Opal Mulitplex IHC Assay Development Guide

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Table of Contents

What is Phenoptics? 4

Phenoptics Product Line-Up 4

Slide Definitions 5

High-level Flow Chart of Panel Development 5

Ingredients 6

Typical Opal Staining Cycle 8 Microwave Settings 9

Monoplex Protocol Development 10

Panel Design 10

Optimizing Antigen Retrieval and Primary Incubation Parameters 11

Instructions for Acquiring Monoplex images for Staining Assessment 14

Monoplex Quantitative Assessment and Optimization 15

Library Development and Assessment of Unmixing 16

General Recommendations for Imaging Opal Library Slides with Mantra or Vectra 16

Use inForm Build Library Function to Create Unmixing Library 18

Library Assessment 19

Opal Multiplex Assay Development 20

Setting Acquisition Exposures for Imaging Opal 21

Assessing Signal Balance 22

Assessing Interference 23

Assessing Crosstalk 23

Image Analysis 24

Tissue Segmentation 24

Cell Segmentation 25

Phenotyping 26

Validation 26

Opal Automation 27

References 28

Supplemental Information 28

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Preface

This document offers recommendations for performing manual application of multiplex Opal labeling based on what has worked in our laboratory and what we have heard from the field. It encompasses the entire Phenoptics workflow, from Opal assay development through imaging and analysis. It is intended as a starting point for our customers and not a locked-down set of rules. We encourage users to build on what we have provided here and also to share any feedback or ideas on how to improve this process.

We are continuing to develop the Opal method by performing experiments to understand its limitations, improve performance and extend its capabilities. This is a living document that will be continually updated with new insights. Please check our website regularly for updates.

This guide assumes that you are familiar with the software functions in Vectra and Mantra image acquisition software and with inForm image analysis software.

We recommend that research laboratories adopting this approach be proficient with conventional immunohistochemistry assay development, fluorescence imaging (preferably on the Vectra or Mantra platforms), and image analysis programs. Advanced measurements like spatial relationship calculations require familiarity with R, TIBCO Spotfire®, or similar data analysis programs.

Performing Opal multiplexed staining manually can take as long as five days and has many steps. In our lab it has been helpful to confirm that each step meets criteria for success before proceeding to subsequent steps because once the assay is completed and if staining is not satisfactory, it can be hard to determine when in the process mistakes were made or issues arose.

For evaluation of more than six IHC targets in one sample, two panels may be developed that include one or more common markers such as a tumor marker.

Last updated: October, 2017

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Phenoptics Product Lineup

What is Phenoptics? Akoya’s Phenoptics workflow enables imaging and analysis of up to six immunofluorescence markers within intact tissue sections (Figure 1). Quantitative assessment of cellular phenotype and activity becomes possible in a way that is similar to flow cytometry, while simultaneously providing tissue context and information about cell-to-cell interactions that is difficult or impossible to obtain by other methods. The Phenoptics workflow incorporates three essential elements.

• Opal ™ is a method for multiplex fluorescent immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) tissue. It allows use of standard unlabeled primary antibodies, including multiple antibodies raised in the same species. The basic approach was inspired by a protocol published by Zsuzsanna Tóth and Éva Mezey1. The current method involves detection with Opal reactive fluorophores that covalently label the epitope. After labeling is complete, antibodies are removed in a manner that does not disrupt the Opal fluorescence signal. This allows the next target to be detected without fear of antibody cross reactivity. Opal enables development of multiplexed assays with balanced, quantitative signal for rare and abundant targets.

• Multispectral imaging eliminates fluorophore crosstalk and interference from tissue autofluorescence, allowing precise measurement of each fluorescence signal within a tissue sample. Vectra®, Vectra® Polaris and Mantra® multiplexed biomarker imaging systems from Akoya Biosciences are recommended for unmixing of spectrally overlapping fluorophores as well as tissue autofluorescence. Separation of all of the fluorescence signals within the sample is essential for quantitative measurement.

• inForm™ image analysis software determines per-cell and per- subcellular compartment intensity values. This information is used in combination with user-trainable machine learning algorithms to phenotype cells, recognize morphologic regions of the tissue (e.g., tumor, dermis, stroma, inflammation), and provide cell counts and densities within each region. Data may be exported from inForm for additional analysis such as distance mapping between selected cellular phenotypes. In addition to fluorescent composite images, inForm provides simulated brightfield monoplex IHC from the same data for improved visual interpretation.

Figure 1, Akoya's Phenoptics Research Solutions for Quantitative Pathology Imaging and Analysis

Slide Definitions

Typically for each assay developed, we generate three different types of slides.

• Monoplex. Control tissue and representative study samples are labeled for one markerand counterstained with DAPI. These are necessary for assessment of stainingperformance and comparison to established standards. Monoplex slides should bedeveloped in such a way that the appropriate number of microwave treatments (MWT)is applied for each target. (e.g., your monoplex slide for your third biomarker insequence should experience antigen retrieval, two MWT before the addition of yourantibody, secondary, and Opal, and four MWT after staining.)

• Library. Spectral library slides each stained with a single fluorophore and made fromreliable positive control tissue (i.e. tonsil) will be necessary for creating an accurateunmixing library for analysis of monoplex and multiplex slides. Once a set of libraryslides is created, it can be re-used. We are presently assessing shelf life of library slides. Aset of library slides consists of:• One control tissue slide stained for each Opal fluorophore, without DAPI. Typically, we use an antibody marking an abundant epitope for each Opal fluorophore(e.g., CD20 ontonsil for all 6 Opal fluorophores.)• One control tissue slide stained with DAPI alone.• One unstained representative study sample, for collection of an autofluorescencespectrum. The unstained slides should be processed in the same way as the other slides,omitting both the Opal fluorophore and DAPI.

• Multiplex. Control tissue and representative study samples are labeled for all of themarkers in the multiplex panel and counterstained with DAPI. These slides allowassessment of the multiplex assay for interference and crosstalk

High-Level Flow Chart of Assay Development.

Page | 5 Figure 2, Overview of the 4 high-level steps in the Opal Multiplex IHC Assay

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Ingredients Figure 3, Opal fluorophores

Opal kits are available with up to seven colors, including DAPI counterstain. The Opal staining protocol is similar in many respects to standard immunohistochemistry (IHC) that uses diaminobenzidine (DAB). Tissue is incubated with unlabeled primary antibodies. These are detected with HRP-conjugated secondary antibody and a detection substrate. For this reason, many of the reagents and materials used in this process will be familiar to anyone who performs IHC currently.

Catalog numbers for Opal kits and some ancillary reagents may be found in the Supplementary Information at the end of this document or at www.perkinelmer.com/opal.

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Suggested Reagents • Opal Multicolor IHC Kit. Kits include DAPI, the Opal polymer secondary

antibody (anti-mouse and anti-rabbit cocktail), 1X Plus amplification diluent,blocking buffer, and AR6 (antigen retrieval, pH 6) buffer that is optimized forthe protocol. Please note that Opal reagents come as a lyophilized powder andmust be reconstituted to a stock solution using 75 uL of the provided DMSO.Opal reagent packs are available separately for building custom multiplexes.

• Primary antibodies for targets of interest:o Any species, unlabeled.o Validated for IHC when available. Usually, they will work as well or

better with Opal.In some cases, antibodies that do not work forstandard IHC will work with Opal.

o For unknown targets, it may be worthwhile to screen multiple clonesto determine which will provide the best performance in the assay.

• AR9 (antigen retrieval, pH 9) may work better for some targets, particularlymarkers that are expressed within the nucleus.

• Isotype control antibodies• Antibody diluent / block.• Secondary antibody HRP conjugate. Opal reagents are compatible with any HRP

conjugate, accommodating any antibody or tissue species. Opal Polymer HRP Ms + Rb isrecommended for work with human tissue and primary antibodies from mouse or rabbit.

• 10% neutral buffered formalin (NBF).• TRIS-buffered saline with Tween® 20 (TBST) wash solution (0.1 M TRIS-HCl, pH 7.5,

0.15 M NaCl, 0.05% Tween®20).• Ultrapure, peroxidase-free water. Autoclaved Milli-Q® water works for this purpose.

o Other options should be validated.• Mounting medium for fluorescence—without DAPI or other counterstain. Users should

validate independently. ProLong® Diamond from Thermo Scientific has worked in ourlaboratories.

Suggested Materials • Microwave oven with a carousel and 10 power settings, rated at 1000-1200 watts, if

possible.• Hellendahl type staining jars are critical for MWT because they hold enough AR buffer to

ensure that slides do not dry out. Opal Slide Processing Jars from Akoya are non-breakable and hold up to 14 slides.

• Baths and solvents for deparaffinization and rehydration of FFPE tissue. Xylene is recommended for deparaffinization. Histological grade ethanol is required forrehydration.

• Slide incubation/humidity tray for incubation steps.• Hydrophobic barrier pen.• Glass coverslips, #1.5.• Control tissues.• Charged slides.• Library slides provided with the Vectra Polaris instrument

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Typical Opal Staining Cycle

Before developing your first panel, it’s probably helpful to understand the typical Opal staining cycle:

Figure 4, Opal staining cycle.

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Microwave Settings

Before starting your first panel development, it is recommended to determine microwave heating parameters, as the microwave cycle differs from microwave-to-microwave. Most conventional home microwaves work well for MWT in the Opal process. Panasonic® products with Inverter® technology have more precise power control and are recommended if purchasing a new microwave oven. Users should validate microwave performance independently as outlined below. We strongly recommend developing your Monoplex slides with the appropriate number of microwave treatments built in to assess the robustness of your antigen and the intensity of the Opal fluorophore. Therefore, it is important to perform your microwave optimization prior to beginning your Monoplex slide development. Note: Do not operate the microwave oven unattended and keep the oven chamber clean and clear of debris, as this is a fire risk.

• Microwave treatment (MWT) can be used to quench endogenous peroxidase activity, forantigen retrieval, and to remove antibodies after a target has been detected with an OpalTSA-fluorophore. Timing for each step in the procedure may require adjustment basedon the microwave oven used. Slides are placed vertically in an Opal Slide ProcessingJar, which is then filled to the top (~140 mL) with AR6 or AR9 buffer and covered loosely.

• One jar at a time is placed in the microwave, near the edge of the carousel to ensureeven distribution of energy

• The microwave procedure consists of two steps:1. Bring the sample to a boil at 100% power. Determine empirically the amount

of time your microwave takes to reach 100 °C using a probe thermometer in amicrowave-safe antigen retrieval jar containing AR6 or AR9 buffer and blankslides at 100% power. The time it takes to reach 100 °C will vary dependingon the make, model, and wattage of your microwave. The time to 100 °C at100% power will be the first step.

2. The second step will be to microwave the slides at 20% power for 15-minutes. Ifafter 20 minutes, buffer level is below the neck in the bottle and there is exposedtissue area on the slide, either reduce power or reduce time until buffer does not gobelow the neck.

Figure 5, Slide processing jars before (left) and after (right) MWT. Although some evaporation has occurred, the tissue is still covered with AR solution.

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Monoplex Assay Development

The goal of this step is to determine individual staining parameters for each marker in the multiplex panel, leading to a set of Monoplex slides. Of the four high level steps (Monoplex, Library, Multiplex, Analysis), this is often the most challenging due to the usual issues associated with IHC. Once you are beyond the Monoplex stage, assay development is usually rather straightforward.

Start with control tissues such as tonsil to get parameters approximately correct, but finish with representative study samples that are likely to be positive for the markers in the panel. The main reason for this is that control tissues often express at higher or at least different levels than diseased tissue samples.

Panel Design

Assign Opal fluorophores to antibodies:

1. Choose which Opal fluorophores to use for each marker. We use the following rules-of-thumb:

a. For co-expression, choose fluorophores that are not spectrally adjacent.b. The table below (Figure 6) compares the relative brightness of Opal

fluorophores given the same concentration of fluor on the Vectra 3 and VectraPolaris. Lower expressers should be assigned or detected with the brightestOpal fluorophores, leaving those with lower brightness for more abundanttargets.

Figure 6, Opal brightness comparison, all things being equal. This is a starting point for choosing your Opal fluorophore-antibody pairing based on expected target expression levels. Your Opal brightness ranking may vary depending on target and sample expressions.

2. Choose order of staining, and prepare monoplex protocols that include theappropriate number of MWTs before and after Opal detection, to mimic tissueexposure that will occur in the multiplex protocol. We have found including theseMWT steps during this mononplex optimization step greatly decreases theamount of rebalancing and adjustments needed for the multiplex protocol.

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a. The order of immunostaining can change signal intensity. Some epitopesbecome more exposed after successive rounds of MWT and for that reasonprovide higher signal when detected later in a multiplex protocol (e.g., PDL1,Ki67). Some epitopes may be degraded by multiple exposures to MWT andshould be detected early in the process .

b. Some Opal fluorophore signal intensity can also be affected by MWT (i.e.,attenuation of Opal 520 and Opal 570). Designing your monoplexes with thecorrect number of MWT can help determine any corrections needed on Opalconcentration and assay order prior to running the multiplex.

c. If possible, arrange the order so sequential antibodies do not colocalize inthe same cellular compartments within the same cells. This can help debugissues such as incomplete stripping, contaminated antibody aliquots, etc.

Optimizing Antigen Retrieval, Primary Antibody Dilution, Incubation Parameters, and Opal Reagent Concentration

Once you have designed your panel, you can begin the Monoplex optimization process. Optimizing antigen retrieval and primary antibody incubation parameters for an Opal multiplex aligns with traditional IHC methodology. Antibody concentration should be empirically determined based on appropriate staining patterns, and adjustments for brightness or signal intensity are achieved by changing Opal reagent concentration. This is aligned with recent thinking that maximizing the number of HRP molecules per type of epitope leads to less need for amplification and thus less interference, or ‘umbrella’ effect. To this end, it is often worthwhile to experiment with antigen retrieval methods to determine which yields the highest staining intensity, using serial sections to compare. Commonly used methods include pressure cookers, conventional household microwaves, HIER platforms like the PT Module, and laboratory-grade microwaves that include temperature probes. We have now heard from many labs that laboratory-grade microwaves (e.g., EZ Retriever from Biogenex and Biowave from Pelco) provide the highest signal levels, sometimes by factors of 2 to 4.

Approaches we’ve used and seen in the field to determine optimum primary antibody concentration:

1. Use conventional IHC and visual assessment:

For each antibody, optimize and stain positive control slides, as you would normally in the course of your IHC protocol development, seeking the appropriate staining pattern, which usually means selecting staining parameters that lead to, for example, specific, intense, captures the full dynamic range of expression with continuous levels of intensity, shows complete membrane staining for surface markers, and has low background. Then, stain serial sections using primary antibody concentrations determined for conventional IHC and with the Opal fluor assigned above during panel design, using Opal dilution of 1:100, counterstained with DAPI, thus making Monoplex slides.

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Acquire example images with Mantra or Vectra of both the chromogenic IHC slides and Monoplex Opal slides using all five standard epi-fluorescence filters (DAPI, FITC, CY3, Texas Red, and CY5), and using exposures for all epi-fluorescence filters set to 150 ms. Confirm that ‘Use Saturation Protection’ option is selected so that exposures will be shortened when needed to avoid image saturation.

Unmix each Monoplex image with a library created from Library Slides provided with Vectra Polaris installation, or with a library from previous work.

Use inForm’s Pathology Views to create simulated DAB IHC views of the Opal Monoplex slides. Confirm the conventional DAB IHC looks essentially the same as the ‘simulated IHC’ view generated in inForm If they look the same, then you may proceed to the next step of adjusting Opal detection to achieve signals within target signal range.

Figure 7, inForm views of monoplex CD8 in human tonsil labeled with Opal 520. The unmixed fluorescencecomposite view on the left visualizesCD8 in green and nuclei in blue. Thesimulated IHC view on theright allows for directcomparison of stainingpatterns with establishedstandards.

2. Skip conventional IHC and use manufacturer’s recommended primary antibodyconcentrations (this is approach we use in our internal work)

Create a set of 6 slides for each antibody, each set consisting of 1x, 2x, and 4x titrations of the manufacturer’s recommended primary antibody concentration, and of treatments with pH6 and pH9 AR buffers. (See Fig. 8). Use Opal dilution of 1:100. Image with Vectra or Mantra, as described in (1) above, and assess each set of parameters for staining pattern, also as described in (1). This approach relies on being familiar with conventional IHC staining patterns for each antibody, as conventional IHC staining patterns are generally considered the gold standard. It is often useful to assess using both inForm ‘composite’ views and simulated IHC views. We often find manufacturer’s recommended concentrations work, especially for ‘ready-to-use’ preparations. If there is no significant difference in staining between titrations and/or antigen retrieval approaches, and all contextual specificity remains, we generally choose a dilution 2x greater than the manufacturer’s recommendation, and AR6 for antigen retrieval.

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Figure 8 Antibody optimization matrix. In 6 DAB slides, you should be able to determine the appropriate antibody concentration and retrieval parameters for your assay.

3. Use measured fluorescence intensity from a titration series to determine primaryantibody concentration,

For each primary and associated Opal label, create a titration set, using control tissue and Opal dilution of 1:100. For example, you might use primary antibody concentrations of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600. Acquire and unmix images as described in (1) above. Create the titration curve by plotting intensity, as measured with inForm’s info curser, against primary antibody concentration. For each antibody, determine the primary antibody concentrations corresponding to intensity just below where the intensity flattens out, suggesting saturation. Confirm that this concentration still yields a satisfactory simulated IHC view of the stain.

4. Use calculated signal-to-background ratio to pick primary antibody concentration

Probably the most rigorous approach is to create a titration curve as in (3) above, but also measure from the same images the level of background, by using the inForm info curser to measure signal from areas of tissue where there is apparent non-specific signal that could potentially be detected as false positive staining. At each concentration, calculate a signal-to-background ratio and plot as a function of primary antibody concentration. Pick the concentration for which the ratio is a peak but also yields a satisfactory simulated IHC view of the stain.

The images corresponding to primary antibody concentrations as determined by these approaches are used below as starting points for the next step – optimizing Opal dilution to achieve signal levels in target range. At this point, all monoplex slides have been labeled with Opal dilution of 1:100.

The above list of approaches to optimizing primary antibody concentration includes approaches we’ve tried and seen customers try. There may be other equally or more effective ways to optimize primary antibody concentration. We welcome the opportunity to learn about others.

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Instructions for Acquiring Monoplex Images for Staining Assessment

The target signal range is below 30 inForm ‘normalized counts’ with a generally favorable count between 10-20 (assuming that the Vectra3 or Mantra lamp is set to 10%; the Vectra Polaris is fixed). Low expressing phenotypes can be below 10, however the ideal signal to background ratio should preferably be greater than 10:1.

Step-by-step approach to adjust fluorescence intensity levels

1) Create an autofluorescence sample. This slide is a representative study slide processed inthe same manner as the monoplex slides, without Opal reagent or DAPI applied.

2) Create an inForm project with the image from each of the Monoplex slides created abovethat represents the optimum antibody concentration, and an image of the autofluorescencesample.

3) Create an unmixing library in inForm utilizing library slides provided with the Vectra Polaris.You may also use an already acquired library.

4) Collect an autofluorescence spectrum.

5) For each Monoplex image, select the library spectrum for DAPI, the library spectrum for theOpal label associated with the Monoplex image, and the autofluorescence spectrum. Donot include the other Opal labels when assessing a Monoplex image.

6) Unmix and determine signal level in typical positive cells, using the inForm info curser (seeFig. 9.)

7) Repeat for each of the Monoplex images, remembering to select appropriate library Opalspectrum and making sure the other library Opal spectra are not selected. You willprobably find at this point that signals are anywhere from a few counts to 100s of counts.

8) With a goal of achieving signal intensities below 30 normalized counts (with a desiredrange of 10-20), the next steps are to adjust Opal dilutions to increase or decreasefluorescence signals so that they in the target range. Since intensity is roughly directlyproportional to Opal concentration, it is straightforward to adjust Opal dilution to achieveintensities in the target range of 10 to 30 normalized counts. For example, if intensity for anOpal fluorophore is 50 normalized counts, increasing the Opal dilution form 100:1 to 200:1will probably result in approximately 25 normalized counts. Typically, we will try threedilutions, one that should give the right target intensity and two others that bracket it, to givea range of intensities to assess. In the example above, we might try 150:1, 200:1, and250:1. If this does not give the desired signal level, then continue to try more dilutions.

9) Sometimes Opal detection needs to be diluted substantially beyond 1:100. We have seendilutions a much as 1:300, 1:400 and in one case 1:2000 worked very well. At this point, weagain recommend not using dilutions less than 1:50.

10) If signals are too low with Opal dilution at 1:100, here are additional things to try:

a) Dilute Opal less, but never less than 1:50. Using Opal at 1:50 can lead to umbrellaeffect. If you are using Opal at 1:50, you will want to be on the watch for umbrellaeffects. Since Opal 690 is a relatively less intense fluorophore, if we use an Opal fluor

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with 1:50 dilution, it’s typically Opal 690.

b) Alternate antigen retrieval strategies. For example, PDL1 seems to be retrieved tohigher levels by more aggressive antigen retrieval methods.

c) Longer incubation times for the primary antibody, even overnight at 4 °C. Note that thismay increase non-specific signal

d) Additional rounds of MWT – some antigens become more exposed under theseconditions. This is often observed in tissues with mucin like lung and colon. Or considermoving the detection later in the staining order to expose the tissue to larger numbersof MWTs.

e) Increase the primary antibody concentration. Note that this may also increase non-specific signal.

f) Consider other antibody clones. Consult the literature for recommendations.

g) Ensure your control tissue is appropriate for your targets. Consult sources such as theHuman Protein Atlas.

11) Once signal is in the correct range, assess signal-to-background ratio to confirmapproximately 10:1 or higher. To determine signal-to-background ratio, we use the inForminfo curser to measure signal levels from areas of tissue where there is apparent non-specific signal that could potentially be detected as false positive staining. If background istoo high (>10% of positive signal) consider these steps:

a) Add additional blocking steps, such as hydrogen peroxide blocking in order to quenchpreviously bound or endogenous HRP (3%) or serum/protein blocking reagents such asgoat IgG containing antibody diluents.

b) If a low ratio is caused by inherent antibody non-specificity, lowering background maybe challenging. In such cases, looking at alternate clones or reducing primary antibodyconcentrations can help. If ratio is determined by the antibody specificity, the onlyoptions may be to search for another clone, or develop an image analysis strategy thataccommodates the lower ratio.

Monoplex Quantitative Assessment and Optimization

The next step we suggest is to apply the Monoplex protocols (with the additional microwave steps) to actual study samples, since expression levels in control samples such as tonsil can be significantly higher, or in some cases lower than levels in study material. If you find signals much lower in study samples, we suggest changing Opal TSA dilution to restore signals. If this is not sufficient, you may want to reassess antigen retrieval approaches, such as switching from pH 6 to pH 9, or increasing primary concentration. In rare cases, the positive controls are so different from the actual study samples, requiring you to revisit the first step.

Alternatively, if you are pleased with your monoplexes and have appropriately incorporated your MWT, you can test the protocol as a full multiplex on a representative study sample. The outcome should be able to tell you if any one of your target/Opal fluorophore combinations will need to be re-optimized for representative expression.

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Figure 9, inForm information cursor showing signal and background intensity for Opal 650 monoplex stain. Opal 650 is visualized in red and DAPI in blue. Positive signal (shown on the left) should be at least 10 times higher than background (shown on the right). Signal intensity for all targets should usually be between 10 and 30 for positive control tissue under optimized monoplex IHC conditions.

Library Development and Assessment of Unmixing

The goal of this step is to create a set of Library slides, each stained with only one fluorophore (Opal or DAPI). Also included should be one slide with no fluorophores to represent autofluorescence. The autofluorescence reference can be the same created previously during the Monoplex optimization step. These slides will be used to create a spectral unmixing library.

Up until recently, we have been recommending the creation of Library slides by running the Monoplex protocols developed above, but without DAPI. While this still works generally well, we are exploring and expecting to transition to an approach the uses the same antibody for all Opal fluorophores, which allows for picking an abundant epitope and a tissue that has morphology that makes it easy to pick areas for library image acquisition, to support reliable extraction of emission spectra using inForm’s ‘Build Libraries’ interface. Our favorite at this point is using CD20 and tonsil, since CD20 is ubiquitous, uniform, and highly expressing. Emission spectra collected from slides like this are more pure and less subject to issues related to sparse and/or low signal levels, which can happen with some antibodies. Another significant benefit of this approach is that the set of library slides can be reused and also support standardization across studies, which is a goal for translational research.

Below are suggestions for assessing unmixing performance of the library.

General recommendations for imaging Opal Library slides with Mantra or Vectra Imaging Systems • Acquire 2 or 3 images using all five standard epi-fluorescence filters (DAPI, FITC, CY3,

Texas Red, and CY5), and using exposures for all epi-fluorescence filters set to 150

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ms. Confirm that ‘Saturation Protection’ is active so that exposures will be shortened when needed to avoid image saturation.

• We recommend making new Library slides for each study, at least until you havecompleted a few studies and have sets to compare. We do not think emissionspectra are affected by the type or condition of the tissue. Once you have a fewprojects completed, you might find that library slides can be re-used, except for theautofluorescence slide which provides the spectrum specific to the tissue type of thestudy. The autofluorescence slide should continue to be created with every study.

• With the Vectra 3, you will need to collect new libraries as the lamp ages and itsspectral properties change. We have noted that beyond 1,500 hours it changesmore rapidly. Upon replacing a bulb, immediately collect a new library andreferences.

• The spectra from each stain can be stored in the ‘Stain Store’ in the inForm program• Always enable saturation protection by checking ‘Use Saturation Protection’ (Figure

12).This protects against unexpectedly bright signals by reducing exposure whennecessary. InForm adjusts for differences in exposure times at each wavelength inunmixing, ensuring that results may be compared between samples.

• Use the lamp at 10% power forimaging OPAL slides on Vectra 3. The VectraPolaris is pre- optimized and not adjustable.

• Certain PAP pens exhibit intrinsicfluorescence that can interfere with whole slide scanning. This problem may be addressed by removing the PAP pen mark before imaging or circling the tissue to be scanned with a blue Sharpie marker.

Figure 10, ‘Use Saturation Protection’ in Vectra should always be checked. Note: for the Vectra Polaris, do not use DAPI as the autofocus channel for your library slides. You will need to choose the channel to the fluor you are imaging.

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Use inForm Build Library Function to Create Unmixing Library

1. Acquire images from the Library slides2. Extract each Opal fluorophore spectrum using inForm 2.1 (or later) automated tools in

Build Libraries tab (Figure 13). Vectra Polaris uses inForm 2.3 or later.

Figure 11, Spectral library slide image for a single fluorophore. Spectrum is extracted into the stain store.

3. Save to ‘Store’ each fluorophore spectrum, preferably with a color that will make iteasier to discern in composite view and with a meaningful group tag (Figure 14).

Figure 12, Saving Opal fluorophore spectrum to the library, with a meaningful group tag.

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Library Assessment

Test the library by unmixing images of the spectral library slides, looking for crosstalk:

• In Manual Analysis, load images of the spectral library slides, includingthe autofluorescence (AF) unstained sample.

• Select the library spectra created earlier.• Collect the autofluorescence signature from the AF unstained sample with the AF

sampling tool. Capture auto- fluorescence only from areas with tissue, avoidingareas without tissue. (Figure 15) Include red blood cells in your autofluorescencesamples if present. In some cases, we find that capturing a pure red blood cellfluorescence spectrum leads to more effective unmixing. Be careful not to crossthe autofluorescence line as you mark your area of interest.

Figure 13, Collection of tissue autofluorescence signal.

• Unmix and inspect the composite images for crosstalk, in FL view with AFdisplay unchecked.

• For each library sample in turn, disable that fluorophore’s channel – it should goblack.

• The autofluorescence sample should be completely dark with AF channel off -toggle on/off to see localization of autofluorescence.

• Signals in other channels may be observed when the extracted spectra in inFormare inaccurate. Inaccurate spectra may be caused by non-specific staining or excesstissue autofluorescence. If this occurs, please refer to ‘General recommendations forimaging Opal Library slides with Mantra or Vectra Imaging Systems’ above.

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Opal Multiplex Assay Development

The next step is to combine Monoplex protocols into a Multiplex protocol. The Multiplex workflow is essentially the concatenation of the Monoplex protocols, keeping a single DAPI application until the end. After the last MWT in each Monoplex protocol (Step 8 below), the slides may be incubated in blocking buffer again before introduction of the primary antibody of the next Monoplex protocol (Figure 14).

Figure 14, The Opal Multiplex IHC workflow.

Here is some advice on multiplex method development:

• 6- or 7-color assays may require leaving slides overnight while in process. Here arethree options for the overnight step, in order of preference:

o Post-microwave cooling in AR buffer at room temperature.o Overnight primary antibody incubation at 4 °C, where verified in monoplex assay

development.o Storing in blocking solution at 4 °C.

• It is rare that the order of AR6 and AR9 together in an Opal multiplex IHC assayimpacts the outcome.

• We find using a paper checklist and marking each step with a pen after completion isvery helpful to avoid errors, since the process is long and contains many steps. (Seeexample of Opal Detection Workflow Checklist in Supplemental Information, below).

• When developing an Opal assay panel, it can be helpful to review staining under themicroscope after application of each Opal fluorophore. The best time to do this is

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immediately after MW T for antibody removal, using a coverslip mounted with water. If there is a problem with the staining, it may possible to repeat at this point. At later stages, it will become more difficult to evaluate staining without spectral unmixing, because more than one fluorophore may be visible in a given epi-illumination cube.

Setting Acquisition Exposures for Imaging Opal

Multiplex Samples

• When setting exposure times for acquisition, one would like to have example slides thatcontain expression levels for all of the markers at least close to the representative mid-range signal across the set of slides. Since it’s hard to know which slide in a set will havethe optimal dynamic range before all are scanned, we will typically grab a handful, andmanually search for appropriate areas for each marker across the handful of slides.

• For each marker, move to an area that is positive; use the autoexpose button to determineexposure for the epi-fluorescence filter corresponding to emission maximum.

• Repeat for all filters until all exposures are determined.• These exposure times can now be used for acquiring images of multiplexed study

samples.• Please refer to the user manuals for the Vectra 3 or Vectra Polaris for more in depth

discussion on acquisition exposures.

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Assess Signal Balance

• When transitioning from Monoplex to multiplex fluorescence, signals can increase ordecrease individually, and can become unbalanced. If you have added microwavetreatments to the Monoplex protocols to simulate actual exposure to microwave, thenchanges that occur when going from Monoplex to Multiplex protocols should be small.

• As mentioned above, ideally, signal levels should be within a factor 3 of each other,particularly for spectrally adjacent fluorophores. Large disparities (>5-fold differences)in signals for spectrally adjacent fluorophores can lead to problems with interferenceand cause unmixing artifacts.

• The best way to check signal balance is to unmix images in inForm and use the infocursor to hover over bright areas of each label and compare the values (Figure 15).Check for the typical expression range for your marker, and assess the valueagainst a bright spot. These bright areas do not have to be in the same field. Ifsignal levels of any fluorophores have increased or decreased to be more than 3times larger or smaller than an adjacent fluorophore, you will want to do somerebalancing, usually by adjusting the signal of the outlier fluorophore.

• Keep in mind outliers in signal can occur for a variety of reasons, such as tissue folds orfixation artifacts. Not all anomalies are caused by unmixing issues.

• Reasons we have seen unexpected changes in signal during the creation of the multiplexprotocol after a robust monoplex development include unforeseen physiology (i.e., twomarkers co-localizing unexpectedly creating cross-talk) and high variability in studysamples. This can impact the order of staining, and staining sequence should be re-assessed.

Figure 15, inForm information cursor showing signal intensity for Opal 620 in a multiplex tissue image.

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Assess Interference

• Overstaining with an Opal fluorophore can block later application of other Opalfluorophores, interfering with multiplexed results. For that reason, signal levels shouldusually remain below 30 (when imaged with the lamp at 10% for Vectra 3) for multiplexcontrols. There are two ways to check for interference.o After spectral unmixing, visually confirm by turning layers off and on in inForm,

using the view editor. This requires knowing what the staining patterns should looklike, and looking for obvious loss of signal in one plane due to the presence ofsignal in another. This is often observed as a "hole" in the staining pattern that isfilled by the interfering signal.

o OPTIONAL: For rigorous, quantitative assessment of interference, stain serialsections of a tissue microarray (TMA) with monoplex and multiplex protocols. UseinForm to measure signal levels within each core in appropriate cells and cellcompartments. Plot average monoplex against multiplex signals for the samecores. The fit of a straight line through the scatter plot should yield an R-squaredvalue of at least 0.8

Assess Crosstalk

• Crosstalk can come from inadequate stripping of antibodies during MWT. This problemis extremely rare when using MWT during a manual Opal multiplex assay and may berelated to unusually high affinity primary antibodies. There are two ways to check forcrosstalk.

o Visually confirm by turning layers off and on in inForm, using the view editor.This requires knowing what the staining patterns should look like, and lookingfor obvious addition of signal in one plane corresponding to localization inanother plane.o OPTIONAL: For rigorous, quantitative assessment of crosstalk, stain serialsections of a TMA with monoplex and multiplex protocols. Use inForm tomeasure signal levels within each core in appropriate cells and cellcompartments. Plot average monoplex against multiplex signals for the samecores. The fit of a straight line through the scatter plot should yield an R-squaredvalue of at least 0.8.

• Spectral crosstalk is also a possibility. Ensure your staining order separates spectrallyadjacent dyes in both sequence and expression (i.e., we would not recommend stainingCD8 with Opal 540 followed in sequence immediately by CD3 with Opal 570 as this wouldlead to a high probability of crosstalk.) To assess for spectral crosstalk:

1. Hover over your multiplex image and your appropriate library image. Confirm thespectra is the same.

2. Compare your most recent library with the library slides shipped with the VectraPolaris. The spectra should be close. If not, this can indicate a poor spectralextraction.

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Image Analysis

Tissue Segmentation

inForm Tissue Finder includes a user-trainable algorithm for tissue segmentation based on morphology as well as specified markers. Here are some suggestions for tissue segmentation.

• Training is usually performed most efficiently using quick iterations and makingadjustments until optimal results are obtained. Make sure to draw your trainingregions carefully so you don’t overlap categories (i.e., including some stromaltissue in a tumor training region.) Draw one or two training areas for each categoryand evaluate segmentation result. Refine by adding new training areas to addressany miscategorized tissue.

• Use only those ‘Components for Training’ that are informative in detecting tissuecategories of interest. For example, DAPI, auto- fluorescence and cytokeratin should beused for differentiating certain tumors from stroma.

• Use the minimum number of tissue categories needed to accomplish your scientificobjective. You can put multiple tissue architectures into one ‘other’ category (e.g.,stroma, necrosis, fat, etc.) that in a particular case you might not be interested in.

• Training regions don’t need to cover the whole tissue area, only enough to capture thepattern.

• It is often helpful to have a few training regions that go right up to and follow the edge oftissue categories. Training regions do not need to be in contact with one another (Figure17).

• There is a trade-off between pattern scale, segmentation accuracy, and resolutionof segmentation. Evaluate changes to these parameters to find optimalsegmentation result.

• Segmentation accuracy is affected by the pattern scale. A higher accuracy does notnecessarily mean better results. Segmentation resolution does not affect the training(i.e., you can change it without needing to re-train, you would only need to re-segment,although changing it does remove any phenotyping training). We typically train atMedium because it’s faster, but then switch to fine before proceeding to cellsegmentation (it looks nicer and generally provides a more accurate boundary).

• It may be worth noting that sometimes retraining on the same regions can improvesegmentation results (even if the accuracy value doesn’t change). However, as soon asyou change any settings above the Recent Trainings box, previous segmenters arecleared (so it’s worth saving a copy of a project if your segmentation is close but you aretrying to perfect it).

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• Segmentation accuracy (Training %) may never reach 100%; technologist appraisal ofcorrect segmentation is true value. Best way to achieve this is trial and error throughmanipulation of pattern scale and tissue regions selection.

• Segmentation options can be adjusted after correct segmentation to refine boarders andresolution.

• Maximum Segment Size and Trim Edges functions can be quite helpful for filtering outsmall segmentation errors and fine tuning segmentation to the edge of tumor areas.• Try to capture full range of tissue morphology in training regions, for each

category, including areas with weak and strong expression of tissue markers.• If samples are very diverse, it may be necessary to create different segmentation

algorithms for different samples. (For example, if the algorithm is getting mostthings correct, but the user finds that additional training on a problematic sampleresults in the segmenter getting stuck at a low level of accuracy)

Cell Segmentation

Here are the recommendations for inForm cell segmentation analysis of Opal slide images.

• Use the Counterstain-Based approach.• Segmentation of all cellular compartments (i.e., nuclei, cytoplasm, membrane)

supports better phenotyping.• Start by only selecting nuclei and optimize those parameters. This aids in visual

assessment. Then move on to cytoplasm and membrane segmentation.• Checking the “Use Membrane Signal To Aid Segmentation” typically improves

segmentation results when there is good membrane signal.• Familiarize yourself with all parameters in the Counterstain-Based approach editor by

adjusting and observing the effect of each on segmentation .• The default settings (for inForm 2.1 or later) are a good place to start, with the

exception of lowering the minimum size to 50 pixels and setting the splitting slidertoward the middle. Note that if splitting is an issue and you have a reliable membranemarker, the membrane can be used to aid in cell segmentation

• Optimize parameters as much as you can before starting phenotype training. Changingany of the tissue or cell segmentation parameters erases phenotype training cellsections. Because of this, it is highly recommended to review the tissue and cellsegmentation algorithms for all of the images to be analyzed with before proceeding tophenotyping. This can be done within an inForm project for a small number of images,but may require batching for a large number of images. If problematic images arediscovered, they can either be discarded or pulled into the inForm project for furthertraining.

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Phenotyping

Here are recommendations for inForm cell phenotyping analysis of Opal slide images.

• Only phenotype what is needed to achieve your measurement goal – don’t createunnecessary phenotypes. Include an ‘other’ phenotype category for all remainingcell types. Use one phenotype per project and utilize Excel or R to combine thedata.

• Make sure tissue and cell segmentation parameters have been adequatelyoptimized before selecting phenotyping training cells. Make sure to check the cellsegmentation of cells included in the phenotype training set to avoid training onpoorly segmented cells, which can skew the results (such as training on negativecells that neighbor positive cells since the shared membrane can result in falsepositives). Any pre-existing phenotype training will be lost if tissue or cellsegmentation parameters are changed.

• For cancer immunology studies across multiple samples, we have found that 5 – 30examples of each cellular phenotype are necessary for reliable phenotyping to start.This may involve an initial training with 5 – 10 for reach phenotype, followed by 3 – 5iterations to assess results. New cells are added during each iteration to improvephenotyping. With a well stained, abundant sample, our training set ends upanywhere between 50-150 examples of each, with a smaller training set beingoptimal.

o We recommend separating out diseases states and only phenotyping onedisease state per inForm project.

Validation

Human interpretation of the imagery is still the gold standard for confirming stain localization and specificity, tissue segmentation, cellular phenotypes and cellular compartments. It is very important to have staining and segmentation results reviewed by someone like a pathologist who can recognize tissue architectures and morphologies. It is also very useful to reference against images from peer-reviewed publications as well as online resources like the Protein Atlas3 and GeneCards4.

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Opal Multiplex IHC Assay: Automation

The basic premise of the Opal Multiplex assay does not change when performing it on an automated platform. It is still necessary to perform all of the appropriate assay development steps, including antibody optimization (running DAB single stains for antibody titration and antigen retrieval buffer parameters, antibody-Opal fluorophore pairings, running monoplexes with the correct number of antibody stripping steps incorporated into your protocol, etc.) The guidance above for assay development is applicable for all platforms.

The largest difference between running the Opal Multiplex assay manually and via an automated platform is the method used to strip previously bound antibodies from the tissue. Therefore, there is an additional set of controls we suggest you run when optimizing Opal Multiplex on an automated platform.

Note: If you intend to utilize your automated platform as your primary method of staining, it is preferable to accomplish all of your optimization activities automated. Optimized manual assays are not “plug-and-play” into automated protocols. Manual and automated concentrations for both primary antibodies and Opal fluorophores can be, and frequently are, different.

• Drop-out controls: Automated antibody stripping is not always 100% efficient. It isimportant to empirically determine if previously bound antibodies are removed/denaturedappropriately, as to ensure signal is not cross-talk. Please note, the following protocolsare assuming the use of a cocktailed secondary polymer.

You will need to run 5 control slides, with the following parameters:• Slide 1: First complete sequence, denature. Second sequence,

without primary antibody (but with secondary and detection.)• Slide 2: Second complete sequence, denature. Third sequence,

without primary antibody (but with secondary and detection.)• Slide 3: Third complete sequence, denature. Fourth sequence,

without primary antibody (but with secondary and detection.)• Slide 4: Fourth complete sequence, denature. Fifth sequence,

without primary antibody (but with secondary and detection.)• Slide 5: Fifth complete sequence, denature. Sixth sequence,

without primary antibody (but with secondary and detection.) If any signal is present in the channel without a primary antibody,

denaturing time or temperature needs to be increased. We recommendtrying both 97 C and 100 C for denaturing.

• Create 6-plex based on above empirical findingso Minding antibody application order (as determined by running Monoplexes with the

correct number of antibody stripping steps written in), antibody concentration,Opal-antibody pairing, Opal reagent concentration, and denaturing parameters foreach antibody in the panel

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References

1. Toth, Zsuzsanna E., and Eva Mezey. "Simultaneous visualization of multiple antigenswith tyramide signal amplification using antibodies from the same species." Journal ofHistochemistry & Cytochemistry 55.6 (2007): 545-554. (http://jhc.sagepub.com/content/55/6/545.short)

2. Stack, Edward C., Chichung Wang, Kristin A. Roman, and Clifford C. Hoyt. "Multiplexedimmunohistochemistry, imaging, and quantitation: A review, with an assessment ofTyramide signal amplification, multispectral imaging and multiplex analysis." Methods70, no. 1 (2014): 46-58. (http://www.sciencedirect.com/science/article/pii/S1046202314002837)

3. Uhlén, Mathias, et al. "Tissue-based map of the human proteome." Science 347.6220(2015): 1260419. (http://www.proteinatlas.org/)

4. GeneCards®: The Human Gene Database (http://www.genecards.org)

Supplemental Information

Akoya Bioscience, Inc. Phenoptics MultiSpectral Imaging Systems

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Ordering Information

Opal Multiplex IHC Kits

Opal 4-Color Automation IHC Kit 50 slides NEL820001KT

Opal 7-Color Automation IHC Kit 50 slides NEL821001KT

Opal 4-Color Manual IHC Kit 50 slides NEL810001KT

Opal 7-Color Manual IHC Kit 50 slides NEL811001KT

Opal Cancer Immunology IHC Panels

Opal 7 Tumor Infiltrating Lymphocyte (TIL) Panel A Kit* (CD4, CD8, CD20, FOXP3, CD45RO, panCK)

50 slides OP7TL3001KT

Opal 7 TIL Panel B Kit* (CD4, CD8, CD20, FOXP3, CD68, panCK) 50 slides OP7TL4001KT

Opal 7 Immunology Discovery Panel Kit* (CD4, CD8, CD68, +3 open channels)

50 slides OP7DS2001KT

Opal 4-color Lymphocyte Panel Kit (CD4, CD8, CD20) 50 slides OP4LY2001KT

Opal IHC Panel kits include primary antibodies, Opal Polymer HRP, Antibody Diluent, Opal fluorophores, DAPI, AR6 & AR9 buffer. *Opal 7-color kits require multispectral imaging.

Opal Reagent Packs

Opal 520 Reagent Pack FP1487001KT

Opal 540 Reagent Pack FP1494001KT

Opal 570 Reagent Pack FP1488001KT

Opal 620 Reagent Pack FP1495001KT

Opal 650 Reagent Pack FP1496001KT

Opal 690 Reagent Pack FP1497001KT

Ancillary Reagents and Accessories

1X Plus Amplification Diluent 1 X 50 mL FP1498A

AR6 buffer (10X) 4 x 250 mL AR6001KT

AR6 buffer (10X) 250 mL AR600250ML

AR9 buffer (10X) 4 x 250 mL AR9001KT

AR9 buffer (10X) 250 mL AR900250ML

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Antibody Diluent / Block 100 mL ARD1001A

Opal Polymer HRP Ms + Rb 50 mL ARH1001A

Spectra DAPI FP1490A

Opal Slide Processing Jars STJAR4 Opal IHC Detection kits include Opal fluorophores, DAPI and AR6 buffer.

Opal Detection Workflow Checklist • Check off every step once completed.• If less than 6 antibodies are used, draw a line through the unused antibody columns on page 2and write “N/A” over column to indicate steps were not performed.• In the second row of the table, list the antigen for each antibody.• In the third row of the table, list the species of the primary antibody.• In the fourth row of the table, complete dilution factor for each antibody.• In the fifth row of the table, complete the incubation time for each antibody.'• In the sixth row of the table, complete the incubation temperature for each antibody.• In the seventh row of the table, list the Opal fluorophore to be used for the antigen.• In the eighth row of the table, circle AR6 or AR9 to indicate antigen retrieval conditions foreach antibody

Multiplex Assay Panel Name:

Antigen Antibody Type Clone # Vendor Catalogue # Lot #

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Preliminary Steps 1. Bake slides for at least 1 hour or overnight at 65 °C

2. Wash slides in xylene for 10 minutes, 3 times

3. Wash slides in 100% Ethanol for 10 minutes

4. Wash slides in 95% Ethanol for 10 minutes

5. Rinse slides in 70% Ethanol

6. Rinse slides in distilled Water

7. Incubate slides in NBF for 20 minutes (some tissues may need longer times)

8. Rinse slides in distilled Water

9. Rinse slides in AR 6 or 9 (use the AR solution that will be used for the first antibody)

10. Repeat step 9Opal Detection

Antibody 1 Antibody 2 Antibody 3 Antibody 4 Antibody 5 Antibody 6

Antigen

Species

Dilution

Inc. Time

Inc. Temp.

Opal fluor

AR6 / AR9 Fill jar AR 6 or 9 Fill jar AR 6 or 9 Fill jar AR 6 or 9 Fill jar AR 6 or 9 Fill jar AR 6 or 9 Fill jar AR 6 or 9 Micro 45" high Micro 45" high Micro 45" high Micro 45" high Micro 45" high Micro 45" high Micro 15' 20% Micro 15' 20% Micro 15' 20% Micro 15' 20% Micro 15' 20% Micro 15' 20% Cool at least 15' Cool at least 15' Cool at least 15' Cool at least 15' Cool at least 15' Cool at least 15' Rinse water Rinse water Rinse water Rinse water Rinse water Rinse water Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST PAP pen encircle PAP pen encircle PAP pen encircle PAP pen encircle PAP pen encircle PAP pen encircle Block 10' Block 10' Block 10' Block 10' Block 10' Block 10' Antibody Antibody Antibody Antibody Antibody Antibody Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' HRP 10' HRP 10' HRP 10' HRP 10' HRP 10' HRP 10' Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' Opal 10' Opal 10' Opal 10' Opal 10' Opal 10' Opal 10' Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST Rinse TBST TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2' TBST 2'

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Use AR6 or AR9 as specified in

next col. Rinse AR 6 or 9 Rinse AR 6 or 9 Rinse AR 6 or 9 Rinse AR 6 or 9 Rinse AR 6 or 9 Rinse AR 6 or 9

Final Steps • Fill jar with AR solution pH 6• Heat slides in microwave for 45 seconds at high power, solution should boil• Heat slides in microwave for 15 minutes at 20% power• Allow slides to cool on bench for at least 15 minutes• Rinse slides in distilled Water• Rinse slides in TBST• Incubate slides with DAPI solution for one minute• Wash slides in TBST for two minutes• Wash slides in distilled Water for two minutes• Allow slides to dry• Apply fluorescent mounting medium to the slides• Apply coverslip to the slides

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