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• Immunocytochemistry and In-Situ Hybridisation on bone marrow trephine biopsy
  Lynne Doverty
  Published on: 18 April 2016

• Published on: 18 April 2016

  Immunocytochemistry and In-Situ Hybridisation on bone marrow trephine biopsy

  • Lynne Doverty, Chief Biomedical Scientist

  Dear Editor

  The world literature clearly recognises the role of the bone marrow trephine (BMT) biopsy in the investigation of haematological disorders and its flexibility in providing both diagnostic and prognostic information, an example of which appeared in the August edition of this journal This article gives an excellent account of the methodology employed and the accompanying illustrations clearly demonstrate th...

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  Dear Editor

  The world literature clearly recognises the role of the bone marrow trephine (BMT) biopsy in the investigation of haematological disorders and its flexibility in providing both diagnostic and prognostic information, an example of which appeared in the August edition of this journal This article gives an excellent account of the methodology employed and the accompanying illustrations clearly demonstrate the results of the wide array of investigations undertaken.

  The authors talk about the use of resin embedding media noting that ‘resin embedding and semi-thin sections provide the best morphology; there have been questions on preservation of antigens and nucleic acids. However, RNA preservation and suitability for ISH analyses of mRNA is not clear’. They also discuss the need for specialised technology and skill and the additional associated costs. For these reasons the authors felt that the wax embedding would be their method of choice, the principles of their approach being that the results gave excellent morphology without compromising preservation of antigens and nucleic acids. They also required the method to be simple and integrated with standard processing schedules and for it to be inexpensive and non-toxic.

  We have been able to meet all of these criteria with the added benefit of superior morphology by using Methyl Methacrylate (MMA) as the embedding medium. The reagent is cheap (<£8.00 for 500ml), easy to dispose of safely as an inert solid, and easily sectioned using disposable glass knives. There are several advantages to this practice in that the need for decalcification is greatly reduced and greater support of the tissue facilitates the routine production of 1-2ìm sections, thereby improving the quality of cell morphology on histological sections, within 24 hours of receipt.

  The choice of resin is critical. Methyl methacrylate provides a stable and reliable embedding medium which has the added benefit of being readily removed from sections and thus does not inhibit the use of either conventional or immunohistochemical staining methods. We reciprocate in regular use, the range of antibodies demonstrated by Naresh et al. We also can demonstrate kappa and lambda light chains using ISH in trephines embedded in MMA.

  

  As they authors rightly conclude, the ideal method for handling BMT specimens should be worked out at each institution. In Aberdeen we have found that initial fixation in 10% NBF followed by six hours in EDTA-NBF provides excellent morphology. Specimens are dehydrated overnight on an automated tissue processor, which is also has the facility to process specimens for EM, and embedded the following morning in fresh MMA. Polymerisation takes three hours thus specimens received late afternoon are embedded, cut and sectioned by the following lunch time. Urgent requests for ICC can be dealt with the same day and the results available within forty eight hours.

  We also find that technical staff easily adapt to sectioning resin embedded material and the staining methods, dye based and immunohistological, are identical to those employed for wax embedded tissue. Antigen retrieval methods do not need to be changed and the ISH techniques required only minimal adaptation to the manufacturer’s instructions.

  The results of both the Hammersmith protocol and that used in Aberdeen are comparable; the biggest advantage of our method must be the reduction in turnaround time of BMT specimens.

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  Conflict of Interest:
  None declared.

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