Methods to Evaluate the Potential Clinical Significance of Antibodies to Red Blood Cells

Abstract

Immune-mediated red blood cell (RBC) destruction due to antibodies is an ongoing problem in transfusion medicine for the selection of the safest blood. Serological testing often revealed incompatibility with donors' RBCs. When this incompatible blood was transfused, destruction was due mostly to extravascular-mediated phagocytosis of the antibody-opsonized RBCs; however, intravascular hemolysis was sometimes observed without explanation. Based on serology, antibodies with potential for clinical sequalae could not be ascertained; thus, antigen-negative blood was usually selected for transfusion to avoid problems. Antibodies to antigens having very high frequency in the general population (>95%), however, made selection of antigen-negative blood difficult and sometimes impossible. Some patients, who were sensitized by previous transfusions or by pregnancy, developed multiple antibodies, again creating a problem for finding compatible blood for transfusion, without the ability to discern which of the antibodies may be clinically irrelevant and ignored. Transfusion medicine scientists began searching for an in vitro means to determine the in vivo outcome of transfusion of blood that was serologically incompatible. Methods such as chemiluminescence, monocyte-macrophage phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC) were described. Over the years, the monocyte monolayer assay (MMA) has emerged as the most reliable in vitro assay for the prediction of the clinical relevance of a given antibody. ADCC has not been fully studied but has the potential to be useful for predicting which antibodies may result in intravascular hemolysis. This article captures the protocols for the implementation and readout of the MMA and ADCC assays for use in predicting the clinical significance of antibodies in a transfusion setting. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Monocyte monolayer assay (MMA) Basic Protocol 2: Antibody-dependent cellular cytotoxicity assay (ADCC).

Keywords: ADCC; MMA; antibody-dependent cellular cytotoxicity; clinically significant RBC antibodies; monocyte monolayer assay.

Figure 1

General experimental flow diagram of the monocyte monolayer assay (MMA). PBMCs isolated from whole human blood are seeded onto chamber slides to allow monocyte adherence for 1 hr. Opsonized RBCs are then added to the monocytes for the phagocytosis to occur. After 2 hr incubation, the chamber slides are washed, and the phagocytic index is determined by counting phagocytosis events under the microscope. Results are considered significant when the phagocytic index exceeds 5 phagocytosed RBCs in 100 monocytes. Abbreviations: PBMCs, peripheral blood mononuclear cells; RBCs, red blood cells.

Figure 2

Specific lysis of human RBCs mediated by peripheral blood mononuclear cells (PBMCs) and NK cells. (A) Determination of the required number of cells (30,000 cells, circled in red) for detection of RBC lysis with a maximal release of ⁵¹Cr. RBC after cells were lysed with 1 N HCl and radioactivity measured in a MicroBeta² microplate counter. (B) Cytotoxicity of PBMCs compared to NK cells against RBC opsonized or not with an anti‐D antibody (WinRho), at the indicated effector/target (E/T) ratios, as assessed in a ⁵¹Cr release assay. Average of at least three independent donors are shown.

Figure 3

General experimental flow diagram of the antibody dependent cellular cytotoxicity assay (ADCC). NK cells are isolated from whole fraction PBMCs by negative selection. In parallel, RBCs are opsonized with antibodies of interest and loaded with radioactive ⁵¹Cr. Thereafter, NK cells and radiolabeled RBCs are co‐cultured for 4 hr allowing NK cell‐mediated lysis of the RBC. The supernatant containing the radioactive ⁵¹Cr released from lysed RBC is collected and dried on LumaPlates for posterior quantification of the released radioactivity. Abbreviations: PBMCs, peripheral blood mononuclear cells; RBCs, red blood cells.