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.