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15.1 Glutaraldehyde is the ‘gold standard’ fixative for electron microscopy.
a. Why is this?
• Two aldehyde groups produce superior fixation with good ultrastructural morphology. No other fixative produces this level of ultrastructural detail.
b. If it is such a good fixative, why is it not used for light microscopy?
• Glutaraldehyde will only penetrate 1–2 mm into
tissue. This fixative would only preserve the outer layers of the
majority of light microscopy samples, leaving the centre
unfixed.
• All antigenic sites would be bound
irreversibly in glutaraldehyde-fixed tissue, making any subsequent
immunocytochemistry impossible. Tinctorial methods may also be
affected.
• Glutaraldehyde is a hazardous chemical and would present an increased health and safety risk to anyone handling the fixative or tissue fixed in this chemical.
c. Can other fixatives be used in electron microscopy?
• Other aldehyde-based fixatives or fixatives designed and
evaluated for electron microscopy can be used but ultrastructural
morphology may be compromised.
• While morphological preservation may not be ideal, formalin-fixed tissue will yield ultrastructural information that may inform the diagnostic process.
• Alternative fixatives may be necessary for specialised applications such as immunoelecton microscopy.
15.2 Why do samples for electron microscopy have to be so small and what difficulties can this introduce to interpretation?
• Design of the electron microscope produces a column that
is only 5 mm wide.
• Resolution of the electron microscope is so great that a very small sample can be magnified to a very large image. (typical EM magnifications used on diagnostic materials would be ×25,000, while maximum magnification is considerably higher).
• Small sample size introduces difficulties of ensuring the sample selected for examination is
• the correct area (ie a glomerulus in the kidney)
• adequately preserved (ie not an area of necrotic tissue)
• not an area of ‘normal’ tissue surrounding a tumour
• representative of the pathology – this can be difficult if the pathology is focal and not diffuse in nature.
15.3 Electron microscopy is a vital part of the evaluation of renal disorders. Why is this the case?
• Electron microscopy offers specific information that can only come from the ultrastructural examination of the renal biopsy; for example:
• the precise location of abnormalities such as immune complexes
•identification of small amounts of proteins such as amyloid when levels are too low to allow identification by tinctorial methods
• it is the only technique that can demonstrate podocyte effacement within a glomerulus
• can provide measurement of basement membrane thickness
• Examination of the ultrastructure will demonstrate low levels of pathology that may be equivocal by light microscopy.
• It will also confirm and support information gathered by other techniques
15.4 What could electron microscopy add to the work-up of a sample of lung tissue from a patient whose chest X-ray shows a mass?
• EM would be used alongside other techniques (eg tinctorial methods) and immunocytochemistry to produce a diagnosis
• Careful sample selection would be needed to ensure the tissue examined provides the maximum amount of diagnostic information.
• EM could help to identify microorganisms in the sample if an infective process is considered possible.
• If a malignancy is suspected, EM could demonstrate ultrastructural features that would assist in identifying tumour type and origin
• Consider malignancies that are common to the lung and what ultrastructural features might help to differentiate these tumours.