Amyloid
Richard Williams

Introduction
First named by Virchow in 1853, amyloid is the term used for the predominantly extracellular deposition of a group of chemically diverse, abnormal proteins which are arranged to a greater or lesser extent in specific beta-pleated fibrils.

Amyloidosis refers to a heterogeneous group of disease complexes characterised by amyloid deposition in body tissues which may either be localised or diffuse. Amyloid deposition is often an incidental finding during routine examination of biopsy material, or it may be suspected clinically in association with chronic disease processes including non-specific inflammatory disorders and malignant conditions such as multiple myeloma. Rarely, familial amyloidosis syndromes are encountered.

The physical nature of amyloid
Until recently the fibrils of amyloid, apart from those in intracerebral deposits, were all thought to have the same ultrastructural appearance and could be specifically identified by their configuration. This (purportedly) unique structure comprises non-branching fibrils of indefinite length, 7.5-10 nm in diameter and in a b -pleated antiparallel configuration which can be demonstrated using X-ray diffraction. Polypeptide chains are orientated at right angles to the axis of the fibrils. Recent studies, on wet gel preparations, of AA fibrils (see later) suggest that these comprise a globular protein of only two strands making up a single b -sheet running perpendicular to the fibril axis. Thus these fibrils are made up of a stack of subunits with only some areas of b -sheet structure. The paired helical filaments seen in neurofibrillary tangles differ from other amyloid and are distinctive biochemically.

Although the structure of the major fibrillary subunits making up different forms of amyloid and the putative precursor proteins has become better understood over the last two decades, the factors which precipitate formation of the fibril components remain unknown.

Classification of amyloidosis, chemical composition and clinical associations
There are many, often overlapping, classifications of amyloidosis. The simplest early classifications recognised a 'primary' form of amyloidosis, affecting mainly the tongue and heart, and a secondary type, with deposits in the spleen, liver and other organs, associated with chronic inflammatory disorders. Familial forms of amyloidosis, with differing patterns of organ involvement, were also included whilst a so-called 'isolated organ amyloidosis' comprised a fourth group of disorders. With characterisation of the chemical composition of amyloid, this classification became obsolete, and in 1980 Glenner¹ classified amyloidosis into (a) acquired systemic amyloidosis, (b) organ limited amyloidosis and (c) localised deposition amyloidosis.

Systemic Amyloidosis
Systemic amyloidosis is a disease process which affects tissues throughout the body. This situation can occur in association with chronic inflammation/infection (AA amyloid - purportedly derived from serum amyloid A protein (SAA), an acute phase reactant); as a complication of immunocyte dyscrasias, where the fibrils are derived from immunoglobulin light chains (AL amyloid); as one of a group of heredofamilial disorders; and in aged individuals where the fibrils are derived from prealbumin in plasma.²

Systemic amyloidosis occurs in association with longstanding infection or autoimmune disease (commonly tuberculosis and rheumatoid arthritis). Cases are associated with the precursor serum protein (SAA), which is subsequently enzymatically hydrolysed to form a tissue protein (AA). Once deposited in an organ the tissue protein is neither metabolised nor excreted and (potentially) keeps accumulating (especially within the kidney, liver and spleen) if the initiating disorder is not arrested. This is despite the fact that AA fibrils may apparently be degraded in vitro by unidentified components in human serum from normal subjects, but not by serum from those with amyloidosis.

Systemic amyloid deposition may also be associated with immunocyte dyscrasias including multiple myeloma and the macroglobulinaemias. In these cases the fibrils are derived from immunoglobulin light chains (AL amyloid).

There is a group of well documented, but rarely encountered, heredofamilial disorders presenting as systemic amyloidosis. Four of the sets of familial amyloidosis syndromes are associated with neuropathies, all inherited as autosomal dominant traits, and some of which have fibril proteins derived from a pre-albumin variant and many have been described.²

Amyloidosis is also associated with aging and amyloid deposits may be demonstrated in cardiac muscle, skeletal muscle, renal parenchyma, lungs and other organ parenchyma in greater than 25% of individuals over the age of 60 years.

Localised Amyloidosis
Amyloid deposition may be limited to, or have a predilection for, a particular organ or tissue with amyloid in other areas being difficult to detect. Clinically each of these instances represents a localised form of amyloidosis. The many different situations in which localised amyloidosis occurs include: nodular amyloidosis, in which nodules of amyloid are found in individual organs (for example the lung or urinary bladder) and cause symptoms by their localised mass effect; cardiac amyloid which occurs with increasing frequency in individuals over sixty years of age but rarely causes significant cardiac impairment; localised amyloidosis may also be seen in skin, diseased joints, some malignant tumours (notably the neuroendocrine group of tumours) and brain.

Cerebral amyloid
Cerebral amyloid is unique in its structure, and is found in association with Down's syndrome, Alzheimer's disease and the transmissible spongiform encephalopathies. It is found centrally in the plaques (neurofibrillary tangles) and also as paired helical filaments in the neuronal cell bodies of plaques. Cerebral amyloid is the only form so far recognised in humans which does not contain the non-fibrillar, amyloid P component.

WHO-IUIS classification system
The most recent attempt to standardise the classification of amyloidosis is that of Husby³ which was later expanded by the WHO-IUIS Nomenclature subcommittee (1993).⁴ The essential feature of this approach is that "the basis for nomenclature and classification of a case of amyloidosis under investigation should be the fibril making up the amyloid deposits".⁴ In many cases there are fibril protein precursors in the serum of the subject, and the classification includes these identified precursors as a second set of data (see TABLE). Variations in the type of amyloid protein constitute a third set of data, and a fourth adjunct to the classification is a description of the disease process affecting the individual. This classification includes only those fibril proteins integrated into the amyloid fibre which have been characterised by their amino acid sequence and extra fibrillar proteins, including the amyloid P-component protein (AP), are excluded. The WHO recommended classification avoids any attempt to distinguish between localised and systemic amyloidosis, based on the rationale that some apparently localised depositions of amyloid may merely represent a predilection site of deposition in cases of systemic amyloidosis.

In the abbreviation system adopted A (for amyloid) is used as the first letter for all amyloid subtypes and this is followed by the precursor protein designation. The protein precursor is that from which the amyloid fibril protein is thought to be derived, and is the protein which can be demonstrated in serum (with the exception of the b protein precursor). The protein type or variant represents many classes and subgroups of immunoglobulins, and whilst the table shows only one example of variant for each amyloid protein, the recommendation for cases where there are multiple protein types or variants is that "sub-tables" should be designed to include all those known at the present time.

Diagnostic procedures
Macroscopic Staining
At autopsy, whole tissues containing amyloid will stain blue if immersed in an iodine solution, followed by dilute sulphuric acid.

Tissue Biopsy
If amyloidosis is suspected the diagnosis must be confirmed by biopsy. In many cases the biopsy will be directed and organ specific, but in other instances a 'non-specific' diagnostic biopsy will be attempted. Many sites have been suggested for the non-specific biopsy, including gingival tissue, subcutaneous fat in the periumbilical region (obtained by fine needle biopsy⁵), and rectum (endoscopic biopsy). In practice the rectal biopsy site appears to be the most reliable with up to 90% diagnostic accuracy. There are several potential pitfalls with the non-specific organ biopsy as follows:

• the rectal biopsy may still fail to reveal the amyloid in 10% of cases.
• in instances where there is minimal deposition of amyloid, thick sections (approximately 10 µm) are more likely to show amyloid with the Congo Red stain. (A negative result based on the staining of 3 to 4 µm thick sections should be repeated using 10 µm thick sections).
• Amyloid deposition may rarely be mimicked by elastofibromatous change in rectal biopsies in H&E stained sections.⁶ Differentiation between these conditions can be made on the basis of staining reactions with elastic and Congo red stains.
• Some methods used to demonstrate amyloid, such as the Thioflavin T reaction, are not specific and various other procedures may need to be used.

For routine identification of amyloid, the Congo Red stain remains the method of first choice. It can be used to identify all forms of amyloid in tissue sections, provided that thick (10 µm) sections are used and staining solutions are fresh. A characteristic apple-green birefringence can be demonstrated after congo red staining. Formalin fixed, paraffin embedded material from a case of proven amyloidosis provides control tissue.

In situations where there is little amyloid present in the biopsy, electron microscopic examination of appropriately processed tissue may be necessary to demonstrate the characteristic fibrils. Ultimately immunostaining techniques using antibodies directed at specific components of different forms of amyloid will supersede the current methods, but at the present time such antibodies are not universally available.

Other (Non-histological) Techniques
Ultrasonography and echocardiography are useful adjuncts in demonstrating enlargement of organs in amyloidosis but do not provide a definitive diagnosis of the disease type. Molecular biology methods to identify carriers are presently being developed.

Staining methods

Congo red stain (after Benhold)⁷
SECTION PREPARATION
Tissue is fixed in 10% buffered neutral formalin. Paraffin sections cut between 6 to 12 µm give optimal results.

In practice a 6 µm section is examined initially, especially if the tissue biopsy is small. If the results are negative or equivocal a 12 µm section is then used. Always include a known positive control.

REAGENTS REQUIRED
1 1% Congo red solution
Congo Red (CI 22120) 1.0 g
Distilled water 100 ml
(For optimal results this solution should be freshly made).

2 1% aqueous sodium hydroxide solution
Sodium hydroxide 1.0 g
Distilled water 100 ml

3 Alkaline alcohol solution
1% Sodium hydroxide solution 1.0 ml
50% Ethyl alcohol 99.0 ml

4 Mayer’s haematoxylin solution

METHOD
1 Dewax and rehydrate sections to distilled water.
2 Stain in filtered Congo Red solution for 1 hour.
3 Rinse in distilled water.
4 Rapidly differentiate in the alkaline alcohol solution.
5 Wash in running tap water for 5 minutes.
6 Counterstain in Mayer's haematoxylin for 5 minutes.
7 Wash in running tap water for 15 minutes.
8 Dehydrate, clear and mount in synthetic resin.

RESULTS
Amyloid (transmitted brightfield illumination) - pink to red
Amyloid (polarised light)- apple green birefringence
Nuclei - blue

TECHNICAL NOTE
Askanas, King Engel and Alvaraz⁸ have suggested using 12 - 15 µm sections and an enhanced fluorescence technique to increase the sensitivity of detection of amyloid stained with Congo Red in freshly frozen muscle biopsies in cases of suspected inclusion body myositis, and in paraffin embedded sections of brain from subjects with Alzheimer's disease. In this method, sections are examined by epifluorescence illumination with filter combinations of either:

This modification is suggested where only small amounts of amyloid may be present.

Crystal violet stain^7,9
SECTION PREPARATION
Tissue is fixed in 10% neutral buffered formalin. Paraffin sections are cut at 4 to 5 µm. Always include a known positive control.

REAGENT PREPARATION
1 Crystal Violet Stock Solution
Crystal Violet 14.0 g
95% Ethyl Alcohol 100.0 ml

2 Crystal Violet Working Solution
Crystal Violet Stock Solution 10.0 ml
Distilled Water 300.0 ml
Concentrated Hydrochloric Acid 1.0 ml

METHOD
1 Dewax and rehydrate sections to distilled water.
2 Stain in the crystal violet working solution for 5 hours.
3 Wash in running tap water for 15 minutes.
4 As the stain may lose its metachromasia in dehydration or clearing procedures using xylene, an aqueous mounting medium should be used and the preparation examined immediately before the stain fades. If, however, an aqueous mounting medium is not available, the stain may be dipped in xylene after a period of air drying (minimum 15 minutes) and mounted in a resinous medium.

RESULTS
Amyloid - purple/violet colouration
All other tissue elements - blue

Sirius red stain^7,10
SECTION PREPARATION
Tissue is fixed in 10% neutral buffered formalin. Paraffin sections are cut at 4 to 5 µm. Always include a known positive control.

REAGENT PREPARATION
1 1% Sodium Hydroxide Solution
Sodium hydroxide 1.0 g
Distilled water 100.0 ml

2 Alkaline Alcohol Solution
1% Sodium hydroxide Solution 1.0 ml
80% Ethyl alcohol 99.0 ml

3 Sirius Red Solution
Sirius red F3BA (CI 35780) 1.0 g
Distilled water 100.0 ml

The Sirius red should be dissolved in the water, then add:
Sodium chloride 0.5 g

The solution must stand overnight before use, and should not be filtered.

4 0.1 M Borate Buffer Solution (pH 9.0)
Sodium borate 38.1 g
Distilled water 1 l

5 Mayer's haematoxylin solution

METHOD
1 Dewax and rehydrate sections to distilled water.
2 Wash in running tap water for 5 minutes.
3 Place in buffered neutral formalin solution overnight.
4 Wash in running tap water for 15 minutes.
5 Place in alkaline alcohol solution for 1 hour.
6 Rinse in distilled water for 10 seconds.
7 Place in Sirius Red solution (preheated to 60°C) and incubate at 60°C for 90 minutes.
8 Rinse for ten seconds each in two changes of the borate buffer solution.
9 Wash in running water for 5 minutes.
10 Stain in Mayer's haematoxylin solution for 5 minutes.
11 Wash in running water for 15 minutes.
12 Dehydrate, clear rapidly and mount in a resinous medium.

RESULTS
Amyloid - pink to red
Nuclei - blue

TECHNICAL NOTE
Elastin also stains making this a less than optimal method in actinic damaged skin.

Thioflavin T reaction¹¹
This reaction is not specific and the sections must be mounted in a non-fluorescing mounting medium. Variations to the pH of the thioflavin T reaction have been proposed as a means of increasing stain selectivity.¹¹

SECTION PREPARATION
Tissue is fixed in 10% neutral buffered formalin. Paraffin sections are cut at 4 to 5 µm thick. Always include a known positive control.

REAGENT PREPARATION
1 1% aqueous Thioflavin T

2 1% aqueous acetic acid

3 Mayer's haematoxylin solution

METHOD
1 Dewax and rehydrate sections to distilled water.
2 Stain in haematoxylin solution for 2 minutes.
3 Wash in tap water for 2 minutes.
4 Stain in thioflavin T solution for 3 minutes.
5 Rinse in distilled water for 2 minutes.
6 Remove excess fluorochrome from background by washing in 1% acetic acid solution for 20 minutes.
7 Wash in running tap water.
8 Dehydrate and mount in a non-fluorescing medium (glycerine-saline 1:9).

RESULTS
Amyloid and other tissue components
With ultraviolet light source, a UG1 Exciter filter, BG38 red suppression filter and K430 barrier filter - silver-blue fluorescence
With blue light fluorescence, BG12 exciter filter and K530 barrier filter - yellow fluorescence (Fig 14.4).

Specialised immunostaining
Monoclonal antibodies directed against the amyloid P component are available as are anti AA serum, anti calcitonin and anti insulin antibodies. Antibodies to Alzheimer neurofibrillary tangles have also been developed.^12,13

None of these reagents are specific for all amyloid sub types and are therefore of limited diagnostic use.

References
1 Glenner GG. Amyloid deposits and amyloidosis. The b -fibrilloses. New England J Medicine 1980; 302: 1283 - 1292

2 Pepys MB. Amyloidosis. Chapter in: Weatherall DJ, Ledingham JGG and Warrell DA. Eds. Oxford Textbook of Medicine. 2nd Edition. Volume 1. Oxford Medical Publications. 1989

3 Husby G. Nomenclature and classification of amyloid and amyloidoses. J Internal Medicine 1992; 232: 511 - 512

4 WHO-IUIS Nomenclature Sub-Committee.. Nomenclature of Amyloid and Amyloidosis. Bulletin of the World Health organisation 1993; 71: 105 - 108

5 Libbey CA, Skinner M, Cohen AS. Use of Abdominal Fat Tissue Aspirate in the diagnosis of systemic amyloidosis. Archives of Internal Medicine 1983; 143: 1549 - 1552

6 Goldblum JR, Beals T, Weiss SW.. Elastofibromatous change of the rectum: A lesion mimicking Amyloidosis. American Journal of Surgical Pathology Path 1992; 16: 793-795.

7 Armed Forces Institute of Pathology. Laboratory Methods in Histochemistry. Prophet EB, Mills B, Arrington JB and Sobin LH. Eds. American Registry of Pathology. Washington DC. 1992

8 Askanas V, King Engel W, Alvarez RB.. Enhanced detection of Congo-red-positive amyloid deposits in muscle fibres of inclusion body myositis and brain of Alzheimer's disease using fluorescence technique. Neurology 1993; 43; 6 -

9 Lieb E. Permanent stain for amyloid. Am J Clinical Pathology 1947; 17: 413 -

10 Sweat F, Puchtler H.. Demonstration of amyloid with direct cotton dyes. Archives of Pathology 1965; 80: 613

11 Theory and Practice of Histological techniques. 3rd Edn. Bancroft JD, Stevens A. Eds. Churchill Livingstone. Edinburgh. 1990

12 Al-Mutlac H, Wheeler J, Robertson H, Watchorn C and Morley AR. Tissue distribution of amyloid P component as defined by a monoclonal antibody produced by immunisation with human glomerular basement membranes. Histochemical Journal 1993; 25: 219 - 227

13 Dickson DW, Kress Y, Crow A, Yen S-H. Mononuclear antibodies to Alzheimer neurofibrillary tangles: ll. Demonstration of a common antigenic determinant between ATN and neurofibrillary degeneration in progressive supranuclear palsy. American Journal of Pathology 1985; 120: 292 - 303