Bone Marrow as a Source of DNA in Forensic Genetics: An Optimized Nucleic Acids Extraction Protocol.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs. These data clearly demonstrate that smaller PCR amplicons provide an attractive alternative to mitochondrial DNA for forensic analysis of degraded DNA.

Postmortem perfusion is a procedure which provides in-vivo fixation of the human body and prevents organ and tissue decomposition after biological death occurs. Formaldehyde-based embalming solutions influence nucleic acid degradation, which reduces the quality and quantity of DNA extracted and the effectiveness of short tandem repeat (STR) typing. This research is a 1-year study aimed at determining the timeframe within which viable DNA profiles can be generated from different tissue types, post-embalming. Samples from the bone marrow (tibia), trapezius and quadriceps muscles, liver, and brain tissues were collected before embalming and at seven time points post-embalming. DNA extraction and quantification were performed on each sample to assess whether sufficient DNA was available for testing. Samples with DNA quantities over 0.05 ng/μL underwent amplification and STR analysis to produce DNA profiles. Quantification values and profile quality were compared across tissue types and time points. Results showed that prolonged exposure to embalming solutions significantly reduced DNA quality, leading to less discernible profiles. Statistically significant differences were detected among all tissue types at all time periods. Liver and brain samples retained more DNA than bone marrow, trapezius, and quadricep muscle samples. Qualitative evaluation of ski slope patterns in liver STR profiles demonstrated increasing signal degradation over time and was consistent across loci, suggesting predictable fragment length-dependent decline. This study suggests that after embalming, the liver is the preferential tissue for recovering DNA for forensic analysis, as the liver samples were the most robust and the only sample type to yield DNA profiles up to 1 year.

Bone marrow (BM) harvesting is one of the essential sources of stem cells for haematopoietic stem cell transplantation. In 2019, commercial BM collection kits became unavailable in Europe. Consequently, we created an in-house BM collection kit as an alternative. We compared two groups of BM collections. The first collections were taken using an in-house kit from June 2022 through February 2023 and the second with a commercial kit from February 2021 through May 2022. These all took place at seven collection centres (CC). We analysed the harvest quality (cell blood count, CD34+ cells, viability, potency and sterility), the incidents occurring with each kit and the time to neutrophil and platelet engraftment in recipients. A total of 23 donors underwent BM harvesting with the in-house kit and 23 with the commercial one. Both cohorts were comparable regarding donor characteristics, CC and time to procedure. No statistical differences were found in harvest quality between the in-house and commercial kits. A new transfusion set was required in three BM harvests (13%) with the in-house kit because of filter clogging. The median time to neutrophil and platelet engraftment was 21 days for both cohorts and 29 days (in-house) and 33 days (commercial), p = 0.284, respectively. The in-house BM collection kit offers a real approach to solve the diminished supply of commercial kits. A higher risk of filter clogging was observed compared with commercial kits due to the lack of 850 and 500 μm filters.

Touch DNA evidence plays a pivotal role in forensic investigations, particularly in the absence of visible biological fluids. However, trace DNA recovery is influenced by multiple factors, including substrate type, collection technique, extraction method, and environmental exposure. Despite widespread adoption of both cotton swabbing and tapelifting methods, little is known about their comparative performance under real-world environmental stressors such as sand contamination, which is common in arid and desert regions. This study evaluated the efficiency of two collection methods Copan cotton swabs (CS) and SceneSafe Fast™ minitapes (MT) and two DNA extraction kits PrepFiler Express BTA™ and QIAamp® DNA Investigator Kit in recovering Touch DNA from 100 fabric-based items (face masks, T-shirts, and caps) exposed to sandy outdoor environments. DNA concentration, STR profile completeness, and total allele count were analyzed using standardized protocols. Statistical methods included factorial ANOVA, chi-square analysis, and Pearson correlation to assess method performance and inter-variable relationships. Cotton swabs consistently outperformed minitapes in DNA yield and profile integrity across all fabric types. The combination of cotton swabs with Prep Filer extraction produced the highest DNA concentrations (mean >2.0 ng/μL) and yielded full STR profiles in a majority of cases. In contrast, minitape sampling, particularly when combined with QIAamp extraction, resulted in lower recovery and an increased rate of partial or mixed profiles. A strong positive correlation (r > 0.8) was observed between DNA concentration and STR profile completeness. These findings indicate that adhesive-based sampling methods are substantially hindered by sand interference, reducing their forensic value in contaminated conditions. The results emphasize the need for adaptive forensic DNA protocols that account for environmental contaminants such as sand. Cotton swabbing in combination with Prep Filer Express BTA™ extraction represents a robust and reliable strategy for maximizing trace DNA recovery from wearable items in sandy or dusty outdoor settings. This research provides critical guidance for improving DNA evidence collection in real-world casework and supports the development of optimized trace DNA workflows for challenging environments.

Background/Objectives: Obtaining reliable DNA profiles from archival tissue preserved as formalin-fixed, paraffin-embedded (FFPE) samples remains a major challenge in both forensic and medical evaluations. The quality of DNA isolated from FFPE material is frequently compromised due to formalin-induced fragmentation and chemical modifications. These limitations are particularly relevant in cases of suspected medical malpractice related to cancer diagnosis or treatment, where retrospective molecular analyses may provide critical evidence. The aim of this study was to evaluate the performance of the Maxwell® RSC Xcelerate DNA FFPE Kit (Promega) in generating DNA profiles from archival FFPE tissue blocks of endometrial cancer and to identify the limitations associated with this approach. Methods: Archival FFPE blocks of endometrial cancer were analyzed using the Maxwell® RSC Xcelerate DNA FFPE Kit. DNA yield, purity, and degradation indices were assessed using standard real-time PCR-based quantification methods. Short tandem repeat (STR) profiling was performed with forensic genotyping kits, and the completeness, allele balance, and reliability of obtained profiles were evaluated. The obtained results were compared with reference quality thresholds commonly used in forensic practice. Results: The Maxwell® RSC Xcelerate Kit allowed for recovery of relatively high DNA yields with consistently low degradation indices, confirming good extraction efficiency from FFPE samples. Nevertheless, despite favorable quantitative values, the generation of complete STR profiles was often unsuccessful. Partial or incomplete profiles were frequent, characterized by allele dropout and imbalance, which substantially reduced their evidentiary value. These findings suggest that DNA fragmentation and fixation-related artifacts impair amplification efficiency and limit the usefulness of STR analysis. Conclusions: This study emphasizes the persistent challenges of DNA profiling from FFPE tissue in forensic-medical contexts. Although the Maxwell® RSC Xcelerate Kit demonstrated effective DNA recovery, the ability to generate complete and interpretable STR profiles remained limited. Further refinement of extraction protocols, as well as improved interpretative strategies, are required to enhance the reliability and evidentiary significance of molecular analyses based on archival FFPE material.

Feasibility and Safety of Adoptive Immunotherapy with CIK Cells after Cord Blood Transplantation

Patients and clinicians often raise concerns about radiation exposure to various organs during computerized tomography-based imaging. We evaluated radiation exposure during standard and low-dose imaging protocols for non-contrast computerized tomography, computerized tomography angiography and computerized tomography perfusion of the head. Whether reducing the radiation dose affected the image quality was also evaluated. Radiation data were retrieved for computerized tomography-based imaging studies performed for acute ischemic stroke patients during 2015. The volume-weighted computerized tomography dose index, dose-length product, scan length, effective dose and whole-body integral dose for brain, skin, eye, thyroid and red bone marrow were extracted from dose-tracking software. Dose metrics for low-dose protocols data were compared with standard protocols. The calculated effective doses for non-contrast computerized tomography, computerized tomography angiography and computerized tomography perfusion were 2.56 ± 0.67 mSv, 4.45 ± 2.5 mSv, and 4.47 ± 0.85 mSv, respectively for 391 acute ischemic stroke patients. Corresponding radiation exposures for low-dose protocol (n = 31) were non-contrast computerized tomography (2.36 ± 0.65 mSv), computerized tomography angiography (1.57 ± 0.74 mSv) and computerized tomography perfusion (2.20 ± 0.55 mSv). Overall, the effective dose for one complete stroke imaging protocol (non-contrast computerized tomography + computerized tomography angiography + computerized tomography perfusion) for the standard-dose protocol was 11.48 mSv, which was reduced to 6.13 mSv (46.6% reduction) using a low-dose protocol (p < 0.001). Reduced radiation exposure was noted for other radiosensitive organs. Radiation exposures of sensitive organs are within acceptable limits with standard neuroimaging protocols for acute ischemic stroke. Lower-dose computerized tomography imaging protocols reduced the radiation doses without appreciable deterioration in image quality.

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.

BackgroundDNA profiling is an integral part of forensic work. Enough and good samples for DNA quantification and profiling are mandatory. Aim of the studyTo quantify and profile DNA from siwak and toothbrushes and study the effect of time on this process. MethodologyThe present study included DNA profiling from siwak and effect of time on extracted DNA. Then; obtained data were compared to those extracted from toothbrushes. For this purpose, 25 uncovered siwaks, 4 covered siwaks, and 25 toothbrushes were used. Samples were divided into 8 groups; first group (4months; 4 volunteers); second group (3months, 5 volunteers); third group (2months, 4 volunteers); fourth group (1month, 5 volunteers); fifth group (1week, 3 volunteers); sixth group (the same day, 4 volunteers and covered siwak was added); seventh group (reference samples) and group 8 (positive and negative control samples). Extraction of DNA was done using Promega kit and then PCR was used for amplification and DNA profiling was done. ResultsConsiderable quantity of human genomic DNA was successfully extracted from siwaks. There was no proof of the existence of any substance in siwak or its components that may interfere with amplification of DNA by PCR or interfere with obtaining DNA profiles. Siwak proved to be a good and reliable source of human genomic DNA that is enough for DNA analysis. There has been no effect of time on DNA analysis and DNA profiling in this study (within the targeted period which is 4months). ConclusionSiwak contains enough quantity of DNA, and retained good DNA profiling; and when compared to toothbrushes, siwak is a reasonable source of DNA profiling when found at the scene of crime. In addition, time of storage up to 4months had no or little effects on results.

A recurring problem with ancient specimens from wildlife animals is that their preserved tissues contain small amounts of DNA in a degraded state. The specific objectives of the present study were: (1) To determine DNA yields from different animal tissues; (2) to compare traditional (manual) DNA extraction protocols with commercial procedures and (3) to assess the success of PCR amplification of Inter-Simple Sequence Repeats (ISSR) loci in degraded animal samples. Liver, stomach and muscle samples were extracted from coyote (Canis latrans) and long-tailed weasel (Mustela frenata) for this research. Manual protocols for DNA extraction were compared to a commercial kit procedure (Qiagen DNeasy kit). Genomic DNA in different states (intact, apoptotic and degraded) were amplified using a panel of ISSR primers. No DNA was recovered from coyote stomach samples using the manual extraction protocol. DNA concentrations in stomach and liver samples from coyote were 10.31 ng/μL and 15.8 ng/μL, respectively using the Qiagen extraction kit. In general, the kit extraction method yielded more DNA than the manual extraction procedure but it is more expensive. Intact and apoptotic genomic DNA were successfully amplified by PCR resulting in a similar profile. Artificially degraded DNA showed partial amplification. Thus, the ISSR marker system is suitable for animal population genetics when only limited and/or degraded animal DNA is available.

Autosomal short tandem repeat (STR) population data collected from a well characterized population are needed to correctly assigning the weight of DNA profiles in the courtroom and widely used for ancestral analyses. In this study, allele frequencies for the 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR® Identifiler® plus kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA) were obtained by genotyping 332 unrelated individuals of Ghanaian origin. Statistical tests on STR genotype data showed no significant departure from Hardy-Weinberg equilibrium (HWE). The overall match probability, combined power of exclusion and combined power of discrimination for these loci were 1 in 3.85 × 1017, 0.99999893 and 0.99999998, respectively. Polymorphic information content (PIC) greater than 0.70 was observed for all loci except TH01 and D13S317. These statistical parameters confirm that this combination of loci is valuable for forensic identification and parentage analysis. Our results were also compared with those for 20 other human populations analyzed for the same set of markers. We observed that the Ghanaian population grouped with other African populations in two-dimensional principal coordinate (PCO) and a neighbor-joining (N-J) data mapping and placed closest to Nigerians. This observation reflects cultural similarities and geographical factors, coupled with the long history of migration and trading activities between Ghana and Nigeria. Our report provides what we believe to be the first published autosomal STR data for the general Ghanaian population using 15 loci genotyped using the AmpFlSTR® Identifiler® plus kit methodology. Our data show that the loci tested have sufficient power to be used reliably for DNA profiling in forensic casework and help to elucidate the genetic history of people living in the country.

Background: DNA profiling with sets of highly polymorphic autosomal short tandem repeat (STR) markers has been applied in various aspects of human identification in forensic casework for nearly 20 years. However, in some cases of complex kinship investigation, the information provided by the conventionally used STR markers is not enough, often resulting in low likelihood ratio (LR) calculations. In these cases, it becomes necessary to increment the number of loci under analysis to reach adequate LRs. Recently, it has been proposed that single nucleotide polymorphisms (SNPs) could be used as a supportive tool to STR typing, eventually even replacing the methods/markers now employed. Methods: In this work, we describe the results obtained in 7 revised complex paternity cases when applying a battery of STRs, as well as 52 human identification SNPs (SNPforID 52plex identification panel) using a SNaPshot methodology followed by capillary electrophoresis. Results: Our results show that the analysis of SNPs, as complement to STR typing in forensic casework applications, would at least increase by a factor of 4 total PI values and correspondent Essen-Möllerʼs W value. Conclusions: We demonstrated that SNP genotyping could be a key complement to STR information in challenging casework of disputed paternity, such as close relative individualization or complex pedigrees subject to endogamous relations.

Nucleic acid extraction from complex environmental and ancient tissue material is prone to co‐extract inhibitory substances that make further molecular analysis difficult or impossible. This co‐extraction occurs in both solid‐phase and liquid‐phase/organic nucleic acid extraction protocols. Currently, the widely used method to overcome inhibition includes the addition of bovine serum albumin (BSA) to the downstream enzymatic reactions or the dilution of the nucleic acid extracts. BSA, however, seems to reduce the inhibitory effect of certain compounds only, and excessive dilution may change the original DNA composition. In this study, we introduce an innovative new method using linear polyacrylamide (LPA) to efficiently precipitate and purify nucleic acids extracted from complex environmental and ancient tissue samples in one working step. The LPA method replaces the precipitation step in classic liquid‐phase/organic extraction protocols or can be easily applied as an additional post‐extraction step on impure DNA extracts. As a proof of concept, we experimented with this method on different ancient human mummy samples (bones, soft tissues and gut contents) from different time periods (5000 BC–1800 AD), as well as on complex environmental samples (e.g. soil, activated sludge and animal faeces) known to contain inhibitory compounds. We demonstrated that LPA precipitates nucleic acids, even in an aqueous ethanol solution without the addition of chaotropic salts, resulting in the recovery of highly pure DNA from all tested samples that displayed inhibition with previously published extraction protocols. Compared to the current, most widely used silica‐based extraction method for ancient and sedimentary DNA, our LPA method resulted in comparable DNA qualities and overall DNA compositions (human endogenous content and microbial diversity). In conclusion, our LPA method with its high purifying capacity provides an important alternative to the commonly used DNA extraction protocols in the environmental and ancient DNA (aDNA) fields.

Forensic short tandem repeats (STR) profiling on touch DNA samples has emerged as a primary method for human identification. The stability and uniqueness of STR combination from the targeted locus in each individual make it a precision marker for human identification. Touch DNA samples can be found in traces of biological material shed from a person. This work aimed to identify the lowest concentration limit required for generating an interpretable DNA profile and the sensitivity of the STR loci applied. Touch DNA samples were collected from donors who were asked to hold a rope for 5 min. A double swab technique was used to lift the touch samples from the rope. These samples are subjected to DNA extraction and quantification. Two STR amplification cycles, 29 and 34 cycles, were used. DNA concentration greatly influences the success of amplifying the target allele at each STR locus to be interpreted into a complete DNA profile, shown by its allele peak. Touch DNA concentration &gt;0.25 ng can produce a complete DNA profile. LCN method successfully amplified touch DNA with a concentration 0.0625–0.25 ng/µL. Limit detection of touch DNA analysis is 0.25 ng/µL. Low-copy DNA can still be analyzed within 0.0625–0.25 ng/µL.