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Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types.

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

Base Sequence↗

Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.

Version 3.0 of the NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD) includes crystal and crystallization data on all forms of biological macromolecules which have produced crystals suitable for X-ray diffraction studies. The data include summary information on each of the macromolecules, crystal data, crystallization conditions and comments about the crystallization procedure if it varies from the traditional methods employed for crystal growth. The database-management software maintains continuity with previous versions providing similar search procedures and displays. Version 3.0 of the BMCD includes protocols and results of crystallization experiments undertaken in space. These new data are comprised of both the NASA Protein Crystal Growth Archive, which includes information on all NASA-sponsored protein crystal growth experiments, and data describing other internationally sponsored microgravity macromolecule crystallization studies. The entries for the space growth crystallization experiments contain the crystallization protocols, apparatus descriptions, flight summary data, indication of success or failure of the experiments, references, etc. Other new features of the BMCD include the addition of crystallization procedures for small peptides and cross references to other structural biology databases.

Journal Article↗

Nuclear accumulation of p53 protein correlates with mutations in the p53 gene on archival paraffin-embedded tissues of human breast cancer.

Fifty invasive ductal carcinomas of the breast were analyzed by immunohistochemistry, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing after microdissection of conventional formalin-fixed, paraffin-embedded tissues. A highly significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (P < 0.0001). Of 13 tumors that demonstrated p53 gene mutations, 11 (84.6%) showed nuclear accumulation of p53 protein. However, of 37 tumors in which gene mutations were not detected, only 5 (13.5%) showed nuclear accumulation of p53 protein. There was a statistically significant association between the nuclear accumulation of p53 protein and a higher histological grade (P < 0.001) or mitotic index (P < 0.01). In addition, gene mutations had a statistically significant association with a higher histological grade (P < 0.05) or mitotic index (P < 0.0001). Therefore, p53 abnormalities might be associated with an aggressive phenotype in breast cancer. We conclude that the immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in archival paraffin-embedded tissues, and that this method is useful for rapid screening of p53 abnormalities.

Base Sequence↗

Assessing HER-2 status in fresh frozen and archival cytological samples obtained by fine needle aspiration cytology.

Determination of HER-2 status is an essential prerequisite in considering a patient's eligibility for anti-HER-2 therapy; few studies have focused on its evaluation on cytological material. We present a new method of assessing HER-2 status on cytological samples, by fluorescence in situ hybridization (FISH) with an automated detection system, using fresh frozen (FF) and May-Grunwald-Giemsa (MGG) stained smears, and we evaluate the reliability of HER-2 determination on fine needle aspiration cytology (FNAC). The pre-treatment protocol for FF smears is easier and faster than for MGG stained slides. However, with the described procedure, FISH is also feasible on archival MGG stained slides. We conclude that with this method, cytological samples obtained by FNAC, either FF or MGG, are a reliable option for assessing HER-2 status.

Biopsy, Fine-Needle↗

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

B-Lymphocytes↗

Detection of HTLV-I and -II in Scottish blood donor samples and archive donations.

BACKGROUND AND OBJECTIVES: Positive samples identified during routine serological screening for HCV (hepatitis C virus), HBV (hepatitis B virus) and HIV (human immunodeficiency virus) are confirmed by nucleic acid testing in the SNBTS (Scottish National Blood Transfusion Service) PCR Reference laboratory. Serological screening for HTLV-I (human T-cell lymphotropic virus type I) and -II was implemented in Scotland in November 2002, at which time a PCR assay was not available for confirmation. Our aim was to develop a real-time PCR assay that could be used for the confirmation of samples showing HTLV-I serological positive or indeterminate reactivity and to investigate whether a serologically silent carrier status exists ('Tax' only) in the Scottish donor population. MATERIALS AND METHODS: A real-time HTLV PCR was devised using a lymphoblastoid cell line which has HTLV-I sequence integrated in the genome (C8166 cells). These were spiked into peripheral blood mononuclear cells. The assay was evaluated on archived serologically confirmed HTLV-positive samples and new positives identified since implementation of screening. RESULTS: HTLV-I and -II were detected in cells and plasma from stored donations and a serological positive donation identified in routine screening. HTLV DNA can also be amplified from the plasma obtained from plasma preparation tubes. There was no evidence of a carrier status ('Tax' only) in 100 serologically negative blood donors tested. The PCR assay developed is reliable and sensitive, capable of identifying one copy of HTLV-I. CONCLUSIONS: The HTLV PCR is a useful addition for HTLV confirmation, especially in serologically indeterminate samples and for look-back studies. HTLV PCR confirmation will provide additional useful information for donor medical staff for counselling donors.

Blood Donors↗

The Protein Data Bank. A computer-based archival file for macromolecular structures.

The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.

Computers↗

Introduction of picture archiving and communication system at The Townsville Hospital.

The performance of a medical imaging department (MID) can be judged by the timing and availability of an image and its report to the treating physician, as this can impact on the patient's treatment, as well as the length and cost of a patient's hospitalization. The use of digital imaging has dramatically improved report turnaround time. In October 2001, The Townsville Hospital (TTH) was opened as a 460 bed greenfield site and as part of the installation a picture archiving and communication system, including web distribution of images and reports to wards and clinics, was included. This retrospective analysis of the MID at TTH is the first data on departmental productivity and individual workload and how these have changed since the hospital's opening 2 years ago, with some ideas for further improvement.

Efficiency, Organizational↗

A modified yeast assay used on archival samples of localized prostate cancer tissue improves the detection of p53 abnormalities and increases their predictive value.

OBJECTIVE: To determine the frequency and predictive value of p53 mutations in localized prostate cancer, comparing the accuracy of detection using immunohistochemistry (IHC) with a modified yeast assay, on archival tissue samples. MATERIALS AND METHODS: Prostate cancer tissue was obtained from 98 patients who had >/= 2 years of clinical follow-up after radical prostatectomy. DNA sequencing was used to verify the presence of p53 mutations in samples that were immunopositive or that gave evidence for p53 alterations using the yeast assay. The IHC and yeast findings were compared with patient outcome to determine the predictive value of these two test types. RESULTS: Fifty-five tumours (57%) were immunopositive, and 58 (59%) were positive using the yeast assay. Sequence-confirmed p53 mutations occurred in 44 (45%) cases. The IHC protocol generated 49% (27/55) false-positive and 36% (15/42) false-negative results, and was 65% sensitive and 50% specific, with an overall accuracy of 57%. The yeast assay resulted in 24% (14/58) false-positive results with a specificity of 74% and an accuracy of 86%. When the p53 status of these patients was correlated with their clinical outcome, patients who had sequence-confirmed p53 mutations had a 2.6-fold greater failure rate (P = 0.026) and a 2.5-fold greater risk of dying from prostate cancer (P = 0.05). Notably, mutations in exon 6 predicted a six-fold increase in treatment failure (P = 0.043) and a 5.3-fold increase in the chance of dying from prostate cancer (P = 0.009). Abnormal yeast-assay findings gave similar predictive results to those obtained for DNA sequencing, while immunopositivity did not correspond to patient outcome. CONCLUSIONS: Mutations of p53 occurred in 45% of localized prostate cancers. These alterations have important prognostic implications. The yeast assay was more accurate for detecting p53 mutations than the IHC protocol used and, unlike IHC, the results of the yeast assay were predictive of patient outcome.

Adult↗

Encephalitozoon hellem in two eclectus parrots (Eclectus roratus): identification from archival tissues.

Members of the phylum Microspora are obligate, intracellular, single-celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach-faced, masked, and Fischer's lovebirds (Agapornis roseicollis, A. personata, and A. fischeri, respectively), budgerigars (Melopsittacus undulatus), and a double yellow-headed Amazon parrot (Amazona ochrocephala). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot (Eclectus roratus). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.

Animals↗

Acute promyelocytic leukaemia: epidemiology and risk factors. A report of the GIMEMA Italian archive of adult acute leukaemia. GIMEMA Cooperative Group.

Acute promyelocytic leukaemia (APL) exhibits peculiar epidemiological, clinical, cytogenetic and molecular features, compared to the other acute myeloid leukaemias (AML). Data on epidemiology and occupational risk factors for APL desumed from the GIMEMA archive are reported and compared with those of the other AML. An exploratory case-case study was designed on AML patients from 56 haematology centres in Italy. Overall, 4296 patients older than 15 yr with a new diagnosis of acute leukaemia were recorded between July 1992 and July 1997. Of these, 335 were classified as APL, and 2894 as other AML. The median age of APL patients was 43 compared to 59 yr for the other AML (p < 0.00001). In order to identify peculiar risk factors for APL development, different parameters were compared in the 2 groups. After adjusting by age no significant differences were observed with regard to education, lifetime prevalence of cancer among siblings and previous diseases in the patient's history. Occupational exposure as a possible risk factor for APL showed no increased risk compared to other AML among farmers, builders and leather workers. A significant association was found in electricians (OR=4.4, 95% CI=2.0-9.7) and a weak association was found in wood workers (OR=3.2, 95% CI=0.8-10.8). The proportion of APL with respect to other AML was significantly higher in the north east of Italy compared to the rest of the country (OR=1.7, 95% CI=1.3-2.2). These data confirm the younger age of APL patients compared to the other AML. A possible role of electromagnetic fields is suggested by the higher risk of APL in electrical workers and in the more industrialized areas of the country.

Adolescent↗

MTS1 gene mutations in archival oral squamous cell carcinomas.

Multiple tumor suppressor gene 1 (MTS1) has been found mutated or deleted in a variety of human cancers. Our purpose was to identify and characterize MTS1 gene mutations in primary oral squamous cell carcinomas (SCCs) in each of the three exons of the MTS1 gene. Seventeen archival samples of oral SCC were evaluated for the presence of MTS1 mutations using single strand conformation polymorphism (SSCP) and DNA sequencing. Three of 17 tumors exhibited MTS1 gene mutations: one tumor exhibited a mutation in exon 2 and two tumors exhibited mutations at the splice site junction of intron 2 and exon 3. Three tumors also exhibited a common base change in the 3' untranslated region of exon 3, which is interpreted as a likely polymorphic variant. An examination of the three tumors exhibiting MTS1 point mutations revealed no unique characteristics relative to p53 immunohistochemical activity, mitotic frequency, or degree of histologic differentiation. This study indicates that MTS1 gene mutations may be involved in at least a minor proportion of oral SCCs.

Adolescent↗

Molecular detection of EWS-FLI1 chimeric transcripts in Ewing family tumors by nested reverse transcription-polymerase chain reaction: application to archival paraffin-embedded tumor tissues.

Chromosomal translocations generating unique chimeric genes are highly characteristic of specific sarcomas, and their use as diagnostic markers has been suggested. From a diagnostic pathologic point of view, detection of such cytogenetic or molecular aberrations applicable to routinely processed archival tissue specimens is considered a powerful tool for tumor diagnosis. To assess the feasibility and reliability of the molecular detection of the transcript originating from the chimeric gene in paraffin-embedded tumor specimens, we performed a nested reverse transcription-polymerase chain reaction (RT-PCR)-based assay to detect the EWS-FLI1 chimeric message in a series of Ewing family tumors. Of 24 paraffin-embedded tumor specimens from 23 cases analyzed, the chimeric message was detectable in 20 (83%) specimens from 20 cases (87%) by this nested RT-PCR assay, whereas none of 7 small round cell tumors not from this family (3 alveolar rhabdomyosarcomas, 2 neuroblastomas, 2 malignant lymphomas) showed detectable chimeric messages. In the sequence analysis of the PCR products, the amplified chimeric messages contained the junctions between exon 7 of the EWS gene and any one of exons 5, 6 and 8 of the FLI1 gene. The detection process was usually completed within 3 days, except for the subseqent sequence analysis. Our results endorse the use of this molecular assay as an ancillary technique in the diagnosis of Ewing family tumors using paraffin-embedded material.

Adolescent↗

An automated patient recognition method based on an image-matching technique using previous chest radiographs in the picture archiving and communication system environment.

An automated patient recognition method for correcting "wrong" chest radiographs being stored in a picture archiving and communication system (PACS) environment has been developed. The method is based on an image-matching technique that uses previous chest radiographs. For identification of a "wrong" patient, the correlation value was determined for a previous image of a patient and a new, current image of the presumed corresponding patient. The current image was shifted horizontally and vertically and rotated, so that we could determine the best match between the two images. The results indicated that the correlation values between the current and previous images for the same, "correct" patients were generally greater than those for different, "wrong" patients. Although the two histograms for the same patient and for different patients overlapped at correlation values greater than 0.80, most parts of the histograms were separated. The correlation value was compared with a threshold value that was determined based on an analysis of the histograms of correlation values obtained for the same patient and for different patients. If the current image is considered potentially to belong to a "wrong" patient, then a warning sign with the probability for a "wrong" patient is provided to alert radiology personnel. Our results indicate that at least half of the "wrong" images in our database can be identified correctly with the method described in this study. The overall performance in terms of a receiver operating characteristic curve showed a high performance of the system. The results also indicate that some readings of "wrong" images for a given patient in the PACS environment can be prevented by use of the method we developed. Therefore an automated warning system for patient recognition would be useful in correcting "wrong" images being stored in the PACS environment.

Biophysical Phenomena↗

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium.

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.

Amino Acid Substitution↗

DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures.

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.

Biological Specimen Banks↗