Is Histologic Thrombus Composition in Acute Stroke Linked to Stroke Etiology or to Interventional Parameters?

Preinterventional Imaging

Unenhanced brain CT and contrast-enhanced CTA of the supra-aortic brain-supplying arteries (Somatom Definition Flash or Definition AS+ scanner; Siemens, Erlangen, Germany) were performed in all patients either in our center or in the referring hospital. All available unenhanced brain CT scans were retrospectively re-analyzed for the presence or absence of the dense artery sign, for thrombus density calculated as the average of 2 Hounsfield unit measurements in the occlusion indicated by CTA and/or the dense artery sign, and for early ischemic changes according to the ASPECTS in cases of middle cerebral artery ischemia.¹⁰

Mechanical Thrombectomy

MT using an aspiration catheter and/or a stent retriever was performed with the patient under general anesthesia in a biplanar DSA system (Allura Xper FD20; Philips Healthcare, Best, the Netherlands). By means of the Seldinger technique, a guiding catheter was inserted transfemorally, the large vessel proximal to the suspected occlusion was probed, and selective DSA was performed using Ultravist 300 as contrast agent (iopromide; Bayer HealthCare, Berlin, Germany). After identification of the arterial occlusion, a distal access or aspiration catheter was introduced coaxially (5F Sofia distal access catheter or 6F Sofia Plus aspiration catheter, MicroVention, Aliso Viejo, California; 3MAX/4MAX/5MAX or ACE 64 reperfusion catheter, Penumbra, Alameda, California) over a microwire (Traxcess14, MicroVention; Transend EX, Stryker, Kalamazoo, Michigan) and, if possible, docked onto the thrombus. Subsequently, direct contact thrombus aspiration was performed using a VacLok syringe (Merit Medical, South Jordan, Utah). If recanalization was not sufficient, the aspiration maneuver was repeated or, as an alternative, a stent retriever (Solitaire FR, Medtronic, Minneapolis, Minnesota; Trevo XP Provue System, Stryker; EmboTrap II Revascularization Device, Cerenovus, Galway, Ireland; Separator 3D System, Penumbra) was inserted, positioned in the thrombus, and 1 or multiple stent-retrieving maneuvers were then performed under constant aspiration. The thrombi were collected in a sterile manner, and those thrombi suitable for histopathologic analysis were immediately preserved in formalin. The records of the endovascular procedures were re-analyzed for interventional parameters, including the time interval between stroke onset and MT, the duration of MT, the endovascular devices/technique used, the number of retrieving maneuvers, and interventional complications. The achieved global recanalization success was quantified using the modified TICI (mTICI) scale.¹¹

Histology and Immunohistochemistry

All investigations were performed on 1-μm sections. Histochemical and immunohistochemical staining was used to determine the quantitative composition of the thrombi. The stains used were hematoxylin-eosin (H&E) for RBC (appears reddish), Ladewig trichrome stain for fibrin deposits (appears brick red), Elastica van Gieson stain for collagen fibers (appears reddish), Von Kossa stain for calcifications (stains black, On-line Fig 2), naphthol AS-D chloroacetate stain for granulocytes (detected by their esterase content, On-line Fig 3), and Prussian blue staining for iron ions in siderophages. In addition, immunohistochemical staining for CD68 (positive for Ki-M1P, macrophages, and activated platelets, their size allowing exact quantification) and for CD45 (positive for leukocyte common antigen and lymphocytes) was performed (On-line Fig 3). Pretreatments and antibody dilutions were performed as described in Table 1. In brief, the endogenous peroxidase activity was first blocked by incubating the sections in 3% H₂O₂ in phosphate-buffered saline. This step was followed by a blocking step with 10% fetal calf serum in phosphate-buffered saline for 10 minutes at room temperature, followed by incubation with the primary antibody for 1 hour at room temperature. The sections were then incubated with the secondary antibody (biotin-labeled antibody). Because the sections were stained using the Dako-Autostainer-Plus (Agilent Technologies, Santa Clara, California), the ZytoChemPlus horseradish peroxidase polymer system (mouse/rabbit) (REF:POLHRP-100, Dako, Hamburg, Germany) was used for detection. Cell nucleus counterstaining was performed with hematoxylin.

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Fig 2.

Differences between the different stroke subtypes in the proportions of platelets and macrophages (subunits A and B) and differences between the individual thrombi in the proportions of platelets and the number of fragments that were ultimately retrieved by contact aspiration alone, stent retrieval alone, or a combination of the 2 techniques (subunits C and D).

The stained sections were digitized using a Leica slide scanner (Leica Biosystems, Wetzlar, Germany). From the scanned files, either the whole thrombus area (H&E for RBC quantification, Ladewig trichrome for fibrin quantification) or the whole thrombus area divided into subareas with an edge length of 1000 μm (CD68 for monocytes/macrophages and activated platelets, CD45 for leukocytes) was extracted and analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland).¹² After we adjusted the hue, saturation, and brightness, we adjusted the “color threshold” so that colored particles or colored areas could be determined using the “Analyze Particles” function in ImageJ.¹³ Regions with densely packed RBC or platelets and areas of dense fibrin deposition were included in the evaluation as coherent areas (On-line Fig 4).

The applied staining methods also allowed an age estimation of the thrombi. Because the Prussian blue stain, indicating iron ions in siderophages, turns positive approximately 72 hours after coagulation, the absence of any blue coloration indicated a thrombus age of <72 hours. In addition, the intermingling of thrombi with collagen fibers, reddish in Elastica van Gieson staining, would argue for their reorganization, a finding that does not occur in fresh thrombi (<72 hours) (On-line Fig 2). Furthermore, calcifications (visualized by the Von Kossa staining) are not found in fresh thrombi (On-line Fig 2).

The areas occupied by fibrin, platelets, RBC, and WBC (including macrophages, lymphocytes, and granulocytes) were quantified as percentages. Furthermore, we investigated the presence of siderophages, collagen fibers, calcifications, and the number of histologic fragments of the thrombi.

Statistical Analysis

Statistical analysis was performed with the SPSS software package (Version 25.0; IBM, Armonk, New York). Normal distribution was determined with the Kolmogorov-Smirnov test. Data are given as frequency counts and percentage values, medians and ranges, and means and SDs, as appropriate. The Kruskal-Wallis test was used to evaluate overall group differences between stroke subtypes in thrombus composition (fibrin, platelets, RBC, WBC, macrophages, lymphocytes, granulocytes, number of thrombus fragments). In case of significant overall group differences, pair-wise comparisons of stroke subtypes were performed with the Kruskal-Wallis 1-way ANOVA for multiple samples with a Bonferroni correction. To investigate a possible influence of thrombus composition on MT, we performed a Kruskal-Wallis test (with post hoc Bonferroni correction for the second step of analysis) or a Spearman rank correlation analysis, as appropriate. Finally, a Mann-Whitney U test was used to determine whether rtPA administration resulted in differences in fibrin content or fragmentation of the thrombi. A P value < .05 was considered statistically significant.