Assessing Composition of Coronary Thrombus in STEMI Patients

A Multiscale Approach to Charaterize Samples Obtained by Catheter Aspiration

Francesco Tessarolo

1,2

, Emiliana Bonomi

, Federico Piccoli

, Fabiola Morat

, Marta Rigoni

Iole Caola

, Mattia Barbareschi

, Patrizio Caciagli

, Simone Muraglia

, Roberto Bonmassari

and Giandomenico Nollo

1,2

IRCS project, Bruno Kessler Foundation, via Sommarive 18, Trento, Italy

Department of industrial Engineering, University of Trento, via Mesiano 77, Trento, Italy

Department of Laboratory Medicine, Azienda Provinciale per i Servizi Sanitari di Trento, via Degasperi 79, Trento, Italy

Division of Cardiology, Santa Chiara Hospital, l.go Medaglie d’oro 9, Trento, Italy

Division of Pathology, Santa Chiara Hospital, l.go Medaglie d’oro 9, Trento, Italy

Keywords: Thromboaspiration, ST-Segment Elevation Myocardial Infarction, Scanning Electron Microscopy,

Histology.

Abstract: Percutaneous catheter thrombectomy allows collecting coronary thrombi from patients with ST-segment

elevation myocardial infarction, preserving most of the sample characteristics available for both visual

assessment and compositional analysis. This study aimed at identifying the association between thrombus

macroscopic aspect and composition and, secondly, correlations between composition and ischemic time.

Aspirated thrombi were grouped into “white”, “red” or “mixed” according to their macroscopic appearance.

Platelets, RBCs and fibrin were quantified on thrombus surface by SEM and on thrombus sections by a

modified Carstairs histochemical staining. Fifty-three samples from 51 patients were included. The median

[inter-quartile range] ischemic time was 210 [190-265] min. Seven (13%) “white”, 19(36%) “mixed” and

27(51%) “red” thrombi were macroscopically identified. Median thrombus composition assessed by SEM

was 23[11-41]%, 43[26-62]%, and 24[11-37]% for platelets, RBCs and fibrin respectively. Median

histological analysis was 10[5-26]%, 45[31-64]% and 30[18-49]% for the same components. Significant

difference in composition were found between “white” and “red” thrombi, showing respectively a higher

amount of platelets (p=0.003) and fibrin (p=0.007). No significant correlations were found between

composition and ischemic time suggesting that plaque instability and thrombus formation can occur before

the symptom onset.

1 INTRODUCTION

Thrombus aspiration (TA) is a recommended

technique in the treatment of myocardial infarction

(MI) allowing thrombus removal from the culprit

artery via a specific catheter (Keeley, 2003; Van der

Werf, 2008, Steg, 2012). A reduced mortality in

patients affected by myocardial infarction with ST-

segment elevation (STEMI) has been associated to

the use of TA in conjunction with primary

percutaneous intervention (PCI) (De Luca, 2008;

Vlaar, 2008; Burzotta, 2009).

TA allows collecting the biological aspirate in a

minimally invasive way, preserving most of the

original thrombus characteristics that are available

for direct visual assessment and complementary

laboratory analysis. The retrieved thrombus can

varies in size, shapes and colour, giving readily

available information to the interventional

cardiologist (Quadros, 2012). Histological methods

are also available for the characterization of

thrombus morphology and for assessing thrombus

age (Kramer, 2008) and scanning electron

microscopy (SEM) has been proposed as a valid

method for assessing composition of aspirated

material (Silvain, 2011). These methods have the

potentials of tracing the evolution of the pathology

from the plaque rupture to the vessel recanalization.

However, histopathological and microstructural

analyses are time consuming and the macroscopic

Tessarolo F., Bonomi E., Piccoli F., Morat F., Rigoni M., Caola I., Barbareschi M., Caciagli P., Muraglia S., Bonmassari R. and Nollo G..

Assessing Composition of Coronary Thrombus in STEMI Patients - A Multiscale Approach to Charaterize Samples Obtained by Catheter Aspiration.

DOI: 10.5220/0005144600050012

In Proceedings of the 2nd International Congress on Cardiovascular Technologies (CARDIOTECHNIX-2014), pages 5-12

ISBN: 978-989-758-055-0

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

aspect of the thrombus remains the most accessible

information to use at the bedside (Quadros, 2012).

We aimed at developing and applying a

multiscale approach by integrating the macroscopic

evaluation of the aspirated material with

compositional data obtained from both histological

and SEM analysis on a series of coronary thrombi

collected during PCI with TA on STEMI patients.

Data will be analyzed in order to identify firstly the

association between macroscopic aspect of the

thrombus and its composition and, secondly,

possible associations between thrombus aspect and

composition with ischemic time.

2 METHODS

Coronary thrombi were prospectively collected

between November 2012 and November 2013 from

all the STEMI patients admitted at the Division of

Cardiology of the Santa Chiara Hospital in Trento,

Italy, within 12 hours from the beginning of the MI

symptoms and treated with catheter thrombus

aspiration (Export 6F, Medtronic, Santa Rosa,

California) before PCI. Procedures were performed

according to the ESC guidelines (Steg, 2012).

Aspirated thrombi were collected on the

disposable filter, washed with sterile saline to

remove excess of blood and immediately fixed in

2.5% glutaraldehyde in 0.1M phosphate-buffered

solution. Specimens were then stored in the fixative

solution at 4°C until they were analyzed.

Demographics, risk factors, blood biomarkers on

admission, ischemic time (defined as the time lag

between the onset of the AMI symptoms and the

reperfusion therapy), and antithrombotic treatment

were recorded on a data collection form.

2.1 Macroscopic Evaluation

In order to make the macroscopic evaluation of the

aspirated material standardized and blinded to

patients’ characteristics and clinical conditions,

thrombi were not evaluated immediately after

aspiration. After a minimum fixation time of 48h, a

photographic documentation of the aspirated

material was performed by collecting one image per

sample at 10x magnification with a stereo-

microscope equipped with a colour digital camera

under constant illumination conditions. Collected

images were pooled and independently evaluated by

two senior interventional cardiologists blinded to

patient details and clinical characteristics. Aspirated

thrombi were grouped into “white”, “red” or

“mixed” according to their macroscopic appearance

and colour (Figure 1).

Figure 1: Macroscopic classification of aspirated coronary

thrombi. Representative image of a “white” (a), “mixed”

(b), and “red” (c) sample. Bar is 5 mm.

2.2 Scanning Electron Microscopy and

Compositional Analysis

Before SEM analysis, samples were washed once in

0.1 M phosphate buffer and trice in distilled water,

de-hydrated in ascending hydro-alcoholic solutions,

dried in a laminar flow cabinet, mounted on a

sample holder with double-sided conductive carbon

tape, and sputter-coated with gold. SEM analysis

was conducted on the thrombus surface with a

XL30, ESEM FEG scanning electron microscope

(FEI, Philips, Nederland).

To perform an operator-independent choice of

the SEM fields of view, 20 point of interest were

previously determined by superimposing a squared

grid to the stereomicroscopic colour picture. A set of

20 electron-micrographs per each sample was than

obtained in the same point of interest at a

magnification of 2000x. The composition of the

thrombus was determined by performing a semi-

quantitative analysis of the SEM images, modifying

the procedure previously proposed by Lucas et al.

(Lucas, 2013). Briefly, a squared grid of 10μm

element size was superimposed to each SEM image

to obtain a total of 400 point of interest per sample.

The observation of each point of interest allowed to

quantify the following micro-morphological

features: a) “platelets” (2 μm wide globules

occurring as aggregates or adherent to the fibrin

strands), b) “erythrocytes” (RBCs) (biconcave disc-

shaped cells of about 7 μm in diameter), c) “fibrin”

(fibrin network or fibrin fibres), d) “other” (other

morphologies not included in the previous features).

Representative SEM micrographs of the different

micro-morphological features are reported in Figure

SEM features quantification was considered

feasible if “other” occurred in less than 50% of the

investigated points. After the exclusion of points

CARDIOTECHNIX2014-InternationalCongressonCardiovascularTechnologies

associated to “other”, the percentage of occurrence

for each feature of interest was finally computed for

each thrombus.

Figure 2: SEM images of the aspirated thrombus.

Representative fields of view with a prevalence of

platelets (a), RBCs (b), fibrin (c), and other features (d).

Bar is 10 microns.

2.3 Histochemical Staining and Feature

Quantification

After SEM characterization, thrombi were released

from the carbon tape by adding a drop of absolute

ethanol on the stub surface, carefully transferred on

histological cassettes and re-hydrated by immersion

in 70% ethanol in water for 24h and in distilled

water for 48h. After this post-SEM conditioning,

samples were processed for permanent histology by

immersion in ascending hydro-alcoholic solutions,

100% ethanol and 100% xylene. After paraffin

impregnation and embedding, 3 µm thick sections

were cut with a rotary microtome.

Two consecutive sections per sample were

stained respectively with haematoxylin-eosin stain

(H&E) and a modified Carstairs stain (Figure 3). We

adapted the original Carstairs staining method

(Carstairs, 1965) to identify RBCs, platelets and

fibrin in the thrombus sections. Staining times and

fixation solutions were optimized for a better

differentiation of thrombus components as described

elsewhere (Lucas, 2014). Briefly, paraffin sections

were hydrated, placed in 5% ferric alum for 10

minutes, rinsed in running tap water, stained in

Mayer’s haematoxylin for 5 minutes, and then rinsed

again in running tap water. Sections were placed for

4 minutes in picric acid-orange G solution (20 mL

saturated aqueous picric acid, 80 mL saturated picric

acid in ethanol, and 0.2 g orange G) and then rinsed

in distilled water. Sections were then placed in

ponceau-fuchsine solution (0.5 g acid fuchsine, 0.5 g

ponceau 2R, 1 mL acetic acid, and distilled water to

100 mL) for 2 minutes and then rinsed in distilled

water. Sections were treated with 1%

phosphomolybdic acid for 3 minutes, rinsed in

distilled water, stained with aniline blue solution (1

g aniline blue in 100 mL 1% acetic acid) for 45

minutes, decoloured in 1% aqueous acetic acid for 3

minutes, rinsed in several changes of distilled water,

dehydrated, cleared, and mounted with acrylic

medium. On sections obtained from thrombi fixed

for 48 hours or longer, Carstairs method produces

differential staining of fibrin (bright red), platelets

(grey-blue to navy), and RBCs (yellow) (Carstairs,

1965).

Platelet, RBCs and fibrin were quantified on

Carstairs stained sections by setting specific colour

thresholds in the L, a*, b* colour space (Figure 4).

Pixel number for each feature was computed and

percent occurrence over the whole section area was

considered for the statistical analysis.

Figure 3: Histological features of a mixed thrombus.

Consecutive sections stained with haematoxylin-eosin (a)

and modified Carstairs stain (b). Bar is 1 mm.

Figure 4: Histological analysis on a mixed thrombus

section stained with Carstairs method (left).

Representative colours associated to the three components

of interest are shown. Binary images after specific

threshold for platelets (a), RBCs (b) and fibrin (c). Inset d)

reports about components not recognized in the three

previous features and excluded from the calculation of

compositional percentages. Bar is 1 mm.

AssessingCompositionofCoronaryThrombusinSTEMIPatients-AMultiscaleApproachtoCharaterizeSamples

ObtainedbyCatheterAspiration

Table 1: Patients characteristics.

Demographics

Age, years 63.0 ±13.1

Male 41 (84.3%)

Risk factors

Hypertension 58%

Active smoker 61%

Dyslipidemia 26%

Diabetes mellitus 13%

Familiarity 41%

Time delays

Symptom onset to ASA, min 95 [50-140]

Symptom onset to ADP inhibitor,

min

120 [90-180]

Symptom onset to PCI, min 210 [190-265]

Biomarkers on admission

Troponin I, µg/ml 0.325±1.293

Platelet, mm

-3

252±82

CRP, mg/l 7.9±14.8

PT, INR 1.02±0.10

Creatinine clearance, ml/min 88.9±39.3

Antithrombotic treatment

ASA 52 (98.1%)

ADP receptor inhibitor 53 (100%)

Clopidogrel 21 (39.6%)

Prasugrel 32 (60.4%)

GP IIb/IIIa inhibitor 25 (47.2%)

Pre-admission 0 (0%)

Catheterization laboratory 25 (47.2%)

Infarct related artery

LAD 32 (60.4%)

RCA 17 (32.1%)

Cx 4 (7.5%)

CABG 0 (0.0%)

Values are mean ± SD, n (%), or median [interquartile range].

ASA : acetylsalicylic acid; ADP: Adenosine diphosphate; CABG:

coronary artery bypass grafting; CRP: C-reactive protein; Cx:

circumflex coronary artery; GP: glycoprotein; LAD: left anterior

descending coronary artery; RCA: right coronary artery.

2.4 Statistical Analysis

Categorical variables were expressed as percentages.

The results for normally distributed continuous

variables are expressed as mean±standard deviation,

and continuous variables that did not have a normal

distribution are presented as median and inter-

quartile interval (IQR).

The intra and inter observer correlations in the

macroscopic analysis were assessed by weighted

kappa coefficients considering that macroscopic

categories were ordered from “white” to “mixed”

and “red”. Kappa coefficient was interpreted as

reported by Altman et al. (Altman, 1991).

The non-parametric Kruskal–Wallis test was used to

compare three groups. If a significant difference was

found with the Kruskal–Wallis test, then a Mann–

Whitney U test was used to compare each pair of

groups. Wilcoxon non-parametric test was used to

compare distributions with paired data obtained

from SEM and histological analysis. Categorical

variables were compared using Fisher exact test.

Spearman correlation coefficients were calculated to

assess the relationships between platelets, RBCs and

fibrin contents vs. ischemic time.

Multivariate linear regression analysis was

performed to identify independent factors associated

with the thrombus composition (age, sex and infarct

related artery, antithrombotic treatment). All

analyses used two-sided tests with a significance

level of p<0.05. Data were analysed using STATA

version 13.0 for Windows (STATACorp. College

Station, TX, USA).

3 RESULTS

Sixty-three thrombi were obtained from 61 patients.

Among them, six cases were excluded for

incomplete clinical records and one sample failed

histological preparation. Fifty-six aspirated thrombi

were subjected to compositional analysis.

SEM characterization was not feasible according

to the adopted criterion (“other” > 50%”) in 3

samples, leaving 53 samples from 51 patients who

were eventually considered for reporting and

statistical analysis. Patients’ characteristics are

summarized in Table 1.

3.1 Thrombus Composition

Macroscopic classification gave 7(13%) “white”,

19(36%) “mixed” and 27(51%) “red” thrombi.

Intra-observer agreement was “good” (K= 0.785)

with 84.9% of agreements. Inter-observer agreement

was “moderate” (K=0.437) with 60.4% of

agreements. “Red” and “white” thrombi were

generally categorized in the same way by the raters,

but discrepancies were frequent in evaluating mixed

thrombi.

Features quantification on SEM images showed a

median [IQR] composition of 23[11-41]%, 43[26-

62]%, and 24[11-37]% for platelets, RBCs and fibrin

respectively. At the histological analysis, the same

samples showed a median [IQR] composition of

10[5-26]%, 45[31-64]% and 30[18-49]% for

platelet, RBCs and fibrin respectively. Data obtained

from SEM and histological methods were consistent

and a paired non-parametric analysis showed no

significant differences between the compositional

distributions determined by the two methods.

CARDIOTECHNIX2014-InternationalCongressonCardiovascularTechnologies

To elicit possible associations between

compositional data and macroscopic thrombus

characteristics, results from SEM and histology were

sub-grouped according to the macroscopic

classification. Results are summarized in Figure 5.

Sub-group analysis of SEM compositional data

showed a higher amount of platelets (p=0.003) and a

lower amount of fibrin (p=0.007) in “white” thrombi

in respect to “red” thrombi. No significant difference

Figure 5: Composition of aspirated thrombi sub-grouped

according to macroscopic categories. Values are indicated

as percent of platelets, RBCs and fibrin determined by

SEM (a) or histological (b) analysis.

Figure 6: Number of aspirated thrombi (sub-grouped by

macroscopic characteristic) plotted according to ischemic

time.

was found for the amount of RBCs within different

macroscopic sub-groups, although a trend to higher

median values of RBCs was found from “white” to

“red” thrombi. Intermediate features were found for

“mixed” thrombi but no significant compositional

difference was present between “mixed” and “red”

or “white” and “mixed” thrombi.

Differently from SEM compositional data,

histological composition showed no significant

compositional difference among the three

macroscopic sub-groups.

3.2 Thrombus Composition Vs.

Ischemic Time

Ischemic time for the 53 samples collected in this

study ranged from 110 to 720 min with a median

value of 210 min. According to previously proposed

criteria (Silvain, 2011), ischemic time was

categorized into three intervals (≤3 h, 3 to 6 h, ≥6 h).

The number of “white”, “mixed” and “red” thrombi

grouped according to the ischemic time intervals is

reported in Figure 6. No statistically significant

difference was observed, but a large variability in

ischemic time was associated to “red” thrombi.

Figure 7: Composition of aspirated thrombi (percent of

platelets, RBCs and fibrin) according to the three ischemic

time intervals. Compositional data were obtained from

SEM (a) and histological (b) methods.

AssessingCompositionofCoronaryThrombusinSTEMIPatients-AMultiscaleApproachtoCharaterizeSamples

ObtainedbyCatheterAspiration

Differently, all “white” thrombi were collected from

patients with an ischemic time from 3 to 6 h.

The amount of platelets, RBCs and fibrin

according to the three ischemic time intervals is

reported in Figure 7 for compositional data obtained

from SEM and histological methods. No significant

difference in composition among the three sub-

groups was found both on data obtained from SEM

and from histology.

A univariate linear regression was performed on

platelets, RBCs, and fibrin amount according to

ischemic time, showing no linear association

between composition and ischemic time.

A multivariate linear regression model was also

performed to include in the analysis the potential

confounding variables (age, sex, infarct related

artery, risk factors, blood biomarkers, antithrombotic

therapy) showing no significant effects on the

thrombus composition.

4 DISCUSSION

The investigation of coronary thrombus

characteristics and composition deserves high

interest for defining the pathogenetic mechanism of

acute coronary syndromes, for optimizing the

therapeutic treatment according to thrombus

characteristics and for having additional prognostic

data on patients’ mortality. Thrombi obtained by an

aspiration device during the PCI treatment can give

a rapid and informative feedback to the operator by

the direct visual observation of the collected

material. Moreover, the same biological aspirate is a

good candidate for additional analysis on

microstructure and composition. This work presents

a new histological method using trichromic

histochemical staining and colour image analysis for

quantifying platelets, RBCs and fibrin on thrombus

sections. The amount of the same features on the

thrombus surface was also determined, in an

independent way, by SEM and semi-automatic

image analysis. Macroscopic features of the

aspirated samples were also determined blindly to

patients’ clinical details by acquiring a low

magnification picture under controlled illumination

conditions. This approach represents a first example

of integrated multiscale analysis that was not

reported before in literature according to authors’

knowledge. Results were obtained from 53 thrombus

samples in a fully paired fashion, starting from a

macroscopic qualitative classification to a

quantitative determination of fibrin, platelets and

RBCs by both histochemical and SEM methods.

Different macroscopic classifications were

previously proposed to classify the colour of

aspirated thrombi. Quadros et al. proposed a

dichotomous classification into “white” and “red”

thrombi (Quadros, 2012). They found that “white”

thrombi occurred in one third of STEMI cases and

were associated with lower ischemic time when

compared with “red” thrombi (Quadros, 2012).

Uchida et al. proposed a larger number of classes for

the angioscopic in-situ classification, including also

“transparent”,” frosty glass–like”, “light red”,

“mixed”, and “brown” thrombi (Uchida, 2011). In

accordance with other authors (Kirchof, 2003) and

considering the inhomogeneities of colour within

many aspirated samples, we considered three

macroscopic categories, grouping thrombi into

“white”, “mixed” and “red”. However, this

classification showed a lower inter-observer

agreement than those reported by Uchida and co-

workers (Uchida, 2011). Differently from other

studies where the macroscopic classification was

performed during the PCI procedure, in this study

the assignment of the macroscopic category was

realized in a completely blinded fashion by

evaluating only the low magnification image of the

aspirated material captured with a digital camera.

The difference in the agreement rate could be in part

related with this methodology.

The compositional analysis was performed by a

non-destructive SEM analysis of the outer surface of

the thrombus. We adapted a previously presented

protocol for SEM preparation (Silvain, 2011) in

order to limit sample damage and allowing the

subsequent histological analysis on the same

aspirated material. Median compositional results

obtained here were partly in agreement with those

previously observed (Silvain, 2011). Specifically,

we obtained a lower amount and fibrin and a higher

amount of RBCs than those previously reported.

Values for the amount of platelets are super

imposable with those reported by Silvain et al.

Surprisingly, although RBCs in clots are the

origin of the word “red” (usually referred as “old”)

thrombi as opposed to platelet-rich “white” (usually

referred as “young”) thrombi, we did not find

significant differences in RBCs among different

macroscopic sub-groups. Similarly, Silvain and co-

workers failed to note a significant correlation

between RBCs content and ischemic time by the

SEM compositional analysis (Silvain, 2011).

Although the present study included a higher

number of samples we did not evidence the same

significant association previously reported between

fibrin and platelets amount vs. ischemic time

CARDIOTECHNIX2014-InternationalCongressonCardiovascularTechnologies

(Silvain, 2011). In this study we included the total

volume of retrieved fragments in both SEM and

histological analysis. Differently, the compositional

results of SEM analysis from Silvain et al. were

limited to the main aspirated piece of the thrombus.

This could partly explain differences in composition

and time course between the two datasets, but the

SEM exclusion criterion we applied could also have

introduced a bias in sample selection.

Difficulties in evidencing significant differences

in thrombus composition among the sample groups

with different ischemic times could also be related to

the inadequacy of the ischemic time in properly

defining the thrombus age. Previous

histopathological studies conducted on larger cohort

of STEMI patients evidenced that intracoronary

aspirated material by thrombectomy is frequently

heterogeneous in terms of thrombus age (Rittersma,

2005, Kramer 2009). In 51% of cases, older thrombi

(>1 day) were reported, which suggests an important

discrepancy between the time of onset of the

thrombotic process and the occurrence of acute

clinical symptoms (<12 h in patients treated with

TA) (Rittersma, 2005). The authors concluded that

plaque instability frequently occurs days or even

weeks before occlusive coronary thrombosis

(Kramer, 2009).

The time between plaque rupture and thrombus

formation is still unpredictable. Sudden coronary

occlusion is often preceded by a variable period of

plaque instability and thrombus formation, initiated

days or weeks before onset of symptoms. The

aspirated thrombus may be older than expected from

the duration of the ischemic time (Kramer, 2009),

and younger thrombus could be superimposed onto

an older thrombus, thereby potentially confusing the

observations.

5 CONCLUSIONS

A multiscale analytical approach to characterize

samples obtained by catheter aspiration in STEMI

patients has been realized by integrating qualitative

information coming from the visual assessment of

the coronary thrombus with compositional

quantitative data obtained from histological and

SEM analysis. Method here presented deserves high

potential for understanding the mechanisms of

thrombus formation in STEMI and for investigating

correlations between composition and thrombus age.

Significant differences in composition were

found, showing a higher amount of platelets and

fibrin respectively for “white” and “red” thrombi.

No significant correlations were found between

composition and ischemic time, supporting

previously reported data showing that plaque

instability and thrombus formation can occur within

longer time interval before AMI symptom onset.

Possible associations between thrombus

composition determined with a multiscale approach

and thrombus age assessed by histopathological

methods should be investigated in future studies.

Eventually, specific analysis could be performed

to elucidate potential association between thrombus

composition and antithrombotic drug treatment.

ACKNOWLEDGEMENTS

Authors are grateful to the staff of the Cardiology

Division of the Trento Hospital for sample

collection.

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