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At least 415 records · Page 23Linked to original sources
PubMed2004

Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue.

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (kappa = 0.661) and HPV18 (kappa = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.

Base Sequence↗
PubMed2025

Cerebrospinal fluid microbiome revisited: no evidence of resident bacteria in archived samples.

UNLABELLED: DNA from oral bacteria has been detected in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease and related dementias (AD/ADRD). We hypothesized that examination of archived CSF samples from donors with variable cognitive status would reveal evidence of a resident microbiome. 176 CSF samples harvested from community-dwelling individuals (77% between 61 and 80 years old) were analyzed; 57% originated from donors with impaired cognitive status. DNA was extracted after adding microbial spike-in controls, and libraries were prepared and sequenced on an Illumina-MiSeq platform. 16S rRNA sequences were processed, and a taxonomic classification was performed. Spike-in bacteria were consistently detected, and Streptococcus pneumoniae was found in a positive control sample from a patient with bacterial meningitis. However, very few reads mapping to other bacterial taxa were detected across samples, suggesting a negligible bacterial content consistent with occasional contamination or sequencing errors. CSF is a privileged, sterile environment that does not harbor a resident microbiome in elderly people with various morbidities, including AD/ADRD. IMPORTANCE: Recent reports have suggested that DNA from oral bacteria has been found in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease and related dementias (AD/ADRD). We hypothesized that examination of archived CSF samples from donors with variable cognitive status would reveal evidence of a resident microbiome. We thus analyzed 176 CSF samples harvested from community-dwelling individuals including donors with impaired cognitive status. While our findings suggested the presence of occasional bacterial contamination, they provided no evidence of a resident microbiome. We thus conclude that the CSF is indeed a privileged, sterile environment that does not harbor a resident microbiome in elderly people with various morbidities including AD/ADRD.

Humans↗
PubMed1995

Molecular detection and serotypic analysis of enterovirus RNA in archival specimens from patients with acute myocarditis.

OBJECTIVE: To determine whether enterovirus RNA can be demonstrated in archival necropsy material in acute myocarditis. DESIGN: Analysis of paraffin embedded myocardial tissue from cases of acute myocarditis. SETTING: University virology department. METHODS: Extraction of RNA from tissue followed by polymerase chain reaction (PCR) and DNA sequence analysis. PATIENTS: Six patients with histologically proven myocarditis and eight controls. RESULTS: Enterovirus RNA was identified in 5 of 6 patients with myocarditis and in none of the controls. The nucleotide sequences of the PCR products showed greatest similarity to group B coxsackieviruses, particularly coxsackievirus B3. CONCLUSION: This study indicates that archival tissue samples, even histologically stained tissue sections, can be used to study the role of enteroviruses in myocardial disease using molecular detection techniques. If a predominant role for coxsackievirus B3 in myocarditis is confirmed by further study, this may have implications for the development of a specific vaccine.

Acute Disease↗
PubMed1992

Use of paraffin wax embedded bone marrow trephine biopsy specimens as a source of archival DNA.

AIMS: To evaluate the use of DNA extracted from paraffin wax embedded trephine biopsy specimens as a source of archival nucleic acid for Southern hybridisation studies and polymerase chain reaction (PCR) amplification. METHODS: DNA was extracted simultaneously from paraffin wax embedded bone marrow trephine and lymph node biopsy specimens after incubation of tissue sections for one to five days in lysis mix and proteinase K with periodic sampling. DNA from 10 trephine biopsy specimens was subjected to PCR amplification using HLA-DPB primers to determine whether the extracted nucleic acid was of sufficient quality to permit amplification. RESULTS: For most specimens the greatest yield of high molecular weight DNA was seen after five days' incubation. Unlike lymph node material the quality of extracted nucleic acid and the quantity obtained from trephines was insufficient for Southern blot analysis. PCR amplification using HLA-DPB primers yielded positive results in six out of 10 trephine biopsy specimens. CONCLUSIONS: DNA extracted from paraffin wax embedded trephine biopsy specimens is largely degraded and unsuitable for Southern analysis but serves as a useful source of archival nucleic acid for PCR amplification.

Biopsy↗
PubMed1995

HLA typing for DR3 and DR4 using artificial restriction fragment length polymorphism PCR from archival DNA.

AIM: To develop polymerase chain reaction based artificial restriction fragment length polymorphism (artificial RFLP PCR) assays for DR3 and DR4 alleles of the multiallelic DRB1 locus and to apply them to paraffin wax embedded archival material. METHODS: Sixty five samples from DRB1 typed cell lines were analysed using the artificial RFLP PCR method to determine the specificity and sensitivity of the system. RESULTS: The artificial RFLP PCR method for typing the DRB1 locus showed 100% accuracy in the 65 samples previously typed using allele specific PCR and serology. The samples included 18 combinations of alleles that included DR3, 18 that included DR4, four that were DR3/DR4 heterozygotes, and 10 samples that were neither DR3 nor DR4. Typing of 10 paraffin wax embedded samples using artificial RFLP PCR was in complete agreement with previous typing at the DRB1 locus. CONCLUSION: The application of artificial RFLP PCR for the analysis of multiallelic loci, such as those of the HLA system, in archival DNA samples has been achieved. Artificial RFLP PCR is a robust, easily implemented, non-isotopic system and may be useful for large retrospective studies.

Alleles↗
PubMed2004

Obtaining explicit consent for the use of archival tissue samples: practical issues.

BACKGROUND: Over the past few years, research ethics committees have increasingly demanded explicit consent before archival tissue samples can be used in research projects. Current UK guidance in this area requires an assessment of whether it is "practical" to obtain explicit consent. Ethics committees have little experience or evidence to help them to judge what is "practical" in this context. METHODS: We attempted to obtain general consent for research use of surplus tissue from renal transplant biopsies from the entire patient population of the renal transplant unit in Leicester. The nature of this patient population would be expected to facilitate this task. RESULTS: A total of 495 letters were sent. Attempts were made to contact non-responders when they attended the outpatient clinic. One year after the initiation of the project, the opinions of 26% of the patients had still not been ascertained. CONCLUSIONS: The results confirm that the vast majority of patients are happy for "surplus" biopsy material to be used for research; the situation does not parallel the use of autopsy tissue. A requirement to obtain explicit consent for the study of archival tissue is likely, however, to block or at least seriously delay research, which is contrary to the public interest and specifically may harm the interests of the patients concerned. In the UK, the problem of tissue being used against the wishes of the donor has now been largely replaced by the problem of prohibition of tissue use against the wishes of the donor.

Biopsy↗
PubMed2003

Stored human tissue: an ethical perspective on the fate of anonymous, archival material.

The furore over the retention of organs at postmortem examination, without adequate consent, has led to a reassessment of the justification for, and circumstances surrounding, the retention of any human material after postmortem examinations and operations. This brings into focus the large amount of human material stored in various archives and museums, much of which is not identifiable and was accumulated many years ago, under unknown circumstances. Such anonymous archival material could be disposed of, used for teaching, used for research, or remain in storage. We argue that there are no ethical grounds for disposing of the material, or for storing it in the absence of a teaching or research rationale. Nevertheless, with stringent safeguards, it can be used even in the absence of consent in research and teaching. Regulations are required to control the storage of all such human material, along the lines of regulations governing anatomy body bequests.

Anonymous Testing↗
PubMed2003

PCR analysis in archival postmortem tissues.

BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.

Apolipoproteins E↗
PubMed1992

Picture archiving and communication systems: an overview.

Organizational techniques that enable small departments to function efficiently often fail as departments become larger. With the recent growth in imaging technology, the capacity of film-based systems to meet the increasing needs of radiology departments has decreased. Electronic picture archiving and communication systems (PACS) have been developed in an attempt to provide economical storage, rapid retrieval of images, access to images acquired with multiple modalities, and simultaneous access at multiple sites. Input to a PACS may come from digital or analog sources (when the latter have been digitized). A PACS consists primarily of an image acquisition device (an electronic gateway to the system), data management system (a specialized computer system that controls the flow of information on the network), image storage devices (both short- and long-term archives), transmission network (which serves local or wide areas), display stations (which include a computer, text monitor, image monitors, and a user interface), and devices to produce hard-copy images (currently, a multiformat or laser camera). The goals of PACS are to improve operational efficiency while maintaining or improving diagnostic ability.

Radiology Information Systems↗
PubMed2004

PHOTOPROBE biotin: an alternative method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin-fixed, paraffin-embedded (FFPE) tissue. A well-known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non-enzymatic, PHOTOPROBE biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

Biotin↗
PubMedDate not supplied

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.

Adolescent↗
PubMedDate not supplied

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples.

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.

AIDS-Related Opportunistic Infections↗
PubMedDate not supplied

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.

Adenocarcinoma, Mucinous↗
PubMed2000

Approximating the nondominated front using the Pareto Archived Evolution Strategy.

We introduce a simple evolution scheme for multiobjective optimization problems, called the Pareto Archived Evolution Strategy (PAES). We argue that PAES may represent the simplest possible nontrivial algorithm capable of generating diverse solutions in the Pareto optimal set. The algorithm, in its simplest form, is a (1 + 1) evolution strategy employing local search but using a reference archive of previously found solutions in order to identify the approximate dominance ranking of the current and candidate solution vectors. (1 + 1)-PAES is intended to be a baseline approach against which more involved methods may be compared. It may also serve well in some real-world applications when local search seems superior to or competitive with population-based methods. We introduce (1 + lambda) and (mu + lambda) variants of PAES as extensions to the basic algorithm. Six variants of PAES are compared to variants of the Niched Pareto Genetic Algorithm and the Nondominated Sorting Genetic Algorithm over a diverse suite of six test functions. Results are analyzed and presented using techniques that reduce the attainment surfaces generated from several optimization runs into a set of univariate distributions. This allows standard statistical analysis to be carried out for comparative purposes. Our results provide strong evidence that PAES performs consistently well on a range of multiobjective optimization tasks.

Algorithms↗
PubMed2001

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

Ammonia↗
PubMed2003

Violence by psychiatric patients: the impact of archival measurement source on violence base rates and risk assessment accuracy.

OBJECTIVE: This study examined the impact of using various archival sources of violence data on the recorded base rate of violence and on the accuracy of violence risk assessments in a sample of civil psychiatric patients. METHOD: Violence in the community, after release from involuntary hospitalization, was measured with 3 types of archival data: criminal records, psychiatric hospital readmission records, and readmission records from multiple general hospitals. RESULTS: Findings indicated that the different measurement sources each identified unique incidents of violence, which were correlated moderately or not at all with one another, and that the estimated base rate of violence varied substantially as a function of outcome source. Not surprisingly, using a combination of sources identified the highest base rate of violence. Predictive validity estimates also varied widely under different measurement procedures and were more stable when a combination of sources was used. CONCLUSIONS: It is important in practical and research terms for studies of psychiatric patient violence to use multiple sources of outcome data.

Adult↗
PubMed2002

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells.

BACKGROUND: A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy. RESULTS: Extensive and variable losses and a few gains of catabolic functions were observed by this standardized method. Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates. CONCLUSION: While the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment. Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog system.

Bacterial Proteins↗
PubMed2005

Evidence of HIV exposure and transient seroreactivity in archived HIV-negative severe hemophiliac sera.

BACKGROUND: Approximately 25% of hemophiliacs that were frequently exposed to blood clotting factor concentrates (CFCs) contaminated with human immunodeficiency virus (HIV) are presently HIV seronegative. In this study, we sought to determine if some of these individuals were at any time transiently HIV seropositive. In the early to mid-1980s the majority of severe hemophilia patients were exposed to CFCs contaminated with HIV. Although many of these hemophiliacs became HIV-positive, a small percentage did not become infected. To determine if some of these individuals successfully resisted viral infection, we attempted to document the presence of transient HIV reactive antibodies in archived plasma samples (1980-1992) from currently HIV-negative severe hemophiliacs who had a high probability of repeated exposure to HIV contaminated CFC. Archived plasma samples were retrospectively tested using an FDA approved HIV-1Ab HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA) and a HIV-1 Western blot assay (Wb), neither of which were commercially available until the late 1980s, which was after many of these samples had been drawn. RESULTS: We found that during the high risk years of exposure to HIV contaminated CFC (1980-1987), low levels of plasma antibodies reactive with HIV proteins were detectable in 87% (13/15) of the haemophiliacs tested. None of these individuals are presently positive for HIV proviral DNA as assessed by polymerase chain reaction (PCR). CONCLUSION: Our data suggest that some severe hemophiliacs with heavy exposure to infectious HIV contaminated CFC had only transient low-level humoral immune responses reactive with HIV antigens yet remained HIV-negative and apparently uninfected. Our data supports the possibility of HIV exposure without sustained infection and the existence of HIV-natural resistance in some individuals.

AIDS Serodiagnosis↗