The overall goal of this in vitro functional assay is to assess various aspects of FC-mediated phagocytosis. This method can help answer key questions in the immunohematology and blood transfusion medicine fields such as what is the clinical significance of auto-and allo-antibodies to red blood cells. This technique provides a functional biological assay that can be performed in vitro for predicting the outcome of transfusion in those patients with preformed antibodies against red blood cells.

Demonstrating the procedure will be Cindy Tong, a graduate student from my laboratory. Now don't forget that working with human blood can be hazardous and that precautions such as wearing protective gowns and gloves should always be taken while performing this procedure. After obtaining one to two 10 milliliter whole blood samples from a healthy donor in vacutainer tubes containing acid citrate dextrose by venipuncture, the tubes are placed into a class II biosafety cabinet and the blood diluted at a one-to-one ratio in warm complete medium.

Next, slowly pipette the blood cells down the sides of a centrifuge tube onto room temperature density gradient, taking care not to mix the layers. Separate the cells by centrifugation. Then, discard the top plasma layer and use a glass Pasteur pipette to transfer the PBMC containing buffy coat into a new 15 milliliter tube.

Wash the isolated PBMCs three times in PBS, resuspending the cells in three to seven milliliters of medium after third centrifugation. After counting, dilute the cells to 1.75 times 10 to the sixth cells per milliliter concentration in complete medium and seed 400 microliters of cells into each well of an eight-chamber slide for a one-hour incubation at 37 degrees Celsius and 5%CO2 with full humidity. To opsonize R2R2 red blood cells, first wash the red blood cells three times in PBS.

Dilute the R2R2 pellet after the third centrifugation at a one-to-one ratio with polyclonal anti-D antibodies from human serum for a one-hour incubation at 37 degrees Celsius with intermittent mixing. At the end of the opsonization, remove the cells from the incubator and then wash them three more times in PBS as just demonstrated. Resuspend the pellet to a 1.25%volume to volume in complete RPMI medium.

Next, replace the supernatant from each well of the eight-chamber slide with 400 microliters of the opsonized R2R2 cells for two hours at 37 degrees Celsius. At the end of the incubation, use the slide adapters to remove the chambers, dabbing the excess R2R2 with a paper towel. Now, fill a 100 milliliter beaker with PBS and slowly dip each slide 30 to 40 times in the salt solution to remove the majority of the unphagocytosed R2R2.

After drying, fix the slides in 100%methanol for 45 seconds and allow the cells to dry before mounting the samples with coverslips. The next day, load each slide onto a phase contrast microscope with a 40X objective lens and manually count at least 200 monocytes and the number of phagocytosed R2R2 within each monocyte using one counter in each hand to simultaneously quantify the number of monocytes and the number of phagocytosed R2R2 cells per sample. Intravenous immunoglobulin binds to and blocks FC-receptors inhibiting phagocytosis in a dose-dependent manner with a nearly 100%inhibition observed starting from 200 microgram concentrations of the intravenous immunoglobulin per milliliter and with almost no inhibition observed at concentrations below 0.5 micrograms of the inhibitor.

When the phagocytic indices are normalized to the R2R2 positive control as 0%inhibition, an inhibition curve with an IC 50 of three micrograms per milliliter can be determined. With experience, phase contrast microscopy can be used to distinguish monocytes from contaminating red blood cells and vacuoles from phagocytosed red blood cells. Dense cell clusters and debris should be avoided during quantification as well as over opsonization of the R2R2 red blood cells which can lead to a heightened phagocytosis that overcrowds the monocyte interior and interferes with an accurate phagocytic analysis.

Once mastered, this technique can be completed in six to eight hours if it is performed properly. Following this procedure, other methods like immunocytochemistry or immunofluorescence using confocal microscopy can be performed to answer additional questions about phagocytic protein expression, red blood cell interactions and compartmentalization. With its initial development and further optimization, this technique will inform the way researchers in the fields of transfusion medicine and cell biology explore opsonization systems.

After watching this video, you should have a good understanding of how to perform a monocyte monolayer assay to assess the types of antibody interaction that provoke phagocytosis.