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      Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples


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          The purpose of this study was to explore the turnaround times, section and image quality of a number of more “difficult” specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples.

          A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO 4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 °C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization.

          The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens.

          Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.

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          Most cited references49

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          Formaldehyde fixation.

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            Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

            We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.
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              Effect of formalin tissue fixation and processing on immunohistochemistry.

              Although immunohistochemistry is routinely performed by many pathology laboratories, its standardization still lags behind. A major cause of variation in the reproducibility of immunohistochemical staining is induced by tissue fixation and, to a lesser degree, tissue processing. This report, stemming from the first meeting of the International Consensus Group on Standardization and Quality Control (ICGSQC) in Nice, France, summarizes the problem and suggests solutions to begin to achieve standardization of fixation and processing. Most laboratories use neutral-buffered formalin (10%) for tissue fixation which introduces cross-links, whereas coagulative fixatives are less popular. Problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly. Solutions to these problems could be to start fixation soon ( 24-48 hrs). For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all solutions freshly every week, depending on the volume of tissue processed. If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains.

                Author and article information

                Micron (Oxford, England : 1993)
                Elsevier Ltd.
                6 January 2006
                August 2006
                6 January 2006
                : 37
                : 6
                : 577-590
                [a ]Central EM Laboratory, Pathology Department, University Hospital Regensburg, Franz-Josef-Strauss Allee 11, D-93053 Regensburg, Germany
                [b ]Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany
                [c ]Central Institute of the Federal Armed Forces Medical Service, Andernacherstraße 100, D-56070 Koblenz, Germany
                [d ]EM Laboratory, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Straße 74, D-20359 Hamburg, Germany
                [e ]Milestone srl Microwave Lab. Systems, Via Fatebenefratelli 1/5, 24010 Sorisole (BG), Italy
                Author notes
                [* ]Corresponding author. Tel.: +49 941 944 6636; fax: +49 941 944 6602. josef.schroeder@ 123456klinik.uni-regensburg.de
                Copyright © 2005 Elsevier Ltd. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 21 October 2005
                : 25 November 2005
                : 26 November 2005

                Microscopy & Imaging
                electron microscopy,same-day diagnosis,microwave-assisted processing,rapid processing,tissue embedding,clinical urgent samples,emerging infectious diseases,bioterrorism


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