Blood footwear impression depletion: comparative morphological and biochemical analysis of continuous and interrupted...

Blood-bearing footwear impressions represent an important source of information in Bloodstain Pattern Analysis (BPA), yet the dynamics of blood depletion during consecutive walking sequences remain poorly characterized. This proof-of-concept pilot study compared two blood depletion models generated by contact between a patterned shoe sole and a passive blood pool on a non-porous surface: a continuous walking sequence and an interrupted partial-drying sequence incorporating a 2-3-minute period of partial drying after the first step. Both models consisted of an initial passive pooling phase (Step 0) followed by ten consecutive transfer events. Each experimental condition was independently performed twice (n = 2 complete sequences per condition; four complete sequences overall). High-resolution calibrated digital images were acquired for each step and analyzed using custom Python/OpenCV-based morphometric algorithms. Parameters evaluated included blood transfer area, spatial distribution of residual microdeposits, and progressive degradation of the tread pattern. Residual blood reactivity was additionally assessed using the Combur Test on fresh and dried traces before and after rehydration with physiological saline solution. In the continuous sequence, transfer area decreased markedly during the first transfer events, declining from approximately 160 cm² at Step 0 to 60-70 cm² at Step 1, 20-30 cm² at Step 2, and 10-20 cm² at Step 3. Despite this substantial quantitative reduction, the overall footwear geometry remained recognizable for a relatively large number of consecutive transfer events. In contrast, the interrupted partial-drying sequence exhibited earlier morphological degradation. Following partial drying, residual blood deposits became more localized and less spatially dispersed; however, footprint interpretability declined sharply around Step 4, which was treated as an experiment-specific transition and not as a universal threshold. Beyond this stage, the tread pattern progressively lost interpretability despite the continued presence of detectable blood residues. Combur Test results were generally negative on completely dried traces after approximately 24 h. However, rehydration with physiological saline restored test positivity even during advanced stages of depletion and on the shoe outsole itself, indicating persistence of presumptive heme reactivity even after substantial loss of footprint interpretability. Overall, the findings suggest that depletion of blood-bearing footwear impressions is a multifactorial morphodynamic and biochemical process whose observed expression differed between the two experimental conditions. Persistence of biologically detectable blood does not necessarily coincide with persistence of the footprint as a morphologically interpretable entity. In particular, the interrupted partial-drying condition showed an apparent dissociation between biochemical persistence and morphological persistence, with marked loss of footwear recognizability beyond Step 4 despite the continued presence of detectable blood residues. These findings suggest that apparently degraded or non-identifiable footwear impressions may retain forensically relevant biological evidence, highlighting the importance of integrating morphological and biochemical approaches in the interpretation of blood-bearing footwear traces.Trial registration: not applicable.