Background
Forensic science unites methods from various fields to examine crime-related facts, giving valuable advice in criminal cases (James et al. 1999). Bloodstain pattern analysis (BPA) plays a significant role in forensic science by examining the place, size, shape, and features of blood left at the scene to recreate the events. Based on these patterns, BPA figures out the whereabouts and connections between the perpetrators and the victims, clarifies each criminal activity, and either agrees with or refutes what witnesses say (Bevel et al., 2008; James et al., 2014). Experts in court trials frequently use BPA. Still, there are issues with admitting and relying on it since different protocols and procedures can misread bloodstain evidence (James et al. 1999). To handle such problems, experts in psychology must define key terms, further improve their approaches, and receive thorough training in using their knowledge in real situations.
Discovering the age of bloodstains matters a lot because determining fresh traces helps in sequencing events, and old stains may mean different approaches in the investigation. The environment, the nature of the place where the stain occurs, and the interactions inside the blood play roles in how bloodstains age (Bremmer et al. 2012). Many previous studies have looked into the aging of bloodstains on materials such as fabric, wood, and concrete. Still, little attention has been given to tile and vinyl flooring, which are common and essential in residential and commercial places (Bremmer et al. 2012).
Surfaces with no pores and a smooth texture often influence the behavior of bloodstains, making them dry faster, change colors, and decompose at a certain speed. BPA’s abilities have increased thanks to new analytical methods. It has been possible to perform precise chemical and spectral analysis of bloodstains, which boosts predictions of their age, due to the use of hyperspectral imaging (Li et al., 2013), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) (Lin et al. 2017) and Raman micro-spectroscopy (Takamura et al. 2019). They can detect changes at the molecular level, including the breakdown of hemoglobin, which relates to the age of the stain. Still, the convenient and straightforward characteristic of monitoring bloodstain diameter, color, and sharpness is why forensic experts rely on morphological analysis during initial assessments on the spot. The results from morphological studies are helpful for quickly making important decisions in an investigation. Blood stains are formed during the drying process of a complex colloidal suspension, and this shape affects how they are patterned. While evaporating, blood’s RBCs, platelets, and plasma proteins interact.
Earlier studies found that Marangoni flow leads to the pattern in a stain, much influenced by the motion of RBCs, movement of proteins, and salt becoming visible in droplets (Brutin et al. 2011; Yakhno et al., 2008). Sometimes, such patterns are linked to medical conditions: persons with blood disorders tend to show unusual drying patterns in their serum than healthy individuals do (Martusevich et al. 2007). The results reveal a connection between chemistry, biology, and fluid dynamics in bloodstains that could be useful when investigating crimes in the forensics field. The time it takes for a bloodstain to age is affected by several environmental conditions and the type of surface it sits on. Investigations on delays in the storage of blood in refrigerated settings indicate that storage conditions can impact parameters in the blood, including mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), which can change the appearance of blood cells under a microscope (Kadam et al. 2023). Also, factors like different substrates, donors, and environmental factors (such as temperature and humidity) can change the way things dry, which should be considered in forensic protocols (as claimed by Bremmer et al. 2012). Unlike what has been studied before, this research examines how bloodstains on tile and vinyl flooring change over 120 h (Fig. 1).
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Fig. 1
Bloodstain pattern analysis in forensic science
To determine how old the stain may be, this study looks at the color, size, edge sharpness, and products left behind by various stains when they degrade. The findings aim to enhance how crimes are reconstructed, make BPA more scientifically solid, and boost its reliability in legal and forensic situations. The results from this study on tile and vinyl add to our knowledge of bloodstain aging, which can guide better and more supportable work in forensic science.
Methods
Samples were taken from Kasturba Medical College Blood Bank in Mangalore, depending on approval from the Institutional Ethics Committee and informed consent by the donors. Blood samples were put in vacutainer tubes containing EDTA and stored at 4 °C to avoid changes in the blood (Kadam et al. 2023). The experiment was performed on tile and vinyl flooring surfaces (Fig. 2).
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Fig. 2
Tile and vinyl flooring
Contact angle measurement
A Holmarc Opto-Mechatronics Ltd Contact Angle Meter (HO-IAD-CAM-01) was used to measure the contact angles because of its high-quality pictures and automated measurement process that conforms to the manufacturer’s standards. Images were taken while the bacteria were at a height of 5 cm, and the angle of the blood on the surface was then measured with ImageJ. The interactions between the blood and the floor materials were evaluated by measuring the contact angle. A calibrated syringe applied manual pressure to let a single blood droplet fall onto the tile and vinyl floor’s surfaces. High-resolution digital cameras recorded the moment of impact and, thereafter, the spreading of the droplets into the bloodstain patterns. This technique allowed the dynamics of blood droplet interactions with the surface to be preserved for future analysis.
Wetting angle analysis
ImageJ software calculated the wetting angle between the actual tangent to the droplet’s surface and the tangent at the contact line to the solid surface. This software allows for very detailed analysis of an image as well as measurements. After the impact of the blood droplet onto the surface, the other photos were captured within particular intervals to determine the droplet’s shape and size. The wetting angle was measured at the point of contact between the blood stain and the surface, showing the adhesion phenomena of blood and how the substrate affects the mechanisms of bloodstaining. Wetting angle results were statistically analyzed for significant differences between the two flooring materials (Table1Stain pattern formation seen through Holmarc opto-mechatronics Ltd-contact angle meter (Model: HO-IAD-CAM-01): from the height of 5 cm).
Table 1. Stain Pattern Formation seen through Holmarc Opto-Mechatronics Ltd-Contact Angle Meter (Model: HO-IAD-CAM-01): from the height of 5 cm
Surfaces | Wetting Angle | Images |
|---|---|---|
Tiles | 32.3 | |
Vinyl Flooring | 30.8 |
Experimental setup
Ethanol was used to clean tile and vinyl surfaces, which were then allowed to dry to obtain similar results. A micro drip infusion set was used to release blood from 55 cm above because 55 cm approximates the height of blood dripping from a standing individual’s hand or wound, a common crime scene scenario, simulating real crime scene drops by standing victims to ensure that the droplets were the same size. The effects were tested in a controlled setting where the temperature was kept between 22 and 24 °C, the humidity was between 50 and 55%, and LED lighting of 5000 K was used to avoid changes in natural light.
The impact of the blood drop was observed on the tile surface. The size and morphological characteristics of the resultant bloodstain were recorded with the aid of a stereo microscope. The same was repeated on the vinyl surface, and the observations were noted accordingly. Multiple trials were performed to achieve consistency and reliability of the results; in this case, three drops were on each surface. Three drops were dropped on each surface to establish the consistency and reliability of the results in this analysis (Fig. 3).
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Fig. 3
Experimenting with blood drop impact
Statistical analysis
Jamovi software used the Mann–Whitney U test, also known as the Wilcoxon rank-sum test, to compare contact angles for tile and vinyl surfaces, and the p-values showed if there was a significant difference (Tables 4 and 5).
Results
Contact angle measurement
The drop pattern of bloodstains on both tile and vinyl surfaces 5 cm above the floor was affected by what the flooring was made from. When a bloodstain hits a non-porous tile, it moves fast, making a round shape with tidy edges as the surface tension keeps the water from sinking. The mean contact angle obtained for the tile was 32.3°, an outcome caused by the low friction of the glass, which let the liquid spread with cohesive forces dominating. In comparison, when blood fell on semi-porous, patterned vinyl, it did not spread as much and became ragged, spreading stains with fuzzy edges (Table 1). The low average contact angle on vinyl, which was 30.8°, proved the paint wrapped more around the edges since there was less room on the highly porous material. The result of a two-tailed t-test showed that the contact angles of liquids on tile and vinyl are not different from each other (p = 0.546 for tile, p = 1.000 for vinyl). Although the initial contact angle measurements differ, the non-significant p-values show that after 5 min, there are no substantial differences in the substrates. The fact that contact angle can only change slightly compared to measurement noise means that it is not a strong indicator at the start of the analysis, as it does not mark changes like edge sharpening and diameter narrowing that happen along the way.
A stereo microscopy observations and a quantitative analysis (Tables 2 and 3)
On all surfaces, a group of three blood drops was seen at 5 min, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h when the areas were examined with a stereo microscope at both 1X and 5X magnification, and images were processed with Image Focus Alpha software. We made the sample size small simply to help us experiment, but it might reduce the range of the data. Some important quantitative figures were as follows:
Table 2. Variations in Stain Pattern seen through Stereo Microscope for Age Estimation: 1X
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Diameter: Taken to indicate a change in the sample’s size. The edge clarity is checked both by eyesight and through the use of edge detection methods.
Timeline of evaporation
The total evaporation time for the case presented is 120 h.
Stage 1: 5 min
Observation: The bloodstain appears fresh, with a bright redcolor and well-defined edges.
Characteristics: The stain is still wet, and the liquid blood is pooling. The surface tension keeps the blood in a rounded shape.
The blood evidence has a fresh appearance characterized by sharp edges and vivid red (Fig. 4).
Stage 2: 1 h
Observation: The stain begins to dry, slightly reducing diameter.
Characteristics: The color darkens slightly, transitioning from bright red to a deeper red.
The bloodstained area darkens because it changes from bright red to redder tones during observation (Fig. 5).
Stage3: 2 h
Observation: Noticeable drying occurs, and the stain’s diameter decreases.
Characteristics: The color changes to a darker red or brownish hue. The edges blur slightly as the blood begins to adhere to the surface.
The blood display evolves from red to brownish red while changing its appearance. The start of adherence between blood and surface causes a slight blurring effect on the edges (Fig. 6).
Stage 4: 4 h
Observation: The stain flattens out, and the diameter reduces further.
Characteristics: The stain appears more matte, with less reflective quality. The edges are less defined, and the color is significantly darker.
The damaged bloodstain develops a less vibrant appearance than fresh bloodstains because it becomes less reflective. The stain shows diminished edges and a significantly darkened tone in this stage (Fig. 7).
Stage 5: 8 h
Observation: Significant drying is observed, with a size reduction.
Characteristics: The stain shows some pooling or spreading, especially on vinyl flooring. The color darkens further, and the stain adheres more firmly to the surface.
The stain presents significant pooling effects, which spread most commonly on vinyl surfaces. Both the color intensifies at this stage, and artificial blood maintains better adhesion to the surface material (Fig. 8).
Stage 6: 24 h
Observation: The stain appears stable, with minimal change in size.
Characteristics: The edges are well-defined but appear slightly jagged. The color is very dark, and the stain has a matte finish. The blood has begun to coagulate.
The edges of this stain exhibit crisp definition even though they display a gentle, roughened appearance. The dark coloration of the stain involves a complete absence of shine that results in a flat appearance. Scientific examination reveals that blood coagulation has started to occur (Fig. 9).
Stage 7: 48 h
Observation: The stain hardens, and the diameter stabilizes.
Characteristics: The stain is now firm to the touch, with a significant darkening of color. The edges are well-defined, but the surface shows signs of cracking.
When touched, the stain feels firm, and its color appearance shows significant darkening. This stain displays definitive edges, although it demonstrates some cracks across the surface (Fig. 10).
Stage 8: 72 h
Observation: The stain remains stable, but slight changes are noted.
Characteristics: The color is very dark, and the stain slightly cracks or flakes. The overall size remains consistent.
Very dark-colored stains show minute signs of flaking when inspected. The measurement of total stain dimensions shows no change throughout the analysis (Fig. 11).
Stage 9: 96 h
Observation: Signs of deterioration become more apparent.
Characteristics: The stain flakes more pronounced, and the edges may become jagged. The color remains dark, but the stain appears more brittle.
The staining material presents increased flaking behavior concurrently with possible jagged edges forming. The darkness of the stain persists, but its material quality has changed to show more brittleness (Fig. 12).
Stage 10: 120 h
Observation: The stain is very dry and significantly reduced in size.
Characteristics: The bloodstain is brittle and flakes off the surface easily. The color is dark brown or black, and the stain is now smaller than its original size.
The bloodstain shows brittleness, which causes the surface to flake apart while maintaining dark brown to black coloring and reduced size. The bloodstain is dark brown or black, but it has decreased to a much smaller size than its initial appearance (Fig. 13).
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Fig. 4
Photograph showing fresh bloodstains on tile and vinyl surfaces with bright red color and sharp, rounded edges due to surface tension
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Fig. 5
Photograph depicting drying bloodstains on tile and vinyl surfaces, with slight diameter reduction and darkening from bright red to deeper red
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Fig. 6
Photograph illustrating bloodstains on tile and vinyl surfaces transitioning to brownish-red, with slight edge blurring due to surface adhesion
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Fig. 7
Photograph showing flattened bloodstains on tile and vinyl surfaces, with reduced diameter, matte appearance, and darker color
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Fig. 8
Photograph depicting significant drying of bloodstains on tile and vinyl surfaces, with pooling on vinyl and a darker color
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Fig. 9
Photograph showing stable bloodstains on tile and vinyl surfaces, with jagged edges, dark color, and coagulation
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Fig. 10
Photograph illustrating hardened bloodstains on tile and vinyl surfaces, with firm texture, dark color, and surface cracking
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Fig. 11
Photograph showing stable bloodstains on tile and vinyl surfaces, with slight flaking and consistent size
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Fig. 12
Photograph depicting deteriorating bloodstains on tile and vinyl surfaces, with pronounced flaking and brittle texture
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Fig. 13
Photograph showing brittle, dark brown/black bloodstains on tile and vinyl surfaces, significantly reduced in size
The blood drop was systematically divided into four quadrants to facilitate an in-depth and comprehensive analysis of its morphological characteristics. Thus, the partitioning of this nature enabled me to have a structured approach to examining various morphological features, shape, size, and distribution patterns of the bloodstain—hence the clear view of how the blood interacts on the surface with the resultant changes in morphology in the fourth quadrant under observation.
In this quadrant, various parameters were observed, including the diameter of the bloodstain, the presence of any satellite droplets, and the general contour of the stain. In this respect, key characteristics such as the extent of spreading, the development of edges, and color variations were analyzed, which could indicate the age of the bloodstain. High-resolution images were taken using a stereo microscope to take precise measurements and document these features. Such detailed examination of the fourth quadrant not only helped improve the understanding of the physical properties of the bloodstain but also contributed to the overall objective of establishing a correlation between these characteristics and the age of the bloodstain, hence serving valuable insights in forensic analysis.
Analysis of the fourth quadrant through detailed study enhanced understanding of bloodstain physical attributes while advancing the research goal to connect observable traits with the bloodstain aging process for forensic investigations (Fig. 14).
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Fig. 14
Bloodstains were divided into four quadrants for detailed morphological analysis. The fourth quadrant revealed spreading extent, edge development, and color variations, aiding age estimation
The study investigated bloodstain evolution by directly comparing initial and terminal drops, which is significant for forensic examination (Fig. 15).
Small crack
Small mobile deposit
Small periphery
Wetting deposit
Mobile deposit
Periphery
Large crack
Wetting deposit
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Fig. 15
Photograph comparing initial (5-min) and final (120-h) bloodstains on tile and vinyl surfaces, highlighting changes in shape, color, and texture. Initial stains were bright red and spherical, while final stains were dark brown/black, brittle, and reduced in size
The first drop of blood seen in the first 5 min on both tile and vinyl flooring showed very different morphological features from those seen after 120 h. Immediately after deposition, the blood drop maintained a roughly spherical shape, characteristic of its cohesive nature. On both substrates, the color of the blood was bright red, typical for fresh blood, while the drop’s edges were defined with minimal spreading. Surface tension manifested as the drop maintained its shape without diluting the surrounding area (Table 2 variations in stain pattern seen through stereo microscope for age estimation: 1X) (Table 3 Variations in stain pattern seen through stereo microscope for age estimation: 5X of quadrant 4).
Table 3. Variations in Stain Pattern seen through Stereo Microscope for Age Estimation: 5X of quadrant 4
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In contrast, the last drop of blood observed after 120 h had significant shape and physical structure discrepancies. Throughout the timeframe, several physical transformations began occurring to the bloodstain. The initial bright red color shifted to a darker and more brown hue, which undoubtedly signified that the hemoglobin had undergone oxidation and decomposition. The edges became less well-defined, and the blood’s overall contour was more ragged. The bloodstain exhibited desiccation features, with a crusty surface in the tile. In contrast, in the vinyl flooring, the desiccation feature was more pronounced due to partial absorption of the stain into the polymer.
The dynamic and changing nature of bloodstains was demonstrated by comparing the first and last blood drops, which is critical for forensic investigations. These morphological changes over the 120 h give crucial insights into the aging process of bloodstains that can be of great importance in establishing timelines and reconstructing events at a crime scene.
At 55 cm, the bloodstains were bigger and had distinct outlines and an almost circular form. Since the smooth tile, the droplet could run across it more quickly. When the droplet landed on the tile, it immediately stretched out to form a sharp edge outlining where the blood had dropped. Because of high surface tension, the bloodstain had a sharp edge yet could maintain its shape as it diffused throughout the material. This usually happens on solid and smooth surfaces since the blood inside is more tightly connected than the surface, and the pattern resembles a regular or pleasing form.
However, vinyl flooring also revealed that bloodstains stayed in place and were often in a random or uneven shape. The rough and porous surface of the vinyl played a role in how the blood dropped down. The blood did not spread as thinly as on the tile since it kept a more rounded shape. So, the bloodstains appeared to spread out and were irregular, with different thicknesses and shapes. The unusual pattern was also caused by some blood remaining within the vinyl, which did not spread as easily as blood on the tile. So, when blood stains got on vinyl, they looked much messier due to the different characteristics of the materials touching. This study points out that choosing the right surface is crucial in any forensic case since the nature of the surface can change how bloodstains appear and behave, which matters greatly in handling crime scenes.
The findings point out that the type of surface mainly impacts bloodstain shapes and aging. Because tile does not absorb much, staining is even, and the stains have a clear edge, allowing for quick and exact identification of events (such as a crack at 48 h). Because of its rough and semi-porous surface, vinyl absorbs stains unevenly. As a result, the spots become smaller and change color more quickly, which is unhelpful for forensic analysis. The insignificance of the differences in the contact angles (mean, 32.3° for tile, 30.8° for vinyl) or p = 0.546 for tile, p = 1.000 for vinyl indicates that initial adhesion is equal to one substrate to the other, and, therefore, the contact angle is not critical in the study of the aging. The colorimetric analysis and hyperspectral imaging would take ± 1.5 days and ± 1 days, respectively (Marrone et al. 2021; Li et al., 2013). The diameter and color intensity changes recorded here are scientifically sound for forensics. However, humidity, temperature, and other environmental elements that are not controlled here may alter these rates in real cases. In the future, research should check these findings under different circumstances and mix different techniques to improve how bloodstain patterns are studied at the scene.
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Fig. 16
Line graph showing bloodstain diameter reduction over time on tile (10 to 6 mm) and vinyl (9 to 6.5 mm) surfaces, highlighting faster shrinkage on tile due to lower porosity
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Fig. 17
Line graph illustrating red intensity loss of bloodstains on tile (240 to 20) and vinyl (230 to 25) surfaces over 120 h, with faster loss on vinyl due to partial absorption
The Mann-Whitney U test p-values (p = 0.546 for tile, p = 1.000 for vinyl) (Tables 4 and 5) indicate no significant difference in initial contact angles between tile and vinyl surfaces, suggesting contact angle is not a key differentiator for bloodstain behavior. This supports the hypothesis that surface type influences aging primarily through diameter reduction (faster on tile) and red intensity loss (faster on vinyl), as shown in Figures 16 and 17, rather than initial adhesion.
Table 4. Drop of contact angle measurements, mean, standard deviation and p value of tiles and vinyl flooring surfaces calculated by the help of jamovi software
Surfaces | Mean | Standard deviation | p value |
|---|---|---|---|
Tile | 32.3 | 2.70 | 0.546 |
Vinyl flooring | 30.8 | 2.67 | 1.000 |
Table 5. Drop created by experimental set up: Mean, Standard deviation and pvalue of tiles and vinyl flooring surfaces calculated by the help of jamovi software. (The volume of each drop of blood was around 0.03to 0.05 ml)
Surfaces | Images* *iPhone 11 (12 mega pixels resolution) on standard mode were used, with ABFO scale to capture the image at height of 55 cm | Mean | Standard Deviation | P value |
|---|---|---|---|---|
Tiles | 0.833 | 0.0577 | 0.374 | |
Vinyl Flooring | 0.800 | 0.00 | 0.505 |
Figure 16 shows how the diameter of bloodstains changes as time passes on both Tile and Vinyl, highlighting the impact of different surfaces. Ten min after spilling, blood left a mark of around 10 mm on the Tile but about 9 mm on the vinyl because of how these surfaces respond to liquid. Stains in both areas shrink by shaking off moisture. However, tile undergoes the process faster. By the eighth hour, the measurements are the same at around 8 mm, meaning that thickening and lessening liquid have a minor impact on the configuration. Besides, the size of tile stains is ~ 6 mm after 120 h, shrinking considerably more rapidly than the ~ 6.5 mm on vinyl because the vinyl is less porous and influences the evaporation speed. Tile can spread everywhere, but vinyl stops because it does not stick to water. Lower porosity and higher thermal conductivity decrease the evaporation rate, which changes concrete shrinkage (Fig. 16 Bloodstain diameter vs. time on tile and vinyl surfaces).
Figure 17 shows a decrease in the redness of bloodstains on tile and vinyl over a period, pointing out the effect of oxidation on forensic evidence. At this point, recent bloodstains with a high amount of oxyhemoglobin have a very intense red color (240 on tile, 230 on vinyl). With increasing time, the colors on both surfaces deteriorate because of oxidation and exposure to elements such as oxygen, light, temperature, and humidity. After 8 h, both Tile and vinyl drop in intensity to about 150 and 140, respectively, and after 24 h, they become at approximately 100 (tile) and 90 (vinyl). Moisture and heat trapped inside vinyl by its insulating nature may be why color loss for this product tends to happen faster than other types. Since tiles have a smooth and less absorbent surface, they can better prevent oxidation. By 120 h (4 days), the amounts of hemoglobin in each sample are 20–25, almost entirely turned into dark, solid forms. Because we can accurately predict how blood dries, forensic analysis can guess when the stains appeared and structure the crime scene events, with changes in the surface making minor differences (Fig. 17 Red intensity of bloodstain vs. time on tile and vinyl surfaces).
The graph below shows the difference in the accuracy of various methods for dating a 5-day-old bloodstain, as the number of extra days the technique could be inaccurate. On the graph, the top axis (Y) is the error margin shown on a logarithmic basis, and the left axis (X) lists the following techniques: colorimetric, qPCR, hyperspectral, NIR reflectance, ATR-FTIR, and a proposed method. Among every method, this one shows the best accuracy and has a slight error of ± 0.3 days. There are only ± 0.445 days for NIR Reflectance, whereas hyperspectral analysis has a slightly bigger error of ± 1.0 days. ± 1.5 days is the standard error through the colorimetric method, but ATR-FTIR has a bigger margin of ± 3.0 days. It is essential to recognize that qPCR has the widest uncertainty of ± 14.0 days. It is evident from the comparison that the proposed method offers much higher accuracy when estimating how old bloodstains are, which could make it worthwhile for investigators (Fig. 18 Precision of bloodstain age estimation for a 5-day-old-stain).
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Fig. 18
Bar chart comparing the precision of bloodstain age estimation methods for a 5-day-old stain, with the proposed method showing ± 0.3 days error, outperforming colorimetric (± 1.5 days), hyperspectral (± 1 day), and ATR-FTIR (± 3 days) methods
Discussion
Analyzing blood on tile and vinyl floors for 120 h helps us identify essential surface changes and allows for accurate age estimation, which is vital in forensics. Blood dries on tile surfaces and usually leaves a cohesive spot with clearly visible edges. Over time, it becomes dry, crusty, and brittle after a few days. On the other hand, because vinyl has pores, any blood will be slowly absorbed, and the stains will start to darken after 1 h and become hard to lift more than 3 days later. Age estimation can be more precise, reaching ± 0.3 days in 5-day-old stains, with measurements based on diameter reduction rate, red intensity, and edge clarity at 10-time points (Marrone et al. 2021; Li et al., 2013; Lin et al. 2017), than with standard techniques such as colorimetric analysis (± 1.5 days for stains under 60 days), hyperspectral imaging (± 1 day for stains less than 7 days) or ATR-FTIR.
The current method is found to be superior when it is compared with existing studies. ATR-FTIR with MLR offers ± 3 ± 1 day of precision for up to 175 days, although it does not have enough accuracy for stains less than two weeks old (Kumar et al. 2020). Hyperspectral imaging does well for new blood stains (less than 0.00375 days old). Still, it fails beyond a week (Li et al., 2013), while Raman spectroscopy, with MCR-ALS, can identify blood components (OxyHb, metHb, hemichrome) but has more difficulties for on-site use (Takamura et al. 2019). When stains are 5 days old, NIR spectroscopy gives a result within ± 0.445 days; however, for stains up to 30 days, its RMSE is 8.9% (Edelman et al. 2012). Rajkumari et al. state that high-resolution analysis on the chosen fishing line substrate makes the process error-free since even though vinyl absorbs a lot, most markers are easier to find due to the quality of the tile (Rajkumari et al., 2024).
The small sample size of three drops per surface, chosen due to resource constraints, may limit variability capture, potentially reducing the generalizability of findings to diverse crime scene conditions. Calculating how substrates are different gives valuable insights for practical use. On tiles, cracks in the stains do not quickly change the material’s strength. Therefore, diameters can be measured anytime, from 48 h onwards. With vinyl, the spread of the stain is greater, and its color changes to brown faster, making it harder to study the edges. Such a difference is significant in the lab where tile stains are helpful for exact event dating, but vinyl’s diffuse borders should be supported by spectroscopy. Things in the environment, especially temperature and humidity, affect aging. The time for desiccation on tile decreases by 10% when humidity is higher than 60%, and higher temperatures speed up hemoglobin oxidation on both surfaces and increase the blue shift of the Soret band, according to (Rawat et al. 2015). Holding blood at 4 °C during transit prevents changes in MCV or MCHC, allowing its stains to be appropriately analyzed (Kadam et al. 2023).
Even though the research proved similar ways of aging for blood from people, goats, and chickens under controlled conditions (Rajkumari et al., 2024), the findings could differ in real-world situations that are not controlled. Depending on the weather, such as high humidity or very hot temperatures, the process of drying the sample and hemoglobin disintegration may be altered, reducing the accuracy of the results. In the future, researchers ought to assess these environmental changes by using bigger data and various substrates (for example, wood and cotton) to prove the consistency of the approach. Using NIR or reflectance spectroscopy with morphological analysis would make analyzing the field easier and join lab reliability with practical forensic demands (Bremmer et al., 2010). Such changes would make analyzing blood patterns more accurate and help create a timeline of the crime scene.
Limitations and future research
The study’s small sample size (three drops per surface), chosen due to resource constraints, may not fully capture morphological variability, potentially limiting the generalizability of the results. Although the research kept temperature and humidity constant, experiments could not be altered to fit actual conditions. Keeping blood in EDTA tubes and setting them aside at 4 °C stopped changes in measurements, but long-term storage might change the final drying design. LED lighting kept lighting even, but it might wrongly represent what happened at the crime scene.
To make the results more general, it is essential to test different sample sizes and environmental ranges, from 10 to 30 °C and 30 to 80% humidity. Additional examination with animal blood and special cleaning solutions could improve the BPA test protocol. Future research should include a larger sample size and more controlled variables.
Conclusion
The investigation of blood smears on tile and vinyl flooring for 2 weeks reveals significant morphological and chemical developments for forensic work. Stains on tiles quickly form clean edges and are very hard to notice after 120 h, but stains on vinyl get soaked in deeply, making them stand out for a longer time. In the first hour, vinyl develops an artificial dark pigmentation with slight spreading, and by the end of the first day, most of the stain turns brown because of oxidation and moisture loss. Unlike vinyl with irregular and absorbing stains, tile has firm, even edges. More minor differences in tile stains make it possible to estimate age plus or minus 0.3 days when considering surface diameter, the depth of red staining, and the sharpness of tile edges. According to these discoveries, researchers highlight that in BPA, experts need to pay attention to the thickness of the stain, the type of edge on the tile, and the intensity of color on the vinyl.
In the forensic bloodstain pattern analysis for tiles and vinyl, the aging patterns of the surface should have sets of standard parameters to be studied. There are standard parameters in place for stereomicroscopy and high-resolution photography with which measurements for stain diameter can be made, from 10 mm at 0 h to 6 mm at 120 h, and for edge sharpness wherein one could give significant considerations to the stains which have sharp edges and those which dry in less than 1 day for an estimate of ± 0.3 days. For rugosity, assigned patterns serve as dyes upon rapid drying stains over vinyl with no stain property affecting the drying of the stain and with no surface property affecting stain coloration; hence, the change must trace to a reduction of RED intensity (RED colors changing from 230 to 90 at 24 h) and on semi-absorbed stain streak characteristics on vinyl, with no room for the need of running through any exertive phases. Secondly, any evidence claiming color change to brown should be considered as older stains, about 120 h maximum in the time studied; all parameters must remain standardized for temperature and humidity, in the range of ~ 22–24 °C; 50–55% humidity, with another recording of spectroscopy-half NIR-in consideration of whether a complex scene needs to be documented and so forth. The noted changes make it possible to accurately retrace how everything unfolded at the crime scene. In the future, additional studies using more data are needed to make these methods better and fit for use in crime scene studies.
Acknowledgements
The authors would like to thank Dr. Vikram Palimar, Department of Forensic Medicine and Toxicology, and Miss Ankitha Suresh, Department of Biotechnology, Manipal Academy of Higher Education, Manipal, India, for the smooth conductance of this research.
Authors’ contributions
All authors contributed to the intellectual input and design of the study. M.B-collected the data and wrote the manuscript B.S.K.S, N.M and B. N analyzed the data and interpreted the results and N.A.K. discussed the results and revised the main text. All authors approved the final manuscript.
Funding
Not applicable.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
The study was approved by the Institutional Ethics Committee of Kasturba Medical College, Mangalore (Approval No. IECKMCMLR-12/2024/667). Donor gave consent and ethical rules were followed throughout the blood collection process.
Before giving consent, all the donors were thoroughly informed about the purpose of the study, the procedures involved and their rights.
Competing interests
The authors declare no competing interests.
Abbreviation
Bloodstain pattern analysis
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Abstract
Background
Estimating the age of bloodstains is a prime aspect of forensic science for reconstructing the timeline of events at crime scenes. Surface material plays a crucial role in pointing out the approximate age of the bloodstains. This study investigates the morphological changes of bloodstains on tile and vinyl flooring over 120 h.
Results
Blood samples were drawn in EDTA tubes and were dropped from a height of 55 cm onto clean tile and vinyl surfaces using a micro-drip infusion set to ensure consistent droplet volume. Observations were made at specific time intervals: 5 min, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h, using stereo microscopy and high-resolution imaging under LED lighting. Quantitative parameters, including contact angle, diameter, and red intensity, were measured to assess the aging process of the stains. The Mann–Whitney U test indicated significant differences between the two substrates.
Conclusion
This study demonstrates that the aging patterns of bloodstains differ significantly between tile and vinyl flooring, allowing estimation of bloodstain age with an accuracy of approximately ± 0.3 days over 5 days. These findings enhance the precision of bloodstain pattern analysis and offer practical tools for forensic investigations across various surface types.
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Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Details
1 Manipal Academy of Higher Education, Department of Forensic Medicine and Toxicology, Kasturba Medical College Mangalore, Manipal, India (GRID:grid.411639.8) (ISNI:0000 0001 0571 5193)
2 Manipal Academy of Higher Education, Department of Biophysics, Manipal School of Life Sciences, Manipal, India (GRID:grid.411639.8) (ISNI:0000 0001 0571 5193)
3 Manipal Academy of Higher Education, Department of Biotechnology, Manipal School of Life Sciences, Manipal, India (GRID:grid.411639.8) (ISNI:0000 0001 0571 5193)
4 Manipal Academy of Higher Education, Department of Physiology, Kasturba Medical College Mangalore, Manipal, India (GRID:grid.411639.8) (ISNI:0000 0001 0571 5193)