Virtual multiplex immunohistochemistry: Application on cell block of effusion and aspiration cytology

With the continuous development of new antibodies, the use of immunohistochemistry (IHC) is becoming more often a requirement. IHC is frequently necessary for establishing cancer diagnosis and making therapeutic decisions. However, cytology specimens such as effusion fluid and fine-needle aspiration are highly variable in cellularity and contain background inflammatory and mesothelial cells. Compared to biopsy or excision specimens, tissue exhaustion and levels of sections not matching are more commonly encountered. We present a method of integrating whole-slide cell block image of multiple antibodies by digital image processing and reconstructing a virtual multiplex IHC image for enhanced interpretation.

Historic archived cell block preparations of carcinomas (n = 19) and melanoma (n = 1) and IHC performed with 3,3'-diaminobenzidine chromogen were reviewed. The slides were digitized by a whole-slide image scanner. Using ImageJ and self-developed code, the slides were aligned by image registration, layered, and recolored to reconstruct a virtual multiplex image, simulating a multiplex preparation with multiple chromogens. To quantity the performance of the image registration, the mean distance between the same cell clusters in aligned images were measured.

All 20 cases were successfully registered. The mean distance between cell clusters after image registration was 8.40 +/- 5.52 μm. The reconstructed images were able to demonstrate coexpression of membranous, cytoplasmic, and nuclear antibodies.

With virtual multiplex IHC, we were able to visualize coexpression of multiple antibodies without the added cost of multiplex IHC. Routine and historic slides, without additional tissue consumption, can be retrieved for image reconstruction. This technique is a low-cost adjunct to diagnosis in cytology for more efficient and accurate assessment of antibody coexpression and histochemical stains.