Classification of four chemically different amyloid types in routine endomyocardial biopsies by advanced immunohistochemistry

The identification of proteins causing cardiac amyloidosis is of paramount importance for further clinical management. Since the first therapeutic approaches for amyloidosis were developed, it has been mandatory to identify the particular amyloid type in order to select the most efficient therapy for each patient. Endomyocardial biopsies from 275 patients with a suspected diagnosis of storage disease, hypertrophic, or restrictive cardiomyopathy were additionally analyzed for the presence of amyloid using Congo red staining. The results revealed the presence of amyloid in 31/275 (11.3%) of cases. The classification of 25 of these 31 patients using special immunohistochemistry resulted in 25 correct diagnoses, including 4 different amyloid classes. The prevalence was ATTR (14/25; 56%), ALl (8/25; 32%), ALk (2/25; 8% ), and AA (1/25; 4%) demonstrating amyloid-type specific cardiotropy and the usefulness of immunohistochemistry validated by various stringent controls on fixed paraffin-sections for amyloid typing as a method, which can be used in every routine laboratory today. Introduction: Cardiac amyloidosis, which is associated with interstitial and vascular deposition of amyloidotic proteins in the myocardium, may cause restrictive cardiomyopathy that can result in a poor prognosis. Of the three major functional categories of the cardiomyopathies (dilated (DCM), hypertrophic (HCM), restrictive (RCM)), restrictive cardiomyopathy is the least common form in adults and in children. Patients present with symptoms of heart failure, a decreased volume of both ventricles, biatrial enlargement, and an increased wall thickness on echocardiogram are suspicious of amyloidosis. Because of the frequency of treatable disorders, endomyocardial biopsy (EMB) is a diagnostic tool to elucidate distinct causes of heart failure associated with unexplained restrictive cardiomyopathy [1]. EMB is a relatively safe and established tool for primary diagnosis and clinical follow-up of cardiomyopathies [2]. In contrast to imaging techniques such as magnetic resonance imaging, histological examination of EMBs allows the direct detection, localization, and classification of amyloid deposits in myocardial tissue. Increasing numbers of amyloid diseases are challenging the currently applied pathohistological diagnostics. Conventional staining with Congo red is indispensable for the diagnosis of amyloid. However, since amyloid does not represent a single disease but is divided into many different disease entities that can be differentiated through the identification of a particular pathologic protein, various methods have been used to this end. Amyloidosis has to be classified by identifying the chemical nature of the amyloidogenic protein in each patient since every protein causes a different disease with its specific variants, and every amyloid type, with more than 27 different amyloid diseases known to date, has a different prognosis and requires a different therapy or no therapy at all [3]. Novel approaches for amyloid typing are based on mass spectrometry (MS) without [3] or with [4] laser microdissection sampling of amyloid areas (LMD-MS) for proteomic analysis [3,4]. The improved immunohistochemical typing of amyloidosis, a cost efficient application for routine diagnostics as employed here, has been proven to be more suitable on a larger number of patients [5,6] in a comparison with MS [6] and has been successfully utilized here on amyloid in endomyocardial biopsies. Methods: From 3925 patients with heart failure symptoms, EMBs were taken for differential diagnostics of heart muscle tissue using histological examinations on paraffin sections, immunohistochemical analysis of intramyocardial inflammation, and molecular pathological detection of the most frequent cardiotropic viruses [7]. EMBs of 275 patients with suspected diagnosis of storage disease, HCM, or RCM were additionally examined for the presence of amyloid in myocardial tissue. For this purpose, the formalin-fixed biopsies were paraffin embedded and serially sectioned (4 mm thickness). The sections were stained with Congo red and counterstained with hematoxylin according to standard procedures and evaluated in triple illumination, that is in bright light, in cross-polarized, and fluorescent light (Leica Microscope DMRE) [6]. When amyloid was detected, tissue specimens underwent immunohistochemical routine typing at the Center of Amyloid Diseases in Martinsried using a panel of the 10 following validated antibodies: monoclonal murine antiAA(mc4,13,29), polyclonal rabbit (as all the following) anti-ALl(HAR), anti-ALl(ULI/LAT), anti-ALk(SIN), anti-ALk(KRA/KUN), anti-AHg (SOL, mix), anti-ATTR(TIE), anti-Ab2M(WOE), anti-FibAa, anti-ApoAI. This panel named ‘amYkit’; (www.amymed.de) can distinguish reliably the 76