Hidden Auricular Amyloid Deposits and Aortic Stenosis: Clinical and Prognostic Implications in Aortic Valve Replacement Surgery
Conditions: Aortic Stenosis, Amyloid
Sponsor: Germans Trias i Pujol Hospital

HIDDEN AURICULAR AMYLOID DEPOSITS AND AORTIC STENOSIS: 
 CLINICAL AND PROGNOSTIC IMPLICATIONS IN AORTIC VALVE REPLACEMENT SURGERY 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
February 20, 2023 

Study Protocol 
The Aor(c-Ru( cohort is a prospec(ve single center study of cardiovascular pa(ents diagnosed 
with severe AS and with indica(on for isolated SAVR. Pa(ent management was decided by the 
physicians, following recommended guidelines. The study also included a compara(ve analysis 
of two subgroups stra(ﬁed by the presence or absence of amyloid deposits in intraopera(ve 
biopsy samples. 
 
From November 2020 and November 2021, a total of 110 pa(ents over 18 years of age, 
diagnosed with severe aor(c stenosis by echocardiographic criteria, with indica(on for isolated 
aor(c valve replacement and elec(ve surgery, were included. The echocardiographic criteria 
for the diagnosis of severe AS were the following: average gradient greater than 40mmHg 
and/or maximum speed greater than 4m/s. Aor(c valve area calculated by the con(nuity 
equa(on: the severity cut-oﬀ point established by the guidelines is less than 1cm2 or set by the 
body surface index <0.6cm2/m2. Those pa(ents undergoing emergency surgery or combined 
surgery, with extra-cardiac disease with a prognosis of less than 365 days, with moderate-
important alcoholism and abnormal laboratory values, were excluded. 
 
During the surgery, atrium and ventricular septum biopsies were collected for histological and 
Visium Spa(al Gene Expression analysis. Blood samples for biomarker analysis were obtained 
before the surgery, at discharge and at one month and one year of follow-up, centrifuged, and 
then serum and EDTA-plasma were stored at -80ºC un(l assay. 
 
Clinical and demographic characteris(cs were recorded at baseline, discharge, 1 month and 1 
year. 
 

Variables were collected prospec(vely. Intraopera(ve histological results were recorded, and a 
dedicated biomarker database was built, incorpora(ng baseline and follow-up serum values 
collected at each visit. 
Data were organized into ﬁve datasets per sub-cohort: Baseline, Follow-up, Biomarkers, 
EuroQuol Quality of Life test (EQ-5D-5L), and Histology. 
Follow-up data analysis focused on one-year post-surgery results, except for EQ-5D-5L scores, 
where a mean of the three global scores (discharge, 1 month, and 1 year) was calculated per 
pa(ent and compared between sub-cohorts. 
 
Blood samples were collected at admission (n=105), discharge (n=84), one month (n=88), and 
one year (n=94) post-surgery. Serum and EDTA plasma were stored at −80 °C. Auricular and 
septal (ssue samples were obtained during surgery in 70 pa(ents and ﬁxed in 4% 
paraformaldehyde before paraﬃn embedding. 
 
Paraﬃn-embedded (ssue sec(ons (4 µm) were stained using Congo Red, Sirius Red, and 
Trichrome techniques. For Congo Red staining, sec(ons were treated with haematoxylin and a 
Congo Red solu(on, and posi(vity was conﬁrmed by apple-green birefringence under polarized 
light. Sirius Red staining involved incuba(on with picrosirius to visualize collagen types I and III, 
appearing red/orange and apple green, respec(vely. Trichrome staining included ﬁxa(on in 
Bowin’s solu(on and sequen(al staining steps to diﬀeren(ate (ssue components, evaluated 
with a LEICA DMI6000B microscope. 
 
Paraﬃn-embedded sec(ons (4 µm) were deparaﬃnized, rehydrated, and blocked with 5% 
horse serum in PBS-Tween and Triton X-100. Sec(ons were incubated overnight at 4°C with 
primary an(bodies against TTR (1:200), ANP (1:200), and troponin I (1:50). Aler washing, 
secondary an(bodies (1:500) were applied for 45 minutes at room temperature. Following PBS 

rinses, Congo Red staining and moun(ng were performed. Imaging was carried out using a 
Leica TCS SP5 confocal microscope. 
 
Midregional Proatrial Natriure0c Pep0de (MRproANP). MRproANP serum levels were 
measured with Midregional ProAtrial Natriure(c Pep(de ELISA Kit (ABIN6975642; an(bodies 
online; lot W17065241). The detec(on range and the minimum detectable dese are 39pmol/L 
– 2500pmol/L and 9.8pmol/L, respec(vely. 
Meteorin-like (Mtrnl). Serum Mtrnl levels were measured with the Human Meteorin-
like/METRNL DuoSet ELISA (DY7867-5; R&D; lot P300680). The detec(on range and the 
minimum detectable dese are 15.6 pmol/L – 1000 pg/mL. 
 
Four (ssue samples (Posi(ve Congo Red, n=2; Nega(ve Congo Red, n=2) were analyzed using the 
Visium Spa(al Gene Expression system at the Josep Carreras Leukaemia Research Ins(tute (IJC). 
RNA sequencing data was pre-processed and normalized using the SC Transform (SCT) method 
to correct for technical variability. Samples were integrated to iden(fy spa(ally dis(nct clusters 
across condi(ons. Diﬀeren(al gene expression analysis was performed to classify cell 
popula(ons, mapping them to known cell types such as ﬁbroblasts, cardiomyocytes, endothelial 
cells, and immune cells. Data visualiza(on was conducted using UMAP dimensionality reduc(on, 
and spa(al cluster annota(on was based on marker gene expression. 
 
 
 
 
 
 
 

StaCsCcal Analysis Plan 
Categorical variables were summarized as counts and percentages, and con(nuous variables as 
mean ± standard devia(on or median (interquar(le range), as appropriate. Changes from 
baseline to 1-year follow-up in clinical, imaging, and biomarker variables were assessed using 
linear regression models, with and without interac(on terms for Congo Red status (posi(ve vs. 
nega(ve). Es(mated marginal means were used to compare adjusted follow-up values. 
Associa(ons between Congo Red status and clinical outcomes, including hospital readmission 
and atrial ﬁbrilla(on, were evaluated using condi(onal Poisson regression models adjusted for 
sex, age, LVEF, and baseline NT-proBNP. Lel atrial size was addi(onally included in models 
assessing atrial ﬁbrilla(on. 
Further linear regression models were used to analyze the rela(onship between Congo Red 
status and log-transformed NT-proBNP, Metrnl, and EQ-5D-5L quality of life scores at 1 year, 
adjus(ng for key covariates. Model assump(ons were veriﬁed, and 95% conﬁdence intervals 
were reported. Analyses were performed using R solware (v4.4.0).