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 methemoglobin (met-Hb) and hemichrome (HC). The fractions of HbO 2, 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

Cremains, what remains

Salvaging burned human remains can be a challenging task for the forensic specialists, and interpreting evidence from thermally altered skeletal elements can be difficult. Based on the results presented in this dissertation more is possible than was previously known. In this dissertation, novel ways of finding and analyzing burned human remains are presented to the field of forensic anthropology. This thesis introduces new methods to collect and differentiate cremated bone from construction debris from a fire scene, and estimate the exposure temperature that bones have been exposed to – which is important for forensic investigation. Further, this dissertation provides the reader with new knowledge on heat induced biophysical changes and characteristics that aids the interpretation of skeletal damage that occurred before, during or after the fire. It is important for new methods in forensic practice to comply with legal standards. Accordingly, the value of the presented methods for practice in both the field and the courtroom are thoroughly discussed to inform both the forensic practitioner as well as the legal experts

Phosphorescence of thermally altered human bone

Bone has photoluminescent characteristics that can aid the analysis of thermally altered human skeletal remains as part of the forensic anthropological investigation. Photoluminescence stands collectively for fluorescence and phosphorescence. Because the difference in lifetime between fluorescence and phosphorescence is usually in the range of nano- to microseconds, it is only possible to visually determine whether bone phosphoresces when the lifetime is long enough to be observed. For this study, a distinction was made between long-decay and short-decay phosphorescence. So far, it was unknown whether (thermally altered) human bone emits long-decay phosphorescence after being illuminated and, thus, whether phosphorescence contributes to the observed photoluminescence. If so, whether the observable phosphorescence is dependent on temperature, exposure duration, surrounding medium, bone type, skeletal element, and excitation light and could aid the temperature estimation of heated bone fragments. In this study, bone samples were subjected to heat in the range of from room temperature to 900 °C for various durations in either air or adipose as surrounding medium. In addition, different skeletal elements of a human cadaver were recollected after cremation in a crematorium. Both sample collections were illuminated with light of different bandwidths and visually inspected for phosphorescence and photoluminescence. The samples were scored by means of a scoring index for the intensity of long-decay phosphorescence and photographically documented. The results show that thermally altered human bone fragments do phosphoresce. The observed phosphorescence is more dependent on temperature than on exposure duration, surrounding medium or skeletal element. Of the used wavelength bands, ultraviolet light provided the most temperature-related information, showing changes in both phosphorescence intensity and emission spectrum. Long-decay phosphorescence and fluorescence with short-decay phosphorescence coincide; however, there are also temperature-dependent differences. It is therefore concluded that phosphorescence contributes to the observable photoluminescence and that the visibly observable phosphorescent characteristics can aid the temperature estimation of cremated human skeletal fragments

Phosphorescence of thermally altered human bone

Bone has photoluminescent characteristics that can aid the analysis of thermally altered human skeletal remains as part of the forensic anthropological investigation. Photoluminescence stands collectively for fluorescence and phosphorescence. Because the difference in lifetime between fluorescence and phosphorescence is usually in the range of nano- to microseconds, it is only possible to visually determine whether bone phosphoresces when the lifetime is long enough to be observed. For this study, a distinction was made between long-decay and short-decay phosphorescence. So far, it was unknown whether (thermally altered) human bone emits long-decay phosphorescence after being illuminated and, thus, whether phosphorescence contributes to the observed photoluminescence. If so, whether the observable phosphorescence is dependent on temperature, exposure duration, surrounding medium, bone type, skeletal element, and excitation light and could aid the temperature estimation of heated bone fragments. In this study, bone samples were subjected to heat in the range of from room temperature to 900 degrees C for various durations in either air or adipose as surrounding medium. In addition, different skeletal elements of a human cadaver were recollected after cremation in a crematorium. Both sample collections were illuminated with light of different bandwidths and visually inspected for phosphorescence and photoluminescence. The samples were scored by means of a scoring index for the intensity of long-decay phosphorescence and photographically documented. The results show that thermally altered human bone fragments do phosphoresce. The observed phosphorescence is more dependent on temperature than on exposure duration, surrounding medium or skeletal element. Of the used wavelength bands, ultraviolet light provided the most temperature-related information, showing changes in both phosphorescence intensity and emission spectrum. Long-decay phosphorescence and fluorescence with short-decay phosphorescence coincide; however, there are also temperature-dependent differences. It is therefore concluded that phosphorescence contributes to the observable photoluminescence and that the visibly observable phosphorescent characteristics can aid the temperature estimation of cremated human skeletal fragments

Mechanical or thermal damage:differentiating between underlying mechanisms as a cause of bone fractures

To investigate the differences between pre- and post-fire fractures, 30 human forearm bones were subjected to either blunt-force impact, burning, or both. Bones, covered in soft tissue and wrapped in clothing, were burned in a reconstructed house fire. The burning context and dynamics led to differential burning, that was equal amongst the three groups. To evaluate the damage to the bones, a data collection sheet was developed based on the current literature on fracture features. To analyze the relation between exposure temperature and fracture class and occurrence, color was measured to estimate the exposure temperature. Observable and measurable changes on bone, and fracture surfaces, were macro- and microscopically analyzed. Many features overlapped between the three groups, and thus are not usable for differentiation. Fractures caused by blunt force impact (post-mortem, pre-fire) showed a rough fracture surface with smoothness in curved/slanted regions near the margin after burning, while heat-induced bone fractures showed a smooth fracture surface. The margins and surface of bone fractures that originated after the fire (indirect heat-induced) were evenly discolored, whereas heat-induced bone fractures showed uneven discoloration of the fracture margin and surface. Although there were generally more heat-induced fractures in the 450-700 °C range, no other distinctive trend was observed between exposure temperature and class of fractures

Colourimetric analysis of thermally altered human bone samples

At this moment, no method is available to objectively estimate the temperature to which skeletal remains have been exposed during a fire. Estimating this temperature can provide crucial information in a legal investigation. Exposure of bone to heat results in observable and measurable changes, including a change in colour. To determine the exposure temperature of experimental bone samples, heat related changes in colour were systemically studied by means of image analysis. In total 1138 samples of fresh human long bone diaphysis and epiphysis, varying in size, were subjected to heat ranging from room temperature to 900 °C for various durations and in different media. The samples were scanned with a calibrated flatbed scanner and photographed with a Digital Single Lens Reflex camera. Red, Green, Blue values and Lightness, A-, and B-coordinates were collected for statistical analysis. Cluster analysis showed that discriminating thresholds for Lightness and B-coordinate could be defined and used to construct a model of decision rules. This model enables the user to differentiate between seven different temperature clusters with relatively high precision and accuracy. The proposed decision model provides an objective, robust and non-destructive method for estimating the exposure temperature of heated bone samples