Advanced VirtualMultiplexing: Your Questions Answered


We have recently hosted a successful webinar on VirtualMultiplexing™, presented by Dr. Regan Baird. During the webinar we had many questions, so we decided to release these for everyone to see in this FAQ blog post.

If you were unable to attend the webinar, you can still access the recording, simply mail us here to receive the link.


Q: What is Virtual Multiplexing?

A: Virtual Multiplexing is resultant of co-registered serial tissue section images that are differentially stained for one or more biomarkers using Visiopharms patented Tissuealign™ module and then analyzed as a single slide for quantitation.

Q: What is Virtual Double Staining?

A: Virtual Double Staining is a clinically relevant technique that utilizes Visiopharm's patented Tissuealign™ module to co-register adjacent serial sections from a patient to augment pathologists in otherwise difficult diagnostic assessments

Q: How many serial sections can Tissuealign co-register?

A: There is no true limit to the number of images that CAN be co-registered using Tissuealign™, as the workflow aligns the second image to the first, the third image to the second, and so forth. However, there is a limit to the number of adjacent sections that SHOULD be co-registered.If the same section is simply restained several times, then there is a high confidence that the detected signals should be in the same location between images. This confidence decreases proportionally to the distance between serial sections (Breast Cancer Res Treat. 2016 Jul;158(1):11-9.), thus it important to understand the resolution required for the final analytics.

Q: Can Tissuealign™ be used for three-dimensional reconstruction?

A: No, Tissuealign™ intentionally eliminates 3D information and treats the serial sections as co-planar. This allows the virtual image to have the same labels but distinguished by the section they were derived.

Q: Can I use Tissuealign™ for three-dimensional counts or other measurements?

A: No. Tissuealign intentionally removed 3D information to create a planar image from differentially stained serial images.3D quantitation should be done using stereology (for more information please see Visiopharm's Stereotopix).

Q: How does Tissuealign™ handle folds and rips in tissue samples?

A: Areas within the tissue sample that have imperfections like rips, tears, and folds can either be excluded from the Tissuealign workflow or can be corrected manually using virtual pins. These areas will have a greater alignment uncertainty and should be analyzed after careful consideration.

Q: Can Tissuealign™ be used with Tissue Micro Arrays?

A: Yes. Tissuealign™ can be included in the Tissuearray workflow after each core is identified from the serial TMA images.Co-registration will then occur on a core by core basis automatically.

Q: Given all the options, what is the best method for histological multiplexing?

A: There are multiple methods for multiplexing within tissue including spectrally separated dyes, serial rounds of staining, imaging and washing, and virtual multiplexing of serial tissue sections. Each has its own advantages and disadvantages. Chromogenic multiplex assays scan quickly but may be limited to two or three detectable colocalization combinations.Fluorescent multiplex assays can detect multiple biomarker colocalizations, but require expensive equipment and lengthy scan times.Virtual Multiplexing utilizing Tissuealign™ can be combined with any of the tissue staining techniques to empower higher order analysis. For example, the co-registration of three serial 5-plex tissue sections would result in a virtual 15-plex.Visiopharm's image analysis tools streamline the analysis of any highly multiplexed tissue samples including maturing technologies like imaging mass cytometry, digital barcoding, and other emerging techniques.

Q: Would there be utility in multispectral or hyperspectral imaging in advancing multiplex analysis?

A: Potentially. The results of these imaging techniques could be co-registered using Tissuealign to cross-reference routine and special tissue stains.


If you have any further questions, please do not hesitate to contact us - you can send your questions to us via email  here or submit them through our contact form here.