Easy, Robust, and Automated Tumor/Stroma Separation

VirtualDoubleStaining™ (VDS) is a novel and patent protected method for automated, robust and verifiable tumor/stroma separation. The method has been developed and validated in collaboration with the NordiQC and thoroughly tested in practical clinical environments at major
diagnostic pathology labs across Europe.

The Devil is in the Data
Important clinical decisions are made by pathologists based on the assessment of biomarkers. It is well known that there is significant intra- and inter reader variability associated with diagnostic reading and interpretation of biomarker expression. There is an urgent need to reduce or eliminate this type of variability for biomarkers to be used as prognostic and predictive markers. Data from thousands of tissue samples supports that image analysis for tumor area detection, combined with quantification of biomarker expression, provide significant improvements in both reproducibility and accuracy.

Virtual double staining is done in a few easy steps
VDS builds on aligning two digitized serial sections; one stained with a tumor marker e.g. PCK, H&E, CK5/CK7+p63 etc. and another with a cancer biomarker e.g. Ki-67, ER, PR, etc. It can be used with single biomarker stains as well as cocktail biomarker stains.

The two images are aligned
The Visiopharm’s patented alignment software is used to create an overlay outlining the tumor cells.

The tumor region is identified using image analysis
The outlined tumor region is then transferred to the section stained with the analytical biomarker to be analyzed with the relevant image analysis APP.

The Visiopharm APPs for Breast Cancer Ki-67, ER and PR are validated for in vitro diagnostic use (CE IVD) in Europe utilizing the PCK VirtualDoubleStaining™ capability (ref. 1). All other VirtualDoubleStaining™ applications are for Research Use Only.

Benefits of VirtualDoubleStaining™

Improved data quality:

  • Objective, verifiable detection of tumor to exclude proliferating lymphocytes
  • Detect and exclude Ductal Carcinoma In Situ
  • Can be based on manual outlining as well as fully automated quantitative image analysis

Fully automated Tumor/Stroma separation:

  • Avoid time-consuming manual outlining of tumor areas
  • Can be performed by lab technicians
  • Pathologist can confirm or edit the result


Virtual Triple Staining

The tumor marker can also be virtually aligned with two slides creating a sandwich around the tumor marker. Allowing Virtual Triple Staining.


1. Package Inserts for Visiopharm Ki-67 APP, Breast Cancer, ER APP, Breast Cancer and PR APP, Breast Cancer.