Expression of CD47, CD35, CD55 and CD59 on Red Cells from Patients with Warm Autoimmune Hemolytic Anemia.
Authors: Barros Melca O. , Yamamoto Mihoko , Figueiredo Maria S. , Kimura Elisa Y. , Bordin Jose Orlando 
Published: October 2019

Abstract
Autoimmune hemolytic anemia (AIHA) is defined as an increased destruction of red cells (RBC) in the presence of anti-RBC autoantibodies. CD47 is an integrin-associated protein expressed on all cells including RBCs. Animal models show that CD47 deficiency contributes to accelerated development of AIHA, while CD35 (CR1- complement receptor 1), CD55 (decay accelerating factor), and CD59 (membrane inhibitor of reactive lysis) are complement inhibitory proteins. Using flow cytometry analysis, in this study we evaluated the expression of CD47, CD35, CD55, and CD59 on RBCs of patients with warm AIHA before any treatment had been initiated. The study population consisted of 12 patients with active AIHA [M:F = 6:6, median age: 32 yrs (3 – 73)], and 20 healthy controls [M:F = 9:11, median age: 36 yrs (25 – 71)]. Ten patients presented idiopathic AIHA while 2 subjects had secondary AIHA (systemic lupus erythematosus and non-Hodgkin’s lymphoma). At presentation the median Hb level was 6.6 mg/dL (range: 2.9 to 10 mg/dL), and the median absolute reticulocyte count was 324 × 109/L (range: 215 to 756 × 109/L). At the time of the analyses, all 12 patients had a positive direct antiglobulin test (DAT), 12 (100%) had IgG on their RBCs, 5 (41.7%) had IgG plus C3, and none had C3 alone. The strength of agglutination of all positive DATs showed a strong reaction. The RBC eluates prepared by a dichloromethane technique from the cell samples were positive in all 12 patients, but the retrieved autoantibodies were pan-reactive showing no specific reactivity. The mean fluorescence intensity (MFI) of the expression of CD47, CD35 and CD55 on RBCs of AIHA patients and healthy individuals were not statistically different (CD47 = 464.4 and 464.4; CD35 = 186.8 and 194.3; CD55 = 396.9 and 381.1, respectively). Four patients with life-threatening AIHA were treated with high dose of steroids and RBC transfusions, but 3 patients evolved to death. Two patients who died presented low CD55 expression on their RBCs at diagnosis. AIHA patients showed significant lower CD59 expression on RBCs than healthy controls (MFI = 512.3 ± 28.0 and 553.7 ± 36.6, P = .03). Although CD59 expression in patients that evolved to remission was not significantly different from healthy controls (MFI = 538.5 ± 14.4 and 553.7 ± 36.6), the expression of CD59 on RBCs of 3 AHAI patients who died were significantly lower than that seen on RBCs of healthy controls (MFI = 433.6 ± 69.6 and 553.7 ± 36.6, P = .0001). Although experimental studies have suggested that CD47 has a profound influence on the severity of AIHA in mice, our preliminary data on 12 patients with AIHA did not demonstrate difference on the expression of CD47 on RBCs of patients with warm AIHA or healthy indibiduals. On the other hand, complement regulatory proteins (CD35, CD55, and CD59) may play an important role in protecting RBC destruction through the activation of complement. Our results suggest that patients with life-threatening warm AIHA may present significant CD59 deficiency on their RBCs that may increase the susceptibility of cells to complement-mediated lysis resulting in severe clinical hemolysis.