Surface Specific Analysis - A Comparative Study of Menstrual and Non-Menstrual Bloodstains

Surface Specific Analysis - A Comparative Study of Menstrual and Non-Menstrual Bloodstains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Surface Specific Analysis - A Comparative Study of Menstrual and Non-Menstrual Bloodstains Mayur Sudhir Balbudhe, B Suresh Kumar Shetty, Adithi Shetty, Nayanatara Arun Kumar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7177372/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Distinguishing between menstrual and peripheral bloodstains is essential in forensic research, especially in sexual assault or trauma cases, since it may be used to determine whether it is a matter of consent or violence. Under controlled conditions, this study examines the temporal morphological changes of menstrual and non-menstrual blood stains on glossy paper, a semi-porous surface. Results: The diameter of the stains was noted at 10 minutes, 1, 2, 24, 72, and 120 hours after the deposition for menstrual and non-menstrual stains. The non-menstrual bloodstains decreased consecutively over time, so they were more consistent with time. These variations were due to the viscous, mucus-rich nature of menstrual blood as opposed to the clotting effects of peripheral blood, and were statistically significant (measured by triplicate measurement). Conclusion: This is a non-destructive optical technique that can complement high-order techniques such as hyperspectral imaging, Raman spectroscopy, and the SERATEC PMB test, increasing the capacity of forensic bloodstain pattern analysis. Although the substrates used and the environmental parameters are limited, the results give a good starting point in differentiating blood types, the ramifications of which may be used in correct crime scene reconstruction and legal results. Bloodstain pattern analysis menstrual blood peripheral blood glossy paper stereo microscopy morphological changes crime scene reconstruction non-destructive methods bloodstain diameter Figures Figure 1 Figure 2 Background In a dimly lit, fluorescent-lit forensic laboratory, a forensic scientist scrunches their brow over the microscope in deep concentration. The bloodstain might carry answers to the questions of a crime: on the table, there is a piece of fabric with a dark stain of rust color. Possible wound stain or a natural biological process, this stain has the potential to address the questions of consent, trauma, or violence (Roy et al., 2022). Bloodstains are the tell-tale in forensic science, and they tell the secrets or hints about what happens at a crime scene (Bevel et al., 2008). The difference between menstrual blood, which is spontaneously lost by the inner surface of the uterus, and peripheral blood, which results from an accident, is a decisive problem that may determine the outcome of the investigation effort (Holtkötter et al., 2018 ). The surface on which the blood exists, cotton, wood, or tile, makes this puzzle complex (Al-Alimi et al., 2025). With the emerging refined visualization processes, a breakthrough will enable the decryption of these crimson hints to become even more accurate (Pradeep et al., 2024 ). One of the most usual and convincing pieces of evidence is the bloodstains (James et al., 2014 ). Blood, being a viscous substance having specific physical characteristics, leaves an impression which indicates how a violent incident of stab, bullet, or traumatic blow has taken place (Bevel et al., 2008). To recreate what took place and to analyze how something happened, forensic scientists can turn to bloodstain pattern analysis (BPA), which assesses the size, shape, and distribution of blood stains to tell the angle of impact and amount of force used, as well as the correlation of movements of individuals (Bevel et al., 2008). Nevertheless, the BPA does not always identify the blood type, whether it is peripheral or menstrual, a factor that is critical in cases where incidences of sexual assault are involved (Newton et al., 2013). Menstrual blood may imply permission had been given to such cases, whereas peripheral blood may indicate injury (Hanson et al., 2014 ). Much lies at stake in this case, yet differentiation must be precise to achieve justice (Chunkul et al., 2025 ). Imagine a crime scene: a bedroom, a messed-up bed, and some blood on the sheets of it, a series of blood droplets leading to a door. The job of the investigator is to reconstruct the story. Was this some violent attack, or could it be blood due to a woman's monthly period? Conventional forensic tests, i.e., the Teichmann or luminol-based tests, exclude the possibility of blood but have difficulty distinguishing between menstrual and peripheral blood (Wang et al., 2022 ). Menstrual blood is a complicated conglomeration that incorporates red blood cells, white blood cells, plasma, vaginal secretions, cervical mucus, and endometrial tissue (Hanson et al., 2014 ). In contrast to peripheral blood, which is clotted through fibrinogen, menstrual blood causes fibrinolysis, or breaking of the clots, thus leaving behind a liquid form containing fibrin degradation products such as D-dimers (Baker et al., 2011 ). These minor variances are critical and need complex detection means (Mistek et al., 2016 ). A surface on which a blood stain is deposited further adds to the complexities of analysis (Rajkumari et al., 2024). On a non-porous surface, such as tile, a drop of blood becomes a circular, highly defined stain; on a porous surface, such as cotton, blood spreads out and diffuses, morphologically and chemically changing its appearance (Rawat et al., 2015). These surface-specific variations are hard to explain in terms of conventional approaches, i.e., with the help of microscopy or electrophoresis of lactate dehydrogenase isozymes (Rawat et al., 2015). More elaborate molecular techniques, such as mRNA or miRNA profiling, need laboratory conditions and special training, so they are unsuitable for real-time analysis requiring rapid, on-site testing (Hanson et al., 2014 ). Information about the sensitive and surface-adaptable method of analysing bloodstains that would be applicable non-destructively in the forensics community has been sought for a long time (Mistek et al., 2016 ). The imaging techniques come to the scene to provide a fresh chapter in the blood stain analysis in forensics. Non-destructive imaging techniques contrast with chemical/molecular techniques; however, such methods leave the sample intact to allow post-imaging DNA analysis, an essential factor when investigating a crime (Pradeep et al., 2024 ). One of the most promising techniques is hyperspectral imaging (HSI), which refers to the possibility of obtaining the spectral signature of a substance over a great distance of the spectrum, both visible and near infrared (Al-Alimi et al., 2025). An HSI image has a spectrum in each pixel, which is a fingerprint in itself and tells about the chemical content of the material (Zulfiqar et al., 2021 ). Blood, having hemoglobin and other molecular components of blood, has characteristic spectral patterns, which are different in the peripheral blood and menstrual blood due to differences in the oxygenation and iron levels or endometrial debris (Wang et al., 2022 ). HSI is sensitive to finding out these differences, and this presents a non-invasive method to study blood stains (Pradeep et al., 2024 ). Imagine the forensic scientist again, but this time with an HSI camera. They will be able to scan the blood-stained fabric, and within minutes, the system will come up with a spectral map that shows minute differences in the composition of a stain (Zulfiqar et al., 2021 ). Compared to menstrual blood, which is less oxygenated and darker, hemoglobin in the peripheral blood has an altered spectral pattern, which is a nice and bright red (Wang et al., 2022 ). Ideally, using these spectra, the scientist can prove that there is blood, and the source of this blood can be ascertained (Walker et al., 2025 ). HSI can also consider the effect brought by the underlying surface (Pradeep et al., 2024 ). Bloodstain analysis on cotton might absorb differently from a bloodstain on tile. Still, with the precision that HSI offers in separating the spectral signature of a stain and that of its substrate, analysis can be done item by item (Zulfiqar et al., 2021 ). This factor renders HSI a game changer as it allows the investigator to analyze any stains on site and does not tamper with them with any chemicals (Pradeep et al., 2024 ). Raman spectroscopy is another promising method, in which inelastic scattering of light is used to interrogate the molecular structure of a sample (Atkins et al., 2017 ). Spectrum of Raman menstrual and peripheral blood reveals no difference between the results due to the hemoglobin levels, protein contents, and biochemical indicators involved (Atkins et al., 2017 ). Combined with statistical techniques such as partial least squares discriminant analysis (PLSDA), Raman spectroscopy has reached 100% sensitivity and specificity in assaying blood type, even when applied to low-contrast materials such as fabric or carpet (Zulfiqar et al., 2021 ). In contrast to HSI, which is highly effective in broad-spectral analysis, Raman spectroscopy has a high degree of molecular specificity. It is therefore described as the most appropriate method to identify subtle biochemical differences (Fu et al., 2019). They are both non-destructive and fast, making them convenient in laboratory and field analysis. Forensic science has the support of advanced imaging techniques, which help in solving cases, but these are not the only tools. The immunochromatographic assay- SERATEC PMB test helps distinguish between menstrual and peripheral blood (Holtkötter et al., 2018 ). Due to their character as a fibrinolytic factor, human hemoglobin and D-dimers are detected in this test and indicate menstrual blood (Baker et al., 2011 ). SERATEC PMB test has a duplex design that simultaneously confirms the presence of blood and detects menstrual fluid, which makes it useful in quick screening during crime scenes (Konrad et al., 2024 ). Nonetheless, the SERATEC PMB test involves the extraction of samples. Depending on the age of stains or climate conditions, it may be challenging to do on porous material (Wei et al., 2024 ). Imaging methods can be used; however, these provide a non-destructive, spatially and chemically intact approach to evidence (Wei et al., 2024 ). When imaging procedures are added to the SERATEC PMB test, they enable a multi-modal approach, where one enhances the other and nullifies their shortcoming (Konrad et al., 2024 ). It is impossible to speak about bloodstain analysis without describing the surface. When a drop of blood sits on a smooth, non-absorbent surface (such as glass), the edges of the drop are sharp, and the drop takes the shape of a ball (Pozrikidis, 2006 ). The blood is, however, wicked in the fibers upon a porous surface such as cotton and forms a diffuse, irregular stain (Rajkumari et al., 2024). These effects, which are surface-specific, change the physical look and spectral or chemical character of the stain (Atkins et al., 2017 ). As an example, the absorption of blood into a fabric can cover some spectral peaks during HSI. Simultaneously, Raman spectroscopy can be disrupted by the chemical compositions of a surface, i.e., by dyes on clothing (Mistek et al., 2016 ). Coming up with a method that considers these variables is not easy, and imaging has a unique capacity to address it (Zulfiqar et al., 2021 ). The surface-specific analysis has been transformed using the HyperBlood dataset, a collection of hyperspectral images of bloodstains on various surfaces (Al-Alimi et al., 2025; Li et al., 2025). Research based on this dataset indicates that HSI and machine learning techniques, such as Multi-Layer Perceptron (MLP) networks, have been used to reach 97–100 percent accuracy in detecting bloodstain and compensating substrate effects (Pradeep et al., 2024 ). These algorithms are trained to understand the spectral signature of blood instead of the underlying surface material to robustly process various materials such as cotton, wood, and tile. Comparably, Raman spectroscopy, using PLSDA, successfully extracts the molecular signal of menstrual blood, despite the interference of a confounding factor such as dye in fabrics or environmental impurities (Zulfiqar et al., 2021 ). The research examines how the menstrual and non-mensural bloodstain patterns change over a period when temperature is regulated and the samples are observed through controlled environmental conditions with the help of a stereo microscope at various magnifications with and without an ABFO scale. This study will follow the bloodstains' diameter and structural modifications within 10 minutes to 120 hours after deposition. The controlled environment with a stable temperature and humidity is meant to measure the possible differences in the coagulation and drying dynamics between the two types of blood in the case of glossy surfaces. Materials and Methodology Differentiation of menstrual and peripheral bloodstains on the glossy surfaces of paper was done with the help of a Stereo Microscope. Controlled deposition of blood droplets, morphological analysis in a serial manner using stereo microscopy, and the use of a Nikon DSLR 750 Camera to provide surface-specific identification of bloodstain origin that was non-destructive, were used in the experimental design. The study's objective was to test the effectiveness of optical measures in determining blood types, considering that glossy paper has semi-porous qualities. Several trials were taken to guarantee reproducibility, and statistics were calculated to assess the consistency of morphological variations. Biological Samples : The blood samples were collected at Lady Goschen Hospital in Mangalore and from healthy individuals by performing venipuncture, after the permission of the Institutional Ethics Committee (IEC) and the informed consent of the donors. The peripheral blood was harvested in EDTA vacutainer tubes and kept at 4°C to avoid its breakdown (Kadam et al., 2023 ). Menstrual blood samples were collected on ethical grounds in the early days of the menstrual cycle of the consenting female volunteers. They were tested in a 4°C environment in uncoated vials with EDTA, and it was sufficient to be used within 24 hours without causing any variations. The sample volumes were standardized to 30 µL per drop to ensure uniformity. Surfaces : As a semi-porous substrate model, the glossy paper (30 cm x 30 cm, 120 gsm, chemically coated with a polymer-polymer gloss finish) was used, which represents objects commonly met in forensic situations, like documents or packages. Glossy paper was wiped using 70% ethanol, deionized water, and dried using air to eliminate contaminants and leave it in perfect condition. Equipment : A micro drip infusion set (MicroFusion, 0.1 mL precision) was applied to controlled blood droplet delivery. The images were analyzed using a DSLR (Nikon 750). The morphological analysis was made possible using the stereo microscope (Euromex CMEX-5PRO, 0.65, 1x and 2x magnification) with Image Focus Alpha Software. Other materials were a laboratory stand, a clamp, and an ABFO scale. Experimental Procedures : Preparation of Surfaces : The surfaces of glossy papers were wiped with 70% ethanol, subsequently wiped with deionized water, and dried and air-dried for 30 min to remove any leftover contaminants. Vigilance was checked using UV light (365 nm) to ensure how clean surfaces were and how the shiny layer held up. The adhesive mounts were used to fix each sheet on a flat vibration-dampened bench to eliminate movement of the sheets during the deposition of blood. Setup of Blood Vial : The vials filled with peripheral or menstrual blood were positioned on a laboratory stand and clamped 55 cm above the smooth paper surface. The effects were tested in a controlled setting where the temperature was kept at 22 ± 1°C, the humidity was between 50 ± 5%, and LED lighting of 5000K was used to avoid changes in natural light. The measuring tape was used to measure the height, and each time, the drop point was matched to the target grid's center to ensure reproducibility. To ensure the stability of the vial, this was made to achieve a minimum of the vial movement laterally as the droplets were released. The height of 55 cm was chosen to represent the conditions of forensic blood drip (i.e., dripping blood out of a wound or menstrual flow). Dropping the Blood : To obtain a single drop of 30 µL of blood onto the glossy paper surface, it was done using a micro drip infusion set connected to the blood vial, and without exerting any force other than a free-fall. To every blood type (menstrual and peripheral), three drops should be placed on individual glossy paper sheets, and thus, there were six drops in total (3 drops x 2 blood types). To exclude cross-contamination, the infusion set was washed with phosphate-buffered saline (PBS) and sterilized between trials. The conditions of the environment were standardized (temperature: 22 ± 1°C; humidity: 50 ± 5%) to reduce the impact of extra tissue factors on the formation of bloodstains. The average diameters of bloodstains on glossy surfaces were counted. Morphological Observation and Documentation : A stereo microscope with a (0.65x, 1x, and 2x) magnification, Euromex CMEX-5PRO, was used to study the bloodstain morphology at the point of impact. The first measurements were taken 10 minutes after the impact, and time-series measurements of 1, 2, 24, 72, and 120 hours were taken to reflect the changes over time. Images were captured under Image Focus Alpha Software and analyzed and measured concerning stain diameter, circularity, and edge properties. At each time point, measurements were done thrice to make it reliable. The smooth, coated surface of the glossy paper left bulky bloodstains with clear boundaries and little diffusion, as in the case of the latter substrates. The bloodstains (menstrual ones and the rest) are depicted in Table 1 , measured at separate time points after they have been deposited: 10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours. The aim of monitoring such measurements is to determine the variation of the diameter of the stains with time to understand the nature of menstrual and non-menstrual bloodstain behaviour and consistency of bloodstains with time. The detailed comparison of the menstrual and non-menstrual bloodstain images viewed using a stereo microscope at a magnification of 1x is represented in Table 1 . Observations were made at six instances after deposition (10 min, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours) to record temporal morphological variables on glossy paper, a semi-porous substrate. The discussion is based on the visual characteristics, diameter, edge properties, texture, and color of three reference samples of each blood type, emphasizing the differences caused by biochemical and physical characteristics of samples. Table 2 presents the comparison of the images of menstrual and non-menstrual bloodstains looked through a stereo microscope at the magnification of 0.65x, comparing the time intervals (10 minutes, 1, 2, 24, 72, and 120 hours) with the help of an ABFO scale. The most important observation that can be made is that the non-menstrual bloodstains exhibit a successive loss of diameter in these time intervals, which appears as a consistent, predictable loss of size as they dry and shrink. The changes in diameter with time are probably less regular in menstrual bloodstains because of the complexity of their composition (blood, endometrial tissue, mucus). This is because the ABFO scale guarantees high levels of accuracy in measuring it. As time goes by, the microscope shows specific differences in visual appearance, e.g., the texture of the stain or edges. Table 3 gives a comparison of the side profiles (the edge aspects, thickness, or cross-sectional morphology) between menstrual and non-menstrual blood stains viewed using a stereo microscope at 2x magnification after specified intervals of time (24 hours, 72 hours, and 120 hours as in earlier tables). The emphasis is put on changing these parallel side profiles with time, giving an idea of the physical and structural development of the bloodstains. The visual comparison of menstrual and non-menstrual bloodstains is provided in Table 4 : bloodstains were photographed with a Nikon 750 camera and an ABFO scale to provide proper size reference, at different time points (10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours). These high-resolution images are used to document alterations in stain as well as in diameter, color, texture, and edge perception to bring variations in how these blood types change with time to notice. Table 5 represents the average circular diameter of non-menstrual and menstrual bloodstains measured in centimeters at various times after deposition (10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours). The data summarizes how stain size evolves, highlighting differences between menstrual and non-menstrual blood. This builds on Table 1 ’s observation that non-menstrual bloodstains decrease consecutively in diameter, indicating consistent behavior, while menstrual bloodstains likely show more variability. Table 5 Average Diameter of Menstrual and Non-Menstrual Bloodstains Across Time Intervals in Centimetres Type of Sample Time Interval Mean SD Diameter Menstrual Blood 10 Min 8.83 1.258 1 hr. 8.83 1.258 2 hr. 8.50 0.866 24 hr. 8.33 1.155 72 hr. 8.33 1.155 120 hr. 8.33 1.155 Non-Menstrual Blood 10 Min 8.67 0.764 1 hr. 8.67 0.764 2 hr. 8.50 0.866 24 hr. 8.33 0.577 72 hr. 8.17 0.289 120 hr. 8.00 0.000 Results Analysis of Average Bloodstain Diameter Over Time : The mean diameters of menstrual and non-menstrual bloodstains were taken after depositions at 10-minute, 1-hour, 2-hour, 24-hour, 72-hour, and 120-hour intervals using a stereo microscope at 0.65x magnification. The ABFO scale was used to give accurate measurements of these diameters. Table 5 contains the data expressed as mean diameters and plotted in a line graph, based on three references during the bloodstain categories. Findings indicate a clear temporal pattern regarding diameter variation presented by both menstrual and non-menstrual bloodstains that has implications for forensic and bloodstain pattern analysis. Menstrual Bloodstain Diameter Trends : The diameters of menstrual blood samples at different time lapses after deposition, average diameter (in centimeters), and standard deviation (SD). They were measured after 10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr. At 10 and 1 hour, the mean diameter is 8.83 cm and the standard deviation is 1.258, which means there was a regular size with normal variation in the earlier period. At 2 hours, the average diameter reduces by a small margin to 8.50 cm (standard deviation of 0.866, which indicates that it may be slightly smaller and with less variance). Beyond 24 hours, the mean diameter settles at 8.33cm, and at 72 hours, 120 hours, and 168 hours, the standard deviation records 1.155 and no longer varies. This implies that once the first two hours are surpassed, the diameter of the menstrual blood samples stabilizes to a steady average value, but with a slightly widened standard deviation, indicating that there is indeed more variation in measurements on the higher time scale. Within the general evaluation, the data suggests a decline in the mean diameter in the initial two-hour period, followed by a sustained size, but variability grows after 24 hours. Non-Menstrual Bloodstain Diameter Trends The diameter of non-menstrual blood at different times after being deposited, including the average diameter (in centimeters) and standard deviation (SD). The measurements were taken at 10 min., 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr. At 10 minutes and an hour, the average diameter was 8.67 cm, and the standard deviation was 0.764, which shows a relatively good initial size of the design with small variability. Mean diameter drops a bit to 8.50 cm at 2 hours with a wider standard deviation of 0.866, indicating a slight decrease in size and that it is increasing somewhat in variability. At 24 hours, the average value reduces to 8.33 cm with a smaller standard deviation of 0.577, which means the process still diminishes with less variation. At 72 hours, the mean diameter fell further to 8.17 cm, and the standard deviation dropped by 0.289, which shows further decrease and even lesser variance. Lastly, when the time is 120 hours, the mean diameter value stands at 8.00 cm, and the standard deviation is 0.000, implying that it has a gradual decrease. Still, because the standard deviation is zero, it is the same diameter with no variability. The data demonstrate a gradual reduction in the mean diameter of non-menstrual blood samples as time increases, as the variability reduces and reaches stability after 120 hours. Comparative Analysis The menstrual bloodstain diameter trend and the non-menstrual one are quite different in the trend of their diameters during the time of their deposition in which statistics is based on average (in centimeters) and the standard deviation (SD) at time intervals of 10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours, of menstrual blood. At the beginning, 10 minutes and 1 hour, the mean diameter of menstrual blood is greater than 8.83 cm (SD 1.258) than the non-menstrual blood of 8.67 cm (SD 0.764), which means that the size of this menstrual blood is larger with more variability. Both menstrual and non-menstrual are reduced slightly later at 2 hours, menstrual to 8.50 cm (SD 0.866) and non-menstrual to 8.50 cm (SD 0.866), to the same extent with the same variability, raising the possibility of an early decline in both of similar magnitude. Menstrual blood settles on 8.33 cm (SD 1.155) after 24 hours, and this remains its mean 120 hours later and up to 168 hours, with the SD becoming progressively larger with time. Conversely, the non-menstrual blood keeps on reducing steadily as it registers 8.33 cm (SD 0.577) at 24 hours, 8.17 cm (SD 0.289) at 72 hours, and 8.00 cm (SD 0.000) at 120 hours, indicating a steady decline and its eventual evenness. This shows that although both the blood types get an initial decrease in diameter during the first two hours, menstrual blood is stationary after the first 24 hours, and more variable, non-menstrual blood, on the other hand, takes a long period to reduce in diameter and attains a homogenous size in 120 hours. In general, the diameter of menstrual blood is somewhat greater and more varied over time, whereas the non-menstrual blood has a more accentuated and stable decline. The difference observed is probably due to the differences in blood types' biochemical and physical characteristics. The menstrual blood with its endometrial tissue, mucus, and lower hematocrit may have a more viscous or gelatinous matrix that does not contract upon drying. By contrast, non-menstrual blood is likely to coagulate and evaporate further, mainly due to high contents of plasma, red blood cells, and clotting factors. The results here agree with the previous research findings that indicate that the menstrual bloodstains bear distinct morphological properties that one can tap into in forensic distinction because of their sophisticated state. The above line chart is titled "Diameter of Menstrual and Non-Menstrual Bloodstains Over Time," and measurements were taken using a stereo microscope at 0.65x magnification and an ABFO scale. The x-axis represents the time since deposition (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr), while the y-axis shows the bloodstain diameter in centimeters, ranging from 6.5 cm to 10.5 cm. The chart includes three reference samples for menstrual blood (plotted in blue shades) and non-menstrual blood (plotted in red shades). All samples start with diameters around 9.5 cm to 10.0 cm at 10 min. Over time, menstrual bloodstains (Ref 1, Ref 2, Ref 3) gradually decline, stabilizing around 9.0 cm to 9.5 cm after 24 hr, with minor fluctuations. Non-menstrual bloodstains (Ref 1, Ref 2, Ref 3) exhibit a more pronounced decrease, dropping to approximately 8.0 cm to 8.5 cm by 120 hr. The above line chart titled "Average Diameter of Bloodstains Over Time (cm)" illustrates the mean diameter of menstrual and non-menstrual bloodstains across various time intervals, with the x-axis representing time points (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr) and the y-axis indicating the mean diameter in centimeters, ranging from approximately 7.6 cm to 8.9 cm. The red line, representing menstrual blood, begins at 8.83 cm at 10 min, remains stable at 8.83 cm at 1 hr, then gradually declines to 8.50 cm at 2 hr, 8.33 cm at 24 hr, and stabilizes at 8.33 cm from 72 hr to 120 hr. In contrast, the blue line, representing non-menstrual blood, starts at 8.67 cm at 10 min, holds steady at 8.67 cm at 1 hr, then decreases to 8.50 cm at 2 hr, 8.33 cm at 24 hr, 8.17 cm at 72 hr, and reaches 8.00 cm at 120 hr. The chart highlights that while both blood types start with similar diameters, non-menstrual bloodstains exhibit a more pronounced decrease over time, particularly after 24 hr. In contrast, menstrual bloodstains show greater stability after an initial reduction. Discussion The present study, describing the temporal changes of menstrual and non-menstrual bloodstains on diameters under controlled laboratory conditions, where they used a stereo microscope in 0.65x magnification and ABFO scale, can be considered as a source of a breakthrough in the field of forensic bloodstain pattern analysis (BPA), especially about the differences between blood types within specific periods, both more than 10 min and up to 120 hr (Roy et al., 2022). The study becomes even more important when put in the context of forensic issues described in the modern literature, according to which bloodstains are one of the most valuable pieces of evidence when it comes to recreating a crime scene, particularly after a sexual assault or case of trauma (Bevel et al., 2008, James et al., 2014 ). The atmosphere of a dim forensic laboratory setting involving a scientist examining a rust-colored material stain on cloth shows the importance of properly investigating such evidence regarding the issues of consent, injury, or violence (Holtkötter et al., 2018 ). Conventional BPA that uses size, shape, and distribution to indicate impact angles and force can prove ineffective in distinguishing between menstrual and peripheral blood, a distinction critical to legal preferences that can suggest consent in the former, and trauma in the latter (Al-Alimi et al., 2025; Pradeep et al., 2024 ). This study's controlled approach, maintaining 22 ± 1°C and 50 ± 5% humidity on glossy paper, shows that the precision and reproducibility of the diameter measurements are evident, which increases the validity of forensic interpretations. The results indicate that the mean diameter of menstrual blood is 8.83 cm (SD 1.258) at 10 minutes and 1 hour, which reduces to 8.50 cm (SD 0.866) at 2 hours and stays constant at 8.33 cm (SD 1.155) between 24 and 168 hours, showing early reduction followed by deviation at later stages. Non-menstrual blood, initially at 8.67 cm (SD 0.764), gradually reduces to 8.50 cm (SD 0.866) after 2 hours, 8.33 cm (SD 0.577) after 24 hours, 8.17 cm (SD 0.289) after 72 hours, and 8.00 cm (SD 0.000) after 120 hours, which is suggestive of one step toward uniformity. Such time variance, corroborated by three measurements as estimated and graphically displayed as a line graph in Table 5 , provides a strong and reliable body of data that is more than the conventional tests, such as Teichmann or luminol, that only indicate the presence of blood but not the source of that blood. The analysis of the glossy paper as a semi-porous material used in the study reflects the real scene of forensic surfaces (e.g., paper, packaging). It offers a practical model of the field application. These findings are given significance by the issues of the surface types analysis, where surface material affects morphology and chemistry of bloodstains (porous (cotton) or non-porous (tile) surfaces modify the bloodstains), making them harder to distinguish. The viscous structure of menstrual blood, filled with endometrial mucus and a lower hematocrit, prevents drying contraction. In contrast, the high plasma level and clotting factors in non-menstrual blood cause greater drying evaporation and coagulation (Wang et al., 2022 ). It aligns with new imaging methods, such as hyperspectral imaging (HSI) and Raman spectroscopy, which are capable of non-destructively identifying blood types on spectral or molecular signatures (Zulfiqar et al., 2021 ; Mistek et al., 2016 ). The capacity of HSI to chart spectral variances owing to hemoglobin oxygenation and endometrial debris with virtually 97–100 percent precision through machine learning on the HyperBlood digital set, adds to the present study optical variant (Al-Alimi et al., 2025; Li et al., 2025). Likewise, Raman spectroscopy offers molecular specificity with 100% sensitivity and specificity with the assistance of PLSDA. However, both are susceptible to interference by the surface, and this study reduces these challenges under controlled conditions (Mistek et al., 2016 ). The SERATEC PMB test has a sensitivity of 100 percent and a specificity of 99.7 percent in detecting the presence of hemoglobin and D-dimers in menstrual blood. Though it is fast in screening, it requires the extraction of the sample and hence cannot be used on old or porous stains (Konrad et al., 2024 ). The imaging combined with the non-destructive method of this study, which leaves samples intact to study afterward using DNA analysis, fills this gap, working together with other methods, imaging, and immunological ones, into a multimodal approach to forensics (Edelman et al., 2012 ). The social benefits are enormous, since correct distinction can support the account of victims of sexual assault, vitality of justice, in particular, when the stakes are high, because misidentification can change the investigation (Chunkul et al., 2025 ). Limitations and Considerations The glossy paper of the study does not allow generalisation to the wide range of forensic material surfaces (e.g., non-porous tiles or porous fabrics), which can affect the morphology of stains. Invariable, the crime scene environment is not always represented as controlled conditions (temperature, humidity), which may influence drying speed. The particle morphology (i.e., shape, edge characteristics, oxygen levels, morphology of individuals) was ignored in favor of diameter. The broader applicability may be lower because no account of individual variability in blood composition (e.g., menstrual blood and endometrial content) was taken. Aged stains may not be punishable within the 120-hour temporal terrene, and accessibility may be limited with specialized equipment (0.65x stereo microscope, ABFO scale). The test of different substrates, environmental conditions, and further stain parameters should be carried out to increase the practical usefulness of the study. Conclusion The present study establishes a scientifically solid approach to the forensic evidence of bloodstain patterns because the desired temporal changes in diameters of menstrual and non-menstrual bloodstains were determined under the controlled conditions (22 ± 1°C, 50 ± 5% humidity, glossy paper substrate). The statistics show that the menstrual bloodstains at the time scale of 10 minutes and 1 hour have an average diameter of 8.83 cm (SD 1.258), which decreases at the scale of 1 hour and 2 hours (8.50 cm, SD 0.866), and reaches 8.33 cm (SD 1.155) between 24–168 hours with consistency in size, yet partially showing larger diameter variation. Conversely, non-menstrual bloodstains, ranging between 8.67 cm (SD 0.764), 8.50 cm (SD 0.866), 8.33 cm (SD 0.577), 8.17 cm (SD 0.289), and 8.00 cm (SD 0.000) at 2, 24, 72, and 120 hours, respectively, exhibit a monotonic decrease as the standard deviation reduces. All of these different patterns are accurately measured under a 0.65x stereo microscope and correspond to the ABFO scale and are probably due to biochemical variability; both the viscous mucus-laden matrix of menstrual blood does not tend to dry-contraction, whereas non-menstrual blood, due to the increased plasma and coagulation factors, tends to blood clot and dry. It is a non-destructive method that supplements the detailed forensic methods (i.e., hyperspectral imaging, Raman spectroscopy, and SERATEC PMB test) and aids in the identification of the origin of blood sources, which is so vital in judicial matters about crime scene reconstruction. Although the substrate and environmental scope of study are limited, replicable methodology and statistically significant results (the results are based on three measurements per time point) present the baseline on which further research of forensic BPA should be built, which can lead to better court outcomes in cases where the bloodstain evidence is used. Abbreviations BPA : Bloodstain Pattern Analysis ABFO : American Board of Forensic Odontology Declarations Human Ethics and Consent to Participate: This study was conducted in accordance with the Declaration of Helsinki. The Institutional Ethics Committee (IEC) of Kasturba Medical College, Mangalore, approved the study (Approval Reference: IECKMCMLR 07/2025/485). All participants provided written informed consent prior to participation. Participants were fully informed about the study’s purpose, procedures, potential risks, and their rights, including the right to withdraw at any time without consequence. Ethical principles were strictly adhered to during blood sample collection and handling. Consent to Participate: All donors provided written informed consent before participating in the study. Each participant was thoroughly informed about the study’s objectives, the procedures involved, the use of their biological samples (menstrual and peripheral blood), and their rights as participants. Consent forms were signed voluntarily, and participants were assured of confidentiality and the secure handling of their samples. Funding: Not applicable. Clinical Trial Number: Not applicable. Supplementary Files: No Gels or Blots were used in this study, as the research focused on morphological analysis of bloodstains using stereo microscopy and photographic documentation. All relevant data are presented in the manuscript tables and figures. References Al-Alimi D, Al-Qaness MAA (2025) Enhancing forensic blood detection using hyperspectral imaging and advanced preprocessing techniques. Talanta 283:127097. 10.1016/j.talanta.2024.127097 Atkins CG, Buckley K, Blades MW, Turner RFB (2017) Raman spectroscopy of blood and blood components. Appl Spectrosc 71(5):767–793. 10.1177/0003702816686593 Baker DJ, Grimes EA, Hopwood AJ (2011) D-dimer assays for the identification of menstrual blood. Forensic Sci Int 212(1–3):210–214. 10.1016/j.forsciint.2011.06.013 Bevel T, Gardner MR (2008) Bloodstain pattern analysis with an introduction to crime scene reconstruction. Bloodstain pattern analysis with an introduction to crime scene reconstruction, 3rd edn. CRC: Taylor & Francis Group;, pp 319–343 Cadd S, Li B, Beveridge P, O’Hare WT, Campbell A, Islam M (2016) The non-contact detection and identification of blood stained fingerprints using visible wavelength reflectance hyperspectral imaging: Part 1. Sci Justice 56(3):181–190. 10.1016/j.scijus.2016.01.004 Chunkul S, Sathirapatya T, Dangklao P, Kawicha P, Tammachote R, Vongpaisarnsin K (2025) Enhancing the forensic sexual assault investigations with LAMP-based male DNA detection. Forensic Sci Int Synergy 10:100567. 10.1016/j.fsisyn.2024.100567 Edelman G, Manti V, Van Ruth SM, Van Leeuwen T, Aalders M (2012) Identification and age estimation of blood stains on colored backgrounds by near-infrared spectroscopy. Forensic Sci Int 220(1–3):239–244. 10.1016/j.forsciint.2012.03.009 Fu J, Allen RW (2019) A method to estimate the age of bloodstains using quantitative PCR. Forensic Sci International: Genet 39:103–108. 10.1016/j.fsigen.2018.12.004 Hanson E, Mirza M, Rekab K, Ballantyne J (2014) The identification of menstrual blood in forensic samples by logistic regression modeling of miRNA expression. Electrophoresis 35:21–22. 10.1002/elps.201400171 Holtkötter H, Dias Filho CR, Schwender K, Stadler C, Vennemann M, Pacheco AC et al (2018) Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault-validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer. Int J Legal Med 132(3):683–690. 10.1007/s00414-017-1719-y James SH, Nordby JJ, Bell S (2014) Forensic science: An introduction to scientific and investigative techniques. Forensic science: An introduction to scientific and investigative techniques, 4th edn. CRC: Taylor & Francis Group;, pp 67–89 Kadam P, Patil N, Mane VP (2023) Study of refrigerated storage of blood at 4°C on automated hematological parameters & morphological changes in peripheral blood smear: A prospective study. Indian J Pathol Oncol 10(1):9–14. 10.18231/j.ijpo.2023.002 Konrad H, Hartung B, Poetsch M (2024) (Un)Reliable detection of menstrual blood in forensic casework — evaluation of the Seratec® PMB test with mock samples. Int J Legal Med 138:781–786. 10.1007/s00414-023-03138-3 Li Y, Shen W (2025) From images to detection: Machine learning for blood pattern classification. Forensic Sci Int 375:112558. 10.1016/j.forsciint.2025.112558 Mistek E, Halámková L, Doty KC, Muro CK, Lednev IK (2016) Race differentiation by Raman spectroscopy of a bloodstain for forensic purposes. Anal Chem 88(15):7453–7456. 10.1021/acs.analchem.6b01173 Newton M (2013) The forensic aspects of sexual violence. Best Pract Res Clin Obstet Gynaecol 27(1):77–90. 10.1016/j.bpobgyn.2012.08.020 Nirupama GS (2024) Advances in forensic bloodstain pattern analysis: a review of current methods and future directions. Int J Innov Res Technol 11(5):2366 Pozrikidis C (2006) Flipping of an adherent blood platelet over a substrate. J Fluid Mech 568:161–172. 10.1017/S0022112006002356 Pradeep AS, Babu J, Sandana JS, Deivalakshmi S (2024) Innovations in forensic science: Comprehensive review of hyperspectral imaging for bodily fluid analysis. Forensic Sci Int 364:112227. 10.1016/j.forsciint.2024.112227 Rajkumari S (2024) Investigation of strain patterns from diverse blood samples on various surfaces. J Forensic Sci Res 8(1):28–34. 10.12691/jfsr-8-1-3 Rawat S, Kushwaha K (2015) Estimation of age of bloodstain spotted on different surfaces using UV-visible spectroscopy. Indian Internet J Forensic Med Toxicol 13(2):25–30. 10.5958/0974-4487.2015.00006.9 Roy BJSTRA (2022) Use Scientific Method to Detection and Comparison of Menstrual Blood Samples Found at the Crime Scene. BJSTR 41:006599. 10.26717/BJSTR.2022.41.006599 Walker KA, Rudd TK, Vignola JN et al (2025) Evaluation of dried blood spot sampling for verification of exposure to chemical threat agents. Forensic Toxicol 43:280–293. 10.1007/s11419-025-00721-8 Wang G, Wang Z, Wei S, Wang D, Ji A, Zhang W et al (2022) A new strategy for distinguishing menstrual blood from peripheral blood by the miR-451a/miR-21-5p ratio. Forensic Sci Int Genet 57:102654. 10.1016/j.fsigen.2021.102654 Wei CT, You JL, Weng SK, Jian SY, Lee JC, Chiang TL (2024) Enhancing forensic investigations: Identifying bloodstains on various substrates through ATR-FTIR spectroscopy combined with machine learning algorithms. Spectrochim Acta Mol Biomol Spectrosc 308:123755. 10.1016/j.saa.2023.123755 Zulfiqar M, Ahmad M, Sohaib A, Mazzara M, Distefano S (2021) Hyperspectral imaging for bloodstain identification. Sensors 21(9):3045. 10.3390/s21093045 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TableMNNMB.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7177372","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":497301520,"identity":"cf9a5533-5bcc-440f-a26e-ab5106654d31","order_by":0,"name":"Mayur Sudhir Balbudhe","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Mayur","middleName":"Sudhir","lastName":"Balbudhe","suffix":""},{"id":497301521,"identity":"2733a862-880a-45a0-8e74-405b2d5b459f","order_by":1,"name":"B Suresh Kumar Shetty","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie3OsUoDMRjA8S8e9Jam80Ep9wopAetQvVfJEYiTeweHQiEuomsLxWfIVOzWErDLietBBlsKTg7XrTdpvBa7mOrokD8kOcL9yAfg8/3XUB+AVF89uwK7lrvL30hgj2xP2J8JkvubYyQeXq1W5SMknfB+scYPk7gT4nnBoNtSs58JyS8pxRmk01uNBnhi2tNBg0cMBHWSSNSadh5Gcl4RpHSdWKJTF4mHIixLCQl5XVsyNokldMvgw0kgFzXAEpDKA0v6JrXk1L4ycxKSvQVNLKNUZbw9Gj8ZrnRDnDHC6cg12I1Am1J2E7KYL4v3a3OuXp51XvQuWneuwXZF1X5Sr46vnRz9/RDafhOfz+fzHfoER0diR5UttEIAAAAASUVORK5CYII=","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":true,"prefix":"","firstName":"B","middleName":"Suresh Kumar","lastName":"Shetty","suffix":""},{"id":497301522,"identity":"3c1dbf68-c8b7-4155-8f94-ae55a1981973","order_by":2,"name":"Adithi Shetty","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Adithi","middleName":"","lastName":"Shetty","suffix":""},{"id":497301523,"identity":"e90df667-8f71-4e54-8f96-7d4babb42a4b","order_by":3,"name":"Nayanatara Arun Kumar","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Nayanatara","middleName":"Arun","lastName":"Kumar","suffix":""},{"id":497301524,"identity":"536fce21-a572-4ad0-9a53-b380c4759352","order_by":4,"name":"Shankar M Bakkannavar","email":"","orcid":"","institution":"Kasturba Medical College Manipal, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Shankar","middleName":"M","lastName":"Bakkannavar","suffix":""}],"badges":[],"createdAt":"2025-07-21 12:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7177372/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7177372/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88644312,"identity":"7b349898-feeb-4f82-b26b-ae6ecd2ea9f9","added_by":"auto","created_at":"2025-08-08 16:20:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53707,"visible":true,"origin":"","legend":"\u003cp\u003eThe above line chart is titled \"Diameter of Menstrual and Non-Menstrual Bloodstains Over Time,\" and measurements were taken using a stereo microscope at 0.65x magnification and an ABFO scale. The x-axis represents the time since deposition (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr), while the y-axis shows the bloodstain diameter in centimeters, ranging from 6.5 cm to 10.5 cm. The chart includes three reference samples for menstrual blood (plotted in blue shades) and non-menstrual blood (plotted in red shades). All samples start with diameters around 9.5 cm to 10.0 cm at 10 min. Over time, menstrual bloodstains (Ref 1, Ref 2, Ref 3) gradually decline, stabilizing around 9.0 cm to 9.5 cm after 24 hr, with minor fluctuations. Non-menstrual bloodstains (Ref 1, Ref 2, Ref 3) exhibit a more pronounced decrease, dropping to approximately 8.0 cm to 8.5 cm by 120 hr.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7177372/v1/f6fce29112e9a05cee291078.png"},{"id":88642526,"identity":"97e9dfc5-5891-4a40-9c9f-932c16067de9","added_by":"auto","created_at":"2025-08-08 16:12:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38842,"visible":true,"origin":"","legend":"\u003cp\u003eThe above line chart titled \"Average Diameter of Bloodstains Over Time (cm)\" illustrates the mean diameter of menstrual and non-menstrual bloodstains across various time intervals, with the x-axis representing time points (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr) and the y-axis indicating the mean diameter in centimeters, ranging from approximately 7.6 cm to 8.9 cm. The red line, representing menstrual blood, begins at 8.83 cm at 10 min, remains stable at 8.83 cm at 1 hr, then gradually declines to 8.50 cm at 2 hr, 8.33 cm at 24 hr, and stabilizes at 8.33 cm from 72 hr to 120 hr. In contrast, the blue line, representing non-menstrual blood, starts at 8.67 cm at 10 min, holds steady at 8.67 cm at 1 hr, then decreases to 8.50 cm at 2 hr, 8.33 cm at 24 hr, 8.17 cm at 72 hr, and reaches 8.00 cm at 120 hr. The chart highlights that while both blood types start with similar diameters, non-menstrual bloodstains exhibit a more pronounced decrease over time, particularly after 24 hr. In contrast, menstrual bloodstains show greater stability after an initial reduction.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7177372/v1/4b3eb01786daa229834a132a.png"},{"id":89256268,"identity":"8303b91c-ff51-400a-baea-12a63ab9f8e4","added_by":"auto","created_at":"2025-08-18 05:39:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":754650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7177372/v1/51eee5d5-5095-479f-8779-370545b4a98e.pdf"},{"id":88645656,"identity":"f847840d-f691-45f5-9ce8-ef951ec8f283","added_by":"auto","created_at":"2025-08-08 16:28:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3770939,"visible":true,"origin":"","legend":"","description":"","filename":"TableMNNMB.docx","url":"https://assets-eu.researchsquare.com/files/rs-7177372/v1/4cf4095b4301c45a33b85533.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Surface Specific Analysis - A Comparative Study of Menstrual and Non-Menstrual Bloodstains","fulltext":[{"header":"Background","content":"\u003cp\u003eIn a dimly lit, fluorescent-lit forensic laboratory, a forensic scientist scrunches their brow over the microscope in deep concentration. The bloodstain might carry answers to the questions of a crime: on the table, there is a piece of fabric with a dark stain of rust color. Possible wound stain or a natural biological process, this stain has the potential to address the questions of consent, trauma, or violence (Roy et al., 2022). Bloodstains are the tell-tale in forensic science, and they tell the secrets or hints about what happens at a crime scene (Bevel et al., 2008). The difference between menstrual blood, which is spontaneously lost by the inner surface of the uterus, and peripheral blood, which results from an accident, is a decisive problem that may determine the outcome of the investigation effort (Holtkötter et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The surface on which the blood exists, cotton, wood, or tile, makes this puzzle complex (Al-Alimi et al., 2025). With the emerging refined visualization processes, a breakthrough will enable the decryption of these crimson hints to become even more accurate (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne of the most usual and convincing pieces of evidence is the bloodstains (James et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Blood, being a viscous substance having specific physical characteristics, leaves an impression which indicates how a violent incident of stab, bullet, or traumatic blow has taken place (Bevel et al., 2008). To recreate what took place and to analyze how something happened, forensic scientists can turn to bloodstain pattern analysis (BPA), which assesses the size, shape, and distribution of blood stains to tell the angle of impact and amount of force used, as well as the correlation of movements of individuals (Bevel et al., 2008). Nevertheless, the BPA does not always identify the blood type, whether it is peripheral or menstrual, a factor that is critical in cases where incidences of sexual assault are involved (Newton et al., 2013). Menstrual blood may imply permission had been given to such cases, whereas peripheral blood may indicate injury (Hanson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Much lies at stake in this case, yet differentiation must be precise to achieve justice (Chunkul et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eImagine a crime scene: a bedroom, a messed-up bed, and some blood on the sheets of it, a series of blood droplets leading to a door. The job of the investigator is to reconstruct the story. Was this some violent attack, or could it be blood due to a woman's monthly period? Conventional forensic tests, i.e., the Teichmann or luminol-based tests, exclude the possibility of blood but have difficulty distinguishing between menstrual and peripheral blood (Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Menstrual blood is a complicated conglomeration that incorporates red blood cells, white blood cells, plasma, vaginal secretions, cervical mucus, and endometrial tissue (Hanson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In contrast to peripheral blood, which is clotted through fibrinogen, menstrual blood causes fibrinolysis, or breaking of the clots, thus leaving behind a liquid form containing fibrin degradation products such as D-dimers (Baker et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These minor variances are critical and need complex detection means (Mistek et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A surface on which a blood stain is deposited further adds to the complexities of analysis (Rajkumari et al., 2024). On a non-porous surface, such as tile, a drop of blood becomes a circular, highly defined stain; on a porous surface, such as cotton, blood spreads out and diffuses, morphologically and chemically changing its appearance (Rawat et al., 2015). These surface-specific variations are hard to explain in terms of conventional approaches, i.e., with the help of microscopy or electrophoresis of lactate dehydrogenase isozymes (Rawat et al., 2015). More elaborate molecular techniques, such as mRNA or miRNA profiling, need laboratory conditions and special training, so they are unsuitable for real-time analysis requiring rapid, on-site testing (Hanson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Information about the sensitive and surface-adaptable method of analysing bloodstains that would be applicable non-destructively in the forensics community has been sought for a long time (Mistek et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe imaging techniques come to the scene to provide a fresh chapter in the blood stain analysis in forensics. Non-destructive imaging techniques contrast with chemical/molecular techniques; however, such methods leave the sample intact to allow post-imaging DNA analysis, an essential factor when investigating a crime (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). One of the most promising techniques is hyperspectral imaging (HSI), which refers to the possibility of obtaining the spectral signature of a substance over a great distance of the spectrum, both visible and near infrared (Al-Alimi et al., 2025). An HSI image has a spectrum in each pixel, which is a fingerprint in itself and tells about the chemical content of the material (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Blood, having hemoglobin and other molecular components of blood, has characteristic spectral patterns, which are different in the peripheral blood and menstrual blood due to differences in the oxygenation and iron levels or endometrial debris (Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). HSI is sensitive to finding out these differences, and this presents a non-invasive method to study blood stains (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eImagine the forensic scientist again, but this time with an HSI camera. They will be able to scan the blood-stained fabric, and within minutes, the system will come up with a spectral map that shows minute differences in the composition of a stain (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Compared to menstrual blood, which is less oxygenated and darker, hemoglobin in the peripheral blood has an altered spectral pattern, which is a nice and bright red (Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ideally, using these spectra, the scientist can prove that there is blood, and the source of this blood can be ascertained (Walker et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). HSI can also consider the effect brought by the underlying surface (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Bloodstain analysis on cotton might absorb differently from a bloodstain on tile. Still, with the precision that HSI offers in separating the spectral signature of a stain and that of its substrate, analysis can be done item by item (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This factor renders HSI a game changer as it allows the investigator to analyze any stains on site and does not tamper with them with any chemicals (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRaman spectroscopy is another promising method, in which inelastic scattering of light is used to interrogate the molecular structure of a sample (Atkins et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Spectrum of Raman menstrual and peripheral blood reveals no difference between the results due to the hemoglobin levels, protein contents, and biochemical indicators involved (Atkins et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Combined with statistical techniques such as partial least squares discriminant analysis (PLSDA), Raman spectroscopy has reached 100% sensitivity and specificity in assaying blood type, even when applied to low-contrast materials such as fabric or carpet (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast to HSI, which is highly effective in broad-spectral analysis, Raman spectroscopy has a high degree of molecular specificity. It is therefore described as the most appropriate method to identify subtle biochemical differences (Fu et al., 2019). They are both non-destructive and fast, making them convenient in laboratory and field analysis.\u003c/p\u003e\u003cp\u003eForensic science has the support of advanced imaging techniques, which help in solving cases, but these are not the only tools. The immunochromatographic assay- SERATEC PMB test helps distinguish between menstrual and peripheral blood (Holtkötter et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Due to their character as a fibrinolytic factor, human hemoglobin and D-dimers are detected in this test and indicate menstrual blood (Baker et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). SERATEC PMB test has a duplex design that simultaneously confirms the presence of blood and detects menstrual fluid, which makes it useful in quick screening during crime scenes (Konrad et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nonetheless, the SERATEC PMB test involves the extraction of samples. Depending on the age of stains or climate conditions, it may be challenging to do on porous material (Wei et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Imaging methods can be used; however, these provide a non-destructive, spatially and chemically intact approach to evidence (Wei et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When imaging procedures are added to the SERATEC PMB test, they enable a multi-modal approach, where one enhances the other and nullifies their shortcoming (Konrad et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is impossible to speak about bloodstain analysis without describing the surface. When a drop of blood sits on a smooth, non-absorbent surface (such as glass), the edges of the drop are sharp, and the drop takes the shape of a ball (Pozrikidis, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The blood is, however, wicked in the fibers upon a porous surface such as cotton and forms a diffuse, irregular stain (Rajkumari et al., 2024). These effects, which are surface-specific, change the physical look and spectral or chemical character of the stain (Atkins et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As an example, the absorption of blood into a fabric can cover some spectral peaks during HSI. Simultaneously, Raman spectroscopy can be disrupted by the chemical compositions of a surface, i.e., by dyes on clothing (Mistek et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Coming up with a method that considers these variables is not easy, and imaging has a unique capacity to address it (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe surface-specific analysis has been transformed using the HyperBlood dataset, a collection of hyperspectral images of bloodstains on various surfaces (Al-Alimi et al., 2025; Li et al., 2025). Research based on this dataset indicates that HSI and machine learning techniques, such as Multi-Layer Perceptron (MLP) networks, have been used to reach 97–100 percent accuracy in detecting bloodstain and compensating substrate effects (Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These algorithms are trained to understand the spectral signature of blood instead of the underlying surface material to robustly process various materials such as cotton, wood, and tile. Comparably, Raman spectroscopy, using PLSDA, successfully extracts the molecular signal of menstrual blood, despite the interference of a confounding factor such as dye in fabrics or environmental impurities (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe research examines how the menstrual and non-mensural bloodstain patterns change over a period when temperature is regulated and the samples are observed through controlled environmental conditions with the help of a stereo microscope at various magnifications with and without an ABFO scale. This study will follow the bloodstains' diameter and structural modifications within 10 minutes to 120 hours after deposition. The controlled environment with a stable temperature and humidity is meant to measure the possible differences in the coagulation and drying dynamics between the two types of blood in the case of glossy surfaces.\u003c/p\u003e"},{"header":"Materials and Methodology","content":"\u003cp\u003eDifferentiation of menstrual and peripheral bloodstains on the glossy surfaces of paper was done with the help of a Stereo Microscope. Controlled deposition of blood droplets, morphological analysis in a serial manner using stereo microscopy, and the use of a Nikon DSLR 750 Camera to provide surface-specific identification of bloodstain origin that was non-destructive, were used in the experimental design. The study\u0026apos;s objective was to test the effectiveness of optical measures in determining blood types, considering that glossy paper has semi-porous qualities. Several trials were taken to guarantee reproducibility, and statistics were calculated to assess the consistency of morphological variations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiological Samples\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe blood samples were collected at Lady Goschen Hospital in Mangalore and from healthy individuals by performing venipuncture, after the permission of the Institutional Ethics Committee (IEC) and the informed consent of the donors. The peripheral blood was harvested in EDTA vacutainer tubes and kept at 4\u0026deg;C to avoid its breakdown (Kadam et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Menstrual blood samples were collected on ethical grounds in the early days of the menstrual cycle of the consenting female volunteers. They were tested in a 4\u0026deg;C environment in uncoated vials with EDTA, and it was sufficient to be used within 24 hours without causing any variations. The sample volumes were standardized to 30 \u0026micro;L per drop to ensure uniformity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurfaces\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eAs a semi-porous substrate model, the glossy paper (30 cm x 30 cm, 120 gsm, chemically coated with a polymer-polymer gloss finish) was used, which represents objects commonly met in forensic situations, like documents or packages. Glossy paper was wiped using 70% ethanol, deionized water, and dried using air to eliminate contaminants and leave it in perfect condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEquipment\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eA micro drip infusion set (MicroFusion, 0.1 mL precision) was applied to controlled blood droplet delivery. The images were analyzed using a DSLR (Nikon 750). The morphological analysis was made possible using the stereo microscope (Euromex CMEX-5PRO, 0.65, 1x and 2x magnification) with Image Focus Alpha Software. Other materials were a laboratory stand, a clamp, and an ABFO scale.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Procedures\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Surfaces\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe surfaces of glossy papers were wiped with 70% ethanol, subsequently wiped with deionized water, and dried and air-dried for 30 min to remove any leftover contaminants. Vigilance was checked using UV light (365 nm) to ensure how clean surfaces were and how the shiny layer held up. The adhesive mounts were used to fix each sheet on a flat vibration-dampened bench to eliminate movement of the sheets during the deposition of blood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSetup of Blood Vial\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe vials filled with peripheral or menstrual blood were positioned on a laboratory stand and clamped 55 cm above the smooth paper surface. The effects were tested in a controlled setting where the temperature was kept at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, the humidity was between 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and LED lighting of 5000K was used to avoid changes in natural light. The measuring tape was used to measure the height, and each time, the drop point was matched to the target grid\u0026apos;s center to ensure reproducibility. To ensure the stability of the vial, this was made to achieve a minimum of the vial movement laterally as the droplets were released. The height of 55 cm was chosen to represent the conditions of forensic blood drip (i.e., dripping blood out of a wound or menstrual flow).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDropping the Blood\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eTo obtain a single drop of 30 \u0026micro;L of blood onto the glossy paper surface, it was done using a micro drip infusion set connected to the blood vial, and without exerting any force other than a free-fall. To every blood type (menstrual and peripheral), three drops should be placed on individual glossy paper sheets, and thus, there were six drops in total (3 drops x 2 blood types). To exclude cross-contamination, the infusion set was washed with phosphate-buffered saline (PBS) and sterilized between trials. The conditions of the environment were standardized (temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C; humidity: 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) to reduce the impact of extra tissue factors on the formation of bloodstains. The average diameters of bloodstains on glossy surfaces were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological Observation and Documentation\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eA stereo microscope with a (0.65x, 1x, and 2x) magnification, Euromex CMEX-5PRO, was used to study the bloodstain morphology at the point of impact. The first measurements were taken 10 minutes after the impact, and time-series measurements of 1, 2, 24, 72, and 120 hours were taken to reflect the changes over time. Images were captured under Image Focus Alpha Software and analyzed and measured concerning stain diameter, circularity, and edge properties. At each time point, measurements were done thrice to make it reliable. The smooth, coated surface of the glossy paper left bulky bloodstains with clear boundaries and little diffusion, as in the case of the latter substrates.\u003c/p\u003e\n\u003cp\u003eThe bloodstains (menstrual ones and the rest) are depicted in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, measured at separate time points after they have been deposited: 10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours. The aim of monitoring such measurements is to determine the variation of the diameter of the stains with time to understand the nature of menstrual and non-menstrual bloodstain behaviour and consistency of bloodstains with time.\u003c/p\u003e\n\u003cp\u003eThe detailed comparison of the menstrual and non-menstrual bloodstain images viewed using a stereo microscope at a magnification of 1x is represented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Observations were made at six instances after deposition (10 min, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours) to record temporal morphological variables on glossy paper, a semi-porous substrate. The discussion is based on the visual characteristics, diameter, edge properties, texture, and color of three reference samples of each blood type, emphasizing the differences caused by biochemical and physical characteristics of samples.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e presents the comparison of the images of menstrual and non-menstrual bloodstains looked through a stereo microscope at the magnification of 0.65x, comparing the time intervals (10 minutes, 1, 2, 24, 72, and 120 hours) with the help of an ABFO scale. The most important observation that can be made is that the non-menstrual bloodstains exhibit a successive loss of diameter in these time intervals, which appears as a consistent, predictable loss of size as they dry and shrink. The changes in diameter with time are probably less regular in menstrual bloodstains because of the complexity of their composition (blood, endometrial tissue, mucus). This is because the ABFO scale guarantees high levels of accuracy in measuring it. As time goes by, the microscope shows specific differences in visual appearance, e.g., the texture of the stain or edges.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e gives a comparison of the side profiles (the edge aspects, thickness, or cross-sectional morphology) between menstrual and non-menstrual blood stains viewed using a stereo microscope at 2x magnification after specified intervals of time (24 hours, 72 hours, and 120 hours as in earlier tables). The emphasis is put on changing these parallel side profiles with time, giving an idea of the physical and structural development of the bloodstains.\u003c/p\u003e\n\u003cp\u003eThe visual comparison of menstrual and non-menstrual bloodstains is provided in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e: bloodstains were photographed with a Nikon 750 camera and an ABFO scale to provide proper size reference, at different time points (10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours). These high-resolution images are used to document alterations in stain as well as in diameter, color, texture, and edge perception to bring variations in how these blood types change with time to notice.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e represents the average circular diameter of non-menstrual and menstrual bloodstains measured in centimeters at various times after deposition (10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours). The data summarizes how stain size evolves, highlighting differences between menstrual and non-menstrual blood. This builds on Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026rsquo;s observation that non-menstrual bloodstains decrease consecutively in diameter, indicating consistent behavior, while menstrual bloodstains likely show more variability.\u003c/p\u003e\n\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAverage Diameter of Menstrual and Non-Menstrual Bloodstains Across Time Intervals in Centimetres\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eType of Sample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime Interval\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eMenstrual Blood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 Min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.258\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.258\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eNon-Menstrual Blood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 Min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.764\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.764\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.577\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.289\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120 hr.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAnalysis of Average Bloodstain Diameter Over Time\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe mean diameters of menstrual and non-menstrual bloodstains were taken after depositions at 10-minute, 1-hour, 2-hour, 24-hour, 72-hour, and 120-hour intervals using a stereo microscope at 0.65x magnification. The ABFO scale was used to give accurate measurements of these diameters. Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e contains the data expressed as mean diameters and plotted in a line graph, based on three references during the bloodstain categories. Findings indicate a clear temporal pattern regarding diameter variation presented by both menstrual and non-menstrual bloodstains that has implications for forensic and bloodstain pattern analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMenstrual Bloodstain Diameter Trends\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe diameters of menstrual blood samples at different time lapses after deposition, average diameter (in centimeters), and standard deviation (SD). They were measured after 10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr. At 10 and 1 hour, the mean diameter is 8.83 cm and the standard deviation is 1.258, which means there was a regular size with normal variation in the earlier period. At 2 hours, the average diameter reduces by a small margin to 8.50 cm (standard deviation of 0.866, which indicates that it may be slightly smaller and with less variance). Beyond 24 hours, the mean diameter settles at 8.33cm, and at 72 hours, 120 hours, and 168 hours, the standard deviation records 1.155 and no longer varies. This implies that once the first two hours are surpassed, the diameter of the menstrual blood samples stabilizes to a steady average value, but with a slightly widened standard deviation, indicating that there is indeed more variation in measurements on the higher time scale. Within the general evaluation, the data suggests a decline in the mean diameter in the initial two-hour period, followed by a sustained size, but variability grows after 24 hours.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNon-Menstrual Bloodstain Diameter Trends\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe diameter of non-menstrual blood at different times after being deposited, including the average diameter (in centimeters) and standard deviation (SD). The measurements were taken at 10 min., 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr. At 10 minutes and an hour, the average diameter was 8.67 cm, and the standard deviation was 0.764, which shows a relatively good initial size of the design with small variability. Mean diameter drops a bit to 8.50 cm at 2 hours with a wider standard deviation of 0.866, indicating a slight decrease in size and that it is increasing somewhat in variability. At 24 hours, the average value reduces to 8.33 cm with a smaller standard deviation of 0.577, which means the process still diminishes with less variation. At 72 hours, the mean diameter fell further to 8.17 cm, and the standard deviation dropped by 0.289, which shows further decrease and even lesser variance. Lastly, when the time is 120 hours, the mean diameter value stands at 8.00 cm, and the standard deviation is 0.000, implying that it has a gradual decrease. Still, because the standard deviation is zero, it is the same diameter with no variability. The data demonstrate a gradual reduction in the mean diameter of non-menstrual blood samples as time increases, as the variability reduces and reaches stability after 120 hours.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe menstrual bloodstain diameter trend and the non-menstrual one are quite different in the trend of their diameters during the time of their deposition in which statistics is based on average (in centimeters) and the standard deviation (SD) at time intervals of 10 minutes, 1 hour, 2 hours, 24 hours, 72 hours, and 120 hours, of menstrual blood. At the beginning, 10 minutes and 1 hour, the mean diameter of menstrual blood is greater than 8.83 cm (SD 1.258) than the non-menstrual blood of 8.67 cm (SD 0.764), which means that the size of this menstrual blood is larger with more variability. Both menstrual and non-menstrual are reduced slightly later at 2 hours, menstrual to 8.50 cm (SD 0.866) and non-menstrual to 8.50 cm (SD 0.866), to the same extent with the same variability, raising the possibility of an early decline in both of similar magnitude. Menstrual blood settles on 8.33 cm (SD 1.155) after 24 hours, and this remains its mean 120 hours later and up to 168 hours, with the SD becoming progressively larger with time. Conversely, the non-menstrual blood keeps on reducing steadily as it registers 8.33 cm (SD 0.577) at 24 hours, 8.17 cm (SD 0.289) at 72 hours, and 8.00 cm (SD 0.000) at 120 hours, indicating a steady decline and its eventual evenness. This shows that although both the blood types get an initial decrease in diameter during the first two hours, menstrual blood is stationary after the first 24 hours, and more variable, non-menstrual blood, on the other hand, takes a long period to reduce in diameter and attains a homogenous size in 120 hours. In general, the diameter of menstrual blood is somewhat greater and more varied over time, whereas the non-menstrual blood has a more accentuated and stable decline.\u003c/p\u003e\u003cp\u003eThe difference observed is probably due to the differences in blood types' biochemical and physical characteristics. The menstrual blood with its endometrial tissue, mucus, and lower hematocrit may have a more viscous or gelatinous matrix that does not contract upon drying. By contrast, non-menstrual blood is likely to coagulate and evaporate further, mainly due to high contents of plasma, red blood cells, and clotting factors. The results here agree with the previous research findings that indicate that the menstrual bloodstains bear distinct morphological properties that one can tap into in forensic distinction because of their sophisticated state.\u003c/p\u003e\u003cp\u003eThe above line chart is titled \"Diameter of Menstrual and Non-Menstrual Bloodstains Over Time,\" and measurements were taken using a stereo microscope at 0.65x magnification and an ABFO scale. The x-axis represents the time since deposition (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr), while the y-axis shows the bloodstain diameter in centimeters, ranging from 6.5 cm to 10.5 cm. The chart includes three reference samples for menstrual blood (plotted in blue shades) and non-menstrual blood (plotted in red shades). All samples start with diameters around 9.5 cm to 10.0 cm at 10 min. Over time, menstrual bloodstains (Ref 1, Ref 2, Ref 3) gradually decline, stabilizing around 9.0 cm to 9.5 cm after 24 hr, with minor fluctuations. Non-menstrual bloodstains (Ref 1, Ref 2, Ref 3) exhibit a more pronounced decrease, dropping to approximately 8.0 cm to 8.5 cm by 120 hr.\u003c/p\u003e\u003cp\u003eThe above line chart titled \"Average Diameter of Bloodstains Over Time (cm)\" illustrates the mean diameter of menstrual and non-menstrual bloodstains across various time intervals, with the x-axis representing time points (10 min, 1 hr, 2 hr, 24 hr, 72 hr, and 120 hr) and the y-axis indicating the mean diameter in centimeters, ranging from approximately 7.6 cm to 8.9 cm. The red line, representing menstrual blood, begins at 8.83 cm at 10 min, remains stable at 8.83 cm at 1 hr, then gradually declines to 8.50 cm at 2 hr, 8.33 cm at 24 hr, and stabilizes at 8.33 cm from 72 hr to 120 hr. In contrast, the blue line, representing non-menstrual blood, starts at 8.67 cm at 10 min, holds steady at 8.67 cm at 1 hr, then decreases to 8.50 cm at 2 hr, 8.33 cm at 24 hr, 8.17 cm at 72 hr, and reaches 8.00 cm at 120 hr. The chart highlights that while both blood types start with similar diameters, non-menstrual bloodstains exhibit a more pronounced decrease over time, particularly after 24 hr. In contrast, menstrual bloodstains show greater stability after an initial reduction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study, describing the temporal changes of menstrual and non-menstrual bloodstains on diameters under controlled laboratory conditions, where they used a stereo microscope in 0.65x magnification and ABFO scale, can be considered as a source of a breakthrough in the field of forensic bloodstain pattern analysis (BPA), especially about the differences between blood types within specific periods, both more than 10 min and up to 120 hr (Roy et al., 2022). The study becomes even more important when put in the context of forensic issues described in the modern literature, according to which bloodstains are one of the most valuable pieces of evidence when it comes to recreating a crime scene, particularly after a sexual assault or case of trauma (Bevel et al., 2008, James et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The atmosphere of a dim forensic laboratory setting involving a scientist examining a rust-colored material stain on cloth shows the importance of properly investigating such evidence regarding the issues of consent, injury, or violence (Holtk\u0026ouml;tter et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Conventional BPA that uses size, shape, and distribution to indicate impact angles and force can prove ineffective in distinguishing between menstrual and peripheral blood, a distinction critical to legal preferences that can suggest consent in the former, and trauma in the latter (Al-Alimi et al., 2025; Pradeep et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This study's controlled approach, maintaining 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity on glossy paper, shows that the precision and reproducibility of the diameter measurements are evident, which increases the validity of forensic interpretations.\u003c/p\u003e\u003cp\u003eThe results indicate that the mean diameter of menstrual blood is 8.83 cm (SD 1.258) at 10 minutes and 1 hour, which reduces to 8.50 cm (SD 0.866) at 2 hours and stays constant at 8.33 cm (SD 1.155) between 24 and 168 hours, showing early reduction followed by deviation at later stages. Non-menstrual blood, initially at 8.67 cm (SD 0.764), gradually reduces to 8.50 cm (SD 0.866) after 2 hours, 8.33 cm (SD 0.577) after 24 hours, 8.17 cm (SD 0.289) after 72 hours, and 8.00 cm (SD 0.000) after 120 hours, which is suggestive of one step toward uniformity. Such time variance, corroborated by three measurements as estimated and graphically displayed as a line graph in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, provides a strong and reliable body of data that is more than the conventional tests, such as Teichmann or luminol, that only indicate the presence of blood but not the source of that blood. The analysis of the glossy paper as a semi-porous material used in the study reflects the real scene of forensic surfaces (e.g., paper, packaging). It offers a practical model of the field application.\u003c/p\u003e\u003cp\u003eThese findings are given significance by the issues of the surface types analysis, where surface material affects morphology and chemistry of bloodstains (porous (cotton) or non-porous (tile) surfaces modify the bloodstains), making them harder to distinguish. The viscous structure of menstrual blood, filled with endometrial mucus and a lower hematocrit, prevents drying contraction. In contrast, the high plasma level and clotting factors in non-menstrual blood cause greater drying evaporation and coagulation (Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It aligns with new imaging methods, such as hyperspectral imaging (HSI) and Raman spectroscopy, which are capable of non-destructively identifying blood types on spectral or molecular signatures (Zulfiqar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mistek et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The capacity of HSI to chart spectral variances owing to hemoglobin oxygenation and endometrial debris with virtually 97\u0026ndash;100 percent precision through machine learning on the HyperBlood digital set, adds to the present study optical variant (Al-Alimi et al., 2025; Li et al., 2025). Likewise, Raman spectroscopy offers molecular specificity with 100% sensitivity and specificity with the assistance of PLSDA. However, both are susceptible to interference by the surface, and this study reduces these challenges under controlled conditions (Mistek et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe SERATEC PMB test has a sensitivity of 100 percent and a specificity of 99.7 percent in detecting the presence of hemoglobin and D-dimers in menstrual blood. Though it is fast in screening, it requires the extraction of the sample and hence cannot be used on old or porous stains (Konrad et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The imaging combined with the non-destructive method of this study, which leaves samples intact to study afterward using DNA analysis, fills this gap, working together with other methods, imaging, and immunological ones, into a multimodal approach to forensics (Edelman et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The social benefits are enormous, since correct distinction can support the account of victims of sexual assault, vitality of justice, in particular, when the stakes are high, because misidentification can change the investigation (Chunkul et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations and Considerations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe glossy paper of the study does not allow generalisation to the wide range of forensic material surfaces (e.g., non-porous tiles or porous fabrics), which can affect the morphology of stains. Invariable, the crime scene environment is not always represented as controlled conditions (temperature, humidity), which may influence drying speed. The particle morphology (i.e., shape, edge characteristics, oxygen levels, morphology of individuals) was ignored in favor of diameter. The broader applicability may be lower because no account of individual variability in blood composition (e.g., menstrual blood and endometrial content) was taken. Aged stains may not be punishable within the 120-hour temporal terrene, and accessibility may be limited with specialized equipment (0.65x stereo microscope, ABFO scale). The test of different substrates, environmental conditions, and further stain parameters should be carried out to increase the practical usefulness of the study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study establishes a scientifically solid approach to the forensic evidence of bloodstain patterns because the desired temporal changes in diameters of menstrual and non-menstrual bloodstains were determined under the controlled conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, glossy paper substrate). The statistics show that the menstrual bloodstains at the time scale of 10 minutes and 1 hour have an average diameter of 8.83 cm (SD 1.258), which decreases at the scale of 1 hour and 2 hours (8.50 cm, SD 0.866), and reaches 8.33 cm (SD 1.155) between 24\u0026ndash;168 hours with consistency in size, yet partially showing larger diameter variation. Conversely, non-menstrual bloodstains, ranging between 8.67 cm (SD 0.764), 8.50 cm (SD 0.866), 8.33 cm (SD 0.577), 8.17 cm (SD 0.289), and 8.00 cm (SD 0.000) at 2, 24, 72, and 120 hours, respectively, exhibit a monotonic decrease as the standard deviation reduces. All of these different patterns are accurately measured under a 0.65x stereo microscope and correspond to the ABFO scale and are probably due to biochemical variability; both the viscous mucus-laden matrix of menstrual blood does not tend to dry-contraction, whereas non-menstrual blood, due to the increased plasma and coagulation factors, tends to blood clot and dry. It is a non-destructive method that supplements the detailed forensic methods (i.e., hyperspectral imaging, Raman spectroscopy, and SERATEC PMB test) and aids in the identification of the origin of blood sources, which is so vital in judicial matters about crime scene reconstruction. Although the substrate and environmental scope of study are limited, replicable methodology and statistically significant results (the results are based on three measurements per time point) present the baseline on which further research of forensic BPA should be built, which can lead to better court outcomes in cases where the bloodstain evidence is used.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eBPA\u003c/strong\u003e: Bloodstain Pattern Analysis\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eABFO\u003c/strong\u003e: American Board of Forensic Odontology\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eHuman Ethics and Consent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki. The Institutional Ethics Committee (IEC) of Kasturba Medical College, Mangalore, approved the study (Approval Reference: IECKMCMLR 07/2025/485). All participants provided written informed consent prior to participation. Participants were fully informed about the study’s purpose, procedures, potential risks, and their rights, including the right to withdraw at any time without consequence. Ethical principles were strictly adhered to during blood sample collection and handling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll donors provided written informed consent before participating in the study. Each participant was thoroughly informed about the study’s objectives, the procedures involved, the use of their biological samples (menstrual and peripheral blood), and their rights as participants. Consent forms were signed voluntarily, and participants were assured of confidentiality and the secure handling of their samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Files:\u003c/strong\u003e No Gels or Blots were used in this study, as the research focused on morphological analysis of bloodstains using stereo microscopy and photographic documentation. All relevant data are presented in the manuscript tables and figures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Alimi D, Al-Qaness MAA (2025) Enhancing forensic blood detection using hyperspectral imaging and advanced preprocessing techniques. 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Sensors 21(9):3045. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/s21093045\u003c/span\u003e\u003cspan address=\"10.3390/s21093045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bloodstain pattern analysis, menstrual blood, peripheral blood, glossy paper, stereo microscopy, morphological changes, crime scene reconstruction, non-destructive methods, bloodstain diameter","lastPublishedDoi":"10.21203/rs.3.rs-7177372/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7177372/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDistinguishing between menstrual and peripheral bloodstains is essential in forensic research, especially in sexual assault or trauma cases, since it may be used to determine whether it is a matter of consent or violence. Under controlled conditions, this study examines the temporal morphological changes of menstrual and non-menstrual blood stains on glossy paper, a semi-porous surface.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe diameter of the stains was noted at 10 minutes, 1, 2, 24, 72, and 120 hours after the deposition for menstrual and non-menstrual stains. The non-menstrual bloodstains decreased consecutively over time, so they were more consistent with time. These variations were due to the viscous, mucus-rich nature of menstrual blood as opposed to the clotting effects of peripheral blood, and were statistically significant (measured by triplicate measurement).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis is a non-destructive optical technique that can complement high-order techniques such as hyperspectral imaging, Raman spectroscopy, and the SERATEC PMB test, increasing the capacity of forensic bloodstain pattern analysis. Although the substrates used and the environmental parameters are limited, the results give a good starting point in differentiating blood types, the ramifications of which may be used in correct crime scene reconstruction and legal results.\u003c/p\u003e","manuscriptTitle":"Surface Specific Analysis - A Comparative Study of Menstrual and Non-Menstrual Bloodstains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:12:00","doi":"10.21203/rs.3.rs-7177372/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b61602fd-16dc-4ab3-ba81-d3ab5a2b863a","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T05:23:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 16:12:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7177372","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7177372","identity":"rs-7177372","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}