Amyloid deposits can be subtle and difficult to differentiate from hyaline on H&E stained sections (Fig. 2.9a). We routinely perform Congo red stain on both cryostat and FFPE sections of all nerve biopsies to evaluate for amyloidosis. Amyloid tends to accumulate within or around epi- or endoneurial blood vessels or in the subperineurial regions. On Congo red stained section, amyloid deposits appear orange red under regular light (Fig. 2.9b) and yellow-green birefringence under polarized light (Fig. 2.9c). A thioflavin S or T special stain is more sensitive than Congo red but requires fluorescence scope to view the amyloid deposits (Fig. 2.9d). Amyloid is also prominent on crystal violet special stain (Fig. 2.9e) and strongly accumulate terminal complement complex detectable by C5b-9 immunostain (Fig. 2.9f). On plastic section (Fig. 2.9g) and EM, the amyloid deposits are composed of haphazardly arranged fibrils. The diameter of the fibrils vary widely and range from 8–24 nanometers depending on the types of amyloid (Fig. 2.9h, i). The most common form of primary acquired amyloid neuropathy is due to immune light chain (AL) deposition that can be highlighted by Kappa or Lambda immunohistochemistry. These patients usually are over 50 years of age and have lymphoproliferative disorders, plasma cell dyscrasias or monoclonal gammopathies. Secondary amyloidosis due to infection or chronic inflammation (AA) usually does not cause polyneuropathy [35]. Patients with hereditary amyloidosis usually present early in their 30–40s but can be later. Vast majority is caused by transthyretin point mutations. The amyloid deposits are negative for immunoglobin light chains but positive on transthyretin (prealbumin) immunostain (Fig. 2.9j). Other rare forms of hereditary amyloidosis involve mutations in gesolin, apolipoprotein A1, fibrinogen A alpha chain, or lysozyme. Amyloid classification can be determined by liquid chromatography-mass spectrometry [36] followed by genetic testing.

Toluidine blue stained 1.5 μm plastic section from resin embedded nerve blocks provides an accurate assessment of the number of myelinated axons and provides higher contrast and finer detail in myelin and axon morphology than frozen or FFPE specimens. Thick sections, in combination with EM when necessary, is the most important tool in determining whether the dominant pathology of a nerve is axonal degeneration, demyelination or mixed. Features of axonal degeneration and regeneration are myelin ovoids (Fig. 2.10) and regenerating clusters (Fig. 2.11), respectively, which are readily identifiable on toluidine blue stained thick sections. They will be discussed in more detail in the EM section. Features of demyelination include naked axons, thinly myelinated axons, and segmental demyelination. Naked axons may be difficult to appreciate on light microscopy and often requires electron microscopy. Thinly myelinated axons (Fig. 2.12a, b) can be seen in regenerating clusters or regenerating axons, thus not a reliable feature of demyelination. Segmental demyelination is best appreciated on teased fiber analysis but can also be seen on longitudinally embedded toluidine stained thick sections. It should be noted though segmental demyelination is not specific for primary demyelination and can be seen in many primarily axonal processes such as diabetic neuropathy, porphyria, uremic neuropathy [37], even vasculopathies [38], these are referred to as “secondary segmental demyelination”. The hallmark of chronic demyelination is the presence of true onion bulbs (Fig. 2.12c, d), which results from repeated cycles of demyelination and re-myelination that leads to the buildup of multiple concentric layers of Schwann cell processes and their basal lamina. In practice, determination of primary axonal degeneration versus demyelination often relies on the dominant pathology of the nerve, e.g. occasional thinly myelinated fibers in a background of frequent myelin ovoids and regenerating clusters are likely axonal, while abundant thinly myelinated axons or onion bulbs with minimal associated axonal degeneration supports primary demyelination. When comparable amount of axonal and myelin alterations are present, a descriptive “mixed axonal and demyelinating features” is rendered. This is a nonspecific but relatively common finding in patients with diabetic peripheral neuropathy.