Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm², with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.     

Comparison of Four Saliva Detection Methods to Identify Expectorated Blood Spatter

Blood spatter analysis is an important step for crime scene reconstruction. The presence of saliva in blood spatter could indicate expectorated blood which is difficult to distinguish from impact spatter. In this study, four saliva test methods (SALIgAE^®, Phadebas^® sheet, RSID^™-Saliva kit, and starch gel diffusion) were compared to identify the best method for detecting expectorated blood spatter. The RSID^™-Saliva kit showed the highest sensitivity even when saliva was mixed with blood, and was not inhibited by the presence of blood. The SALIgAE^® test provided easy and rapid results, but the yellow color of a positive reaction was overwhelmed by the red color of the blood. The starch gel diffusion method and the Phadebas^® sheet exhibited relatively low sensitivity and the assay took a long time. When using the RSID^™-Saliva kit for identifying saliva in blood, results should be read within 10 min.     

Alpha-amylase kinetic test in bodily single and mixed stains

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.     

Detection and visualization of human tears using alternate light sources for forensic purposes

The current scientific techniques for locating body fluids focus on quick and effective methodologies for easy and reliable identification. Efficient detection and identification of body fluids play a vital role in establishing the ‘corpus delecti’ of a crime. Non-destructive techniques such as the use of Alternate Light Sources (ALS) have been exploited for crime scene searches over large areas and detection of body fluids such as blood, semen, vaginal secretions, and saliva on a range of substrates. Tears are rarely found but can be considered as potential body fluid evidence due to their unique biochemical and molecular properties. Tears are secreted in response to physical or emotional stimuli. Due to the small volume of secretions, they are often overlooked in the crime scene. Tears may be found on surfaces such as clothing, bedding, tissue, handkerchief, or balaclava. The use of ALS to locate tears on tissue paper and fabric surfaces was tested which were not apparent to the naked eye. Tears stains were successfully detected on surfaces of forensic interest with varying sample ages up to three months with a broad excitation spectrum between 254 nm and 410 nm. Dried stains on tissue paper and fabric substrates were better detected with sharp margins, clear stain pattern visibility, and fluorescence intensity in comparison with moist and fresh stains. Tears stains can hence be detected with the use of ALS and suitable filter combinations under normal conditions and do not require any specific settings to locate them. These findings are suggestive for easy and quick identification of tears on large surfaces and as a presumptive test for forensic casework evidence examination.     

False‐Positive Results with Amylase Testing of Citrus Fruits

In a case of robbery in which the criminals passed through the garden adorned with calamondin trees (Citrus madurensis), the investigators found in the grass six calamondin fruits, some undamaged, while others apparently bitten. The fruits were collected and sent to the laboratory for DNA analysis to verify the presence of saliva and robbers' DNA profile. A specific immunochromatographic strip test for saliva confirmed the presence of human salivary α‐amylase, but similar positive results were also observed for intact calamondin and other citrus fruits. Further analysis with a specific automated amylase test confirmed the absence of amylase activity. DNA quantification and typing using a specific forensic kit revealed no human DNA presence in any fruits. This case report demonstrates for the first time the occurrence of false positives when human saliva is sought on citrus fruits.     

Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2 h and 30 min with ≤0.8 bp allele typing accuracy. The minimal amount of DNA required for a complete DNA profile is 100 copies. To critically evaluate the capabilities of our portable microsystem as well as its compatibility with crime scene investigation processes, real-time STR analyses were carried out at a mock crime scene prepared by the Palm Beach County Sheriff's Office (PBSO). Blood stain sample collection, DNA extraction, and STR analyses on the portable microsystem were conducted in the field, and a successful “mock” CODIS hit was generated on the suspect's sample within 6 h. This demonstration of on-site STR analysis establishes the feasibility of real-time DNA typing to identify the contributor of probative biological evidence at a crime scene and for real-time human identification.     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

A Rapid,Confirmatory Test for Body Fluid Identification,

We have developed a technique that allows investigators to confirm the presence of blood, semen, and/or saliva in a crime scene sample. It is a confirmatory test where multiple samples can be processed in less than an hour, and it is potentially portable, permitting samples to be processed at the crime scene. Samples at a scene giving a positive result can be further processed while those failing to do so may be ignored. There is a large and growing backlog of DNA evidence in the USA, slowing down the criminal justice system. This backlog has continued to grow despite an increase in the ability to process evidence faster. This technique uses quantum dot molecular beacons to test for tissue‐specific RNA species, identifying particular body fluids. We have demonstrated the tissue specificity of molecular beacons for blood, semen, and saliva.     

Advantage of ForensiX Swabs in Retrieving and Preserving Biological Fluids

This study compares two novel swabs (forensiX) with a standard cotton swab (EUROTUBE) for the collection of saliva stains on glass slide for STR analysis. ForensiX collection tubes are a standard cotton swab in an “active drying” tube, where swab sample is soon dried by its innovative tube surface of the wall. The other is forensiX Nylon Flocked Swab. The study is two phases: The first “phase” assesses swab types regarding to retrieve ability of saliva. The second “phase” compares the drying ability of each swab to assess how crime samples would fare when left in storage. The main result showed that “active drying” is effective to store swabbed sample. The forensiX swabs generally are effective for higher (twofold to fourfold) DNA yield compared to delta lab swab (around 750 pg and 250 pg from 0.5 μL of saliva), respectively. These findings demonstrate the importance of drying performance in the preservation of DNA and swab selection.     

DNA investigative lead development from blood and saliva samples in less than two hours using the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System produces full STR DNA profiles (sample in, results out) in 90 min. Here we present results from a new protocol on the RapidHIT System, designed for crime scene samples. Data are presented for blood and saliva samples, with results for a range of samples and substrates. Success rates and sensitivity display a high level of performance for the sample types evaluated in the study and demonstrate the efficacy of the new protocol for crime scene samples.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

Comparative performance of the Phadebas® Forensic Press Test at room temperature and 37 °C for the detection of saliva stains on fabric exhibits

The Phadebas® Forensic Press Test (PFPT) is an enzyme-based colorimetric test used to visualise and locate latent saliva stains on forensic exhibits. The test relies upon the presence of the enzyme α-amylase which is present in high levels in saliva. Even though the optimal in vitro temperature for α-amylase activity is 37 °C, the PFPT manufacturer’s protocol specifies that the PFPT should be carried out at room temperature (RT). In this study, we compared the performance of the PFPT at RT and 37 °C using combinations of four fabric types (cotton, polyester, acrylic and a cotton/polyester blend), three saliva dilutions (neat, 1:10 and 1:100) and stains aged for four time periods (1 day, 1 week, 1 month and 3 months). The intensity of the PFPT colour reactions at RT and 37 °C were not statistically different across all fabric types, saliva concentrations and stain ages, indicating that maximum sensitivity and performance of the PFPT can be achieved at RT.     

DNA in the Criminal Justice System: The DNA Success Story in Perspective,

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high‐volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high‐volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high‐volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high‐volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool.     

Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.     

Powdered Activated Carbon: An Alternative Approach to Genomic DNA Purification

Forensic evidence samples are routinely found as stains on various substrates, which may contain substances known to inhibit polymerase chain reaction (PCR). The goal of this study was to evaluate post‐Chelex^®100 purification using powdered activated carbon (PAC). Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the DNA recovery and inhibitor removal using PAC with those of the Amicon^®Ultra 100K. For extracted bloodstains on soil and wood substrates, PAC and Amicon^®Ultra 100K generated similar DNA yield and quality. Moreover, the two methods significantly decreased the concentration of humic substances and tannins compared to nonpurified extracts (p < 0.001). In instances where extracts contained indigo dye (bloodstains on denim), Amicon^®Ultra 100K performed better than PAC due to improved amplifiability. Efficient adsorption of humic substances and tannins, which are common inhibitors, indicates PAC's potential application in the purification of high‐template DNA extracts.     

Developmental Validation of RSID™-Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Abstract:  Current methods for forensic identification of saliva generally assay for the enzymatic activity of α-amylase, an enzyme long associated with human saliva. Here, we describe the R apid S tain ID entification (RSID™-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID™-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID™-Saliva detects less than 1 μL of saliva. The sensitivity of RSID^TM-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID™-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID™-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.