Role of cardiac biopsy in the diagnosis and management of cardiac disease

Role of Cardiac Biopsy in the Diagnosis Cardiac Disease

and Management

of

Robert E. Fowles and Jay W. Mason

ARDIAC BIOPSY has been available since the 1950s but the technique has only C become popular during the past ten years. The recent increasing use of heart biopsy has resulted from the development of safer, easier methods and from new information about the indications for the procedure. It has long been a fundamental tenet of medical science that illness can be better understood through knowledge of the structural aberrations in diseased tissue. In the case of the myocardium, it currently appears that microscopic examination may yield clinically useful information only in certain disorders. Because the myocardium seems to respond similarly to a variety of insults, it may be true that biopsy will prove more revealing in the early than in the late stages of disease. The diagnostic usefulness of myocardial biopsy continues to be debated and clarified; however, there are several applications of the technique that have gained general acceptance. Our purpose in this article is to describe the evolution and current practice of heart biopsy, to explain the procedure’s risks, to review the diagnoses attained by myocardial biopsy, to suggest its clinical indications, and to outline several new areas of investigation using this invasive tool. HISTORICAL DEVELOPMENT OF CARDIAC BIOPSY

In 1956 human heart biopsy performed via limited thoracotomy using a Vim-Silverman needle was described by Sutton.’ Since then a number of percutaneous needle techniques have been reported, which failed to gain acceptability due to a relatively high complication rate (mainly involving pneumothorax and cardiac tamponade).2*3 In 1962 Sakakibara and Konno developed a biopsy catheter, or “bioptome,“4 revolutionizing the field of heart biopsy. Transvascular endomyocardial biopsy is much safer than needle techniques and is more convenient to both patient and physician. Patient discomfort and anxiety are greatly reduced and the entire test can be done in a few minutes; at Stanford myocardial biopsy is often done on an outpatient basis. Progmw

in Cardiovascular

Diseases,

Vol XXVII.

No 3 (November/December).

Technical advances in catheter biopsy since 1962 have retained the principal features of Konno and Sakakibara’s design: transvascular introduction of a catheter with movable cutting jaws. In 1972 Caves and associates at Stanford designed a modified bioptome that could be inserted via the right internal jugular vein and did not require a guiding catheter or long sheath.’ This instrument was used for the diagnosis of cardiac transplant rejection, now an undisputedly valuable clinical application of heart biopsy. Subsequent refinements in the bioptome catheter have been accompanied by broader diagnostic use.6 Brooksby and coworkers as well as Richardson and his associates have demonstrated the efficacy of a modified bronchoscope biopsy forceps in obtaining tissue from both right and left ventricles.‘.’ Miniaturized bioptomes have been used safely in children and infants as young as four months.9~‘0 Kawai and Kitaura have designed a new biopsy catheter with a flexible tip controlled by the operator, facilitating entry into the left ventricle and potentially thereby enhancing the ability to obtain samples from different sites.” INSTRUMENTS

Primarily because of safety, but also due to the other virtues mentioned above, the transvascular catheter technique has become the standard method for nonoperative myocardial biopsy. Three general approaches for endomyocardial biopsy are currently used. The bioptome developed by Caves and associates at Stanford and modified by Mason is predominantly used in the United States. The Konno instrument has probably been most widely employed in the world, and From the Cardiology Division, Stanford University School of Medicine. Veterans Administration Medical Center, Palo Alto, Calif, and the Cardiology Division, University of Utah Medial Center, Salt Lake City. Address reprint requests to Robert E. Fowles. MD, Cardiology Division, University of Utah Medical Center, 50 North Medical Dr. Salt Lake City, UT 84132. o 1984 by Grune & Stratton, Inc. 0033-0620/84/2703~002$0S.00/0 1994:

pp 153-

172

153

FOWLES

Fig 1. Closeup view of the Stanford bioptome handle. The drive wire within the shaft is moved by opening and closing the modified clamp device, with the amount of tension being varied by an adjustable spring mechanism.

the King’s College bioptome has been popularized in Europe. The Stanford instrument is a relatively short (50 cm) bioptome that can reach the right ventricle from the right internal jugular or subclavian vein (Fig I ). The abbreviated length of the bioptome markedly improves the operator’s ability to maneuver the catheter and direct it toward the appropriate endocardial region. The Stanford bioptome’s hemispherical cutting jaws usually remove samples 1 to 3 mm in diameter: one of the jaws swivels open. the other is stationary. The moderately large specimen size afforded by an instrument that can be selectively directed toward multiple sites enhances adequate histologic inspection. Mason’s modification of the original Caves design involves installation of an adjustable spring mechanism allowing the operator to vary the cutting force of the movable jaw.

MASON

Other biopsy instruments are used in similar fashion. The Konno bioptome.J IO0 cm long, can be introduced via arm veins, but has a relatively stiff shaft, diminishing maneuverability. Its jaws are available in two sizes. 7.5 mm and 3.5 mm. outer diameter. Both jaws move on a doubleswivel mechanism. The King’s bioptome” is a modified Olympus bronchoscope forceps (Olympus Corp, New Hyde Park, NY) with a jaw diameter of I .8 mm. As such. it is the smallest of the currently used instruments. It may be inserted through an 8 French sheath admitted directly to the ventricle. A commercially available bioptome similar to the King’s instrument has been recently introduced by Cordis in the US (Cordis Laboratories. Inc. Miami. FL). with the unique quality 01 disposability. TECHNIQUES

OF

ENDOMYOCARDIAL

BIOPSY

In nearly all cases, the right internal jugular vein is used for access to the right ventricle. The skin overlying the right internal jugular vein between the medial and lateral heads of the zternocleidomastoid muscle and approximateI> 3 cm ccphalad from the superior border of the clavicle is prepared in a sterile fashion, draped. and intiltrated with local anesthetic. A puncture site is made with a pointed scalpel and widened with a small hemostat in the usual manner for percutaneous catheterization. A I .5 inch, 71. gauge probing needle attached to a saline-filled syringe is directed at an angle of 30’ to 4S”

Fig 2. Technique of transvenous right ventricular endomyocardial biopsy, slightly curved, allowing maneuverability. Panel 1: the bioptome is introduced atrium, with the tip curved laterally. From the lower third of the right etrium toward the right ventricle. Panel 2: the catheter bioptome is placed across the onto the endocardial trabeculated surface, catheter is in the right Panel 5: the jaws are

AND

using the Stanford bioptome. The bioptome is from the right internal jugular vein into the right the tip is then rotated medially, pointing now tricuspid valve into the right ventricle, advanced

surface and then withdrawn approximately 1 cm. Panel 3: with the tip free the bioptome’s jaws are opened. Panel 4: the bioptome is advanced gently. ventricular apex and appears to be below the left hemidiaphragm due to the closed, and in Panel 6, the bioptome and the biopsy specimen it now contains

of the endocardium and its In this view, the tip of the anteroposterior projection. are removed.

CARDIAC

BIOPSY

caudal from the vertical and loo to 20” toward the right shoulder and advanced gently with mild syringe suction until free-flowing blood is drawn back from the internal jugular vein. With the syringe removed, the probe needle is left in place as a marker. An 18-gauge thin-walled needle, again attached to a saline-filled syringe, is then advanced through the skin wound alongside the probe needle and introduced also into the vein, whereupon the probe needle is removed and standard Seldinger technique is used to advance a 9 French sheath and dilator set into the right internal jugular vein. The sheath must have an occluding flexible diaphragm or other device to prevent air embolism from occurring when the bioptome is not in place. The bioptome is inserted through the sheath into the lower third of the right atrium under fluoroscopic visualization, with the curved tip of the instrument initially pointed laterally (Fig 2). The tip is then rotated medially, advanced across the tricuspid valve. pointed toward the interventricular septum, and advanced toward the ventricular apex. Right ventricular location (rather than coronary sinus, for example) is confirmed by the observation of premature ventricular beats, the sensation of myocardial contraction transmitted via the bioptome, and the fluoroscopic image of the catheter position. Two dimensional echocardiography may provide in some cases further verification of

155

bioptome position (Fig 3). Bioptome tip location is important because of the relative thinness of the right ventricular free wall compared to that of the septum. We have always preferred the short, torque-controllable bioptome because of this. Two-dimensional echocardiography may be an adjunctive aid, but does not yet replace fluoroscopy in our view. After the apical septal biopsy site is contacted by the closed-jaw tip, the bioptome is withdrawn 1 cm, the jaws opened, and the instrument readvanced gently against the interventricular septum. The jaws are then closed and the bioptome removed, usually requiring a gentle tug. This procedure is repeated as needed to obtain a sufficient number of specimens, usually three to five. Adequate samples are obtained in 98% of procedures with this technique, in our laboratory (Fig 4). Left Ventricular

Endomyocardial

Biopsy

Left ventricular bioptomes can be introduced via the brachial or femoral artery as originally reported by Konno, or through a long sheath.’ The sheath is positioned in the left ventricle over a regular pigtail angiography catheter; once in place, the sheath will admit the bioptome multiple times for repeated biopsies. As noted above, Kawai and Kitaura have recently designed a “steerable” biopsy catheter that is more easily guided into the left ventricle without a sheath, but requires a straight stylet to be advanced through the catheter to provide enough stiffness for the jaws to excise a sample of endomyocardium.” Other Methods

Percutaneous transthoracic needle biopsy of the heart has been largely replaced by the cathe-

Fig 3. An illustration of echo-guided biopsy. In this two-dimensional echocardiographic short axis view, the tip of the bioptome is indicated by the large black arrow. The bioptome itself can be seen as a curvilinear echo within the right ventricle W. Note that the bioptome tip curves onto the endocardial surface of the interventricular septum (ivs) rather than the thinner right ventricular free wall Mwj. The left ventricle (LVj is seen in the lower portion of the image. A dimensional scale is given to the right: “ant,” anterior.

Fig 4. Photograph of the open jaws of the Stanford bioptome, showing the endomyocardial specimen just obtained. Note that only one of the jaws opens, preventing too large a sample from being taken, and affording some stability to the direction of the tip.

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156

ter methods. Needles with outer diameters as large as 17 gauge were inserted through the chest wall close to the cardiac apex in order to traverse as little pleural and lung tissue as possible, and to avoid large epicardial vessels. Most samples were more than l-cm long and I -mm thick, representing pericardium, epicardium, myocardium, and endocardium. COMPLICATIONS

AND

RISKS

Catheter biopsy of the endomyocardium is quite safe when performed by experienced physicians. Occasionally the patient may experience unpleasant chest sensations or even pain at the instant of biopsy, but this is only momentary and rarely if ever followed by serious problems. The procedure is performed in the cardiac catheterization suite with local anesthesia. We use systemic heparinization for left ventricular biopsies to avoid arterial thromboembolization. The needle biopsy method is the most hazardous of the techniques described. In his series of 198 patients, Shirey observed an overall complication rate of 10% with an 8%) incidence of tamponade, half requiring emergency thoracotomy.’ In contrast to the complication rate for needle biopsy, the incidence of complications for catheter biopsy in the Stanford series of over 4,000 cases is less than 1% (Table 1). Cardiac tamponade has occurred in only four patients (less than 0.14%). none of whom required thoracotomy. The only other complication referrable to the catheter biopsy procedure itself has been the occurrence of atria1 fibrillation in three patients; although frequent premature ventricular contractions are common, we have induced a sustained ventricular arrhythmia in only one 1.

Complications

and

Risks

SPECIFIC

Pneumothorax

Pneumothorax Arrhythmias

(ventricular)

Epicardial

damage,

including

artery

laceration

coronary

muscle

hepatic

injury

Hemoparicardium, ade

(8%)

or splenic,

Cervical

tissue

trauma,

Cardiac Allograft

Rejection

Since the early 1970s endomyocardial biopsy has been a mainstay at Stanford for the diagnosis of transplant rejection. The Stanford bioptome was. in fact, developed for this purpose. The success of modern cardiac transplantation is in part due to early and reliable detection of rejection, which has in the last decade become a 2.

Specific

Disorders

Endomyocardiel Cardiac

allograft

rejection

Myocarditis (Adriamyclnl

hematoma

Cardiac

sarcoidosis

Air embolism

Cardiac

hemochromatosls

Endocardial (atria1

ventricular

Hemopericardium, tamponade Stanford

fibrilla-

Fabry’s

ectopy)

Carcinoid

cardiac (0.12%,

series)

Diagnosed Biopsy

amyloidosis

Arrhythmias

BY

BIOPSY

Several disorders have been specifically diagnosed by endomyocardial biopsy. These include the list given in Table 3. Additionally, the nonspecific histologic and ultrastructural abnormalities seen in a variety of cardiac illnesses have been identified or verified by biopsy, and will be discussed later below.

Doxorubicln

soft

ATTAINABLE

CARDIAC

Cardiac

tion, tampon-

DIAGNOSES

Biopsy

Catheter Biopsy (Overall Rate. 1%)

Needle Biopsy: (Overall Rate. 10%)

Potential

of Cardiac

MASON

patient. Complications related to the internal jugular vein approach have included pneumothorax in three patients, uncomplicated air embolism in six patients, transient right recurrent laryngeal nerve paresis in two patients, and a Horner’s syndrome in one patient. There have been no fatalities in our series. In an unpublished survey, Dr E.G.J. Olsen with Dr U. Baandrup of the National Heart Hospital in London found a complication rate of I .5S% in 3,097 reported catheter biopsy cases from Europe. Sekiguchi has assembled by questionnaire the complications from 6,739 worldwide cases, finding an overall incidence of I. 17% .I’ This includes 28 cases of perforation of the right or left ventricle (0.42%) and two deaths (0.03%). These rates of complication are less than those for routine cardiac catheterization.

Table Table

AND

fibrosis disease

of the heart

disease

Irradiation

injury

Glycogen

storage

Cardiac

cardlotoxiclty

tumors

disease

by

CARDIAC

BIOPSY

157

histologic rather than a clinical diagnosis, as it was in earlier days. Histologic findings on biopsy correlate well with recently developed immunologic measures of rejection. It is rare in the Stanford experience to find sampling error in rejection: multiple simultaneous samples almost uniformly agree, and autopsy specimens reflect those obtained by bioptome in life. During the initial postoperative period of one to two months, transplant recipients undergo biopsy weekly or more frequently if any clinical or histologic sign suggests acme rejection. Experience with transplant patients has shown that serially repeated right ventricular septal biopsies can be obtained via the jugular transvenous route easily and without adverse sequelae. Many recipients have undergone more than 20 biopsies. Three to five samples are gathered at each procedure. Good histologic correlation between the biopsy obtained and postmortem findings has been observed in our program. Others have confirmed that multiple specimens from different ventricular sites are essential to avoid sampling error.13 Specimens are routinely stained with hematoxylin and eosin, Masson’s trichrome, and methyl green pyronine. The histopathologic features of acute cardiac

text

Fig 5. Mild for histologic

cardiac allograft description.

rejection:

sea

transplant rejection correlate with clinical severity and are graded according to the following scale: Grade I = mild acute rejection. Endocardial and interstitial edema with scanty perivascular lymphocyte infiltrate. These lymphocytes, or immunoblasts, have been determined to be of T cell origin, and take up methyl green pyronin stain, indicating high RNA turnover (Fig 5). Grade 2 = moderate acute rejection. A more substantial endocardial, interstitial, and perivascular infiltrate is seen; focal degenerative myocytolysis may be evident in heavily infiltrated areas (Fig 6). Grade 3 = severe rejection. Marked infiltrate with large, pyroninophilic lymphocytes and polymorphonuclear leukocytes; frank myocyte necrosiscan be seen,aswell asinterstitial hemorrhage (Fig 7). Resolving rejection. Active, new fibrosis with a few nonpyroninophilic interstitial lymphocytes; lipochrome pigmentation. With appropriate therapy even the more severe changesin rejection may begin to resolve within 24 hours. This resolution is monitored by biopsy; complete reversal may be noted in subsequent samplesas early as 72 hours later.

158

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Fig 6.

We have observed a marked decrease in the incidence of acute rejection since the institution of routine cyclosporine administration in transplant recipients. This agent seems to cause a slower onset of rejection when such occurs, with a likewise slower resolution time. Without rejec-

Fig 7.

Severe

rejection

(see

text].

Moderate

rejection

AND

(see

MASON

text)

tion. a baseline “normal” sparse interstitial intiltrate of poorly staining mononuclear cells is often present. not indicative of rejection. Additionally. cyclosporine administration appears to be associated with fine, diffuse interstitial fibrosis unrelated to previous rejection injury.

CARDIAC

BIOPSY

Doxorubicin

159

Cardiotoxicity

Doxorubicin hydrochloride (Adriamycin), a potent, wide-spectrum antitumor agent, is limited in its usefulness by dose-related cardiotoxicity. Many clinicians administer the drug up to a maximum total dose of 500 mg/m*. However, some patients may suffer cardiotoxic effects at lower cumulative doses, particularly in the setting of previous chest radiotherapy, preexisting cardiac disease, or age greater than 70 years. On the other hand, some patients tolerate doses well beyond the arbitrary limit and would often benefit from extended treatment. In our experience, traditional noninvasive methods such as systolic time intervals and echocardiography have not proven highly reliable for the detection of doxorubicin-induced cardiac dysfunction during chemotherapy.14 We have found that the degree of myocardial injury, as assessed by invasive hemodynamic measurement, can be estimated by histologic grading of biopsy specimens.rs Biopsy is an effective means

Fig 8. An example of histologic lesions points to a cardiac myocyte with myofibrillar cell with vacuolixation.

in anthracycline loss. A few

other

of monitoring patients receiving doxorubicin, actually permitting the administration of more doxorubicin, without emergence of congestive heart failure.16 The doxorubicin-induced lesion appears to have a characteristic histopathologic and ultrastructural pattern: two types of myocyte change are induced, myofibrillar loss and cytoplasmic vacuolization (Fig 8). The first, myofibrillar loss, causes cells to appear shrunken, with a homogeneous, pale cytoplasm on light microscopy (Fig 8). Ultrastructurally, myofibrils are nearly totally lost, often despite normal appearing nuclei. The second type of lesion, cytoplasmic vacuolization, is due to a coalescence of dilated sarcoplasmic reticulum (Figs 9 and 10). The histologic grading of doxorubicin cardiotoxicity is divided into three degrees of severity. Grade 0 is normal myocardial morphology. Grade 1 includes scattered, single myocytes affected with partial myofibrillar loss or distended sarcotubular system, usually with completely normal appearing neighboring cells; this focal finding

cardiotoxicity. such cells

csn

In this photomicrograph, be seen in the field.

The

the small, straight arrow curved arrow points to a

160

FOWLES

Fig tion cells are

tin, with

Fig 9. Cellular vacuolization Adriamycinl cardiotoxicity. extensive vacuole formation.

in anthracyciine The arrow

points

(doxorubito a cell

permits continued administration of doxorubicin. Grade 2 has clusters of unequivocally. more severely affected cells; at this point, doxorubicin treatment can be extended cautiously if hemodynamic performance is satisfactory. Grade 3 shows diffuse myocyte damage. including cell necrosis; no further doxorubicin should be given until the abnormality resolves. Intermediate grades between categories are signified by “grade I .5, ” “grade 2.5,” etc. Since 1978 more than 100 patients have been monitored at Stanford with endomyocardial biopsy and right heart catheterization. There have been only two cases of cardiomyopathy. Some patients, who have undergone biopsy after each 150- to 200-mg/m’ dose increment above a total of 450 mg/m’, have gone on to receive greater than 1,000 mg/m’ cumulatively. Other workers have reported valuable experience in monitoring doxorubicin patients with radionuclide angiography.‘7,‘x This method appears helpful in identifying patients who have developed cardiotoxicity, or those who already have cardiac dysfunction and thus are at risk for cardiotoxicity.

10.

Electron

in anthracycline are affected; abnormal,

with

photomicrograph

showing

AND

MASON

vacuoliza-

cardiotoxicity. in milder degrees,

In this field, adjacent only occasional cells

neighboring

unaffected.

cells

Myucardial biopsy provides the sole accurate method for antemortem diagnosis of myocarditis. In the past, myocarditis has been clinically suspected or inferentially “diagnosed” in patients with sudden onset congestive heart failure, sometimes associated with a viral or other infectious illness, myalgias, and nonspecific clcctrocardiogram (ECG) changes. We have found. however, that fewer than 25q of such patients actuallv have inflammatory cellular infiltrates on endomyocardial biopsy (Fig I I ). It is thcrefort not surprising that in former experience. patients with clinically suspected myocarditis have not been clearly helped by corticosteraid therapy. In a recent series of patients with biopsy proven myocarditis we found that immunosuppressive therapy with prednisone and azathioprine was strikingly associated with improvement or stabilization of cardiac function.!” Furthermore, our study suggested that humorally mediated inflammatory myocardial disease ( IgG or complement deposition without marked cellular infiltration) may be relatively common and can respond to immunosuppressive therapy. Tapering or withdrawing therapy too early or too quickly has appeared to cause relapse, confirmed by repeated myocardial sampling. Biopsy is therefore justified when myocarditis is suspected, and is also helpful in assessing response to therapy. We emphasize that because of the relatively low rate of true inflammatory myocar-

CARDIAC

well this

BIOPSY

Myocarditis. Fig 11. as polymorphonuclear unequivocally positive

161

In this biopsy-obtained specimen, extensive infiltration by mononuclear cells (mostly leukocytes can be seen. Evidence of myocyte necrosis is also present. Note the example of myocarditis in a severe form.

dial disease, and due to the risks of immunosuppressive therapy this treatment should be reserved for patients with biopsy-confirmed myocarditis. Richardson and coworkers at King’s College Hospital in London have substantiated these findings, Fenoglio et al have suggested that myocarditis can be divided into clinically meaningful histologic subsets,” but this classification is derived from relatively small patient groups and may be confounded by cloudy histologic criteria used to distinguish true myocarditis from cardiomyopathy. Opinion differs between pathologists regarding exact definitions for myocarditis, some requiring extensive, diffuse lymphocytic infiltration with evidence of cardiac myocyte damage, others having more liberal criteria, often allowing focal patches of mononuclear cells (Fig 12). A quantitative histologic system may be a helpful step in the right direction.*’ A multicenter cooperative trial has been initiated to test the efficacy of immunosuppressive therapy in myocarditis. Hopefully, this will serve also to better define the relation between histologic appearance and disease, and which

lymphocytes) diffuse nature

as of

patients if any are best treated with immunosuppressive drugs. Cardiac Amyloidosis Myocardial amyloid deposits are readily detected by biopsy” (Fig 13). The amyloid material can be easily seen enveloping individual myocytes in advanced cases, even without special techniques. Positive identification can be made with Congo red, thioflavin-T, or other stains. In early involvement, electron microscopy may be the most sensitive method of examination, showing amyloid deposits in the interstitium and blood vessels as well as around myocardial cells. Whether some forms of systemic or cardiac amyloidosis are treatable with chemotherapy is yet unproven. Heart biopsy plays a further diagnostic role in differentiating cardiac amyloidosis from an entity that it may mimic hemodynamitally, pericardial constriction. Cardiac Sarcoidosis Although the lesions of cardiac sarcoidosis are nonconfluent (increasing the hazard of false neg-

162

Fig 12. Another biopsy sample (note the endocardium (arrows). Higher power views (upper right, inset1 shows specimen was interpreted as showing myocerditis, focal cardiomyopathy as well.

FOWLES

at lower these infiltration

left). Focal infiltration cells to be lymphocytes. by lymphocytes and

Fig nosed power amyloid cardiac

AND

MASON

by small mononuclear ceils is seen Although this particular biopsy other cells can be seen in d&ted

13. Myocardial by endomyocardial view showing the material farrows) myocytes.

amyloidosis diagbiopsy. High typical amorphous surrounding the

CARDIAC

163

BIOPSY

ative biopsy findings), sarcoid granulomas have been detected by endomyocardial biopsy, which has led to lifesaving therapy with corticosteroids.23 Most patients with cardiac sarcoidosis have clinically apparent systemic or pulmonary involvement; heart failure or arrhythmias can, however, be the first sign of the disease, and may occur without apparent extracardiac involvement. Granulomas are usually found in greatest density in the interventricular septum, so right ventricular biopsy is the most appropriate approach. When cardiac sarcoidosis is suspected it is particularly important to obtain multiple specimens from several sites to enhance the chance of positive diagnosis. Clearly, a negative biopsy does not rule out myocardial sarcoidosis. Cardiac Hemochromatosis

Cardiac biopsy is not usually required to diagnose cardiac hemochromatosis, since it is invariably accompanied by manifest involvement of other organs (Fig 14). However, when irondepleting therapy is undertaken, it is helpful to

Fig 14. Myocardial Prussian Blue dye, the

hemochromatosis, characteristic

iron

cardiac deposits

examine the myocardium to ascertain whether iron deposition there is eliminated, which may not happen until well after hypoferremia and iron deficiency anemia have occurred.24 Further, iatrogenic cardiac hemochromatosis might be avoided during the treatment of hematologic disorders such as the thalassemias, using cardiac biopsy as a monitoring tool. Some investigators have concluded, however, that catheter biopsy of the endomyocardium may be relatively insensitive due to variations in the extent of iron deposition between epi- and endocardium, or between different myocardial regions.25 Endocardial

Fibrosis

A certain amount of endocardial fibrous buildup can be found in most cardiomyopathies. The term endocardial fibrosis is reserved for a specific disorder characterized by a markedly fibrous endocardium, often without involvement of the remaining myocardium and epicardium. At least two such entities have been described, Loeffler’s endocarditis with eosinophilia and

biopsy specimen. can be seen diffusely

In this black and white scattered throughout

photo of a slide stained the myocytes in the field.

with

164

Davies’ tropical endomyocardial fibrosis, which may be variations of the same disease. The patient will present with the usual signs of resistance to ventricular filling, as in constrictive pericardial disease. Contrast ventriculography can often reveal the fibrotic obliteration of the cavity, whether left- or right-sided. Endocardial fibrosis can be diagnosed by catheter biopsy,” although it may be difficult to cut the fibrous tissue with the jaws of the instrument. Additionally, the fibrosis may be limited to one ventricle, with focal distribution. Typically, the apex and body of the right ventricle, and inferior inflow region of the left ventricle are the sites of involvement. Aside from distinguishing endocardial fibrosis from pericardial constriction, biopsy can assist specific therapy: in some patients the pannus of fibrous tissue on the endocardium can be surgically removed, much as a thickened pericardium can be “stripped” from the epicardial surface. Fabry ‘s Disease Cardiac involvement is common in Fabry’s disease (angiokeratoma corporis d~flhsum universale), with death often resulting from congestive heart failure. Endomyocardial biopsy from patients with Fabry’s disease shows changes that may be specific, including a lacework appearance of myocardial fibers on light microscopy with marked perinuclear vacuolization and numerous cytoplasmic inclusion bodies probably containing glycolipid.“~‘” Cardiac biopsy may lead to the diagnosis of Fabry’s disease in cases that have not been recognized in childhood, or in which cornea1 opacities and proteinuria are absent. Several other myocardial diseases can be diagnosed by cardiac biopsy. These include carcinoid disease and glycogen storage disease, which are usually apparent in other organ systems. The discovery of myocardial involvement may nevertheless be helpful in treating these patients. Irradiation injury to the myocardium can likewise be detected in the patient with an appropriate history. Biopsy has discovered cardiac tumors, but usually by chance. Endomyocardial biopsy should not be the primary mode of such a diagnosis, for the tumor is often enveloped by organized thrombus.

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D[j,ftirentiation Between M?wcardial and Pericardial Constriction

AND

MASON

Restriction

Several myocardial disorders can mimic pericardial constriction, although usually only superficially. Many experienced cardiac surgeons have had the unpleasant experience of discovering a normal pericardium in a patient undergoing thoracotomy for “diagnosed” pericardial constriction. Amyloidosis. endomyocardial fibrosis. curcinoid heart disease. generalized myocardial tibrosis. and radiation-induced myocardial fibrosis are among the entities that can resemble pericardial constriction. Constrictive disease is usually diagnosed hemodynamically when the diastolic pressures are the same in the right and left ventricles throughout various physiologic and pharmacologic interventions. Involvement by ;I restrictive process. however. can occasionally produce such diastolic equilibration. In restrictive cardiomyopathy. ventricular contraction ih often impaired: in early cases systolic performance may be normal enough to blur any distinction from constrictive pericarditis. Since pericardial abnormalities arc not always demonstrable by echocardiography or radiography. in ;I small number of cases. therefore. direct histologic examination is necessary. It is our practice to perform endomyocardial biopsy in patients with suspected pericardial constriction, but in whom restrictive myocardial disease is a consideration. under the following conditions: lack of pericardial calcification on chest roentgenogram. no antecedent history of pericarditis. and absence of echocardiographic evidence of pericardial thickening. Patients who have undergone mediastinal irradiation with resultant pericardial constriction should have myocardial biopsy before pericardiectomy. because severe myocardial disease might nullify any potential benetit from operation and may therefore contraindicate surgery. NONSPECIFIC BIOPSY BIOPSY IN VARIOUS

FINDINGS CARDIAC

REVEALED DISORDERS

BY

Nonspecific histologic and ultrastructural changes have been noted with biopsy in many myocardial disorders (Table 3). These findings may confirm the clinical diagnosis, but they are rarely if ever pathognomonic of a specific disease.

CARDIAC

165

BIOPSY

Table

3. Nonspecific Changes Detectable Endomyocardial Biopsy

by

Dilated cardiomyopathy Hypertrophic cardiomyopathy Myotonic dystrophy Methysergide toxicity Hepatolenticular degeneration

(Wilson’s

disease)

Hypothyroidism Hyperthyroidism Mitral valve prolapse

Dilated Cardiomyopathy

Endomyocardial biopsy has contributed disappointingly little information about idiopathic dilated (congestive) cardiomyopathy. This entity cannot be diagnosed by histologic examination, for there are no specific micropathological features. The changes found in dilated cardiomyopathy can be seen in hearts that are failing due to other causes, such as valvular disease. These abnormalities include the following: (a) varying myocyte size, often hypertrophied and occasionally disarrayed; (b) hyperchromatic, enlarged, or odd-shaped nuclei; (c) interstitial fibrosis, occasionally with a scant cellular infiltrate; (d) mitochondrial, myofibrillar, and sarcoplasmic reticulum abnormalities on electron microscopy. Several investigators have attempted to correlate pathology with clinical status in dilated cardiomyopathy. In general, the severity of morphologic changes seems to parallel the degree of clinically determined cardiac dysfunction.29-3’ Some studies have found a correlation between the severity of histologic changes on biopsy with prognosis in dilated cardiomyopathy, those patients with milder abnormalities having a lower mortality rate.32*33Such conclusions have been challenged by Baandrup et al, who found no convincing relationship between functional status, hemodynamic parameters, clinical diagnosis, prognosis and morphologic abnormalities.34 This may be due to topographic variation in biopsy samples from cardiomyopathy patients.35 A more quantitative approach to tissue examination using morphometric methods may yet reveal histologic or ultrastructural features having better clinicopathologic correlation in idiopathic dilated cardiomyopathy.36 As its name indicates, the etiology of idiopathic dilated cardiomyopathy remains elusive. There are probably multiple causes of this disor-

der, but to date cardiac biopsy has not been helpful in clarifying any. Occasionally, biopsy has revealed intracellular inclusions thought to represent virus particles, but virus is very rarely ever isolated from the endomyocardial samples themselves.37*3s Immunofluorescent staining of biopsy tissue has revealed heavy deposition of immunoglobulins or even the presence of viral antigen. This is an area of potential progress, as are several new research techniques mentioned below. As a rule, endomyocardial biopsy is not necessary in the diagnosis and evaluation of idiopathic dilated cardiomyopathy, unless there is reason to suspect one of the specifically diagnosable diseases mentioned above. Biopsy will reveal unexpected findings in only a small proportion of patients with apparent dilated cardiomyopathy, usually 1O%.39 Hypertrophic Hypertrophic

Cardiomyopathy (Idiopathic Subaortic Stenosis)

Myofibrillar and myocyte disarray are found at biopsy in the septal myocardium of patients with hypertrophic cardiomyopathy. These structural abnormalities are not pathognomonic for hypertrophic cardiomyopathy, for they are found in myocardial hypertrophy of any etiology.40 Most investigators agree that left or right septal biopsy cannot be used to include or exclude a diagnosis of hypertrophic cardiomyopathy. It may be that the myofiber disarray is quantitatively greater in hypertrophic cardiomyopathy than in other entities, and that advanced morphometric examination will prove useful in distinguishing this, but such techniques may be difficult to apply in biopsy tissue, where only small fragments rather than carefully selected, large segments are obtained.4’ Alcoholic

Cardiomyopathy

Although some have suggested that there are specific ultrastructural changes in alcoholinduced myocardial disease, this impression is not currently shared by most pathologists. The changes seen in idiopathic dilated cardiomyopathy are similar to those in alcoholic cardiomyopathy (with the possible exception of those induced by cobalt-containing beer).

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166

Myotonic

D+vstrophy

Myocardial changesare associatedwith many of the skeletal myopathies. Myotonic dystrophy is especially notable for its electrocardiographic and conduction system abnormalities. We have recently found myocardial ultrastructural abnormalities in biopsies from myotonic dystrophy patients without clinically evident heart disease.42 Methysergide-induced

Cardiotoxicit?,

The structural similarity between Shydoxytryptamine and the serotonin antagonist, methysergide, appears to account for the finding of endocardial fibrosis, similar to that of carcinoid heart disease,in somepatients taking methysergide. Although it has been thought in the past to involve only the left-sided valves, we have recently found right ventricular septal endomyocardial involvement by biopsy in a methysergidetreated patient.43 The lesion in carcinoid and methysergide heart disease is an endocardial pannus of fibrous tissue that can envelope valve leaflets and extend over the endocardium, sometimes leading to severe valvular and ventricular dysfunction. It may be surgically resectable. Wilson’s

Disease

Wilson’s disease (hepatolenticular degeneration) is usually recognized by liver failure and characteristic ocular findings. Cardiac involvement is rare, so heart failure in patients with Wilson’s diseasecannot be ascribed to cardiac copper deposition without substantiation by biopsy. Myocardial copper deposits can be detected in biopsy-obtained tissue. and can be quantitatively assayed. The amount of myocardial copper in heart failure due to Wilson’s diseasecan be ten times the amount found in the normal heart, or in that of patients with hepatolenticular degeneration without heart failure.4J Disorders

If hypo- or hyperthyroidism has causedsigniticant myocardial dysfunction, nonspecific histologic changes can be seen. Patients with mitral valve prolapse have been thought to have a myocardial disorder becauseof the occurrence of arrythmias and abnormal left ventricular con-

MASON

traction patterns observed in this entity. Recent endomyocardial biopsy studies” have shown focal hypertrophy. increased endocardial and interstitial fibrosis. and mitochondrial degenerative changes in several patients, supporting the hypothesis that a myocardial disorder is present in mitral valve prolapse. INDICATIONS

FOR

MYOCARDiAL

BIOPSY

As myocardial biopsy techniques became more widespread in the late 1970s. controversy developed regarding their clinical usefulness. Ferrans and Roberts have concluded that biopsy has proven valuable chiefly for diagnosing cardiac allograft rejection. but has failed to help much in other situations.” This opinion may be derived from their experience predominantly with dilated and hypertrophic cardiomyopathy. and the continued failure to find any specific “markers” for either illness. Olsen strongly supports the clinical utility 01 myocardial biopsy. basedon experience from an ongoing European multicenter study.” In over 600 patients with dilated cardiomyopathy, for example, Olsen claims biopsy results to be helpful in X5’? of cases,due to confirmation of the suspectedclinical diagnosis in 59%. and discovery of an unsuspected condition in 16%. The value of endomyocardial biopsy in confirming the diagnosisof dilated (congestive) cardiomyopath! would probably be contested by some investigators in North America and Europe However, in some regions (such as certain African countries) endomyocardial fibrosis is ascommon 3s dilated cardiomyopathy, and cannot always be easily differentiated from it by clinical inference. particularly in early cases. Most investigators now agree that, at the current level of knowledge, myocardial biopsy in patients with dilated cardiomyopathy is of cliniTable

4.

Suspected

Other

AND

Indications cardiac

Detectron

and

for

Endomyocardial

transplant

monitoring

Biopsy

rejection of doxorubwn

IAdrlamyclnl

cardlotoxicity Suspected

myocardms

Dlagnosls

of other

myocardial

SIS, hemochromatosls, Confirmation

of myocardial

ders

3)

Research

(see Table appkations

disease etc,

see Table

rwolvement

iCardIac

amylordo

2) In certain

dlsor-

CARDIAC

BIOPSY

tally pivotal importance in only a minority of cases, those with an unsuspected, specifically diagnosable disease. General clinical use of biopsy in all patients with suspected myocardial disease is therefore probably not justified. Current indications for endomyocardial biopsy are listed in Table 4. Myocardial biopsy’s value in the diagnosis of cardiac allograft rejection is well established, and will continue to be important as more centers undertake heart transplantation, and as new techniques and antirejection drugs are introduced. Biopsy should be performed to establish the diagnosis of myocarditis, especially if immunosuppressive therapy is considered. Myocarditis may be suspected in patients with sudden onset of congestive heart failure not attributable to any known cause, and perhaps associated with a viral-like illness, myalgias, ECG changes, and serum muscle enzyme rises. The relatively low prevalence of myocarditis among cases of “garden variety” idiopathic dilated cardiomyopathy is a problem that may be resolved in the future by new noninvasive screening tools or immunopathological techniques. Biopsy should be used where feasible to monitor patients at risk of developing doxorubicin cardiotoxicity, especially those in whom cardiac dysfunction may be caused by other factors such as coronary, valvular, or radiation-induced heart disease. Biopsy assessment of doxorubicin cardiotoxicity is sensitive, specific, and correlates with the degree of cardiac dysfunction. Endomyocardial biopsy should be used to diagnose suspected secondary myocardial disease. For example, cardiac amyloidosis may mimic pericardial constriction or may present with low cardiac output, sinoatrial dysfunction, and electrocardiographic abnormalities. Cardiac involvement in sarcoidosis, hemochromatosis, hepatolenticular degeneration, or other multisystem disorders should be substantiated by histologic evidence through biopsy, especially when therapeutic options may be broadened by a firm diagnosis. The choice between right versus left ventricular endomyocardial biopsy should be made according to the expected safety and efficacy of the two approaches. There has been no large, systematic comparison of biopsies from both ventricles. Some investigators prefer to sample

167

the left ventricle due to its perceived preponderant involvement in cardiac dysfunction, especially in valvular disease. We identify five conditions in which left ventricular biopsy is specifically indicated: (a) certain forms of endomyocardial fibrosis involving the left ventricle as suggested by clinical, hemodynamic, or angiographic data; (b) sclerodermatous heart disease, which predominantly involves the left ventricle (however, endomyocardial biopsy cannot differentiate this from fibrosis due to other causes, and scleroderma may only involve the midwall or epicardium, suggesting the possible need for transthoracic needle biopsy); (c) left heart irradiation, because the left ventricle often received the greatest radiation doses prior to current shielding techniques; (d) cardiac fibroelastosis in infants and newborns, which involves the left ventricle; (e) research applications, such as studies of left ventricular dysfunction in mitral and aortic disease or of various forms of cardiac hypertrophy. Right and left ventricular biopsy techniques are probably comparable with regard to safety, although the risk of grave complications (such as thromboembolism, systemic air embolism, stroke, myocardial infarction, arrhythmia, or death) is probably greater with left ventricular biopsy. As currently applied, the right ventricular technique probably yields more tissue than left-sided biopsy. Further, most of the myocardial processes for which biopsy may be performed involve both ventricles. Endomyocardial biopsy should not be used to “diagnose” dilated or hypertrophic cardiomyopathy, for these disorders have not yet been shown to display any pathognomonic histologic features. Further, there is no consensus or convincing evidence that biopsy can render a meaningful prognosis in cardiomyopathy. The cardiomyopathies undoubtedly have many causes, but biopsy has not yet revealed any markers referrable to a specific etiology. For example, it would be very useful to know whether a case of congestive cardiomyopathy were due to ethanol consumption; biopsy cannot distinguish alcoholinduced myocardial disease from dilated cardiomyopathy. There are few specific contraindications to endomyocardial biopsy. In bleeding disorders or anticoagulation, the chief danger is hemorrhage

168

FOWLES

due to inadvertent cardiac perforation. Another contraindication is ventricular thrombus. Although we have performed biopsies in patients with known right ventricular thrombus, this condition may prevent procurement of adequate endomyocardial tissue. Due to possible systemic embolization, biopsy of a left ventricle containing thrombus should not be done. Since the likelihood of mural thrombus is high after myocardial infarction, and since there may be some thinning of the involved wall, recent or remote myocardial infarction is a contraindication to left ventricular biopsy. The presence of intracardiac shunts is in our opinion a relative contraindication to right ventricular endomyocardial biopsy, due to the risk of paradoxical systemic embolization. RESEARCH APPLICATIONS AND SPECIAL INVESTIGATIONS

Light and electron microscopy remain the standard methods for examination of endomyocardial biopsy tissue. New ways of studying specimensare currently under development and may find greater application in the future. Biochemical Studies Several workers have begun to determine the levels of enzymes and other substancesin endomyocardial biopsy specimensfrom patients with various myocardial diseases.Peters and his associates have found decreased adenosine triphosphatase (ATPase) and increased lactic dehydrogenase (LDH) in biopsied myocardium from valvular heart diseasepatients with poor ventricular function4’ as well as from those with dilated

AND

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cardiomyopathy.” Barrie and coworkers, on the other hand, have measured decreased activities of LDH and other enzymes in such patients.“” Such apparent discrepancies may be methodological in nature. or due to patient heterogeneity. Schultheiss and colleagues have reported increased total LDH in dilated cardiomyopathy. the severity of hemodynamic dysfunction directly related to the LDH-5 level.” Torp has used micromethods to assay lipids in biopsy specimensfor a variety of myocardial disorders, including alcoholic cardiomyopathy.52,53 The validity of biochemical analysis is diminished by problems such as contamination of samplesby blood and connective tissue. Further, the likelihood of sampling error, or of interregional variation within the myocardium makes chemical analysis of cardiac biopsy material dithcult. Unverferth and coworkers have shown. however, that reproducibility can be maintained adequately in their carefully done studies on ATP and cyclic AMP and cyclic GMP in human endomycardial biopsies.” Transmural intraoperative needle biopsies of the myocardium have recently found several biochemical applications, including studies on the subendocardial versus subepicardial content of high-energy phosphate? and on the cellular chemical effects of myocardial protection during cardiopulmonary bypass.‘” Morphomrtric, Ana!)~sis The amount of fibrosis in endomyocardial specimensfrom patients with aortic valve disease or congestive cardiomyopathy has been found by

Fig 15.

Photomicrograph

showing

an optical

technique for morphomatric analysis of myocardial biopsy tissue. A grid assists in quantitative estimation of histologic features, such as amount of fibrosis. In this illustration, for example, the percentage of line intersections occurring within fibrotic areas serving as an index. (Reproduced by permission of the American Heart Association Inc. Hess OM at al: Diastolic function and myocardial structure Circulation

in patients 63~360-371,

with

myocardial 1961 .=I

hypertrophy,

169

morphometric studies (Fig 15) to correlate with myocardial stiffness and clinical status.” These and other methods may be useful in the future for quantifying myocardial abnormalities, rendering more specific diagnoses, evaluating the timing of surgical intervention (such as valve replacement), and investigating the causes and mechanisms of pathological processes. Ultrastructural morphometry has revealed, for example, differences between volume-loaded ventricles in mitral insufficiency and normal tissue, and distinctions between volume-loaded and pressure-loaded ventricles.s8 In ischemic heart disease morphometry on operative biopsy samples has disclosed quantitatively less myocardial fibrosis in segments supplied by collateral vessesls than in those without.59 Other morphometric studies have shown a correlation between ventricular wall segment contractile function and the extent of fibrosis.60 Pharmacologic

Studies

TorpS2353has investigated the mechanism of glucagon’s actions on the heart using endomyocardial biopsy. He found no change in cyclic AMP levels after administration of glucagon, whose myocardial effects therefore appear not to be mediated through the adenylate cyclase system.53 Using biopsy, digitoxin concentration has been found to be considerably higher in myocardium than in serum.6’ Biopsy may in some cases by helpful for detecting more meaningful, endorgan concentrations of cardioactive drugs. Such work is being done in Kate’s laboratory, where myocardial drug concentrations have been related to plasma drug levels, revealing clinically useful information about kinetics.62 Immunologic

Studies

The research application of immunologic methods to myocardial biopsy has focussed largely on investigation of dilated cardiomyopathy, due to its frequent presentation in previously healthy individuals weeks after a viral-like syndrome, with occasional relapses or flare-ups during the course of the illness. Past studies have shown possible participation by the humoral immune system in the genesis of idiopathic myocardial disease,63*64but a convincing causal connection has not been made. We have reported

abnormal cellular immune function in cardiomyopathy patients, their peripheral blood lymphocytes exhibiting suppressor cell dysfunction in vitro.6s Such a finding may correlate with demonstrated cytotoxicity by such patients’ lymphocytes against cardiac target cells.66 Preliminary work from this laboratory indicates that some patients with idiopathic cardiomyopathy display autoimmunelike lymphocyte reactivity against their own, biopsy-obtained heart cells. Further work in this area is promising and is currently active. Such experiments using myocardial biopsy may provide clues for further investigation of dilated cardiomyopathy. CONCLUSIONS

According to current knowledge, the general usefulness of endomyocardial biopsy is limited for several reasons. First, the technique as now applied can sample only those myocardial areas closest to the endocardium; unless a suspected pathological process involves the heart in a diffuse fashion, the biopsy may fail to include the abnormal tissue. A negative biopsy is thus not as helpful as a positive biopsy. Second, accurate interpretation of biopsy findings requires a thoroughly trained, accomplished cardiac pathologist who is well-versed in the various artefacts present on both light and electron microscopy in such tissue.67,68The performance of cardiac biopsy is unwarranted without expert interpretation of results. Moreover, the technique must be sufficiently practiced to minimize the risk of complications; cardiac transplant programs or biopsyrelated research are probably required to generate enough cases to allow safe, meaningful results. Third, although there are several relatively firm indications for endomyocardial biopsy in which useful information may be expected, there are many areas where biopsy is yet unable to yield specific diagnosis. Expansion into such areas should not proceed with an attitude of pointless curiosity merely because the technique is available, but should rather await validation by carefully done studies. We expect that myocardial biopsy will continue to be a valuable research tool, especially in centers where the technique is performed frequently and safely. It is only through continued scientific investigation that we may gain the

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170

knowledge and new techniques necessary to put histologic information to better use, and that we may discover more specific indications for myocardial biopsy. Biopsy of patients with idiopathic myocardial disease is thus warranted for

AND

MASON

research applications. As with every clinically applied test, cardiac biopsy must be used wisely and prudently. We have found the above indications to be helpful in organizing our own application of this promising invasive tool.

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CARDIAC

BIOPSY

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48. Peters TJ, Brooksby IAB, Webb-Peploe MM, et al: Enzymic analysis of cardiac biopsy material from patients with valvular heart disease. Lancet 1:269-270,1976 49. Peters TJ, Wells G, Oakley CM, et al: Enzymic analysis of endomyocardial biopsy specimens from patients with cardiomyopathies. Br Heart J 39:1333-l 339, 1977 50. Barrie SE, Saad EA, Ubatuba S, et al: Myocardial enzyme activities in congestive cardiomyopathy. Res Commun Chem Pharmacol23:375-381, 1979 51. Schultheiss HP, Bolte HD, Fischer S, et al: Enzymatic analysis and collagen content in endomyocardial biopsy samples of patients with congestive cardiomyopathy of uncertain etiology. Clin Cardiol 3:329-334, 1980 52. Belfrage P, Johanson BW, Torp A, et al: Micromethods for analysis of lipids in endomyocardial biopsy specimens. Acta Med Stand 205:283-286, 1979 53. Torp A: Special investigations in COCM [congestive cardiomyopathy]: Biochemical analysis of cardiac biopsies. Postgrad Med J 54:494-498, 1978 54. Unverferth DV, Fertel RH, Altschuld R, et al: Biochemical measurements of endomyocardial biopsies. Cathet Cardiovasc Diagn 7:55-64, I98 1 55. Jones RN, Peyton RB, Sabina RL, et al: Transmural gradient in high-energy phosphate content in patients with coronary artery disease. Ann Thorac Surg 32:546-553, 1981 56. Cooper J, Bull C, Cankovic-Darracott S, et al: Cellular chemical indices of right ventricular protection in children. Histochemical J 14:739-746, 1982 57. Hess OM, Scheider J, Koch R, et al: Diastolic function and myocardial structure in patients with myocardial hypertrophy. Circulation 63:36&371, 1981 58. Fleischer M, Wippo W, Themann H, et al: Ultrastructura1 morphometric analysis of human myocardial left ventricles with mitral insufficiency. A comparison with normally loaded and hypertrophied human left ventricles. Virchows Arch (Pathol Anat) 389:205-210, 1980 59. Schwarz F, Schaper J, Becker V, et al: Coronary collateral vessels:their significance for left ventricular histologic structure. Am J Cardiol49:291-295, 1982 60. Stinson EB, Billingham ME: Correlative study of regional left ventricular histology and contractile function. Am J Cardiol39:378-383, 1977 61. Storstein L: Studies on digitalis: X. Digitoxin metabolites in human myocardium and relationship between myocardial and serum concentrations of digitoxin in patients on maintenance treatment. Clin Pharmacol Ther 21:395408, 1976 62. Keefe DL, Kates RE: Myocardial disposition and cardiac pharmacodynamics of verapamil in the dog. J Pharmacol Ther 220:91-96,1982 63. Hatle K, Melbye 0: lmmunoglobulins and complement in chronic myocardial disease: A myocardial biopsy study. Acta Med Stand 200:385-389, 1976 64. Bolte HD, Grothey K: Cardiomyopathies related to immunological processes.In Riecker G, Weber A, Goodwin J (eds): Myocardial Failure. Berlin, Springer-Verlag, 1977, pp 266-274 65. Fowles RE, Bieber CP, Stinson EB: Defective in vitro suppressor cell activity in idiopathic congestive cardiomyopathy. Circulation 59:483-491, 1979

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66. Jacobs B. Matsuda Y. Deodhar S. et al: Cell-mediated cytotoxicity to cardiac cells of lymphocytes from patients with primary myocardial disease. Am J Clin Pathol 72: l-4. 1979 67. Adomian GE, Laks MM. Billingham ME: The incidence and significance of contraction bands in endomyocar-

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dial biopsies from normal human hearts. Am Heart J 95:348 351.1978 68. Olmesdahl PJ. Gregory MA. Cameron EWJ: Ultrastructural artefacts in biopsied normal myocardium and their relevance to myocdrdial biopsy in man. Thorax 34:82-90, 1979