Introduction

Determining the etiopathological source of stroke is a cornerstone for correct secondary prophylaxis. The majority of cerebral ischemic strokes are thromboembolic [1]. Although, their etiologic un pathogenetic mechanisms can differ significantly. The main embolic cerebral ischemic stroke causes are arterioarterial embolism from atherosclerotic plaque and cardioembolism [2]. The proportion of thromboembolic infarctions in Latvia is reaching 75% (Pauls Stradiņš Clinical University Hospital local stroke registry). Nevertheless, in up to 40% the precise source of embolism cannot be determined even after extensive evaluation [3]. For clinicians cryptogenic strokes are a major problem and are an important research focus [4,5]. Several studies have shown high rates of recurrent stroke episodes of ≤30% in the first year after suffered cryptogenic stroke [6].

With the development of endovascular therapy, it became possible to analyze the thrombus material histologically, thus providing additional diagnostic opportunities [7]. Some authors have hypothesized that it is histologically possible to differentiate between cardioembolus and arterioarterial embolus using the classical hematoxylin-eosin staining method [2]. The pattern of each thrombus component distribution often differs between patients with cardiac thrombi and those with arterial thrombi, and the efficacy of endovascular thrombectomy is different according to the thrombus composition [8]. Thus, it can be possible to clarify cryptogenic stroke etiology basing on thrombus histology. Although it may be possible, that histopathology of retrieved thrombi may be affected in patients who received intravenous thrombolysis procedure before endovascular mechanical thrombectomy was performed.

There is also little data on the correlation of radiological results with the histological qualitative structure of the thrombus. It is proposed that the presence of hyperdense artery sign (HAS) indicates more red blood cells (RBC`s) components and better revascularization outcomes [9]. The presence of thrombus in the heart and its impact on histopathology of cardioembolic thrombi has never been investigated, although it can provide an important clinical information about the thrombus composition on acute pretreatment stage.

However, only a few studies have analyzed the outcome of endovascular therapy recanalization and the neurological clinical outcome in correlation with the histological composition of the thrombus. Successful reperfusion is routinely achieved in 70-80% of cases [10]. That’s why a major and unanswered question in vascular neurology is about the poor clinical outcome of stroke patients with endovascular therapy. Of course, there are several already known factors such as time, collaterals, and vascular anatomy, however, it cannot be ruled out that thrombus structure is also important. For instance, it has been proposed that thrombi with a high white blood cells (WBC`s) fraction are related to more organized thrombi of cardioembolic origin associated with less favorable recanalization and clinical outcome in acute ischemic anterior circulation stroke [11]. A better understanding of thrombus type and properties can significantly improve stroke treatment outcomes.

The aim of this study was to investigate histopathological features of different stroke emboli subtypes retrieved during mechanical thrombectomy and compare radiological findings and stroke outcomes with histological profile of thrombi.

Materials and Methods

The study is classified as a single-center analytical observational study. Forty patients with cerebral infarction and large magistral artery occlusion, during a period of three months (13 January 2021 – 1 July 2021), have received acute reperfusion therapy with endovascular thrombectomy in Pauls Stradiņš Clinical University Hospital (Riga, Latvia). Immediately after clot retrieval, thrombus material was fixed in phosphate-buffered (10%) formalin. The total of 24 included patients were treated with intravenous recombinant tissue-type plasminogen activator (IV tPA) before endovascular thrombectomy.

Study Population

Patient data (age, gender, stroke subtype) were extracted from the medical records. Major clinical characteristics of patients included in the study could be found in Table S1 (see supplementary materials).

Table S1: Major clinical characteristics of patients included in the study.

Pre-interventional Imaging

Unenhanced brain Computed Tomography (CT) and contrast-enhanced Computed Tomography Angiography (CTA) of the brachiocephalic arteries were performed in all patients either in our center or in the regional hospital. Brain Computed Tomography Perfusion (CTP) imaging was performed, if time from symptoms onset to hospital arrival exceeded 4,5 hours (26 patients). Unenhanced brain CT retrospectively was analyzed for presence or absence of the HAS. HAS was determined by an increased radiodensity of an artery and Hounsfield (HU) value was not specifically used for measurement of clot density. 29 patients during the acute stroke CT protocol have received unenhanced thoracic CT examination. Unenhanced thoracic CT was retrospectively analyzed for the presence or absence of intracardiac thrombi.

Endovascular Techniques

All procedures were performed using arterial access under sedation or general anesthesia according to present clinical status and stroke severity. The procedures were performed in a dedicated angio-suite (biplane Artis Zee, Siemens). Femoral access was obtained with an 8F vascular sheath and 6F long vascular sheath (Neuro max 088, Penumbra Inc.) The guide was advanced distally, as far as safely possible, into the internal carotid or in the vertebral artery, according to previous imaging findings. The aspiration catheter (Ace 60 or 68, Penumbra) was advanced to the level of the thrombus over any microcatheter and microwire the operator chooses. Under road map guidance, the wire and microcatheter were navigated to or past the thrombus. Over this platform, the larger aspiration catheter was delivered and positioned immediately adjacent to the site of occlusion. The microcatheter and wire were removed, and aspiration was applied via the aspiration pump that is part of the Penumbra separator system. If aspiration failed, the Solumbra technique as rescue switching strategy was performed advancing a stent-retriever (Solitare, Covidien) through the 0.025-inch microcatheter distal to the clot with an aspiration catheter (Ace 60 or 68, Penumbra) at the clot face. The microcatheter was then removed till stent retrieval proximal markers. Intermediate catheter was connected to a negative pressure suction pump (Penumbra) for continuous aspiration. Three to five minutes later, the aspiration catheter was slowly withdrawn until the blood flow velocity returned to normal in the connecting pipe of the negative pressure pump.

Histological and Immunohistochemical Analysis of Retrieved Thrombi

Histopathologically 40 thrombi were investigated. The size of each thrombus was registered. The formalin-fixed specimens were embedded in paraffin, cross-sectioned at 4-µm thickness and stained with haematoxylin-and-eosin (H&E) with Dako Autostainer link 48.

Subsequently, based on H&E staining, the percentage of erythrocytes, leukocytes and fibrin was measured in investigated thrombi, as well as microscopically was measured the area of these cells in mm2. The area of the thrombus components was measured with Nikon’s program Nis Elements D. From the H&E image of each thrombus, each component was outlined and the area obtained, which was then converted to a percentage of the total thrombus area. All thrombi were stained immunohistochemically, according to manufacturer`s (Dako) instructions, for CD3 positive T cells, CD68 positive monocytes, von Willebrand factor positive thrombocytes and CD15 positive leukocytes. Their amount was categorically measured ranging “+, “++” and “+++”. Where “+” presence of cells occupying < 25% of the tissue, “++” presence of cells occupying 25 to 50% of the tissue and “+++” presence of cells occupying > 50% of the tissue.

Stroke Subtypes with Characteristic Histopathological Profile

Ischemic stroke patients, included in this study, were divided into three groups – cardioembolic, atheroembolic and cryptogenic group. In our study groups were defined as follows: cardioembolic group – patients with atrial fibrillation and/or proved thrombi in left atrium appendage. Atheroembolic group – patients with large artery atherosclerosis and/or artery dissection. Cryptogenic pathogenesis group – patients with ischemic stroke of unknown etiology – either fully investigated without any pathological findings or incompletely investigated. Histopathological findings of retrieved thrombi were compared between three mentioned groups.

The distribution of ischemic stroke patients by pathogenetic groups was as follows: cardioembolic (26 patients; 65,0%), atheroembolic (7 patients; 17,5%) and cryptogenic group (7 patients; 17,5%), see Figure 1.

Figure 1: Distribution of Ischemic Stroke Subtypes in Included Patients.

The hystologic composition of each trombi group analysed by Hematoxylin and Eosin technicque and their size is present in Table S2 (see supplementary materials).

Table S2: The hystologic composition of each trombi group analysed by Hematoxylin and Eosin technicque and their size.

Immunohystochemical profile of each ischemic stroke group is represented in Table S3 (see supplementary materials).

Table 3: Immunohystochemical analysis of composition of thrombi in each ischemic stroke group.

The Histologic Composition of each Group of Thrombi Analysed by Hematoxylin and Eosin Technique and their Size

Statistical Analysis

Statistical analysis was performed using SPSS® (IBM SPSS Statistics for Windows, Version 25.0). All tests were statistically significant if p value was less than 0,05. As the data did not fit the normal distribution, only non-parametric statistical methods were used. For the analysis of dependencies between nominal and scale data, were applied Mann-Whitney U test and Kruskal-Willi’s test. For finding correlations between nominal data was used Fisher`s exact test. Standard deviation as an additional descriptive parameter was not applied, as data did not fit a normal distribution. As additional descriptive parameters were chosen median with quartiles. Results

Impact of IV tPA on Thrombus Composition

The total of 24 patients have received IV tPA before mechanical thrombectomy. Only 16 patients underwent mechanical thrombectomy procedure alone. There were no statistically significant differences in thrombus composition comparing data in both groups (with and without IV tPA )– see Table S4 (see supplementary materials). From those, who received IV tPA, 14 had cardioembolic thrombi, 4 had atheroembolic thrombi and 6 had cryptogenic thrombi.

Table S4: Differences in composition of thrombi in patients with and without performed intravenous thrombolysis before mechanical thrombectomy.

Although there was a tendency to be less T lymphocytes in the thrombi of patients, who did not receive IV tPA (p = 0,056), see Table 5.

Table 5: Distribution of thrombi by amount of T lymphocytes depending on received intravenous (IV) thrombolysis therapy with recombinant tissue plasminogen activator (rt-PA).

Histopathological Differences in Thrombus Composition in Different Ischemic Stroke Groups

Cardioembolic, atheroembolic and cryptogenic thrombi groups were classified as erythrocyte rich or fibrin rich according to their predominant composition. Histological examination revealed, that all of the investigated atheroembolic and cryptogenic thrombi were fibrin rich, whereas among cardioembolic thrombi 11,5% (n=3) were erythrocyte rich (see Table 6). Even so, using such simplified classification of retrieved thrombi, some differences were found in their histopathological composition (Bonferroni correction was not applied for the tests).

                                                                                                       Fibrin rich thrombi                                         Erythrocyte rich thrombi

Table 6: Distribution of thrombi type depending on the cerebral infarction pathogenesis.

Thrombi in patients with cryptogenic strokes had slightly larger leukocytes area (median - 4,0 mm2) comparing with cardioembolic thrombi (median – 1,6 mm2; p=0,099), although this result was not statistically significant. No differences were found between cryptogenic and atheroembolic thrombi (median – 1,3 mm2; p=0,165), (Figure 2), as well as between cardioembolic and atheroembolic thrombi (p=0,590).

Figure 2: Leukocytes area in mm2 in different thrombi types.

Cryptogenic thrombi had statistically significantly lower fibrin/leukocytes ratio than atheroembolic thrombi (median - 5,8 and 14,0 respectively; p=0,026), the same tendency, but without statistical significance, comparing cryptogenic thrombi and cardioembolic thrombi (median - 5,8 and 11,5 respectively; P=0,067). No statistically significant differences were found comparing cardioembolic and atheroembolic thrombi (p=0,330), see Figure 3.

Figure 3: Fibrin to leukocytes ratio in different thrombi types.

Atheroembolic thrombi had a higher percentage of the fibrin area versus total thrombi area than cryptogenic thrombi (median - 85,0% and 79,0% respectively; P=0,038). The same tendency, but not statistically significant was found between atheroembolic and cardioembolic thrombi (median - 85,0% and 82,5% respectively; P=0,099). There were no any statistically significant differences comparing both cryptogenic and cardioembolic thrombi (p=0,288), see Figure 4.

Figure 4: Fibrin area in percentages in different thrombi types.

There were no statistically significant differences in the proportion of other thrombus components between three mentioned groups, see Table S7 (see supplementary materials).

Table S7: Comparison of the histological composition between etiological stroke groups.

Impact of Radiological Findings on Thrombus Histopathology

In total, 23 patients had HAS on non-enhanced head CT and 17 patients did not. Histopathological findings in both patients` groups, that is with and without HAS, is seen in Table S8 (see supplementary materials). The mentioned information shows, that HAS did not correlate with thrombus size, histological composition of thrombus, as well as immunohistochemical profile. There was no statistically significant correlation between HAS and outcomes.

Table S8: Composition of thrombi in patients with and without hyperdense artery sign (HAS).

In total, 29 patients have performed non-enhanced thoracic CT, and 11 patients did not receive this examination. From those, who had this investigation performed, 21 had thrombus in heart, whereas 8 had not.

Patients with visualised thrombus on CT in heart had larger erythrocyte area in thrombus, than those without visualised thrombus in heart (p=0,047). Other parameters did not differ statistically significantly and may be seen in Table S9 (see supplementary materials).

Table S9: Composition of thrombi in patients with (21 patients) and without (8 patients) CT visualised thrombi in heart.