The risk for stroke is increasing yearly due to the aging population on a global level and the accumulation of risk factors.1 Stroke is the second leading cause of death and the main cause for decreased quality of life.2 Time is of the essence when it comes to treating the stroke, where thrombolytic treatment, usually with systemic administration of recombinant tissue plasmin activator (rt-PA), and mechanical thrombectomy are the main approaches; however new experimental treatments are also under consideration.3
The main cause of stroke is the acute cerebral artery occlusion, which leads to ischemic stroke, where the oxygen supply is cut off.4 The occlusion can either be thrombotic or embolic, originating from various locations, which results in various thrombi structures. The structure can also give us information on the age of the thrombi.5 On a microscopic level, thrombi are composed of different levels of red and white blood cells intertwined with fibrin meshwork.6,7 The structure can strongly influence the permeability which is important for the success of the thrombolytic treatment.8
The diagnosis of stroke is a multiple step process, which consists of clinical evaluation and diagnostic imaging. The clinical evaluation includes the assessment of the effect of the stroke, which can be estimated using the modified Rankin Scale (mRS). This is a seven-level stroke scale ranging from 0, which stands for no symptoms to 6, which stands for death.9 Another scale serving the same purpose, however, with more grades is National Institutes of Health (NIH) severity stroke scale (NIHss).10
In diagnostics of stroke, computed tomography (CT) is the main tool in selection of patients that are suitable candidates for stroke treatment: firstly, to exclude hemorrhage, to exclude large areas of hypodense brain tissue, suggesting irreversible ischemia and to exclude stroke mimics. Native CT is complemented by CT angiography (CTA), which provides high diagnostic value in the detection of occlusion in high degree of stenosis as well as CT perfusion (CTP), which provides high specificity in the detection of ischemia and infarcted brain tissue. However, the most accurate assessment for acute stroke involving the site of occlusion, infarction core and salvageable brain tissue is a combination of different CT procedures involving CTA and CTP. In addition, CT scanning is fast, it is widely accessible and its price per scan is relatively low. However, sensitivity of CT to soft tissues cannot match with that of MRI.11,12 While it has been proven that CT can be used to determine some characteristics of the thrombi14, the field of connecting CT characteristics to clinical and procedure parameters has yet to be investigated.
The aim of this study is to demonstrate that CT images provide more information than currently used in routine stroke diagnostics. More specifically, mainly geometrical and macroscopic parameters of CT images are used, while information on the HU values of the thrombus causing the ischemic stroke, are usually overlooked or considered not important. In this study we want to show that this information is relevant for assessment of the thrombus microscopic structure and that these parameters correlate with some clinical parameters and can be therefore used for better treatment planning.
This study was performed on
Experimental data of patients qualified for the study, which include CT, histological, clinical and procedure parameters
CT parameters |
Histology | Clinical parameters |
Procedure parameters |
|||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
# | HU avg |
HU var |
Stroke etiology | Therapy before stoke | NIHSS |
mRS |
||||||||||||||
occl | nor | diff | diff |
occl | nor | diff | diff |
L |
RBC |
strt | end | diff | strt | end | diff | Rd |
# pass | |||
1. | 29.1 | 32.4 | -3.3 | -11.4 | 4.20 | 4.42 | -0.22 | -5.0 | 20.8 | 11.5 | AT | / | 21 | 1 | 20 | 4 | 1 | 3 | 42 | 1 |
2. | 42.0 | 29.2 | 12.8 | 30.5 | 4.58 | 8.98 | -4.40 | -96.3 | 16.3 | 45.1 | CE | / | 17 | 8 | 9 | 5 | 4 | 1 | 62 | 2 |
3. | 38.5 | 35.1 | 3.4 | 8.9 | 3.56 | 2.75 | 0.80 | 22.6 | 17.0 | 12.0 | CE | / | 23 | 7 | 16 | 4 | 3 | 1 | 65 | 1 |
4. | 36.9 | 34.3 | 2.5 | 6.8 | 11.66 | 4.58 | 7.08 | 60.7 | 17.5 | 41.4 | CE | AA | 18 | 3 | 15 | 5 | 2 | 3 | 77 | 1 |
5. | 43.7 | 44.2 | -0.5 | -1.2 | 3.55 | 3.86 | -0.31 | -8.7 | 17.9 | 65.9 | AT | / | 26 | 6 | 20 | 5 | 3 | 2 | 97 | 3 |
6. | 35.8 | 30.0 | 5.9 | 16.4 | 4.17 | 2.23 | 1.95 | 46.7 | 16.3 | 61.0 | CE | / | 7 | 0 | 7 | 3 | 0 | 3 | 38 | 3 |
7. | 31.8 | 36.0 | -4.2 | -13.2 | 2.77 | 3.26 | -0.49 | -17.8 | 22.2 | 57.3 | AT | / | 13 | 3 | 10 | 4 | 1 | 3 | 90 | 1 |
8. | 37.8 | 38.9 | -1.1 | -2.9 | 2.82 | 4.45 | -1.63 | -58.0 | 26.9 | 50.3 | CE | / | 14 | 3 | 11 | 4 | 3 | 1 | 77 | 1 |
9. | 32.0 | 24.0 | 8.1 | 25.1 | 9.50 | 4.90 | 4.60 | 48.4 | 22.7 | 56.3 | CE | ACAA | 5 | 2 | 3 | 3 | 2 | 1 | 115 | 2 |
10. | 38.5 | 36.5 | 2.0 | 5.1 | 3.20 | 2.80 | 0.40 | 12.5 | 19.7 | 52.4 | CE | / | 26 | 42 | -16 | 5 | 6 | -1 | 82 | 4 |
11. | 38.3 | 39.4 | -1.1 | -2.8 | 5.49 | 3.66 | 1.83 | 33.4 | 17.1 | 19.1 | AT | / | 12 | 0 | 12 | 3 | 0 | 3 | 60 | 1 |
12. | 35.7 | 33.9 | 1.7 | 4.9 | 3.56 | 5.39 | -1.83 | -51.2 | 13.9 | 31.8 | CE | AA | 6 | 3 | 3 | 3 | 1 | 2 | 69 | 1 |
13. | 44.3 | 31.9 | 12.3 | 27.8 | 4.66 | 5.11 | -0.46 | -9.8 | 22.5 | 74.7 | AT | / | 19 | 12 | 7 | 5 | 4 | 1 | 108 | 2 |
14. | 44.1 | 37.5 | 6.6 | 15.0 | 2.60 | 3.92 | -1.32 | -50.9 | 19.3 | 38.9 | CE | / | 18 | 20 | -2 | 4 | 5 | -1 | 63 | 5 |
15. | 36.0 | 35.4 | 0.6 | 1.6 | 3.48 | 2.67 | 0.81 | 23.4 | 17.9 | 48.8 | CE | / | 42 | 42 | 0 | 5 | 6 | -1 | 75 | 2 |
16. | 38.1 | 36.4 | 1.7 | 4.5 | 4.16 | 5.45 | -1.30 | -31.2 | 18.6 | 21.3 | AT | / | 15 | 4 | 11 | 5 | 4 | 1 | 77 | 1 |
17. | 42.5 | 40.7 | 1.8 | 4.2 | 3.11 | 3.74 | -0.63 | -20.4 | 13.5 | 14.8 | CE | AA | 14 | 2 | 12 | 5 | 3 | 2 | 53 | 1 |
18. | 38.7 | 23.3 | 15.4 | 39.8 | 5.66 | 4.79 | 0.87 | 15.3 | 19.1 | 42.4 | CE | AC | 22 | 3 | 19 | 5 | 4 | 1 | 43 | 1 |
19. | 34.7 | 30.9 | 3.8 | 11.0 | 5.61 | 4.43 | 1.18 | 21.1 | 20.9 | 37.7 | AT | / | 3 | 1 | 2 | 1 | 0 | 1 | 60 | 1 |
20. | 41.2 | 32.4 | 8.8 | 21.4 | 5.14 | 4.58 | 0.56 | 10.9 | 19.3 | 56.0 | AT | AC | 11 | 40 | -29 | 4 | 6 | -2 | 76 | 3 |
21. | 37.2 | 28.5 | 8.7 | 23.5 | 3.29 | 5.27 | -1.99 | -60.4 | 29.2 | 79.1 | AT | / | 19 | 10 | 9 | 5 | 5 | 0 | 61 | 1 |
22. | 35.2 | 32.5 | 2.6 | 7.5 | 3.02 | 2.79 | 0.24 | 7.9 | 13.6 | 9.3 | CE | / | 16 | 9 | 7 | 5 | 5 | 0 | 95 | 3 |
23. | 41.5 | 34.5 | 6.9 | 16.7 | 5.14 | 4.47 | 0.67 | 13.0 | 21.5 | 80.4 | AT | / | 16 | 3 | 13 | 4 | 2 | 2 | 65 | 2 |
24. | 28.9 | 23.2 | 5.6 | 19.5 | 3.52 | 4.72 | -1.20 | -34.1 | 12.1 | 57.1 | CE | / | 13 | 3 | 10 | 4 | 1 | 3 | 55 | 1 |
25. | 40.2 | 33.4 | 6.8 | 17.0 | 2.91 | 4.30 | -1.39 | -47.8 | 18.7 | 46.8 | AT | AA | 42 | 42 | 0 | 5 | 6 | -1 | 90 | 4 |
AA = antiaggregation; AC = anticoagulant; ACAA = both types of drugs; AT = atherothrombotic; CE = cardioembolic; diff = absolute difference; diff [%] = relative difference in %; HU avg = average Hounsfield units; HU var = variability of Hounsfield units; L [mm] = CT length of the thrombi; mRS = modified Rankin score; NIHSS = NIH Stroke Scale; nor = normal artery; occl = occlusion; RBC [%] = percentage of red blood cells in the thrombi; Rd = duration of mechanical recanalization; # pass = number of passes
The patients for the study were admitted to the Neurology Clinic of University Medical Center in Ljubljana for urgent neurological symptoms suggesting brain stroke. These patients were managed according to the standard steps of acute ischemic stroke management in our tertiary center. Firstly, an urgent clinical examination was performed, which was followed by a CT scan (non-contrast enhanced CT scan, CT perfusion and CT angiography) on a Siemens Sensation Open 40 CT scanner, where ischemic stroke caused by the occlusion of the middle cerebral artery, was confirmed. The protocol continued with standard full dose of rt-PA (0.9 mg/kg, maximum 90 mg) systemic thrombolytic treatment. In all studied patients clinical stroke signs persisted after the thrombolytic treatment, therefore further therapy was done by the mechanical thrombectomy. This was performed by skilled interventional neuroradiologist, using the standard mechanical recanalization procedure with the thrombectomy device (Trevo®stent retriever, 4 x 20 mm, Stryker Neurovascular, Kalamazoo, MI). The retrieved thrombi were preserved and additionally examined through histological analysis.
The protocol of the study was approved by the Institutional Review Board and the Ethical Committee of the National Ministry of Health of the Republic of Slovenia, approval No. 0120-99/2021/7. The study was performed in agreement with the informed-consent policy.
Stroke patients with qualifying conditions for the study underwent urgent CT scanning of the brain which included non-contrast enhad (NCE) sequential CT scans and CTA scans. The NCE CT scan is a sequential scan consisting of two parts-the skull base (120 kV, 265 mAs, matrix 1024×1024, slice thickness 3 mm, collimation 20×0.6, rotation time 1 s, window width 90-190, window center 38) and the cerebral part (120 kV, 310 mAs, matrix 1024×1024, slice thickness 4.8 mm, collimation 24×1.2, rotation time 1 s, window width 80, window center 38). Acquired CT images were further analyzed by the ImageJ program (NIH, Bethesda MD, USA) to obtain relevant CT data on brain thrombi of the patients. CTA images were specifically used to determine the position of the thrombi. This information was then used to stack three slices from NCE CT scan containing the thrombus to correctly position the line along the thrombus on the stacked image as well as the symmetrical non-occluded MCA segment on the opposite side of the brain. Special care was taken to center the lines in the middle of the vessel in order to avoid signals from the vessel wall tissue and therefore reduce a possible partial volume effect and also an increased Hounsfield Units (HU) values due to vessel calcifications. Along the lines the HU intensity profiles were measured in the NCE CT images. NCE CT images were more suitable for the thrombus analysis than CTA images, because they have no contrast enhancement due to the contrast agent that could alter the HU of thrombi. Measured HU intensity profiles were further analyzed by determining average HU value of the profile
During every interventional procedure, recanalization time and number of passes with the thrombectomy device were registered as the procedure parameters. The recanalization time was considered as the time between the first contact of the thrombectomy device with the thrombus to the successful recanalization through the occluded artery with complete removal of the thrombus.
For clinical parameters, modified Ranking Scale (
Histological analysis was done to determine the percentage of red blood cells (RBC) in the retrieved thrombi (RBC%). The thrombi samples were fixed in 10% buffered formaldehyde for 48 h. After the fixation, they were cut longitudinally as 5-μm-thick cross-sections and embedded in paraffin. The cross-sections were stained with monoclonal antibodies Anti-Human Glycophorin A (GPA) for RBC content and with anti β-3 integrin Anti-Human CD61 (DakoCytomation, Denmark) for platelet content.
Micro-photography of the stained cross-sections was performed, using a Nikon Eclipse E600 optical microscope (Nikon, Düsseldorf, Germany) equipped with a Nikon 4x Plan Fluor objective and with a high-resolution CCD camera Nikon DS-Fi1. The micro-photography system was controlled by the Nikon NIS Elements software package. The exposure time yielding the optimal image contrast was equal to 10 ms while the in-plane image resolution was equal to 10 μm (imaging matrix was 1024×1124 and field of view (FOV) 10.24×11.24 mm2).
Histological (hematoxylin-eosin) images of the central cross-section along the thrombi were examined for the RBC proportion by the analysis encompassing the following steps. First, each image was corrected for uneven illumination (vignetting).15 Then the corresponding intensity histogram was calculated and used to determine the optimal threshold for the discrimination between the RBC-rich and the platelet-rich regions. The RBC proportion was determined as the ratio between the thresholded RBC area and the total thrombi area.
Possible correlations between different groups of data (CT image parameters, histological parameters, clinical parameters and procedure parameters) were tested. Univariate linear regression was the statistical method of choice, where
Histological sections of selected representative retrieved cerebral thrombi are shown in Figure 1. Histological slices were stained by hematoxylineosin and then analyzed for RBC proportions using digital image processing of the thrombi images acquired by using optical microscopy of the histological sections. All retrieved thrombi had a distinctly heterogeneous, laminated (multilayer) structure. The structure involves the interweaving of compacted erythrocyte-rich (red) regions with thinner (pink) coatings containing a combination of complementary-linked platelets and a fibrin network. The laminations are often folded and twisted, which is probably due to blood flow turbulence in the environment where the thrombi were formed and their turning as they traveled along the vessel. Among the retrieved thrombi there were no homogeneous single-layered (red only) thrombi. Which can be explain by their high susceptibility to thrombolysis.
Figure 2 shows an example of a CT image of a stroke patient with a clearly visible MCA segment on both hemispheres of the brain. In the example the MCA segment on the right hemisphere is occluded by the cerebral thrombus, while the MCA segment on the opposite hemisphere is normal. Exact location of each thrombus was determined from its CTA image first and then this location was used to obtain HU values along the thrombus from the corresponding CT image. Yellow line on the image is drawn along the thrombus and the symmetrical position of the normal MCA segment. Graphs in Figure 2A correspond to HU intensity profiles along both yellow lines: along the cerebral thrombus in Figure 2B and along the symmetrical section of the normal MCA segment in Figure 2C. For each HU, intensity profile was then calculated by average HU value
Correlation between different pairs of data groups from Table 1 were tested by the Pearson correlation coefficient and the univariate linear regression analysis. Results of this analysis for the pairs with the highest correlation is shown by linear regression graphs in Figure 3 and their corresponding correlation and linear regression parameters are formulated in Table 2. The linear regression statistical analysis showed significant differences (
Linear regression and Pearson correlation coefficient analysis of data group pairs with statistically most significant correlations and linear regression parameters
Data group pair | Linear regression |
Pearson coefficient | |||
---|---|---|---|---|---|
HU avg occl | mRS end | 0.227 ± 0.086 | 0.015 | 0.233 | 0.483 |
HU avg occl | # passes | 0.119 ± 0.052 | 0.031 | 0.186 | 0.432 |
HU avg occl | mRS diff | -0.140 ± 0.067 | 0.049 | 0.158 | -0.398 |
HU avg diff | RBC [%] | 1.646 ± 0.809 | 0.053 | 0.153 | 0.391 |
HU avg occl | mRS start | 0.087 ± 0.045 | 0.065 | 0.140 | 0.374 |
HU avg diff | mRS diff | -0.104 ± 0.059 | 0.093 | 0.118 | -0.343 |
HU avg = average Hounsfield Units; occl = occluded MCA segment; diff = absolute difference; mRS = modified Rankin score ; RBC [%] = percentage of red blood cells in the thrombi
CT was already employed to study properties of artificially made blood clots. In a study by Kirchhof
The correlation between the number of passes with the thrombectomy device and average HU of the thrombi can also be associated with the thrombi organization. More organized (hard, fibrin-rich) thrombi can attach strongly with the arterial wall, which may result in the more difficult retrieval of such thrombus during the interventional procedure.22 This suggest, that if higher average HU values in the MCA occlusion are found on the CT scan, it is getting more likely that more than one pass will be needed to successfully retrieve the thrombus during the interventional procedure.
When treating patients with stroke, time is essential.23 The longer time taken till reestablishment of the normal cerebral blood flow, the less oxygen is present in the brain, which may result in brain cell and nerve connection loss. This contributes to more serious symptoms and a higher mRS classification over time.23 Outcome after mechanical thrombectomy can be more dependent on the capacity of collateral circulation than on the time from stroke onset.24 However, time still plays an important role in saving tissue at risk (penumbra). In addition, the longer time taken till resolving the occlusion, the thrombus can retract further and therefore becomes more compact with a low serum content. Clot retraction is an important contributing factor for the correlation that was found between the initial mRS (mRS_start before the treatment) and the average HU of the thrombus (HU_avg_occl). This factor may also explain the correlation between the average HU of the thrombus (
In addition to average HU in the occluded MCA segment (
Positive correlation between RBC proportion (
Pretreatment thrombus evaluation can be of great clinical importance. Failure of thrombolytic treatment can be expected in fibrin rich thrombi. Thrombus composition can also have an impact on interventional treatment planning (mechanical thrombectomy). Optimal technique (stent retriever
Major drawback of this study is limited number of cases (
Routinely acquired CT images of stroke patients provide also information on HU values of thrombi that is usually ignored. In this study a relation between the HU value of a thrombus and its composition was confirmed. As more compact thrombus may represent a bigger problem for the interventional procedure a priori assessment of thrombus compaction is very important for good intervention planning. The present study also provides foundations for further studies where the accuracy of study could be improved by increasing the number of samples (patients and thrombi) and having more accurate spiral CT images of patients with stroke that would allow improve HU value determination in the targeted region.