Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

Currently, it is challenging to stabilize the analysis within a certain range. It is also difficult to maintain the quality control of the experiment. Our research can help the patients with abnormal endometrial sensitivity in immune perspective to find the cause of reproductive failure.

The local proportion of immune cells in the endometrial can be stably and repeatedly detected. Our findings may promote the research progress of the endometrial sensitivity evaluation in patients with repeated implantation failure. To begin, use tweezers to transfer the endometrial tissue from the formalin onto the embedding paper.

With a ruler, measure the dimensions of the endometrial tissue. Now wrap the tissue in the embedding paper and place it into an embedding cassette. Transfer the cassette into the tissue basket for dehydration.

To dehydrate the endometrial tissue, place the tissue basket in the reaction chamber of the dehydrator and initiate the procedure for routine tissue dehydration. Once the dehydration is complete, open the reaction chamber of the dehydrator and remove the tissue basket. To begin, select an appropriate embedding mold that matches the size of the dehydrated endometrial specimen.

Fill it with paraffin wax heated at 70 degrees Celsius. Immediately place the dehydrated human endometrial tissue in the mold. Use forceps to carefully adjust the tissue to ensure it is placed centrally.

Now transfer the mold to the cooling plate and gently press the tissue with tweezers as the paraffin at the bottom solidifies. Place the embedded cassette over the mold and top it with more wax. Then place the mold back on the cooling plate.

Once the paraffin has solidified completely, remove the block and its attached cassette from the mold. For sectioning, insert the block onto the sample clip of the microtome. Place the blade into the holder and adjust the angle between the block's plane and the blade.

Now adjust the thickness of the section to four micrometers. Then turn the hand wheel to start slicing. Cut thin sections from the block to expose the appropriate tissue surface.

Use the brush to gather the continuous and complete sections. Once enough sections have been obtained, use tweezers to remove the unqualified sections at the front. With a brush, transfer the sections into a warm water bath, heated to 42 degrees Celsius.

When the sections have fully flattened out, use anti detachment slides to lift them. Next, bake them in a slide warmer at 65 degrees Celsius for over 60 minutes before removing. To begin, transfer the diluted antibody solution into a special reagent bottle.

Insert the detection kit into the reagent compartment of an automatic immunohistochemical standing instrument. Next, set the slides with the sectioned endometrial tissue on a slide holder and cover them with a special cover tile. Then insert the holder into the experimental reaction compartment of the instrument.

Once the instrument automatically recognizes the information on the reagent and slide, press the start button to initiate the immunohistochemical staining. After staining, take out the slide holder. Put the stained slide into the slide holder to wash off any remaining dye with clean water.

Now, place the slide holder onto an automated cover slipper and run the dehydration and sealing procedure. Remove the slides once dehydration and sealing are complete. To start, place the slides with the immunohistochemically stained endometrial sections on the slide rack of a panoramic pathological image scanner.

Then place the rack in the instrument's slide scanning compartment to initiate scanning. Once scanning is complete, launch the image analysis software and create a new folder. Import the images that require analysis.

To build a tissue classifier, mark several tissues along with a blank annotation to establish a classifier that identifies the tissue and blank areas. Use real-time tuning to observe the software's recognition capacity during the mark annotation. To build an analysis algorithm, select the standard multiplex IHC algorithm in the software and choose the negative and positive pixels to set the color parameters of cell recognition.

Establish the parameters of the nucleus, cytoplasm, and cell membrane until the optimal parameters for the image are found. Now, set the positive cell recognition threshold and optimize it until an appropriate threshold is obtained. Next, select the tissue classifier in the algorithm.

Check the tissue part within it to identify cells based on tissues. Select the analysis area and then use the established algorithm to analyze the images. Once the analysis is complete, manually check the accuracy of tissue recognition, negative and positive recognition.

If the image recognition is inaccurate, adjust the algorithm and parameter thresholds again. Run the image analysis until it is successful. Then, export the results of the analysis.

Set the different analysis parameters according to the expression of different endometrial immune markers. Use the software to calculate the proportion of endometrial immune cells. Following calculation, use the values to assess the levels of various immune cells in the endometrium of patients with recurrent miscarriage.

Different immune cells were immunohistochemically stained based on their individual expression of different markers. The brown stain represents the positive immune cells and the blue represents the nucleus. Different immune cells were expressed in women with recurrent miscarriages.

CD56 cells appear to be predominant at 16.76%with CD1a and Foxp3 cells making up 0.11 to 0.12%of the immune cell population.