Cystic Fibrosis Newborn Screening in Austria Using PAP and the Numeric Product of PAP and IRT Concentrations as Second-Tier Parameters

In Austria, newborns have been screened for cystic fibrosis (CF) by analyzing immunoreactive trypsinogen (IRT) from dried blood spots (DBS)s for nearly 20 years. Recently, pancreatitis-associated protein (PAP) analysis was introduced as a second-tier test with the aim of reducing recalls for second DBS cards while keeping sensitivity high. For 28 months, when IRT was elevated (65–130 ng/mL), PAP was measured from the first DBS (n = 198,927) with a two-step cut-off applied. For the last 12 months of the observation period (n = 85,421), an additional IRT×PAP cut-off was introduced. If PAP or IRT×PAP were above cut-off, a second card was analyzed for IRT and in case of elevated values identified as screen-positive. Above 130 ng/mL IRT in the first DBS, newborns were classified as screen-positive. IRT analysis of first DBS resulted in 1961 (1%) tests for PAP. In the first 16 months, 26 of 93 screen-positive were confirmed to have CF. Two false-negatives have been reported (sensitivity = 92.8%). Importantly, less than 30% of families compared to the previous IRT-IRT screening scheme had to be contacted causing distress. Adding IRT×PAP caused a marginally increased number of second cards and sweat tests to be requested during this period (15 and 3, respectively) compared to the initial IRT-PAP scheme. One case of confirmed CF was found due to IRT×PAP, demonstrating an increase in sensitivity. Thus, the relatively simple and economical algorithm presented here performs effectively and may be a useful model for inclusion of CF into NBS panels or modification of existing schemes.

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A product of immunoreactive trypsinogen and pancreatitis-associated protein as second-tier strategy in cystic fibrosis newborn screening

Journal of Cystic Fibrosis ◽

10.1016/j.jcf.2016.07.002 ◽

2016 ◽

Vol 15 (6) ◽

pp. 752-758 ◽

Cited By ~ 6

Author(s):

Sophia Weidler ◽

Konrad H. Stopsack ◽

Jutta Hammermann ◽

Olaf Sommerburg ◽

Marcus A. Mall ◽

...

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Second Tier ◽

Pancreatitis Associated Protein ◽

Immunoreactive Trypsinogen

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Concurrent Confirmation and Differential Diagnosis of Congenital Adrenal Hyperplasia from Dried Blood Spots: Application of a Second-Tier LC-MS/MS Assay in a Cross-Border Cooperation for Newborn Screening

Hormone Research in Paediatrics ◽

10.1159/000439380 ◽

2015 ◽

Vol 84 (5) ◽

pp. 311-318 ◽

Cited By ~ 9

Author(s):

Péter Monostori ◽

Pál Szabó ◽

Otilia Marginean ◽

Csaba Bereczki ◽

Eszter Karg

Keyword(s):

Differential Diagnosis ◽

Congenital Adrenal Hyperplasia ◽

Newborn Screening ◽

Dried Blood Spots ◽

Adrenal Hyperplasia ◽

Blood Spots ◽

Cross Border ◽

Second Tier ◽

Cross Border Cooperation

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Newborn Screening for Cystic Fibrosis

PEDIATRICS ◽

10.1542/peds.87.6.954 ◽

1991 ◽

Vol 87 (6) ◽

pp. 954-955

Author(s):

IAN C. T. LYON ◽

DIANNE R. WEBSTER

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Elevated Level ◽

Meconium Ileus ◽

Screening Tests ◽

Initial Screening ◽

False Negatives ◽

Immunoreactive Trypsinogen

To the Editor.— The report on newborn screening for cystic fibrosis1 illustrates the need for continued evaluation of such programs. The authors state that the identification of cases of cystic fibrosis (CF) by an elevated level of immunoreactive trypsinogen (IRT) in second (follow-up) samples from infants with positive initial screening tests could result in false negatives in 27% of cases of cystic fibrosis without meconium ileus (MI). We have screened 401 122 infants using the method originally reported.2

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Second-Tier Test for Quantification of Alloisoleucine and Branched-Chain Amino Acids in Dried Blood Spots to Improve Newborn Screening for Maple Syrup Urine Disease (MSUD)

Clinical Chemistry ◽

10.1373/clinchem.2007.098434 ◽

2008 ◽

Vol 54 (3) ◽

pp. 542-549 ◽

Cited By ~ 65

Author(s):

Devin Oglesbee ◽

Karen A Sanders ◽

Jean M Lacey ◽

Mark J Magera ◽

Bruno Casetta ◽

...

Keyword(s):

Amino Acids ◽

Newborn Screening ◽

Dried Blood Spots ◽

Branched Chain Amino Acids ◽

Maple Syrup ◽

Blood Spots ◽

Urine Disease ◽

Second Tier ◽

Branched Chain ◽

Positive Results

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

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Newborn Screening for Cystic Fibrosis by Use of a Multiplex Immunoassay

Clinical Chemistry ◽

10.1373/clinchem.2009.132480 ◽

2010 ◽

Vol 56 (3) ◽

pp. 445-450 ◽

Cited By ~ 16

Author(s):

Barbara A Lindau-Shepard ◽

Kenneth A Pass

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Screening Program ◽

Pancreatic Disease ◽

High Rate ◽

Dna Testing ◽

Second Tier ◽

Sample Set ◽

Positive Results ◽

Immunoreactive Trypsinogen

Abstract Background: Since its beginnings, newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high rate of false-positive results (screen positive, diagnosis negative), despite attempts to reduce this rate by use of altered cutoffs and second-tier DNA testing. IRT exists as 2 isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Assay standardization between programs is a continuing problem because the IRT assays currently in use variously recognize either 1 or both isoforms. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. Methods: Using 2 different Luminex bead sets, we developed assays for each IRT isoform separately and then combined them. Using the sum of IRT1 and IRT2 values (IRT1+IRT2), we compared the results with a CF kit currently in use. Results: In a sample set consisting of 16 cases confirmed positive for CF, we established a cutoff at >97 μg/L total IRT. Seven of 8 carriers with 1 CF mutation screen-positive by the standard method were also screen-positive by IRT1+IRT2. Of 32 cases screen-positive by standard IRT, 11 were screen-negative by IRT1+IRT2. None of these 11 cases had CF mutations identified by the screening program. Conclusions: These data indicate that the multiplex method with specificity for 2 isoforms of IRT has performance comparable to that of a standard IRT method and the advantage of improved standardization by detection of the 2 isoforms.

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Combining Immunoreactive Trypsinogen and Pancreatitis-Associated Protein Assays, a Method of Newborn Screening for Cystic Fibrosis that Avoids DNA Analysis

The Journal of Pediatrics ◽

10.1016/j.jpeds.2005.05.017 ◽

2005 ◽

Vol 147 (3) ◽

pp. 302-305 ◽

Cited By ~ 49

Author(s):

Jacques Sarles ◽

Patrice Berthézène ◽

Christian Le Louarn ◽

Claude Somma ◽

Jean-Marc Perini ◽

...

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Dna Analysis ◽

Protein Assays ◽

Pancreatitis Associated Protein ◽

Immunoreactive Trypsinogen

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Next-generation sequencing as a second-tier diagnostic test for newborn screening

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2018-0088 ◽

2018 ◽

Vol 31 (8) ◽

pp. 927-931 ◽

Cited By ~ 2

Author(s):

Xiaomei Luo ◽

Ruifang Wang ◽

Yanjie Fan ◽

Xuefan Gu ◽

Yongguo Yu

Keyword(s):

Next Generation Sequencing ◽

Newborn Screening ◽

Diagnostic Test ◽

Genomic Dna ◽

Sanger Sequencing ◽

Dried Blood Spots ◽

Next Generation ◽

Blood Spots ◽

Second Tier ◽

Generation Sequencing

Abstract Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.

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Initial evaluation of a biochemical cystic fibrosis newborn screening by sequential analysis of immunoreactive trypsinogen and pancreatitis-associated protein (IRT/PAP) as a strategy that does not involve DNA testing in a Northern European population

Journal of Inherited Metabolic Disease ◽

10.1007/s10545-010-9174-7 ◽

2010 ◽

Vol 33 (S2) ◽

pp. 263-271 ◽

Cited By ~ 22

Author(s):

Olaf Sommerburg ◽

Martin Lindner ◽

Martina Muckenthaler ◽

Dirk Kohlmueller ◽

Svenja Leible ◽

...

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Sequential Analysis ◽

European Population ◽

Initial Evaluation ◽

Dna Testing ◽

Northern European ◽

Pancreatitis Associated Protein ◽

Immunoreactive Trypsinogen

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Initial Evaluation of Prospective and Parallel Assessments of Cystic Fibrosis Newborn Screening Protocols in Eastern Andalusia: IRT/IRT Versus IRT/PAP/IRT

International Journal of Neonatal Screening ◽

10.3390/ijns5030032 ◽

2019 ◽

Vol 5 (3) ◽

pp. 32

Author(s):

Sadik ◽

Algaba ◽

Jiménez ◽

Benito ◽

Blasco-Alonso ◽

...

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

False Positive ◽

Good Alternative ◽

Safety Net ◽

Strategy Versus ◽

Second Tier ◽

Post Hoc ◽

Positive Results ◽

Immunoreactive Trypsinogen

Identifying newborns at risk for cystic fibrosis (CF) by newborn screening (NBS) using dried blood spot (DBS) specimens provides an opportunity for presymptomatic detection. All NBS strategies for CF begin with measuring immunoreactive trypsinogen (IRT). Pancreatitis-associated protein (PAP) has been suggested as second-tier testing. The main objective of this study was to evaluate the analytical performance of an IRT/PAP/IRT strategy versus the current IRT/IRT strategy over a two-year pilot study including 68,502 newborns. The design of the study, carried out in a prospective and parallel manner, allowed us to compare four different CF-NBS protocols after performing a post hoc analysis. The best PAP cutoff point and the potential sources of PAP false positive results in our non-CF newborn population were also studied. 14 CF newborns were detected, resulting in an overall CF prevalence of 1/4, 893 newborns. The IRT/IRT algorithm detected all CF cases, but the IRT/PAP/IRT algorithm failed to detect one case of CF. The IRT/PAP/IRT with an IRT-dependent safety net protocol was a good alternative to improve sensitivity to 100%. The IRT × PAP/IRT strategy clearly performed better, with a sensitivity of 100% and a positive predictive value (PPV) of 39%. Our calculated optimal cutoffs were 2.31 µg/L for PAP and 167.4 µg2/L2 for IRT × PAP. PAP levels were higher in females and newborns with low birth weight. PAP false positive results were found mainly in newborns with conditions such as prematurity, sepsis, and hypoxic-ischemic encephalopathy.

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