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Detection of Epstein-Barr virus in archival Hodgkin's disease specimens
  1. K J Flavell1,
  2. J A Linford1,
  3. J R Flavell1,
  4. P G Murray1,
  5. L S Young1,
  6. K Scott1
  1. 1School of Health Sciences, University of Wolverhampton, Wolverhampton WV1 1DJ, UK

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    The Epstein-Barr virus (EBV) is associated with several malignancies, including endemic Burkitt's lymphoma, undifferentiated nasopharyngeal carcinoma, post-transplant lymphoproliferative disease, and Hodgkin's disease. The “gold standard” for the detection of EBV infection in clinical tissues is RNA in situ hybridisation that targets the abundantly produced Epstein-Barr virus early RNAs (EBERs).1 This approach is effective in the detection of latent EBV infection in routinely processed, paraffin wax embedded histological material, and has been widely used to analyse the association of EBV with a variety of malignant and non-malignant diseases. In addition, a range of monoclonal antibodies directed against latent EBV proteins has enabled viral gene expression to be investigated in EBV associated diseases. For example, in Hodgkin's disease, the latent membrane protein 1 (LMP1) is consistently expressed in EBV associated cases and can be detected using the CS1-4 monoclonal antibody reagent.2

    Although these approaches are effective on optimally processed histological material, little is known about their ability to detect EBV in tissues stored for many years, or those that may have been processed before the introduction of automated paraffin wax embedding. Therefore, we wished to investigate whether these methods could be used to detect EBV in Hodgkin's disease specimens stored for 50 years or more. In addition, these specimens were processed before the advent of improved tissue embedding procedures.

    Paraffin wax embedded tissue blocks were retrieved from 14 patients diagnosed with Hodgkin's disease between 1943 and 1952. Specimens were reviewed by haematoxylin and eosin staining in all cases. All histologically confirmed Hodgkin's disease specimens were subjected to EBER in situ hybridisation and immunohistochemistry for LMP1, as described previously.34

    LMP1 was detected in five of 14 Hodgkin's disease specimens, where staining was confined to the cytoplasm of Hodgkin-Reed-Sternberg cells (fig 1). Four of these five cases were also positive for EBER in situ hybridisation (fig 2).

    These results indicate that archival specimens stored for periods in excess of 50 years, or those that were subject to less than optimal tissue processing, are still viable for the detection of EBV. These approaches will allow the collection of valuable comparative information about whether EBV prevalence in certain tumour types, such as Hodgkin's disease, has altered over time.5

    Figure 1

    Immunohistochemical staining for latent membrane protein 1 in an archival Hodgkin's disease specimen, with staining confined to the cytoplasm of Hodgkin-Reed-Sternberg cells.

    Figure 2

    In situ hybridisation for Epstein-Barr virus early RNAs. Positive result seen in an archival Hodgkin's disease specimen.