healthmedicinet

Study protocol and population

Consecutive patients scheduled to undergo surgery for either severe aortic stenosis,
severe aortic or severe mitral regurgitation at our institution (Comité éthique hosptalo-facultaire
des Cliniques Universitaires St. Luc, Brussels, Belgium), were considered eligible
for inclusion in the study. Recruitment was performed between May 2013 and May 2014.
Exclusion criteria were: history of prior myocardial infarction; presence of significant
coronary artery disease on preoperative angiography; and contraindications to CMR
(ferrometallic cerebral aneurysm clips, pacemaker or implantable defibrillator, or
severe claustrophobia), or to injection of Gd based contest agent (ie allergy to contrast
media or renal insufficiency with GFR-MDRD??30 ml/min/1.73 m2). The study protocol
consisted of a Gd-enhanced MR prior to cardiac surgery followed by myocardial biopsy
at the time of surgery. The protocol was IRB approved and patients were included after
giving written informed consent to participating in this study. 31 participants fulfilled
inclusion criteria and consented to participating in the study (Table 1). Scan-rescan reproducibility of pre-, post- T1 and ECV was performed in 11 other
subjects: 5 healthy volunteers (3 males 44?±?11 years), 4 patients with aortic stenosis
(3 males, mean age 60?±?20 years) and 2 patients with mitral regurgitation (2 male,
mean age 65?±?7 years).

Table 1. Patients characteristics

Cardiac MR

Cardiac MR was performed using a 3 Tesla system (Ingenia, Philips Medical Systems,
Best, the Netherlands). To assess LV myocardial function and mass, 10 to 12 consecutive
short-axis images and 2-, 3- and 4-chamber long axis image of the LV were acquired
using a cine steady state free precession sequence. Then, mid-ventricular short axis
Modified look locker (MOLLI) images were acquired for T1 determination using an 11
image, 18 heart-beat 3-(3)-3-(3)-5 SSFP sequence. Imaging parameters were: Field of
view: 340 mm, slice thickness 8 mm, TR: 2.6 ms TE 1.03 ms, matrix 172?×?150 pixels
resulting in a resolution of 2×2.6 mm, Sense Factor 2, trigger delay end-diastole,
inversion times ranging from 150–3287 ms. Then, a total dose of 0.2 mmol/kg gadobutrol
(Gadovist, Schering) was injected in a 2 phase protocol: 3 mL gadobutrol were infused
as bolus pushed by a 15 mL saline at 3 mL/s, 15 s later, the remaining contrast dose
followed by 20 mL saline were infused at a slower rate of 1 mL/s. Ten to 15 min after
contrast injection, short- and long-axis 2D inversion recovery LGE images were acquired
with an inversion-recovery gradient-echo imaging sequence to evaluate focal myocardial
fibrosis, as previously described 11]. Finally, post-contrast (15 min post contrast) MOLLI T1 mapping was repeated in identical
prescription as pre-contrast T1 mapping.

For scan-rescan reproducibility, after acquisition of the first baseline T1 mapping,
the patient was removed from the scanner and asked to stand up, then was put back
in the scanner for the acquisition of the second set of images. A different scanner
operator, starting from new scout images, acquired images for the second scan. After
contrast administration, the same procedure was performed for post-contrast T1 maps.
Post-contrast images were repeated without additional contrast agent administration
between both scans.

CMR data analysis

CMR images were anonymized and analyzed in double by two experienced (CD with 6 years
of CMR experience, MA with 4 year’s CMR experience level and 3 Euro-CMR certification)
observers blinded to clinical data.

Pre and post-contrast MOLLI images were processed using the open-source software MRmap v1.4 12] under IDL. Images were corrected for respiratory motion when needed, and T1 maps
were generated by fitting pixels to the equation s(t)?=?a – b exp (t/T1*), and T1?=?T1*((b/a-1),
where a and b are constants, t is time and s(t) signal intensity at time t. The generated
pre- and post-contrast T1 maps were stored in DICOM format and imported into Osirix
software (Pixmeo; Switzerland; version 5.8). Pre and post contrast blood T1 times
were measured on a region of interest manually drawn in the center of the blood pool.
Pre and post myocardial T1 times were measured in a segment corresponding approximately 
to the site of the biopsy (depending on the patient either the anterior or antero-septal
wall). To evaluate the influence of segmental heterogeneity on measurements we also
measured T1 times, in 6 different regions of the myocardium (anterior-, anterolateral,
inferolateral, inferior, inferoseptal, anterosepal) and one global region of interest
encompassing the entire left ventricular wall. The partition coefficient lambda (?)
and ECV were computed as: 

ECV=? (1-Hematocrit). Late enhanced CMR short axis images were analyzed using the freely available software software Segment
v1.9 (http://www.medviso.com/products/segment) with a fully automated method 13] validated in an animal model of experimental infarction. The method automatically
computes mean and standard deviation of signal intensity in 5 sectors per slice. The
region with the lowest mean signal intensity is considered ‘remote’ myocardium. LGE
regions are considered??2.4 SD of remote after correction for partial volume effects.
Isolated LGE regions??1.5 ml, unless being 1 % of LV volume or being the largest
LGE area in the image volume were deducted. The pattern of LGE was assessed by 2 independent
observers (BG- 15 years of CMR experience, and MA 4 years CMR experience, both EuroCMR
level 3 certified) who were blinded to the clinical and histopathological data. Discordant
findings were resolved by consensus.

Left ventricular biopsy

In each patient, 2–4 myocardial biopsy samples weighing approximately 25–75 mg were
gathered, of which the largest sample was analysed for histopathology, and the remaining
were preserved for other use. In patients undergoing open-chest aortic valve replacement
for aortic valve stenosis, or aortic valve repair for aortic regurgitation, biopsies
were sampled for full width of myocardium, under direct vision by means of a Tru-Cut®
biopsy needle (CareFusion, Waukegan, IL) in the anterior wall. Patients undergoing
mitral valve repair for mitral valve regurgitation by either Port-Access or Da-Vinci
minimal-access had samples taken under endoscopic vision taken by a surgical scissor
from the anterior septum. Samples were immediately fixed in 10 % buffered formalin,
embedded in paraffin, sectioned, and stained with picrosirius red. Stained sections
were digitalized with a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany).
Quantification was performed using TissueIA software (Leica Biosystems, Dublin, Ireland).
After elimination of artifacts and perivascular fibrosis, area occupied by interstitial
fibrosis was expressed a percentage of total endomyocardial area. Four different histological
slices were analyzed per patient and the average of the quantification of the different
specimens was considered the final value of fibrosis for the patient.

Statistical methods

Statistical analyses were performed using SPSS version 20.0 software (IBM Inc, Chicago,
IL). Continuous variables were expressed as mean?±?one standard deviation (SD) or
medians [quartiles] if not normally distributed; categorical variables were reported
as counts and percentages. All tests were 2 sided and p value ?0.05 was considered
statistically significant.

To compare baseline characteristics, T1 times and ECV of patients with different type
of valve disease, ANOVA or Chi square tests were used. The Pearson correlation coefficient
was employed to examine the relationship between percent LGE, pre and post contrast
T1 times and ECV by CMR versus quantitative interstitial fibrosis by histopathology.
Intra and inter observer and scan-rescan reproducibility of T1 times and ECV and histopatholoy
were assessed with Bland-Altman methods and intraclass correlation coefficient.