Abstract
Background and aim
Mechanical thrombectomy (MT) for large vessel occlusion often requires multiple passes to retrieve the entire thrombus load. In this multi-institutional study we sought to examine the composition of thrombus fragments retrieved with each pass during MT.
Methods
Patients who required multiple passes during thrombectomy were included. Histopathological evaluation of thrombus fragments retrieved from each pass was performed using Martius Scarlet Blue staining and the composition of each thrombus component including RBC, fibrin and platelet was determined using image analysis software.
Results
154 patients underwent MT and 868 passes was performed which resulted in 263 thrombus fragments retrieval. The analysis of thrombus components per pass showed higher RBC, lower fibrin and platelet composition in the pass 1 and 2 when compared to pass 3 and passes 4 or more combined (P values <0.05). There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05).
Conclusion
Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in the first passes were associated with lower fibrin and platelet composition compared to fragments retrieved in passes three and four or higher. Also, thrombus fragments retrieved after failed pass were associated with higher fibrin and platelet components.
Keywords: Stroke, thrombectomy, thrombus composition, per pass analysis
Introduction
The successful treatment of AIS requires timely restoration of blood flow to the brain by recanalization of the occluded blood vessel. In patients with AIS due to LVO, recanalization can be achieved by endovascular thrombectomy that physically removes thrombus. Recently, many studies have investigated the influence of number of mechanical thrombectomy (MT) attempts or passes on patient outcome. 1 It has been shown that achieving successful revascularization on first pass (FPE) is associated with better functional outcome and lower mortality rate, however only around 25% of patients achieve FPE. 2 It has also been shown that thrombus composition could be a key factor in recanalization success in stroke patients. Certain thrombus features can affect thrombectomy outcomes such as rate of successful reperfusion or procedural time and can theoretically influence the rate of FPE. Higher RBC component in thrombi has been shown to be associated with higher rate of successful revascularization and lower number of passes required for retrieval however compositional characteristics of thrombus fragments retrieved in each pass has not been investigated widely.3–6
Understanding the per pass histological composition of clots which required multiple passes to retrieve is important for a number of reasons. First and foremost, if we were able to identify the typical composition of components that required multiple passes to retrieve, determining the correlation between thrombus composition and number of passes required for successful revascularization could be an important step toward developing newer devices and technologies for retrieving emboli in the first pass. And second, if we were able to identify the typical composition of these difficult components, this could guide targeted thrombolytic therapy in the future as well (i.e. antiplatelet versus fibrinolytic, etc).
The objective of this study was to explore the histological features from large samples of thrombus that required multiple pass thrombectomy treatment in patients with AIS due to LVO.
Methods
Patient selection
This multi-center study was performed as a part of the STRIP registry (Stroke Thromboembolism Registry of Imaging and Pathology). Following IRB approval at participating centers, thrombi samples retrieved from AIS patients were sent to our center for histopathological analysis. A structured abstract form was filled out by centers for each patient which included demographic variables, stroke etiology based on TOAST criteria, 7 image findings, and procedural details. For the purpose of this study, we only included patients who underwent thrombectomy and had multiple passes to retrieve the thrombus load. Patients younger than 18 were excluded from this study.
Histopathological analysis
Thrombus fragments for each pass were collected and fixed in separate 10% NBF (neutral buffered formalin) containers. If multiple fragments were retrieved in one pass, all fragments were put in a single 10% NBF container. All samples were processed and then embedded in paraffin. Paraffin-embed formalin fixed blocks were then cut into 3 µm sections. Two sections were prepared for each block and then each slide was stained with Martius Scarlett Blue (MSB). Quantification of clot components was performed using Orbit Image Analysis software. 8 The percentage of red blood cells (RBCs), white blood cells (WBCs), platelet and fibrin was calculated for each sample. 9 All analysis was performed by a single reviewer blinded to clinical data.
The area of thrombus fragments were measured using Image J software. The fractional contribution of each component in a pass to whole thrombus material load was calculated if thrombus was retrieved in multiple passes. We used equations proposed by Duffy, et al. for calculation of overall and fractional contributions. 10
Statistical analysis
All statistical analyses were conducted using SPSS version 23 for windows. Continuous variables were presented as mean (±SD) and categorical variables were reported as percentage. Independent T-test and one-way ANOVA with post-hoc tests including Tukey and LSD were used to interpret the data. A p-value less than 0.05 was considered statistically significant.
Results
Clinical and interventional data
A total of 263 thrombus fragments were collected from 154 patients. r-TPA was administrated in 43 patients. The mean (±SD) number of passes was 5.6 (±2.7). Cardio-embolic (40.3%) and large artery atherosclerosis (19.5%) were the most prevalent etiologies. Combination of contact aspiration and stent retriever was the most common thrombectomy technique (61%) followed by stent-retriever (22.1%), and contact aspiration (16.9%). The most common locations of occlusion were M1 (64.3%), M2 (26.6%), and ICA terminus (18.8%). TICI 2 b or higher was achieved in 77.6% of patients.
Comparison of thrombus composition with clinical and interventional data
The components of full thrombus load from patients were not significantly different between the main four etiology groups including cardioembolic, large artery atherosclerosis, other, and unknown (P values >0.05). r-TPA administrations did not show an effect on clot compositions (P values >0.05).
Per pass thrombus characteristics
Figure 1 depicts the RBC, WBC, Fibrin, and platelet components of thrombus fragments in each pass. Of 868 passes, 605 failed to retrieve thrombus materials.
Figure 1.
Bar graph representing percentage of each component for every pass.
Table 1 summarizes the mean of each thrombus component per pass. The analysis of thrombus components per pass (Figure 1) shows that RBC composition in passes 1 and 2 was significantly higher when compared to pass 3 and passes 4 or more combined (P values <0.05). The inverse pattern was noted for fibrin and platelet components, with thrombus fragments in passes 1 and 2 associated with a significantly lower fibrin (P values <0.05) and platelet (P values <0.05) components when compared with thrombus fragments retrieved in passes 3 and passes 4 or more combined. There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05).
Table 1.
The mean (±SD) of each component per pass.
| Pass | No of samples | RBC | WBC | Fibrin | Platelet | Collagen |
|---|---|---|---|---|---|---|
| 1 | 87 | 48.9 (24.5)% | 3.9 (2.4)% | 24.5 (16.8)% | 21.9 (16.8)% | 0.6 (3.3)% |
| 2 | 96 | 46.5 (23.4)% | 3.5 (2.1)% | 26.3 (16.5)% | 22.9 (17.3)% | 0.6 (6.5)% |
| 3 | 41 | 33.3 (23.1)% | 3.3 (2.1)% | 32.3 (14)% | 30.3 (19.6)% | 0.5 (3.3)% |
| ≥4 | 39 | 31.9 (26.5)% | 3.0 (1.9)% | 32.6 (18.3)% | 30.5 (24)% | 1.7 (5.1)% |
When seventy-six cases with thrombus fragments retrieved in one pass only out of multiple passes (with and without prior failed attempt) were excluded from the analysis similar findings were observed (Table 2, Figure 2).
Table 2.
The mean (±SD) of each component per pass excluding cases with only one pass thrombus retrieval.
| Pass | No of samples | RBC | WBC | Fibrin | Platelet | Collagen |
|---|---|---|---|---|---|---|
| 1 | 63 | 45.5 (23.9)% | 3.6 (2.3)% | 27.7 (15.4)% | 22.4 (15.7)% | 0.8 (3.9)% |
| 2 | 69 | 44.2 (23.7)% | 3.3 (2.0)% | 28.9 (16.4)% | 23.3 (17.6)% | 0% |
| 3 | 26 | 31.2 (24.9)% | 3.0 (2.2)% | 31.9 (15.3)% | 32.9 (19.8)% | 0.8 (4.1)% |
| ≥4 | 29 | 31 (26)% | 3.1 (1.7)% | 32.3 (19.1)% | 31 (23.3)% | 2.3 (5.8)% |
Figure 2.
Bar graph representing percentage of each component for excluding cases with only one pass thrombus retrieval.
We also compared the composition of clots on first two passes among thrombectomy techniques. We found that stent-retriever group had higher fibrin fragment compared to aspiration group (31.8% vs 22.6%, P = 0.029). No other association was found between clot components and thrombectomy techniques (Table 3).
Table 3.
The mean (±SD) of each component for the first two passes by thrombectomy techniques.
| Thrombectomy technique | RBC | WBC | Fibrin | Platelet | Collagen |
|---|---|---|---|---|---|
| Aspiration | 53.09% (±25.72) | 3.66% (±2.75) | 22.66% (±16.65) | 19.72% (±18.90) | 0.89% (±3.42) |
| Stent-retriever | 43.12% (±20.05) | 4.31% (±2.32) | 31.8% (±16.78) | 20.75% (±11.66) | 0.003% (±0.02) |
| Combination | 41.97% (±23.97) | 3.56% (±2.10) | 28.42% (±16.31) | 25.26% (±17.64) | 0.77% (±6.34) |
Discussion
Our study had number of clinically-relevant findings. First, we found that the composition of thrombus fragments retrieved in consecutive passes differed significantly. Within the first two passes, higher RBC and lower fibrin and platelet components were noted when compared with thrombus fragments retrieved in three or more passes. Second, thrombus fragments retrieved after failed attempts were associated with higher fibrin and platelet fractions. These findings confirm the contribution of fibrin and platelet to overall resistance of thrombus to removal. Our study provides evidence on the effect of thrombus composition on the complexity of thrombectomy procedure by evaluating per pass thrombus fragments in patients suffering from AIS.
Currently, as our knowledge regarding the thrombus composition before thrombectomy is limited, there are uncertainties about whether we should undertake similar treatment approach for all thrombi. Greater understanding of thrombus composition could be a determining factor for successful thrombectomy. Numerous studies have histologically evaluated thrombus composition and have documented marked heterogeneity.8,11–13 At present, only one study evaluated the per pass histopathological features of thrombus fragments. In line with our findings, Duffy et al 10 reported that the portion of the thrombus fragments removed in the first two passes tended to have higher RBC components and the later passes were mostly associated with fibrin-rich thrombus fragments. Authors also shown that thrombus fragments retrieved after failed attempt have higher fibrin-content. Our results confirm these findings with the advantage of having larger number of thrombus samples included in the analysis. We have also found that thrombus load would have a higher fibrin and platelet content if only one pass out of multiple passes had resulted in thrombus retrieval. This finding indicates that regardless of thrombus fragmentation, achieving better revascularization outcome is difficult if the thrombus is associated with higher fibrin and platelet content.
As shown in our study, later passes are associated with higher fibrin content of retrieved thrombus fragments. Some studies have highlighted the significance of fibrin-rich thrombus in achieving successful thrombectomy. In a study by Gunning et al, 14 authors reported that fibrin-rich clots have a greater coefficient of friction than RBC-rich clots which may propose a greater resistance to retrieval of fibrin-rich clots. Yoo et al 15 described that the compression of fibrin-dominant thrombi could lead to higher coefficient of friction and the consequent difficulty of thrombus retrieval when multiple passes are utilized.
Based on our findings and those of other groups, we hypothesize that the reason why clots retrieved in the first two passes were more RBC rich and less fibrin and platelet rich is the fact that the clot retrieval device has to overcome a lower coefficient of friction in order to retrieve RBC rich clots. More passes may be required to retrieve the fibrin rich components of a clot in order to overcome the static coefficient of friction between the clot and the endothelial cell lined vessel wall. This hypothesis could be studied in future in vitro studies.
Several studies have reported the association of thrombus composition with imaging characteristics. Hyperdense artery sign on CT scan and susceptible vessel sign on MRI are suggestive of RBC-rich thrombi, while the absence of these signs has been shown to be indicative fibrin-rich thrombi. Impervious thrombi also have higher fibrin fraction compared to pervious thrombi 16 and platelet-rich thrombi appear to be iso-dense on non-contract CT scan. 9 Correlating imaging characteristics of thrombus with its composition specifically with per-pass composition could help in selecting proper thrombectomy device or technique and predicting recanalization outcome.
This study has limitations. The use of different devices and thrombectomy techniques are confounding factors that could impact the thrombus load retrieved in each pass. We did not have any limitation on the number of thrombectomy passes. Such limitation would have provided us with a better understanding of differences between thrombus fragments in each pass. We also did not account for other contributing factors to failed attempts including length of the thrombus, variations in the vascular architecture, and vessel tortuosity. A greater understanding into per-pass thrombus composition would help to realize why successful recanalization is not achieved in all cases and, in turn, to help in the development of devices that could effectively retrieve difficult thrombi with fewer attempts. More studies with larger sample size and controlling for confounding factors are warranted to deepen our knowledge regarding the per pass thrombus composition.
Conclusion
Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in passes one and two were associated with lower fibrin composition compared to fragments retrieved in passes three and four or higher. Our results could be useful in developing new devices to retrieve difficult thrombi with fewer attempts.
Footnotes
Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: This work was supported by the National Institutes of Health grant number (R01 NS105853).
Ethical statement: Institutional Review Board approval (Mayo Clinic, Rochester, MN) was obtained prior the study.
Patient consent: Not required.
Data sharing statement: Data can be made available by contacting the corresponding author via email.
ORCID iDs: Mehdi Abbasi https://orcid.org/0000-0001-6978-2563
Biraj M Patel https://orcid.org/0000-0002-2830-954X
Seán Fitzgerald https://orcid.org/0000-0001-6634-092X
Daying Dai https://orcid.org/0000-0003-4051-6450
Ramanathan Kadirvel https://orcid.org/0000-0002-6786-9953
David F Kallmes https://orcid.org/0000-0002-8495-0040
Yasha Kayan https://orcid.org/0000-0002-7747-0188
Pouya Nazari https://orcid.org/0000-0003-1135-1095
Alhamza Al-Bayati https://orcid.org/0000-0001-8103-1930
Waleed Brinjikji https://orcid.org/0000-0001-5271-5524
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