Measuring free thyroxine levels in neonatal heel-prick samples.

The Dutch neonatal screening scheme for Congenital Hypothyroidism (CH) is primarily based on the determination of thyroxine (T4) in filter paper blood spots. In the lowest 5% of T4 values, thyroxine binding globulin (TBG) is measured in order to be able to correct for occasional low TBG levels. However, because the commercial TBG kit has been withdrawn from the market, alternative strategies are needed to be explored including the assessment of free T4. We evaluated the Neonatal Free Thyroxine (fT4) enzyme immunoassay (EIA) kit of Bio-Rad. FT4 as measured in a daily run of random samples correlated with T4. We also observed a correlation between fT4 and T4, and between fT4 and T4/TBG ratio in blood spots with low T4 concentrations. The correlation between fT4 and T4 in blood spots of proven CH-patients was highly significant. ROC curves were constructed for the fT4 assay and the T4/TBG ratio based on 27 CH patients and 215 controls with a complete set of data. The curves of both assays seemed to be rather similar. We conclude that the validity of the fT4 and the T4/TBG-approach seems to be the same. A study with a larger sample size giving the same or even more favorable results for the fT4-approach is necessary before we will change the present CH protocol.

The influence of sex, gestational age, birth weight, blood transfusion, and timing of the heel prick on the pancreatitis-associated protein concentration in newborn screening for cystic fibrosis.

BACKGROUND: Pancreatitis-associated protein (PAP) is currently discussed as a marker in newborn screening (NBS) for cystic fibrosis (CF). However, it is not known if PAP concentrations are influenced by sex, gestational age, birth weight, blood transfusion or time of collection and what this would mean for NBS for CF. METHODS: In 2008 all newborns in part of the Netherlands were screened for CF by an IRT/PAP protocol. PAP concentration was determined by the MucoPAP ELISA (DynaBio), which was modified to a Dissociation Enhanced Lanthanide Fluoroimmunoassay (DELFIA) method following a protocol of PerkinElmer. RESULTS: In healthy newborns, the median PAP concentration was 0.5 µg/l (Interquartile range (IQR 0.3-0.8) whereas this was 3.2 µg/l (IQR 2.0-12.5) in CF infants. PAP concentrations were lower in premature infants 0.94 and 0.91 times for 25 to 31 + 6 weeks GA and 32 to 36 + 6 weeks respectively. A higher PAP concentration was observed in low-birth-weight infants (<2500 gram)(p = 0.001), per 100 gram birth weight gained the PAP concentration decreased with 0.1 %. PAP levels were higher after a blood transfusion, the 95th percentile increased from 1.3 to 3.6 µg/l leading to a higher false-positive rate. The PAP concentration increased when newborn screening was performed more than 168 hours (day 7) after birth (ß = 1.63), the 95th percentile increased from 1.3-1.6 µg/l to 4.0 µg/l after 168 hours (72,874 newborns were screened). CONCLUSION: Sex, birth weight, and gestational age lead to small differences in PAP concentrations without consequences for the screening algorithm. However, blood transfusion as well as performance of the heel prick after 168 hours (7 days) lead to clinically significant higher PAP levels and to a higher risk on a false-positive screening test result.

Two-component cluster analysis of a large serodiagnostic database for specificity of increases of IgG antibodies against pertussis toxin in paired serum samples and of absolute values in single serum samples.

Measuring IgG antibodies against pertussis toxin (IgG-Ptx) with an enzyme-linked immunosorbent assay (ELISA) can be used to diagnose pertussis infection; however, the cutoff points are not unanimously defined. To determine the diagnostic specificity of increases of IgG-Ptx in paired sera and of absolute values in single serum samples, we applied a two-component cluster analysis to serum samples of patients suspected for pertussis, whose sera had been submitted to a routine diagnostic laboratory between 2003 and 2009, and had been assayed with an in-house IgG-Ptx ELISA calibrated with the international FDA lot 3 IgG-Ptx reference serum. Children eligible for the acellular pertussis vaccination were excluded to avoid interference from a vaccine-induced IgG-Ptx rise. Binary distribution mixtures were fitted to the data. Receiver operating characteristic (ROC) curves were calculated for absolute values in single samples (n = 14,452) and increases in paired samples (n = 2,455). For both parameters, two subpopulations could be identified: a population with high reactivity (persons with pertussis infection) and a population with low reactivity (persons without pertussis infection). For absolute values in single samples, the area under the curve (AUC) of the ROC curve was 0.993 and the optimum cutoff (with the highest cumulative value of specificity plus sensitivity) was 67.7 IU/ml (95% confidence interval, 63.9 to 74.1; sensitivity, 96.4%; specificity, 95.7%). A previously determined diagnostic cutoff of 125 IU/ml was associated with a sensitivity of 88.1% and a specificity of 98.8%. For increases in paired sera, the AUC was 0.999 and the optimum cutoff was 3.1-fold (95% CI, 2.8 to 3.4; sensitivity, 99.6%; specificity, 99.2%). Given the methodology of this study, estimates of sensitivity probably are overrated (because pertussis patients without IgG-Ptx response are not detected), but estimates of specificities can be considered very accurate.

Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: data from a cohort study.

BACKGROUND: Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes. METHODS: We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007-2010. Clinical, molecular, and enzymatic data were integrated. RESULTS: Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210-1/11,130). CONCLUSIONS: Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and >10% residual MCAD enzyme activity.

Novel strategies in newborn screening for cystic fibrosis: a prospective controlled study.

CONTEXT: Newborn screening for cystic fibrosis (CF) is included in many routine programmes but current strategies have considerable drawbacks, such as false-positive tests, equivocal diagnosis and detection of carriers. OBJECTIVE: To assess the test performance of two newborn screening strategies for CF. DESIGN, SETTING AND PARTICIPANTS: In 2008 and 2009, CF screening was added to the routine screening programme as a prospective study in part of The Netherlands. INTERVENTIONS: Two strategies were performed in all newborns. In the first strategy, concentrations of immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) were measured. In the second method, samples with IRT ≥60 µg/litre were analysed for 36 CFTR mutations, followed by sequencing when a single mutation was detected. Tests were positive only with two identified CFTR mutations. MAIN OUTCOME: Sensitivity, specificity and positive predictive value (PPV) of both screening strategies. RESULTS: 145,499 infants were screened. The IRT/PAP approach showed a sensitivity of 95.0%, a specificity of 99.897% and a PPV of 12.3%. Test properties for the IRT/DNA/sequencing strategy were respectively 100%, 100% and 64.9%. Combining both strategies (IRT/PAP/DNA/sequencing) led to a sensitivity of 95.0%, a specificity of 100% and a PPV of 87.5%. CONCLUSION: In conclusion, all strategies performed well. Although there was no statistically significant difference in test performance, the IRT/DNA/sequencing strategy detected one infant that was missed by IRT/PAP (/DNA/sequencing). IRT/PAP may be the optimal choice if the use of DNA technology must be avoided. If identification of carriers and equivocal diagnosis is considered an important disadvantage, IRT/PAP/DNA/sequencing may be the best choice.

The Proline/Citrulline Ratio as a Biomarker for OAT Deficiency in Early Infancy.

Deficiency of ornithine-δ-aminotransferase (OAT) in humans results in gyrate atrophy. Early diagnosis may allow initiation of treatment before irreversible damage has occurred. However, diagnosis is commonly delayed well into adulthood because of the nonspecific character of initial symptoms. Here, we report findings in a neonate who was evaluated because of a positive family history of OAT deficiency. The reversed enzymatic flux in early infancy resulted in borderline low ornithine concentration - evoking urea cycle disturbances - and increased proline. In addition, plasma citrulline was low. Consequently, the proline/citrulline ratio in plasma was increased compared to controls. To find out whether amino acid profiling in neonatal dried blood spots is suitable to detect OAT deficiency, we evaluated the original newborn dried blood spots of two affected patients and compared it with a database of >450,000 newborns tested in Minnesota since 2004. Proline concentrations (777 and 1,381 µmol/L) were above the 99 percentile (776 µmol/L) of the general population, and citrulline concentrations (4.5 and 4.9 µmol/L) only just above the 1 percentile (4.37 µmol/L). The proline/citrulline ratio was 172.9 and 281.8, respectively. This ratio was calculated retrospectively in the normal population, and the 99 percentile was 97.6. Applying this ratio for NBS could lead to early and specific detection of neonatal OAT deficiency, with no additional expense to newborn screening laboratories quantifying amino acids. Given that early diagnosis of OAT disease can lead to earlier treatment and prevent visual impairment, further studies are indicated to evaluate whether newborn screening for OAT deficiency is warranted.

Implementing neonatal screening for haemoglobinopathies in the Netherlands.

BACKGROUND: The birth prevalence of severe haemoglobinopathies such as sickle cell disease (SCD) in the Netherlands has been estimated to be at least 50 newborns per year. Neonatal screening for SCD was added to the Dutch screening programme in January 2007. We here evaluated three high performance liquid chromatography (HPLC) systems for application in neonatal screening for haemoglobinopathies, and present the results of a subsequent pilot screening programme. METHODS: The Variant NewBorn Screening (Vnbs) HPLC system (Bio-Rad) was validated by analysing 131 blood samples and blood mixtures. Subsequently, the performance of the G7 (Tosoh BioScience) and Ultra (Primus Corporation) was compared with the Vnbs. The three HPLC analysers were tested in a pilot screening programme on 21,969 dried blood spot samples from the routine Dutch neonatal screening programme. RESULTS: The pilot screening resulted in 188 abnormal patterns. The three HPLC devices presented comparable within- and between-run precision and detected the abnormal samples similarly. The high throughput, sampling systems, presentation of results, and integration of the chromatograms, however, were different. CONCLUSION: All three analysers detected the same abnormal haemoglobins satisfactorily, but integrated the chromatograms with variable imprecision. Comparison of the results suggested that the Bio-Rad Vnbs was the preferred system. However, software adjustments were required to improve the diagnostic potential of this device for screening for beta- and alpha-thalassaemia.

Seroprevalence of Bordetella pertussis infection during pregnancy measured by IgG antibodies against pertussis toxin.

Bordetella pertussis infection may cause severe illness in newborns. Mothers with B. pertussis infection during delivery can infect newborns. The seroprevalence of B. pertussis infection in pregnancy was measured in pregnant women by detection of immunoglobulin G against pertussis toxin; 6.3% had serological evidence of infection. Maternal vaccination should be considered to prevent pertussis in newborns.

Cutoff levels of 17-alpha-hydroxyprogesterone in neonatal screening for congenital adrenal hyperplasia should be based on gestational age rather than on birth weight.

OBJECTIVE: In newborn screening programs for congenital adrenal hyperplasia, 17-alpha-hydroxyprogesterone (17OHP) cutoff levels are based on birth weight (BW) or on gestational age (GA). We investigated which approach would result in the greatest specificity and sensitivity. STUDY DESIGN: For the determination of 17OHP, a neonatal 17OHP assay was used in filter paper blood of 9492 newborns. The relationships between 17OHP and BW and between 17OHP and GA were studied by regression analysis. Reference curves with a specificity of 99.95% were constructed with the method that summarizes the distribution by three smoothed curves representing the skewness (L curve), the median (M curve), and the coefficient of variation (S curve). Median cutoff levels for BW and for GA according to the 99.95% reference curves were calculated. RESULTS: Regression analysis showed that GA is a better predictor of 17OHP than BW (R(2) was 50.6 vs. 35.8%, respectively). At a specificity of 99.95%, the calculated median 17OHP cutoff level was lower for GA [12.6 microg/liter (38 nmol/liter)] than for BW [17.6 microg/liter (54 nmol/liter)], thus leading to a greater sensitivity. CONCLUSION: This study demonstrates that GA is a better predictor of 17OHP in newborns and will result in greater specificity than BW despite the fact that the determination of GA might be less reliable than BW.