Thrombus Histology Suggests Cardioembolic Cause in Cryptogenic Stroke

Up to 39% of acute ischemic strokes cannot be assigned to a definite cause despite intensive diagnostic workup^1,2 and are, therefore, classified as cryptogenic. To the clinician, cryptogenic strokes pose multiple unresolved problems, most of all in regard to secondary stroke prevention. Several studies show high rates of recurrent stroke episodes of ≤30% in the first year after cryptogenic stroke.^3,4 Accordingly, the so-called cryptogenic stroke is a major health issue and an important research focus,^5,6 calling for multidisciplinary approaches.

With the introduction of thrombus-extracting devices into acute stroke therapy in the mid-2000s, human thrombus material became available for histopathologic analysis. The subsequent development of highly efficient stent retrievers and the increasing implementation of mechanical thrombectomy in clinical practice led to much higher numbers of analyzable thrombus samples. Recent preliminary studies evaluated basic thrombus morphology and categorized main thrombus components, such as fibrin/platelet (F/P) conglomerates and red and white blood cells (RBCs and WBCs, respectively).^7–13 In preceding studies, we, and others, have observed differences in thrombus composition between different stroke subtypes, with higher F/P and leukocyte counts but smaller RBC fraction in cardioembolic stroke compared with other stroke causes.^10,11 The present study assesses the hypothesis that histopathologic clot composition might provide relevant information about stroke cause in the so-called cryptogenic strokes.

The study was approved by the Institutional Review Board, and informed consent of patients was obtained. A total of 145 intracranial thrombi were collected between October 2010 and September 2012 during endovascular recanalization therapy in patients with acute stroke (including all patients of our first published series [n=34]¹⁰).

As primary end points, we investigated the relationship between stroke cause and clot composition, expressed as percentage of the main components (F/P, RBCs, and WBCs).

Quantitative analysis of these components was done using semiautomated color-based segmentation (Adobe Photoshop CS4, Adobe Systems, San Jose, CA), defining their relative fractions as previously described.¹⁰ As hematoxylin and eosin staining does not allow to adequately distinguish fibrin and platelets, F/P aggregations are labeled as F/P. Thrombus size was estimated by using the totalized pixel count per specimen.

Stroke cause according to the international Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification¹⁴ was determined based on all diagnostic and clinical information available for each patient, including cerebral computed tomography (CT), CT angiography and magnetic resonance imaging, transcranial and extracranial duplex sonography, coagulation tests, long-term electrocardiography recording, and transthoracic or transesophageal echocardiography.¹⁰ Complete diagnostic workup was defined as at least long-term electrocardiography, any imaging of the brain (CT or magnetic resonance imaging) and the extracranial vessels (angiography, CT angiography, or duplex sonography), and any form of echocardiography (transthoracic or transesophageal echocardiography). All TOAST assignments were additionally verified by an experienced senior neurologist (H. Poppert).

In a second step, we prespecified interventional and outcome variables that are potentially associated with stroke cause: time to reperfusion (time from symptom onset to recanalization), recanalization time (time from first angiographic series in the target vessel to final run), number of retraction maneuvers, preinterventional National Institutes of Health Stroke Scale (NIHSS) scores, NIHSS scores at discharge, and modified Rankin Scale (mRS) scores within 90 days. NIHSS scores on admission and at discharge were determined by the respective responsible neurologist, and mRS scores were assessed by personal clinical examination, by phone interview, or by evaluating the medical reports of the rehabilitation centers.

Besides age and sex, basic clinical and interventional data assessed included relevant vascular risk factors, site of occlusion, time to treatment (time from symptom onset to groin puncture), and application of intravenous alteplase, which are summarized in Table 1 and Table I in the online-only Data Supplement.

Between-group differences, especially on the different TOAST groups, were assessed by nonparametric Kruskal–Wallis tests. Boxplot diagrams were generated to illustrate group differences and similarities. All statistical analyses were performed or verified by an experienced statistician (F. Scheipl) using R (http://www.r-project.org) and SPSS Statistics version 23.0 (SPSS Inc, IBM, Ehningen, Germany).

Further information on interventional procedures, thrombus processing, and analysis can be found in the Methods section in the online-only Data Supplement.

A total of 145 thrombi were collected, and of these, 137 clot samples of sufficient quality were used for histological processing. The clinical characteristics of the remaining 137 patients are summarized in Table 1. The main occlusion site was the middle cerebral artery in >50% of all patients. Vertebrobasilar occlusions accounted for 11% of patients. Successful recanalization, defined as Thrombolysis in Cerebral Infarction 2b or 3, was reached in 89.7% of cases.

Workup was incomplete in 12 of the 36 patients with strokes classified as cryptogenic. In 7 of these cases, this was because of early death, and in 4 cases, it was because of early transfer to other hospitals. In 1 case, echocardiography was not performed for unknown reason. To exclude possible bias related to patients with incomplete workup, additional subgroup analyses were performed restricted to patients with complete workup. In these, transesophageal echocardiography was performed in 58.3% of cases and additional coagulation tests in 29.2%.

Of the 11 patients classified as TOAST 4 (other determined cause), 8 patients were with dissections, 1 with an inflammatory process infiltrating the internal carotid artery at the skull base, 1 with a radiogenic stenosis, and 1 with a local thrombus after clipping of an middle cerebral artery aneurysm.

Overall, thrombus composition showed comparable amounts of F/P and RBCs with a slightly higher amount of F/P. There were no significant differences in thrombus composition between anterior and posterior circulation stroke and between patients with or without thrombolysis. As expected, thrombus size decreased with the size of the occluded vessel (internal carotid artery>M1/BA>M2/A2). There were no significant differences between TOAST categories concerning thrombus size. All interventional and histological data are summarized in Table I in the online-only Data Supplement.

These data show (1) that cardioembolic thrombi have histopathologic characteristics that are distinct from those of noncardioembolic thrombi and (2) that cryptogenic thrombi have the same or at least similar characteristics as those of cardioembolic thrombi, but they are, again, distinct from those of noncardioembolic thrombi. These observations are based on the—to our knowledge—largest published series of histologically analyzed stroke thrombi to date. In contrast to attempts that aim at a clinical differentiation of stroke subtypes^16–18 or trying to improve and escalate poststroke diagnostic efforts,^19,20 we used a distinctly different approach by analyzing the different TOAST groups trying to infer stroke cause from histological and also interventional and clinical outcome parameters. The data strongly support the notion that most cryptogenic strokes are cardioembolic.

The F/P fraction was the dominant clot component in the apparently more organized cardioembolic thrombi, whereas RBCs preponderated in noncardioembolic thrombi. This is in line with our own previous results,¹⁰ with the findings of Niesten et al,¹¹ and with some restrictions also to the findings of Simons et al¹² but stands in contrast to recently published results by Kim et al,⁷ questioning the traditional concept of cardioembolic being mostly red, erythrocyte-rich clots. This discrepancy may be related to differences in methods (2 different stainings, thereby including platelet content; possibly different quantification method and component assignment) and to the much smaller sample size in this latter study with only 8 clots defined as arterioembolic. Furthermore, our results seem to be in contrast to the imaging study by Cho et al²¹ that describes a relationship between the blooming artifact of the vessel-occluding thrombus in magnetic resonance imaging with cardioembolic stroke cause. On the other hand, they concur perfectly with a CT study,²² showing a clear relationship between the hyperdense artery sign (which undoubtedly reflects a higher erythrocyte content) and noncardioembolic stroke cause.

Nevertheless, as our study addresses basic differences and similarities in clot composition, possible discrepancies with some of these studies do not affect the derived conclusions.

In addition, cardioembolic thrombi were associated with different interventional and outcome characteristics when compared with noncardioembolic thrombi (Figure 2B and 2C). The higher number of retraction maneuvers required for the extraction of cardioembolic thrombi may be because of a higher organizational degree of these thrombi, which is congruent with our histopathologic findings. Worse outcome values, reflected by higher mRS and NIHSS scores in these groups, are also in line with published findings: several studies report worse (also long-term) outcome and higher NIHSS values of patients with cardioembolic strokes, especially if caused by atrial fibrillation.^{16–18,23,24}

Thrombi from cryptogenic stroke patients showed the same basic pattern as cardioembolic thrombi, with higher proportions of F/P and smaller fractions of RBCs, in clear distinction from noncardioembolic thrombi (arterioembolic and TOAST 4 other determined cause; Figures 1 and 2). Analogous similarities between cryptogenic and cardioembolic and differences to noncardioembolic strokes were also observed regarding interventional and outcome parameters (Figure 2B and 2C). Differences between cryptogenic and noncardioembolic strokes were somewhat less pronounced than those between cardioembolic and noncardioembolic strokes, but still they are significant for F/P and most of the prespecified clinical and interventional parameters. Importantly, the histological differences were even more distinct when patients with incomplete diagnostic workup were excluded, providing strong support for a genuine similarity between cryptogenic and cardioembolic thrombi.

These findings suggest that the majority, although not all, of cryptogenic strokes have a cardioembolic origin. This is in line with recent reports, indicating that the detection rate of atrial fibrillation in cryptogenic stroke patients increases substantially when these patients are examined by multimodal long-term recording.²⁵

Advanced age is associated with poorer clinical outcome, possibly with less favorable technical thrombectomy results and also with a higher prevalence of atrial fibrillation and therefore higher incidence of cardioembolic events.^26,27 Conversely, patients with cardioembolic strokes have a higher mean age than patients with strokes of other causes.^23,24 The observed associations of technical and clinical outcome parameters and stroke causes might, therefore, reflect a confounding influence of age. However, in line with other reports,²⁸ the mean age of cardioembolic stroke patients in our sample was significantly higher than that of all other TOAST groups, including cryptogenic strokes. Likewise, TOAST 4 patients were significantly younger than patients in all other TOAST groups, including TOAST 1 (arterioembolic). Thus, the similarities in technical and clinical outcome parameters of cryptogenic and cardioembolic strokes on the one hand and of TOAST 1 and TOAST 4 patients on the other hand cannot be explained by overlapping age distributions.

As the application of intravenous lysis (recombinant tissue-type plasminogen activator) may directly influences thrombus composition by interfering with the coagulation system, differences in histological clot composition in patients with and without intravenous thrombolysis are conceivable. However, as we did not observe any histological differences between these patient groups, such a possible bias can be ruled out.

Taken together, our congruent histopathologic, clinical, and interventional data support the hypothesis that cardioembolic and so-called cryptogenic strokes have overlapping causes but are distinct from the TOAST groups 1 and 4 (Figure II in the online-only Data Supplement).

Without doubt, secondary prevention measures are more effective if they are specifically adapted to identified stroke causes.^29,30 Two thirds of our cryptogenic cases fulfill the criteria of embolic strokes of undetermined source (ESUS).¹⁵ In these patients, the decision of anticoagulant versus antiplatelet therapy as secondary prevention still remains a matter of debate. Two large studies comparing direct anticoagulants with standard therapy of antiplatelet therapy in ESUS have recently been initiated (https://clinicaltrials.gov/ct2/show/NCT02313909 and https://clinicaltrials.gov/ct2/show/NCT02239120).

Presently, the worldwide standard of secondary prevention in cryptogenic stroke relies on platelet inhibition,³¹ a strategy that might be revisited, at least for the group of patients with large-vessel occlusions.

The previously discussed findings are mainly based on group comparisons, accounting for the considerable high interindividual variabilities of the evaluated parameters. However, it would obviously be desirable, if thrombus histology would allow to make inferences to stroke causes also in individual patients.

The receiver operating characteristic analysis with a selected cutoff value of 60% F/P content resulted in a positive predictive value of 80.7% for distinguishing cardioembolic strokes. Accordingly, the diagnostic benefit of such an approach may be substantial even for individual patients. Thrombus histology alone may not allow to make treatment decisions in individual patients. Nevertheless, it may be worthwhile to keep and conservate thrombus specimens retrieved during endovascular stroke therapy (rather than to simply discard them) for subsequent analysis in cases in which the routine workup does not clarify stroke cause.