Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

All tissues used in this protocol were obtained under the Singapore NHG Domain Specific Review Board B study 2014/00693.

1. Selection and Validation of Antibodies

NOTE: Before proceeding with the establishment of any multiplexed panel, ensure that all antibodies stain robustly, identifying only the target antigen of interest. The aim is to select antibodies that specifically recognize the antigen of interest in tissue sections.

1. For an antibody with a well-established research use, or routine clinical use in IHC, confirm conditions such as epitope retrieval and antibody dilution by performing conventional IHC^5,13 on positive and negative control tissue sections. For tissue sections of human origin, ensure that the appropriate ethics clearances are in place prior to initiating experiments.
   NOTE: Positive controls are tissues that are expected to express the antigen of interest, and negative controls are those that do not. Benign tonsil tissue is chosen as a good tissue control for lymphoma antigens because it contains a mixture of immune cells including B cells, T cells, and antigen-presenting cells, as well as stromal and epithelial cells. The latter serve as useful negative internal controls.
2. For unknown targets or for commercial antibodies with insufficient published data, perform antibody validation by creating matched positive and negative control FFPE cell blocks¹⁴, using CRISPR knock-out or siRNA knock-down of the antigen of interest in an appropriate cell line through standard molecular biology techniques. Use these FFPE cell blocks in lieu of positive and negative control tissues for conventional IHC as per step 1.1.
   NOTE: HeLa or 293T cells are commonly used for antigens that are not cell-type specific, as lymphoma cell lines are difficult to transfect. For antigens that are lymphocyte specific, lymphoma cell lines can be used with viral transduction or electroporation as the mode of gene delivery (albeit with low efficacy).

2. Planning the Sequence of Antibodies and Fluorophores for the Multiplex Panel

1. Plan the sequence of reagents for the final multiplex staining. For example, here the sequence for the multiplex protocol was initially planned as first, Bcl-6; second, Bcl-2; third, C-Myc; fourth, CD20; fifth, Ki67.
   NOTE: To decide on the sequence, antibodies with a weak affinity requiring higher concentrations should be stained first in the final multiplex staining. High-affinity antibodies that are likely to be resistant to multiple rounds of microwave stripping are applied last in the multiplex protocol to avoid nonspecific staining.
2. Plan the fluorophore partner for each antibody in the panel. See Table 1 and Table of Materials for the choices in this example.
   NOTE: To decide on the fluorophore partner for each antibody, choose spectrally distinct fluorophores for antibodies with similar patterns of localization within the cell and within tissue. This will minimize spectral overlap and difficulty with unmixing.

3. Monoplex Tyramide-based IF in a Simulated 5-plex Multiplex Panel

NOTE: In this example, the protocol for CD20 is discussed, which is planned as the fourth antibody in a multiplex sequence described above. The number of additional stripping steps will differ for the position of the antibody in the sequence.

1. Cut 3 µm-thin sections of positive and negative control tissue using a microtome and place the sections on poly-L-lysine-coated microscope glass slides.
2. Dewax the sections using an appropriate clearing solution 3x for 4 min each. Rehydrate the slides in 100% alcohol, 90% alcohol, and 70% alcohol, 4 min each. Put the slides in distilled water for 2 – 3 min.
3. Place the slides in a microwave-safe glass jar in a standard antigen retrieval buffer (commercially available) to immerse the slides. Perform heat-induced epitope retrieval (HIER) using a suitable microwave, in pH9 antigen retrieval solution at 98 °C for 25 min.
   NOTE: Any microwave with a pressure ranging from 800 – 1,100 Watts can be used, and HIER can be optimized accordingly. In this case, a multifunctional microwave tissue processor (open to air) was used for HIER and stripping. The initial settings for the retrieval of a particular epitope are based on the prior knowledge from conventional IHC.
4. Perform additional rounds of microwave stripping (three in this case, for the fourth antibody in a panel), each round with 100% power to 98 °C and 20% power for 10 min to maintain at the  same temperature. Cool down the slides in distilled water for at least 10 min.
   NOTE: Perform multiple rounds of microwave stripping during the monoplex IF step to expose the target antigen to the same number of heating steps as in the proposed multiplex. Since CD20 is planned as the fourth antibody, do microwave stripping 3x before and one time after doing the primary antibody incubation.
5. Check and replenish the buffer after every 5 min during additional microwave stripping. Importantly, do not let slides dry out during the multiple stripping procedures. When taking the slides out of the microwave, use forceps and heatproof gloves to place them into a separate jar of distilled water. Wait for the antigen retrieval solution to cool naturally.
6. Block the tissue peroxidase activity by using a commercial peroxidase block for 10 min (3% hydrogen peroxide can be used as an alternate reagent). Wash with Tris-buffered saline (TBS) and a nonionic detergent for 5 min.
   NOTE: TBS is composed of 50 mM Tris-Cl, pH 7.5, and 150 mM NaCl.TBS to make TBS-D.
7. Block with antibody diluent or Bovine serum albumin for 15 min followed by incubation with the primary antibody of interest (e.g., CD20, 1:2,000 dilution in antibody diluent, see Table of Materials) at room temperature for 30 min. Wash 3x with TBS-D buffer for 5 min each time. There should be sufficient TBS-D buffer to immerse the slide completely in all washing steps.
   NOTE: Bovine serum albumin can be used as an alternate antibody diluent. The volume of antibody diluent depends on the size of the tissue, ranging from 50 µL (for biopsy samples) to 400 µL (for excision samples or tissue microarray samples).
8. Incubate with an appropriate horseradish peroxidase (HRP)-labelled secondary antibody, chosen on the basis of the species of origin of the primary antibody for 15 min at room temperature. Wash 3x with TBS-D buffer for 5 min each time.
9. Apply an appropriate tyramide-based fluorescent reagent (1:100 in amplification diluent) and incubate at room temperature for 5 min, to allow fluorophore conjugation to the tissue sample at the sites of primary antibody binding. Wash 3x with TBS-D buffer for 5 min each time.
10. Perform an additional microwave-based stripping to remove the primary and secondary antibody. Repeat as required based on the position of the antibody in the sequence.
11. Place the slide in distilled water to cool down. Incubate with DAPI (1:100 dilution in antibody diluent) for 10 min, followed by wash 3x with TBS-D buffer for 5 min each time. Dry the area on the slide without the tissue with wipes and mount the slide with appropriate mounting media.
12. Image the slide (see sections 6 and 7 of this protocol) and determine the appropriateness of the staining (as defined by clear discrimination of the positive and negative control tissue).
    NOTE: If the staining pattern is incorrect, redo the monoplex staining after adjusting one or more of the following parameters: the number of heat retrieval steps, the amount of heat retrieval, the incubation period/concentration of antibodies (primary and secondary), the position of antibody in the sequence, and the choice of fluorophore. The monoplex staining step typically requires multiple rounds of optimization to obtain a suitable set of parameters for appropriate staining. It is advisable to test a range of fluorophores with each primary antibody, as this will provide flexibility in deciding the final multiplex set.

4. Repetition of the Monoplex for Each Antibody in the Multiplex Protocol

1. Repeat the monoplex staining for the other antibodies in a similar way by adjusting the number of microwave stripping steps, based on the position of the antibody in the sequence. For instance, when performing monoplex staining for the third antibody in a six-plex multiplex panel, perform two microwave stripping steps before and three microwave stripping steps after the primary antibody incubation.
   NOTE: It is important to appreciate that the microwave-based stripping of antibodies also exposes the sample to HIER. If a specific antibody requires a different epitope retrieval strategy, this needs to be taken into account when planning the multiplex sequence. In this example, Ki67 epitope retrieval requires pH 9, for 30 min. Since 25 min of HIER will be done prior to KI67 staining in the sequential protocol, only an extra 5 min of HIER are needed.

5. Multiplex Staining Protocol

NOTE: Proceed with the multiplex staining protocol only after all the components have been optimized using monoplex IF staining. Review the results of the monoplex staining and design a table showing the final layout of the order of multiplex and the choice of fluorophore for each antibody. The details of antibody concentration, the duration of staining, and the sequence and nature of heat retrieval for each antibody used here is provided in Table 1.

1. Cut 3 µm-thin sections of positive and negative control tissue, as well as the target tissue of interest (here, FFPE samples of lymphoma), using a microtome, and place the sections on poly-L-lysine-coated microscope glass slides (see Table of Materials).
   NOTE: Inclusion of the positive and negative controls for each antibody in the panel is advisable, along with the actual samples for multiplex. This allows for a confirmation that the staining protocol was performed correctly.
2. Dewax, perform heat-induced epitope retrieval (HIER), and block tissue peroxidase activity as in the monoplex protocol steps 3.3 – 3.5.
3. Incubate with the first primary antibody of the optimized multiplex sequence, as previously determined using the monoplex IF step. In this example, BCL-6, at a 1:30 dilution in antibody diluent at room temperature for 60 min (see Table of Materials), was the first step of the multiplex protocol. Wash with TBS-D buffer for 5 min.
4. Incubate with an appropriate HRP-labelled secondary antibody based on the species of the primary antibody used in the prior step (see Table of Materials) at room temperature for 10 min. Wash with TBS-D buffer for 5 min.
5. Apply the optimized tyramide-based fluorescent reagent (Cy5 in this example, 1:100 in amplification diluent, see Table of Materials) with incubation at room temperature for 5 min. After the incubation, wash with TBS-D buffer for 5 min.
   NOTE: The choice of the tyramide-based fluorescent reagent is based on the optimized monoplex IF.
6. Check the efficiency of staining of the first antibody using an appropriate microscope (see Table of Materials).
   NOTE: Interim imaging checks can be done with ease if the sample is a TMA or single slide. If, however, the stain is being performed on multiple slides, this may be impractical, and this step may be omitted.
7. Perform microwave stripping for the second antibody in the protocol, using conditions optimized in the monoplex step. Then, proceed with the staining of the second primary antibody (here, BCL2) with the HRP-labelled secondary antibody and tyramide-based fluorescent reagent (here, 520, see Table of Materials). Check the efficiency of staining under a fluorescent microscope after the completion of the second round of staining.
8. Similarly, repeat the procedure using the third, fourth, and fifth antibodies in the sequence (here, c-Myc, CD20, and ki67, respectively, with fluorophores 570, 540, and 620, respectively). Perform imaging checks in between each step if feasible.
9. Add a nuclear counterstain (DAPI, 1:100 dilution in antibody diluent) for 10 min and, then, wash 2x in TBS-D for 5 minutes each.
10. Mount the slides using an appropriate mounting reagent.

6. Preparation of Spectral Library Slides

NOTE: Sections 6 – 8 of this protocol are unique to multiplexed experiments that are imaged using a spectral camera.

1. Create library slides (single-stain reference images) for each fluorophore, DAPI, and autofluorescence on the same control tissue, to be used for multispectral image analysis.
   1. Cut 2 x 3 µm-thin sections of the tissue type of interest (here, a lymphoma sample or a tonsil as control) using a microtome and place the sections on poly-L-lysine-coated microscope glass slides.
   2. Process both slides using the monoplex protocol (with all stripping and washing steps), but without antibody or fluorophore addition.
   3. Stain one slide with DAPI as per step 5.9 and leave one slide unstained (for the generation of the tissue autofluorescence spectrum).
      NOTE: The optimized monoplex slides (with a single fluorescence dye, without DAPI) can be used as spectral library slides to generate spectra of each fluorescence dye.
2. Scan these set of slides using appropriate filters and upload them into the image analysis library.

7. Spectral Imaging

1. Scanning monoplex slides
   1. Choose the filters from available standard epi-fluorescence filters (DAPI, FITC, CY3, Texas Red, and CY5) appropriate for the employed fluorophore.
      NOTE: The recommended filter cubes for specific fluorophores used in this example are shown in Table 2¹⁵.
   2. Examine each marker in its corresponding fluorescence channel to identify a suitable exposure time to obtain a clean signal. Focus on the tissue component that is supposed to have the strongest signal for the marker.
   3. Determine a fixed exposure time for each analyte (antibody-fluorophore combination), to allow cross-sample comparison of pixel intensity (although some commercial platforms have normalization strategies to account for differences in exposure).
      1. To decide on a fixed exposure time for a given channel, use a live camera setting to adjust the exposure time until there are no overexposed areas in the live camera image, and repeat this for all channels.
   4. After scanning the monoplex slides, generate simulated brightfield images (if the function is available in the software used) to visually compare with the normal IHC pattern and to determine if the staining pattern is correct (Figure 1 and Figure 2).
2. Scanning multiplex samples (TMA slides/multiple samples)
   1. Set up the optical parameters as described for scanning monoplex images (step 6.1). First, adjust the focus manually or automatically (follow the instructions of the specific image scanning machine). Secondly, adjust the exposure time for each fluorescent channel to ensure that all the cores on a TMA, or all areas on a slide, are appropriately exposed.
      NOTE: The area selected to adjust the exposure time is important. Users need to choose the strongest signal region for each filter (i.e., the Ki67 signal is stronger in the tonsil germinal center region than in the nongerminal center region; hence, users should use the germinal center region set at an appropriate exposure time). Users can also adopt an oversaturation correction function of the imaging system if available.
   2. Scan TMA slides under a TMA scanning mode if available in the imaging system, with an appropriate autofocus algorithm.
   3. For whole-tissue section slides, get the slides reviewed by a qualified pathologist to select optimal images from the most representative tumor areas.
      NOTE: The protocol described here is based on a defined microscope/image analysis system (see Table of Materials). There are other machines and image analysis software that can scan and analyze multiplex IHC slides⁷.

8. Data Analysis

1. Preanalysis assessment and planning
   1. Use the reference slide for autofluorescence to subtract autofluorescence from the images scanned for analysis.
   2. Review the images before analysis to ensure that they are in-focus and without staining artifacts.
   3. Use a tumor marker (e.g., CD20 in this example) to identify cells of interest and to proceed with cell segmentation, scoring, and batch analysis approaches. Select CD20-positive cells for the analysis.
      NOTE: For example, in the DLBCL samples analyzed here, the setting and parameters that are defined for cell segmentation are based on nuclear size and intensity but are specific to the image analysis software used (e.g., a DAPI mean pixel intensity of at least 0.05; size in between 80 – 320 pixels, with a splitting sensitivity at 2 [this is a software-specific parameter which relies heavily on the morphology of the tumor: for small tumor cells, splitting of 0.7 – 1 is appropriate; for large tumor cells, splitting can be adjusted up to 4]).
   4. Request for qualified pathologist to review individual images and decide whether tissue segmentation is required to select tumor cell enriched regions and exclude regions with necrosis arearegion orand abundant reactive cells. If additional tissue segmentation is required, then select appropriate control regions (such as tumor cells/stroma/necrosis) and check that the image analysis software can correctly identify such regions
   5. Proceed with cell segmentation after tissue segmentation (if any), request a qualified pathologist to review the cell segmentation map to ensure the fidelity of the intended segmentation approach⁵.
   6. Determine the most biologically/clinically appropriate method of analysis for a given biomarker of interest (e.g., percentage positivity, or mean intensity per cell).
   7. Select a cut-off value for each marker (for percentage positivity).
   8. Determine the optical intensity positive cut-off value for each marker in conjunction with a pathologist. Generate histograms by analyzing the frequency distribution of the marker intensity/cell in appropriate statistics software (see Table of Materials).
      NOTE: An intensity value histogram can offer an overview of the distribution of the signal intensities.
   9. Determine an approximate cut-off from the histogram and verify this with a pathologist review, to correlate with manually determined cut-offs on selected images. In some situations, a uniform single cut-off will not be possible due to variability in staining, and a manual cut-off value for each sample will be required.
      NOTE: Section 8.1 should be done in image analysis software (see Table of Materials) unless otherwise specified.
2. Marking positive and negative cells
   1. For each marker of interest, according to the cut-off number determined (through histograms or manually), use an “IF” or similar logic formula to mark positive and negative cells with marked value: number 1 for positive cells, number 0 for negative cells.
   2. For the positivity of all the markers, multiply the marked value of each marker (either 1 or 0) with the products in separate columns. If the product equals 1, it means the cell is positive for all markers. For the positivity and negativity of a specific marker, use an IF logic algorithm.
      NOTE: Section 8.2 needs to be done in statistics software (see Table of Materials).
3. Generating percentage data and numeric data using a pivot table
   1. Generate percentage data for specific markers of interest, within defined cells (e.g., CD20-positive cells, or CD20-negative cells) (Figure 6).
   2. Insert a pivot table into the data sheet.
   3. To calculate the positivity percentage of a single marker, such as CD20, select the SUM function under the Value part of the pivot table to count the total number of CD20-positive cells using the sum of the CD20⁺ cells divided by the total cell number. The result is the CD20⁺ cell percentage within one core or one study number (depends on the selection of the pivot table row).
   4. Calculate the positivity percentage of multiple markers using the same method (Figure 3).
   5. Generate numeric data. Extract the median normalized count for each marker of each core or each study number by obtaining the mean intensity of each marker of interest in all the cells studied within a sample (Figure 4).
      NOTE: The median value cannot be derived in the pivot table directly. It can be derived using this formula: MEDIAN (IF (column of study number=specific study number, column of normalized value)).
4. Plotting the data in a suitable graph
   1. Plot the results of the percentage positivity or median intensity per marker in a cell type of interest in an appropriate manner for further statistical testing and presentation.
   2. Create dot plots to provide a visualization of numbers and distribution.
      NOTE: Representing data with bar graphs does not convey information on distribution. Estimation plots are also recommended as a good method for data representation, with emphasis on the magnitude of difference between samples¹⁶.