We performed comparative investigation in visible-spectrophotometric methods for determining carboxyhemoglobin (CO-Hb) in blood samples. About 58% carboxyhemoglobin-saturation (%CO-Hb) of blood samples (sample H) was prepared from control blood by carbon monoxide bubbling and this sample was diluted to be 4/11 and 3/25 with control human blood to prepare moderate and weak CO-Hb saturated samples (sample M and L, respectively). We measured %CO-Hb of four samples, samples H, M, L and control human blood (relative %CO-Hb were 1.00, 0.36, 0.12 and 0, respectively), by four different methods in five different forensic science laboratories. By summing up the measurement results, the method (1), which is described in “Standard method of chemical analysis in poisoning (edited by the Pharmaceutical Society of Japan)”, gave %CO-Hb values that reflected the relative %CO-Hb of the four samples. The method (2), which is an isosbestic point method (developed by Department of Forensic Medicine, Kagawa University) gave higher values compared to the expected ones. The method (3), which is performed with a strong alkaline condition, gave higher values for low %CO-Hb samples by Katsumata's formula (method (3)-1). But the values calculated using the formula improved by Forensic Science Laboratory, Hokkaido Prefectural Police H.Q. (method (3)-2), gave the values reflecting the relative %CO-Hb. The method (4), which is also performed with a strong alkaline method, gave values which reflected the relative %CO-Hb when the Fukui's formula was used for calculation. But the formula modified by Aoki (method (4)-2) gave higher values for the blood samples of low %CO-Hb. In comparison of the three methods that gave the values reflecting the relative %CO-Hb, the method (1) and (3)-2 gave similar values but the measured values obtained by method (4)-1 was lower than the values obtained by method (1) and (3)-2. On the other hand, the method (3)-2 and method (1) showed the large dispersion in the measured values among the laboratories, but the dispersion by the method (4)-1 was small.
Mitochondrial DNA (mtDNA) analysis is a useful tool to analyze forensic samples. MtDNA exists as numerous molecules in a single cell in contrast to nuclear DNA, therefore mtDNA analysis can provide successful results in analyzing minute DNA samples. In our current PCR protocol of mtDNA analysis, hypervariable region 1 (HV1) and hypervariable region 2 (HV2) are divided into 5 regions (region A to E), and each region is amplified separately. It requires labor and long PCR time. In this paper, we study multiplex PCR of mtDNA and high-speed PCR using high-speed DNA polymerase to reduce labor and time of mtDNA analysis.
Forensic biologists are increasingly likely to identify an individual using three-dimensional imaging equipment. Existing equipment requires taking the subjects to the place where the equipment is located. In recent years, a non-contact, hand-held three-dimensional color scanner has been developed that is compact and lightweight. Therefore, we already use it for facial recognition and cranio-facial superimposition. The three-dimensional scanner reconstructed skull images precisely, and the anthropological measurements obtained from the images were comparable to those obtained from actual skulls (the differences in measurements were less than 1mm). Furthermore, the skull images produced by the scanner corresponded with high accuracy to the three-dimensional images reconstructed using computed tomography (concordance rates were approximately 95%) and to the two-dimensional facial photographs of the same person (the differences were less than the standard value 2.5mm for the reciprocal point-to-point matching). In addition to the skull images, the three-dimensional scanner precisely reconstructed facial images of living people. The three-dimensional facial images approximately corresponded to the two-dimensional facial photographs of the same people taken from both the vertical direction and from a bird's-eye view (the difference were less than the standard values, 0.9mm for the outline matching and 2.5mm for the reciprocal point-to-point matching). In conclusion, the current study confirmed that the non-contact, hand-held three-dimensional color scanner can provide forensic biologists with precise three-dimensional images of both skulls and living people's faces and that the images are of sufficiently good quality to be put to practical use as the quality of conventional stationary type.
The aim of this study was to develop a practical method to analyze tetrodotoxin (TTX), quantitatively, from postmortem specimens, not only blood and urine, but also organs. Extraction was achieved with 2% acetic acid and the use of an anion-exchange solid-phase extraction (SPE) cartridge. The quantitation method was a standard addition method with a calibration curve consisting of at least 3 points and an internal standard, voglibose (VOG). Separation by LC-MS/MS was achieved using a Luna HILIC (Phenomenex) column. The mobile phase was acetonitrile: 5 mM ammonium formate buffer (95:5), delivered at 0.2 mL/min. The selected reaction monitoring (SRM) transitions for TTX and VOG were m/z 320>302 and m/z 268>92, respectively. Cleaner extracts were achieved by using a lipid removal cartridge and washing with heptane. The addition of steps to remove interfering components that are prominent in postmortem samples aided in successful analysis. The HILIC column improved the retention of TTX to greater than 2 min to avoid the area where ion suppression has its greatest effect. Also, the use of anion-exchange SPE lessened the influence of acetic acid used during extraction. By using this method, we were able to quantitate low levels of TTX in postmortem specimens.