Investigation of the Effects of Environmental Insults on Presumptive and Confirmatory Tests and DNA Degradation

Abstract

The correct identification of body-fluid samples such as blood, semen and saliva is an integral part of the forensic analysis, which determines the course of downstream genetic analysis. Body-fluid samples are routinely found to be affected by local environmental insults such as UV radiation, high temperatures and humidity, compromising the quality of the samples and the DNA profiles generated. This problem can be exacerbated when the environmental conditions are harsh. It is widely understood that environmental insults affect the efficacy of presumptive and confirmatory tests and can have a direct impact on the quality and quantity of amplifiable DNA present. However, little published data exists detailing the empirical effects of environmental insults on presumptive and confirmatory tests. Some studies have assessed the persistence of DNA in biological samples, but none of the published research was based in environments that are routinely exposed to extremely high temperature and UV irradiation.
To understand the impact of the environment data were collected from the Police Forensic DNA Unit in Ras Al Khaimah, in the United Arab Emirates. Analysis of forensic samples received from the year 2012 to 2014 identified items containing body-fluids to constitute over 95% of the total casework samples. The success rate of obtaining STR genetic profiles from these body-fluid samples was assessed: over 80% of blood samples generated full STR profiles, whereas 71.8% of semen and 46% of saliva samples resulted in full STR profiles. This was comparable to data published in the literature and illustrated the potential impact of environmental insult on body fluids, especially saliva. However, data were not available to investigate why particular samples had failed and so the effect of the environment in leading to failed profiles could only be inferred.
In order to get a better understanding of the impact of the environment a series of experiments have been undertaken to empirically investigate the effect of the local environment on biological fluids commonly found at crime scenes, i.e. blood, semen and saliva. Samples were deposited on cotton, glass, and metal and exposed to the environment in direct sunlight. They were then collected over a period of 51 days in the summer of 2014 at intervals of 48 to 72 hours. Each collected sample was subjected to presumptive and confirmatory tests and DNA was extracted and assessed. The four presumptive tests were: the Kastle-Mayer and Hemastix® (Bayer Diagnostics) for blood, the Phosphatesmo KM® (Macherey-Nagel) for semen and Phadebas® Tablet Test (Megal LifeSciences) for saliva. Whereas the four confirmatory tests were: the Hexagon® OBTI test (Gesellschaft fur Biochemica und Diagnostic) and the RSID™-Blood (Independent Forensics) for blood, the RSID™-Semen and the RSID™-Saliva.
Before undertaking the main experiment the DNA extraction methods available were assessed so that the most appropriate method could be used. The methods used were: Chelex-100, Phenol/chlorophorm, QIAamp® DNA Investigator Kit (Qiagen), InnuPREP® forensic extraction kit (Analytikjina), the EZ1® system using the investigator card (Qiagen), the AutoMate Express™ System using the PrepFiler™ Express kit (Applied Biosystems), the Maxwell® 16 Forensic instrument with the DNA IQ™ chemistry (Promega) and the InnuPure® C16 using the innuPREP forensic DNA kit-IPC16 (Analytikjina). One-way analysis of variance (ANOVA) showed significant differences in DNA yields when comparisons of eight extraction methods were conducted on body-fluid samples (p=2.0e-16 for blood, p=5.98e-16 for semen and p=2.82e-10 for saliva samples). Pair-wise analysis using the Tukey’s (HSD) test in addition to Box-plot graphical representations identified the AutoMate Express™ System and the Chelex-100 method as giving significantly higher DNA yields. However, DNA quality was compromised when extracting blood samples using the Chelex-100 (mean IPC= 39 Ct). Based on these data Chelex-100 was chosen as the optimum method, when also taking cost into consideration.
To investigate the effects of the environment on the biological fluids, the sensitivity of four presumptive and four confirmatory tests were evaluated. Presumptive tests for blood and saliva were shown to be more sensitive in identifying their respective body-fluids than their counterpart confirmatory tests. However, this was not true for semen where the confirmatory RSID™-Semen test was shown to be twice as sensitive as the presumptive Phosphatesmo KM® test. In addition, all the screening tests were able to identify their respective body-fluids even when the DNA amounts present were less than recommended for STR analysis, i.e. 1 ng.
Following environmental insult ANOVA analysis on the outcome of the presumptive and confirmatory tests showed that material types did not play a significant role in the identification of environmentally insulted body-fluid samples (all p-values were above 0.05). However, significant differences were observed between the presumptive and confirmatory groups for blood samples placed on either metal or cloth (p=0.004 and p=0.002 respectively), with presumptive tests detecting blood after longer period of exposure than confirmatory tests. Semen samples on cloth also showed significant differences between the presumptive and confirmatory tests conducted (p=0.008), in this case the confirmatory tests detected semen for a longer period of environmental insult.
DNA persistence varied widely between different sample types. Saliva was the most susceptible to degradation, with no DNA detected after nine days. On the other hand, full STR profiles were obtained from blood samples on cloth after 51 days of exposure (2201.5 ADD) whereas partial profiles were observed from blood recovered from metal after 18 days (724.5 ADD) and glass after 30 days (1250.25 ADD). Allelic drop-outs began to appear in semen samples after 9 days (338.5 ADD) on all substrates.
Comparisons of DNA quantification results between three different DNA quantification methods, Quantifiler® Human (Applied Biosystems), Quantililer® Trio (Applied Biosystems) and Quantus™ Fluorometer (Promega) showed no significant differences when applying the techniques to environmentally insulted body-fluid samples (all p-values were above 0.05). However, some differences were observed when using the Quantus™ where DNA quantities were either too high or too low such as blood on cloth (p=0.0567) and saliva on metal (P= 0.001).
Environmental insults were observed to have diverse effects on both presumptive and confirmatory tests, as well as to DNA quantities available for down-stream DNA analysis. The level of these effects was more profound in the DNA quantity and quality than body-fluids presumptive and confirmatory tests, where the tests for body fluids were positive, but no DNA profile could be generated. However, an improved knowledge of the behavior of the different tests can help with the appropriate selection of screening tests, extraction techniques and accurate quantification methods will improve the recovery of valuable information which will ultimately lead to better success rate in the process of STR genetic profiling in our Forensic Unit.