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diphosphonate (HMDP)] and negative search in those with plasma cell dyscrasia by serum-free light chain
assay, serum, and urine protein electrophoresis. The importance of ruling out AL-CA is highlighted by
99m
studies that demonstrated cardiac uptake by TC-DPD also in patients with AL-CA, with cardiac uptake
associated with poorer cardiac function and outcomes [26,27] .

BIOPSY DIAGNOSIS
Diagnosis of CA can be reached by extracardiac biopsy coupled with typical imaging features of CA by
[28]
echocardiography or CMR, in the absence of an alternative cause for increased LV wall thickness . A so-
called “screening biopsy” of the rectum or salivary gland may be performed, but fatty tissue biopsy or
aspiration is actually the easiest method to obtain a sample for amyloid diagnosis. The procedure of
abdominal fat pad fine needle aspiration requires Congo red staining and is very simple and cheap.
However, although the reported specificity is high, the diagnostic sensitivity varies widely among different
studies [29-31] . In particular, the use of fatty tissue aspiration is highly supported in systemic AL amyloidosis
but demonstrates its limitations for diagnosis of ATTR amyloidosis, particularly ATTRwt amyloidosis, and
generally cannot be used to exclude amyloidosis.

However, demonstration of amyloid fibrils in myocardial tissue through endomyocardial biopsy (EMB)
remains the gold standard for the diagnosis of CA . The first step of CA diagnosis is the visual recognition
[28]
of a homogenous, eosinophilic substance within the myocardial interstitium by hematoxylin-eosin staining.
Although Congo red is commonly applied for histological analysis when clinical suspicion of CA is present,
it requires the use of polarized light microscopy and a certain degree of expertise; moreover, the difficulty of
detecting the diagnostic “apple green birefringence” of the dye can cause a low sensitivity. In addition to
Congo red, probably the most widely used histochemical stain to detect amyloid by pathologists worldwide,
other stains can be helpful in formalin-fixed paraffin-embedded (FFPE) tissues, such as thioflavin T and S,
[32]
which bind with the amyloid proteins and exhibit fluorescence in dark-field microscopy , and a slightly
more complex to realize but highly specific histochemical dye, Sulfated Alcian Blu, which stains the
mucopolysaccharide matrix associated with amyloid and gives a bright green appearance to the deposits .
[33]
If available, confocal laser microscopy can increase sensibility and specificity of amyloid detection in Congo
red- and thioflavin T-stained tissues [Figure 1] .
[34]
Amyloid typing
After histological confirmation, amyloid typing is essential and can be obtained with different methods:
antibody-based techniques and mass spectrometry are the most widely used. Both can be applied on FFPE
tissue, avoiding the need for special handling of endomyocardial biopsy samples. Antibody-based methods
consist of immunofluorescence (IF), immunohistochemistry (IHC), and immunoelectron microscopy. All
three techniques require a broad panel of antibodies to accurately detect the abnormal protein. In addition,
each technique has its own flaws: IHC has been reported as technically complex and difficult to interpret, IF
requires fresh tissue, and immune electron microscopy is expensive and requires a longer turnaround
time . This latter method is still used in highly specialized centers in Italy and is applied through the so-
[35]
called immunogold labeling. This technique is useful for identifying biomarkers in tissues and is applicable
for transmission electron microscopy and scanning electron microscopy.

Another technically complex but very specific technique is the proteomic method of the combination of
laser microdissection with tandem mass spectrometry . This method, first used at the Mayo Clinic in the
[36]
United States, utilizes FFPE specimens as source and is highly sensitive and specific in identifying the
amyloid proteins. In addition, some reports demonstrated the possibility of detecting also the underlying
genetic alterations .
[37]