Detailed histological analysis of a thrombectomy-resistant ischemic stroke thrombus: a case report

A 63-year old female was admitted in the late evening to a primary stroke center at a peripheral hospital with an acute left sensorimotor hemi-syndrome and visual extinction, during the same day she had noticed some transient hemianopia. An initial non-contrast-enhanced computed tomography (NCCT) revealed no intracerebral hematoma or other abnormalities but as the exact time of symptom onset was unknown, no intravenous thrombolysis was administered. The patient was treated with aspirin and low-molecular heparin, after which the symptoms disappeared spontaneously overnight.

Early next morning the patient experienced an acute recurrence of the left sensorimotor hemi-syndrome and repeated imaging with NCCT showed a hyperdense right middle cerebral artery (MCA) sign (average 138 HU), with a partial infarction of the basal ganglia and no signs of intracranial hemorrhage (Fig. 1a). CT-angiography confirmed distal M1 and inferior branch M2 right-sided MCA occlusions with good pial collateralization (Fig. 1b).

The patient was transferred to AZ Groeninge Hospital in Kortrijk, Belgium, for endovascular therapy, arriving 2.5 h after symptom onset. At arrival, the patient had a score of 11 at the National Institute of Health Stroke Scale (NIHSS). Her medical history included a successful treatment in 2001 of an invasive, hormone-receptor positive ductal breast carcinoma but no cardiovascular risk factors except a minor dyslipidemia, for which she was treated with rosuvastatin.

After a brief discussion in the multidisciplinary stroke team, an endovascular procedure was performed under general anesthesia. Vascular access was achieved 25 min after arrival with insertion of an 80 cm long 8French (F) femoral arterial sheath (Arrow, Morrisville, US) into the right common carotid artery. A 95 cm 8F balloon guide catheter (BGC; FlowGate, Stryker Neurovascular, Fremont, California, USA) was inserted through the sheath and positioned with the tip in the distal cervical segment of the internal carotid artery. A 0.021-in. microcatheter (Prowler Select Plus, Codman Neurovascular J&J, Raynham, Massachusetts, US) was advanced over a 0.014″ microwire (Transend-EX Platinum, Stryker Neurovascular, Fremont, California, USA) through the occluded distal M1- and inferior M2-segments of the right MCA. The microwire was exchanged for a 5 × 33 mm stent retriever thrombectomy device (EmboTrap II, Cerenovus – J&J, Wokingham, UK), which was subsequently deployed in the occluded parts of the M1-M2 segments. Under temporary BGC-occlusion but without an intermediate aspiration catheter, the stent retriever was slowly retracted in three consecutive unsuccessful attempts to retrieve the clot. Technically identical procedures where then performed another three times but with a 4 × 30 mm Trevo-XP Provue stent retriever (Stryker Neurovascular, Fremont, California, US). These retrieval attempts were also unsuccessful with an unchanged complete occlusion. The initial stent retriever strategy was then changed to direct contact aspiration utilizing a 125 cm 5F Sofia intermediate catheter (MicroVention, Aliso Viejo, California, US), inserted through the BGC over the microcatheter and microwire, reaching the occluded segment of the right MCA. Three attempts with manual aspiration were made with a 20 cc syringe, but the occlusion remained unchanged with no clot removal. Finally, the Sofia intermediate catheter was replaced by the slightly larger Catalyst-6 (Stryker Neurovascular, Fremont, California, US) aspiration catheter and another two attempts were made with a combination PROTECT^PLUS technique [10] in conjunction with the EmboTrap stent retriever. At the second of these two attempts, focusing on holding the clot by a steady negative pressure in the aspiration catheter during withdrawal of the stent retriever, microcatheter and aspiration catheter simultaneously under BGC-induced carotid flow-arrest, a successful retrieval of a complete clot was achieved in the eleventh removal attempt, resulting in a TICI 2c revascularization (Fig. 1c). Macroscopically, the thrombus appeared unusually hard with a clear white coloring (Fig. 1d).

The day after the endovascular treatment a brain magnetic resonance imaging (MRI) investigation was performed including diffusion-weighted- (DWI), fluid-attenuated inversion recovery (FLAIR) and contrast enhanced T1-weighted images. DWI demonstrated restricted diffusion in the right basal ganglia and several small cortical and subcortical lesions (Fig. 1e), consistent with acute small embolic infarctions. Magnetic Resonance Angiography (MRA-TOF) showed a complete recanalization of all branches of the right middle cerebral artery (Fig. 1f).

A screening was made with an implantable loop recorder in collaboration with the Cardiology Department to search for an underlying cardioembolic source and the patient was thoroughly investigated regarding a potential atherothrombotic source or an underlying coagulopathy, but no causative pathology could be found.

After thrombus removal, the thrombus was fixed in 4% paraformaldehyde, embedded in paraffin and sectioned in 5 μm sections. To obtain a general overview of the thrombus structure, a hematoxylin and eosin (H&E) staining was performed (Fig. 2a-c). H&E analysis showed various regions that included fibrin/platelet aggregations (pink), red blood cells (RBCs; red) and nuclear material (dark blue) (Fig. 2a). Whereas one fragment was more RBC-rich (Fig. 2b; red), the majority of the thrombus stained largely dark blue or purple, indicative of nucleic acid material with little RBCs (Fig. 2c; blue smears). To confirm the presence of extracellular DNA, a highly specific DNA stain (Feulgen’s reaction) was performed. High amounts of extensive DNA networks were found throughout the majority of the thrombus (Fig. 2d, pink). To specifically identify fibrin and RBCs, a Martius Scarlet Blue (MSB) staining was performed, which confirmed the absence of RBCs in the DNA-rich thrombus parts (Fig. 2e). Fibrin (red on MSB staining) was predominantly found on the outer rim of the thrombus but not in the DNA-rich inner core (Fig. 2e). Similarly, immunohistochemical platelet staining showed the presence of platelets only on the outer shell of the thrombus (Fig. 2f; purple). We previously identified von Willebrand factor (VWF) as an important constituent of stroke thrombi, conferring resistance to fibrinolytic therapy in mice [11]. Strikingly, using an immunohistochemical VWF staining, very high amounts of von Willebrand factor-positive smears were identified in the DNA-rich area (Fig. 2g, purple).

Given our recent findings showing the presence of neutrophil extracellular DNA traps (NETs) in stroke thrombi, we stained for citrullinated histone H3 (H3Cit), a marker of NETosis [12]. H3Cit-positive staining was found within nuclei and also extracellularly as smears (Fig. 2h, purple). Immunostaining for the leukocyte marker CD45 showed an abundant and dense signal in the DNA-rich areas (Fig. 2i, purple). Together, the staining results for DNA, H3Cit and CD45 suggest that leukocytes could be the origin of extracellular DNA, of which a significant portion is decondensed, as seen in NETs. The uncommon structure is further underlined by the von Kossa staining, which revealed the exceptional presence of high amounts of microcalcifications (Fig. 2j, black).