Direct Tissue Evaluation via Immunofluorescence

Abstract

Multiple precursor proteins have been shown to cause cardiac amyloidosis. The most common forms are due either to immunoglobulin light chains or to transthyretin proteins (either wild-type or mutant forms). Correct subclassification of the amyloid is paramount because treatment differs in accordance with the type of amyloidosis. Indirect diagnostic methods, including serologic analysis, can lead to misdiagnosis. Definitive diagnosis often requires analysis of amyloid in the tissue. We present a case of a woman who was diagnosed with hereditary transthyretin cardiac amyloidosis by means of immunofluorescence and genetic analysis. This case highlights the importance—in the diagnostic algorithm of cardiac amyloidosis—of direct evaluation of the tissue with immunofluorescence and of genetic testing.

Amyloidosis is a relatively rare disease characterized by the extracellular deposition of precursor proteins. Cardiac involvement can be especially devastating due to symptoms of diastolic dysfunction and right-sided heart failure.¹ As a result, prompt diagnosis and subclassification of the amyloid is essential so that correct treatment can be initiated.² The following case of a woman diagnosed with hereditary transthyretin (TTR) cardiac amyloidosis illustrates the importance of direct evaluation of the tissue with immunofluorescence and genetic testing, and it also draws attention to the pitfalls associated with indirect methods of testing.

Case Report

In March 2010, a 66-year-old Taiwanese woman with a history of essential thrombocytosis and demyelinating polyneuropathy presented at our hospital with palpitations and dyspnea. Approximately 4 months before presentation, she had developed fevers, malaise, and muscle aches after receiving the seasonal influenza vaccine. She was given antibiotics and her symptoms improved. Shortly thereafter, she began noticing mild edema in her feet, as well as exertional dyspnea. She then developed palpitations and associated chest pressure. Because of these symptoms, she sought medical care.

On initial examination, the patient was in no acute distress. Her pulse was regular but tachycardic, ranging from 130 to 150 beats/min. Her blood pressure was 98/53 mmHg, and her oxygen saturation was 99% on room air. Jugular venous pulsation was at 7 cm of water without a Kussmaul sign. Her lungs were clear to auscultation, and on cardiac examination an S₄ gallop was present. She also had trace lower-extremity edema. Her electrocardiogram revealed atrial flutter with variable block. Initial laboratory studies showed normal electrolytes and an elevated platelet count consistent with her known essential thrombocytosis. Thyroid-stimulating hormone and troponin T levels were both normal; however, N-terminal pro-brain natriuretic peptide was elevated at 5,525 pg/mL.

As part of the diagnostic evaluation, she underwent echocardiography, which revealed normal left ventricular function and concentric hypertrophy in which both the interventricular septum and posterior wall measured 15 mm (Fig. 1). The tissue Doppler profile indicated grade 3 diastolic dysfunction. These data raised the possibility of an infiltrative cardiomyopathy. The patient also underwent cardiovascular magnetic resonance imaging, which showed diffuse extensive late gadolinium enhancement without associated edema, consistent with a cardiac infiltrative process (Fig. 2). Serum protein electrophoresis (SPEP) performed at another hospital showed an IgA (immunoglobulin A) lambda M-spike, which suggested paraproteinemia. It was of interest, however, that a repeat SPEP performed at our institution failed to show an M-spike, and a quantitative free-light-chain assay was also unremarkable.

Fig. 1 Transthoracic echocardiograms in parasternal A) long-axis and B) short-axis views show hypertrophy of the left ventricle (LV). Ao = aorta; LA = left atrium; RV = right ventricle

Fig. 2 Cardiovascular magnetic resonance in A) short-axis and B) long-axis views shows global subendocardial late gadolinium enhancement (arrows) consistent with amyloidosis. LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle

Given these findings, we performed a right ventricular endomyocardial biopsy. Amyloid deposits were identified on Congo red staining (Figs. 3A and 3B) and were also seen on electron microscopy. Fresh frozen endomyocardial tissue was then examined by means of immunofluorescence. The technique used to identify the amyloid subtype has been described previously in detail. Briefly, 2-mm-thick frozen sections (routinely obtained at our institution) were evaluated by means of immunofluorescence with a panel of antibodies including lambda, kappa, serum amyloid A, and transthyretin.² In this particular patient, the tissue staining was highly positive for only the transthyretin antibody (Figs. 3C–F).

Fig. 3 Endomyocardial biopsy specimen. Congo-red-stained section shows A) interstitial amyloid deposition, which with plane-polarized light shows B) apple-green birefringence. Evaluation of frozen tissue by immunofluorescence for C) kappa light chain, D) lambda light chain, E) serum amyloid A, and F) transthyretin reveals that the amyloid is derived from transthyretin.

Although tissue immunofluorescence identified the amyloid subtype as TTR, genetic testing was required to determine whether the TTR amyloid was a wild type or a mutant form. A TTR germline mutation analysis was performed on DNA extracted from white blood cells in whole blood. All TTR coding regions and splice sites were amplified using primer sets flanking each of the 4 exons. These polymerase chain reaction products were sequenced using an ABI 3730. This technique, which is more than 99.9% accurate in detecting DNA sequence variants,^3–5 identified a known heterozygous mutation in exon 4: c.349G>T (Ala117Ser) in our patient.

Clinically, the patient did well: she spontaneously converted to sinus rhythm within 24 hours of her hospitalization. She was ultimately discharged on a β-blocker, furosemide, and warfarin, with improvement in her symptoms. As of January 2012, she was enrolled in an investigational trial of diflunisal, a nonsteroidal anti-inflammatory agent that has been shown to stabilize the TTR protein, thereby preventing amyloidogenesis.⁶ Our patient was also under evaluation for possible liver transplantation.

Discussion

Amyloidosis is a generic term applied to a group of diseases characterized by the extracellular deposition of proteinaceous material, which disrupts normal tissue architecture by forming insoluble fibrils.¹ When viewed under the electron microscope, all amyloid deposits have an extended β-sheet conformation. Because of this structure, amyloid deposits stained with Congo red dye have a characteristic apple-green birefringence when viewed under polarized light. Despite these common features, the clinical and pathologic manifestations of the amyloid diseases vary greatly. Amyloidosis is classified on the basis of the precursor protein that forms the fibrils; currently at least 27 distinct forms have been identified, and 12 of these have been shown to affect the heart and great vessels.²

The 3 most common forms of systemic amyloidosis affecting the heart are acquired immunoglobulin light chain amyloidosis (AL), amyloidosis due to hereditary TTR mutations (ATTRm, with more than 80 mutations identified), and systemic senile amyloidosis derived from the wild-type TTR protein.⁷ Amyloid fibrils may be found in all regions of the heart, including the atria, ventricles, valves, conduction system, and blood vessels.¹ Correct diagnosis is essential, because the appropriate treatment depends on the type of amyloidosis. Whereas treatment of AL amyloidosis includes chemotherapy with possible bone marrow and heart transplantation, treatment of the TTR-derived amyloidoses involves liver and heart transplantation.⁸ In addition, hereditary TTR amyloidosis is typically transmitted in an autosomal dominant pattern, which has important implications for the subsequent testing of other family members.^1,9

Approaches to the diagnosis of amyloidosis include laboratory and genetic testing, imaging, and the histologic examination of tissue. Unfortunately, the potential for misdiagnosis is relatively high with the traditional tests, and this can lead to delays in initiating appropriate treatment. For example, patients with amyloidosis often demonstrate low QRS voltage on electrocardiography and low voltage-to-mass ratio on echocardiography. In patients with ATTRm amyloidosis, however, neither of these findings is common.⁷ While cardiovascular magnetic resonance imaging has shown a characteristic pattern of global subendocardial late gadolinium enhancement in patients with amyloidosis, its role in the diagnostic evaluation of cardiac amyloidosis is still uncertain, and many abnormal findings are nonspecific.^1,10

Laboratory testing to determine the presence of a circulating monoclonal paraprotein is an important part of the diagnostic evaluation of amyloidosis. Serum or urine electrophoresis might not reveal a small amount of circulating paraprotein; immunofixation and serum free-light-chain assays are more sensitive.^1,11 Unfortunately, reliance on these tests without additional confirmation has led to the misdiagnosis of amyloidosis in patients who actually have a hereditary form. In addition to incomplete penetrance and variable expressivity for inherited forms of amyloidosis, the de novo mutation rates for disease caused by TTR mutations are unknown, so patients will not always have a family history of the disease. Furthermore, both monoclonal gammopathies and hereditary amyloidoses are more common in older patients, so the presence of a circulating paraprotein may have no relationship to the underlying amyloidosis. Conversely, the absence of circulating paraprotein does not necessarily rule out AL amyloidosis.^9,12

Tissue diagnosis via immunohistolabeling is most often used for the definitive diagnosis of amyloidosis. Customarily, this is done by staining formalin-fixed tissue with immunoperoxidase^13,14—a method that unfortunately can lead to misdiagnoses, often due to high background staining.^15,16 Therefore, additional techniques, including immunofluorescence, have been developed to improve diagnostic accuracy.¹⁷ Because fresh frozen tissue is required, immunofluorescence is not widely used for the evaluation of cardiac biopsy specimens. A recent study,² however, has shown this technique to be an excellent method to diagnose and subtype AL amyloidosis in cardiac specimens. Success rates were similar to those previously reported in kidney specimens, and there were no instances of misdiagnosis. Because there were no cases of known ATTRm amyloidosis in that study, the usefulness of immunofluorescence in that particular population was not established.² At our institution, we now routinely freeze native endomyocardial biopsy specimens; we suggest that all institutions do the same, in order to facilitate subtyping in cases of suspected amyloidosis.

This case illustrates that ATTRm amyloidosis can be successfully diagnosed by using the immunofluorescence technique on an endomyocardial biopsy specimen. Confirmation of this finding was made by genetic analysis, specifically by identifying the Ala117Ser mutation in the transthyretin gene—a known mutation with particular prevalence in Chinese and Taiwanese patients.^3–5 On the basis of these recent data, we believe that immunofluorescence should be considered an acceptable method to aid in the diagnosis of cardiac amyloidosis.