Evaluation workflow of patients with hemolytic anemia. (A) Proposed algorithm for the laboratory evaluation of a patient presenting with symptoms and signs of hemolysis. Of note, anemia may be well compensated, as in many cases of mild HS and most cases of PIEZO1-associated HX. Evaluation for autoimmune or, especially in an infant, alloimmune hemolytic anemia with direct and indirect antiglobulin test (DAT and IAT) is the first priority, since such a diagnosis typically requires immediate action. Consideration of the possibility of microangiopathic hemolytic anemia (MAHA) and paroxysmal nocturnal hemoglobinuria (PNH) may also be necessary. Blood smear review of the patient and parents, with attention to the RBC indices, including MCV, MCHC, and red blood cell distribution width (RDW), along with hemolytic markers (bilirubin, lactate dehydrogenase, and haptoglobin [the last one reliable after 6 months of life, since earlier, it may be low due to decreased production by the infant’s liver]), ferritin, and transferrin saturation to consider iron-loading inefficient erythropoiesis, can guide the differential diagnosis. Flow cytometry with eosin-5′-maleimide (EMA) binding of band 3 and Rh-related proteins is a rapid screening test for RBC membrane disorders that are characterized by membrane loss.^96,97  Osmotic fragility is increased in HS and often decreased in HX. Osmotic gradient ektacytometry, which evaluates the deformability of RBCs as they are subjected to constant shear stress in a medium of increasing osmolality in a laser diffraction viscometer, is the reference technique for differential diagnosis of erythrocyte membrane and hydration disorders when a recent transfusion does not interfere with phenotypic evaluation of the patient.^98-100  When the patient is recently or chronically transfused, as it is typically the case in young children with congenital severe hemolytic anemia, options for phenotypic evaluation are limited. In such cases, genetic evaluation with clinically available next-generation sequencing panels may provide an accurate diagnosis necessary for appropriate management decisions.^12,101,102  aHUS, atypical hemolytic uremic syndrome; HUS, hemolytic uremic syndrome; TTP, thrombotic thrombocytopenic purpura. (B) Osmotic gradient ektacytometry. The ektacytometry curve is determined by the RBC structural features.⁹⁹  The points indicated in red are the following: O_min corresponds to the value of the hypotonic osmolality at which 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface to volume ratio of the RBCs. A shift to the right reflects a decrease in the surface area to volume ratio (ie, increased osmotic fragility). EI_max corresponds to the maximum deformability of the RBC, and its value is affected by the cytoskeleton mechanics. The hypertonic descending part of the curve is represented by O_hyp, the osmolality value at which the cells’ average maximum diameter is half of EI_max. The value of O_hyp correlates with the initial intracellular viscosity of the cell sample. A shift to the left reflects increased intracellular viscosity of the erythrocyte caused by increased intracellular concentration of hemoglobin, typically due to dehydration of the cell; a shift to the right may represent an overhydration state of the cell in overhydrated stomatocytosis or, most commonly, a decreased intracellular concentration of hemoglobin, such as in iron deficiency. (C) Typical osmotic gradient ektacytometry curves for various RBC membrane disorders (in red) in comparison with a normal control curve run concurrently (in green). (i) HS characterized by increased O_min and decreased EI_max. (ii) HE/HPP characterized by decreased EI_max and a trapezoid shape of the curve. (iii) HX with decreased O_min and decreased O_hyp. (iv) SAO with severely decreased deformability and decreased O_min.