Recommended policies for uses of human tissue in research, education, and quality control. Ad Hoc Committee on Stored Tissue, College of American Pathologists.

As recipients of tissue and medical specimens, pathologists and other medical specialists regard themselves as stewards of patient tissues and consider it their duty to protect the best interests of both the individual patient and the public. The stewardship of slides, blocks, and other materials includes providing, under appropriate circumstances, patient materials for research, education, and quality control. The decision to provide human tissue for such purposes should be based on the specific (ie, direct patient care) and general (ie, furthering medical knowledge) interests of the patient and of society. The same standards of responsibility should apply to all medical professionals who receive and use specimens. This document proposes specific recommendations whereby both interests can be fostered safely, ethically, and reasonably.

Clinical assessment of DNA analysis for toxicology.

The impact of discoveries in biotechnology-particularly those dealing with DNA--will be among the most significant factors changing the world in the next decade. Toxicologists will not escape this change; they, too, will be significantly impacted by the new genetics.

Real-time microchip PCR for detecting single-base differences in viral and human DNA.

This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.

Rapid PCR for identity testing using a battery-powered miniature thermal cycler.

A microfabricated, battery-powered thermal cycler was implemented in PCR-based DNA typing for human identification. HLA DQ alpha and an STR triplex were PCR amplified using a device known as the Miniature Analytical Thermal Cycling Instrument (MATCI). The extremely efficient heating properties of the MATCI enabled thermal cycling to be completed in as little as 21 min. In addition, the feasibility of using the real-time fluorescent detection system of the MATCI was demonstrated. The successful application of this portable, prototype device to forensic identity testing is a significant milestone towards the eventual development of a completely integrated DNA testing instrument that would also incorporate sample preparation and allele detection.

Postmortem identifications of remains.

Forensic pathologists must identify the bodies and remains falling under their purview in the normal course of death investigation and death certification. Accurate identification is critical and can at times be daunting challenge, especially in mass disaster situations. Many methods can be employed from various scientific disciplines and otherwise, resulting in positive or presumptive identification. Then, proper identification must be maintained and supported where challenged.

A high observed substitution rate in the human mitochondrial DNA control region.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II.

In 1991, nine sets of skeletal remains were excavated from a mass grave near Yekaterinburg, Russia which were believed to include the Russian Tsar Nicholas II, the Tsarina Alexandra, and three of their daughters. Nuclear DNA testing of the remains verified such a family group, and mitochondrial DNA (mtDNA) sequences of the presumed Tsarina matched a known maternal relative, Prince Philip. mtDNA sequences from bone of the presumed Tsar matched two living maternal relatives except at a single position, where the bone sample had a mixture of matching (T) and mismatching (C) bases. Cloning experiments indicated that this mixture was due to heteroplasmy within the Tsar; nevertheless, the 'mismatch' fueled a lingering controversy concerning the authenticity of these remains. As a result, the official final report on the fate of the last Russian Royals has been postponed by Russian authorities pending additional, convincing DNA evidence. At the request of the Russian Federation government, we analysed the skeletal remains of the Tsar's brother Georgij Romanov in order to gain further insight into the occurrence and segregation of heteroplasmic mtDNA variants in the Tsar's maternal lineage. The mtDNA sequence of Georgij Romanov, matched that of the putative Tsar, and was heteroplasmic at the same position. This confirms heteroplasmy in the Tsar's lineage, and is powerful evidence supporting the identification of Tsar Nicholas II. The rapid intergenerational shift from heteroplasmy to homoplasmy, and the different heteroplasmic ratios in the brothers, is consistent with a 'bottleneck' mechanism of mtDNA segregation.

Forensic DNA tests.

DNA testing has revolutionized the forensic sciences. Forensic testing has continued to evolve, and now several methods are in use. The predominant methods of forensic DNA analysis are described, and their application in casework is summarized. The legal acceptance of this testing also is discussed.

Further validation of a quadruplex STR DNA typing system: a collaborative effort to identify victims of a mass disaster.

The relatively new, PCR-based technique of Short Tandem Repeat (STR) profiling has been used in the identification of the victims of a mass disaster. The analysis relied upon a recently developed multiplex reaction and the use of automated fluorescence technology to simulataneously analyse four tetrameric STR loci. The performance of the 'quadruplex' test was assessed by use of a collaborative study incorporating a blind trial and was demonstrated to be accurate, reliable and robust. Furthermore, the system proved to be highly successful despite the fact that many of the samples from the mass disaster scene were extremely degraded. The high success rate coupled with the discrimination power of the system enabled many severely decomposed human remains to be positively identified.

A systematic approach to the sampling of dental DNA.

As investigations into the forensic aspects of DNA analysis continue, the human tooth will play a dual role in identification. Dentin and enamel provide a protective enclosure for genomic and mitochondrial DNA as well as providing the basis for radiographic, biochemical, and ultrastructural forensic studies. The purpose of this investigation is to establish technical guidelines, based on histology and experimental evidence, for the management and sampling of dental DNA. The anatomic location of dental DNA is discussed with emphasis on the conservation of tooth structure during sampling. Ten pairs of maxillary right and left third molars were sampled for DNA following storage for 18 weeks at ambient temperature and humidity. Right third molars were crushed, whereas the left third molars were sectioned conservatively prior to sampling the DNA. The quantity and quality of human DNA obtained from each tooth was compared, as well as the radiographic appearance of remaining hard tissue and the overall simplicity of each approach. DNA typing was performed, both sequence and length based analyses, comparing teeth from the same individual and teeth from different donors. The results of this study suggest that the odontologist will maximize the dental DNA yield by crushing the entire specimen but that substantial yields of human DNA can be obtained by using a conservative technique that preserves the tooth structure. In addition, the method of sampling does not affect the ability to perform DNA typing analyses.

Forensic DNA testing.

Forensic deoxyribonucleic acid (DNA) testing has revolutionized criminal investigations. Deoxyribonucleic acid testing is superseding traditional serologic testing due to its discriminatory power, universal application to biologic materials, and resistance to environmental insults, among other advantages. Its acceptance is becoming commonplace, and it is being put into widespread use. Forensic DNA testing technology and its application is continuing to evolve.

Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War.

Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

Short tandem repeat loci: application to forensic and human remains identification.

The short tandem repeat (STR) locus ACTBP2 (common name SE33) was analyzed for its potential use in forensic and human remains identification. PCR amplification conditions were determined, and an allele-specific ladder was generated so that discrete alleles could be scored. The allele frequency distributions were determined for both Caucasian and Black populations. The frequency data meets Hardy-Weinberg expectations, and the allele distributions were similar from one racial group to another and between ethnic groups. SE33 analysis was subsequently used to confirm the identification of human remains for the Office of the Armed Forces Medical Examiners.

Extraction, evaluation, and amplification of DNA from decalcified and undecalcified United States Civil War bone.

Deoxyribonucleic acid (DNA) was extracted from documented skeletal specimens of U.S. Civil War soldiers to determine the need for decalcification prior to extraction. The polymerase chain reaction (PCR) was performed to determine if the calcification state had an effect on the ability to amplify the extracts and to determine how successful amplification would be with these aged specimens. Bone samples were pulverized to a fine powder and divided into two sets. One set of samples was decalcified and the other set left undecalcified. Both sets were extracted using an organic procedure. The results demonstrate that decalcification is not a necessary step in the extraction process and that the yield of DNA is generally two times greater when decalcification is omitted. Furthermore, the calcification state had no effect on the ability to perform the PCR. Although the extracted DNA was very degraded, a 410 base pair (bp) segment of the mitochondrial DNA (mtDNA) control region was amplified. These results suggest that DNA can be extracted and amplified from 125 year old bone without decalcification, which may assist in the identity of modern and historic forensic specimens.

Electrocution: a review of 155 cases with emphasis on human factors.

A review of 155 cases of electrocution were investigated to determine the role of human factors, such as carelessness and intoxication, as contributions. The cases were retrieved by computer coding from the repository of the Armed Forces Institute of Pathology, covering the period 1955-1988. The cases are predominantly of military origin. Cases were divided into low-voltage electrocution (N = 47), high-voltage electrocution (N = 79), lightning strikes (N = 16), and unclassified (N = 13). For each group, data is presented on the circumstances of the incident and the pathologic findings. Blatant carelessness, misuse or improper maintenance of equipment, and intoxication are analyzed as contributory factors.

Patient personal injury litigation against dermatology residency programs in the United States, 1964-1988. Implications for future risk-management programs in dermatology and dermatologic surgery.

A national survey reviewing patient injury litigation against US dermatology residency programs revealed that 50% of the respondents had experienced at least one lawsuit between 1964 and 1988. The northeast region reported the most legal activity. Fifty percent of the lawsuits related to therapeutic or surgical complications. Plaintiffs were successful in 37.9% of the lawsuits. The mean award was $26,505, and the largest reported award of $200,000 was for failing to diagnose herpes simplex in an immunocompromised patient. In view of several recent trends in dermatology, the amount of litigation against dermatologists may increase.