GITAD proficiency test recognition
The Y Chromosome Haplotype Reference Database (YHRD), whose goals are to generate a reliable Y-STR haplotype frequency database, assess the worldwide male population stratification, and provide tools for research in Y-STRs and Y-SNPs, has established on its webpage two preconditions for submitting an haplotype: (1) a laboratory accreditation certificate and (2) having passed a proficiency test that includes YSTR analysis, with GITAD one of the accepted proficiency tests . Furthermore, participating in and succeeding with the GITAD proficiency test is a prerequisite for adding a genetic profile to the Brazil Federal Police genetic database (Rede Integrada de Bancos de Perfis Genéticos, RIBPG) .
Number of participating laboratories
The number of participating laboratories has been constantly growing since the implementation of the proficiency test in 1999, when there were 8 participating laboratories. This number was doubled in 2005 and again in 2010 to 40 participating laboratories, a number that has remained fairly steady in the following 5 years, reaching a total of 58 laboratories in 2019 and 2020, the highest number in the proficiency test’s history. The growing number of applicant laboratories suggests their interest and satisfaction with the proficiency test.
DNA typing evolution
One of the aims of this article was to call attention to the evolution of the various DNA typing techniques employed in Ibero-America, which is very close to the evolution of forensic DNA analysis. Despite the lower use of preliminary tests, there was a tendency to use a wider range of techniques for various biological substances (blood, saliva, and semen). Although the use of a preliminary test depends on the forensic sample, human blood preliminary tests are the most common among the various exercises, especially the Hexagon OBTI kit and Adler and Teichmann methods. Although less frequently, semen and saliva detection are still employed. Surprisingly, quite obsolete methods such as the Adler (which uses a benzidine reaction with blood in the presence of hydrogen peroxide) and Teichmann tests (hemin crystals observed through microscopy) are commonly used. However, the number of participating laboratories using any presumptive test is still low, or at least it has not been reported. Presumptive tests have a major role in forensic sciences in general and in forensic genetics in particular, because the association between a DNA profile and a body fluid is not fully associated, even if a preliminary test yields a positive result. The augmented sensitivity of current DNA typing laboratories makes it possible to obtain a touch DNA profile previously deposited by a body fluid stain at the crime scene; therefore, analysts need to consider all DNA and non-DNA evidence . An example of this is 2016 M5, a non-human bloodstain in a human touch DNA background, topic that will be covered later.
DNA extraction has evolved from manual, time-intensive, and laborious protocols to automated DNA extraction and commercial kits, aimed at fast, reliable, and efficient solutions for optimal extraction. DNA extraction is the most important step in DNA typing because it has the greatest impact on the ultimate goal of obtaining a good DNA profile, which is possible only by obtaining the largest quantity and highest quality of DNA. Classical DNA extraction methods such as Chelex® and phenol–chloroform are still employed by forensic scientists for certain samples; however, manual and automated DNA extraction kits (developed in the early 2000s) are the most commonly used. DNA extraction from forensic samples, unlike clinical samples, is not completely automated or standardised, since forensic samples differ in the type of body fluid, nature of the tissue, substrate, nature of the crime, quantity of biological material, and source. The chosen method should therefore yield a high amount of DNA, removing or reducing PCR inhibitors that may have an impact on downstream analyses .
Many of the laboratories reported no DNA quantification step given that automated systems with a sample normalisation step or FTA® purification reagents are used. The use of various commercial kits has been reported, but there has been a tendency to use TaqMan® chemistry and kits that offer a variety of targets. The main usefulness of DNA quantification with casework samples is the optimisation of DNA input in the various STR amplification kits, thereby providing information on the quantity and quality of samples.
DNA amplification is probably the most variable stage in the DNA typing process, with several strategies available, each with a variety of commercial kits. However, there is a clear tendency to use more markers, and Y-STRs analysis has become a routine technique, despite mitochondrial DNA analysis; X-STRs studies have been less often reported. In addition to the aim of using more strategies, we should note the introduction of next-generation sequencing in the laboratories, given that it might become a common approach in the future.
DNA visualisation has evolved with the various available systems, although there is a clear preference for more fluorochromes (which translates into more markers in one run), more flexibility (the required maintenance for genetic analysers is increasingly flexible), and more capillary tubes so that more samples can be analysed. The use of data analysis software, however variable, shows a clear tendency towards specific forensic applications. In contrast, freeware and open-source options are preferred for statistics software.
Concordance problems among the various commercial kits are another subject of discussion. NIST and several researches have conducted concordance testing to establish the presence of concordance problems among the various available kits, obtaining percentages > 99.0%. The observed problems are normally reported to the commercial vendors so that they can improve their kits .
The most widely used statistics applications (in order to give a probability to the result given by the laboratories) were PATCAN (from 2003 to 2011) and Familias (from 2012 to the present). Statistic software is used mostly in order to solve the theoretical exercise, yet some of the laboratories give the obligatory and forensic exercise results with a probability or LR obtained with those programs if any biological relationship was observed among samples. Finally, 83% of the participant laboratories reported the use of freeware software, while 14% of them reported the use of commercial software (the rest reported calculations performed by hand).
GITAD Forensic exercises
Several of the exercises with forensic samples during the past 14 years they were conducted deserve a mention. The human bone fragment and bone powder samples (2007 and 2008 M5) that were sent the first years were very well evaluated by the participating laboratories. They were interesting samples because they probed the laboratories’ capacity for dealing with challenging samples; however, these samples are significantly more difficult to send than regular samples due to customs regulation and prohibitions; in addition, homogeneity is more challenging to achieve with the growing number of participant laboratories. Nevertheless, it was an appreciated matrix and its re-implementation is nowadays under evaluation by the proficiency test organisation staff.
The mixture exercises were the most common forensic sample, many of which were composed of samples from genetically related individuals, which made the analyses and interpretation even more complex. This type of experience helps the laboratories to gain awareness to the inherent difficulties in the analysis and interpretation of mixtures. When comparing results, this experience helps evaluate and compare strategies so that a more successful mixture interpretation could be achieved. In 2013 M5 (bloodstain mixture of M4 and M4 subject’s daughter), for instance, many laboratories detected the mixture completely, some of them detected the alleles with some drop-out or drop-in, and a few laboratories did not report any mixture. In 2014 (bloodstain mixture of M4 and M4 subject’ sister) and 2015 (M4 and his/her brother), transcription and stutters were the most common errors observed; however, it was difficult to assess given that many of the laboratories did not send the electropherograms, an issue that needed to be corrected. For 2018 M5 (woman hair soaked in M4 blood) preliminary tests, it was expected positive in human blood and negative for any other fluid, the identification of two different genetic profiles (M4 in hair wash and MX in hair) and no family relationship between M4 and MX. However, four types of results were reported: the same as expected (type 1), mixture profiles in different proportions (type 2), the obtention of only M4 genetic profile (type 3), and no results or contamination results (type 4). In the end, conditional transposition in LR calculation was observed, different hypotheses were proposed, and divergent results and conclusions were achieved.
The forensic sample with pork blood in a human touch DNA background (2016 M5), as well as its discussion, exposed the participating laboratories to the challenges and issues regarding DNA transferences and the importance of performing preliminary detection and orientation tests on biological samples, particularly for determining whether or not the sample has a human source. In this exercise presumptive tests, positive results for blood (not specific) but negative results for human blood or any other human fluid were expected (here lies the importance of immunochromatographic tests for screening samples given their specificity for detecting human blood), and it was also expected not only the obtention of only one genetic profile as background DNA, but also the identification of Sus scofra species by mitochondrial DNA analysis, so no association between genetic profile and body fluid was anticipated. The challenge of this forensic sample was precisely the dangerousness of associating a genetic profile to a certain body fluid or origin, an issue that has already being evaluated . The reported results could be classified in three groups: negative, partial profile and complete profile, and the laboratories who reported the performance of a preliminary test noticed that there was blood, however not human. A common error in this exercise was DNA excess in amplification, linked to the fact that DNA quantification was poorly reported.
Theoretical exercises have become a very much appreciated instance in proficiency test programs of different scientific groups, since they allow to detect statistical problems and software errors in routine reports of forensic genetics laboratories . Since the theoretical exercise was implemented, almost half of the participant laboratories perform it, with varying results. In general, three are the main committed mistakes: conditional transposition, hypothesis considered, and conclusions. Conditional transposition is also called “prosecutors’ fallacy”, well known among forensic scientists , an issue that should be avoided in laboratory reports.
Thus, there have been interesting conclusions to these exercises since its implementation, for example, 2013 theoretical exercise (half siblings) including an incompatibility in one single DNA marker, a challenge that laboratories assumed in three different ways: eliminating the marker from calculations, including it with its correspondent mutation rate, or giving results without any explanation.
In the mixtures of theoretical cases (2014), the electropherogram information (peaks and RFU height) is given as well as the genetic profiles and the population genetic database. Questions about the number and the genetic profiles of contributors and the difference between stutters and alleles are asked. Besides, there is a huge variability in answers: there was a wide range of answers when giving the proportion of the mixture and a huge variability when deducing the genotypes from electropherograms. However, the majority of the laboratories noticed correctly the mixture and present alleles.
Other exercises such as 2015 theoretical case (putative father and son in a possible incest case) offer an extra challenge to participant laboratories. In this case in particular, no mother genetic profile was given, so participants were forced to use different statistical calculations to the routine ones, and once again, a huge variability was observed in reports. The majority of the participant laboratories did not use any special formula, however being warn of the possibility of an incest case. The most common error was, once again, conditional transposition.
Finally, in 2016 exercise (sisterhood), the most common error was an inadequate hypothesis contrast, since not all the possibilities were taken into account. Furthermore, conditional transposition error was widely distributed among answers, and the same can be said about 2018 exercise (grandparenthood). Disaster Victim Identification (DVI) theoretical exercise in 2019, however being correctly performed by almost every participant laboratory, showed a wide range of results. The different family profiles that were given in the exercise were close family members: father and mother, spouse, daughter/son, or siblings.
The evolution of the theoretical exercise performance has stated the importance of statistics, calculations, hypotheses formulation, and conclusions formulation in forensic genetics, so those topics has been emphasised in several GITAD annual meetings by discussing them in a general assembly or by offering specific workshops related to these issues.
Challenges sending the samples
There have been several problems in sending the samples to the participating laboratories. First, biological samples are usually classified as dangerous goods by most countries and are therefore particularly treated by the customs officials of the various receiving countries. Second, custom offices present a common problem given that shipments are often returned from foreign countries. This problem was magnified during the last exercise due to COVID-19 restrictions for receiving packages from foreign countries, with most of the letters subject of quarantine for several days before delivery.
Participant laboratories improvement
GITAD exercise is not just about participating in a proficiency test: collaboration and improvement are two of its main pillars. Indeed, participant laboratories may have changed in one or more of the following aspects.
Firstly, laboratories have gained in partnership: being part of a forensic genetics network with colleges from their surroundings countries, attending meetings that stress on the latest advancements or issues, workshops, information exchange between colleges, or protocol unification are some examples of this kind of collaboration.
Secondly, discrepancies may be useful to participants since they may reveal a critical point in their protocols or procedures, so they are able to change them in order to improve them. Participants may also discover malfunctions in their instruments and equipment, for example, a participant realised they had electrophoretic mobility problems or lack of resolution for distinguish allelic variants with their proficiency test results. Other laboratories may change their protocols when they notice an error, for instance, when participants observed transcription errors as one of the main cause of discrepancies, they changed their protocols so now results are peer-reviewed before being reported. In addition, as many laboratories are already accredited under ISO 17025, a formal discrepancy protocol may be activated, so it promotes revisions, actions, and changes. Communication between discrepant laboratories and the proficiency test organisers is the best indicator of improvement, since discrepant laboratories usually request advice for managing the discrepancies causes and, later, they inform about their improvements.
Statistic calculations, conclusions, and in the end, reports emitted by participants have also enhanced. The theoretical exercise usefulness relies on illustrate how participants bring their laboratory results to a report that is going to be read by law enforcement agents such as judges and magistrates, attorneys, lawyers, or even other experts. As it has been widely stated, report conclusions are as important as the employed techniques, and GITAD exercise has also done its part to improve them. For instance, some participants changed their conclusions from a categorical statement to a hypothesis contrast.
Finally, collaboration and improvement have also worked together during the 20 years of GITAD. An example of this is the possibility of its members to request courses and workshops about a specific topic, and they are organised during GITAD meetings so they are given by other members with an expertise or demonstrated experience in that field.
GITAD organises a meeting in a different participating country every year: Brazil (Belo Horizonte, 1999; Curitiba, 2001; Brasilia, 2007; Sao Paulo, 2019; Salvador, 2011), Uruguay (Montevideo, 2000), Chile (Santiago, 2002), Mexico (Mexico City, 2005 and 2014; Veracruz, 2013), and Guatemala (Guatemala City, 2010, 2015 and 2018; Online, 2020).
Every GITAD proficiency test exercise involves holding a meeting in one of the participating countries, and those meetings have provided a forum for discussing solutions to the various problems and discrepancies observed, especially transcription errors and lack of resolution, which, as explained earlier, have been the main source of error. Over the last 20 years, the participating laboratories have felt a growing awareness of the quality of their results and have therefore committed to the continuing training of their staff. During GITAD meetings topics related to the work group are treated (assemblies, work commissions), as well as conferences and workshops about topics of interest in forensic genetics.
A number of aspects can be considered for improving GITAD proficiency test exercises. First, and most important, is the accreditation under ISO 17043:2010, Conformity assessment-General requirements for proficiency testing, which specifies the general requirements of providers of proficiency test exercises and thus for the operation and development of those proficiency tests, requirements that are supposed to be the basis for particular field applications. Nevertheless, ISO 9001 is already obtained, ISO 17025 is under obtention, and ISO 17043 guidelines for item preparations are undertaken: homogeneity, same matrix, routine items match, sufficient number of items are prepared for participant laboratories and if they are lost or damaged during distribution, and a procedure for acquisition, collection, handling, storage, and disposal of items is already followed by the prepared laboratory. Moreover, most of the items contained in ISO 17043 are met: both technical requirements (personnel, equipment, facilities, program design, planning, method choice, instructions to participants, manipulation and storage of items, packing, labelling, and distribution of items, data analysis and records, performance, reports, communication with participants, and confidentiality) and organisation requirements (almost shared with ISO 17025 requirements of organisation, management system, document control, etc.).
Secondly, some improvements when sending the exercise samples can be made to ensure their reception by the participating laboratories. With that aim, international private courier and package delivery enterprises have been contemplated for next year’s exercise. Lastly, advances in the data collection step could be conducted, elaborating a more homogenous form that could be filled online via a specially designed website.