The effect of the anti‐coagulant EDTA on the deposition and adhesion of whole blood deposits on non‐porous substrates

An investigation into whether the addition of a commonly used anti‐coagulant agent like ethylenediaminetetraacetic acid (EDTA) has an impact on the adhesion potential of blood to non‐porous substrates was conducted. Two non‐porous substrates (aluminum and polypropylene) exhibiting six different surface roughness categories (R1–R6) were used as test substrates upon which either whole blood or blood treated with EDTA was deposited. Samples were exposed to different drying periods (24 hours, 48 hours, and 1 week) before undergoing a tapping agitation experiment in order to evaluate the adhesion to the surface. Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA. Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre‐agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week. Both EDTA‐treated and EDTA‐untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51–1.50 μm surface roughness). Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered. Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

evaluate the adhesion to the surface.Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA.Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre-agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week.Both EDTA-treated and EDTA-untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51-1.50 μm surface roughness).Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered.Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

K E Y W O R D S
blood adhesion, blood deposits, EDTA-treated blood, persistence, transfer, whole blood

Highlights
• Differences in adhesion strength between blood treatments were observed.
• Blood deposited on aluminum dislodged more than on polypropylene.
• Blood mixed with EDTA dislodged more readily than untreated blood.

| INTRODUC TI ON
Indirect transfer of DNA within biological materials through contact with a secondary surface, including within packaging during the transport of exhibits containing biomaterial, is of great interest to forensic practitioners and the judiciary alike when estimating the probability for DNA transfer to occur given a particular activity [1][2][3].Blood is one of these biological materials of forensic relevance and can be of value in a range of different crime scenarios.However, the natural ability of blood to coagulate in response to injuries or once removed from the body creates a challenge for researchers looking to effectively replicate blood in laboratory-based experiments.To overcome this challenge, many non-forensic researchers utilize blood that has been treated with an anti-coagulant such as EDTA, citrate, and/or heparin [4][5][6][7][8][9] to aid their experimental methodology.By adding the anti-coagulant, changes are made to the whole blood and thus the study subject, in order to prevent the premature clotting [10].Forensic researchers conducting DNA transfer studies have also sought to overcome these challenges by using blood treated with anti-coagulants [11][12][13]; however, it is important to consider the potential impacts these changes have on the interactions of blood with the receiving surfaces relevant to DNA transfer studies.
The fluid dynamics of blood is governed by non-Newtonian characteristics where the viscosity of blood changes upon the exposures to stresses such as sheer force and is able to adapt its viscoelasticity when undergoing deformation [14].This natural function of blood is important for the regulation of blood pressure, so that blood can become thinner as the blood vessels constrict and thus, increase the sheer strain [15,16].Along with the ability of blood to change its viscosity and elastic properties, the corpuscles within blood have a strong influence on how blood interacts with a surface it is deposited on.Platelet aggregation for example is a key factor in inducing fibrinogen adhesion to a surface (e.g., thrombogenic surfaces [17], biocompatible materials [18], nanoparticle functionalized materials [19]) in order to initiate the coagulation cascade that creates a thrombus.Since EDTA inhibits this cascade process by calcium ions chelation, EDTA blood used for forensic studies evaluating indirect transfer probabilities may produce lower than normal adhesion potentials due to reduced protein adsorption capabilities.
Thus, this study is a preliminary exploration into possible effects compromising the blood adhesion potential to aluminum and polypropylene substrates of various roughness levels, over time post-deposition, brought on by the anti-coagulant EDTA, to evaluate whether caution should be given to perceived estimates of indirect DNA transfer probabilities obtained via EDTA-treated blood.

| MATERIAL S AND ME THODS
An investigation into whether the addition of the anti-coagulant agent EDTA has an impact on the adhesion potential of blood to non-porous substrates was conducted.Evaluating the differences between the whole blood and EDTA-treated blood was studied to assess the likelihood of dried blood to become dislodged upon movement and/or agitation, and what these observations may signify for DNA transfer studies relevant to forensic science investigations.

| Substrate selection and treatment groups
Two non-porous substrates (aluminum and polypropylene) were selected according to previous studies conducted by , both exhibiting six different surface roughness categories (Table 1) to evaluate whether changes to the surface roughness influenced the level of dried blood dislodgement from the substrate surface.Each treatment group (whole blood vs EDTA-treated blood) entailed four replicate samples, resulting in a total sample size of n = 48 per substrate.

| Collection of whole blood and EDTA blood
This study was conducted under institutional ethics approval with informed consent of the volunteer.Whole blood and EDTA-treated blood were collected from the same individual by a qualified phlebotomist on the same collection day and transported back to the laboratory within 20 minutes for immediate use.This was repeated on three occasions to acquire fresh blood for each of the three drying duration tests to be conducted.
The EDTA-treated blood was the first sample to be collected into a 4 mL K 2 EDTA sample tube (Vacutainer®, Becton Dickinson, UK).Whole blood was collected second to limit the total waiting time before deposition, in accordance with the method applied by Hughes et al. [23], where blood was collected into a 9 mL EDTA-free tube (Vacutest® Kima s.r.l., Arzergrande, Italy), which was then kept warm with a self-activating heat pad (Kobayashi, HotHands Toe Warmers, Dalton, USA) and maintaining a gentle side-to-side motion to prevent the premature clotting of the blood.

| Substrate DNA decontamination and post-deposition cleaning procedure
Before the deposition of blood, the substrates were decontaminated according to Hughes et al. [20][21][22][23][24], using 1% sodium hypochlorite solution for polypropylene, and absolute ethanol for aluminum, followed by a rinse with deionized water.The substrates were left to dry overnight before blood deposition the next day.
TA B L E 1 Surface roughness categories R1-R6, representing average surface roughness (Sa) measurements in μm.After deposition and the completion of individual experiments (the three different drying duration tests), the substrates were cleaned according to Hughes et al. [20,22,23], by submerging and cleaning the samples in an anionic soap (Earth Choice, Australia) bath followed with a rinse in deionized water, before repeating this washing process a second time and then drying the samples with sterile KimWipe™.Subsequent DNA decontamination as outlined above was then again performed 1 day prior to deposition.

| Blood deposition
Whole blood was deposited first by immediately pipetting 10 μL of blood onto the center of the substrate disc, before lightly spreading the blood with the pipette tip as per procedure used by Hughes et al. [20,23].The collection tube was recapped and agitated between sets to limit the exposure of the blood to oxygen in the air and to prevent blood serum separation.After completion of whole blood deposition, the deposition of EDTA blood was performed as per the procedure outlined above, without the need for blood sample agitation between sets.Sample sets were then left to dry at room temperature on a laboratory bench within a restricted no-access laboratory, for either 24 h, 48 h, or 1 week, respectively, according to treatment group requirements.

| Blood agitation experiment and sample photography
Four replicate samples of each roughness category and treatment group were carefully placed into their own sterile biospecimen jar (20 mL) for transportation to the dark room photography laboratory.Each replicate sample was then photographed pre-and postagitation using a digital camera (Nikon, D850, Japan) coupled with a 60 mm focal lens (Nikon, AF Micro lens 1:2.8 D, Japan), with the use of a white light source using a Polilight (Rofin, PL 550 XL, Australia).
The agitation experiment involved placing each individual sample disc into a sealable test tube (10 mL) using tweezers, sealing the tube, and tapping (×3) the tube with sample moderately hard against the laboratory bench.This process was repeated for each sample of both treatment groups and substrate variety.All samples were then gently removed from the tube, returned to their specimen jar, and rephotographed (post-agitation step).

| Blood dislodgement scoring
The blood dislodgement scoring was conducted using the ImageJ (v. 1.53 k) area mapping tool.The calibration scale was set to the sample disc diameter (Ø 10 mm), before mapping the total sample area, total blood deposit area, and finally the total area of blood dislodgement.
The final score was then converted into percentage values to express the amount of dislodgement observed.

| Data analysis
General linear model (GLM) univariate analysis was conducted to evaluate differences in the dislodgement tendency of whole blood versus EDTA-treated blood exposed to different periods of total drying time before agitation.One-way ANOVA analysis was conducted to evaluate whether changes to the substrate surface roughness had an impact on the dislodgement of blood.

| Aluminum
After a drying time of 24 h (Figure 1), whole blood had a significantly (p = 0.027, F = 5.191, df = 1) greater dislodgement tendency upon agitation than EDTA-treated blood.This was observed across all roughness categories (F (5, 18) = 1.199, p = 0.349) with EDTA-treated blood also displaying significant (F (5, 18) = 3.759, p = 0.017) differences in dislodgement across roughness categories.Although whole blood has shown to have a greater dislodgement tendency than EDTA blood after a 24 h drying duration, the substrate impact on the dislodgement trend showed consistency in both blood deposits.
A drying time of 48 h before agitation revealed a shift in dislodgement tendency, with whole blood now showing reduced dislodgement compared to EDTA-treated blood in four out of six roughness categories (Table 2).This change was substantial although not statistically significant (p = 0.147, F = 2.181, df = 1).The dislodgement of whole blood did not appear to be influenced by a change in substrate surface roughness (F (5, 18) = 0.692, p = 0.636), while a more noticeable dislodgement tendency across roughness categories was observed for EDTA-treated blood (F (5, 18) = 1.919, p = 0.141).
A drying time of 1 week before agitation resulted in a further increase in dislodgement of whole blood, and a significantly (p = 0.002, F = 10.900,df = 1) lower overall dislodgement tendency than EDTA blood across all roughness categories (Table 2).Blood treated with EDTA displayed a significant (F (5, 18) = 7.800, p < 0.001) increase in dislodgment tendency across all roughness categories after a drying time of 1 week (Table 2).

| Polypropylene
After a drying time of 24 h (Figure 2) before agitation, both sample types have shown a strong adherence potential to polypropylene (p = 0.999, F = 0.000, df = 1), with little to no dislodgment observed as the surface roughness of the substrate increased.Whole blood has shown to exhibit a lower dislodgement tendency compared to EDTA blood overall.
Interestingly, although whole blood displayed a high tendency for dislodgement at a substrate roughness of 0.51-1.00μm, no dislodgement was observed thereafter except for 1.1% outlier at R5. Dislodgement (7.9%) was still observed for EDTA blood at R2 (1.01-1.50μm) before ceasing any further dislodgment thereafter.

| Aluminum
The dislodgement distribution of whole blood as well as EDTAtreated blood after a 24 h drying time indicated a decline as the substrate surface roughness increased, suggesting that the dislodgement potential on smooth aluminum surfaces is greater than that on rough surfaces.This may be attributed to the overarching substrate effects, where blood deposited on hydrophilic substrates TA B L E 2 Total blood dislodgement observed post agitation after different drying durations, and the differences observed between each.

Roughness category (μm)
Avg. change over time (%) Difference in change (%) F I G U R E 1 Blood dislodgement tendency of whole blood (WB) versus EDTA-treated blood over time (24 h, 48 h, and 1 week), with evaluations conducted across six aluminum surface roughness categories (R1-R6).
like aluminum produce distinct morphological changes that induce the formation of radial and ortho-radial cracks during the drying phase [25], providing a preferential prospect for dislodgement (Figure 3).
Greater variability in dislodgement without a clear trend was observed for whole blood samples of a 48 h drying time.These observations may indicate that blood is undergoing compositional changes over time where the dried blood deposit will undergo Blood dislodgement tendency of whole blood (WB) and EDTA-treated blood over time, evaluated across six polypropylene surface roughness categories (R1-R6).

F I G U R E 3
Representation of blood dislodgement tendencies of whole blood (WB) and EDTA-treated blood on aluminum at a surface roughness of 1.01-1.50μm; illustrating the effect of prolonged drying periods on the deposit adhesion strength.
not only physical changes between 24 and 48 h but also chemical changes.Bergmann et al. [4] discussed the compositional changes in blood once exposed to air, induced by oxidative reactions triggered by the exodus of blood from the body, where deoxyhemoglobin (Hb) is converted into methemoglobin (metHb) and finally denatured to hemichrome (HC).This denaturation process occurs in a fixed pattern (between 5 and 100 h) and thus may be used to estimate the level of denaturation of Hb over time for blood aging estimations, confirming that compositional changes are detectable over time.However, the probability that dislodgement tendencies were induced by compositional changes within blood will need to be investigated further with the aid of chemical analysis of blood using FTIR or Raman spectroscopy [26][27][28][29][30][31][32].For EDTAtreated blood, the overall trend here showed again an increased dislodgement on smooth surfaces and a decreased dislodgement on rough surfaces.The decreased adhesion potential on smooth surfaces may be attributed to the inability of EDTA blood to initiate and maintain a fibrinogen network capable of anchoring to the aluminum substrate, due to the calcium ion chelation effect that hinders the coagulation cascade [4,33].After a drying time of 1 week, the dislodgments increased with data suggesting that the substrate is either too smooth for blood to maintain a strong adhesion potential or too rough to effectively create sufficient intermolecular bonds to maintain a strong adhesion potential; instead, it appears that the deposit remains on the upper surface layer of the substrate.This lack of adhesion potential was also documented by Hughes et al. [20,22,23], where blood had shown to have the lowest persistence on aluminum compared to touch, semen, and/or salivary deposits.Again, difference in persistence as well as the high dislodgment tendency may be driven by the formations of radial cracks (Figure 3) in the drying blood drop, due to stresses created by the diffusion of solvent molecules during the solvent evaporation phase [34].

| Polypropylene
It was apparent that both sample types had a strong adherence potential to polypropylene indicating a clear substrate effect.
Polypropylene further has a tendency to create a more fibrous surface finish the rougher the substrate surface became, subsequently increasing the total substrate surface area and thus, the potential to form deposit substrate interactions.This aligns with observations made by Nonckreman et al. [19] who discussed the influence of substrate topography and material composition on protein adsorption including for platelets and fibrinogen.Interestingly, the longer the deposit dried on polypropylene, the greater the dislodgment tendency, with EDTA-treated blood on polypropylene alike on aluminum still showing a greater increase in dislodgment tendency than whole blood.This may be attributed to the increased adsorption affinity of fibrinogen to hydrophobic substrates, with whole blood to the contrary of EDTA-treated blood able to convert fibrinogen to fibrin networks as part of the coagulation cascade [19].Nevertheless, blood adhesion potentials were a lot higher on polypropylene than that observed on aluminum and would align with findings by Roach et al. [18] who stated that a higher protein adsorption affinity could be observed on CH 3 (like polypropylene)-terminated substrate layers than on OH-functionalized substrate layers.The findings of this study will need to be considered when interpreting and using the findings of DNA transfer, persistence, prevalence, and recovery (DNA-TPPR)-related studies, such as those that utilized EDTA blood [11,12] or lithium heparin blood [13] as their source of blood, or did not provide details on blood collection methods used [35][36][37][38].

| Further considerations
The finding of different levels of dislodgement depending on substrate material, substrate roughness and drying time, may also be relevant when considering the probability of particular types of DNA profiles being generated from the collected sample given alternative activity scenarios associated with the sample [1][2][3].
While this study was primarily undertaken to consider the relevance of using blood treated with anti-coagulants on DNA transfer outcomes, it may also have bearing on the interpretation of research outcomes in the area of bloodstain pattern analyses.

| CON CLUS ION
Clear differences in dislodgement and adhesion potential were observed between whole blood and EDTA blood.Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre-agitation, while whole blood thereafter (drying time of 48 h and 1 week) presented with a stronger adhesion strength instead.On polypropylene, the overall blood dislodgement tendency was low in both types of blood, only really differing on smooth surfaces.Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene, said differences need to be considered.Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflicts of interest to declare.