Moderate to marked spherocytosis is diagnostic of IHA. In this condition, spherocytes are produced when antibody coating red cells bind to the Fc portion of macrophages, resulting in partial phagocytosis of the red cell. The remnant has a reduced surface area to volume ratio and assumes a sphere shape rather than a disc. A positive direct Coombs' test (also called direct antiglobulin test or DAT) or detection of spontaneous three-dimensional clumping of red cells in blood (autoagglutination) confirms the presence of antibody on red cells.
Low numbers of spherocytes can be seen in conditions other than IHA, therefore the presence of spherocytes (especially if in low numbers) is not always indicative of IHA. These conditions include fragmentation anemias, oxidative injury to red blood cells (although the cells are actually pyknocytes, see eccentrocytes), coral snake envenomation, pyruvate kinase deficiency in Basenjis (spheroechinocytes) and disorders associated with abnormal macrophage function (hemophagocytic syndrome, malignant histiocytosis). Remember that stored red blood cells in blood bags will lose surface area with storage and, when transfused, will appear as spherocytes in blood smears from the recipient.