Saliva Traces Research Articles

Cytological Assessment of Barr Bodies Using Aceto-Orcein and Papanicolaou Stains in Buccal Mucosal Smears and Their Sex Estimation Efficacy in an Indian Sample

Objective: Buccal epithelial cells in saliva traces found at a crime scene can be used for sex determination by examining the presence of Barr bodies in the nucleus. Papanicolaou (PAP) and fluorescent stains, among others, have been used in the past. Aceto-orcein (AO) is a relatively unexplored staining technique for this purpose. This study aims to assess the efficacy of sex determination using AO and PAP stains for the detection of Barr bodies in buccal mucosal scrapes. Study Design: Buccal scrapings were collected from 120 healthy individuals (60 males and 60 females). They were stained with AO and PAP. Fifty cells in each sample were analyzed for identification of Barr bodies. Samples with a presence of Barr bodies ≤5% were recorded as male and those with >5% were recorded as female. The percentage accuracy in determining sex using both stains was evaluated. Results: The percentage of Barr bodies in AO-stained slides ranged from 5 to 32 among females and from 0 to 8 in males, while with PAP the ranges recorded were 4-20 in females and 0-5 in males (p < 0.001). The sensitivity and specificity of AO for detecting sex accurately was around 98.3 and 95% for PAP. Conclusion: Sex determination using Barr bodies in the buccal cells is a simple method that provides up to 95-98% accuracy; making it a significant accessory for sex determination. AO proved to be a better stain than PAP for visualizing nuclear details, and its staining time was remarkably shorter. It also demonstrated enhanced sex estimation efficacy compared to PAP.

Hartmannella vermiformis can promote proliferation of Candida spp. in tap-water

Candida yeasts colonize humans' oral cavities as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with saliva traces mainly because of dysfunction of anti-retraction valves. Free-living amoebae (FLA), like Hartmannella vermiformis, are frequently found in aquatic environments and they have also been already isolated from dental unit waterlines. They can be implicated as reservoir for pathogens or directly in infections. This work deals with the survival of three species of Candida (Candida albicans, Candida glabrata and Candida parapsilosis), in co-cultivation with FLA in tap-water. One strain of each Candida species was studied. Microbiological and microscopic approaches were used; amoebae–yeasts co-cultivation assays were performed at different temperatures of incubation. Results have shown that H. vermiformis were able to internalize Candida yeasts and promote their proliferation in tap-water with or without saliva traces (2% v/v). Amoebae interact differently with Candida depending on the temperature used and the studied species of yeasts. In conclusion, this study emphasizes the survival of yeasts and/or FLA in tap-water. The ability of yeasts and amoebae to interact should be taken into account because it could lead to candidiasis infection for the frailest patients after a dental treatment.

Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence

In forensic casework analysis it is often necessary to attempt to obtain DNA profiles from microscopic amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA’ evidence, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Although a genetic profile from trace biological evidence is routinely obtained, the tissue source of the profile is rarely known. This merely perpetuates the ‘mystery’ of the nature of ‘touch DNA’ evidence allowing the significance or meaningfulness of genetic profiles obtained from these samples to be challenged. Numerous reports state that the tissue source of origin of ‘touch DNA’ evidence cannot be determined due to the small amount of biological material present, while others conclude that the DNA profiles are obtained from shed skin cells (as opposed to, say, buccal epithelial cells present in saliva traces) without any scientific basis for this assertion. Proper identification of the biological material present might be crucial to the investigation and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Thus far, research has failed to provide forensic scientists with feasible, definitive methods to identify the tissue origin of ‘touch DNA’.In the present work, we sought to identify novel highly specific and sensitive messenger RNA (mRNA) biomarkers for the identification of skin. Gene candidates were identified using both literature searches and whole transcriptome deep sequencing (RNA-Seq). Utilizing this dual approach, we identified and evaluated over 100 gene candidates. Five mRNA markers were identified that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully detect and identify skin using as little as 5–25pg of input total RNA from skin and, significantly, in swabs of human skin and various touched objects. One of the markers, LCE1C, is particularly highly sensitive and was detected in the majority of skin samples tested including touched objects. We have been successful in incorporating the five skin biomarkers into two multiplex systems. Although further work is needed to optimize the assay for routine casework, the initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible.

DNA Identification of Biological Traces on Cigarettes: Vices Reveal

ABSTRACTIsolation of cell material from biological traces on forensic evidence is often a serious challenge to solving forensic cases. Successful isolation of high-grade DNA from cell material even in critically low quantity could be achieved in examination of traces which are more than two years old.We present two cases representative of a group of 28 cases with cigarette butts that were from 10 days to 2 years old. The proper collecting and storage of the material is very important for successful DNA typing from saliva traces and epithelial cells from the lips and oral cavity. Meeting these conditions would increase the chances for successful DNA profiling of biological traces on evidence of an earlier date.The result of our examination confirms the importance of the worldwide accepted approach for setting up a database of DNA profiles. In the presented forensic cases the opportunity for solving the crime was given by the vices of the suspects.

Multidimensional Raman Spectroscopic Signatures as a Tool for Forensic Identification of Body Fluid Traces: A Review

The analysis of body fluid traces during forensic investigations is a critical step in determining the key details of a crime. Several confirmatory and presumptive biochemical tests are currently utilized. However, these tests are all destructive, and no single method can be used to analyze all body fluids. This review outlines recent progress in the development of a novel universal approach for the nondestructive, confirmatory identification of body fluid traces using Raman spectroscopy. The method is based on the use of multidimensional spectroscopic signatures of body fluids and accounts for the intrinsic heterogeneity of dry traces and donor variation. The results presented here demonstrate that Raman spectroscopy has potential for identifying traces of semen, blood, saliva, sweat, and vaginal fluid with high confidence.

Saliva promotes survival and even proliferation of Candida species in tap water

Candida yeasts colonize the human oral cavity as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with traces of saliva mainly because of the dysfunction of antiretraction valves. This study deals with the growth ability of Candida albicans, Candida glabrata and Candida parapsilosis in tap water with saliva (0-20% v/v). Results show that C. glabrata is the most susceptible species in tap water. Furthermore, saliva promotes both survival and proliferation of the three studied Candida species in tap water.

Discriminant Analysis of Raman Spectra for Body Fluid Identification for Forensic Purposes

Detection and identification of blood, semen and saliva stains, the most common body fluids encountered at a crime scene, are very important aspects of forensic science today. This study targets the development of a nondestructive, confirmatory method for body fluid identification based on Raman spectroscopy coupled with advanced statistical analysis. Dry traces of blood, semen and saliva obtained from multiple donors were probed using a confocal Raman microscope with a 785-nm excitation wavelength under controlled laboratory conditions. Results demonstrated the capability of Raman spectroscopy to identify an unknown substance to be semen, blood or saliva with high confidence.

Use of non-CODIS miniSTR markers: Creation of a database in Argentina

The use of miniSTR markers is a valuable tool in the forensic laboratory since it allows high-quality genetic profiles to be obtained on low copy number (LCN) or degraded DNA taken from different samples, such as cadaveric material, paraffin-embedded tissue, chewing gum, traces of saliva, pieces of cloth, cigarette butts, bone samples, etc. In addition, it is useful to have additional STR loci to solve paternity cases with one or more inconsistencies or to increase the amount of genetic information for incomplete family studies, where the genetic profile of the alleged father has to be reconstructed. Genetic frequencies and forensic parameters were calculated for 12 non-CODIS miniSTR loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, D17S1290, D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) from a total of 506 unrelated individuals from various provinces in the central region of Argentina (Cordoba, Buenos Aires, Salta, Entre Rios and Santa Fe). The application of these miniSTR markers allowed LR > 1000 values to be obtained in kinship cases with incomplete families whose results had not been significant when using commercial kits. In conclusion, a database with Argentine population frequencies from these 12 miniSTR markers may be very useful in adding supplementary information for the forensic genetic laboratory.

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags)

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3-week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.

Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids

Raman spectroscopy was used to compare body fluids commonly found at crime scenes in a nondestructive manner. The dry traces of semen, vaginal fluid, sweat, saliva, and blood were analyzed using confocal Raman microscopy with a 785-nm excitation. The results show that the five fluids can be differentiated from one another by visual comparison of their Raman spectra, and that the laser radiation does not damage the sample. The Raman signature of each body fluid is specific and correlates with the known composition of the fluid. Dry traces of human and canine semen exhibited distinctly different Raman signatures. Overall, this preliminary study demonstrates the great potential of Raman spectroscopy for nondestructive, confirmatory identification of body fluids for forensic purposes.

Canine-specific STR typing of saliva traces on dog bite wounds.

Forensic investigations in dog attacks usually involve the examination of bite marks and toothprints, the dog's stomach and pathological methods. For identification of the offending dog we evaluated canine STR typing of saliva traces on dog bite marks. The specificity of 15 canine-specific STRs was tested on human-canine DNA mixtures prior to an applied study in which 52 cases of dog bites were investigated. The first-aid wound bandages as well as swab samples from the surrounding area of the wound were used for DNA analyses. Generally, it was possible to obtain a canine-specific STR profile from the dog's saliva left on the wound area, even when high background of human DNA was present (blood). Interestingly, we found canine STR typing to be more successful when the bandages and swabs showed high amounts of human blood, i.e. when the dog bite was severe. Canine saliva was then sometimes visible as white-coloured secretion on the human blood surface. Less severe bite cases, which did not result in bleeding wounds, showed less success in obtaining useful STR results, probably due to the fact that the surface of the wounds may have been treated before the victims consulted medical aid which therefore removed the canine cells.

Identifying the culprit from LCN DNA obtained from saliva and sweat traces linked to two different robberies and use of a database

This work refers to the identification of a criminal who committed, during a period of 6 months, two robberies in two different towns in Northern Italy. In the first robbery, we analyzed traces on a pillowcase used to carry the stolen goods. As the pillowcase showed no visible traces, we made a specific search for latent traces by means of a Crimescope CS-16 tunable forensic light source used in a special darkroom at a wavelength ranging from 415 to 490 nm: it revealed some interesting luminescent areas possibly due to biological traces (saliva). Subsequently, the latent stains were analysed with the alpha-amylase test, which gave positive results. The evidence available for the second robbery consisted in a pair of glasses left by the criminal in a bank. The glasses showed no visible traces, however, the surface in contact with the nose and ears were sampled using FTA-Paper to collect any residual sweat. The use of the database for the management of DNA profiles allowed us to link the two crimes that had occurred in two different places and at different times. In fact, the same complete male profile for 15 STRs was obtained both from the traces of saliva identified on the pillowcase used to carry the stolen goods (first robbery) and from the glasses frame surface which had been in contact with the nose (second robbery). Moreover, these results enabled the extraction of low copy number (LCN) DNA from uncommon evidence left at the crime scene, increasing the possibilities to identify the culprits.

A look at forensic dentistry – Part 2: Teeth as weapons of violence – identification of bitemark perpetrators

Teeth are often used as weapons when one person attacks another or when a victim tries to ward off an assailant. It is relatively simple to record the evidence from the injury and the teeth for comparison of the shapes, sizes and pattern that are present. However, this comparative analysis is often very difficult, especially since human skin is curved, elastic, distortable and undergoing oedema. In many cases, though, conclusions can be reached about any role a suspect may have played in a crime. Additionally, traces of saliva deposited during biting can be recovered to acquire DNA evidence and this can be analyzed to determine who contributed this biological evidence. If dentists are aware of the various methods to collect and preserve bitemark evidence from victims and suspects it may be possible for them to assist the justice system to identify and prosecute violent offenders. This paper reviews the recognition and recovery of this evidence and provides insight into modern methods used to investigate bitemark evidence from heinous crimes.

A look at forensic dentistry--Part 2: teeth as weapons of violence--identification of bitemark perpetrators.

Teeth are often used as weapons when one person attacks another or when a victim tries to ward off an assailant. It is relatively simple to record the evidence from the injury and the teeth for comparison of the shapes, sizes and pattern that are present. However, this comparative analysis is often very difficult, especially since human skin is curved, elastic, distortable and undergoing oedema. In many cases, though, conclusions can be reached about any role a suspect may have played in a crime. Additionally, traces of saliva deposited during biting can be recovered to acquire DNA evidence and this can be analyzed to determine who contributed this biological evidence. If dentists are aware of the various methods to collect and preserve bitemark evidence from victims and suspects it may be possible for them to assist the justice system to identify and prosecute violent offenders. This paper reviews the recognition and recovery of this evidence and provides insight into modern methods used to investigate bitemark evidence from heinous crimes.

Mastication and swallowing: an overview.

The cycles of jaw and tongue movement during feeding produce not only the breakage of food but its intra-oral transport; which activity predominates depends upon the physical characteristics of the food. When hard food is eaten and tooth-food-tooth contact is made during jaw closure, the velocity of closing is suddenly reduced, producing two clearly different phases of closure; during the second phase the activity of the jaw closing muscles is much increased. Conversely, in cycles with a mainly transport function (eating soft food), the antero-posterior movements of the tongue are much greater; this alters the time and rate at which the jaw opens. The pattern of jaw movement during closing and during opening consequently varies with food consistency. The evidence suggests that sensory input controls the form of the cyclical tongue and jaw movements. However the basic plan of movement is produced by the activity of a brainstem pattern generator which receives input from both cerebro-cortical and peripheral sources. The swallow that occurs in normal feeding consists of the equivalent of the classical second stage of swallowing inserted into the occlusal or initial jaw opening phase of an otherwise standard cycle. Although leakage of traces of food or saliva into the vallecula appears to be a peripheral sensory input of major importance in inducing such a swallow, the execution of the swallow is due to a pattern generator in the brainstem.

PCR-based typing of DNA extracted from cigarette butts

Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.