**3.2 Endomyocardial biopsy**

Despite the improvement of imaging techniques, endomyocardial biopsy still represents the gold standard for the diagnosis of myocarditis as allow the recognition and the

setting of myocarditis presenting with either arrhythmia or heart failure reported a sensitivity and specificity of more then up to 90% (Mahrholdt et al., 2004). Moreover, CMR was able to differentiate active from healing inflammation in patients presenting with

In particular, De Cobelli and colleagues studied patients presenting with chronic myocarditis assessed with EMB and CMR; they found areas of high signal intensity in T2 weighted images in 36% of patients with histological evidence of active myocarditis, but not in patients with borderline myocarditis. They also found areas of late gadolinium enhancement in 84% and 44% of patients with active and borderline myocarditis, respectively. A midwall pattern of late enhancement was a frequent finding in patients with both active myocarditis and borderline myocarditis, whereas a subepicardial pattern was only observed in patients with histological evidence of active myocarditis (De Cobelli et al., 2004). Taken together, these data support the need to include sequences for the detection of myocardial inflammation when CMR is performed in the non-invasive diagnostic work-up of patients presenting with ventricular arrhythmias (Figure 2). In addition CMR represents an important noninvasive tool in the follow-up of patients with a diagnosis of myocarditis.

Fig. 2. CMR findings in a patient with chronic myocarditis presenting with ventricular arrhythmias. ECG shows repetitive polymorphic ventricular ectopic beats (A). CMR shows

Despite the improvement of imaging techniques, endomyocardial biopsy still represents the gold standard for the diagnosis of myocarditis as allow the recognition and the

sub-epicardial late enhancement in the posterior and postero-lateral walls (B) and transmural edema in the same location (C). Histology shows the presence of active

chronic myocarditis.

myocarditis (D).

**3.2 Endomyocardial biopsy** 

characterization of the myocardial inflammatory process, the assessment of myocyte necrosis and fibrosis as well as the possible detection of viral genomes.

A recent statement from AHA/ACC/ESC (Cooper et al., 2007) aimed to define the role of endomyocardial biopsy in the management of cardiovascular disease, identified 14 clinical scenarios in which the incremental diagnostic, prognostic and therapeutic value of endomyocardial biopsy could be estimated and compared with the procedural risks. According to this statement, in the absence of randomized clinical trials or multicenter studies, endomyocardial biopsy may be considered in the setting of unexplained ventricular arrhythmias (Clinical scenario 13) only in exceptional cases in which the perceived likelihood of meaningful prognostic and therapeutic benefit outweighs the procedural risks (Class of recommendation IIb, Level of evidence C).

Nevertheless there is growing evidence that endomyocardial biopsy may be crucial to clarify the pathological substrate and therefore the cause of otherwise unexplained ventricular arrhythmias, with a possible impact on both treatment and prognosis as detailed further in this chapter.

Accordingly a recent document from the Italian federation of Cardiology (Leone et al., 2009) suggested indications to endomyocardial biopsy different from the above-mentioned statement, in particular with regard to patients with ventricular arrhythmias. The authors graded clinical indications according to the following scheme:


According to this grading, taking into account the potential diagnostic usefulness of endomyocardial biopsy in different myocardial disorders, the authors identified several clinical scenarios. In the presence of sustained and/or life-threatening ventricular arrhythmias, when a myocarditis is suspected or is a possible diagnosis, the grade of recommendation to perform endomyocardial biopsy is 1. Similarly in the presence of AV blocks associated with a clinical context consistent with a diagnosis of myocarditis, recommendations are Grade 2A and 2B in the presence or in the absence of left ventricular dysfunction respectively. Therefore the Italian Federation of Cardiology document seems to more adequately address the diagnostic and therapeutic issues that are frequently faced by cardiologists in everyday clinical practice when dealing with patients with severe ventricular arrhythmias or atrioventricular conduction disturbances and no evidence of ischemic heart disease or overt valvular or myocardial disease.

Both documents do also indicate how many samples are needed in different clinical conditions, how to handle and process myocardial samples, and which studies are necessary to obtain the desired diagnostic information. In the case of clinically suspected myocarditis 5 to 10 samples must be obtained to perform histology and immunohistochemistry studies and molecular bioloy studies to detect the presence of viral genomes. Samples for molecular biology must be flash-frozen in liquid nitrogen and stored at -80°C. Endomyocardial biopsy should better not be performed in patients with clinically suspected myocarditis, if immunohistochemistry and virology studies are not available.

Myocarditis Presenting with Ventricular Arrhythmias:

(amplitude >0.5 and <1.5 mV) (Figure 3).

For colours interpretation see text.

other studies.

Role of Electroanatomical Mapping-Guided Endomyocardial Biopsy in Differential Diagnosis 375

obtained by CARTO showed that small structures such as microaneurysms, can be missed by the mapping catheter and therefore are not properly visualized. These limitations would be partially overcome by importing CT and CMR images and merging them with 3D maps

Similar to CARTO the NavX system is a mapping and 3D nonfluoroscopic intracardiac browsing system that can reconstruct in real time geometry as well as the electric activation of the heart chamber. NavX technology makes use of six surface patch electrodes that generate, once correctly positioned, an electric filed. Within this electric field it is possible to locate in real time the position of every electrophysiology catheter employed during the procedure, including the cardiac bioptome when electically connected to the mapping system. We tested both systems in guiding endomyocardial biopsy execution. Although a comparison between the systems exceeds the scopes of this chapter, CARTO system probably offers a more detailed reconstruction of the ventricular chambers, while NavX system has the advantage of direct real time visulization of the bioptome in the 3D map. In this chapter we will refer to the CARTO system as it was more frequently used in our and

High-density mapping must be obtained in sinus rhythm (reference channel: QRS complex) by sampling at least 100 points in each chamber, uniformly distributed. The voltage maps are then edited setting the point density (fill threshold) at 15 mm and manually eliminating intracavitary points. According to current literature "electroanatomic scar" is defined as an area including at least 3 adjacent points with bipolar signal amplitude <0.5 mV; the reference value for normal endocardium is usually set at 1,5 mV. In the CARTO system the color display to identify normal and abnormal voltage myocardium ranges from red (electroanatomic scar tissue; amplitude <0.5 mV), to purple (electroanatomic normal tissue; amplitude ≥1.5 mV). Intermediate colors represent the electroanatomic border zone

Fig. 3. Example of electroanatomic maps obtained with CARTO (A) and NavX (B) systems.

so that during mapping the catheter can be visualized inside the anatomical images.

The main criticism against a wider use of endomyocardial biopsy in the diagnostic approach to patients with ventricular arrhythmias is represented by the possible sampling error in the presence of a focal myocarditis. In fact, as previously mentioned, the myocardial inflammation in patients presenting with arrhythmias may present a patchy distribution and therefore myocardial samples drawn from a same conventional site (the right side of interventriculrar septum or the left ventricular apex) may not include myocardial tissue involved by the inflammatory process. Increasing the number of samples obtained or arbitrarily changing the site of biopsy can in part minimize sampling error, but these tricks may increase the risks of the procedure. To overcome the sampling error and increase the diagnostic sensistivity of endomyocardial biopsy, we recently demonstrated that in patients presenting with ventricular arrhythmias, electroanatomical mapping may guide the execution endomyocardial biopsy identifying the segments of ventricular wall presenting an abnormal voltage, suggesting an abnormal histological substrate (Pieroni et al, 2009; Casella & Dello Russo, 2009). This technique represents an important innovation in the field of endomyocardial biopsy as reduces sampling error and increases the sensitivity of biopsy, thus possibly reducing the number of samples needed for a complete study of myocardial tissue and therefore reducing the risks of the procedure.

#### **3.3 Electroanatomic mapping**

Electroanatomic mapping allows operators to record intracardiac electrical activation in relation to anatomic location in a cardiac chamber of interest, even during arrhythmia mapping. Several 3D-EAM systems are currently available that accomplish these tasks. When applied properly, such technology allows one to accurately determine the location of arrhythmia origin, define cardiac chamber geometry in three dimensions, delineate areas of anatomic interest, and allow catheter manipulation and positioning without fluoroscopic guidance. These systems often simplify mapping efforts and can enhance procedural success, particularly in cases in which complex arrhythmias and unusual cardiac anatomy are encountered.

The CARTO mapping system (Biosense, Diamond Bar, CA, USA) is a three-dimensional nonfluoroscopic mapping system that utilizes a low-level magnetic field (5 x 10-6 to 5 x 10-5 Tesla) delivered from three separate coils in a locator pad beneath the patient. The magnetic field strength from each coil is detected by a location sensor embedded proximal to the tip of a specialized mapping catheter. The strength of each coil's magnetic field measured by the location sensor is inversely proportional to the distance between the sensor and coil. Hence, by integrating each coil's field strength and converting this measurement into a distance, the location sensor (and therefore, catheter tip location) can be triangulated in space. The mapping catheter has proximal and distal electrode pairs, and a tip electrode capable of radiofrequency energy delivery. This catheter can be moved along a chamber's surface to record local endocardial activation times for arrhythmia mapping, while simultaneously recording location points to generate 3D chamber geometry. Validation studies have shown CARTO to have substantial accuracy in navigating to single points, in returning to prior ablation sites, and in creating a desired length of ablation line. Electrograms recorded from the specialized mapping (NaviSTAR) catheter showed excellent correlation with recordings from standard EP catheters. Moreover human validation studies have shown a good level of spatial precision and accuracy, and realistic reconstruction of chamber geometry and electroanatomic activation during arrhythmia mapping. Nevertheless in our experience, the comparison between biplane right ventricular angiography and the 3D reconstruction

The main criticism against a wider use of endomyocardial biopsy in the diagnostic approach to patients with ventricular arrhythmias is represented by the possible sampling error in the presence of a focal myocarditis. In fact, as previously mentioned, the myocardial inflammation in patients presenting with arrhythmias may present a patchy distribution and therefore myocardial samples drawn from a same conventional site (the right side of interventriculrar septum or the left ventricular apex) may not include myocardial tissue involved by the inflammatory process. Increasing the number of samples obtained or arbitrarily changing the site of biopsy can in part minimize sampling error, but these tricks may increase the risks of the procedure. To overcome the sampling error and increase the diagnostic sensistivity of endomyocardial biopsy, we recently demonstrated that in patients presenting with ventricular arrhythmias, electroanatomical mapping may guide the execution endomyocardial biopsy identifying the segments of ventricular wall presenting an abnormal voltage, suggesting an abnormal histological substrate (Pieroni et al, 2009; Casella & Dello Russo, 2009). This technique represents an important innovation in the field of endomyocardial biopsy as reduces sampling error and increases the sensitivity of biopsy, thus possibly reducing the number of samples needed for a complete study of myocardial

Electroanatomic mapping allows operators to record intracardiac electrical activation in relation to anatomic location in a cardiac chamber of interest, even during arrhythmia mapping. Several 3D-EAM systems are currently available that accomplish these tasks. When applied properly, such technology allows one to accurately determine the location of arrhythmia origin, define cardiac chamber geometry in three dimensions, delineate areas of anatomic interest, and allow catheter manipulation and positioning without fluoroscopic guidance. These systems often simplify mapping efforts and can enhance procedural success, particularly in cases in which complex arrhythmias and unusual cardiac anatomy

The CARTO mapping system (Biosense, Diamond Bar, CA, USA) is a three-dimensional nonfluoroscopic mapping system that utilizes a low-level magnetic field (5 x 10-6 to 5 x 10-5 Tesla) delivered from three separate coils in a locator pad beneath the patient. The magnetic field strength from each coil is detected by a location sensor embedded proximal to the tip of a specialized mapping catheter. The strength of each coil's magnetic field measured by the location sensor is inversely proportional to the distance between the sensor and coil. Hence, by integrating each coil's field strength and converting this measurement into a distance, the location sensor (and therefore, catheter tip location) can be triangulated in space. The mapping catheter has proximal and distal electrode pairs, and a tip electrode capable of radiofrequency energy delivery. This catheter can be moved along a chamber's surface to record local endocardial activation times for arrhythmia mapping, while simultaneously recording location points to generate 3D chamber geometry. Validation studies have shown CARTO to have substantial accuracy in navigating to single points, in returning to prior ablation sites, and in creating a desired length of ablation line. Electrograms recorded from the specialized mapping (NaviSTAR) catheter showed excellent correlation with recordings from standard EP catheters. Moreover human validation studies have shown a good level of spatial precision and accuracy, and realistic reconstruction of chamber geometry and electroanatomic activation during arrhythmia mapping. Nevertheless in our experience, the comparison between biplane right ventricular angiography and the 3D reconstruction

tissue and therefore reducing the risks of the procedure.

**3.3 Electroanatomic mapping** 

are encountered.

obtained by CARTO showed that small structures such as microaneurysms, can be missed by the mapping catheter and therefore are not properly visualized. These limitations would be partially overcome by importing CT and CMR images and merging them with 3D maps so that during mapping the catheter can be visualized inside the anatomical images.

Similar to CARTO the NavX system is a mapping and 3D nonfluoroscopic intracardiac browsing system that can reconstruct in real time geometry as well as the electric activation of the heart chamber. NavX technology makes use of six surface patch electrodes that generate, once correctly positioned, an electric filed. Within this electric field it is possible to locate in real time the position of every electrophysiology catheter employed during the procedure, including the cardiac bioptome when electically connected to the mapping system. We tested both systems in guiding endomyocardial biopsy execution. Although a comparison between the systems exceeds the scopes of this chapter, CARTO system probably offers a more detailed reconstruction of the ventricular chambers, while NavX system has the advantage of direct real time visulization of the bioptome in the 3D map. In this chapter we will refer to the CARTO system as it was more frequently used in our and other studies.

High-density mapping must be obtained in sinus rhythm (reference channel: QRS complex) by sampling at least 100 points in each chamber, uniformly distributed. The voltage maps are then edited setting the point density (fill threshold) at 15 mm and manually eliminating intracavitary points. According to current literature "electroanatomic scar" is defined as an area including at least 3 adjacent points with bipolar signal amplitude <0.5 mV; the reference value for normal endocardium is usually set at 1,5 mV. In the CARTO system the color display to identify normal and abnormal voltage myocardium ranges from red (electroanatomic scar tissue; amplitude <0.5 mV), to purple (electroanatomic normal tissue; amplitude ≥1.5 mV). Intermediate colors represent the electroanatomic border zone (amplitude >0.5 and <1.5 mV) (Figure 3).

Fig. 3. Example of electroanatomic maps obtained with CARTO (A) and NavX (B) systems. For colours interpretation see text.

Myocarditis Presenting with Ventricular Arrhythmias:

3D ventricular map.

safety of the procedure (Avella et al., 2008).

**3.4.2 Safety** 

Role of Electroanatomical Mapping-Guided Endomyocardial Biopsy in Differential Diagnosis 377

Fig. 4. 3D-EAM-guided EMB. With the CARTO system (A) the long sheath with the bioptome inside (arrow) is positioned near to the mapping catheter (arrowhead). With the NavX system (B) the bioptome tip (green point and arrow) can be directly visualized in the

The execution of endomyocardial biopsies drawing myocardial samples from non conventional sites, including the right ventricular free-wall and outflow tract do not increase the risks of the procedure. Since 2006 we perfomed 3D-EAM-guided endomyocardial biopsy in more than sixty patients with about 400 samples obtained. In order to evaluate the safety of this new approach we prospectively analysed the rate of major and minor complications through continuous ECG monitoring during the procedure, ECG and 2D-echocardiography at the end the procedure and after 3 and 6 hours. Major complications included pericardial tamponade with need for pericardiocentesis, hemo- and pneumopericardium, permanent atrioventricular block requiring permanent pacemaker implantation, myocardial infarction, transient cerebral ischemic attack and stroke, severe valvular damage, and death, whereas minor complications included transient chest pain, transient ECG abnormalities, transient arrhythmias, transient hypotension, and small pericardial effusions. The major complication rate was 0% and the minor complication rate was 4.5%: minor complications were represented by small pericardial effusions in 2 patients and chest pain in 1. These rates are in line with those observed in a recent large two-centers study including 755 procedures (right, left and biventricular endomyocardial biopsy) and reporting a major and minor complication rate of 0.82% and 5.1% respectively for right ventricular endomyocardial biopsy (Yilmaz et al., 2010). In literature another study in which 3D-EAM-guided endomyocardial biopsy was performed in 22 patients, reported a higher rate of major complications (1.1%) and minor complications (5.7%), thus suggesting that the expertise of the operators besides the clinical condition and the underlying disorder may influence the

Adequate catheter contact should be confirmed by concordant catheter tip motion with the cardiac silhouettes on fluoroscopy and by adherence of voltage map to angiographic right ventricular shape. To avoid low voltage recordings due to poor contact, the following tools can be used: 1) the signal has to satisfy 3 stability criteria automatically detected by CARTO system in terms of cycle length, local activation time and beat-to-beat difference of the location of the catheter (<2%, <3 ms, and <4 mm, respectively); 2) both bipolar and unipolar signals are simultaneously acquired to confirm true catheter contact through the analysis of local electrogram (in particular the shape of the unipolar electrogram); 3) in the presence of a low voltage area, at least 3 additional points should be acquired in the same site to confirm the reproducibility of the voltage measurement. The anatomical distribution of the pathological areas is evaluated dividing the right ventricular voltage map into five segments: outflow tract, free (anterolateral) wall, inferior and posterior basal segments, apex, and interventricular septum.

#### **3.4 Electroanatomic mapping-guided endomyocardial biopsy**

The main limitation of endomyocardial biopsy to provide a specific diagnosis in patients with ventricular arrhythmias caused by focal myocardial diseases, (i.e. myocarditis or initial forms of arrhythmogenic right ventricular cardiomyopathy), is represented by the sampling error due to the lack of an effective guide in selecting ventricular areas where to perform biopsies. In the last years, after the development of 3D-EAM systems, we introduced a new technique for the execution of endomyocardial biopsies in patients with ventricular arrhythmias (Pieroni et al., 2009).

#### **3.4.1 Technique**

The new approach is aimed to perform endomyocardial biopsies in the ventricular segments presenting electrical abnormalities at electroanatomical mapping. When using the CARTO system, once the electroanatomical map is completed, the mapping catheter is placed in a region of interest of the ventricular wall and the preformed sheath is positioned closed to the catheter tip (Figure 4). Endomyocardial biopsy is then performed in the area with abnormal electrical properties as showed by the map. As previously mentioned, another approach we tested requires the electrical connection of the bioptome to the mapping system: the presence of the metallic jaws makes the bioptome similar to a mapping catheter and it is therefore visualized in the 3D electroanatomical map of the ventricle. In this case, the site of biopsy can be chosen "live", directly mapping the ventricular wall with the bioptome (Figure 4). The latter technique can be adopted when using the NavX mapping system. With both systems we usually performed ventricular angiography before the execution of ventricular mapping, in order to improve the anatomical accuracy of the map, as angiography still represents the gold standard to detect wall motion abnormalities and small aneurysms of the right venticle.

Although no data on specificity and sensitivity of 3D-EAM-guided vs. conventional technique are currently available in literature, it is reasonable that obtaining myocardial samples from areas of the ventricular wall presenting electrical abnormalities will probably reduce the sampling error and the need for multiple biopsies from the same patient (See below). Moreover the combination of 3D-EAM with other imaging tools such as cardiac MRI would further improve our ability to obtain samples from selected regions that present structural, functional and electrical abnormalities.

Fig. 4. 3D-EAM-guided EMB. With the CARTO system (A) the long sheath with the bioptome inside (arrow) is positioned near to the mapping catheter (arrowhead). With the NavX system (B) the bioptome tip (green point and arrow) can be directly visualized in the 3D ventricular map.
