Alternative primers for DYS391 typing: advantages of their application to forensic genetics

The amplification of the STR DYS391, using the primers described in the Genome Data Base (GDB: G00-365-251), shows not only an additional band to the Y-specific one in males with a size range of 26 bp less than those of DYS391 locus alleles, but also a polymorphic pattern in females in the same size range as the additional band observed in males. The DYS391 pattern in families reflects a Y-specific linked locus and also a polymorphic X locus with an X-linked pattern of inheritance. A first screening in the X homologous locus allowed the identification of five different alleles. Allele frequencies were explored in different population groups for both the Y locus and the homologous locus in the X chromosome showing a similar allele distribution pattern in the X and Y homologous loci. An alternative reverse primer was designed to amplify the Y-chromosome specific STR in order to improve the specificity and applicability of this system to forensic genetics. Comparative results of the amplification with the new and the previously described primers proved that with this new primer there is a substantial increase in the specificity of the amplification. Moreover, a smaller fragment is amplified with a size out of the range of the alleles of the other Y-STRs usually used in forensic applications, therefore simplifying its inclusion in multiplex systems.     

Statistical examination of hair color as a potential biasing factor in hair analysis

We review eight different data sets in this paper for the purposes of assessing the possibility that reported color of hair can produce a systematic bias in the interpretation of hair assays. We review studies or data sets that include heroin and its metabolites, cocaine and its metabolites, MDMA and its analogs, and amphetamine and methamphetamine. The studies have utilized a variety of different degrees of color categorization, ranging from the simple dichotomy of brown and black, to a high of 12 categories. The mean number of categories reported approaches 6 (mean = 5.875). There are a total of 2791 data points in this analysis. We utilize two major statistical techniques for assessing significance; one-way analysis of variance, and Tukey's Honestly Significant Difference procedure. In circumstances were only dichotomous contrasts are possible, one-way analysis of variance is used. In contrasts involving three or more categorical groups, Tukey's procedure is used. In circumstances where the homogeneity of group variances is not sustained by the Levene statistic, we use the Tamahane procedure, allowing an assessment that assumes unequal variances. The analysis of this data fails to discern a significant color effect. We speculate that it may be that variance is large in many domains affecting analyte recovery from hair. In large groups these variations tend to regress towards a typical or mean value. Thus the data here show that while there are group or aggregate differences in these 'typical' values, they are not great when considered in relation to the within-group variations which exist for those values. It is our view that color may play a role in the accumulation of drugs in hair, however it is likely to account for only a very small part of the complex process of drug accumulation.     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.     

A biomechanical study of the human vertebral artery with implications for fatal arterial injury

We studied the biomechanical behaviour of ring and strip specimens from along the length of 18 vertebral arteries taken from 16 subjects aged 28-90 years, in order to consider some of the factors which may play a role in vertebral artery rupture. The method was chosen to allow a comparison between circumferential distension (ring samples) and longitudinal extension, (strip samples). The samples were extended between the jaws of a tensile testing apparatus until the specimen broke and a number of biomechanical parameters were derived. These were the percentage extension to break, the tensile strength, Young's modulus and the peak load. There were a number of important findings. The vertebral artery was shown to be susceptible to longitudinal stretching with a number of strip samples breaking when extended by as little as 16-20%. The tensile strength and load at peak of the strip specimens were correspondingly lower than for the ring samples. Marked intersubject variations were shown for all these parameters and prominent changes in behaviour occurred along the vertebral artery. This study indicates that the artery may be susceptible to head and neck movements which cause the vessel to stretch, and intersubject variations in behaviour may be one important explanation for the marked differences in outcome which appear to exist in subjects who suffer broadly similar head and neck insults.     

Computer-assisted facial image identification system using a 3-D physiognomic range finder

This system consists of a 3-D physiognomic range finder and a computer-assisted facial image superimposition unit. The 3-D range finder is composed of a detector for measuring facial surface and its control computer. The detector has two sinusoidal grating projection devices and two CCD cameras. The computer-assisted facial image superimposition unit consists of a host computer including a proprietary software, a flat surface color display and a color image scanner for inputting 2-D facial images of a criminal. The 3-D facial shape and texture of a suspect is obtained by using the range finder. To make the comparison between the 3-D facial image and the 2-D facial image, the 3-D facial image is first reproduced on a display of the host computer from a MO disk and then the 2-D facial image is taken with the color image scanner and reproduced on the display. The 3-D facial image is exactly adjusted to match the orientation and size of the 2-D facial image under the fine framework mode, and then the fine framework mode of 3-D facial image is converted to the fine texture image. The shape and positional relationships of facial components between the 3-D and 2-D facial images are examined by the fade-out or wipe image mode. The distance between the selected two points and angle among the selected three points on the 3-D and 2-D facial images are automatically measured for the assessment of anthropometrical data between both images. For evaluating the fit between the anthropometrical points on the 3-D and 2-D facial images, the reciprocal point-to-point difference between both images is compared.     

Post-mortem analysis of apoptotic changes associated with human skin bruises

The estimation of the age of skin bruises is of importance in forensic medicine, especially in child abuse cases. Time-dependent changes in bruise colour and/or associated histological features have been used with a limited degree of success. An increased rate of apoptosis in the injured tissue has been considered as a novel time-dependent marker of cell death, by injury inflicted in a rat model. The object of the present study was to apply the TUNEL method of DNA end labelling to identify and enumerate apoptotic cells in bruised and normal skin in order to study the relationship of apoptotic cell density with the age of the bruise. A commercially available DNA end labelling kit, TUNEL method, was standardised, validated and used for this purpose. Twenty unselected post-mortem cases with bruises due to a variety of causes were studied. The apoptotic cells stained with TUNEL reaction were counted in 10 high power fields in the epidermis, as well as in the dermis of formaldehyde-fixed paraffin-embedded skin specimens. The mean positive cell densities (+/- 1 S.E.) were compared with respect to the age of the bruise. In the epidermis, the mean apoptotic cell count was statistically significantly greater in the bruised skin compared to normal skin in 2- to 6-day-old bruises; whilst in the dermis the same was true in 3- to 8-day-old bruises. The overall findings suggest that there is a quiescent period prior to the increase in the apoptotic cell activity that is seen following skin bruising. This is so provided the post-mortem skin samples were collected within a lapse of 6 days or less between the time of death and formalin fixation and paraffin embedding to avoid the bias made by the difference of length of post-mortem interval.     

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.