Tissue biopsy

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To find amyloidosis you have to know how to look for it

Biopsy is a procedure where a small sample of tissue is obtained, processed and examined under the microscope.  In order to detect amyloidosis in a biopsy, special techniques are necessary at both the processing and the examination stages. If these techniques are not used, or are used incorrectly by inexperienced laboratory staff, the diagnosis of amyloidosis may be missed. Sometimes the diagnosis is missed even if the procedure was performed correctly. For this reason, an SAP scan may be recommended to rule out amyloidosis, even if a biopsy is negative.

Kidney tissue, viewed using specialist laboratory techniques to pick up amyloid deposits (stained with Congo Red dye and viewed under cross-polarised light)
Reproduced with permission from Int J Biochem Cell Biol, Dec 2003;35:12;1608-1613

A biopsy sample may be taken from almost any organ in the body and it may be performed either because:

1. There is suspicion of amyloidosis.
or:
2. There is unexplained deterioration in the function of an organ.

If there is suspicion of systemic amyloidosis, the biopsy may be taken from a variety of sites. The procedure is simple, quick, safe and relatively painless. Usually a small piece of fat tissue is taken from either the skin of the stomach area (abdominal fat biopsy) or the rectum.

Abdominal fat biopsy proceeds as follows:

  1. A small area of the skin in the stomach area is numbed with local anaesthetic.
  2. A needle is inserted into the numb area to take out some fat cells from underneath the skin.
  3. The fat cells are preserved and sent to the laboratory for analysis.

If there is systemic amyloidosis it is often detectable in such a sample. However, in some patients with amyloidosis, it may just happen that there are no amyloid fibrils in the small tissue sample that was examined. So an SAP scan my be recommended to rule out amyloidosis, even if a biopsy is negative.

A biopsy may also be taken from a particular organ, in order to try to detect the reason why it is not functioning normally. In such cases, amyloidosis may be one of a long list of possible diagnoses, so it is very important for the doctor who examines the tissue under a microscope to use appropriate techniques when preparing and examining the sample.

Amyloid may be detected on biopsy of the:

  • kidneys
  • liver
  • heart
  • bowel
  • nerves
  • lymph nodes
  • skin
  • thyroid gland
  • bone marrow

Since hospitals often preserve the biopsy slides, it is usually recommended that NAC histopathologists (doctors who specialise in examining material on microscope slides) should review them if possible.

Biopsy – how is amyloid detected?

Histopathologists are doctors who are expert at looking at slides of body tissues under the microscope. They can look at very small tissue samples and identify which organ they were taken from by recognising the characteristic appearance of the different cell types. They can also make diagnoses by observing the damage caused to the body cells by different diseases. They can gain additional insights into various disease processes by examining tissue samples after staining them with certain dyes.

Amyloid deposits viewed on biopsy
(a) shows appearance on slide before special treatment techniques.
(b) shows appearance of the same tissue sample using special techniques to detect amyloid deposits (stained with Congo Red dye and viewed under cross-polarised light)
Reproduced with permission from Curr Op Pharm 2006;6:214-220

This is important because amyloid deposits are very specifically identified by staining with a particular dye, called “Congo red”. If there is suspicion of amyloidosis, Congo red dye is applied to the tissue sample on the slide and this must be done properly according to a very precise method. The Congo red dye molecules attach themselves to the amyloid fibrils and can then be detected there, and distinguished from staining of any other structures in the body, by microscopic examination under very strong illumination using polarised light. When this is done correctly, and a special filter for polarised light is rotated on the microscope, amyloid deposits stained with Congo red dye uniquely show up as red changing to a very characteristic “apple green” colour, and back again.This result, known as birefringence or dichroism, is the “gold standard” for diagnosis of amyloid. It is the best test and all other tests are measured against it. However it is crucial that all stages in the procedure are performed carefully and correctly by experienced laboratory staff. Any errors in the preparation, staining or viewing of the slides may lead to a missed or incorrect diagnosis.

Distinguishing between different types of amyloidosis

Different types of amyloidosis require very different treatments. Once the diagnosis of amyloidosis has been confirmed, correct identification of the type of amyloid fibril present is necessary so that appropriate treatment can be started.

The laboratory techniques discussed below are used  in order to determine the type of amyloid fibril present. Immunohistochemistry and proteomics are techniques applied to tissue biopsy samples. Genetic testing, discussed at the bottom of this page, is performed on blood samples taken from the patient’s vein.

Amyloid fibril analysis

Immunohistochemistry:

This is a laboratory technique in which antibodies may be helpful in identifying the type of amyloid fibril present. These tests are performed on tissue from biopsy samples.

Antibodies and the proteins they bind (antigens) can be viewed as similar to keys and locks. A number of different keys may be similar in many ways, yet each of them fits only one lock. Similarly, different antibody molecules bind (adhere to) particular antigens in a very specific way. It is possible to create antibodies for use in testing which specifically recognise and distinguish between every single type of amyloid fibril protein and they are routinely used for this purpose.

This approach is particularly helpful in identifying

  • AA amyloid fibrils
  • β2-microglobulin amyloid fibrils
  • ATTR amyloid fibrils

First the diagnosis of amyloid must always be confirmed, using Congo red staining and birefringence under polarised light. Then different antibodies may be applied to the tissue containing amyloid.

If AA amyloid fibrils are present, antibodies to serum amyloid A (SAA) protein will always stick to the fibrils, so AA amyloidosis can be diagnosed.

If β2-microglobulin amyloid fibrils are present, antibodies to β2-microglobulin will always stick to the fibrils, so β2-microglobulin amyloidosis can be diagnosed.

If ATTR amyloid fibrils are present, antibodies to TTR will always stick to the fibrils, so ATTR amyloidosis can be diagnosed.

However, this technique is only helpful in about half of all cases of AL amyloidosis, due to the wide range of different light chains that may form amyloid fibrils. Each patient with AL amyloidosis has their own unique, monoclonal, protein forming the amyloid fibrils, and standard antibodies to light chains fail to stick to some of them.

Proteomics:

This method is relatively new and is very complex and expensive to set up.  Where it is available it is now considered the gold standard test to identify the type of amyloid fibrils. This means that this is the best test available, and that the accuracy of other tests is measured in comparison to this test.

The method is applied to tissue from biopsy samples.

It involves techniques called laser microdissection and tandem mass spectrometry.

Laser microdissection: a section of tissue containing amyloid fibrils is examined in the microscope and just the amyloid deposits are cut out of it using a laser and then further prepared for analysis.

Tandem mass spectrometry: the prepared amyloid deposit fragments are analysed in a machine called a spectrometer which allows identification of the type of protein and protein fragments present.

Using these techniques, the type of amyloid fibril present can be identified in very close to 100% of cases, including cases where other techniques have not led to a definite diagnosis.

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