Abstract
Amyloidosis is a group of disorders caused by extracellular deposition of insoluble fibrillar proteins, leading to progressive organ dysfunction. Cardiac amyloidosis is clinically significant, as myocardial infiltration results in restrictive cardiomyopathy, arrhythmias, and heart failure. The main subtypes are light-chain (AL) and transthyretin (ATTR) amyloidosis, while AA and isolated atrial amyloidosis (IAA) are less common. Accurate subtype identification is crucial for management and prognosis. Diagnosis requires a multimodal approach combining imaging and tissue-based techniques. Echocardiography is usually first-line, showing increased wall thickness, biatrial enlargement, and apical sparing. Cardiac magnetic resonance (CMR) provides superior tissue characterization through late gadolinium enhancement and elevated extracellular volume. Nuclear scintigraphy with 99mTc-labeled tracers enables non-invasive ATTR detection, while amyloid-specific PET tracers show potential for early diagnosis. Histochemical confirmation remains essential. Congo Red staining with apple-green birefringence under polarized light is the diagnostic gold standard, supported by Thioflavin T, PAS, and Alcian Blue stains. Immunohistochemistry and mass spectrometry aid amyloid typing, while electron microscopy provides ultrastructural confirmation. Integrating imaging, histochemical, immunohistochemical, and proteomic techniques enhances early recognition and precise classification, improving therapeutic strategies and patient outcomes.
Keywords:amyloidosis; histochemistry; immunohistochemistry; typing; cardiology
1. Introduction
Amyloidosis is a heterogeneous group of diseases characterized by the extracellular deposition of insoluble fibrillar proteins, known as amyloid, in various tissues and organs. These fibrils are formed when normally soluble proteins misfold, adopt a beta-sheet structure, and aggregate into highly ordered, resistant fibers. Over time, amyloid deposits disrupt normal tissue architecture and organ function, leading to progressive organ damage. The disease can be systemic, affecting multiple organs, or localized, confined to a single tissue. The organs most commonly involved are the heart, kidneys, liver and peripheral and autonomic nervous system. Amyloidosis is classified according to the type of precursor protein involved. More than 30 different proteins have been identified that can form amyloid [1].
Cardiac amyloidosis is a condition characterized by the extracellular deposition of misfolded proteins, known as amyloid, within the heart tissue. These deposits can lead to structural and functional abnormalities, resulting in clinical manifestations such as heart failure, unexplained left ventricular hypertrophy, and, in some cases, severe aortic stenosis. The disease is most commonly caused by two major types of amyloid: immunoglobulin light chain (AL) amyloidosis and transthyretin (ATTR) amyloidosis, although other types, including AA and isolated atrial amyloidosis (IAA), can also affect the heart [2,3].
2. Amyloidosis
Light chain (AL) amyloidosis is a rare disorder, occurring in 5–10 people per million annually. It arises from plasma cell dyscrasias that produce misfolded immunoglobulin light chains. These light chains deposit mainly around cardiac cells, sometimes comprising more than 40% of the myocardial tissue. Deposition is often accompanied by inflammatory infiltrates, primarily T lymphocytes and macrophages, which exacerbate tissue damage and contribute to cardiac dysfunction [4,5].
Transthyretin (ATTR) amyloidosis results from the deposition of misfolded transthyretin, a tetrameric protein synthesized in the liver responsible for transporting thyroid hormone and retinol. It can be hereditary (hATTR), caused by mutations in the transthyretin gene, or wild-type (wtATTR), occurring without genetic mutations. Over 120 transthyretin mutations have been identified, with Val30Met being the most common, while Ile68Leu and Leu111Met are associated with a heart-dominant phenotype. Deposits of transthyretin typically appear as irregular nodules in the interstitium or along vascular structures. In wtATTR, the reasons for protein instability remain unclear, though age-related factors are considered contributors [1,6].
Serum amyloid A protein (AA) amyloidosis, also called secondary or reactive amyloidosis, is linked to chronic inflammatory conditions such as rheumatoid diseases. The amyloid precursor in AA amyloidosis is serum amyloid A (SAA), an acute-phase protein produced by hepatocytes in response to inflammatory cytokines. Cardiac involvement is rare, occurring in about 1% of cases, but when it does, it often manifests as right ventricular failure and indicates a poor prognosis. The kidneys are the most frequently affected organ, typically leading to proteinuria and hypertension [7].
Isolated atrial amyloidosis IAA results from the excessive production of atrial natriuretic peptide (ANP), a hormone involved in blood pressure regulation, natriuresis, and inhibition of the renin–angiotensin–aldosterone system. ANP and its precursor accumulate in the atria, particularly in conditions such as valvular heart disease, persistent atrial fibrillation, or after mitral valve replacement. While generally confined to the atria, these deposits can contribute to atrial structural remodeling and dysfunction [8,9,10,11].
3. Pathophysiology of Cardiac Amyloid Involvement
Cardiac involvement in amyloidosis results from the progressive extracellular deposition of misfolded protein fibrils within the myocardium, vasculature, and conduction system, leading to structural, metabolic, and electrical dysfunction [12]. In AL amyloidosis, myocardial injury arises from both physical infiltration of amyloid fibrils and the direct cardiotoxicity of circulating light chains, which induce oxidative stress, mitochondrial dysfunction, and activation of stress-related signaling pathways such as p38 MAPK and JNK, ultimately impairing contractility and promoting apoptosis; this dual mechanism explains the rapid clinical deterioration typical of AL disease [13]. In contrast, ATTR amyloidosis primarily causes myocardial stiffening through massive interstitial accumulation of transthyretin fibrils, which progressively thicken the ventricular walls, reduce compliance, and impair diastolic filling without the marked biochemical toxicity observed in AL [14,15]. Amyloid infiltration of intramural coronary arterioles produces microvascular dysfunction and reduced myocardial perfusion reserve, contributing to ischemia, myocyte degeneration, and worsening heart failure [16]. Involvement of the atrioventricular node and His–Purkinje system leads to conduction disturbances, including atrioventricular block, sinus node dysfunction, and atrial arrhythmias, particularly prominent in wild-type ATTR [17]. As deposition progresses, the extracellular matrix undergoes fibrotic remodeling with increased collagen deposition and expansion of the extracellular volume, further exacerbating stiffness and restrictive physiology [18]. Ultimately, the combined effects of mechanical infiltration, cellular toxicity, microvascular ischemia, and conduction system involvement culminate in severe diastolic dysfunction, low-output heart failure, and arrhythmic risk, defining the complex pathophysiology of cardiac amyloidosis [12].
4. Non-Invasive Diagnosis
Clinical diagnosis of cardiac amyloidosis relies on a multimodal imaging approach that integrates anatomical, functional, and molecular assessments. Transthoracic echocardiography is typically the first-line investigation due to its accessibility and ability to detect proper features, including increased left ventricular wall thickness, biatrial enlargement, restrictive diastolic filling, and pericardial effusion. Advanced techniques, such as longitudinal speckle-tracking strain imaging, can reveal the characteristic “apical sparing” pattern of global longitudinal strain, aiding differentiation from other hypertrophic phenotypes. Cardiac magnetic resonance imaging (CMR) offers superior tissue characterization, with diffuse subendocardial or transmural late gadolinium enhancement, prolonged native T1 times, and elevated extracellular volume serving as strong indicators of amyloid infiltration. CMR also supports disease staging and therapy monitoring. Cardiac scintigraphy with bone-seeking tracers labeled with 99mTc, such as 99mTc-DPD or 99mTc-PYP, demonstrates high myocardial uptake in most cases of transthyretin amyloidosis, enabling non-invasive diagnosis and subtype distinction without the need for biopsy in certain scenarios. Positron emission tomography (PET), although largely confined to research, utilizes amyloid-specific tracers like 11C-Pittsburgh Compound B, 18F-florbetapir, and 18F-florbetaben to visualize and quantify amyloid burden in vivo, offering promise for early detection and therapeutic response evaluation. Together, these modalities complement histological and proteomic methods, forming a comprehensive diagnostic framework that improves accuracy, facilitates amyloidosis subtype identification, and guides optimal clinical management [19,20].
5. Image-Guided Biopsy Strategy
While endomyocardial biopsy (EMB) remains the gold standard for definitive diagnosis, its sensitivity is intrinsically limited by the spatial heterogeneity of amyloid deposition. Standard transvenous biopsies are typically restricted to the right ventricular (RV) septum; however, amyloid infiltration can be patchy, particularly in early-stage disease or specific subtypes, leading to a risk of sampling error and false-negative results [21]. To mitigate this “blind” approach, pre-procedural imaging plays a critical role in predicting diagnostic yield. Cardiac Magnetic Resonance (CMR) provides a detailed non-invasive “roadmap” of amyloid distribution, specifically, late gadolinium enhancement (LGE) and T1 mapping can confirm whether the RV septum the intended biopsy site is actually involved [22]. If CMR reveals that amyloid deposition is confined to non-septal regions or is absent in the RV, the cardiologist can anticipate a low yield from a standard RV biopsy and may opt for alternative diagnostic strategies or direct the biopsy to more involved segments if technically feasible [23]. Thus, integrating CMR findings prevents futile procedures and enhances the precision of tissue sampling.
6. Biopsy-Based Diagnosis
Mass spectrometry (MS) is a powerful technique used to identify and characterize amyloid proteins, playing a crucial role in the diagnosis of amyloidosis. By measuring the mass-to-charge ratio of ionized molecules, MS provides precise information about the protein composition of amyloid deposits, confirming the presence of amyloid and helping to identify specific amyloid proteins, such as immunoglobulin light chains in AL amyloidosis or transthyretin in ATTR amyloidosis. Tissue samples are typically processed through laser capture microdissection or liquid chromatography before being analyzed by MS. While endomyocardial biopsy provides the most direct assessment, MS can also be successfully performed on specimens from surrogate sites, including abdominal subcutaneous fat, salivary glands, or the kidney and liver in systemic disease. This flexibility allows for precise typing even when cardiac tissue is not available, provided that amyloid deposits are present in the sampled surrogate tissue [24,25]. MS offers several advantages over traditional methods, such as its ability to detect multiple amyloid types in a single sample and its high sensitivity to small amounts of amyloid fibrils. However, its application in clinical diagnostics is limited due to the need for specialized equipment and technical expertise, making it more common in research settings or specialized centers.
Recent advancements in MS, such as tandem mass spectrometry (MS/MS) and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight), have improved its accuracy and efficiency in identifying amyloid proteins. While MS provides definitive information about the protein composition of amyloid deposits, it is typically used alongside other techniques like Congo Red staining or immunohistochemistry to ensure comprehensive diagnostic evaluation, especially in complex cases such as cardiac amyloidosis [26,27]. While mass spectrometry (MS) represents the definitive method for amyloid typing at the molecular level, non-invasive imaging techniques play a pivotal role in the diagnosis, phenotyping, and longitudinal assessment of organ involvement, particularly in cardiac amyloidosis.
7. Histochemistry Techniques
Congo red (CR) staining (Figure 1) is a histochemical technique used to detect amyloid deposits in tissue. The dye binds specifically to the β-sheet structure of amyloid fibrils, producing red staining under brightfield illumination and characteristic apple-green birefringence when viewed under polarized light between a crossed polarizer and analyzer. Tissue must be formalin-fixed, paraffin-embedded, sectioned (6–10 µm), deparaffinized, and rehydrated before staining. Hematoxylin may be added for contrast [28,29]. CR is highly specific for amyloid but cannot identify the protein subtype or quantify deposition. It may miss small deposits and occasionally produces background staining. For definitive diagnosis, CR is often combined with immunohistochemistry or mass spectrometry. Sensitivity can be improved using enhanced protocols, such as alkaline CR staining and digital analysis