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Vyšlo v časopise: Čes-slov Pediat 2009; 64 (4): 210-220.

89. Ascertaining Parental Consent to Storage of Newborn Bloodspot Cards

Mackenzie J., Brown A., Fitch M., Estell A., McCaw G., Stewart A., Aitken D.

Scottish Newborn Screening Laboratory, Institute of Medical Genetics, Glasgow, Scotland

Newborn bloodspot screening for Phenylketonuria (PKU), Congenital Hypothyroidism (CHT) and Cystic Fibrosis (CF) is available for all babies born in Scotland. Testing is carried out in the Scottish Newborn Screening Laboratory (SNSL) in Glasgow. It has been policy since 1968 to store all bloodspot cards after testing. The cards are available for retesting if necessary, and research. In 2003 this policy was changed with parents being asked for written consent for testing and storage of the bloodspot card beyond an initial 12 months retention period. In 2004 a tick box was introduced on the card to assist the monitoring of parents declining storage. The number of parents declining storage before during and after the introduction of the tick box was audited. There was a marked increase in the number of parents who did not agree to storage when a tick box was introduced to the bloodspot cards (from 0.06% to 1.10%) and a decrease when these were removed (0.55%). Parents who had initially declined were contacted prior to expiry of the initial 12 months retention period to see if they still wished the card destroyed. Of the 840 responses received 270 parents (32%) changed their mind allowing their card to be placed in long-term storage. The addition of a tick box on the newborn screening card appears to be associated with an increase in the number of parents who declined long term storage of their baby’s card. The number who changed their mind is of some concern suggesting that 5-6 days immediately after giving birth is not the best time to make decisions about card storage. The single tick box, which offers only the opportunity to decline storage, may have influenced the way midwives broach this issue with parents.

90. A high frequency of private mutations in the Swedish galactosemia population

Ohlsson A., Hunt M., Guthenberg C., von Dobeln U.

Karolinska Institute, Dept of Laboratory Medicine, Stockholm, Sweden

Classical galactosemia (GAL) is caused by impaired galactose-1-phosphate uridyltransferase and leads to the accumulation of galactose, galactose-1-phosphate and galactitol in blood and tissues. If untreated, poor feeding, bilateral cataracts, jaundice, hepatic failure, sepsis and death may occur. Screening for GAL using the Beutler transferase activity measurement method (cut-off level 15%) and as second tier determination of galactose (cut-off level >0.5 mmol/L) and galactose-1-phosphate (cut-off level >1.5 mmol/L) offers a rapid and secure method to detect affected newborns. Using this combination, patients with the Duarte-variant of galactosemia are avoided. Essentially no false positive (0,3 infants per 100,000) or false negative cases have been found/reported. Since 1967, 41 patients have been detected by screening (incidence approximately 1/100 000). Treatment was initiated at an average age of 7.5 days. Mutation analyses confirmed the diagnosis in all patients. A total of 17 mutations were detected. 80% of the patients are either homozygous or heterozygous for the common p.Gln188Arg mutation (allele frequency 61%). Only four other mutations are found in more than one patient p.Met142Lys, p.Lys285Asn, p.Leu195Pro and p.Leu37Arg. Nine mutations are unique for our population, p.Arg25Pro, p.Leu37Arg, p.Pro109Pro, p.Glu160del, p.Lys334fsX25, c.377+1G>A, c.820+5G>A, c.1059+56C>T, g.1476_1479del. Three patients have mild galactosemia with almost normal secretion of galactitol in urine. They have genotype p.[Gln188Arg]+[Arg201His], p.[Arg25Pro]+ +[Gln188Arg] and p.[Pro109Pro]+[Pro109Pro] respectively. In summary screening for GAL with the Beutler method and follow up with galactose and galactose-1-phosphate in dried blood spots as described picks up only patients with classical galactosemia caused by mutations resulting in phenotypes which need treatment. This study confirms that galactosemia is caused by a heterogeneity of disease causing mutations.

91. Newborn screening for sickle cell disease in France

Bardakdjian-Michau J.

Centre Hospitalier Universitaire, Biochimie et Génétique, Creteil, France

Association Française pour le Dépistage et la Prévention de l’Handicap de l’Enfant (AFDPHE)

Newborn screening for sickle cell disease (SCD) has been performed since 1987 in selected areas. Initially universal, since 1995 it has turned to a targeted to at risk newborn screening. In 1996 the screening was made part of the national screening programs with the National Association for Neonatal Screening (AFDPHE) and has been extended to the whole country in 2000 for only at risk newborns. Sickle cell disease is a public health problem in mainland France and overseas departments. Overall incidence of SCD in France is 1/2065 newborns (2007). In the Paris area the overall incidence is 1/781. The laboratory procedure is the following: Isoelectricfocusing (IEF) from PERKIN ELMER (Agarose), Citrate Agar electrophoresis, BECKMAN (Paragon) and/or high performance liquid chromatography BIO-RAD (Variant) for confirmation of abnormal Hb. We have performed an evaluation of three automates for this screening.CE HPLC Bio Rad Variant nbs, Sebia Capillarys, Perkin Elmer AutoDelfia (PE). PE reveals only Hb A and Hb S. CE HPLC and Capillarys electrophoresis show all the common hemoglobins heterozygous, homozygous and composite heterozygotes. These two last techniques allow therefore prevention. Bio Rad Variant nbs: Robust/Strong and adapted to neonatal screening activity (high volume). Possibility to work the night (cooling sample module). An “expert“ software assigns automatically a phenotype on the patient report (FA, FS, FSC,...), we cannot add a samples microplate during the system is already runing. Sebia Capillarys: Software friendly handling: Different colors for normal or abnormal patterns. Easy validation with several graphs on the same screen. Actually low volume of reagents (a lot of replacement, not possible to run the night). Perkin Elmer AutoDelfia Fully automated, high throughput Only Hb S is detected (heterozygotes or SCD).

92. Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry

Boemer F.1, Ketelslegers O.2, Minon J.-M.2, Bours V.3, Schoos R.1

1Research associate, Centre de Génétique Humaine, CHU, University of Liège, Belgium

2Service de Biologie Clinique, CHR Citadelle, Liège, Belgium

3Centre de Génétique Humaine, CHU, University of Liège, Belgium

Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or high performance liquid chromatography to detect hemoglobin variants. However, since tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing this technology for hemoglobinopathy screening. We minimized sample preparation and analysis times by avoiding prior purification, derivatization or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C and Hb E) and β-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, four transitions with good signal intensities were selected for each specific peptide and, for each one, variant/Hb A ratios were calculated. A method validation was achieved, evaluating intra- and inter- series variability, carry-over and limit of detection. We also performed a comparative study with isoelectric focusing results on 2,082 specimens. Intra-assay imprecision values (CVs) varied between 2.5 and 30.7%. Inter-assay CVs fluctuated between 6.3 and 23.6%. Carry-over was less than 0.03% and limit of detection was fixed at 1% of hemoglobin S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E and β-globin production defects), the comparative study did not generate any discrepancy between the two techniques involved. MS/MS is a reliable method for hemoglobinopathy neonatal screening.

93. Results of the first year of universal neonatal screening for sickle cell disease in the Netherlands

Bouva M.1, Breuning-Boers J.2, Elvers B.1, Peters M.3, Loeber J. G.1

1National Institute for Public Health and the Environment, Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, The Netherlands

2TNO Quality of Life, Department of Prevention and Health, Leiden, The Netherlands

3Academic Medical Center, Emma Children’s Hospital, Department of Pediatric Hematology, Amsterdam, The Netherlands

On January 1 2007 the neonatal screening program in the Netherlands was expanded from three (PKU, CH, CAH) to 17 diseases. Besides 13 metabolic diseases, the screening on sickle cell disease (SCD) was added to the program. The HPLC technique used for SCD also detects other hemoglobin variants and carriers, but only results indicative for SCD, β-thalassemia major, HbH disease and sickle cell trait are reported. The aim is to discuss the results of the first year of screening for SCD. Dried blood spots were analyzed on the Bio-Rad Variant Newborn Screening System. Blood from a 3 mm punch was eluted in 230 µl deionized water and analyzed according to the manual. Of 182,303 screened children, 64 were referred to a pediatrician with an abnormal hemoglobin pattern (0.35‰) i.e. 41 children were diagnosed with SCD, four with β-thalassemia major and one child with HbE homozygosity, resulting in a β-thalassemia phenotype. Eighteen children were referred with a FAST peak ≥20%, indicative for HbH disease. Of these children, one had HbH disease and 13 α-thalassemia minor. The diagnosis in four children is not yet known. Another 806 children were reported to the general practitioner with presumed sickle cell trait. Also 947 children had unexpected high percentage hemoglobin A (≥40%). For 267 children (1.46‰) the high HbA could not be explained by blood transfusion or advanced age. The number of referrals for hemoglobinopathies was according to expectations. The current screening for α-thalassemia resulted predominantly in referral of children with a mild form of α-thalassemia. A study is being conducted to see if adaptation of the cut-off value will result in referral of HbH-patients only. Furthermore, 267 children had an unexpected high HbA. This could be caused by a γ-thalassemia. Further investigation will be performed to determine the cause of the high HbA levels in these newborns.

94. Evaluation of nine years AABR hearing screening in Dutch NICU’s

Verkerk P. H.1, van Dommelen P.1, van Straaten H. L. M.2

1TNO, Prevention and Health, Leiden, The Netherlands

2Isala Clinics, Neonatology, Zwolle, The Netherlands

Aim: Evaluation of the nationwide Automated Auditory Brainstem Response (AABR) hearing screening program in Dutch Neonatal Intensive Care Units (NICU) in the period 1998–2007.

Design: As part of a nationwide hearing screening program two stage AABR hearing screening has been gradually introduced in all Dutch NICU’s (1998–2001). From 1998–2007 graduates with >1 risk factors according to the 2000 Joint Committee on Infant Hearing statement were included.Central registration of results facilitates screening, tracking, follow-up after abnormal screening results and quality assurance of the program were established. Endpoints are prevalence of uni/bilateral hearing loss (HL) and referral rate after the 1st and 2nd test. Quality indicators are: participation rate 1st test (goal >98%), 2nd test (goal >95%), diagnostic examination (goal >95%), and percentage of neonates with an age corrected for gestational age at the 1st test <30 days (goal >90%), 2nd test <5 weeks (>90%), diagnosis <13 weeks (goal >90%).

Results: In total 29,110 neonates have been screened. 9.2% of the neonates did not pass at the 1st test. After the 2nd test, 2.7% (n=778) was referred for diagnostic examination. Bilateral HL was diagnosed in 61% of the referrals (prevalence 1.6%) and unilateral HL in 17% (prevalence 0.5%). The participation rates over years varied between 98.0%–99.6% (1st test), 88.5%–94.4% (2nd test), 88.6%–96.5% (diagnostic examination). 96% of the neonates had their 1st test <30 days and 77% of the referred neonates had their 2nd test <5 weeks. 67% of the referred neonates were diagnosed <13 weeks.

Conclusion: The AABR neonatal hearing screening program in Dutch NICU graduates is highly effective in terms of participation and referral rate. That not all goals of the high standard quality indicators were attained may be due to interference with severe concomitant pathology. The positive predictive value of the two stage program is 79%.

95. Newborn screening long-term follow up – case study: Improving outcome for PKU use of drug Tx with diet liberalization

Adams J.1, Adams J.2

1Canadian PKU and Allied Disorders Inc., Toronto, ON, Canada

2OZ Systems International, SARL, Toronto, ON, Canada

We report significant improvements in PKU management and outcome as a result of the first-ever drug therapy for BH4-responsive phenylketonuria. This case study is about a 22-year-old male with mild PKU after newborn screening who volunteered in an FDA-approved study and continues on therapy. Compliance with the traditional PKU diet, especially for older children, teens and adults is difficult and non-compliance at older ages is common. The key biomarker in PKU management is the plasma phenylalanine level. Elevated Phe blood levels are toxic to the central nervous system. This patient started the drug therapy on October 3, 2007 and recorded a reduction of the key biomarker of 75% after two days of treatment. The outcomes to date include persistent better control of phenylalanine levels in plasma, incremental liberalizations of the protein-restricted diet to the point of no restrictions and in May, 2008 the elimination of the burden of daily intake of medical food in the form of synthetic amino acid substitute. The patient reported, “I feel sharper and more clearheaded.” Subsequent DNA mutation analysis identified G272X and E390G. E390G has been reported as BH4-responsive. A literature search found no reports of this mutation combination before. This patient received the drug Kuvan, a synthetic form of BH4, which is required as co-factor for the key enzyme in PKU. The US FDA granted orphan drug designation to Kuvan, fast tracked its consideration and granted approval to market in December 2007. Japan approved the drug for PKU in mid 2008. Approval to market throughout Europe was issued in December 2008. Clinical trials showed that not every PKU patient responds to this drug but there is not yet any established correlation to phenotype or genotype. The only way to determine individual responsiveness is to try the drug for an extended period of several weeks.

96. Education about newborn screening dried bloodspot tests. A programme to improve the quality of dried blood spots and improve the knowledge of staff taking the specimen

Asplin D., Salmons E.

Birmingham Children’s Hospital, NHS Foundation Trust, Newborn Screening Centre, Birmingham, England

To introduce a revised teaching programme to:

  • improve the quality of dried blood spots received at the Newborn Screening Centre in Birmingham, West Midlands, United Kingdom.
  • expand the knowledge of staff taking the specimen about the conditions tested for and the process of the test.
  • increase the confidence of the staff taking the specimen
  • educate staff collecting the samples, following the implementation of new standards developed by UKNSPC.

Sessions, which included basic descriptions of laboratory methods, capillary sampling techniques and information about the conditions screened for, were held at Birmingham Children’s Hospital and local Hospitals within the West Midlands. Staff taking newborn screening dried blood spots were invited. Included were hospital and community midwives, health visitors, community children’s nurses, neonatal nurses and others. The teaching was undertaken by a nurse, working with laboratory staff, who also takes samples, in the hospital and the community.

The quality of samples received at the laboratory improved. Evaluation forms showed that midwives and others, were more aware of the need for good quality specimens, the reasons for repeat tests, implications for and treatment of babies with positive screening test results. Staff reported that they felt more confident to take the test.

The quality of specimens received at the laboratory improved following a teaching programme including methodologies used for the screening test. Anecdotal evidence showed that parents who had a baby with a positive test result were less anxious if they had been given accurate information about the test, prior to it being taken.

The teaching programme will continue to be evaluated and revised.

Annual updates for this group of staff are now recommended.

97. Evaluation of the neonatal screening for sickle cell disease using the adapted integration software for Variant NewBorn Screening System

Bouva M.1, Mohrmann K.2, Brinkman H.2, Verheul F.2, Elvers B.1, Loeber J. G.1

1National Institute for Public Health and the Environment, Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, The Netherlands

2IJsselland Hospital, General Clinical Laboratory, Capelle a/d IJssel, The Netherlands

In 2005/2006 the Bio-Rad Variant Newborn Screening System was evaluated for the neonatal screening for sickle cell disease in the Netherlands. In a subsequent pilot screening a bi-phasic distribution was noticed when the percentages of the fetal hemoglobin (HbF) and adult hemoglobin (HbA) were plotted against gestational age. The bi-phasic distribution of the hemoglobin variants made it difficult to set a cut-off value. The two populations would require two different cut-off values, depending on the integration used. The use of two types of integration, depending on the peak height, seemed to be the cause. On our request Bio-Rad changed the software for the Dutch systems so only valley-to-valley integration was used. A normal distribution is necessary to set a cut-off value, which makes it possible to screen for thalassemias. A cut-off value for the FAST peak, containing Hb Bart’s, facilitates screening for α-thalassemia. A lower limit for HbA, gestational age dependent, makes screening for β-thalassemia possible. The aim of the study was to see whether the adapted software, only using valley-to-valley integration, results in a normal distribution of the peak percentages.

Data from the pilot screening, performed in 2006, were recalculated with the new software and once again plotted against gestational age.

The new software resulted in a standard valley-to-valley integration with a normal distribution and thus the ability to set a cut-off value. Further analysis of the data will determine which cut-off values should be applied to screen for thalassemias.

98. Detection at birth of hearing-impaired children

Dauman R.1, Roussey M.2

1CHU et Universitte Bordeaux, Service ORL et Audiologie, Bordeaux, France

2AFDPHE, Newborn Screening, Paris, France

This report is based on a cohort of 192,716 live births, eligible to screening for permanent hearing impairment in the framework of a program supported by the national Health care system. The AFDPHE, a national association for screening and prevention of childhood handicap, was entrusted with carrying out the screen. Six population centres, each with an average of 12,000 annual births were selected (Bordeaux, Lille, Paris, Marseille, Toulouse and Lyon). Automated auditory brainstem response (AABR) was the test chosen, all infants being screened with the same equipment, an Algo3i® (Natus). A two-stage screening procedure was employed, infants with an initial “positive” result being screened a second time, 12 hours at least after the first test. Infants with a second positive test were referred to the regional audiological centre for diagnostic ascertainment. Two categories of infants were distinguished. Those who, due to a health condition, were transferred from the regular nursery to a specialized area, constituted the first group (4.5% of the whole population). They were transferred into a conventional neonatology unit (60%) or admitted to a neonatal intensive care unit (40%). The infants able to stay in a well-baby nursery constituted the non-transferred group.

The purpose of this report was: (1) to investigate to what extent the status of being transferred or not influenced screening coverage, false-positive rate, and incidence of bilateral permanent hearing impairment; (2) to summarize the ethical issues that were recently raised about neonatal auditory screening by the national ethics committee.

99. Early detection of sickle cell anemia and others hemoglobinopathies in neonates in the Basque country. Pilot study in anonymous not related population

Espada M., Valle A., Marcos E., Elorduy J.

Public Health Laboratory, Basque Government, Bilbao, Spain

Objective: Sickle cell anemia is a hereditary disease wich, as a result of migration, constitutes one of the most frequent genetic disorders in northwest Europe. To determine the incidence of sickle cell anemia and other hemoglobinopathies in the neonatal population of the Basque Country and to determine the need for a screening program.

Methods: The study was performed with the same blood spots specimen dried on filter paper used for Congenital Hypothyroidism, Phenylketonuria and Medium-chain acylCoA dehydrogenase deficiency. Collection of blood samples on filter paper: Blood samples were obtained at two to three days of life by lancet puncture of the heel, spotted on thick filter paper (Whatman 903) and allowed to dry at room temperature. All neonates born in the public and private hospitals of the Basque Country were included and universal-type screening was performed. High-performance liquid chromatography (HPLC) was used to detect variant hemoglobins. The variant automated system was used to separate and identify hemoglobin F, A, S, C and D.

Results: A total of 9,159 specimens were screened and 36 cases of heterozygote carriers of different pathological hemoglobins (HbS, HbC, HbD and HbE) were detected. The overall incidence was 1/254. There were one case of sickle cell anemia (phenotype FSC) with an incidence of 1/9159 and 25 cases of sickle cell traits (1/366).

Conclusions: These results confirm the need to include screening for sickle cell disease and other hemoglobinopathies in our neonatal program. The universal screening program is expected to reduce morbidity and mortality in the first years of life.

100. MALDI-TOF-MS based screening test for newborn detection of sickle cell disease: what throughput for what results

Hachani J.1, Duban-Deweer S.1, Pottiez G.1, Cecchelli R.1, Renom G.1, Périni J.-M.2, Flahaut Ch.1

1Faculté des Sciences Jean Perrin, Université d’Artois, Lens, France

2Laboratoire de dépistage néonatal, CHRU de Lille, Lille, France

Sickle cell anemia (SCA) is the most common form of sickle cell diseases and is the first inherited disorder that the genetic cause have been elucidated. SCA is an inherited abnormality that affects the hemoglobin, the most abundant protein of the red blood cells. SCA is an autosomal recessive genetic disorder. In other word, the homozygosity for the mutation, which consists to the replacement of an adenine by a thymine on the beta-globin gene (chr 11), causes the disease. Therefore, people with SCA have red blood cells that contain mostly an abnormal hemoglobin, named hemoglobin S (Hb S). When O2 partial pressure is low, these red blood cells become sickle-shaped, have diffilculty passing through small blood vessels and depriving downstream tissues of oxygen. SCA is a chronic and longlife disease where its early detection signifcantly increases the quality and time of people life. Today, all over the world, the SCA screening is performed at 3 days of life from dried blood samples collected according to the Guthrie’s process and consists to detect Hb S by in gel isoelectrofocusing from Guthrie’s eluats [ Lubin et al., 1991]. In case of positive result, a second determination is required and HPLC is then used. As reported by recent papers [Wild et al., 2004; Kiernan et al., 2002], mass spectrometry seems to be a particularly well-adapted analytical tool to develop the SCA high-throughput screening correlated with a high-specificity and a high-sensitivity. In this poster, we present the different steps of the automatization of a MALDI-TOF-MS based screening test where 4 x 1000 summed spectra are used to classify samples from homozygosity Hb A (Hb normal), heterozygosity (Hb AS) and homozygosity Hb S. The proof of concept have been conducted as a retrospective study onto 2000 blood samples.

101. Automated instrument for dried blood spot assays

Furu P., Kimppa K., Hakala H., Karunen J., Korpi J., Karvinen J.

PerkinElmer, LAS, Wallac Oy, Turku, Finland

The number of samples to be analyzed in a typical neonatal screening laboratory are already high. This brings up capacity issues with instruments and laboratory space. Thus the automation of screening laboratories and the reliability of instruments and results are of interest. The GSP (Genetic Screening Processor) was developed to meet these demands. The GSP system combines multiple technologies and high sample capacity with a low instrument foot print to meet the needs of medium to large neonatal screening laboratories. The GSP instrument combines time resolved fluorescence, fluorescence and absorbance measurement technologies to support its use with the DELFIA® immunoassays and other enzymatic assays on a single instrument platform. The GSP is designed for continuous loading with a capacity of up to 26-96-well plates simultaneously. The plates are processed in parallel. As soon as the processing of each plate ends, the results are sent to calculation, and the plate is unloaded from the instrument. The GSP instrument includes cooled storage for reagents, and thus the on-board stability of reagents is up to 5 days. Assay processing areas are temperature controlled so that any variation in the results due to external conditions is minimized. On board stability data for different reagents is presented, along with information about processing times for different assay combinations.

102. False results in newborn bloodspot screening

Hall K., Finnerty D.

Birmingham Children’s Hospital, West Midlands Newborn Screening Centre, Birmingham, England

Many factors are involved in producing valid newborn screening test results from bloodspots which health professionals need to be aware of.

This study aims to increase awareness of pre-analytical, analytical and post-analytical factors which can falsify results or their interpretation. This knowledge may facilitate improvement in all aspects of the quality of newborn bloodspot screening.

Incidents which had or had potentially resulted in incorrect bloodspot test results were investigated including

  1. blood spot collection quality
  2. bloodspot contamination
  3. incorrect demographics recorded on the card
  4. missing demographics on the card such as date of birth, date of specimen and date of blood transfusion
  5. unusual amounts of adult compared to foetal haemoglobin

Findings included

Bloodspots compressed to fill the circle may alter the measured result

Bloodspot size differences compared to the calibrators may alter the measured result

Bloodspots contaminated e.g. with Aspartame or EDTA may give false positive PKU and false negative CHT screen results respectively

Bloodspots presumptively contaminated with faeces may give false positive CF screen results

Inaccurately named bloodspot cards may lead to the omission of testing a child

An unusually high percentage of adult blood may represent a recent blood transfusion diluting the blood or that the bloodspots are not from a baby

Errors in newborn bloodspot screening may arise from a wide variety of sources due to misunderstanding, lack of education, fear of failure and emotional bonds to the baby. With feedback to health practitioners and education, errors can be eliminated or significantly reduced.

103. Development of a new Galactose kit from Ani Labsystems

Hao W., Pitkanen E.-M., Carrard G.

Ani Labsystems, Research and Development, Vantaa, Finland

Ani Labsystems Neonatal Galactose kit is intended for the quantitative determination of total galactose concentrations from dried blood spot samples as an aid in screening newborns for galactosemia. The test is based on the traditional reaction scheme which involves alkaline phosphatase and galactose dehydrogenase. Stability of the kit is achieved with lyophilized components and other enzyme stabilizer. Precision of the assay is increased through optimized buffer components and operating procedure. The end result is a user friendly and robust performing kit. The kit consists of only two enzymes compared to three from competitor’s kit, while still able to distinguish between free and total galactose. On the other hand, the whole kit is stored at +4°C during the entire shelf life while components from competitor’s kit must be stored at -20°C.

104. Extended immunoassay for hemoglobinopathy screening: moving further with a true screening assay

Huhtinen P.1, Hurskainen P.2, Seppala J.2

1PerkinElmer, Genetic Screening, Turku, Finland

2PerkinElmer, Chemistry R&D, Turku, Finland

Hemoglobinopathies (HBO) are the most common genetic disorders globally. According to WHO (2006) roughly 5% of the global population carries a gene for HBO and there are around 300 000 infants born with a major HBO annually. To specify a great majority of these infants have sickle cell disease (SCD). The prevalence of SCD together with the benefits achieved with preventive medication have been the main motivators for including especially SCD in the newborn screening programs, although other HBOs are often included too. Earlier we developed a screening assay, AutoDELFIA® Hb Immunoassay (AD Hb), specific for HbS to fulfill the primary requirements of most neonatal HBO screening programs. Still, the secondary requirements of the programs support a widening of assay’s scope. Here we present an extension of immunoassay by increasing the number of detection antibodies in the current assay. The extended assay has all the same characteristics as the current assay, i.e. europium and samarium-labelled antibodies together with time-resolved fluorometry are utilised in generating the qualitative result. The assay performance has been evaluated in a small-scale study with controls and retrospective neonatal heel prick samples. The results were as expected: all the samples containing HbS, -C, or -E, and the ‘F only’ samples were screened positive. Moreover all ‘FA’ samples were screened negative. So the sensitivity, specificity and method agreement were all 100 %. The turnaround time for a 4-plate run with 384 tests was roughly 2½ h, making the time per test <25 s. The extended immunoassay enables the detection of HBOs essential in neonatal screening, i.e. HbS containing cases. Further, the assay detects several other common and relevant HBOs, like HbE and -C and the most severe thalassemias. The technical features, like throughput and automation, make the assay a applicable alternative for neonatal HBO screening.

105. A case for mandatory expanded screening

John K.1, Hart C.2, Meng Goh D. L.3, Shien Tan E.4

1KK Hospital, National Expanded Newborn Screening Programme, Singapore

2KK Hospital, Biochemical Genetics, Singapore

3National University Hospital, Paediatric Medicine, Singapore

4KK Hospital, Paediatric Medicine, Singapore

Screening in Singapore was implemented in June 2006 and during 2008 screened 18,500 babies, approximately half of the current birth rate. Screening is not mandatory in Singapore, but is dependent on the individual hospital awareness and testing procedures. During 2008, two unscreened patients born during the time screening has been available, were diagnosed with inborn errors of metabolism that could have been identified by expanded newborn screening and which are amenable to treatment. A 5 month old girl was admitted to A&E with acidosis and encephalopathy. Blood spot acylcarnitines showed raised propionyl carnitine and urine organic acids revealed a gross excretion of methylmalonic acid with 3-hydroxy propionate and methylcitrate, consistent with a diagnosis of methylmalonic aciduria. Second patient was a 13 month old boy admitted to ICU with tachypnea, drowsiness and poor feeding. Blood spot acylcarnitine showed a raised C5:1 (tiglylcarnitine) and C5-OH (2 methyl 3 hydroxy butyric acid), confirmed on organic acids as beta ketothiolase deficiency. These two babies were born in the same hospital and highlight the need for mandatory expanded newborn screening for metabolic disorders. Both were fortunate enough to survive their first decompensation event without long term sequelae, but these events could have been avoided had they been screened. Currently, to an extent, screening in Singapore is a lottery and depends on where the baby is born. While there is good coverage from the public sector hospitals, those babies born in the private sector are less likely to be screened.

106. Improved total galactose assay for galactosemia screening in newborns

Karvonen H.1, Makinen M.-L., Ronnmark S., Tuomola E., Hietala A.2, Torresani T.3, Seppala J.1

1PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland

2Minnesota Department of Health, St. Paul, United States

3Kinderspital Zurich, Zurich, Switzerland

The objective was to improve the calibration, stability and precision of PerkinElmer Neonatal Total Galactose (TGal) assay used for determination of quantitative concentrations of galactose and galactose-1-phosphate in dried blood specimens (DBS).

Total Galactose concentrations in the blood spots that were used as primary calibrators were established by determining the total galactose concentrations with HPLC-MS/MS using USP reference galactose standards. The stability of the product was improved by drying the substrate, HPPA (3-(p-hydroxyphenyl)propionic acid) and storing the kit calibrators and controls at -20°C. In the assay, sample disks are punched into V-bottom plate followed by elution of galactose and galactose-1-phosphate using zinc sulphate - ethanol solution (1h at RT). The eluate is diluted and transferred to a white plate and the reconstituted enzymes and substrate are added to the reaction mixture. After one hour incubation at RT stop solution is added and the fluorescence is measured using VICTOR® Fluorometer or equivalent. Shelf-life and precision were determined for the Neonatal Total Galactose kit. Routine newborn specimens from Europe and USA were analyzed to determine the median and 99.5th percentile. Also five retrospective true positive specimens were analyzed. The kit was successfully calibrated against an HPLC-MS/MS method. The shelf-life for the kit was one year (previously 3 months). In two studies, 1867 routine DBS in Europe and 2109 in USA (medians 1.9 and 1.7 mg/dL respectively) were assayed. The 99.5th percentiles for the studies were determined 8.8 mg/dL and 9.9 mg/dL respectively to represent the cut-offs. All five retrospective true positive specimens were detected positive using our method. The total variation around the cut-off values was 12 %CV. The updated version of PerkinElmer TGal kit calibrated against an HPLC-MS/MS method has an improved assay performance and prolonged shelf life when compared to the previous kit.

107. A pilot newborn screening programme for congenital toxoplasmosis in the Republic of Ireland

Mayne P.1, Guy E.2, Finnegan N.1, Ferguson W.3, Butler K.4, Cafferkey M.5

1Children’s University Hospital, National Newborn Screening Laboratory, Dublin, Ireland

2Singleton Hospital, Toxoplasma Reference Laboratory, Swansea, Wales

3The Rotunda Hospital, Paediatrics, Dublin, Ireland

4Our Lady’s Children’s Hospital, Infectious Diseases, Dublin, Ireland

5The Rotunda Hospital, Microbiology, Dublin, Ireland

Congenital toxoplasmosis (CT) is caused by toxoplasma infection acquired by susceptible women during pregnancy. Studies indicated that the toxoplasma seroprevalence rate in young women was 25%, ranging from 20 to 41% by geographic area, suggesting that up to 75% of women were susceptible to acquiring toxoplasma infection during pregnancy. Early detection through prenatal or neonatal screening programmes could optimize management and improve outcome. This study approved by Research Ethics Committees was designed to determine the incidence of CT by screening all newborns as part of the newborn screening programme and to develop a treatment strategy to improve outcome of those with CT. All newborn screening sample-takers attended workshops before the programme started; written consent was obtained from parents at the time of heal-prick sample collection (day 3 to 5). Samples were collected from 144,564 newborns born during a two year recruitment period; 363 (0.25%) parents opted out. Thirty-four samples were ISAGA IgM (bioMérieux) positive following a raised bloodspot autoDELFIA IgM (PerkinElmer) >4 IU/mL. Following serology testing on samples from mother and infant, 15 infants were confirmed positive and 19 false positive, giving an overall incidence of 1 in 10,000. Complications at presentation included unilateral or bilateral chorioretinal scaring (6/15), moderate or high frequency hearing loss (2/15), focal or multiple intracranial calcification on brain imaging (4/15) and hydrocephalus in one infant that had not been detected clinically and which required VP shunting. Fourteen infants were started on Pyrimethamine, Sulfadiazine and Folinic acid for one year at a median age of 4.5 wk (range 3 wk–17 wk). All patients developed uncomplicated mild to severe neutropenia. The infant with hydrocephalus has mild motor delay; there has been no progression of any of the eye lesions. No false negative cases born during the recruitment period have been identified clinically.

108. Comparison of different filter papers for the use in newborn screening

Müller C.1, Stopsack M.2, Blankenstein O.3

1University of Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany

2Universitätsklinikum Carl Gustav Carus, Childrens Hospital, Dresden, Germany

3Charité-Universitätsmedizin Berlin, Experimental Paediatric Endocrinology, Berlin, Germany

Dried blood spots are widely used in newborn screening. Most newborn screening assays are standardized using Whatman 903® filter paper. Recently, two new brands of filter paper have been announced with FDA approval for the use in newborn screening assays. As differences in filter papers affect the quantitative results of the analyses, we compared the three types of filter paper due to physicochemical properties and differences in usability and results in screening assays. Tests were performed with filter paper from Whatman, Munktell and ID. 30 µl- resp. 50 µl- aliquots whole blood were dispensed, were allowed to diffuse and to dry. 3.1 mm punch-specimen were tested emptily and with dried blood to measure homogeneity and blood uptake. Measurements ofTSH, 17-OHP, Biotinidase- and GALT-activity by conventional methods as well as acylcarnitines and amino acids by TMS analysis were compared. We tested qualitatively for spot size and form and for pollution of the orifice of an ABI 2000 MS/MS. We found differences in the three filter papers due to blood uptake and homogeneity reflected in most of the analytes. Generally the blood uptake was highest in ID 226 filter paper followed by Whatman 903 and Munktell TFN. Qualitative differences were found in form and size of the blood spots and concerning the pollution of the MS/MS machine. The differences between the three types of filter papers were significant in some analytes. In no case the detection of true positive cases would fail. Differences between healthy and affected individuals are much higher than those detected in our study and they did not surpass the variability specified by the FDA for filter paper. All three types of filter papers were within the limits for filter paper and can be used in newborn screening. Specific differences might influence the choice for each laboratory.

109. A novel software algorithm for the quantitative calculation of mass spectral ion intensities for inborn errors of metabolism

Nagtalon D.1, McClure T.2

1Thermo Fisher Scientific, LC-MS Clinical Research and Toxicology, San Jose, United States

2Thermo Fisher Scientific, Software Research, San Jose, United States

Objective: Predicated on making the tandem mass spectrometer more user-friendly, a customizable software application was developed to facilitate ease-of-use of the system for accurate data processing, review, and reporting of the data acquired for quantification experiments utilizing ion intensities like those for the determination of inborn errors of metabolism.

Methods: “ICALQ” or “Intensity Calculating Quantification” application was developed to facilitate inline/offline processing of spectral data acquired using a Thermo Scientific triple quadrupole mass spectrometer with the Xcalibur data system for routine quantification analyses using ion intensities. It consists of three modules. The sequence module allows for importing of acquisition sequence files and selection of a processing parameters file to be used in processing the sample sequence acquired. A default file can be configured for automatic post-acquisition processing. The processing module allows for creation of the processing file with user-specified target/internal standard masses, internal standard concentrations, data flagging using threshold ranges, spectral filter averaging, and peak detection features. Each processing file can contain more than one processing algorithm or “test” to be used in processing each sample. The reporting module allows for quick data review and report output. All calculated results in the sequence are grid-displayed with each “test” results displayed and data-flagged accordingly. It is also conducive to batch reprocessing and samples can be further interrogated using Xcalibur’s Qual Browser. Reports are generated in CSV format for LIS compatibility.

Results: ICALQ is currently operating successfully in a major neonatal screening center in Asia. Data will be presented on the software’s performance and next steps for improvement for development.

Neonatologie Pediatrie Praktické lékařství pro děti a dorost

Článek vyšel v časopise

Česko-slovenská pediatrie

Číslo 4

2009 Číslo 4
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