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Background: Renal amyloidosis (RA) has a worldwide incidence of 5–13 cases per million person‐years and is expected to rise in upcoming years due to growing awareness, plus improvement of diagnostic modalities. Diagnosing RA remains challenging, especially when encountering very small, focal, or early amyloid deposits. Since delays in diagnosis portends poor prognosis, high morbidity, and mortality, it is crucial to evaluate the performance of commonly used diagnostic modalities. This is the first study that presents a full picture of the diagnostic performance of fluorescence microscopy (FM) with a tetramethylrhodamine isothiocyanate (TRITC) filter to diagnose RA in general and stratified by compartments. Materials and methods: A retrospective double‐blind diagnostic accuracy study of FM‐TRITC filter was performed. The presence or absence of amyloid in the vascular, interstitial, and glomerular compartments was established in 316 representative Congo red‐stained core biopsies with an FM‐TRITC filter. This was contrasted with polarized microscopy (PM) showing apple‐green birefringence as the gold standard. Sensitivity, specificity, positive and negative predictive values, likelihood ratios, and the receiver operating characteristic (ROC) curve were obtained using STATA13. Results: The prevalence of RA was 6.01%, comparable with that reported in the literature. Reciprocity with regard to the location and pattern of fluorescence and birefringence between the two diagnostic modalities was seen. The FM‐TRITC filter has a sensitivity of 100%, specificity of 97.64%, and a positive and negative predictive value of 73.08% and 100%, respectively. The positive likelihood ratio was 42.37, and the negative was 0.00. Overall accuracy was 97.78%. The area under the ROC curve was 0.98. The Diagnostic performance of the FM‐TRITC filter stratified by compartments is shown in Table 1. The area under the ROC curve was 0.99, 0.98, and 0.99 for the vascular, interstitial, and glomerular compartment, respectively. All patients with RA (n = 19) were correctly identified; this included one new case, one case with small and focal amyloid, and two early cases with less dense amyloid where birefringence was ambiguous by PM. Discussion: The FM‐TRITC filter is a highly accurate, sensitive, specific, with excellent predictive values, time‐efficient, easy to perform, and suitable to reproduce diagnostic modality for RA. It can accurately rule out RA in all compartments, and in most cases concomitant use of PM should not be indispensable. The diagnosis of vascular, interstitial, and glomerular amyloid deposits can be done using only the FM‐TRITC filter with Congo red‐stained slides. Exceptionally, a few cases of interstitial amyloidosis could be overdiagnosed due to interferences (e.g. artefacts), these cases could be further assessed with a second diagnostic modality if positive fluorescence is seen. Routine use of the FM‐TRITC filter can aid in the diagnosis of early RA, even when the deposits are inconspicuous by PM. [ABSTRACT FROM AUTHOR]