Algorithm for the diagnostic evaluation of PK deficiency. An accurate diagnosis of PK deficiency is key for managing patients with disease-targeted treatments. Diagnostic testing should be considered in patients of all ages with chronic hemolytic anemia. After hemolysis has been established (low hemoglobin, increased reticulocyte count and indirect bilirubin level), autoimmune hemolytic anemia, hemoglobinopathies, and membranopathies should be excluded. The peripheral blood film in PK deficiency often does not have specific red cell morphology except polychromasia. Once the more common causes of hemolysis are excluded, a red cell enzyme panel or a hemolytic anemia gene panel may be pursued. Falsely normal PK enzyme activity levels may occur with recent red cell transfusions, reticulocytosis, and/or sample contamination with other blood cells. PK enzyme activity is red cell age dependent and will be low in comparison to other red cell age dependent enzymes, such as hexokinase. In those with a low PK:hexokinase ratio on enzyme testing, PKLR genetic testing should be pursued given that low PK activity can also be found in carriers of PK deficiency and in those with mutations in KLF1.³⁸  Up to 20% of patients currently tested will be found to have a PKLR variant of uncertain significance.¹²  In these patients, a low PK:hexokinase ratio can confirm the diagnosis. EMA, eosin-5’-maleimide.