Presentation number: FG 1
VALIDATION OF SERATEC HEMDIRECT ON ANIMAL BLOOD
Mia Belobrajdić1, Dženita Vugdalić1, Jadranko Boras2, Damir Skok2, Antonela Paladin3, Livia Slišković1, Ivan Jerković1, Josip Crnjac1
1University Department of Forensic Sciences, University of Split, Split, Croatia, 2Zagreb Zoo, Zagreb, Croatia, 3Faculty of Science, University of Split, Split, Croatia
The validation study of the SERATEC HemDirect blood test has been performed and the test is validated for forensic use. Initial validation test animal blood but all animal blood samples used in this study were from mammalian species and animals from other systematic groups were not included. This study aimed to determine whether the SERATEC HemDirect immunochromatographic test is highly specific for forensic identification of human blood, and whether there are non-mammalian species whose blood would show a false positive result on this test. The preliminary study included 8 samples of venous blood, human and 7 different animal species. All animal blood samples were obtained from Zagreb Zoo during the regular examination of animals. We used the blood of 7 animals who are commonly involved in car accidents or illegal hunting. All blood samples were analyzed by the SERATEC HemDirect test. A dilution set (1/4, 1/8, 1/10, 1/16, 1/20, 1/24) was prepared from whole blood by adding double-distilled water. A 50 µL sample of whole blood and each dilution were placed on a white cotton cloth and left to dry at room temperature. After drying, 1/4 of the sample was cut out and extracted in HemDirect buffer for 30 minutes. After extraction, three drops of each sample were added to the test well and the results were read and recorded after 10 minutes. The SERATEC HemDirect test showed positive results in all analyzed human blood samples (whole blood and dilutions 1/4, 1/8, 1/10, 1/16, 1/20, 1/24). In the analyzed blood samples of all animal species, the test gave negative results except in one case where a diluted blood sample of Rook (Corvus frugilegus L.) showed a weak positive result. The one weak positive result is ground for extending the study to more species of birds and reptiles since those can be found as pets in homes and can be involved in forensic investigations.
Key words: HemDirect, validation, forensics, blood test
Presentation number: FG 2
GENERATING HUMAN Y-STR HAPLOTYPES FROM MEDICINAL LEECHES
Elizabeth Knapp, Reena Roy
The Pennsylvania State University, Schreyer Honors College and Forensic Science Program, University Park, Pennsylvania, USA
Leeches are annelids which have been used for many centuries in the field of medicine. These worms convey an anticoagulant, known as hirudin, in their salivary glands. Once they hook onto human flesh, they make a small incision, and ingest 5 mL to 15 mL of blood from one human. In this study, 420 samples from 35 leeches were analyzed using Copan microFLOQ® Direct swabs, and 4N6 FLOQSwabs® Crime Scene collection devices, as well as the Yfiler® Plus, and the PowerPlex® Y23 amplification kits. The Yfiler® Plus amplification kit is a 27-plex Y-STR system which includes seven rapidly mutating Y-STR loci which allows for discrimination among related individuals, while the PowerPlex® Y23 System includes 23 Y-STR loci. North American medicinal leeches, Macrobdella Decora, obtained from a commercial source, were fed human blood meal from a male donor, and euthanized by freezing at specific times from 0 hour to 24 hours. The first method involved collecting minute amount of blood on the tip of Copan microFLOQ® Direct swab from the midgut of these organisms and the amplification of the blood while the swab remained in the reagents during thermal cycling. In the second method, 5 µL of blood from each midgut was concentrated to approximately 0.5 µL to 2.0 µL. The microFLOQ® Direct swabs were used to collect the concentrated blood and amplified directly. Finally, the 4N6 FLOQSwabs® Crime Scene swabs were utilized to collect the blood from the midgut, extracted with the DNA Investigator Kit from Qiagen. Extracted samples were quantified with the Quantifiler Trio DNA Quantification Kit. Y-STR profiles were obtained from known blood samples (reference samples) using the same methods. The Y-STR profiles generated from the blood ingested by the medicinal leeches using the three methods were consistent with the reference profiles of the donors. The results of this research reveal that the Copan microFLOQ® Direct swab is an excellent way to generate Y-STR profiles from the blood ingested by medicinal leeches. The extracted samples using 4N6 FLOQSwabs® Crime Scene swabs also yielded consistent and concordant profiles within and between samples and thus can be utilized to extract ingested blood. The direct amplification process bypasses the time-consuming, labor-intensive extraction and quantitation steps.
Key words: Copan microFLOQ® Direct swabs, Y-STR, Leech
Presentation number: FG 3
THE ROLE OF GENETICS IN FATAL PULMONARY THROMBOEMBOLISM FORENSIC DIAGNOSIS
Giuseppe Davide Albano, Stefania Zerbo, Ginevra Malta, Antonina Argo
PROMISE Department, University of Palermo, Palermo, Italy
The study aims to review the most recent and best evidence provided in the literature regarding the application of genetics methodology in the forensic diagnosis of fatal pulmonary thromboembolism (PTE) cases and the medico-legal issues involved. This review was conducted by performing a systematic literature search on online resources (PubMed Central database and Google Scholar) until 31st March 2022, using the following key terms: “((Genetics) OR (Forensic genetics)) AND ((pulmonary thromboembolism) OR (fatal pulmonary thromboembolism) OR (pulmonary thromboembolism diagnosis) OR (pulmonary thromboembolism deaths)).” Reviews, abstracts, animal studies, articles regarding surviving subjects, and articles in which the correlation between forensic genetics and pulmonary thromboembolism diagnosis is not discussed were excluded. Only human studies of fatal PTE with genetic analyses were included. The characteristics of the identified articles will be summarized. Most of the articles were retrospective studies on autopsy samples. Pulmonary thromboembolism was identified as the cause of death in all cases. Epidemiological data and clinical history were available in all studies. The most frequently used samples for genetic analyses were blood and postmortem tissues. Genetic testing for common prothrombotic variants included FV Leiden, FII G20210A, and methylenetetrahydrofolate reductase (MTHFR). Trauma and immobilization were the most frequent risk factors for PTE, and in most cases, the clinical and epidemiological analysis showed patients’ risk conditions. However, the identified articles show the importance of thrombophilia genetic screening in PTE cases with no significant risk factors. Moreover, unique characteristics were observed among different gender, age, and ethnic groups. This study highlights an association between genetic differences in different loci and PTE risk. Clinicians must be aware of the role of genetics in PTE epidemiology to undertake the proper preventive measures. In the forensic field, genetic testing is mandatory in selected fatal PTE cases, especially in medical malpractice cases. Genetic screening in selected cases should become a routine diagnostic test for PTE prevention.
Key words: forensic genetics, fatal pulmonary thromboembolism, pulmonary thromboembolism deaths, forensic diagnosis
Presentation number: FG 4
MASTR: AN EFFECTIVE PROBABILISTIC GENOTYPING TOOL FOR INTERPRETATION OF 2, 3, AND 4-PERSON STR MIXTURES ASSOCIATED WITH DIFFERENTIALLY DEGRADED DNA
Mitchell Holland1, Teresa Tiedge2, Abigail Bender3, Sidney Gaston-Sanchez4, Katherine Marino5, Sofia Canlas6, Jennifer McElhoe1
1Pennsylvania State University, Biochemistry & Molecular Biology Department, Forensic Science Program, University Park, PA, USA, 2Department of Population Health and Pathology, North Carolina State University, Raleigh, NC, USA 3Texas Department of Public Safety, Austin Crime Laboratory, Austin, TX, USA 4Armed Forces Medical Examiner System, Dover, DE, USA 5Baptist Medical Center South, Montgomery, AL, USA 6Massachusetts State Police Crime Laboratory, Maynard, MA, USA
The goal was to evaluate the impact of differentially degraded 2, 3, and 4-person STR mixtures on likelihood ratios (LRs) generated by the probabilistic genotyping software, MaSTR (SoftGenetics, LLC). Mixtures were prepared from donors with known Fusion 6C profiles. Ratios considered were 1:1, 1:3, 1:6, and 1:10 for 2-person (n=144), 1:1:1, 1:1:3, 1:3:6 and 1:6:6 for 3-person (n=102), and 1:1:1:1, 1:1:1:3, 1:1:3:3, 1:3:3:6, 1:3:6:6, and 1:6:6:6 for 4-person mixtures (n=52), with combinations of pristine and degraded sources of DNA rotated between major and minor contributors. Shearing of buccal DNA extracts was achieved mechanically using a Covaris S220; 150 and 250 bps to simulate severe and moderate levels of DNA degradation, respectively. Data were analyzed using GeneMarker HID (SoftGenetics, LLC). Results were imported into MaSTR and analysis performed using a model panel of Fusion 6C data run on a 3130xl Genetic Analyzer. A subset of LRs were generated using 40,000 versus 10,000 iterations per chain (eight chains total, with a burn-in of 8,000 iterations), and with or without a conditioning profile. MaSTR performed as expected. The log(LR) values for mixture samples containing high quantities of pristine sources of DNA were at optimal levels. Lower-quality mixture data associated with sources of DNA at <0.05 ngs for each contributor resulted in peak imbalance and allelic dropout which reduced the weight in support of a contributor. This was exacerbated by higher levels of degradation, with some instances resulting in log(LR) values in support of an exclusion. As expected, LRs were lower when a known contributor was not provided, especially for samples containing degraded DNA. There was no appreciable difference in LRs when comparing 10,000 and 40,000 iterations per chain for 2-person mixtures. In all cases, findings were consistent with expectations associated with CE-based profile information. Overall, MaSTR proved to be a reliable tool for the analysis of STR mixtures of differentially degraded sources of DNA. The points of view in this abstract are those of the authors and do not reflect the views of their respective agencies. In addition, this abstract in no way reflects an endorsement of products, instruments, or software.
Key words: probabilistic, genotyping, STR, mixtures, degraded