**5. Diagnostic evaluation**

The diagnostic evaluation begins with a complete history and physical examination, mainly considering both cardiac and non-cardiac signs and symptoms or "red flags" that suggest high suspicion for the condition to delineate a diagnostic approach using a combination of serum biomarkers as well as imaging studies (**Table 1**).


*HF, heart failure; CAVB, complete atrioventricular block; AF, atrial fibrillation; AS, aortic stenosis; CMR, cardiac magnetic resonance; ECV, extracellular volume; N-Pro-BNP, N-terminal-pro hormone-brain natriuretic peptide.*

#### **Table 1.**

*Red flags for cardiac amyloidosis.*

#### **5.1 Serum biomarkers**

#### *5.1.1 ATTR*

Witteles et al. propose considering evaluation of ATTR cardiomyopathy in patients older than 65 years (females >75 years) with a diagnosis of heart failure and ≥ 1 clinical finding indicative of ATTR such as suggestive imaging tests results, and/or chronic elevation in biomarkers (troponins, BNP, pro-BNP) [39–41]. Appropriate assessment include evaluation of absence of monoclonal gammopathy using serum free light chains (FLC), serum, and urine immunofixation electrophoresis as serum protein electrophoresis is insensitive leasing to false negative results.

### *5.1.2 AL cardiac amyloidosis*

Pro-BNP concentration is found to be increased in patients with heart failure and in patients with AL amyloidosis before the onset of clinical heart failure. This peptide is considered a marker for cardiac involvement. A report where pro-BNP was measured in patients with AL amyloidosis, found that its concentration was elevated despite the presence of clinical heart failure. Moreover, the diagnostic utility of pro-BNP study in 152 patients identified with amyloidosis where a plasma N-terminal pro-BNP concentration of 152 pmol/L detected cardiac involvement with a sensitivity of 93% and specificity of 90% [42].

### **5.2 Electrocardiogram (ECG)**

Low voltage electrocardiogram QRS (<0.5 mV on limb leads, <1 mV on precordial leads, and Sokolow index defined as the sum of amplitudes of the S-wave in V1 and the R-wave in V5-6 < 1.5 mV) has always been thought to be pathognomonic of cardiac amyloidosis (**Figure 2**). Nevertheless, this finding has been shown to have low sensitivity and its prevalence varies markedly according to the etiology of cardiomyopathy. Low voltage QRS is found in around 60% of AL-type amyloidosis and in close to 20% of ATTR type amyloidosis [43]. However, when present it has been associated with poor survival regardless the type of cardiac amyloidosis [43]. Moreover, a Sokolow index <1.5 mV was shown to be predictive of a combined outcome of time to hospitalization, heart transplant, or death in both AL and ATTR cardiomyopathies [44]. On the other hand, some studies have suggested that an increase in LV mass to EKG voltage relationship (mass-voltage ratio) might be more specific to CA than low voltage alone perse [45, 46]. Left ventricular hypertrophy changes have been described in precordial leads in small group of patients with very uncommon hypertrophy changes noted in limb leads [43].

Other electrocardiographic evaluation using signal-averaged ECG (SAECG) shows that late potentials were significantly more frequents on patients with echocardiographic evidence of cardiac amyloidosis (31% vs. 9% in those with normal echocardiograms) and was independently predictive of an increased risk of sudden cardiac death [47].

#### **5.3 Transthoracic echocardiography**

Transthoracic echocardiography is cornerstone in the diagnostic evaluation of patients with heart disease and is considered the standard of care in cases in

#### **Figure 2.**

 *Low voltage ECG 12-lead electrocardiography depicting the typical changes observed in cardiac amyloidosis. Low voltage limb leads (orange arrowhead) and poor precordial leads "R" wave progression with pseudo infarct pattern (green arrowhead) secondary to amyloid infiltration at the level of the left ventricular myocardium. Also, there is evidence of sinoatrial and atrioventricular conduction defects in this case there is a 1st degree AV block secondary to amyloid infiltration into the conduction system.* 

which CA is suspected. Cardiac amyloidosis has very distinctive echocardiographic features such as small LV cavity with associated increased left ventricular thickening (>12 mm) ( **Figures 1** and **3** ), biatrial enlargement, increased thickness of right ventricle, interatrial septum, and atrioventricular valves ( **Figure 3** ) with near normal LV systolic performance. The often-described increased echogenicity characterized as granular or sparkling texture pathognomonic of cardiac amyloidosis is not very sensitive and only present in a minority of patients especially when disease is advanced ( **Figure 3** ) [ 48 , 49 ]. However, this changes which are often referred to as hypertrophy are inaccurate since it is caused by a progressive infiltrative process rather than myocyte hypertrophy as it occurs in other forms of cardiomyopathy. AL and ATTR have overlapping echocardiographic features, although in general ATTR is characterized by thicker walls, owing to the more insidious nature of deposition and late diagnosis versus the toxic aspect of light chains in AL facilitating apoptosis and earlier recognition. However, asymmetric left ventricular thickness, mimicking hypertrophic cardiomyopathy, has been described in patients with familial amyloidosis [ 50 ]. Studies have demonstrated that there is a correlation between severity of LV thickness, a higher frequency of associated echocardiographic abnormalities such as left atrial enlargement or granular sparkling appearance and more common reduced systolic function with a decrease survival (median of 1.1 years) in these patients [ 51 ]. RV involvement assessed with TAPSE <14 mm is associated with events such as worsening of heart failure, increased mortality and heart transplant ( **Figure 3** ) [ 48 , 52 ]. Diastolic dysfunction to different degrees is common and is present in all patients. In advanced CA, doppler mitral flow evaluation shows restrictive pattern characterized with short deceleration time of E wave with decreased A wave velocity ( **Figure 3** ).

#### **Figure 3.**

*Transthoracic echocardiogram with characteristic findings of cardiac amyloidosis (A) PLAX & SAX Markedly thickened myocardium with severe concentric hypertrophy and abnormal texture described as ground glass appearance. Prominent and thickened MV leaflets and severe LA enlargement. Associated posterior pericardiac effusion (B) mitral valve inflow velocities with shortened deceleration time of the "E" wave (98 ms) and absent "A" wave with an E/A > 2 consistent with severe diastolic impairment also known as restrictive pattern. MVI TDI with lateral velocities with 5-5-5 pattern (s'—systolic, e'—early diastolic, and a'—late-atrial-diastolic) with TDI velocities <5 cm/s. (C) Echocardiogram with evidence of increased RV thickening (88 mm) and dilated cavity, reduced RVs' TDI and TAPSE suggestive of RV dysfunction. (D) Left ventricular showing a decrease in global longitudinal deformation (absolute value <−15. Cherry-on-top sign (yellow arrow) on the STE "Bulls" map due to a relative reduction of the middle and basal segments of the left ventricle compared to the apical segments. There is an Apical to Basal/mid segments >2.1.*

Diminished A wave velocity is due to progression of restrictive disease and intrinsic atrial dysfunction.

Tissue Doppler, strain, and strain rate imaging allows early diagnosis of diastolic dysfunction in patients with cardiac amyloid and helps distinguish cardiac amyloidosis from other restrictive cardiomyopathies etiologies such as constrictive pericarditis. Usually, a mitral annular diastolic velocity (E') <8 cm/s is a good discriminator for restrictive physiology [51, 52]. In patient with more advanced presentation of CA, the "5-5-5" sign with TDI velocities are <5 cm/s (**Figure 3**). Other signs of advanced CA besides tissue velocities include increased isovolumetric contraction time (IVCT) and isovolumetric relaxation time (IVRT) and decreased ejection time. However, regional strain has shown long-axis dysfunction in early cardiac amyloidosis and impairment of longitudinal contraction despite preserved fractional shortening. Reduction in global measures of systolic function, such as left ventricular (LV) ejection fraction, are late manifestations and characteristics of advanced disease. In CA, overall global longitudinal strain (GLS) is < −15 with a characteristically depressed longitudinal
