Macrocytic anemia and indirect hyperbilirubinemia suggest hemolysis, which in this patient is likely due to IgM antibodies which may follow Mycoplasma infections and which cross-react with RBC surface molecules of the I/i system These antibodies are termed cold-reacting antibodies because they react at temperatures less than 37degC (98degF). Examination of the peripheral blood smear is the first step in evaluation of hemolytic anemia. The young red cells (which would show up as reticulocytes when properly stained) are much larger than mature RBCs, accounting for the macrocytosis (the MCV can be as high as 140 with vigorous reticulocytosis). The presence of micro spherocytes suggests immune-mediated hemolysis, while the presence of fragmented RBCs or schistocytes suggests a mechanical cause of hemolysis, as seen in the microangiopathic hemolytic anemias. Serum protein electrophoresis is useful to diagnose multiple myeloma, which is rarely associated with hemolysis, but this would not be the best initial test; the anemia in multiple myeloma is normocytic. Flow cytometry can detect surface proteins such as CD55, CD59 on granulocytes and red blood cells in paroxysmal nocturnal hemoglobinuria (a rare cause of hemolysis), but again is not the best first test. Glucose-6-PD levels might be useful once hemolytic anemia is established by a peripheral smear and negative Coombs test. Bone marrow biopsy would show erythroid hyperplasia but is usually not required to diagnose hemolytic anemia.