A definitive diagnosis of ARVC/D relies on histological demonstration of fibro-fatty replacement of ventricular myocardium and it is listed among the major Task Force diagnostic criteria .
In vivo, the pathological specimens are obtained through EMB. Right ventricular EMB is a well-established procedure in the diagnosis of heart muscle disease and seems to be particularly helpful in sus pected ARVC/D because of the peculiar topographic and histologic features of the disease. The fibro-fat-ty replacement is almost transmural exclusively in the right ventricular free wall, whereas the septum is usually spared and the left ventricular free wall, when affected, shows involvement of the subepicardial-mid layers. To avoid false negative results, biopsies should not be taken from the septum, but the bioptome should be directed to the free wall, with the potential risk of perforation and hemopericardium. However, clinically relevant complications from EMB sampling are rare and are estimated at 1%-2%, with death as an exceptionally rare event (less than 0.2%) .
In the 1994 Task Force diagnostic criteria only a qualitative analysis of EMB samples was mentioned in terms of fibro-fatty replacement . However, we should recognize that the histopathologic findings of fatty or fibrous tissue in the myocardium is far from specific, since fatty infiltration frequently occurs in the right ventricle in the elderly and obese population , and fibrosis can be observed in many cardiomy-opathic and noncardiomyopathic conditions [71-75].
The key to diagnosis pertains to the site and quantity rather than to quality of myocardial tissue replacement . Quantitative diagnostic parameters have been established by histomorphometric analysis of EMBs from patients with suspected ARVC/D, dilated cardiomyopathy and controls. Angelini et al. [76, 77] obtained the following diagnostic parameters in ARVC/D: myocardial atrophy with residual myocytes less than 45% of the EMB cross sectional area, fibrous tissue >40% and fatty tis-sue>3%, with a 67% sensitivity and a 92% specificity for at least one parameter. It should be emphasized that the morphometric analysis was a mean of the evaluation of all the specimens drawn from the different sites of the right ventricle. It is known that there is a difference in the percentage of tissue parameters among the different specimens, since the disease is frequently segmental and, at times, only one specimen can be informative (Figs. 6.9, 6.10).
In 1992, Pinamonti et al.  reported moderate-severe fatty infiltration in 50% of EMBs taken from 25 ARVC/D patients, and it was associated with fi-brosis in about half of the cases. No fat was detected in six left ventricular EMBs, but fibrosis associated with hypertrophy and myocyte attenuation was evident. Similar features were found in the Mayo Clinic experience, who reported right ventricular EMB findings with adequate tissue sampling in 13 affected patients . Since the authors considered the presence of abnormal amounts of adipose tissue as diagnostic in either EMB specimens or explanted hearts, only 54% of patients had characteristic histo-
logic features of ARVC/D, while in the remaining 46% only myocyte hypertrophy and interstitial fi-brosis were detected. In four patients who had left ventricular EMB, no fatty infiltration was evident, and only myopathic changes were present.
In the series by Wichter et al. , abnormal fibro-fatty or fatty tissue infiltration exceeding an area of 25% in one or more biopsy samples, taken from different regions of the right ventricle, was considered a diagnostic feature of ARVC/D. Based upon this definition, EMB resulted diagnostic in 66% of patients, whereas it was normal in 28% and nonspecific in 6%.
Daliento et al.  compared EMB findings in 12 young vs. 17 adult patients with ARVC/D. By strictly applying the histomorphometric criteria , only 25% of the young and 35% of the adult patients would have had a histopathologic diagnosis at EMB. No difference was found as far as the amount of myocardium was concerned, whereas in the younger group a greater amount of fibrous tissue and a lower amount of fatty tissue were found. In ten young patients there were no adipocytes. The low prevalence of diagnostic EMBs on the basis of histomorphome-tric data may be the result of either sampling error in segmental forms of ARVC/D or of the fact that, in the early stages of the disease, histological abnormalities may be restricted to subepicardial regions which are not accessible through EMB.
In a clinical-pathologic study correlating EMB findings with cardiac imaging and electrocardio-graphic data, fibrous substitution (>30%) was a significant univariate predictor of late potentials in the signal-averaged electrocardiogram and of reduced right ventricular ejection fraction, whereas fatty tissue, traditionally considered a more specific histo-morphometric parameter, did not correlate with any clinical feature . This is in contrast with the findings by Chimenti et al. , who demonstrated more evident fatty replacement in the EMB samples taken from the areas with more prominent morphofunc-tional abnormalities.
To clarify the issue, we recently carried out an in vitro EMB study by simulating endocardial sampling in archival heart specimens in different sites of the right ventricle as well as in the left ventricle . According to our data, a <64% of residual myocardium in right ventricular samples should be considered as a diagnostic cut-off for ARVC/D, with a sensitivity of 80% and a specificity of 90%. Mean fatty tissue is a less reliable diagnostic histologic parameter, since there are no statistically significant differences when comparing ARVC/D vs. the adipositas cordis and the elderly, whereas fibrous tissue is in creased both in ARVC/D and in dilated cardiomy-opathy. When considering the sampling site from the right ventricular apex, the amount of fatty tissue in ARVC/D is higher than in dilated cardiomyopathy, but no significant differences are found vs. controls, adipositas cordis and the elderly, thus indicating that fatty tissue is mostly nonspecific, particularly at the apex, whereas the residual myocardium and fibrous tissue appear the most relevant diagnostic parameters. Finally, no significant differences were found among the groups when considering the septum and the left ventricle, clearly confirming that septal and left ventricular EMB are not useful for diagnostic purposes in ARVC/D. Although histomorphometric evaluation is mandatory, a careful qualitative assessment of residual myocytes as well as of characteristics of fibrosis and adipocytes may be also of help and sometimes crucial to the final correct diagnosis (Fig. 4.4).
EMB for Differential Diagnosis Between ARVC/D and Other Arrhythmias with Right Ventricular Origin
EMB can aid in the differential diagnosis between ARVC/D and other diseases that can mimic ARVC/D, such as dilated cardiomyopathy, myocarditis, and id-iopathic right ventricular outflow tract tachycardia.
Nava et al.  first demonstrated the role of EMB in revealing concealed forms of ARVC/D as a cause of apparently idiopathic ventricular arrhythmias, since they observed myocardial atrophy with fibrous-fatty substitution by right ventricular EMB. Recently, by correlating abnormal voltage mapping with myocardial atrophy and fibrofatty replacement at EMB, we also confirmed an underlying segmental form of ARVC/D at risk of life-threatening events in a subgroup of patients with apparently idiopathic right ventricular outflow tract tachycardia .
Chimenti et al.  found that only 30% of patients with sporadic ARVC/D had diagnostic histo-logic features of ARVC/D at EMB, while the majority had myocarditis according to Dallas criteria. That myocarditis can mimic the clinical picture of ARVC/D was also reported recently by our group in a study comparing CARTO electroanatomic mapping with EMB features . The differential diagnosis is important, since prognosis and treatment are different.
Sarcoidosis deserves special mention because affected patients may present with clinical and morphological features of ARVC/D, including typical ECG features like epsilon wave and left bundle branch block ventricular tachycardia [87-89]. Recently, Koplan et al.  reported a series of eight pa tients with recurrent monomorphic ventricular tachycardia due to cardiac sarcoid, two of whom had a previous presumptive diagnosis of ARVC/D. A differential diagnosis by EMB is important, since treatment with corticosteroids may benefit patients with sarcoidosis, although results of this therapy remain controversial.
Idiopathic giant cell myocarditis may be confused with sarcoidosis, although the former is a much more fulminant disease. Okura et al.  recently compared the two entities and found that both conditions frequently had ventricular tachycardia, although a longer duration of symptoms and the presence of heart block should suggest sarcoidosis.
EMB to Validate Cardiac Imaging Techniques for Tissue Characterization
EMB is the only tool available for in vivo tissue characterization of the right ventricular free wall. However, it is an invasive procedure which carries a certain risk of complications, thus raising ethical concerns. For these reasons, efforts have been made to identify an alternative diagnostic tool able to provide an in vivo tissue characterization and EMB has been used as the reference standard to validate the sensitivity and specificity.
Among imaging techniques, cardiac magnetic resonance has been first employed for differentiating fatty tissue from normal myocardium, through spin echo T1-weighted pulse sequences, but its diagnostic accuracy remains uncertain . Our group first compared the diagnostic accuracy of EMB vs. spin echo cardiac magnetic resonance in patients with the clinical diagnosis of ARVC/D and we found that EMB sensitivity was higher than that of spin echo magnetic resonance (89% vs. 56%) in diagnosing ARVC/D . More recently, the additional diagnostic value of cardiac magnetic resonance has been shown using delayed-enhancement after contrast medium gadolinium administration in order to evaluate the presence of fibrous tissue replacement. To this regard, Tandri et al.  reported an excellent correlation between delayed-en-hancement magnetic resonance and histopatholog-ic findings on EMB. This resulted in the superiority of the former technique in diagnosing ARVC/D, since it can reveal segmental forms, which could be not be detected by EMB due to sampling error, as well as left ventricular involvement. The latter finding is important, since left ventricular involvement may not be detected by EMB due to subepicardial-midwall location of fibro-fatty replacement.
Finally, we demonstrated the value of three-dimensional electro-anatomic voltage mapping for the diagnosis of ARVC/D, by showing that right ventricular fibro-fatty myocardial replacement at EMB corresponds to positive CARTO mapping with low voltage areas .
In conclusion, EMB is a relatively safe interven-tional procedure, which is useful to assess and diagnose ARVC/D. However, since it is an invasive procedure, we must consider ethical and legal issues when advising the procedure. EMB can be useful in patients presenting with ventricular arrhythmias when a diagnosis of ARVC/D is suspected but not established with other noninvasive techniques. Both the clinician and the pathologist should appreciate that the interpretation of EMB specimens requires knowledge of the patient's clinical history as well as the location of the biopsy. The recognition of ARVC/D and the differential diagnosis with other diseases, such as myocarditis and sarcoidosis, influences the type of therapy, risk stratification, and prognosis.
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