Analytical verification and validation of immunohistochemical (IHC) tests and their equipment are common practices for today’s anatomic pathology laboratories. Few references or guidelines are available on how this should be performed. The study of Sciensano (the Belgian national competent authority regarding licensing of medical laboratories) performed in 2016, demonstrated a significant interlaboratory variation in validation procedures of IHC tests among Belgian laboratories. These results suggest the unavailability of practical information on the approach to the verification and validation of these tests. The existing Belgian Practice Guideline for the implementation of a quality management system in anatomic pathology laboratories has been reviewed to meet this demand and, in addition, to prepare the laboratories for the EU—IVD revised regulations (IVDR). This paper describes Belgian recommendations for the verification and validation of IHC tests before implementation, for ongoing validation, and for revalidation. For each type of test (according to the IVDR classification and the origin) and its intended use (purpose), it addresses how to perform analytical verification/validation by recommending: (1) the number of cases in the validation set, (2) the performance characteristics to be evaluated, (3) the objective acceptance criteria, (4) the evaluation method for the obtained results, and (5) how and when to revalidate. A literature study and a risk analysis taking into account the majority of variables regarding verification/validation of methods have been performed, resulting in an expert consensus recommendation that is a compromise among achievability, affordability, and patient safety. This new consensus recommendation has been incorporated in the aforementioned ISO 15189:2012–based Practice Guideline.

CONTEXT.—:Errors in laboratory medicine could compromise patient safety. Good laboratory practice includes identifying and managing nonconformities in the total test process. Varying error percentages have been described in other fields but are lacking for molecular oncology.OBJECTIVES.—:To gain insight into incident causes and frequency in the total test process from 8 European institutes routinely performing biomarker tests in non-small cell lung cancer and colorectal cancer.DESIGN.—:All incidents documented in 2018 were collected from all hospital services for pre-preanalytical entries before the biomarker test, as well as specific incidents for biomarker tests.RESULTS.—:There were 5185 incidents collected, of which 4363 (84.1%) occurred in the pre-preanalytical phase (all hospital services), 2796 of 4363 (64.1%) related to missing or incorrect request form information. From the other 822 specific incidents, 166 (20.2%) were recorded in the preanalytical phase, 275 (33.5%) in the analytical phase, and 194 (23.6%) in the postanalytical phase, mainly due to incorrect report content. Only 47 of 822 (5.7%) incidents were recorded in the post-postanalytical phase, and 123 (15.0%) in the complete total test process. For 17 of 822 (2.1%) incidents the time point was unknown. Pre-preanalytical incidents were resolved sooner than incidents on the complete process (mean 6 versus 60 days). For 1215 of 5168 (23.5%) incidents with known causes a specific action was undertaken besides documenting them, not limited to accredited institutes.CONCLUSIONS.—:There was a large variety in the number and extent of documented incidents. Correct and complete information on the request forms and final reports are highly error prone and require additional focus.

En 2002, des fouilles menees par Marie-Cecile Truc (Inrap) a Saint-Dizier ont permis la decouverte de trois tombes d’une richesse exceptionnelle – une femme, deux hommes et un cheval – datees du deuxieme quart du VIe siecle. Ces tombes presentent des caracteristiques qui tranchent avec les rites funeraires habituellement observes dans la region : chambres de type Morken, inhumation de cheval, epees a anneaux, etc. Ces elements incitent a rattacher les tombes de Saint-Dizier au facies archeologique des tombes dites « de chefs francs » du debut du VIe siecle, interpretees comme des temoins archeologiques de l’expansion franque. Les inhumes de Saint-Dizier sont-ils des Francs emigres ou bien des Gallo-Romains de souche s’etant eleves dans la hierarchie guerriere et assimiles aux coutumes franques ? La question ne se pose pas forcement en termes ethniques mais plutot en termes d’identite sociale et politique. Le faste et le rituel de ces inhumations refletent une culture materielle particuliere et revelent la volonte de mettre en valeur l’appartenance de ces defunts a une elite sociale qui suit une mode bien precise dans la mort.

There is limited knowledge on the biological relatedness between citizens and on the demographical dynamics within villages, towns and cities in pre-17th century Western Europe. By combining Y-chromosomal genotypes, in-depth genealogies and surname data in a strict genetic genealogical approach, it is possible to provide insights into the genetic diversity and the relatedness between indigenous paternal lineages within a particular community at the time of the surname adoption. To obtain these insights, six Flemish communities were selected in this study based on the differences in geography and historical development. After rigorous selection of appropriate DNA donors, low relatedness between Y chromosomes of different surnames was found within each community, although there is co-occurrence of these surnames in each community since the start of the surname adoption between the 14th and 15th century. Next, the high communal diversity in Y-chromosomal lineages was comparable with the regional diversity across Flanders at that time. Moreover, clinal distributions of particular Y-chromosomal lineages between the communities were observed according to the clinal distributions earlier observed across the Flemish regions and Western Europe. No significant indication for genetic differences between communities with distinct historical development was found in the analysis. These genetic results provide relevant information for studies in historical sciences, archaeology, forensic genetics and genealogy.

Y-chromosomal short tandem repeats (Y-STRs) are often used in addition to Y-chromosomal single-nucleotide polymorphisms (Y-SNP) to detect subtle patterns in a population genetic structure. There are, however, indications for Y-STR haplotype resemblance across different subhaplogroups within haplogroup R1b1b2 (R-M269) which may lead to erosion in the observation of the population genetic pattern. Hence the question arises whether Y-STR haplotypes are still informative beyond high-resolution Y-SNP genotyping for population genetic studies. To address this question, we genotyped the Y chromosomes of more than 1000 males originating from the West-European regions of Flanders (Belgium), North-Brabant and Limburg (the Netherlands) at the highest resolution of the current Y-SNP tree together with 38 commonly used Y-STRs. We observed high resemblance of Y-STR haplotypes between males belonging to different subhaplogroups of haplogroup R-M269. Several subhaplogroups within R-M269 could not be distinguished from each other based on differences in Y-STR haplotype variation. The most likely hypothesis to explain this similarity of Y-STR haplotypes within the population of R-M269 members is a recent radiation where various subhaplogroups originated within a relatively short time period. We conclude that high-resolution Y-SNP typing rather than Y-STR typing might be more useful to study population genetic patterns in (Western) Europe.

The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.

The analysis of human short tandem repeats of the Y-chromosome (Y-STRs) provides a powerful tool in forensic cases for male sex identification, male lineage identification and identification of the geographical origin of male lineages. As the commonly used 12 and 17 Y-STR multiplexes do not discriminate between some unrelated males, additional Y-STRs were implemented in the PowerPlex® Y23 System to supplement the existing commercial Y-STR kits. Until today, the forensic value of a (near) 23 versus 17 Y-STR haplotype match between an unknown DNA donor and a certain biological sample in a database is not yet well studied. This will be of huge interest for cases where an autosomal DNA profile yields no match to a DNA database and the database is used for familial searching (male relative(s) of the offender) or for the estimation of the geographical origin of the offender. In order to value (near) 23 Y-STR haplotype matches in a local sample from Western Europe, we selected the region of Flanders (Belgium) due to the already present knowledge on its Y-chromosomal variants. Many Y-chromosomes of this region were previously genotyped with Y-SNPs at a high resolution of the most recently updated Y-chromosomal tree and the deep-rooted genealogy of each DNA donor was already established. By comparing (near) matches of 23 versus 17 Y-STR haplotypes between patrilineal-unrelated males, a substantial lower number of uninformative (near) 23 Y-STR haplotype matches has been observed compared to 17 Y-STR haplotypes. Furthermore, the use of SNP data was informative to discriminate >60% of unrelated males with an (near) identical 17 Y-STR match while SNP data was only necessary to discriminate about 10% of unrelated males with a 23 Y-STR haplotype that differed at only two Y-STRs. This shows the higher value of the Y23 haplotype within familial DNA searching and the estimation of the geographical origin of a DNA donor. Therefore, the use of the PowerPlex® Y23 System instead of the commonly used 12 and 17 Y-STRs by the forensic community is recommended as it will increase the efficiency of Y-STRs in forensic casework.

Many Y-chromosomal lineages which are defined in the latest phylogenetic tree of the human Y chromosome by the Y Chromosome Consortium (YCC) in 2008 are distributed in (Western) Europe due to the fact that a large number of phylogeographic studies focus on this area. Therefore, the question arises whether newly discovered polymorphisms on the Y chromosome will still be interesting to study Western Europeans on a population genetic level. To address this question, the West-European region of Flanders (Belgium) was selected as study area since more than 1000 Y chromosomes from this area have previously been genotyped at the highest resolution of the 2008 YCC-tree and coupled to in-depth genealogical data. Based on these data the temporal changes of the population genetic pattern over the last centuries within Flanders were studied and the effects of several past gene flow events were identified. In the present study a set of recently reported novel Y-SNPs were genotyped to further characterize all those Flemish Y chromosomes that belong to haplogroups G, R-M269 and T. Based on this extended Y-SNP set the discrimination power increased drastically as previous large (sub-)haplogroups are now subdivided in several non-marginal groups. Next, the previously observed population structure within Flanders appeared to be the result of different gradients of independent sub-haplogroups. Moreover, for the first time within Flanders a significant East-West gradient was observed in the frequency of two R-M269 lineages, and this gradient is still present when considering the current residence of the DNA donors. Our results thus suggest that an update of the Y-chromosomal tree based on new polymorphisms is still useful to increase the discrimination power based on Y-SNPs and to study population genetic patterns in more detail, even in an already well-studied region such as Western Europe.

Zooarcheological evidence suggests that pigs were domesticated in Southwest Asia 8,500 BC. They then spread across the Middle and Near East and westward into Europe alongside early agriculturalists. European pigs were either domesticated independently or more likely appeared so as a result of admixture between introduced pigs and European wild boar. As a result, European wild boar mtDNA lineages replaced Near Eastern/Anatolian mtDNA signatures in Europe and subsequently replaced indigenous domestic pig lineages in Anatolia. The specific details of these processes, however, remain unknown. To address questions related to early pig domestication, dispersal, and turnover in the Near East, we analyzed ancient mitochondrial DNA and dental geometric morphometric variation in 393 ancient pig specimens representing 48 archeological sites (from the Pre-Pottery Neolithic to the Medieval period) from Armenia, Cyprus, Georgia, Iran, Syria, and Turkey. Our results reveal the first genetic signatures of early domestic pigs in the Near Eastern Neolithic core zone. We also demonstrate that these early pigs differed genetically from those in western Anatolia that were introduced to Europe during the Neolithic expansion. In addition, we present a significantly more refined chronology for the introduction of European domestic pigs into Asia Minor that took place during the Bronze Age, at least 900 years earlier than previously detected. By the 5th century AD, European signatures completely replaced the endemic lineages possibly coinciding with the widespread demographic and societal changes that occurred during the Anatolian Bronze and Iron Ages.

Genetic analysis strongly increases the opportunity to identify skeletal remains or other biological samples from historical figures. However, validation of this identification is essential and should be done by DNA typing of living relatives. Based on the similarity of a limited set of Y-STRs, a blood sample and a head were recently identified as those belonging respectively to King Louis XVI and his paternal ancestor King Henry IV. Here, we collected DNA samples from three living males of the House of Bourbon to validate the since then controversial identification of these remains. The three living relatives revealed the Bourbon's Y-chromosomal variant on a high phylogenetic resolution for several members of the lineage between Henry IV and Louis XVI. This 'true' Bourbon's variant is different from the published Y-STR profiles of the blood as well as of the head. The earlier identifications of these samples can therefore not be validated. Moreover, matrilineal genealogical data revealed that the published mtDNA sequence of the head was also different from the one of a series of relatives. This therefore leads to the conclusion that the analyzed samples were not from the French kings. Our study once again demonstrated that in order to realize an accurate genetic identification of historical remains DNA typing of living persons, who are paternally or maternally related with the presumed donor of the samples, is required.

Zooarcheological evidence suggests that pigs were domesticated in Southwest Asia ∼8,500 BC. They then spread across the Middle and Near East and westward into Europe alongside early agriculturalists. European pigs were either domesticated independently or more likely appeared so as a result of admixture between introduced pigs and European wild boar. As a result, European wild boar mtDNA lineages replaced Near Eastern/Anatolian mtDNA signatures in Europe and subsequently replaced indigenous domestic pig lineages in Anatolia. The specific details of these processes, however, remain unknown. To address questions related to early pig domestication, dispersal, and turnover in the Near East, we analyzed ancient mitochondrial DNA and dental geometric morphometric variation in 393 ancient pig specimens representing 48 archeological sites (from the Pre-Pottery Neolithic to the Medieval period) from Armenia, Cyprus, Georgia, Iran, Syria, and Turkey. Our results reveal the first genetic signatures of early domestic pigs in the Near Eastern Neolithic core zone. We also demonstrate that these early pigs differed genetically from those in western Anatolia that were introduced to Europe during the Neolithic expansion. In addition, we present a significantly more refined chronology for the introduction of European domestic pigs into Asia Minor that took place during the Bronze Age, at least 900 years earlier than previously detected. By the 5th century AD, European signatures completely replaced the endemic lineages possibly coinciding with the widespread demographic and societal changes that occurred during the Anatolian Bronze and Iron Ages.

Recent evidence suggests that seeking out extra-pair paternity (EPP) can be a viable alternative reproductive strategy for both males and females in many pair-bonded species, including humans. Accurate data on EPP rates in humans, however, are scant and mostly restricted to extant populations. Here, we provide the first large-scale, unbiased genetic study of historical EPP rates in a Western European human population based on combining Y-chromosomal data to infer genetic patrilineages with genealogical and surname data, which reflect known historical presumed paternity. Using two independent methods, we estimate that over the last few centuries, EPP rates in Flanders (Belgium) were only around 1–2% per generation. This figure is substantially lower than the 8–30% per generation reported in some behavioural studies on historical EPP rates, but comparable with the rates reported by other genetic studies of contemporary Western European populations. These results suggest that human EPP rates have not changed substantially during the last 400 years in Flanders and imply that legal genealogies rarely differ from the biological ones. This result has significant implications for a diverse set of fields, including human population genetics, historical demography, forensic science and human sociobiology.

Patrilineal heritable surnames are widely used to select autochthonous participants for studies on small-scale population genetic patterns owing to the unique link between the surname and a genetic marker, the Y-chromosome (Y-chr). Today, the question arises as to whether the surname origin will be informative on top of in-depth genealogical pedigrees. Admixture events that happened in the period after giving heritable surnames but before the start of genealogical records may be informative about the additional value of the surname origin. In this context, an interesting historical event is the demic migration from French-speaking regions in Northern France to the depopulated and Dutch-speaking region Flanders at the end of the sixteenth century. Y-chr subhaplogroups of individuals with a French/Roman surname that could be associated with this migration event were compared with those of a group with autochthonous Flemish surnames. Although these groups could not be differentiated based on in-depth genealogical data, they were significantly genetically different from each other. Moreover, the observed genetic divergence was related to the differences in the distributions of main Y-subhaplogroups between contemporary populations from Northern France and Flanders. Therefore, these results indicate that the surname origin can be an important feature on top of in-depth genealogical results to select autochthonous participants for a regional population genetic study based on Y-chromosomes.

We present an automated method to generate DNA profiles from replicate PCRs by combining advantages of the composite and consensus method by a system of brackets in which an allelic balance threshold is used as a variable to separate DNA-profiles of major from minor donors. Through the analysis of artificial low (125 pg) and high (250 pg) template three-person mixtures with low (1:1.5:3) and high (1:5:10) donor ratios we demonstrate the usefulness of a tool to determine the optimal allelic balance threshold within a locus. The automated extraction of dominant profiles saves considerable amounts of time when producing composite-consensus profiles. Drop-in/drop-out rates are produced and a comparison is made with an alternative open source script to evaluate the dominant profiles generated. By introducing this script into the forensic community we hope to increase awareness of much needed collaborative efforts with bioinformaticians and statisticians to develop forensic open source software scripts.

The archaeological site of Sagalassos is located in Southwest Turkey, in the western part of the Taurus mountain range. Human occupation of its territory is attested from the late 12th millennium BP up to the 13th century AD. By analysing the mtDNA variation in 85 skeletons from Sagalassos dated to the 11th–13th century AD, this study attempts to reconstruct the genetic signature potentially left in this region of Anatolia by the many civilizations, which succeeded one another over the centuries until the mid-Byzantine period (13th century BC). Authentic ancient DNA data were determined from the control region and some SNPs in the coding region of the mtDNA in 53 individuals. Comparative analyses with up to 157 modern populations allowed us to reconstruct the origin of the mid-Byzantine people still dwelling in dispersed hamlets in Sagalassos, and to detect the maternal contribution of their potential ancestors. By integrating the genetic data with historical and archaeological information, we were able to attest in Sagalassos a significant maternal genetic signature of Balkan/Greek populations, as well as ancient Persians and populations from the Italian peninsula. Some contribution from the Levant has been also detected, whereas no contribution from Central Asian population could be ascertained.

The nine Belgian laboratories, officially recognized by the Belgian Minister of Justice for performing DNA analysis in criminal cases, have jointly decided to use a common local population database for statistical calculations. A first database has been set up for the loci covered in AmpFlSTR® Identifiler™ and PowerPlex® 16 [1], [2]. With the decision of ENFSI and EDNAP to extend the current ESS loci (D3S1358, vWA, D8S1179, D21S11, D18S51, THO1 and FGA) with new loci (D1S1656, D2S441, D10S1248, D12S391 and D22S1045), there was a need to obtain also data for these loci within the Belgian population [3], [4]. Here, we present the allele frequencies and forensic efficiency data for the new ESS loci and one additional miniSTR, D1S1677.

Recent genetic studies of the Tuareg have begun to uncover the origin of this semi-nomadic northwest African people and their relationship with African populations. For centuries they were caravan traders plying the trade routes between the Mediterranean coast and south-Saharan Africa. Their origin most likely coincides with the fall of the Garamantes who inhabited the Fezzan (Libya) between the 1st millennium BC and the 5th century AD. In this study we report novel data on the Y-chromosome variation in the Libyan Tuareg from Al Awaynat and Tahala, two villages in Fezzan, whose maternal genetic pool was previously characterized. High-resolution investigation of 37 Y-chromosome STR loci and analysis of 35 bi-allelic markers in 47 individuals revealed a predominant northwest African component (E-M81, haplogroup E1b1b1b) which likely originated in the second half of the Holocene in the same ancestral population that contributed to the maternal pool of the Libyan Tuareg. A significant paternal contribution from south-Saharan Africa (E-U175, haplogroup E1b1a8) was also detected, which may likely be due to recent secondary introduction, possibly through slavery practices or fusion between different tribal groups. The difference in haplogroup composition between the villages of Al Awaynat and Tahala suggests that founder effects and drift played a significant role in shaping the genetic pool of the Libyan Tuareg.