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\begin{document}

\title{Optical profilometry for forensic bloodstain imaging}



\author[1]{Theresa Stotesbury}%
\author[1]{Brayden Vale}%
\author[2]{Amanda Orr}%
\author[1]{Colin Elliott}%
\affil[1]{University of Ontario Institute of Technology Faculty of Science}%
\affil[2]{Trent University}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Understanding the physical, chemical and biological changes that occur
during the drying of a bloodstain is important in many aspects of
forensic science including bloodstain pattern analysis and time since
deposition estimation. This research assesses the use of optical
profilometry to analyze changes in the surface morphology of degrading
bloodstains created using three different volumes (4, 11, and 20 \selectlanguage{greek}µ\selectlanguage{english}L) up
to four weeks after deposition. We analyzed six surface characteristics,
including surface average roughness, kurtosis, skewness, maximum height,
number of cracks and pits, and height distributions from the
topographical scans obtained from bloodstains. Full and partial optical
profiles were obtained to examine long-term (minimum of 1.5 hour
intervals) and short-term (5 minute intervals) changes. The majority of
the changes in surface characteristics occurred within the first 35
minutes after bloodstain deposition, in agreement with current research
in bloodstain drying. Optical profilometry is a non-destructive and
efficient method to obtain surface profiles of bloodstains, and can be
integrated easily into additional research workflows including but not
limited to time since deposition estimation.%
\end{abstract}%



\sloppy


\textbf{Optical profilometry for forensic bloodstain imaging}

Brayden Vale\textsuperscript{a}, Amanda Orr\textsuperscript{b}, Colin
Elliott\textsuperscript{c}, Theresa Stotesbury\textsuperscript{d*}

\textsuperscript{*}Corresponding
author:\emph{theresa.stotesbury@ontariotechu.ca}

\textsuperscript{a} Forensic Science Undergraduate Program, Ontario Tech
University, 2000 Simcoe St N, Oshawa, ON, Canada, L1G 0C5

\textsuperscript{b} Environmental and Life Sciences Graduate Program,
Trent University, 1600 West Bank Drive, Peterborough, ON, Canada, K9J
0G2

\textsuperscript{c} Applied Bioscience Graduate Program, Faculty of
Science, Ontario Tech University, 2000 Simcoe St N, Oshawa, ON, Canada,
L1G 0C5

\textsuperscript{d} Faculty of Science, Forensic Science, Ontario Tech
University, 2000 Simcoe St N, Oshawa, ON, Canada, L1G 0C5

\textbf{ORCID:} AO (0000-0002-2245-8448); CE (0000-0001-5883-0291); TE
(0000-0001-6452-4389)

\textbf{Data Availability Statement:} Scans are available by request and
data is available through
https://gitlab.com/f4301/bloodstain-profilometry.

\textbf{Funding Statement:} We are thankful for the funding support to
conduct this research. This work was supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC) Discovery and
Discovery Accelerator Supplement Grants (RGPIN-2020-05816) awarded to
TS. Authors CE and AO are supported through the NSERC Canada Graduate
CGS-M (MSc) and CGS-D (PhD) Scholarship programs respectively.

\textbf{Conflict of Interest Disclosure:} The authors have no conflicts
of interests to declare.

\textbf{Acknowledgements:}The authors would like to formally acknowledge
Mitchell Tiessen, Kgalalelo Rampete, Julia Harvey, Dhrumik Patel, and
Laurianne Lagace, who assisted BV with fluid property measurements
throughout his undergraduate thesis. A most special thank-you goes to
Dr. Iraklii Ebralidze, of the Ontario Tech University Materials
Characterization Facility for training BV on the optical profilometer.

\textbf{Abstract:}

Understanding the physical, chemical and biological changes that occur
during the drying of a bloodstain is important in many aspects of
forensic science including bloodstain pattern analysis and time since
deposition estimation. This research assesses the use of optical
profilometry to analyze changes in the surface morphology of degrading
bloodstains created using three different volumes (4, 11, and 20 \selectlanguage{greek}µ\selectlanguage{english}L) up
to four weeks after deposition. We analyzed six surface characteristics,
including surface average roughness, kurtosis, skewness, maximum height,
number of cracks and pits, and height distributions from the
topographical scans obtained from bloodstains. Full and partial optical
profiles were obtained to examine long-term (minimum of 1.5 hour
intervals) and short-term (5 minute intervals) changes. The majority of
the changes in surface characteristics occurred within the first 35
minutes after bloodstain deposition, in agreement with current research
in bloodstain drying. Optical profilometry is a non-destructive and
efficient method to obtain surface profiles of bloodstains, and can be
integrated easily into additional research workflows including but not
limited to time since deposition estimation.

\textbf{Keywords:}

Drying bloodstains, surface profile, time since deposition (TSD),
forensic science, crack formation

\section*{Research Highlights:}

{\label{research-highlights}}

\begin{itemize}
\tightlist
\item
  Optical profilometry is a non-contact tool to scan bloodstains in
  ambient conditions
\item
  Drying phases are observable in small drip bloodstains
\item
  Significant surface morphology changes occur within 35 minutes after
  deposition
\end{itemize}

\textbf{Graphical Abstract:}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{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
(\textasciitilde{}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 \selectlanguage{english}30 min (24 \selectlanguage{ngerman}°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.

\textbf{2. Materials and Methods}

\emph{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).

\subsection*{\texorpdfstring{\emph{2.2 Drip Bloodstain
Creation}}{2.2 Drip Bloodstain Creation}}

{\label{drip-bloodstain-creation}}

Two different experiments were completed: long-term and short-term time
experiments. For the long-term experiments, three
90\textsuperscript{º}drip bloodstains were created using a 10 \selectlanguage{greek}µ\selectlanguage{english}L
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 \selectlanguage{greek}µ\selectlanguage{english}L 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 \selectlanguage{greek}µ\selectlanguage{english}L, which was deposited
by a gas-tight syringe, 11 \selectlanguage{greek}µ\selectlanguage{english}L, and 20 \selectlanguage{greek}µ\selectlanguage{english}L, both of which were deposited
using a micropipette. A gas-tight syringe was used to deposit 4 \selectlanguage{greek}µ\selectlanguage{english}L
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 \selectlanguage{ngerman}± 2°C).

\subsection*{\texorpdfstring{\emph{2.3 Optical
Profilometry}}{2.3 Optical Profilometry}}

{\label{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 \selectlanguage{greek}µ\selectlanguage{english}m for 4 \selectlanguage{greek}µ\selectlanguage{english}L, to 1.6 mm x 2.7 mm x
100 \selectlanguage{greek}µ\selectlanguage{english}m for the 11 \selectlanguage{greek}µ\selectlanguage{english}L and 20 \selectlanguage{greek}µ\selectlanguage{english}L). 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 \selectlanguage{greek}µ\selectlanguage{english}m.

\subsubsection*{\texorpdfstring{\emph{2.4 Image Analysis and Data
Processing}}{2.4 Image Analysis and Data Processing}}

{\label{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 (\emph{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 \selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{2} 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 \selectlanguage{greek}µ\selectlanguage{english}L, 6.94\% for 11 \selectlanguage{greek}µ\selectlanguage{english}L and 4.77\% for 20 \selectlanguage{greek}µ\selectlanguage{english}L of the total stain area.
A scaling factor of 1.2 and 1.3 was applied to the 4 \selectlanguage{greek}µ\selectlanguage{english}L and 20 \selectlanguage{greek}µ\selectlanguage{english}L
respectively data for comparison.

\section*{3. Results}

{\label{results}}

\subsection*{\texorpdfstring{\emph{3.1 Long-Term
Experiment}}{3.1 Long-Term Experiment}}

{\label{long-term-experiment}}

In the long-term experiments, the bloodstain scans showed little
variation between 1.5 hours and four weeks (Figure 1A). For the
long-term experiments, kurtosis values showed slight variation over the
four weeks (Supplemental S3), although the values were always less than
three, indicating platykurtic distributions, or height profiles with
multiple heights of similar frequencies. Skewness values showed similar
timewise fluctuations but were influenced by individual bloodstain
variation. For example, bloodstain 1 had overall negative skewness
values, while bloodstains 2 and 3 had positive skewness values. Surface
roughness slightly increased within the first day, but then plateaued
over the tested timeframe. Maximum height was measured from the
horizontal and vertical slices taken from the long-term bloodstains,
where minimal change was observed in the height profile over time,
except for the earlier time points (1.5 and 6 hours) which had lower
heights at the centre of the bloodstain.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\par\null

\textbf{Figure 1.} Topographical scans of the top and side of one 4 \selectlanguage{greek}µ\selectlanguage{english}L
bloodstain at varying times since deposition up to 4 weeks. The scans
show the height (\selectlanguage{greek}μ\selectlanguage{english}m) against the X-distance (\selectlanguage{greek}μ\selectlanguage{english}m) on the plate. The
corresponding height profile is taken from the vertical/horizontal slice
of each scan over time is also shown (bottom right).

\subsection*{}

{\label{section}}

\subsection*{\texorpdfstring{\emph{3.2 Short-Term
Experiment}}{3.2 Short-Term Experiment}}

{\label{short-term-experiment}}

Topographical scans for the short-term experiments indicate that in the
first \textasciitilde{}15 minutes, the bloodstain changes from a liquid
sessile drop to a soft-solid as more of the bloodstain can be detected
by the profilometer (Figure 2). After the bloodstain had dried, the
formation of the rim was observed, indicating that the majority of the
RBCs had moved to the outside of the bloodstain (Figure 2, 15 minutes).
The formation of cracks occurred between \textasciitilde{}20-30 min, and
after \textasciitilde{}35 minutes after deposition, there were no
significant visual changes in the subsequent scans (Figure 2).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\par\null

\textbf{Figure 2} . Topographical scans of the right side of a 4 \selectlanguage{greek}µ\selectlanguage{english}L
bloodstain. Scans were taken every 5 minutes after deposition up to 120
minutes. Scans of the 11 \selectlanguage{greek}µ\selectlanguage{english}L and 20 \selectlanguage{greek}µ\selectlanguage{english}L bloodstains can be found in
Figures S1 and S2 of the supplemental.

A correlation matrix was used to assess the relationship between each
surface characteristic in the short-term experiments. RMS roughness and
surface roughness were found to be highly correlated (\emph{r} = 0.99).
RMS roughness was also found to have a greater correlation with maximum
height (\emph{r} = 0.93) compared to surface roughness (\emph{r} =
0.89); therefore, surface roughness was chosen for data interpretation.
Some early data points were not included, as sections of the bloodstain
were still liquid and could not be accurately measured by the
profilometer, particularly in the larger blood volumes. Each volume of
blood displayed similar general trends for each surface characteristic
assessed, with differences between timepoints scalable to the volume of
blood used (Figure 3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} Average values (n=3) for surface average roughness
(\selectlanguage{greek}µ\selectlanguage{english}m), maximum height (\selectlanguage{greek}µ\selectlanguage{english}m), skewness, and kurtosis of the right side of
degrading bloodstains of various volumes at varying time points after
deposition. Inlet images show topographical scans from 4 \selectlanguage{greek}µ\selectlanguage{english}L bloodstain
(stain \#2), 11 \selectlanguage{greek}µ\selectlanguage{english}L bloodstain (stain \#1) and 20 \selectlanguage{greek}µ\selectlanguage{english}L bloodstain (stain
\#1) at 10 minutes and 120 minutes after deposition.

As the bloodstain desiccated, the surface average roughness decreased
until approximately 20 minutes, and then increased slightly until
plateauing at about 35-40 minutes (Figure 3, d). The maximum height of
the bloodstains typically decreased until 20 minutes, which remained
consistent until 120 minutes (Figure 3, b). A sharp decrease in height
occurred as the bloodstain began to flatten out, and the rise around
\textasciitilde{}15 minutes corresponded with the formation of the RBC
`donut' (Figure 3). This trend was more difficult to confirm in the
larger volumes of blood due to the lack of time points prior to 10
minutes. Kurtosis increased until approximately 20 minutes, and also
plateaued at about 40 minutes (Figure 3, a). Interestingly, 11 \selectlanguage{greek}μ\selectlanguage{english}L
displayed the greatest kurtosis values at almost every time point in the
short-term experiments. The skewness values decreased up to 20 minutes,
before increasing up to 40 minutes, where the values then plateaued
(Figure 3, c). Similar to the kurtosis trends, the 11 \selectlanguage{greek}μ\selectlanguage{english}L bloodstains had
higher relative skewness values. Kurtosis and skewness displayed
opposite trends within the first 40 minutes, which was corroborated by
the correlation coefficient (\emph{r} = -0.70). Lastly, height
distributions for the short-term experiments were assessed. The height
of the bloodstain was higher at earlier time points and decreased
sharply around 25-30 minutes (Figure 4). Interestingly, we observed
variance in size distributions between volumes and replicates, with the
maximum frequency ranging between approximately 30 -- 60 \selectlanguage{greek}µ\selectlanguage{english}m.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4.} Height distribution from the right side of the
degrading bloodstains at various timepoints (in minutes). The designated
bin width was set to 5 \selectlanguage{greek}µ\selectlanguage{english}m.

Additionally, we observe a high degree of variance in the number of
observed cracks and pits; generally, for all stains, they increase
rapidly for the first 30 minutes and then they plateau (Figure 5).
Typical minute cracks around the edge of the bloodstain were not
detected by the profilometer, making it difficult to assess when crack
formation and propagation began, but it can be approximated to be
\textasciitilde{}15-20 minutes after deposition. Larger cracks located
towards the centre of the bloodstain appeared around 20-25 minutes after
deposition; surface average roughness values at these times slightly
increased. Earlier time points also contained more cracks and pits with
smaller areas which subsequently increased over time, leading to cracks
with larger areas at later time points (Supplemental S4). There does not
appear to be a volume dependence in this study as there is a high degree
of variance observed in these data.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 5} . Total number of cracks and pits observed in the
degrading bloodstains at various timepoints (in minutes).

\textbf{4. Discussion}

Overall, our findings corroborate and are supported by the literature on
bloodstain drying, suggesting that optical profilometry is a viable
technique to observe and characterize bloodstain drying and degradation.
In the short-term experiments, scans showed significant changes within
the first 35-40 minutes after deposition, after which the surface
characteristics plateaued. The pre-gelation phase begins as soon as the
droplet is deposited onto the plate, however, many of the surface
characteristics could not be obtained immediately due to the
liquid-state of the bloodstain. Topographical scans could detect the
edge of bloodstains five minutes after deposition, signifying the
beginning of drying. Within the first 15-20 minutes of drying, height
profiles and maximum height observations showed a significant change,
with differences as large as 60 \selectlanguage{greek}µ\selectlanguage{english}m within a 10-minute timespan. The
change(s) in surface roughness, skewness, kurtosis and maximum height of
the bloodstain over time is primarily driven by RBC movement
{[}10,23{]}. As the RBCs gather along the edge of the bloodstain to form
the rim, the maximum height will increase and the skewness and kurtosis
values will change. The height distributions corroborate this and show
that there is a decrease, then an increase in overall height as the rim
of the bloodstain begins to form. The surface average roughness values
could be correlated to RBC movement during drying. At earlier time
points (before 20 minutes), the bloodstains contained no cracks, and the
maximum height was in the centre of the bloodstain (Figure 2). As the
RBCs moved towards the outside of the bloodstain and the surface
temporarily flattened out, the surface average roughness value
decreased. Then, as the rim began to form, the surface average roughness
increased and cracks and pits formed. Cracks continued to form until
\textasciitilde{}30-35 minutes after deposition; all surface
characteristics and height profile data then displayed a plateau,
suggesting that no further changes occurred. The analysis of the area of
cracks and pits over time was completed as a quantitative complement to
the qualitative observations made using the topographical scans.
Importantly, the profilometer will not interpolate a crack or pit. If a
crack is present, but only one surface point is detected, it might
interpolate the crack as much smaller than it is and, in some cases, it
might only appear as one pixel after the scan is interpolated. This
means that some cracks are larger than what the analysis determined;
however, we decided rather than focus on the absolute value of a single
crack/pit, the trend in the size of cracks and pits over time is more
relevant. Similar to our work, Brutin et al. (2011) {[}23{]} found the
drying time of approximately 10 \selectlanguage{greek}µ\selectlanguage{english}L of human blood to be 36 minutes
(temperature = 22 \selectlanguage{ngerman}ºC), while Ramsthaler et al. {[}8{]} found the drying
time of \textasciitilde{}25 \selectlanguage{greek}µ\selectlanguage{english}L of human blood to be 34 minutes
(temperature = 24 \selectlanguage{ngerman}ºC). Although outside the scope of this study, these
findings suggest that different blood sources (bovine vs. human) display
comparable drying mechanisms. The inclusion of an anticoagulant also can
account for differences in drying time.

From an imaging and spectroscopy perspective, it is important to
understand the surface profiles of bloodstains for TSD estimates. Where
the light source is focused could have spectral implications -- for
example, unwanted scattering. Having an idea of surface roughness
changes and cracks and pits formation over time may help explain
observed spectral variance beyond inter- and intra-specific variation
between bloodstain samples. While there is a significant effect of
spectral signatures with time, the variation between TSD models is also
high due to substrate-dependent interactions and environmental effects.
However, given the large observed variance in surface topography of the
dried bloodstain, we wonder how much this also influences models,
particularly those that use solid-state imaging techniques.

\par\null

\textbf{5. Conclusion}

Optical profilometry provided detailed topographical scans of degrading
whole bloodstains, an area not yet explored in the forensic literature.
We were able to characterize the drying of three different bloodstain
volumes through the analysis of relevant surface characteristics.
Importantly, this work investigated the timewise degradation of whole
blood, expanding on previous work that explored the drying of blood and
other biocolloidal fluids. Using this method, we observed that most of
the timewise changes to drying bloodstains occur within the first 35
minutes after deposition. Over a longer time period, the overall surface
morphology shows little variation, currently limiting the use of optical
profilometry as a standalone technique in TSD estimates. Optical
profilometry has previously been paired with SEM {[}24{]} and AFM
{[}25{]} for imaging purposes; combining these two techniques could be
useful for a more detailed analysis of crack and pit formation. In
addition, factors such as viscosity, temperature, PCV\%, and blood
diseases affect the drying of bloodstains {[}8,9,11,26{]}. Further
research into these factors should be undertaken to understand their
effects on the topography of degrading bloodstains {[}8,9,11,26{]}.
Additionally, the influence of topography on spectral changes for TSD
estimation should also be considered. Finally, various substrates, such
as those with rougher surfaces, may lead to interesting crack formation
and timewise trends with bloodstain degradation that could be captured
using optical profilometry.

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