Rapid and Reagent-Free Analysis of Dried Blood Spot by Paper Spray Mass Spectrometry Reveals Sex: Implications in Forensic Investigations

Identifying sex from an unknown dried blood spot (DBS), especially when the corpse remains undiscovered, often provides valuable evidence in forensic casework. While DNA-based sex determination is a reliable method in forensic settings, it requires expensive reagents and is time-consuming. To develop a rapid reagent-free blood test for sex, we employed paper spray ionization mass spectrometry (PSI-MS) to capture sex-discriminatory lipid profiles from 200 DBS samples comprising 100 males and 100 females. We conducted a supervised machine learning (ML) analysis on all detected lipid signals to hunt biomarkers of sex within the data set. This analysis unveiled significant differences in specific sphingomyelin and phospholipid species levels between male and female DBS samples. Using the parsimonious set of 60 lipid signals, we constructed a classifier that achieved 100% overall accuracy in predicting sex from DBS on paper. Additionally, we assessed three-day-old air-exposed DBS on glass and granite surfaces, simulating the typical blood samples available for forensic investigations. Consequently, we achieved ∼92% overall sex prediction accuracy from the holdout test data set of DBS on glass and granite surfaces. As a highly sensitive detection tool, PSI-MS combined with ML has the potential to revolutionize forensic methods by rapidly analyzing blood molecules encoding personal information.