Introduction:
Kidney is among the commonest organs involved by amyloid deposits in the AL (light chain amyloidosis), AA (serum amyloid A amyloidosis), and several other forms of amyloidosis. The accurate typing of amyloid deposits is necessary as the management of these cases varies greatly depending on the fibrillar protein deposited. The main aim of the study was to characterize the subtype of amyloid deposits using immunohistochemistry (IHC) and to explore the utility of Western blot in the classification of amyloid deposits
Methods:
All cases of renal amyloidosis diagnosed by kidney biopsy in our Institute over a period from January 2019 to July 2024 were included. Necessary clinical details and laboratory findings were obtained from the histopathology requisition form. For IHC, the slides were stained for antibodies specific for serum amyloid A (Thermo, PA5102456,1:100), kappa light chain (Thermo, BS-3800R, 1:100), lambda light chain (Thermo, BS-3801R, 1:100), leukocyte cell-derived chemotaxin-2 (LECT-2) (R & D system, MAB722, 1:50), fibrinogen (Thermo, MA5-37943, 1:500), and transthyretin (Thermo, MA532634, 1:200) as per the specified protocol. Western blotting was performed from the protein extracted from the formalin fixed paraffin embedded tissue using Qproteome FFPE tissue kit (Cat. no.37623). The extracted protein was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide Gel electrophoresis) and transferred to the mini-size nitrocellulose membrane, followed by immunoblotting against the antibodies specific for SAA, kappa, lambda, LECT-2, fibrinogen and transthyretin. The result was visualized under chemiluminescence (Syngene G Box). The study was approved by the Institutional ethics committee (Certificate number- AIIMS/IEC/2022/3770).
Results:
A total of 32 cases of renal amyloidosis was diagnosed during this period. The mean age of the patient was 53 years with M:F ratio of 1.9:1. The mean serum creatinine and 24 hr urine protein were 3.19mg/dl (range 0.4-11.29) and 8.28gm/24hr (range 0.4-26.4gm/24hr), respectively. Immunohistochemical studies, performed in 28 cases, revealed AA amyloidosis in 21 cases and AL (all lambda restricted) in 3 cases, while 4 cases were inconclusive due to staining for more than one antibody. Tuberculosis was the most common predisposing factor, and was noted in 61.9% cases of AA amyloidosis. Western blotting was performed in 16 cases due to limited tissues in remaining cases. SDS-PAGE revealed several strong protein bands in the molecular weight region from 10kD to 45 kD. However, immunoblot analysis was interpretable in 12 cases of which AA amyloidosis was reported in 11 cases and AL lambda in one case. For 4 cases, where the IHC result was inconclusive, Western blot was performed in 2 cases, and both cases showed inconclusive results possibly related to poor protein quality.
Conclusions:
AA amyloidosis is the most common form of amyloidosis in our study, followed by AL amyloidosis. Non-specific staining is commonly observed in immunohistochemistry, and noted in 14.3% cases. Western blot could not increase diagnostic sensitivity in amyloid typing, when combined with immunohistochemistry. Further, it was noted that more tissue needed for performing Western blot. Advanced shotgun proteomics approach using liquid chromatography mass spectrometry may be a more preferable approach for typing amyloid deposits in limited tissue samples.
I have potential conflict of interest to disclose.
The study was funded by the grant from Science and Engineering Research board, India (approval letter- SRG/2021/001073) dated 10 Nov 2021. However, the funding agency does not have any influence on the study plan or its execution.
I did not use generative AI and AI-assisted technologies in the writing process.