Pb2546: diagnostic power of erythrocyte and reticulocyte automatic parameters in the screening for congenital hemolytic anemias

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Congenital hemolytic anemias (CHAs) are a group of disorders caused by defects of red cell membrane or metabolism, or by abnormal hemoglobin. Due to their rarity and heterogeneity, diagnosis is often challenging, and requires specific tools not always available outside reference centers; as a consequence, many cases may remain undiagnosed or misdiagnosed. The availability of additional parameters for complete blood count has emerged in recent hematology analyzers; however, a few studies have been conducted on advanced RBC parameters and hemolytic anemias. Diagnostic algorithms for screening of hereditary spherocytosis (HS) have been reported using Sysmex XE and Sysmex XN analyzers. Aims: We investigated the use of Sysmex parameters, the percentage of microcytes (MicroR) and hypochromic red blood cells (Hypo-He), as well as the immature fraction of reticulocytes (IRF) in combination with complete blood and reticulocyte count, for screening HS and pyruvate kinase deficiency (PKD) with the final aim to develop a diagnostic algorithm for differential diagnosis of CHAs. Methods: Complete blood count and reticulocytes from 319 patients with a confirmed diagnosis of CHAs were analyzed (In particular, HS: 81; PKD: 18; autoimmune hemolytic anemia: 50; congenital dyserythropoietic anemia: 9 patients; PIEZO1 gene mutations/ stomatocytosis: 9; thalassemia and sickle cell disease: 118 patients; and the other CHAs: 34 patients) on Sysmex XN analyzer. To further increase number of cases, data were merged with that of 94 other CHAs patients, including 61 HS, available from literature and analyzed on the same instruments (F. Mullier et al. 2011; F. Persijn et al. 2012; V. Bobée et al. 2018; Sottiaux et al. 2020). Patients were stratified according to the severity of anemia (Hb <8g/dL; Hb 8-12g/dL and Hb >12g/dL) and ROC curve analysis was performed for each parameter and condition to establish optimal cut-off limits. A control group of 149 patients with hematological disorders of different etiology, and an extended database of 11.194 (blood count results) cases has been used to establish algorithm disease specificity. Results: All 142 HS patients had RET>75x109/L and RET/IRF>7.7 (“pre-screening” condition) and were characterized by reticulocytosis without an increase in IRF, increased MicroR without significant increase of Hypo-He. Instead, PKD patients showed increased IRF (>25% and >15,6% in non-anemic and anemic, respectively) accompanied by reticulocytosis, MicroR and Hypo-He reduction. Based on ROC curves analysis results, by combining parameters (Hb, RET, IRF, MicroR, Hypo-He) a diagnostic algorithm was developed for screening of HS and PKD showing a sensitivity and specificity of 97.9% and 92.6% for HS and 94.4% and 99.2% for PKD respectively (figure 1). The algorithm showed a superior diagnostic performance when all patients were tested in parallel by using all the previous algorithms reported in literature. Summary/Conclusion: The presented algorithm proposes an easy, economic and efficient approach to detect HS and PKD using Sysmex analyzers, definitely contributing to an early diagnosis and a better management of these patients.Keywords: Automation, Hereditary spherocytosis, Hemolytic anemia, Pyruvate kinase deficiency