High-gradient magnetic separation chip-based small extracellular vesicle isolation for noninvasive subtyping of primary aldosteronism.

Abstract

Primary aldosteronism (PA), a leading cause of secondary hypertension, remains vastly underdiagnosed due to unreliable screening tools. We developed an integrated platform combining a high-gradient magnetic separation three-dimensional (HGMS-3D) chip and locked nucleic acid (LNA)-enhanced droplet digital polymerase chain reaction (ddPCR) for noninvasive detection of potassium inwardly rectifying channel subfamily J member 5 (<i>KCNJ5</i>) mutations in plasma small extracellular vesicles (sEVs). The HGMS-3D chip uses a nickel mesh-based stereoscopic immunoaffinity capture system, achieving a vesicle isolation efficiency 4.4-fold higher than ultracentrifugation. Coupled with LNA-ddPCR, the platform detects <i>KCNJ5</i> hotspot mutations [p.Gly151Arg, (G151R); p.Leu168Arg (L168R)] at minor allele frequencies of ≤0.05% (<i>R</i>² ≥ 0.99), overcoming plasma-derived noise. Clinical validation in 106 patients with PA demonstrated 64.58% sensitivity and 96.88% specificity for sEV-based mutation profiling. The assay identified one aldosterone-producing adenoma (APA) case missed by tissue genotyping, achieving area under the receiver operating characteristic curve (AUC) values of 0.767 ~ 0.852 across mutations. This noninvasive approach could enable curative treatment for millions with undiagnosed PA, advancing precision management of endocrine hypertension through sEV-based liquid biopsy.