Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.

Identification of Peripheral Blood and Menstrual Blood Based on the Expression Level of MicroRNAs and Discriminant Analysis.

ABSTRACT: Objective To construct a discriminant analysis model based on the differential expression of multiple microRNAs （miRNAs） in two kinds of blood samples （peripheral blood and menstrual blood） and three non-blood samples （saliva, semen and vaginal secretion）, to form an identification solution for peripheral blood and menstrual blood. Methods Six kinds of miRNA （miR-451a, miR-144-3p, miR-144-5p, miR-214-3p, miR-203-3p and miR-205-5p） were selected from literature, the samples of five kinds of body fluids commonly seen in forensic practice （peripheral blood, menstrual blood, saliva, semen, vaginal secretion） were collected, then the samples were divided into training set and testing set and detected by SYBR Green real-time qPCR. A discriminant analysis model was set up based on the expression data of training set and the expression data of testing set was used to examine the accuracy of the model. Results A discriminant analysis statistical model that could distinguish blood samples from non-blood samples and distinguish peripheral blood samples from menstrual blood samples at the same time was successfully constructed. The identification accuracy of the model was over 99%. Conclusion This study provides a scientific and accurate identification strategy for forensic fluid identification of peripheral blood and menstrual blood samples and could be used in forensic practice.

Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

Evaluation of three commercial kits effective identification of menstrual blood based on the D-dimer.

Blood or bloodstains are encountered frequently in forensic investigations. Presumptive and more confirmatory tests for peripheral blood are well established, however, similar methods for menstrual blood identification are less so. D-dimer is a fibrin degradation product that occurs at high concentration in menstrual blood and therefore a potential target to screen for this body fluid. We evaluated three rapid tests to determine if they can discriminate menstrual blood from peripheral remote from a laboratory setting. Their sensitivity, specificity and robustness were also assessed. The assays were: a latex agglutination (Dade Dimertest Latex Assay), SERATEC PMB test and OneStep D-dimer RapidCard InstaTest, both of which are based on lateral flow immunochromatographic analysis. Of the three, greater sensitivity was observed using the OneStep D-dimer RapidCard InstaTest, regardless of whether liquid or a stain was used. This test also detected a result using the smallest volume of menstrual blood, 0.003125 µL. Specificity testing was based on six different body fluids (urine, saliva, peripheral blood, semen, sweats and vaginal fluid) resulting in all 30 samples testing negative for the D-dimer using the OneStep D-dimer RapidCard InstaTest. Mixtures at ratios 1:1, 1:3 and 1:9 (menstrual blood: the other biofluid or PBS) were tested and the results showed that D-dimer could be detected for all samples using either the Dade Dimertest Latex Assay or the OneStep D-dimer RapidCard InstaTest. The body fluids were exposed to environmental stresses such as various temperature (-20 °C, 4 °C, room temperature and 37 °C for 30, 90, 180 and 360 days) and fluctuations in humidity (42%, 76% and 100% humidity at room temperature for 1, 3, 5, 10 and 20 days): all samples were D-dimer positive using the OneStep D-dimer RapidCard InstaTest though the strength decreased relative to the increase of storage time and temperature or humidity. All 6 postmortem blood samples gave a positive result for D-dimer using the OneStep D-dimer RapidCard InstaTest and 2 samples gave a positive response using the Dade Dimertest Latex Assay and the SERATEC PMB test; peripheral blood postmortem samples can show an increase in D-dimer. Menstrual blood was recovered from the pads under the sample wells after testing using the two immunochromatographic assays from which STR alleles could be amplified successfully. The results presented here support the application of these commercial kits for effective identification of menstrual blood.

Analysis and comparison of machine learning methods for blood identification using single-cell laser tweezer Raman spectroscopy.

Raman spectroscopy, a "fingerprint" spectrum of substances, can be used to characterize various biological and chemical samples. To allow for blood classification using single-cell Raman spectroscopy, several machine learning algorithms were implemented and compared. A single-cell laser optical tweezer Raman spectroscopy system was established to obtain the Raman spectra of red blood cells. The Boruta algorithm extracted the spectral feature frequency shift, reduced the spectral dimension, and determined the essential features that affect classification. Next, seven machine learning classification models are analyzed and compared based on the classification accuracy, precision, and recall indicators. The results show that support vector machines and artificial neural networks are the two most appropriate machine learning algorithms for single-cell Raman spectrum blood classification, and this finding provides essential guidance for future research studies.

Microbiome-based body site of origin classification of forensically relevant blood traces.

Human blood traces are amongst the most commonly encountered biological stains collected at crime scenes. Identifying the body site of origin of a forensic blood trace can provide crucial information in many cases, such as in sexual and violent assaults. However, means for reliably and accurately identifying from which body site a forensic blood trace originated are missing, but would be highly valuable in crime scene investigations. With this study, we introduce a taxonomy-independent deep neural network approach based on massively parallel microbiome sequencing, which delivers accurate body site of origin classification of forensically-relevant blood samples, such as menstrual, nasal, fingerprick, and venous blood. A total of 50 deep neural networks were trained using a large 16S rRNA gene sequencing dataset from 773 reference samples, including 220 female urogenital tract, 190 nasal cavity, 213 skin, and 150 venous blood samples. Validation was performed with de-novo generated 16S rRNA gene massively parallel sequencing (MPS) data from 94 blood test samples of four different body sites, and achieved high classification accuracy with AUC values at 0.992 for menstrual blood (N = 23), 0.978 for nasal blood (N = 16), 0.978 for fingerprick blood (N = 30), and 0.990 for venous blood (N = 25). The obtained highly accurate classification of menstrual blood was independent of the day of the menses, as established in additional 86 menstrual blood test samples. Accurate body site of origin classification was also revealed for 45 fresh and aged mock casework blood samples from all four body sites. Our novel microbiome approach works based on the assumption that a sample is from blood, as can be obtained in forensic practise from prior presumptive blood testing, and provides accurate information on the specific body source of blood, with high potentials for future forensic applications.

Effects of PCR inhibitors on mRNA expression for human blood identification.

Detection of body fluid-specific mRNAs, such as those specific for blood, using real-time polymerase chain reaction (PCR) has become a useful tool in forensic science. Blood stains often contain PCR inhibitors that may be co-extracted with RNA and adversely affect PCR. The effects of inhibitors on the detection of mRNA markers for blood identification, namely, hemoglobin beta (HBB) and actin beta, were examined herein. Inhibitors were added to a real-time PCR mix, reverse transcription mix, and blood samples before RNA extraction, and the following parameters: Ct, delta Ct (dCt), and melting temperature (Tm) values, were monitored. Hematin, humic acid, indigo carmine, and tannic acid were used as PCR inhibitors. The results showed that Ct values for HBB in samples containing inhibitors in their real-time PCR mix increased in a concentration-dependent manner, and were undetectable at higher concentrations. Moreover, Ct values for HBB in tannic acid-spiked samples reached a maximum once, and inhibition decreased at higher concentrations. dCt values increased in hematin-spiked samples, but decreased in tannic acid-spiked samples. Tm values decreased following the addition of each inhibitor. The reverse transcription reaction was scarcely inhibited at concentrations that markedly affected real-time PCR. The complete removal of inhibitors added to blood was difficult. However, the observed inhibitory effects were weaker than those when inhibitors were added to the PCR cocktail. PCR inhibition was effectively reduced by repurification of complimentary DNA with DNA extraction kits. These results will assist examiners in deducing contaminating inhibitors and selecting an appropriate method to remove them.

Kastle-Meyer blood test reagents are deleterious to DNA.

The Kastle-Meyer (KM) test is a quick and easy chemical test for blood used in forensic analyses. Two practical variations of this test are the KM-rub (indirect) test and the more sensitive KM-direct test, the latter of which is performed by applying reagents directly to a suspected blood stain. This study found that sodium hydroxide present in the KM reagents eliminated the potential to generate a DNA profile when applied directly to small quantities of blood. A modified approach to the KM-rub test that increases its sensitivity is presented as a method to replace destructive KM-direct testing.

Evaluation of a blood-specific DNA methylated region and trial for allele-specific blood identification from mixed body fluid DNA.

The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations.

Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False-negative by Immunochromatography.

Forensic laboratories are often faced with cases in which methamphetamine hydrochloride-mixed blood is unable to be identified as human blood by immunochromatography against human hemoglobin A0. The application of mRNA expression analysis to samples that showed a false-negative with immunochromatography was investigated as an alternative approach that did not depend on the antigen-antibody reaction. Real-time PCR was used to examine the expression levels of blood markers such as glycophorin A, spectrin beta, and hemoglobin beta. Hemoglobin beta was the only marker that was specifically detected in blood, while glycophorin A was useful for determining human specificity. Hemoglobin beta showed good detection sensitivity and was detectable in 37-year-old blood stains. Hemoglobin beta was exclusively detectable in methamphetamine hydrochloride-mixed blood stains. Detergents and disinfectants did not significantly influence mRNA markers. The proposed mRNA expression analysis was suitable for human blood identification as an alternative method to immunochromatography.

The Detection and Species Identification of Blood $mdash; A Bibliography of Relevant Papers from 1980 to 1995.

The comprehensive review of the biochemical forensic literature by Gaensslen covered much of the subject until about 1980. This review focuses on two aspects of forensic serology and reports on the progress and research in the identification of blood and its species determination during the period 1980 to 1995. The development of ELISA techniques and the explosive developments in DNA technology have and will change the course of forensic science. The usefulness of these and other techniques has been extended to archaeology as well as other disciplines. While borrowing heavily from the forensic literature, these researchers have produced their own literature which, in turn, is of interest to the forensic scientist.