DNA analysis is a widely used tool in the forensic field to identify the perpetrator of the crime. Nevertheless, the recovery of a DNA profile from the perpetrator does not reveal the time and circumstances in which the crime has been conducted. Body fluid stains recovered at crime scenes or on the victim's body are important types of evidence to forensic investigators: particularly, the identification of a body fluid is essential, because the nature of the fluid is very important to insure the correct handling of the sample and it could be a very important tool for reconstructing the event dynamics. Each body fluid has a unique composition, and the presence of specific components in one fluid versus another is the basis of its identification.1 Nevertheless, the inability to determine the tissue source of forensically relevant biological fluids in sexual assault cases could result in a failure to provide crucial information necessary to the investigation, especially in cases where the victims are not able to furnish information about the dynamics of the event.2 In recent years, messenger RNA (mRNA) and microRNA analysis have been identified as a promising method for the identification of body fluids,3 resulting in a trend to bypass the conventional approaches. In fact, RNA can be isolated simultaneously with DNA, therefore avoiding sample and time loss.4,5 The specificity of mRNA markers for the most forensically relevant body fluids has been identified on the basis of functional differences of the cells and tissues involved, also with the purpose to avoid cross-reactivity among them. In cases of sexual assault, the identification of menstrual blood is important in discriminating menstruation from vaginal trauma producing injuries with consequent recovery on the victim of traces of peripheral blood.
Menstrual secretions comprised of a complex mixture of different tissue types: blood, degraded endometrial tissue, and epithelial cells. In various studies of mRNA-based body fluids identification, matrix metalloproteinase (MMP) 7 and MMP11 were reported as specific markers for menstrual blood,4,6,7 thus permitting to distinguish peripheral blood from menstrual one.
The first publication on the possible use of mRNA markers for the identification of menstrual blood belongs to Bauer and Patzelt7 in 2002, which evaluated for this purpose the matrix metalloproteinase mRNA marker (MMP). They were able to detect MMP in endometrium but not in blood and other epithelia.7 Haas et al4 in 2009 demonstrated that the menstrual blood samples revealed high levels of MMP7 and MMP11. In the vaginal secretion, samples MMP7 and MMP11 signals were weak, but nonetheless present. All blood markers were rather weak in the menstrual blood samples.4 The efficiency of MMP11 was confirmed by Ferri et al6 in the application of a real forensic case and by Fleming and Harbison3 including this marker in an mRNA multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for identification of 5 body fluids.
Tissue-specific mRNA detection offers crucial advantages in forensic casework: high sensitivity due to the possibility of PCR amplification, high specificity due to the pattern of gene expression, unique for the functional status of cells and organs, simultaneous DNA isolation without material loss, using extraction methods that concurrently isolate RNA and DNA from the same stain extract, and, last but not the least, the mRNA stability in forensic stains.5
The advantages of applying this analysis are that mRNA profiling has now evolved to a multiplex PCR platform, thus providing information about multiple gene expression. Also, mRNA profiling could easily be combined with DNA profiling from the same exact sample, using a coextraction method.8,9 In this way, analysis on the mRNA extracts will yield information regarding the origin of the stain, and the DNA analysis will reveal the donor's identity. There are commercially available coextraction kits that allow the simultaneous isolation of DNA and RNA and minimize sample consumption, reducing the amount of time required to perform the analysis.
The patient was a healthy 18-year-old woman. She was found unconscious by police officers outside a club where a concert was being held and was rescued and transported with the ambulance to the nearest gynecological emergency department, where she was admitted at 5 AM. An obstetric gynaecology physician completed the medical history and the physical examination. Once the victim regained consciousness, she was not able to give information about the dynamics of the assault, because she was under the effect of a high amount of alcohol (2.4 g/L). She reported having spent the night with a friend in the club and that during the concert she had met a young man who offered her some drinks; she remembered having seen vaginal bleeding after spending some minutes in the bathroom with the said young man, but she was not able to disclose what happened inside the bathroom. She did not recall forced penile-vaginal or penile-anal penetration, nor forced penile-oral sex. She affirmed to be virgin.
The patient was examined, and blood traces were present on the slip wore by the victim. Two anal and 2 vaginal swabs were collected for DNA analysis and sent to the Forensic Genetics Laboratory, together with all the clothes the victim was wearing. Vaginal swabs were also collected to test for gonorrhoea, chlamydia, and trichomonas, and serum was collected for baseline human immunodeficiency virus, hepatitis (B, C), and syphilis status.
As reported in the medical record of the emergency department, genital inspection of external and perianal areas showed the presence of an abrasion at the level of the posterior fourchette, and the hymenal right remnant appeared to be injured. The examination of the vagina with the speculum revealed regular vaginal walls, poorly visualized portio, and the presence of dark brown residue in the vagina.
The analysis of the Forensic Genetics Laboratory was aimed at identifying the biological material/s, first of all to detect the presence of semen and then to identify other fluids sources with an mRNA multiplex, verifying the presence of male biological material and then establishing the nature of the bloody-like traces present on the slip and on the vaginal swab.
MATERIALS AND METHODS
Two vaginal and two rectal swabs were collected and brought to the Forensic Genetic Laboratory, where they were stored at −20°C. Clothes, worn by the victim during the aggression, were also delivered to the Forensic Genetic Laboratory. In particular, we found small blood traces on the slip.
The genetic analyses were performed on 1 vaginal and 1 rectal swab and on the traces on the slip. The RNA extraction was performed using the column-based extraction QiagenAllPrep DNA/RNA Micro kit, according to the manufacturer's instructions. Samples were denatured with some adaptations: 366.9 μL of lysis buffer including 345 μL of Quiagen RLT plus buffer, 6.9 μL 1,4-dithiothreitol (2 M), 10 μL of Qiagen proteinase K, and 5 μL of Carrier-RNA (310 ng/μL) in an Eppendorf tube, incubated at 56°C for up to 3 hours to improve the extraction. The samples were eluted with 15 μL of RNAase-free water.
The removal of DNA contaminant was performed with the Ambion Turbo DNA-free. Ten microliters of RNA was heated with 0.1 volume of DNase turbo buffer 10× and 1 μL of turbo DNase at 37°C for 30 minutes. The inactivation of the DNase was obtained adding 0.1 volume of inactivating resin for 5 minutes at room temperature. After a centrifugation of 10,000 g for 1.5 minutes, supernatants were collected and ready for quantification through NanoDrop One and then for reverse transcription (RT).
RNA samples (10 μL) were subjected to RT using Ambion RETROscript Reverse Transcription kit. RNA carrier was used for the extraction, the RT protocols was performed according to the manufacturer's instruction, using random decamers, and complementary DNA (cDNA) was obtained in a final volume of 20 μL.
Multiplex Polymerase Chain Reaction
A multiplex with the primers for ALAS2, CD93, HBB, HTN3, STATH, BPIFA1, KLK3, CYP2B7P1, SEMG1, PRM1, MUC4, MMP7, MYOZ1, MMP10, MMP11, CDSN, LCE1C, ACTB, and 18S-rRNA was adopted following what was reported in literature for the primer sequences and concentrations,10,11 as well as for the housekeeping genes ACTB and 18SrRNA as endogenous controls.
In the multiplex reaction, up to 3.75 μL of cDNA was amplified in the presence of 6.25 μL of 2× QIAGEN Multiplex master mix and 2.5 μL 5× primer mix, resulting in a total volume of 12.5 μL. A GeneAmp9700 PCR System (Applied Biosystems) was used with the following cycling conditions: 95°C for 15 minutes, 33 cycles of 94°C for 20 seconds, 60°C for 30 seconds, 72°C for 40 seconds, and a final step at 60°C for 45 minutes.
Two types of negative controls and a positive control (included with the RT kit) were included with all runs. The minus-RT negative control was obtained adding non–reverse transcripted residual RNA in the multiplex PCR, whereas minus-template negative control was composed of all the multiplex PCR reagents except cDNA.
According to what was described by Lindenbergh et al,12 4 PCR replicates of the RNA samples were performed.
Amplified fragments were detected with the ABI Prism 3130 Genetic Analyzer capillary electrophoresis system (Applied Biosystems by Life Technologies). For each reaction, 1 μL of PCR product was added to 15 μL of master mix (14.5 μL of HiDiformamide and 0.5 μL of GeneScan 500 LIZ size standard [Applied Biosystems by Life Technologies]). Negative control composed only of master mix was included. Samples were processed using dye-set G5 and analyzed with GeneMapper Analysis Software version 3.2 (Applied Biosystems by Life Technologies). A peak detection threshold of 50 relative fluorescence units was applied.
DNA STR Profiling
DNA Extraction, Quantification, Amplification, and Detection
Microscopic examinations on the vaginal and rectal swabs were performed and no sperm cells were observed; nevertheless, in the hypothesis that few sperm cells were present and we had not seen them through microscopic examination, DNA was coextracted with the QiagenAllPrep DNA/RNA Mini kit buffer, following the manufacturer's instructions and including 6.9 μL of 1,4-dithiothreitol (2 M) and finally eluted in 20 μL of qiagen elution buffer. DNA extracts were quantified using the NanoDrop One (Applied Biosystems), according to the manufacturer's instructions. This spectrophotometer allows for the quantification of DNA in general, without discriminating between male and female DNA.
Two nanograms of total DNA were amplified with the IdentifilerPlus multiplex Kit (Applied Biosystems) in a total reaction volume of 25 μL on a GeneAmp PCR System 9700 (Applied Biosystems) according to the manufacturer's protocol.
Polymerase chain reaction products were detected using the same procedure in section Capillary Electrophoresis.
To identify the profile of the sexual offender, short tandem repeat analysis was performed in the DNA extracted from vaginal and rectal swabs and from blood spots on the underwear. No DNA mixture was identified; indeed, the electropherograms corresponded perfectly with the victim's profile.
The mRNA analysis was performed to reveal the nature of the presumed blood trace on the slip worn by the victim and on the vaginal swabs and to exclude or confirm the presence of sperm.
The rectal swab was not further investigated since bloody-like traces were not observed and male biological material was not identified (no mixed short tandem repeat profile detected nor sperm cells evidence on microscopic examination).
RNA data interpretation was performed using the 4 PCR replicates according to the x = n/2 rule as described by Lindenbergh et al,12 which compares the number of observed peaks (x) to the number of theoretically possible peaks (n) in all replicates. A body type fluid is scored observed when at least half of the possible peaks are detected (x ≥ n/2), denoted sporadically observed when less than half of the possible peaks are observed (0 < x < n/2), and scored not observed when no peaks are detected (x = 0).
Sperm markers (PRM1 and SEMG1) were not observed in all replicates.
Blood (HBB and ALAS2), vaginal (MYOZ1), and skin (CDSN and LCE1C) markers were scored observed in the samples obtained from slip traces and vaginal swab. Figure 1 represents one of the electropherograms.
The housekeeping markers (ACTB and 18S-rRNA) were present, thus demonstrating the good efficiency of the analysis.
The peak disequilibrium of the different markers is due to the major robustness of the HBB, CYP2B7P1, and the 2 housekeeping markers with respect to others.11
The absence of metalloproteases markers MMP7, 10, and 11 confirmed the peripheral nature of the blood, excluding the menstrual origin of the biological fluid recovered on the victim's slip and vaginal swab.
The case regards a young woman found unconscious and believed to be the victim of sexual assault. Once the victim regained consciousness in the hospital, she was not able to provide information concerning the dynamics of the event, because she was under the effect of high amounts of alcohol.
The identification of certain body fluids can be useful for reconstructing the occurred event, in particular in case of sexual assault. For this purpose, we have analyzed the traces with an mRNA multiplex to identify the biological material/s, first of all to detect the presence of semen and then to identify other fluids sources. The electropherogram showed the absence of semen-specific markers (PRM1 and SEMG1). Furthermore, the DNA profiling analysis confirmed the absence of male biological material, showing only the victim's genetic profile.
Consequently, it became important to identify the nature of the blood found in the slip and vaginal swab. The purpose was to discriminate between the possibility that the assault had produced the vaginal injuries and thus vaginal bleeding under the prosecutor's hypothesis, or that the presence of vaginal bleeding was due to the menstrual cycle and was not related to violent vaginal penetration under the defense hypothesis.
Thanks to the use of mRNA analysis, it was possible to exclude the hypothesis that the blood found on the victim's samples was menstrual blood. The identification of peripheral blood markers has made it possible to define this finding as compatible with the presence of lesions in the hymen and on the posterior fourchette; thus, the origin of the peripheral blood was consistent with anatomical findings and with the hypothesis of traumatic injuries (due to a foreign body insertion or to a sexual intercourse) in the perineal area.
This is important to set aside the defense hypothesis that the observed anatomical lesions occurred before the fact and that the bleeding was due to the menstrual cycle and not to the assault, supported also by the fact that no other injuries were detected on the victim's body.
This second step could also be reached through the use of the SERATEC PMB test, which has been proved to be a helpful tool in interpreting samples containing human menstrual and/or peripheral blood.13 Nevertheless, our approach to this real casework was to first of all identify all possible biological fluids in the stains and swabs, not only the differentiation between menstrual and peripheral blood. The usefulness of the multiplex mRNA approach for real caseworks is that it may lead to the detection not only of blood but also of semen or saliva traces, thus helping in reconstructing how the event happened.
The performed forensic genetic analysis supports the proposition that the victim had a sexual intercourse, violent or not, in the absence of male biological material, in a case where the victim, altered by high blood alcohol levels, was unable to provide information.
In conclusion, the great improvement given by RNA technology to the forensic genetic laboratory is the ability, in forensic casework, to detect the origin of the body fluids and represent a confirmatory test that can be applied in this field to an unknown stain, because it is able to identify any of the body fluids that might be present. Furthermore, as seen in this case, the possibility of DNA and RNA coextraction from the same sample and the ability to multiplex mRNA markers for one or more body fluids' identification may overcome conventional identification methods, thus avoiding sample loss.