A fast and safe non-bleaching method for forensic skeletal preparation

Over the last three decades, forensic anthropologists increasingly have consulted on fleshed human remains cases in which the examination of skeletal elements is critical in answering questions of identification and the circumstances of death. This was certainly the case at the Human Identification Laboratory in Tucson, Arizona. As the caseload increased, it became clear that a method for defleshing human remains was needed in order to expeditiously expose the osseous surfaces for analysis, yet at the same time, preserving the evidentiary nature of the material. As a result, a fast, safe and economical method for defleshing human remains and producing high quality, degreased skeletal elements was developed. This non-bleaching cooking method utilizes chemicals that are easily obtained and inexpensive standard household ingredients that can be purchased at most grocery stores.     

A rapid screening technique for the detection of spermatozoa

Phase contrast microscopy has been used for some time to search for and identify spermatozoa. An enhancement to the technique using xylene in conjuction with phase contrast microscopy is discussed. The method has been found to save time by allowing identification of spermatozoa with xylene-enhanced phase contrast microscopy in many cases that would have been unsuccessful using the normal "dry" phase contrast microscopy techniques. This eliminates a staining and reexamination step.     

Application of Lamendin's adult dental aging technique to a diverse skeletal sample

Lamendin et al. (1) proposed a technique to estimate age at death for adults by analyzing single-rooted teeth. They expressed age as a function of two factors: translucency of the tooth root and periodontosis (gingival regression). In their study, they analyzed 306 singled rooted teeth that were extracted at autopsy from 208 individuals of known age at death, all of whom were considered as having a French ancestry. Their sample consisted of 135 males, 73 females, 198 whites, and 10 blacks. The sample ranged in age from 22 to 90 years of age. By using a simple formulae (A = 0.18 x P + 0.42 x T + 25.53, where A = Age in years, P = Periodontosis height x 100/root height, and T = Transparency height x 100/root height), Lamendin et al. were able to estimate age at death with a mean error of +/- 10 years on their working sample and +/- 8.4 years on a forensic control sample. Lamendin found this technique to work well with a French population, but did not test it outside of that sample area. This study tests the accuracy of this adult aging technique on a more diverse skeletal population, the Terry Collection housed at the Smithsonian's National Museum of Natural History. Our sample consists of 400 teeth from 94 black females, 72 white females, 98 black males, and 95 white males, ranging from 25 to 99 years. Lamendin's technique was applied to this sample to test its applicability to a population not of French origin. Providing results from a diverse skeletal population will aid in establishing the validity of this method to be used in forensic cases, its ideal purpose. Our results suggest that Lamendin's method estimates age fairly accurately outside of the French sample yielding a mean error of 8.2 years, standard deviation 6.9 years, and standard error of the mean 0.34 years. In addition, when ancestry and sex are accounted for, the mean errors are reduced for each group (black females, white females, black males, and white males). Lamendin et al. reported an inter-observer error of 9+/-1.8 and 10+/-2 sears from two independent observers. Forty teeth were randomly remeasured from the Terry Collection in order to assess an intra-observer error. From this retest, an intra-observer error of 6.5 years was detected.     

The effects of skeletal preparation techniques on DNA from human and non-human bone

The forensic pathologist increasingly relies on the forensic anthropologist to be the consulting expert in human identification. Likewise, if identification is not possible from visual inspection of skeletal remains, the forensic biologist may be called upon to conduct DNA analysis. The possibility of downstream DNA testing needs to be considered when skeletal preparation techniques are employed to deflesh human remains, as they have the potential to strongly impact genetic analyses and subsequent identification. In this study, three cleaning techniques, boiling bone in water, in bleach, and in powdered detergent/sodium carbonate, were tested for their effect on nuclear and mtDNA recovery from a variety of human and non-human bones. A statistically significant reduction in DNA yields occurred in non-human bones cleaned with bleach, and DNA degradation was apparent electrophoretically. The human bones also showed much lower yields from bleach cleaning, while the detergent/carbonate method allowed the largest segments of DNA to be amplified, indicating it may have a less degradative effect on bone DNA than either of the other cleaning processes.     

A fast preparation of skeletal materials using enzyme maceration

The current study investigates the removal of soft tissues from mice and rats by the use of three different proteases and one lipase from Novozymes A/S. The results demonstrate the enzyme maceration to be remarkably fast (1-3 h) compared to the traditional warm-water procedure, which requires up to several days. In addition, the enzyme maceration eliminates the odor problem associated with the traditional procedure. It is shown that stirring of the enzyme maceration bath is the main factor which determines the speed of the maceration. For mice, the time required for enzyme maceration can vary from 1 to 8 h depending on the stirring speed. The method investigated here allows preparation of skeletal material in an essentially odorless way within a matter of hours, making the method useful in particular for forensic science, private conservation workshops, and educational purposes.     

A hybrid ampholyte focusing technique for esterase D subtyping of evidentiary material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).     

A simple technique for age estimation in adult corpses: the two criteria dental method.

A method for age determination of adults from single rooted teeth is presented. It is based on the measurement of two dental features: periodontosis height times 100/root height (P) and transparency of the root height times 100/root height (T). These measurements are made on the labial surface of the entire tooth without section and do not require special equipment or training. The application of multiple regression analysis to a working sample of 306 teeth of known age, sex and race provided the following equation: Age (years) = 0.18 x P + 0.42 x T + 25.53. The mean error between the actual and estimated age was +/- 10 years on the working sample and +/- 8.4 years on a control sample made of 45 forensic science cases. Upper incisors showed a better precision than the other single rooted teeth and accuracy was not sex related. A comparison of the Gustafson and Lamendin methods on a control sample of 39 teeth resulted in an advantage of the latter considering the mean error on the estimation (14.2 +/- 3.4 years for Gustafson versus 8.9 +/- 2.2 for Lamendin). The Lamendin method can be practical interest for any forensic pathologist or dentist as it is fast, easy to use, and reasonably accurate except for cases of individuals under age 40 where other methods must be preferred.     

Sex allocation of skeletal material by analysis of the proximal tibia.

In this study we contrasted the confidence with which individuals may be grouped and then reallocated on the basis of a set of measurements from the proximal tibia. The data were derived from 100 Caucasoid (50 male) and 102 Negro (50 male) tibia housed in the R.A. Dart Collection. Multivariate discrimination was effected by means of canonical and stepwise discriminant analyses, whilst probabalistic models were used to allocate individuals. High levels of correct classification (84.62-92%) were matched by high levels of reallocation, suggesting that, in contrast to dental or craniobasal measurements, those of the proximal tibia may be usefully employed to allocate an individual on the basis of sex.     

A simple method for preparing human sketetal material for forensic examination

A method using dilute sodium hypochlorite for the cleaning of human skeletal material from decomposed bodies is described. With this simple procedure, which requires minimal expenditure of time and money, bones may be cleaned in a manner that produces specimens satisfactory for forensic examination, documentation of injuries, and jury presentation.     

Differentiation of fetal and adult bloodstains by pyrolysis-gas-liquid chromatography

A pyrolysis-gas-liquid chromatographic method has been developed for the differentiation of adult and fetal bloodstains. In a blind-coded study, five adult and three fetal bloodstains were correctly identified on the basis of the pyrograms of stain extracts. The differentiation between adult and fetal bloodstains is based on the peak height ratio of two long-retention-time peaks appearing in their pyrograms. The first of these peaks has been tentatively identified as indole derived from the pyrolysis of tryptophan, while the second peak is an as-yet unidentified molecular fragment produced by the pyrolysis of some component of the hemoglobin molecule other than the amino acids tryptophan, tyrosine, and phenylalanine.     

An ideal material for the preparation of known toolmark test impressions

Traditionally, toolmark test exemplars are produced by applying a tool's working surface to a piece of soft metal such as lead. Soft, pliable metals are primarily used for this purpose because they will replicate the microscopic grooves present on a tool's working surface without damaging the tool. In this paper the authors present an alternative material for the preparation of test toolmarks. Jewelry modeling or carving waxes are utilized in this study. These waxes are designed for the jewelry modeling industry to create very fine, highly detailed wax models of jewelry pieces that will be cast in various metals utilizing the lost wax casting method. Jeweler's waxes have been found to be ideal for preparing test toolmarks from exemplar tools. The test tool's working surface is applied to a piece of the appropriate wax in a manner consistent with the tool's design. The replicas obtained are exact, highly detailed, 1:1, negative impressions of the exemplar tools working surface, have a long shelf-life, and are suitable for use in toolmark examination and comparison cases.     

Tests of genetic markers on aborted fetal material

Women who conceive as a result of rape often elect to abort the fetus. We describe twelve cases where genetic markers were tested on the aborted fetal material to provide evidence of the genetic constitution of the rapist. Two cases are presented in detail, and the problems encountered with the testing are discussed.     

Advanced technique for the preparation of bone and tooth tissue for molecular-genetic expert tests

Laboratory schemes related with the procedure of purifying the osseous and dental samples by applying the dental drilling machine, dental technical carborundum shapes, fogging discs and hard-alloy dental drills were made use of to improve the quality and effectiveness of the mentioned schemes used prior to molecular-and-genetic expertise tests.     

A validation study for the extraction and analysis of DNA from human nail material and its application to forensic casework.

A validation study was conducted to demonstrate that deoxyribonucleic acid (DNA) could be successfully extracted from human nail material and analyzed using short tandem repeat (STR) profiling and/or mitochondrial DNA (mtDNA) sequencing. This study involved the development of a DNA extraction protocol that includes a cleaning procedure designed to remove external contaminants (e.g., biological, chemical). This protocol was used to test human nail material that had been soaked in whole blood from a second donor and coated with gold-palladium to simulate scanning electron microscopic analysis. The results showed no indication of a mixture and were consistent with that of the nail donor. Fresh human nail material usually yielded both STR profiles and mtDNA sequence information; however, aged human nail material (approximately eight years old) yielded only mtDNA sequence information. Upon completion of the validation study, the extraction protocol was used for the analysis of a torn fingernail fragment recovered from the scene of a violent homicide in 1983. A partial STR profile and mtDNA sequence information indicated that the fingernail fragment was excluded as originating from the suspect and was, in fact, consistent with originating from one of the victims.     

A sandwich enzyme immunoassay for human muscle-specific beta-enolase and its application for the determination of skeletal muscle injury

A sensitive sandwich enzyme immunoassay for human beta-enolase was developed and used to examine beta-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human beta-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human beta-enolase IgG were incubated with human beta-enolase and then with anti-human beta-enolase Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human beta-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of beta-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that beta-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing beta-enolase was recovered well after storage for 60 days at room temperature. The ratio of beta-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of beta-enolase in bloodstains from traumatic blood was obviously elevated (10-30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of beta-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of beta-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that beta-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

A simple technique for imaging the human skeleton using a flatbed scanner

A simple technique for imaging the human skeleton with a flatbed scanner is presented using the auricular surface of the ilium as an example. A flatbed scanner with resolution capabilities of 600 dpi or greater allows for images of human bones. The auricular surface of the ilium was selected to demonstrate this technique as it is a fairly three-dimensional area that can be difficult to record photographically. Fifty left ilia of various ages at death from the Athens Collection were selected from which three observers (SCF, CE, and IM) scored the morphology of the auricular surface using a well-established aging method. Observations were taken of the dry bone, of digital photographs of the bone, and of scanned images of the bone, and in that sequence. Results indicate that scores of scanned images are equivalent or better than digital images of the same ilia. This technique allows for sharing data electronically with ease.