Comparative Analysis of Microvessel Density in Xenograft Tumors: Manual and Automated Methods for Qu

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Comparative Analysis of Microvessel Density in
Xenograft TumorsManual and Automated Methods
for Quantitating Changes in Tumor
VasculatureBelinda Cancilla, PhD
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Outline

• Spectrum Setup
• Organization of digital slides in research
  environment
• Aperio MVD Algorithm Optimization
• Standard CD31 xenograft images
• Overstained CD31 xenograft images
• Understained CD31 xenograft images
• MVD Analysis of Xenograft Studies
• Manual MVD analysis
• Semi-automated analysis using ACIS
• Semi-automated analysis using Aperio

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Spectrum Setup
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Spectrum Setup Projects
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Spectrum Setup Specimens
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Spectrum Setup Digital Slides
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Project Structure
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Specimen Structure
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Spectrum Setup TMAs
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TMA Specimen
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Aperio MVD Algorithm

• CD31 in MCF7 Xenograft

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Aperio MVD Algorithm Optimization
Tuning vessel stain
Analysis vessels
Analysis vessels (show excluded vessels)
Tuning vessel thresholding
Analysis lumen and vascular cells
Analysis lumen and vascular cells Include
incomplete vessels
Segmented vessel
18 Vessels (include incomplete vessels)
7 Vessels (exclude incomplete vessels)
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Aperio MVD Algorithm

• Overstained CD31 in MCF7 Xenograft

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Aperio MVD Algorithm Optimization
Tuning vessel stain
Tuning vessel thresholding
Tuning vessel thresholding
Dark stain threshold 160 Light stain threshold
215
Dark stain threshold 150 Light stain threshold
185
Analysis lumen and vascular cells Include
incomplete vessels
Segmented vessel
Analysis lumen and vascular cells Include
incomplete vessels
Segmented vessel
Smoothing 2 20 Vessels
Smoothing 5 9 Vessels
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Aperio MVD Algorithm
Understained CD31 in A549 Xenograft
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Aperio MVD Algorithm Optimization
Tuning vessel stain
Tuning vessel thresholding
Tuning vessel thresholding
Dark stain threshold 160 Light stain threshold
210
Dark stain threshold 210 Light stain threshold
235
Analysis lumen and vascular cells Include
incomplete vessels
Analysis lumen and vascular cells Include
incomplete vessels
Enlarged lumen
Smoother vessel
8 Vessels
Adjusted vessel parameters
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MDA-MB-231 Xenograft StudyCD31 Manual Counts









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MDA-MB-231 Xenograft StudyCD31 ACIS Analysis





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MDA-MB-231 Xenograft StudyCD31 Aperio Analysis





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Comparison of Results
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Alternative to MVD counts
CD31 in Angiosarcoma
Analysis lumen and vascular cells Include
incomplete vessels
289 Vessels
Tuning vessel thresholding
Positive Pixel Algorithm
Total Analysis Area 230081.6 um2 Total Stain
Area 51012.1 um2 CD31 staining ( area) 22
CD31 staining ( area) 17
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Summary Aperio MVD Algorithm

• Pros
• Identifies CD31 stained vessels in well stained
  xenograft images
• Identifies CD31 stained vessels in overstained
  xenograft images
• Identifies CD31 stained vessels in understained
  xenograft images
• Analysis allows semi-automated batch analysis
• Analysis is comparable to manual and other
  semi-automated methods
• Cons
• Uses lumen to define vessels not all vessels
  have nice round lumens
• Over-segmenting vessels based on lumen parameters
• Undercounting some samples need to re-adjust
  parameters for some samples within a batch
• MVD unit conversion need data in mm2 not µm2