Early Postoperative Immunothrombosis of Bioprosthetic Mitral Valve and Left Atrium: A Case Report

Figures

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  Figure 1. General examination of the excised bioprosthetic valve. (A) A massive thrombus located on the inflow side of the excised BHV; (B) Thrombosis-free outflow side of this BHV; (C) Polymorphonuclear leukocytes (indicated by red arrows) heavily infiltrated the thrombus (top) but did not invade the prosthetic leaflets (bottom). Hematoxylin and eosin staining, magnification ×200. Scale bar = 100 µm; (D) Left atrial thrombus also contained numerous polymorphonuclear leukocytes. Hematoxylin and eosin staining, magnification ×200. Scale bar = 100 µm.

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  Figure 2. Ultrastructural analysis of thrombotic masses and prosthetic tissues. (A) Neutrophil-free BHV (pericardium) as compared to the neutrophil-rich clot (thrombus). Right image represents a close-up of the left image (demarcated by a red contour). Yellow arrows at the left image demarcate thrombus and bioprosthetic bovine pericardium. Yellow arrows at the right image indicate neutrophils. Magnification ×500, scale bar = 100 µm (left image) and magnification ×2500, scale bar = 20 µm (right image); (B) Neutrophil-rich left atrial clot. Right image represents a close-up of the left image (demarcated by a red contour). Yellow arrows at the right image indicate neutrophils. Magnification ×500, scale bar = 100 µm (left image) and magnification ×2500, scale bar = 20 µm (right image). Backscattered scanning electron microscopy (EM-BSEM method) [15], accelerating voltage 10 kV.

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  Figure 3. Neutrophil invasion and abundant NETs indicate immunothrombosis of BHV. (A) Immunohistochemical staining of the prosthetic thrombus identified platelets (CD41-positive staining), fibrin, and neutrophils (ELA2- and MPO-positive staining). CD45 staining was inconsistent. Macrophages (CD68), T cells (CD3) and B cells (CD19) were not detected. Magnification ×200. Scale bar = 100 µm; (B) Immunofluorescence staining showed the co-localization of citrullinated histone H3 with neutrophil elastase (ELA2) and nuclear dye (4′,6-diamidino-2-phenylindole, DAPI) within the thrombus indicating the formation of NETs. Sections without the primary antibody staining but still counterstained with DAPI were used as a negative control. Magnification ×200. Scale bar = 50 µm.

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  Figure 4. Neutrophil invasion and NETs in the left atrial thrombus confirm immunothrombosis. (A) Immunohistochemical staining of the left atrial thrombus identified platelets (CD41-positive staining), fibrin, and neutrophils (ELA2- and MPO-positive staining). CD45 staining, macrophages (CD68), T cells (CD3) and B cells (CD19) were not detected. Magnification ×200. Scale bar = 100 µm; (B) Immunofluorescence staining showed the co-localization of citrullinated histone H3 with neutrophil elastase (ELA2) and nuclear dye (4′,6-diamidino-2-phenylindole, DAPI) within the thrombus indicating the formation of NETs. Sections without the primary antibody staining but still counterstained with DAPI were used as a negative control. Magnification ×200. Scale bar = 50 µm.

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  Figure 5. Immunothrombosis of BHV is not related to prosthetic valve endocarditis. (A) Negative Gram staining of the clot at the prosthetic surface. Magnification ×400. Scale bar = 100 µm; (B) Negative Gram staining of the clot and underlying prosthetic tissues. Magnification ×400. Scale bar = 100 µm; (C) Positive control for Gram staining (bacterial vegetations on the leaflet excised from the patient with prosthetic valve endocarditis). Black arrows indicate bacteria. Magnification ×400. Scale bar = 100 µm; (D) Positive control for Gram staining (bacteria within the microthrombi on the leaflet surface excised from the patient with prosthetic valve endocarditis). Black arrows indicate bacteria. Magnification ×400. Scale bar = 100 µm; (E) Negative periodic acid–Schiff staining of the clot and underlying prosthetic tissues. Magnification ×400. Scale bar = 100 µm; (F) Positive control for periodic acid–Schiff staining (intact bovine pericardium intentionally contaminated with the fungi). Black arrows indicate the fungi. Magnification ×400. Scale bar = 100 µm.