A method validation for the hemoglobin chemical reagent test strip with the addition of ethylenediaminetetraacetic acid to increase the selectivity of this presumptive test for residual blood

Abstract

Tetramethylbenzidine based chemical reagent test strips are often used in forensic science as a presumptive test for blood. These tests are designed as urinalysis test strips and include brands such as Combur®, HENSOTest®, Hemastix®, MultiStix® and Chemstrip®. They are used because they are simple to apply, stable, temperature tolerant and cost effective. The addition of a chelating agent, ethylenediaminetetraacetic acid increases the selectivity of this presumptive test for blood. This is a method validation for the hemoglobin chemical reagent test strip with EDTA. A range of substances, metal compounds, chemical solutions, blood and mixtures were tested in this method validation. The chelation with EDTA successfully prevented non-blood (false) positive results from all the substances tested and consistently produced a positive result for blood on a variety of surfaces. This study has shown that this method is capable of discriminating a blood stain on copper metal surfaces and eliminate the positive results generated by clean-up solutions such as hydrogen peroxide, which usually produce a positive result for most other presumptive tests for blood. This modified method is a simple, effective and reliable test for blood stains. A variety of variations were evaluated in this study. The simplest method of application was spraying the surface of the stain with a 0.5 M EDTA solution and testing the surface of the stain, and only requires a spray bottle of 0.5 M EDTA and the chemical reagent test strip. This spray approach is rugged and can be applied to horizontal, vertical and underside surfaces and requires little additional training. Overall, this study provides the forensic science community with an improved method more easily used, stored, transported and selective for blood, than luminol and safer than TMB.