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

Lymphomas are histologically complex tumors. Therefore, multiplexed fluorescent immunohistochemistry offers advantages over conventional chromogenic immunohistochemistry to study markers of interest in the tumor cells and the microenvironment. This technique has the potential for standardizing scoring of antibody based staining in histological lymphoma samples, a process that has historically been challenging due to the complexity of the tumor microenvironment.

Specifically, multiplexed fluorescent immunohistochemistry and spectral imaging allows the quantitative measurement of multiple stains within a single slide, thereby allowing the assessment of antigen co expression and spacial relationship within clinical material. Begin by immersing the de-waxed and rehydrated slides in a standard antigen retrieval buffer in a microwave save glass jar and performing heat-induced epitope retrieval in a suitable microwave. Perform additional rounds of microwave stripping based on the position of the antibody in the final multiplex sequence.

For example, the fourth antibody requires three additional rounds of microwave stripping prior to staining. After completion of the stripping process, use forceps and heat-proof gloves to transfer the slides to distilled water at room temperature for an at least 10 minute cool down. When the antigen retrieval solution has cooled, block the tissue peroxidase activity with a commercial peroxidase block for 10 minutes followed by a five minute wash in tris-buffered saline, or TBS, and a non ionic detergent.

Block any nonspecific staining with a 10 minute incubation in blocking buffer followed by incubation with the primary antibody of interest at room temperature for 30 minutes and three five minute washes with TBS-D buffer. Next, incubate the samples with an appropriate horseradish peroxidase conjugated secondary antibody for 15 minutes at room temperature, followed by three washes in TBS-D buffer as demonstrated. After the last wash, apply an appropriate tyramide based fluorescent reagent to the slides for a five minute incubation at room temperature followed by three five minute TBS-D washes.

Perform an additional microwave based stripping to remove the primary and secondary antibodies in fresh antigen retrieval solution as demonstrated. At the end of the retrieval, cool the slides in distilled water and dry the areas on the slides without tissue with lab wipes. Incubate slide with dappy for 10 minute at room temperature followed by three TBS-D washes.

Then, carefully dry the slides with lab wipes without contacting the tissues and mount the samples with an appropriate mounting medium for imaging. For monoplex slide scanning, select the appropriate filters from the fluorophores used for labeling the samples and examine each marker and its corresponding fluorescence channel to identify a suitable exposure time for obtaining a clean signal, focusing on the tissue component that should have the strongest signal for the marker. To allow cross-sample comparison of the pixel intensity, use the live camera setting to adjust the exposure time in each individual channel until there are no overexposed areas in the live camera image.

When all of the slides have been scanned, acquire simulated bright field images to visually compare with the normal immunohistochemical pattern and to determine if the staining pattern is correct. To scan tissue microarray slides, use the tissue microarray scanning mode in the imaging system and an appropriate auto focus algorithm. For whole tissue section slides, have the slides reviewed by a qualified pathologist to select optimal images from the most representative tumor areas.

Before beginning the analysis, select a tumor marker to identify the cells of interest for the analysis. Have a pathologist review the images and decide whether tissue segmentation is required. If required, select the appropriate control regions for segmentation and check whether the image analysis software can correctly identify such regions.

After the cell segmentation, have the pathologist review the segmentation map to ensure the fidelity of the intended segmentation approach and review individual images to determine whether the cell segmentation is adequate or if additional tissue segmentation is required to select regions enriched for tumor cells, stroma, and or necrosis. Then determine the optical intensity positive cutoff value for each marker in conjunction with the pathologist and generate histograms in an appropriate statistic software program to analyze the frequency distribution of the marker intensity per cell. To generate percentage data for specific markers of interest within defined cells, insert a pivot table into the datasheet and select Sum to calculate the positivity percentage of a single marker of interest.

The total number of marker positive cells divided by the total number will be calculated. The result is the marker positive cell percentage with one core, or one study number. To generate numeric data, obtain the mean intensity of each marker of interest in all of the cells study within a sample to extract the median normalized count for each marker of each core or study number.

Here, representative multiplex fluorescent immunohistochemical images for a diffuse large B-cell lymphoma sample with c-MYC and BCL2 gene rearrangement are shown. Simulated bright field immunohistochemical images demonstrate a similar tumor marker staining pattern. The application of multiplex fluorescent immunohistochemical images to a T-cell panel in angioimmunoblastic T-cell lymphomas reveals the cell heterogeneity of the tumor sample.

Here, the optimization of a tonsil control sample and the resulting data analysis are shown. A variety of image analysis software programs can be used after the unmixing step to quantitate marker expression and localization in tumor cells and the tumor microenvironment. This technique paves the way for researchers to study the co expression of multiple markers within specific cell types in single tissues sections of lymphoma.

Take care to prevent heat-induced injury during the microwaving steps and be aware of fall risks during the scanning steps that are performed in the deck.