Stable isotope analysis of human hair and nail samples: the effects of storage on samples

When submitting samples for analysis, maintaining sample integrity is essential. Appropriate packaging must be used to prevent damage, contamination or loss of sample. This is particularly important for stable isotope analysis by isotope ratio mass spectrometry as this technique is capable of detecting subtle differences in isotopic composition with great precision. In a novel study, scalp hair and fingernail samples were placed in five different types of packaging, routinely used in forensic laboratories and stored for 6 weeks and 6 months. Samples were subsequently cleaned and submitted for (13)C/(12)C, (15)N/(14)N, (2)H/(1)H and (18)O/(16)O analysis. Results from (13)C analysis indicate that type of packaging can cause slight changes in (13)C abundance over time. Differences were noted in the (15)N isotope signatures of both hair and nail samples after 6-week storage, but not after 6 months. This apparent discrepancy could be a result of the packaging not being properly sealed in the 6 weeks study. Fewer differences were noted when analyzing samples for (2)H and (18)O abundance.     

A study of ten red cell enzymatic markers in the Naples' population. Report of a new GPT variant phenotype

A sample of the population of Naples has been examined for several red cell enzyme markers. About 2,000 newborn have been analyzed for ACP, GLO I, and UMPK; 1,000 of them were also analyzed for PepA and PepB, and 500 for PGM1 and PGM2. In addition about 400 school children have been typed for the PGD and PGP polymorphisms. The observed gene frequencies for the polymorphic systems are: ACPA = 0.293, ACPB = 0.667 and ACPC = 0.040; GLO1 = 0.372; GPT2 = 0.462; UMPK2 = 0.029; PGM21 = 0.279; PGDC = 0.037; PGP1 = 0.953, PGP2 = 0.038 and PGP3 = 0.009. Moreover during the screening of PepA, PepB and GPT markers, some rare alleles have been encountered, one of which, at the GPT locus, has never been reported before. We propose for it the name GPT10.     

Simultaneous phenotyping of genetic markers for paternity testing

Time-and cost-saving methods for paternity testing are described. Seventeen genetic systems were divided into six groups: (1) transferrin (Tf), factor B (Bf), and phosphoglucomutase 1 (PGM1); (2) group-specific component (Gc) or alpha 1-antitrypsin (PI) and alpha 2HS-glycoprotein (HSGA); (3) complement components C6 and C7, factor 13B (F13B), and plasminogen (PLG); (4) haptoglobin (Hp), C8 alpha-gamma chain (C81), and factor I (IF); (5) red cell acid phosphatase (ACP), esterase D (ESD), and glutamic-pyruvic transaminase (GPT); and (6) 6-phosphogluconate dehydrogenase (PGD) and glyoxalase I (GLO). Each group of systems was typed simultaneously by electrophoresis or isoelectric focusing (IEF) followed by staining or immunoblotting. These methods are very practical because they afford a considerable saving of time, work and expense, and facilitate semipermanent preservation of electrophoretic patterns.     

Surface-enhanced Raman spectroscopy enables confirmatory detection of dyes on hair submerged in hypolimnion water for up to twelve weeks

Difficulties in the localization of bodies of homicidal or drowning victims in natural water result in their submergence for weeks if not months. Water insects and microbes drastically change the body's appearance, which significantly changes the determination of a victim's identity. DNA analysis is commonly used for identifying the decedent; however, this PCR-based approach is time-consuming and destructive of the evidence. Considering that nearly half of the people in the world dye their hair with a variety of permanent and semi-permanent dyes, one can expect that confirmatory identification of dyes on the body's hair can be used to shed light on the victim's identity. A growing body of evidence suggests that surface-enhanced Raman spectroscopy (SERS) can be used to detect and identify hair dyes. In this study, we investigated the extent to which SERS could be used to detect black and blue, permanent and semi-permanent dyes on hair submerged in hypolimnion water for up to twelve weeks. We found that SERS enabled 100% accurate identification of analyzed dyes on hair submerged in hypolimnion water for up to 8 weeks, whereas, on average, 87% accurate identification of the hair dyes could be achieved on hair exposed for 10 weeks and 50% for hair exposed 12 weeks in hypolimnion water. We also found that the aqueous environment caused progressive fading of some dyes, whereas other dyes showed substantial spectral transformations after prolonged submergence. Finally, we found that changes in the intensity of vibrational bands of dyes could be used to predict the duration of submergence of colored hair in hypolimnion water.     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

液氮冷冻单根毛发根直接PCR进行DNA分型

首次用液氮冷冻单根毛发根，再经三次94℃高温处理，直接PCR扩增，从68根毛发根中，成功地检测了62根毛发根的人类vWF基因40内含子VNTR，检出率达91．2％。本方法简便、快速、实用，在法医学检验及群体遗传学研究中有重要应用价值。     

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.     

Hair testing for drugs of abuse: evaluation of external cocaine contamination and risk of false positives.

In some laboratories hair testing may be the main method for the evaluation of individual's drug history, however, compelling evidence supports the possibility that the presence of a small amount of drug in hair can derive from external contamination. The aim of the present study is to verify if a single external contamination with a small amount of cocaine will last sufficiently long to make a contaminated subject indistinguishable from active users, and if normal washing practices together with the decontamination procedures are sufficient to completely remove the external contamination. The results obtained using the decontamination methods suggested in literature demonstrate that significant concentrations of cocaine (>1 ng/mg) and moderate quantities of benzoylecgonine (generally <0.5 ng/mg) are still detectable up to 10 weeks after contamination. These results question the reliability of hair testing. In fact, even using the most sophisticated decontamination procedures it is not possible to distinguish a drug-contaminated subject from an active user. Thus, while a negative result excludes both chronic use and "contact" with drugs, a positive result cannot and must not be interpreted as a sure sign of drug addiction, but should be further confirmed by urine analysis.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.     

Ofloxacin in human hair determined by high performance liquid chromatography.

A procedure is presented for quantitating ofloxacin (OFLX) in human scalp hair by high performance liquid chromatography (HPLC) with a fluorescence detector. An octadecylsilane (ODS) column was used and the mobile phase was a mixture of potassium phosphate buffer (pH 2.6) and acetonitrile. The recovery of OFLX was 90.9-93.8% and within- and between-run precisions were 0.35-1.41% and 1.41-5.49% as the coefficient of variation (CV), respectively, when 5-50 ng OFLX was added to 1 mg blank hair. The calibration curve was linear in the range of 0.5-50 ng/tube (0.5 ml). Interference with other quinolone derivatives could be avoided according to the difference in their retention times or fluorescence spectra. Several pieces of hair were obtained from each of twelve healthy male volunteers, who had taken OFLX (100, 300 or 900 mg total dose) over a 1-3 day period 2 weeks before the hair sampling. In all hair samples except one obtained from a volunteer, who had taken the lowest dose, the 2-cm long segments nearest the scalp contained OFLX (5-45 ng/mg hair), while the upper segments did not. A highly significant positive correlation was observed between the total dose and the concentration of OFLX in the 2-cm long hair segments. Such a positive correlation was also revealed in rat hair sampled after repeated i.p. administration of OFLX over a 5-week period. These results suggest that the measurement of OFLX in hair by the present method would be useful for testing patient compliance in clinical pharmacology as well as for application to forensic science.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

The role of variations in growth rate and sample collection on interpreting results of segmental analyses of hair

Segmental analysis of hair for drugs, metabolites, and poisons has been widely reported in the scientific literature over the past two decades. Two fundamental assumptions in interpreting results of such analyses are (1) an average linear growth rate of head hair of 1cm/month and (2) that sample collections occur with the hair being cut directly next to the scalp. The purpose of this study was to evaluate the variability associated with growth rate of human head hair, as well as the ability to uniformly collect hair next to the scalp. The results were used to determine how these factors affect the interpretation of results generated in segmental analysis of hair. A thorough literature review was conducted to assess the range of linear growth of human head hair from the vertex posterior and occipital regions. The results were compiled to establish the average (1.06cm/month), as well as the range of possible growth rates of head hair. The range was remarkable and suggests that conclusions based on the 1-cm/month growth rate could be significantly skewed. A separate study was undertaken to evaluate collection of hair next to the scalp. Fourteen individuals were provided oral instructions, as well as a written standard collection procedure for head hair. The experience levels among the collectors varied from novice to expert. Each individual collected hair from dolls with short- and long-hair. Immediately following each collection, the sampling area was evaluated to determine how close to the scalp the cuts were made, as well as the variability in the lengths of hair remaining at the sampled area. From our collection study, we determined that 0.8±0.1cm of hair was left on the scalp after cutting. When taking into account the amount of hair left on the scalp after collecting, the use of a growth rate of 1.06cm/month, and the assumption that it takes two weeks for newly formed hair in the follicle to reach the scalp, we find that the first 1-cm segment of hair typically corresponds to hair formed 1.3±0.2 to 2.2±0.4 months (95% confidence) earlier. The impact of these findings as it relates to the corresponding time for each additional segment is demonstrated. As a result, we recommend that hair collection be delayed 8 weeks after a suspected ingestion to ensure that the sample fully represents the exposure period. The results of this study suggest that the variability in the growth rate of human head hair, as well as the inconsistent collection of hair, significantly affect the interpretation of results from segmental analysis of hair.     

Mismatched multiplex PCR amplification and subsequent RFLP analysis to simultaneously identify polymorphisms of erythrocytic ESD, GLO1, and GPT genes

ESD (esterase D), GLO1 (glyoxalase I), and GPT (glutamate pyruvate transaminase) are human erythrocytic isoenzymes and have previously been applied in forensic medicine caseworks. The molecular bases of the polymorphic gene expression products have been demonstrated to be because of SNPs in respective coding regions. However, it has not been revealed whether the SNPs conferring the polymorphisms to the aforementioned erythrocytic isoenzymes could be simultaneously detected by using a simple PCR method. In this study, we used mismatched primers to simultaneously amplify three common isoenzyme loci so that all amplified products contained the same Hph I cleavage sites. The products were then digested with Hph I and electrophoretically separated and stained so that alleles were identified. The accumulated values for the probability of discrimination power and excluding the probability of paternity to the aforementioned systems attained 90.41% and 41.72%, respectively, in the Chinese Han population. This assay could be extremely valuable for future forensic medicine practices.     

Pharmacological criteria that can affect the detection of doping agents in hair

When positive drug results are reported, a common interpretive question posed is whether or not it is possible to put a quantitative finding into context. A standard answer to this inquiry is that a positive hair testing result can be interpreted as meaning that the donor has chronically or repetitively used the drug identified in the hair, but that chronic or repetitive are not defined in the same way for all individuals. The Society of Hair Testing published on June 16, 1999, a consensus opinion on the use of hair in doping situations. However, although accepted in most courts of justice, hair analysis is not yet recognised by the International Olympic Committee. To be considered as a valid specimen for doping control, some issues still need to be addressed. The scientific community has demonstrated significant concern over the proper role that hair drug testing should serve in toxicological applications. Among the unanswered questions, five are of critical importance: (1) What is the minimal amount of drug detectable in hair after administration? (2) What is the relationship between the amount of the drug used and the concentration of the drug or its metabolites in hair? (3) What is the influence of hair color? (4) Is there any racial bias in hair testing? (5) What is the influence of cosmetic treatments? The present report documents scientific findings on these questions, with particular attention to the applications of hair in doping control.     

Detection of hereditary enzyme characteristics in vaginal secretion

Upon investigation of semen- and blood-free vaginal swabs using starch gel electrophoresis the Phosphoglucomutase type was clearly identified in about 40%. Using cellulose acetate membrane electrophoresis PGM could not be demonstrated. In all cases the results correspond with those obtained in blood. No relation was found between secretor type (determined in saliva) and PGM typing. In vaginal material the following could not be determined: Adenylatkinase (AK) using starch gel electrophoresis, Esterase D (EsD) using cellulose acetate membrane electrophoresis, and Glyoxalase I (GLO) using agarose gel thin-layer electrophoresis.     

A review of major factors contributing to errors in human hair association by microscopy.

Forensic hair examiners using traditional microscopic comparison techniques cannot state with certainty, except in extremely rare cases, that a found hair originated from a particular individual. They also cannot provide a statistical likelihood that a hair came from a certain individual and not another. There is no data available regarding the frequency of a specific microscopic hair characteristic (i.e., microtype) or trait in a particular population. Microtype is a term we use to describe certain internal characteristics and features expressed when observing hairs with unpolarized transmitted light. Courts seem to be sympathetic to lawyer's concerns that there are no accepted probability standards for human hair identification. Under Daubert, microscopic hair analysis testimony (or other scientific testimony) is allowed if the technique can be shown to have testability, peer review, general acceptance, and a known error rate. As with other forensic disciplines, laboratory error rate determination for a specific hair comparison case is not possible. Polymerase chain reaction (PCR)-based typing of hair roots offer hair examiners an opportunity to begin cataloging data with regard to microscopic hair association error rates. This is certainly a realistic manner in which to ascertain which hair microtypes and case circumstances repeatedly cause difficulty in association. Two cases are presented in which PCR typing revealed an incorrect inclusion in one and an incorrect exclusion in another. This paper does not suggest that such limited observations define a rate of occurrence. These cases illustrate evidentiary conditions or case circumstances which may potentially contribute to microscopic hair association errors. Issues discussed in this review paper address the potential questions an expert witness may expect in a Daubert hair analysis admissibility hearing.     

alpha-L-fucosidase phenotyping in human tissues, dental pulps and hair roots

Phenotypes of alpha-L-fucosidase (Fu) were demonstrated from tissue samples of spleen, liver, lung and kidney stored for a few weeks at room temperature. Activity was fairly low in pancreas, heart, muscle, skin and brain, and typing was not reliable after 1 week storage. Fu types were detectable from dental pulp tissue of up to 1 week storage. Activity was present in hair root cells, but was extremely low. The results show that the Fu typing may be applicable to individual identification of organ tissues and teeth.     

DNA的apoB位点扩增片段长度多态性的检测及其在法医学中的应用

用PCR方法对100例无关个体血样的apoB位点扩增片段进行研究,已发现有11个等位基因,片段长度分布于600～1100bp之间,基因频率为0.005～0.525,杂合度为70％。家系分析及对人体血液、血斑、精液、精斑、混合斑、带毛囊毛发及其它各种有核细胞组织的研究表明,该技术在个人识别及亲子鉴定上均能发挥重要作用。     

Drugs in prehistory: chemical analysis of ancient human hair

Concern about drug abuse in modern populations has led to the development of specific methods for identification of cocaine, opiates and cannabis in human hair. Drug use in prehistory can provide indirect evidence of interpopulational contact and social stratification. This paper reports drug evaluation in nineteen ancient hair samples from archaeological sites in northern Chile. Each sample was tested for the presence of traces of cocaine, opiates and cannabis, in order to establish a standard methodology for studies of drug use among prehistoric groups. Although results are negative, this absence of evidence could be due to two main causes: (1) the individuals evaluated did not use any drugs, which does not mean that other members of their cultural group did, or (2) the wide range of known drugs studied did not consider some group specific drugs, derived from local or imported plants, thus meaning that a greater drug range must be tested. In any case, our study confirms that drug testing in prehistoric samples is viable. However, in order to determine what kind of substances were used in prehistoric times new patterns that incorporate all drugs which are not part of the western pharmacopeia must be created. Finally, a methodology for the study of drug use among prehistoric groups using ancient hair samples is described.     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.