What Does the Histological Composition Tell Us About the Origin of the Thrombus?
Background and Purpose—The introduction of stent retrievers allows for a complete extraction and histological analysis of human thrombi. Ischemic stroke is a major health issue, and differentiation of underlying causes is highly relevant to prevent recurrent stroke. Therefore, histopathologic analysis of the embolic clots after removal may provide valuable information about underlying pathologies. This study analyzes histological clot composition and aims to identify specific patterns that might help to distinguish causes of ischemic stroke.
Methods—Patients with occlusion of the carotid-T or middle cerebral artery who underwent thrombectomy at our university medical center between December 2013 and February 2016 were included. Samples were histologically analyzed (hematoxylin and eosin, Elastica van Gieson, and Prussian blue), additionally immunohistochemistry for CD3, CD20, and CD68/KiM1P was performed. These data, along with additional clinical and interventional parameters, were compared for different stroke subtypes, as defined by the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification.
Results—One hundred eighty-seven patients were included, of these, in 77 patients, cardioembolic; in 46 patients, noncardioembolic; and in 64 patients, cryptogenic pathogenesis was determined. Cardioembolic thrombi had higher proportions of fibrin/platelets (P=0.027), less erythrocytes (P=0.005), and more leucocytes (P=0.026) than noncardioembolic thrombi. We observed a strong overlap of cryptogenic strokes and cardioembolic strokes concerning thrombus histology. The immunohistochemical parameters CD3, CD20, and CD68/KiM1P showed no statistically noticeable differences between stroke subtypes.
Conclusions—Histological thrombus features vary significantly according to the underlying cause and may help to differentiate between cardioembolic and noncardioembolic stroke. In addition, our study supports the hypothesis that most cryptogenic strokes have a cardioembolic cause.
See related article, p 2040
Background and Purpose
The introduction of stent retrievers allows for a complete extraction and histological analysis of embolic thrombus material. Currently, after the diagnosis of embolic stroke, patients undergo long diagnostic cascades to determine underlying risk factors and potential pathogenesis to provide the best possible therapy and minimize the risk of recurrent stroke events.1 However, the pathogenesis remains unclear in ≈40% of patients.2 Additional tests, such as histological clot analysis, seem promising to improve diagnostic accuracy and simplify diagnostic cascades.
Initial studies deal with the possible impact of histological clot composition on the effect of recanalization and clinical outcome; however, they only evaluated basic thrombus morphology and categorized main thrombus components (fibrin/platelet conglomerates and red and white blood cells [WBCs]).3–9 Moreover, first results about the differentiation of cardioembolic versus noncardioembolic stroke suggest that cryptogenic stroke is mostly of cardiac origin.8,10 However, evidence is limited, especially for immunohistochemical parameters.11
Our study, therefore, aimed to evaluate further the theory of histological determination of thrombus origin and additionally included immunohistochemical parameters that may help to specify the thrombus origin.
We prospectively included a cohort of 198 consecutive ischemic stroke patients with occlusion of the carotid-T or middle cerebral artery in whom thrombectomy with stent retriever was performed. Only patients with complete diagnostic and histological workup were included for further analysis. We primarily investigated on the relationship between stroke causes according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification12 and clot composition, expressed as percentage of the main components (fibrin, red blood cells [RBCs], and WBCs). Second, we performed immunohistochemical tests, including analysis of clusters of differentiation coreceptors (CD3, CD20, and CD68/KiM1P).
Stroke cause was determined according to the TOAST classification using computed tomography and magnetic resonance imaging, duplex sonography of the cervical arteries, coagulation tests, long-term electrocardiography, and transthoracic or transesophageal echocardiography.
Additionally, baseline information, including age, sex, application of intravenous tPA (tissue-type plasminogen activator), and interventional parameters, was obtained from patients’ clinical records and imaging on admission (Table 1).
The study was approved by the Ethics Committee of the University of Muenster and the Westfalian Chamber of Physicians, Muenster, Germany. All study protocols and procedures were conducted in accordance with the Declaration of Helsinki.
Interventional thrombectomy was performed by experienced neuroradiologists under general anesthesia. In all patients, we used a biplane neuro-X-ray system (Allura Xper FD20/20; Philips, Best, the Netherlands). Interventional therapy included positioning of a 6F guiding catheter in the cervical segment of the internal carotid artery. In most of the patients, a pRESet stent retriever device (Phenox, Bochum, Germany) with a size of 20×4 or 30×6 mm was used. All angiograms were reviewed postinterventionally and graded using the modified thrombolysis in cerebral infarction score.
Histology and Immunohistochemistry
The complete specimen was macroscopically inspected, formalin fixed, and six 5-mm sections were analyzed (hematoxylin and eosin, Elastica van Gieson, Prussian blue, and immunohistochemical stainings). The observer was completely blinded to the TOAST classification.
For immunohistochemical staining, sections were placed on slides, deparaffinized, rehydrated, and then washed in Aqua Dest. If necessary, for antigen retrieval, slides were microwaved twice for 7 minutes each and then washed in a buffer of phosphate buffered saline, bovine serum albumin, and Triton X-100 3 times for 5 minutes each. The slides were blocked in 20% goat serum diluted in PBT for 30 minutes and then incubated with the following antibodies: CD3 (mouse monoclonal, 1:25, pH 6.1; Dako, Glostrup, Denmark) and CD20 (mouse monoclonal, 1:700, pH 6.1; Dako). For CD68 staining supernatant from KiM1P hybridoma cells, kindly provided by Prof Klapper, Institute of Pathology, Kiel (1:5000, mouse monoclonal) was used. After washes in PBT, slides were incubated with a biotinylated goat anti rabbit secondary antibody (E0432; 1:500 dilution; Dako) for 45 minutes at room temperature after incubation with the ABC kit (SK6100, Vectastain avidin–biotin complex–horseradish peroxidase; Vector Laboratories, Burlingame, CA) for 45 minutes. The signal was developed using a 3,3′-diaminobenzidine substrate kit (SK4100; Vector Laboratories), and the sections were counterstained with hematoxylin.
Quantification of Stainings
Using an Olympus BX43 microscope and digital camera, thrombus materials were photographed (magnification ×40). For quantification of erythrocytes, fibrin and other cellular components, ImageJ software (ImageJ 1.47n; National Institute of Health, Bethesda, MD) was used. Quantification of fibrin, RBC, and WBC was performed manually, whereas for quantification of immunohistochemical stainings, the pictures were converted to gray scale (8-bit), threshold was set, and particles were analyzed automatically (areas covered by the respective cells [%] were measured).
Univariable distribution of metric variables is described by median and interquartile range. For categorial data, absolute and relative frequencies are given. The stroke subgroups arterioembolic (TOAST 1) and other determined causes (TOAST 4) were composed to the group noncardioembolic strokes (TOAST 1 and 4). The patient groups noncardioembolic (TOAST 1 and 4), cardioembolic (TOAST 2), and cryptogenic strokes (TOAST 5) were pairwise compared by Mann–Whitney U test for metric variables and by chi-quadrat test for categorical variables. Boxplot diagrams were induced to illustrate group differences and similarities.
All statistical analyses were performed using SPSS, version 22 (IBM Software, Chicago, IL). P values ≤0.05 were considered statistically significant.
Of 198 patients in whom thrombus material was collected, 187 patients with complete histological processing were included for further analysis (f=89 and m=98). In 77 patients, cardioembolic (TOAST 2); in 35 patients, noncardioembolic (large artery atherosclerosis; TOAST 1), and in 11, another determined pathogenesis (6 as tumor associated, 3 after cervical artery dissection, and 2 radiogenic stenosis; TOAST 4) was diagnosed. Sixty-four patients were classified as having cryptogenic strokes (TOAST 5). Overall, thrombus composition showed higher amounts of fibrin compared with RBC. Successful recanalization, defined as thrombolysis in cerebral infarction 2b or 3, was reached in 94% of cases with a median number of maneuvers of 1. The clinical, interventional, and histological characteristics of the 187 patients are given in Table 1. Additionally, categorial data of all patients where thrombectomy was attempted is given in Table 1.
Primary Target Variables
Cardioembolic (TOAST 2) Versus Dichotomized Noncardioembolic (TOAST 1 and 4) Strokes
All main thrombus components showed significant differences in their percentages between cardioembolic and noncardioembolic stroke causes. Cardioembolic thrombi consisted of higher median proportions of fibrin (60.0% versus 51.5%; P=0.027), less RBCs (28.0% versus 42.0%; P=0.005), and more WBCs (8.0% versus 5.0%; P=0.026) than noncardioembolic thrombi.
All predefined interventional and clinical parameters showed no significant differences between these groups as summarized in Table 2.
Cryptogenic (TOAST 5) Versus Dichotomized Noncardioembolic (TOAST 1 and 4) Strokes
In patients with cryptogenic strokes, the median proportion of fibrin was almost identical to that in cardioembolic stroke patients (63.5% versus 60.0%; P=0.916) but significantly larger than in noncardioembolic stroke patients (63.5% versus 51.5%; P=0.002). Likewise, the RBC proportion was about as high in cryptogenic strokes as in cardioembolic strokes (26.0% versus 28.0%; P=0.767) but significantly lower than in noncardioembolic strokes (26.0% versus 42.0%; P=0.002). Also, the WBC proportion was statistically noticeably higher in thrombi of cryptogenic compared with noncardioembolic stroke patients (10.0% versus 5.0%; P=0.016).
All predefined interventional and clinical parameters showed no significant differences between the groups (Table 3).
Secondary Target Variables
Second, we performed immunohistochemical tests, including analysis of CD3, CD20, and KiM1P. Analysis revealed that the CD3 (P=0.609) and CD20 (P=0.560) showed no statistically noticeable differences between noncardioembolic (TOAST 1 and 4) and cardioembolic patients (TOAST 2), but the immunohistochemical parameter CD68/KiM1P was substantially, although nonsignificantly, higher in cardioembolic strokes compared with noncardioembolic strokes (3% versus 1%; P=0.061). The groupwise comparison between cryptogenic and noncardioembolic strokes revealed no statistically noticeable differences regarding immunohistochemical parameters (CD3 [P=0.388], CD20 [P=0.158], and CD68/KiM1P [P=0.711]).
Our results show that histopathologic assessment can become a valuable option to differentiate between cardioembolic and noncardioembolic thrombus pathogenesis. In our study, erythrocyte-rich thrombi were significantly associated with noncardioembolic stroke cause, whereas fibrin-rich thrombi showed a significant correlation with cardioembolic stroke cause.
This supports recent studies by Boeckh-Behrens et al,8,10 Niesten et al,13 and Simons et al,7 that report about an association of high fibrin and low RBC proportion in cardioembolic thrombi and which are contrary to the concept of red and erythrocyte-rich cardioembolic thrombi (Figure 1). The contradictory findings of Kim et al14 may be related to differences in methods and study population. The patient collective of Kim et al, on the one hand, was too small to draw a strong conclusion because especially the number of patients with atherosclerosis of large arteries was small (8 patients) compared with the group of patients with cardioembolism. On the contrary, clot composition might have been affected by intravenous r-tPA (recombinant tissue-type plasminogen activator) application.
As reported by Boeckh-Behrens et al,10 we also found a strong association of cryptogenic strokes with cardioembolic strokes compared with noncardioembolic strokes, which further strengthens the theory that strokes where the pathogenesis after complete diagnostic workup is classified as cryptogenic are mostly of cardiac origin.
Moreover, our study assessed immunohistochemical parameters (CD3, CD 20, and CD68/KiM1P) to test whether lymphocyte or macrophage activity could present any additional information on thrombus origin. Even though we did not find any significant associations with cardioembolic or noncardioembolic causes, CD68/KiM1P was substantially higher in cardioembolic compared with noncardioembolic strokes (P=0.061). Recently, Dargazanli et al reported that the CD3+T-cell count in intracranial thrombi was significantly higher in atherothrombotic origin strokes.11 These different findings might partly be explained by different histological stainings as Dargazanli et al analyzed the whole thrombus with respect to CD3, whereas we tried to establish a standardized histological workup that is applicable in clinical routine and, therefore, used six 5-mm thin sections to cover stainings for hematoxylin and eosin, Elastica van Gieson, Prussian blue, and for three immunohistochemical markers. Interestingly, Dargazanli et al quantified CD3 only in the slices with the highest CD3-content, which is a completely different approach compared with ours. Additionally, the patient collective of Dargazanli et al is considerably smaller (only 10 patients considered as atherothrombotic origin), and the classification as atherothrombotic stroke might not be correct in any case because it relies on likelihoods (stenosis or occlusion of a cervical or ipsilateral intracranial artery). Future studies will have to clarify whether combinations of KiM1P, CD3, and other immunohistochemical parameters might add to an even more specific diagnostic workup.
This study has some limitations partly attributed to the single-center retrospective design with an inherent selection bias. Naturally, the use of intravenous r-tPA might have altered the specimens. The retrieved thrombus material might not always reflect the whole thrombus, and a bias toward more stable clot components cannot be ruled out. The manual segmentation of the thrombus components may be associated with an operator bias. Finally it cannot be ruled out that manipulation with catheters might have produced some thrombi; however, thromboembolic complications are reported in only 1% to 2% of cerebral angiographies.15 Strengths of our study include the large number of patients and the complete and homogenous histological and diagnostic workup in the included patients.
Histological thrombus composition in embolic stroke is an important feature that may help to differentiate between cardioembolic and noncardioembolic stroke causes. It remains a promising attempt to further specify stroke pathogenesis and an interesting research question. In addition, our study supports the hypothesis that most cryptogenic strokes have a cardioembolic cause.
We thank Andrea Rothaus for her expert technical assistance.
Consulting Editor for this article was Ajay K. Wakhloo, MD, PhD.
- Received January 8, 2017.
- Revision received March 3, 2017.
- Accepted March 31, 2017.
- © 2017 American Heart Association, Inc.
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