Histopathological evaluation of thrombus in patients presenting with stent thrombosis. A multicenter European study: a report of the prevention of late stent thrombosis by an interdisciplinary global European effort consortium

Abstract

Background: Stent thrombosis (ST) is a rare but serious complication following percutaneous coronary intervention. Analysis of thrombus composition from patients undergoing catheter thrombectomy may provide important insights into the pathological processes leading to thrombus formation. We performed a large-scale multicentre study to evaluate thrombus specimens in patients with ST across Europe.

Methods: Patients presenting with ST and undergoing thrombus aspiration were eligible for inclusion. Thrombus collection was performed according to a standardized protocol and specimens were analysed histologically at a core laboratory. Serial tissue cross sections were stained with haematoxylin-eosin (H&E), Carstairs and Luna. Immunohistochemistry was performed to identify leukocyte subsets, prothrombotic neutrophil extracellular traps (NETs), erythrocytes, platelets, and fibrinogen.

Results: Overall 253 thrombus specimens were analysed; 79 (31.2%) from patients presenting with early ST, 174 (68.8%) from late ST; 79 (31.2%) were from bare metal stents, 166 (65.6%) from drug-eluting stents, 8 (3.2%) were from stents of unknown type. Thrombus specimens displayed heterogeneous morphology with platelet-rich thrombus and fibrin/fibrinogen fragments most abundant; mean platelet coverage was 57% of thrombus area. Leukocyte infiltrations were hallmarks of both early and late ST (early: 2260 ± 1550 per mm(2) vs. late: 2485 ± 1778 per mm(2); P = 0.44); neutrophils represented the most prominent subset (early: 1364 ± 923 per mm(2) vs. late: 1428 ± 1023 per mm(2); P = 0.81). Leukocyte counts were significantly higher compared with a control group of patients with thrombus aspiration in spontaneous myocardial infarction. Neutrophil extracellular traps were observed in 23% of samples. Eosinophils were present in all stent types, with higher numbers in patients with late ST in sirolimus-and everolimus-eluting stents.

Conclusion: In a large-scale study of histological thrombus analysis from patients presenting with ST, thrombus specimens displayed heterogeneous morphology. Recruitment of leukocytes, particularly neutrophils, appears to be a hallmark of ST. The presence of NETs supports their pathophysiological relevance. Eosinophil recruitment suggests an allergic component to the process of ST.

Keywords: Eosinophils; Histopathology; Neutrophil extracellular traps; Neutrophils; Platelets; Stent thrombosis; Thrombus aspiration.

Figure 1

Frequency of stent types and timing of stent thrombosis. (A) Percent distribution of thrombus aspirates retrieved from bare metal (white bar) and drug-eluting (black bars) stents (n = 245), sirolimus, paclitaxel, everolimus, zotarolimus, biolimus; (B) percent distribution of early vs. late stent thrombosis in drug-eluting stents (n = 166); (C) percent distribution of early vs. late stent thrombosis in bare metal stents (n = 79); (D) percent distribution of early vs. late stent thrombosis in first and second-generation drug-eluting stents. Sirolimus (n = 31), paclitaxel (n = 22), everolimus (n = 59), zotarolimus (n = 23).

Figure 2

Histological analysis of thrombus aspirated from intracoronary stents. (A) Representative Carstairs stainings of thrombus aspirates (n = 11). Upper row: overview image (left bar, 50 µm; other bars 100 µm). Bottom row: insets of the overview images (left bar, 25 µm; other bars 50 µm); platelets are stained in grey blue to navy, fibrin/fibrinogen in red and erythrocytes (RBC) in yellow; (B) representative image of platelet aggregation area (CD41 positive) in thrombus aspirates (n = 7). Nuclei were counterstained with Hoechst. Bar, 100 µm; (C) fibrin/fibrinogen immunofluorescence staining (n = 34). Nuclei were counterstained with Hoechst. Bar, 100 µm; (D) Coverage of whole thrombus area (%) with RBC, platelet (CD41) and fibrin/fibrinogen rich areas. Due to co-localization, overall coverage exceeds 100%; data are shown as mean + SD.

Figure 3

Leukocyte accumulation in stent thrombus specimens. (A) Leukocyte accumulation in human stent thrombus specimens. Left images: Haematoxylin–eosin staining (n = 253). Arrows indicate granulocytes, arrowheads indicate mononuclear cells. Right images: immunofluorescence staining of neutrophil elastase to identify neutrophils (n = 229). Nuclei are counterstained with Hoechst. Bars, 200 µm (upper row) and 50 µm (bottom row); (B) Quantification of leukocytes and neutrophils in early (n = 67) vs. late (n = 162) stent thrombosis (leukocytes: P = 0.44; neutrophils: P = 0.81); (C) Leukocytes and neutrophils in stent thrombosis from drug-eluting stents (n = 149) and bare metal stents (n = 73) and in thrombi aspirated from patients with spontaneous myocardial infarction (spont. myocardial infarction; n = 104) (P < 0.05 for drug-eluting stents vs. spont. myocardial infarction and bare metal stents vs. spont. myocardial infarction). Shown are mean + SD, each symbol in (B) and (C) represents one individual patient.

Figure 4

Detection of neutrophil extracellular traps in stent thrombus specimens. (A) Immunofluorescence images of neutrophil extracellular traps stained for neutrophil elastase and DNA (Hoechst). Extracellular DNA originates from neutrophil elastase positive neutrophils. Arrowheads, nuclei; arrows, neutrophil extracellular trap fibres. Bars, 5 µm; (B) number of neutrophil extracellular traps in early (n = 23) vs. late (n = 37) stent thrombosis (P = 0.75); (C) quantification of neutrophil extracellular traps in thrombi derived from drug-eluting stents (n = 36), bare metal stents (n = 23), and spontaneous myocardial infarction (spont. myocardial infarction) (n = 25) (P = 0.13); data are shown as mean + SD, each symbol in (B) and (C) represents one individual patient.

Figure 5

Eosinophil accumulation in stent thrombus specimens. (A) Eosinophils in human thrombi were identified by Luna staining. Arrowheads indicate eosinophils (red-brown colour). Bars, 100 µm (left) and 50 µm (right); (B) Number of eosinophils in early (n = 71) vs. late (n = 146) stent thrombosis (P = 0.63); (C) Quantification of eosinophils in thrombi derived from drug-eluting stents (n = 143), bare metal stents (n = 66) and spont. myocardial infarction (n = 93) (P = 0.85); (D) Number of eosinophils in very late ST according to drug-eluting stents type; SES (n = 24), PES (n = 16), EES (n = 27), ZES (n = 6) (P = 0.14); (E) Number of eosinophils according to drug-eluting stents polymer type; bioabsorbable polymer (BP drug-eluting stents): n = 8, durable polymer (DP drug-eluting stents): n = 120 (P = 0.58); data are shown as mean + SD, each symbol in (B–E) represents one individual patient.