2011 Consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology

Cardiovascular Pathology 21 (2012) 245 – 274

Original Article

2011 Consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology Ornella Leone a,⁎, John P. Veinot b,⁎, Annalisa Angelini c , Ulrik T. Baandrup d , Cristina Basso c , Gerald Berry e , Patrick Bruneval f , Margaret Burke g , Jagdish Butany h , Fiorella Calabrese c , Giulia d'Amati i , William D. Edwards j , John T. Fallon k , Michael C. Fishbein l , Patrick J. Gallagher m , Marc K. Halushka n , Bruce McManus o , Angela Pucci p , E. René Rodriguez q , Jeffrey E. Saffitz r , Mary N. Sheppard g , Charles Steenbergen n , James R. Stone r , Carmela Tan q , Gaetano Thiene c , Allard C. van der Wal s , Gayle L. Winters r a

Bologna, Italy Ottawa, Ontario, Canada c Padua, Italy d Hjoerring, Denmark e Stanford, CA, USA f Paris, France g London, UK h Toronto, Ontario, Canada i Rome, Italy j Rochester, MN, USA k New York, NY, USA l Los Angeles, CA, USA m Southampton, UK n Baltimore, MD, USA o Vancouver, BC, Canada p Pisa, Italy q Cleveland, OH, USA r Boston, MA, USA s Amsterdam, The Netherlands b

Received 3 August 2011; received in revised form 28 September 2011; accepted 7 October 2011

Abstract The Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology have produced this position paper concerning the current role of endomyocardial biopsy (EMB) for the diagnosis of cardiac diseases and its contribution to patient management, focusing on pathological issues, with these aims: • Determining appropriate EMB use in the context of current diagnostic strategies for cardiac diseases and providing recommendations

for its rational utilization

⁎ Corresponding authors. O. Leone is to be contacted at U.O. di Anatomia ed Istologia Patologica, pad. 18, Azienda Ospedaliero-Universitaria S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna. Tel.: +39 051 6364511; fax: +39 051 6364571. J.P. Veinot, Department of Pathology and Laboratory Medicine, The Ottawa Hospital, Ottawa, ON, Canada K1H 8L6. Tel.: +1 613 737 8291; fax: +1 613 737 8712. E-mail addresses: [email protected] (O. Leone), [email protected] (J.P. Veinot). 1054-8807/11/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2011.10.001

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• Providing standard criteria and guidance for appropriate tissue triage and pathological analysis • Promoting a team approach to EMB use, integrating the competences of pathologists, clinicians, and imagers. © 2012 Elsevier Inc. All

rights reserved. Keywords: Biopsy; Consensus; Heart; Molecular biology; Myocardium

1. Introduction Endomyocardial biopsy (EMB) is a commonly performed procedure for the evaluation of cardiac tissue for transplant monitoring [1,2], myocarditis [3–5], drug toxicity, cardiomyopathy (CMP) [3,6–8], arrhythmia [9–14], and secondary cardiac involvement by systemic diseases [8], and for the diagnosis of cardiac masses [15–21]. Others have highlighted EMB utility from prognostic–therapeutic [22–24] and pathogenetic view points [25]. Pathologists should assume responsibility for providing input concerning the utility of the cardiac biopsy for specific diseases and provide guidance for tissue triage and standards of morphologic evaluation [26]. This implies considering both the recent developments in myocardial and viral molecular biology and advances in imaging, electrophysiology, and genetics, and understanding how these techniques can complement traditional histopathology. The invaluable expertise of the pathologist in morphological tissue assessment may be essential in outlining appropriate diagnostic strategies in collaboration with clinicians, imagers, molecular biologists, and geneticists. Referral of the tissue to expert pathologists and molecular biologists is warranted in many cases, given the complexity of the diagnostic tests. Moreover, pathologists should endeavor to use uniform reference criteria and language in their reports, including: •

degrees of diagnostic certainty: for example, certain– definite, probable, possible, nonspecific; • biopsy sample adequacy evaluation: for example, optimal, suboptimal. These guidelines should be viewed as recommendations and not as mandatory requirements. They represent an overall consensus of the committee members and the opinions expressed are not necessarily those of all members of the Association for European Cardiovascular Pathology and/or the Society for Cardiovascular Pathology. 2. Recommendations, limits, and precautions The modern view of diagnostic EMB requires the pathologist to have specific professional training [27], use accurate specimen processing [28], and support when warranted the traditional histological examination with histochemical, immunohistochemical (IHC), molecular, or ultrastructural tests (Tables 1 and 2) [24,29–45] and apply

standardized diagnostic histopathologic criteria to minimize EMB reporting variability [2,46,47]. As EMB is usually a nontargeted biopsy procedure and false-negative results are possible, especially when used for multifocal or microfocal or localized diseases [36,48,49], some general rules can optimize diagnostic accuracy: ■ ■

EMB timing appropriate to specific disease [24,50,51], adequate bioptic sampling with multiple specimens from different sites (guided by imaging techniques when indicated) [26,36,52,53].

3. Techniques and risks EMB involves percutaneous insertion into the heart of a fluoroscopically or ultrasound-directed sheath, which allows safe and rapid insertion of the bioptome and facilitates obtaining multiple specimens. Bioptic samples can be taken from the right ventricle, via the venous route through jugular, subclavian, or femoral veins, or from the left ventricle with transseptal puncture or by direct access through a peripheral artery, usually the femoral or brachial artery [5,54,55]. For better anatomic definition of the specimen site, noninvasive techniques of cardiac imaging may usefully be associated, such as gadolinium magnetic resonance imaging (MRI) in targeting bioptic specimens in suspected myocarditis [56] and electroanatomic mapping guided techniques in arrhythmogenic right ventricular cardiomyopathy (ARVC) [10,11,57]. Echocardiography allows accurate intracardiac insertion of the bioptome without the use of ionizing radiation [58,59] and may be useful in biopsy of cardiac masses. The principal complications of EMB include hematoma, arteriovenous fistula, vasovagal reaction, pneumothorax, Table 1 Technical procedures for light microscopy examination Routine procedure Rapid procedure for transplant and urgent diagnostic biopsies Cutting (routine and rapid)

Fixation in buffered formalin for 15–30 min Processing in automatic processor overnight Microwave fixation Processing in microwave or vacuum-prepared automatic processor (maximum 30–40 min) Serial and numbered sections of paraffinembedded biopsies using at least two thirds of the samples Half the cut sections are stained with hematoxylin–eosin; the others are mounted on sialinized slides (or on slides with polylysine or electrostatic charge) for possible further stains.

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Table 2 Stains on paraffin-embedded and frozen tissue Stains on formalin-fixed and paraffin-embedded sections Histomorphological and ▪ Masson or Mallory trichrome, Movat pentachrome, Weigert–Van Gieson for collagen and elastic fibers histochemical stains ▪ PAS with and without diastase for glycogen storage (better on frozen sections) ▪ Congo red, sulfate alcian blue, or S/T thioflavin for amyloid ▪ Perls' iron IHC stains ▪ CD45, CD20, CD3, CD4, CD8, CD68/PGM1, HLA-DR. HLA-ABC for inflammatory and cellular infiltrate assessment ▪ Transthyretin, kappa and lambda chains, apolipoprotein, amyloid A component for amyloid typing ▪ Appropriate antibodies for neoplasm characterization ▪ Dystrophin, lamin A/C a, desmin, a plakoglobin, a N-cadherin a for some genetic CMPs Stains on frozen sections Histomorphological and histochemical stains Histoenzymatic stains Immunofluorescence stains

Sudan black, oil red 0, PAS with and without diastase SDH and COX Many immunostains listed above, including amyloid subtypes and desmosomal proteins, may be performed on frozen tissue. Frozen tissue may be better than immunohistochemistry for dystrophin (−COOH and NH2 ends, intermediate domain) and HLA-ABC.

PAS = periodic acid-Schiff; SHD = succiante dehydrogenase; COX = cytochrome oxidase. a

In the course of validation for diagnostic use.

arrhythmia, heart block, infection, tricuspid valve damage, pulmonary embolism or systemic embolism during left ventricular biopsy, and cardiac chamber perforation with possible hemopericardium and cardiac tamponade [60,61]. In skilled hands, EMB is a safe procedure [55]. The overall complication rate is low (1%–2%) [5]. In particular, the risk of cardiac perforation was estimated at 0.4% in the largest case records in the world published in 1980 [62] and at 0.12% in more recent data regarding a large number of patients (3048 cases) [63].

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brady- or major ventricular tachyarrhythmias to identify or exclude possible myocardial causes of arrhythmias (e.g., myocarditis, ARVC, sarcoidosis, etc.), chronic heart failure to evaluate myocardial diseases with dilated–hypokinetic or hypertrophic–restrictive phenotype, acute exacerbation of chronic CMP, investigation of cardiac masses, and assessment of cardiac transplant biopsy specimens.

5. Pathological section 4. Patient selection and clinical indications 5.1. General sampling and handling Considering the cost–benefit ratio and possible procedural risks, careful patient selection is appropriate [24], using these criteria: 1. Clinical setting: EMB should be considered in the context of a sequential diagnostic process, after other appropriate basic clinical–instrumental tests have excluded various diseases and focused on the possible diagnoses [26,64]; 2. EMB effects on patient clinical management [26,38,65–74]: ■ obtain a definite diagnosis (or exclude diseases) for therapy, clinical management, and prognosis; ■ monitor the disease clinical course and therapeutic efficacy; ■ reduce misdiagnosis and improve risk management. 3. Different diagnostic potential of EMB in various cardiac diseases [26] (see part on “specific diseases” and summary Table 3). The main clinical scenarios for use of EMB are [3,6,8,24,26,73]: ■ new-onset b6 months unexplained heart failure,

Pathological analysis of EMB is complex and requires standard protocols [26,75]. Therefore, it is vital that the pathologist be provided with clinical information as an aid for diagnosis and to guide the most appropriate technical choices. 5.1.1. Optimal specimen procurement and triage ■ At least three, preferably four, endomyocardial fragments, each 1–2 mm [3] in size, should immediately be fixed in 10% buffered formalin at room temperature for light microscopic examination. In expected focal myocardial lesions, additional sampling is recommended. ■ If indicated, one or two specimens may be snap frozen in liquid nitrogen and stored at −80°C for possible molecular tests or specific stains. Otherwise, fragments may be stored in RNA-later (a solution preventing RNA degradation) at room temperature. ■ If indicated, one fragment should be fixed in 2.5% glutaraldehyde or Karnovsky solution for ultrastructural tests.

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Table 3 Endomyocardial biopsy diagnostic potential in cardiac diseases with grading of recommendation EMB diagnostic potential and histological notes

Technical aspects and tissue triage

Grading of recommendation

Myocarditis/inflammatory cardiomyopathy

Definite diagnosis: lymphocytic, granulocytic, polymorphous, eosinophil, necrotizing eosinophilic, giant cell, granulomatous myocarditis, with or without associated myocyte damage/ necrosis. Histological findings alone or together with molecular techniques may be able to specify the etiology of the inflammatory disease.

Recommended: In addition to formalin fixation, two fragments (or one if greater than 3 mm2) could be snap-frozen or preserved in RNA-later for virus molecular investigation. One peripheral blood sample (5–10 ml) collected in EDTA or sodium citrate tubes. NB: Timing and number of EMB and serial histological sections are important for sensitive detection of myocarditis.

S

Cardiac sarcoidosis

Definite diagnosis: noncaseous granulomatous myocarditis. Histological findings pathognomonic or highly suggestive of the disease.

Possible increased utility with guided biopsy procedure.

S

Myocardial diseases due to drugs and chemical/“toxic” substances

Probable/possible diagnosis: hypersensitivity myocarditis (histological findings suggestive of the disease), toxic damage.

Glutaraldehyde or Karnowsky solution fixed fragment for electron microscopy may be useful for cardiotoxicity evaluation of some drugs.

S: hypersensitivity myocarditis M: cardiotoxicity

Peripartum cardiomyopathy

Probable diagnosis: myocarditis/borderline myocarditis. Histological findings give some indications of probable disease.

Fresh sample may be useful for virus molecular biology. Important: EMB timing

M

Cardiac amyloidosis

Definite diagnosis: amyloid infiltration. Histological findings+histomorphological stains are pathognomonic.

Recommended: Congo red, modified sulfated alcian blue, or thioflavin T stains. IHC, IF, immunoelectron microscopy, protein sequencing, and/or mass spectrometry to establish the type of amyloid. Frozen tissue is required for IF and protein sequencing

S

Iron overload

Definite diagnosis: iron intracellular deposition. Histological findings+iron staining are pathognomonic.

Iron staining is advisable for all diagnostic EMBs from patients with unexplained DCM.

S

Glycogen storage diseases: Type II glycogenosis (Pompe disease) Type III glycogenosis (Cori disease) Type V glycogenosis (Andersen disease) Danon disease PRKAG2 mutations

Probable diagnosis: diffuse intracellular glycogen storage. Histological findings may be suggestive, but not specific, for the diagnosis.

Recommended: Fresh sample for histochemical stains and glutaraldehyde or Karnowsky solution fixed fragment for electron microscopy are useful to assess the nature of intracellular deposits.

S

Anderson–Fabry disease

Probable diagnosis: histology: hypertrophic vacuolated cells with dislocation of contractile elements to the periphery of myocytes. Histological findings may be suggestive, but not specific for diagnosis.

Recommended: Glutaraldehyde or Karnowsky solution fixed fragment may be useful for electron microscopy to assess electron dense concentric lamellar bodies.

S

Desmin cardiomyopathy

Definite diagnosis based on ultrastructural findings: abnormal granulofilamentous aggregates of desmin-type intermediate filaments in the cytoplasm of cardiomyocytes (interfibrillar areas) and at the Z band level. Nonspecific light histological findings.

Recommended: Glutaraldehyde or Karnowsky solution fixed fragment for electron microscopy necessary. Immunoelectron microscopy confirms that these aggregates are formed by desmin.

S

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Clinically suspected disease

Definite diagnosis in Duchenne muscular dystrophy: absence of dystrophin in myocyte sarcolemma. Probable diagnosis in Becker muscular dystrophy: extensive irregularities and discontinuities of dystrophin in myocyte sarcolemma. Nonspecific histological findings.

Recommended: dystrophin IHC or IF stain in which dystrophin is markedly diminished or absent in affected individuals. Fresh sample is required for IF and may be required for IHC.

M

Laminopathies (lamin A/C)

Nonspecific diagnosis: interstitial/replacement fibrosis, myocyte hypertrophy and vacuolization, enlarged and irregular-shaped nuclei. Nonspecific histological findings.

Data on diagnostic use of IHC staining for lamin A/C in human EMB are not available. In centers performing IF, fresh samples are required.

N

Mitichondrial cardiomyopathies

Probable/possible diagnosis: morphologically altered mitochondria by electron microscopy Nonspecific histological findings: enlarged myocytes with extensive cytoplasmic vacuolization

Recommended: Fresh sample is needed for histoenzymatic stainings. Glutaraldehyde or Karnowsky solution fixed fragment is needed for ultrastructural tests.

M (especially in isolated MIC)

Arrhythmogenic right ventricle cardiomyopathy

Probable diagnosis: fibrous or fibrofatty replacement and myocardial atrophy. Nonspecific histological findings.

IF or IHC for plakoglobin and other cellular junction proteins is promising test (being validated). EMB from the interventricular septum may not be informative. Diagnostic accuracy increases if EMB sampling site is guided by imaging (MRI) or electrophysiological (electroanatomic mapping) techniques.

S (in selected cases having no clear-cut diagnosis with noninvasive and other invasive procedures)

Loeffler fibroplastic endocarditis/ endomyocardial fibrosis

Definite diagnosis in the acute phase: endomyocardial infiltrates rich in degranulated eosinophils and endocardial thrombosis. Specific histological findings. Possible diagnosis in the chronic phase: evidence of endocardial fibrous thickening and subendocardial myocyte abnormalities. Nonspecific histological findings.

EMB timing is important as the utility of biopsy probably decreases with disease time course.

S in the acute–subacute phase

Cardiac tumors

Definite diagnosis

IHC useful for tumor typing

S

Hypertrophic cariomyopathy (sarcomeric mutations)

Possible diagnosis: myocyte hypertrophy, interstitial and/or substitutive fibrosis, disarray, and possible small vessel disease. Nonspecific histological findings.

M: The clinical diagnosis of HCM usually relies on noninvasive diagnostic tools, and EMB is not recommended in the diagnostic workup. However, in selected cases, EMB may be helpful in excluding infiltrative or storage diseases with variable diagnostic degrees of certainty.

Idiopathic restrictive cardiomyopathy

Possible diagnosis: normal myocardium, and/or fibrosis and/or disarray. No other identifiable diseases causing restrictive phenotype. Nonspecific histological findings.

M: EMB is unable to give a specific diagnosis. However, in selected cases, EMB may be helpful in excluding infiltrative or storage diseases. Useful in the workup of constriction versus restriction.

Idiopathic dilated cardiomyopathy

Possible diagnosis: myocyte hypertrophy, nuclear alterations, perinuclear halo, with or without fibrosis. Nonspecific histological findings.

M: EMB is unable to give a diagnostic picture, but may be helpful in excluding other diseases.

Heart transplantation

Definite diagnosis: acute cellular rejection, some infectious disease, PTLD, and nonrejection posttransplant findings. Specific histological findings.

Recommended: C4d IHC/IF staining for AMR. Fresh sample is required for IF staining.

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Dystrophin cardiomyopathy

S

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DCM, dilated cardiomyopathy; EDTA, ethylenediaminetetraacetic acid; EMB, endomyocardial biopsy; IF, immunofluorescence; IHC, immunohistochemistry; MRI, magnetic resonance imaging; M, mixed; N, not supported; PTLD, posttransplant lymphoproliferative disorders; S, supported.

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A sample of peripheral blood (5–10 ml) in EDTA or citrate from patients with suspected myocarditis allows for molecular testing for the same viral genomes sought in the myocardial tissue, and a sample from patients with genetic CMPs allows for genetic tests. 5.1.2. Pathological analysis • Light microscopic examination is routinely performed on formalin-fixed and paraffin-embedded tissue (Table 1) and includes, when appropriate: • multiple and numbered hematoxylin–eosin section examination; • additional histochemical, histomorphologic, and IHC stains performed in the majority of cases on paraffin-embedded fragments or, in limited cases, on frozen sections; • morphometric analysis on sections cut for histochemical, histoenzymatic, and IHC tests. • Molecular tests, such as polymerase chain reaction (PCR), quantitative or qualitative, and reverse transcriptase-PCR on the fragments frozen in liquid nitrogen or stored in RNA-later. • Transmission electron microscopy examination on fragments fixed in glutaraldehyde or Karnowsky solution and embedded in resin (Epon). Ultrastructural analysis is preceded by examination of semithin sections stained with toluidine blue. Table 2 details the stains which may be performed, according to histological picture and clinical information. 6. Grading/evidence of recommendation Existing recommendations face limitations since, at present, they are not supported by controlled and randomized clinical studies [26,64]. Only control–case record series; registries; single-center case records, often limited in number; and expert opinions are available, and moreover, the majority of publications are pathological diagnosis focused. A cardiologist should not perform EMB “in the dark,” but only after consideration of the clinical presentation suggests a possible diagnosis and restricts the field of possible diseases. We strongly suggest using the following recommendation grading, based on published peer-reviewed evidence on various expected diseases. ■

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■

S: published peer-reviewed evidence SUPPORTS the utility of the test. M: published peer-reviewed evidence is MIXED concerning the utility of the test. N: there is NO published peer-reviewed evidence assessing the utility of the test.

Table 3 summarizes EMB diagnostic potential and grading of recommendation in cardiac diseases, detailing the histological picture and special technical notes.

7. EMB in specific diseases 7.1. Myocarditis/inflammatory CMP 7.1.1. Evidence of recommendation: S Myocarditis is an inflammatory disease of the myocardium associated with cardiac dysfunction. Three forms of inflammatory CMP are recognized: infectious, autoimmune, and idiopathic. Due to the variable clinical manifestation from latent to very severe clinical forms, such as acute congestive heart failure, life-threatening arrhythmias, and sudden death, the prevalence of myocarditis is still unknown and probably underestimated. In spite of the development of various diagnostic modalities, early and definite diagnosis of myocarditis still depends on the detection of inflammatory infiltrates in EMB specimens according to the Dallas criteria (Table 4) [46,76–79]. Fig. 1 shows two cases of viral lymphocytic myocarditis (in Fig. 1A–C, myocarditis is caused by enterovirus; in Fig. 1D–F, it is caused by adenovirus). Giant cell myocarditis may present as sudden or chronic heart failure. When it presents as an acute disease, EMB may detect it. This is important as the disease is sensitive to therapy with immunosuppressants [80]. Fig. 2 demonstrates the typical histological findings of giant cell myocarditis. In addition, in immune inflammatory diseases, cardiac involvement may occur in the course of systemic connective tissue diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, ankylosing spondylitis, dermatopolymyositis, and scleroderma. Cardiac involvement is more frequent in SLE and in dermatopolymyositis, and includes a

Table 4 EMB diagnosis of myocarditis: the Dallas criteria [77] Definition of idiopathic myocarditis: “an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of the ischemic damage associated with coronary artery disease” Classification First biopsy • Myocarditis with/without fibrosis • Borderline myocarditis (repeat biopsy may be indicated) • No myocarditis Subsequent biopsy • Ongoing (persistent) myocarditis with or without fibrosis • Resolving (healing) myocarditis with or without fibrosis • Resolved (healed) myocarditis with or without fibrosis Descriptors

Distribution Extent Type

Inflammatory infiltrate

Fibrosis

Focal, confluent, diffuse Mild, moderate, severe Lymphocytic, eosinophilic, granulomatous, giant cell, neutrophilic, mixed

Endocardial, interstitial Mild, moderate, severe Perivascular, replacement

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Fig. 1. (A–C) Viral active lymphocytic myocarditis in 35-year-old male. (A–B) EMB showing a multifocal moderate lymphomonocytic inflammatory infiltrate (A, hematoxylin–eosin, 25×); at higher magnification, myocyte damage (arrow) associated with inflammatory infiltrate is evident (B, hematoxylin–eosin, 200×). (C) PCR gel electrophoresis for enterovirus. Line 1: DNA marker (VIII factor); line 2: positive control for enterovirus (180 bp); line 3: EMB positive for enterovirus; line 4: negative control (reagents without DNA). (D–F) Viral active lymphocytic myocarditis in 5-year-old female. (D–E) EMB showing a diffuse lymphomonocytic infiltrate (D, hematoxylin–eosin, 100×) associated with myocyte damage (arrows) (E, hematoxylin–eosin, 200×). No viral cytopathic effects are present. (F) PCR gel electrophoresis. Line 1: DNA marker (VIII factor); line 2: adenovirus positive control (308 bp); line 3: EMB positive for adenovirus; line 4: beta-globin amplification (housekeeping gene, 269 bp); line 5: negative control (reagents without DNA).

range of clinical expressions, from conduction diseases to progressive cardiac dysfunction with the clinical features of dilated cardiomyopathy (DCM). The EMB may be helpful in distinguishing between SLE-related myocarditis and cardiotoxic effects of chloroquine, a drug for treatment of the disease. Furthermore, the pathologic involvement of the heart can occur in many other immunologic systemic diseases including Churg–Strauss disease, Wegener granulomatosis, sarcoidosis, Takayasu arteritis, and idiopathic inflammatory

bowel disease. Such examples further emphasize the relevance of receiving updated clinical information. Cardiac histopathology in these immune diseases can be polymorphous depending on the type or the evolutive phase of the disease; however, EMB frequently reveals a myocardial inflammatory picture and/or vasculitis indicating heart involvement [82,83]. Myocarditis is found in about 10% of cases of clinical DCM, so may play a role in the pathogenesis of the DCM group [84,85]. Myocarditis may evolve into DCM as the

Fig. 2. (A) Giant cell myocarditis in 43-year-old female. Histology: EMB shows a severe polymorphous inflammatory infiltrate (lymphocytes, macrophages, eosinophil granulocytes, multinucleated giant cells) associated with diffuse myocyte damage (necrosis and degeneration) (hematoxylin–eosin, original magnification 100×). (B) Detail of picture (A) showing multinucleated giant cells at higher magnification (arrows) (hematoxylin–eosin, 400×). From Leone et al., modified [81].

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initial viral infection may lead to myocyte destruction, followed by a chronic immunological attack of the myocytes with autoantibodies and involvement of the cellular immune system.

principal antibodies used for immunophenotype characterization are CD45, CD45RO, CD3, CD20, CD4, CD8, and CD68/PGM1. Additional stains relevant to immune activation include HLA-ABC and HLA-DR.

7.1.2. EMB diagnostic potential EMB is a key diagnostic tool [86,87]. Imaging techniques (echocardiography, nuclear imaging, and magnetic resonance) can be useful for determining the best place to biopsy, but these cannot replace EMB for a definitive diagnosis of myocarditis. EMB can provide:

7.1.4.1. Molecular analysis for viral detection. The introduction of new molecular techniques, particularly amplification methods like PCR or nested-PCR, allows the detection of low-copy viral genomes from even an extremely small amount of tissue such as in an EMB specimen [88–91]. Molecular techniques may be helpful as an ancillary diagnostic tool only with other mandatory investigations (clinical and morphological) and should be performed by those with this specific expertise. Molecular testing may be done in a special laboratory separate from the histology laboratory. Peripheral blood analysis should accompany the molecular tissue analysis so that the results may be interpreted in the correct context. Knowledge of the viral findings of the patient's blood and of the incidence of the virus in the patient's community is an important datum to be aware of before interpretation of the myocardial specimen molecular results. Appropriate quality assurance and control tissues for molecular biology are important. The pathologist's role is to gather the available histological and molecular data and provide an interpretation. The principal viruses to be considered are adenovirus, cytomegalovirus, enterovirus, Epstein–Barr virus, hepatitis C virus, herpes simplex virus 1 and 2, human herpes virus 6, influenza viruses A and B, parvovirus B19, and rhinovirus [35,36]. The Dallas criteria have proven useful in the histological assessment of myocarditis, but some investigators feel that these criteria have some limitations including lack of definition and quantification of myocyte damage, lack of quantification of the inflammatory infiltrate, lack of characterization (site/topography) and quantification of fibrosis, and lack of an etiological characterization [76]. These criticisms are variably accepted. The diagnosis of myocarditis by EMB is thus in evolution, as every medical test and procedure go through. Data are limited and conflicting, so the acquisition of reliable data is essential to determine the optimal means for diagnosis. From this standpoint, we suggest that pathological diagnosis of myocarditis may report histological findings including the cell type, immunophenotypic characterization of the inflammatory infiltrate, and molecular viral data, if these are available. An EMB registry might be useful. If EMB for suspected myocarditis were to be routinely performed following a standardized protocol, important and extremely helpful information could well be obtained on this complex and varied disease, including epidemiology as well as etiology and prevalence, since this latter is very likely underestimated.

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Definitive diagnosis of inflammatory myocarditis Identification of the likely etiology (both infective and immunological) Monitoring of inflammatory CMP Confirmation of cardiac involvement (myocarditis or vasculitis) in the context of systemic immune diseases

To assess immune etiology, cardiac autoantibodies in the blood should also be investigated. At histology, routine (hematoxylin and eosin) stained serial sections should be evaluated for the presence of: ■

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the inflammatory infiltrate: immunotype and semiquantitative or quantitative analysis myocyte damage type: myocytolysis, apoptosis, or other myocyte alteration fibrosis: type and semiquantitative or morphometric analysis; special stains may be helpful

Immunotyping and quantitation of the inflammation and fibrosis are controversial, with strong opinions regarding the utility of these. In view of this, both Pathologist Groups support quantification, as a research tool, to obtain data to assess its utility. 7.1.3. Specific technical aspects If viral genome detection is being pursued, it is recommended to have: ■

■

two fragments (or one if greater than 3 mm 2), snapfrozen or preserved in RNA-later, for molecular investigation one peripheral blood sample (5–10 ml) collected in EDTA or sodium citrate tubes

NB: Timing and number of EMB specimens are important for sensitive detection of myocarditis. 7.1.4. IHC and molecular aspects Immunohistochemistry is a useful corollary to identify and characterize inflammatory infiltrates in EMB. The

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7.2. Idiopathic/primary DCM

7.3. Dystrophin CMP

7.2.1. Evidence of recommendation: M DCM is a disease of the myocardium characterized by dilatation and impaired systolic contraction of one or both ventricles [92]. DCM is a common CMP and may affect patients of any age. The wall thickness may be reduced due to stretching of the myocytes as the ventricle dilates. There may be atrial or ventricular thrombus in all chambers and on both sides. DCM may be complicated by biventricular congestive heart failure; arrhythmias, both supraventricular and ventricular; thromboemboli; and sudden death. To reach a diagnosis of idiopathic/primary DCM, clinical information is very helpful, including knowledge of other illnesses and systemic diseases, and exclusion of significant valvular heart disease, coronary artery disease, and systemic arterial hypertension. Although the histological findings are nonspecific, EMB diagnosis may be valuable in the workup of heart failure (active myocarditis versus acute exacerbation of a chronic disease) or arrhythmias to exclude specific diseases.

7.3.1. Evidence of recommendation: M Mutations in the dystrophin gene can result in DCM. The phenotype may be exclusively progressive cardiac failure (Xlinked DCM), or more commonly, it also involves skeletal muscle, causing concurrent Duchenne or Becker muscular dystrophies. Becker-type dystrophy can be associated with mild functional skeletal involvement and more profound myocardial involvement. Dystrophin gene, found on the X chromosome (Xp21), is maternally inherited, and the phenotype is only seen in males. Dystrophin mutations have been found in up to 7% of males with DCM, representing a minor but not insignificant cause of “idiopathic” DCM [41]. Diagnosing dystrophin-induced DCM is complex. An elevated serum creatinine phosphokinase has been reported in N80% of affected men, suggesting that this serum test may be useful as an initial screening test.

7.2.2. EMB diagnostic potential and IHC aspects Nonspecific histology includes fibrosis, myocyte nuclear enlargement, and often sarcoplasmic degenerative changes such as a perinuclear halo, and myocyte clearing or vacuolization. Myofiber attenuation and stretching may be marked. Nuclei may be irregular in shape and hyperchromatic. Fibrosis, most commonly pericellular, endocardial and substitutive, may also be perivascular in location. Immunohistochemistry for muscle fiber abnormality may be attempted in CMPs associated with skeletal myopathy. However, skeletal muscle biopsy may be easier in such patients. 7.2.3. Genetic aspects Familial DCM represents approximately 30% of cases of DCM [93–95]. Nonfamilial and familial forms of DCM cannot be distinguished by routine histology or imaging [96].

7.3.2. EMB diagnostic potential and IHC aspects Routine light microscopy may identify nonspecific findings including interstitial/replacement fibrosis, fibrofatty replacement, myocyte hypertrophy, vacuolization, and myocytolysis (Fig. 3A). The most useful analysis is a dystrophin IHC stain in which dystrophin is markedly diminished or absent in affected individuals (Fig. 3B). However, this is best done in skeletal muscle biopsies along with IHC for spectrin, a positive control for membrane staining. By ultrastructural examination, membrane irregularities including discontinuation of the sarcolemma have been described, although this is not a specific finding. Ultimately, genetic mutation screening is necessary to make a definitive diagnosis and should be considered the primary diagnostic tool. 7.3.3. Genetic aspects We suggest genetic testing as a first-line diagnostic tool when dystrophin CMP is strongly suspected based on clinical findings and mode of inheritance. However, in cases of “idiopathic” DCM, dystrophin IHC and serum creatinine phosphokinase can be used to entertain this diagnosis and

Fig. 3. A 36-year-old male patient with Becker muscular dystrophy. (A) Histology: marked interstitial fibrosis, myocyte sarcoplasmic vacuolization, hypertrophy, and focal atrophy (Mallory trichrome, 200×). (B) Immunohistochemistry shows loss or discontinuity of sarcolemmal stain for dystrophin molecule (200×).

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focus genetic testing. In Becker disease with solely or predominantly cardiac involvement and in X-linked CMP, EMB may be the only diagnostic tool able to raise the question of a dystrophin-gene-related disease. 7.4. Mitochondrial cardiomyopathies (MICs) 7.4.1. Evidence of recommendation: M (especially in isolated forms) MICs can occur both in the context of multiple organ syndromes and as the sole or predominant feature of respiratory chain dysfunction. MICs may occur at any age, and their frequency in patients with mitochondrial disease is high. Because of the dual genetic control of respiratory chain, MICs can be caused by mutations in either the mitochondrial or the nuclear genome. Thus, MICs can be maternally inherited, autosomal dominant or recessive, Xlinked, or sporadic. The phenotype is usually hypertrophic, nonobstructive type. Biventricular dilation and congestive heart failure also can occur in the natural history of the disease [97,98]. 7.4.1.1. EMB diagnostic potential. When MICs are associated with multisystemic disease, the diagnosis of mitochondrial etiology relies upon skeletal muscle biopsy, biochemical analysis, and genetic tests. However, in isolated MICs, when the skeletal muscle biopsy is unremarkable, EMB can be useful. At histology, myocytes are enlarged, with extensive cytoplasmic vacuolization (Fig. 4A). Histoenzymatic stains on frozen sections for succinate dehydrogenase (SDH) and cytochrome oxidase (COX) are required for the diagnosis. The presence of COX-negative myocytes and an increased staining for SDH demonstrate the respiratory chain defect and mitochondrial proliferation (Fig. 4B). At ultrastructure, there is diffuse mitochondrial proliferation (Fig. 4C), often with giant mitochondria, mitochondrial inclusions, abundant cytoplasmic glycogen, lipid droplets, and contractile myofibril loss. 7.4.2. Molecular aspects and genetics In isolated MICs, we recommend storing frozen myocardial tissue to check for mutations, deletions, or depletion of mitochondrial DNA in homogenate tissue, which may pass unnoticed when using DNA extracted from peripheral blood. Mutations of most genes coding for structural and functional components of mitochondria can be appropriately assessed using peripheral blood.

Fig. 4. Maternally inherited MIC due to a point mutation in the gene encoding for isoleucine tRNA. (A) Histology: extensive myocyte vacuolization (hematoxylin–eosin, 40×). (B) Combined histoenzymatic stain for nuclear-encoded SDH (in blue) and mitochondrial-encoded COX (brown). Several myocytes are stained in blue, indicating proliferation of mitochondria lacking COX activity (100×). (C) Ultrastructural view of interfibrillar mitochondria proliferation (5000×).

7.5. Lamin A/C-related CMP 7.5.1. Evidence of recommendation: N Lamins are intermediate filaments that play a structural role in the formation of the nuclear lamina that lines the inner core of the nuclear envelope of cells. The proteins lamin A and lamin C are the products of alternative splicing of the gene LMNA. The genes LMNB1 and LMNB2 encode

lamins B1 and B2, respectively. Mutations in the LMNA gene produce clinical disease with several distinct syndromes such as Charcot–Marie–Tooth disease type 2, mandibuloacral dysplasia, familial partial lipodystrophyDunnigan type, restrictive dermopathy, and premature aging syndromes (Hutchinson–Gilford progeria and atypical Werner syndrome) as well as CMP.

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Fig. 5. A 32-year-old female patient with lamin AC deficiency DCM. (A–B) EMB showing interstitial/replacement fibrosis, myocyte hypertrophy and vacuolization, and enlarged and irregular-shaped nuclei. (A: Mallory trichrome 250×; B: hematoxylin–eosin 400×). (C) Immunohistochemical staining for lamin A/C shows discontinuity or loss in perinuclear staining (400×). (D) Normal IHC control with regular and strong perinuclear staining (400×).

Cardiovascular manifestations in patients with mutations in lamin A/C include conduction system abnormalities early in the disease with pacemaker requirement in up to 44% of the patients over 30 years of age. These patients may show evidence of delayed intracardiac conduction. Their electrocardiograms show low-amplitude P waves and prolonged PR interval with normal QRS complex. Atrial fibrillation appears later with thromboembolic complications. Prognosis is poor, with congestive heart failure as the most common clinical evolution [99]. 7.5.2. EMB diagnostic potential Pathologic examination shows nonspecific features of DCM: interstitial/replacement fibrosis, myocyte hypertrophy and vacuolization, and enlarged irregularly shaped nuclei (Fig. 5A, B) [100,101]. 7.5.3. IHC and molecular aspects Data on diagnostic use of IHC staining for lamin A/C in human EMBs are not available, although promising research studies are ongoing (Fig. 5C). Ultrastructural nuclear changes have been reported including large nuclear blebs (not folds) [102]. These reports consist mostly of nonspecific features of hypertrophy, nuclear contour irregularity, and degeneration of the cardiac myocytes. At this juncture, the diagnosis should be established by genetic testing.

7.6. Arrhythmogenic right ventricle cardiomyopathy 7.6.1. Evidence of recommendation: S (in selected cases having no clear-cut diagnosis with noninvasive and other invasive procedures) ARVC is a rare primary heart muscle disease with a 1:2000–1:5000 prevalence in the general population. Genetic defects have been identified in genes mostly encoding for desmosomal proteins. The clinical presentation includes ventricular arrhythmias [103]; sudden death, particularly in the young and athletes; and also, less commonly, mechanical dysfunction. With the latter presentation, such systolic dysfunction may mimic DCM [104]. From the pathology viewpoint, ARVC is characterized by progressive atrophy of the ventricular myocardium with fibrofatty replacement. The septum is rarely involved. The clinical diagnosis is a major challenge since there is no single gold standard and it requires fulfilment of major and minor diagnostic criteria (scoring system) [9]. 7.6.2. EMB diagnostic potential If noninvasive and other invasive procedures do not allow a clear-cut diagnosis of ARVC, right ventricle EMB may serve as a complementary diagnostic tool (tissue characterization diagnostic criterion). Moreover, in sporadic forms (not familial), EMB can be useful to exclude phenocopies (i.e., myocarditis, sarcoid, etc.).

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Fig. 6. EMB findings in a proband affected by a diffuse form of ARVC. The three biopsy samples are from different regions of the right ventricle free wall. In each, there is extensive fibrofatty tissue replacement with myocardial atrophy, which is a major criterion (i.e., residual myocytes b60% by morphometric analysis or b50% if estimated) (Mallory trichrome, 60×). From Marcus et al., modified [105].

Histological examination (on hematoxylin–eosin- and connective-tissue-stained slides) shows fibrous or fibrofatty replacement and myocardial atrophy (Fig. 6), which are not specific (see, for example, muscular dystrophies). Fatty tissue alone is not considered diagnostic for ARVC since fat is normally found in the myocardium, particularly at the anterolateral apical right ventricular free wall. Myocyte hypertrophy and vacuolization as well as inflammatory infiltrates can be present. Histomorphometry evaluation of right ventricle EMB is mainly supported as a research tool to gather data. However, according to the updated diagnostic criteria, fibrous or fibrofatty replacement with b60% residual myocardium in at least one EMB sample is a major criterion, and 60%–75% residual myocardium is a minor criterion for ARVC [12,105]. 7.6.3. Specific technical aspects EMB samples from the septum may not be that helpful. If possible, consideration to biopsying the affected area, including the right ventricle free wall, may be considered. Diagnostic accuracy increases if the EMB sampling site is guided by imaging (MRI) or electrophysiologic (electroanatomic mapping) techniques. 7.6.4. Notes on IHC and molecular aspects Immunofluorescence for plakoglobin and other cellular junction proteins is promising as it may decrease the need for free wall biopsy since it works on morphologically preserved myocardium and thus on septal bioptic samples. Attention to specific antibody dilutions and technique is important. However, this test needs validation before entering routine practice and cannot at present replace morphologic diagnosis [106]. 7.7. Sarcomeric hypertrophic cardiomyopathy (HCM) 7.7.1. Evidence of recommendation: M HCM is a primary heart muscle disease with a 1:500 prevalence in the general population [107,108].

The clinical diagnosis is based upon instrumental demonstration of left ventricular hypertrophy (electrocardiogram and echo) and requires exclusion of other causes of ventricular hypertrophy. HCM is most commonly due to a mutation in one of the genes encoding sarcomeric proteins. From the pathology viewpoint, HCM is characterized by increased wall thickness/mass with variable distribution, most commonly with asymmetric hypertrophy, involving the basal interventricular septum. Histological features include myocyte hypertrophy, interstitial and/or replacement fibrosis, myocardial disarray, and thickening of the walls of the small mural vessels. 7.7.2. EMB diagnostic potential The clinical diagnosis of HCM usually relies on noninvasive diagnostic tools, and EMB is not recommended in the diagnostic workup. However, in selected cases, EMB may be helpful in excluding infiltrative or storage diseases (such as Fabry and PRKAG disease) with variable diagnostic degrees of certainty [109]. 7.8. Idiopathic/primary restrictive cardiomyopathy (RC) 7.8.1. Evidence of recommendation: M RC is a primary heart muscle disease characterized by impaired filling of the ventricles in the setting of normal wall thickness and systolic function. Formerly believed to be of idiopathic nature, once inflammatory, infiltrative, or systemic diseases were excluded, it is now known to be associated with mutations in sarcomeric contractile protein genes, like HCM. Patients may develop severe symptoms of heart failure over a short period of time, even requiring heart transplantation. The clinical diagnosis is based on clinicoinstrumental demonstration of restriction and requires exclusion of other causes of constriction and restriction. EMB may be of utility in the distinction between restrictive myocardial disease and constriction from pericardial disease [110].

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Fig. 7. Eosinophilic disease. (A) A 44-year-old man affected by Loeffler disease in the subacute thrombotic stage. EMB shows endocardial thrombus and endomyocardial infiltrates rich in eosinophils. (A) Fresh thrombus (hematoxylin–eosin, 200×); (B) organizing thrombus (hematoxylin–eosin, 100×); (C–D) typical eosinophil-rich endomyocardial inflammation (hematoxylin–eosin, C, 400×; D, 250×).

7.8.2. EMB diagnostic potential EMB is unable to give a diagnostic picture as histological findings (i.e., interstitial fibrosis, myocardial disarray, or even normal myocardium) are not specific. However, in selected cases, EMB may be helpful in excluding infiltrative or storage diseases [110,111]. 7.9. Loeffler fibroplastic endocarditis/endomyocardial fibrosis—also known as “obliterative cardiomyopathy” 7.9.1. Evidence of recommendation: S (in the acute–subacute phase) This section addresses heart diseases with thickening of endocardium lining the heart cavities and progressive obliteration of the ventricular cavities. Loeffler endocarditis (also known as eosinophilic endomyocardial disease or fibroplastic parietal endocarditis) is typical in the temperate zone, with a male/female ratio of 9:1, and usually manifests in the fourth decade of life. It appears to be a subcategory of the hypereosinophilic syndrome in which the heart is predominantly involved. It is an uncommon myocardial disease, thought to be secondary to damage from eosinophils. At gross examination, there is diffuse deposition of mural thrombus over the ventricular endocardium incorporated into a whitish fibrous plaque. On histological examination, there may be eosinophilic myocarditis, endocardial damage, thrombotic deposition infiltrated by eosinophils, and dense myocardial fibrous

scar (Fig. 7). Three stages can be distinguished along the time course: a necrotic (rarely symptomatic) stage, a thrombotic stage (with risk of thromboembolism), and a fibrotic stage with cardiac and atrioventricular valvular dysfunction [112,113]. Tropical (Davies) endomyocardial fibrosis is typical in the tropical areas, with a male/female ratio of 1:1, usually occurring in children and young adults. Eosinophilia is infrequent and reflects a parasitic infection. Tropical endomyocardial fibrosis is commonly associated with nutritional deficiency, hypersensitivity to malaria, viral and parasitic infections, magnesium deficiency, and high cerium intake. Pathologically, there is fibrotic endocardial thickening at the apex and inflow of right or both ventricle, with atrioventricular valve apparatus entrapment, endocardial calcification, and mural thrombosis, as well as variable degrees of subendocardial necrosis or degeneration of the underlying myocardium [114–116]. 7.9.2. EMB diagnostic potential EMB can allow a “definite” diagnosis only in the acute phase, with the demonstration of endomyocardial infiltrates rich in degranulated eosinophils and eosinophil cationic protein, and endocardial thrombosis. A “probable/possible” diagnosis can be reached in the chronic phase of endomyocardial fibrosis, with evidence of endocardial fibrous thickening and subendocardial myocyte abnormalities. Clinical features and laboratory evidence of past/present hypereosinophilia are useful for final diagnosis.

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Fig. 8. Cardiac sarcoidosis in a 53-year-old female. (A) Histology: a giant cell granulomatous inflammatory infiltrate is visible with$2 absence of caseous necrosis. In the cytoplasm of a giant cell, an asteroid body (arrow) should be noted (hematoxylin–eosin, 200×). (B) Immunohistochemistry: the macrophages and the multinucleate giant cells are positive with the CD68/PGM1 antibody (200×). (C) Subsequent control EMB showing the persistence of nodular polymorphous inflammatory infiltrate (hematoxylin–eosin, 100×). (D) Subsequent chronic evolution with interstitial fibrosis and sparse inflammatory infiltrate (Masson trichrome, 100×). From Leone et al. [26].

7.10. Cardiac sarcoidosis

7.11. Peripartum cardiomyopathy (PPCM)

7.10.1. Evidence of recommendations: S Sarcoidosis is a systemic granulomatous disease of unclear etiology (Fig. 8). Assessment of cardiac involvement is important as the cause of death in half of sarcoidosis patients is cardiac in origin. In some cases, an unexpected diagnosis of sarcoidosis is made on biopsy of patients with ventricular tachycardia, suspected ARVC, or RC. Sarcoid enters the differential in the investigation of unexplained arrhythmias [117–119].

7.11.1. Evidence of recommendations: M PPCM is often defined as the onset of unexplained left ventricular dysfunction in the last trimester of pregnancy or within 5 months postpartum. The etiology of this rare disorder remains unclear. Borderline myocarditis and active myocarditis, as well as healing myocarditis, have all been demonstrated in EMB from some of these women, with a prevalence of myocarditis on EMB ranging from 8.8% to 62%, depending upon the diagnostic criteria used and the timing of biopsy with respect to onset of symptoms.

7.10.2. EMB diagnostic potential Due to the focal nature of the infiltrates, EMB has been reported to have poor sensitivity in systemic sarcoidosis with presumed cardiac involvement, being positive in 19%–25% of cases [120,121]. A positive biopsy, however, may be reflective of more extensive disease and is associated with worse survival [121]. More importantly, a positive biopsy guides therapeutic management of these patients with corticosteroids and potential prophylactic use of a defibrillator. To increase the sensitivity of the procedure, electrophysiologic (electroanatomic mapping) or image-guided biopsy procedures have been proposed.

7.11.2. EMB diagnostic potential The role of EMB in the diagnosis of PPCM is controversial since there may be no difference in clinical outcome between those with and without myocarditis on biopsy [122]. Spontaneous resolution of myocarditis has been observed in some patients, while others require immunosuppressive therapy or progress to DCM.

7.10.3. Special technical aspects Special stains for microorganisms are important to rule out infectious granulomatous diseases.

7.11.3. Molecular aspects Investigations into the presence of viral genomes have yielded inconsistent results [123]. 7.12. Cardiac amyloidosis 7.12.1. Evidence of recommendations: S The amyloidoses comprise a group of disorders characterized by the deposition of abnormally folded protein with an extended beta-sheet conformation.

O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 Table 5 Summary of the main forms of amyloidosis that affect the heart Nomenclature

Precursor of amyloid fibril

Systemic (S) or localized (L)

AL ATTR

Immunoglobulin light chain Familial (mutant) and senile systemic (wild type) transthyretin Mutant apolipoprotein Serum amyloid A Atrial natriuretic peptide

S S

AApoA1 AA AANP

S/L S L

7.12.2. EMB diagnostic potential EMB is well recognized as an important modality in the workup of patients with cardiac amyloidosis. At least 11 types of amyloidosis may involve the heart [124]. Table 5 summarizes the main forms of amyloidosis that affect the heart [70]. In hematoxylin–eosin-stained sections, the amyloid appears as a homogeneous, slightly eosinophilic, amorphous, and nonstructured substance (Fig. 9A, B, D). It is recommended that nontransplant biopsies be stained for the presence of amyloid using Congo red (deposits appear with a characteristic apple-green birefringence under polarized light; Fig. 9 E), modified sulfated alcian blue, or thioflavin T. Frozen tissue is necessary if the amyloid is to be typed by immunofluorescence. 7.12.3. IHC and molecular aspects EMB has been proven useful for establishing the type of cardiac amyloid using techniques such as immunohisto-

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chemistry [125], immunofluorescence (Fig. 9C, F) [124], immunoelectron microscopy [126], protein sequencing [127], and mass spectrometry [128]. Thus, it is recommended that efforts be made to subtype amyloid deposits in EMBs. Subclassification of amyloid deposits is nontrivial, and the misclassification of amyloid in pathologic specimens has been reported (reviewed by Collins et al. [124]). Mistyping of amyloid can have catastrophic effects, as the prognosis is very different and treatment options are specific for the type of amyloid present [70]. Amyloid subtyping should be performed in a center with experience using techniques that have been validated to be reliable in that center. 7.13. Iron overload 7.13.1. Evidence of recommendations: S The heart normally contains no iron, and therefore, finding stainable iron in the cardiomyocytes indicates the presence of pathology. Heart failure induced by iron deposition is a leading cause of death in patients with transfusion-dependent anaemia, notably beta-thalassemia [129]. Cardiac iron deposition is also a feature of hereditary hemochromatosis, but is seldom a direct cause of death [130]. Beta-thalassemia CMP may have a dilated phenotype with impaired contractility or a restrictive pattern with pulmonary hypertension and right heart failure [129]. In cardiac biopsy studies, there is a good correlation between

Fig. 9. (A–C) Late-onset systemic familial transthyretin-related amyloidosis (mutation: Ile68Leu) in a 62-year-old male. The EMB (A) shows diffuse interstitial deposits of amyloid (25×). (B) In the hematoxylin–eosin routine section (400×), amyloid appears as a homogeneous, amorphous, and eosinophilic substance. (C) Immunohistochemistry: amyloid deposits show a diffuse and intense brown positivity for the anti-transthyretin antibody (400×). (D–F) Right ventricular EMB from an 84-year-old man. Hematoxylin–eosin (D) and Congo red stain (E) show interstitial amyloid deposition. Subtyping of the amyloid by immunofluorescence (F) shows specific staining for transthyretin consistent with senile systemic amyloidosis (wild-type transthyretin). (D–F) All images are at 400× magnification.

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serum ferritin levels and the degree of iron deposition [131]. Recently, MRI has proven to be effective in quantifying iron in the heart and liver [132]. In a comparative study of EMB and MRI, patients with stainable iron in more than 50% of myofibers were shown to have lower T2 relaxation times than those with less iron on biopsy [133]. Iron tends to initially deposit subepicardially and may not always be found in the right ventricle septal region; thus, the possibility of sampling error exists [134].

diagnosis, but in selected cases with no clear-cut diagnosis, EMB is recommended. EMB from patients with unexplained DCM should be assessed for the presence of iron, and an iron stain should be performed when necessary. Biopsy may be also useful in monitoring of patients to verify therapeutic results. Genetic testing on blood is commonly utilized to make the diagnosis of hereditary hemochromatosis.

7.13.2. EMB diagnostic potential The histological appearance of iron storage on hematoxylin–eosin-stained sections is that of intramyocytic granular brownish deposits (Fig. 10A), which are blue on a pink field by blue Prussian (Perls) staining (Fig. 10B). It is not possible to histologically distinguish primary from secondary iron overload. Magnetic resonance imaging may contribute to

7.14.1. Evidence of recommendations: S DRM is a clinically heterogeneous group of diseases with progressive skeletal and/or cardiac myopathy, which results from mutations in the intermediate filament desmin (DES) gene [45,135,136]. Cardiac phenotypes related to DRM include dilated, restrictive, and occasionally ARVC-like [137].

7.14. Desmin-related cardiomyopathy (DRM)

Fig. 10. Storage diseases. (A–B) Hemochromatosis in a 60-year-old male with sudden onset of refractory cardiac failure. (A) The EMB shows numerous intracytoplasmic granules in the cardiomyocytes (hematoxylin–eosin: 400×). (B) Specific staining for iron highlights the intracytoplasmic granules in blue (Perls blue, 100×). (C–D) Fabry disease. (C) Hematoxylin–eosin-stained section (200×) showing hypertrophied vacuolated myocytes. (D) Transmission electron micrograph (10.000×) showing lamellar bodies within the cytoplasm of cardiac myocytes. (E–F) Glycogenosis type II (Pompe's disease). (E) PAS-stained section (400×) showing cytoplasmic glycogen deposits. (F) Transmission electron micrograph (27,500×) showing lysosomal bound glycogen surrounded by mitochondria; some extralysosomal glycogen is also present.

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7.14.2. EMB diagnostic potential EMB may be helpful to provide a definitive diagnosis in suspected cases, confirm cardiac involvement in patients with positive genetic testing, and rule out other processes. The histological picture is nonspecific (fibrosis and sometimes a small increment in myofiber size). Electron microscopy shows abnormal aggregates of desmin-type intermediate filaments (granulofilamentous aggregates) in the cytoplasm of cardiomyocytes (interfibrillar areas and at Z band level). 7.14.3. IHC aspects Immunoelectron microscopy may confirm that these aggregates are formed by desmin. IHC for desmin on histological sections shows abnormal distribution and, in advanced cases, large aggregates of desmin filaments, although these IHC findings are not entirely specific for DRM.

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cient glycoprotein syndromes, and Sandhoff's disease). All of these diseases may lead to cardiac hypertrophy. 7.16.2. EMB diagnostic potential Microscopically, there are hypertrophied vacuolated myocytes on EMB [140,141]. Special stains (periodic acidSchiff [PAS], alcian blue) may be used to assess the nature of the storage products, preferably on frozen sections (as glycogen may wash out with routine processing for paraffin sections). Electron microscopy is used to assess the nature and location of the intracellular deposits. Enzyme replacement therapy is utilized for lysosomal storage diseases and mucopolysaccharidoses; thus, the use of EMB in monitoring may be applicable, but skeletal muscle biopsy is more often employed [142,143]. 7.17. Cardiac tumors 7.17.1. Evidence of recommendation: S

7.15. Anderson–Fabry disease 7.15.1. Evidence of recommendations: S Anderson–Fabry disease is an X-linked inborn metabolic defect caused by a deficiency of alpha-galactosidase A, resulting in the accumulation of glycosphingolipid, which may cause conduction disturbances, valve disease, and left ventricular hypertrophy [138]. Long-term treatment with recombinant human alphagalactosidase A may halt the progression of microvascular pathology and prevent the clinical manifestations [139]. EMB may be very informative for the assessment of unexplained left ventricular hypertrophy, particularly in carrier women that develop heart disease later in life and in patients that have mutations associated with residual enzyme activity and a milder disease phenotype.

7.17.1.1. Primary cardiac tumors. Most primary cardiac tumors (90% in surgical series) are benign [144–147]. In autopsy series, the frequency of primary cardiac tumors was 1/1000, whereas cardiac metastases (mainly from lung, breast, or kidney, or from malignant melanoma) were far more prevalent (1/100) [147]. The most common primary cardiac neoplasm is the cardiac myxoma, a benign tumor that has no microscopic counterpart in other organs and tissues [148,149], whereas the most frequent malignant tumors are represented by sarcomas, which have the same classification as soft tissue sarcomas [150]. According to the World Health Organization classification, primary cardiac tumors are categorized

7.15.2. EMB diagnostic potential EMB may be a useful guide for clinicians and pathologists who diagnose and follow these patients [139]. At histology, hypertrophic vacuolated cells are evident, with dislocation of contractile elements to the periphery of myocytes (Fig. 10C). Electron microscopy reveals electron dense concentric lamellar bodies, which are typical, although not exclusive to the disease (Fig. 10D). 7.16. Other inherited storage diseases 7.16.1. Evidence of recommendations: S Cardiac manifestation of systemic metabolic diseases caused by deficiencies of various enzymes in metabolic pathways includes glycogen storage diseases [e.g., glycogenosis type II (Pompe's disease, Fig. 10E–F), glycogenosis type III, Danon disease, and PRKAG2 mutations] and lysosomal storage diseases (e.g., Niemann–Pick disease, Gaucher disease, I-cell disease, mucopolysaccharidoses, GM1 gangliosidosis, galactosialidosis, carbohydrate-defi-

Fig. 11. Angiosarcoma: biopsy of a right atrium tumor. At low magnification, spindle cell tumor with hemorrhage and thrombus (hematoxylin–eosin 50×). At higher magnification (insert A, hematoxylin–eosin 400×), pleomorphic and atypical nuclei are conspicuous, and some tumor cells show intracytoplasmic vacuoles containing red blood cells (arrows). Immunohistochemistry (insert B, 400×) showing strong positivity for factorVIII-related antigen.

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Fig. 12. Right atrium fibrosarcoma. (A) Transvenous endocardial biopsy specimen of mass (hematoxylin–eosin stain, 120×). (B) At higher magnification, histologic examination of mass shows spindle-shaped cells arranged in fascicles at acute angles with frequent mitoses (hematoxylin–eosin stain, 480×). (C) Diffuse positivity for vimentin at immunohistochemistry (insert, 480×) and negative immunostaining with other tested antibodies. From Basso et al., modified [16].

as benign tumors and tumor-like lesions, malignant tumors, and pericardial tumors [151]. For more detailed information and diagnostic criteria, extensive literature is available [146,148,152–154]. Tumors reported in the literature that have been diagnosed by EMB are numerous, and mainly malignant or metastatic [8,21,155–170]. Figs. 11 and 12 show two examples of biopsied cardiac primary tumors, and Fig. 13 shows two cases of biopsied cardiac metastases.

EMB is a valuable tool for preoperative diagnosis of intracardiac masses. It is mainly indicated for the investigation of right-sided masses showing an infiltrative or obstructive growth pattern and for the differential diagnosis of sarcomas, lymphomas, and metastatic tumors. Unresectable cardiac masses may benefit from EMB to help plan therapy or palliation [15,16,21,172,173]. Table 6 lists some biopsied malignant and metastatic neoplasms that have been reported.

7.17.2. Indications for EMB

7.17.2.2. Limits. False-negative results are possible due to sampling error but can be minimized by procurement of multiple specimens guided by echocardiography. Inspecting the biopsy specimens for a pale color may also be useful. Left-sided EMB is possible but sometimes avoided because of the potential for systemic embolism.

7.17.2.1. Indications. Clinical diagnosis of cardiac masses is mainly done by echocardiography, computerized tomography, and/or MRI. Histology remains crucial for differentiating neoplastic from nonneoplastic masses and benign from malignant, and for subtyping the neoplasms. It therefore provides critical information for treatment and prognosis, which are largely dependent upon tumor histotype and biological behavior [171].

7.17.3. Specific technical aspects At least three to five formalin-fixed and paraffinembedded biopsy samples (2–3 mm 2 each) are advisable.

Fig. 13. Anaplastic large cell lymphoma and carcinoma cardiac metastasis. (A) Transvenous EMB specimen taken from the right ventricle showing a diffuse neoplastic myocardial infiltration by atypical lymphoid cells (hematoxylin–eosin 100×). (B) CD20 IHC staining reveals a B-cell lymphoma infiltrate (100×). (C) Transvenous right ventricular EMB showing a cardiac breast carcinoma metastasis (hematoxylin–eosin 200×).

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Table 6 Malignant primary or metastatic cardiac tumors reported in the literature as diagnosed by EMB

7.18. Hypersensitivity reactions and cardiac toxicity due to drugs and chemical/toxic substances

Malignant primary tumors

Metastatic tumors

Angiosarcoma Fibrosarcoma Granulocytic sarcoma (chloroma) Leiomyosarcoma Lymphoma Malignant fibrous histiocytoma Rhabdomyosarcoma Sarcoma NOS Synovial sarcoma

Adenocarcinoma Cervical carcinoma Endometrial carcinoma Lymphoma Melanoma Squamous cell carcinoma

7.18.1. Evidence of recommendation: hypersensitivity myocarditis: S; toxic cardiopathies: M (depending on the drug) Drug-related cardiac toxicity and hypersensitivity reactions represent, for physicians, the most worrying aspect of the wide panel of substances capable of injuring the heart since many prescribed drugs can be associated with cardiac toxicity or hypersensitivity reactions [176–178]. It is expressed in two clearly distinct entities: hypersensitivity myocarditis and toxic “damage.”

7.17.4. Immunohistochemistry The type of antibodies to be utilized depends upon tumor type. An initial useful suggested panel includes vimentin, factor-VIII-related antigen, CD31, CD34, epithelial membrane antigen, wide-spectrum cytokeratins, S-100 protein, smooth muscle actin, desmin, myogenin, HMB-45 molecule, CD45, CD20, and CD3. For detailed diagnosis of lymphomas, a wide panel of lymphocytic antibodies is usually required [174].

7.18.2. Hypersensitivity myocarditis This is probably the most common form of drug-induced cardiac dysfunction and may occur with many usual therapeutic agents from many classes (Table 7). It is unpredictable; is independent of drug dosage; and, as with “direct toxicity,” is likely due to sensitization after previous safe use. The most common pattern of hypersensitivity myocarditis is pancarditis with a perivascular mixed inflammatory infiltrate that is rich in eosinophils. Lymphocytes, macrophages, giant cells, and neutrophils may be present in variable numbers, and the process may be associated with the development of small granulomas. The inflammation has a tendency for an interstitial or perivascular pattern of distribution, with little in the way of myocyte injury (Fig. 14).

7.17.5. Molecular biology Fluorescence in situ hybridization and PCR tests are commonly used, at least in most referral centers, for the evaluation of sarcomas and lymphomas [e.g., reciprocal translocation t(x;18)(p11.2;q11.2) in synovial sarcoma and t(2;13)(q35;q14) in rhabdomyosarcoma] [175].

7.18.3. Toxic damage This is due to substances usually known for their potential to cause direct cardiac toxicity (Table 7). However, the development of new drugs with undetermined capacities for cardiotoxicity can occur. Cardiac toxicity is dose dependent, possibly has a cumulative effect, and is often potentiated by other antineoplastic therapies such as radiation therapy.

One to two additional snap-frozen samples for molecular biology may be useful for diagnosis of sarcomas and lymphomas.

Table 7 Selected drugs and other chemical substances associated with cardiac toxicity and their most frequent histological picture Histological picture

Drugs

Hypersensitivity myocarditis

Miscellaneous

Toxic damage

Antineoplastic drugs

Toxic damage

Miscellaneous

Toxic damage Toxic damage

Drugs of abuse Poisoning

Acetazolamide, amitriptyline, aminophylline, amphotericin B, ampicillin, azathioprine, benzodiazepines, bumetanide, captopril, carbamazepine, cephalosporins, chloramphenicol, chlorthalidone, clozapine, cyclosporine, digoxin, dobutamine, furosemide, heparin, hydralazine, hydrochlorothiazide, indomethacin, interleukin-2, isoniazid, isosorbide dinitrate, methyldopa, metolazone, nitroprusside, oxyphenbutazone, para-aminosalicylic acid, penicillin, phenindione, phenylbutazone, phenytoin, spironolactone, streptomycin, sulfadiazine, sulfamethoxypyridazine, sulfisoxazole, sulfonylureas, tetracyclines, triazolam, tricyclic antidepressants. Amsacrine, cyclophosphamide, anthracyclines (doxorubicin, daunorubicin, epirubicin), 5-fluorouracil, interferon-alpha, interleukin-2, mitomycin C, mitoxantrone, tyrosine kinase inhibitors (trastuzumab antiHER2/neu, cetuximab and erlotinib anti-EGFR, bevacizumab and sunitinib anti-VEGF, imatinib anti-cKIT) Amphetamine-derived compounds (benfluorex, type 2 diabetes modulator), antimony compounds (antileishmaniasis treatment), antiretroviral agents, catecholamines, chloroquine, hydroxychloroquine, dopamine agonists (Parkinson's disease), emetine (antiamoebiasis treatment), lithium, phenothiazines, serotonin-derived compounds (fenfluramine/phentermine, appetite suppressants) Alcohol, amphetamine and related compounds (methamphetamines, ecstasy), anabolic steroids, cocaine Arsenicals, cobalt, lead

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Fig. 14. (A) EMB showing eosinophilic myocarditis from patient with history of allergies, who was on multiple medications (hematoxylin–eosin 400×). (B) Drug-induced hypersensitivity myocarditis. EMB in a heart transplant patient treated with horse antilymphocyte serum for rejection in the early 80s. He developed serum sickness with cutaneous rash, pulmonary infiltrate, and high eosinophil count at bronchoalveolar lavage, and a typical pattern of eosinophilic myocarditis. All the signs resolved with steroids (hematoxylin–eosin 400×).

Cardiotoxicity may be reversible after discontinuation of the medication, allowing for its reintroduction later [179] or for avoidance of drug-related heart failure [180–182]. Recently, new drugs have revealed cardiac toxicity, including new antineoplastic drugs [179,180,183–185], appetite suppressants [186], and drugs used in type 2 diabetes [187] or in Parkinson's disease (Table 7) [188]. Currently, cases of cardiac toxicity due to street drugs or illicit substances and alcohol, and industrial or environmental chemicals have been overshadowed by the frequency of cocaine cardiotoxicity (Table 7) [189–191]. The pathology of cardiac toxicity [177,178] is quite variable and often lacks specificity so that the implication of a drug can be difficult. This is compounded by the fact that many patients are often on a polypharmacy of drugs with potential cardiotoxicity. The gross appearance of the heart varies from no structural changes to a pattern of “dilated cardiomyopathy” in the later stages of some cumulative drug exposures. Histology can show three main general histological patterns: ■

■

■

Myocardial necrosis usually without inflammatory infiltrate: patchy eosinophilic changes in myofibers, focal myofibril degeneration or focal coagulative necrosis, and prominent contraction bands which may be associated with fibrin deposition, interstitial edema, and hemorrhagic extravasation. “Toxic myocarditis,” characterized by multiple areas of cell death of different ages, surrounded by a mixed inflammatory infiltrate of macrophages, neutrophil granulocytes, and a small number of lymphocytes. Interstitial edema and hemorrhagic extravasation and myocyte coagulative necrosis may be present to a variable degree. Cyclophosphamide-induced cardiac toxicity targets endothelial cells and results in microthrombi, hemorrhagic edema, and myocyte necrosis [192]. DCM in the chronic phase, with nonspecific histological alterations (interstitial fibrosis and myofiber alterations). In some cases, such as with

chloroquine toxicity, the myocytes may have a vacuolated appearance, and lamellar lysosomal inclusions may be identified by electron microscopy. In other cases, such as with Adriamycin toxicity, dilation of the sarcoplasmic reticulum may be seen on electron microscopy. 7.18.4. EMB diagnostic potential Drug-induced hypersensitivity myocarditis may be suggested using EMB when myocarditis is clinically suspected. As with all forms of myocarditis, sampling error may lead to false-negative diagnosis. In toxic CMPs, the role of EMB is variable depending upon the specific drug. For drugs with a well-established cardiac toxicity, for example, anthracyclines (including Adriamycin) [193], EMB has been replaced by noninvasive techniques such as stress or strain echocardiography, radionuclide angiography, MRI, and troponin I level monitoring [176,191]. On the other hand, in recently marketed drugs, EMB is important in order to document cardiac toxicity, to study the mechanisms of cardiac toxicity, and to establish the imputability of the drug. Even with the use of the EMB, it may still be difficult to pinpoint one specific drug as the culprit. 7.18.5. Specific technical aspects For toxic cardiopathies, transmission electron microscopy may sometimes be performed, implying special fixation of EMB samples in glutaraldehyde. 7.19. Transplantation 7.19.1. Evidence of recommendation: S 7.19.1.1. Acute cellular rejection. In 1990, an international grading system for cardiac allograft biopsies was adopted by the International Society for Heart and Lung Transplantation

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Table 8 1990 and 2004 ISHLT grading systems for cellular and antibody-mediated rejections (AMR) [2] 2004 ISHLT revised standardized cardiac biopsy grading Grading of acute cellular rejection Grade 0 Grade 1R mild Grade 2R moderate Grade 3R severe

Grading of acute AMR Grade 0 Grade 1

1990 ISHLT standardized cardiac biopsy grading

No rejection Interstitial and/or perivascular inflammatory infiltrate with up to one focus of myocyte damage Two or more foci of inflammatory infiltrates with associated myocyte damage Diffuse inflammatory infiltrates with multifocal myocyte damage±edema±hemorrhage±vasculitis

Grade 0 Grades 1A, 1B, 2 Grade 3A Grades 3B and 4

Negative for acute AMR No histologic or immunopathologic features of AMR Positive for AMR Histologic features of AMR: myocardial capillary injury (endothelial swelling, intravascular macrophages), interstitial edema and hemorrhage, neutrophils in and around capillaries, intravascular thrombi, myocyte necrosis Positive immunofluorescence or immunoperoxidase staining for AMR: immunoglobulin (IgG, IgM, and/or IgA) and complement (C3d, C4d, and/or C1q) deposition in capillaries by immunofluorescence on frozen sections; macrophages (CD68) in capillaries (CD31 or CD34) and complement (C4d) deposition on capillary endothelium by paraffin immunohistochemistry

(ISHLT) [1]. The grading system was widely adopted and served the transplant community well for over a decade. In 2004, under the direction of the ISHLT, a multidisciplinary review of the grading system was undertaken to address challenges and inconsistencies in its use, to consider changes in rejection incidence and treatment practices, and to incorporate advances in medical knowledge [194–206]. The outcome of the consensus conference was revision of the 1990 working formulation for cellular rejection as outlined below with “R” after the rejection grade indicating the 2004 revision (Table 8) [2]. In Fig. 15, 2004 ISHLT cellular rejection grades are shown. 7.19.1.2. Acute antibody-mediated rejection (AMR). AMR is an evolving issue in cardiac transplantation [207–210]. While mentioned only briefly in the 1990 grading system, the 2004 revision (Table 8) recommends reporting whether AMR is absent (AMR 0) or present (AMR 1). AMR has been recognized as a clinicopathologic entity that has been characterized by (a) cardiac dysfunction in the absence of cellular rejection or ischemic injury, (b) characteristic histological features on EMB, (c) positive immunostaining of capillaries for the complement component C4d, and (d) presence of circulating donor-specific antibodies (DSAs). AMR often occurs in sensitized patients (including those with previous transplantation, transfusion or pregnancy, and previous ventricular assist device placement) and is associated with a worse graft survival [210,211]. Increasingly AMR is being detected late after transplantation both in presensitized patients and in patients developing de novo DSAs [212]. The incidence of AMR is highly variable

between transplant centers, but most agree that the incidence is low (b5%), especially since high-risk patients may be treated prophylactically before transplantation. The histological features of AMR include interstitial edema; endothelial cell swelling; intravascular macrophages; and, less frequently, interstitial hemorrhage, mixed inflammatory infiltrates, capillary fragmentation, and endothelial cell pyknosis (Fig. 16A). There should be immunophenotypic evidence of immunoglobulin and complement deposition, especially C4d (and perhaps C3d), in capillaries (Fig. 16B). However, several studies since 2005 have raised the question of asymptomatic AMR [213], questioned the sensitivity and specificity of histological criteria for screening biopsies (as stated in the 2004 grading system revision) [214,215], shown positive histology (in the kidney) for AMR without C4d capillary deposition [216], and shown C4d capillary deposition without positive histology or intravascular macrophages [217,218]. This suggests that there is a morphologic and immunophenotypic spectrum of AMR-related changes, which should be regarded as a clinical–pathologic continuum that starts with a latent humoral response of circulating antibodies alone and progresses through a silent phase of circulating antibodies with C4d deposition, without histological or clinical alterations; to a subclinical stage with circulating antibodies, and histological and immunopathologic findings; and to symptomatic AMR with clinical manifestations. Furthermore, although C4d is the most widely used immunohistologic marker for AMR, there is no uniformity of approach to frequency of testing or to what constitutes a positive result in terms of distribution and intensity of staining [219–222]. Lastly, ACR and AMR may coexist in

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in 2011 focused solely on AMR specifically to address (a) appropriate testing and interpretation of histological findings and immunohistochemistry (at present, C4d immunofluorescence and immunoperoxidase detection seem to be more or less equivalent) [223,224]; (b) use of serologic data, specifically donor specific antibodies; and (c) correlation of allograft dysfunction and histopathologic/immunophenotypic/serologic findings. In both consensus conferences, it was agreed that the diagnosis of AMR should be made according to specific pathologic features. The combination of histopathologic and immunopathologic findings should be reported as the “pathological diagnosis of AMR” and should be designated by pAMR (Table 9). Descriptors for immunologic and clinical presentation, such as DSA and cardiac dysfunction, would assist in diagnosis and management [223,224]. Uncertainty still remains as to the best treatment for both symptomatic AMR, currently defined as positive histology and C4d staining on EMB and positive serum antibodies in a hemodynamically compromised patient [225], and asymptomatic AMR in a patient who is clinically doing well with negative histology yet has positive C4d staining on EMB with or without positive DSA [213].

Fig. 15. (A) Cellular rejection, ISHLTR 1R. Perivascular and/or interstitial lymphocytic infiltrate with no more than one focus of myocyte damage (hematoxylin–eosin, 400×). (B) Cellular rejection, ISHLTR 2R. Two or more foci of lymphocytic infiltrates with associated myocyte damage (hematoxylin–eosin, 200×). (C) Cellular rejection, ISHLTR 3R. Diffuse infiltrate with multifocal myocyte damage and edema. In some cases, hemorrhage and vasculitis may be present (hematoxylin–eosin, 200×).

the same biopsy, but given the above challenges we face in diagnosing AMR, its frequency and clinical significance are unknown. Due to the many unanswered questions regarding AMR, the heart transplantation section of the Tenth Banff Conference on Allograft Pathology in August 2009 and two Consensus Conferences held in conjunction with the ISHLT Annual Meeting in Chicago in 2010 and San Diego

7.19.1.3. Nonrejection posttransplant biopsy findings. Early perioperative ischemic injury is an extremely common finding up to 6 weeks (or more) posttransplant, characterized by myocyte necrosis which often extends to the endocardial surface. As healing ensues, a mixed inflammatory infiltrate including neutrophils as well as lymphocytes, macrophages, and eosinophils must be distinguished from acute rejection (Fig. 17A) [226,227]. Late ischemic injury resulting from allograft coronary disease may be detected by secondary myocardial changes since vessels large enough to demonstrate this process are rarely present in biopsy material. Myocyte vacuolization and/or microinfarcts are characteristic of this process and may be helpful in determining the cause of late cardiac failure or, more importantly, ruling out other potentially treatable etiologies [228,229]. Quilty effect (nodular endocardial infiltrates) (Fig. 17B) may be confined to the endocardium (1990 ISHLT Quilty A) or extend into the underlying myocardium where associated myocyte damage may be present (ISHLT 1990 Quilty B) [230–234]. However, because the subtyping of Quilty A and B was never shown to have clinical significance, the designations “A” and “B” were eliminated in the 2004 revision. 7.19.1.4. EMB diagnostic potential. Despite many advances in immunosuppression therapy, acute rejection still occurs, most commonly during the first posttransplant year [235]. The EMB has been the “gold standard” for the diagnosis of allograft rejection, and its routine use has been credited as a factor in improved recipient survival [236]. No established noninvasive marker of rejection is available, and no indicator of heart allograft rejection has been proven more sensitive,

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Fig. 16. Antibody-mediated rejection (AMR). (A) Histology of AMR: endothelial cell swelling with intravascular macrophages and perivascular edema (hematoxylin–eosin 400×). (B) Immunohistochemical staining for C4d complement factor is diffuse and strong in capillary endothelial cells (100×).

Table 9 2011 ISHLT preliminary nomenclature scheme for pathologic AMR [223] pAMR 0: pAMR 1 (H+): pAMR1 (I+): pAMR 2: pAMR 3:

Negative for pathologic AMR: Both histologic and immunopathologic studies are negative. Histopathologic AMR alone: Histological findings are present, and immunopathological findings are negative. Immunopathologic AMR alone: Histological findings are negative, and immunopathological findings are positive. Pathologic AMR: Both histologic and immunopathologic findings are present. Severe pathologic AMR: This category recognizes the rare cases of severe AMR with histopathologic findings of interstitial hemorrhage, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis and/or karyorrhexis, and marked edema. The reported experience of the group was that these cases are associated with profound allograft dysfunction and poor clinical outcomes.

Fig. 17. (A) Ischemic injury: coagulative myocyte necrosis with mixed inflammatory infiltrate representing early healing (hematoxylin–eosin, 200×). Note that, in contrast to cellular rejection, the degree of myocyte damage is more extensive than the cellular infiltrate. (B) Quilty effect: endocardial infiltrate, often with prominent vascularity, which may extend into the underlying myocardium (hematoxylin–eosin 100×).

specific, or temporally useful. The sensitivity of the EMB is high (generally 85%–100%), and the complication rate is low [237–239]. The use of gene expression profiling in which peripheral blood specimens are analyzed is the most recent challenge to the EMB for rejection surveillance. However, the recent Invasive Monitoring Attenuation through Gene Expression trial was limited to recipients more than 6 months posttransplant (when rejection frequency is lower) with an end point of no increased risk of serious clinical outcome (not the number of rejection episodes

detected), confirming that, to date, the EMB remains the “gold standard” for diagnosis of acute allograft rejection [240,241]. Today, new knowledge regarding AMR and its more accurate pathologic diagnostic criteria also confirms this role. EMB is useful in differential diagnosis between acute cellular rejection and myocardial posttransplant infections (e.g., Cytomegalovirus, Toxoplasma gondii) and may play an important role in the diagnosis of posttransplant lymphoproliferative disorders (Fig. 18) [242,243].

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Fig. 18. Three years posttransplant EMB from a 34-year-old woman affected by a posttransplant lymphoproliferative disorder. (A) Hematoxylin–eosin stain with the diffuse inflammatory cell infiltration (200×). (B) Immunohistochemistry for antibody against B lymphocytes (CD20) (200×). (C) PCR amplification of variable diversity joining (VDJ) regions of heavy chain immunoglobulins showing the monoclonality for B cells.

7.19.1.5. Technical considerations. Routine diagnosis and grading of cardiac allograft cellular rejection are performed on formalin-fixed tissue prepared for light microscopy examination [2]. Staining with hematoxylin and eosin are sufficient for diagnosis of ACR. A connective tissue stain such as Masson's trichrome may be helpful for assessing myocyte damage, interstitial fibrosis, and Quilty lesions. Assessment of AMR or mixed ACR and AMR may be performed by immunohistochemistry on paraffin-embedded tissue, which enables assessment of staining localization and distribution on a deeper level of all pieces of submitted endomyocardial tissue. An alternative approach utilizes immunofluorescence on fresh/frozen tissue. While this technique allows assessment of staining intensity and comparison with previous studies, it is usually performed on only one fragment of frozen tissue. 8. Conclusion EMB is a common procedure for the evaluation of cardiac tissue for transplant monitoring, myocarditis, drug toxicity, CMPs, arrhythmia, secondary cardiac involvement by systemic diseases, and cardiac masses. It is the gold standard for the diagnosis of cardiac rejection, myocarditis, and infiltrative/storage disorders and can be crucial for differential diagnosis in heart failure and ventricular arrhythmias. Its role in differential diagnosis, i.e., in excluding other diseases, should be stressed. EMB can also be strategic, both in unexplained pathological conditions to focus diagnosis and the subsequent diagnostic workup and in pathological conditions where, despite a clinical diagnostic orientation, a definite diagnosis and specific etiological data for therapy, clinical management, and prognosis (e.g., amyloidosis, hemochromatosis, Anderson–Fabry disease, myocarditis, etc.) are required.

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