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Rapid, sensitive diagnosis of hemolytic anemia using antihemoglobin antibody in hypotonic solution
Wonbae Lee1, Yonggoo Kim, Jihyang Lim
1Department of Clinical Pathology and Pediatrics, Catholic University Medical College, Seoul, Korea.
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 microl) 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 > or =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.
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 microl) 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 > or =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.