Novel automatic electrochemical-mechanical polishing (ECMP) of metals for scanning electron microscopy.

A low-stress automated polishing device was developed for preparing titanium and nickel alloys for scanning electron microscopy imaging. The system used pulsed electrochemical reactions within an alkaline electrolyte to generate a thin passivation layer on the surface of the sample, which was removed by the mechanical vibration of the system. The passivation layer development and removal were documented for Ti-6Al-4V and IN718 samples subjected to varying electrical potential cycles and polishing times. Results indicated that the applied cyclic potentials removed material faster than typical removal techniques. In addition, electron back scatter diffraction data showed a decrease in subsurface damage using the developed electrochemical-mechanical process compared to standard mechanical polishing techniques.

Optimizing Storage and Handling of DNA Extracts.

Nucleic acid sample storage is of paramount importance in forensic science as well as in epidemiological, clinical, and genetic laboratories. Millions of biological samples, including cells, viruses, and DNA/RNA, are stored every year for diagnostics, research, and forensic science. PCR has permitted the analysis of minute sample quantities. Samples such as bone, teeth, touch samples, and some sexual assault evidence may yield only low-quality and low-quantity DNA/RNA. Efficient storage of the extracted DNA/RNA is needed to ensure the stability of the sample over time for retesting of the CODIS STRs, mtDNA, YSTRs, mRNA, and other future marker-typing systems. Amplification of some or all of these markers may fail because the biological material has been highly degraded, contains inhibitors, is too low in quantity, or is contaminated with contemporary DNA. Reduction in recovery has been observed with refrigerated liquid DNA extracts and also those exposed to multiple freeze-thaw cycles. Therefore, the development of optimal storage and amplification methods is critical for successful recovery of profiles from these types of samples since, in many cases, retesting is necessary. This review is divided into three sections. The Introduction and Background covers forensic DNA storage, factors that influence DNA stability, and a brief review of molecular strategies to type non-optimal DNA. Section I covers the importance of DNA extract storage in forensic and non-forensic DNA databanks and the mechanisms responsible for loss during storage. Finally, Section II covers strategies and technologies being utilized to store DNA.

Statistical analyses of 14 short tandem repeat loci in Brazilian populations from Rio de Janeiro and Mato Grosso do Sul states for forensic and identity testing purposes.

This work describes the statistical features of a database for two Brazilian populations (one from the Rio de Janeiro State (southeast region), and one from the Mato Grosso do Sul State (central western region) using fourteen short tandem repeat loci (STR).

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.

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system.

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.

Analysis and interpretation of short tandem repeat microvariants and three-banded allele patterns using multiple allele detection systems.

The Palm Beach County Sheriffs Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented analysis of short tandem repeat (STR) sequences on casework using silver staining kit and SYBR Green I detection systems and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO 100 instrument. Concurrently, the Broward County Sheriff's Office (BSO) Crime Laboratory is validating the ABI Prism310 Genetic Analyzer capillary electrophoresis STR detection system (ABI CE310) from Perkin Elmer Applied BioSystems. During the course of analyzing over 10,000 individuals for the STR loci CSF1PO, TPOX and THO1 (CTT) using silver staining for allele detection, 42 samples demonstrated alleles that were "off ladder," contained three-banded patterns at a single locus, or exhibited an apparent THO1 "9.3,10" allele pattern. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the allele patterns observed in these 42 samples using the following allele detection systems: (1) manual silver staining, (2) SYBR Green I staining, and/or (3) fluorescently tagged amplified products separated by polyacrylamide gel electrophoresis or capillary electrophoresis followed by laser detection. Regardless of the CTT allele detection system utilized, concordant results were obtained for 41 of the 42 samples. The only exception was a sample in which a wide band within the THO1 locus was identified as a THO1 "9.3, 10" genotype by silver staining kit and SYBR Green I staining but was verified to be a THO1 "9.3" homozygote by all other allele detection systems. Manual allele detection could readily identify microvariants, as a visual assessment of stained gels clearly shows that alleles do not migrate coincident with well-characterized allele size standards. As would be predicted, however, the manual detection systems did not provide adequate resolution to approximate the basepair size for off-ladder variants. All fluorescent software program systems were consistent in designating alleles "not in range" or "off ladder," thereby indicating true microvariants. All single-locus three-banded patterns were detected using all of the STR multiplex systems. In addition, individual locus-specific primers verified multiplexed amplified products were specific for the locus in question.

Confirmation of PM typing protocols for consistent and reliable results.

A recent report in the Perkin Elmer "Forensic Forum" bulletin described a modification to the previously published PM typing protocol indicating that in order to obtain consistent and reliable PM and DQA1 typing results, disodium EDTA should be added to the post-amplification mixture before denaturation of the DNA fragments. The analysis and validation of this suggestion is described in the accompanying paper. We report the evaluation of this additional step when typing for PM alleles and conclude that the standard operating procedures currently enforced at the Palm Beach County Sheriff's Office and Indian River crime laboratories do not necessitate the need for the addition of disodium EDTA to the PM amplified products prior to the heat denaturation step. Further, depending on an individual laboratory's PM protocol, the recommendation by Perkin Elmer to add disodium EDTA to PM amplified products before typing has merit and should be carefully considered when determining laboratory PM typing protocols.

Investigation of species specificity using nine PCR-based human STR systems.

Several eukaryotic genomes contain polymorphic markers consisting of trimeric and tetrameric short tandem repeats (STR). Recent reports have demonstrated the variability of short tandem repeat (STR) polymorphisms at a variety of loci among several human population groups. Currently, there are nine commercially available STR PCR systems from Promega Corporation that may be utilized for human identification. We report here the analysis of 23 different species DNA's using these nine STR primer systems to assess their specificity for human euchromatin. The STR systems tested include, CSF1PO, TPOX, THO1, HPRTB, FESFPS, vWF and F13A01 as single systems and as triplex systems (CSF1PO/TPOX/THO1 and HPRTB/FESFPS/vWF). There were no STR PCR products observed for seventeen of the twenty-three species regardless of the STR system. Amplified STR fragments were detected in rhesus DNA for CSF1PO, TPOX and HPRTB systems. STR PCR products were detected for human, gorilla, chimpanzee, and orangutan DNAs using eight of the nine systems. FESFPS primers did not amplify DNA fragments from any of the species tested. Most of the STR PCR products detected from primate DNAs electrophoretically migrated outside of the human allelic ladder fragments and as a result, allele designations were not possible.

Analysis of HLA DQ alpha allele and genotype frequencies in populations from Florida.

HLA DQ alpha allele and genotype frequencies for Caucasian, African American, Haitian, and Hispanic populations in Florida have been estimated. The Florida laboratories involved in these studies collected donor samples from a variety of sites including clinical laboratories, victim and suspect standards, blood banks, county jail detainees, and laboratory personnel. We have determined that the Caucasian and African American DQ alpha genotype frequencies do not deviate significantly from Hardy-Weinberg expectations and as a result of this heterogeneity analyses, data from the four Florida Caucasian populations may be combined and data from the four Florida African American populations may be combined to form two large HLA DQ alpha genotype frequency databanks. Further, data from the Florida Haitian population may be combined with the Florida African American population. Comparison of the combined Florida Caucasian populations, combined Florida African American populations, the Palm Beach Sheriff's Office (PBSO) Hispanic, and PBSO Haitian population with other databases does not support combination because allele frequency distributions are heterogeneous.

Analysis and interpretation of the HLA DQ alpha "1.1 weak-signal" observed during the PCR-based typing method.

The Perkin-Elmer (PE) AmpliType DQ alpha Forensic Kit is currently available for amplification and typing of a polymorphic region in the Human Leukocyte Antigen (HLA) DQ alpha DNA sequence. Following amplification of the DQ alpha region with the PE kit, typing strips are processed. These strips contain immobilized DNA probes designed to distinguish six possible HLA DQ alpha alleles. It has been observed in this laboratory and others that in a single source DNA sample, it is possible to detect a weak signal on the 1.1 specific allele dot when the samples' genotype clearly does not contain the 1.1 allele. It has been suggested that a potential source of this weak-signal is the non-specific amplification of a HLA DX alpha gene sequence. To demonstrate the relationship of the DX alpha gene to the 1.1 nonspecific signal, we designed biotinylated DX alpha PCR primers specific for a 178 bp region in which the amplified product spans the homologous DQ alpha region encompassing the DNA probes present on the typing strips. DX alpha DNA sequences from various DQ alpha genotypes were amplified and hybridized to DQ alpha typing strips. We have demonstrated that DX alpha PCR products do not always hybridize to the 1.1 probe on the typing strips. Sequence analysis of DX alpha PCR products show that this region is polymorphic which may explain why the occurrence of the "1.1 weak-signal" is unpredictable. We have further analyzed the effect of DNA template concentration for the DQ alpha amplification protocol and have shown that regulation of PCR input DNA optimizes the amplification and typing protocols for HLA DQ alpha alleles and minimizes the potential observation of the "1.1 weak-signal."

Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures.

Sonication procedures for the extraction of DNA from forensic-type semen specimens have been developed, which, when compared to currently utilized sperm DNA extraction techniques, are simple, rapid and result in comparable DNA yields. Sperm DNA extraction by sonication was performed on whole semen, seminal stains, buccal swabs and post-coital specimens. Ultrasound disruption of sperm cells and their ultimate release of cellular DNA has been conducted in the presence of sperm wash buffers followed by organic extraction or Chelex 100 with little or no compromise to DNA quality, quantity or amplifiability. Two advantages of sonication over currently used forensic techniques to extract sperm DNA include 1) sperm DNA extraction that occurs within five minutes of sonication compared with an hour or greater for water bath incubations in classic enzyme digestion DNA extractions and 2) one less preparatory step with the Chelex/sonication protocol and three less steps with the sonication/organic protocol compared with other procedures thus eliminating potential sample-to-sample cross-contamination. Sperm DNA extracted by optimum sonication procedures was used for forensic HLA DQ alpha typing and restriction fragment length polymorphisms analysis without any adverse effects on typing results.

Epstein-Barr virus and the lacrimal gland pathology of Sjögren's syndrome.

The lacrimal gland (LG) immunopathology of Sjögren's syndrome (SS) consists of a proliferation of B and CD4 lymphocytes surrounding epithelial structures (Pepose JS, et al: Ophthalmology 1990, 97:1599-1605). Based on the detection of EBV genomes in a greater percentage of SS than normal LG biopsies, we previously postulated that Epstein-Barr virus (EBV) is a risk factor for LG lymphoproliferation in SS (Pflugfelder SC, et al: Ophthalmology 1990, 97:976-984). The purpose of this study was to determine the cellular site(s) of infection, virus type, and antigen expression of EBV infecting normal and SS LGs. EBV DNA was detected by in situ hybridization in intraductal epithelia in 13-33% of lobules in 21% of normal LGs and in cells in areas of B lymphoproliferation as well as the majority of epithelia in 86% of SS LGs. EBV genomic sequences were amplified from 36% of normal and 88% of SS LG biopsies by polymerase chain reaction. Only type 1 EBV sequences were amplified in SS LGs; in contrast EBV nuclear antigen 2-deleted but not type 1 sequences were amplified in normal LGs. Immunohistochemistry with EBV-specific monoclonal antibodies was performed on normal and SS LGs. No EBV antigens were detected in normal LGs. In contrast, latent antigens (latent membrane protein, EBV nuclear antigen 2) were detected in lymphocytes in areas of B lymphoproliferation, and early and late lytic cycle antigens were observed in epithelia in SS LGs. These studies suggest that EBV may play a role in the LG B lymphoproliferation and epithelial pathologic changes observed in SS.

Cytoskeletal antigen expression in ocular mucosa-associated lymphoid tissue.

The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial cell populations in normal human ocular mucosa-associated lymphoid tissue (MALT; LG, conjunctiva, and cornea) based on their immunohistochemical staining patterns with anticytoskeletal monoclonal antibodies (MoAbs) reactive with cytokeratins (AE-1, AE-2, AE-3, AE-5, AE-14, PKK1, and 34 beta E12), muscle-specific actin (HHF35), and vimentin. AE-1 stained LG (acini, ducts, and myoepithelia) and the full thickness of corneal and conjunctival epithelia. It stained only the superficial and basal limbal epithelium. AE-2 weakly stained all epithelia, except LG acini and proximal intralobular ducts. AE-3 and 34 beta E12 MoAbs had strong immunoreactivity with all MALT epithelia. AE-5 strongly stained the inner cells (suprabasal) of LG central intra- and interlobular ducts and the suprabasal epithelial layers of the cornea. It weakly stained LG myoepithelia and the superficial conjunctival epithelium. AE-14 stained the outer (basal) cells of LG central intra- and interlobular ducts, LG myoepithelia, basal epithelial layers of the limbus and conjunctiva, and all corneal epithelia. PKK1 stained all epithelia, except the basal limbus. HHF35 and the antivimentin MoAbs stained only the LG myoepithelia. The results of these studies indicate that the different epithelia in human ocular MALT may be differentiated by specific patterns of immunoreactivity with anticytoskeletal MoAbs. These MoAbs may be useful molecular markers for identifying ocular MALT epithelia.

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.

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 Epstein-Barr virus genomes in normal human lacrimal glands.

Epstein-Barr virus (EBV) has been implicated in several ocular diseases; however, detection of the EBV genome in ocular tissues has not been documented. We report the detection of amplified EBV genomic sequences in 11 of 26 normal lacrimal gland DNA samples by using the polymerase chain reaction. Serum was available for 19 of the lacrimal gland donors. All 19 were EBV seropositive, although of the 19 lacrimal gland-seropositive patients, EBV sequences were detected in only 10 of the samples. Further, amplified EBV sequences were not detected in circulating lymphocyte DNA from normal seropositive volunteers, most likely because of the low frequency of circulating EBV-infected B cells. Amplification of EBV from cadaver lacrimal gland DNA was possible with minute quantities of DNA, whereas peripheral blood mononuclear cell DNA from normal volunteers did not amplify EBV sequences. Interestingly, the peripheral blood mononuclear cell polymerase chain reactions contained approximately 100 times more DNA than the lacrimal gland polymerase chain reactions. We conclude that the lacrimal gland may be a site for EBV persistence and that positive EBV serology is not an indicator of which individuals may have EBV harbored within their lacrimal glands.

Detection of the complement (CD21)/Epstein-Barr virus receptor in human lacrimal gland and ocular surface epithelia.

CD21, the receptor for the C3d complement fragment, has been reported to be the Epstein-Barr virus (EBV) receptor in B lymphocytes and cultured squamous epithelial cells. This receptor has previously been found to be expressed in the epithelia of nonocular mucosal-associated lymphoid tissues (MALT) which are also sites of persistent EBV infection. We recently found evidence of persistent EBV infection in human ocular MALT and hypothesized that the epithelia in these tissues may also express the complement (C3d)/EBV receptor. To test this hypothesis, histologic sections of human lacrimal gland, ocular surface tissue (conjunctiva, limbus, and cornea), and conjunctival impression cytology specimens were stained by immunohistochemical techniques using three different anti-CD21 monoclonal antibodies (HB-5, B2, OKB7). HB-5 stained lacrimal gland ductal and all ocular surface epithelia, except limbus. B2 stained lacrimal gland ductal and limbal epithelia, and OKB7 stained only the limbal and corneal epithelia. The intensity of limbal and corneal epithelial staining with all anti-CD21 antibodies correlated with the level of epithelial differentiation, with the weakest staining noted in the cells which are thought to be the stem and transient amplifying cells of the cornea. These results suggest that external ocular tissues, similar to other MALT, have CD21-positive epithelia which may be potential targets for EBV infection.