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In the winter of 2011 a corpse of a teenager was found in a fallow field after three month of environmental exposure. The autopsy showed no sign of rape but the presence of several cuts on the victim’s body. Despite exposure to weathering and animals, the clothes from the waist up were well preserved, whereas the trousers were extensively torn and the underpants were clearly cut. Searching evidence from the perpetrator was difficult since the traces present on the victim’s clothes and underpants were subjected to morphological and chromatic modifications due to the environmental exposure. Initially, sample collection was based on an integrated approach that involved circumstantial information and inspection with the forensic light. The use of this light source allowed us to identify some areas with more clearly fluorescence, also visible to the naked eye, that were the first to be collected. The accurate inspection led us to perform approximately 300 samplings on the clothes. In one of these areas (called 31-2) located on the underpants, the results of the sampling showed the presence of alleles unrelated to the victim. As a result, an extensive sampling through the application of a virtual grid was undertaken in this area to confirm and improve the male profile and obtain serological information. Due to the complete absence of other relevant clues on the perpetrator, when a full male profile -Unknown Male #1 (UM#1)- was found on the victim’s clothes, a huge mass screening that involved the collection and analysis of over 16,000 reference samples was carried out, until a full match was obtain.

Here, we illustrate how statistical methods can help extract information from mixed DNA profiles pertaining to an Italian case, referred to by the media as The murder of Yara Gambirasio. We base the analysis on a model for DNA mixtures that takes fully into account the peak heights and possible artefacts, like stutter and dropout that might occur in the DNA amplification process. We show how to combine the evidence from multiple samples and from different marker systems all within the model framework. The combined evidence is used for deconvolution, where the focus is to find likely profiles for the donors to the sample. We also show how a mixture can be used to establish familial relationships between a reference profile and a donor to the mixed DNA sample. We compare results based on a single mixed DNA profile, combination of replicates, combinations of different samples and combinations of different kits. Based on the Yara case, we discuss just a few of the plethora of possibilities of combining evidential information.

Hemp cultivation is living a period of renewed interest worldwide after long years of opposition and abandonment. The European Union (EU) allows and subsidizes the growing of fiber and oilseed cultivars of Cannabis sativa L. with respect to the THC content limit of 0.2%. The EU method for the quantitative determination of Δ9-tetrahydrocannabinol (THC) content in hemp varieties provides to apply a tolerance of 0.03 g of THC per 100 g of sample concerning compliance assessment to that limit. However, the method does not report any precision data, especially useful as a function of THC content to evaluate measurement uncertainty and therefore to establish the conformity of hemp at different THC legal limits. Measurement uncertainty of the method by both bottom-up and top-down approach, besides repeatability and reproducibility, was investigated and estimated in the THC concentration range 0.2–1.0%, which includes the different legal limits set out for hemp around the world. We proposed Decision Rules for conformity of hemp showing that a non-compliant declaration beyond reasonable doubt should be stated when the THC content, as a mean result on a duplicate analysis, exceeds the limit by at least 11–15%, depending on THC limit. We highlighted other issues concerning practical aspects of hemp analysis, from sampling to evaluation of results, as well as the need to carry out collaborative studies on the EU method. Graphical abstract

DNA extraction is arguably the most critical stage in the process of forensic profiling. If done poorly, the sample can be lost and all downstream processing steps will be fruitless as they are dependent on the quality of the extract. The main factors to take into consideration to ensure a good quality of extracted DNA are: the efficiency of the extraction method and the absence of cross-contamination between samples. In this article we will discuss the implementation and validation of a fully automated DNA extraction method that involves the use of Qiagen® QIAsymphony® DNA Investigator® kit being processed on a Hamilton® Microlab STAR® automated platform in conjunction with Autolys® technology (barcoded Autolys® tubes used for the initial lysis, and subsequent filtration of lysates). Forensic trace samples are first placed on the Hamilton® Microlab STAR® Autolys® in Autolys® tubes by the operator. After the lysis and extraction process, the DNA extracts are collected into tubes with unique two-di...

The forensic application of the luminol chemiluminescence reaction is reviewed. Luminol has been effectively employed for more than 40 years for the presumptive detection of bloodstains which are hidden from the naked eye at crime scenes and, for this reason, has been considered one of the most important and well-known assays in the field of forensic sciences. This review provides an historical overview of the forensic use of luminol, and the current understanding of the reaction mechanism with particular reference to the catalysis by blood. Operational use of the luminol reaction, including issues with interferences and the effect of the luminol reaction on subsequent serological and DNA testing is also discussed.

Allele frequencies for the 15 autosomal STR loci included in the AmpFlSTR((R)) IdentifilerTM PCR Amplification Kit panel from Applied Biosystems (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, CSF1PO, D19S433, D2S1338, D16S539) and several statistical parameters were estimated from a sample of 103 unrelated individuals, mostly Shia and Sunni Arabs, living in most of central and southern Iraq provinces. We compared the allele frequency spectrum detected in the Iraqi population to allele frequencies from 11 other data sets from published studies of individuals from Turkey, Iraqi-Kurdistan, Saudi Arabia, Arab Emarates, Oman, Iran, Syria, and Jordan. Significant global differences in allele frequencies were detected in 9 of the 11 comparisons following sequential Bonferroni corrections. Comparisons with the two independent panels from Saudi Arabia were not significant after applying Bonferroni corrections, however, low P-values (P0.05) associated with these two contrasts nonetheless suggested that at least slight genetic differences between populations may exist.

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.

SummaryThe evaluation of nuclear DNA evidence for identification purposes is performed here taking account of the uncertainty about population parameters. Graphical models are used to detail the hypotheses being debated in a trial with the aim of obtaining a directed acyclic graph. Graphs also clarify the set of evidence that contributes to population inferences and they also describe the conditional independence structure of DNA evidence. Numerical illustrations are provided by re-examining three case-studies taken from the literature. Our calculations of the weight of evidence differ from those given by the authors of case-studies in that they reveal more conservative values.

Background In forensic science there are many types of crime that involve animals. Therefore, the identification of the species has become an essential investigative tool. The exhibits obtained from such offences are very often a challenge for forensic experts. Indeed, most biological materials are traces, hair or tanned fur. With hair samples, a common forensic approach should proceed from morphological and structural microscopic examination to DNA analysis. However, the microscopy of hair requires a lot of experience and a suitable comparative database to be able to recognize with a high degree of accuracy that a sample comes from a particular species and then to determine whether it is a protected one. DNA analysis offers the best opportunity to answer the question, ‘What species is this?’ In our work, we analyzed different samples of fur coming from China used to make hats and collars. Initially, the samples were examined under a microscope, then the mitochondrial DNA was tested for species identification. For this purpose, the genetic markers used were the 12S and 16S ribosomal RNA, while the hypervariable segment I of the control region was analyzed afterwards, to determine whether samples belonged to the same individual. Results Microscopic examination showed that the fibres were of animal origin, although it was difficult to determine with a high degree of confidence which species they belonged to and if they came from a protected species. Therefore, DNA analysis was essential to try to clarify the species of these fur samples. Conclusions Macroscopic and microscopic analysis confirmed the hypothesis regarding the analyzed hair belonging to real animals, although it failed to prove with any kind of certainty which actual family it came from, therefore, the species remains unknown. Sequence data analysis and comparisons with the samples available in GenBank showed that the hair, in most cases, belonged to the Canidae family, and in one case only to Felidae.

One hundred and fifty individuals have been sampled across Central-South Italy and genotyped for Y chromosome STRs by PowerPlex® Y system. Comparison with previous Italian databases revealed that majority of Y chromosome variation still need to be sampled. Identification of locus duplications, distribution of genetic variation and firstly identified alleles point to the necessity of more focused sampling strategies for reference databases.

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

The sarcophagus containing the remains of Federico II, located in the Cathedral of Palermo (Sicily, Italy), was opened on 1998 to perform a multidisciplinary survey [1]. Next to the remains of Federico II and in close contact with them were laying two other skeletons belonging, according to historical records, to Pietro II di Aragona and to an anonymous person (“The Third Individual”), probably a woman. The bones appeared severely deteriorated. Chemical analysis performed on bone samples excluded that the bodies underwent some kind of embalming process. The analysis of mtDNA from bone samples taken from the three skeletons was successful in only one of the two labs involved. The HVR1-mtDNA sequence (region: from nt 16,035 to nt 16,395), obtained from the bone samples of Federico II and “The Third Individual” appear identical but bear double peaks at the same nucleotide positions, suggesting mixing (i.e. contamination) of different mtDNA types. The HVR1 sequence obtained from the bone sample of Pietro II di Aragona does not present double peaks and differ from the Cambridge Reference Sequence (CRS) at six nucleotide positions. Cloning experiment of the Federico II amplicon demonstrated that the mixed mtDNA types are only two: one identical to CRS, the other identical to the sequence of Pietro II di Aragona. A reconstruction of these data are proposed in the Discussion. Due to the problematic context in which this study was carried out (mixed and deteriorated biological material, failure to replicate results in two different labs), our results and reconstruction can only be offered on a tentative basis. It is hoped that the data presented in this study will reveal useful, for future comparison, if further molecular genetics research will be carried out on the royal dynasties that ruled Sicily in the early centuries of the past millennium.

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

Sir—Adrian Linacre (May 31, p 1841) is an enthusiastic proponent of the UK’s National DNA Database (NDNAD). We wish to offer a more critical perspective of the database. In 2002, a young girl was murdered in a Tuscan village by a foreigner. From a bloodstain left at the crime scene, the suspect’s DNA profile was produced and circulated on the Interpol net. In February, 2002, P N Hankin, a barman in England was identified from the NDNAD and charged with the murder. He denied the charge and protested that he had been at his workplace on the day of the murder, which was corroborated by several eye-witnesses. He was, however, arrested. Eventually, British officers reported a fresh profile from a sample volunteered by Hankin, with one previously unseen mismatch. He was subsequently cleared of all charges. This case highlights some of the limitations of DNA profiling. Inferring a person has committed a crime on the basis of a profile match alone is problematic. The rest of the evidence should not be overlooked in favour of the DNA match. The use of DNA databases does not prevent forensic scientists from misrepresenting the population associated with a crime (the forensic population). A forensic population is not an abstract category, but a small group of individuals identified by background evidence. In the past, concerns have been raised about the existence of genetic isolates and the forensic use of gene frequency multiplication rule. These hypotheses have been strenuously and effectively criticised. But informing a person’s guilt probablility by comparison with the national (or whatever other abstract) population is a flawed approach that has little to do with population genetics axioms and their exceptions. Linacre’s statement that “such high probabilities lead the investigator to place the person at the crime scene” reiterates an old cliche—technically known as the fallacy of transposed conditional—here embodied in the desultory idea that every man on earth can pass by a Tuscan village on a certain day and commit a crime. Databases contain undetected errors—the larger the database, the more prone it is to errors. As it turns out, hidden errors are part of forensic evidence, in the sense that we can modify our beliefs according to the chance of their occurrence. We believe national DNA databases offer an opportunity to study errors and their real-scale effects. Why overstate the importance of these databases when the real population of criminal suspects is so small and the risk of misclassification with DNA from a national database so tangible. The forensic community needs to adopt a more circumspect attitude towards the use of DNA evidence from national databases in criminal cases and recognise the problems inherent in such an approach. *Vince L Pascali, Giampietro Lago, Marina Dobosz