FORENSIC SCIENCES Implementation of Forensic DNA Analysis on Casework Evidence at the Palm Beach County Sheriff's Office Crime Laboratory: Historical Perspective

Palm Beach County is the largest of the 64 counties in the state of Florida, USA, with most of the area uninhabited and the population concentrated near the coastal region. The Serology/DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory serves a community of approximately one million residents, and an additional million tourists visit Palm Beach County every year. In addition to the unincorporated county regions, there are thirty-four city police agencies, the Florida State Highway Patrol, several university security agencies, the local Federal Bureau of Investigation, and the county Medical Examiners Office that all use the PBSO Serology/DNA Laboratory for the analysis of casework evidence. The purpose of this manuscript is to provide laboratories that are in the process of initiating DNA analysis on casework with practical information regarding the decision-making processes that occurred during the development of the DNA testing program at PBSO. Many of the concerns addressed in the early 1990's are still a guide to the development of a quality forensic DNA analysis program in the year 2001. Issues, such as personnel, laboratory space, internal standard operating procedures, implementation of DNA analysis on casework evidence, and building a relationship with law enforcement personnel are discussed. The decision to implement a forensic DNA analysis program in a crime laboratory must take into account several important factors, such as the type of DNA technology to be employed, the location and the layout of the facilities, the number of personnel, training protocols, and annual budgetary needs. In 1992, Palm Beach County Sheriff's Office (PBSO) elected not to implement the technology of restriction fragment length polymorphism (RFLP) but to develop a polymerase chain reaction-based (PCR) analysis for casework evidence. RFLP was the DNA technology of choice in 1992 and finding information regarding construction of a forensic PCR DNA program was challenging. At this time, the only forensic PCR-based DNA typing genetic marker that had been validated and commercially available in a kit format was the HLA-DQA1 locus (previously DQalpha) provided by Roche Molecular Systems (1). Results of an HLA-DQA1 analysis are presented as "blue dots" on a nylon membrane. This first PCR-based test allowed the forensic community to become acquainted with the nuances of the PCR process and the import of training DNA analysts in all aspects of DNA typing, including preparation of DNA extracts, conducting PCR in designated areas of the laboratory, DNA profile interpretation issues, reporting DNA profile frequencies, and reporting findings through court testimony. The HLA-DQA1 test was an important contribution to the forensic community. Even though the eventual implementation of a second "blue dot" forensic DNA typing kit called "PM/DQA1", which tests for six genetic markers, was initiated in many laboratories, the genetic markers were of low polymorphism and DNA mixture interpretation was challenging (2). By 1990, the international community was evaluating genetic markers within the human genome that demonstrated high polymorphism, were amenable to PCR analysis, and could easily be multiplexed in a single reaction, thereby conserving biological stain materials (3,4). These genetic markers, known as microsatellite or "short tandem repeat" (STR) DNA sequences, have become the standard in forensic DNA typing. STRs are genetic regions containing repetitive sequences, usually between 3-5 base pairs in length, thereby allowing for separation of the PCR STR fragment lengths. As a result, interpretation of DNA mixtures is not as difficult as the "blue dot" typing method. The purpose of this manuscript is to provide the forensic community with issues encountered by the PBSO DNA laboratory while initiating a PCR-based forensic DNA program. In addition, it is hoped that the resources presented will act to guide laboratories through the challenges of meeting the needs of the citizens being served, as well as maintaining a high quality DNA program capable of withstanding courtroom scrutiny. www.cmj.hr 24

Detection of herpes viral genomes in normal and diseased corneal epithelium

Herpetic ocular disease is one of the major causes of corneal blindness. Clinical diagnosis of corneal disease is based principally on corneal appearance. However, abnormal morphology of the corneal epithelium (CE) is not an indicator for the presence of a herpes virus. Further, it has not been established if herpes viruses are present in normal corneal epithelial tissue. In these studies, the polymerase chain reaction was used to evaluate normal and diseased corneal epithelium for the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) genomic sequences. Thirty-two normal corneal epithelium specimens obtained from cadavers shortly after death were analyzed for HSV-1, EBV and CMV genomic sequences. Three of the 32 normal CE specimens were positive for amplified EBV DNA, 1 was positive for HSV-1 DNA, and none was positive for CMV DNA. We also tested eight herpetic dendritic lesions of which 3 were HSV-1 culture and PCR positive. The remaining five dendritic lesions were HSV-1 culture and PCR negative. Since these lesions were not evaluated for other herpesviruses, the etiology of these dendritic lesions is unknown. Six corneal epithelium samples from HIV-infected donors were negative for EBV, CMV and HSV-1 amplified sequences. Positive EBV, CMV and HSV-1 serology on all normal donors and on donors with clinically apparent disease did not correlate with positive PCR results. The results of these studies suggest that EBV and HSV-1 DNA can be amplified from a small percentage of apparently normal corneal epithelium

Detection of Herpesvirus DNA in Vitreous and Aqueous Specimens by the Polymerase Chain Reaction

• Members of the herpesvirus family, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV), have been recognized as causal agents of chorioretinal inflammatory diseases. We investigated the use of the polymerase chain reaction for the detection of CMV, HSV, and EBV genomes in aqueous, subretinal fluid, and vitreous specimens in patients with clinically diagnosed CMV retinitis. Cytomegalovirus but not HSV or EBV genomic sequences were detected in all of these clinical specimens. We also investigated 18 normal aqueous and eight normal vitreous specimens obtained from patients undergoing cataract or vitrectomy surgery. Cytomegalovirus, HSV, and EBV DNA were not detected in any of the normal aqueous specimens. There was one weakly positive CMV normal vitreous, but none was HSV or EBV positive by the polymerase chain reaction. These results indicate that the polymerase chain reaction may be useful as a rapid and sensitive diagnostic technique to aid in the confirmation of clinical observations