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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015
236
AFRICA
perfusion. Before releasing the aortic cross-clamp, warm
reperfusion was given (37°C) until the patient’s body temperature
was 35–37°C. Heparin was neutralised with protamine in a ratio
of 1:1.5 within 10 minutes of the end of CPB.
Blood sample collection and measurement of
cardiac markers
Blood samples were drawn after atrial cannulation, just before
aortic cross-clamping (pre-ischaemic sample), and within 15
minutes of aortic declamping (reperfusion sample). Blood
samples were collected from the arterial line of the bypass circuit
(arterial sample) and from the pressure-monitoring line of the
coronary sinus perfusion catheter (coronary sinus sample).
Blood samples were collected into an evacuated serum-
separator clot-activator tube (Vacuette
®
, Greiner Bio-One,
Kremsmunster, Austria) and a 2.0-ml dipotassium (K
2
) ethylene
diamine tetra-acetic acid (EDTA) vacuum tube (BD Vacuteiner
®
BDPlymouth, UK) for creatine kinase-MB isoenzyme (CK-MB),
high-sensitivity cardiac troponin T (hs-cTnT), creatine kinase-
MB isoenzyme mass (CK-MB mass) and cardiac troponin I
(cTnI) measurements. The tubes were centrifuged at 1 500
×
g
for 15 minutes within one hour to obtain serum samples for the
measurement of CK-MB and hs-cTnT concentrations.
Whole blood samples, which were collected in the K
2
EDTA
tubes, were not centrifuged and CK-MB mass and cTnI
concentrations were measured in the whole blood samples on
the same day as the surgery.
Serum CK-MB activities were measured with the immuno-
inhibition method on a Roche Cobas c501 analyser (Roche
Diagnostics GmbH, Mannheim, Germany). The reference range
of CK-MB activity measured by this method was
<
25 U/l.
Serum hs-cTnT concentrations were measured by
electrochemiluminescence immunoassays (ECLIA) on a Roche
Cobas e 601 analyser (Roche Diagnostics GmbH, Mannheim,
Germany). In healthy subjects, the upper reference limit for
hs-cTnT concentrations was 14 ng/l (99th percentile) and the
measurement range was 3–10 000 ng/l.
CK-MB mass and cTnI were measured with the time-
resolved fluorescence method on a radiometer AQT90 FLEX
(Radiometer Medical ApS, Brønshøj, Denmark). In healthy
subjects the upper reference limit for cTnI concentrations was
0.023
µ
g/l (99th percentile) and the measurement range was
0.010–25
µ
g/l. In healthy subjects the upper reference limit for
CK-MB mass concentrations was
<
7.2
µ
g/l (99th percentile) and
the measurement range was 2–500
µ
g/l.
Atrial tissue sample collection and histopathological
examinations
Using a sharp scalpel, myocardial biopsy samples from the same
site of the right atrial appendage were taken from each patient
within 15 minutes of aortic declamping. The area of the biopsy
sample in contact with the forceps was removed. Particular care
was taken to avoid possible ischaemic areas caused by surgical
manipulation. Tissue samples were fixed in 10% formalin,
embedded in paraffin, sectioned (4
µ
m), placed on slides, stained
with haematoxylin and eosin (H&E), and examined under a light
microscope (Olympus BX51, Tokyo, Japan) by a pathologist who
was blinded to the study design.
The slides were graded histopathologically, according to
the severity of myocardial injury, using a previously described
scoring system.
12
Histological changes (oedema, leukostasis, cell
necrosis and focal bleeding) were scored from 0 to 3 as follows: 0
=
no changes; 1
=
slight changes: focal myocyte damage or small
multifocal degeneration with slight degree of inflammation; 2
=
moderate changes: extensive myofibrillar degeneration and/or
diffuse inflammatory process; 3
=
severe changess: necrosis with
diffuse inflammatory process.
In situ
detection of myocardial apoptosis
We used an
in situ
TUNEL (terminal deoxynucleotidyl trans-
ferase-mediated deoxyuridine triphosphate nick end-labelling)
assay to assess the degree of myocardial apoptosis. Formalin-
fixed sections were deparaffinised in xylene and rehydrated
through graded concentrations of ethanol to water.
DNA fragmentation during apoptosis was detected using a
commercially available kit (ApopTag
®
peroxidase
in situ
apoptosis
detection kit, Millipore, Billerica, MA, USA) according to the
manufacturer’s instructions. Processed samples were examined
under a light microscope (Olympus BX51, Tokyo, Japan). For
quantitative analysis, TUNEL-positive cells were counted in six
random fields per section (80–120 cells per field). The apoptotic
index was calculated as the mean of apoptotic (positive-stained)
cells.
Statistical analyses
Statistical analyses were performed using GraphPad Prism
version 6.05 (GraphPad Software, Inc, CA, USA). All data sets
were tested for normality using the Shapiro–Wilk test. Data
were presented as median and interquartile ranges (IQR) and
non-parametric statistical tests were used, as the values were
not normally distributed. The net release of cardiac markers
was quantified as the arteriovenous difference (coronary sinus
concentration minus arterial concentration).
The comparison of cardiac marker values between pre-ACC
(just before aortic cross-clamping) and post-ACC (within 15
minutes of aortic declamping) periods was analysed using
the Mann–Whitney
U
-test. The correlation between apoptotic
index (TUNEL), histopathological myocardial injury score,
intra-operative data and cardiac marker values in the post-ACC
period was analysed using Spearman’s correlation analysis. An
r
-value > 0.5 indicated a strong correlation, 0.35–0.5 a moderate
correlation, and 0.2–0.34 a weak correlation.
13
A
p
-value
<
0.05
was considered statistically significant.
Results
Demographic, pre-operative and intra-operative data of the patients
are shown in Table 1. In the histopathological examinations, our
resultsshowedthatCABGsurgerywithCPBandACCcausedslight-
to-moderate myocardial injury and moderate-to-severe apoptosis
in all cases (Table 1). Acute ischaemic changes with interstitial
oedema, myofibrillar thinning and wavy pattern consistent with
reperfusion injury were observed in histopathological sections of
atrial tissue. In addition, neutrophilic-to-mixed inflammatory cell
infiltration and transmigration indicating reperfusion injury were
observed (Figs 1–4).