May 1, 1997

Electron Microscopy of Paraffin Embedded Tissue

Case Presentations

Diagnosis

Discussion

Optimal tissue preservation for electron microscopy (EM) is achieved with rapid fixation of one millimeter cube pieces of tissue in a glutaraldehyde based fixative. However, in some instances, it is necessary to reprocess formalin fixed, paraffin embedded tissue for electron microscopy ^{1, 2}. These three cases of Langerhan's Cell Histiocytosis (Eosinophilic Granuloma) illustrate the utility of paraffin embedded material for EM. In Cases 1 and 3, retrospective examination of paraffin embedded tissue was used to confirm the diagnosis of Langerhan's Cell Histiocytosis. In Case 2, paraffin embedded tissue had to be used for diagnosis because the initial tissue sample that had been submitted for electron microscopy consisted only of blood.

Most advocates of diagnostic electron microscopy would state that EM of paraffin embedded tissue should only be done as a last resort. Ultrastructural examination of paraffin embedded tissue is associated with loss of membrane clarity, disruption of membranes and varying degrees of tissue degradation. Since Birbeck granules represent intracytoplasmic membranes that are separated by "zipperlike" junctions ³, it is likely that these granules would be too degraded to be recognized in paraffin embedded tissue that had been reprocessed for EM ¹. Examination of paraffin embedded tissue from these three cases of Langerhan's Cell Histiocytosis suggest otherwise because Birbeck granules were easily recognized in the diagnostic cells in each case. The Birbeck granules in tissue reprocessed from deparaffinized tissue for electron microscopy had "smudgy" and more homogenized membranes and the striated, central zipperlike junctions were more electron dense (Fig 1). Optimally preserved Birbeck granules have crisp membranes and a central region of cross striations ³.

Fig 1. Ultrastructural features of Birbeck Granules in a glutaraldehyde based fixative and paraffin embedded tissue that is deparaffinized for EM.
Trump's Fix - Tissue fixed in Trump's fixative ( a glutaraldehyde based fixative) and processed for electron microscopy. Paraffin - Tissue fixed in 10% neutral buffered formalin then retrieved from paraffin blocks and reprocessed for electron microscopy.

Birbeck granules are not the only organelles that can be recognized in the EM of paraffin material ^{1, 4, 5}. A list of other organelles that are also recognizable in paraffin embedded tissue that is reprocessed for EM are shown in Table 1. Mitochondria, lipid, glycogen, polyribosomes, microtubules and Weibel Palade bodies are exhibit marked degradation in reprocessed paraffin embedded tissue.

Table 1. EM Structures that are recognizable in deparaffinized tissue.
Desmosomes	Intermediate filaments
Infectious agents	Tonofilaments
Sarcomeres of rhabdomyosarcomas	Electron dense granules - endosecretory granules, zymogen granules
Glanudular differentiation in malignant tumors

The ultrastructural features of paraffin embedded tissue that is reprocessed for EM is quite variable. Fixation rather than paraffin embedding seems to be the more important variable that affects the quality of the ultrastructural features. Paraffin embedded tissues that have optimal initial fixation in 10% neutral buffered formalin will have preservation of ultrastructural features that approach those of tissue that is optimaly fixed in a glutaraldehyde based fixative. The ultrastructural study of paraffin embedding tissue can be optimized by avoiding paraffin blocks that are prepared from poorly fixed tissues and selection of tissue adjacent to areas of necrosis.

In addition to the processing artifact that is present in the reprocessed paraffin embedded tissue, laborious techniques for reprocessing paraffin embedded tissue would also tend to dissuade the electron microscopist from using this source of material for EM. The initial methods that were used for preparation of paraffin embedded tissue for EM had processing times that could last for up to one week. The majority of this time was spent in deparaffinization of the tissue sample. These lengthy processing methods have been replaced by simpler methods that can have a processing time as short as thirty minutes ^{4, 5}.

Paraffin embedded tissue that is reprocessed for EM will never have the crisp preservation of rapidly fixed one millimeter cube tissues that are initially fixed in a glutaraldehyde based fixative. However, with careful attention to tissue fixation, the processing of paraffin embedded tissue for EM need not become a choice of last resort and can provide useful, diagnostic information..

References

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