Reproducible Biochemical Clusters Embedded Within a Continuous Neurochemical Landscape of Autism Spectrum Disorder Revealed by NeuroCLAD

BackgroundAutism Spectrum Disorder (ASD) is marked by pronounced biological heterogeneity, yet most neurochemical studies have relied on single-analyte comparisons that cannot capture coordinated variation across neurotransmitter systems. Whether ASD blood neurotransmitter profiles reflect discrete subtypes, a continuous landscape, or something in between remains unresolved. MethodsWe applied NeuroCLAD, a structured multivariate analytical framework, to peripheral blood neurotransmitter profiles from 261 children with ASD (mean age 6.98 {+/-} 3.13 years; 78.5% male). The pipeline incorporated z-score normalisation, natural cubic spline residualisation for age and sex, principal component analysis, k-means clustering, consensus stability assessment, Gaussian mixture modelling, Cohens d enrichment analysis, and clinical symptom mapping. Cross-compartment consistency was explored using urine neurotransmitter profiles from the same cohort. ResultsTwelve reproducible biochemical cluster patterns were identified, each characterised by distinct pathway-level fingerprints spanning trace amines, monoamines, catecholamine turnover, histamine signalling, and excitatory-inhibitory amino acid balance. Cluster stability was confirmed across 200 bootstrap iterations. Gaussian mixture modelling showed that most individuals were assigned with high confidence, while a subset occupied transitional positions between clusters, consistent with stable biochemical modes embedded within a continuous landscape. Descriptive behavioral mapping revealed graded symptom tendencies across biochemical modes, particularly for aggressiveness, self-injurious behaviour, and picky eating. LimitationsThe findings are based on peripheral blood measurements, which indirectly reflect central neurochemical activity. The study is cross-sectional, lacks a neurotypical comparison group, and behavioural associations are exploratory given cluster sizes. External replication in an independent cohort has not yet been performed. ConclusionsBlood neurotransmitter biology in ASD is neither uniform nor discretely partitioned, but organised into reproducible biochemical modes within a continuous multivariate landscape. These findings support a dimensional view of ASD neurochemistry and provide a foundation for pathway-informed, individualised approaches to biological characterisation.