Assessment of histological characteristics, imaging markers, and rt-PA susceptibility of ex vivo venous thrombi

Figures

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  Figure 1. The fractional area of collagen, fibrin, platelets, and erythrocytes (RBC) present in (top) all analyzed VTE specimen (26 samples), (bottom left) pulmonary emboli (14 samples), and (bottom right) deep vein thrombi (12 samples). Components were assessed using histochemical staining. Colorimetric analysis was conducted in ImageScope (Leica, Biosystems, Germany) to identify pixels associated with each components. Horizontal red lines indicate median values. The top and bottom portions of the blue box represent the 25th and 75th percentiles, respectively, and whiskers extent to the data points not considered outliers. Red crosses indicate outliers, which correspond to datapoints greater than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{q}}_{75} + 1.5 \times \left( {{{q}}_{75} - {{q}}_{25} } \right)$$\end{document}q75+1.5×q75-q25 or less than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs}…

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  Figure 2. Observed patient-to-patient variability in collagen observed in ex vivo thrombus samples. Deep vein thrombi (DVT) are shown in the left column, pulmonary emboli (PE) are shown in the right column. Collagen appears blue in the Masson’s trichrome stain and compromises 60% (top left) and less than 1% (bottom left) for DVT, and 14% (top right) and less than 1% (bottom right) for PE by area. Colorimetric analysis was conducted in ImageScope (Leica Biosystems, Germany) to identify pixels associated with collagen. Color thresholds were evaluated and accepted by a board-approved pathologist. The scale bars indicate 1 mm.

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  Figure 3. Trends in thrombus composition for fibrin, collagen, red blood cells (RBCs), and platelets for all samples (N = 26 total). The Pearson’s correlation coefficient (R) and p-values are reported for each respective pair.

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  Figure 4. Histological analysis for acute (less than 7 days old, N = 12) and chronic (greater than or equal to 7 days old, N = 14) thrombi. None of the 26 samples were excluded from analysis. Red crosses indicate outliers and horizontal red lines indicate median values. The top and bottom portions of the blue box represent the 25th and 75th percentiles, respectively, and whiskers extent to the data points not considered outliers. Outliers correspond to datapoints greater than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_{75} + 1.5 \times \left( {q_{75} - q_{25} } \right)$$\end{document}q75+1.5×q75-q25 or less than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_{25} - 1.5 \times \left( {q_{75} - q_{25} } \right)$$\end{document}q25-1.5×q75-q25, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym}…

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  Figure 5. Representative multiplexed immunofluorescent images collected from VTE sample. (A) DAPI (nucleated cells) in blue, (B) erythrocytes (pink), (C) CD61 (platelets, red), (D) collagen, types i, ii, and iii (orange), (E) fibrin (green), (F) PAI-1 (cyan), and (G) VEGR-1 (yellow). The scale bar in the lower left corner corresponds to a 20 µm distance (40 × magnification). Antibody information can be found in Supplemental Table 2. A total of eight different antibodies were used for analysis, with five shown here. Autofluorescence was used to visualize erythrocytes. No cross reactivity was observed for antibodies as assessed with human tonsil control samples.

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  Figure 6. (Top) Representative multiplexed thrombus image indicating nucleated cells assessed with DAPI (blue), PAI-1 (yellow) and CD31 (red). Orange arrows indicate representative locations for cells associated with revascularization (co-expression of DAPI and CD31). Magenta arrows indicate representative areas for co-expression of PAI-1 and CD31. The white bar is a 10 µm distance (40 × magnification). Multiplex IHC staining was performed on 5 micron FFPE tissue sections using the Opal 7-Color Manual IHC Kit (NEL861001KT; Akoya Biosciences) following the manufacturer instructions. All slides were counterstained with Spectral DAPI (1:10, Akoya Biosciences) nuclear stain and mounted with Slowfade Diamond Antifade mountant (Thermo Fisher Scientific). The slides were then scanned using the Vectra Polaris whole slides scanner (Akoya Biosciences), the regions of interest were selected using the Phenochart software (Akoya Biosciences) and analyzed using Inform software (Akoya Biosciences). Antibody information can be found in Supplemental Table 2. The individual channels for each components are shown in the lower three panels.

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  Figure 7. Comparison of sample thrombolytic susceptibility (mass loss) and magnetic susceptibility (QSM analysis). Error bars represent the range of positive QSM values observed within the sample. Separate segments of each specimen were subjected to the clot mass loss assay and imaging analysis. One sample was highly heterogenous (red cross), and gross observation indicated strong variation between the portion of that specimen that was subjected to rt-PA and the portion subjected to QSM analysis. The dashed yellow line is a least-squares best fit to the data. A significant correlation was observed between the thrombolytic and magnetic susceptibility of the sample (Spearman’s rho = 0.90, p < 0.01, N = 8 samples) The heterogenous sample (red cross) was excluded from analysis.