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PubMed1997

Ribonucleases may limit recovery of ribonucleic acids from archival human temporal bones.

Messenger ribonucleic acid (mRNA) for actin was detected in celloidin-embedded archival human temporal bone sections with reverse transcription polymerase chain reaction (RT-PCR). Actin mRNA was detected in 10% of sections analyzed. One possible reason for this modest detection incidence is enzymatic degradation of RNA by exogenously introduced ribonucleases (RNases). We have identified steps of the temporal bone processing protocol for archival storage in which exogenous RNases could be introduced to the tissue, and have verified that the bone sections are exposed to these enzymes. We have demonstrated that implementing precautions to minimize exogenous RNase contamination during processing improves recovery of intact RNA. This study indicates that although gene expression analysis of archival human temporal bones may be limited by enzymatic degradation of RNA, simple modification of processing protocol can improve yield of informative data.

Actins↗

PubMed2002

Application of unbiased stereology on archival human temporal bone.

OBJECTIVE: The current report presents unbiased stereological sampling and counting strategies for estimating the total number of neurons in the spiral and vestibular ganglia of the archival human temporal bone. STUDY DESIGN: Application of unbiased stereology using archival human temporal specimens. Observational study. METHODS: The practical sampling and counting strategies for estimating the number of neurons are presented. To illustrate the method described, the total number of neurons in spiral and vestibular ganglia from archival human temporal bone was estimated. RESULTS: The study found, on average, 41,643 (coefficient of variation = 0.12) spiral ganglion neurons in the subjects with an average age of 35.6 years (age range, 22-60 y) and, on average, 27,635 (coefficient of variation = 0.10) vestibular ganglion neurons in the subjects with an average age of 28.6 years (age range, 2-58 y). CONCLUSIONS: The total number of primary auditory and vestibular ganglion neurons from the current study differs significantly from the previously published data using assumption-based methods. The unbiased stereological technique presented in the current report thus would be invaluable for future studies on such specimens, including the study of the changes of neurons during aging and various pathological conditions.

Cell Count↗

PubMed1997

Detection of gene fusions in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction assay of archival samples.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue tumor that is often difficult to distinguish from other small round-cell tumors. The PAX3-FKHR and PAX7-FKHR gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival material, we used reverse transcriptase-polymerase chain reaction and oligonucleotide hybridization methodology to develop an assay capable of identifying PAX3-FKHR and PAX7-FKHR fusion transcripts in formalin-fixed, paraffin-embedded tissue. Use of a control assay for wild-type FKHR mRNA indicated that RNA was successfully isolated, reverse-transcribed, and amplified in 15 of 16 archival cases. Comparison of assay results for the PAX3-FKHR and PAX7-FKHR fusions with standard molecular assays of paired frozen material revealed that all eight cases of known fusion-positive rhabdomyosarcoma were correctly identified and distinguished as PAX3-FKHR or PAX7-FKHR. The seven cases of known fusion-negative rhabdomyosarcoma showed no evidence of either product. These results indicate that we have developed a molecular assay that accurately identifies the fusion transcripts characteristic of alveolar rhabdomyosarcoma in archival samples. This assay will be useful for diagnosis and for retrospective clinicopathologic correlative studies.

Base Sequence↗

PubMed2006

Detection of small pulmonary nodules using direct digital radiography and picture archiving and communication systems.

PURPOSE: To evaluate the detection of small pulmonary nodules, in the diameter range of 5.4 to 15 mm, using direct digital chest imaging and soft copy interpretation on picture archiving and communication systems. MATERIALS AND METHODS: The results of clinical computed tomography (CT) scans of the thorax were retrospectively reviewed from our radiology information system and picture archiving and communication systems archives. Patients with CT studies containing between 1 and 6 nodules, who also had a digital chest examination within 1 month of the CT scan were selected. Thirty patients with suitable nodules and 30 without nodules were included and form the data base for this study. The nodules were between 5.4 and 15 mm in average diameter. Four separate observers independently viewed the frontal and lateral chest studies of the 60 patients. The presence or absence of nodules was determined. Data were analyzed with Kappa, McNemar and Fischer exact tests for agreement and differences between observers, nodule size, and nodule zone. RESULTS: A total of 42 nodules between 5.4 and 15 mm were present. The overall detection rate for the 4 observers was 41.7%. For nodules between 5.4 and 8 mm the detection rate was 26.2%. Agreement between observer's detection was poor to moderate. Differences between observers for both nodule size and zone were not significant. Only 1 observer had a relationship between nodule detection and nodule size. CONCLUSIONS: Observer detection of pulmonary nodules in the range of 5 to 15 mm using current digital radiography systems is not reliable in the confusing environment of the lung. Additional modification of these systems is required to increase nodule conspicuity.

Adult↗

PubMed1991

Immunoelectron microscopic localization of elastic tissue components in archival tissue samples.

Tissue samples that have been stored for many years, in different media and under a variety of conditions, have been examined by modern techniques of immunoelectron microscopy, using antibodies against elastic tissue components. A range of postembedding restorative procedures has been identified, which will allow reliable immunolocalization of antibodies against the elastic tissue component of such specimens. These methods have been applied successfully to autopsy-derived material, fixed in buffered formaldehyde, to archival material stored frozen at -70 or -20 degrees C, to specimens fixed for electron microscopy and stored for many years in buffer, and even to archival material from formaldehyde-fixed, paraffin-embedded blocks, reprocessed for electron microscopic examination. The successful restorative methods included pre-treatment of the sections with 6 M guanidine hydrochloride, or 1 M Tris/saline, each containing 100 mM dithiothreitol (a reducing agent) followed by alkylation with 220 mM iodoacetamide. The application of these techniques allowed reliable study of elastic tissue antibody distributions in archival tissues that could not be obtained again, as well as comparative studies with tissues processed many years previously.

Animals↗

PubMed2004

The microextraction of RNA from archival cardiac allografts embedded in paraffin.

One of the difficulties encountered in studying rejection in patients is the availability of tissue. The goal of our study was to isolate RNA from archival allograft tissue, and to demonstrate that it is of suitable quality for further molecular experimentation. Thirty-two paraffin embedded cardiac and five renal allograft archival samples were obtained after IRB approval, from a total of 18 transplant patients (13 cardiac/five renal transplant patients) from a search of the University of Tennessee's teaching hospitals. RNA was extracted from the paraffin blocks and amplified by reverse transcription-polymerase chain reaction (RT-PCR). Grade 3A and higher and non-rejection samples were tested. In addition, normal mouse liver tissue was isolated for comparison. Negative control samples were also included. RT-PCR amplification of 18s RNA, 324 bp target sequences, revealed readily detectable bands. Only one block that was 3 years old did not yield detectable RNA secondary to presumed degradation. The negative control showed no bands at 324 bp. We conclude that RNA from archived allograft tissue can be used for further experiments. The use of this tissue offers some distinct advantages when studying correlation of gene expression with clinical outcome and therapeutic response.

Adolescent↗

PubMedDate not supplied

Use of NADP archive samples to determine the isotope composition of precipitation: characterizing the meteoric input function for use in ground water studies.

Stable oxygen and hydrogen isotopes have been used in ground water studies to investigate recharge, mixing, ground water/surface water interaction, advective-diffusive transport, paleohydrogeologic interactions and to estimate ground water ages. Such studies require that the isotopic composition of precipitation be known, as precipitation is a major input to ground water and surface water systems. As oxygen-18 and deuterium data for precipitation are lacking across much of the United States, there is need to establish additional local meteoric water lines as isotope input functions across the region, as well as to develop better understanding of the isotopic climate linkages that control oxygen and hydrogen isotope ratios in precipitation. In the absence of long-term monitoring stations, one possible solution to this problem is to determine the delta 18O and delta 2H values of precipitation using archive samples collected at monitoring stations managed by the National Atmospheric Deposition Program (NADP). This study describes and interprets the seasonal delta 18O and delta 2H composition of archived precipitation samples collected in eastern Nebraska near the town of Mead during the years 1992-1994. Values for delta 18O range from -23.6 to -0.7@1000. Values for delta 2H range from -172 to 0@1000. Yearly arithmetic mean delta 18O and delta 2H values for the Mead station are -8.1@1000 and -53@1000, respectively. Weighted yearly means for delta 18O and delta 2H were -7.4@1000 and -48@1000, respectively. Mead values show a strong isotopic enrichment between winter and summer precipitation, and a strong delta 18O-T correlation (r2 = 0.91) for mean monthly values of about 0.5@1000 per degree Celsius. The local meteoric water line for the Mead site is delta 2H = 7.40 delta 18O + 7.32. Deuterium excess values suggest that most of the moisture across the region is derived primarily from a Gulf of Mexico source. The results of this study demonstrate that in the absence of long-term monitoring stations such as those operated globally by the International Atomic Energy Association, NADP archive samples can be used to determine the isotopic composition of precipitation, to characterize the local meteoric water line and establish the various climatic relationships, and define the meteoric input function for use in ground water studies.

Deuterium↗

PubMed2004

Diversity of phage types among archived cultures of the Demerec collection of Salmonella enterica serovar Typhimurium strains.

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.

Alleles↗

PubMed2006

Rapid and sensitive detection of single cryptosporidium oocysts from archived glass slides.

In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.

Animals↗

PubMed2004

Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples.

AIMS: To investigate the use of different molecular analyses that can identify distinct strains of human pathogenic mycobacteria in formalin fixed and paraffin wax embedded archival tissue samples to see whether it is possible to differentiate between the members of the Mycobacterium tuberculosis complex (M tuberculosis, M bovis, M africanum, M microti, or M canettii) and/or substrains in a high number of samples. This would be of interest for identifying individual infection traits and superinfection by different mycobacterial strains. METHODS: Forty nine archival tissue samples with clinically and/or histologically suspected tuberculosis infection were subjected to molecular DNA analysis. RESULTS: The molecular analysis revealed the presence of M tuberculosis complex DNA in 20 samples, whereas acid fast bacilli could be detected by Ziehl-Neelsen staining in only eight samples. All IS6110 positive samples were further characterised by spoligotyping and seven cases provided M tuberculosis specific signatures, whereas M bovis specific signatures were obtained in four cases. The analysis of mtp40, oxyR, and pncA partial gene sequences confirmed the presence of M tuberculosis in six cases and M bovis in one case. The amplification and sequencing of four further genetic regions (katG, gyrA, TbD1, RD9) characterised six "modern" M tuberculosis strains belonging to genetic groups 2 or 3. CONCLUSION: This study provides clear evidence that archival paraffin wax embedded material can be used for further studies on the strain identification of M tuberculosis complex strains and can therefore unequivocally be used for the study of the epidemiology and evolution of tuberculosis pathogens.

Adult↗

PubMed2006

Depositing archived paraffin tissue core biopsy specimens in paraffin tissue microarrays using a paraffin tissue punch modified with a countersink.

Paraffin tissue microarrays (PTMAs) introduced by Kononen et al in 1998 have become a widely used technique in routine pathology and even more so in research. Kononen used a tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI, USA) to take paraffin tissue core biopsy specimens (PTCBs) of 0.6-2 mm in diameter from routine paraffin tissue blocks and transfer them to another paraffin block with up to 1000 holes. As pointed out by Mengel et al, however, it is not possible to use the Kononen/Beecher system to construct PTMAs out of archived PTCBs. To overcome this drawback in the extremely popular Beecher system, the paraffin tissue punch was modified by incorporating a conical 4 mm deep countersink. This countersink was milled with a conical precision cutter that can be bought in an ordinary hardware store (cost <5 US dollars). The countersink facilitates the insertion of an archived PTCB into the paraffin tissue punch and enables the construction of PTMAs with previously archived PTCBs using the widely distributed Beecher system. Moreover, this paraffin tissue punch can be used for other systems to create PTMAs, such as the low-budget systems designed by Vogel.

Biopsy↗

PubMed1995

Human papillomavirus in false negative archival cervical smears: implications for screening for cervical cancer.

AIM: To assess the value of detecting human papillomavirus (HPV) DNA in false negative archival cervical smears in population based screening programmes for cervical cancer. METHODS: Cytomorphologically classified false negative archival Pap smears (n = 27) taken from 18 women up to six years before cervical cancer was diagnosed were blindly mixed with 89 smears from hospital patients with a variety of gynaecological complaints and tested for HPV by the polymerase chain reaction (PCR). Corresponding cervical cancer biopsy specimens were also available for HPV analysis. Neither the examining cytopathologist nor the molecular biologist was aware of the study design. RESULTS: HPV DNA was detected in the smears of 16 patients with cervical cancer missed previously by cytology. HPV 16 and 18 were found predominantly in those smears taken up to six years before the diagnosis of cervical cancer. The smears of the two remaining patients were reclassified as inadequate for cytology or contained no suitable DNA for PCR. In 15 patients the same HPV type could be found in the smears and the cervical cancer biopsy specimens. CONCLUSIONS: The results indicate that high risk HPV types can be detected in archival smears classified as false negative on cytology and that cytological screening errors may be reduced if combined with PCR testing for HPV.

DNA, Viral↗

PubMed1992

Software suite for image archiving and retrieval.

The efficient operation of a clinical picture archiving and communication system (PACS) requires a fully functional image archive. The archive should not only be able to store and retrieve images reliably but should also work in concert with software that optimizes the flow of image-related information throughout the PACS. The authors devised a software suite that serves to improve the flow and content of information and the integrity of the data. The software was developed by translating the functions performed by a conventional film library. Types of transactions possible with this system include scheduling, canceling, rescheduling, and prestaging examinations; changing demographic information; merging patient information; and generating reports. The authors believe such a software suite is essential for a clinically useful PACS.

Data Display↗

PubMed1998

Incremental cost of department-wide implementation of a picture archiving and communication system and computed radiography.

PURPOSE: To determine the incremental cash flows associated with department-wide implementation of a picture archiving and communication system (PACS) and computed radiography (CR) at a large academic medical center. MATERIALS AND METHODS: The authors determined all capital and operational costs associated with PACS implementation during an 8-year time horizon. Economic effects were identified, adjusted for time value, and used to calculate net present values (NPVs) for each section of the department of radiology and for the department as a whole. RESULTS: The chest-bone section used the most resources. Changes in cost assumptions for the chest-bone section had a dominant effect on the department-wide NPV. The base-case NPV (i.e., that determined by using the initial assumptions) was negative, indicating that additional net costs are incurred by the radiology department from PACS implementation. PACS and CR provide cost savings only when a 12-year hardware life span is assumed, when CR equipment is removed from the analysis, or when digitized long-term archives are compressed at a rate of 10:1. CONCLUSION: Full PACS-CR implementation would not provide cost savings for a large, subspecialized department. However, institutions that are committed to CR implementation (for whom CR implementation would represent a sunk cost) or institutions that are able to archive images by using image compression will experience cost savings from PACS.

Academic Medical Centers↗

PubMedDate not supplied

Informatics in radiology (infoRAD): A complete continuous-availability PACS archive server.

The operational reliability of the picture archiving and communication system (PACS) server in a filmless hospital environment is always a major concern because server failure could cripple the entire PACS operation. A simple, low-cost, continuous-availability (CA) PACS archive server was designed and developed. The server makes use of a triple modular redundancy (TMR) system with a simple majority voting logic that automatically identifies a faulty module and removes it from service. The remaining two modules continue normal operation with no adverse effects on data flow or system performance. In addition, the server is integrated with two external mass storage devices for short- and long-term storage. Evaluation and testing of the server were conducted with laboratory experiments in which hardware failures were simulated to observe recovery time and the resumption of normal data flow. The server provides maximum uptime (99.999%) for end users while ensuring the transactional integrity of all clinical PACS data. Hardware failure has only minimal impact on performance, with no interruption of clinical data flow or loss of data. As hospital PACS become more widespread, the need for CA PACS solutions will increase. A TMR CA PACS archive server can reliably help achieve CA in this setting.

Computer Communication Networks↗

PubMedDate not supplied

Detection of trisomy 12 by fluorescence in situ hybridization on archival cytopathologic material in chronic lymphocytic leukemia/small lymphocytic lymphoma.

OBJECTIVE: To evaluate fluorescence in situ hybridization (FISH) for the detection of trisomy 12 in archival cytologic specimens of chronic lymphocytic leukemia/small lymphocytic lymphoma. STUDY DESIGN: The cytopathology database was searched for all cases of chronic lymphocytic leukemia/small lymphocytic lymphoma. Six cases of chronic lymphocytic leukemia/small lymphocytic lymphoma obtained by fine needle aspiration and one case of small lymphocytic lymphoma with plasmacytoid features were analyzed for trisomy 12 by FISH. These cases had been archived between 1 week to 16 months prior to analysis. RESULTS: We detected trisomy 12 in four of the six cases of small lymphocytic lymphoma/chronic lymphocytic leukemia. The case of small lymphocytic lymphoma with plasmacytoid features was negative for trisomy 12. CONCLUSION: Detection of trisomy 12 by FISH can be effectively performed on routinely prepared, stained and coverslipped archival cytologic material.

Aged↗

PubMedDate not supplied

Polymerase chain reaction amplification of human papillomavirus DNA from archival, Papanicolaou-stained cervical smears.

OBJECTIVE: To test the applicability of four protocols in recovering DNA suitable for amplification with the polymerase chain reaction (PCR) in archival, Papanicolaou-stained cervical smears. STUDY DESIGN: The most efficient method was used to isolate DNA from 11 archival, Papanicolaou-stained smears with cytopathic changes due to human papillomavirus (HPV) infection to confirm the presence of HPV DNA. RESULTS: beta-Globin was successfully amplified in all smears, while HPV DNA was detected in 6 of 11. Four of the four HPV DNA-negative smears were classified as high grade squamous intraepithelial lesions. Failure to detect HPV DNA might have been due to the low copy number of HPV DNA or deletion of the L1 region. CONCLUSION: High cellularity and the method of recovering DNA from the smear are important determinants of successful amplification of HPV DNA in archival cervical smears.

Beta-Globulins↗

PubMed2001

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.

Acrylic Resins↗