Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system

Abstract

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Introduction

In recent years, it has become evident that the type, density and/or spatial distribution of immune cells in tumour specimens is potentially valuable for predicting cancer prognosis and response to treatment (Schnell et al., 2018). In colorectal cancer, tumour-infiltrating T cell density can add important prognostic information over traditional pathological staging (Mlecnik et al., 2011) and efforts are underway to standardise T cell assessment for inclusion in routine pathology review (Galon et al., 2014; Pages et al., 2018). However, chromogenic immunohistochemistry (IHC), the gold standard in pathology for analysing proteins in tissues, can only assess one or two markers concurrently and is therefore unlikely to yield sufficiently detailed insights into complex interactions between multiple cells. This has driven the development of different multiplexed technologies that allow for simultaneous assessment of multiple markers on the same tissue section (Parra et al., 2019).

Multiplex immunofluorescence (IF) staining techniques using tyramide-signal amplification (TSA) are gaining popularity worldwide, as they allow researchers to use primary antibodies from the same species and thereby select antibodies solely on the basis of their performance (Stack et al., 2014; Blom et al., 2017; Feng et al., 2017; Parra et al., 2017; Gorris et al., 2018). Traditionally, multiplex IF or IHC has relied on using primary antibodies raised in different species to avoid cross-reactivity on application of fluorescently-labelled secondary antibodies (van der van der Loos, 2008). The range of host species in which commercially-available primary antibodies are produced therefore limits the value of this technique (most anti-human antibodies being raised in mouse, rabbit or goat). Multiplex IF using TSA involves sequential staining and stripping steps whereby deposited fluorophores remain on the tissue surface but antibody complexes are removed, allowing for the use of multiple primary antibodies raised in the same species in a single staining panel (Toth and Mezey, 2007; Zhang et al., 2017; Buchwalow et al., 2018). While multiplex staining with primary antibodies of the same species can be achieved using TSA-free techniques, such as those using Fab fragments (Brown et al., 2004) or antibodies of different subclasses (Ijsselsteijn et al., 2019), TSA is an attractive alternative given the added benefits of signal amplification, allowing detection of low expressed antigens.

Many commercial TSA-based multiplex IF staining kits, such as the Perkin Elmer OPAL kits, are recommended for use with a multispectral microscopy system for image acquisition and analysis. These systems capture many narrow bandwidths of light and use algorithms to separate spectral curves of multiple fluorophores. However, many research groups have limited access to such systems. Traditional fluorescence detection using band pass filters can be used to distinguish multiple markers (albeit not as many) and are more widely available, but their utility for analysing multiplex IF staining is relatively unexplored. Since clinical research projects often involve assessment of large tissue areas, such as tissue microarrays (TMAs), or multiple whole tissue sections, use of a scanning system rather than a single field of view fluorescence microscope is usually preferable (Isse et al., 2012).

Here we describe our experience optimising two 5-colour immunofluorescence panels (four IF markers plus DAPI) combining TSA and indirect immunofluorescence for the assessment of tumour-infiltrating lymphocytes (TILs) in formalin-fixed, paraffin-embedded (FFPE) colorectal tissue (TMAs and whole tissue sections), for imaging on a fluorescence slide scanner with standard bandpass filters. We discuss some of the major steps and key considerations in optimising a TSA-based assay and developing multiplex assays.