Preliminary Validation of a Multispectral Image Analysis Application for Confirmation of Isolated Tumor Cells in Axillary Lymph Nodes from Breast Cancer Patients
Jeffrey L Fine ; University of Pittsburgh;
Content:
When examining axillary lymph nodes for breast cancer, small metastases can be difficult to confirm utilizing traditional immunohistochemistry (IHC) methods, as the focus may not be present on the stained slide. Although alternative methods exist (e.g., destaining), morphologic context of the original hematoxylin and eosin (H&E) stain may be lost. Multispectral image analysis is a technique which permits demixing of multiple stains on a slide. This study details preliminary validation of a special IHC procedure that generates virtual H&E and IHC images for the ordering pathologist.
Technology:
Multispectral image analysis was carried out using the Nuance system (CRi,Woburn, MA). IHC staining for cytokeratin AE1/AE3 (Dako, Carpinteria, CA) was performed using a Benchmark XT automated stainer (Ventana, Tucson, AZ).
Design:
H&E recuts were ordered on 5 axillary lymph nodes with macro-metastases. IHC was performed directly on these H&E recuts, and additional stains were performed: traditional IHC, diaminobenzidene (DAB) only IHC (e.g. IHC without counterstain), negative IHC controls, hematoxylin only, and eosin only. The eosin, DAB, and hematoxylin only slides were used to build a spectral library for image analysis. Composite H&E/IHC slides were spectrally de-mixed then recombined as false-color virtual H&E and IHC images.
Results:
All IHC stains on all five metastases were validated manually. Composite H&E/IHC stains did not retain sufficient eosin for high quality virtual H&E image reconstruction. Re-application of eosin permitted virtual H&E and IHC stain images to be generated; images were validated against H&E and IHC slides. Even with all three stains (hematoxylin, eosin, and DAB) composite H&E/IHC slides did not require image analysis for validation.
Conclusion:
This is a preliminary validation of our procedure to multiplex H&E and a single IHC stain as a clinical application. Following further validation with 20 known axillary breast cancer metastases, this service will be offered to pathologists for clinical use. Due to the ease of manual validation, this is an ideal introduction for this technology. Future related applications will focus on other very small foci that are difficult to stain (e.g., microinvasion) and multiplexing of multiple IHC stains.
