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

Summary

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Abstract

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) > 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) > 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

Introduction

Predicting transfusion reactions remains a significant challenge in the field of transfusion medicine. Over the past 4 decades, the monocyte-monolayer assay (MMA), pioneered by Tong and Branch^1,2, has served as a valuable in vitro cellular assay for predicting the clinical outcome of hemolysis in blood transfusion patients¹. Indeed, this assay has been instrumental in distinguishing between clinically significant and insignificant red blood cell (RBC) antibodies². While monocytes have traditionally been the standard leukocyte used in this assay, our research aims to explore the potential benefits of using monocyte-derived macrophages as an alternative. These cells may enhance the assays’ ability to assess the clinical relevance of red cell alloantibodies.

In the historical MMA, monocytes, which are the precursors to macrophages, the immune cells responsible for the clearance and destruction of red blood cells during an adverse transfusion reaction, are introduced in an in vitro assay along with RBCs and antibodies^{1,2,3,4,5,6,7}. Phagocytosis is then assessed visually by counting phagocytosed RBCs within monocytes. A phagocytic index (PI) of < 5 phagocytosed RBCs per 100 monocytes counted indicates the patient is at a reduced risk of experiencing an adverse transfusion reaction, and the antibody is deemed clinically insignificant^4,5,6,7. Preliminary experiments demonstrate using peripheral blood-derived monocytes may not be ideal for determining clinical significance as they have a lower phagocytic capacity than activated monocytes and certain macrophages.

Monocytes are a subset of cells found in the blood, spleen, and bone marrow and account for 10% of the total leukocytes in humans⁸. These cells typically circulate for 1-2 days before being recruited by different tissues, where they go on to differentiate into macrophages⁸. This typically happens during hematopoiesis, in which the bone marrow produces monocytes that are released into circulation to become tissue macrophages that reside in the spleen and the liver². Known as the first line of defense against foreign pathogens, macrophages are large phagocytic mononuclear cells that play a role in adaptive and innate immunity⁹. Among the intricate and complex roles of the immune system, understanding and characterizing macrophage phenotypes presents a formidable challenge that is yet to be fully understood. Over the past two decades, the notion of macrophage polarization has garnered increasing recognition, with recent studies employing the use of single-cell RNA sequencing to discern the spectrum in which these macrophages exist.

Classically activated M1 and M1-like macrophages arise in inflammatory environments dominated by Toll-like receptors (TLRs)¹⁰. These cells may be involved in autoimmune diseases and arteriosclerosis and present surface markers such as MHC-II, CD80, and CD86^11,12. Anti-inflammatory M2 and M2-like macrophages are found in environments dominated by Th2 responses, lack expression of CD80, and present surface markers such as CD209 and CD206^11,12. M1/M2 macrophages may be cultured in vitro from peripheral blood mononuclear cells, with lipopolysaccharide (LPS) and cytokines such as GM-CSF and IFNγ (M1) and M-CSF and IL-4 (M2) stimulating their polarization^10,12.

This manuscript and associated studies aim to demonstrate that M2 macrophages exhibit increased sensitivity for phagocytosis compared to M1 macrophages and monocytes. Investigating the phagocytic activity of M1/M2 macrophages versus monocytes in the context of red cell alloantibodies and transfusion medicine is an area that is yet to be explored. Here, we describe current ongoing work for the generation of M1/M2 macrophages and compare the classic monocyte monolayer assay (MMA) to the novel monocyte-macrophage assay, using the acronym M-MA to distinguish this macrophage assay from the monocyte assay, to improve the predictive value of in vitro phagocytosis assays.

Protocol

This research was performed in compliance with institutional guidelines for conducting ethical research involving human subjects. Ethics approval was granted from the Canadian Blood Services Research Ethics Board (REB), approval CBSREB#2023.008. All steps of this protocol are to be carried out in a biosafety cabinet under sterile conditions.

1. Isolation of PBMCs

1. Obtain whole human blood from a donor in an ACD tube. Store the blood at room temperature (18 °C–22 °C) for up to 36 h.
2. Transfer the whole blood ACD tubes to 50 mL centrifuge tubes (a single 50 mL tube for every two ACD tubes). Add RPMI-1640 complete medium at room temperature to a final volume of 35 mL.
3. Add 15 mL of room temperature density gradient medium to a new 50 mL tube. Carefully layer the diluted whole blood on top of the density gradient medium, minimizing mixing at the interface for optimal separation of blood.
4. Centrifuge the layered mixture at 700 x g for 30 min with brakes OFF. The density gradient centrifugation will separate the mixture into the following layers from top to bottom: Plasma, Buffy coat (containing PBMCs), density gradient material, Granulocytes, and red blood cells. 
5. Aspirate and discard the majority of the top layer (plasma). Using a transfer pipette, carefully retrieve the buffy coat (PBMCs) layer, avoiding bringing any additional material. Transfer the retrieved PBMCs into a new 50 mL tube.
6. Wash the isolated buffy coat layer 1x with pH 7.4 PBS solution for 10 min at 350 x g with full brakes ON.
7. Use a 70 µM cell strainer to eliminate debris or unwanted material from the PBMCs before transferring them to a 15 mL conical tube. Wash 2x with pH 7.4 PBS solution for 10 min at 350 x g with full brakes ON.
8. Optional: Lyse any RBCs carried over with ACK lysis buffer. Add 5-10 mL of ACK lysis buffer, depending on pellet size, and incubate at room temperature for up to 3 min. After incubation, top up with pH 7.4 PBS and centrifuge for 10 min at 350 x g with full brakes ON and wash 1x. Perform this step if the number of RBCs is too high.
   NOTE: It always is better to avoid the use of ACK for better results. Alternatively, water can be used to eliminate non-phagocytosed RBCs by incubating with water for no longer than 15 seconds, then immediately add 1X PBS to wash.
9. Reconstitute PBMC pellet in 2-3 mL (use 0.5 mL per initial ACD tube) of RPMI-1640 complete medium. Count PBMCs with a hemocytometer after preparing a 1:1 mixture with trypan blue. Only counts cells not stained with trypan blue. Reconstitute PBMCs to 2 x 10⁶ cells/mL in RPMI-1640 complete medium.

2. Culture of M1/M2 macrophages

1. Day 0 seeding of monocytes
   1.  Prepare a 25 mL cell culture flask for each macrophage population. Add 5 mL of poly-D-lysine (50 mg/500 mL) to each and leave it in the hood for at least 1 h. Rinse the flask with PBS to get rid of the residual poly-D-lysine.
   2. Isolate PBMCs from Buffy coat or patient samples, as usual (see step 1). After a pellet of PBMCs was generated, resuspend it in 10 mL of pre-warmed RPMI-1640 complete medium.
   3. Determine the cell number and begin the monocyte isolation with the Monocyte isolation kit. To run the kit, use at least 50 x 10⁶ cells.
   4. Resuspend the cells at 50 x 10⁶ cells/mL in an isolation medium. According to the cell number obtained, use the purple (0.25-2 mL) or gray (0.5-8.5 mL) magnet recommended in the monocyte isolation kit.
   5. Follow the kit instructions and add the sample to the required propylene tube (5 mL or 14 mL). Add enrichment cocktail to sample at 50 µL/mL of sample.
   6. Pipet up and down or vortex to mix the sample and then incubate at 2-8 °C for 10 min in an ice bucket. Meanwhile, vortex magnetic particles for 30 s. After incubation, add magnetic particles to the sample: 100 µL/ml of sample.
   7. Pipet up and down or vortex to mix the sample and then incubate at 2-8 °C for 5 min. Top up with isolation medium to 2.5 mL or 10 mL, accordingly using a graded pipette. Pipette up and down 2x-3x to mix. 
   8. Place the tube (without lid) into the magnet and incubate at room temperature for 2.5 min. Pick up the magnet and, in one continuous movement, invert the magnet and tube, pouring the cell suspension into a new tube, 5 mL or 14 mL. 
   9. Resuspend cells in RPMI-1640 medium and count them. Do this step as soon as possible. Seed between 1 x 10⁶ – 5 x 10⁶ monocytes into each 25 mL flask previously precoated with poly-D-lysine. Bring the volume to 5 mL with RPMI-1640 if necessary.
   10. Incubate at 37 °C, 5% CO₂, for at least 2 h. Meanwhile, prepare M1 and M2 differentiation media. Prepare enough for Day 0 (10 mL) and Day 4 (2 mL) top-up.
       NOTE: M1 differentiation medium: RPMI-1640 + 2.5 ng/mL GM-CSF; M2 differentiation medium: RPMI-1640 + 50 ng/mL M-CSF.
   11. After the incubation time, wash each flask 1x with PBS and 2x with complete RPMI-1640. Add 10 mL of differentiation medium to each flask according to the cell type to differentiate. 
   12. Let cells differentiate for 6 days in the incubator at 37 °C, 5% CO₂. Due to evaporation, top up with differentiation medium on Day 4.
2. Day 4 - Top-up
   1. Add 2 mL of each differentiation medium to the respective flask. Add medium directly to the flask.
3. Day 6 - Macrophage polarization
   1. Prepare M1 and M2 polarization media. For M1 polarization media, prepare 5 mL of M1 polarization medium to add to the corresponding flask. The final concentrations should be 50 ng/mL IFNγ, 10 ng/mL LPS, and 2.5 ng/mL GM-CSF in RPMI-1640. The flask at the end will have 15 mL of total volume (10 mL already in + 5 mL to be added), do the calculations accordingly.
   2. For M2 polarization media, prepare 5 mL of M2 polarization medium to add to the corresponding flask. The final concentrations should be 20 ng/mL IL-4 and 50 ng/mL M-CSF in RPMI-1640. Note that the flask at the end will have 15 mL of total volume (10 mL already in + 5 mL to be added); do the calculations accordingly.
   3. Add 5 mL of M1 or M2 polarization medium to each flask. Let the macrophages polarize for at least 2 days and not more than 4 days in the incubator at 37 °C, 5% CO₂.
4. Day 8 - Harvest and flow cytometry
   1. Before harvesting the M1 or M2 macrophages, collect the supernatant into 1.5 mL tubes for further use (if needed). Add 1 mL of cell detachment solution to the flask and leave it in the incubator at 37 °C, 5% CO₂ for 5 min.
   2. To stop the reaction, add 3 mL of complete RPMI-1640. Collect media into a 15 mL tube. Add 3 mL of complete RPMI-1640 to the flask and use a cell scraper to detach the cells from the bottom of the flask.
   3. Collect cells in a 15 mL tube. Place the flask under the microscope at 10x and watch the cells to ensure no more cells are attached to the bottom of the flask.
   4. Wash the cells in PBS 2x. Resuspend them in complete RPMI-1640 and count using the hemocytometer.
   5. To analyze the quality and quantity of M1/M2 macrophages, run a flow cytometry assay. Use 0.5 x 10⁶ cells per tube and stain as follows: M1: CD80+ CCR7+ CD209- M2: CD206+ CD209+ CD80-. Gating strategy used in this assay: #1 Forward Scatter (FSC-A) vs. Side Scatter (SSC-A) Dot Plot, #2 Forward Scatter Height (FSC-H) vs. Forward Scatter Area (FSC-A) Dot Plot for Doublet Discrimination, #3 Side Scatter (SSC-A) vs. DAPI-A Dot Plot for Live/Dead Cell Discrimination, #4 Side Scatter (SSC-A) vs. Single Parameter Dot Plot or Histogram (i.e., SSC-A vs. FITC-A), #5 Double Parameter Dot Plot (i.e., FITC-A vs. APC-A).

3. MMA using M1/M2 macrophages

1. After obtaining the M1/M2 macrophages, count them using a hemocytometer in a 1:1 staining ratio with trypan blue. Reconstitute M1/M2 to 1 x 10⁶ cells/mL in RPMI-1640 complete medium.
2. Seed 400 μL (400,000 cells) of cell suspension using a micropipette into each well of the 8-well chamber slide and incubate at 37 °C, 5% CO₂ for at least 1.5 h in a fully humidified tissue culture incubator. Each test should be performed using a minimum of triplicate wells.
3. Opsonization of testing samples and RhD+ R2R2 red blood cells
   1. Wash RhD+ R2R2 RBCs in pH 7.4 PBS 3x by centrifugating at 350 x g for 5 min each time. We prefer to use R2R2 RhD+ RBCs as our control due to it having a higher D-antigen density, but any RhD+ RBCs could be substituted. Opsonize the testing RBC sample (e.g., patient or donor) RBCs using antibodies of interest. Use a 5% RBC suspension by adding 15 μL of packed RBCs to 300 μL of antibody mixture. Opsonize the R2R2 RBCs with Anti-D by adding 15 µL of packed R2R2 RBCs to 300 µL of Anti-D antibody mixture (100 ng/mL).
   2. Incubate at 37 °C, 5% CO₂ for 1 h. Carry out intermittent reconstitution of RBCs settled at the bottom (e.g., vortex every 15 min). Wash opsonized RBCs with pH 7.4 PBS 3x by centrifugation at 350 x g for 5 min each time.
   3. To check for RBC opsonization, perform an indirect antiglobulin test (IAT). Add a secondary opsonizing anti-human antibody to the primary opsonizing antibody. Hemagglutination or clumping of RBCs can be observed within 30 s and interpreted as a successful opsonization¹³.
   4. Reconstitute washed opsonized RhD+ R2R2 RBCs in 1.25% (v/v) with RPMI-1640 complete medium. Add 1,200 μL of medium to 15 μL of RBCs to achieve 1.25% (v/v) per well.
4. Fc receptor-mediated phagocytosis
   1. After the 1.5 h incubation of the M1/M2 macrophages, to allow these cells to adhere to the wells of the chamber slide, aspirate the supernatant medium and discard, following a gentle technique, ensuring the pipette tip only touches the corner of the wells. The macrophages should be adhered to the wells. Avoid touching the middle of the wells with the pipette tip.
   2. Gently wash wells 1x with pH 7.4 PBS. Add 400 μL of the appropriate 1.25% (v/v) opsonized RBC mixture to each well of the triplicate. Incubate at 37 °C, 5% CO₂ for 2 h undisturbed.
   3. After incubation, prepare two beakers with 100 mL of pH 7.4 PBS in each one.
   4. Remove the 8-well chambers using the manufacturer’s adaptors. Dab off excess on a paper towel while keeping slides moist.
   5. Submerge the slide into the beaker with the discarded supernatants and wash it by moving it back and forth slowly (20-30 strokes) to remove any remaining non-phagocytosed RBCs.
   6. Then submerge the slide in the second beaker and wash slowly for 20-30 strokes more. Remove the slide from PBS and dab off the excess on a paper towel. Avoid touching the front of the chambers. At this point, dip the slide into water for 1 min to remove any adherent, non-phagocytosed RBCs, but it may not be necessary if you can distinguish phagocytosed from adherent RBCs. Air dry.
   7. Submerge the slides in 100% methanol for 45 s to fix them, and then air dry. Mount the slide using an in-house prepared mounting medium and add coverslips (24 x 75 mm). Allow it to dry overnight before quantification. 
5. Quantification of phagocytosis
   1. Using a phase contrast microscope, 40x objective lens, and a manual cell counter, quantify phagocytosis.
   2. With the help of two manual cell counters, one in each hand, count phagocytosed RBCs with one counter and the total number of monocytes/macrophages with the other. Count 300 monocytes/macrophages per well.
   3. Calculate the average phagocytic index (PI) per test (across triplicates) by dividing the number of phagocytosed RBCs by the number of total monocytes counted and multiplying by 100. Express the data as average PI ± standard error of the mean (SEM).
      PI = (phagocytosed RBCs / 300 macrophages) x 100

Results

The results in Figure 1 are consistent with the literature and indicate a successful polarization of macrophages from their M0 state to their subsequent M1/M2 state. M1 and M2 macrophages were cultured for 8 days (6 days with growth factors and 2 days of polarization), and anti-D or anti-k opsonized RBCs were tested (Figure 2). M2 macrophages demonstrate a high phagocytic index compared to M1s with RBCs opsonized by either anti-D (control) or an...

Discussion

To ensure the method’s success, one must adhere to the following critical steps: 1) successful M1/M2 polarization, 2) generation of the macrophage layer and RhD+ control 3) quantification of phagocytic index. While our methods state to use isolated monocytes for the cell culture, PBMCs may be used, but we recommend using purified monocytes. It is known that PBMCs contain various cell types, with these cells secreting multiple different cytokines and mediating factors. This may have an impact on the differentia...

Materials

Name	Company	Catalog Number	Comments
1X PBS, pH 7.4, without Ca2+/Mg2+	Wisent Bioproducts	311-425-CL	Store at 4 degrees or room temperauture
AccutaseTM	STEMCELL Technologies	7920	Cell detachment solution
ACK Lysis Buffer	STEMCELL Technologies	07850, 07800	Store at 4 degrees
Anti-Human Globulin	NOVACLONE, Immunocor.	N/A	NOVACLONE Anti-igG for IAT testing
Anti-Rh(D) (WinRho. SDF CDN)	Saol Therapeutics	1003092	Any commerical source of Rh immune globin will suffice
Cell Scraper	UofT Medstore	83.395	cell detachement
Cell Strainer 70uM nylon	Falcon	352350	filter of cells
Chamber slide Nunc. Lab-TekTM II with Cover, RS Glass Slide Sterile	Thermo Fisher Scientific	154534	chamber slides for MMA
Coverslips	VWR	48393-081	24 x 50 mm
Cytiva Ficoll Paque Plus, density 1.077 g/L	Thermo Fisher Scientific	17-1440-03	sepeation of PBMCS from whole blood; density gradient medium
Elvanol Mounting Medium	N/A	N/A	Dulbecco’s PBS (D-PBS) without Ca2+/Mg2+, 15% (w/v) polyvinyl resin, and 30% (v/v) glycerine.
Fresh whole blood (ACD tube) or Buffy coat	Canadian Blood Services		May rest at room tempruatre for up to 36 hours
Human Recombinant GM-CSF	STEMCELL Technologies	78015.1	Cytokine for polarization of M1 macrophages
Human Recombinant IFN-gamma	STEMCELL Technologies	78020	Cytokine for polarization of M1 macrophages
Human Recombinant IL-4	STEMCELL Technologies	78045.1	Cytokine for polarization of M2 macrophages
Human Recombinant M-CSF	STEMCELL Technologies	78057.1	Cytokine for polarization of M2 macrophages
ID-CellStab	Bio-Rad	005650 05740	RBC cell storage/stabilization solution
Isolation Medium	N/A	N/A	PBS Ca2+ and Mg2+ free + FBS 2% + 1mM EDTA
Lipopolysaaracide (LPS)	Sigma Aldrich	L3024-5MG	Cytokine for polarization of M1 macrophages
Methanol (100%)	N/A	N/A	Fixing of slides
Monocyte Isolation Kit STEM-cells EasySep Human Monocyte Enrichment Kit without CD16 Depletion	STEMCELL Technologies	19058	Isolation of monocytes from PBMCs
Poly-D-ysine	UofT Medstore	P6407	Cell attachment solution
Rh(D) positive R2R2 RBCs	Canadian Blood Services	N/A	Also commerically avaiable
RPMI-1640	Wisent Bioproducts	350-000-CL	supplemented with 10% heat-inactivated FBS,1 mM GlutaMAX supplement, 1 mM HEPES, and 1%penicillin/streptomycin. Store at 4 degrees.
Trypan Blue solution	Thermo Fisher Scientific	15250061	Cell counting solution