Evaluation of the effect of ozone disinfection on forensic identification of blood, saliva, and semen stains.

Good laboratory practice minimizes the biological hazard posed by potentially infectious casework samples. In certain scenarios, when the casework sample is contaminated with highly contagious pathogens, additional safety procedures such as disinfection might be advised. It was previously proven that ozone gas treatment does not hamper STR analysis, but there is no data on how the disinfection affects other steps of the forensic analysis. In this study, we aimed to assess the interference of ozone disinfection with forensic tests used to identify biological stains. A dilution series of blood, saliva, and semen samples were pipetted onto cotton fabric and let completely dry. Half of the samples were subjected to ozone treatment, while the rest served as controls. All the samples were tested with specific lateral flow immunochromatographic assays and for specific RNA markers with quantitative real-time PCR. Additionally, luminol test was carried out on blood spots, Phadebas® Amylase Test on saliva stains, and semen stains were examined with STK Lab kit and light microscope following Christmas Tree or Hematoxylin-Eosin staining. Ozone treatment had no detrimental effect on the microscopic identification of sperm cells. Undiluted blood samples were detected with luminol and immunoassay, but at higher dilution, the sensitivity of the test decreased after disinfection. The same decrease in sensitivity was observed in the detection of semen stains using STK Lab kit from STK® Sperm Tracker, and in the case of the immunoassay specific for prostate-specific antigen (PSA). Ozone treatment almost completely inhibited the enzymatic activity of amylase. The sensitivity of antibody-based detection of amylase was also greatly reduced. RNA markers showed degradation but remained detectable in blood and semen samples after incubation in the presence of ozone. In saliva, the higher Ct values of the mRNA markers were close to the detection limit, even before ozone treatment.

Effect of ozone disinfection on forensic STR profiling.

While good laboratory practice in a forensic lab minimises the chance of infection of laboratory personnel, in certain cases, possible contamination of pieces of evidence with highly contagious pathogens might call for additional precautions. The number of potential disinfection methods that might be suitable for forensic genetics are surprisingly limited. First and foremost, the ideal technique should not inhibit DNA amplification, it should be effective against a host of pathogens, and it should be applicable on porous surfaces. We examined ozone treatment on extracted DNA samples and mock casework samples. Ozone-treated and control specimens were amplified with Qiagen Investigator ESSplex SE QS kit. Detected allele counts were compared between the treated and untreated sample groups. Following disinfection, concentration of ozone-treated DNA was about half of the control samples, but full STR profiles were recovered. In the case of mock casework samples (disposable surgical masks), there was no significant difference (p = 0.513) between the detected allele counts of control and ozone-treated samples. Sampling location of surgical masks (earloop, nosepiece) showed a statistically significant difference (p = 0.011), though. Comparing the effect of contributors on STR profiling, a significant difference (p = 0.001) was observed, which could be explained with the differences between individuals including shedding capacity, head size or shape. According to our pilot study, ozone treatment does not encumber the routine forensic DNA analysis, the sampling position or the contributor affected the allele counts more than the ozone treatment.

Sternal aspirate sampling of Bacillariophyceae (diatoms) and Cyanobacteria in suspected drowning cases.

A diagnosis of drowning is not always possible based on the traditional autopsy findings. The most widely used ancillary methods are based on the detection of diatoms and other waterborne organisms in the organs of the systemic circulation by light microscope or polymerase chain reaction (PCR). One of the greatest concerns is sample contamination. Bone marrow is a favourable source because the compact bone protects the sample from water ingress in the case of advanced decay. In our pilot study, we aimed to adopt sternal bone marrow aspiration - which is a widely used technique in haematology - for postmortem sampling. Control experiments of non-drowning victims showed that cleaning the skin over the sternum can prevent external contamination. Sternal aspirate samples were taken from seven suspected drowning victims along with lung, spleen, and femoral bone marrow samples. All specimens were examined for the presence of diatoms by light microscope and Cyanobacteria-specific DNA by PCR. We were able to obtain bone marrow aspirates from all cases without complications. In four of the sternal samples both diatoms and cyanobacterial DNA were detected, while one additional sternum sample was tested positive with PCR, but no diatom shells were detectable. Sternal bone marrow aspiration is simple and quick, which can be performed at the beginning of an autopsy, minimizing the chance of contamination. We have shown that this sampling method can be adopted for postmortem diatom testing. This minimally invasive technique might be used in virtual autopsy (postmortem computed tomography, PMCT) settings without opening body cavities.