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.

The Prognostic Value of Anti-PLA2R Antibodies Levels in Primary Membranous Nephropathy.

Anti-PLA2R antibodies (Ab) are a diagnostic and prognostic biomarker in primary membranous nephropathy (PMN). We assessed the relationship between the levels of anti-PLA2R Ab at diagnosis and different variables related to disease activity and prognosis in a western population of PMN patients. Forty-one patients with positive anti-PLA2R Ab from three nephrology departments in Israel were enrolled. Clinical and laboratory data were collected at diagnosis and after one year of follow-up, including serum anti-PLA2R Ab levels (ELISA) and glomerular PLA2R deposits on biopsy. Univariable statistical analysis and permutation-based ANOVA and ANCOVA tests were performed. The median [(interquartile range (IQR)) age of the patients was 63 [50-71], with 28 (68%) males. At the time of diagnosis, 38 (93%) of the patients had nephrotic range proteinuria, and 19 (46%) had heavy proteinuria (≥8 gr/24 h). The median [IQR] level of anti-PLA2R at diagnosis was 78 [35-183] RU/mL. Anti-PLA2R levels at diagnosis were correlated with 24 h proteinuria, hypoalbuminemia and remission after one year (p = 0.017, p = 0.003 and p = 0.034, respectively). The correlations for 24 h proteinuria and hypoalbuminemia remained significant after adjustment for immunosuppressive treatment (p = 0.003 and p = 0.034, respectively). Higher levels of anti-PLA2R Ab at diagnosis in patients with active PMN from a western population are associated with higher proteinuria, lower serum albumin and remission one year after the diagnosis. This finding supports the prognostic value of anti-PLA2R Ab levels and their possible use in stratifying PMN patients.