A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.
Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4′,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
Immunoprofiling analysis of FFPE tumor tissue samples has become an essential component of immuno-oncology studies, particularly for the discovery and validation of novel predictive biomarkers for cancer immunotherapy in the context of clinical trials1,2. Chromogenic IHC, using chemical chromogens such as diaminobenzidine, remains the standard technique in diagnostic pathology for the immunolabeling of biopsy tissue3. Standard IHC can also be used for cancer tissue immunoprofiling, including the quantitation of subpopulations of tumor-associated lymphocytes and the assessment of expression levels of immune checkpoints such as programmed cell death ligand 1 (PD-L1)4,5. Standard IHC is limited, however, in that only one antigen can be labeled per tissue section. Because immunoprofiling studies typically require the analysis of the combined expression of several markers, the use of standard IHC would require the staining of multiple tissue sections, each stained with a single marker, and would, therefore, be substantially limited for the analysis of small tissue samples such as core needle biopsies. Standard IHC methods are also limited for the assessment of markers that are coexpressed by diverse cell populations, as is common with immune checkpoint markers such as PD-L1, which is expressed by both tumor-associated macrophages and cancer cells. This limitation has been reported in, for instance, the use of standard monoplex IHC by pathologists for the quantitative analysis of an IHC marker expressed by diverse cell types6. The development of multiplex chromogenic IHC techniques employing diverse colored chromogens on the same tissue section represents an advancement over the standard IHC monoplex method,7 although they remain limited by the immunolabeling of just a few markers and also present an important technical challenge for the proper evaluation of markers expressed in the same subcellular compartments of the same cell populations.
The aforementioned caveats regarding tissue availability from clinical samples, as well as the limitations of multiplex chromogenic IHC techniques, have given rise to the need to develop improved multiplex methods for immuno-oncology studies based on fluorescent labeling combined with imaging systems that can effectively separate the signals of multiple fluorophores from the same slide. One such technique is based on tyramide signal amplification (TSA) combined with multispectral microscopy imaging for efficient color separation8. A commercially available TSA-based kit employs fluorophores optimized for multispectral imaging8 (see Table of Materials). A critical advantage of this system is its compatibility with the same unlabeled primary antibodies that have already been validated and optimized for standard chromogenic IHC9,10,11. This allows not only faster optimization but also flexibility in the optimization and panel modifications incorporating new targets. Furthermore, the multiplex immunofluorescence (mIF) TSA method can be optimized for commercially available automated IHC stainer systems, allowing for a straightforward transfer from monoplex chromogenic IHC to mIF.
Here we present a protocol for an mIF panel for immuno-oncology studies that is based on automated mIF TSA staining and uses a multispectral scanner for imaging. This protocol can be adapted and modified by any laboratory user with access to the described instrumentation and reagents. The protocol includes a panel of six primary antibodies for the immunoprofiling of carcinomas: PD-L1, PD-1, CD68 (as a pan-macrophage marker), CD8 (T-cytotoxic cells), Ki-67, and AE1/AE3 (pan-cytokeratin, used as an epithelial marker for the identification of carcinoma cells). A recent study describes the optimization of a manual TSA mIF protocol by using chromogenic IHC as a standard reference to validate the multiplex staining12. The updated method presented here has been developed by using a commercially available, seven-color TSA kit optimized in an automated stainer, drastically shortening the staining time from 3–5 days to 14 h, while also improving the consistency of the staining. In addition to the detailed main protocol presented here, a Supplemental Materials section includes the "drop-control" method, an additional quality control process to evaluate a new mIF panel, as well as technical notes for the optimization, troubleshooting, and development of new multiplex panels to help the laboratory user to set up and optimize the mIF TSA method for customized mIF panels.
NOTE: The protocol presented here describes how to perform immunoprofiling of an mIF panel by using TSA for six antibodies (CD68, ki67, PD-L1, PD-1, CD8, and AE1/AE3) on an automated stainer (see Table of Materials). The protocol also describes how to perform the drop controls for a quality control of a new mIF panel (see Supplemental Materials). In this protocol, staining is performed with eight unstained FFPE slides from human tonsil (positive control) and eight unstained slides from human lung adenocarcinoma. The first slide is used for full multiplex staining with all six markers, the second slide for the isotype control in which no primary antibodies are utilized, and the remaining six slides for the drop controls (see Supplemental Materials). An additional control for tissue autofluorescence is highly recommended and should always be included in a multiplex study (see Supplemental Materials). However, investigators can employ other tumor types and controls according to their own project goals. Laboratory users without previous experience with the mIF TSA method and multispectral scanner techniques should read the Multiplex IHC Development Guide (available online at http://info.perkinelmer.com/2016-lp-Opalassaydevelopmentguide-lp). Although this guide describes a manual protocol, it also provides a good introduction to the mIF staining method. All tissue sections employed in this protocol were anonymised and approved according to the Declaration of Helsinki.
1. Tissue Samples
2. Creation of a New mIF Staining Program on an Automated Stainer: Registration of the Reagents
NOTE: Reagents must be added to the reagent list in the automated stainer software before they are available for use in protocols. See the Table of Materials for information on the automated stainer model and software version.
3. Automated Stainer: Registration of the Containers
4. Automated Stainer: Creation of an mIF Protocol Program
5. Automated Stainer: Addition of Drop Controls, and Isotype Control Protocol
6. Automated Stainer: Slide Preparation Protocol
7. Automated Stainer: Preparation of the Reagents
8. Automated Stainer: Sample Setup and Multispectral Staining
9. Coverslipping of the Multispectral Slides
10. Multispectral Scanner
11. Multispectral Scanner: Multispectral Imaging
12. Spectral Unmixing
NOTE: The spectral unmixing step is required for channel separation and image analysis of the slides. Before spectral unmixing is performed in the inForm software, the spectral library must be built by using lung adenocarcinoma slides stained with each fluor alone and with DAPI alone. This procedure is also described in the aforementioned Multiplex IHC Assay Development Guide.
13. Evaluation of Fluorescence Intensity and Signal Attenuation
NOTE: The counts tool, which appears as an arrow with a small brown box, is the most important tool for multiplex optimization and should be used frequently (see Supplemental Materials). Using the counts tool in the spectral unmixing software, assess the signal-to-noise ratio, which at a minimum should exceed 10:1 (see Supplemental Materials for troubleshooting and for the evaluation of the multiplex staining with the drop controls).
14. Multispectral Image Analysis
The protocol described here will provide results like those shown in Figure 2. Start with an evaluation of the staining in the tonsil control, beginning with the surface squamous cell epithelium. The histology of the tonsil sample can be reviewed with a pathologist, using the H&E slide as a reference. If chromogenic IHC sections are performed with the same markers on the same tissue block, then these can be used to confirm the density and distribution of each marker on the mIF slide. As shown in Figure 2A, the tonsil tissue should provide clearly defined AE1/AE3 cytokeratin staining in the tonsil surface squamous epithelium (Figure 2A; labeled as red pseudocolor), without background in the lymphoid tissue. The staining should be cytoplasmic, occasionally with membrane accentuation, and the nuclei must be negative. The reticulated epithelium in the crypts should be positive for both cytokeratins (red) and PD-L1 (Figure 2A; green pseudocolor). The follicular germinal centers of the tonsil should, then, be identified. The germinal centers should be easily recognizable and should be rich in Ki-67-positive lymphocytes (Figure 2A; yellow pseudocolor). Ki-67 staining should always be nuclear. The macrophages present in the germinal centers should also be easily recognizable, sometimes as larger cells (also called "tingible bodies"). They will show positive staining for CD68 as a granular cytoplasmic stain (Figure 2A; orange pseudocolor). A variable proportion of CD68 cells outside the germinal centers is also to be expected. Macrophages may show membrane staining for PD-L1. This is typically paler in intensity than staining for PD-L1 in the reticulated epithelium. There should be no nuclear CD68 staining. CD8 should stain lymphocytes with a T-cell distribution (fewer in the germinal centers and a greater proportion in the interfollicular areas; see Figure 2A, cyan pseudocolor). CD8+ lymphocytes are usually small cells with scant cytoplasm, so it is practically impossible to distinguish membrane versus cytoplasm in the lymphocytes. PD-1 will strongly stain small lymphocytes in the germinal center area, usually clustered at the periphery of the germinal centers (Figure 2A; magenta pseudocolor), as well as scattered lymphocytes in the interfollicular region, usually showing a lower staining intensity than those in the germinal center area. As with CD8, it is not possible to clearly distinguish membrane and cytoplasmic staining in small lymphocytes stained with PD-1. Because a variable proportion of CD8 cells coexpress PD-1, it is recommended for quality-control purposes that the staining pattern and distribution of each marker that is stained in the same tissues be compared with chromogenic IHC reference slides, as suggested in previously published protocols12.
After the expected staining pattern has been confirmed in the tonsil tissue control, proceed to evaluate the staining in the lung cancer sample (Figure 2B). Because tumor tissues are expected to show a variable and heterogeneous expression of the markers, it is very important during the optimization of a new mIF panel to compare the staining pattern for each individual marker observed in the mIF method and its expression observed in the standard chromogenic IHC slide, evaluating the quantity and distribution of the positive cells as previously described12. Nevertheless, the basic staining pattern should be preserved; for example, cytokeratins should be observed in the cytoplasm of epithelial cells and never in a nucleus; CD68 should appear as granular cytoplasmic staining in macrophages, etc. Importantly, in some markers, the staining intensity may change from the tonsil control to the cancer tissue; for example, PD-1 and PD-L1 can show lower-intensity staining in the tumor tissue as compared with the very high expression observed in tonsil control. It is, therefore, important to perform the evaluation and optimization with the counts tool in the inForm software, using examples of tumor tissues rather than the tonsil controls.
Finally, isotype-negative controls and drop controls need to be evaluated, not only for the detection of background and autofluorescence but also for umbrella effects and spectral bleed (Figure 3 and Figure 4). Isotype controls should not demonstrate any immunostaining across the slide; if any staining is observed, then the imaging or staining procedure must be revisited. Drop controls are important to evaluate potential artifacts in the staining, such as spectral bleed or umbrella effects, due to the mIF method during the optimization of a new multiplex panel (see Figure 5, Supplementary Figure S2, Supplementary Figure S3, and Supplemental Materials). Fluorescent monoplex and drop controls should be evaluated and compared with the full multiplex panel. Each drop control should show the same staining pattern except for the single primary antibody, which should be removed on each specific control. Further details are provided in the Supplemental Materials section.
Figure 1: inForm Counts Tool. The inForm software counts tool is activated by selecting the beige box icon. Normalized counts are corrected for bit depth, exposure time, gain, and binning. The images were taken at 20X magnification; the scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 2: Representative results. (A) Tonsil and (B) nonsmall-cell lung carcinoma were stained with PD-L1 (520 TSA, green), CD8 (540 TSA, cyan), Ki67 (570 TSA, yellow), CD68 (620 TSA, orange), cytokeratin (650 TSA, red), and PD-1 (690 TSA, magenta). Individual channels are represented separately below in small panels. The marker is indicated in the upper right of each image. The images were taken at 20X magnification; Scale bar = 50 (or 250 µm). Indicated in panel A are 1) lymphoid follicle, 2) germinal center, 3) interfollicular area, and 4) stratified squamous epithelium. Please click here to view a larger version of this figure.
Figure 3: TSA blocking/umbrella effect. This is an example of 690 TSA deposited by a PD-1 antibody-blocking CD3 signal in a manner dependent on the amount of TSA present. The brightest 690 TSA signal blocks the 620 TSA signal most effectively (nonsmall-cell lung carcinoma stained with anti-PD-1 D4W2J for 30 min at 0.52 µg/mL or isotype followed by a 10 min 690 TSA detection at 1:100; then, anti-CD3 F7.2.38 for 30 min at 0.14 µg/mL followed by a 10 min 620 TSA detection at 1:100). The images were taken at 20X magnification; the scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: Spectral bleed. Nonsmall-cell lung carcinoma tissue was stained with the Ki-67 drop-control protocol (complete multiplex protocol with Ki-67 antibody omitted). The 540 TSA channel (CD8) and the 570 TSA channel (Ki-67) are shown. No nuclear staining pattern is apparent, yet a diminished copy of the CD8 staining pattern is observed in the 570 TSA channel. This is best addressed by ensuring comparable staining intensities in all channels. In this case, the addition of a robust Ki-67 signal (normalized counts between 10 and 20) will correct the spectral bleed. The images were taken at 20X magnification; the scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 5: Drop controls. This figure shows a composite image of a full multiplex compared with the same region of sequential slides of lung adenocarcinoma stained with drop controls in which the indicated primary antibody was omitted. The images were taken at 20x magnification; Scale bar = 50 µm. Please click here to view a larger version of this figure.
Name | Abbr. Name | Type | Container | Group |
PKI Blocking Buffer | OPALBLOK | Ancillary | Open 30 mL | General |
Opal Polymer HRP | OPAL 2AB | Ancillary | Open 30 mL | General |
OPAL 1x TBS | OPAL1TBS | Ancillary | Open 30 mL | Det. Kit |
CD68 0.30 µg/mL PG-M1 Mus | OPALCD68 | Ancillary | Open 7 mL | General |
ki67 0.61 µg/mL MIB-1 Mus | OPALki67 | Ancillary | Open 7 mL | General |
PD-L1 0.20 µg/mL SP263 Rab | OPALPDL1 | Ancillary | Open 7 mL | General |
PD-1 0.52 µg/mL D4W2J Rab | OPAL PD1 | Ancillary | Open 7 mL | General |
CD8 0.70 µg/mL SP239 Rab | OPAL CD8 | Ancillary | Open 7 mL | General |
CK 0.24 µg/mL AE1/AE3 Mus | OPAL CK | Ancillary | Open 7 mL | General |
Mouse IgG | OPAL MUS | Ancillary | Open 7 mL | General |
Rabbit IgG | OPAL RAB | Ancillary | Open 7 mL | General |
OPAL Peroxidase Block | OPALPERX | Ancillary | Open 7 mL | General |
Spectral DAPI | OPALDAPI | Ancillary | Titration 6 mL | General |
Opal 520 Reagent | OPAL 520 | Ancillary | Titration 6 mL | General |
Opal 540 Reagent | OPAL 540 | Ancillary | Titration 6 mL | General |
Opal 570 Reagent | OPAL 570 | Ancillary | Titration 6 mL | General |
Opal 620 Reagent | OPAL 620 | Ancillary | Titration 6 mL | General |
Opal 650 Reagent | OPAL 650 | Ancillary | Titration 6 mL | General |
Opal 690 Reagent | OPAL 690 | Ancillary | Titration 6 mL | General |
Table 1: Leica Bond RX Protocol, Opal Reagents
Reagent | Time (h) | Temperature (degrees Celsius) | |
1 | No Reagent | 120:00 | 60 |
2 | Bond Dewax Solution | 0:30 | 72 |
3 | Bond Dewax Solution | 0:00 | 72 |
4 | Bond Dewax Solution | 0:00 | Ambient (room temperature) |
Table 2: OPAL Preparation Protocol
Target | µg/mL | Clone | Source | Lot | Stock µg/mL | µL Diluent | µL AB |
CD68 | 0.3 | PG-M1 | Dako | 20043031 | 30 | 2376 | 24 |
ki67 | 0.61 | MIB-1 | Dako | 20049476 | 46 | 2368.17 | 31.83 |
PD-L1 | 0.2 | SP263 | Vent. | F09591 | 1.61 | 2101.86 | 298.14 |
PD-1 | 0.52 | D4W2J | CST | 1 | 100 | 2387.52 | 12.48 |
CD8 | 0.7 | SP239 | Ventana | 160318 | 70 | 2376 | 24 |
CK | 0.24 | AE1/AE3 | Dako | 10129428 | 179.5 | 2396.79 | 3.21 |
Mus IgG | 0.61 | ||||||
Rab IgG | 0.7 |
Table 3: Primary Antibodies Preparation.
Opal Fluor | Slides | Dilution | µL Diluent | µL Opal Fluor |
Opal 520 | 16 | 1:200 | 2736.3 | 13.8 |
Opal 540 | 16 | 1:700 | 2746.1 | 3.9 |
Opal 570 | 16 | 1:150 | 2731.7 | 18.3 |
Opal 620 | 16 | 1:100 | 2722.5 | 27.5 |
Opal 650 | 16 | 1:350 | 2742.1 | 7.9 |
Opal 690 | 16 | 1:150 | 2731.7 | 18.3 |
Table 4: OPAL Fluor Preparation.
Supplemental Material Word Document: Please click here to download this document.
Supplemental Figure 1: Please click here to download this figure.
Supplemental Figure 2: Please click here to download this figure.
Supplemental Figure 3: Please click here to download this figure.
Supplemental Table 1: Leica Bond RX Opal Multiplex Protocol. Please click here to download this table.
Supplemental Table 2: Summary and comparison of full multiplex, drop controls, and full isotype controls. Please click here to download this table.
The ongoing cancer immunotherapy revolution is opening novel and promising therapeutic options for cancer patients13. Advances in the field of immuno-oncology will require increased knowledge of the inflammatory tumor microenvironment, not only to understand the biology of the immunological mechanisms involved in carcinogenesis but also to find predictive biomarkers for new immunotherapy-based treatments1,2. Due to the complex biology of cancer immunology, interrogation of tumor tissue samples is usually required to develop a growing list of immune markers, which interact with each other and are frequently coexpressed by diverse cell populations in the tissue1,2,5. Clinical trials typically employ small biopsies, such as core needle or endoscopic biopsies, which are collected at different points of therapy to evaluate and monitor patient response. Such biopsies provide limited amounts of tissue, which in turn limits the number of tissue sections that can be employed for cancer immunoprofiling. This limitation is particularly onerous when traditional techniques, such as standard monoplex IHC, are used. There is, therefore, a clear need for new methods for cancer immunoprofiling that make use of multiplex techniques with the capacity to simultaneously evaluate the coexpression of different biomarkers and to make more efficient use of valuable tissue specimens.
In the multiplex staining and multispectral imaging methods described in detail here, an mIF panel is used for immuno-oncology studies on FFPE tissues such as biopsies from a clinical trial. This method has been previously described, validated, and successfully applied to cancer immunoprofiling8,9,10,11,12,14,15. Previously published protocols have described similar mIF staining methods with TSA-based systems, such as the seven-color kit, which is time-consuming and requires approximately 4 days of work by a dedicated laboratory scientist12,15. In response to these shortcomings, this protocol has been optimized by using a commercially available automated stainer, dramatically shortening the staining time to 14 h and eliminating the variability introduced by manual operators. Imaging is conducted on a multispectral scanner capable of separating the spectra of the seven or more fluorescent signals in the multiplex slides, including autofluorescence, which can be efficiently removed without degrading the signal quality. This protocol can be replicated, modified, and performed by any laboratory user with access to the instruments and reagents detailed in the Table of Materials.
Critical steps in this protocol are sections 7, 10, 11, 12. Section 7 pertains to the preparation of reagents. A careful preparation of the reagents for multiplex staining is essential, requiring the focused attention of a laboratory scientist or technician to avoid operator-dependent variation. For instance, one of the major issues with the multiplex method is staining variability, which can be reduced by the careful preparation of the dilutions of the reagents, specifically the TSA dyes. Sections 10 and 11 are related to image acquisition. An important risk is the overexposure or oversaturation of the images, which may lead to spectral bleed, an artifact in which the signal from another channel interferes with the channel being imaged (see Supplemental Materials). Carefully regulating the exposure times can help to prevent oversaturation and spectral bleed. Spectral unmixing is conducted in section 12. This relies on the preparation of the spectral library (described in the Multiplex IHC Assay Development Guide, available online). In this step, the inForm software is "trained" to specifically recognize the colors for each TSA dye. The spectral library is created by using sections from a control tissue, such as human tonsil, stained with a uniformly expressed marker, such as CD20, coupled with each individual TSA dye on individual slides. In addition, autofluorescence slides (also described in the Multiplex IHC Assay Development Guide) are needed to train the inForm software to recognize and subtract tissue autofluorescence. Autofluorescence control slides should be prepared by using samples from the same tissues being studied (such as lung cancer, etc.), treated in the same way as the mIF slides, and incubated with primary and secondary antibodies but without the TSA dye. Only a single autofluorescence spectral profile can be selected, so care must be taken to select a representation of the observable autofluorescence which may come from collagen, fibrosis, red blood cells, endogenous pigments, and other sources. Creating a new spectral library at the beginning of each new project and using an autofluorescence spectrum which is representative of the tissue study specimens, as well as running the same tissue blocks as the positive control for each batch within the same project, are valuable methodological steps that will help to improve the consistency of the spectral unmixing across different samples within the same project.
The multiplex method described in this protocol offers several troubleshooting tools, including a signal-to-noise evaluator (counts tool), drop controls (Supplemental Materials), and a pathology view for quality control of the staining. The counts tool helps to evaluate the intensity of the staining and the level of background. If the intensity and/or background levels are high, the first approach is to reduce the dilution of the TSA dye. If the problem persists, the chromogenic IHC slides used for the optimization of that particular marker should be reviewed with a pathologist, looking for the presence of background. The pathology view is generated by the inForm software, using a diaminobenzidine-like pseudocolor representation of the fluorescence from the TSA-linked fluor, as well as a faux hematoxylin representation generated from all fluorescent signals, including DAPI. Images from the pathology view help with the visual evaluation of the staining patterns by a pathologist for improved staining. These tools should always be employed during the optimization of a new panel and as quality control steps during the staining workflow.
The mIF method requires the use of primary antibodies that have been properly validated and optimized for standard chromogenic IHC on formalin-fixed tissues. For instance, one of the major advantages of the mIF TSA method is its compatibility with the use of primary antibodies that have been validated and optimized in the anatomic pathology laboratory for clinical use12. This advantage means that the multiplex panels can be customized by the laboratory user to answer specific cancer immunology questions without the need for preconjugated antibodies, providing rapid and dynamic panel development. In this regard, the actual workflow for the optimization of a new panel begins with the validation and optimization of the antibodies with standard chromogenic IHC, looking for the best dilution and staining conditions that will provide clean and consistent staining. The next step, using the same dilutions optimized for IHC, is to transfer the staining by using TSA fluors instead of diaminobenzidine and to compare the results of monoplex immunofluorescent TSA with the chromogenic IHC as reference. Finally, the antibodies can be combined in mIF staining, always using the chromogenic IHC staining pattern as a control reference to adjust the mIF TSA staining parameters, mainly the TSA dye dilutions and the staining sequence. During this process, collaboration with a pathologist is instrumental to evaluate the quality of the staining.
Limitations of this technique include the time and effort required for the optimization and validation of new panels and the immunophenotyping analysis of mIF panels. The main concern, however, is the proper validation of mIF methods. The lack of proper validation of these techniques carries the risk of generating invalid results, which may add confusing data to the literature, thereby hindering attempts to gain a better understanding of the biology of cancer immunology16. It is, therefore, incumbent on the investigator to make every effort to properly validate multiplex methods, including ensuring close participation and evaluation by pathologists with training and experience in validation and evaluation of tissue histopathology and IHC-based techniques17,18,19,20. The protocol presented here includes some of the tools that can help with the validation of a new multiplex panel, including the pathology evaluation of H&E slides before staining and analysis, the use of chromogenic IHC as a reference to optimize the mIF TSA staining, the drop control method for quality control of a new multiplex panel, and the use of isotype, autofluorescence, and drop controls. Despite the technical complexity of mIF methods, technologies such as multispectral imaging with spectral unmixing, as well as other novel multiplex platforms, are opening a new era for the analysis of tissue specimens from patients and the generation of new forms of data that is not possible with standard IHC alone. Therefore, the development and application of mIF techniques will continue to grow, becoming powerful tools for the cancer immunoprofiling of biopsies for clinical trials and helping to identify new multiparametric predictive biomarkers for immunotherapy, benefiting, above all, the cancer patient.
The authors have nothing to disclose.
Editorial support was provided by Deborah Shuman of MedImmune.
"InForm 2.4.2" Software for Spectral Unmixing and Image Analysis | PerkinElmer | CLS151066 | Called "spectral unmixing software" in text |
"Phenochart 1.0.9" QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing | PerkinElmer | CLS151067 | Called "QPTIFF software" in text |
#1.5 Coverslips | Sigma Aldrich | 2975246 | |
200 Proof Ethanol | Koptec | V1001 | |
20x Tris-Buffered Saline | VWR | J640-4L | |
Antibody Diluent | DAKO | S2203 | |
Anti-CD68 Mouse Monoclonal | DAKO | M087601-2 | Clone PG-M1 |
Anti-CD8 Rabbit Monoclonal | Ventana | M5392 | Clone SP239 |
Anti-CK Mouse Monoclonal | DAKO | M351501-2 | Clone AE1/AE3 |
Anti-ki67 Mouse Monoclonal | DAKO | M724001-2 | Clone MIB-1 |
Anti-PD-1 Rabbit Monoclonal | Cell Signaling | #86163 | Clone D4W2J |
Anti-PD-L1 Rabbit Monoclonal | Ventana | 790-4905 | Clone SP263 |
Bond Dewax Solution | Leica | AR9222 | Called "dewax solution" in text |
Bond Epitope Retrieval Solution 1 | Leica | AR9961 | Called "ER1" in text |
Bond Epitope Retrieval Solution 2 | Leica | AR9640 | Called "ER2" in text |
Bond Open Containers, 30 mL | Leica | OP309700 | Called "30 mL open containers" in text |
Bond Open Containers, 7 mL | Leica | OP79193 | Called "7 mL open containers" in text |
Bond Polymer Refine Detection | Leica | DS9800 | Called "chromogenic detection kit" in text |
Bond Research Detection Kit | Leica | DS9455 | Called "research detection kit" in text |
Bond Titration Kit | Leica | OPT9049 | Called "titration kit" in text |
Bond Universal Covertile Novocastra | Leica | S21.2001 | Called "covertiles" in text |
Bond Wash Solution 10X Concentrate | Leica | AR9590 | Called "10x wash solution" in text |
BondRX Autostainer | Leica | Called "automated stainer" in text | |
BondRX Software Version 5.2.1.204 | Leica | Called "automated stainer software" in text | |
Opal 7-Color Automation IHC Kit | PerkinElmer | NEL801001KT | Called "multispectral staining kit" in text |
Peroxidase Block | Leica | RE7101 | |
ProLong Diamond Antifade Mountant | Thermo | P36965 | Called "slide mountant" in text |
Starfrost Slides | Fisher | 15-183-51 | |
Vectra Polaris Multispectral Microscope with "Vectra 3.0.5" Software for Multispectral Microscope Control | PerkinElmer | CLS143455 | Called "microscope control software" in text |