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Journal of Applied Sciences Research, 5(10): 1425-1435, 2009 © 2009, INSInet Publication Preliminary Results of Egypt Experience for Use of Tandem Mass Spectrometry for Expanded Metabolic Screening 1 Fayza Hassan M.D. 1Fatma El Mougy M.D, 1 Iman Mandour M.D, 2Sawsan Abdel Hady MD, Laila 2 Selim M.D, 1 Sahar Abdel Atty M.D, 1Marian Morgan M.D, 2 Fadia Salem 2 M.D, Azza Oraby M.D 1Dina Mehaney MSc, 1 Mohammed Abdel Monem MSc, 3Ibrahim El Nekhely, MSc and 3Nadia Moharam MD 1 From the clinical and chemical pathology and 2pediatrics departments, 3 Faculty of Medicine, Cairo University and Ministry of Health and Population Abstract: It is suspected that metabolic disorder rates among Egyptian newborn has high incidence due to the prevalence of consanguinity (38%), where NBS for metabolic disorders is neither mandatory nor available. This pilot study is the first attempt to elicit prevalence of metabolic disorders in Egypt by tandem mass spectrometry (MS/MS). Subjects and Methods: 16000 neonates (3-7 days) and 550 children (3 months to 15 years) presenting to the neuro-metabolic outpatient (OP) clinic, Cairo University Children Hospitals (CUCH) underwent expanded metabolic screening by MS/MS. Dried blood spots samples –were extracted, derivatized and submitted to MS/MS analysis. Amino acids & acylcarnitine values were calculated. Method validation was achieved through participation in CDC NBSQA program and our own QC samples. Results: From the 16000 screened neonates, 10 cases were detected (1/1600). Four PKU (1/4000); 1case MSUD (1/16000), 5 cases with abnormal acylcarnitine profile approximately (1/3000). Accordingly, the first Egyptian reference values for expanded screening by MS/MS were calculated. From 550 patients presenting to the neurometabolic clinic, 24 patients with metabolic disorders were detected approximately (1/23). Fourteen PKU cases (1/39), 2 MSUD (1/275), 1 patients with citullinemia(1/550) , 2 homocystinuria (1/275) and 5 cases with abnormal acylcarnitine profile (1/110). Conclusion: MS/MS technology was effective in identifying several metabolic disorders. Overall incidence was found to be high particularly among disabled children, who could have been saved from disabilities if expanded NBS was offered. The study provided the first Egyptian reference values for amino acids and acylcarnitines measured by MS/MS for newborns. Key words: TMS, newborn screening, amino acids, acylcarnitines, metabolic disorders, pilot study. INTRODUCTION Newborn screening, for the detection of inborn errors of metabolism (IEM), has long been recognized as an essential, life-saving, and effective preventive public health service. In many cases, detecting these disorders spells the difference between life and death for these babies; in other instances, identifying newborns with a disorder means that they can be treated and thus not face lifelong disability or cognitive impairment. Now, with the advent of this new screening technology, babies can be tested-and treated- for many more disorders than was previously possible. Newborn screening clearly is “more than a test” because it involves follow-up care, counseling, and other important services. Yet a critical piece of any comprehensive newborn screening program begins with the test itself and with its reliability. Laboratories, pediatricians and parents must be confident that test results are accurate and that disorders are not missed [1]. A breakthrough in technology particularly the use of MS/MS have allowed for the development of fast and inexpensive protocols to quantify amino, organic and fatty acid disorders from a single dried blood spot[2]. Developing of a comprehensive neonatal screening program was one of the major objectives of the program for prevention, early detection and early intervention in Egyptian children with genetic disability and children at risk, a project financed by the European commission (EC). Corresponding Author: Iman Mandour M.D., From the clinical and chemical pathology Iman Mandour; (+2)-0123-954-879; E-mail: [email protected] 1425 J. Appl. Sci. Res., 5(10): 1425-1435, 2009 An increasing number of reports on the use of the new technology; tandem mass spectrometry (MS/MS), as a technique for newborn screening[3] have stimulated national interest in adding MS/MS to the current newborn screening program [4]. This technology, which uses a tiny amount of blood, can rapidly identify and measure the concentrations of a large number of amino acids, fatty acids, and organic acids. This method has the potential to detect >30 rare genetic metabolic disorders [5]. This Pilot Study Aims At: 1- Setting the first Egyptian reference intervals for NBS from dried blood spots by MS/MS and the first database registry for inborn errors of metabolism (IEM) in Egypt. 2- Early detection, via newborn screening, of treatable and preventable heritable disorder s to ensur e appropriate and timely intervention. 3- Finding the prevalence of IEM detectable by MS/MS screening among children presenting with different types of disabilities (high risk group) 4- Gaining practical experience in using the new technology and to evaluate the overall effectiveness of the new test. Subjects and Methods: The sample of this study was composed of sixteen thousand neonates aged 3- 7 days and 550 infants and childr en attending the neurometabolic clinic of Cairo University Children Hospital (CUCH). Blood samples were taken by heel prick and spotted on Whatman filter paper cards and sent to the laboratory for screening by MS/MS. The blood spots were analyzed for acylcarnitines and amino acids by triple-quadruple tandem mass spectrometer (Quattro LC; Micromass, Mancheste r, UK) with a positive electrospray ionization probe. Using a BSD 400 automated Puncher, a single 3-mm disk (3 μl blood) was punched in the center of the dried blood spot, placed in flat bottomed 96-well polypropylene microtitre plate for extraction of the blood with 200 μl of methanol, containing known concentrations of a mixture of isotopically labeled internal standards of acylcarnitines and amino acids, provided by Cambridge Isotope Laboratories (Woburn,Mass., USA). The concentrations of the deuterium labeledworking internal standards per liter of methanol were as follows: 2.5 μmol/L ([2H3]-Leu); [2H3]-phenylalanine ([2H5]-Phe) ; [2H3]-methionine ([2H3]-Met); [2H4]-tyrosine ([2H4]Tyr); [2H6]-ornithine ([2H6]-Orn); [2H2]- citrulline ([2H2]-L-Cit); [2H4]-[13C1]-arginine ([2H4]-[13C1]-LArg)and 12.5 μmol/L for 15N2 glycine (15N2Gly); [2H9]-carnitine( [2H9 )C0), 0.76 μmol/L; [2H3 ]1426 acetylcarnitine 2H3C2, 0.19 μmol/L. And 0.04 μmol/L [2H3]- pro pio nylcarnitine ([2H3]-C3 ); [2H3]octanoylcarnitine ([2H3]-C8); ([2H 3]myristoylca rnitine ([2H 3]C14 while[ 2H3] palmitoylcarnitine ([2H3]-C16), 0.08 μmol/L. After 20 min of shaking, the solvent was transferred to U bottom microtiter plate, gently evaporated under a stream of nitrogen and the dried residue was derivatized with 80 μl of butanolic HCl and heated at 65°C for 15 min. The solvent was evaporated again and the residue was finally reco nstituted in 80 μl of the mobile phase (acetonitrile/water, 80/20 v/v). A 20μl sample was directly injected into the MS at a flow rate of 0.2 ml/min. A Waters 2695 HPLC with an autosampler (Waters, Milford, Mass., USA) was used for automatic injection. The run cycle time for each sample was 2 min from injection to injection. Principle of MS/MS: A mass spectrometer is a device that ionizes molecules and separates the charged products, or ions according to their mass-to-charge ratios (m/z). The instrument has two mass analyzers separated by a reaction cell. The latter contains an inert gas (argon), whose molecules collide with ions entering the cell from the first analyzer. These collisions cause fragmentation of ions. The second mass analyzer in turn separates and detects the charged products according to the chemistry of the metabolite. Electrospray ionization used is a soft ionization method which simplifies the analysis – by reducing sample preparation steps and eliminating matrix interference – as it produces predominantly intact molecular ions from the mixture [6]. ESI-MS/MS was applied to the analysis of amino acids after extraction from bloo d spots and derivatization to butyl esters. The butylated AA species give a common neutral loss (NL) of 102D upon fragmentation. This corresponds to loss of butylformate from their protonated molecular ions. It is noteworthy that glycine produces a very weak signal which can be easily missed. Six AA including asparagine, glutamine, arginine, citrulline, lysine and ornithine lose ammonia first followed by butyl formate, resulting in a total NL of 119 D [7] . Therefore to get more complete picture of AA in the sample, four constant NL scan functions were used at 102 116 119 and 56 generating a separate profile for each scan function. Normal profile at 102 and at 119 NL is shown in figures 3 and 5. Multiple reaction monitoring (MRM) experiment was performed for Phenyalanine and Tyrosine (Fig. 1). As for acylcarnitines, they were chemically J. Appl. Sci. Res., 5(10): 1425-1435, 2009 converted to their butyl esters. Electrospray ionization produces mostly intact molecular ions that undergo fragmentation in the reaction cell to produce a common fragment of m/z 85. Fixing the second mass analyzer to transmit only ions of m/z 85 and scanning the output of the first analyzer over the mass range 200500, which includes the molecular weights of all the acylcarnitines, allows a computer to generate a special type of mass spectrum that consists of the molecular masses of individual acylcarnitines and their relative abundance (Fig. 7). Data interpretation by the system used was facilitated by the use of the provided Neolynx program, where interpretation process was an objective efficient and automated process. This program allowed the introduction of m/z ratio for each analyte and its labeled form, and concentrations of internal standards added during sample preparation, whereby metabolite concentrations are automatically calculated. If the analyte is not available in the labeled form, the nearest homologue was used as a substitute for ratio calculations. The cutoff levels were first selected according to values reported by several published data (1 and 8). All sample results together with newborn or infant demographic data were stored in a database. Analytes exceeding the assigned cut off values were flagged. Butanolic HCl was obtained from Regis Technologies (Morton Grove,Ill.,USA). HPLC-grade methanol and acetonitrile were purchased from Merck. W ater wa s pur ified b y M illi-Q d evice (Millipore,Bedford, Mass.,USA). Whatmann filter paper cards (S and S 903; Schleicher and Schull, Dassel, Germany) were used to collect blood samples. RESULTS AND DISCUSSION Discussion: Inborn errors of metabolism are common in Egypt, presumably because of the high rates of consanguinity (38%) (10). This is the first study to be carried out to estimate the exact prevalence of IEM among neonates in 3 governorates in Egypt. The establishment of metabolic screening unit to diagnose and treat IEM was supported by a project funded by the EC. The introduction of ESI/MS/MS as a diagnostic tool in our metabolic unit has allowed diagnosis of aminoacidopathies and organic acidemias among both neonates and sick children referred to the metabolic clinic. ESI/MS/MS allowed high sample throughput and efficient neonatal screening method. About 360 samples were run daily. Automation of the whole process was facilitated by the use of 96-well plates, automated punching and automated data processing by Neolynx 1427 software ; however, more efficient performance could have been achieved by the introduction of barcode readers and computerized microplate mapping. At the start of our work reference ranges and cutoff values used were those used by CDC and from the literature [8,11,12,13]. One of our goals was to establish our own population-based cutoff values from a large number of normal samples recruited from an ongoing neonatal screening program. The values obtained in the present study from 16000 normal neonates are presented in table (1 and 2). Values for amino acids and acylcarnitines are very similar to those reported by Sander et al. [14] and in partial agreement with Rashed et al., [11]. Minor elevations of acylcarnitines especially calculated ratios were responsible for >50% of our flagged results. In reality these increases are of no pathological significance and fine adjustments of the cutoff values to be based on our population would minimize these false flags [11]. Proper interpretation of results was achieved by examination of the reason of flagged results and visually inspecting the spectra, thus allowing proper data analysis. For some of the flagged results retesting was done if it seemed appropriate and for abnormal profiles denoting disease. Samples were reanalyzed in the next batch to assure reproducibility of initial results. Among 16000 neonates examined through this screening program 10 cases of IEM were detected (1/1600), 5cases with aminoacidopathies (1/3200) and 5 cases with organic acidemias (1/3200), this frequency is higher than that reported by Applegarth et al., 2000 (1/4160) in Caucasian population as well as Schulze and coworkers, 2003 (1/3800) and Torres-Sepúlveda and coworkers, 2008 (1/5000) and Hostetler, 1999 (1/2500) [15,8,16,17]. This can be explained by the ethnic differences between populations studied. However our prevalence of IEM was less than that reported by Rashed and coworkers, 1997 (1/740) [11]. This may be due to the higher rate of consanguinity among the Saudi population (56%) [18] as compared to that of Egypt. Among 550 OP cases presenting to the neurometabolic clinic, 24 cases with IEM were detected (1/ 23; 4.3%) 19 cases with aminoacid disorders (1/28) and 5 cases with organic acidemias (1/110). Similar results were reported by Kumta, 2005 in India who detected 5.75% IEM among mentally retarded children [19] . Slightly higher values were reported by Xie and coworkers, 20 08 in China (11%) [20]. In Omani population, Joshi et al., 2002 detected 32% IEM by MS/MS in the metabolic unit [21]. The authors reported that parental consanguinity was twice as frequent in the study patients as compared to the general population. J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Table 1: Egyptian Reference values for AA by MS/MS MEDIAN 99th percentile 5th percentile ------------------------------------------------------------------------------------------------------------Aminoacid (µmol/L) Tyrosine (TYR) 97.89 396.3 45.4 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Phenylalanine (PHE) 45.03 84.7 30.7 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------phe/tyr 0.45 1.32 0.18 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Leucine/Isoleucine (LEU/Ile) 159.03 305.5 96.9 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Valine (VAL) 152.2 321.2 94.8 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methionine (MET) 15.9 34.16 9.6 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citrulline (CIT) 11.9 40.7 3.3 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Alanine (ALA) 253.1 511.8 160.4 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glycine (GLY) 71.14 236.4 38.4 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Proline (PRO) 7.99 18.54 4.6 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glutamine (GLU) 225 727 101.8 Table 2: Egyptian Reference Values for AC by MS/MS MEDIAN 99th percentile 5th percentile ------------------------------------------------------------------------------------------------------------Acylcarnitine (µmol/L) Free carnitine (C0) 24.38 59.22 13.76 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Acetylcarnitine (C2) 19.55 62.5 10.3 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Propionylcarnitine C3 1.94 7.02 0.84 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C3/C2 0.1 0.39 0.05 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Malonylcarnitine (C3DC) 0.06 0.16 0.02 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Butyrylcarnitine (C4) 0.29 0.86 0.12 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovalerylcarnitine (C5) 0.3 0.88 0.16 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methylcrotonylcarnitine (C5:1) 0.04 0.17 0.01 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hydroxyisovalerylcarnitine (C5:OH) 0.25 0.89 0.11 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Glutarylcarnitine (C5DC) 0.04 0.16 0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hexanoylcarnitine (C6) 0.08 0.37 0.04 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octanoylcarnitine (C8) 0.07 0.21 0.03 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Decanoylcarnitine (C10) 0.1 0.31 0.04 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C3DC/C10 0.57 2.69 0.12 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Decenoylcarnitine (C10:1) 0.08 0.22 0.03 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Tetradecanoylcarnitine ( C14) 0.2 0.49 0.04 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Tetradecenoylcarnitine (C14:1) 0.07 0.25 0.03 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hexadecanoylcarnitine (C16) 2.67 6.55 1.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Hydroxyhexadecanoylcarnitine (C16:OH) 0.05 0.13 0.01 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octadecanoylcarnitine (C18) 0.96 2.3 0.39 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Octadecenoylcarnitine (C18:1) 1.14 2.47 0.39 1428 J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Fig. 1: Normal MRM scan Fig. 2: A case of PKU as seen in a MRM scan mode Fig. 3: Normal AA profile Fig. 4: A case of MSUD Fig. 5: Normal Scan in NL 119 Fig. 6: A case of Citrulinemia 1429 J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Fig. 7: Normal AC profile Fig. 8: A case of MMA showing high C3 (46.4 µmol/L) Fig. 9: A case of IVA Fig. 10: A case with elevated C5OH Fig. 11: Urinary Argininosuccinic Acid Profile by MS/MS 1430 J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Fig. 12: Urinary Acylcarnitine Profile by MS/MS showing high methylmalonyl carnitine m/z 374.5 Cases of aminoacidopaties detected in the present study were 10 (NBS) and 19 (OP). Diseases detected were PKU, MSUD, Citrulinemia and Homocystinuria (table 3) The profile in Fig. 2 corresponds to a case of PKU where a prominent ion at m/z 222 corresponding to phenylalanine was observed, in the MRM mode, and phenylalanine/tyrosine ratio was elevated. These results are clearly consistent with the diagnosis of PKU. The profile in Fig. 4 shows a prominent ion at m/z 188 corresponding to leucine/isoleucine, a finding strongly diagnostic of maple syrup urine disease MSUD Fig. (13) shows the initial Leucine/isoleucine and valine levels among the three cases of MSUD detected. Fig. 6 shows an abnormal profile for a patient suspected of having a urea cycle disorder. The profile o f NL of 119Dshowed an abundant ion at m/z ratio 232 corresponding to butyl ester of citrulline .The level of citrulline in this patient was 646 µmol/L a finding strongly suggestive of citrullinemia. Diagnosis of citrulinemia was confirmed by argininosuccinic acid in urine (Fig. 11). An elevated methionine level of 339 µmol/L was detected in one case in the NL 102 profile with a prominent ion at m/z 206, a finding diagnostic of homocystinuria. As for the acylcarnitine profile 10 cases from the two studied groups were detected. The disorders detected were MMA, PA, isovaleric acidemia and methylcrotonylglycinuria. The distribution of diagnosed cases among the two groups is presented in table 3. The acylcarnitine profile presented in Fig. 9 is for a case of isovaleric acidemia affected with disorders of 1431 branched chain aminoacid catabolism, the profile reveals a prominent ion at m/z ratio 302 corresponding to isovalerylcarnitine with elevated C5 7.46µmol/L and C5/C2 ratio 0.67. The decision criteria for positive testing for isovaleric acidemia where C5> 2 and C5/C2> 0.06 according to Schulze et al., [8]. This preliminary diagnosis was further confirmed by urine organic acid analysis (GC/MS) where highly elevated isovalerylglycine (mono and di-derivatives) were detected. Propionylcarnitine (C3), with a prominent ion at m/z 274.5, was elevated in 6 cases, 3 of them were found to have methylmalonic acidemia after confirmation by urinary organic acid analysis by GC/MS. Urinary acylcarnitine profile with very high methylmalonylcarnitine at m/z 374.5 is shown in figure 12. One case was found to have propionic acidemia, confirmed by urinary acylcarnitine (m/z 274.5) by MS/MS and two cases from NBS were not confirmed. This was due to failure of recall of these cases for further testing. Two cases with elevated C5OH at m/z 318.2 (Fig. 10) were found to have elevated urinary 3 methyl crotonyl glycine by GC/MS, diagnosed as 3 methylcrotonyl Co-A carboxylase deficiency. It is noteworthy that no cases of fatty acid oxidation (FAO) defects in the form of MCAD, LCAD, VLCAD and MADD were found. This stands in contrast with other population based studies in-UK, Germany and USA [22,23,24] and indicate the rarity of these disorders in our country, or possibly late presentation of these disorders. J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Table 3: Cases detected among screened newborns (n=16000) and outpatient clinic patients (n=550) from January to August 2008 Detected disorders Elevated Marker Range (µmol/L) Screened Newborns OP ClinicClinical presentation Amino Acids PKU Phenylalanine (458- 2047) 4 14 Microcephaly, MR, seizures, Phe/tyr (6.3-39.5) fair complexion --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MSUD Leu/Ileu (1945- 3930) 1 2 Poor feeding, vomiting, Valine (460-900) muscle hypertonicity, seizures --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citrulinemia Citrulline (678) 1 Hyperammonemia, vomiting, diarrhea, lethargy, seizures --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Homocystinuria Methionine (149- 339) 2 Skeletal and ocular problems, MR Organic Acidemia MMA and PA C3 (10.7- 46.4) 4 2 Failure to thrive, MR, hyperammonemia, lethargy, vomiting --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovaleric Acidemia C5 (7.46- 12.89) 1 1 Poor feeding, acidosis, seizures --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methylcrotonylglycinuria C5OH (9.59- 60.1) 2 Hypoglycemia, severe metabolic acidosis --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Amino Acidemias Total 5 19 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Organic Acidemias Total 5 5 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Total Screened 16000 550 Table 4: Precision from 23 replicates of one QC CDC sample (AA and AC) Analyte CDC mean Mean (SD) mg/dl CV% Aminoacids (mg/dl) Tyrosine 13.8 11.12 (0.74) 8.6 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Phenylalanine 11.3 10.65(0.93) 8.7 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Valine 10.2 11.3(1.0) 11.5 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Leu/Ileu 14.6 11.9 (0.68) 9.3 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Methionine 5.7 4.77 11.7 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Citruline 4.8 4.2 (0.69) 17.3 Acylcarnitines (µmol/L) Free carnitine C0 251 200 (13.6) 6.8 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Acetylcarnitine C2 97.5 111.2 (12.3) 11.0 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Propionylcarnitine C3 14.25 18.6 (2.5) 13.5 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Isovalerylcarnitine C5 3.55 3.2 (0.14) 11.87 Fig. 13: MSUD Cases Detected (2 OP and 1 NBS) 1432 J. Appl. Sci. Res., 5(10): 1425-1435, 2009 Fig. 14: Participation of Egypt, represented by our lab, in the Laboratory Quality Improvement of Newborn Screening by MS/MS (Quoted from Rinaldo, 2008) (9) Fatty acid oxidation and organic acid disorders represent a group of inborn errors of mitochondrial ? oxidation and inborn errors of carnitine cycle which were not detected by conventional screening methods [8] . It was reported that the number and favorable outcome of these newborns provide the strongest argument for improved outcomes with expanded screening by MS/MS. To assure and validate the results obtained in our study participation in the Center for Disease Control (CDC) newborn screening quality assurance program (NBSQAP) where both quality control (QC) and proficiency testing (PT) dried blood spots samples were delivered to our lab and results submitted before the deadline. Replicates for intra and inter-assay precision were analyzed from the same spots of CDC QC and PT samples (table 4). CDC evaluation for our lab PT samples indicated 100% satisfactory results with no misclassification Furthermore, spiked blood samples from normal subjects with aminoacid standards phenylalanine, tyrosine, leucine and valine and samples from confirmed PKU patients were prepared and used as internal quality control samples. Our laboratory also participated in the Laboratory Quality Improvement collaborative project of Newborn Screening By MS/MS among 100 participants labs worldwide [9]. This project collects data from all participant labs to implement universal screening and confirmatory testing of newborns for inborn errors of metabolism of aminoacids, organic acids and fatty acid oxidation defects. Effective management of detected cases is established through referral of the cases to the metabolic clinic for assessment and treatment. Due to unavailability of GC/MS in our laboratory, contacts with several metabolic centers in Kuwait and USA were established to perform the confirmatory tests required for some of the abnormal acylcarnitine profiles. 1433 Conclusions and Recommendations: Difficulties encountered during the implementation of this work included the delay in DBS card receipt. This may be solved by assigning separate DBS cards for the metabolic lab to be sent promptly. PKU, a condition that silently causes neurological devastation but is amenable to early detection and effective prevention with diet control, needs raising public awareness for hazards of non compliance especially in rural areas and among low socioeconomic individuals. The high rejection rate for poor blood spots encountered (30%), calls for the necessity of training personnel on proper sample collection. It is to be noted that due to incomparable value of expanded screening by MS/MS for detection of a wide range of metabolic disorders with a high degree of reliability and high throughput and the high prevalence detected in our newborns and patients with mental and physical disabilities expanded metabolic screening by MS/MS is highly recommended and every effort should be done to implement it in our country. A back up plan for instrument downtimes should be established. The plan should include ready access to additional instruments such as MS/MS and GC/MS. ACKNOWLEDGEMENT We express our deepest gratitude to Prof. Dr. Mohammed Abdel Hamid, Faculty of Pharmacy Kuwait University; Prof. Dr. Mohamed Rashed, King Faisal Hospital, Riyadh; and Prof. Dr. Johannes Sander, Hannover, Germany for their continuous support and help throughout our work by offering their experiences and collaboration. We also would like to thank Prof. Dr. Piero Rinaldo, Mayo Clinic, USA for sincere help with establishing our reference ranges and always offering a helping hand. Our deepest gratitude also goes to Prof. Dr. Ibrahim El Nekhely and his team at the MOHP for facilitating supply of DSB samples for neonates included in this work. We are extremely J. Appl. Sci. Res., 5(10): 1425-1435, 2009 grateful to our poor affected patients who have been the greatest motive for our efforts to implement this work. Last but not least we are grateful for the administr ati on, clinicia ns, and nurse s of our participating hospitals and for our colleagues and chemists without whom this work would have not existed. 8. 9. Footnotes: Nonstandard abbreviations: MS/MS, tandem mass spectrometry; PKU, phenylketonuria; MCAD, medium chain acyl-co A dehydrogenase; QC, quality control; CDC, centre of disease control; C3, propionylcarnitine; C5, isovalerylcarnitine ; C5OH, 3-hydroxydecanoylcarnitine; PA, propionic acidemia; MMA, methylmalonic academia; FAO, fatty acid oxidation; MSUD, maple syrup urine disease; MADD, multiple acyl-coA dehydrogenase deficiency; LCAD, long chain acyl-coA dehydrogenase;; GC/MS, gas chromatography/mass spectrometr y; ESI/MS/MS, electrospray ionization/tandem mass spectrometry; MOHP, ministry of health and population; CH, congenital hypothyroidism; IEM, inborn errors of metabolism; EC, European Commision, CUCH, Cairo University Children Hospital; NBSQAP, newborn screening quality assurance program. 10. 11. 12. REFERENCES 1. 2. 3. 4. 5. 6. 7. CDC: Every Child Deserves a Healthy Start in Life! NCEH Pub November, 2005. 05-1037. 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