Desarrollo de metodología de cribado y optimización de extracción de fluidos corporales a partir de polímeros superabsorbentes (SAP) en casos de agresión sexual

Resumo

This Doctoral Thesis entitled “Development of methods of screening and extraction of bodily fluids in superabsorbent materials prior to genetic profiling in sexual aggression cases” aimed to develop a spectral non-invasive method to detect and identify different bodily fluids in superabsorbent materials to improve the screening efficiency for confirmatory bodily fluid detection and, secondly, to optimize the posterior extraction of bodily fluids from those materials, particularly semen, to further obtain the genetic profile of interest in the sexual aggression sample. Firstly, a bibliographic context was compiled in order to introduce the current methods of identification and extraction of bodily fluids, and to enumerate several challenges of those techniques. One of the discussed challenges was the analysis of superabsorbent sanitary pads as evidence in sexual aggression cases. Superabsorbent pads consist on different layers with different composition and different absorption capacity, being the lower layer the most absorbent. Due to their composition, superabsorbent pads retain bodily fluids, and can be used for forensic laboratories to retrieve DNA for a genetic profile. However, due to their complexity, the identification and extraction of bodily fluids from superabsorbent pads certainly presents a current challenge in the forensic laboratories. Next, in Chapter 1, a method for non-destructive screening of bodily fluid samples was developed and described. In this study, semen, vaginal fluid and urine stains placed on superabsorbent pads including sanitary napkins, panty-liners and diapers were nondestructively detected by Attenuated Total Reflectance (ATR) Fourier Transform Infrared spectroscopy (FTIR). In spite of the higher absorbent capacity of the lower layers, this technique was able to detect the three fluids on the upper layer of all pads, showing that bodily fluids are distributed within all layers. Additionally, mixtures of these bodily fluids prepared on superabsorbent pads and cotton fabric substrates were studied, since real forensic investigations involving sexual assault cases usually deal with mixtures of different fluid types. Due to their IR marked protein region (1800 – 1480 cm-1), semen and vaginal fluid were easily distinguished from urine. However, since semen and vaginal fluid have both a high protein composition, the region of their IR signatures was quite similar, except for slight visual differences. In order to differentiate bodily fluids without damaging the sample, the infrared spectra of fluids coming from sexual evidence was correlated with those previously recorded from undamaged stains of individual bodily fluids. With the definition of different thresholds, this method could be applied to an initial screening to collect all stains containing semen; and afterwards, it could be used to perform a more accurate discrimination of the stains with higher concentration of semen. In this way, the forensic practitioner would be able to select the best samples for posterior extraction of DNA prior to the genetic profiling in an attempt to identify the perpetrator. The described method provides qualitative and reliable results, without requiring human interpretation. Therefore, the proposed method opens a door for a low-cost, fully automated and efficient system for non-destructive screening of semen, which could be easily and massively implemented. On the other hand, in Chapter 2, an extraction protocol from the lower core and the other layers of superabsorbent polymer (SAP) sanitary pads was developed for semen analysis. The current procedure for extracting this bodily fluid from the pads consists in the incubation with water of the SAPs-free upper layers. Besides, there were no studies of efficiency regarding the recapture of semen from the different layers, and a real quantitative assessment was necessary in this respect. Therefore, the subchapter 2.1 was the first approach to characterise different superabsorbent sanitary pads and to assess the efficiency of semen recovery from their SAPs-free layers. Semen was detected up to 1:25000 dilutions for all substrates in the upper layers with maximum yields of 8% for male cell DNA and 4% for sperm cells quantifications. A simulation of the lower-absorbent core was performed creating sodium polyacrylate hydrogels embedded with semen. Different chemical treatments followed by filtration with nylon and polyester (PETE) membranes installed in NAO® Baskets allowed the extraction of semen from the hydrogels. The 100% isopropanol incubation presented the highest sperm cell recovery (6.0%). Previous results showed a need to develop and optimise the protocol for semen extraction from the lower core, in contrast with the common procedure that only considers the upper layers. Thus, a follow-up study was performed to analyse the efficiency of different chemical and physical treatments to extract semen from the lower core of pads. The lower core is usually composed of sodium polyacrylate SAPs. SAPs generate a hydrogel that retains bodily fluids, hindering the possibility of obtaining cells and hence their genetic material. A comparison of both chemical and physical shredding treatments was studied for maximising sperm and male cell DNA recovery from superabsorbent substrates, as sanitary napkins, panty-liners or diapers. With different treatments, it was possible to obtain genetic profiles from the solution extracted from the lower core of pads, without chemical or SAPs interference. Entire sections of the substrates incubated with different solutions obtained enhanced sperm and male cell DNA recaptures than when just eluting the upper layers of the pads. From the present Thesis, it is recommended to induce a dewaterisation of the hydrogels in order to maximise the extraction of bodily fluids. Despite the crucial impact these types of evidence might have in sexual assault cases, due to their capability of retain bodily fluids and thus maintain the integrity of DNA, there are a low number of published studies focused on optimizing the elution of bodily fluids from superabsorbent sanitary pads.