2. Materials and Methods
2.1 Blood Source
Whole bovine blood with 12.5% volume by volume (v/v) acid citrate dextrose anticoagulant solution A (ACD-A) was used to create the drip bloodstains. Whole bovine blood from three biological replicates was collected from Windcrest Meat Packers (Port Perry, Ontario, Canada) and mixed with ACD-A. The ACD-A anticoagulant was created by dissolving 0.3072 g of citric acid monohydrate, 0.8448 g of sodium citrate dehydrate, and 0.8550 g of D (+) glucose (Sigma Aldrich, Ontario, Canada) into 62.50 mL of Millipore water. The drip bloodstains were created within 48 hours of blood collection. Fluid properties of the whole blood, including density, surface tension, viscosity, and PCV% were measured in ambient conditions on the same day as the blood collection (see Table S1).

2.2 Drip Bloodstain Creation

Two different experiments were completed: long-term and short-term time experiments. For the long-term experiments, three 90ºdrip bloodstains were created using a 10 µL gas-tight syringe (Hamilton 80300, 701 N) in triplicate 1.5 hours apart from each other. The syringe was held by a retort stand 30 cm above a 15 cm x 15 cm polished aluminum plate. The gas-tight syringe was used to collect 4 µL of blood, which was deposited onto the plate as a single droplet. The syringe was rinsed with MilliQ water and dried between generation of each droplet. For the short-term experiments, three different volumes of blood were investigated: 4 µL, which was deposited by a gas-tight syringe, 11 µL, and 20 µL, both of which were deposited using a micropipette. A gas-tight syringe was used to deposit 4 µL instead of a micropipette because the diameter of the micropipette tip was too large for the blood to drop without using additional force. All experiments were completed at ambient temperatures (22 ± 2°C).

2.3 Optical Profilometry

Immediately after bloodstain deposition, a Filmetrics Profilm3D Optical Profiler was used to scan the bloodstains. Scans of the bloodstains were taken with a 20x objective lens (1.0 x 0.85 mm field of view) with a 10% overlap to produce a topographic scan through ProFilm 3D. In the long-term experiments, scans were taken of the entire bloodstain, which took approximately 90 minutes. Scans were captured at 1.5 hours, 6 hours, 10.5 hours, 24 hours, 48 hours, 72 hours, 120 hours, 1 week, 2 weeks, 3 weeks, and 4 weeks after deposition. In the short-term experiments, to achieve faster scan times, scans of the right side of the bloodstain were taken. Scans were taken every five minutes up to two hours after deposition; these scans included a portion of the plate, as well as the rim of the bloodstain. For the rim of the bloodstain to be visualized, the scan area was progressively increased as volume increased (from 1.6 mm x 1.6 mm x 80 µm for 4 µL, to 1.6 mm x 2.7 mm x 100 µm for the 11 µL and 20 µL). Topographic scans for every experiment were processed in the same manner using the Profilm3D software. All profiles were first scale corrected and manually levelled, then invalid data points (surface points that were not detected by the profilometer) were interpolated using the “Fill In Invalids” tool. Topographical scans for each short-term bloodstain at each time point were taken after each processing step to produce a timelapse of the bloodstain over two hours, with a scale of 80 µm.

2.4 Image Analysis and Data Processing

In Profilm3D, the ‘Area Roughness’ function was used to determine the surface average roughness, root mean square (RMS) roughness, skewness, and kurtosis of the entire bloodstain; the “Crop” function was used to remove as much of the aluminum plate as possible from the scans before analysis. The ‘Line Profile’ function was used to collect two height profiles from the bloodstain, one running from North to South (vertical slice), the other running East to West (horizontal slice), with both running through the centre of the bloodstain. Each bloodstain produced during the short-term experiments was also analyzed to determine the number of cracks present using FIJI (v. 2.30/1.53q). Like the long-term experiments, the “Area Roughness” function was used to determine the same surface characteristics and the maximum height of the bloodstain present within the scan. Surface average roughness was determined by calculating the average distance of surface points from the mean height plane [22]. The full, uncropped scans were exported as .txt files for analysis and figure conception in R Studio (V. 4.2.0). Pearson’s correlation coefficients (r ) were computed between pairs of surface characteristics to quantitatively evaluate their relationship.
To standardize the height profiles, the average height of the polished aluminum surface was recorded for each scan and subtracted from the maximum height in order to account for differences in surface height between time points. Heights were collected from overall bloodstains using horizontal and vertical slices, OriginPro (V. 9.7) was used to do a baseline subtraction to generate a corrected height profile. Points on the left and right of the bloodstain, as well as any cracks that reached the surface, were selected as anchor points. FIJI was used to analyze cracks and pits. After uploading the scans to FIJI and setting a scale, a colour threshold was created to only select surface colours. The image was converted to binary and subsequently cropped to remove the aluminum surface and any artefacts along the edge of the scan. The “Analyze Particles” function with a 8.5 μm2 size filter was then used to measure the area of each crack and pit. It is important to note that the scans only surveyed a portion of the bloodstains; 5.52% for 4 µL, 6.94% for 11 µL and 4.77% for 20 µL of the total stain area. A scaling factor of 1.2 and 1.3 was applied to the 4 µL and 20 µL respectively data for comparison.