**2. Materials and Methods**

#### *2.1. Sample Preparation*

Remnant, de-identified tissues from kidney biopsies performed for diagnostic purposes (IRB approval: IRB00090103) were used for this study, as illustrated in Figure 1. The biopsied tissues of AA, AL, and NA amyloidosis from 6 patients were prepared as a frozen tissue block. Fresh frozen blocks were sectioned by a cryostat, and thin-sliced kidney tissue sections were placed on quartz and glass microscope slides for Raman measurements and histological evaluation, respectively. Tissue sections for Raman measurements remained unstained and were prepared on quartz slides to avoid spectral interference with the biochemical fingerprints of the tissue sample. Consecutive slices from each tissue block were used to detect and identify amyloid fibrils through histological evaluation (Figure 2).

**Figure 1.** The overall workflow for amyloid identification and subtyping. (**A**) Tissue preparation steps. Biopsied tissues were frozen and sectioned for evaluation. (**B**) Raman spectroscopic data acquisition and analysis. Fresh frozen tissue sections were prepared on quartz slides to minimize spectral interference, and employed for Raman measurements, which were subjected to machine learning analysis. (**C**) Histopathologic validation. Consecutive tissue sections used in (**B**) were utilized for the gold standard, immunohistochemistry evaluation. (Created with BioRender.com (accessed on 23 February 2023)).

**Figure 2.** Renal amyloidosis. (**A**) Glomerular and arteriolar deposits of amyloid identified as Congo red-positive material (magnification 400×). (**B**) Glomerular amyloid Congo red-positive deposits

showing birefringence under polarized light (magnification 400×). (**C**) AA amyloidosis: the immunohistochemical stain for amyloid A is strongly positive in the glomerulus and in the arterioles (magnification 400×). (**D**) AL amyloidosis: by immunofluorescence, a glomerulus containing Congo red-positive material (not shown) shows a positive stain for the kappa light chain (magnification 400×). (**E**) The immunofluorescence stain for lambda light chain is negative in the same glomerulus (magnification 400×). (**F**) The immunohistochemical stain for amyloid A is negative in the glomeruli containing deposits of AL amyloid.
