VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations
René Hägerling, Dominik Drees, Aaron Scherzinger, Cathrin Dierkes, Silvia Martin-Almedina, Stefan Butz, Kristiana Gordon, Michael Schäfers, Klaus Hinrichs, Pia Ostergaard, Dietmar Vestweber, Tobias Goerge, Sahar Mansour, Xiaoyi Jiang, Peter S. Mortimer, Friedemann Kiefer
René Hägerling, Dominik Drees, Aaron Scherzinger, Cathrin Dierkes, Silvia Martin-Almedina, Stefan Butz, Kristiana Gordon, Michael Schäfers, Klaus Hinrichs, Pia Ostergaard, Dietmar Vestweber, Tobias Goerge, Sahar Mansour, Xiaoyi Jiang, Peter S. Mortimer, Friedemann Kiefer
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Clinical Research and Public Health Dermatology Vascular biology

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations

Abstract

BACKGROUND. Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS. VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy–based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS. Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION. VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING. Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

Authors

René Hägerling, Dominik Drees, Aaron Scherzinger, Cathrin Dierkes, Silvia Martin-Almedina, Stefan Butz, Kristiana Gordon, Michael Schäfers, Klaus Hinrichs, Pia Ostergaard, Dietmar Vestweber, Tobias Goerge, Sahar Mansour, Xiaoyi Jiang, Peter S. Mortimer, Friedemann Kiefer

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Figure 4

Quantitative parameters distinguish the lymphatic vascular network in control and patient skin biopsies following automated extraction of vessel shape and connectivity characteristics.

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Quantitative parameters distinguish the lymphatic vascular network in co...
Parameters that distinguished the control sample (n = 1) from the lymphedematous (n = 1) and lymphangiomatosis sample (n = 1) were calculated from automatically extracted vessel shape and connectivity data. (A–F) The line in the box-and-whisker plots in represents the median, the boxes represent the upper and lower quartile, and the end of the whiskers represent the 1.5-fold interquartile range. (G and H) Results of the connectivity analysis for (G) total branching point degree and (H) the higher per-edge branching degree. The total branching point analysis is cantered on the spheres forming each node, while the higher degree per-edge analysis for each segment considers the maximum branching degree of both connected nodes. A total branching point degree of zero therefore represents spherical vascular elements, whereas a higher per-edge branching degree of one represents the number of elongated, nonconnected, nonbranched vascular elements. Note the decreased segment length and distance in lymphedema- and lymphangiomatosis-affected samples as well as the increased number of spherical and elongated, nonbranched vessel elements in the lymphedematous sample. Supplemental Figure 3 provides an overview of the definitions of the extracted parameters depicted in Figure 4. Mann-Whitney U test with a Bonferroni correction for multiple comparison was used to compare data between groups. **P < 0.01, ***P < 0.001.