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PubMed2004

Evidence of genotypic resistance diversity of archived and circulating viral strains in blood and semen of pre-treated HIV-infected men.

OBJECTIVE: To study the genetic diversity of drug-resistant HIV strains present in blood and in semen, especially those archived in peripheral blood mononuclear cells (PBMC) and non-sperm cells (NSC). METHODS: Paired blood and semen samples were collected from twenty heavily pre-treated HIV-infected men. HIV RNA in blood plasma (BP) and seminal plasma (SP), as well as proviral DNA in PBMC and NSC were quantified and used for resistance genotyping. Phylogenetic analysis of protease gene clones was used to explore the diversity of the viral quasi-species. RESULTS: Median BP HIV RNA, PBMC proviral DNA, SP HIV RNA and non-sperm cell proviral DNA loads were respectively: 4.77, 3.65, 3.16 and 1.77 log10 copies per ml or per 10 cells. Resistant HIV strains were found in the BP and PBMC of all the patients, in the SP of 14 patients, and in the NSC of five patients. Overall, the blood and genital compartments exhibited different genotypic resistance patterns in six patients (30%), with additional resistance mutations in the semen of four patients. Phylogenetic analysis of clones of HIV protease gene showed that viral strains in SP originated not only from passive diffusion from BP, but also from local production in semen. The storage of archived proviruses differed according to the anatomic reservoir. CONCLUSION: HIV resistant strains are frequent (70%) in the semen of heavily pre-treated men, and the diversity of genotypic resistance pattern confirms HIV compartmentalization. Thus, the risk of sexual transmission of resistant strains can only be partly predicted by standard tests applied to BP.

Adult↗
PubMed1990

Potential use of a 100-mm film digitizer and image processor for film archiving.

The present study was undertaken to examine experimentally and clinically whether a 100-mm roll-film digitizer and image processing device could preserve or increase the diagnostic accuracy of 100-mm roll films used in the mass screening of patients for lung cancer and provide picture archives of high enough image quality to replace the original films. The quality of the processed digitized CRT image was found to be only slightly less than that of the original films. There was only a slight decrease in diagnostic accuracy, and the system was judged to be potentially useful as a miniature picture archiving and reference system.

Equipment Design↗
PubMed2003

Picture archiving and communication systems (PACS).

Over the past 2 decades, groups of computer scientists, electronic design engineers, and physicians, in universities and industry, have worked to achieve an electronic environment for the practice of medicine and radiology. The radiology component of this revolution is often called PACS (picture archiving and communication systems). More recently it has become evident that the efficiencies and cost savings of PACS are realized when they are part of an enterprise-wide electronic medical record. The installation of PACS requires careful planning by all the various stakeholds over many months prior to installation. All of the users must be aware of the initial disruption that will occur as they become familiar with the systems. Modern fourth generation PACS is linked to radiology and hospital information systems. The PACS consist of electronic acquisition sites-a robust network intelligently managed by a server, multiple viewing sites, and an archive. The details of how these are linked and their workflow analysis determines the success of PACS. PACS evolves over time, components are frequently replaced, and so the users must expect continuous learning about new updates and improved functionality. The digital medical revolution is rapidly being adopted in many medical centers, improving patient care and the success of the institution.

Radiology Information Systems↗
PubMed1996

Identification of p53 mutations in archival prostate tumors. Sensitivity of an optimized single-strand conformational polymorphism (SSCP) assay.

To correlate molecular changes with clinical information in prostate tissue, it is necessary to have accurate methods for screening for mutations in clinically available specimens. We have refined the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere with SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samples are heterogeneous in containing tumor, normal tissue, and hyperplastic tissue. To address the first issue, the method included an initial selection of PCR products using exonuclease I, followed by a second-step selection using nested PCR. This step ensures adequate amplification of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue, in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restriction enzyme digestion of products prior to SSCP analysis. Detection of mutations in heterogeneous tissue was evaluated by determining the minimal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous clinical samples, we performed study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and two metastases) and detected abnormally migrating products in six of the prostate cancer specimens (four primaries and two metastases). In five of these samples, there was sufficient DNA to perform sequencing, which disclosed single-base change mutations in all five samples.

DNA Mutational Analysis↗
PubMed1998

RNA from decades-old archival tissue blocks for retrospective studies.

The validity of molecular studies using DNA and RNA extracted from decades-old formalin-fixed and paraffin-embedded tissue blocks has been demonstrated. The quality and usability of DNA and RNA from archival tissues are modified by various factors, such as the fixative, the fixation time, and the postmortem time. However, in contrast to DNA, there are no comprehensive studies quantitatively addressing the feasibility of RNA from old (more than 10 years) archival samples. This study examined the integrity of RNA extracted from 738 autopsy liver and 63 autopsy thyroid cancer tissue blocks procured during a span of nearly four decades, beginning in 1952 and ending in 1989, from the atomic bomb survivors. The integrity of RNA was assessed by amplification of c-BCR messenger RNA (mRNA) between two sequential exons with an intervening intron by reverse-transcription polymerase chain reaction (RT-PCR). The integrity of RNA was influenced by the age of the samples and the postmortem time, but not by the formalin-fixation period. It was possible to amplify more than 60% of the samples. Using these RNAs, the HCV genome in liver cancers and the H4-RET gene in thyroid cancers were detectable. This study illustrates the possibility of molecular studies using RNA from routinely prepared paraffin blocks stored for long periods and provides the statistics and critical factors to consider in assessing the feasibility of such contemplated studies.

Drosophila Proteins↗
PubMed1999

Use of the same archival papanicolanou smears for detection of human papillomavirus by cytology and polymerase chain reaction.

An optimal method for the processing of archival cervical Papanicolaou (pap)-stained smears for the amplification of human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) was developed. This methodology was then applied to a series of 44 pap smears designated as HPV positive or negative (on the basis of both major and minor cytological criteria) or cervical intraepithelial neoplasia (CIN)-cancer. For the detection of HPV DNA, each sample was tested with the consensus GP5/6 primers, and when negative, with CPI-IIG primers. The HPV DNA was detected in 100% (8 of 8) of CIN-cancer smears using the GP5/6 primers. In smears with cytological evidence of HPV without CIN. the use of both sets of primers yielded positive results in 100% (19 of 19) of the samples. Direct sequence analysis of PCR products showed that 16 of the 27 HPV-positive samples contained more recently described HPV types. When tested with both primer combinations, all 17 cytologically negative smears were positive for beta-globin but negative for HPV DNA. The findings show the value of using archival pap smears for further investigations to address issues such as latency, but they indicate that cytological criteria and DNA technology will be critical factors in the reliability of the results.

Cytodiagnosis↗
PubMed1999

Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues.

The reciprocal translocation t(17;22)(q22;q13) and a supernumerary ring chromosome, r(17;22), derived from the translocation, have been shown to be highly characteristic of dermatofibrosarcoma protuberans (DFSP). Its consequence is a fusion of two genes, a collagen type I alpha 1 gene (COL1A1) and platelet-derived growth factor B-chain gene (PDGFB). The COL1A1-PDGFB fusion gene, is expected to be a diagnostic molecular assay. However, previous studies on this subject were mostly based on frozen tissue specimens or cultured tumor cells. In this present study, the investigators conducted a reverse transcription (RT)-polymerase chain reaction (PCR) assay to detect the COL1A1-PDGFB fusion transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from 12 patients with DFSP. To amplify the fusion transcripts, a specific COL1A1 forward and PDGFB reverse primers were designed for single step PCR. The COL1A1-PDGFB fusion transcripts could be detected in 10 of 12 paraffin-embedded DFSP tumor specimens (83%). Subsequent sequence analysis using the PCR products confirmed that the detected messages were derived from gene fusions composed of PDGFB exon 2 and different regions of the COL1A1 gene (exon 8, 10, 22, 24, 32, 38, 45 or 46). Two samples of Bednar tumor included in this series also contained the fusion transcripts. In sample of DFSP with fibrosarcomatous transformation, the COL1A1-PDGFB could not be detected in the fibrosarcoma areas of the third recurrence, though the chimeric transcripts were identified in the ordinary DFSP areas of the first recurrence. No fusion transcripts could be amplified in non-DFSP lesions, including 10 dermatofibromas and 9 malignant fibrous histiocytomas. These results indicate that this molecular assay could be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a diagnostic aid for DFSP.

Adolescent↗
PubMed2000

Novel polymerase chain reaction approach for full-coding p53 mutation detection in microdissected archival tumors.

Evidence suggests that up to 25% of p53 mutations are outside of exons 5-8 and that insertions, deletions, and polymorphic sites in the p53 gene may play a significant role in the process of carcinogenesis. A novel polymerase chain reaction (PCR) approach for the analysis of the entire p53 coding and splice site regions from microdissected, formalin-fixed, paraffin-embedded tumor tissues has been developed which allows multiple genetic analyses to be performed from one primary amplification reaction. The method was initially evaluated using well-characterized cell lines. In addition to confirming the published p53 mutations for HT29, Molt 4, A431, and HN5, a 16 base pair (bp) duplication within intron 3 was detected in both the A431 and HT29 cell lines. Analysis of archival samples of ovarian cancer identified the same 16-bp duplication and coding region variations. In all samples, using GenBank submission U94788 as a reference, a C-insertion was detected at nucleotide positions 11818 and 11874 within intron 2. At nucleotide position 14168, within intron 7, a T-to-G base change was found. This novel PCR approach has the potential to reduce the amount of clinical material required by up to 95%, thus facilitating retrospective studies on archival tumor collections. Furthermore, a wider analysis of the p53 gene, including splice sites and intronic regions, may yield additional information regarding cancer predisposition, response to therapy, and progression.

Base Sequence↗
PubMed2006

A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.

All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.

Base Sequence↗
PubMed2002

Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER2 oncogene in archival human breast carcinoma.

The high incidence of HER2 overexpression on the cell surface of breast cancer cells and the recognized prognostic and potentially predictive value of HER2 render this cell surface receptor a novel and important therapeutic target. Although immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH; PathVysion and INFORM)-both approved by the Food and Drug Administration-have emerged as the most viable assays for evaluation of HER2 status in routine clinical practice, there is still no consensus on which is the best method for assessing HER2 status. Therefore, our specific objective was to establish a chromogenic in situ hybridization (CISH) assay for the detection of HER2 amplification on a cohort of 173 archival invasive breast carcinomas. Results were compared with HercepTest, which is the most frequently used method for detecting HER2 alteration. Additionally, HER2 gene copy number was investigated using differential PCR (dPCR) as a testing system. HER2 overexpression was found by IHC in 24.3%; HER2 amplification was found by CISH in 19.1% and by dPCR in 9.2% of the tumors. The overall concordance rate was 95.9% between CISH and IHC and 85.0% between dPCR and IHC. Kappa statistics revealed an excellent agreement between IHC and CISH (kappa = 0.878), but only a moderate agreement was found between IHC and dPCR (kappa = 0.482). Discrepant cases between CISH and HercepTest and all IHC-positive cases (+2 and +3), a total of 42 cases, were analyzed with the FISH PathVysion (Vysis) assay. Among 25 HercepTest-positive cases (score +3), 2 showed no gene amplification by FISH or CISH. Four of 13 tumors with weak HER2 overexpression (score +2) were negative with both FISH and CISH. Concordance between CISH and FISH was 100% for the 38 cases analyzed. The current study showed that CISH represents a practical and simple assay for evaluating HER2 gene amplification in archival material, offering a promising alternative to IHC or FISH for the routine diagnostic setting.

Breast Neoplasms↗
PubMed2005

INK4-ARF and p53 mutations in metastatic cutaneous squamous cell carcinoma: case report and archival study on the use of Ink4a-ARF and p53 mutation analysis in identification of the corresponding primary tumor.

So far, histopathologic, immunohistochemical and molecular properties of metastatic cutaneous squamous cell carcinomas (CSCCs) are relatively unexplored. In patients with multiple CSCCs, as for instance renal transplant recipients (RTRs), it might prove difficult to identify the primary tumor responsible for metastasis. We report a case of an RTR with multiple CSCCs, one of which metastasized. By using p53 and INK4a-ARF mutation analysis, we identified the responsible primary tumor due to an identical mutation in exon 2 of the INK4a-ARF locus. Archival study yielded 14 cases of metastatic CSCC (present case included). In only 8 of 14 metastases, DNA quality was sufficient to perform PCR reactions. In 7 of 8 metastases, either an INK4a-ARF (6 of 8 cases) and/or p53 (3 of 8 cases) mutation was present. In 6 of 7 cases, the corresponding primary could be identified by an identical mutation in p53 and/or INK4a-ARF. In conclusion, molecular analysis using a combination of p53 and INK4a-ARF mutation analysis can identify the corresponding primary skin tumor in case of CSCC metastases in the majority of cases. This is facilitated by the high frequency of these mutations in metastatic CSCC when compared with frequency spectra reported in the literature in primary CSCCs. The major limitation was formed by insufficient DNA quality in archival tissue.

ADP-Ribosylation Factor 6↗
PubMed1994

An improved method for semiquantification of gene amplification from archival material.

A differential PCR-based assay is presented that increases the accuracy of quantification of C-erbB-2 gene-copy number in DNA extracted from archival tumors. The C-erbB-2 gene is amplified in a high percentage of human adenocarcinomas arising at numerous sites, including breast, lung, and stomach. A number of studies have correlated C-erbB-2 with poor prognosis. Gene copy number may be relevant in identifying patients with different clinical outcomes. In this study a target gene and a single copy reference gene were coamplified in the same reaction tube. The level of target gene amplification was reflected by the ratio of the two resulting PCR products. Cell lines exhibiting variable copies ranging from 1 to > 8 of the C-erbB-2 gene were used as quality controls. This technique can reliably show a single copy difference between cell lines and can be used to semiquantitatively estimate gene copy number in DNA extracted from archival paraffin-embedded samples.

Adenocarcinoma↗
PubMed2004

Factors predicting successful DNA recovery from archival cervical smear samples.

Polymerase chain reaction (PCR)-based DNA testing of archival cervical smear slides is a useful method of retrospectively establishing the presence of the human papillomavirus (HPV) in cervical cells. A cellular DNA recovery test is performed in parallel to HPV DNA testing to ensure that sufficient cells are present and purification of sample DNA has been successfully performed. Previous studies have not comprehensively assessed DNA recovery rates in slides older than 13 years. We undertook a study to determine the factors impacting DNA recovery in 436 UK slides dating from 11 to 33 years prior to testing. Overall, a low cellular DNA recovery success rate of 29% was obtained but a strong trend was observed with increasing recovery rates the older the slides (P < 0.001). Recovery rates increased from 22% in the most recent slides collected from 1988 to 1992, to 61% in the oldest slides, collected in 1970-72. It is likely that fixation compounds incorporating acetic acid, introduced in the UK through the 1980s, have compromised subsequent attempts at PCR amplification. These findings emphasize the importance of the original fixation method in the success of DNA recovery from archival smear samples.

DNA, Viral↗
PubMed1994

Effect of formalin fixation and long-term storage on the detectability of bovine viral-diarrhoea-virus (BVDV) RNA in archival brain tissue using polymerase chain reaction.

Detection of DNA or RNA in formalin-fixed, paraffin-embedded tissues using polymerase chain reaction (PCR) may be hindered by degradation of nucleic acids during tissue collection, preparation and archivation. This study describes investigations on the effect of formalin fixation and prolonged storage of paraffin-embedded tissues on bovine viral-diarrhoea (BVD)-virus RNA as a model system. Brain tissues from eight persistently BVDV-infected calves containing high amounts of the virus were fixed in 5% neutral-buffered formalin or 10% non-buffered formalin for different fixation times, respectively, and paraffin embedded. Subsequent detection of an 803 bp fragment from single tissue sections using nested PCR after reverse transcription (nested RT-PCR) demonstrated a loss of detectability of viral RNA after more than 10 days (10% non-buffered formalin) and 3 months (5% neutral-buffered formalin) of fixation. Additional studies with 280 initially BVDV-positive brain tissues from 25 persistently BVDV-infected calves after storage of up to 10 years revealed a loss of detectable RNA after more than 1 year of storage. For estimation of the higher sensitivity of nested RT-PCR compared to single step RT-PCR, serially diluted BVD virus suspensions were examined using both methods. Nested RT-PCR was found to be about 100-fold more sensitive than single-step RT-PCR, and is therefore recommended as the appropriate technique for archival studies.

Animals↗
PubMed2005

Modified fluorescence in situ hybridization methods using pathology archives.

Brain tumors, like many other common tumors, are frequently associated with chromosomal numerical abnormalities. However, as the identification of abnormal characteristics by conventional cytogenetic or molecular methods has been hampered by technical difficulties, minimal information has been available about specific chromosomal or locus-specific gene alterations. Recently, fluorescence in situ hybridization (FISH) has emerged as a powerful clinical and research tool for the assessment of genomic instability within interphase nuclei. Here, we developed a modified FISH protocol including short-term microwave treatment to analyze specimens from the pathology archives that had been routinely processed and stored. The FISH signals obtained using this modified method showed a significant improvement compared with those obtained using the standard FISH method. This new technique thus enables the analysis of various paraffin-embedded tissue sections of intracranial tumors obtained under inappropriate fixation conditions. We highlight the advantages of this modified FISH procedure on a tissue microarray of archival materials for current diagnostic and investigative neuropathology applications.

Brain Neoplasms↗
PubMed2005

Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archives.

A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype-phenotype analysis of individual cells.

Antigens, Surface↗
PubMed1997

Immunohistochemical detection of p53 in archival formalin-fixed tissues of lip and intraoral squamous cell carcinomas from Norway.

We investigated the expression of p53 in 82 formalin-fixed, paraffin-embedded archival tissue specimens of lip and intraoral squamous cell carcinomas (SCCs) from the period 1930-1995, by immunohistochemistry using three monoclonal antibodies (MAbs DO-7, DO-1 and 1801). Before incubation, sections were pretreated with 0.1% Protease enzyme at 37 degrees C for 10 min followed by 5 + 5 min microwave oven heating at 700 W and 425 W, respectively. Formalin-fixed tissues of 10 carcinomas of the uterine cervix positive for p53 were used as controls. With one or more of the three MAbs, p53 was expressed in 73% of the 82 SCCs examined. With only protease enzyme pretreatment or microwave oven heating, p53 was expressed in 9/82 and 12/82 of the SCCs, respectively. Of the 82 SCCs, 60%, 45% and 23% expressed p53 with DO-7, DO-1 and 1801, respectively. The kappa coefficient indicated poor agreement between these results for the antibodies, and for lip and intraoral SCCs, except for p53 expression in intraoral SCCs demonstrated by DO-1/1801, which showed fair agreement. The present study suggests that combined protease pretreatment and microwave oven heating of tissue sections improved unmasking of p53 antigenic sites in archival material stored for up to 65 years.

Adult↗
PubMed1998

Evaluation of a commercially available reverse transcription-PCR assay for diagnosis of enteroviral infection in archival and prospectively collected cerebrospinal fluid specimens.

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

Central Nervous System Diseases↗