Forensic Application of Atomic Force Microscopy for Age Determination of Bloodstains

Forensic determination of the age of a bloodstain with little temporal error can provide important information in a judicial inquiry. Atomic force microscopy could provide estimations for bloodstain ages based on temporal changes in the elasticity of red blood cells (RBCs). This technology enables RBC nano-indentations with the tip of a cantilever resulting in force measurements and high resolution mapping of nanomechanical cell properties like the Young's modulus of elasticity. This study applied atomic force microscopy to investigate the elasticity of randomly selected RBCs from the peripheral zone of 4- to 8-day-old bloodstains (controlled laboratory conditions). Special attention was paid to the condition of the silicon probes when continuously used to indent RBCs. The elasticity of 6 RBCs from a 5-day-old bloodstain appeared homogenous over the cell with a mean Young's modulus of 1.6 ± 0.4 GPa. Data spreading was for only 6.5% caused by the choice of the RBC. Upon ageing of a bloodstain, 4 to 8 days, a significant age effect was observed in RBC elasticity (4 days: 0.8 ± 0.1 GPa; 5 days: 1.7 ± 0.9 GPa; 6 days: 2.3 ± 0.6 Gpa; 7days: 4.5 ± 0.6 GPa; 8 days: 6.0 ± 1.8 GPa; probe spring constants 25.16 - 67.48 N/m). The spreading between the data (4 bloodstains; 3 RBCs/stain) enabled a bloodstain age determination with a 24 h precision only for 6- to 7-day-old stains. In other cases a 48 h temporal accuracy was obtained. The condition of the silicon tip was regularly checked using scanning electron microscopy and an increasing bluntness noticed after 4 to 6 cell indentations. A correction factor for the increase in tip radius was therefore applied for data processing. Particularly for crime scene investigations a more robust tip is required thereby increasing both precision and accuracy of the elasticity data.