Complement C3d Deposition on Erythrocytes as a Candidate Biomarker of Extravascular Hemolysis in Paroxysmal Nocturnal Hemoglobinuria: An Exploratory Single-Center Study

Objective To evaluate complement component 3 (C3) deposition on paroxysmal nocturnal hemoglobinuria (PNH) cells by flow cytometry and its clinical relevance as a marker of significant extravascular hemolysis (EVH). Patients and Methods From March 2024 until February 2025, we evaluated erythrocyte C3d deposition by direct antiglobulin test (DAT) and high-sensitivity flow cytometry in 27 patients with PNH (14 hemolytic, 13 myelodysplastic syndrome [MDS]/aplastic anemia [AA]-associated), untreated (4) or on terminal (16), proximal (6), or combined inhibitors (1). Results C3d+ DAT was positive in 6 of 27 patients, who showed higher reticulocytes (197 vs 119×10 9 /L; P =.02; Holm-adjusted P =.35), and comparable hemoglobin ( P =.11). By flow, an additional 29% of DAT-negative patients showed C3d + >5%. With this threshold, patients with higher deposition had lower hemoglobin (10.4 vs 12.7 g/dL, unadjusted P =.006, Holm-adjusted P =.07) and higher reticulocytes (198 vs 95×10 9 /L, unadjusted P <.001, Holm-adjusted P =.002). C3d+ correlated positively with reticulocytes (Spearman rho=0.83, P <.001) and negatively with hemoglobin (rho=−0.49, P =.03); by ROC analysis C3d deposition discriminated clinically significant EVH with an area under the curve of 0.69 (95% CI, 0.42-0.91), and an exploratory cut off of 2.4% C3d+/CD59− erythrocytes, associated with reticulocytes ≥120×10 9 /L, neutrophil clone ≥65% and monocyte clone ≥71%. DAT had a lower sensitivity of 0.25 and a specificity of 0.69. In the few patients with AA/MDS-PNH, the corresponding analysis indicated a higher cutoff for C3d deposition (≥5%) associated with lower reticulocytes (≥111×10 9 /L) as possible indicators of clinically significant EVH. Conclusion Flow C3d quantification is a sensitive marker of complement deposition that may add to the clinical assessment of EVH and residual anemia in PNH.