Investigation of Choice for Phenylketonuria (PKU) in Newborns
Newborn screening for PKU is performed using tandem mass spectrometry (MS/MS) on dried blood spots collected in the first days of life, followed by confirmatory quantitative plasma amino acid analysis when the screen is positive. 1
Initial Screening Method
Tandem mass spectrometry is now the primary screening method for PKU in the United States, having largely replaced older methods. 1 The three main laboratory methods historically used include:
- Tandem mass spectrometry (MS/MS) - current standard 1, 2
- Guthrie bacterial inhibition assay - older method 1
- Fluorometric analysis - older method 1
MS/MS offers superior accuracy because it simultaneously quantifies both phenylalanine (Phe) and tyrosine (Tyr), allowing calculation of the Phe/Tyr ratio, which dramatically reduces false-positive rates compared to fluorometric methods. 3 Research demonstrates that MS/MS reduces false-positives almost 100-fold when using a Phe/Tyr molar ratio cutoff of 2.5. 3
Confirmatory Diagnostic Testing
When newborn screening is positive, quantitative plasma amino acid analysis is the standard confirmatory method. 1 This analysis must include:
- Quantitative phenylalanine level 1
- Phenylalanine-to-tyrosine (Phe:TYR) ratio 1
- Complete amino acid profile to exclude generalized aminoacidemia 1
Samples should be collected before dietary phenylalanine restriction begins. 1 By the time confirmatory testing occurs, newborns with PAH deficiency typically show phenylalanine concentrations higher than the original screening sample. 1
Critical Additional Testing
Before initiating treatment, tetrahydrobiopterin (BH4) deficiency must be excluded in all newborns with elevated phenylalanine. 1 This is essential because BH4 deficiencies require different treatment approaches. The American College of Medical Genetics recommends:
- Pterin analysis (neopterin and biopterin) measured in urine or blood 1
- Erythrocyte dihydropteridine reductase activity measured on whole blood spotted on filter paper 1
- Quantitative urinary neopterin and biopterin assay to confirm filter paper results 1
Abnormal pterin levels should prompt enzyme testing for deficiencies of GTP cyclohydrolase, 6-pyruvoyl-tetrahydrobiopterin synthetase, dihydropteridine reductase, or pterin carbinolamine-4α-dehydratase. 1
Genotyping for Treatment Planning
PAH genotyping is indicated for improved therapy planning after biochemical diagnosis is confirmed. 1 Genotyping helps determine:
- The extent of dietary phenylalanine restriction needed 1
- Likelihood of response to sapropterin (BH4) cofactor supplementation 1
- Biochemical phenotype severity 4
Timing Considerations
Treatment must begin within the first week of life, with blood phenylalanine in the treatment range by 2 weeks of age to prevent irreversible neurological damage. 1, 2 This requires:
- Prompt newborn screening (first days of life) 1, 2
- Rapid follow-up of positive screens 1, 2
- Immediate confirmatory testing 1
- Treatment initiation when phenylalanine exceeds 360-605 μmol/L (approximately 6-10 mg/dL) 1
Common Pitfalls to Avoid
Early discharge specimens collected before 24 hours of age have higher false-positive rates with older fluorometric methods, though MS/MS with Phe/Tyr ratio significantly mitigates this issue. 3
Failure to screen for BH4 deficiency before starting dietary treatment is a critical error, as these patients require neurotransmitter replacement therapy rather than dietary restriction alone. 1
Significant variation exists in screening protocols between states, including different cutoff values for positive screens and varying follow-up procedures, which can delay diagnosis. 1, 2