Authors: Carla Coltharp, Yi Zheng, Rachel Schaefer, Ryan Dilworth, Chi Wang, Kristin Roman, Linying Liu, Kent Johnson, Cliff Hoyt, Peter Miller


Issue: Digital Pathology Association: Visions 2018 Tradeshow Poster

Background

Fluorescence imaging enhances quantitation in digital pathology by 100 µm providing linear readouts of multiple marker expressions. However, conventional fluorescence IHC is typically limited to 3-4 markers and can be confounded by tissue autofluorescence. Multispectral imaging expands the number of distinguishable markers and can robustly remove autofluorescence. However, to date, field-based rather than whole slide imagery and extended acquisition times have been disadvantages compared to conventional digital pathology. Here, we demonstrate and validate a novel, high-throughput method that can acquire a multispectral scan of a 1x1.5 cm tissue section in ~6 minutes, providing an unmixed digital slide that distinguishes up to 6 markers and counterstain with autofluorescence removal. This streamlined workflow enables assessment of cell phenotypes and functional states across the entire digital slide, enabling investigations of spatial relationships from the scale of cell-to-cell interactions to macroscopic tissue architecture.

Methods

Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents. Conventional and multispectral digital scans were acquired on a Vectra™ Polaris® automated imaging system and analyzed with inForm® and MATLAB® software.


Multiplex Staining with Opal™ Reagents

Opal™ reagents allow multiplex fluorescence IHC staining with signal amplification and any combination of mouse and/or rabbit primary antibodies.



Fig 1. Opal Detection. The Opal HRP polymer amplifies IHC detection by covalently depositing multiple Opal fluorophores near the detected antigen. Then, antibodies are stripped to allow for sequential labeling of multiple markers.


The schematic below outlines the steps in an Opal multiplex staining protocol. Up to 8 antigens can be labeled sequentially with distinct Opal dyes.





Multispectral Imaging on Vectra Polaris

Fig. 2. Multispectral imaging on the Vectra Polaris is built upon an epifluorescence light path (below, left). Different combinations of agile LED bands, bandpass excitation filters, bandpass emission filters, and a liquid crystal tunable filter (LCTF) are used to select narrow spectral bands that reach the imaging sensor.



For each spectral band, an image is acquired and added to a ‘data cube’ that contains up to 40 spectral layers (above, right). The data from all spectral layers is then linearly unmixed using previously-determined pure emission spectra for each fluorophore using inForm® software. Intensity values in the resulting ‘unmixed’ image are directly related to the amount of each dye present.


Novel High-speed Multispectral Scanning Method

Typical multispectral imaging workflows can accommodate a wide range of fluorophores, but can be time consuming as they require up to 40 spectral layers to unmix 7 fluorophores, and often require exposure times in the hundreds of milliseconds.

Here, we have developed a high-throughput multispectral scanning approach by optimizing a multispectral workflow for a specific set of 7 fluorophores:

• We applied computational modeling to determine a minimal set of spectral bands to unmix 7 optimized fluorophores and tissue autofluorescence.

   • This includes two new Opal™ fluorophores: Opal 480 & Opal 780

• We minimized the number of mechanical filter movements using agile LED illumination and multiband filters.

• We decreased exposure times down to tens of milliseconds with efficient filter pairings and Opal™ amplification. This arrangement provides robust unmixing of all 7 fluorophores from tissue autofluorescence, and from one another.


Results: 7-color Whole Slide Scans, Conventional vs. Multispectral


Fig 3. Whole slide scans of lung cancer FFPE tissue section captured in 6 minutes. Top) Conventional narrowband scan acquired with bandpass filters optimal for Opal fluorophores. Bottom) Unmixed multispectral scan that removes crosstalk and autofluorescence. Arrows indicate autofluorescence contamination; asterisks indicate crosstalk from a spectrally adjacent band.


Autofluorescence Removal: Improved Limit of Detection & Increased Dynamic Range



Fig 4. Limit of detection was determined in two ways. A) By unmixing an unstained tissue and determining the average signal attributed to each fluorophore in this negative control. B) For an example fluorophore (Opal 520, green fluorescent), tissues were stained against PD-L1 in a titration series with varying amounts of Opal dye. Whole slide imagery from these tissues was divided into small ROIs that contained 300-400 cells each. In the scatterplot, each point represents the average PD-L1 membrane signal in cells within one ROI. The intersection of the correlation plot reveals the improved limit of detection with unmixing.


Crosstalk Reduction: Improved Signal Accuracy

Crosstalk between Opal fluorophores was assessed by imaging tonsil sections stained with single fluorophores and unmixing using library spectra from all fluorophores simultaneously.



Table 1. Crosstalk in each channel for each fluorophore calculated as a percentage of the intensity in the proper channel within positively stained pixels. As a performance comparison, values from well-established field-based multispectral imaging are also shown.


Conclusions


High-throughput multispectral scanning and unmixing outperformed conventional scanning by:

• Reducing autofluorescence contributions for all immune markers, lowering the limit of detection and extending the dynamic range of some channels by more than 30-fold (Fig. 4).

• Reducing crosstalk from more than 8% to under 3% (typically <0.5%), thereby reducing false colocalization between non-colocalized markers (Table 1).

The novel multispectral scanning method described here overcomes limitations imposed by crosstalk and autofluorescence, expanding the number of probed targets and improving analytical performance.

This streamlined workflow enables multiplexed studies at the throughput required for translational studies of cellular phenotypes and interactions across an entire slide, and provides the ability to quickly re-analyze imagery as new biological understanding emerges.