A Bibliometric and Visual Analysis of the Current Status and Trends of Forensic Mixed Stain Research.

OBJECTIVES: To explore the context and hotspot changes of forensic mixed stain research through bibliometric approach. METHODS: The literature of forensic mixed stain included in the core collection of Web of Science database from 2011 to 2022 were collected as the study object, and the annual publication number, countrie (region), institution, journal, keywords, etc. were bibliometrically and visually analyzed using the R-based Bibliometrix 1.1.6 package and VOSviewer 1.6.18 software. RESULTS: A total of 732 articles on forensic mixed stain were included from 2011 to 2022, with the annual number of articles published and the annual citation frequency showing a steady increase year by year. Among the 59 countries (regions) with the most published articles, the United States ranked first with 246 articles, followed by China with 153 articles. The literature came from 104 journals, and the total number of articles published in the top 10 journals was 633. FORENSIC SCI INT GENET ranked first with 307 articles. Visual analysis using VOSviewer software showed that keywords could be divided into four research clusters, namely the genetic marker development group (blue), the mixed stain typing analysis theory group (red), the sequencing analysis group (yellow), and the case sample research group (green). It can be divided into four development stages in terms of different time periods: early development (2011-2013), middle development (2014-2016), rapid development (2017-2020) and latest development (2021-2022). CONCLUSIONS: The number of publications by domestic and foreign scholars in the study of mixed stain in forensic science is showing a relatively stable trend. Machine learning, next generation sequencing and other research have been the hottest topics that have attracted the most attention in recent years, which is expected to further develop the theory of mixed stain typing and sequencing analysis in forensic mixed stain research.

A Bibliometric and Visual Analysis of the Current Status and Trends of Foren-sic Mixed Stain Research / 法医学杂志

Objective To explore the context and hotspot changes of forensic mixed stain research through bibliometric approach.Methods The literature of forensic mixed stain included in the core col-lection of Web of Science database from 2011 to 2022 were collected as the study object,and the an-nual publication number,countrie(region),institution,journal,keywords,etc.were bibliometrically and visually analyzed using the R-based Bibliometrix 1.1.6 package and VOSviewer 1.6.18 software.Re-sults A total of 732 articles on forensic mixed stain were included from 2011 to 2022,with the an-nual number of articles published and the annual citation frequency showing a steady increase year by year.Among the 59 countries(regions)with the most published articles,the United States ranked first with 246 articles,followed by China with 153 articles.The literature came from 104 journals,and the total number of articles published in the top 10 journals was 633.FORENSIC SCI INT GENET ranked first with 307 articles.Visual analysis using VOSviewer software showed that keywords could be divided into four research clusters,namely the genetic marker development group(blue),the mixed stain typing analysis theory group(red),the sequencing analysis group(yellow),and the case sample research group(green).It can be divided into four development stages in terms of different time peri-ods:early development(2011-2013),middle development(2014-2016),rapid development(2017-2020)and latest development(2021-2022).Conclusion The number of publications by domestic and foreign scholars in the study of mixed stain in forensic science is showing a relatively stable trend.Machine learning,next generation sequencing and other research have been the hottest topics that have attracted the most attention in recent years,which is expected to further develop the theory of mixed stain typing and sequencing analysis in forensic mixed stain research.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

Selection and Characterization of DNA Aptamers Targeting hLCN6 Protein for Sperm Capture.

It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science. In this project, we developed a DNA aptamer-based system for sperm separation and purification from mixed stain samples by targeting sperm surface proteins. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and associated with sperm maturation. Using systematic evolution of ligands by exponential enrichment (SELEX) technology, aptamers that bind with high affinity and specificity to hLCN6 were screened from a random single-stranded DNA (ssDNA) library using magnetic bead-bound hLCN6 as target. The enriched library was obtained after 15 SELEX rounds. Of hLCN6-binding aptamer variants, 19 were further classified into one of the four groups based on their N60 random sequence regions, wherein one representative from each group was characterized. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Binding capability of selected aptamers was investigated by quantitative PCR, and aptamer H2 was found to be the most specific aptamer to sperm cells. The dissociation constant (Kd) of H2 aptamer was calculated as 3.21 ± 0.75 nM. Furthermore, H2 aptamer-coupled magnetic beads can recognize and capture sperm cells, which establishes the foundation of an approach for rapidly isolating sperm cells from mixed stains based on nucleic acid-protein interaction.

Screening and Identification of Nucleic Acid Aptamers Specifically Binding to Human Lipocalin 6 / 中国生物化学与分子生物学报

It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science. Nucleic acid aptamers targeting sperm cell-surface proteins can be used for the separation and purification of sperms from mixed stain samples. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and is associated with sperm maturation. Using the systematic evolution of ligands by the exponential enrichment (SELEX) technique, magnetic bead-bound hLCN6 was used as the target molecule to screen for aptamers with high affinity and specificity to hLCN6 from a random single-stranded DNA (ssDNA) library. Through 15 rounds of positive selection and 3 rounds of negative selection, 24 clones were selected and subjected to sequence analysis. Subsequently, 4 candidate aptamers were selected and further examined for their binding affinity and specificity by enzyme-linked oligonucleotide adsorption (ELONA) and cell binding assays. One aptamer (H2) against hLCN6 with a high affinity and specificity was isolated and investigated by dot blotting and immunofluorescence staining. The result revealed that the candidate aptamer H2 with a dissociation constant of (3. 21 ± 0. 75) nmol/ L was able to recognize and specifically bind to hLCN6. The aptamer H2 also showed high affinity and specificity to human sperms in vitro, which establishes the foundation for the separation of sperm cells from mixed stain based on nucleic acid-protein interactions and provides a new scheme.

Application Prospect of Massively Parallel Sequencing in Mixed Stain Detection.

Mixed stains is the common biological sample in sexual crime cases. Its analysis and DNA profiles interpretation are one of the difficulties in forensic examination. The current genetic marking of mixed stain detection mainly rely on capillary electrophoresis （CE） separation technology, and the analysis methods of the results are mainly inclusion rate and likelihood methods. Because CE has limited resolution and is not able to exploit the efficacy of each genetic marker, its ability to split mixed stain is limited. In recent years, the emerging massively parallel sequencing （MPS） technique not only can obtain the base sequence information of genetic markers, but also is capable of detecting multiple genetic markers simultaneously, and thus derives new analytical methods, bringing new opportunities for forensic detection and analysis of mixed stain. This paper intends to review the application prospects of conventional mixed stain analyses and MPS technique, therefore to provide references for later research and practice.

[Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads].

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻9 mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 104/mL. When the number of sperm cells was 10³/mL, 104/mL and 105/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Application Prospect of Massively Parallel Sequencing in Mixed Stain Detection / 法医学杂志

Mixed stains is the common biological sample in sexual crime cases. Its analysis and DNA profiles interpretation are one of the difficulties in forensic examination. The current genetic marking of mixed stain detection mainly rely on capillary electrophoresis （CE） separation technology, and the analysis methods of the results are mainly inclusion rate and likelihood methods. Because CE has limited resolution and is not able to exploit the efficacy of each genetic marker, its ability to split mixed stain is limited. In recent years, the emerging massively parallel sequencing （MPS） technique not only can obtain the base sequence information of genetic markers, but also is capable of detecting multiple genetic markers simultaneously, and thus derives new analytical methods, bringing new opportunities for forensic detection and analysis of mixed stain. This paper intends to review the application prospects of conventional mixed stain analyses and MPS technique, therefore to provide references for later research and practice.

Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads / 生物工程学报

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Extraction of DNA from Sperm Cells in Mixed Stain by Nylon Membrane Bushing Separation Technique.

OBJECTIVES: To establish a novel method for the separation of sperm cells in mixed stain, and to evaluate its application value. METHODS: Totally 40 mixed stain samples were collected from sexual assault cases. Sperm cells were separated by the conventional differential lysis method and the nylon membrane bushing separation technique, respectively. The DNA of sperm cells was extracted with the silicon membrane kit （Forensic DNA Extraction Kit for Soft Tissues）. The PCR amplification was performed using AmpFℓSTR® Identifiler® Plus kit, and the products were electrophoresed by 3500xL genetic analyser. The results of two separation methods were then compared. RESULTS: Complete and single-source male STR genotypes could be obtained from all the 40 mixed stain samples except three samples with minimal residual of female DNA by the nylon membrane bushing separation technique. The STR genotypes of sperm cells could not be detected in 25 samples, which were obtained in 15 samples （seven were of incomplete male STR genotypes, six with residual of female DNA, two were complete and single-source STR genotypes of sperm cells）. CONCLUSIONS: The nylon membrane bushing separation technique developed in present study can be used in the separation of sperm cells in mixed stain, especially for the extraction of a small amount of sperm from a large quantity of female cells, which is inexpensive, rapid and simple.

Extraction of DNA from Sperm Cells in Mixed Stain by Nylon Membrane Bushing Separation Technique / 法医学杂志

OBJECTIVES@#To establish a novel method for the separation of sperm cells in mixed stain, and to evaluate its application value.@*METHODS@#Totally 40 mixed stain samples were collected from sexual assault cases. Sperm cells were separated by the conventional differential lysis method and the nylon membrane bushing separation technique, respectively. The DNA of sperm cells was extracted with the silicon membrane kit （Forensic DNA Extraction Kit for Soft Tissues）. The PCR amplification was performed using AmpFℓSTR® Identifiler® Plus kit, and the products were electrophoresed by 3500xL genetic analyser. The results of two separation methods were then compared.@*RESULTS@#Complete and single-source male STR genotypes could be obtained from all the 40 mixed stain samples except three samples with minimal residual of female DNA by the nylon membrane bushing separation technique. The STR genotypes of sperm cells could not be detected in 25 samples, which were obtained in 15 samples （seven were of incomplete male STR genotypes, six with residual of female DNA, two were complete and single-source STR genotypes of sperm cells）.@*CONCLUSIONS@#The nylon membrane bushing separation technique developed in present study can be used in the separation of sperm cells in mixed stain, especially for the extraction of a small amount of sperm from a large quantity of female cells, which is inexpensive, rapid and simple.

DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

Application of Differex(TM) system and FTA(R) technology to separation of sperm DNA from mixed stains / 대한법의학회지

Specimens from sexual crimes are generally mixed stains consisted of sperm cells(from suspect) and virginal cells(from victim). We have combined two new methods - Differex(TM) system and FTA(R) technology- to overcome shortcomings of method that has been used before to separate sperm DNA from mixed stains. This methods have shown additional benefits and similar quality than using the only Differex(TM) system to the experiment. The result of our experiment represents the possibility that Differex(TM) system and FTA(R) technology would be useful methods for DNA analysis related to sexual crimes because this system can save time, labor and contamination for experiments.