Utility of immunofluorescence and electron microscopy in endomyocardial biopsies from patients with unexplained heart failure

Cardiovascular Pathology 19 (2010) e99 – e105

Original Article

Utility of immunofluorescence and electron microscopy in endomyocardial biopsies from patients with unexplained heart failure Rosemary C. She a,⁎, Elizabeth H. Hammond a,b a Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA Department of Pathology, LDS Hospital, Intermountain Healthcare, Salt Lake City, UT, USA

b

Received 11 August 2008; received in revised form 7 March 2009; accepted 14 April 2009

Abstract Background: With our increasing understanding of inflammatory heart disease, the relevance of Dallas criteria has come into question. Immunofluorescence (IF) and electron microscopy (EM) can potentially identify immune reactants and ultrastructural changes not visible by light microscopy (LM), particularly in cases not meeting Dallas criteria. Methods: This was a retrospective, descriptive study of native endomyocardial biopsies (MBx) performed 1981 to 2006, undertaken to assess the utility of these methods. All patients had decreased cardiac function but normal coronary angiographic studies. LM identified cases as myocarditis (Dallas+), borderline myocarditis (Dallas+/−), or cardiomyopathy (Dallas−). IF studies (human leukocyte antigen (HLA)-DR, IgG, IgM, IgA, C3d, C1q, and fibrinogen) reported interstitial, capillary, or heart-reactive, antibody-like staining patterns. EM findings were also reviewed. Results: Of 472 records from 429 patients (6 months–78 years old), 44 were Dallas+, 47 Dallas+/−, and 381 Dallas−. Significant IF and/or EM findings were identified in 421 cases (89%). By IF, 142 (37%) Dallas− cases had significant capillary HLA-DR expression. Thirty-four of 37 cases with vascular immune complex deposition were Dallas−. LM commonly failed to detect myofilament loss (138 cases) and endothelial cell changes (126 cases) that were observed by EM. Conclusions: IF is a useful strategy for defining inflammatory phenomenon as it revealed significant immune-related heart disease not demonstrable by LM. EM better defined myofilament loss, a finding previously found to be associated with adverse clinical outcome. We strongly recommend that a portion of tissue obtained from all MBx be routinely frozen for IF and fixed appropriately for EM studies. Future studies characterizing the inflammatory molecular profile of myocardial tissues may better define myocarditis. © 2010 Elsevier Inc. All rights reserved. Keywords: Myocarditis; Electron microscopy; Immunoglobulin; Complement

1. Introduction Myocarditis, as defined by the Dallas criteria established in 1986, should consist histologically of mononuclear cellular inflammation with myocardial necrosis or degeneration [1]. Since establishment of the Dallas criteria, additional reports have defined the role of viruses and autoimmune phenomena in myocarditis, broadening our understanding of the nature of these inflammatory heart diseases. Autoimmune T-cell-mediated damage to myocytes due to persistent T-cell activation is now believed to lead to dilated cardio⁎ Corresponding author. ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA. Tel.: +1 801 583 2787; fax: +1 801 584 5207. E-mail address: [email protected] (R.C. She). 1054-8807/09/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2009.04.004

myopathy after viral infection [2–4]. Autoreactive antibodies, developed because of cross-reactivity of viral or other infection-related antigens with myocardial proteins, may also play a role in this cardiomyopathic process [3,5–11]. Persistent autoimmune activation, incited by macrophage activation, can further contribute to development of dilated cardiomyopathy [2,12,13]. Viral nucleic acid has also been detected in myocardial biopsies not meeting Dallas criteria but from patients with a clinical history compatible with myocarditis [14,15]. The relevance of Dallas criteria has therefore come into question due to its low sensitivity for inflammatory processes in the heart and the lack of correlation with treatment outcomes [16]. Few studies have examined the utility of immunofluorescence (IF) or electron microscopy (EM) in myocardial

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biopsies even though they are potentially useful studies, given our present understanding of the spectrum of inflammatory processes involving the myocardium. IF detection of immune reactants is a useful method to detect autoimmune phenomena in tissues including up-regulation of the major histocompatibility complex (MHC) antigens as well as deposition of immune reactants like immunoglobulin (Ig), complement components, and clotting factors. EM allows better visualization of changes within the vasculature and myocytes which may potentially add diagnostic and prognostic information. In this study, we retrospectively reviewed myocardial biopsies in which light microscopy (LM) was routinely supplemented with IF and EM studies in order to assess the value of these pathologic test methods for defining inflammatory myocardial processes that lead to cardiomyopathy. 2. Methods Pathology records of native endomyocardial biopsies (MBx) performed 1981–2006 at Intermountain Healthcare hospitals in the Salt Lake City, UT, region and the University of Utah Health Sciences Center were retrospectively reviewed. MBx were taken from patients who had decreased cardiac function in the presence of normal coronary angiographic studies. Only cases in which both LM and IF were performed were included in this study. EM was done on the majority of the biopsies as well. 2.1. Light microscopy LM identified cases as myocarditis (Dallas+), as borderline, persistent, or resolving myocarditis (Dallas+/−), or as cardiomyopathy (Dallas−) based on Dallas myocarditis criteria [1]. Staining with Congo red for amyloidosis and with iron stains for hemochromatosis was performed when appropriate, and cases were excluded when positive. 2.2. Immunofluorescence Frozen sections of myocardial biopsy tissue were airdried and washed in phosphate-buffered saline (PBS). They were then incubated with unconjugated antibodies directed against Ia (human leukocyte antigen (HLA)-DR-like) antigen (Hybritech), IgG, IgM, IgA, C3d, C1q, fibrinogen, and albumin (CBA-Cappel Laboratories, West Chester, PA, USA) at optimal concentrations for 30 min at 25°C. Sections were washed in PBS and incubated with fluorescein labeled IgG fractions of rabbit antimouse antibodies (BCA-Cappel Laboratories or Coulter Laboratories) for 30 min at 25°C. The slides were washed in PBS and coverslipped with polyvinyl alcohol. Appropriate positive and negative control slides were included in each run. Slides were examined under a Zeiss immunofluorescence microscope (Carl Zeiss, Thornwood, NY, USA) and examined for interstitial, capillary, or heart-reactive antibody (HRA)-like staining

patterns. HRA-like was defined as staining of sarcolemmal membranes, myofilaments, or myocyte nuclei. HLA-DR staining of interstitial regions consisted of staining of inflammatory cell surfaces only. All staining intensity was graded from 0 to 3+ and only considered significant (1+ or greater) if the intensity was brighter than or distribution was different from albumin. Positive and negative controls were routinely examined and studies were repeated if the controls exhibited inappropriate staining patterns. 2.3. Electron microscopy One microgram of toluidine blue-stained sections were chosen for EM studies based on review by LM. EM was performed using a Jeol 100S electron microscope (Jeol USA, Peabody, MA, USA). Myofilament loss was defined as decreased myofilament content in some proportion of the cells examined. If all myocytes lacked myofilaments, this process was deemed severe. If actin or myosin filaments were lost in irregular proportions, this finding was included in a comment. Capillary endothelial cell changes were defined as endothelial cytoplasmic swelling. Such swelling included increased amounts of cell membrane tortuosity or increased amounts of pinocytotic vesicles which was sometimes accompanied by basement membrane reduplication. Nonspecific findings that were noted included interstitial cell debris, interstitial fibrosis, and findings of myocyte degeneration (myocyte vacuolization, lysosomal accumulation, mitochondrial accumulation, and fat accumulation). Each of these features was graded 0 (none), 1 (focal), or 2 (diffuse). 3. Results Four-hundred seventy-two records from 429 individual patients were identified in which LM and IF were performed. Ages ranged from 6 months to 78 years. Pediatric cases (age ≤18 years) accounted for 79 cases (16.7%). EM was performed in 443 (93.9%) of the cases. Of the 472 total cases, 421 (89.2%) had significant IF and/or EM findings. 3.1. Light microscopy LM identified 44 cases (9.3%) as myocarditis (Dallas+), 47 cases (10.0%) as borderline, persistent, or resolving myocarditis (Dallas+/−), and 381 cases (80.7%) as cardiomyopathy not satisfying Dallas criteria (Dallas−). Five of the Dallas+ cases were from four patients with giant cell myocarditis. One Dallas+ case was diagnostic of eosinophilic myocarditis. 3.2. Immunofluorescence studies Overall, IF demonstrated evidence of inflammatory markers (HLA-DR-positive capillaries or interstitial inflammatory cells, capillary or intersitital deposits of immuno-

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Table 1 Pattern of immunofluorescence staining based on classification of cases by Dallas criteria

IF+ capillary IF capillary and HRA-like+ IF+ interstitial only IF interstitial and HRA-like+ only IF HRA-like+ only IF− Total

LM Dallas+

LM Dallas+/−

LM Dallas−

Total

29 1 7 2 2 3 44

28 5 5 1 2 6 47

179 10 37 1 11 143 381

236 16 49 4 15 152 472

Positive IF was considered 1+ staining or greater.

globulin, complement components, and/or fibrinogen) in 320 (67.8%) of the 472 cases (Table 1). 3.2.1. Myocarditis (Dallas +) Among the 44 Dallas+ cases of active myocarditis, all but three cases (93.2%) showed significant presence of inflammatory markers. Twenty-three (52.2%) demonstrated significant HLA-DR staining on blood vessels, whereas five cases demonstrated HLA-DR staining only in the interstitium (inflammatory cell membrane staining) (Table 2). These cases also had findings suggestive of capillary damage, since they also showed interstitial fibrinogen, and in two of the cases, 1-2+ interstitial staining for IgG, IgM, and/or C3d was also found. Five (11.4%) Dallas+ cases showed 1+ or greater staining for IgG and/or C3d in an HRA-like distribution (Table 1). 3.2.2. Borderline Myocarditis (Dallas+/−) Similar proportions of the borderline cases had inflammatory markers. Of the 47 cases that were Dallas+/− by LM, IF detected inflammatory markers in 41 cases (87.2%), including 33 cases showing capillary staining and six cases showing only interstitial staining. HLA-DR was detected on the endothelium in 27 (57.4%) of the 47 Dallas+/− cases. Four other cases showed interstitial HLA-DR inflammatory cell membrane staining only. Eight (17.0%) Dallas+/− cases had significant HRA-like staining for IgG, IgM, and/or C3d (Table 1). 3.2.3. Negative for Myocarditis (Dallas−) Approximately two thirds of the negative cases also showed inflammatory markers by IF. The most common feature in the 381 Dallas− cases was significant capillary

HLA-DR up-regulation, found in 142 patients (37.3%). In 50 of Dallas− cases, vascular HLA-DR staining was the only significant finding on IF. Vascular immune complex deposition was detected in 34 cases (8.9%) negative for myocarditis by Dallas criteria. Five of the 34 cases had historical reasons for such immune complex deposits: one patient had a history of hepatitis A, one patient had a history of hepatitis C, and three cases were consistent with collagen vascular disease (Fig. 1). Of the 381 Dallas− cases, three were diagnosed as consistent with rheumatic heart disease based on IF findings and clinical history. HRA-like distribution of IgG, IgM, C3d, and/or C1q was seen in 22 Dallas− cases (5.8%). 3.3. Electron microscopy EM demonstrated significant findings in 418 (94%) of 443 cases (Table 4). Myofilament loss and endothelial cell changes on EM were noted in all categories of cases. EM identified 138 cases (33.0%) with myofilament loss which LM failed to detect. In 126 cases (30.1%), EM demonstrated endothelial cell swelling and/or basement membrane reduplication that were not evident by LM. Eighty-nine percent (33/37) of biopsies with immune complex deposition identified by IF had associated endothelial cell changes by EM. 4. Discussion Endomyocardial biopsies remain a valuable tool for establishing a diagnosis in patients presenting with unexplained cardiomyopathy [17]. However, few studies have examined the utility of additional studies to detect

Table 2 HLA-DR expression detected by IF in all cases sorted by Dallas criteria findings on LM a

HLA-DR vascular+ only HLA-DR interstitial+ only HLA-DR vascular and interstitial HLA-DR− Total a

LM Dallas+

LM Dallas+/−

LM Dallas−

Total

20 5 3 16 44

25 4 2 16 47

131 10 11 229 381

176 19 16 261 472

HLA-DR staining in interstitial regions consists entirely of cell membrane staining of inflammatory cells.

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Fig. 1. Findings from a 17-year-old female demonstrating humoral inflammation without cellular infiltrates, most consistent with lupus. (A) Trichrome-stained section highlights pronounced perivascular fibrosis. No inflammatory cell infiltrate was identified (100×). (B) Note endothelial cell swelling and focal myocyte hypertrophy (H&E, 40×). (C) IF shows 2+ IgG vascular staining (100×), and 1+ C3 vascular staining (200×) (D). (E) EM shows endothelial cell swelling and focal myofilament loss (2500×). The myofilament loss is evident in one myocyte (asterisk), which is in contrast to the two adjacent myocytes demonstrating normal myofilament content.

inflammatory markers such as immunoglobulin, complement components, and HLA-DR in heart tissues, and even fewer studies have evaluated the utility of EM for MBx [18–20]. Here, we studied a histologically diverse group of cases, collected over a 25-year period to assess the utility of those markers in further defining the nature of the pathologic processes. Since the Dallas criteria defining myocarditis have been shown to be inadequate for patient stratification into inflammatory and noninflammatory heart disease categories [16], new diagnostic criteria are needed to define the spectrum as they are currently understood. The World Health Organization has defined a category of inflammatory cardiomyopathy [21], but diagnostic criteria were not formally outlined. Clearly, mononuclear infiltrates as required by the Dallas criteria are not always present on MBx in patients with inflammatory cardiomyopathy. Autoimmune mechanisms, the complement pathway, and cytokine release, in addition to cell-mediated immunity,

have been found to play important roles in myocardial injury [12–16,22]. Our study has demonstrated that a high proportion (85– 95%) of biopsies with inflammatory infiltrates of any magnitude (myocarditis or borderline myocarditis by Dallas criteria) as well as two fifths of those with only cardiomyopathic features possessed markers of inflammation. In most cases, this marker of inflammation was at least HLA-DR staining of the capillary endothelium. The up-regulation of HLA-DR observed in the vasculature is consistent with an underlying inflammatory process, as HLA-DR up-regulation in endothelial cells has been shown to be induced by interferon-gamma [23]. Increased expression of MHC antigens is seen in tissues affected by autoimmune and viral diseases and has been described to occur in the myocardial vasculature in active myocarditis [24,25]. Thus, our study confirms that it may serve as a useful marker of myocardial inflammation.

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Autoimmune mechanisms have been implicated in the progression of myocarditis to cardiomyopathy. Autoantibodies directed against the myolemma, sarcolemma, myosin heavy chain, and other cardiac antigens have been found in several studies, although it is controversial as to whether they represent a phenomenon secondary to myocyte injury or a mechanism of myocyte damage [3,6–8,10,11,13]. Nonetheless, it has been reported that approximately one-fourth of patients with dilated cardiomyopathy have detectable autoantibodies to myocyte antigens in the sera, as opposed to less than 3% of normal controls or patients with other cardiac disease, as reviewed elsewhere [7]. In this study, we found HRA-like distribution of IgG, IgM, C3d, and C1q on MBx regardless of presence of an inflammatory infiltrate (Tables 1 and 3). Overall, 7.4% of our cases demonstrated the HRA-like staining pattern, supporting the idea that autoantibodies directed against cardiac myocytes are involved in at least a subset of patients with inflammatory heart disease. Observing this pattern on IF can also serve as a marker for inflammatory heart disease, especially in cases without LM evidence of inflammation. Serologic studies examining for cardiac autoantibodies may be a useful adjunct to MBx and can be considered in future testing of patients with unexplained heart failure. Our study confirms that HLA up-regulation and HRAs can be demonstrated in patients without histologic evidence of myocarditis by the Dallas criteria. Both these findings may predict a better response to immunosuppressive therapy, regardless of identification of Dallas criteria [25–27]. Performance of IF may therefore yield information important in identifying an inflammatory process and predicting response to treatment and clinical outcome. Our observations, although merely descriptive, suggest methods that can be applied prospectively to further define criteria for inflammatory heart disease. Immune complex deposition, while well-described in lupus erythematous myocarditis [28] and animal models of myocarditis [29,30], has not been established as a diagnostic marker for myocarditis [9,20]. It has, however, been shown to be involved in the pathogenesis of both active myocarditis

Table 3 Number of cases with 1+ or greater immunofluorescence staining listed by specific immune reactant and staining pattern IgG Capillary Interstitial Capillary and interstitial Arterial/arteriolar HRA-like b Total a

IgM

IgA

C3d

C1q

HLA-DR a

61 74 19

56 5 5

8 2 0

82 32 2

54 15 2

176 19 16

0 28 182

0 6 72

0 0 10

8 11 136

0 2 73

0 0 211

HLA-DR staining in interstitial regions consists entirely of cell membrane staining of inflammatory cells. b HRA-like is defined as anti-sarcolemmal, anti-myofibrillary, or antinuclear staining pattern.

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and dilated cardiomyopathy [29,31] and is seen infrequently in healthy controls [32]. In this study, we found that immune complex deposition was common, seen in 37 cases (7.8% of total cases), 34 of which were Dallas−. As immune complexes are known to activate leukocytes and complement where they are deposited [10], their presence in myocardial biopsies is not only likely to be directly related to endothelial cell damage but also to myocardial dysfunction observed clinically in this study. Thus, the detection of immune complexes on MBx points to a potentially important mechanism of myocardial injury in patients presenting with idiopathic heart failure. Complement is believed to play an important role in inflammatory cardiomyopathy [29,33,34]. Cardiac myosin, among other antigens, can trigger the complement cascade. C3 byproducts are generated with complement activation and have been shown to regulate proinflammatory cytokine production [2,29]. Interstitial immunoglobulin deposits, found in cardiomyopathy, can also activate complement and lead to tumor necrosis factor production [34]. The distribution of complement in native human heart tissue, on the other hand, has not been studied in detail previously [20]. We show that C3d was detected in a significant percentage (28.8%) of MBx, mainly in a capillary or interstitial distribution, but rarely both, and occasionally in an HRA-like pattern (Table 3). C1q was similar in distribution but seen in a smaller percentage of cases (15.5%). We and others have previously reported the importance of these inflammatory markers (immune complexes including complement) in elucidating antibody mediated rejection in human heart and kidney transplant biopsies [19,35,36]; this current study shows that such staining patterns may also be significant in native human heart biopsies as they are in kidney biopsies. EM expanded on the data obtained by IF, providing some useful information in at least 94% of cases in which it was used. The EM findings suggest that LM routinely missed subtle abnormalities in MBx material even though pathologic changes were present at the ultrastructural level. EM served to confirm the presence of immune complexes and highlight the capillary endothelial damage in most cases in which inflammatory markers were demonstrated by IF. In addition, myofilament loss was observed in patients presenting with unexplained cardiomyopathy, whether or not Dallas criteria were met (Table 4). This feature has been correlated with adverse clinical outcome in patients with cardiomyopathy with or without active myocarditis [19,37] and patients with idiopathic dilated cardiomyopathy [38]. In one study, it was shown to have stronger correlation with patient mortality than clinical indicators such as ejection fraction and duration of symptoms [19]. We found that LM alone is insufficient to demonstrate myofilament loss, as EM identified 138 cases in which it was not detected by LM. Myofilament loss can serve as an indicator of disease severity, but further studies are needed to assess myofilament loss as a predictor of response to treatment.

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Table 4 Electron microscopy findings based on classification of cases by Dallas criteria LM Dallas+ LM Dallas+/− LM Dallas− Total (n=39) (n=44) (n=360) (N=443) EM capillary 22 changes EM myofilament 14 loss EM nonspecific 10 findings only EM no pathology 1

done, the availability of frozen tissue from cardiomyopathic biopsies will make microarray analysis of inflammatory heart disease possible. In the future, study of the genomic expression profile of patients with inflammatory cardiomyopathy may contribute more knowledge to the underlying mechanisms of myocyte injury as well as elucidate novel therapeutic targets [42].

32

168

222

26

205

245

3

68

81

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23

25

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One limitation of this study is the lack of a control group due to difficulty in obtaining myocardial tissue from healthy individuals. However, immunoglobulin deposits, HLA molecule up-regulation, and cellular inflammation have been shown to be present only rarely in control patients [22,24,39,40]. A unique aspect of the current study is the performance of IF and EM on myocardial biopsies from native hearts, a practice that is not routine in other laboratories. This was largely a descriptive study of the pathologic findings seen in inflammatory heart disease. Follow-up data on patients were therefore not obtained, but future studies investigating clinical outcome based on the measures discussed in this study should be performed. We have shown that inflammatory cardiomyopathy can be more clearly stratified by the additional application of IF and EM to the examination of MBx. The value of these modalities was apparent in a high proportion of biopsies, as shown in this descriptive study. Such evaluations are routine for kidney biopsies because of their diagnostic utility. In heart biopsies, defining the underlying immune mechanism may be important in predicting response to treatment as well as defining prognosis in a case of inflammatory cardiomyopathy. Particularly in Dallas− cases, IF can suggest whether the pathogenic mechanism contributing to the patient's heart failure is related to an autoimmune response (e.g., heart-reactive antibody staining pattern), complement activation (e.g., interstitial or capillary C1q or C3d deposition), and/or vascular immune complex deposition. Once inflammation is identified, an infectious, autoimmune, or toxin-mediated cause can be sought using new molecular methods, and this will become increasingly useful as more anti-inflammatory therapies become available [27,41]. EM can contribute information on disease severity and help predict clinical outcome based on degree of myofilament loss [19]. We recommend that, in all native heart MBx, tissue should be saved for IF and EM studies in addition to routine light microscopy. If only one ancillary study is possible, IF is preferred as it provides more specific information on disease mechanism than EM. Further studies should be done to specifically characterize the inflammatory processes involved and validate the stratification of patients into these subgroups. When that is

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