Aurora Valenzuela

The matrix metalloproteinase gelatinase A in human dentine

A dentine protein extraction protocol was modified in order to identify matrix metalloproteinase gelatinolytic activities in the non-mineralized and mineralized phases of human dentine. Dentine proteins from 24 individual permanent molars from patients aged 15-73 years were sequentially extracted, first with guanidinium chloride (G1 extract), then EDTA (E extract), and after this demineralization step, again by guanidinium chloride (G2 extract) to dissociate collagen-associated proteins. Extracts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the gels were processed by Western blotting and zymography to detect gelatinolytic activities. Active and latent forms of gelatinase A were identified in the non-mineralized dentine fraction (G1 extract) of 58% of the teeth. Other gelatinolytic species were also detected by zymography with apparent M(r) of 92, 54 and 30 kDa. Although gelatinase A was detected in the G1 extracts of teeth from all ages, indicating more recent synthesis and remodelling of the predentine, gelatinase A was never detected in any E extract or in the G2 extracts of patients older than 41 years. The presence of the active form of gelatinase A in mineralized human dentine implicates this enzyme in dentine mineralization.

An Improved Method to Recover Saliva from Human Skin: The Double Swab Technique

Human bite mark evidence is often found in violent crimes. Due to the difficulties of physically comparing an injury site on elastic and curved skin surfaces to the teeth of a suspect, the authors have considered using salivary DNA evidence to identify the bite perpetrator. Several techniques were evaluated to determine the best method of recovering saliva from human skin before extracting genomic DNA from the collection substrate. A classical stain recovery technique using a wet cotton swab was tested against one utilizing a wet filter paper. Additionally, a new method, referred to as the double swab technique, using a wet cotton swab followed by a dry cotton swab was also evaluated. After recovering a dried saliva stain, DNA was extracted using the modified Chelex method, quantified using the slot-blot procedure, and amplified at three polymorphic loci. The double swab technique showed the highest percentage recovery of saliva from human skin among the three methods studied. This technique is suggested as an improvement over the classical single wet cotton swab technique.

Increasing DNA extraction yield from saliva stains with a modified Chelex method

Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

The application of dental methods of identification to human burn victims in a mass disaster

Abstract This paper deals with the usefulness of dental identification in the case of 28 human burn victims of a bus accident in Spain. Postmortem forensic procedures for identification were used including general external examination, routine photographs and radiographs and complementary biological methods. Dental identification was also used and a description of the method is presented. Dental identification was established in 57% of the cases. When victims were less than 20 years of age, the success rate of identification by dental methods was higher (76% of victims in this age group). The assessment of dental age allowed the establishment of identity of four victims. We recommend that dental procedures be used in human identification after mass disasters. Odontological and radiographic procedures are powerful methods when dealing with burn victims.

PCR-Based DNA Typing of Saliva Stains Recovered from Human Skin

Human bites in cases of homicide, sexual assault, and abuse are often distorted due to the elasticity and curvature of the skin. Physical comparison of a bite mark to a suspects teeth is sometimes difficult. Saliva, which is usually deposited during biting, can be collected and analyzed to identify the perpetrator. Using simulated bite mark situations in two experimental series, three samples of 40 microL of whole saliva were deposited on the skin of 27 cadavers (at 33 sites) and three samples of 100 microL of whole saliva were deposited on the skin of 5 cadavers (at 12 sites). Saliva was collected using the double swab technique at t = 5 min, t = 24 h, and t = 48 h. DNA was extracted using the modified Chelex method and submitted to PCR-based typing at two short tandem repeat loci. Results indicate that the concentration of DNA in saliva recovered from skin varies as a function of time since deposition. There is a significant decrease in concentration in the first 24 h but the concentration remains stable from 24 to 48 h. The success of PCR amplification is independent of the time since deposition or the concentration of DNA in the saliva sample. Contamination from the DNA of the cadaver was not found in any of the cases studied.

Computer-based production of comparison overlays from 3D-scanned dental casts for bite mark analysis.

Bite mark analysis assumes the uniqueness of the dentition can be accurately recorded on skin or an object. However, biting is a dynamic procedure involving three moving systems, the maxilla, the mandible, and the victims reaction. Moreover, bite marks can be distorted by the anatomic location of the injury or the elasticity of the skin tissue. Therefore, the same dentition can produce bite marks that exhibit variations in appearance. The complexity of this source of evidence emphasizes the need for new 3D imaging technologies in bite mark analysis. This article presents a new software package, DentalPrint (2004, University of Granada, Department of Forensic Medicine and Forensic Odontology, Granada, Spain) that generates different comparison overlays from 3D dental cast images depending on the pressure of the bite or the distortion caused by victim-biter interaction. The procedure for generating comparison overlays is entirely automatic, thus avoiding observer bias. Moreover, the software presented here makes it impossible for third parties to manipulate or alter the 3D images, making DentalPrint suitable for bite mark analyses to be used in court proceedings.

Objective measurement of dental color for age estimation by spectroradiometry

Age-related changes in tooth color have been described previously, however, the use of dental color for age estimation in forensic odontology has been limited due to the difficulty of measuring color objectively. This study presents an objective method for determining dental color to estimate the age of an individual. Dentine color in 250 teeth from patients ranging in age from 10 to 89 years was determined by spectroradiometry. Color measurements were performed as suggested in the CIE 1931 (International Commission on Illumination). Chromaticity coordinates (x, y, z), luminance (Y), whiteness index (WIC, Z%, WIC) and yellowness index (YI) were obtained. Correlations between these colorimetric variables and aging were established by linear regression analyses. All the variables fit the mathematical model with correlation coefficients ranging from 0.53 to 0.75. This method of color measurement produced an expected associated error of calibration averaging 13.7 years about the mean estimated values, at a 70% level of confidence. Two different multiple regression models for dental age estimation were tested, and variables that made the greatest contributions to age calculation were identified. To determine the effect of postmortem interval on tooth color and its influence in age estimation, 37 teeth obtained from human skeletal remains buried during an interval ranging from 21 to 37 years were also studied. In this material, the correlation between age and dental color measured by spectroradiometry was weaker than in fresh extracted teeth. It is concluded that determination of dentine color by spectroradiometry is a potentially useful objective method to estimate age in forensic studies in combination with other methods.

Differences in Morphological Age-Related Dental Changes Depending on Postmortem Interval

Morphological methods for age estimation from teeth have been developed and applied to samples without taking the postmortem interval into consideration. We studied differences in morphological age-related changes between fresh extracted teeth and teeth from human skeletal remains in order to develop appropriate dental age estimation methods according to the time after death. Forty-three permanent teeth from dental patients were compared to 37 teeth obtained from human skeletal remains with a postmortem interval from 21 to 37 years. Morphological age-related changes were investigated by measuring variables on intact and half-sectioned teeth. A new computer assisted image analysis procedure to avoid subjectivity was developed to measure variables in sectioned specimens. Dental color, translucency length, attrition, cementum apposition, and secondary dentin showed higher values in teeth from human skeletal remains than in fresh extracted teeth. Variables obtained by morphometric analysis of computer-generated images (tooth length, tooth width, root length, and root area) showed higher values in fresh extracted teeth than in teeth from skeletal remains. The postmortem interval affects age-related morphological changes, and therefore different methods should be used for teeth of unknown postmortem interval.

Sudden cardiac death: A comparative study of morphological, histochemical and biochemical methods

The study deals with the comparison of morphological, histochemical and biochemical methods applied to the detection of myocardial infarction in 150 medico-legal autopsies performed at the Institute of Forensic Pathology in Copenhagen. The study also included an NBT (formazan) test of cardiac cross-sections, and light microscopy and fluorescence microscopy of acridine orange-stained specimens from four different sites of the cardiac musculature. Specimens of myocardium from the same four sites and pericardial fluid were analysed biochemically at the Institute of Legal Medicine in Granada. The K+/Na+ ratio was determined in the myocardial tissue and total creatine phosphokinase activity, creatine phosphokinase isoenzymes (MM, MB and BB) and myoglobin were assayed in pericardial fluid. When the results from Copenhagen and Granada were compared, there was absolute concordance in 96 cases, discrepancy in 53 and one case was inconclusive. After studying the circumstances of death, the number of discrepancies were reduced to 20, so that concordance was reached in 86% of all the cases. The results show that the combination of different methods leads to a diagnosis of myocardial infarction in far more cases than with morphological or biochemical methods alone.

Effectiveness of Comparison Overlays Generated with DentalPrint© Software in Bite Mark Analysis*

ABSTRACT: Validation studies of the new DentalPrint© software were carried out with experimental bite marks in pigskin. The bite marks were digitally photographed according to the ABFO guidelines for evidence collection. Dental casts used in the experiment were scanned in 3D and 2D, and comparison overlays were generated using DentalPrint© and Adobe® Photoshop® software, respectively. Digitized photographs of the experimental bite marks and the biting edges obtained in the overlays were compared by two different examiners to analyze the impact of training and experience with the two methods. Receiver operating characteristic (ROC) analysis, sensitivity, specificity, and 95% confidence intervals for each cutoff point were calculated. The expert examiner using DentalPrint© obtained the best results, with an area under the ROC curve of 0.76 (SE=0.057; CI at 95%=0.652–0.876). Fairly high specificity values were found for DentalPrint©, and the best results were obtained for the cutoff value that discriminated between the examiners response “biter” and the rest of the possible (specificity 97.9%, CI at 95%=93.2%–99.6%). Therefore, the results presented here indicate that DentalPrint© is a useful, accurate tool for forensic purposes, although further research on the comparison process is needed to enhance the validity of bite mark analysis.