U.S. flag

An official website of the United States government, Department of Justice.

Development and Testing of a Rapid Multiplex Assay for the Identification of Biological Stains

NCJ Number
Date Published
250 pages
The goal of this research was to develop and test a fast, accurate, and sensitive multiplex assay for the simultaneous identification of saliva, semen, urine, peripheral blood, menstrual blood, and vaginal secretions, since current antibody-based and enzyme activity-based assays used by forensic practitioners for biological stain identification produce only presumptive results.

The results of the conducted experiments populated a database that contained 30 body-fluid-specific protein biomarkers with candidates linked to each target body fluid. These include biomarker candidates to identify semen, saliva, urine, peripheral blood, menstrual blood, and vaginal fluid. The researcher advises, however, that although these results are encouraging, the discovery process was performed on the sample from only five individuals per bodily fluid; thus, these proteins can be considered only candidate biomarkers. This is because of the possibility that some candidate biomarkers might be secreted into non-target fluids, particularly with the less-documented proteins associated with vaginal fluid. Consequently, a larger scale study must be completed in order to provide a more rigorous confirmation of these candidates as being body-fluid-specific. The study identified just over 1,000 proteins by using 3 approaches. The biomarker-identification phase used multidimensional protein separation technologies, bioinformatics tool, and tandem mass spectrometry in developing a database of fluid-specific candidate markers for each targeted body fluid. The biomarker-verification phase used the most promising candidates from the biomarker-identification phase in developing a targeted multiplex assay on a Quadruple Time-of-Flight mass spectrometer in verifying the specificity of these candidate biomarkers with single-source laboratory samples, as well as single- and mixed-source casework-type samples. The prototype-validation phase developed and tested a case working laboratory prototype assay on a triple quadrupole mass spectrometer, using single- and mixed-source casework-type samples. 42 figures, 20 tables, 215 references, and appended supplementary data

Date Published: January 1, 2013