Influence of anticoagulant on the spectroscopic analysis of drying bovine blood pools

Rapidly, untreated whole blood undergoes a clotting cascade, making forensic research that investigates “fresh” bloodletting events difficult. In bloodstain pattern analysis research, whole blood treated with anticoagulant is often used to prolong the usability of the blood and allow for transport and experimentation to simulate pattern formation using “fresh” clot-free blood. Anticoagulants bind to clotting components, making them unavailable to participate in coagulation, preventing the formation of clots. Herein, we investigate the spectral implications of anticoagulant addition for time since deposition (TSD) estimation methods, particularly of larger volume bovine blood pools. We characterized the differences in spectral profiles of blood pools with and without a citrate-based anticoagulant (ACD-A) using visible absorbance, attenuated total reflection-Fourier transform infrared (ATR-FTIR), and X-ray photoelectron (XPS) spectroscopies. Across all methods, notable spectral differences were observed, namely the red-shift in the Soret peak maxima (visible), delayed increase in the 1532 cm⁻¹ peak (ATR-FTIR), and increased accessibility of iron (XPS) in pools treated with ACD-A. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to assess the variation in the visible absorbance and ATR-FTIR spectra over time. The blood pools differed most significantly in the first week following deposition due to the addition of water in the anticoagulant, slowed desiccation, and lack of clotting in the treated blood pools. At timepoints exceeding 1 week following deposition, the spectral profiles of the pools regained similarity. In summary, the inclusion of anticoagulants is an important consideration during experimental design and TSD estimation method development.