Sex determination from plucked human hairs without epithelial root sheath. II. Depigmentation of melanin in the hair cortex before Feulgen reaction

In hair roots devoid of the epithelial root sheath, an attempt was made to decolorize the melanin granules without affecting the Feulgen reaction for the sex chromatin. The hair samples were treated with 0.25% potassium permanganate for 1 hour, 0.3% hydrogen peroxide for 1 minute and 5% oxalic acid for 5 minutes, and then stained with Feulgen. The frequency of sex chromatins ranged from 22% to 47% (average 32%) in female samples and from 0% to 8% (average 5%) in male samples. Thus, the frequency distributions of the male and female samples were completely independent of each other. The sex chromatins in dried female hairs were detectable at a frequency of 16 – 26% several weeks after plucking. The depigmentation procedure almost completely bleached the melanin granules in the hair cortex, and produced no harmful effect on the Feulgen reaction that followed.     

Sex determination of human blood micro-stains and single hairs using specific DNA amplification with polymerase chain reaction

Amplification of Y chromosome specific DNA in vitro enables a rapid and reliable sex determination of human minute traces such as blood stains and hairs. In presence of male DNA a band of 154 bp is visualized by agarose gel electrophoresis after amplification, this band is lacking in case of female DNA alone. Amplification of a sex independent DNA locus (such as a fragment from the alcohol dehydrogenase gene) generates identical reaction products for both sexes. This shows that the absence of a band is not due to the lack of trace DNA. It is possible to perform this technique with as little as 0.5 microliters of blood or with a single hair.     

单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

Sex determination from the talus of Koreans by discriminant function analysis

The aims of this study were to investigate the sex discriminating potential of the talus in Koreans and compare this with other analyses in different populations. Statistical analyses were performed using data from nine measurements acquired from 140 tali (70 men, 70 women). The talus of Koreans is dimorphic between sexes in all measurements (p < 0.01). Discriminant function equations were generated by univariate, multivariate, and stepwise methods with a range of accuracy from 67.1 to 87.1%. Stepwise equations of other populations did not discriminate the sex of the Korean sample as accurately as each equation's own accuracies. The variables with high accuracy in this study are useful for sex determination of Koreans on the basis of confirmation of population specificity.     

Sex determination from the radius and ulna in a modern South African sample

With a large number of unidentified skeletal remains found in South Africa, the development of population specific osteometric standards is imperative. Forensic anthropologists need to have access to a variety of techniques to establish accurate demographic profiles from complete, fragmentary and/or commingled remains. No research has been done on the forearm of African samples, even though these bones have been shown to exhibit sexual dimorphism. The purpose of this paper is to develop discriminant function formulae to determine sex from the radius and ulna in a South African population. The sample consisted of 200 male and 200 female skeletons from the Pretoria Bone (University of Pretoria) and Raymond A. Dart (Witwatersrand University) collections. Sixteen standard anthropometric measurements were taken from the radius (9) and ulna (7) and subjected to stepwise and direct discriminant function analysis. Distal breadth, minimum mid-shaft diameter and maximum head diameter were the best discriminators of sex for the radius, while minimum mid-shaft diameter and olecranon breadth were selected for the ulna. Classification accuracy for the forearm ranged from 76 to 86%. The radius and ulna can be considered moderate discriminators for determining sex in a South African group. However, it is advised that these formulae are used in conjunction with additional methods to determine sex.     

Sex determination from the distal part of the femur in a French contemporary population

Until now, determining the sex of a recently deceased individual using the measurement of the bicondylar breadth of the femur (also known as condylar width, epicondylar breadth and distal epiphyseal breadth) raised some concerns as to accuracy because no sample of contemporary French subjects was available. In this study, a sample of 88 female and male femurs taken from recently deceased elderly French people was studied. The bones were collected from subjects who had donated their bodies to the Medical School of Nice. The mean value of the male bicondylar breadth was found to be greater than that of females (84.3mm versus 74.8mm), confirming the sexual dimorphism of this parameter. Furthermore, the results showed a 95.4% accuracy rate for sexing individuals. To date, in the French population, as in some other samples, epicondylar breadth is the single most accurate measurement of sex determination, ahead even of head diameter. A discriminant function is presented to allow sex determination from remains of the distal femur. With regard to the data available in the literature, sexual dimorphism is probably the result of both genetic and environmental factors. The comparison of our results with those of other populations shows that there are inter-population variations of the bicondylar breadth, and also intra-population variations that account for the differences in the accuracy rate of this variable for the purposes of sex determination. These findings underscore the need to re-evaluate bone measurements within various contemporary populations.     

Sex determination and estimation of stature from the long bones of the arm

The determination of sex and the estimation of stature from bones play an important role in identifying unknown bodies, parts of bodies or skeletal remains. In medico-legal practice statements on the probable sex of a decomposed body or part of a body are often expected even during autopsy. The present study was, therefore, restricted to few easily accessible dimensions from bones which were prepared only by mechanically removing soft tissues, tendons and ligaments. The specimens came from the Anatomical Institutes in Munich and Cologne from the years 1994-1998 including a total of 143 individuals (64 males and 79 females). The mean age was 79 years (46-108), the mean body height 161cm (134-189). The following measurements were taken: maximum humeral length (mean: 33.4cm in males; 30.7cm in females), vertical humeral head diameter (mean: 5.0cm in males, 4.4cm in females), humeral epicondylar width (mean: 6.6cm in males; 5.8cm in females), maximum ulnar length (mean: 26.5cm in males, 23.8cm in females), proximal ulnar width (mean: 3.4cm in males, 2.9cm in females), distal ulnar width (mean: 2.2cm in males; 1.8cm in females), maximum radial length (mean: 24.6cm in males; 22.0cm in females), radial head diameter (mean: 2.6cm in males, 2.2cm in females) and distal radial width (mean: 3.6cm in males; 3.2cm in females). The differences between the means in males and females were significant (P<0.0005). A discriminant analysis was carried out with good results. A percentage of 94.93% of cases were correctly classified when all measures of the radius were applied jointly, followed by humerus (93.15%) and ulna (90.58%). Applied singly, the humeral head diameter allowed the best distinction (90.41% correctly grouped cases), followed by the radial length (89.13%), the radial head diameter (88.57%) and the humeral epicondylar width (88.49%). The linear regression analysis for quantifying the correlation between the bone lengths and the stature led to unsatifactory results with large 95%-confidence intervals for the coefficients and high standard errors of estimate.     

Sex determination from the head of the femur of South African whites and blacks

The current practice whereby criminals dismember the remains of their victims in an attempt to make their identification difficult requires that simple methods of sex determination from fragmented skeletal remains are available to forensic anthropologists and skeletal biologists. The head of the femur is an example of such bone fragments. Identification and demarking points have been derived from the diameters of the head of the femur and used to determine the sex of individuals. It has been shown, however, that the numerical values of these parameters that permit sex identification vary between races. The objectives of the present study were therefore to establish the standard numerical values of the identification and demarking points for sex determination in South African whites and blacks and to see if these standards are different in the two races. A total of 520 femurs of white (160 males and 100 females) and black (160 males and 100 females) South Africans were obtained from the Raymond Dart Skeletal Collection in the Department of Anatomical Sciences of the University of the Witwatersrand, Johannesburg, South Africa. The vertical and transverse diameters of the heads of the femurs were measured by means of a stainless steel vernier caliper. Identification and demarking points were derived from the values of these diameters. The head diameter identification point and demarking point were found to be sexually dimorphic in both white and black South Africans. The mean head diameter of the male femur was significantly greater than the mean head diameter of the female femur in both population groups (significant at P<0.001). These values were correspondingly greater in the white than the black population. The numerical values of the male identification and demarking points were higher than the corresponding female values in the two population. In both sexes, these values were greater in the whites than the blacks South Africans. It is concluded that the diameters of the head of the femur and the identification and demarking points that are derived from them are sexually dimorphic in South African white and black populations. However, the numerical values of these sex-determining bone parameters defer between the two population groups. Therefore, it is necessary to determine race-specific standards of these parameters.     

Studies on the frequencies of PGM1, PGM3 and Es-D types from hair roots in Japanese subjects and the determination of these types from old hair roots

The frequencies in Japanese subjects are reported of hair roots type PGM₁, PGM₃ and Es-D, and the determination of these types from old hair roots. The gene frequencies were: PGM₁¹, 0.762; PGM₁², 0.230; PGM₁⁷, 0.008; PGM₃¹, 0.621; PGM₃², 0.379; Es-D¹, 0.625; and Es-D², 0.375. The old hair roots were analysed after storage at 25 °C, 4 °C and ?80 °C; the enzyme activities were detected and typed at 25 °C within PGM₁ 10 days, PGM₃ 4 days, and Es-D 4 days.     

Preparation and application of a fortified hair reference material for the determination of methamphetamine and amphetamine

Quality assurance is one of the major issues in forensic analytical laboratories, where the need for a reference material (RM) has rapidly increased. RMs are very useful for method development and validation, internal quality control or proficiency tests. In the present study, we prepared a RM using drug-free hair for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) according to the recommendations of ISO Guide 35. The concentrations of MA and AP were determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC/MS) after derivatization with trifluoroacetic anhydride (TFAA). The assignment of values was conducted through the homogeneity study and characterization of the material. Furthermore, an internal proficiency test was performed with the prepared RM, of which the results were compared with those of the authentic hair RM prepared in our previous study. As a result, a hair RM containing MA and AP was prepared at the level of 4.86+/-0.69 ng/mg and 4.63+/-0.44 ng/mg, respectively. Most participants showed satisfactory performances in the internal proficiency test with the both RMs. The hair RM prepared in this study demonstrated its suitability for quality assurance in forensic laboratories.     

Detection of methamphetamine and amphetamine in a single human hair by gas chromatography/chemical ionization mass spectrometry

A detailed procedure of an extremely sensitive method for quantitation of methamphetamine and amphetamine in human hair by gas chromatography (GC)/chemical ionization (CI) mass spectrometry (MS) is presented. N-methylbenzylamine was used as an internal standard. The samples, after extraction with an organic solvent, were derivatized with trifluoroacetic anhydride before the GC/MS analysis. Quantitation was made with quasi-molecular ions of the derivatives by selected ion monitoring in the CI mode. The detection limit was about 10 pg in an injected volume. The high sensitivity enabled us to measure both stimulants in a single human hair in actual cases.     

Solid-phase microextraction for the determination of cocaine and cocaethylene in human hair by gas chromatography-mass spectrometry

A method for the simultaneous determination of cocaine (COC) and cocaethylene (CE) in human hair was developed, using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) as analytical technique to identify and quantify the drugs. Selected ion monitoring (SIM) mode was used to obtain higher sensitivity. The deuterated-labeled analogues were used as internal standards. The detector response was linear for the drugs studied over the range 0.4-15 ng/mg, with correlation coefficients higher than 0.995. The coefficients of variation oscillated between 0.65% and 14.18% and the accuracy was in the range from 0.73% to 11.20%. The limits of quantitation and detection were found to be acceptable. Finally, this method was applied to 15 hair samples from cocaine users, obtaining positive results in all cases. The mean concentrations were 5.39 ng/mg (range: 0.43-8.98 ng/mg) for cocaine and 1.11 ng/mg (range: 0.42-2.23 ng/mg) for cocaethylene.     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.     

Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC-MS

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC-MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-micron polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0-50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.     

Enhancement of an insufficient dye-formation in the ninhydrin reaction by a suitable post treatment process

A study of the reaction between ninhydrin and alanine has been carried out taking into account that, adhered on the surface of a dry porous medium such as paper, a quasi-heterogeneous reaction has to take place. Instead of amino acids released from human sweat glands, aqueous solutions of alanine were taken to deliver a given amount of amino acid to the sample. The dye density, obtained after using a standard development process, could noticeably be increased by setting a drop of water on the dye dot, thus indicating that not all the alanine had been used for dye formation during the usually applied process. The incomplete reaction can be explained by the problem of bringing the reactants into contact with each other when both are in the solid phase in the porous surrounding. The temporary presence of water allows a reorientation of the insoluble reactants. With fingerprints an increase in both the rate of development and the final dye density could be obtained when the sample was post treated after the developing process with the blank solvent, thus also the background coloration could be decreased. The ideas presented in this paper may form the basis for a modification of developing processes with ninhydrin in order to increase the proportion of amino acids present (in the sample) used in dye formation without data loss.     

Elucidation of the effect of heat exposure on hair colored by permanent and semipermanent colorants using surface-enhanced Raman spectroscopy

Confirmatory identification of hair colorants can be used to establish a connection between a suspect and the crime science or demonstrate the absence of such connections. A growing body of evidence shows that surface-enhanced Raman spectroscopy (SERS) could be a confirmatory, minimally destructive, and fully noninvasive analysis of hair colorants. In SERS, a signal that provide the information about the chemical structure of both permanent and semipermanent dyes present on hair is enhanced by a million-fold using noble metal nanostructures. However, it is unclear whether the information of hair colorants can be revealed if hair was contaminated or exposed to harsh environments such as sunlight and heat. In this work, we determine the effect of a short- and long-term heat exposure on SERS-based analysis of hair colored with blue and red permanent and semipermanent dyes. We found that short and especially long-term heat exposure at 220°C could alter chemical structure, and consequently SERS spectra, of permanent and semipermanent colorants. This thermal degradation of permanent dyes complicates their direct identification using SERS. We also found that partial least squares discriminant analysis can be used to overcome this issue allowing for highly accurate identification of both permanent and semipermanent dyes on colored hair that was exposed to 220°C for 6–12 min. These results show that heat exposure of colored hair should be strongly considered upon their SERS-based examination to avoid both false positive or false negative identification of chemical dyes.