A fluorometric erythrophagocytosis assay using differentiated monocytic THP‐1 cells to assess the clinical significance of antibodies to red blood cells

Abstract

Background and Objectives The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry‐based erythrophagocytosis assay was established using phorbol 12‐myristate 13‐acetate (PMA)‐treated THP‐1 cells and RBCs labelled with PKH26 to assess allo‐ and autoantibodies to RBCs. Materials and Methods THP‐1 cells were differentiated into macrophage‐like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA‐treated THP‐1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. Results We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti‐D and anti‐K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti‐Ch) or autoantibodies from patients and pregnant women without haemolysis. Conclusion The flow cytometry‐based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Intelligent berberine-loaded erythrocytes attenuated inflammatory cytokine productions in macrophages

“…Afterward, macrophages were resuspended in the phagocytosis medium after being washed once with the RPMI-1640 medium. The data were analyzed using FlowJo software (version 7.6.1) in comparison to the control samples 56 . Wright-Giemmsa was also used to stain stratified macrophages.…”

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

“…Although, these in vivo assays were useful, involvement of radioactivity or having to transfuse incompatible blood without any preconception of outcome is dangerous; so, in the early 1980s, investigators began in earnest to try and address the clinical significance of detected RBC antibodies by designing in vitro cellular assays to mimic the in vivo environment (Branch, Gallagher, Mison, Sy Siok Hian, & Petz, 1984; Conley et al., 1982; Gallagher, Branch, Mison, & Petz, 1983; Hunt, Beck, Hardman, Tegtmeier, & Bayer, 1980; Schanfield, Schoeppner, & Stevens, 1980; Stevens, Schanfield, & Braley, 1976). Assays to assess the potential for an antibody to cause hemolysis of transfused red blood cells in patients having the corresponding alloantibody included a chemiluminescence test (CLT; Downing, Templeton, Mitchell, & Fraser, 1990; Hadley, Wilkes, Poole, Arndt, & Garratty, 1999; Lucas, Hadley, Nance, & Garratty, 1993), monocyte‐macrophage assays (MMAs; Tong & Branch, 2017; Tong, Burke‐Murphy, et al., 2016; Zupanska, 1985), flow cytometry (Balola, Mayer, Bartolmas, & Salama, 2021), and less characterized assays such as antibody‐dependent cellular cytotoxicity (ADCC; Barcellini, 2015). Herein, we provide detailed protocols for the MMA and ADCC assays for use to determine the clinical significance of antibodies in patients requiring transfusion of serologically incompatible donor blood.…”