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SUPPLEMENTARY MATERIAL
ABBREVIATIONS
cTFC= corrected TIMI frame count
EMP = endothelial derived microparticles
MBG = myocardial blush grade
PMP = platelet derived microparticles
pPCI = primary percutaneous coronary intervention
PPP = platelet poor plasma
QuBE = quantitative blush evaluator
TS = thrombus score
ΣSTR = ST-Segment Resolution
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SUPPLEMENTARY METHODS
Angiographic analysis of thrombotic burden
Thrombus score (TS) 0 corresponded to no angiographic evidence of thrombotic material; TS 1
corresponded to possible thrombus, appearing as a convex, hazy lesion with irregular contours at
the site of total occlusion; TS 2 corresponded to definite thrombus ≤ 1/2 the vessel diameter; TS 3
corresponded to definite thrombus > 1/2 but < 2 vessel diameters; thrombus grade 4 corresponded
to definite thrombus ≥ 2 vessel diameters; TS 5 corresponded to inability of thrombus burden
assessment due to persistent total occlusion of the culprit vessel after guidewire passage. (1)
Corrected TIMI frame count (cTFC)
cTFC was measured at the end of pPCI by counting, in a final long angiographic run at 30
frame/second, the number of cineframes required for contrast to first reach standardized distal
coronary landmarks in the infarct-related artery starting from the frame in which the dye fully enters
the artery producing a column of nearly full dye across the entire width of the origin of the artery
with antegrade motion (2). We used a power-injector (Avanta, Medrad Inc., Indianola, PA, USA)
with injection of 6 ml of Iomeprol (Iomeron 350, Initios Medical AB, Goteborg, Sweden) at rate of
4 ml/sec and with a pressure limit of 300 psi for both left and right coronary arteries.
Myocardial Blush Grade (MBG)
MBG was scored as follows: 0, no myocardial blush or contrast density or persisting myocardial
blush (“staining”); 1, minimal myocardial blush or contrast density; 2, moderate myocardial blush
or contrast density but less than that obtained during angiography of a contralateral or ipsilateral
non–infarct-related coronary artery; and 3, normal myocardial blush or contrast density, comparable
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with that obtained during angiography of a contralateral or ipsilateral non–infarct-related coronary
artery. (3)
Quantitative Blush Evaluator (QuBE)
At the end of each pPCI procedure, a long final run was acquired with the same settings used for
MBG assessment, with the only exception of frame-rate acquisition that was modified at 15
frames/second. QuBE score was calculated offline by a separate observer blinded to clinical data. A
manual region-of-interest was drawn in the vascular perfusion bed of the infarct-related artery, and
the translational movement of the heart was manually corrected on each frame. The QuBE
algorithm performs an automatic pixel analysis, by filtering all large-scale structures, dividing all
the selected pixels in blocks and, considering the average of the darkest few pixels in each block,
calculating the QuBE value as the average of the best 50% of pixel blocks. This process is repeated
for 125 frames (8.3 seconds of acquisition at 15-frames/second), and a perfusion curve is generated.
The QuBE score, expressed in arbitrary units, equals the maximum increase plus the maximum
decrease in this curve. This methodology has been described in detail elsewhere and the software
released as open-source code with explicit permission for other groups to use, redistribute and
modify (4, 5).
ST-Segment Resolution (ΣSTR)
Each electrocardiogram was analyzed by a blinded assessor and summed ST-segment elevation was
calculated as the sum of elevation in V1–6, I, and aVL for anterior infarction and as the sum of
elevation in leads II, III, aVF, V5, and V6 for non-anterior infarction. ΣSTR was defined as the
percent reduction in the summed ST-segment elevation score between electrocardiograms obtained
prior to PCI and at 90 minutes. ΣSTR was classified as complete (≥70%), partial (30 to 70%), and
absent (< 30%). (6)
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Microparticles levels measurement
All blood samples were collected in citrate-buffer tubes. All samples were analysed on-line
immediately after the collection in the catheterization laboratory (time interval from blood sample
collection to completed sample processing: 40±20 minutes; time for flow cytometry: 15±5 minutes)
in order to prevent any sort of bias deriving from freezing and/or storing (7). In order to avoid
platelet contamination, platelet-poor plasma (PPP) was obtained by centrifuging blood samples at
1500g for 15 minutes at room temperature and then by immediately centrifuging the supernatant at
13000g for 2 minutes. (8) For platelet derived microparticles (PMP) and endothelial derived
microparticles (EMP) detection, 50 µl of PPP were incubated for 20 minutes at room temperature in
the dark with 5 µl of PE -conjugated monoclonal antibody against CD31 (clone 1F11, cat 2409,
Beckman Coulter, Miami, USA) and 5 µl of FITC-conjugated monoclonal antibody against CD42b
(clone SZ2, cat PN IM0648U, Beckman Coulter, Miami, USA). EMP were defined as particles
positively labelled for CD31 and negatively for CD42b (CD31+/CD42-), while PMP were defined
as particles positively labelled for CD31 and CD42b (CD31+/CD42b+). 5 µl of appropriate
fluorochrome-conjugated isotype-matched mAb were used as control for background staining
(IgG1-FITC, clone 679.1MC7, cat A07795, Beckman Coulter, Miami, USA; IgG1-PE, clone
679.1MC7, cat A07796, Beckman Coulter, Miami, USA). After incubation, samples for PMP and
EMP detection were resuspended in 500 µl of PBS and analyzed in a EPIC XL-MCL (Beckman
Coulter, Miami, USA) flow cytometer using EXPO32 software (Beckman Coulter, Miami, USA);
forward scatter and sideward scatter were set on a logarithmic gain, to best cover a wide size range.
In order to assess the power of our cytometer in discriminating between 0.5 µm and 0.9 µm events
(now considered mandatory before starting a microparticles measurement protocol by a recent
consensus conference), (9) and to evaluate whether our results were comparable to those from other
studies, standardization was achieved using a blend of monodisperse fluorescent beads (Megamix,
BioCytex) of three diameters (0.5, 0.9 and 3 µm). Forward scatter (FS) and side scatter (SS)
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parameters were plotted on logarithmic scales to best cover a wide size range. Single staining
controls were used to check fluorescence compensation settings and to set up positive regions. (10)
To enumerate MP, EPIC XL-MCL flow cytometer was set at “low” rate, and actual flow rate was
calculated using Trucount beads (BD Biosciences) in an initial series of 20 samples. Consistency
was checked monthly. The mean flow rate was 10.0 µl/min, with a variation coefficient of 5.04%.
The volume sample analyzed was calculated with the formula: 10/60*T, where T was the time of
analysis expressed in seconds, 10 was the calculated mean volume in µl analyzed by the cytometer
in the unit of time (1 minute) and 60 was the conversion factor from minutes to seconds. 2500
events were collected and acquired. The time necessary for counting 2500 events was determined
and MP concentration calculated as: (absolute number per microliter, n/µl) (n/l) = (Number of
events/Volume sample analyzed)*(Total volume of the sample/Amount of PPP), where the total
volume of the sample was 500 µl, and 50 µl was the amount of PPP. (11)
Sample collection validation protocol
To exclude that differences in MP levels from intracoronary and aortic samples could be related to
collection strategy (guiding catheter vs thrombectomy device), ten additional patients with
admission diagnosis of Non ST Elevation Myocardial Infarction (mean age 67±14 years)
undergoing PCI were enrolled, and, after informed consent, MP levels in blood samples collected
from the ascending aorta before PCI were compared. Briefly, with the guiding catheter still in the
ascending aorta, a first sample of 30 ml was obtained from the guiding catheter lumen, then the
thrombectomy device was inserted and, without exiting the guiding catheter tip, a further 30 ml
were obtained from its lumen.
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SUPPLEMENTARY RESULTS
Microparticles, microvascular obstruction and thrombus score
Intracoronary PMP levels continued to be related to MVO after correcting for TS. In higher TS
classes, the presence of combined angiographic and electrocardiographic MVO (MBG< 2 and
absent ST resolution) was significantly related to higher levels of intracoronary PMP and EMP.
Indeed, intracoronary PMP were 5238.0 (3726.9-7924.7) vs 2747.2 (2678.8-4688.1), p: 0.04 for TS
5; 1061.3 (686.2-2249.0) vs 725.5 (258.2-898.7), p:0.02 for TS 4; 155.1 (84.0-230.9) vs 117.4
(77.4-245.5), p: 0.14 for TS 3; 32.2 (5.9-67.4) vs 41.5 (4.0-94.4) p: 0.32 for TS 0/1/2. The same
trend was observed for intracoronary EMP : 1050.0 (763.6-2731.8) vs 530.5 (513.5-806.3) p: 0.02
for TS 5; 419.9 (48.4-1431.2) vs 322.5 (45.3-781.3) p: 0.04 for TS 4; 136.7 (85.0- 613.5) vs 102.3
(74.9-134.1) p: 0.09 for TS3; 73.5 (70.0-125.0) vs 70.2 (65.4-93.2) p: 0.10 for TS 0/1/2, but not for
aortic PMP and EMP [Supplementary Figure 3].
Effect of blood sampling by thrombus aspiration device on aortic microparticles levels
No significant differences were observed between both PMP and EMP levels in blood samples
collected from ascending aorta by guiding catheter and by thrombectomy device (161.0 (119.0328.7) vs 172.5(125.7-344.2) respectively, p: 0.13 for PMP and 88.5 (68.5-109.0) vs 85.7 (73.7102.0) respectively, p: 0.78 for EMP). Moreover, both PMP and EMP levels in blood samples
obtained with the two collection methods were strongly related (r: 0.9; p<0.001 for both aortic PMP
and EMP) [Supplementary Figure 4].
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SUPPLEMENTARY MATERIAL REFERENCES
1.
Sianos G, Papafaklis MI, Serruys PW. Angiographic thrombus burden classification in
patients with ST-segment elevation myocardial infarction treated with percutaneous coronary
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2.
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assessment of myocardial reperfusion in patients treated with primary angioplasty for acute
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Zijlstra F, Koch KT. Feasibility and applicability of computer-assisted myocardial blush
quantification after primary percutaneous coronary intervention for ST-segment elevation
myocardial infarction. Catheter Cardiovasc Interv. 2010;75:701-6.
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Megamix Bead Manufacturer's Instructions
http://www.milananalytica.ch/_downloads/spec_sheet/7801MegaMix/MegaMix_package_inse
rt.pdf. (27 November 2011)
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van Ierssel SH, Van Craenenbroeck EM, Conraads VM, Van Tendeloo VF, Vrints CJ,
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SUPPLEMENTARY FIGURES
Figure 1. Study Flow Chart. The figure summarizes the main steps for selection of patients
included in the study. (pPCI = primary percutaneous coronary intervention, MP= microparticles,
pts= patients, STEMI= ST elevation myocardial infarction)
Figure 2. Correlations of intracoronary and aortic PMP and EMP levels and cTFC and QuBE
score. Scatter plots show the relationship between intracoronary and aortic PMP and EMP levels
with continuous indexes of MVO. Higher MP levels are related to higher cTFC and lower QuBE
values. Data are presented as scatter plot. R= Person's correlation coefficient.
Figure 3. Relationship between intracoronary PMP and EMP and combined
electrocardiographic (incomplete ΣSTR) and angiographic (MBG <2) MVO according TS
classes. Figure clearly shows how in higher TS classes both intracoronary PMP and EMP are
related to increased occurrence of combined MVO, defined as a combination of incomplete ΣSTR
and MBG < 2. (* stands for p < 0.05)
Figure 4. Relationship between PMP and EMP in blood samples from ascending aorta
obtained by guiding catheter and thrombus aspiration device (Diver CE Max, Invatec, Italy).
Scatter plots describe the strong relationship between PMP and EMP levels in blood samples
collected from ascending aorta by two different sampling strategies: guiding catheter versus
thrombus aspiration device. (Data are presented as scatter plot. R= Person's correlation coefficient).
PMP: platelet derived microparticles
EMP: endothelial derived microparticles
MBG: myocardial blush grade
ΣSTR: ST resolution
MVO: microvascular obstruction
TS: thrombus score
QuBE: quantitative blush evaluator
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cTFC: corrected TIMI frame count