The German Stain Commission: recommendations for the interpretation of mixed stains.

In the course of forensic DNA analysis, the interpretation of DNA profiles of mixed stains, i.e. cell material from more than a single donor, has become increasingly more important. The German Stain Commission, a joint commission of Institutes of Forensic Science and Legal Medicine, has therefore developed guidelines aiming to harmonize the evaluation of mixed stains in German criminal cases.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

DNA Commission of the International Society for Forensic Genetics (ISFG): recommendations regarding the role of forensic genetics for disaster victim identification (DVI).

The ISFG membership consists of scientists and medical professionals specialized in using genetic testing for kinship analysis and the individualization of biological material. This expertise makes the forensic geneticist a resource of advice to international and national organizations dealing with human identifications and causes many DNA laboratories to get involved in DVI tasks. The present recommendations are meant to educate more forensic geneticists about their potential involvement in mass fatality preparedness and possible DVI efforts, as well as to provide practical guidance for each of the laboratories' individual tasks. The idea to work on DNA-specific recommendations was born after a round table discussion dealing with the 2004 Tsunami disaster in south east Asia during the 21st congress of the International Society for Forensic Genetics on the Azores, Portugal, in September 2005. The ensuing discussion between scientists and pathologists that had been involved in the International Center in Khao Lak, Thailand, revealed the need for the scientific community to be better prepared to answer the local authorities' questions by formulating generally acceptable scientific standards for the most efficient use of DNA-based victim identification methods. These recommendations, as well as the many cited references, are intended to provide guidance on establishing preparedness for the forensic genetics laboratory, on collecting and storing ante-mortem and post-mortem samples suitable for DNA analysis, on DNA extraction and genetic typing strategies, on data management, and on issues related to the biostatistical interpretation and reporting of results.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories. Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male). All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data. As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland. Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.

STR typing of human telogen hairs--a new approach.

A new approach to short tandem repeat (STR) typing of DNA extracted from telogen shed hairs is presented. Newly designed primer pairs with annealing positions close to the repeat units of the STR loci HUMFES, HUMTH01 and HUMTPOX were used for amplification. The typing results were compared to those obtained by the commonly used primer pairs by means of success rates. The primer pairs capable of producing very short amplicons (< 106 bp with HUMFES, < 86 bp with HUMTH01 and < 87 bp with HUMTPOX) described in this study significantly increased the success rates when typing telogen hairs.

A brief history of the formation of DNA databases in forensic science within Europe.

The introduction of DNA analysis to forensic science brought with it a number of choices for analysis, not all of which were compatible. As laboratories throughout Europe were eager to use the new technology different systems became routine in different laboratories and consequently, there was no basis for the exchange of results. A period of co-operation then started in which a nucleus of forensic scientists agreed on an uniform system. This collaboration spread to incorporate most of the established forensic science laboratories in Europe and continued through two major changes in the technology. At each step agreement was reached on which systems to use. From the beginning it was realised that DNA databases would provide the criminal justice systems with an efficient way of crime solving and consequently some local databases were created. It was not until the introduction of the amplification technology linked to the analysis of short tandem repeats that a sufficiently sensitive and robust system was available for the formation of efficient and effective DNA databases. Comprehensive legislation enacted in the UK in 1995 enabled forensic scientists to set up the first national DNA database which would hold both personal DNA profiles together with results obtained from crime scenes. Other countries quickly followed but in some the legislation has severely restricted the amount and type of data which can be retained and, therefore, effectiveness of the databases is limited. The widespread use of commercially produced multiplex kits has produced a situation in which nearly all European laboratories are using compatible systems and there is, therefore, the potential for the introduction of a pan-European DNA database. However, the exchange of results between countries is hampered by the various legislations which currently exist.

Results of collaborative study regarding the standardization of the Y-linked STR system DYS385 by the European DNA Profiling (EDNAP) group.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control sample. Furthermore, population data from eight different European countries with samples sizes between 91 and 150 male individuals were collected. The results confirm previous observations that DYS385 is one of the most informative Y-linked STR loci. It could also be demonstrated that reproducible results can be obtained independently from the electrophoretic separation and detection methods used. Thus DYS385 may serve as a useful complementation to the routinely used autosomal STR systems in special cases.

ACTBP2-nomenclature recommendations of GEDNAP.

A system of nomenclature is proposed for the complex STR system ACTBP2 (SE33) in order to facilitate data exchange between laboratories. The nomenclature conforms to the ISFH recommendations as far as it is possible for such complex systems. A blind trial was carried out between up to 20 laboratories to ascertain the reproducibility of the nomenclature under working conditions. The population studies carried out have established that there are minimal regional differences in the allele frequencies and that the system of nomenclature is robust.

Report of the European DNA Profiling Group (EDNAP)--an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

Report of the European DNA profiling group (EDNAP): an investigation of the complex STR loci D21S11 and HUMFIBRA (FGA).

This paper describes a collaborative exercise which was intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using two complex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosen because they are commonly used by different European laboratories. D21S11 has approximately 14 common alleles (f > 0.001), whereas HUMFIBRA has 19 common alleles. Laboratories were asked to test seven blood stains, one of which was a known control, and to report the results to the coordinating laboratory. The exercise demonstrated that complex STRs were amenable to standardisation.

Report on the third EDNAP collaborative STR exercise. European DNA Profiling Group.

This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designated to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, the laboratories were also requested to report the results obtained from any other loci which would normally be used in crime case investigations. A comparison of these results showed some inter-laboratory variation.

[Gc subtyping in blood stains by isoelectric focusing in immobilized pH gradients]. / Gc-Subtypisierung in Blutspuren durch isoelektrische Fokussierung in immobilisierten pH-Gradienten.

The applicability of isoelectric focusing in immobilized pH gradients for the Gc-subtyping in the forensic examination of bloodstains was tested. It is shown that due to the excellent separation of the Gc 1S and 1F bands subtyping of bloodstains can be done with high reliability.

[The use of ultrathin isoelectric focusing in the study of traces of blood and secretions]. / Die Anwendung der Ultradünnschicht-isoelektrischen Fokussierung (UDIEF) bei der Untersuchung von Blut- und Sekretspuren.

Four examples of the applicability of ultrathin layer isoelectric focusing in the investigation of bloodstains and stains of body secretions are given: -- differentiation of stains of human and animal blood; -- determination of fetal hemoglobin in bloodstains; -- subtyping of PGM1 isoenzymes in extracts of blood and semen stains; -- Gc typing of bloodstains using an immunofixation technique. For the visualisation of PGM1 subtypes a modified sandwich technique using a celluloseacetate membrane as separator between the focused gel and the visualisation agar is described.