Diagnosis of pyruvate kinase deficiency (PKD) remains challenging in clinical practice. The pyruvate kinase (PK) to hexokinase (HK) activity ratio (PK/HK) was proposed to reduce the confounding effect of reticulocytosis on PK activity measurement. However, decreased PK activity and PK/HK ratios have also been observed in other anemias, raising doubts about their diagnostic value. We assessed the diagnostic accuracy of PK/HK ratio versus PK activity in differentiating PKD from other hereditary anemias. This study included 41 patients with molecularly confirmed PKD and 62 patients with other anemias. We also evaluated the influence of reticulocytosis and transfusions on erythrocyte PK activity. The PK/HK ratio showed 73% specificity, while PK activity alone achieved 95%. In PKD patients, reticulocytosis did not affect PK activity because reticulocyte PK activity was already markedly reduced (23-fold) compared with controls. In other anemias, decreases in PK activity were present in both reticulocytes and erythrocytes, but to a lesser extent. Transfusions contribute more to the false-normal result of PK activity than reticulocytosis. Measuring reticulocyte-specific PK activity during regular transfusions provided reliable results, as only patient-derived reticulocytes are present in the blood. PK activity demonstrates higher specificity than PK/HK ratio in diagnosing PKD. Reticulocytosis is not a confounder, while transfusions remain the main limitation. Reticulocyte-specific PK activity measurement may improve diagnostic accuracy in transfused patients.

Diagnosis of pyruvate kinase (PK) deficiency remains challenging in clinical practice. To avoid the influence of reticulocytosis when measuring PK activity, was proposed to measure PK:hexokinase (HK) enzyme activity ratio. However, decreased PK activity and PK:HK ratio were observed in patients with other anemias, so use of this ratio in differential diagnostics remains questionable. Here, we assessed the specificity and sensitivity of this ratio in the differential diagnosis of PK deficiency (PKD) and other anemias. Under these conditions, the specificity of the ratio was 73% while the specificity of PK activity was 95%. In a cohort of PKD (n=41) and other anemias (n=62) patients with molecularly confirmed anemias, we demonstrated the absence of PK activity dependence on reticulocytosis in PKD patients and showed the reason for this absence: PK activity is already significantly (23-fold) reduced in reticulocytes in these patients. A decrease in PK activity in both reticulocytes and erythrocytes was also found in patients with other anemias, but to a much lesser extent. Transfusions contribute more to the false-normal result of PK activity than reticulocytosis and we suggested measuring specific PK activity in reticulocytes during regular transfusions, since after the transfusion the patient's blood contains only his own reticulocytes.