Sensitivity assessment of the modified ABAcard® HemaTrace® and p30 immunochromatographic test cards.

Operational forensic laboratories routinely perform immunological assays for detecting various body fluids. The ABAcard® p30 and HemaTrace® immunochromatographic tests from Abacus Diagnostics are used for detecting the p30 enzyme in human semen and human haemoglobin present in blood respectively. In early 2023, manufacturer modifications to the ABAcard® p30 and HemaTrace® tests resulted in a reduction in card size and volume of sample extract used in the recommended protocol. This change in card design and/or the reduced volume of sample extract may alter the sensitivity of the test. This study established and compared the limit of detection (LOD) for the old and newly modified ABAcard® p30 and HemaTrace® test cards. The LOD values showed that the new test cards were approximately 2.4-fold (HemaTrace® test) and 3.4-fold (p30 test) more sensitive than the old cards. Additionally, it was found that the new HemaTrace® test cards were more susceptible to the high dose hook effect. In response to the increased sensitivity, existing data pertaining to the reactivity of these test cards to non-target body fluid and substances warrants re-investigation to ensure positive results are interpreted correctly.

A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry, hair and eye colour, and Y chromosome lineage.

Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.

A mini-multiplex SNaPshot assay for the triage of degraded human DNA.

Short Tandem Repeat (STR) genotyping is currently the primary DNA-based method for human identification, however it can have limited success when applied to degraded human remains. Massively parallel sequencing (MPS) provides new opportunities to obtain genetic data for hundreds of loci in a single assay with higher success from degraded samples. However, due to the extra requirement for specialised equipment, expertise and resources, routine use of MPS may not be feasible or necessary for many forensic cases. Here we describe the development of a mini-multiplex SNaPshot screening tool (Miniplex) for human samples which allows the qualitative comparison of short mitochondrial and nuclear DNA targets, as well as the interrogation of biogeographic ancestry, lineage, and phenotype single nucleotide polymorphisms (SNPs). This tool is useful to triage samples based on sample quality prior to downstream identification workflows and provides broad biological profile data for intelligence purposes.