Amyloid Typing in Cardiac Amyloidosis Using Western Blotting.

BACKGROUND: Cardiac amyloidosis (CA) is characterized by the extracellular deposition of misfolded protein in the heart. Precise identification of the amyloid type is often challenging, but critical, since the treatment and prognosis depend on the disease form and the type of deposited amyloid. Coexistence of clinical conditions such as old age, monoclonal gammopathy, chronic inflammation, or peripheral neuropathy in a patient with cardiomyopathy creates a differential diagnosis between the major types of CA: amyloidosis light chains (AL), amyloidosis transthyretin (ATTR) and amyloidosis A (AA). OBJECTIVES: To demonstrate the utility of the Western blotting (WB)-based amyloid typing method in patients diagnosed with cardiac amyloidosis where the type of amyloid was not obvious based on the clinical context. METHODS: Congo red positive endomyocardial biopsy specimens were studied in patients where the type of amyloid was uncertain. Amyloid proteins were extracted and identified by WB. Mass spectrometry (MS) of the electrophoretically resolved protein-in-gel bands was used for confirmation of WB data. RESULTS: WB analysis allowed differentiation between AL, AA, and ATTR in cardiac biopsies based on specific immunoreactivity of the electrophoretically separated proteins and their characteristic molecular weight. The obtained results were confirmed by MS. CONCLUSIONS: WB-based amyloid typing method is cheaper and more readily available than the complex and expensive gold standard techniques such as MS analysis or immunoelectron microscopy. Notably, it is more sensitive and specific than the commonly used immunohistochemical techniques and may provide an accessible diagnostic service to patients with amyloidosis in Israel.

Amino acid sequence homology of monoclonal serum free light chain dimers and tissue deposited light chains in AL amyloidosis: a pilot study.

OBJECTIVES: Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs). METHODS: Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M) and dimers (D) were analyzed by Western blotting (WB) and mass spectrometry (MS). RESULTS: WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M-D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases. CONCLUSIONS: This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease.

Diagnostic Challenges and Solutions in Systemic Amyloidosis.

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice.

Kidney disease and plasma cell dyscrasias: ambiguous cases solved by serum free light chain dimerization analysis.

BACKGROUND: Plasma cell dyscrasias (PCD) comprise a wide spectrum of disorders, which may adversely affect the kidney. However, in some PCD cases associated with kidney disease, the routine laboratory tests may be incapable to determine precisely the form of PCD, i.e., benign or malignant. Moreover, the kidney biopsy needed for precise diagnosis may be risky or declined. To overcome these limitations, we have developed and reported a new non-invasive technique based on serum free light chains (FLC) monomer (M) and dimer (D) pattern analysis (FLC MDPA), which allowed differentiation between malignant and benign PCD forms. The objective of our retrospective study was to demonstrate the utility of FLC MDPA in solving ten puzzling PCD cases complicated with kidney disease (patients 1-10). METHODS: Ten patients with uncertain form of PCD or with a questionable response to treatment were studied. In addition to routine laboratory tests and clinical evaluation of the PCD patients, our previously developed FLC MDPA in sera and biochemical amyloid typing in biopsy tissues were applied. RESULTS: The FLC MDPA aided the diagnosis of the PCD underlying or accompanying the kidney disease in patients 1-5, and helped to interpret properly the response to treatment in patients 1, 6-10. The FLC MDPA findings were confirmed by a biochemical analysis of tissue amyloid deposits and subsequently by the outcome of these patients. CONCLUSIONS: FLC MDPA is a non-invasive diagnostic test useful in the management of ambiguous cases of PCD associated with kidney disease.

The use of serum free light chain dimerization patterns assist in the diagnosis of AL amyloidosis.

The discrimination between benign and malignant forms of plasma cell dyscrasia (PCD) is often difficult. Free light chain monomer-dimer pattern analysis (FLC-MDPA) may assist in solving this dilemma and distinguish between AL amyloidosis and benign PCD. Serum samples of patients with AL amyloidosis and benign PCD were analysed in a blinded manner. Quantitative Western blotting was performed to estimate dimerization and clonality indices, and thereby determine the source of the tested samples, as derived either from benign or malignant PCD. The findings obtained by the FLC-MDPA were compared with the actual diagnosis. Of 37 samples from patients with active AL amyloidosis, 34 (91·9%) fulfilled dimerization criteria for diagnosis of AL amyloidosis. Of the 45 samples from patients with benign PCD, 10 (21·2%) tested falsely positive or gave an inconclusive result. Thus, the sensitivity of the analysis was 92·5% with a remarkable negative predictive value of 91·9%. In addition, of 20 patients who were in complete or very good partial remission, only one tested positive. By multivariate analysis, FLC-MDPA was the best independent marker predicting AL amyloidosis (odds ratio of 84). The FLC-MDPA offers a highly effective tool in the diagnostic assessment of patients with PCD.

Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive.

Immunoglobulin free light chain dimers in human diseases.

Immunoglobulin free light chain (FLC) kappa (κ) and lambda (λ) isotypes exist mainly in monomeric and dimeric forms. Under pathological conditions, the level of FLCs as well as the structure of monomeric and dimeric FLCs and their dimerization properties might be significantly altered. The abnormally high fractions of dimeric FLCs were demonstrated in the serum of patients with multiple myeloma (MM) and primary systemic amyloidosis (AL), as well as in the serum of anephric patients. The presence of tetra- and trimolecular complexes formed due to dimer-dimer and dimer-monomer interactions was detected in the myeloma serum. Analysis of the amyloidogenic light chains demonstrated mutations within the dimer interface, thus raising the possibility that these mutations are responsible for amyloidogenicity. Increased κ monomer and dimer levels, as well as a high κ/λ monomer ratio, were typically found in the cerebrospinal fluid from patients with multiple sclerosis (MS). In many MS cases, the elevation of κ FLCs was accompanied by an abnormally high proportion of λ dimers. This review focuses on the disease-related changes of the structure and level of dimeric FLCs, and raises the questions regarding their formation, function, and role in the pathogenesis and diagnosis of human diseases.

Free light chains in plasma of patients with light chain amyloidosis and non-amyloid light chain deposition disease. High proportion and heterogeneity of disulfide-linked monoclonal free light chains as pathogenic features of amyloid disease.

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders accompanied by a monoclonal gammopathy. Monoclonal free light chains (FLCs) are precursors of the pathological light chain tissue deposits that are fibrillar in AL and granular in NALCDD. However, direct biochemical examination of plasma FLC precursors, which would allow comparison and better understanding of these two diseases, is still lacking. In this study, we examined FLCs in plasma of patients with AL and NALCDD by employing separation on Sep-PaK C18 cartridges, micro-preparative electrophoresis, Western blotting and mass spectrometry. Comparative analysis of AL versus NALCDD and control plasma samples showed new evidence of increased level and heterogeneity of circulating disulfide-bound FLC species in AL. In addition to full length monomers comprising the disulfide-linked FLCs, the monoclonal disulfide-bound FLC fragments were typically revealed in AL plasma. We hypothesized that enhanced disulfide binding of FLCs in AL interferes with their normal clearance and metabolism, which in turn might play a role in amyloid formation. The applied methods might be useful to diagnose or predict the pathological form of the disease and shed light on the mechanisms involved in light chain aggregation in tissues.

Isolation and biochemical characterization of plasma monoclonal free light chains in amyloidosis and multiple myeloma: a pilot study of intact and truncated forms of light chains and their charge properties.

BACKGROUND: The presence of monoclonal immunoglobulin free light chains (FLC) in the serum is commonly associated with the gammopathies, including multiple myeloma, systemic light chain amyloidosis and non-amyloid light chain deposition disease. Although a sensitive nephelometry-based assay is used for quantification of serum FLC and patient follow-up, this method does not provide information regarding the biochemical properties of these proteins. The present study focused on the development of the procedure for isolation and biochemical characterization of monoclonal FLC in small plasma specimens from patients with these disorders. METHODS: Methods used in this study were ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein elution from gel and support membranes, dialysis, lyophilization, isoelectric focusing (IEF) and Western blotting. RESULTS: The isolation, concentration and partial purification of FLC was based on micro-preparative SDS-PAGE employing analytical scale gels. For the determination of the isoelectric point of FLC, the developed protocol included consecutive IEF, electrotransfer of IEF-separated proteins onto and elution from support membranes, and their analysis by SDS-PAGE-based Western blotting. The procedures, which require only 20-50 microL of starting plasma, allow biochemical characterization of the monomeric, dimeric and truncated forms of FLC, including their charge properties. CONCLUSIONS: The developed procedure may be applied to reveal distinguishing chemical features of FLC in serum, which could be important in predicting the pathologic form of disease, and in yielding information to better understand the mechanism(s) involved in the deposition of light chains in tissues.

Transthyretin amyloidosis in a patient of Iranian-Jewish extraction: a second Israeli-Jewish case.

BACKGROUND: We present a patient with late-onset progressive polyneuropathy and a Congo-red positive staining of sural nerve biopsy, where routine immunohistochemical analysis failed to determine the type of amyloid deposited. Since precise determination of the amyloid type has crucial therapeutic implications, we employed our new biochemical micro-techniques, together with molecular biology methods, to characterize the amyloid protein deposited. METHODS: We used a micro-method for extraction of amyloid proteins, amyloid typing by Western blotting, DNA sequencing, and restriction enzyme site analysis. RESULTS: Our new micro-technique for biochemical analysis of fat aspirate demonstrated transthyretin (TTR) amyloid (ATTR) deposition in the patient's tissue. Sequence analysis of the TTR-encoding DNA identified a single mutation, causing a valine to alanine substitution (V32A). CONCLUSIONS: Although there are approximately 100 known TTR variants associated with peripheral neuropathy, in Israel only one patient with familial amyloid polyneuropathy (FAP), a patient of Ashkenazi origin with ATTR due to an F33I mutation, has been reported so far. Our study identified a second case of ATTR in the Israeli population, this time in a patient of Iranian-Jewish extraction. The study also emphasizes the importance of our new biochemical micro-techniques in elucidating the type of amyloid protein.

Charge differences between in vivo deposits in immunoglobulin light chain amyloidosis and non-amyloid light chain deposition disease.

Immunoglobulin light chain amyloidosis (AL) and non-amyloid light chain deposition disease (NALCDD) are different forms of protein aggregation disorders that may occur in plasma cell dyscrasias with dysproteinemia. In systemic AL, the deposits are fibrillar and patchy in distribution, within and amongst different organs, whereas in NALCDD, the deposits are granular and diffusely distributed in systemic basement membranes, suggesting different mechanisms of aggregation and deposition. Previous evidence, that charge differences between the light chains in AL and NALCDD might account for their different phenotypes, prompted the present study, which compared the isoelectric points (pIs) of AL and NALCDD protein deposits extracted from human tissues. The pI profiles (5.2-8.8) of polypeptides in AL deposits were heterogenous in four cases, with a spread of both anionic and cationic isoforms; in contrast, in three of NALCDD the pI profiles (8.2-8.8) were homogeneous and restricted in the cationic range. These in vivo findings in human disease, together with other reported in vitro and in vivo experimental data, suggest that the fibrillar deposits in AL may form by electrostatic interaction between oppositely charged polypeptides, whereas the granular deposits in NALCDD form by the binding of cationic polypeptides to anionic proteoglycans sites in basement membranes.

Gastrointestinal beta2microglobulin amyloidosis in hemodialysis patients: biochemical analysis of amyloid proteins in small formalin-fixed paraffin-embedded tissue specimens.

We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. A new microtechnique was applied for extraction and immunochemical/chemical characterization of amyloid proteins in small amounts of fixed tissue, thus allowing precise identification of beta2microglobulin amyloid (Abeta2M) in both cases studied. The molecular mass of the identified amyloid beta2M was close to that of intact beta2M (12 kDa), with no evidence of the products of proteolytic fragmentation of these molecules. The isoelectrofocusing of the purified Abeta2M demonstrated a shift to more acidic pI as compared to the normal beta2M analyzed under the same experimental conditions. The obtained data suggest that the intestinal amyloid deposits in dialysis-related amyloidosis contain disease-specific beta2M isoforms, which could play a role in the pathogenesis of amyloid disease. The new methodology used might be useful in obtaining precise diagnosis of amyloidosis that is necessary for appropriate therapy, and also provide new important information on the chemical structure of amyloid proteins.

Co-deposition of amyloidogenic immunoglobulin light and heavy chains in localized pulmonary amyloidosis.

Localized pulmonary amyloidosis is a rare condition whose pathogenesis is insufficiently understood. In the present study, we report a case of localized pulmonary amyloidosis associated with lung-restricted lymphoplasmacytoid lymphoma, monoclonal for immunoglobulin (Ig) G lambda (lambda). Biochemical microtechniques have been applied for extraction, purification, and characterization of amyloid proteins. Surprisingly, chemical analysis of these proteins revealed a not-previously-described case of combined deposits containing Ig fragments of gamma heavy chain (variable domain) and lambda light chain (constant domain). In view of the absence of circulating monoclonal Ig, this case supports the hypothesis that localized amyloid is formed by local plasmacytoid cells.

Biochemical subtyping of amyloid in formalin-fixed tissue samples confirms and supplements immunohistologic data.

The systemic amyloidoses are a heterogeneous group of congophilic fibrillar protein deposition diseases that should be subtyped chemically by immunohistologic methods. Biochemical methods sometimes are required to confirm or identify the amyloid type in unfixed or informalin-fixed tissue samples. We report the results of formic acid extraction and immunochemical and biochemical characterization of deposits informalin-fixed tissue samples from 10 cases of amyloidosis and 3 from nonamyloid monoclonal immunoglobulin light chain deposition disease. The results in 11 of 13 cases demonstrated concordance with the previous immunohistochemical and/or biochemical data obtained in unfixed tissue samples from the same specimens, and in 2 of 13, the protein deposits that previously could not be classified by standard immunohistochemical methods were identified by amino acid sequence. An additional new finding of constant-region rather than variable-region fragments as the major constituent protein in 1 case of lambda light chain amyloidosis demonstrated the value of the method and its importance for future applications.