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Licordari et al. Vessel Plus 2022;6:12 https://dx.doi.org/10.20517/2574-1209.2021.86 Page 3 of 10

METHODS
In total, 119 patients (60 men and 59 women; mean age 48 ± 11 years), belonging to seven unrelated families
with TTR gene mutation (Glu89Gln, Phe64Leu, and Thr49Ala) followed at the Department of Cardiology of
University Hospital of Messina, were prospectively enrolled from June 2010 to February 2018. None of the
included patients had evidence of monoclonal protein in the serum or urine, a monoclonal population of
plasma cells in the bone marrow, or other diseases that could be responsible for secondary amyloidosis.

All patients underwent the following examinations: two-dimensional standard echocardiography, strain
99m
echocardiography, and Tc-DPD scan. Patients with New York Heart Association (NYHA) III and IV
and/or previous cardiac disease were excluded.

The study was approved by our institutional review board. Informed consent was obtained from all patients.

Standard echocardiography data acquisition and analysis
Standard echocardiographic examinations were performed in all patients using a commercial ultrasound
machine (My Lab ALFA, Esaote, Florence, Italy) equipped with a 2.5 MHz phased-array transducer.
Parasternal short-axis views at the basal, mid, and apical levels and three standard apical views (4-chamber,
2-chamber, and LV outflow long-axis) were acquired. The same cardiologist performed all examinations.
The following measurements were obtained according to recommendations of the American Society for
Echocardiography: diastolic thickness of the LV basal anterior septum (LVST), basal posterior wall
thickness, LV volumes (end-diastolic volume and end-systolic volume), EF, and LV diastolic function
quantified by the ratio between the E-wave velocity of the pulsed-wave Doppler mitral flow image and the
early diastolic velocity of the septum at the mitral annulus level (E’ wave) on tissue Doppler imaging [10,11] .

Strain acquisition and analysis
A dedicated software package (XStrain™, Esaote, Florence, Italy) was used for an offline quantification of
right and left strain. A 16-segment model was used to divide the LV. Left ventricular longitudinal strain was
acquired on 4- and 2-chamber apical views. GLS was obtained from the average longitudinal strain of the 16
segments on apical views; apical longitudinal strain (ALS) as the average of 4 apical segments and
mediobasal longitudinal strain (MBLS) as the average of the 12 medial and basal segments. Relative regional
strain ratio (RRSR) was calculated by dividing the average apical LS by the sum of the basal and mid-LS
[9]
values . Strain analysis was performed by a single trained cardiologist, blinded to patient’s clinical data.
Later, another trained cardiologist, blinded to the previous measurements, performed the same analysis on
16 patients to test the inter-observer reliability.

99m
Tc-DPD data acquisition and analysis
Whole-body scans (anterior and posterior projections) were obtained 5 min and 3 h after the intravenous
injection of 740 MBq of Tc-DPD using a dual-headed gamma camera (MillenniumVG, GE Healthcare,
99m
Milwaukee, Wisconsin, USA) equipped with low-energy, high-resolution collimators.

The whole-body scans were visually evaluated by consensus of two experienced nuclear medicine physicians
who searched for cardiac radiotracer accumulation; readers were blinded to echocardiographic data.

A semi-quantitative score was obtained based on the results of planar images as previously described by
Perugini . Briefly, a score of 0 signified absence of cardiac uptake, 1 indicated mild uptake less than bone, 2
[12]
indicated moderate uptake equal to the bone, and 3 indicated strong uptake with mild or absent bone
uptake.