Quantitative evaluation of endometrium-expressed mRNAs for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness.

Development and comparison of forensic interval age prediction models by statistical and machine learning methods based on the methylation rates of ELOVL2 in blood DNA.

Age estimation can be useful information for narrowing down candidates of unidentified donors in criminal investigations. Various age estimation models based on DNA methylation biomarkers have been developed for forensic usage in the past decade. However, many of these models using ordinary least squares regression cannot generate an appropriate estimation due to the deterioration in prediction accuracy caused by an increased prediction error in older age groups. In the present study, to address this problem, we developed age estimation models that set an appropriate prediction interval for all age groups by two approaches: a statistical method using quantile regression (QR) and a machine learning method using an artificial neural network (ANN). Methylation datasets (n = 1280, age 0-91 years) of the promoter for the gene encoding ELOVL fatty acid elongase 2 were used to develop the QR and ANN models. By validation using several test datasets, both models were shown to enlarge prediction intervals in accordance with aging and have a high level of correct prediction (>90 %) for older age groups. The QR and ANN models also generated a point age prediction with high accuracy. The ANN model enabled a prediction with a mean absolute error (MAE) of 5.3 years and root mean square error (RMSE) of 7.3 years for the test dataset (n = 549), which were comparable to those of the QR model (MAE = 5.6 years, RMSE = 7.8 years). Their applicability to casework was also confirmed using bloodstain samples stored for various periods of time (1-14 years), indicating the stability of the models for aged bloodstain samples. From these results, it was considered that the proposed models can provide more useful and effective age estimation in forensic settings.

Potential application of Staphylococcus species detection in the specific identification of saliva.

When found at crime scenes, saliva constitutes forensically relevant evidence. Although several tests have been developed to effectively identify saliva in such circumstances, most cannot discriminate between saliva and nasal secretion. Recently, studies have developed saliva tests involving oral bacteria as salivary markers. Although the specificity of such tests has been evaluated on most biological specimens, their specificity for nasal secretion samples remains to be tested. Herein, to improve the specificity of the saliva detection tests for nasal secretion samples, we reanalyzed a public microbiome dataset and conducted inhouse 16S rRNA sequencing to identify a new marker to distinguish between saliva and nasal secretions. The sequencing data indicated the existence of oral bacteria such as Streptococcus in nasal secretion samples, which may be responsible for the false positives in the saliva tests. Furthermore, we found that including the 16S rRNA gene of the genus Staphylococcus as a nasal secretion marker may improve the specificity of PCR-based saliva tests for nasal secretion samples. In addition, we assessed the specificity of previously developed salivary bacteria detection tests for nasal secretion samples and oral bacterial markers were detected in two of eight nasal secretion samples, which led to the false positive results for saliva detection. Thus, the specificity of such tests can be improved by adding Staphylococcus as a nasal marker, as revealed by our sequencing analysis.

Validation of a novel fluorescent probe-based real-time PCR assay to detect saliva-specific unmethylated CpG sites for saliva identification.

The identification of saliva from forensic samples is often important to establish what happened at a crime scene, especially in sexual assault cases. Recently, CpG sites that are specifically methylated or unmethylated in saliva have been reported as markers for saliva identification. In this study, we designed a fluorescent probe-based real-time polymerase chain reaction (PCR) assay for analyzing the methylation status of two neighboring CpG sites, which we previously found were saliva-specifically unmethylated. Specificity analysis using various types of body fluid/tissue samples demonstrated a probe detecting the unmethylation of the two CpG sites reacted only to saliva DNA, indicating this probe as an all-or-nothing marker for the presence of saliva DNA. Sensitivity analysis demonstrated that the detection limit was 0.5 ng saliva DNA as input for bisulfite conversion, while we confirmed a negative effect of larger amounts of non-saliva DNA on sensitivity in the analysis of saliva-vaginal DNA mixtures. We finally validated the applicability of this test to swabs from licked skin and bottles after drinking as mock forensic samples in comparison with other saliva-specific markers. We confirmed the potential usefulness of this test for skin samples, from which a saliva-specific mRNA was not detected reliably, while the ingredients in several beverages might affect methylation analysis. Given the simplicity of real-time PCR as well as the high specificity and sensitivity of the test, we believe the developed method is suitable for routine forensic analysis and can play an important role in saliva identification.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes.

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration.

Sexual assault with anal penetration is closely related to child sexual abuse or male victims. However, it is difficult to prove such an act by using biological samples collected from the surface of a suspected object because procedures for identifying rectal mucosa have not been developed sufficiently. Therefore, for the specific identification of rectal mucosa, mRNA markers reported to be characteristically expressed in the rectum were screened and a multiplex RT-PCR procedure was developed for the simultaneous determination of those candidate markers. The detectability and specificity of rectal mucosa candidate markers were evaluated using rectal mucosa samples and forensically relevant body fluids. Diluted or mixed samples were also tested to evaluate the applicability of this procedure for forensic casework. As a result, simultaneous amplification and determination of the selected candidates (PHGR1, MUC13, CLCA1, MEP1A, CDX1, and ZG16) and reference gene were successfully performed using a multiplex RT-PCR assay combined with capillary electrophoresis and fragment analysis. Applying the cutoff values, none of the other body fluids cross-reacted with rectal mucosa candidate markers. Because the low sensitivity and detectability of some candidate markers could be compensated for by their simultaneous detection, all six candidate markers were considered to be applicable as rectal mucosa markers. Besides, the developed assay should not be performed on suspicious fecal samples directly because these markers could be positive in the fecal samples themselves. The developed multiplex RT-PCR procedure might not be suitable for minute or diluted samples; however, it might be resistant to contamination with sexual assault-related body fluids. In conclusion, the simultaneous determination of selected rectal mucosa markers with a biological sample collected from the surface of a suspected object could be beneficial for criminal investigation of sexual assault with anal penetration.

A new approach for forensic analysis of saliva-containing body fluid mixtures based on SNPs and methylation patterns of nearby CpGs.

Saliva samples obtained from crime scenes often contain body fluids from other people, which makes it difficult to not only interpret the obtained DNA profiles, but also interpret saliva identification test results. α-amylase activity, an indicator of most saliva identification methods, can be slightly detected in other body fluids. This study aimed to overcome these difficulties. Here, we identified 13 saliva-specific methylated regions and five saliva-specific unmethylated regions neighboring common single nucleotide polymorphisms (SNPs) by array-based genome-wide methylation analysis of pooled saliva, blood, semen, or vaginal swab samples. Bisulfite sequencing by massively parallel sequencing (MPS) technology was then performed using individual body fluid samples to evaluate the saliva-specificity of each CpG of the three regions selected from the identified candidates. Although no single CpG demonstrated complete saliva-specificity, we found that the reads that were simultaneously (un)methylated at the selected neighboring two to three CpGs of each region were highly specific for saliva DNA. Based on these findings, we then designed MPS-based bisulfite sequencing assays for each region to analyze the selected CpGs and SNP(s) on the same read. These assays could identify the saliva of a target person from body fluid mixtures of known contributors (individual-specific saliva identification) by calculating the ratios of simultaneous (un)methylation at the selected CpGs within the reads containing SNP alleles unique to the target person. Moreover, these assays could indicate the SNP types of saliva DNA (saliva-specific genotyping) from body fluid mixtures by analyzing the alleles of the reads simultaneously (un)methylated at the selected CpGs, while careful attention should be paid to interpret the results of heterologous genotypes. Although further regions should be identified, especially for saliva-specific individual identification, the CpG-SNP approach may be an effective method to interpret the complicated results obtained from saliva-containing body fluid mixtures.

A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification.

The identification of vertebrate species is important in numerous fields including archaeology, ecology, as well as food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.

Evaluation of CRISPR Diversity in the Human Skin Microbiome for Personal Identification.

The highly personalized human skin microbiome may serve as a viable marker in personal identification. Amplicon sequencing resolution using 16S rRNA cannot identify bacterial communities sufficiently to discriminate between individuals. Thus, novel higher-resolution genetic markers are required for forensic purposes. The clustered regularly interspaced short palindromic repeats (CRISPRs) are prokaryotic genetic elements that can provide a history of infections encountered by the bacteria. The sequencing of CRISPR spacers may provide phylogenetic information with higher resolution than other markers. However, using spacer sequencing for discrimination of personal skin microbiome is difficult due to limited information on CRISPRs in human skin microbiomes. It remains unclear whether personal microbiome discrimination can be achieved using spacer diversity or which CRISPRs will be forensically relevant. We identified common CRISPRs in the human skin microbiome via metagenomic reconstruction and used amplicon sequencing for deep sequencing of spacers. We successfully reconstructed 24 putative CRISPR arrays using metagenomic data sets. A total of 1,223,462 reads from three CRISPR arrays revealed that spacers in the skin microbiome were highly personalized, and conserved repeats were commonly shared between individuals. These individual specificities observed using CRISPR typing were confirmed by comparing the CRISPR diversity to microbiome diversity assessed using 16S rRNA amplicon sequencing. CRISPR typing achieved 95.2% accuracy in personal classification, whereas 16S rRNA sequencing only achieved 52.6%. These results suggest that sequencing CRISPRs in the skin microbiome may be a more powerful approach for personal identification and ecological studies compared to conventional 16S rRNA sequencing.IMPORTANCE Microbial community diversity analysis can be utilized to characterize the personal microbiome that varies between individuals. CRISPR sequences, which reflect virome structure, in the human skin environment may be highly personalized similar to the structures of individual viromes. In this study, we identified 24 putative CRISPR arrays using a shotgun metagenome data set of the human skin microbiome. The findings of this study expand our understanding of the nature of CRISPRs by identifying novel CRISPR candidates. We developed a method to efficiently determine the diversity of three CRISPR arrays. Our analysis revealed that the CRISPR spacer diversity in the human skin microbiome is highly personalized compared with the microbiome diversity assessed by 16S rRNA sequencing, providing a new perspective on the study of the skin microbiome.

Current Methods for Body Fluid Identification Related to Sexual Crime: Focusing on Saliva, Semen, and Vaginal Fluid.

Although, DNA typing plays a decisive role in the identification of persons from blood and body fluid stains in criminal investigations, clarifying the origin of extracted DNA has also been considered an essential task in proving a criminal act. This review introduces the importance of developing precise methods for body fluid identification. Body fluid identification has long relied on enzymatic methods as a presumptive assay and histological or serological methods as a confirmatory assay. However, because the latest DNA typing methods can rapidly obtain results from very small and even old, poorly preserved samples, the development of a novel corresponding body fluid identification method is required. In particular, an immunochromatographic method has been introduced to identify saliva and semen from sexual crimes. In addition, for vaginal fluid identification, attempts have been made in the past decade to introduce a method relying on body fluid-specific mRNA expression levels. At present, the development of molecular biological methods involving microRNA, DNA methylation, and resident bacterial DNA is ongoing. Therefore, in criminal investigations, body fluid identification is an essential task for correctly applying the results of DNA typing, although further research and development are required.

A Proposed Procedure for Discriminating between Nasal Secretion and Saliva by RT-qPCR.

In forensic casework, nasal secretion can be a good source of DNA. Moreover, saliva can prove useful in cases of sexual assault. However, discriminating between these body fluids is often difficult because of cross-reactivity between them on presumptive and confirmatory tests. Therefore, an RT-qPCR procedure was developed to discriminate between nasal secretion and saliva. Characteristic genes in nasal secretion and/or saliva (BPIFA1, STATH, HTN3, and PRH2) were selected as candidates. Discrimination criteria were established based on the expression levels of these markers in various body fluids. In addition, a flowchart was proposed and used to discriminate among nasal secretion, saliva, and other body fluids in various forensic samples. BPIFA1 was highly expressed in nasal secretion but was also expressed in saliva, semen, and vaginal fluid at trace levels. STATH was expressed in nasal secretion and saliva but not in other body fluids. HTN3 was specifically expressed in most of the saliva samples, as reported previously. Unexpectedly, PRH2 was expressed in only a few saliva samples. Using the proposed criteria and flowchart, nasal secretion and saliva were successfully discriminated among the various body fluids tested. The developed procedure could be useful in forensic casework.

Development of a multiplex RT-PCR assay and statistical evaluation of its use in forensic identification of vaginal fluid.

The identification of vaginal fluid from casework samples of sexual assaults provides important probative evidence of vaginal intercourse. The aim of this study was to establish a more specific procedure for identifying vaginal fluids for forensic purposes. Vaginal fluid marker candidates have been evaluated quantitatively and five of these markers (ESR1, SERPINB13, KLK13, CYP2B7P1, MUC4) have been amplified simultaneously by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) procedure. Each amplicon has been separated and quantified automatically using chip electrophoresis. Subsequently, in the present study, detectability and cross-reactivity of the developed multiplex procedure were assessed in detail using various forensically relevant body fluids. Then, a cutoff value for the positive detection of vaginal fluids was set for each marker by Youden index. The ability of the multiplex RT-PCR assay to distinguish between vaginal and other body fluids was evaluated statistically using a likelihood ratio (LR) that was estimated using a Bayesian estimation approach to consider the infrequency of detection. A high LR was obtained when all five markers showed positive results (LR = 4.33 × 109; 95% credible interval, 3.95 × 107 -2.87 × 1012). The developed procedure was validated using vaginal fluid samples under various conditions. High LRs were found for aged vaginal fluid stains, although each amplicon peak was low. It was also able to identify vaginal stains mixed with other body fluids. In conclusion, the multiplex RT-PCR-based procedure followed by the statistical evaluation using LR could be a powerful tool for the objective identification of vaginal fluids.

Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification.

Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.

Immunohistochemical staining of skin-expressed proteins to identify exfoliated epidermal cells for forensic purposes.

The determination of cell type in biological casework samples would be helpful to identify the type of body fluids and interpret the DNA source in forensic laboratories. Exfoliated epidermal cells are considered to be a reasonable source of touch DNA; therefore, we developed and assessed an immunohistochemistry (IHC) procedure for identifying exfoliated epidermal cells as a screening test of touch DNA samples. Among five candidate protein markers investigated in this study, keratin 10 and kallikrein-related peptidase 5 were strongly expressed in the stratum corneum layer of the skin; however, their specificity was insufficient to identify epidermal cells. In contrast, IHC for corneodesmosin (CDSN), desmocollin 1 (DSC1), and filaggrin (FLG) was considered to be applicable because of their detectability and specificity on skin swab samples. Actually, CDSN and DSC1 could be good markers for exfoliated epidermal cells on touched contact traces that were contaminated with many unidentified impurities. Besides, positivity for FLG on mock casework samples appeared to be lower than for the other markers, which might be caused by its instability. Finally, the relationship between positivity for IHC and DNA yield was analyzed using skin swab samples. Although it was difficult to determine these correlations quantitatively because of the heterogeneous distribution of cells and the presence of cell-free DNA, the DNA-quantifiable samples analyzed in this study contained at least some of IHC-positive epidermal cells. In conclusion, IHC detection of skin-enriched proteins, especially CDSN and DSC1, could be useful for screening samples that have been handled or touched by someone before DNA analysis.

Development of a DNA methylation-based semen-specific SNP typing method: A new approach for genotyping from a mixture of body fluids.

Genotyping from samples containing different types of body fluids is a major difficulty in forensic investigations. Recently, CpG sites that are specifically methylated or unmethylated in different types of body fluids have been reported as novel markers for body fluid identification. In this study, we hypothesized that the simultaneous analysis of CpGs and neighboring polymorphic sites on the same molecule could be useful for individual DNA typing from mixed samples. We performed a proof-of-concept study of this approach by searching the genome-wide methylation dataset deposited at the National Center for Biotechnology Information Gene Expression Omnibus repository for semen-specific CpG markers adjacent to common single nucleotide polymorphisms. From the identified candidates, we selected 5 regions on different chromosomes and validated the presence of semen-specific methylation or unmethylation in each region by pyrosequencing analyses. By combining methylation-specific polymerase chain reaction and pyrosequencing technology, we developed a semen-specific DNA typing method for two semen-specific methylated regions and one semen-specific unmethylated region. Finally, the method successfully identified semen-derived alleles from mixed stains, indicating that this methylation-based approach can be applicable to actual forensic samples. Since existing separation techniques physically isolate cells derived from each type of body fluid, this approach may be useful when existing methods cannot be performed due to the degradation of samples.

Soft and Robust Identification of Body Fluid Using Fourier Transform Infrared Spectroscopy and Chemometric Strategies for Forensic Analysis.

Body fluid (BF) identification is a critical part of a criminal investigation because of its ability to suggest how the crime was committed and to provide reliable origins of DNA. In contrast to current methods using serological and biochemical techniques, vibrational spectroscopic approaches provide alternative advantages for forensic BF identification, such as non-destructivity and versatility for various BF types and analytical interests. However, unexplored issues remain for its practical application to forensics; for example, a specific BF needs to be discriminated from all other suspicious materials as well as other BFs, and the method should be applicable even to aged BF samples. Herein, we describe an innovative modeling method for discriminating the ATR FT-IR spectra of various BFs, including peripheral blood, saliva, semen, urine and sweat, to meet the practical demands described above. Spectra from unexpected non-BF samples were efficiently excluded as outliers by adopting the Q-statistics technique. The robustness of the models against aged BFs was significantly improved by using the discrimination scheme of a dichotomous classification tree with hierarchical clustering. The present study advances the use of vibrational spectroscopy and a chemometric strategy for forensic BF identification.

Evaluation of skin- or sweat-characteristic mRNAs for inferring the human origin of touched contact traces.

The source of small amounts of touch DNA, which is transferred from the skin to an object when it is handled or touched, could be an issue in the forensic analysis of criminal cases. Here, we performed an extended evaluation of skin- or sweat-characteristic mRNAs to investigate their usability to infer whether an object has been handled or touched by someone. First, we compared the expression levels of candidate genes between skin swabs and other body fluids by quantitative RT-PCR analysis. Among the analyzed genes, corneodesmosin (CDSN), late cornified envelope 1C (LCE1C), filaggrin (FLG), desmocollin 1, and dermcidin were selected for further analysis on the basis of their specificities and sensitivities. Then, we tried to detect these genes from mock casework samples. As a result, CDSN, LCE1C, and FLG could be good markers because of their detectability. Finally, we determined the correlation between the expression of these genes and DNA yield of skin swabs to assess their adaptability as a screening test for touch DNA samples. However, the detectability of these genes was not correlated with the DNA yield of skin swab samples. In conclusion, gene expression analysis of the skin- or sweat-characteristic mRNAs CDSN, LCE1C, and FLG could be useful for inferring the skin origin of touched contact traces, but the use of the expression levels of these mRNAs for the prediction of DNA yield is problematic. To develop a screening test for touch DNA samples, other markers that have a well-correlated sensitivity with DNA analysis should be investigated.

Practical evaluation of an RNA-based saliva identification method.

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.

Spectral Mining for Discriminating Blood Origins in the Presence of Substrate Interference via Attenuated Total Reflection Fourier Transform Infrared Spectroscopy: Postmortem or Antemortem Blood?

Often in criminal investigations, discrimination of types of body fluid evidence is crucially important to ascertain how a crime was committed. Compared to current methods using biochemical techniques, vibrational spectroscopic approaches can provide versatile applicability to identify various body fluid types without sample invasion. However, their applicability is limited to pure body fluid samples because important signals from body fluids incorporated in a substrate are affected strongly by interference from substrate signals. Herein, we describe a novel approach to recover body fluid signals that are embedded in strong substrate interferences using attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy and an innovative multivariate spectral processing. This technique supported detection of covert features of body fluid signals, and then identified origins of body fluid stains on substrates. We discriminated between ATR FT-IR spectra of postmortem blood (PB) and those of antemortem blood (AB) by creating a multivariate statistics model. From ATR FT-IR spectra of PB and AB stains on interfering substrates (polyester, cotton, and denim), blood-originated signals were extracted by a weighted linear regression approach we developed originally using principal components of both blood and substrate spectra. The blood-originated signals were finally classified by the discriminant model, demonstrating high discriminant accuracy. The present method can identify body fluid evidence independently of the substrate type, which is expected to promote the application of vibrational spectroscopic techniques in forensic body fluid analysis.

Advanced forensic validation for human spermatozoa identification using SPERM HY-LITER™ Express with quantitative image analysis.

Identification of human semen is indispensable for the investigation of sexual assaults. Fluorescence staining methods using commercial kits, such as the series of SPERM HY-LITER™ kits, have been useful to detect human sperm via strong fluorescence. These kits have been examined from various forensic aspects. However, because of a lack of evaluation methods, these studies did not provide objective, or quantitative, descriptions of the results nor clear criteria for the decisions reached. In addition, the variety of validations was considerably limited. In this study, we conducted more advanced validations of SPERM HY-LITER™ Express using our established image analysis method. Use of this method enabled objective and specific identification of fluorescent sperm's spots and quantitative comparisons of the sperm detection performance under complex experimental conditions. For body fluid mixtures, we examined interference with the fluorescence staining from other body fluid components. Effects of sample decomposition were simulated in high humidity and high temperature conditions. Semen with quite low sperm concentrations, such as azoospermia and oligospermia samples, represented the most challenging cases in application of the kit. Finally, the tolerance of the kit against various acidic and basic environments was analyzed. The validations herein provide useful information for the practical applications of the SPERM HY-LITER™ Express kit, which were previously unobtainable. Moreover, the versatility of our image analysis method toward various complex cases was demonstrated.