Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

We are trying to determine the best method to use for the prediction of red blood cell antibody significance. The monocyte-macrophage assay appears to be more sensitive than the monocyte monolayer assay. So the questions we are trying to answer is whether the monocyte-macrophage assay is a better assay for prediction of red blood cell antibody significance, better than the monocyte monolayer assay.

Experimental challenges with both the monocyte monolayer assay and the monocyte-macrophage assay is optimizing their efficiency to enable faster completion, while eliminating the need for manual phagocytosis assessment. In other words, the challenge is to try to semi-automate these assays. The monocyte monolayer assay, which I pioneered actually, in 1980, has been used routinely by Immunohematology Reference Laboratory since 1983 to help decide which red blood cell auto or alloantibodies have potential for causing hemolysis when donor blood is transfused into patients having these antibodies.

So the MMA uses monocytes in the assay, but antibody mediated hemolysis is typically caused by macrophages in the spleen or the liver. So here we are exploring whether the use of primary macrophages in this assay would be better than monocytes as predictors of potential antibody clinical significance. Trying to make the monocyte-macrophage assay more user-friendly, faster, and not requiring light microscopy, but maybe an automated mechanism for reading the phagocytosis would be desirable.

To begin, dilute the whole blood with RPMI-1640 Complete Medium, and layer it onto a density gradient medium for centrifugation. Post centrifugation, retrieve the buffy coat containing peripheral blood mononuclear cells or PMBCs. After pelleting the obtained PBMCs, resuspend them in 10 milliliters of pre-warmed RPMI-1640 Complete Medium.

Determine the cell number using appropriate equipment before starting the monocyte isolation process. Follow the monocyte isolation kit instructions and transfer the sample into propylene tube of appropriate size. Add the enrichment cocktail to the sample at a concentration of 50 microliters per milliliter of the sample.

After pipetting the sample up and down, vortex it to mix thoroughly and incubate the sample at two to eight degrees Celsius for 10 minutes in an ice bucket. Now, vortex the magnetic particles for 30 seconds during this incubation period. After incubation, add magnetic particles to the sample at a concentration of 100 microliters per milliliter of sample.

Vortex the sample and incubate it at two to eight degrees Celsius for five minutes. Then add the isolation medium to top up the volume to 2.5 milliliters or 10 milliliters as required, using a graded pipette, and mix. Next, place the propylene tube without its lid into the magnet and incubate at room temperature for about 2.5 minutes.

Then, pick up the magnet and inverted in one continuous motion to pour the cell suspension into a new 5 or 14 milliliter tube. Resuspend the isolated cells in RPMI-1640 Medium. To begin, add five milliliters of poly-D-lycine solution to a 25 milliliter flask for each macrophage population, M1 and M2, and leave the flasks in the hood for at least one hour.

After determining the cell count, add five milliliters of RPMI-1640 Medium, then seed between one to five times, 10 to the power of six monocytes into each pre-coded 25 milliliter flask. Incubate the flask at 37 degrees Celsius with 5%carbon dioxide for at least two hours. Following the incubation period, wash the flask once with PBS, and twice with complete RPMI-1640 Medium.

Add 10 milliliters of M1 or M2 differentiation medium to the flask according to the desired cell type, and let the cells differentiate in the incubator for six days at 37 degrees Celsius with 5%carbon dioxide. On day six, add five milliliters of M1 or M2 polarization medium to each flask as required. Let the macrophages polarize in the incubator at 37 degrees Celsius with 5%carbon dioxide for at least two days.

Before harvesting M1 or M2 macrophages on day eight, collect the supernatant into a 15 milliliter tube for further use, if needed. Add one milliliter of cell detachment solution to the flask, and incubate. To stop the reaction, add three milliliters of complete RPMI-1640 Medium to the flask and collect the media into a 15 milliliter tube.

After adding another three milliliters of medium to the flask, use a cell scraper to detach the cells from the bottom. Collect the detached cells in a fresh 15 milliliter tube. To determine the quality of M1 and M2 macrophages, wash the cells twice with PBS and resuspend in complete RPMI-1640 Medium at 0.5 times, 10 to the power of six cells per tube.

Then, analyze the two populations of cells using a flow cytometry assay. Count the obtained M1 and M2 macrophages using a hemocytometer in a one-to-one staining ratio with trypan blue. Reconstitute the macrophages to a concentration of 1 times 10 to the power of six cells per milliliter in RPMI-1640 Complete Medium.

Using a micro pipette, seed 400 microliters of the cell suspension into each well of the eight well chamber slide and incubate the slide at 37 degrees Celsius with 5%carbon dioxide for at least 1.5 hours in a fully humidified tissue culture incubator. To wash the RhD positive R2R2 RBCs, add PBS at pH 7.4 to the cells and centrifuge the sample at 350 G for five minutes. After two more such washes, opsonize the test RBC sample with antibodies of interest.

Incubate the antibody opsonized and non-opsonized RBCs at 37 degrees Celsius with 5%carbon dioxide for one hour. Vortex intermittently every 15 minutes to prevent the RBCs from settling. Then wash the opsonize RBCs three times with PBS at pH 7.4 as demonstrated earlier.

To check for RBC opsonization, perform an indirect antiglobulin test with a secondary opsonizing anti-human antibody to the primary opsonizing antibody. After reading the indirect antiglobulin test, reconstitute washed opsonized RHD plus R2R2 RBCs in a 1.25%volume by volume suspension with RPMI-1640 Complete Medium. After the 1.5 hour incubation of the M1 and M2 macrophages, aspirate and discard the supernatant medium gently along the corner of the well.

Then add 400 microliters of the 1.25%opsonized RBC mixture to each well of the triplicate setup. Incubate the sample at 37 degrees Celsius with 5%carbon dioxide for two hours undisturbed. Now pour 100 milliliters of PBS at pH 7.4 into two beakers.

Submerge the slide into the first beaker and move it back and forth slowly for 20 to 30 strokes. Then, transfer the slide to the second beaker and wash for 20 to 30 strokes. Remove the slide from the PBS and dab off excess liquid on a paper towel.

Submerge the slide in 100%methanol for 45 seconds to fix the cells. Air dry the slide and mount it using an in-house prepared mounting medium. Add cover slip and allow the slide to dry overnight before quantification of phagocytosis.

M1 and M2 macrophages were successfully polarized and cultured for eight days with distinct morphological features observed in microscopy images. In the flow cytometry, M1 macrophages displayed about 86.1%expression of CD80 and 0.17%expression of CD209. M2 macrophages displayed 97.3%of CD209 expression and 0.003%of CD80 expression.

Histograms of fluorescence intensity confirmed higher CD80 expression in M1 macrophages compared to M2 macrophages. While M2 macrophages showed significantly higher CD209 expression compared to M1 macrophages. M2 macrophages demonstrated a significantly higher phagocytic index compared to M1 macrophages.

Microscopy images showed clear evidence of increased phagocytosis in M2 macrophages compared to M1 macrophages. This study aimed to evaluate the potential of using macrophages instead of monocytes to predict red blood cell antibody significance. This table shows that M2 macrophages phagocytose better than M1 macrophages or monocytes with antibodies considered to be clinically significant.

Thus, with further study, an assay using M2 macrophages may be used for better prediction of antibody clinical significance.