Comparative Evaluation of Presumptive and Confirmatory Methods for the Detection of Blood at Crime Scenes: Sensitivity, Specificity and Substrate Interference

Abstract

Bloodstains are among the most important forms of biological evidence encountered during crime-scene investigations. Their detection can provide critical information for crime-scene reconstruction and may subsequently enable human blood identification and DNA profiling. However, bloodstains may be present in very small quantities, may be partially cleaned or degraded, and may occur on substrates that interfere with chemical testing. The present study is proposed to comparatively evaluate commonly used presumptive blood-detection methods, including the Kastle–Meyer phenolphthalein test, leucomalachite green test, and luminol-based chemiluminescence, followed by a human-blood specific confirmatory immunochromatographic assay. Blood samples at different dilutions will be deposited on representative crime-scene substrates including cotton, paper, glass, metal, wood and tile. The effects of substrate, dilution, aging and selected interfering substances will be evaluated. Analytical performance will be assessed using sensitivity, specificity, positive predictive value, negative predictive value and misclassification rate. Particular emphasis will be placed on the preservation of biological evidence for subsequent DNA analysis. The study is expected to provide a practical evidence-based framework for selecting blood-detection methods at crime scenes and forensic laboratories. The proposed approach may also help identify circumstances in which presumptive tests produce false-positive or false-negative results and establish an appropriate sequential testing strategy.