Electron microscopy is sensitive to tissue handling procedures. Whilst we recognise and appreciate that non-MFT service users may prefer to determine their own laboratory procedures relating to the fixation and handling of the tissue samples that they send to us for examination, service users are reminded that fixation and handling procedures affording satisfactory light microscopy fixation, may not be suitable or satisfactory for ultrastructural studies.
Therefore, we strongly recommend that all service users meet the following specifications for all tissue samples to be processed for EM. Failure to do this is likely to result in sub-optimal tissue preservation and may place limitations on the ultrastructural interpretation of the sample. Deviations from these recommendations by service users should be discussed and agreed with the Electron Microscopy Lead Biomedical Scientist before specimens are sent.
a. Samples are placed into fixative immediately upon removal from the patient. 2.5% buffered glutaraldehyde is preferred but 10% Neutral Buffered Formalin is acceptable. The transport of samples from theatre or clinic for the application of fixative within the laboratory is not advised.
b. If samples are fixed initially in Formalin, they are transferred to 2.5% buffered glutaraldehyde at the earliest opportunity and fixed at 4 – 20°C for between 24 – 72 hours, after which time the sample must be transferred to 0.1M phosphate buffer (pH 7.4) for transport.
c. Samples fixed in buffered glutaraldehyde are not larger than approx. 1mm3 in size. Strips or cores of tissue up to 1mm thick/diameter are acceptable. However, lengths tissue over 3-4mm are generally to be considered excessive for EM purposes.
d. Tissue must be kept hydrated in an isotonic medium before and during transport to the EM laboratory.
Please note that we do not currently provide external service users with suppliers of fixative and/or buffer
Main factors causing ultrastructural preservation artefact
Below is a summary of the common factors that negatively affect the performance of the examination and/or interpretation of EM results:
- Delayed fixation: Detrimental ultrastructural changes begin immediately upon removal of blood supply. The longer the delay: the more significant the ultrastructural artefact. Note: Although the use of saline may advantageous from the point of view of preventing the tissue drying out, it does not provide fixation and can cause cell swelling. Therefore, it has an overall detrimental effect for electron microscopy.
- Inadequate fixation: This can affect a sample even if immediately placed into fixative and be caused by multiple factors including:
- sample size too large
- fixation time too short
- fixative to weak
- wrong fixative
- Defective buffer: A suboptimal pH of the buffer can lead to significant artefactual changes to tissue ultrastructure. The effect is time dependent.
- Dewaxed samples: The ultrastructure of tissue samples that are embedded in paraffin wax and subsequently dewaxed and reprocessed into resin for electron microscopy is significantly affected by the stresses afforded to it by the process involved. The ultrastructural appearances of dewaxed samples are unreliable and potentially misleading in certain diagnostic scenarios
Storage of fixed samples prior to transport to MFT
Fixed tissue samples must be stored in 0.1M phosphate buffer (pH 7.4) between 4 – 20°C (room temperature). It is recommended that they are sent to us as soon as is feasible to the sender. Prolonged storage may affect ultrastructural detail.
For certain clinical questions, a limited amount of useful information can sometimes be gained by de-paraffinising the tissue and reprocessing for EM. Where EM examination on tissue sent in fixative has not proved possible, the requesting pathologist may choose to send formalin fixed paraffin embedded (FFPE) tissue remaining from light microscopy procedures. However, service users should be fully aware of the limitations of examining de-paraffinised tissue ultrastructurally (with respect to the clinical question) before sending. This can be advised on a case by case basis if necessary.
Again, the quality and reliability of the ultrastructural review is dependent on the quality of initial Formalin fixation and tissue processing. FFPE tissue sent for EM should not be that which may have been frozen and used for immunofluorescence procedures prior to being fixed. Similarly, the sending of FFPE tissue which may have originally travelled from the ward/clinic to the laboratory in saline or similar solution is strongly discouraged.
(Last reviewed November 2020)