Endomyocardial Biopsy in Medicine in the Evaluation of Conditions Leading to Cardiac Transplantation and in the Evaluation of Cardiac Allograft Rejection

Denton A. Cooley's 50th Anniversary Endomyocardial Biopsy in Medicine in the Evaluation of Conditions Leading to Cardiac Transplantation and in the Evaluation of Cardiac Allograft Rejection

Hugh A. McAllister, Jr., MD T hese issues of the journal commemorate the 50th anniversary (1944) of Dr. Denton A. Cooley's practice of medicine, and the 26th year (1968) since his performance of the 1st successful human heart transplant in the United States and the 2nd in the world. It seems appropriate, therefore, to discuss the utility of endomyocardial biopsy in the evaluation of conditions leading to cardiac transplantation and in the evaluation of cardiac allograft rejection after transplantation. Sakakibara and Konno described the technique of transvenous right ventricular endomyocardial biopsy in 1962.' The technique was used sparingly at first, mostly in attempts to distinguish primary from secondary cardiomyopathies. In- terest in the procedure increased as its use in monitoring doxorubicin cardiotoxicity and cardiac allograft rejection was realized.2-' Endomyocardial biopsy is now widely used in the evaluation of patients with unexplained congestive heart failure, arrhythmia, or atypical chest pain. A number of causes of these clinical conditions have been identified, including myocarditis. Indeed, one of the most exciting and challenging areas in endomyocardial biopsy interpretation is the diagnosis of myocarditis and the subsequent monitoring of the effects of treatment on this disease process. This series in recognition of Dr. Cooley's 50th Endomyocardial Biopsies anniversary in medicine is continued from the Procedures used to obtain myocardial tissue include operative resection (infun- December 1994 issue. dibular tissue in patients with muscular obstruction to right ventricular outflow, ventricular septal muscle in patients with hypertrophic obstructive cardiomyop- Key words: Biopsy; athy, and left atrial appendage in patients with mitral valvular disease); operative cardiomyopathy/congestive; cardiomyopathy/hyper- biopsies; and removal by various types of bioptome catheter, which can be used trophic; collagen vascular to obtain samples of ventricular endocardium and adjacent myocardium. In the disease; endocardial fibro- right ventricle, catheter biopsies usually are taken from the septal wall; in the left elastosis; endocardiuml pathology; endocrine heart ventricle, they are taken from the free wall. Multiple samples can be obtained, disease; glycogen storage each measuring about 3x2x2 mm. Because of the risk of perforation of a thin disease; graft rejection! chamber wall, catheter biopsies usually are not taken from the atrial walls or the classification; heart transplantation; ischemia/ right ventricular free wall. Transmural samples of ventricular wall have also been pathology; myocarditis obtained (percutaneously or at open thoracotomy) with biopsy needles. Regardless of the method used to obtain tissue, myocardial biopsies show arti- From: The Departments facts related to the unopposed contraction that the free edges of the tissue un- of Pathology, St. Luke's dergo as a response to cutting. These artifacts are manifested as hypercontraction Episcopal Hospital and Texas bands and are most pronounced in a peripheral zone that extends for 100 to 200 Heart Institute, Houston, Texas 77030 gm into the depth of the tissue. Thus, the artifacts constitute a significant draw- back to the use of needle biopsies, particularly those taken with needles of small internal diameters. Operative biopsies also may show artifacts (especially in mito- Section editors: Grady L. Hallman, MD chondria) related to elective cardiac arrest and cardiopulmonary bypass. Robert D. Leachman, MD Endomyocardial biopsy specimens are to be evaluated systemically to include: John L. Ochsner, MD 1) endocardium (thickness, cell types and number, stroma, contiguity, and thrombus); 2) cardiac myocytes (size, arrangement, degeneration, storage deposits, sar- Address for reprints: coplasmic membrane changes, nuclear changes, organisms such as Toxoplasma Hugh A. McAllister, Jr., MD, or trypanosomes, or cytomegalovirus inclusions); 3) myocardial interstitium (cell Texas Heart Institute, MC 4-265, PO. Box 20345, types and number, stromal composition, storage deposits such as amyloid, and Houston, TX 77225-0345 organisms such as fungi; and 4) blood vessels (endothelium, basement membrane,

R-was Hearl Institute.lournal Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection 5_ wall thickness and composition, thrombus or em- most severe cellular degeneration. Perhaps the most bolic materials, and organisms such as rickettsiae). important degenerative change is myofibrillar lysis, Endomyocardial biopsy specimens need to be prop- which results in loss of myofibrils and usually in- erly oriented to include endocardium in the plane of volves the thick (myosin) filaments to a greater ex- sectioning. For electron microscopic processing this tent than the thin (actin) filaments. Because of this, should be done before tissues are darkened by myofibrillar lysis often leaves in the muscle cells postfixation with osmium tetroxide. numerous actin filaments that are no longer associ- Light microscopic stains routinely used to study ated with myosin filaments. The Z-bands are much formalin-fixed endomyocardial biopsies are hema- wider than normal and may actually become con- toxylin and eosin (H&E) and Masson trichrome. fluent. Some of these Z-bands have a highly orga- Movat pentachrome, periodic acid-Schiff (PAS), nized substructure similar to that seen in skeletal Congo red, Perls' stain for iron, and stains for organ- muscle in nemaline myopathy. In cells undergoing isms are used as needed. For immunohistochemical myofibrillar loss, the sarcoplasmic reticulum can or biochemical studies, unfixed tissue must be undergo proliferation and formation of various types frozen rapidly and kept under appropriate storage of aggregates of the sarcoplasmic reticulum tubules conditions; however, monoclonal antibodies have and cisterns. Other changes indicative of degenera- recently become available that are most useful for tion of cardiac muscle cells include intranuclear tu- the immunohistochemical identification of inflam- bules; intramitochondrial and intranuclear deposits matory cells and subtypes of lymphocytes (common of glycogen (these also can occur in nondegenerated leukocyte antigen-L26 for B cells and UCHL1 and cells); accumulation of tangled masses of intermedi- T12 for T cells), and that can be used with paraffin ate or cytoskeletal (100 A in diameter) cytoplasmic sections of formalin-fixed tissue. Tissue should not filaments; dilatation and disorganization of T tu- be rinsed with saline before fixation, because this bules; formation of electron-dense concentric la- causes severe artifacts. If electron microscopic study mellae (myelin figures); dissociation of intercellular is to be performed, the tissue can be fixed either junctions, and development of unusual (intracyto- with 2.5% to 3% glutaraldehyde or with a mixture of plasmic) junctions formed by 2 parts of the plasma 1% glutaraldehyde and 4% formaldehyde in 0.1 M membrane of the same cell (rather than by the plas- phosphate buffer, pH 7.4 (McDowell's fixative). Fixa- ma membranes of 2 different cells); thickening of tion with this solution allows satisfactory prepara- the basal laminae of the muscle cells; and formation tions to be made for both light and electron micro- of spherical microparticles derived from the plasma scopic study. Staining en bloc with uranyl acetate is membrane, particularly in junctional areas. These not necessary for ultrastructural study and may in- spherical microparticles should not be confused terfere with the staining of glycogen particles. with viral particles. Two types of myocardialfibrosis are recogniz- Cardiac Hypertrophy able. The first, interstitial fibrosis, is associated rela- and Fibrosis tively frequently with myocardial hypertrophy and is characterized by bands of fibrous connective Hypertrophy is evaluated by light microscopy on the tissue that encircle the cardiac muscle cells and basis of the transverse diameters of the muscle cells separate them from adjacent cells. The 2nd type, re- (normally less than 15 jum) and nuclear morphology. placement fibrosis, is associated with the healing of If necessary, additional evaluation is made by elec- muscle cell necrosis and is characterized by patches tron microscopy, which is useful in determining the of fibrous connective tissue in which cardiac muscle presence and severity of degenerative changes that cells are either very scarce or absent. Ultrastrictur- occur in the late stages of hypertrophy. Structural ally, both types are composed of collagen fibrils, alterations resulting from hypertrophy alone include spicules and star-shaped granules of proteoglycan increased size of the nuclei, Golgi complexes, and material, small elastic fibers, and connective-tissue T-tubules; increased degrees of convolution of in- microfibrils. The relative amounts of these compo- tercalated disks; increased numbers of ribosomes; fo- nents are variable. cal accumulations of Z-band material; variability in mitochondrial size; and large accumulations of gly- Hypertrophic cogen granules and mitochondria in perinuclear Cardiomyopathy areas. Degenerative changes in hypertrophied myocar- Several forms of hypertrophic cardiomyopathy are dium may involve practically every type of subcellu- recognized clinically according to whether or not ob- lar organelle and can occur in hypertrophy of any struction to left ventricular outflow and asymmetric cause. The enlarged, dilated atria of patients with hypertrophy of the ventricular septum are present. mitral valvular disease and atrial fibrillation show the The most common of these is concentric hypertro-

54 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection Vblume 22, iVziitibet- 1, 1995 phy of the left ventricle due to hypertension or other pressure overload. Occasionally, concentric hypertrophy may be idiopathic (Fig. 1). Cardiomegaly, generalized hypertrophy, thick ventricular walls, a small, slit-shaped left ventricular cavity, and asymmetric hypertrophy of the ventricular septum are present in the majority of patients with idiopathic hypertrophic subaortic stenosis (asymmetric septal hypertrophy).6 In a minority of patients, the mass of asymmetrically hypertrophied ventricular septal tissue is localized to the apical region of the left ventricle (apical hypertrophic cardiomyopathy); however, in most patients the asymmetric thickening is maximal in the middle third of the ventricular Fig. 2 In asymmetric septal hypertrophy, the ventricular sep- septum. In patients with the obstructive form of tum is hypertrophied in excess of the left ventricular free wall. hypertrophic cardiomyopathy, the mitral valve- Often, there is endocardial thickening overlying the septal of the anterior hypertrophy, caused by contact with the anterior leaflet of particularly the ventricular surface the mitral valve during ventricular systole. leaflet-usually is thickened by fibrous and elastic tissue; a plaque of similar fibroelastotic thickening is present on the endocardium of the septal wall of the left ventricular outflow tract (Fig. 2). This plaque, of the ventricular septum and the left ventricular free thought to result from contact between the septal walls. However, it is most pronounced in the central surface and the anterior mitral leaflet, is removed third of the septum, an area that cannot be sampled during left ventricular myotomy-myectomy. by using an endomyocardial bioptome by a left heart The cardiac muscle shows severe hypertrophy approach. Myocardial fiber disarray is observed very and foci of disarray in which cells are arranged in frequently in the myocardial biopsies of patients whorls instead of in parallel, and their myofibrils with hypertrophic cardiomyopathy; however, it is are oriented in various directions. This disarray is not specific for this disorder in a qualitative sense: present in the ventricular septum and in the anterior it occurs, with involvement of less than 5% of and posterior free walls of the left ventricle. The dis- myocytes in the ventricular septum, in patients with array has been quantified in large histologic sections various other disorders. Therefore, the diagnosis of and found to involve more than 5% of the total area asymmetric hypertrophic cardiomyopathy should not be either made or ruled out only on the basis of the findings in the small areas of tissue included in myocardial biopsies. Dilated Cardiomyopathy The term dilated cardiomyopathy designates a het- erogeneous group of syndromes that are characterized anatomically by marked cardiac dilatation, mild or no thickening of the ventricular walls, mural thrombosis, atrioventricular valvular regurgitation because of displacement of the papillary muscles toward the apex, and variable degrees of fibrosis and myocardial cellular degeneration (Fig. 3). Foci of myocytolysis may also be present. The nonspecific nature of the histologic and ultrastructural changes precludes making the diagnosis of dilated cardiomyopathy on the basis of biopsy findings alone. However, the histologic, immunohistochemical, and ultrastructural findings in dilated cardiomyopathy Fig. 1 In concentric left ventricular hypertrophy, the free wall may be of clinical predictive value. Much attention and septum are uniformly thickened. This type of hypertrophy is being given at the present time to the possibility may be secondary (e.g., with aortic stenosis, coarctation, or systemic hypertension) or may occur as idiopathic primary that in many cases dilated cardiomyopathy develops hypertrophy. as a consequence of myocarditis in which most of the inflammatory reaction has subsided, leaving

Texas Hean Institute.journal Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection 5S rent tachyarrhythmias, which eventually have been fatal in most cases. The etiology of this disorder re- mains unknown. Myocarditis Inflammatory cell infiltrates and myocyte damage or degeneration serve as the basis for the diagnosis of myocarditis," which can be acute (infiltrates composed of polymorphonuclear leukocytes, lymphocytes, or both) or chronic (also including plasma cells, macrophages, and usually some degree of fibrosis). The timing of the endomyocardial biopsy Fig. 3 In dilated congestive cardiomyopathy, all 4 cardiac with respect to the onset of myocarditis is crucial to chambers are dilated out of proportion to myocardial hyper- diagnosis and perhaps to therapeutic outcome. The trophy. Usually, there is diffuse endocardial thickening, and interstitial cell population changes with time, as dem- mural thrombi are common. onstrated by studies of viral myocarditis in animal models. In patients with progressive disease, de- creasing numbers of lymphocytes and increasing myocyte damage and interstitial fibrosis. Dilated car- numbers of fibroblasts have been observed by elec- diomyopathy of unknown cause, with or withlout tron microscopy in serial endomyocardial biopsies.12 lymphocytic infiltrates, has been reported in some Myocarditis can be caused by any one of a het- patients with the acquirecd immune deficiency syn- erogeneous group of disorders that usually present drome (AIDS). in predictable morphologic patterns, including in- Peripartal cardiomvopatby and alcoholic car- flammatory cell infiltrates characterized predomi- diomyopathy are 2 syndromes of dilated cardio- nantly by granulomas, eosinophils, neutrophils, or myopathy that do not have specific microscopic lymphocytes. The morphologic types of myocarditis features,"8 although they are clinically distinctive. and their causes are summarized in Tables I-IV. Alcoholic cardiomyopathy may be complicated by The most common type of myocarditis recognized thiamine deficiency (which can be determined only by endomyocardial biopsy is lymphocytic myocar- by biochemical studies). A significant number of pa- ditis, which is presumed in most cases to be postviral tients thought to have peripartal cardiomyopathy and to be mediated by an aberrant immune re- actually have lymphocytic myocarditis. Endomyo- sponse. However, the histologic criteria for diagnosis cardial biopsy is necessary to establish the proper of lymphocytic myocarditis are the most controver- diagnosis. Anthracycline cardiomiyopathy is a heart sial, primarily because numerous types of mono- muscle disorder induced by the administration of nuclear cells (which are connective tissue cells) in daunorubicin or doxorubicin, 2 antibiotics currently the myocardial interstitium resemble lymphocytes by used in cancer therapy. This type of cardiomyopathy conventional light microscopy (Table V). is characterized microscopically by myofibrillar loss and by striking dilatation of the sarcoplasmic reticu- TABLE 1. Causes of Lymphocytic Myocarditis lum, which imparts a characteristic vacuolated appearance to the affected cells.' The severity of these Infection (viral, fungal, protozoal, rickettsial, bacterial, changes can be assessed in a semiquantitative man- chlamydial, mycoplasmal) ner in myocardial biopsies.' "- This assessment is of value in deciding whether to continue administering Infectious mononucleosis these agents to patients Suspected of developing Aberrant immune response (postviral; Kawasaki disease; anthracycline-induced cardiomyopathy. polymyositis; systemic lupus erythematosus, mixed con- Another type of cardiomyopathy with distinctive nective tissue disease, other collagen-vascular diseases) morphologic features is infantile cardiomyopathy Drug reaction (hypersensitivity, drug-induced lupus, other) with histiocytoid change." Its features consist of yellow nodules composed of large, round or elongated Sarcoidosis (and other causes of granulomatous myocarditis) cardiac muscle cells that have lost practically all their Cardiac allograft rejection contractile elements; are filled with mitochondria, lipid droplets, and glycogen; and show various de- Idiopathic process grees of dissociation of their intercellular junctions. These features have been reported only in small (Adapted from: McAllister HA Jr,12 with permission.) children presenting with the sudclen onset of recur-

56 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection 1,blume 22, Number 1, 1995 The identification of lymphocytes alone is not nosis of lymphocytic myocarditis12 (Fig. 4). Lympho- enough to establish the diagnosis of myocarditis. cytes may be found in the myocardium of apparently The presence of lymphocytes in close approxima- normal individuals"3 and may also be present in such tion to the sarcolemmal membranes of degenerating conditions as drug-associated myocardial damage cardiac myocytes is required to establish the diag- (the consequences of drug hypersensitivity or toxicity),'4 at the periphery of another lesion such as a granuloma, 1517 or in ischemic lesions in the process TABLE II. Causes of Granulomatous Myocarditis of healing. Some disorders (lymphomas, leukemia, etc.) are associated with increased numbers of lym- Collagen-vascular disease (rheumatic fever, rheumatoid phocytes in many tissues of the body, including the arthritis, ankylosing spondylitis, Wegener's granulomatosis) myocardium, without evidence of myocyte damage. Metabolic disorder (Farber's disease, gout, oxalosis, granu- Large numbers of myocardial lymphocytes may be lomatous disease of childhood) present in these disorders but usually indicate a non- inflammatory or neoplastic condition. Proliferative disorders of the mononuclear-phagocyte system An (juvenile xanthogranuloma, Chester-Erdheim disease, malig- intensive search using cultures, serologic stud- nant histiocytosis) ies, and appropriate tissue stains must be made for specific etiologic agents such as viruses, rickettsiae, Infection (bacterial, mycobacterial, fungal, parasitic, rickettsial, or Whipple's disease) bacteria, fungi, or parasites when indicated. Viruses are considered the most frequent cause of myo- Sarcoidosis carditis but seldom are specifically identified. The Hypersensitivity value of viral cultures of myocardial biopsy specimens has not yet been established. Foreign body granulomas Idiopathic process TABLE V. Types of Mononuclear Cells in the (From: McAllister HA Jr,)2 reprinted with permission.) Myocardial Interstitium

Lymphocytes Endothelial cells Plasma cells Perithelial cells TABLE Ill. Causes of Eosinophilic Myocarditis Mast cells Cardiac histiocytes Schwann cells Macrophages Drug hypersensitivity Smooth muscle cells Fibroblasts Disseminated eosinophilic collagen-vascular disease Undifferentiated mesenchymal cells (Loffler's syndrome) Parasitic infestation (From: McAllister HA Jr,2 reprinted with permission.) Wegener's granulomatosis Cardiac allograft rejection - - Idiopathic process ->, M - >" ,-_ --

(From: McAllister HA Jr,12 reprinted with permission.)

TABLE IV. Causes of Neutrophilic Myocarditis v- ___'-,_.w-_.

_ ~ % . t- - : *-~~~~~~O Infarction Infection Direct (bacterial) Indirect (toxic, as in diphtheria, or septic) Leukocytoclastic vasculitis (collagen-vascular disease) Fig.+~e4 Lymphocytic_# myocarditis with myocyte degeneration and replacement fibrosis (H&E orig. x73). Progressive myocyte degeneration results in loss of contractile elements and (From: McAllister HA Jr,12 reprinted with permission.) subsequent heart failure.

Texas Heati Institute.fournal Endomyocardial Biopsy in;~~~~Cardiac Transplantation and Allograft Rejection 57 Rheumatic Myocarditis and Aschoff Nodules !

Aschoff nodules, located in endocardium or in perivascular areas, provide the basis for the diagnosis of the rheumatic process. They undergo a process of evolution, during only part of which they show specific diagnostic features (Aschoff cells and fibrinoid necrosis). Aschoff cells are large, mononucleated or polynucleated cells, contain chromatin bars located in the central third of the nucleus, possess ampho- philic cytoplasm, and have indistinct cytoplasmic borders. These cells differ from Anitschkow cells, which also have nuclear chromatin bars but are small and elongated, have a scanty cytoplasm lacking basophilia, and constitute a totally nonspecific finding.5 A nonspecific lymphocytic myocarditis is often found in patients with rheumatic fever. In the ab- sence of Aschoff nodules in a biopsy specimen, this rheumatic myocarditis cannot be accurately distin- with large areas of myocardium guished from other causes of lymphocytic myocar- Fig. 5 Cardiac sarcoid replaced by confluent granulomas, resulting in congestive ditis. heart failure. Myocardial inflammatory reactions also occur in collagen-vascular diseases and in sarcoidosis. Le- sions in collagen-vascular diseases include nonspecific myocarditis, which occurs in dermatomyositis somes and in the main cytoplasmic compartment. (with lymphocytic infiltrates) and in systemic lupus These deposits are associated with massive cardio- erythematosus (often in association with fibrinoid megaly and with a typical lacework appearance of necrosis, vasculitis, and pericardial and endocardial the muscle cells. In type III, the glycogen is morpho- lesions); fibrosis, which occurs in scleroderma with- logically normal bLut biochemically abnormal, and it out being associated with a significant inflammatory is free in the cytoplasm. In type IV, the glycogen is reaction; rheumatoid nodules and less distinctive both bioclhemically and morphologically abnormal; granulomatous lesions, which are found in rheu- it is basophilic, is free in the cytoplasm, is very matoid arthritis; and myocardial necrosis associated slowly degraded by amylase, and forms fibrils that with vascular lesions, which occur in periarteritis are about 40 to 50 A in diameter and are ultrastruc- nodosa, Wegener's granulomatosis, and thrombotic turally similar to those that occur in cardiac muscle thrombocytopenic purpura.' The lesions in myocar- cells in basophilic degenleration (a frequent, non- dial sarcoidosis consist of noncaseating granulomas specific incidental finding in the hearts of elderly with epithelioid cells and multinucleated giant cells individuals) and in the Lafora type of myoclonic (Fig. 5). Such lesions must be distinguished from epilepsy.'"' One patient with phosphofructokinase other types of myocardial granulomatous processes deficiency, glycogen storage disease, and cardiac in- and from giant cell myocarditis (Table II).'2 volvement has been reported," and 1 patient with glycogen storage disease limited to the heart and Metabolic and associated with deficient activity of cardiac phos- Two Storage Diseases phorylase kinase also has been described.21 necessary notes of caution are that myocardial bi- The diagnosis of glycogen storage disease should be opsy specimens often contain strikingly prominent hased not only on the morphologic demonstration pools of glycogen in perinuclear regions of the cells of increased amounts of glycogen but also on bio- (especially in comparison with necropsy speci- chemical analysis of the glycogen structure and on mens), and that glutaraldelehyde fixation prodluces a identification of the enzymatic defect (in leukocytes, marked artifactuial increase in the intensity of the liver biopsy, or tissue culture of skin fibroblasts). periodic acid-Schiff (PAS) reaction. Myocardial involvement is most severe in the infan- Cardiovascular lesions in the mnucopolyjaccbari- tile form or type II (Pompe's disease), but also oc- dose(s consist of deposits of acid mucopolysaccha- curs in types III and IV. In type I1, the glycogen is rides, and often also of glycolipids (whiclh have not morphologically and biochemically normal. In heart been fully characterized), in pleomorphic (usually muscle, the glycogen is stored both within lyso- vacuolated) inclusions in cardiac muscle cells, and in

58 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection l'ollittie 22, Aumber 1, T995 connective tissue cells and smooth muscle cells in endocardium, valves, and vessels. The large, extra- mural coronary arteries may display severe intimal thickening. Distinction between the cardiovascular morphologic findings in the various types of muco- 4'; polysaccharidoses and mucolipidoses is extremely difficult, and these findings should be closely corre- lated with extracardiac anatomic, clinical, and bio- - _- :- chemical observations. The lesions in these disorders also must be distinguished19 from those in generalized (GMI) gangliosidosis, Sandhoff disease (a type of generalized [GM,] gangliosidosis), and Farber disease (lipogranulomatosis). Other disorders associated with lipid storage phenomena within cardiac muscle cells and cardiac con- Fig. 6 Iron deposits in cardiac myocytes from a patient with hemochromatosis. These deposits are readily demonstrated nective tissue cells include Fabry's disease, in which by the Prussian blue reaction. (Perls' stain x84) the deposits contain glycolipids that show strong birefringence, are soluble in lipid solvents, and form parallel or concentric electron-dense lamellae with a regular periodicity (these deposits also involve cor- terioles) are involved to some extent in most of the onary endothelium and smooth muscle); type I and syndromes of amyloidosis (Fig. 7). Amyloid fibrils in type III hyperlipoproteinemia, in which foam cells myocardium must be distinguished from connective (containing neutral lipids) can form yellow patches tissue microfibrils, which are larger in diameter (120 in endocardium; homozygous type II byperlipopro- to 150 A) than amyloid fibrils (100 A), have a beaded teinemia, in which cholesterol-rich foam cells can appearance, and can be very numerous in fibrotic infiltrate the endocardium and coronary arteries; and hearts. Amorphous deposits of electron-dense mate- Gaucher's disease and Niemann-Pick disease, in rial have been observed ultrastructurally in endo- which foam cells (containing glucocerebroside and cardium, myocardium, and blood vessels in light sphingomyelin, respectively) occasionally can in- chain disease associated with restrictive cardiomy- filtrate the myocardial interstitium.19 The lamellar opathy. Such deposits can be recognized only by deposits that occur in the cytoplasm of cardiac ultrastructural study or by immunohistochemical myocytes in Fabry's disease and other metabolic dis- demonstration of the presence of immunoglobulin orders must be distinguished from those that occur light chains.26 in cbloroquine toxicity. In the latter disorder, lamellar deposits are associated with curvilinear bod- ies.2223 Triglyceride deposits within cardiac muscle cells are found in numerous disorders, including car- nitine deficiency, hypoxia and ischemia, alcoholic cardiomyopathy, Reye's syndrome, thyrotoxicosis, diabetes mellitus, prolonged hypotension, and conditions caused by administration of a variety of toxic agents. Other diseases that must be considered in the dif- ferential diagnosis of myocardial storage disorders are hemochromatosis, hemosiderosis, oxalosis, and amyloidosis. 19.2'.2; In hemocbromatosis and bemosid- erosis, the deposits show positive histochemical reactions for iron (Fig. 6). In the various syndromes of primary and secondary oxalosis, the oxalate crystals can be identified in myocardium by polarization microscopy of routine paraffin sections and by specific histochemical staining. The green birefringence of Congo red-stained amyloid deposits and the identification of amyloid fibrils by electron microscopy Fig. 7 Cardiac amyloid most commonly involves the myo- establish the diagnosis of cardiac involvement in cardium. However, the heart valves may also be involved amvloidosis. Myocardium, endocardium, valves, and by amyloid deposits, as illustrated in this photograph. vessels (mainly intramural coronary arteries and ar-

Texas Heati Instititte.fournal Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection 59 The cardiomyopathy in the Duchenne type of elastic fibers.2 In Loffler's endomyocardialfibrosis, progressive muscular dystrophy is characterized by the endocardial lesions consist of organizing throm- myocardial fibrosis that is preferentially distributed bus containing numerary eosinophils."' Selective in subepicardial areas. Cardiac involvement without calcification of elastic fibers, which often assume a distinctive anatomic changes occurs in numerous characteristic curled appearance, occurs in the mu- other heredofamilial neuromuscular disorders.1'9 ral endocardium and systemic vessels of patients Myocardial fiber atrophy, interstitial edema, and, who have pseudoxanthoma elasticum.1'5 Small, flat rarely, inflammatory cell infiltrates have been de- plaques containing amyloid deposits have been described in obese patients who have had severe, scribed in the mural endocardium in amyloidosis. often fatal ventricular arrhythmias while taking a Fibrous rings present in the left ventricular outflow modified liquid protein diet as part of a weight re- tract of patients with fixed outlet subaortic stenosis duction program. '9 have the usual layered structure of endocardium."' Cardiac morphologic lesions reported in enido- Pacinig catheters inserted into the right ventricle be- crine disorders27 include myocardial hypertrophy come attached to mural endocardium by a small and fibrosis in acromegaly; basophilic degeneration thrombus, which also forms a thin layer over the of the muscle cells in myxedema; focal myocarditis catheter surface. Organization of this thrombus leads and myocytolysis in pheochromocytoma; fiber cal- to the formation of a tightly adherent, white fibrous cification in hypercalcemia; focal vacuolization and sheath composed of collagen, microfibrils, fibrin, hyalinization of the muscle cells in Cushing's syn- and connective tissue cells.' drome (and also in hypokalemia of other causes); and hypertrophy in hyperthyroidism. Dilated cardio- Myocardial lschemia myopathy has been reported in association with diabetes mellitus, but morphologic findings have been Anatomic changes involving the myocardium in variable and nonspecific.2 The basement mem- ischemic heart disease comprise 1) cardiac muscle branes of myocardial capillaries have been reported cell damage, necrosis, and associated inflammatory to be thicker in diabetic and myxedematous patients reaction; 2) myocardial fibrosis; and 3) complica- than in control patients.28 tions such as perforation of the ventricular septum or a ventricular free wall, various forms of papillary Mural Endocardium muscle dysfunction (which show variable degrees of necrosis, fibrosis, atrophy. and calcification), ven- Mural endocardium in biopsy or surgical material tricular aneurysms, and embolic phenomena related should be evaluated with respect to 1) overall thick- to mural thrombi. Necrosis occurs in 2 main forms: ness; 2) layered arrangement; 3) type and number 1) coagulation necrosis, which is basically limited to of cells present; 4) presence of overlying thrombus, central regions of infarcts and is characterized by a and 5) relative amounts of extracellular components relaxed appearance of the sarcomeres and by intra- of connective tissue (collagen, elastic fibers, and mitochondrial flocculent deposits, and 2) necrosis proteoglycans). The pathologic reactions of mural with contraction bands, which is found in peripheral endocardium to injury generally lead to endocardial regions of infarcts and is characterized by deeply thickening, of which several types are recognized on eosinophilic hypercontraction bands, intramitochon- the basis of the predominant changes in the cellular drial calcific deposits, and progression to myocytol- and extracellular components of the endocardium.' vsis.3" Necrosis with contraction bands is thought Regional differences in normal endocardial thickness to arise from reperfusion of the ischemic area sur- and layered structure must be taken into account: rounding the central zone of coagulation necrosis. endocardium is much thicker and contains a much This type of necrosis is also observed in patients more developed layer of smooth muscle cells in the dying soon after cardiac operations (circumferential left atrium than elsewhere. hemorrhagic necrosis), and it is characteristically In congenital endocardialfibroelastosis, the thick- severe in the stone heart syndrome. However, this ness of ventricular and atrial endocardium is mark- necrosis is a nonspecific lesion that can also be seen edly increased, and the elastic fibers are very numer- in other conditions, including prolonged hypo- ous and larger than normal. Acquired endocardial tension, toxicity of endogenous or exogenous cat- fibroelastosis can be diffuse (a nonspecific finding in echolamines, accidental and iatrogenic electrical many conditions associated with ventricular dilata- injury, and myocarditis. The association of contraction) or focal (friction lesions, contact lesions, or jet tion band necrosis with coagulation necrosis in a lesions); elastic fibers in these lesions are small, and myocardial biopsy specimen should suggest the pos- the relative amounts of elastic fibers and collagen sibility of acute myocardial infarction, particularly are variable.' In contrast, the endocardial fibrous when lymphocytes are not a significant feature of the thickening in carcinoid heart disease is devoid of inflammatory infiltrate. Fibrosis associated with the

60 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection Iblume 22, A'tiiiibei- 1. 1995 healing of myocardial infarcts may lead to formation nothing but endocardium and visceral pericardium of ventricular aneurysms. These and resected left (Fig. 9), are characteristically found in Chagas' dis- ventricular papillary muscle (from patients un- ease;3' such aneurysms also occur in idiopathic di- dergoing mitral valve replacement for papillary lated cardiomyopathyy32 and, rarely, in hypertrophic muscle dysfunction) are the most common surgical cardiomyopathy.3334 pathology specimens related to ischemic heart dis- A pseudoaneurvsm results when the myocardial ease. wall ruptures, but the rupture is contained by adherent thrombus or by pericardial adhesions. The diag- Other Conditions Leading nosis is based on the following findings: 1) the to Cardiac Transplantation orifice by which the pseudoaneurysm communicates with the cardiac chamber is small compared with the The term cardiac aneurysm denotes a spectrum of size of the pseudoaneurysmal cavity; and 2) myo- gross and microscopic changes associated with an- cardial fibers are not present in the wall, which is a eurysmal bulging of a segment of the ventricular fibrous sac derived from parietal pericardium, organ- wall. These changes consist of overall thinning of the izing thrombus, or both. The latter distinction is ventricular wall, endocardial thickening by fibrous best found by examining the borders of the resected and elastic tissue, decrease in the amount of cardiac specimen. The most common causes of cardiac muscle, and corresponding increase in the amount pseudoaneurysm are 1) transmural myocardial in- of fibrous connective tissue in the wall. In their most farction with cardiac rupture; 2) cardiac operations severe form, these changes lead to the formation of in which incisions are made in the left ventricular large aneurysmal sacs, which are thin-walled, con- wall (or even in the ventricular septum, as in the tain very little cardiac muscle in their walls, may be myotomy-myectomy procedure for hypertrophic partially filled by laminated thrombi, and commu- obstructive cardiomyopathy); and 3) various forms nicate by a large orifice with the main portion of the left ventricular cavity. Calcific deposits may be present in the aneurysmal wall. In less severe forms, the affected areas of the wall show lesser degrees of thinning, fibrosis, and endocardial thickening and contain more numerous cardiac muscle cells. The latter often show degenerative changes. The transi- tion between fibrous aneurysmal wall and unin- volved myocardial wall may be gradual, and the borders of the ventricular aneurysm may show poor demarcation. The most common cause of true cardiac aneurysms is infarction of the anterior wall of the left ventricle (Fig. 8). However, apical aneurysms, formed by progressively severe thinning of the ventricular wall so that the aneurysmal wall contains practically

Fig. 8 Large aneurysm secondary to myocardial infarction. Fig. 9 Apical aneurysm with marked myocardial thinning in a The aneurysm contains organizing thrombus. heart from a patient with Chagas' disease.

Texas Heatl Instititte.journal Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection *,1 of trauma. Pseudoaneurysms are much more likely sarcomas with low metastatic potential, such as fi- to rupture than are true cardiac aneurysms. brous histiocytoma, may also be treated by trans- Valvular heart disease resulting in advanced myo- plantation.36 cardial failure may be an indication for cardiac transplantation (Figs. 10 and 11). Any of the cardiac Endomyocardial Biopsy and valves may be causative; however, aortic valve dys- Evaluation for Acute Rejection function is the most common. Evaluation of pulmonary arterial pressure is especially important in the During the endomyocardial biopsy procedure, 4 to pretransplant evaluation of these patients. 8 pieces of endomyocardium ranging from 1 to 4 Patients with certain types of congenital heart mm are obtained from the right ventricular septum. disease not amenable to correction may be candi- This number of biopsy specimens reduces the dates for cardiac transplantation. Hypoplastic left possibility of false-negative biopsy results due to heart syndrome is the most common condition in sampling error and also reduces the possibility of this category. Again, evaluation of pulmonary arte- false-positive biopsy results due to inflammation in rial pressure is important in the pretransplant eval- previous biopsy sites. Evaluating multiple versus uation. single biopsy specimens also increases the accuracy Intramural cardiac tumors, especially fibromas, of biopsy grading. Spiegelhalter and Stovin37 used a that interfere with myocardial function may necessi- statistical model to determine the number of unal- tate transplantation35 (Fig. 12). Some primary cardiac tered biopsy specimens required to produce a level of certainty regarding the precise degree of allograft rejection. They found that 3 unaltered specimens are 'ARI* I% . sufficient if a 5% chance of inadequate sampling is I f, ., acceptable. Four unaltered specimens are needed to reduce the chances of sampling error to 2%. For light microscopy evaluation, the biopsy specimens are fixed in 10% buffered formalin and pro- cessed for paraffin embedding. The usual fixation time is 2 hours. After fixation, tissue processing is expedited by use of a rapid tissue processor. Mul- tiple sections of the specimens are subsequently prepared. The sections on 2 slides are stained with H&E, and the sections on a 3rd slide with Masson trichrome. The evaluation of the degree of cardiac allograft rejection is based on examination of the slides that Fig. 10 Mitral stenosis secondary to chronic rheumatic heart have been stained with H&E. The Masson trichrome disease. The left atrium is markedly dilated.

Fig. 11 Bicuspid aortic valve with marked stenosis. Stenosis of bicuspid aortic valves rarely is due to commissural fusion. Fig. 12 The right ventricle of the child's heart is filled by a Rather, there is fibrocalcific thickening of the base and mid- large rhabdomyoma, resulting in almost total obstruction of portion of the valve. the pulmonary valve.

62 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection l1blitine 22, Number 1, 1995 stain, which is used as an adjunct to diagnosis, al- Grades 4 to 8 represent interstitial mononuclear lows closer evaluation of the extent of interstitial cells with cardiac myocyte degeneration of increas- fibrosis, and it also "highlights" cardiac myocyte de- ing severity (Fig. 15). In grade 4, only occasional car- generation.38 Electron microscopy is not useful in the diagnosis of acute cardiac allograft rejection, because a longer processing period is required for this method than for others.38'39 However, if ultrastructural examina- Ar._~A i;A tion is needed, the tissue is fixed immediately in l4r.~~ ~ ~~~~ 2.5% glutaraldehyde. Electron microscopy and im- munoelectronmicroscopy may be useful research tools in understanding the mechanisms of myocyte injury and in identifying cell populations involved in the rejection process. We have also found electron microscopy to be useful in distinguishing cardiac Chagas' disease from Toxoplasma by differences in organism ultrastructure. Either electron microscopy or immunoperoxidase techniques are useful for et...I' ' -9 olqllE distinguishing cytomegalovirus inclusions from re- Fig. 13 THI grade 2 cardiac allograft rejection. There are peri- active nuclei near previous biopsy sites. vascular accumulations of mononuclear cells. (H&E orig. x250) Two major systems have been used to measure cardiac allograft rejection: qualitative (using the terms mild, moderate, or severe to describe the degree of rejection)38'40 and quantitative (assigning a numerical value to describe the degree of rejection).3941-43 The primary disadvantage of the qualitative system is the potential for wide variation in the interpretation of qualitative terms. Such differences in interpretation may result in miscommunication among physicians or between physicians and their patients. In the case of cardiac allograft rejection, the consequences could be devastating. At the Texas Heart Institute (THI), a numerical value of 0 is given if there is no evidence of cardiac allograft rejection (Table VI). A grade of 1 or 2 is assigned if there are only perivascular aggregates of Fig. 14 THI grade 3 cardiac allograft rejection. The mono- mononuclear cells (Fig. 13). Grade 3 indicates that nuclear cells extend into the interstitium without myocyte in addition to perivascular aggregates, mononuclear degeneration. (H&E orig. x250) cells are extending into the interstitium (Fig. 14).

TABLE VI. Texas Heart Institute's Evaluation of Cardiac Allograft Rejection by Endomyocardial Biopsy

0 No evidence of rejection 1-2 Perivascular aggregates of mononuclear cells 3 Perivascular aggregates of mononuclear cells with extension into the interstitium 4-8 Interstitial mononuclear cells with cardiac myocyte degeneration of increasing severity 9-10 Extensive cardiac myocyte degeneration, interstitial mononuclear cells, and polymorphonuclear leukocytes Fig. 15 THI grade 4 cardiac allograft rejection. The mononuclear cells extend into the interstitium with rare foci of (From: McAllister HA Jr, et al,39 reprinted with permission.) myocyte degeneration. (H&E orig. x250)

Texas Heart InstituteJournal Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection 63 diac myocyte degeneration exists, whereas in grade Because of the differences between quantitative'3 8, cardiac myocyte degeneration is frequent and and qualitative-- systems, the International Society multifocal. Simply stated, grade 4 represents occa- for Heart Transplantation, in 1989, convened an in- sional myocyte degeneration (all pieces must be ex- ternational meeting of pathologists from large, estab- amined to detect it); grades 5 and 6 represent lished transplantation centers to establish a universal scattered myocyte degeneration (readily found, but grading system for biopsy interpretation.'' The pur- not in every microscopic field); and grades 7 and 8 pose was not necessarily to change the grading sys- signify multifocal degeneration (present in almost tems in individual centers but to find one to which all high-power fields). This larger category ranging most grading systems could be extrapolated for use from grades 4 to 8 corresponds to the Stanford grad- in publications and multicenter drug trials. A gener- ing system for moderate cardiac allograft rejection. alized comparison of the Stanford, THI, and Inter- We believe that subdividing the "moderate" range national Society for Heart Transplantation grading of severity of rejection allows treatment to be more systems of cardiac allograft rejection is illustrated in closely tailored to individual patient needs. A grade Figure 17. Such a comparison must be generalized 8 level of rejection, for example, would require more for several reasons. The Stanford system uses myo- aggressive treatment than would a grade 4. cyte necrosis (cell death) as a criterion for distin- Grades 9 and 10 indicate extensive cardiac myo- guishing between moderate and severe rejection, cyte degeneration with polymorphonuclear leuko- whereas, we recognize the degree of myocyte de- cytes in addition to the interstitial mononuclear cells. generationi as the basis criterion to distinguish be- Interstitial hemorrhage secondary to vascular dam- tween grades 4 and 8. We believe that this type of age, occasionally with microthrombi, may be present cell degeneration is usually reversible if the patient in the higher grades of allograft rejection (Fig. 16). It is appropriately treated. We have rarely observed is important, however, not to over-interpret extra- true necrosis associated with the rejection process vasation of erythrocytes and leukocytes that result below grades 9 or 10. Myles and associates-" pre- from the mechanical trauma of the biopsy proce- sented ultrastructural evidence that myocyte necro- dure. The prime determinant of higher grades of al- sis is rarely seen in rejection and concluded that cell lograft rejection is the extent of cardiac myocyte injury is potentially reversible. degeneration. Sibley and colleagues4` also questioned the valid- l'lotting the numerical value and date of the pa- ity of myocyte necrosis as a criterion for the diagno- tient's previous endomyocardial biopsies on the 0 to sis of rejection and suggested that the classification 10 scale leads to accurate observations with regard of rejection be based on the number of mononuclear to 1) the degree ofcardiac allograft rejection, 2) the cells. At THI, however, we have observed patients direction ofchange (resolving or progressing), and who had a robuist cellular infiltrate in the perivas- 3) the speed ofchange. We believe that each of these cular and interstitial positions but did not develop variables is important in establishing the patient's evidence of myocyte degeneration or experience immunosuppressive regimen and scheduling the car- clinical rejection. Alternatively, we have seen pa- diac allograft biopsy intervals. tients develop muiltifocal myocyte degeneration and clinical signs of rejection with relatively few mononuclear cells. (These changes should not he confused with ischemic damage, whiclh would not have mononuclear cells intimately associated with lytic damage to myocytes.) This phenomenon of myocyte degeneration and signs of clinical rejection with few

Stanford - Mild Moderate -Severe-

THI -O-|-1-o-2- -3--4- -5--6--7- -8--9- -1O-

4' Fig. 16 THI grade 10 cardiac allograft rejecti'on. Mononu- ISHT -0-- IA -IB- -11- -lIlA- -IIIB- - IV- clear cells diffusely involve the myocardium with multifocal myocyte degeneration accompanied by myocyte necrosis Fig. 17 A generalized comparison of the Stanford, Texas with neutrophils, eosinophils, hemorrhage, and edema. Heart Institute (THI), and International Society for Heart Trans- (H&E orig. x250) plantation fISHT) grading systems of cardiac allograft rejection.

64 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection lWimie 22, Number 1, 1995 mononuclear cells usually occurs within 2 weeks Schreiber SS, eds. Alcohol and abnormal protein biosynthe- after transplantation or later in the clinical course, sis: biochemical and clinical. New York: Pergamon Press, with rapidly accelerating rejection. 1975:139-85. In most biopsy specimens, eosinophils can be 9. Ferrans VJ. Overview of cardiac pathology in relation to an- in thracycline cardiotoxicity. Cancer Treat Rep 1978;62:955-61. found the myocardial interstitium within the 10. Legha SS, Benjamin RS, Mackay B, Ewer M, Wallace S, 1st week after transplantation; however, their signi- Valdivieso M, et al. Reduction of doxorubicin cardiotoxicity ficance is unknown. They do seem to increase in by prolonged continuous intravenous infusion. Ann Intern number and frequency of degranulation in advanced Med 1982;96:133-9. degrees of rejection (grade 7 and above). Increased 11. Ferrans VJ, McAllister HA Jr, Haese WH. Infantile cardiomyopathy with histiocytoid change in cardiac muscle cells. graft eosinophilia has been described as a sensitive Report of six patients. Circulation 1976;53:708-19. indicator of graft rejection in human liver48 and kid- 12. McAllister HA Jr. Myocarditis: some current perspectives ney49 transplantation. Three patients at THI devel- and future directions. Tex Heart Inst J 1987;14:331-4. oped fatal eosinophilic myocarditis and arteritis 13. Billingham ME. The diagnostic criteria of myocarditis by without advanced features of the classic pattern of endomyocardial biopsy. Heart Vessels 1985;1(Suppl):133-7. 14. McAllister HA Jr, Hall RJ. latrogenic heart disease. In: Cheng lymphocytic allograft rejection.50 No parasites or TO, ed. The international textbook of cardiology. New York: other infective agents were identified, and no pe- Pergamon Press, 1986:871-83. ripheral blood eosinophilia was present in any of 15. McAllister HA Jr, Ferrans VJ. Granulomas of the heart and these patients. The relationship of this type of graft major blood vessels. In: loachim HL, ed. Pathology of granu- destruction to classic rejection is not yet clear; there- lomas. New York: Raven Press 1983:75-123. 16. McAllister HAJr, Ferrans VJ. Eosinophilic and granulomatous fore, the number of intact and degranulating eosin- inflammation of the heart. In: Kapoor AS, Reynolds RD. ophils is now evaluated at THI as an independent Cancer and the heart: a textbook of cardiac oncology. New factor. York: Springer-Verlag, 1986:246-63. In summary, a quantitative scale has been devel- 17. Edwards WD, Holmes DR Jr. Reeder GS. Diagnosis of active oped at THI to provide numerical objectivity as to lymphocytic myocarditis by endomyocardial biopsy: quantitative criteria for light microscopy. Mayo Clin Proc 1982; the degree, direction, and rate of cardiac allograft 57:419-25. rejection and to enhance communication between 18. McAllister HA Jr. Collagen vascular diseases and the cardio- pathologists, surgeons, and cardiologists. Each grade vascular system. In: Silver MD, ed. Cardiovascular pathology. of rejection requires a specific therapeutic approach, 2nd ed. Vol 2. New York: Churchill Livingstone, 1991:1151- especially in the moderate range (THI grades 4 79. 19. Ferrans VJ. Metabolic and familial diseases. In: Silver MD, ed. through 8). The system is simple and reproducible; Cardiovascular pathology. 2nd ed. Vol 2. New York: it has minimal interobserver variability and is easily Churchill Livingstone, 1991:1073-149. learned. With the use of this system during a 9-year 20. Hays AP, Hallett M, Delfs J, Morris J, Sotrel A, Shevchuk MM, period at the Texas Heart Institute, over 10,000 car- et al. Muscle phosphofructokinase deficiency: abnormal diac allograft biopsy specimens have been evaluated polysaccharide in a case of late-onset myopathy. 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66 Endomyocardial Biopsy in Cardiac Transplantation and Allograft Rejection l'ohime 22. Aumber 1. 1995