Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents.

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation. The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated. During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly. Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling.