Amyloid is identified on biopsy by Congo Red staining followed by examination under polarised light
- A biopsy is required to identify all non-cardiac ATTR amyloidoses
- Targeted biopsies of an involved organ is superior to “off-target” biopsies
- Baseline protein typing is performed by TTR, kappa, lambda and AA immunohistochemical staining
- Immunohistochemical subtyping stains only have a positive predictive value of ≈ 60%
- immunohistochemical typing stains cannot be interpreted in isolation and must always be correlated with organ staging, monoclonal gammopathy screening and other typing tests (such as cardiac amyloid scintigraphy)
Amyloid is identified on biopsy by salmon pink Congo Red staining exhibiting apple green birefringence and dichroism effects under polarised light.
Biopsy evidence of amyloid is required to identify all cases of non-cardiac ATTR amyloidosis. Tissue is also required to differentiate between cardiac ATTR and cardiac AL in cases with positive cardiac amyloid scintigraphy and a co-incident monoclonal gammopathy.
For diagnostic and typing purposes, it is recommended to perform targeted biopsies of involved organs rather than “off-target” biopsies of the abdominal fat pad, rectum and buccal mucosa. This is because targeted biopsies are more sensitive for amyloid detection and yield greater amounts of amyloid for subtyping purposes. The involved organs can be identified by using simple organ screening procedures.
Table 2: Fat Pad Biopsy Amyloid Detection Rate
||Detection Rate of Amyloid (%)
||Burden of Amyloid (as determined by SAP scan)
Fat pad biopsies are particularly insensitive in ATTR. 3,4,5. For AL, international reference centres have reported fat pad biopsy detection rates which are dependent on the burden of disease 4. However, in the general Australian setting, lower sensitivity rates are observed when using “off target” biopsies in AL.6
“Look back” or “add-on” Congo Red staining upon archived tissue samples can be a useful way to identify amyloid deposits.
- This is most relevant for ATTRwt where vascular amyloid may be present in tissues (such as the prostate and colon) for years before the onset of cardiac ATTR.
Anatomical Pathology Basics
Amyloid is identified using Congo red staining followed by examination under polarised light. Congo Red stains amyloid deposits salmon pink which then exhibit apple green birefringence and dichroism effects under polarised light. The Congo Red staining technique can be falsely positive or falsely negative when performed in non-reference labs;
- 9% of biopsies referred to an AAN reference lab were falsely negative in one study. Of the biopsies referred to the UK’s National Amyloidosis Centre’s reference lab in 2018, 24% were falsely positive and 12% falsely negative for the presence of amyloid 8
- False positives can occur when the diagnosis is based solely on salmon pink staining of extracellular tissues (a not uncommon finding) without confirmation of apple green birefringence under polarised light. False negatives can occur when the Congo Red stain is not properly maintained
Once amyloid has been identified on a biopsy, it is recommended to perform amyloid typing by immunohistochemical staining for the most common amyloid forming proteins: TTR, kappa, lambda and AA. Although antisera to many other amyloid fibril proteins are commercially available these are not routinely required or easily accessible as;
- Stains for the rare hereditary amyloidosis types have largely been supplanted by mass spectrometry analysis and/or genetic screening
- LECT 2 stains are currently not available in Australia
Select Australian laboratories routinely perform the basic panel of amyloid stains and have acquired the expertise to interpret them.
It is critically important to be aware that immunohistochemical subtyping stains are inherently prone to false negative and false positive results. The positive predictive value of amyloid typing stains is ≈ 60% 9. Even the immunohistochemical stains at the UK National Amyloidosis Centre’s international reference lab only provide a definitive result in 76% of cases 10.
Immunohistochemical typing stains have a low positive predictive value as the anti-sera have been developed against physiologic proteins leading to;
- False positive staining of non-amyloidogenic (or physiologic) proteins and increased background staining
- False negative results as the stains are not specific for variant forms of amyloid proteins (e.g. not specific for misfolded light chain or variant hereditary TTR proteins)
- Kappa and lambda light chain stains have a particularly high false negative rate in the order of 20-30% 11
The immunohistochemical typing result cannot be interpreted in isolation and must always be correlated with organ staging, monoclonal gammopathy screening and other typing tests (such as cardiac amyloid scintigraphy).
In addition, it is not always necessary to type the constituent amyloid protein for all amyloidosis cases e.g.
- Localised mucosal amyloid deposits are almost always derived from light chain and hence a negative vital organ and monoclonal gammopathy screen are all that is required to type as localised AL
- For systemic AL, specific combinations of organ staging, monoclonal gammopathy and cardiac scintigraphy findings can be sufficiently specific to type as systemic AL without the need to type the constituent amyloid protein