Age Of Bloodstains Research Articles

The applicability of fluorescence lifetime to determine the time since the deposition of biological stains

This work demonstrates that fluorescence lifetime measurements can successfully predict the age of human bloodstains.

Determination of Bloodstain Age by UV Visible Integrating Sphere Reflection Spectrum

To establish a method for rapid identification of bloodstain age. Under laboratory conditions （20 ℃, 25 ℃ and 30 ℃）, an integrating sphere ISR-240A was used as a reflection accessory on an UV-2450 UV-vis spectrophotometer, and a standard white board of BaSO₄ was used as reference, the reflection spectrums of bloodstain from human ears' venous blood were measured at regular intervals. The reflection radios R₅₄₁ and R₅₇₇ at a specific wavelength were collected and the value of R₅₄₁/R₅₇₇ was calculated. The linear fitting and regression analysis were done by SPSS 17.0. The results of regression analysis showed that R² of the ratios of bloodstain age to UV visible reflectivity in specific wavelengths were larger than 0.8 within 8 hours and under certain circumstances. The regression equation was established. The bloodstain age had significant correlation with the value of R₅₄₁/R₅₇₇. The method of inspection is simple, rapid and nondestructive with a good reliability, and can be used to identify the bloodstain age within 8 hours elapsed-time standards under laboratory conditions.

Practical Implementation of Blood Stain Age Estimation Using Spectroscopy

Blood is one of the most often encountered and valuable traces found at a crime scene. It not only contains information about what happened, but the chemical composition can also lead to the identification of the donor and even to the time of deposition, an important objective in forensic science. Many techniques have been explored for determination of the time of deposition, but none have made it into forensic practice. We have earlier used optical reflection spectroscopy for the age estimation of blood stains on white cotton, and to make this method suitable for forensic practice, we have adapted it to correct for the influence of the optical properties of the various substrates where blood may be found by extending a one-dimensional light-transport model to a two-layered model. With this model, we are able to calculate the relative amounts of OxyHemoglobin, MetHemoglobin, and Hemichrome in blood stains on colored surfaces, based on their reflectance spectra. We describe here a statistical method to calculate the 95% confidence interval around the calculated age. We demonstrate the applicability to field work and the potential value for crime investigations in a case study of a shooting incident. The absolute and relative ages of different groups of blood stains were measured using our new method. The measured age of blood stains can lead to a more realistic reconstruction of the timeline of events. To our knowledge, this paper presents the world's first implementation in practice of a method for determining the age of blood stains.

Estimation of Age of Bloodstain Spotted on Different Surfaces Using UV-visible Spectroscopy

Estimation of age of bloodstain found at scene of crime is crucial for forensic personnel, which aids in defining time frame in which the individual depositing it was present. Although many methods have been discovered to estimate age of the bloodstain but only few have gained widespread use due to poor time resolution and weak age correlation. The present study used UV-vis spectrophotometric analysis of hemoglobin (Hb) for estimating the age of the bloodstains spotted on different substrates, which are usually encountered at crime scene. The average rate of Hbsoret Band blue-shift rate varied from 0.0075 to 0.0015 nm/h for bloodstain of environmental exposure while for room temperature exposed it varied from 0.0012 to 0.0002 nm/h. Preliminary blood test (benzidine test), serological test (species of origin) and genetic analysis (DNA extraction) on the same bloodstain were also performed. The benzidine test and octerlony double diffusion test was positive for the samples kept at RT for 73 day while DNA could not be extracted.

Experimental Study on Age Estimation of Bloodstains Based on Biological and Toxicological Analysis

The determination of the age of bloodstains in forensic investigations helps in estimating the time elapsed between incidents (such as accidents or violent crimes wherein the blood was shed) and sampling. In this study, bloodstains containing either ethanol or amphetamine sulfate and methamphetamine hydrochloride were prepared and incubated at 25°C and 50% humidity to determine their age, based on temporal changes in the relative levels of microRNA 16 (miR-16) and miR-451 after incubation. The miR-16 and miR-451 levels decreased significantly 5-28 days after incubation in the chamber. The concentration of ethanol in the bloodstains decreased rapidly during storage and became undetectable after 180 min. The concentrations of amphetamine sulfate and methamphetamine hydrochloride in the bloodstains remained almost constant throughout the experimental period. These findings indicate that the age of bloodstains can be calculated simply on the basis of the relative levels of miRNAs. Although it is difficult to utilize the concentrations of ethanol, methamphetamine, and amphetamine sulfate for estimating the age of the bloodstain, these data could provide evidence of the victim or assailant having used these substances at the scene of an accident or crime.

Age estimation of bloodstains using smartphones and digital image analysis

Recent studies on bloodstains have focused on determining the time since deposition of bloodstains, which can provide useful temporal information to forensic investigations. This study is the first to use smartphone cameras in combination with a truly low-cost illumination system as a tool to estimate the age of bloodstains. Bloodstains were deposited on various substrates and photographed with a smartphone camera. Three smartphones (Samsung Galaxy S Plus, Apple iPhone 4, and Apple iPad 2) were compared. The environmental effects – temperature, humidity, light exposure, and anticoagulant – on the bloodstain age estimation process were explored. The color values from the digital images were extracted and correlated with time since deposition. Magenta had the highest correlation (R2=0.966) and was used in subsequent experiments. The Samsung Galaxy S Plus was the most suitable smartphone as its magenta decreased exponentially with increasing time and had highest repeatability (low variation within and between pictures). The quantifiable color change observed is consistent with well-established hemoglobin denaturation process. Using a statistical classification technique called Random Forests™, we could predict bloodstain age accurately up to 42 days with an error rate of 12%. Additionally, the age of forty blind stains were all correctly predicted, and 83% of mock casework samples were correctly classified. No within- and between-person variations were observed (p>0.05), while smartphone camera, temperature, humidity, and substrate color influenced the age determination process in different ways. Our technique provides a cheap, rapid, easy-to-use, and truly portable alternative to more complicated analysis using specialized equipment, e.g. spectroscopy and HPLC. No training is necessary with our method, and we envision a smartphone application that could take user inputs of environmental factors and provide an accurate estimate of bloodstain age.

The age estimation of blood stains up to 30days old using visible wavelength hyperspectral image analysis and linear discriminant analysis

A novel application of visible wavelength hyperspectral image analysis has been applied to determine the age of blood stains up to 30 days old. Reflectance spectra from selected locations within the hyperspectral image, obtained from a portable instrument, were subjected to spectral pre-processing. This was followed by the application of a linear discriminant classification model, making estimations possible with an average error of ±0.27days for the first 7 days and an overall average error of ±1.17days up to 30 days. This is also the first reported study of the determination of the age of fresh blood stains (less than one day old) with an error of ±0.09h. The studies have been made under controlled conditions and represent, at this stage, proof of concept results but also are the most accurate age estimation results for measurements between 0 and 30 days reported to date. The results are consistent with well-established kinetic processes suggesting that the pre-processing stages described are revealing spectroscopic changes which are reliably following the time dependent oxidation of HbO2. The potential for parameterisation of environmental factors to make the method generally applicable at crime scenes is discussed, along with the developments required to further improve classification and to make the instrument genuinely portable.

Gender-related difference in bloodstain RNA ratio stored under uncontrolled room conditions for 28 days

Bloodstain age is a parameter that can be used in crime scene investigations. Bloodstain age can be determined by measuring the 18S rRNA:β-actin mRNA ratio by Reverse Transcription-quantitative PCR (RT-qPCR). Since this ratio is a function of time, it can be used as an estimator of bloodstain age. However, it is important to validate the technique in a variety of scenarios before it can be applied. We investigated 18S rRNA:β-actin mRNA ratio in bloodstains from sixteen Chinese subjects in 28 days under uncontrolled room conditions. The ratio changed in a linear fashion. It was also found that the subjects' gender affected the relationship between time and the RNA ratio.

Hyperspectral imaging for the age estimation of blood stains at the crime scene

The age estimation of blood stains can provide important information on the temporal aspects of a crime. As previously shown, visible spectroscopy of blood stains can successfully be used for their age estimation. In the present study we evaluated the feasibility to use hyperspectral imaging for this purpose. Visible reflectance spectra of blood stains were recorded using a pushbroom hyperspectral imaging system. From these spectra, the relative amounts of oxyhemoglobin, methemoglobin and hemichrome within the blood stains were derived. By comparison of the hemoglobin derivative fractions with a reference dataset, the age of blood stains up to 200 days old was estimated. The absolute error of the age estimation task increased with age, with a median relative error of 13.4% of the actual age. To test the practical applicability of this method, a simulated crime scene was analyzed, in which blood stains of several ages were deposited. Hyperspectral imaging combined with the proposed analysis provided insight in the absolute age of the blood stains. Additionally, the blood stains were clustered based on their hemoglobin derivative fractions, without the use of a reference dataset. Results demonstrated that the order of formation of blood stains can be determined, even under unknown environmental circumstances, when no proficient reference dataset is available. These findings are an important step toward the practical implementation of blood stain age estimation in forensic casework.

Biphasic Oxidation of Oxy-Hemoglobin in Bloodstains

BackgroundIn forensic science, age determination of bloodstains can be crucial in reconstructing crimes. Upon exiting the body, bloodstains transit from bright red to dark brown, which is attributed to oxidation of oxy-hemoglobin (HbO2) to met-hemoglobin (met-Hb) and hemichrome (HC). The fractions of HbO2, met-Hb and HC in a bloodstain can be used for age determination of bloodstains. In this study, we further analyze the conversion of HbO2 to met-Hb and HC, and determine the effect of temperature and humidity on the conversion rates.MethodologyThe fractions of HbO2, met-Hb and HC in a bloodstain, as determined by quantitative analysis of optical reflectance spectra (450–800 nm), were measured as function of age, temperature and humidity. Additionally, Optical Coherence Tomography around 1300 nm was used to confirm quantitative spectral analysis approach.ConclusionsThe oxidation rate of HbO2 in bloodstains is biphasic. At first, the oxidation of HbO2 is rapid, but slows down after a few hours. These oxidation rates are strongly temperature dependent. However, the oxidation of HbO2 seems to be independent of humidity, whereas the transition of met-Hb into HC strongly depends on humidity. Knowledge of these decay rates is indispensable for translating laboratory results into forensic practice, and to enable bloodstain age determination on the crime scene.

Time-dependent surface adhesive force and morphology of RBC measured by AFM

Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.

Age determination of blood spots in forensic medicine by force spectroscopy

We present a new tool for the estimation of the age of bloodstains, which could probably be used during forensic casework. For this, we used atomic force microscopy (AFM) for high-resolution imaging of erythrocytes in a blood sample and the detection of elasticity changes on a nanometer scale. For the analytic procedure we applied a fresh blood spot on a glass slide and started the AFM detection after drying of the blood drop. In a first step, an overview image was generated showing the presence of several red blood cells, which could easily be detected due to their typical “doughnut-like” appearance. The consecutively morphological investigations in a timeframe of 4 weeks could not show any alterations. Secondly, AFM was used to test the elasticity by recording force–distance curves. The measurements were performed immediately after drying, 1.5 h, 30 h and 31 days. The conditions were kept constant at room temperature (20 °C) and a humidity of 30%. The obtained elasticity parameters were plotted against a timeline and repeated several times. The elasticity pattern showed a decrease over time, which are most probably influenced by the alteration of the blood spot during the drying and coagulation process. The preliminary data demonstrates the capacity of this method to use it for development of calibration curves, which can be used for estimation of bloodstain ages during forensic investigations.

Estimation of the age of human bloodstains by electron paramagnetic resonance spectroscopy: Long-term controlled experiment on the effects of environmental factors

In this study, we examined the efficacy and limitations of electron paramagnetic resonance (EPR) for estimating the age of human bloodstains. At 77 K, human bloodstains give four striking EPR signals in the g = 6.2 (g6), 4.3 (g4), 2.27 (H) and 2.005 (R) regions due to ferric high-spin, ferric non-heme, ferric low-spin and free radical species, respectively. We found that plotting double logarithms of the EPR intensity ratio of H/g4 versus days past bleeding gave a linear correlation up to 432 days with an error range within 25% of the actual number of days under controlled conditions. However, environmental factors such as differences of absorbent, light exposure and fluctuations of storage temperature affected the changes of these EPR-active compounds, which result in misestimation of the time since bleeding occurred. Therefore, one should take such factors into account in estimating the period since bleeding by this method.

Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?

In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. We have developed a semi-quantitative duplex reverse transcription-polymerase chain reaction (RT-PCR) assay which, in combination with competitive RT-PCR using an external standard, allows quantification of RNA degradation levels. Using this method, we have investigated 106 bloodstains stored up to 15 years. The distribution of the peak area quotients of standard and target messenger-RNA (mRNA) as measured by laser-induced fluorescence capillary electrophoresis was closely correlated with the age of the samples. Further statistical analysis showed that bloodstains with age differences of 5 years and more exhibit statistical significant variances in peak area quotients of both housekeeping genes included in this study, β-actin and cyclophilin. This can be of value when evidence from old cases is re-investigated. Our data show, that, although RNA continues to be degraded in dried bloodstains, mRNA suitable for RT-PCR can be isolated from samples stored for at least 15 years.

The Estimation of Age of Bloodstains by HPLC Analysis

A new HPLC system is described for estimating the age of bloodstains deposited on clothing. In addition to the decomposition peak designated as "X" and reported earlier in the literature, several other ageing processes were studied and found suitable for estimation of age of the stains. The various processes can be used independently of each other for estimation of bloodstain age when the storing temperature is known. Moreover, the ratio between the different peaks formed by ageing is practically independent of temperature between 0 degree C and 37 degrees C.

Estimation of bloodstain age by rapid determinations of oxyhemoglobin by use of oxygen electrode and total hemoglobin.

Bloodstain age could be estimated from the ratio of oxyhemoglobin/total hemoglobin (fractional oxyhemoglobin) in the bloodstain by this present method, if the temperature at which the bloodstain had been kept was known. The oxyhemoglobin was determined with an oxygen electrode immersed in water in which the oxygen had been depleted, and the total hemoglobin was determined by conventional colorimetry (cyanomethemoglobin method). Ages of prepared bloodstain samples (within 24 h after bleeding) were estimated by this present method, which requires only 20 microliters of bloodstain and only 5 min for the whole analysis.

Quantitative analysis of species identification tests of bloodstains using anti-human serum.

Immunological reactions of bloodstains of human and animals to anti-human serum were analysed quantitatively to determine which technique is the most effective to differentiate human bloodstains from those of animals. The lower threshold of counter-electrophoresis was 10-25 micrograms protein/ml and increased only slightly with increasing age of the bloodstains, while that of the ring test and immunodiffusion decreased greatly. In counter-electrophoresis, absorption of anti-human serum by Japanese monkey serum resulted in a marked decrease in the cross reaction with animals, and still showed no change in the lower threshold for human samples. The present results show that counter-electrophoresis is especially useful for the identification of human bloodstains.

A new marker for estimation of bloodstain age by high performance liquid chromatography

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a μBondasphere C18 column with a two step linear gradient of 10.5–46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as ‘X’. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4°C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios ( R x) and the ages of stains in weeks (W) is ln(1000 · R x) = 1.1084 + 0.3937 · ln(7 · W), and the coefficient of correlation ( r) is 0.9776 ( n = 144, P < 0.001). When stains were stored at 37°C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37°C is ln(1000 · R x) = 2.4477 + 0.0866 · W ( r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.

Identification of fetal hemoglobin and simultaneous estimation of bloodstain age by high-performance liquid chromatography

A method using reverse-phase high-performance liquid chromatography (HPLC) for the identification of fetal hemoglobin (Hb F) and the simultaneous estimation of bloodstain age is described. Umbilical cord and neonatal bloodstains can be differentiated from adult stains by the presence of gamma-globin chains which are characteristic of Hb F. With this method, cord and neonatal blood could be distinguished from adult blood in stains up to 32 weeks old. The age of the stain was estimated from the ratio of the peak area of the alpha-globin chain to that of heme on the same chromatogram. The ratio decreased gradually with an increase in the age of the stain up to 20 weeks old. Studies performed at each time period revealed no significant difference in the ratios of cord and neonatal bloodstains or in the ratios of cord and adult stains. The regression equation calculated from the ratios (y) and the ages of stains in weeks (x) expressed logarithmically is y = 2.5758-0.2497 In (x) and the coefficient of correlation is -0.7491 (n = 252, P less than 0.001). The present method, having the advantages of simplicity, speed and sensitivity, should be of great value to forensic science.

Quantitative determinations of Hp, Pi, Tf and GC in stored blood stains

Quantitative analysis of serum proteins in blood stains by rocket immunoelectrophoresis showed the following results: Long elution times in processing blood stains do not produce a significantly higher yield of serum proteins in stains, and they increase the risk of structural alteration of protein molecules. The amount of proteins detected decreases with increasing age of blood stains. Some proteins are already altered in stains after a short storage time and are no longer useful for phenotyping. Our results confirm that blood stain material should be processed as soon as possible.