubation time and concentration of iTRAQ^® Reagent 115 was also studied. The accuracy of the quantitation is improved by adding a labeled standard to the sample, providing an internal standard for every amine or amino acid in the analysis. Results from analysis of dried blood spots were comparable to results from the analysis of plasma samples. Although plasma and urine samples are usually the preferred matrix for amino acid analysis, this work demonstrates that it is possible to add DBS to the list of physiological matrices tested.

27. Value of tandem mass spectrometry in the diagnosis of organic acidemias

Selim L.¹, Hassan S.², Hassan F.³, Salem F.², El Mogy F.³, Mandour I.³, Sharaf S.³, Morgan M.³, Amin A.¹, Mahmoud I.⁴, El Defrawy M.⁵, Alayat A.⁴, El Badawy A.⁴, Girgis M.⁶, Mancy A.⁶, Moneim M. A.⁷, Mahany D.³

¹Cairo University Children Hospital, Pediatric Neurology & Metabolic Unit, Cairo, Egypt

²Cairo University Children Hospital, Pediatrics & Genetic, Cairo, Egypt

³Cairo University Children Hospital, Clinical & Chemical Pathology, Cairo, Egypt

⁴Cairo University Children Hospital, Metabolic Unit, Cairo, Egypt

⁵Benha Faculty of Medicine, Pediatric Hepatology, Cairo, Egypt

⁶Cairo University Children Hospital, Pediatric Neurology, Cairo, Egypt

⁷Cairo University, Clinical & Chemical Pathology, Cairo, Egypt

Organic acidurias (OA) applies to a group of disorders characterized by the excretion of non-amino organic acids in urine. Expanded newborn screening (NBS) methods by tandem mass spectrometry (TMS) can detect organic acidemias. This is a retrospective study aiming at reviewing the clinical, biochemical and neuroradiological data of patients diagnosed as organic acidemias among 800 patients referred to the Neurometabolic Clinic at Cairo University Children Hospital and to highlight the importance of including organic acidemias in newborn screening programs using TMS. 800 patients were screened by tandem mass spectrometry (TMS). 19/800 patients were diagnosed as OAs, 14/19 had abnormal acyl carnitine profile by TMS. The urine OA profile of all 19 patients was analysed by gas chromatography mass spectrometry (GCMS). 3/19 patients (16.7%) were diagnosed as methyl malonic acidemias(MMA), 3 (16.7%) as b ketothiolase deficiency (BKT), 2 (11.1%) as 3 methyl crotonyl glycinuria(MCG), 3 (16.7%) as biotinidase deficiency), 2 (11.1%) as canavan disease, one (5.6,%) as glutaric aciduria type1, one as D-2 hydroxyglutaric aciduria (5.6%), one case as argininosuccinic aciduria (5.6%), one case(5.6%) as 3-methyl glutaconic aciduria, and one as propionic acidemia. Delayed development was a dominant symptom in 16 (84.2%) metabolic acidosis in 14 (73.6%), vomiting in 10 (52.6%) encephalopathy with disturbed conscious level in 9 (47%) seizures in 8 (42%) namely in MMA, PA & BKT. Central nervous system abnormalities included cranial nerve affection in 6 (31%), extrapyramidal manifestations in 4 (21%) ataxia in 2 (10.5%), long tract signs in 7 (36.8%) and hypotonia in 10 (52.6%) patients predominantly in cerebral organic acidurias.

Conclusion: Including organic acidopathies in NBS would help in rapid and properly timed therapeutic intervention to prevent devastating neurological outcome.

28. Amino acid and acylcarnitine profile testing in newborns by tandem mass spectrometry. An institutional experience by Acibadem Labmed Clinical Laboratories

Serteser M.¹, Elci A.¹, Aslan G.¹, Coskun J.¹, Coskun A.², Unsal I.²

¹Acibadem Labmed Clinical Laboratories, Biochemistry, Istanbul, Turkey

²Acibadem University, School Of Medicine, Dept. of Biochemistry, Istanbul, Turkey

Inborn errors of metabolism (IEM) are uncommon diseases and resulting in significant morbidity and mortality. Early recognition and providing therapeutic measures could improve clinical outcome, decrease morbidity and mortality. The growth of MS/MS in clinical laboratories primarily occured in two areas one of which is human genetics and metabolism, mainly in IEM and newborn screening. Unlike measuring single analyte, MS/MS measures more than 40 analytes by single assay. Acibadem Labmed Clinical Laboratories were established in 2002 and provide laboratory services in a multidisciplinary manner. Newborn screening by MS,MS started in 2006. Between December 2006 and December 2008, 9,911 babies (5,046 males, 4,865 females), aged less than 1 month were screened. Fortysix percent of the babies were screened by using the samples taken 2 days after the birth and screening of 86% of the babies completed in the first week of the birth. Phenyalanine and tyrosine levels were found to be increased above cutoff levels (150 μmol/L and 260 μmol/L) in 12 and in 63 babies respectively. Only 6 babies had increased ratio of Phe/Tyr (>1,7). Branched chain amino acids were found to be higher than those of cut-off levels in 13 babies. On the other hand, acylcarnitine screening revealed possible carnitine uptake defect in 11 babies by means of decreased free carnitine levels. In 9 of them, decreased levels of acetyl and propionyl carnitine levels accompanied to low free carnitine levels. Moreover, isovaleryl, hexanoyl, 3-OH isovaleryl and tetradecenoyl carnitines were the most prominent acylcarnitines that found to be higher than those of upper reference ranges. In the presentation, disorders of amino acid metabolism and fatty acid oxidation will be evaluated and some aspects of pre-analytical issues will be addressed.

29. Evaluation of the commercially available Chromsystems Newborn Screening Kit (non derivatised) for the measurement of acylcarnitines and amino acids

Stopsack M.¹, Merk M.²

¹University Childrens Hospital TU Dresden, Metabolic and Screening Laboratory, Dresden, Germany

²Chromsystems Instruments&Chemicals, Research and Development, München, Germany

Aim of the study was the analytical and diagnostic evaluation of the non derivatised Chromsystems Newborn Screening (NBS) tandem mass spectrometry (TMS) Kit comprising reagents, disposables and dried blood spot controls. TMS is the method of choice for identifying inherited metabolic disorders and for simultaneously profiling of acylcarnitines and amino acids. As NBS requires a high throughput method, sample preparation should be easy and fast. Sample preparation includes dried blood spot extraction and filtration of the eluate in a second microplate. Measurement was performed with Waters Quattro Micro TMS system in MRM mode. We evaluated cut-off levels, diagnostic sensitivity and specificity, interassay variation and accuracy: Phase I “cut off”: Measurement of more than 1,000 random blood samples of newborns and calculation of cut off levels as the 99.9th percentile of the entire value distribution. Exclusion criteria: samples of confirmed patients, samples before 36 hours of life and/or before 32 weeks of gestation Phase II “sensitivity”: Additional 18 true positive samples from known patients were randomly mixed with 1,000 further blood samples and subjected to measurement. Evaluation was performed according the recovery of true positive samples Phase III “specificity”: Positive results from phase II were tested again. Basing on the new results a recall rate was calculated.

Results: 1. The interassay CVs for the screening metabolites were between 7.8 and 17.2%. The accuracy of control measurement was between 88 and 131%. All external quality control samples from CDC Atlanta met required values. 2. The diagnostic sensitivity was 100% for true positive samples from patients with metabolic target defects. 3. The recall rate after repeated positive measurement for one or more metabolites accounts to 0.3%, e. g. a specifity of 99,7% was achieved.

Conclusion: The non derivatised NBS method is easy to handle, reliable, fast and very well suited for routine measurement.

30. Screening of newborns and older children for inborn errors of metabolism by tandem mass spectrometry in Nuevo Leon, Mexico

Torres M. R.¹, Villareal L. E.¹, Ruiz C.¹, Bernal M.¹, Rangel E.¹, Gonzalez R.¹, Villareal P. J. Z.²

¹Depto de Genetica, Fac. de Medicina, Monterrey, Nuevo Leon, Mexico

²Servicios de Endocrinologia, Hospital Universitario, Monterrey, Nuevo Leon, Mexico

Introduction: Acylcarnitines (ac) and aminoacids (aa) profiling from dried blood spots by MS/MS has been recognized recently as a useful tool in the biochemical diagnosis of inborn errors of metabolism (IEM). This report describes the ongoing effort to identify more than 30 metabolic disorders by MS/MS in Monterrey N.L., Mexico since 2003.

Objective: To know the prevalence of IEM in our state.

Methods: Blood spot was collected in newborns between 24 hours and the first week of life, and other samples from children of older than one month old were also received. One punch (3,2 mmID) of dried blood spots was extracted with methanol containing isotopically labeled aminoacids and acylcarnitines internal standards, and derivatized with Butanol/HCL. The butyl esters from aa & ac, were analyzed in a MS/MS API 2000, PerkinElmer Life Sciences. Data were acquired by full scan and some of them by multiple reaction monitoring mode. The diagnosis was confirmed by repeating the aa & ac profile, urine organic acids, or plasma aminoacids analysis, in suspected cases; and in some of them, the molecular testing was also performed.

Results: Approximately 60,000 neonates and children were screened. In neonatal screening 6 organic acidurias, 6 aminoacids, 2 fatty acid oxidation, and 2 carbohydrates disorders were detected. The estimated incidence in neonates was approximately 1:5,000. About 7 aminoacid disorders and 6 organic acidurias were confirmed in children older than one month of age.

Conclusions: The relatively normal development of patients with metabolic disorders among newborns demonstrates the usefulness of newborn screening by MS/MS for early diagnosis and medical intervention, because MS/MS is highly sensitive, reproducible, and suitable for IEM detection.

31. Development of an automated galactose-1-phosphate uridyl transferase (GALT) assay for newborn screening of classical galactosemia

Vaisanen V., Tuomola E., Tamminen J., Karvonen H., Seppala J.

PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland

Objectives: The objective is to develop an automated Neonatal GALT kit to be used with the new Genetic Screening Processor (GSP^™). The assay is intended for the semi quantitative determination of enzymatic activity of galactose-1-phosphate uridyl transferase (GALT) in blood specimens dried on filter paper. The new design is based on the PerkinElmer Neonatal GALT kit NG-1100, but the components have been modified to be GSP compatible and easy-to-use. Additionally, the calibration units have been changed to U/dL blood.

Methodology: The new assay design under development uses a clear microtiter plate to allow both visual and automated verification of the presence of a blood spot sample. Calibrators and controls are provided as individually packed cassettes. A set of assay reagents (buffer and substrate) contains enough components for running four 96-well plates. The kit includes three assay reagent sets (for 12 plates). After punching, the assay is completely automated, and from plate loading to completion. A set of four plates can be run in four hours.

Results: The calibration curve is linear and spans 25 enzymatic units/dL blood. The GALT activity of an adult deficient sample is about 1 U/dl. An adult carrier has a GALT activity of 7 U/dL and adult normal samples up to 20 U/dL. The automated assay has an improved precision and dried blood spot samples typically have a within lot CV below 10%.

Conclusions: The analytical performance of the automated GSP GALT kit is equal to that of the manual Neonatal GALT kit NG-1100. The assay is anticipated to be suitable for its intended use as an aid in screening newborns for classical galactosemia. The changes made to the assay design and calibration allow a linear calibration curve, improved assay precision and shorter incubation time when compared to kit NG-1100.

32. Fluorimetry and mass spectrometry for the determination of lysosomal enzyme activities in dried blood – possibilities for neonatal screening

Lukacs Z.¹, Nieves Cobos P.¹, Keil A.¹, Hartung R.², Mengel E.², Beck M.²

¹Hamburg University Medical Center, Institute of Clinical Chemistry, Hamburg, Germany

²University of Mainz, Villa Metabolica, Mainz, Germany

A few years ago a new method for the determination of lysosomal enzyme activities in dried blood has been established by Chamoles et al. To allow the simultaneous assessment of different enzyme activities a mass spectrometric assay with easily-ionizable substrates has been developed. Recently, the substrates became available through the CDC. In our study, we have validated the mass spectrometric assays and evaluated its application for the diagnosis of Gaucher-, Fabry- and Pompe disease in comparison to its respective fluorimetric counterparts. Finally, a feasibility study for the screening of Pompe disease using the fluorimetric substrate has been carried out. In summary, between-run CVs have been below 20%. All further assay parameters have also been satisfactory. Concerning the diagnosis of patients, the MSMS assay performed very well and all patients have been positively identified. For the fluorimetric Pompe test the same positive results have been obtained. In contrast, one female Fabry patient has been missed in the fluorimetric test while the α-galactosidase activity has been in the upper reference range of the MSMS test (432 pmol/spot*20 h; ref. range >480 pmol/spot*20 h). The fluorimetric test for Gaucher disease failed to pick up several patients. As Pompe disease, in our opinion, qualifies most for neonatal screening, we have used a simplified protocol to screen 3251 newborns in a feasibility study. Less than 0.5% of the samples had to be repeated and no second samples had to be requested. In conclusion, the MSMS assay has proven more sensitive while the fluorimetric tests are adequate for the diagnosis of patients with the exception of Gaucher disease. The fluorimetric test for Pompe disease is a robust assay that can easily be performed in a newborn screening laboratory. Thus, technically newborn screening for Pompe disease is feasible using either of the available methods.

33. Newborn screening for Pompe disease in Taiwan

Hwu W.-L., Chien Y.-H., Lee N.-Ch.

Department of Pediatrics and Medical Genetics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

Pompe disease is a rare lysosomal storage disease. Because of the deficiency of acid alpha-glucosidase (GAA) activity, excessive glycogen storage in the muscular system causes respiratory failure, cardiac failure, and gastrointestinal dysfunction. A recent natural history survey revealed a higher prevalence of infantile-onset Pompe disease in the Taiwanese, probably due to a founder mutation in the GAA gene. Recently, recombinant human GAA is available for the treatment of Pompe disease. Current experiences demonstrated that the treatment effectively rescued the heart, but many infants had poor responses in their skeletal muscles. In order to improve the treatment, we initiated a pilot screening program in Taiwan. The screening employed regular Guthrie card, a fluorescence substrate, and acarbose as an inhibitor. A ratio between GAA and another reference enzyme was helpful to overcome quality issues of the blood spots. Because of a high prevalence of the pseudodeficiency GAA mutation, the diagnosis needed to be confirmed by skin fibroblast GAA activity assay. From 206,088 newborns, five babies were found to have low GAA activity and hypertrophic cardiomyopathy before age one month. Enzyme replacement was immediately started. Six to 24 months after treatment, they all had normalization of cardiac size and muscle pathology with normal physical growth, and age-appropriate gains in motor development. This pilot study proves the value of newborn screening in the treatment of infantile-onset Pompe disease. Pompe disease has been added as an optional newborn screening item in Taiwan since February 2009.

34. Krabbe disease screening in New York State

Orsini J. J., Saavedra C., Helton L., Caggana M.

New York State Dept. of Health, Wadsworth Center, Albany, United States

Purpose: Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Symptoms include irritability, unexplained fever, limb stiffness, and seizures, feeding difficulties, reflux, and slowing of mental and motor development. Most patients have the infantile form, which is generally fatal before two years of age. Currently, the only treatment is cord blood transplantation. The New York State newborn screening program was mandated to implement statewide screening of all newborns for KD in 2006.

Methods: All newborn specimens were assayed for GALC activity using tandem mass spectrometry. All newborns with activities <12% of the daily mean were subjected to molecular analysis. Samples with either very low enzyme activity and/or one or more mutations were referred for follow-up diagnostic testing using a more conventional method and a neurological exam, if warranted.

Results: Activities of newborns ranged from 0.17–175 μmol/h/L (mean = 4.06 μmol/h/L, N = 676,857, σ = 3.13 μmol/h/L) for the period (8/7/06–02/02/09). Of the newborns screened, 291 were subjected to second-tier molecular testing. Of those, 118 were referred for diagnostic testing; seven were considered at high risk for Krabbe. Two infants had enzyme activities and mutations consistent with infantile KD; clinical and neurodiagnostic testing showed significant abnormalities and both underwent cord blood transplantation. Fourteen additional infants were determined to be at risk for late-onset KD and are being followed clinically.

Conclusions: New York’s screen positive rate is 0.017% and the positive predictive value is 14%. To date, the program has identified sixteen infants with or at risk for Krabbe disease.

35. CDC’s quality control program for lysosomal storage disorder newborn screening

De Jesus V. R., Zhou H., Vogt R. F., Hannon W. H., Mei J. V.

Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States

Lysosomal storage disorders (LSDs) are a group of over 40 genetic disorders caused by inborn errors of metabolism. Individuals with LSDs are characterized by low activities of particular enzymes found in the lysosome, resulting in the accumulation of molecules that are meant to be processed by these enzymes. While LSDs are each rare diseases, as a group they are estimated to affect 1 in 7,700 live births worldwide. Most LSDs are progressive and life threatening; if left untreated, they can cause physical debilitation, mental retardation and early death. At present, New York is the only state performing newborn screening for any LSD disorder (Krabbe disease). Other states (Illinois, Washington) plan to conduct population-based pilot studies using tandem mass spectrometry. Several international laboratories are currently evaluating the tandem mass spectrometry-based (MS/MS) assay in their laboratories. To support this important global public health effort, the Newborn Screening Translation Research Initiative at the Centers for Disease Control and Prevention (CDC) provides reagents to be used for newborn screening for selected LSDs by MS/MS. In addition, quality control (QC) dried blood spot (DBS) materials for lysosomal storage disorders were developed at the CDC and evaluated by seven independent laboratories (domestic and international) using the MS/MS method and a fluorometric method to investigate their suitability for LSD assay monitoring. Five laboratories submitted data using the MS/MS method, and two laboratories submitted results using the fluorometric method. Preliminary results show good agreement among data submitted by participating laboratories, with coefficient of variation values less than 15% within methods. However, differences in lysosomal enzymatic activity values were observed when the MS/MS and fluorometric methods were compared. This may be attributed to the use of different calculation algorithms and/or unit differences. We found that the DBS QC materials were suitable for LSD assay monitoring, and they are being certified for use with both analytical methods reported by the participants.

36. Pilot study for neonatal screening for Fabry disease

Colon C.¹, Alonso-Fernandez J. R.¹, Cocho J. A.¹, Moure J.¹, Rana-Diez P.², Barros F.², Fraga J. M.¹

¹Complexo Hospitalario Universitario de Santiago. Universidade de Santiago, Unidade de Metabolopatias. Departamento de Pediatria, Santiago de Compostela, Spain

²Fundacion de Medicina Xenomica, Medicina Molecular, Santiago de Compostela, Spain

Introduction: Fabry Disease is a glycosphingolipids catabolism congenital error due to a deficiency of alpha-galactosidase-A (GLA), coded by the GLA gen located in X chromosome. The bibliography estimated incidence is 1:117,000 newborn. It is defined as the second most prevalent lysosomal lipids storage disease. Recent studies (Spada et al., AJHG July 2006) indicate that Fabry Disease is underdiagnosed demonstrating that neonatal incidence may fluctuate 1:3,100 to 1:4,600.

Material & methods: We adapted the N. Chamoles technique (Clin. Chim. Acta. 2001;308:195–6) to the screening laboratories methodology. So we used blood samples impregnated on Whatman 903 paper, were cut 1.8 Ø discs (about 3.4 µL of blood). Discs were disposed in microtiter plates suitable for reading fluorescence. The cases with low enzyme activity were processed for two-way direct sequencing for the gen GLA 7 exons, obtaining DNA from the same sample used in screening. We have analysed a total of 9,000 neonates.

Results & conclusions: We have found low GLA activity levels in 60 cases (42 males, 18 females). In 4 cases (3 males, 1 female) it was not possible to carry out genetic confirmatory test. From the 56 cases with complete confirmatory study, in 33 cases (20 males, 13 females) we do not found any variation in the gene GLA. In 23 cases (19 males, 4 females) we were found the following variants: A143T 5 (1F, 4M), R118C (2M), D313Y (3M), intronic variants 13 (3F, 10M). In total 23 variants (4F, 19M). The procedure is shown valid and may be acceptable by the screening laboratories because may be adapted to neonatal screening routine, is simple and cheap, with a similar protocol that Cystic Fibrosis screening. If we can confirm that the 7 cases with variants A143T and R118C (associated with Fabry’s disease) have a family history with the disease. We would be faced with a birth prevalence of 1:1,300 newborns or 1:700 male births.

37. The application of activity ratio in screening for Pompe disease using florescence substrate

Hsu L.-W., Chen L.-Ch., Chiang S.-Ch., Hwu W.-L., Chien Y.-H.

National Taiwan University Hospital, Department of Medical Genetics, Taipei, Taiwan

Background: Pompe disease is caused by deficient acid β-glucosidase (GAA) activity. Recent data reveal that newborn screening for infantile-onset Pompe disease (IOPD) could lead to an earlier diagnosis of the patients.

Objective: We aimed to establish a proper cut-off to better detect those patients with IOPD, to minimize the false-positive rate while avoid false-negative result.

Methods: Screening for Pompe disease was conducted since Oct. 2005 using 4-methylumbelliferyl-β-D-glucoside as the substrate and acarbose as an inhibitor to measure GAA activity in DBS for newborns three days of ages. Total neutral glucosidase activity (NAG) was measured with the same substrate in neutral condition. We employed a two-tier assay for the 1st DBS. Confirmatory assays were conducted for highly abnormal 1st DBS, or abnormal data from both the 1st and the 2nd DBS. Data from the 1st DBS were analyzed retrospectively.

Results: From Nov. 2005 to Feb. 2008, 206,426 newborns were screened and 6 cases of IOPD were diagnosed. No false-negative case was noted during this study period. If we employ just GAA activity as the cut-off, a cut-off of 10% of Normal Mean would include all cases while give a false-positive rate of 0.2% and give a positive prediction rate (PPV) as 1%. If we employ both cut-off of GAA activity of 10% of Normal Mean and ratio of 60 in the 1st DBS, we would give a false-positive rate of 0.01% and the PPV as 21%.

Conclusion: The two-dimension cut-off which includes both GAA activity and the ratio between GAA and another unrelated enzyme gives the best result for newborn screening of Pompe disease. The ratio cut-off is more effective than the activity cut-off. Direct detection of cases without the second DBS is possible if only classical cases of IOPD are targeted.

38. Plasma chitotriosidase activity in children with lysosomal storage disorder

Sheth J.

FRIGE’s Institute of Human Genetics, Biochemistry and Molecular Biology, Ahmedabad, India

Chitotriosidase (ChT) is a newly identified enzyme that is selectively elevated in tissue macrophage. It is found to be raised in Gaucher and other lysosomal storage disorders. This makes ChT the potential screening test for certain LSDs. Present study is to demonstrate the utility of ChT as a predictive screening test in the diagnosis of various LSDs. Twenty healthy children in the age range of 10 days to 5 yrs and 129 children in the age range of 2.5 months to 13 yrs with regression of milestones, skeletal dysplasia, neuroregression and hepatosplenomegaly were selected for plasma ChT activity using fluorogenic substrate. Confirmative diagnosis was carried out by specific lysosomal enzyme study from the leukocytes or fibroblasts. Plasma ChT was 55.21 ± 20.81nmol/ml/hr in twenty healthy age matched controls. Raised ChT level was observed in 43 of 129 children [33.33%] ranging from 213.74 to 23,511.40 nmol/ml/hr. They were divided in Group1–IV depending on ChT activities. Further confirmation of LSDs by lysosomal enzyme has shown Morquio-B, Sly Syndrome, Metachromatic Leukodystrophy [MLD], GM1 & GM2 Gangliosidosis, Niemann Pick disease A/B [NPD-A/B] and Krabbe disease. 86 of 129 children [66.66%] showed normal ChT activites, however 14 of these children (10.85%) were found to have LSD’s like Morquio, Pompe, MLD, Maroteaux Lamy, GM2 gangliosidosis and NPD-C. The moderately raised activity of Plasma ChT can be utilized as a positive predictive test for certain LSDs. Those with marked elevated ChT have confirmed NPD-A/B or Gaucher disease making it a specific diagnostic test for this group of disease.

39. Optimization of α-L-iduronidase activity assay in dried bloodspots by tandem mass spectrometry

Zhang K.¹, Chuang W.-L.¹, Elbin C.¹, Cooper S.¹, Olivova P.¹, Keutzer J.²

¹Genzyme Corporation, Therapeutic Protein Research, Framingham, United States

²Genzyme Corporation, LSD Therapeutic, Cambridge, United States

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase (IDUA; EC 3.2.1.76), which is the lysosomal enzyme that hydrolyzes unsulfated α-L-iduronosidic linkages in the glycosaminoglycans dermatan sulfate and heparan sulfate. The Gelb group at the University of Washington developed a mass spectrometry-based assay to measure IDUA activity in dried bloodspots on filter paper (DBS). To optimize enzyme reaction conditions and procedures for the assay, substrate (S) and internal standard (IS) were manufactured at a large scale. To enhance the separation of samples from MPS I patients and healthy adults, we varied the amount of DBS extract, concentration of S and IS, assay incubation time, sample clean-up procedure, and mass spectrometer operation. We will report the validation of a high-throughput multiplex enzyme assay using DBS to measure five lysosomal storage disorders enzyme activities, including Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease. Application of the optimized assay to measure IDUA activity in samples from healthy adults, healthy newborns, and MPS I patients as a single disorder assay and in the multiplex assay format will also be discussed.

40. Screening for congenital hypothyroidism – new evidence towards a European consensus?

Krude H., Blankenstein O., Grüters A.

Inst. for Experimental Paediatric Endocrinology, Charité-Universitätsmedizin Berlin, Germany

Since the ESPE published a guideline about Neonatal Screening programs for Primary Congenital Hypothyroidism in 1999 new technical and medical knowledge is available which might give reason for a re-viewing and updating of that guideline. As new data is available about the clinical impact of central hypothyroidism which might be applicable for other less severe forms of congenital hypothyroidism as well an expansion of the target diseases is to be discussed. On the other hand technical possibilities to widen the spectrum of target diseases have been described and used within large screening programs. New findings for a higher incidence of central hypothyroidism might put questions to those TSH-only based screening programs while the clinical impact is increased because this opens the possibility to detect life-threatening multiple pituitary deficiencies as well. Drawbacks for implementing screening for central hypothyroidism on the technical side are the less advanced assays to detect free T4 as well as the difficulty in the confirmatory diagnostic procedures for central hypothyroidism might be diminished by recent findings from the Netherlands, which argue for the usefulness of a modified TRH-test to confirm the diagnosis of central hypothyroidism. Similar to the experience in the metabolic screening programs new technical possibilities of MS/MS diagnostic are emerging to the field of thyroid diagnostic as well although not yet used in larger screening programs. Whether the available data is sufficient for re-viewing of the guideline or whether more studies are needed to gain evidence will be discussed.

41. Thyroid screening in mothers of children from the Neonatal Thyroid Screening (NTS) Program

Stoeva I.¹, Koleva R.², Antova R.¹

¹University Pediatric Hospital Sofia, Screening and Functional Endocrine Diagnostic, Sofia, Bulgaria

²First Diagnostic and Consultation Center Stara Zagora, Pediatrics, Stara Zagora, Bulgaria

Insufficiently controlled maternal thyroid disease (TD) increases the risk for thyroid disturbances in the child as well. Environmental factors, like iodine deficiency (ID)/supplementation influence also the prevalence of TD in the mothers during pregnancy and after delivery. Our aim was to compare parameters of the thyroid antibody and functional status in a special subgroup – mothers of children with pathological results from the NTS Program during mild ID and after 3 years of iodine sufficiency (declared 2005 by the ICCIDD) in Bulgaria. A total of 420 children born 1993–2008 were picked up by NTS and treated. In 348 (82.9%) of their mothers thyroid function (TSH, T4, fT4 by DELFIA^®) and antibody status (TAT, MAT, by Elisa) were investigated. From 1997–2008 repeatedly 224 (64.4%) of all investigated mothers participated in the study. The proportion of maternal TSH >4.5 mU/l decreased slightly (from 20 to 17%), all other parameters showed an increase comparing 2003 (mild ID) with 2008 (iodine sufficiency): fT4 <9 pmol/l from 1.7 to 2.1%; TAT >100 IU/l from 6.7 to 17%; MAT >100 IU/l from 1.7 to 4.2%. The maternal thyroid disturbances could be explained by: 1. improvement of diagnosis in the preclinical stage; 2. possibility of transient increase of latent existing (because of former ID) autoimmune thyroiditis; 3. combination of other genetic/environmental factors. The maternal prospective study will be continued and extended.

42. The Italian National Register of Infants with Congenital Hypothyroidism: surveillance, research and prevention of the disease

Olivieri A.¹, Medda E.², Fazzini C.¹, Stazi M. A.²

¹Istituto Superiore di Sanità, Dipartimento di Biologia Cellulare e Neuroscienze, Roma, Italy

²Istituto Superiore di Sanità, Centro Nazionale di Epidemiologia, Sorveglianza e Promozione della Salute, Roma, Italy

The Study Group for Congenital Hypothyroidism, Italy

In Italy the nationwide newborn screening programme for congenital hypothyroidism (CH) began in 1977 and the 100% coverage of neonatal population has been achieved since the 90’s thanks to an efficient network of 26 regional and inter-regional screening, diagnosis and follow-up Centres. All the Centres participate in the Italian National Registry of Infants with CH (INRICH) which performs the nationwide surveillance of the disease. The aim of the Register is to monitor efficiency and effectiveness of neonatal screening, to provide disease surveillance and to allow identification of possible aetiological risk factors for the disease. During the past twenty years the active and continuous collaboration between the Register and the Italian Centres for CH allowed to perform a standardization of screening procedures and considerable improvements in the time at starting treatment (median value: 19 days between 2000 and 2004) and in the dose of therapy (median value: 9.6 µg/Kg/day between 2000 and 2004). Moreover, the high number of CH infants with confirmed diagnosis recorded in the INRICH (about 3,800 at the end of 2007) has allowed to perform a robust estimation of the CH incidence in our country. This results to be 1:2,400 live borns (1995–2003) and is based only on cases with permanent CH (40% ectopy, 26% agenesis, 34% eutopic thyroid). Moreover, for the first time it has been possible to estimate the CH incidence in multiple (10.1/10,000 live births) and single deliveries (3.2/10,000 live births) separately, although a low pairwise concordance rate (11.0%; 22% monozygous, 7% dizygous) was found. On the basis of our Italian experience, we are confident that the possible availability of a central European wide database on CH in a near future would represent a potent tool of surveillance, epidemiological research and knowledge on CH, and that the INRICH could contribute to achieve this goal.

43. Congenital hypothyroidism in Czech population: current approach to diagnosis and follow-up

Al Taji E.¹, Kracmar P.¹, Banghova K.², Krude H.³, Hnikova O.¹

¹3rd Faculty of Medicine, Charles University, Dpt. of Paediatrics, Prague, Czech Republic

²2nd Faculty of Medicine, Charles University, Dpt. of Paediatrics, Prague, Czech Republic

³University Children’s Hospital Charité, Institute of Experimental Paediatric Endocrinology, Berlin, Germany

In the Czech Republic, nation-wide neonatal screening for congenital hypothyroidism (CH) was established in 1985. TSH levels has been measured since 1996 (cut-off 15 mIU/l). Otoacoustic emission testing is compulsory for all CH patients till 3 months of age. In our study, we aimed to describe the frequency of CH due to thyroid dysgenesis and dyshormonogenesis, distinguish syndromic and non-syndromic forms, uncover underlying genetic defects, and establish up-to-date algorithms for diagnosis and clinical care of paediatric and adolescent patients affected by CH. Population-based cohort of 182 patients with primary permanent CH detected by neonatal screening in 1985–2002 was collected, thoroughly characterized and analysed for mutations in several candidate genes. The TPO (thyroid peroxidase) gene, PDS/SLC26A4 gene (encoding pendrin), and PAX8, NKX2.1, NKX2.5, FOXE1 and HHEX genes, encoding thyroid transcription factors, were analysed for mutations with a focus on clinically defined subgroups of patients matching the phenotypes of previously described candidate gene mutations. Two patients with thyroid dyshormonogenesis and sensorineural hearing impairment were identified as compound heterozygotes for PDS/SLC26A4 gene mutations. Two patients with non-syndromic goitrous CH were diagnosed as compound heterozygotes for TPO mutations. The very low frequency of genetic defects in a population-based cohort of children affected by CH, even in a phenotype-focused screening study, suggests the pathogenetic role of either non-classic genetic mechanisms or the involvement of so far unknown genes. The establishment of precious clinical diagnosis (including the type and severity of CH, the occurrence of associated congenital malformations and functional impairments), optimally followed by molecular-genetic diagnosis, enables correct treatment and comprehensive long-time follow-up, counselling and long-life psychosocial support.

Acknowledgements: Supported by Research Project MSM 0021620814.

44. High incidence of permanent and transient congenital hypothyroidism in premature babies

Chen H.-Ch.¹, Chang H.-Y.¹, Hsu L.-W.¹, Chien Y.-H.², Hwu W.-L.²

¹National Taiwan University Hospital, Medical Genetics, Taipei, Taiwan

²National Taiwan University Hospital, Medical Genetics and Pediatrics, Taipei, Taiwan

The diagnosis of hypothyroidism in the premature babies is always a dilemma because of the multiple confounding factors like stress, iodine exposure, and immature in organization. Moreover, we have noted a recent increase in the incidence of neonatal transient hypothyroidism (NTH) in newborn screening in Taiwan. We want to inspect the occurrence of hypothyroidism in the premature babies, including permanent hypothyroidism and NTH. From Jan. 2006 to Dec. 2006, 90,157 newborns older than 48 hours of age were screened by the Newborn Screen Center at National Taiwan University Hospital. Thyroid stimulated hormone (TSH) in dry blood spots (DBS) was measured. 2,552 newborns (2.8% of the total screened population) had birth weights less than 2,200 g and were classified as prematurity. Among them, 82 (3.2%) had elevated TSH levels, whereas only 2.43% of newborns with normal birth weights were screened positive (p=0.0136). A 2nd DBS was obtained from those who were positive for the first screening, and 20.73% of the premature babies and 9.7% of normal babies still had TSH elevation (p=0.0021). Confirmatory test showed that 9 premature babies (0.35% of the 2,552 premature newborns) and 67 (0.08%) normal babies had both elevation of TSH and decrease of thyroxine (p <0.0001). However, follow-up of the 9 affected premature babies revealed transient hypothyroidism in 4 (stop thyroxin after 3 years of age) and permanent hypothyroidism in 2. Three babies were still younger than 3 years. In the premature newborns, the incidence of transient hypothyroidism would be 1:365 to 1:638 and the incidence of permanent hypothyroidism would be 1:1,276, both higher than the incidences of hypothyroidism in the normal newborns. Since the differentiation between the two types of congenital hypothyroidism is difficult at the newborn stage, a well-monitored screen and treatment protocol for the premature newborns should be necessary.

45. Neonatal thyroid screening in the Republic of Macedonia (2003–2008)

Anastasovska V., Kocova M.

Pediatric Clinic, Endocrinology & Genetics, Skopje, Macedonia

Congenital hypothyroidism fulfils criteria for nationwide screening. The program for neonatal thyroid screening has started in 2002 as a pilot screening in the capital city of Skopje and two larger town nurseries (Prilep and Bitola). Activities encompassed the initial education of the nurses for sampling and a communication structure with the university hospital where the diagnosis and treatment are performed. International accreditation of the central laboratory for screening has been obtained. Gradually, all 29 nurseries from the entire territory of the Republic of Macedonia were included into the screening program. The program is regularly financed by the Ministry of Health. Here we present the results of the screening during the period 2003–2008. The blood spots were obtained on the day 2–5 after birth on the filter paper Schleicher & Schull 903. TSH levels were determined using DELFIA neonatal hTSH time-resolved fluoroimmunoassay on 1234 DELFIA research fluorometer. Cut-off value was 15 mU/L. During a period of 6 years out of 100,394 newborns 93,683 have been screened. Thirty one newborns with congenital hypothyroidism were detected. The incidence of the disease was 1:3,331, comparable to the surrounding countries. Therapy with levothyroxin was started on the day 12 average (range 4–22 days). The coverage of the screened newborns increased from 88.1% in 2003 up to 98.2% in 2008. The number of nurseries with 100% coverage increased to 48.5% in 2008. It seems that the good organization and communication, as well as the size of the country where the largest distance is 200 km, provide conditions for successful screening. In order to further increase the coverage, activities are undertaken for education of midwifes in the patronage service to cover the babies born at home. The education continues for the doctors, nurses and public through brochures, texts and media.