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Rikke Riber-Hansen MD, PhD Institute of Pathology

Digital image analysis as a catalyst for applied stereology

The digital image analysis

Definition Extraction of meaningful information from digital images by dedicated computer software

HER-21

1 Brügmann et all

Ki67/MART-12

1 Nielsen et all

The digital image analysis process

Preprocess Enhance structures of interest, i.e. nuclei or colour Suppress noise

The digital image analysis process

Segmentation Into the relevant components, i.e. nuclei versus non-nuclei, or IHC positive versus IHC negative

The digital image analysis process

Postprocessing Refining the segmentation

Segmentation

Is often predominantly based on differences in colour

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Segmentation by colour Segmentation by colour

Pitfalls

Holes

Folds

Ink

Unspecific staning

Immunohistochemical requirements

Sensitivity Specificity Optimisation

Stability over time Riber-Hansen et al. APMIS 2012; 120(4):276-89

Begelman et al. IEEE Trans Med Imaging 2009:28;1317-1324

Colour spectrums overlap

DAB

Haematoxylin

Immunohistochemical requirements

Tuominen VJ, Ruotoistenmaki S, Viitanen A, Jumppanen M, Isola J. Breast Cancer Rec. 2010;12:R56

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Why digital image analysis?

Workload reduction

Reduced inter/intraobserver

variation

Quantitiative results

Requirements for new tests

Analytical validity: How accurately does the test measure the feature of interest? Clinical validity: How well does the test predict the outcome of interest?

Example no. 1

Metastasis size: a known prognostic factor in melanoma, and breast cancer

A quick estimate of size

Metastasis diameter

Metastasis diameter and outcome

van der Plough et al. JCO 2011;16:2206-2214

Clinical validity

Can it be reproduced?

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HE S-100 HBM-45

ICC = 0.88

Does this make this analytically valid?

Analytical validity: How accurately does the test measure the feature of interest?

So reproducibility does not guarantee analytical validity

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Problem no. 1

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Solution: Stereology

No. of steps Max

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No. of steps

Riber-Hansen R et al. Cancer 2009: 115; 2177-2187

A quick estimate of size

Metastasis diameter

Clinically valid Analytically invalid

Problem: Workload + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

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Solution

Digital image analysis based on a stereological measurement: the total metastatic volume

Clinical validity – unpublished data

Example no. 2

Nielsen PS et al. Modern Pathology 2012

Ki67/MART-1

Manual stereology

What did the digital image analysis add?

Nielsen PS et al. Arch Pathol Lab Med. 2012:136;627-634

New prognostic markers

Digital image analysis may act as a high-throughput device to examine multiple stereologically sound ways of quantification for a given marker.

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Conclusions

Digital image analysis

Is versatile Requires high quality sections and staining Is sensitive to suboptimal haematoxylin staining Needs to be taken into account from the onset of the study Should be based on stereologically sound estimates