Analysis of linkage between luminescence duration and age of the blood stain on different types of wooden surfaces

 Introduction:

One of the most often found physical evidence in violent crime investigations is blood. Blood samples collected from crime scenes are subjected to forensic examinations, which provide a wealth of information that may be crucial in solving a case(1,2). Blood is a vital component of the human body, serving essential functions such as transporting oxygen and nutrients, regulating temperature, and defending against infections. However, its presence outside the body can often be indicative of criminal activity, accidents, or other traumatic events. Blood at a crime scene can provide valuable information to investigators and aid in crime scene reconstruction (3,4). Like other forensic evidence, it can reveal valuable details about a crime, including the location, physical orientation, weapon or object used, number of blows inflicted, and individualization (4). Due to its significance in forensic investigations, the detection and analysis of blood at crime scenes plays a crucial role in solving crimes and obtaining justice.  The blood stains are usually found at crime scenes, which are identified and collected for advanced analysis in specified forensic laboratories. However, most often the culprits try to hide or destroy the blood evidence from the crime scene. The identification of these latent blood stains at the crime scene is crucial for crime scene reconstruction. To visualize these latent blood stains or their patterns, the luminol reagent-mediated luminescence test is considered the most sensitive test (5). Luminol reagent interacts with blood, chemiluminescence occurs, and through visualizing the resulting lights, the investigator can easily locate the possible presence of blood stains (6). Subsequent analysis of the blood pattern provides information about the movement of the victim as well as the culprit along with the weapon used to commit the crime (7). Luminol (5amino 2,3-dihydro-1,4 phathazine-dione), after reacting with tiny amounts of blood, gives chemiluminescence due to the charge in the excitation state of the electron within the iron group present in the hemoglobin (8). Although Luminol is very sensitive but it may produce false positives in the presence of other oxidants like bleach, plant peroxides, and with other compatible Metal ions(9,10). But despite this, luminol is the most reliable and sensitive presumptive test used for locating latent blood stains.

Outdoor crime scenes present unique challenges for forensic investigations, as environmental factors such as weather conditions, sunlight exposure, and natural decomposition processes can affect the detection and preservation of blood evidence. Bloodstains at outdoor crime scenes may be subjected to degradation due to exposure to sunlight, moisture, and microbial activity, leading to changes in their appearance and composition over time (11-13). Additionally, the presence of vegetation, soil, and other outdoor elements can complicate the collection and analysis of bloodstains (12). The analysis of bloodstains involves understanding how blood behaves when it comes into contact with different surfaces, which can affect the appearance, detection, and interpretation of the evidence (14).

 During a crime scene investigation, the investigator has to locate the blood stains on different types of surfaces, and blood stains present on these surfaces have variable degrees of sensitivity with the luminol reagent. At most of the crime scenes, whether it is outdoor or indoor, woody material is one of the potential deposition sites for blood because these materials are common everywhere. Since the physical and chemical properties of wood depend upon its source, i.e., from which part of the tree, the wood has been obtained, the aim behind this study was to evaluate and compare the sensitivity of Luminol reagent with blood stain present on four different types of wooden material sourced from the same tree (Mangifera indica) with blood stain of different aged.   

2. Material and Method.

2.1. Chemical reagent and blood:  Luminol reagent was purchased from Merck and luminol reagent was prepared by dissolving 0.2 gm of luminol and 15 gm of Potassium Hydroxide (KOH) in 200 ml of water and 3% of Hydrogen Peroxide (H₂O₂). Human blood was collected from the local blood bank by following the proper channel and procedure.

2.2. Procurement of different types of wood: The types of woods i.e. Polished Wood (Wood that has been polished to create a smooth, glossy surface, also protecting it from moisture and abrasion), Barked wood (The bark gives a natural edge but is prone to deterioration and bugs), Debarked wood (Wood that has had its bark removed, revealing only the interior wood, prevents rotting), and heart wood (The dense, interior section of a tree trunk that offers structural stability) included in the study were obtained from the same tree of mango (Mangifera indica).

2.3. Imprinting of blood on wood surfaces: 10 samples of polished wood, barked wood, debarked wood, and heartwood (40 in total) were obtained.  Each of them was properly labeled and cut into uniform measurements of 70×55mm. After taking proper precautions, 100ul of the same blood sample was applied onto the surface of each wood sample using a dropper. On a single piece of wood (from every type of chosen wood type), a total of 21 blood stains were developed to analyze the luminol reaction at different time intervals. These 21 stains were further marked as seven different groups, each group consisting of three blood stains. The imprinted blood stains from these seven groups on each piece of wood were subjected to luminescence reaction at different time intervals i.e.(i) within 5 mins i.e. fresh (ii) after 6 hrs (iii) after 12 hrs (iv) after 24 hrs(v) 48 hrs (vi) 120 hrs (vii) 240 hrs. The rationale behind these ages of blood is that, on average investigating team arrives and completes their crime scene investigation within these selected time intervals.

2.4. Performing chemiluminescence reactions: Luminol reagent was prepared by following the standard protocol as described in section 2.1. This freshly prepared luminol reagent was used to perform the chemiluminescence reactions with a time interval based on different groups of blood stains as described in section 2.3 of this manuscript. The stopwatch was used to record the time of appearance of luminescence as well as to record the total luminescence duration (LD). LD was defined as the duration of the appearance of luminescence light. All the experiments were performed at room temperature and under the indoor settings of laboratories (Fig.1).

2.5. Statistical analysis: As described earlier, at each time interval included in the study, the luminescence reactions were performed in triplicate on each piece of wood. Since a total of 10 pieces of each type of wood were selected for study, the mean of luminescence duration (LD) of each piece of wood (from the same type of wood) was then used for further analysis by using two-way ANOVA and Bonferroni posttests provided by Prism software. A value of significance p<0.05 was considered significant.

3. Results

3.1. Analysis of the appearance of chemiluminescence on different woody substrates through the interaction of luminol reagent with blood stains of different ages.

The observation of this study revealed that chemiluminescence appeared on all the surfaces with blood stains of different ages within a second, except for debarked wood, where the appearance of luminescence started to get delayed with 120 hours and 240 hours old blood stains. This delay was observed with much earlier blood stains (48 hours old) on heartwood (Table 1).

3.2 Analysis of variation in the luminescence duration (LD) with different aged blood on chosen surfaces individually:

When the researcher compares (one-way ANOVA and Tukey’s multiple comparison test) the luminescence duration (LD) of fresh (within 05 mins) with all the time intervals on each separate wood, the decrease in the LD pattern is almost similar on all types of woods (Table 1) but with varying degree of significance (p>0.0001 to p>0.01). When the LD of a 6-hour-old blood stain was compared with different-aged blood stains on different types of wood, the changes were less significant compared to 12-hour and 24-hour-old blood stains, but this difference was very significant with older blood stains, i.e. 120 and 240-hour-old blood stains. Results of the study also reflect that there was no significant change in LD when compare between 12 hours and blood stain with 24hrs old blood stain (except for polished wood and debarked wood) but with increasing age of blood stain the difference of LD with 24-hour-old blood stains was not significant with 48-hours old blood but falls in LD became very significant with older blood i.e. 120-hour and 240-hour-old blood stains. The significant changes occurred only on barked wood when the comparison between the LD of 48 hours old blood stains with 120 hours and 240-hour-old blood stains was performed. The similar trends have been observed when the comparison between the LD of 120 hours old blood with the 240-hour-old blood was done. The polished wood and heartwood were exception with no significant difference in LD of 120 hours old blood and 240 hours old blood (Fig 2).

3.3. Comparative analysis of variation in the luminescence duration (LD) with different aged blood on chosen surfaces:

Comparison of luminescence duration (LD) on all the surfaces when measured within 05 minutes are statistically significant (p>0.0001) through Bonferroni posttests it was evident that the difference in the LD was significant within 05 minutes at all the surfaces but this difference was non-significant when the comparison of the LD between polished wood and heartwood was performed. The trend was similar with 06 hours old blood stains and significant variations in the LD had been observed between the different wood materials. The trends were similar for other different aged blood stains with few exceptions like between barked and debarked wood (at 12 hours). The difference of the LD at different surfaces are remained significant overall (p>0.0001) as well inter-surfaces (p>0.05) (Fig. 3).

Discussion

Spotting the blood at crime scene is crucial. There are various chemical reactions based presumptive tests have been developed to identify the blood spot at the scene of crime. Luminol based presumptive test is supposed to be most sensitive for the identification of blood (6). It has been observed that the sensitivity of luminol towards blood is influenced by the nature of surfaces on which blood get imprinted (15,16). At a crime scene there is a possibility of getting blood spot on various types of material like brick, concrete, metal, fiber or wood. Apart of nature of surfaces the time interval between imprinting of blood and its detection by luminol affect the duration of luminescence caused by luminol reagent (17,18). Fresh blood drops obviously give longer LD with time may be influenced by the factors such as environmental condition and type as well as nature of the surfaces(18-21).Since woody materials are one of the most likely surfaces for possible location of blood spot at the crime scene, this research aimed to elucidate any possible link between the duration of luminescence and age of the blood stain at different kind of woody surfaces. The four types of woods i.e. polished wood, barked wood, debarked wood and heartwood sourced from the same tree (Mangifera indica) have been selected for the study. The results of this study clearly indicate that the trends for differences in the LD with changing age of blood stain is almost similar on all the types of selected wood but the magnitude of changes between different time intervals differs from surface to surface. Fresh blood on all the surfaces gave maximum LD in comparison to other time intervals at respective woody material and as expected, minimum LD were observed with oldest blood stain (240 hours i.e. 10 days old) on all the surfaces. According to the result observed, the changes in the LD was significant just after 06 hours aging of blood stain on heartwood, whereas on other surfaces, this fall in the LD was not very sharp.

Since the environmental conditions, except wood types, were kept similar during the experiments, so changes observed in the LD must be linked with the types of wood. These different woods have different porosity and have different adhesive attraction with blood molecule (22-25). The significant changes in the LD on barked wood occurs only after 12 hours but changes became significant quickly on debarked wood (24 hours’ time interval). Since barked wood have higher porosity naturally (26,27). but the distance between pores of barked wood and debarked wood are relatively longer than the other chosen wood in the study (27-29). This may be one explanation for the observation that decrease in LD is much lesser in other woods in comparison to heartwood. The duration of luminescence on polished wood was minimum with fresh blood in comparison to all wood surface and it may be due to the interference of ethanol and other chemical present in the polished wood with the activity of luminol (30-32). It has been reported that ethanol may act as competing reducing agent or may change the chemical environment, which subsequently can affect the luminol reaction (32-24). Due to this prolonged exposure to polishing material, there are significant difference in LD after 12 hours (almost 50% decrease) which remain constant at subsequent time interval. Since, there is no added chemicals present on barked wood, debarked wood and heartwood the LD with fresh blood is almost similar to these woods.

The heartwood gave a very significant fall in the LD very quickly (at 06 hours 60% decrease). This sharp fall observed at 06 hours of blood age may be due to the presence of various resin, tannins and gums present in the heartwood. The tannic acid is a polyphenol with antioxidant properties that can sufficiently reduce the oxidase activity of hemoglobin present in the blood stain (35-37). These all interferences may be the main reason behind the sharp decrease in luminescence after 06 hrs on heartwood. It has been established that luminescence time depends upon the amount of blood present and specially on the oxidase properties of hemoglobin (38,39).These enzymatic properties of hemoglobin are liable to environmental conditions and this is evident by significant falls in LD on all the surfaces after 240 hours i.e. 10 days.

5. Conclusion:

This study was an attempt to evaluate the link between the luminescence duration (LD) and age of the blood spot on different type of woody surfaces obtained from the same source tree. The type of woody surface chosen in this study are that which is most frequently found on the crime scene. Although this is a very preliminary work but results and observations of this study aptly suggest that types of surfaces may influence the luminescence sensitivity, especially the luminescence duration. The results of this study re-emphasize the importance of the age of the blood stain on LD but also provide insight into the pattern of change in LD with the age of blood spot and its linkage with types of woody surfaces. Findings of this study provide enough preliminary results to promote the workers to research further on this line to provide more information which may be of great use for forensic investigators. The finding of this study may help the investigator in crime scene recreation. By comparing the LD on different woody surfaces, the investigator may estimate the age of the blood stain under scrutiny.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this manuscript, the authors used Grammarly, and Mendeley have been used but only for the grammatical correction, formatting and enhancement of manuscript presentation.

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