1. Introduction
Blood is a versatile type of evidence in forensic science investigations and can provide important information, such as the ‘who’, ‘what’, and ‘how’ as it relates to criminal investigations. For example, blood can be collected and used for DNA analysis, chemically analyzed to identify drugs and other substances, and physically observed to conduct bloodstain pattern analysis (BPA) [1,2]. Whole blood is composed of red blood cells (RBCs), white blood cells (WBCs) – which contain DNA, and platelets, all suspended in a liquid plasma [3]. Blood is a non-Newtonian and shear-thinning fluid, meaning its viscosity is dependent on the amount of applied shear stress, which causes blood to become more liquid-like at higher shear rates [4]. Bloodstains observed at crime scenes display a large variability in appearance. For example, their size, shape, distribution and colour are largely dependent on the mechanism, environmental and surface conditions of their deposition and observed degradation state (more commonly referred to as time since deposition, or TSD) [1,5].
The drying process of blood begins immediately after exiting the body [6] and has been summarized in detail by Sobac and Brutin [7] to occur in three phases; the pre-gelation phase (Phase 0), the gelation phase (Phase 1) and the post-gelation phase (Phase 2). In Phase 0, RBCs begin to migrate toward the edge of the bloodstain where a desiccation line begins to form. In Phase 1, a compaction front forms at the edge of the bloodstain and moves inwards toward the center. Simultaneously, the bloodstain desiccates inward, while a dark red ‘donut’ can be seen; this donut is highly concentrated with RBCs and moves from the center of the bloodstain to the outside. The donut begins to desiccate, and the center of the bloodstain turns a lighter red. By this point, the edge of the bloodstain is almost fully desiccated, and the donut shape has become a solid mass; cracks have begun to form at the edge of the bloodstain and propagate inwards. By Phase 2, the center of the bloodstain begins to dry and cracks begin to form, while the rim becomes fully desiccated. The remainder of the bloodstain then becomes desiccated and no further changes are observed. Laan et al. [7] and Benabdelhalim et al. [6] found a similar drying process analyzing blood pools (~4 mL); however, the first phase consisted of the bloodstain coagulating, and increased colour changes were observed, including the pool changing to a black colour as it desiccated.
Droplet desiccation is influenced by a variety of parameters such as packed cell volume (PCV%, the packed cell volume percentage in a blood sample), surface wettability, temperature, and relative humidity (RH) [8]. Larkin et al. [9] investigated the effects of PCV% on the drying process and corroborated the drying mechanism described by Sobac et al. [10]. In their study, it was found that a decrease in PCV% increased the effects of Marangoni flow due to surface tension differences, but did not influence drying time [9]. RH also plays a key role in the drying process and phase separation of larger bloodstains [6]; as RH increases, the transfer of water between its liquid and gaseous state is limited by the increased concentration of water in the air [11]. This decreased evaporation rate leads to a variation in plaque formation in the rim, which are sections of the rim that separate from the surrounding bloodstain to produce islands of dried blood [11]. Temperature differences also influence the drying time and morphology of bloodstains. Ramsthaler et al. [8] observed an increase in drying times of bloodstains from 30 min (24 °C) to upwards of 120 min (15 °C), and Pal et al. [12] observed sharper-edged rings in bloodstains deposited at greater temperatures (35 and 45°C) compared to 25°C.
Degrading bloodstains have been imaged by techniques such as scanning electron microscopy (SEM) [13], atomic force microscopy (AFM) [14,15], and hyperspectral imaging [16,17]. Surface profilometry is a technique used to measure and analyze the topographies of small surfaces. Optical profilometry provides a contact-free measurement using optical sensors, providing detailed topographical information without touching the bloodstain [18]. As a non-destructive technique, optical profilometry has emerged as a useful tool in forensic science analyses. Alcaraz-Fossoul et al. [19] showed that optical profilometry could visualize latent fingerprints without pre-treating them, and in aging studies, fingerprints were not required to be re-developed before each collection point. Heikkinen et al. [20] used white light interferometry to identify similarities and differences between tool mark samples and further identified firing pins via impression details that could not be identified using 2D imaging. Hertaeg et al. [21] used laser confocal microscopy to collect height profiles of bloodstains with varying concentrations of RBCs suspended in three different solutions: plasma, phosphate buffered saline (PBS), and bovine serum albumin (BSA). Their results corroborated previous findings that higher RBC concentrations increase the amount of RBC deposition at the edge of the bloodstain [21]. From this, we asked whether optical profilometry, using full scan and centre profiles, can also be useful in monitoring time-wise changes in degrading bloodstains. We investigated the changes in bloodstain surface characteristics such as surface height, surface roughness, and number of cracks and pits for small drip bloodstains over the course of four weeks. In addition, we evaluated the influence of small differences in bloodstain volume on the extent of these changes.