Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Summary

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

Abstract

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples.

These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells.

Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

Introduction

Recurrent miscarriage (RM) can be defined as the loss of two or more pregnancies before 24 weeks of gestation¹. This frequent reproductive condition affects up to 1% of couples worldwide^2,3. The pathophysiology is multifactorial and can be divided into embryologically driven causes (mainly due to an abnormal embryonic karyotype) and maternally driven causes that affect the endometrium and/or placental development. This manifestation can result from parental genetic abnormalities, uterine anomalies, prothrombotic conditions, endocrinology factors, and immunological disorders⁴.

In recent years, immune effector cell dysfunction has been implicated in the pathogenesis of early pregnancy loss⁵. This has inspired many investigations into elucidating the specific populations of immune cells in the endometrium during the menstrual cycle, implantation, and early pregnancy, with specific roles in early pregnancy. Among these immune cells, uterine natural killer (uNK) cells play a critical role during embryo implantation and pregnancy, particularly in the processes of trophoblastic invasion and angiogenesis⁶. Studies have shown an increased uNK cell density in the endometrium of women with RM^7,8, although this finding was not associated with an increased risk of miscarriage⁹. However, this stimulated research evaluating the density of other immune cell types (such as macrophages, uterine dendritic cells) in the endometrium in women with RM^{10, 11}. Nevertheless, it remains uncertain whether there is a significant alteration in the immune cell density in the peri-implantation endometrium in women with RM.

One possible explanation for the uncertainty is that evaluation of the endometrial immune cell density might be difficult due to the rapid changes in the endometrium during the window of implantation. During the 24 h timeframe, significant changes in the endometrium alter immune cell density and cytokine secretion, introducing a source of variation in these results¹². In addition, most reports mainly rely on the use of single-cell staining (e.g., traditional IHC methods) that could not examine multiple markers on the same tissue section. Although flow cytometry can be used for detecting multiple cell populations in a single sample, the large amounts of cells required and the time-consuming optimization hinder the popularity and efficiency of this method. Hence, the recent advancement in multiplex IHC staining could solve this problem by immunostaining multiple markers on the same slide to evaluate multiple parameters, including cell lineage and histological localization of individual immune subpopulations. Further, this technology can maximize the information obtained in case of limited tissue availability. Ultimately, this technique can help elucidate the differences in immune cell interactions in the endometrium between fertile women and women with RM.

Two groups of women were recruited from the Prince of Wales Hospital, including fertile control women (FC) and women with unexplained recurrent miscarriage (RM). Fertile control was defined as women who had at least one live birth without any history of spontaneous miscarriage, and RM women were defined as those who had a history of ≥2 consecutive miscarriages before 20 weeks gestation. The subjects from the two groups met the following inclusion criteria: (a) age between 20 to 42 years old, (b) non-smoker, (c) regular menstrual cycle (25-35 days) and normal uterine structure, (d) no use of any hormonal regimen for at least 3 months before the endometrial biopsy, (e) no hydrosalpinx by hystero-salpingogram. In addition, all the subjects recruited had normal karyotyping, normal 3-dimensional ultrasonography hysterosalpingogram, day 2 follicle-stimulating hormone < 10 IU/L, mid-luteal progesterone > 30 nmol/L, normal thyroid function, and tested negative for lupus anticoagulant and anticardiolipin IgG and IgM antibodies.

To better understand the immunological basis of RM, it would be most desirable to simultaneously quantify and localize the major immune cell types present in the endometrium at the time of implantation. This paper describes the entire protocol from sample preparation, the multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis with specific reference to detecting endometrial immune cells. Moreover, this paper describes how to determine the density and clustering of the immune cell types simultaneously in the endometrium.

Protocol

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Informed consent was obtained from the participants before collecting the endometrial biopsies. Refer to the introduction section for inclusion criteria of the control and RM groups.

1. Sample preparation

1. Ensure that all the women in this study undergo a daily urine dipstick test from day 9 of the menstrual cycle onwards to identify the luteinizing hormone (LH) surge to detect ovulation, and time the endometrial biopsies precisely on the 7^th day after the LH surge (LH+7).
2. Obtain a 0.5 cm² fragment of endometrial biopsy using a Pipelle sampler or Pipet Curet from fertile women and women with unexplained RM. Label the container and place the specimen immediately in 10% neutral-buffered formalin (pH 7) for overnight fixation at room temperature.
   NOTE: The volume of the fixative should be 5-10 times that of tissue.
3. Place the tissue in a cartridge for dehydration in a tissue-processing machine before embedding it in molten paraffin wax. Embed the tissues in paraffin at 58-60 °C.
4. Allow the paraffin block to cool overnight at room temperature. Use a microtome to trim the paraffin blocks to a thickness of 3 µm.
   ​NOTE: The use of distilled water aids in proper tissue mounting and adherence throughout multiplex staining.
5. Place the paraffin ribbon in a water bath at 40-45 °C for 30 s.
6. Mount the sections onto poly-L-lysine (0.1% w/v)-coated microscope glass slides. Place the slides with the tissue facing upward and allow to dry at 37 °C overnight. Store the slides in a slide box away from extreme temperatures until further use.

2. Determine the ideal concentration of antibodies for multiplex IHC using conventional IHC.

NOTE: This is important for identifying the expression level and pattern of each immune marker in the endometrium sample, and determine the staining sequence of each marker as well as their associated tyramide signal amplification (TSA) fluorophore pairings.

1. Test the antibodies for their suitability for multiplex IHC by using manual conventional IHC¹³.
2. Use endometrium tissues for single-antibody testing.
3. Include a positive control (e.g., spleen) and negative control (isotype control) for optimizing the staining condition of each antibody.
4. Perform the staining using the dilution recommended by the antibody's datasheet.
5. Perform additional staining using concentrations above and below the recommended dilution used in step 2.3.
   ​NOTE: A clinical pathologist should assess the stained slides blindly to confirm the localization of the antibody probing and cellular integrity.

3. Multiplex staining method

1. Slide preparation and fixation
   1. Lay the slides (from step 1.6) flat with the tissue facing upwards in an oven and bake at 60 °C for at least 1 h.
   2. Remove the slides from the oven and allow them to cool for at least 20 min at room temperature before placing them in a vertical slide rack.
   3. Dewax and rehydrate the formalin-fixed, paraffin-embedded slides with 10 min allotted to each of the following steps: xylene (2x), 100% ethanol (2x), 95% ethanol (1x), 70% ethanol (2x), and distilled water (2x).
   4. Place the rack of slides in a plastic slide box and submerge them in Tris-buffered saline (TBS, pH 7.6).
      NOTE: The slides must remain moist starting from this rehydration step until mounting in the final step.
   5. Fix the samples by submerging the slides in a plastic slide box filled with a mixture of formaldehyde diluted in methanol (1:9) for 30 min in the dark.
   6. Wash the slides twice in deionized water for 2 min and then proceed to antigen retrieval.
2. Epitope retrieval
   1. Place the rack of slides in a heat-resistant box and fill it with citric acid buffer (pH 6.0) to cover the slides.
   2. Place the box in the microwave, and heat the slides for 50 s at 100% power followed by 20 min at 20% power to maintain the same temperature.
   3. Allow the slides to cool for approximately 15 min at room temperature.
   4. Rinse the slides in water for 2 min followed by TBS-Tween 20 (TBST) for 2 min.
      NOTE: TBST is composed of 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 (v/v).
3. Blocking
   1. Block endogenous peroxidase activity in the tissue by immersing the slide onto a jar containing peroxidase blocking solution (see the Table of Materials) for 10 min.
   2. Wash the slides with TBST for 5 min.
   3. Use a hydrophobic barrier pen to mark a boundary around the tissue section on the slide.
   4. Cover the tissue sections with a blocking buffer (see the Table of Materials) or bovine serum albumin (5%, w/v), and incubate the slides in a humidified chamber for 15 min.
4. Antibody and signal application
   1. Remove the blocking reagents.
   2. Incubate with the primary antibody of interest (e.g., CD3, 1:100 dilution in antibody diluent, see Table 1) in a humidified chamber at room temperature for 30 min.
   3. Remove the primary antibody. Wash 3x with TBST for 5 min each time.
   4. Incubate with the polymer horseradish peroxidase (HRP)-labeled secondary antibody (Table 1) for 15 min in a humidified chamber at room temperature. Wash 3x with TBST for 5 min each time.
   5. Apply the Opal fluorophore TSA working solution (1:100 in amplification diluent) and incubate at room temperature for 10 min to allow fluorophore conjugation to the tissue sample at primary antibody binding sites. Wash with TBST in triplicate for 5 min each time.
5. Microwave-based stripping
   1. Rinse with the antigen retrieval buffer (citrate buffer solution, pH 6.0).
   2. Perform microwave-based stripping to remove the primary-secondary-HRP complex to introduce the next primary antibody (e.g., CD20).
   3. Place the slides in antigen retrieval buffer, microwave them at 100% power for 50 s followed by 20% power for 20 min in microwave-safe containers, and cool them at room temperature for 15 min.
   4. Repeat steps 3.2.4 to 3.5.2 until the tissue samples have been probed with all the primary antibodies.
6. Counterstain and mounting
   1. After microwave-based stripping and cooling of the antigen retrieval buffer, rinse the slides in distilled water and TBST.
   2. Incubate with 4', 6-diamidino-2-phenylindole (DAPI) solution (1.0 µg/mL) for 5 min in a humidified chamber at room temperature.
   3. Wash 3x with TBST for 5 min each time. Wash with water once for 5 min.
   4. Air-dry the slide and mount with the appropriate mounting medium (see the Table of Materials).
      NOTE: Counterstaining will not be required for monoplex slides to be used for spectral library development.

4. Image and analysis

1. Preparation of Spectral library slides
   1. Create library slides (single-stain reference images) for each fluorophore, DAPI, and auto-fluorescence with the same control tissue for multispectral image analysis.
   2. Using the slides of the endometrial biopsy samples from women with RM and fertile women, perform steps 1.1 to 3.6.4 for single-antibody detection for each slide (without further antibody or fluorophore addition).
   3. For each antibody detection, stain one of the slides with DAPI (as in step 3.6.2) and leave one slide unstained for the detection of any possible tissue auto-fluorescence in the spectrum.
   4. Use the appropriate filters in the workstation to obtain the image for this set of slides for each antibody and upload them into the image analysis library (as described in step 4.2).
   5. Once the image is captured, select inForm as the Spectral Library Source and build the spectral library.
   6. As fluorophores are used, select Stains/Fluors... from the menu.
   7. While choosing the stains or fluorophores, narrow the choices by selecting one or more Groups. Select All to show all spectra compatible with the images.
2. Spectral imaging
   NOTE: Images were captured using the Mantra Workstation with the spectral library established using the inForm Image Analysis software.
   1. Capture the image of the single-antibody-stained slides with the appropriate epi-fluorescence filters as proposed in Table 2 (e.g., DAPI, fluorescein isothiocyanate [FITC], CY3, Texas Red, and CY5) using the workstation.
      NOTE: Recommended filters for the specific fluorophores used in this protocol are shown in Table 2.
   2. Identify a suitable exposure time for an optimal signal by examining each marker in its corresponding fluorescence channel.
      NOTE: The optimal signal is determined according to the reference on the positivity and localization obtained in the single antibody staining.
   3. Determine a fixed exposure time for each analyte (antibody-fluorophore combination) to standardize a cross-sample comparison.
      NOTE: The determination of the fixed exposure is dependent on the intensity in the sample of interests.
   4. Scan the multiplex stained slides in the appropriate scanning mode with the embedded autofocus algorithm.
      NOTE: The established spectral library would be used to differentiate the multispectral image cube into single individual components (spectral unmixing). This would allow the color-based identification for all markers of interest to be processed using the following two main steps: training session and image analysis session.
3. Image analysis
   1. Cell counting of selected immune cell types in the endometrium
   2. Capture a minimum of 10 fields for analysis under 200x magnification.
      NOTE: The fields were captured by scanning the whole section without any selection. This can ensure the simultaneous capture of all cell components of the endometrium, e.g., the luminal epithelial border, stroma, and glands.
   3. To count the cells, click on the Count Objects button in the step bar to display the Object Counting Settings panel.
   4. Check the Discard Object if Touching an Edge box for the exclusion of any objects touching the edge of the image, process region, or tissue region.
   5. If the tissue has been segmented, select the Tissue Category in which the objects are to be found. Do not count objects outside of the selected tissue category.
   6. Select the desired Approach to identify objects: Object-Based or Pixel-Based (Threshold).
      NOTE: The Pixel-Based (Threshold) approach should be selected in case of a reliable or consistent stain for which the application of a simple threshold will yield the object pixels. The Object-Based approach is recommended when more advanced morphometry-based approaches are required in case of lack of consistency and specificity of staining of objects.
   7. Select the desired Signal Scaling: Auto Scale or Fixed Scale.
      NOTE: Choosing Auto Scale will result in automatic scaling of each component plane before performing object segmentation. The Fixed Scale option is recommended when better segmentation performance is required, and stain signals are consistent and reliable.
   8. Select the Primary component for object segmentation from the drop-down list.
   9. Adjust the Minimum Signal value for the primary component to the desired threshold value.
   10. To automatically fill holes in objects, select Fill Holes.
   11. To detect objects that touch other objects as individual objects, instead of as one object, select the Refine Splitting check box after selecting the Maximum Size (pixels) check box.
   12. To exclude objects based on the roundness of the object, check the Roundness box and specify the desired Minimum Circularity.
   13. Count all stromal cells (CD3⁻/CD20⁻/CD68⁻/CD56⁻, and DAPI⁺), including the cells surrounding the blood vessels.
   14. Count the immune cells separately, including T cells (CD3⁺ and DAPI⁺), B cells (CD20⁺ and DAPI⁺), macrophages (CD68⁺ and DAPI⁺), and uNK cells (CD56⁺ and DAPI⁺).
   15. Express the data as the percentages of the immune cells relative to the total number of stromal cells for each captured image, and report the final cell count as an average of all fields.
   16. Use the View Editor to view the resulting data tables post processing. Export the Count Data table.
   17. Quantification of endometrial immune cell spatial distribution
   18. Under 200x magnification, estimate the L-function using the R program for a range of 0-20 µm considered a cell-cell contact maximum distance¹⁴.
       NOTE: The R language toolbox 'spatstat' was used to measure the L-function.
       1. Denote the level of clustering of different pairs of immune cells based on the area under the curve (AUC) of their L-function.

Results

The overall schematic process of performing a 4-color multiplex assay for the detection of 4 endometrial immune cell types is shown in Figure 1. In brief, the protocol for this multiplex immunofluorescence staining required 8 key steps: 1. Slide preparation, 2. Epitope retrieval, 3. Blocking, 4. Primary antibody application, 5. Secondary antibody application, 6. Signal amplification, 7. Removal of antibody, and 8. Counterstain and mount. Image rendering and analysis were then conducted using...

Discussion

Critical steps within the protocol
It is important to note that multiplex staining requires diligent optimization. Antigen retrieval, using citrate buffer and microwave technology, requires optimization to ensure complete antibody stripping and maintain tissue viability. As TSA reagents covalently bind to sites surrounding the antigen, they can potentially inhibit the binding of a subsequent primary antibody through steric hindrance (also known as the "umbrella effect"). This tends to occur...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amplification Diluent	Perkin Elmer	FP1498	Fluorophore diluent buffer
Antibody diluent	Perkin Elmer	ARD1001EA	Diluting the antibody
CD3	Spring Bioscience	M3072	Primary antibody
CD20	Biocare Medical	CM004B	Primary antibody
CD56	Leica	NCL-CD56-504	Primary antibody
CD68	Spring Bioscience	M5510	Primary antibody
Citrate Buffer Solution, pH 6.0 (10x)	Abcam	AB64214	Antigen retrieval solution
EMSURE Xylene (isomeric mixture)	Merck	108297	Dewaxing
Ethanol absolute	Merck	107017	Ethyl alcohol for rehydration
HistoCore BIOCUT Manual Rotary Leica Microtome	Leica	RM2125RTS	Sectioning of paraffin-embedded tissue
inForm Advanced Image Analysis Software	Perkin Elmer	inForm® Tissue Finder Software 2.2.1 (version 14.0)	Data Analysis software
Mantra® Workstation	Akoya Biosciences	CLS140089	Spectral imaging
Microwave	Panasonic	Inverter	Microwave stripping
Opal 520	Perkin Elmer	FP1487A	Appropriate tyramide based fluorescent reagent
Opal 620	Perkin Elmer	FP1495A	Appropriate tyramide based fluorescent reagent
Opal 650	Perkin Elmer	FP1496A	Appropriate tyramide based fluorescent reagent
Opal 690	Perkin Elmer	FP1497A	Appropriate tyramide based fluorescent reagent
Oven	Memmert	U10	Dewaxing
Peroxidase Blocking Solution	DAKO	S2023	Removal of tissue peroxidase activities
Poly-L-lysine coated slide	FISHER SCIENTIFIC	120-550-15	Slide for routine histological use
PolyHRP Broad Spectrum	Perkin Elmer	ARH1001EA	Secondary antibody
ProLong™ Gold Antifade Mountant	ThemoFisher Scientific	P36930	Mounting
Spatstat	/	Version 2.1-0	Spatial point pattern analysis
Spectral DAPI	Perkin Elmer	FP1490A	Nucleic acid staining
Tissue Processor	Thermo Fischer	Excelsior ES	Tissue processing for dehydration and paraffination
Tris Buffer Saline (TBS), 10x	Cell Signaling Technology	12498S	Washing solution
Tween 20	Sigma-Aldrich	P1370-1L	Nonionic detergent