Long-Term Tissue Preservation at Ambient Temperature for Post-Mass Fatality Incident DNA-Based Victim Identification.

In a mass fatality incident (MFI), effective preservation of tissue samples is the cornerstone for downstream DNA-based identification of victims. This is commonly achieved through freezing of tissue samples excised from bodies/fragmented remains which may be buried or stored in refrigerated containers. This may, however, not be possible depending on the nature of the MFI; in particular, during armed conflict/war where extended periods of electrical outages would be expected. The present study compared the effectiveness of long-term tissue preservation at ambient temperatures using two commercial products (non-iodized kitchen salt and a 40% alcoholic beverage) against a chemical preservative (Allprotect™ Tissue Reagent (Qiagen, Germantown, MD, USA)) and freezing at -20 °C. Bovine muscle tissue, used as a proxy for human tissue, was treated with the four preservation methods and sampled at six different time-points over a 24-month period. All four methods were able to preserve the bovine tissue, generally yielding STR-PCR (Short Tandem Repeat-Polymerase Chain Reaction) amplicons > 200 bp in size even at the end of 24 months. Gel electrophoresis, however, indicated that salt was more effective in preserving DNA integrity with high-molecular-weight DNA clearly visible as compared to the low-molecular-weight DNA smears observed in the other methods. This study also proposes a simple process for the rapid and low-cost preservation of tissue samples for long-term storage at ambient temperatures in support of post-incident victim identification efforts.

Artificial neural network, predictor variables and sensitivity threshold for DNA methylation-based age prediction using blood samples.

Regression models are often used to predict age of an individual based on methylation patterns. Artificial neural network (ANN) however was recently shown to be more accurate for age prediction. Additionally, the impact of ethnicity and sex on our previous regression model have not been studied. Furthermore, there is currently no age prediction study investigating the lower limit of input DNA at the bisulfite treatment stage prior to pyrosequencing. Herein, we evaluated both regression and ANN models, and the impact of ethnicity and sex on age prediction for 333 local blood samples using three loci on the pyrosequencing platform. Subsequently, we trained a one locus-based ANN model to reduce the amount of DNA used. We demonstrated that the ANN model has a higher accuracy of age prediction than the regression model. Additionally, we showed that ethnicity did not affect age prediction among local Chinese, Malays and Indians. Although the predicted age of males were marginally overestimated, sex did not impact the accuracy of age prediction. Lastly, we present a one locus, dual CpG model using 25 ng of input DNA that is sufficient for forensic age prediction. In conclusion, the two ANN models validated would be useful for age prediction to provide forensic intelligence leads.

Optimization of spermatozoa detection using immunofluorescent staining and laser micro-dissection.

The present study evaluated the use of an immunofluorescence-based assay for the microscopic detection of human spermatozoa, following which the fluorescence-labelled spermatozoa could be excised with a laser micro-dissection system. The Sperm Hy-Liter™ PI kit was able to detect spermatozoa from as little as 20nL of semen. No interference or non-specificity were observed when the kit was used on semen mixed with various body fluids such as blood and urine, as well as when semen was spiked onto different types of fabric. Good results could also be obtained with rectal samples which contain auto-fluorescent fecal materials through the use of dual FITC/PI filters. We also developed a method for concurrent testing of two protein biomarkers of semen (semenogelin and prostate-specific antigen) and detection of spermatozoa. This approach would maximize the evidential value from a single piece of sexual assault exhibit. The results also showed that staining by Sperm Hy-Liter™ PI does not interfere with DNA recovery, facilitating the generation of clear male DNA profiles from dissected spermatozoa, thereby making profile interpretation less complex. In summary, Sperm Hy-Liter™ PI staining was demonstrated to be sensitive, robust and specific.