Single-cell strategies for the interpretation of complex forensic DNA mixtures: isolation techniques, genotype data integration, and prospects for application.

Complex forensic DNA mixtures remain challenging to interpret, especially when contributor proportions are highly imbalanced or when the mixture involves the same cell type. Single-cell isolation strategies have provided a novel methodological approach for overcoming these technical limitations. This review provides a summary of recent advances in single-cell isolation techniques for complex forensic DNA mixtures. It covers three major categories of techniques: micromanipulation and laser capture microdissection (LCM); fluorescence‑activated cell sorting (FACS) paired with imaging flow cytometry (IFC); and microfluidic‑based systems represented by the DEPArray™ platform. The performance of these techniques is comparatively evaluated in terms of throughput, sample adaptability, and cost. We further discuss key challenges in single‑cell STR profiling, such as allelic drop‑out (ADO) and allelic drop‑in (ADI), and summarize strategies for multi‑single‑cell clustering, consensus genotype reconstruction, and probabilistic evidence interpretation. Finally, the forensic value of single-cell strategies is emphasized. The future directions include the integration of automation and high‑throughput workflows, artificial intelligence-assisted analysis, as well as the development of standardized interpretation frameworks.