Thrombi With a Higher Erythrocyte Composition Are More Fragile in Acute Stroke

Article information

J Stroke. 2024;26(3):454-457
Publication date (electronic) : 2024 September 4
doi : https://doi.org/10.5853/jos.2024.00787
1Department of Neurology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea
2Department of Neurology, Yonsei University College of Medicine, Seoul, Korea
3Integrative Research Center for Cerebrovascular and Cardiovascular Diseases, Seoul, Korea
4Department of Radiology, Yonsei University College of Medicine, Seoul, Korea
5Department of Neurology, Seoul Hospital, Ewha Womans University College of Medicine, Seoul, Korea
Correspondence: Ji Hoe Heo Department of Neurology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemoon-gu, Seoul 03722, Korea Tel: +82-2-2228-1605 E-mail: jhheo@yuhs.ac
Received 2024 February 22; Revised 2024 April 29; Accepted 2024 June 10.

Dear Sir:

Endovascular thrombectomy (EVT) is the standard of care for acute stroke, wherein achieving complete recanalization may improve clinical outcomes [1]. However, fragile thrombi are susceptible to fragmentation during EVT, causing distal embolization and migration, which diminishes the likelihood of complete recanalization [2-5]. Thrombus fragility may be predominantly affected by its structure and composition. In this study, we investigated the association between thrombus fragility and tumor histology.

This study included consecutive patients who underwent successful thrombus retrieval by EVT between January 2016 and December 2021, had their retrieved thrombi histologically evaluated, and were enrolled in the prospective registry. Eligible participants were patients primarily treated with a stent retriever; we excluded those who underwent only contact aspiration thrombectomy. This study was approved by the Institutional Review Board of Severance Hospital (4-2023-1010). Written informed consent was obtained from the patients or their next of kin for enrollment in the cohort and for utilizing the retrieved thrombi in the study.

EVT was performed under local anesthesia, according to common recommendations (Supplementary Methods). The retrieved thrombi were immediately fixed in 4% paraformaldehyde. All thrombi were immunohistochemically stained for erythrocytes, platelets, fibrin, lymphocytes, neutrophils, monocytes, tissue factors, and neutrophil extracellular traps (Supplementary Methods and Supplementary Table 1) [6]. Images of stained thrombi were acquired using a whole-slide scanner or Stereo Investigator Imaging system (MBF Bioscience, Williston, VT, USA) equipped with a light microscope (Axio Imager D2; Carl Zeiss Co., Ltd., Jena, Germany). The acquired images were analyzed using an Automated Region-of-interest-based Image Analysis software (Supplementary Methods) [7]. Each thrombus composition fraction (%) was determined by calculating the pixel density as a percentage of the total thrombus area.

All conventional angiographic images obtained immediately after each thrombectomy attempt were reviewed. The presence of a fragile thrombus was defined as either the thrombus fragmentation during EVT or the presence of initially multiple intracranial occlusions. Thrombus fragmentation manifests as downstream occlusion subsequent to thrombectomy attempts, characterized by angiographical occlusion distal to the original target site coupled with recanalization of the initial target [2-4]. Furthermore, the presence of initially multiple intracranial occlusions was considered indicative of a fragile thrombus, as it may reflect pre-existing fragmentation during thrombus growth or embolism. Two independent neurointerventionalists (JHB and BMK), blinded to the histological and clinical information, assessed the images (κ-values: 0.79 for thrombus fragmentation, 0.82 for successful recanalization, and 0.91 for first-pass effect). Discrepancies were resolved through consensus.

In a cohort of 376 patients who underwent EVT and had their thrombi retrieved, immunohistochemistry was performed on 340 (90.4%) (Supplementary Figure 1). Finally, 310 patients were included in the study (mean age, 70.9±13.6 years; men, 49.4%) (Table 1). Fragile thrombi were observed in 115 (37.1%) patients. Atrial fibrillation (67.8% vs. 51.3%; P=0.004) and internal carotid artery (ICA) occlusion (40.9% vs. 19.0%; P<0.001) were more frequent in patients with fragile thrombi than in those without (Table 1). Regarding thrombus composition, fragile thrombi were significantly associated with higher fractions of erythrocytes (40.2% [30.7–51.4] vs. 31.0% [16.6–43.3]; P<0.001) (Table 1). A higher fraction of erythrocytes relative to whole common thrombus components (i.e., erythrocytes, platelets, fibrin, and leukocytes) was also significantly associated with a fragile thrombus (44.0% [35.9–54.2] vs. 33.3% [20.2–42.3]; P<0.001). Multivariable analysis identified ICA occlusion (adjusted odds ratio, 2.45; 95% confidence interval, 1.41–4.27; P=0.001) and erythrocyte composition in thrombus (adjusted odds ratio, 1.14 per 5% composition; 95% confidence interval, 1.05–1.23; P<0.001) as independent factors for fragile thrombus (Table 2 and Figure 1). Erythrocyte composition in the thrombus could predict fragile thrombus (area under the receiver operating characteristic curve, 0.655; cutoff, 26.9%; sensitivity, 84.4%; specificity, 42.1%; P<0.001) (Supplementary Figure 2). Platelet composition tended to be lower in a fragile thrombus (8.9% [3.8–17.4] vs. 10.9 [4.5–20.7]; P=0.057).

Comparison of clinical and histological findings according to thrombus fragility

Clinical and histological factors associated with fragile thrombus

Figure 1.

Probability of a fragile thrombus by erythrocyte composition.

In this study, a fragile thrombus was observed in 37.1% of cases, a frequency comparable to 35.2%–57.5%, as stated in previous studies employing similar definitions to ours [2,3]. It was also associated with ICA occlusion and erythrocyte-rich thrombi. ICA occlusion has been suggested to correlate with distal and secondary embolisms [2]. Preclinical studies have shown that erythrocyte-rich thrombi are softer, less stiff, and more prone to fracture, suggesting their fragility [8,9]. Experimental retrieval of erythrocyte-rich thrombi resulted in the generation of more embolic fragments [9]. These findings support the association between fragility and erythrocyte-rich thrombi observed in our study.

Erythrocytes, fibrin, and platelets exhibit different degrees of stability. Platelets aggregate with each other by adhesively binding integrin α2bβ3 to fibrinogen. Consequently, platelets are tightly attached to each other. In contrast, erythrocytes primarily aggregate via passive packing, making them more susceptible to external forces. This may explain why erythrocyte-rich thrombi were more fragile, whereas those with higher platelet content tended to be less fragile.

However, previous clinical studies have reported inconsistent results. While one study demonstrated an association between clot migration and higher erythrocyte content [5], others found associations of secondary embolism with high fibrin and low erythrocyte contents [4], as well as an association between thrombus fragmentation and higher lymphocyte counts [10]. Such disparities may arise from the varying definitions of fragile thrombi and smaller sample sizes in previous studies. Additionally, previous studies utilized hematoxylin-eosin and histochemical stains, such as Martius Scarlet Blue, to detect erythrocytes and fibrin; this study employed immunohistochemistry. Precise differentiation between the major thrombus components may occasionally be challenging with histochemical staining.

Despite our strengths, such as a relatively large sample size, precise analysis of thrombus components through immunohistochemistry, and a semi-automated software program, our study has limitations. First, it focused on patients who underwent EVT primarily using stent retrievers. This was because patients who underwent only contact aspiration thrombectomy were excluded from this study, potentially having different mechanical impacts on thrombus. Second, although thrombus fragmentation may not always result in downstream occlusion, potentially leading to embolism in a new territory, such occurrences are rare and unlikely to significantly impact the outcome of the study. Finally, the definition of a fragile thrombus in this study may not accurately identify all such cases.

In conclusion, we found that thrombi with a higher erythrocyte composition were more fragile.

Supplementary materials

Supplementary materials related to this article can be found online at https://doi.org/10.5853/jos.2024.00787.

Supplementary Table 1.

Primary antibodies for histological components of thrombus

jos-2024-00787-Supplementary-Table-1.pdf
Supplementary Figure 1.

Patient selection flowchart.

jos-2024-00787-Supplementary-Fig-1.pdf
Supplementary Figure 2.

Receiver operating characteristic curves of erythrocyte composition in the thrombus and internal carotid artery (ICA) occlusion to predict a fragile thrombus. The erythrocyte composition in the thrombus could predict a fragile thrombus (area under the receiver operating characteristic curve [AUC], 0.655; cutoff, 26.9%; sensitivity, 84.4%; specificity, 42.1%; P<0.001). ICA occlusion could also predict fragile thrombi (AUC, 0.609; sensitivity, 40.9%; specificity, 81.0%; P<0.001). Considering both erythrocyte composition and ICA occlusion, it could predict fragile thrombi (AUC, 0.642; sensitivity, 39.1%; specificity, 89.2%; P<0.001).

jos-2024-00787-Supplementary-Fig-2.pdf

Notes

Funding statement

This study was supported by a National Research Foundation of Korea (NRF) grant, funded by the Korean government (RNF-2021R1A2C2003658), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2023-00265497).

Conflicts of interest

The authors have no financial conflicts of interest.

Author contribution

Conceptualization: JHB, JHH. Study design: JHB, JHH. Methodology: JHB, IK, JHH. Data collection: all authors. Investigation: all authors. Statistical analysis: JHB. Writing—original draft: JHB. Writing—review & editing: JHB, JHH. Funding acquisition: YDK, JHH. Approval of final manuscript: all authors.

References

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Article information Continued

Figure 1.

Probability of a fragile thrombus by erythrocyte composition.

Table 1.

Comparison of clinical and histological findings according to thrombus fragility

Total (n=310) Fragile thrombus (n=115) No fragile thrombus (n=195) P
Demographics and stroke risk factors
 Age (yr) 70.9±13.6 70.8±13.7 70.9±13.6 0.951
 Male sex 153 (49.4) 57 (49.6) 96 (49.2) 0.955
 Hypertension 224 (72.3) 80 (69.6) 144 (73.8) 0.416
 Diabetes 100 (32.3) 34 (29.6) 66 (33.8) 0.436
 Dyslipidemia 81 (26.1) 24 (20.9) 57 (29.2) 0.106
 Current smoking 40 (12.9) 15 (13.0) 25 (12.8) 0.955
 Coronary artery occlusive disease 46 (14.8) 14 (12.2) 32 (16.4) 0.311
 Atrial fibrillation 178 (57.4) 78 (67.8) 100 (51.3) 0.004
Clinical conditions
 Initial NIHSS score 14.0 [9.0–18.0] 15.0 [10.0–18.0] 12.0 [8.0–18.0] 0.089
 Intravenous tPA administration 114 (36.8) 47 (40.9) 67 (34.4) 0.251
 Location of occlusions <0.001
  Internal carotid artery 84 (27.1) 47 (40.9) 37 (19.0)
  M1 segment of middle cerebral artery 120 (38.7) 41 (35.7) 79 (40.5)
  M2 segment of middle cerebral artery 75 (24.2) 21 (18.3) 54 (27.7)
  Anterior cerebral artery 1 (0.3) 0 (0.0) 1 (0.5)
  Vertebrobasilar artery 26 (8.4) 4 (3.4) 22 (11.3)
  Posterior cerebral artery 4 (1.3) 2 (1.7) 2 (1.0)
 Tandem occlusion 26 (8.4) 10 (8.7) 16 (8.2) 0.880
 Onset-to-puncture time (min) 292.0 [167.0–610.0] 267.0 [138.0–571.0] 308.0 [176.0–624.0] 0.210
 Use of balloon guide catheter* 278 (89.7) 108 (99.1) 170 (99.4) 0.747
Endovascular outcomes
 Recanalization status
  Successful recanalization 287 (92.6) 104 (90.4) 183 (93.8) 0.268
  Complete recanalization 193 (62.3) 37 (32.2) 156 (80.0) <0.001
  First-pass effect 102 (32.9) 5 (4.4) 97 (49.7) <0.001
 Number of passes of stent retriever 2.3±1.4 2.8±1.3 2.0±1.4 <0.001
 Puncture-to-recanalization time (min) 31.0 [20.0–49.5] 33.5 [24.0–53.0] 29.0 [19.0–49.0] 0.128
Thrombus composition (%)
 Erythrocyte 35.6 [20.3–46.7] 40.2 [30.7–51.4] 31.0 [16.6–43.3] <0.001
 Platelet 9.7 [4.3–19.5] 8.9 [3.8–17.4] 10.9 [4.5–20.7] 0.057
 Fibrin 32.3 [21.2–47.2] 31.6 [21.0–48.1] 32.7 [21.4–45.7] 0.875
 Leukocytes 8.5 [4.2–15.7]
  Lymphocyte 0.3 [0.2–0.5] 0.3 [0.2–0.5] 0.2 [0.1–0.4] 0.135
  Neutrophil 0.6 [0.2–2.1] 0.7 [0.2–2.5] 0.6 [0.2–2.0] 0.813
  Monocyte 5.0 [2.2–11.1] 5.1 [2.3–10.7] 4.9 [2.1–11.6] 0.989
 Tissue factor 12.8 [1.5–42.7] 13.9 [1.9–45.1] 11.8 [1.1–41.0] 0.608
 Neutrophil extracellular trap 2.7 [1.1–5.1] 3.0 [1.1–6.4] 2.7 [1.1–4.6] 0.368

Values represent mean±standard deviation, number (%), or median [interquartile range].

NIHSS, National Institutes of Health Stroke Scale; tPA, tissue-type plasminogen activator.

*

The calculation of the percentages of patients using a balloon-guided catheter was limited to those with anterior circulation stroke;

Successful recanalization was defined as achieving final modified Thrombolysis In Cerebral Infarction (mTICI) 2b or 3; Complete recanalization was defined as the attainment of a final mTICI score of 3.

Table 2.

Clinical and histological factors associated with fragile thrombus

Adjusted odds ratio (95% CI) P
Clinical factors
 Atrial fibrillation 1.61 (0.95–2.73) 0.075
 Initial NIHSS score 1.01 (0.97–1.06) 0.494
 Internal carotid artery occlusion 2.45 (1.41–4.27) 0.001
Histological factors
 Erythrocyte, per 5% 1.14 (1.05–1.23) <0.001
 Platelet, per 5% 0.99 (0.90–1.07) 0.725

The analysis included variables with P<0.1 in the univariable analyses.

CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale.