Diagnosis

Suspected ammyloidosis

Suspicion of amyloidosis can occur in many ways. If, for example, someone belongs to a family with a history of hereditary amyloidosis, then minor symptoms may lead to a suspicion of amyloidosis. If someone is known to have a long-term inflammatory illness such as rheumatoid arthritis, then it is a good idea to regularly check the urine for the presence of proteins and to monitor renal function. Suspicion of amyloidosis can also arise due to medically unexplained symptoms such as enlargement of the tongue, liver or spleen, a neuropathy in both legs, serious loss of protein via the kidneys or kidney failure, serious diarrhea, or heart failure with greatly thickened heart walls. The suspicion is greater if combinations of the above unexplained symptoms arise.

Suspicion alone is not enough. The presence of amyloid should be unequivocally established in a tissue sample, known as a biopsy.

The result of such a biopsy is often the first indicator of amyloidosis. Due to the relative rarity of the condition, the very different ways in which it can present itself, and the insidious and very gradual onset of symptoms, in many cases the disease is only detected at a late stage where serious damage has already been caused to one or more organs.

Further assessment in brief
After the discovery of amyloid in a tissue sample, further evaluation should take place to identify the type of amyloidosis, and map its severity and extent. It is important to determine whether the amyloidosis accumulation is localised or systemic, and which organs and tissues may have possibly been damaged by it. This provides the practitioner with a picture of the severity and extent of the amyloidosis and allows a rough estimate of the life expectancy to be made. From this, is can be considered whether treatment is possible and, if so, what the options are.


Biopsy

In order to determine the presence of amyloid, it is necessary to identify it in tissue samples. This can be shown by a positive staining using Congo red with the associated apple-green birefringence in polarised light. An abdominal fat biopsy is the most elegant and least harmful method for this purpose, with a >90% chance of detection of most forms of systemic amyloidosis if the biopsy and the processing of it are carried out correctly. A good alternative is to perform a rectal biopsy. If one of these methods gives a negative result, but a strong suspicion of amyloidosis remains, then it is worthwhile to also carry out the other one (or try the somewhat less positive bone marrow biopsy). However, if all these biopsies are negative yet the suspicion of amyloid nevertheless persists, then a biopsy of the suspected organ or tissue is appropriate.

Below is an example of the Congo red colouring of a suction (aspiration) biopsy of fat tissue. This sample can be taken at an outpatient clinic under local anesthetic, just beneath the skin of the stomach close to the navel (bellybutton). Download the instruction video (approx. 15 MB) here and see the Fat aspiration procedure 2019 (pdf file) for more details.

Fat aspiration – normal light Fat aspiration – polarised light

As can be seen in the above images, the amyloid is visible as red-coloured deposits between the normal, blue-coloured structure of the fat tissue. When this is viewed under polarised light, the red colour changes to green or yellow-green. This green birefringence under polarised light of material dyed with Congo red is characteristic of amyloid.
In the laboratory, the presence and quantity of the precursor proteins, as how these building blocks of the amyloid are called, can then be examined. It is also possible to look under a microscope in tissue pieces of the affected organ on a specimen slide with specific antibodies (directed against the precursor proteins) in order to establish the presence of amyloid irrefutably and to investigate which type of amyloid is involved.


Typing

Clinical prediction of the amyloid type
After confirmation of the presence of amyloid in tissue, the amyloid should be typed. In many cases, a good prediction of the type of amyloid can be made based on history, symptoms and clinical picture. A patient with long-standing rheumatoid arthritis and nephrotic syndrome almost certainly has the AA type. Someone with polyneuropathy who belongs to a family with hereditary amyloidosis is likely to have the ATTR type. And for a patient with the characteristic “shoulder pads” and a significantly enlarged tongue, the AL type is most likely. Blood, urine and bone marrow tests can also help to predict the type of amyloidosis. In case of AL amyloidosis, proteins produced by malignant plasma cells (free light chains) can almost always be detected in the blood and / or urine and malignant plasma cells can be detected in the bone marrow. With AA amyloidosis, elevated acute phase proteins (such as CRP and the precursor protein SAA) can be found in the blood. This is indicative of, but certainly not conclusive for AA amyloidosis. These inflammatory proteins may also be present in the blood at high levels in many other circumstances.

Even though a certain type of amyloid is clinically plausible, confirmation of the amyloid type is necessary. The treatment, prognosis and consequences of the different types of systemic amyloidosis vary considerably.

Typing using biopsy
Immunohistological examination of the biopsy is a technique in which the type of amyloid can be further characterized via specifically targeted antibodies. In the case of AA amyloidosis, this test is sufficient for typing, provided that reliable antibodies are used for this. However, with AL and ATTR amyloidosis, this method fails in some cases because the antibodies used are less reliable and the recognizability of the precursor protein within the amyloid deposits is reduced.

Below are examples of a kidney biopsy of an AA amyloidosis patient where the biopsy is stained with Congo red (where the characteristic effect of the polarization filter can be seen) and on the right the Immunohistochemical staining with the reliable antibody directed against Serum amyloid A (SAA; clone Reu86.2).

Kidney – Congored staining Kidney – Immunohistochemical stainingwith anti-SAA

In Groningen we have the possibility to make the amyloid available through certain solvents from the fat biopsy and to type the amyloid with four laboratory tests, so-called ELISAs. This method is suitable for distinguishing the most common types of amyloidosis (AA, AL and ATTR), but it only works if there is sufficient amyloid in the fat biopsy. If this method fails to characterize the amyloid, it is possible to consider using other biochemical techniques (such as mass spectrometry).


Genetic testing

If there is evidence of ATTR amyloidosis, genetic testing (DNA analysis) is performed to distinguish between the hereditary and wild-type forms. In addition to hereditary ATTR amyloidosis, there are several other types of hereditary amyloidosis. Examples of these are apolipoprotein A-I amyloidosis, apolipoprotein A-II amyloidosis, Gelsolin amyloidosis, Lysozyme amyloidosis, Cystatin C amyloidosis, and fibrinogen Aα chain amyloidosis. Most forms of hereditary amyloidosis have an autosomal dominant pattern of inheritance.

Abbreviation Protein(precursor)
Affected organs
ApoAI Apolipoprotein A-I Heart, liver, kidneys, nerves, larynx
ApoAII Apolipoprotein A-II Kidneys, heart
ApoCII Apolipoprotein C-II Kidneys
ApoCIII Apolipoprotein C-III Kidneys
Beta2M Beta-2 microglobulin Gastrointestinal tract, autonomic nervous system, salivary gland, lacrimal glands (tear ducts)
CST3 Cystatin Brain
FGA Fibrinogen A α-chain Kidneys
GSN Gelsolin Nervous system, skin and eyes
IL31RA Interleukin-31 receptor A Skin
LYS Lysozyme Kidneys, liver, heart, spleen, gastrointestinal tract and salivary gland
OSMR Oncostatin M receptor Skin
TTR Transthyretin Nervous system, heart, eyes, meninges

In brief

  • If amyloidosis is suspected, the amyloid must be proven to be present in a biopsy
  • The type of amyloid must also be determined. Various laboratory techniques are available for this
  • Genetic testing is performed to assess whether there is a hereditary form of amyloidosis