DR. SAURAV SINGH

Endomyocardial Biopsy(EMB) The endomyocardial biopsy remains the gold standarad mode of investigation for diagnosing many primary and secondary cardiac conditions. EMB a diagnostic interventional procedure after development and application of cardiac catheterisation and cardiothoracic surgery. In sophisticated centers, evaluation of cardiomyopathic patients includes a detailed history and physical examination, coronary angiography and EMB.

Importance of EMB to clinicians Differentiating between established dilated cardiomyopathy with no evidence of myocardial inflammation and active myocarditis. Cardiac amyloidosis and myocarditis that can be definitely diagnosed. Used to diagnose specifically various types of myocarditis.

INSTRUMENTS Newer Flexible Bioptomes used due to less complication rates Commonly used bioptomes are: Single use 50cm Novatome Argon endomyocardial biopsy forceps Bipal 7 bioptome Fluoroscopy is the standard imaging modality used to guide the biopsy catheter.

The easily assessable anatomy of venous return to the heart with peripheral access makes the right ventricle an attractive location for tissue sampling. The interventricular septum is the preferred biopsy site due to: Its thickness compared with the right ventrical wall Its continuity with the left ventricle Its location in the natural path of blood flow facilitating vascular access. Myocyte disarray is normally encountered in the region of ventricular apex and its junction with inter-ventricular septum. Free wall perforation and haemopericardium is a major reason for avoiding the free ventricular wall for biopsy.

Left ventricular biopsies(LVB) Infrequently done Performed by needle biopsy at the time of open heart surgery or via a trans-septal approach Procedure performed percutaneously through the femoral artery, retrograde through the aortic valve into the left ventricle. Disease affecting L.V. walls and L.V. masses are the only indications for LVB.

TISSUE HANDLING Proper tissue procurement and handling are essential for optimal diagnostic evaluation Biopsy specimens should be gently extracted from the bioptome with a needle tip. 10% neutral buffered formalin is needed to diagnose: Transplant rejection Unexplained cardiomyopathy Myocarditis Infiltrative cardiomyopathies Cardiac tumors

Zeus fixative or saline (used primarily for immunofluorescence studies to evaluate antigen-antibody rejection) can be used to assess allograft rejection. Trumps fixative or 4% glutaraldehyde used for: Unexplained cardiomyopathy Drug induced cardiotoxicity. Specimen in trumps fixative or 4% glutaraldehyde can be viewed with Transmission electron microscopy(TEM) in assessing: Drug toxicity Metabolic/ storage disorders Light chain deposition disease

Snap freezing tissue is optimal for preserving tissue for molecular analysis like PCR and real-time PCR in evaluation of viral pathogens. For routine diagnostic evaluation, overnight processing and parrafin embedding are sufficient. All of the biopsy pieces should be embedded in the same block.

A minimum of 3-6 slides prepared at 4-5micronM thickness within the paraffin block. Multiple paraffin ribbons are placed on each slide. Routinely stain with hematoxylin and eosin. For emergent cases, a 90min rapid(ultra) processing cycle is available, and slides can be prepared within 2-3hrs. Immunohistochemical, immunofluorescence, and molecular studies are used for specific studies.

Paraffin section immunohistochemistry is used to evaluate for infectious endocarditis by CMV, EBV. In situ hybridization is helpful to demonstrate the presence of EBV or other viral genome. For diagnostic EMB, a sample should always be set aside for transmission electron microscopy(TEM) The sensitivity of detecting transplant rejection can approach 98% with five adequate biopsy fragments.

The adequacy of tissue fragments is very important for correct diagnostic accuracy and interpretation. The greatest limitation to EMB is Sampling error.

Special studies TEM(transmission electron microscopy) are indicated for conditions like: Cardiomyopathy Infiltrative disorders(such as amyloidosis or glycogen storage disorders) Viral myocarditis Drugs cardiotoxicity Universal fixative is recommended, although any 2% glutaraldehyde based fixative is suffice.

Immunofluorescence Allograft rejection Cardiomyopathy Immunohistochemistry Acute allograft rejection Amyloidosis Neoplasm Cardiomyopathy Polymerase chain reaction Viral genome myocarditis

SPECIAL STAINS Congo red or sulfated Alcian blue stain for amyloid fibrils. Prussian blue stain for Iron deposition. Massons trichome or Movat pentachrome stains to confirm the presence of myocyte necrosis or interstitial fibrosis. Gomori methanamine silver for Fungi Gram stains for Bacteria(endocarditis)

ROLE OF ARTIFACTS IN EMB Commonest artifact is contaraction bands in myocyte identical to linear bands on acute ischaemic necrosis and catecholamine (pressor) effect. Intussusception or telescoping of small arteries confused with transplantation-related arteriosclerosis and luminal occlusion by thrombus. Accumulation of fresh platelet/ fibrin rich thrombus may be identified along the endocardial surface of biopsy fragments.

(A) Section showing the typical pattern of myofibre disarray that can be seen at previous biopsy sites.(B) Biopsy artifact showing intussusception of an intramural vessel, not evidence of occlusion/vasculitis (arrow) .

Ventricular perforation is identified by the prescence of mesothelial cells. Other commonest is Crush artifact occur while cutting large specimen. Bioptome-induced tissue distortion or Crush artifact can occur.

Crush artifact

(A) Biopsy artefact showing pinching of the sample at the time of procurement (arrows). Also note the region of fibrosis,represents the site of an earlier biopsy. (B) Tissue fragment from transplant showing endocardial fibrosis, secondary to a healed biopsy site (thick arrow). Also note the region of interstitial fibrosis and mixed inflammatory infiltrate consistent with a vasopressor effect and probably not the site of a healed episode of rejection.

Indication s of EMB: Diagnosis and monitoring of acute transplantatio n rejection Diagnosis of tumors Diagnosis of storage disorders Evaluation of restrictive heart disease such as amyloidosis Classification of myocarditis Grading of drugs(anthrac ycline, adriamycin) cardiotoxicity Evaluation of recent onset heart failure in the absence of coronary artery disease

CARDIAC ALLOGRAFT REJECTION: TYPIFIED BY LYMPHOCYTIC INFILTRATE WITH ASSOCIATED DAMAGE TO CARDIAC MYOCYTES

. Graft rejection reaction: (A)Early rejection: Foci of lymphocytic infiltrates. (B)Severe rejection: Significant lymohoid infiltration and myocyte necrosis

Allograft vasculopathy: Graft coronary arteriosclerosis,shows severe diffuse concentric intimal thickening. The internal lamina(arrow) and media are intact

EMB provides useful information in the following disorders: Idiopathic hypertrophic cardiomyopathy: Shows changes : Myofibrils disarray Myocyte Hypertrophy Interstitial fibrosis Endocardial fibrosis Fibrous changes in intramyocardiac arteries. Commonest finding is basophilic degenerationof myocardium appears as finely granular basophilic.

Hypertrophy myocarditis:disarray, extreme hypertrophy, branching of myocytes as well as characteristic interstitial fibrosis(collagen is blue in this masson trichome stain).

Contd.. Idiopathic dilated cardiomyopathy: Endocardial fibrosis, interstitial fibrosis Hypertrophy of myocardial fibers Degenerative changes of myocardial fibers Leukocytic infiltrates are commonly present Restrictive myocardiopathy: Variable myocyte hypertrophy In active stage, heavy component of eosinophils is present In inactive stage, various nonspecific changes are seen

DCM demostrates variable myocyte hypertrophy and interstitial fibrosis(collagen is highlighted as blue in Masson trichome stain)

Dilated cardiomyocarditis (A)Shows endocardial fibrosis, myocyte hypertrophy, myocyte nuclei, moderate interstitial fibrosis. (B)Shows thickened fibrous endocardium with interstitial fibrosis

RESTRICTIVE MYOCARDITIS

Classification of Myocarditis : Diagnosis of myocarditis requires presence of inflammation infiltrate and myocyte necrosis or degeneration. Etiology can be viral, bacterial, fungal, parasitic, collagen vascular disease, drug and radiation induced or transplant rejection Infiltrate is of lymphocytic in nature admixed with histiocytes. Fibrosis if present should be quantified(mild, moderate, severe) and qualified(interstitial, endocardial replacement)

Contd Hypersensitivity myocarditis: Eosinophilic infiltrate Predominantly perivascular Lesser degree of necrotizing changes. Persistent myocarditis: When both the myocyte damage and the inflammation persist on subsequent biopsies. Resolving or healing myocarditis: When both the myocyte damage and the inflammation are substantialy reduced, and resolved or healed.

HYPERSENSITIVITY MYOCARDITIS: Interstitial infiltrate composed largely eosinophils and mononuclear inflammatory infiltrate.

Contd Giant cell myocarditis: Characterised by multicentric destruction of the cardiac myocytes by cytotoxic cells and the multinucleated cells. Recently described newer form of myocarditis characterised by T lymphocytes that express the gamma-delta receptor and runs a fulminant course. Lymphocytic myocarditis: Active disease: Interstitial inflammatory infiltrate Focal myocyte necrosis. Diffuse, mononuclear, lymphocytic infiltrate

GIANT CELL MYOCARDITIS: Mononuclear inflammatory infiltrate containing lymphocytes, macrophages, extensive loss of muscle and multinucleated giant cells.

LYMPHOCYTIC MYOCARDITIS: Lymphocyte infiltrate with myocyte injury

Contd. CHAGAS DISEASE: Parasitization of myofibrils by trypanosomes Inflammatory infiltrate by neutrophils, lymphocytes, macrophages and occasional eosinophils. Myofibrils distended with trypanosomes infiltration.

CHAGAS DISEASE: Myofibrils distended with trypanosomes(arrow) is present along with inflammation and necrosis of individual myofibrils.

Contd Infiltrative myocardiopathies: It includes various infiltrating conditions like: Amyloidosis Hemosiderosis Hemochromatosis Glycogenosis

AMYLOIDOSIS

AMYLOIDOSIS

AMYLOIDOSIS ON EM

Contd Drug induced and radiation induced cardiomyopathies: Adriamycin shows changes like: Vacuolisation of cardiac myocytes is the earliest change Appearance of adria cell(characterised by loss of cross striations and myofilamentous bundles and basophilic staining) Inflammation is nil or absent, which differentiates it from other myocardinal lesions. Changes are diffuse, dose dependent and tend to occur in subendocardial region.

Contd Cyclophosphamide : Hemorrhagic necrosis Intensive capillary thrombosis Interstitial hemorrhage Fibrin deposition Necrosis of myocardial fibers Radiation induced heart diseases: Constructive pericarditis Myocardial fibrosis Coronary artery lesions

DRUG INDUCED MYOCARDITIS

The evaluation of an endomyocardial biopsy. .

FINDINGS AND IMPORTANCE Surgical pathology report should provide as much as diagnostic information as possible, it includes: Number of pieces of myocardium Appearance of myocyte nuclei(hypertrophied, pkynotic, attenuated, or atrophic) Prescence of cytoplasmic pigments Pattern of necrosis(focal or diffuse)

FINDINGS AND IMPORTANCE Composition of interstitium(e.g., cellularity, fibrosis, edema, amyloid deposits) Prescence of endocardial inflammation

DIFFERENTIAL DIAGNOSIS ENDOCARDIUM FIBROSIS ULCERATION/ NECROSIS Healed myocarditis Cardiomyopathy Drug toxicity Organised thrombus Healed biopsy site Healed acute rejection site Endocardial hyperelastosis Graft procurement injury Healing biopsy site Hypereosinophilic syndrome

MYOCARDIUM HYPERTROPHY Hypertrophic/dilated cardiomyopathy Pressure/volume overloaded ventricle FIBRE DISARRAY Hypertrophic cardiomyopathy Ventricular apex Healed biopsy sites Muscle dystrophies Junction of free wall and interventricular septum

INTERSTITIUM FIBROSIS Healed biopsy site Cardiomyopathy Drug toxicity Healed myocarditis NEUTROPHILS Fulminant endocarditis EOSINOPHILS Parasitic infection Hypersensitivity Hypereosinophilic syndrome LYMPHOCYTES Allograft rejection Bacterial endocarditis Normal allograft Acute allograft rejection Leukaemia Myocarditis

INTRAMURAL VESSELS Systemic arterial hyperten sion Hypertrophic cardiomyopathy Intramural vessels Amyloid osis Allograft vasculopathy

COMPLICATIONS The current complication rate with the intravascular procedure at specialised centres is less than 1%. Sampling error( if focal in nature or limited to L.V) Hemopericardium(most common) Cardiac perforation(most serious) Nerve injuries Hematomas Cardiac arrhythmias Tricuspid valve apparatus damage Pericardial fibrosis/ Thickening Air embolism and pneumopericardium

TAKE HOME MESSAGE The endomyocardial biopsy remains the gold standard mode of investigation, as there is considerable limitation to non-invasive imaging techniques. EMB is used to follow allograft rejection after heart transplantation. EMB is used to diagnose conditions like Cardiomyopathies Myocarditis Infiltrative lesions Arrhythmias Drug toxicities

EMB is used as a research tool to investigate the natural history of disease. EMB is a safe, simple, and effective interventional procedure with a very low rate of morbidity and mortality. Interpretation of EMB specimens requires knowledge of patients clinical history. It also requires appropriate understanding of cardiovascular pathophysiolgy.

An approach to endomyocardial biopsy interpretation -- Cunningham et al_ 59 (2) 121 -- Journal of Clinical Pathology Rosai and Ackermanns Sternberg surgical pathology Internet Robbins

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