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67 Evaluation of precision and robustness of a flexible multiplex immunofluoresce workflow for immune-profiling analysis
  1. Yvette Cajigas,
  2. Cassandra Kysilovsky,
  3. Erin Nicola,
  4. Ruben Cardenes,
  5. Friedrich Preusser and
  6. Angela Vasaturo
  1. Ultivue, Cambridge, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Characterization of the tumor microenvironment is becoming a clinical reality for discovering evidence of novel treatments’ efficacy. Multiplex immunofluorescence (mIF) assays have emerged as potent tools for immune-profiling analysis to examine spatial biology of tissues and determine tumor and immune cell interactions. Though, the successful application of mIF assays requires deep verification and investigation of the accuracy, precision, and reproducibility against standard clinical immunohistology. Validation of mIF staining that allows flexibility in biomarkers selection still represents a barrier for mIF integration into clinical practice while it is widely recognized that mIF assays have several critical implications for clinical translation. Here we describe a simple approach to evaluate sensitivity and reproducibility of InSituPlex (ISP) staining workflow without sacrificing the flexibility required to answer biological questions tailored to specific treatments’ evaluation.

Methods InSituPlex (ISP) mIF assays in different configurations (4-plex vs 8-plex) were performed on consecutive tissue sections of formalin-fixed paraffin-embedded (FFPE) TMAs containing tonsil, lymph node, colon, and melanoma tissues. Slides were stained on the BOND Rx autostainer (Leica) and imaged on the Zeiss Axioscan7 using a locked scan profile. Resulting images were analyzed using Ultivue’s AI based mIF analyzer software v1.6.1 and single marker staining reproducibility assessed as coefficient of variability in signal intensity and cellular density of immune cell subtypes across adjacent slides.

Results Single markers tested in the multiplex ISP assay showed expected localization and expression pattern. Quantitative analysis revealed high reproducibility of the assay in multiple configurations, with each single markers’ coefficients of variance falling within an acceptable range. Targets stained and imaged in Round 1 of the multiplex assay were highly concordant to those in Round 2 of the multiplex assay. Single targets showed a high level of reproducibility irrespective of other targets present the multiplex assay, demonstrating lack of steric hinderance even between targets co-expressed and co-localized.

Conclusions This study demonstrates that the multiplex ISP staining workflow is a reliable and reproducible approach for immune-profiling analysis in the tumor microenvironment. The high level of concordance between targets stained and imaged in any detection order in the staining panel of the multiplex assay highlights the robustness of the technique. Importantly, we observed that changing the biomarker panel composition did not hinder the reproducibility of individual markers, indicating workflow, reagents, and technology robustness that allows the flexibility needed in translational research and supports the integration of ISP into clinical studies for a deeper understanding of the tumor microenvironment.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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