Diagnosis & biomarkers

Newborn screening for MCADD is performed worldwide using tandem mass spectrometry (MS/MS) to analyze acylcarnitines from dried blood spots. The technique allows simultaneous detection of multiple metabolic disorders.

Newborn screening for MCADD has been implemented in many countries, including the United States (expanded to all states), the Netherlands, Denmark, Germany, Austria, and others. The specific start dates vary by country and state. Orphanet notes that MCADD is included in newborn screening programs in many European countries, including the United Kingdom, Germany, the Netherlands, Portugal, and Spain.

Newborn screening uses dried blood spots collected on filter paper (Guthrie cards), typically from a heel prick. The dried blood spots are then analyzed using tandem mass spectrometry (MS/MS), which measures acylcarnitine species. The MS/MS method allows rapid, high-throughput analysis of multiple metabolic markers from a single blood spot.

The acylcarnitine profile measured by MS/MS includes multiple species. In MCADD, the key markers are:

• C8 (octanoylcarnitine) — the primary and most specific marker
• C6 (hexanoylcarnitine) — secondary marker
• C10 (decanoylcarnitine) — secondary marker
• C10:1 (decenoylcarnitine) — also elevated in some cases

Ratios between these markers improve diagnostic specificity:

• C8/C10 ratio — elevated in MCADD
• C8/C2 ratio — elevated in MCADD

C8 (octanoylcarnitine) is the most sensitive and specific marker for MCADD on newborn screening. Elevated C8 is the primary trigger for further diagnostic evaluation.

Specific cutoff values vary by screening program and are not standardized internationally. Each newborn screening program establishes its own cutoffs based on local population data and analytical methods.

Aggregate 99th centile values from North American newborn screening samples (Collaborative Laboratory Integrated Reports) are:

• C8: 2.46 nmol/mL
• C6: 2.44 nmol/mL
• C10: 2.38 nmol/mL
• C10:1: 2.44 nmol/mL
• C8/C2 ratio: 2.43
• C8/C10 ratio: 2.53

Note: 1 nmol/mL is equivalent to 1 µmol/L. Because of differences in sample analysis, aggregate C8 values may not be directly comparable to local program results; each program’s own cutoffs should be used.

Genotype-stratified C8 values (observational data):

• In a retrospective cohort of 90 patients, individuals homozygous for c.985A>G (p.Lys329Glu) had higher NBS C8 (23.4 ± 19.6 vs 6.6 ± 3.0 µmol/L) than those with at least one other pathogenic variant (Anderson et al 2020).
• In a US cohort of 221 newborn-screened neonates, the average C8 on the first screen was 11.2 µmol/L (median 8.6, range 0.36–43.91); higher C8 correlated with symptomatic newborns, and C8 and genotype were significant predictors of neonatal symptoms (Bentler et al 2016).

When newborn screening suggests MCADD, confirmatory testing is required. GeneReviews recommends:

1. Plasma acylcarnitine profile — confirms the elevation pattern.
2. Molecular genetic testing — ACADM gene sequencing to identify disease-causing variants.
3. Urine organic acid analysis — may show elevated dicarboxylic acids (suberic, adipic, sebacic acids).
4. Enzyme activity analysis — in fibroblasts or other tissues, can measure residual MCAD activity (performed in specialized laboratories).

Molecular testing typically includes sequencing of the ACADM coding region and may include deletion/duplication analysis.

ACADM molecular genetic testing is the primary method for confirming MCADD diagnosis. Testing identifies disease-causing variants in the ACADM gene. The K304E (c.985A>G) variant is the most common, particularly in northern European populations.

Targeted testing for common variants may be performed first, followed by full sequencing if needed. When available, deletion/duplication analysis can identify variants not detected by sequencing.

False positive newborn screening results for MCADD can occur due to:

• Maternal MCADD (if the mother has undiagnosed MCADD, her acylcarnitines can elevate the newborn’s result).
• Heterozygous ACADM c.985A>G (p.Lys329Glu) carriers — term, appropriate-for-gestational-age infants heterozygous for the common pathogenic variant can have C8 levels above the 90th centile (Blois et al 2005).
• Other conditions causing elevated C8 (e.g., carnitine palmitoyltransferase I deficiency, medium-chain triglyceride supplementation).
• Prematurity or low birth weight.
• Technical/analytical issues.

False positives for elevated C8 are not common with MS/MS, but can be seen in term heterozygous carriers and premature infants. Second-tier testing (e.g., molecular testing, additional acylcarnitine ratios) helps reduce false positives.

False negative results are less common but can occur. Cases have been reported of:

• MCADD with severe neonatal onset and normal acylcarnitine profile at the time of screening.
• ACADM splice mutations missed by newborn screening.
• Rare genotypes not producing elevated C8 in the early newborn period.
• Low free carnitine levels — newborns with low free carnitine (e.g., infants born to a mother with previously undiagnosed MCAD deficiency, maternal carnitine transporter deficiency, or nutritional carnitine deficiency) can have lower elevations of C8/C6/C10 acylcarnitines, or even normal profiles, leading to false negatives (Leydiker et al 2011; Aksglaede et al 2015; Weiss et al 2023).

This is particularly concerning because false negatives can lead to delayed diagnosis and potentially life-threatening presentations.

Conditions that can present with similar biochemical findings or clinical pictures include:

• Other fatty acid oxidation disorders (e.g., VLCAD deficiency, SCAD deficiency, glutaric acidemia type II).
• Carnitine transporter defects.
• Reye syndrome (differential for clinical presentation).
• Hypoglycemia from other metabolic causes (e.g., glycogen storage diseases).

Biomarker interpretation must be done in clinical context. A single biomarker value should never be treated as independently diagnostic without proper clinical context.

Key considerations:

• C8 levels can vary with age, feeding status, and clinical condition.
• The acylcarnitine profile during an acute metabolic crisis may differ significantly from the well state.
• Residual enzyme activity varies by genotype and may influence biochemical phenotype.
• C8 level and genotype are significant predictors of neonatal symptoms in newborn-screened cohorts; individuals homozygous for the common c.985A>G (p.Lys329Glu) variant have the highest C8 values and are most likely to have neonatal symptoms (Bentler et al 2016; Anderson et al 2020).
• Some infants identified by newborn screening may have biochemical evidence of MCADD but never develop symptoms.