Abstrakta - pokračování

46. Robust and standardised Neonatal hTSH EIA and FEIA assays from Ani Labsystems

Carrard G., Pitkanen E.-M.

Ani Labsystems, Research and Development, Vantaa, Finland

Congenital hypothyroidism (CH) is an endocrine disorder of newborns characterized by the decreased levels of thyroid hormones. Through the negative feedback mechanism the deficiency of thyroid hormones stimulates the secretion of hTSH. An elevation of hTSH concentration in infant blood leads to the presumption of primary CH (or transient thyro-tropinemia). The disease, if undiagnosed and untreated, leads to severe mental retardation, as well as to growth and developmental disturbances. Due to the high incidence of CH worldwide (on average 1/4000 live births) and to the availability of non-expensive and effective replacement therapy by L-thyroxine, this disorder is screened for in all industrialized countries and also in the majority of developing countries. A successful screening program requires the coordination of several different components of the health care system in order to insure homogeneity between test results and screening programs. However, today only a few manufacturers include reference material in their calibration process. Ani Labsystems was one of the first manufacturers to use international reference material for TSH. Indeed, the neonatal TSH test was first calibrated against WHO 3rd IRT 81/565 standard preparation since 1991. As soon as the 1st ISNS-RPNS standard was prepared in 2005, it was instantly added in the production process as calibration standard. We present in this poster the performance of the tests in clinical settings along with the recall rate in two major clinical studies. In addition, the assays were recently updated to provide lower background (improved by 6-fold), longer stability (2-fold) and higher linear calibrating range (0 to 100 mIU/L blood). The sensitivity of the tests is 0.9 mIU/L blood. The recovery with quality rounds samples from CDC is about 100%. Ani Labsystems hTSH reliable and robust assays provide an opened system, to be adapted to all laboratory settings and size.

47. Development of Neonatal T4 assay for congenital hypothyroidism screening using automated GSP system

Hiekkanen A., Meriö L., Lindroos H., Seppälä J.

PerkinElmer, LAS, Wallac Oy, Turku, Finland

The new Genetic Screening Processor (GSP) instrument is a fully automated, high capacity analyzer for the quantitative and qualitative in vitro diagnostic measurements of genetic screening disorders in neonatal screening laboratories. We have developed a GSP Neonatal T4 assay for the quantitative detection of thyroxine (T4) in dried blood spot specimens based on the well-known and sensitive DELFIA^® technology. All kit components are bar-coded and ready-to-use. Calibrators and controls are in cassette format to allow standardization for automated systems. Components were loaded in reagent cassettes into the GSP and stored in a cooled reagent carousel. The ready-to-use reagents in the cooled reagent carousel are stable for up to five days and the same calibration curve can be used for 24 hours. The assays started automatically when punched plates were loaded into the GSP. Data analysis was done with the GSP Workstation software incorporated in the GSP system. The performance of new GSP kit was studied (e.g. precision, interfering factors, linearity). The assay was compared to the corresponding AutoDELFIA^®kit in-house and in two routine screening laboratories using routine screening specimens. The linear measuring range of the GSP Neonatal T4 kit is 1.61–30 µg/dL serum. The precision was comparable to the corresponding AutoDELFIA assay. The within-lot CV %’s of kit controls (2 different studies) were: 11.7–12.2% (C1), 8.6–8.9% (C2) and 9.3–9.5% (C3). The correlation of the GSP Neonatal T4 (y) to the AutoDELFIA B065-112 (x) assay was y=0.80x + 1.35 (n=120, r=0.89). EDTA, citrate and heparin do not interfere with the GSP assay. The GSP Neonatal T4 assay offers an easy-to-use and reliable way of screening for congenital hypothyroidism with high capacity. The performance of the GSP assay is comparable to the commercially available AutoDELFIA method.

48. Development of Neonatal hTSH assay for congenital hypothyroidisms screening using automated GSP system

Hiekkanen A., Airenne S., Furu P., Seppälä J.

PerkinElmer, LAS, Wallac Oy, Turku, Finland

The new Genetic Screening Processor (GSP) instrument is a fully automated, high capacity analyzer for the quantitative and qualitative in vitro diagnostic measurements of genetic screening disorders in neonatal screening laboratories. We have developed a GSP Neonatal hTSH assay for the quantitative detection of human thyroid stimulating hormone (hTSH) in dried blood spot specimens based on the well-known and sensitive DELFIA^® technology. All kit components are bar-coded and ready-to-use. Calibrators and controls are in cassette format to allow standardization for automated systems. Components were loaded in reagent cassettes into the GSP and stored in a cooled reagent carousel. The ready-to-use reagents in the cooled reagent carousel were stable for up to five days and the same calibration curve could be used for 24 hours. The assays started automatically when punched plates were loaded into the GSP. Data analysis was done with the GSP Workstation software incorporated in the GSP system. The performance of new GSP kit was studied (e.g. precision, interfering factors, linearity). The assay was compared to the corresponding AutoDELFIA^® kit in-house,and in two routine screening laboratories using routine screening specimens. The linear measuring range of the GSP Neonatal hTSH kit is 1.31–250 µU/mL blood. The precision was comparable to the corresponding AutoDELFIA assay. The within-lot CV %’s of kit controls (2 different studies) were: 7.5–7.6% (C1) and 5.7–8.5% (C2). The correlation of the GSP Neonatal hTSH (y) to the AutoDELFIA B032-312 (x) assay was y=0.98x + 0.12 (n=103, r=0.99). EDTA, citrate and heparin do not interfere with the GSP assay. The GSP Neonatal hTSH assay offers an easy-to-use and reliable way of screening for congenital hypothyroidism with high capacity. The performance of the GSP assay is comparable to the commercially available AutoDELFIA method.

49. Congenital hypothyroidism (CH) in Moscow city: the results of neonatal screening for 2008 year

Mitkina V., Denisenkova E., Mitkin V.

Moscow Newborn Screening Center, Moscow, Russia

The results of newborn screening for CH in Moscow city for 2008 year reported. It was examined 123,800 newborns with the 63 newborns as a risk group. The cut-off level for DELFIA TSH kit is 15 µU/ml in according 99.5th percentile. Diagnosis of CH confirmed in 40 newborns of risk group, the incidence was 1:3,095. In the other 23 newborns confirmed the diagnosis of transitory hypothyroidism. The number of cases with the concentration of TSH more than 50 µU/ml were 20 and in 19 of them confirmed the CH. Thus confirmed the importance of 50 µU/ml TSH level as the main cause to urgent therapy these newborns. On the other hand the 52% (21) of newborns with CH had the concentration of TSH less than 50 µU/ml with the next distribution: 15 cases from 15 to 30 µU/ml, 6 from 30 to 50 µU/ml. It also confirmed the significance to reevaluate in time for all population in risk group. The incidence of CH increased by compare with the previous year: 1:3,570 for 2007 contrary to 1:3,095 for 2008 year. One of possible explanation is the influence the mild degree of iodine deficiency in Moscow city region, because about 11% samples have the TSH level more than 5 µU/ml.

50. Biochip array technology for measurement of thyrotropin in dried blood spots

Parlato G.¹, Michniewicz A.¹, Martino L.², Saiaci C.³

¹University of Catanzaro, Clinical and Experimental Medicine, Chair of Chemistry Applied to Medicine, Regional Center for Neonatal Screening, Unit of Clinical Chemistry , Catanzaro, Italy

²University of Catanzaro, School of Clinical Biochemistry, Catanzaro, Italy

³Azienda Policlinico Materdomini, Unit of Clinical Chemistry, Catanzaro, Italy

Evidence^® (Randox), an analyzer based on biochip array technology, performs simultaneous immunoassays of TSH, T4 and T3 or TSH, free T4 and free T3 on 9 mm x 9 mm microchip in 100 µl sample. The immuno-complex are detected by a signal, expressed in relative luminescent units (RLU). The neonatal screening for congenital hypothyroidism (CH) reveals all CH forms, when TSH and T4 are separately measured in dried blood spot (DBS). An ideal approach is the simultaneous assay of TSH and T4, but a better strategy should be the simultaneous assay of TSH and free T4, the T4 active form. The feasibility of simultaneous assays was firstly evaluated by verifying correlations between TSH measured with Evidence^® and AutoDELPHIA^® (PerkinElmer), analyzer used for CH screening. One 3-mm disc from DBS was eluted in 300 µL of 0.1 M phosphate buffer, pH 7.4, after a 45 min shacking at room temperature. The procedure was performed to measure by Evidence TSH levels in DBSs from: AutoDELPHIA^® calibrators in range 0.7–256 mU/L of whole blood ; controls of AutoDELPHIA^® neonatal hTSH kit in the range 11–17 and 46–70 mU/L; 55 screened newborns (55 SN); 6 newborns with TSH values around the cut-off (7 mU/L) of CH screening (6 N). Thanks to the strong linear correlation (R=0.99, Ycal=-8.18 + 118.1 Xmeas) between TSH values of calibrators and those measured with Evidence, AutoDELPHIA^®calibrators were used for calibration curve with Evidence. The calibration plot ,YRLU=17.1 X, was linear with Rsquare=0.99. TSH values were 14.3 and 54.5 mU/L for controls, strongly correlated (R=0.91) for 55 SN and practically similar for 6 N (mean ± standard deviation of Evidence and AutoDELPHIA was 9.7±1.77 and 9.6±1.81 mU/L respectively). The use of Evidence to measure TSH in DBS encourage to find conditions for simultaneous assay of TSH and T4 or free T4.

51. The Bulgarian Neonatal Thyroid Screening (NTS) Programme 1993–2008

Stoeva I., Antova R.

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

The NTS started in 1993 (3 years Bulgarian--Swiss project) in a new centralized screening unit at the University Pediatric Hospital in Sofia (PKU logistic since 1978). Blood was taken from newborns (NB) mainly on days 3–5 (2008: 133 maternities, S&S 2992, 903). TSH was determined (DELFIA^®, cut-off initial screening 15 mU/l, quality control DGKL) in 991,448 NB (till 30.11.2008). Data were registered by special software. Following changes occurred: increase of coverage from 58 (1993) to 99.5% (2007, p <0.001); shift to earlier screening-sampling on day 1 and 2 increased from 10.6 (1999) to 24.2 (2005) and 19.9% (2008), but decreased after day 5 from 10.9 to 4.9% (1999 vs 2008); High proportion of late (after day 11) arrived FPC during the whole period: 47 (2000), 17 (2005), 20.6% (2008); Decrease of NB with TSH >15 mU/l (1st FPC): 1.8 to 0.09 (1993 vs 2008, p <0.001). Different forms of primary thyroid disturbancies were diagnosed in 420 NB (1:2,360 screened NB). Treatment with L-T4 was instituted at median age from 18 to 24 (1993 vs 1997) and 13 to14 (2001–2002 vs 2004–2008) days. The whole strategy changed completely. Follow-up studies in screened patients with good compliance showed normal growth and development, according to the individual genetic potential. Systematic thyroid screening in mothers (participation 82.9%) of children with abnormal NTS started in 1998. The NTS was also adapted to monitor the iodine supplementation since 1997: NB with TSH >5 mU/l (3–5 day) increased from 7.8 (1997) to 9.6% (2000), declined below 3% in 2006. Stable improvement of all screening process indicators was achieved by integrating efforts of all participants. The transition of the NTS in a more efficient public health program is a constant challenge and of utmost importance for the future extension of all neonatal screening programmes.

52. The ISNS 17OHP Initiative: Establishing of 17OHP cut-off levels by international collaboration

Blankenstein O.¹, Stopsack M.², Fingerhut R.³, Loeber G.⁴, Torresani T.³

¹Charité-Universitätsmedizin Berlin, Experimental Peadiatric Endocrinology, Berlin, Germany

²Carl-Gustav Carus University, Paediatrics, Dresden, Germany

³University Children’s Hospital, Newborn Screening, Zurich, Switzerland

⁴National Institute for Public Health and the Environment (RIVM), Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, The Netherlands

Re-establishing of cut-off levels for screening assays is often difficult because of an increased risk for false-negative or false-positive results. Cut-off levels generated by a single laboratory are cost-intensive due to longer periods of parallel measurements. To establish generally usable cut-off values for the recently introduced 17α-OH-progesterone (17OHP)-kit (Perkin Elmer B024) using a multi-center approach coordinated by the ISNS. The cut-off values are stratified according to Gestational Age (GA), Birth Weight (BW) and age at sampling (Age), to take account of the different screening protocols used. Routine screening 17OHP results obtained with the B024 Kit were collected from neonatal screening centers in different parts of the world. The following data were collected: GA in weeks, BW in grams, date of birth, date of sampling, 17OHP result in nmol/L blood. Where available also sex and the exact time of birth and sampling were collected. All collected data was fully anonymized. Inclusion criteria: GA: 24–42 wk; Age <10 days. Exclusion criteria: Samples of confirmed CAH cases. Exception in premature babies (GA ≤36 wk): samples with age >10 days were accepted. Parallel measurements with the preceding B048 screening-kit did not show a linear correlation. More than 100,000 results have been collected and evaluated so far. Cut-offs were calculated statistically for term and preterm babies according to Age. Example for cutoff-levels in term newborns:

Age (h) → 17OHP-level (nmol/L blood)

36 – 48 → 38.5

48 – 72 → 32.3

72 – 96 → 28.5

96 – 144 → 23.9

The collaborative approach allowed rapid establishment of 17OHP cut-off values for the new B024 CAH screening kit which can be used generally. These data should help the transition to the new kit, eliminating the need of lengthy and expensive studies performed in each individual laboratory and reducing the rate of false positive results.

53. Newborn screening for congenital adrenal hyperplasia (CAH) by steroid profile analysis using turbulent flow chromatography by tandem mass spectrometry without acetonitrile

Matern D.¹, Lacey J.¹, Magera M.¹, McCann M.², Raymond K.¹, Tortorelli S.¹, Gavrilov D.¹, Oglesbee D.¹, Rinaldo P.¹

¹Mayo Clinic College of Medicine, Biochemical Genetics Laboratory, Rochester, MN, United States

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

Background: Immunoassays are typically used to quantify 17-OHP as a marker for CAH, but are associated with a high false positive rate (FPR). Previously, we have shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) allows for the specific determination of 17-OHP, androstenedione and cortisol simultaneously, thereby increasing the specificity of newborn screening (NBS) for CAH (Clin Chem 2004;50:621-5). We modified this method to include analysis of additional steroids: 11-deoxycortisol and 21-deoxycortisol. Due to an ongoing world-wide shortage of acetonitrile, this method was further modified to eliminate the need for this reagent. In Minnesota (MN), steroid profiling has been applied to NBS for CAH as a 2nd tier test since 6/2004.

Method: Steroids are eluted within 30 minutes from a 3/16-inch DBS punch with water/10% acetone containing d8-17-hydroxyprogesterone, d7-androstenedione, d3-cortisol, d2-11-deoxycortisol and d8-21-deoxycortisol as internal standards. Extraction of steroids from the blood spot eluate is performed by turbulent flow chromatography (ARIA TLX4, ThermoFisher Scientific) and analysis is by electrospray LC-MS/MS (SCIEX API 5000). The concentrations of the various steroids are established by comparison of their ion intensity to that of their respective internal standards.

Results: Since 6/2004, 329,033 babies were screened in MN for CAH. 2,712 had abnormal immunoassay results but only 211 were subjected to follow up after the 2nd tier test was also positive. 19 CAH cases were confirmed. FPR and positive predictive value before and after expansion of the steroid profile were 0.82% and 0.06% as well as 0.7% and 9.0%, respectively. Two CAH patients escaped identification by NBS with one having a normal immunoassay and the other a normal LC-MS/MS result (false negative rate: 0.0003%).

Conclusion: NBS for CAH can be greatly enhanced by steroid profiling. However, neither LC-MS/MS nor immunoassay are 100% sensitive.

54. Proficiency testing for second tier CAH screening: towards harmonization of results

Mei J., De Jesus V., Kennedy M., Schiffer J., Hannon H.

Centers for Disease Control and Prevention, Newborn Screening Quality Assurance Program, Atlanta, GA, United States

The Newborn Screening Quality Assurance Program (NSQAP) at the Centers for Disease Control and Prevention (CDC) provides proficiency testing (PT) dried blood spot materials (DBS) for second tier screening for Congenital Adrenal Hyperplasia (CAH) using tandem mass spectrometry (MS/MS). Specimens were distributed over ten quarters during 2006–2008 to four laboratories performing MS/MS testing on DBS enriched to represent four types of clinical CAH profiles. Specific laboratory protocols for MS/MS second tier analysis varied, but all based the clinical assessments on the ratio of 17-hydroxyprogesterone (17-OHP), androstenedione (4-AD), and cortisol. The DBS contained a range of 17-OHP, 4-AD and cortisol concentrations resulting in different analytical ratios. Laboratories received blinded, quarterly PT DBS and reported results to CDC within a month of sample receipt. Participants reported quantitative values for 17-OHP, 4-AD and cortisol, reflecting slight differences in their analytical methods. Yearly analyte means from participating laboratories showed improvement between the enrichment levels and reported quantitative values. Significant disparities in analyte quantitation in the early PT panels resulted in re-evaluation of the analytical methods. Addition of an internal standard for each analyte resulted in improved harmonization between laboratories in the later PT panels. Average quantitative values for 17-OHP increased from 81% to 101% recovery over the 2.5-year period; cortisol recoveries increased from 61.9% to 87.5%, and 4-AD recoveries decreased from 177% to 80%. The use of labeled internal standards for the three analytes, as encouraged by NSQAP, has resulted in greater data harmonization among laboratories performing the second-tier CAH MS/MS assay. Moreover, the use of an external PT program has fostered communication between laboratories and the CDC, resulting in improved analyte recoveries and enhanced sample preparation methodologies.

55. Prevalence and clinical features of congenital adrenal hyperplasia (CAH) in a multiethnic population without newborn screening

Knowles R. L.¹, Khalid J. M.¹, Oerton J.¹, Hindmarsh P.², Kelnar Ch.³, Dezateux C.¹

¹Institute of Child Health, MRC Centre of Epidemiology for Child Health, London, England

²Institute of Child Health, Paediatric Endocrinology, London, England

³University of Edinburgh, Paediatric Epidemiology, Edinburgh, Scotland

Background: Congenital Adrenal Hyperplasia (CAH) is due to recessively inherited enzyme deficiencies in cortisol production and affects around 1 in 10,000–20,000 live births. Children may present with life-threatening adrenal or salt-wasting crisis in the newborn period, incorrect sex assignment, tall stature or precocious puberty. Girls may be more readily diagnosed due to signs of virilisation. Newborn screening policies require reliable and contemporary information about disease burden.

Objectives: To report, in a multiethnic European country, the prevalence and clinical features at presentation of newly diagnosed CAH in order to inform a review of newborn screening policy.

Methods: Active surveillance, for 2 years from August 2007, of newly diagnosed CAH in any child <16 years with clinical features and elevated 17-OHP.

Results: Data are available for the first 12 months of surveillance. 67 cases were notified: 21-hydroxylase deficiency (n=59), 11β-hydroxylase deficiency (n=3), 3β-hydroxysteroid dehydrogenase deficiency (n=1) [4 children not yet classified]. 19/67 (28%) were Asian ethnicity and 36/67 (54%; 95% CI: 42–65%) were girls. 35/67 (52%; 17 boys) presented in the first year of life: estimated birth prevalence 0.96 (95% CI 0.16–5.59) per 10,000 live births. One boy died aged 9 days with adrenal crisis (2.9% mortality in infancy). Children diagnosed in the first year of life presented by 30 days; 12 (34%; 11 boys) after day 13, the age at which newborn screening results would generally be available.

Conclusion: The UK birth prevalence of CAH is similar to other conditions for which newborn screening is currently offered. Boys and girls appear equally affected but boys present with more severe manifestations in infancy. One third of neonates with CAH may benefit from earlier identification through newborn screening. While mortality is consistent with recent estimates from other countries, deaths may be under-ascertained if based on BPSU notifications alone.

56. Newborn screening for congenital adrenal hyperplasia in the Czech Republic: evaluation of the first 28 months reveals one false negative case

Votava F.¹, Novotna D.², Kracmar P.¹, Vinohradska H.³, Stahlova-Hrabincova E.⁴, Vrzalova Z.⁴, Neumann D.⁵, Matern D.⁶, Eliasova I.⁷

¹Dept. of Pediatrics, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic

²2nd Dept. of Pediatrics, Children´s Medical Centre, Masaryk University, Brno, Czech Republic

³Dept. of Clinical Biochemistry and Hematology, Children´s Medical Centre, Brno, Czech Republic

⁴Center of Molecular Biology and Gene Therapy, Dept. of Internal Medicine-Hematooncology, University Hospital Brno, Masaryk University, Brno, Czech Republic

⁵Dept. of Pediatrics, Faculty of Medicine, Charles University, Hradec Kralove, Czech Republic

⁶Biochemical Genetics Laboratory, Mayo Clinic College of Medicine Rochester, MN, United States

⁷Institute of Biology and Medical Genetics, 2nd School of Medicine and University Hospital Motol, Prague, Czech Republic

From II/2006 through VI/2008 a total of 246,235 newborns were screened. In addition, leftover dried blood spots (DBS) of 33 patients with congenital adrenal hyperplasia (CAH) diagnosed clinically and born outside of this period were analyzed. 17-hydroxyprogesterone (17OHP) levels in dried blood spots were measured by fluoroimmunoassay. Patients´ 17OHP levels in DBS were divided into 4 groups according to genotype: group A-the most severe CAH; group B-severe/moderate; group C-moderate; group D-mild CAH. 15 boys and 8 girls were detected (1:10,705). One girl, birth weight (BW) 2,300 g, group B, escaped NBS (17OHP 32 nmol/l, cut-off 50 nmol/l) and presented with hyponatremia (Na 124 mmol/l). However, retrospective LC-MS/MS analysis of her original DBS disclosed a 17OHP concentration of 28 nmol/l (cut-off 12,1 nmol/l) and steroid ratio (17OHP+androstendion/cortisol) 8,34 (cut-off 2,5). The recall rate in BW ≥2,500 g was 0.29% (17OHP cut-off 40 nmol/l) and in BW < 2,500 g 4.61% (BW related cut-offs 50–200 nmol/l). 53 newborns (BW ≥2,000 g) with significantly elevated 17OHP in DBS (≥90 nmol/l in BW ≥2,500 g; ≥125 nmol/l in BW 2,000–2,499 g) were referred for urgent clinical follow up but CAH was not confirmed. The median of 17OHP levels in group A was 609 nmol/l; in group B 387 nmol/l; in group C 80 nmol/l; and in group D 47 nmol/l. Applying current cut-offs for 17OHP, one, two and one case of group B, C and D, respectively, would have had false negative NBS results. NBS for CAH in CZ is efficient for the most severe form of this disease but will fail some cases with milder variants of CAH. Furthermore, we consider the recall rate and false positive rate to be too high for a NBS test for a single condition. Therefore, NBS must be further improved by steroid profile analysis as a 1st or 2nd tier test or by DNA analysis as 2nd tier test.

Acknowledgements: Supported by Ministry of Health, CZ, project IGA 9981-3 and Ministry of Education, CZ, project MSM0021620814.

57. Demonstration of increased throughput with automated multiplexed immunoassays for newborn screening

Sorette M., Knight K., Sorensen K.

Luminex, R&D, Austin, TX, United States

As the dried blood spot sample from newborn is limited in quantity, and the demand for more testing is increasing, one obvious solution is multiplexing the analysis. This is done routinely with MS/MS for amino acids and other small molecules. Other technologies have been used for hemoglobinopathies. One area which has historically been challenging to multiplex are immunoassays for newborn screening. We demonstrate the successful multiplexing of immunoassays for newborn assays using Luminex xMAP technology which is based on spectrally distinct microscopic beads that each carry an antibody or antigen for a specific analyte. We are in the process of developing a triplex assay that quantitates TSH, T4 and 17-OHP in a single microtiter well. The prototype assay has been tested with CDC proficiency samples as well as standards from commercially available assays for newborn. The prototype assay is conducted in standard 96 well plates, utilizing a single standard 3 mm punch from the DBS. Feasibility studies showing linearity, reproducibility, repeatability, throughput and other standard test parameters will be reported. In parallel to the assay development, standard automated liquid handling platforms have been investigated and their applicability will be presented.

58. Congenital adrenal hyperplasia as cause of sudden infant death – argument for neonatal screening

Strnadova K.¹, Votava F.², Bouska I.³, Lebl J.¹

¹Dept. of Pediatrics, 2nd Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic

²Dept. of Pediatrics, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic

³University Hospital Motol, Forensic Medicine, Prague, Czech Republic

Background: The etiology of sudden infant death syndrome (SIDS) remains unclear. However, a small number of such cases may have been caused by monogenic diseases such as inborn errors of metabolism and long QT syndrome that may lead to sudden unexpected death with no previous symptoms and no autopsy findings. We assume that congenital adrenal hyperplasia (CAH) may lead to sudden unexpected death due to hyperkalemia induced ventricular fibrillation.

Objective and hypotheses: The study was aimed to estimate the number of infants who died of unrecognized congenital adrenal hyperplasia in Austria and the Czech Republic within the past thirteen years, before the introduction of adequate neonatal screening.

Methods: The study was based on retrospective analysis of neonatal screening cards of 242 infants who died suddenly between 7 days and 12 months of age and whose cause of death could not be identified. 17-hydroxyprogesterone (17-OHP) was measured by fluoroimmunoassay and positive samples were subsequently analyzed to detect the nine most common mutations of CYP21 gene.

Results: Three infants out of 242 may have had unrecognized CAH due to CYP21 (steroid 21--hydroxylase) gene defect. Their newborn 17-OHP levels and CYP21 genotypes were 706 nmol/l and del/conv//del/conv, 53 nmol/l and I2//I2 and 811 nmol/l and I2//Gln318Stop, respectively.

Conclusions: CAH due to CYP21 defect can lead to sudden unexpected death without prior symptoms typical for the condition. Hence, newborn screening would have prevented these deaths had it been available. In addition, we have shown that I2 point mutation that is expected to lead to simple virilizing form may lead to fatal outcome.

59. Activity and Consensus of European Cystic Fibrosis Society working group of neonatal screening for cystic fibrosis

Castellani C.

Cystic Fibrosis Centre, Verona, Italy

The spreading of Cystic Fibrosis Neonatal Screening (CF NBS) has prompted the European Cystic Fibrosis Society (www.ecfs.eu/) and the European Coordination Action for Research in Cystic Fibrosis (EuroCareCF, www.eurocarecf.eu/) to organize a dedicated Consensus Conference. The International Society for Neonatal Screening (www.emqn.org/emqn/), the EuroGenTest Network of Excellence (www.eurogentest.org), and the European Molecular Genetics Quality Network (http://www.emqn.org/emqn/) were joined partners of the Consensus Conference, which took place in Garda, Italy, on March 28–29, 2008. Its main purpose was the production of European Best Practice Guidelines for CF Neonatal Screening, but it was also an opportunity for an update on current practice of Cystic Fibrosis Neonatal Screening in Europe and for discussing the possible implementation funding strategies for a collaborative European CF neonatal screening network. Altogether 37 experts in the field and professionals with an interest in Cystic Fibrosis Neonatal Screening were involved in pre-conference consultations and in the drafting of preliminary documents, and 31 attended the meeting. The conference addressed a wide range of issues, including the rationale for CF Neonatal Screening, technical issues, diagnostic criteria, and communication to parents related to CF Neonatal Screening. The presentation will highlight the main result of the Consensus Conference.

Although for a long time a general skepticism about the positive effects of NBS for CF has hampered its universal introduction, there is now wide agreement on its benefits in terms of expression of disease, burden of care, and cost. Risks appear to be mainly associated with disclosure of carrier status and diagnostic uncertainty. When starting a CF NBS program it is important to take precautions in order to minimize avoidable risks and maximize benefits.

In Europe alone more than 25 screening programs have been developed. Given the wide geographic, ethnic, and economic variations, a complete harmonization of protocols seems inappropriate. There is little evidence to support the use of IRT alone as a second tier, without some DNA step. However, if IRT/DNA testing does not lead to the desired specificity/sensitivity ratio, the usage of only biochemical tests in the screening program (IRT/IRT) may be evaluated.

Dosing sweat chloride remains the gold standard for discriminating between NBS false and true positives, but sweat test age-related issues should be taken into account. Programmes should also allow for the fact that a clear diagnosis is not always possible, and adopt strategies for managing these infants.

All newborns identified by NBS should be managed according to internationally accepted guidelines. CF centre care and the availability of necessary medication are essential prerequisites before the introduction of NBS programs.

Clear explanation to families of the process of screening and of the implications of normal and abnormal results is central to the success of any CF NBS program. Effective communication is especially crucial when parents are told their child is affected or carrier.

60. Cystic fibrosis: 7 years of neonatal screening in Catalonia

Marin J. L.¹, Hernandéz J. M^a.¹, Asensio O.², Casals T.³, Cobos N.⁴, Gartner S.⁴, Prats R.⁵, Seculi J. L.⁶, Vidal Mª. J.⁵

¹Newborn Screening Unit, Hospital Clínic de Barcelona, Barcelona, Spain

²Clinical Unit of Cystic Fibrosis, Hospital de Sabadell, Sabadell, Spain

³Center for Molecular Genetic Diagnosis, Fundació IDIBELL de Barcelona, Barcelona, Spain

⁴Clinical Unit of Cystic Fibrosis, Hospital Universitari de la Vall d´Hebron de Barcelona, Barcelona, Spain

⁵Maternal Health Program, Generalitat of Catalonia, Barcelona, Spain

⁶Clinical Unit of Cystic Fibrosis, Hospital Sant Joan de Déu de Barcelona, Barcelona, Spain

Cystic Fibrosis (CF) is the most severe and frequent genetic disease within caucasian population. CF produces multisystemic affectation and is of recessive autosomic inheritance. The calculate frequency is one asympthomatic carrier per 37 newborns. The neonatal screening program for CF in Catalonia was started in year 2000. The protocol used in our program is based in immunoreative trypsin determination (TIR) as a biochemical marker in two capillar blood samples obtained by heel punction and dried on absorbent paper (Whatman 903^®): the first sample is obtained between the 3rd and the 5th day of the life, and if a positive result is obtained, we ask for a second sample between the 25th and 40th day of the life. Since 2000 the number of childbirths per year has presented an increasing progression and 622,570 newborns of the entire catalonian territory have been analyzed. Along these years 111 affected have been diagnosed (0.018%), representing an incidence of 1/5608. During this period we found 13,136 positive results in the first TIR determination (2.11%) representing 99.1% of false positives; and 3,050 positive results in the second TIR determination (0.49%), witch means 96.3% of false positives. For the 111 CF cases, in ninety two (82.88%) the diagnostic was achieved by means of our neonatal screening program, thirteen (11.71%) by the presence of meconial ileus at birth and six (5.40%) were false negatives cases for our biochemical screening (TIR) and were diagnosed by the presence of respiratory clinical symptoms, for exhibit a positive sweat test of because of affected relatives. The molecular analysis showed that F508del is the most prevalent mutation in our population, present in 87 patients (78.3%), 26 homozygote (23.4%). Other relevant mutations were G542X (14, 12.6%), one homozygote (0.9%) and L206W (8, 7.2%).

61. Cystic fibrosis heel prick among a newborn population in the Netherlands: the CHOPIN-study

Vernooij-van Langen A.¹, Loeber G.², Elvers B.², Triepels R.³, Gille H.⁴, Reijntjens S.⁵, van der Ploeg K.⁶, Dompeling E.⁷, Dankert-Roelse J.⁸

¹Atrium Medical Centre – RIVM, Research and Innovation – LIS, Heerlen – Bilthoven, The Netherlands

²National Institute for Public Health and the Environment (RIVM), Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, The Netherlands

³St. Elisabeth Hospital, Clinical Chemnistry Laboratory, Tilburg, The Netherlands

⁴VU Medical Centre, Clinical Genetics, Amsterdam, The Netherlands

⁵Atrium Medical Centre, Research and Innovation, Heerlen, The Netherlands

⁶TNO Quality of Life, Prevention and Health, Leiden, The Netherlands

⁷University Hospital Maastricht, Pediatric Pulmonology, Maastricht, The Netherlands

⁸Atrium Medical Centre, Pediatrics, Heerlen, The Netherlands

Although newborn screening for CF (NBSCF) is included in many routine newborn screening programs, there is no ideal newborn screening test yet. The aim of the study was to assess the test characteristics of 2 new strategies for NBSCF. In 2008 NBSCF was added to routine NBS in the middle and south-eastern parts of the Netherlands. In each collected blood sample screening for CF was performed by 2 strategies. In the 1st strategy concentrations of immunoreactive trypsin (IRT) and pancreatitis-associated protein (PAP) were determined. The test was considered positive with concentrations of IRT ≥50 and PAP ≥1.8 or IRT ≥100 and PAP ≥1.0 (all µg/l). For the 2nd method all samples with IRT concentrations ≥50 were analyzed for 36 CF-mutations. An extended gene-analysis (EGA) by sequencing was performed in samples with 1 CF-mutation. Tests were considered positive only when 2 CF-mutations were identified. To assess the sensitivity a retrospective analysis with the 2 methods was performed in heel prick samples of known CF-patients. In total 72,890 neonates were screened. With the IRT-PAP strategy 125 positive screening tests were found, among those 9 CF patients were identified. With the IRT-DNA-EGA strategy 17 positive tests identified 10 CF-patients. This strategy also revealed 84 carriers and 7 compound heterozygotes with R117H-7T as 2nd mutation. With the retrospective heel prick sample analysis of 18 CF-patients, the IRT-PAP strategy identified 13, the IRT-DNA-EGA strategy 18 patients. Specificity and PPV for the IRT-PAP strategy were 99.97 and 7.2%, while for the IRT-DNA-EGA strategy these were 99.99 and 59%. Sensitivity calculated on both the results of the screening program and the retrospective analysis were 79 respectively 100%. Compared to current NBSCF-strategies, an improved test performance of the IRT-DNA-EGA strategy was found, but not of the IRT-PAP strategy.

62. Newborn screening for cystic fibrosis in England in 2007-2008

Sinclair D.¹, Southern K. W.², Judge B.³

¹Queen Alexandra Hospital, Blood Sciences; Newborn Screening, Portsmouth, England

²Royal Liverpool Children’s Hospital, Alder Hey, Liverpool, England

³Great Ormond Street Hospital, Newborn Screening Programme Centre, London, England

From variable 2007 start dates until March 2008, 589,931 babies were screened using IRT and 3253 (0.55%) samples referred for CF genetics investigations. 604 samples were above the 99.9th IRT centile (0.102%). The algorithm requires a second sample for IRT be taken after the first IRT result is found to need further investigation. We requested 627 second IRT samples, 179 because a single mutation was detected on initial genetics screen and 448 to further investigate an IRT greater than the 99.9th centile but without any CF mutations on the initial 4 mutation screen. From the initial 3253 babies samples sent for genetics, 2521 babies were reported as “CF- Not Suspected” as their IRT values were between the 99.5 and 99.9th centiles but had no mutations detected in the 4 mutation gene panel. Similarly, “CF- Not Suspected” was reported in 393 babies whose first IRT was greater than the 99.9th centile but in whose second sample, the IRT had returned to “normal”. 243 babies were referred to the clinicians as “CF Suspected”; data will be presented on these various categories i.e. two copies of CF genes present or one CF mutation and a persistently raised IRT concentration in a second sample or an initial and subsequent raised IRT etc. 140 babies had one mutation and a second IRT that had returned to “normal” i.e. “CF carriers” unless or until shown otherwise by subsequent clinical signs of CF. 30 babies present clinically before screening with most having subsequent positive screens. Two babies were notified to us who could be considered to be “false negatives” although both had meconium ileus. Screening has achieved its aim of maximising the numbers of clinically relevant CF cases detected but with a relatively low proportion of putative carriers recognized.

63. Biochemical strategy IRT/PAP in Newborn Screening for Cystic Fibrosis

Stopsack M.¹, Hammermann J.², Nilson N.²

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

²University Childrens Hospital TU Dresden, Pulmology, Dresden, Germany

Background: Neonatal screening for cystic fibrosis (CF) is currently based on the immunoreactive trypsinogen (IRT) assay followed by a 2nd tier assay (repeat IRT or genetic analysis). Ethical doubt is directed against CFTR gene mutation analysis. We have performed IRT/CFTR screening from 1996–2007 and found 38 patients among 160,675 screened.

Objective and hypotheses: As shown by SARLES, the use of Pancreatits associated Protein (PAP) as second step after IRT elevation could minimize the problems with low IRT specifity, delayed diagnosis after 2. IRT and unwanted carrier detection.

Methods: IRT measurement was done with AutoDELFIA Kit (PerkinElmer, Turku). To increase sensitivity of PAP measurement, the essential reagents from MucoPAP Kit (DYNABIO, Marseille) were modified for measurement by DELFIA reaction. Detection limit, intra- and interassay precision were established for this version. Blood samples from day 2–3, gestational age >32 weeks, were tested for IRT elevation. For samples exceeding 50 ng/ml PAP measurement in duplicate was added. All newborns with result combinations IRT >50 and PAP >1.8 or IRT >100 and PAP >1.0 or those with IRT >150 alone were referred to pilocarpin iontophoresis.^*)

Results: PAP detection limit accounts for 0.07 ng/ml, intraassay precision was 9.42% and interassay precision was 12.46 %. We tested 19,924 newborn samples. 494 (2.5%) of them had IRT >50 and got PAP measurement, whereof 28 (0.15%) were further elevated in IRT/PAP combination. Additional two newborns had only IRT >150 without PAP elevation. All of them got sweat test. Among screened population three CF patients were clearly found.^*)

Conclusions: Modified PAP test fullfills analytical demands for screening purposes and is comparable to other screening methods. Biochemical CF screening by IRT/PAP leads to acceptable recall rate and avoids undesirable carrier identification.

^{*) all concentrations in ng/ml}

64. The effectiveness of a combined Immunoreactive Trypsin (IRT)/Pancreatitis associated protein (PAP)/DNA Newborn Screening Strategy for Cystic Fibrosis

Ranieri E.¹, Gerace R.¹, Kase M.², Bontemps C.³, Dagorn J. Ch.⁴, Fletcher J.¹

¹SAPathology, Women’s & Children’s Hospital Campus, Genetic Medicine, Adelaide, South Australia, Australia

²PerkinElmer Life & Analytical Sciences, Turku, Finland

³Dynabio S.A., Marseille, France

⁴Inserm U624, Camous de Luminy, Marseille, France

Introduction: A two-tier IRT/DNA neonatal screening strategy for CF has been operating in South Australia (SA) since December 1989. Two major limitations of this strategy are; it identifies healthy CFTR carriers and infants with CF-like phenotypes. Recent reports show that PAP which is elevated in neonates with CF has the potential to improve the performance of newborn screening for CF. The aim of this study is to evaluate an IRT-PAP/DNA CF screening strategy.

Methods: The study measured PAP (MucoPAP; DYNABIO S.A. Marseille, France), with a modification to use europium labeled strepavidin and IRT (NeoIRT; PerkinElmer, Turku, Finland) on 3mm blood-spot samples from SA newborns.

Results: A normal population distribution to establish a cut-off (N=2,250, cohort A) gave a median, 95th and 99th percentile values for; PAP of 0.14 ng/mL, 0.43 & 0.86 ng/mL and IRT of 17, 58 & 73 ng/mL respectively. In addition, PAP was also determined on neonates (N=128) selected with an IRT ≥99th percentile and either with (cohort C) or without (cohort B) a single CFTR mutation. In both cohort B & C the median & 95th percentile values were for; IRT of 87.5, 177 ng/mL & 106, 499 ng/mL and PAP of 0.16, 0.6 ng/mL & 0.27, 2.8 ng/mL respectively. In cohort B, the PAP values were not significantly different from the normal population (p=0.12, Kolmogorov-Smirnov test) and only 5% had a PAP level ≥99th percentile. Whilst in cohort C, identified with an IRT ≥99th, a single CFTR mutation and sweat-test normal, only 35% had a PAP level >99th percentile.

Conclusion: These results show that the addition of PAP to those with an IRT level ≥99th (first-tier) has the potential to significantly reduce the number of neonates requiring CFTR mutational analysis by 65%. A multi-centre Australian study is underway to provide a larger cohort of samples to confirm these preliminary results.

65. Pilot study of newborns screening for cystic fibrosis in Serbia

Grkovic S., Radivojevic D., Lalic T., Minic P., Nikolic R., Cvorkov Drazic M.

Mother and child health care institute of Serbia, pediatric, Belgrade, Serbia

Despite its relative frequency among autosomal recessive disease and the availability of the sweet test, cystic fibrosis (CF) has been difficult to diagnose in early childhood and delays can lead to severe malnutrition, lung disease and death. The purpose of this work was to report pilot study of newborn screening for cystic fibrosis in Serbia. Immunoreactive tripsinogen (IRT) analysis was applied to dried newborn bood specimens for recognation of cystic fibrosis from 2007 and was coupled to DNA based detection of mutations in cystic fibrosis transmembrane regulator (CFTR) gene. We assayed 1,000 dried blood samples for immunoreactive trypsinogen with commercial reagents (Biorad). A total of 3 patients with cystic fibrosis were diagnosed through pilot study in the Mother and Child Health Care Institute of Serbia. All newborns have very high immunoreactive tripsinogen (IRT >200 µg/l) in blood, positive sweet test (chloride >60 mEq/l) and presence of two CF mutations (ΔF508/ΔF508). Our experience confirms that a protocol consisting of a two IRT measurement and an expanded mutation panel is effective for CF newborn screening.

66. Development of an updated Neonatal AutoDELFIA IRT assay (B005-212) for cystic fibrosis screening

Kerokoski P.¹, Kinos R.¹, Suonpaa M.¹, Hiekkanen A.¹, Brokopp Ch.², Hoffman G.², Seppala J.¹

¹PerkinElmer Life and Analytical Sciences, Wallac Oy, Neonatal Reagents Product Development, Turku, Finland

²University of Wisconsin-Madison, Wisconsin State Laboratory of Hygiene, Madison, WI, United States

Quantitative determination of human immunoreactive trypsin(ogen) (IRT) from dried blood spots (DBS) is widely used in screening newborns for cystic fibrosis (CF). The updated B005-212 AutoDELFIA IRT kit will replace the current B005-112 AutoDELFIA IRT kit which is the most commonly used primary CF screening method worldwide. The changes implemented to the updated kit version involve raw material and manufacturing process updates, and also the calibration of the kit (in-house procedures, kit calibrators) has been updated to minimize lot-to-lot variation. The objective of the study was to characterize the precision of the updated kit, and to perform a method comparison study of the updated and current kit using routine newborn screening DBS samples. Precision (total CV%) of the updated B005-212 AutoDELFIA IRT kit was analyzed. In the study, 27 runs were performed during 20 days using three different kit lots, and three instruments (two plates per run, four replicates per plate, three samples). The results were: Sample 1, 24.7 ng/mL, CV% 10.8%; Sample 2, 61.8 ng/mL, CV% 8.6%; Sample 3, 97.8 ng/mL, CV% 8.2%. The correlation of the B005-212 (y) and B005-112 IRT kits (x) was: y=0.93x + 0.43; r=0.97 (n=1370, range 16–330 ng/mL, one lot of both kits). In the range of 16–330 ng/mL, the average level difference of the kits was -4.4%, and in the range of 40–80 ng/mL the difference was -1.1%. Both kits produced similar results on 12 CF positive subjects included in the study, and the IRT population distributions matched closely. In conclusion, the performance of the updated B005-212 kit is equivalent to the current IRT kit (B005-112). The calibration of the new kit has been updated to minimize lot-to-lot variation.

67. The decision making process leading towards implementation of a nationwide newborn screening for cystic fibrosis in the Czech Republic

Skalicka V.¹, Balascakova M.², Zemkova D.¹, Piskackova T.², Holubova A.², Kracmar P.³, Vavrova V.¹, Macek M. Jr.², Votava F.³

¹Charles University, 2nd Medical School and University Hospital Motol, Dept. of Pediatrics, Prague, Czech Republic

²Charles University, 2nd Medical School and University Hospital Motol, Dept. of Biology and Medical Genetics-Cystic Fibrosis Centre, Prague, Czech Republic

³Charles University, 3rd Medical School and University Hospital Kralovske Vinohrady, Dept. of Pediatrics, Prague, Czech Republic

Cystic fibrosis (CF) is the most common autosomal recessive disorder. It affects approximately 1:2,736 newborns in the Czech Republic according to epidemiological studies. CF prognosis is highly influenced by early diagnosis. Decision to implement newborn screening (NBS) for CF as a tool for early diagnosis of CF is based on: 1. A significant delay of age at diagnosis (ADG) that has emerged in last decade due to health care decentralisation. Before 1999 median ADG was 0.58 years (60.2% of cases were diagnosed <1 year), while after 1999 median ADG increased to 1.2 years (47.1% of cases were diagnosed by age 1; p=0.036). Furthermore, increasingly patients are now diagnosed between ages 2 and 6 (27.6% vs 16.5%; p=0.03) by primary care physicians. 2. Completion of successful CF newborn screening (NBS) pilot study (II/2005–XI/2006) by applying IRT/DNA/IRT model: Total of 76,438 newborns were screened, 11 patients were detected. Median of ADG was 37 days (range 26–54). In addition 3 older siblings were diagnosed (ADG 3–13 years) in families with newborn diagnosed through CF NBS. No case of clinical diagnosed CF was reported in referral region from within the period of the project. 3. Justification of CF NBS efficacy by favourable clinical outcome of detected patients: Due to early diagnosis patients diagnosed with NBS have better nutritional status than patients diagnosed according to clinical symptoms (CS): body weight NBS -0.2+/-0.6 SD; CS -1,4+/-1,4 SD (p=0,03); BMI 0,2+/-0,6 vs -0,7+/- 1 SD (p=0,03), including lower costs for medical care. Negotiations with Czech Medical Society of J. E. Purkyne and Czech Ministry of Health are under way for establishing a permanent budget line in the health insurance system and for accompanying legislature for CF NBS. We anticipate the start of nationwide CF NBS during year 2010.

Acknowledgements: Supported by IGA9986-3, VZ64203, MSM0021620814.