שיטות לאבחון עמילואידוזס ולסיווגו
עבודות מחקר בנושא שיטות לאבחון עמילואידוזס וסיווגו ניתן למצוא ברשימת המאמרים המלאה, במאמרים הבאים: 286, 299, 310, 350, 358, 378, 403, 510. (לרשימת המאמרים המלאה). להלן, מובאים תקצירים של 5 מאמרים נבחרים:
Am J Clin Pathol. 2004 Jun;121(6):794-800.
Biochemical subtyping of amyloid in formalin-fixed tissue samples confirms and supplements immunohistologic data.
Kaplan B, Martin BM, Livneh A, Pras M, Gallo GR.
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.
Clin Chem Lab Med. 2007;45(5):625-8.
Transthyretin amyloidosis in a patient of Iranian-Jewish extraction: a second Israeli-Jewish case.
Kaplan B, Shinar Y, Avisar C, Livneh A.
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.
Clin Chem Lab Med. 2008;46(3):335-41.
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.
Kaplan B, Ramirez-Alvarado M, Dispenzieri A, Zeldenrust SR, Leung N, Livneh A, Gallo G.
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.
Br J Haematol. 2009 Mar;144(5):705-15.
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.
Kaplan B, Ramirez-Alvarado M, Sikkink L, Golderman S, Dispenzieri A, Livneh A, Gallo G.
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.
Am J Hematol. 2014 Sep;89(9):882-8.
Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.
Kaplan B, Golderman S, Aizenbud B, Esev K, Kukuy O, Leiba M, Livneh A, Ben-Zvi I.
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.