Rapid, sensitive diagnosis of hemolytic anemia using antihemoglobin antibody in hypotonic solution

We have developed a new and simple flow cytometric method to detect damaged red blood cells (RBCs) using anti-Hb in hypotonic solution. We studied a total of 200 patients, including 62 patients with schistocytosis, 8 postsplenectomy patients, and 108 healthy controls. Peripheral blood (2 μl) was stained with phycoerythrin-conjugated (PE) antihemoglobin antibody (anti-Hb) in 0.6% (w/v) NaCl solution, and analyzed by flow cytometry omitting the washing step. The proportion of RBCs stained by anti-Hb was 0.55% (SD±0.23%) in normal controls and was significantly higher in patients with schistocytosis (2.95±2.95%, p <0.001). Six of 108 blood samples from normal controls and 60 of 62 samples from schistocytosis patients showed ≥ 1.01% stained RBCs (ie, values > mean+2SD of normal controls). The number of schistocytes counted by microscopic examination correlated with the proportion of RBCs stained by anti-Hb (r = 0.637, p <0.001). The proportions of stained RBCs in blood samples with malaria, spherocytosis, and elliptocytosis were also significantly higher than in normal controls. However, the results in postsplenectomy and iron-deficiency anemia (IDA) patients were not significantly different from the normal controls; the number of schistocytes in postsplenectomy patients was not related to the proportion of RBCs stained by anti-Hb. Based on these findings, flow cytometry of damaged RBCs using anti-Hb in hypotonic solution is a simple, sensitive, and accurate method to detect active hemolysis.