Abstract Background In the subtypes of cardiac amyloidosis (CA), transthyretin-related (ATTR) CA has been reported to exhibit a higher prevalence of microcalcifications in the interventricular septum (IVS) compared to other subtypes. Whether microcalcifications in the IVS can stratify CA subtypes remains unclear. Methods A total of 442 patients with CA: 76 light chain/ amyloid A (AL/AA) type; 255 wild type ATTR (ATTRwt) and 48 variant ATTR (ATTRv) and 19 patients with endomyocardial biopsy-confirmed hypertensive heart disease (HHD) were subjected to echocardiographic pixel brightness quantification of the IVS using open-access image analysis software. The IVS Brightness Index (IVSBI) was defined as the ratio of the average pixel brightness in the IVS to that in the left ventricular (LV) wall opposite the IVS (see figure 1). Results The IVSBI from apical 4-chamber view was higher in both the ATTRwt group (median 1.40, IQR 1.22 to 1.67) and the ATTRv group (1.59, 1.32 to 2.00) compared to the HHD group (1.20, 1.02 to 1.37) and the AL/AA group (1.25, 1.11 to 1.46), respectively (P <0.001, see figure 2). In the 273 CA patients undergoing myocardial technetium-99m-pyrophosphate scintigraphy, patients with positive results (1.41, 1.24 to 1.70) exhibited a higher IVSBI compared to those with negative (1.19, 1.09 to 1.46) (P = 0.006). In Cox proportional hazards analysis, age, gender and LV ejection fraction adjusted hazard ratio for the all-cause death of IVSBI (0.5 increase) was 2.0 (95% CI 1.2 to 3.5, P = 0.012) among ATTRwt patients (n=157). Conclusions An IVSBI obtained from the apical 4-chamber view could prove valuable in differentiating ATTR subtypes in CA, potentially revealing subtle septal calcifications. A prognostic value may be derived from the elevation of IVSBI in ATTRwt patients.figure 1figure 2