Poster Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Feasibility of using cryopreserved or formalin-fixed paraffin-embedded (FFPE) stored clinical sample tissues in spectral flow cytometry for immune profiling (24543)

Justin F Tirados 1 , Kirsten Ward-Hartstonge 1 , Fran Munro 2 , John McCall 2 , Roslyn Kemp 1
  1. Microbiology and Immunology, University of Otago, Dunedin, Otago, New Zealand
  2. Surgical Sciences, University of Otago, Dunedin, Otago, New Zealand

A challenge faced by cancer immunology researchers is to study long-term patient outcomes in relation to in-depth immune cell profile, given that many archived patient samples are stored as tissue blocks. An infiltrate of immune cell subsets, including CD3+CD8+ T cells and CD3+CD4+CD25+FOXP3+BLIMP1+ Tregs have been associated with positive patient outcomes in colorectal cancer. Spectral flow cytometry allows the use of fluorochromes in combination with one another in ways that were previously not possible in traditional flow cytometry, opening the possibility to dive deeper into immune cell profiling of cancer patients. Abundant long-term clinical data exists for patient samples stored as tissue blocks however, this cytometric technique is currently only routinely applied to patient samples that are analysed fresh or following storage as viable single-cell suspensions. A recent study showed the feasibility of using mass cytometry to study epithelial cells at a molecular level in FFPE archived tissue blocks. We have attempted to adapt this technique to study immune cells with spectral flow cytometry from either cryopreserved or FFPE tissue blocks of colorectal cancer patients. Our data show that it is challenging to capture a good yield of intact immune cells from FFPE tissue blocks. In addition, anti-human monoclonal antibodies from our team’s standard immune phenotyping panels produced mixed success with respect to specificity and signal strength. We also explored methods to release cells from cryo-preserved tissue blocks for immuno-phenotyping. Maintaining cell viability and minimising cell stress were significant challenges. Results from these studies will be presented.  Archived cryopreserved patient samples represent a rich seam of immunological information from colorectal cancer patients and any progress in accessing this for spectral cytometry immune cell profiling could allow new insights into the immune cell populations present in tissues, allowing links to patient prognosis and survival.