In the mess and confusion of a crime scene, the origin of a particular stain is not always apparent-was it saliva, blood, semen, or vaginal excretion? For investigations that type of knowledge could be vital, but until recently all body-fluid identification testing was expensive, time-consuming, and labor-intensive (and they could not even identify saliva). Now, thanks to breakthroughs from Jack Ballantyne's laboratory, mysterious stains can be discerned quickly, accurately, and efficiently.

When Ballantyne started working on the body-fluid identification problem he had certain criteria that he wanted to meet. Namely, the tests needed to be parallel (that is, all of the body-fluids needed to be checked for at one time), and the new tests needed to be compatible with the current DNA analysis methods.

Messenger RNA analysis held the key. Messenger RNA, unlike DNA or other types of RNA, is expressed differently in each type of bodily tissue. Researchers knew that if the type of messenger RNAs present in a stain or tissue sample could be determined, then it would be possible to definitively identify the tissue or body fluid in question, but RNA was thought to be too unstable to use.

Ballantyne's laboratory team demonstrated that it is possible to isolate a sufficient amount of good-quality RNA from biological stains. (Their experiments proved that it was actually a widely-held misconception that RNA was unstable.) Then, using those techniques, the team devised tests for each type of common body fluid (blood, saliva, semen, and vaginal secretions).

This unique approach to comprehensive and definitive body-fluid identification has stimulated the interest of the broader forensic community. The laboratory is now collaborating with the FBI to validate the messenger RNA analysis methods.

Detection of RNA Expression in Biological Stains

Since it can be important to identify the nature of the body fluids present in a stain recovered at a crime scene, the development of a body fluid identification system that is compatible with current DNA typing procedures is desirable. Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Moreover, for some frequently encountered body fluids no confirmatory technique exists. There is no definitive test, for example, for the presence of saliva or vaginal secretions. In seeking to develop novel multiplex (i.e. parallel) analysis procedures for body fluid identification that are compatible with current DNA analysis procedures, we have considered assays based upon protein and messenger RNA (mRNA) since both are expressed in a tissue specific manner. However, multiplex analysis of complex, partially degraded protein mixtures such as those present in body fluid stains awaits further developments in proteomics. Messenger RNA is considered a better option because the technologies for massively parallel analysis continue to be developed due to the rapidly evolving field of functional genomics.

Terminally differentiated cells, whether they comprise of blood monocytes or lymphocytes, ejaculated spermatozoa, or epithelial cells lining the oral cavity become such during a developmentally regulated program in which certain genes are turned off whereas others are turned on. Thus, a pattern of gene expression is produced that is unique to each cell type, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs could be determined in a stain or tissue sample recovered at the crime scene it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automatic analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies.

Previously, we demonstrated that it is possible to isolate total RNA of sufficient quality and quantity from biological stains to enable subsequent detection of particular mRNA species using the RT-PCR technique. We also identified candidate sets of saliva-specific genes. Since that time, we have also identified and tested candidate sets of blood-, semen- and vaginal secretions-specific genes. We now report the development of a multiplex RT-PCR assay for the definitive identification of all of the body fluids commonly encountered in forensic casework analysis, namely blood, saliva, semen, and vaginal secretions. The octaplex is composed of eight body fluid specific genes and has been optimized for the detection of blood, saliva, semen, and vaginal secretions as single or mixed stains. The methodology is based upon gene expression profiling analysis in which the tissue specific genes are identified by detecting the presence of appropriate mRNA species.

An mRNA based approach, such as the multiplex RT-PCR method described above, could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

For More Information

Jack Ballantyne
Department of Chemistry, CH 117
University of Central Florida
Orlando, FL 32816
Telephone: 407-823-0163
FAX: 407-823-2252
E-Mail: jballant@mail.ucf.edu