Rolf H. Bremmer

Age estimation of blood stains by hemoglobin derivative determination using reflectance spectroscopy

Blood stains can be crucial in reconstructing crime events. However, no reliable methods are currently available to establish the age of a blood stain on the crime scene. We show that determining the fractions of three hemoglobin derivatives in a blood stain at various ages enables relating these time varying fractions to the age of the blood stain. Application of light transport theory allows addressing the spectroscopic changes in ageing blood stains to changes in chemical composition, i.e. the transition of oxy-hemoglobin into met-hemoglobin and hemichrome. We have found in 20 blood stains that the chemical composition of the blood stain with age, called hemoglobin reaction kinetics, under controlled circumstances, shows a distinct time-dependent behavior, with a unique combination of the three hemoglobin derivatives at all moments in time. Finally, we employed the hemoglobin reaction kinetics inversely to assess the age of 20 other blood stains studied, again over a time period of 0-60 days. We estimated an age of e.g. 55 days correct within an uncertainty margin of 14 days. In conclusion, we propose that the results obtained under controlled conditions demand further evaluation of their possible value for age determination of blood stains on crime scenes.

Biphasic Oxidation of Oxy-Hemoglobin in Bloodstains

Background In 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. Methodology The 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. Conclusions The 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.

Remote Spectroscopic Identification of Bloodstains

Abstract:  Blood detection and identification at crime scenes are crucial for harvesting forensic evidence. Unfortunately, most tests for the identification of blood are destructive and time consuming. We present a fast and nondestructive identification test for blood, using noncontact reflectance spectroscopy. We fitted reflectance spectra of 40 bloodstains and 35 nonbloodstains deposited on white cotton with spectroscopic features of the main compounds of blood. Each bloodstain was measured 30 times to account for aging effects. The outcome of the blood measurements was compared with the reflectance of blood‐mimicking stains and various body fluids. We found that discrimination between blood and nonblood deposited on white cotton is possible with a specificity of 100% and a sensitivity of 98%. In conclusion, a goodness of fit between the sample’s reflectance and the blood component fit may allow identification of blood at crime scenes by remote spectroscopy.

Volume determination of fresh and dried bloodstains by means of optical coherence tomography.

The volume of bloodstains found on crime scenes may help forensic investigators reconstruct the location and kinematics of bloodletting events, as stain size, volume, and impact velocity are related. Optical coherence tomography was used as a method to determine the volume and volume ratio of dried and fresh bloodstains on both glass and irregular surfaces or deposited with an impact velocity. The volume of blood drops deposited on smooth glass surfaces was measured within a deviation of 2%. This deviation increased for droplets on irregular surfaces or deposited with an impact velocity. The volume ratio of dried and fresh bloodstains was equal to 19–28% depending on the individual donor and on the use of an anticoagulant. Optical coherence tomography is a good method to determine the volume of fresh and dried bloodstains in laboratory conditions and allows accurate determination of the dry/fresh ratio.

Non-contact spectroscopic determination of large blood volume fractions in turbid media

We report on a non-contact method to quantitatively determine blood volume fractions in turbid media by reflectance spectroscopy in the VIS/NIR spectral wavelength range. This method will be used for spectral analysis of tissue with large absorption coefficients and assist in age determination of bruises and bloodstains. First, a phantom set was constructed to determine the effective photon path length as a function of μa and μs′ on phantoms with an albedo range: 0.02-0.99. Based on these measurements, an empirical model of the path length was established for phantoms with an albedo > 0.1. Next, this model was validated on whole blood mimicking phantoms, to determine the blood volume fractions ρ = 0.12-0.84 within the phantoms (r = 0.993; error < 10%). Finally, the model was proved applicable on cotton fabric phantoms.

Diffuse reflectance relations based on diffusion dipole theory for large absorption and reduced scattering

Abstract. Diffuse reflectance spectra are used to determine the optical properties of biological samples. In medicine and forensic science, the turbid objects under study often possess large absorption and/or scattering properties. However, data analysis is frequently based on the diffusion approximation to the radiative transfer equation, implying that it is limited to tissues where the reduced scattering coefficient dominates over the absorption coefficient. Nevertheless, up to absorption coefficients of 20  mm−1 at reduced scattering coefficients of 1 and 11.5  mm−1, we observed excellent agreement (r2=0.994) between reflectance measurements of phantoms and the diffuse reflectance equation proposed by Zonios et al. [Appl. Opt. 38, 6628–6637 (1999)], derived as an approximation to one of the diffusion dipole equations of Farrell et al. [Med. Phys. 19, 879–888 (1992)]. However, two parameters were fitted to all phantom experiments, including strongly absorbing samples, implying that the reflectance equation differs from diffusion theory. Yet, the exact diffusion dipole approximation at high reduced scattering and absorption also showed agreement with the phantom measurements. The mathematical structure of the diffuse reflectance relation used, derived by Zonios et al. [Appl. Opt. 38, 6628–6637 (1999)], explains this observation. In conclusion, diffuse reflectance relations derived as an approximation to the diffusion dipole theory of Farrell et al. can analyze reflectance ratios accurately, even for much larger absorption than reduced scattering coefficients. This allows calibration of fiber-probe set-ups so that the object’s diffuse reflectance can be related to its absorption even when large. These findings will greatly expand the application of diffuse reflection spectroscopy. In medicine, it may allow the use of blue/green wavelengths and measurements on whole blood, and in forensic science, it may allow inclusion of objects such as blood stains and cloth at crime scenes.