Staining Characteristics Of Amyloid Notes

Staining Characteristics Of Amyloid

1. Stain On Gross:

The oldest method since the time of Virchow for demonstrating amyloid on the cut surface of a gross specimen, or on the frozen/paraffin section is iodine stain.

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Lugol’s iodine imparts a mahogany brown colour to the amyloid-containing area which in addition to dilute sulfuric acid turns blue.

• This starch-like property of amyloid is due to the AP component, a glycoprotein, present in all forms of amyloid.
• Various stains and techniques employed to distinguish and confirm amyloid deposits in tissue sections are given in Table.

2. H and E Amyloid:

Amyloid by light microscopy with haematoxylin and eosin staining appears as extracellular, homogeneous, structureless and eosinophilic hyaline-like material, especially about blood vessels and basement membranes.

However, if the deposits are small, they are difficult to detect by routine H and E stains. Besides, a few other hyaline deposits may also take pink colour.

3. Metachromatic Stains (Rosaline Dyes):

Amyloid has the property of metachromasia i.e. the dye reacts with amyloid and undergoes a color change.

• Metachromatic stains employed are rosaniline dyes such as methyl violet and crystal violet which impart rose pink colouration to amyloid deposits.
• However, small amounts of amyloid are missed, and mucins also have metachromasia; moreover, aqueous mountants are required for seeing the preparation.
• Therefore, this method has low sensitivity and lacks specificity.

4. Congo Red And Polarized Light:

All types of amyloid have an affinity for Congo red stain; therefore this method is used for confirmation of amyloid of all types. The stain may be used on both gross specimens and microscopic sections; amyloid of all types stains pink-red in colour.

• If the stained section is viewed in polarised light, the amyloid characteristically shows maple-green birefringence due to the cross-β-pleated sheet configuration of amyloid fibrils.
• The stain can also be used to distinguish between AL and AA amyloid (primary and secondary amyloid respectively).
• After prior treatment with permanganate or trypsin on the section, the Congo red stain is repeated—in the case of primary amyloid (AL amyloid), the Congo red positivity (zoophilia) persists, while it turns negative for Congo red in secondary amyloid (AA amyloid).

Congo red dye can also be used as an in vivo test (described below).

5. Fluorescent Stains:

Fluorescent stain thioflavin-T binds to amyloid and fluoresces yellow under ultraviolet light i.e. amyloid emits secondary fluorescence. Thioflavin-S is less specific.

Staining characteristics of amyloid.

6. Immunohistochemistry:

The type of amyloid can be classified by immunohistochemical stains in which the corresponding antibody stain is used against the specific amyloid protein acting as an antigen.

• However, for mere confirmation of any type of amyloid, the most useful stain is anti-AP immunostain since the P component is present in all forms of amyloid.
• But for determining the biochemical type of amyloid, various antibody stains against the specific antigenic protein types of amyloid are commercially available such as anti-AA, anti-lambda (λ), anti-kappa (κ,), transthyretin antibody stains etc.

Morphology Of Amyloidosis Of Organs

Although amyloidosis of different organs shows variation in morphological pattern, some features apply in general to most of the involved organs.

Locations of amyloid deposit:

In general, amyloid proteins get filtered from blood across the basement membrane of vascular capillaries into extravascular spaces. Thus, most commonly amyloid deposits appear at the contacts between the vascular spaces and parenchymal cells, in the extracellular matrix and within the basement membranes of blood vessels.

• Grossly:  The affected organ is usually enlarged, pale and rubbery. Cut surface shows firm, waxy and translucent parenchymal which takes positive staining with the iodine test.’
• Microscopically: The deposits of amyloid are found in the extracellular locations, initially in the walls of small blood vessels producing microscopic changes and effects, while later the deposits are in large amounts causing pressure atrophy of parenchymal cells.

Based on these general features of amyloidosis, the salient pathologic findings of major organ involvements are described here.

Amyloidosis Of Kidneys

Amyloidosis of the kidneys is the most common and most serious because of ill effects on renal function.

• The deposits in the kidneys are found in most cases of secondary amyloidosis and about one-third of cases of primary amyloidosis.
• Amyloidosis of the kidney accounts for about 20% of deaths from amyloidosis. Even small quantities of amyloid deposits in the glomeruli can cause proteinuria and nephrotic syndrome.

• Grossly: The kidneys may be normal-sized, enlarged or terminally contracted due to the ischemic effect of narrowing of vascular lumina. The Cut surface is pale, waxy and translucent
• Microscopically: Amyloid deposition occurs primarily in the glomeruli, though it may involve peribulbar interstitial tissue and the walls of arterioles as well
• • In the glomeruli, the deposits initially appear on the basement membrane of the glomerular capillaries, but later extend to produce luminal narrowing and distortion of the glomerular capillary tuft.
  • This results in an abnormal increase in the permeability of the glomerular capillaries to macromolecules with consequent proteinuria and nephrotic syndrome.
  • In the tubules, the amyloid deposits begin close to the tubular epithelial basement membrane.
  • Subsequently, the deposits may extend further outwards into the intertubular connective tissue, and inwards to produce degenerative changes in the tubular epithelial cells and amyloid casts in the tubular lumina.

• • Vascular involvement affects chiefly the walls of small arterioles and venules, producing narrowing of their lumina and consequent ischaemic effects.
  • Congo red staining imparts red pink colour and polarising microscopy shows apple-green birefringence which confirms the presence of amyloid.