Abstract
Sexual assault against women is prevalent, prompting initiatives such as Australia's National Plan To End Violence Against Women. Forensic DNA analysis is key to achieving justice by linking offenders to crime scenes. Differential extraction remains the gold standard for separating victim and perpetrator DNA through selective lysis of cells. Despite widespread adoption of the original differential lysis protocol, results can have mixed success, prompting consideration of inter-operator variability. This study assessed the reproducibility of the 1980s method by having three forensic researchers independently conduct the published method using identical sperm samples (n = 3 replicates each). Their protocol interpretation was recorded, revealing numerous procedural differences. Operator 1 recovered 1.95 ng (range 1.92 - 1.99 ng) of DNA, operator 2 obtained no detectable DNA, and operator 3 resulted in 0.58 ng (range 0.56 - 0.60 ng) of DNA. These findings suggest that the original published protocol contains sections with insufficient detail to reproduce with precision. Forensic research manuscripts must ensure that methods are comprehensive to avoid similar circumstances.