الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Beta-thalassemia is one of most common autosomal recessive disorders worldwide. It is caused by the reduced or absent synthesis of the beta globin chains of the hemoglobin tetramer. Within erythroid precursors and mature red blood cells, the unassembled alpha chains precipitate and lead to oxidative damage of cell membrane and ineffective erythropoiesis.
Nucleated red blood cells are defined as immature erythrocytes which normally present in the bone marrow but not in the peripheral blood of adults. The presence of circulating NRBCs in adults occurs in situations of hematopoietic stress or extramedullary hematopoiesis.
In thalassemia syndromes, the count of NRBCs in the peripheral blood reflects the degree of ineffective erythropoiesis. It was suggested that NRBCs count of less than 5% was an indicator of adequate transfusion therapy in transfusion-dependent thalassemia patients. Therefore, precise detection and counting of NRBCs in those patients is important.
Manual microscopy is commonly utilized tool of NRBCs enumeration in clinical laboratories. This method is relatively simple but laborious, time-consuming and imprecise especially with elevated normoblasts count.
Previously, it was practically difficult to differentiate NRBCs from small lymphocytes by automated hematology analyzers; but with the recent technical development, it was possible to enumerate NRBCs automatically with high reliability and short turnaround time.
The use of flow cytometry for detection and counting of normoblasts in peripheral blood was initiated several years ago; it was more accurate and reproducible in comparison with reference microscopy method.
Soluble transferrin receptor is a single polypeptide chain that can be measured in human serum, derived from transferrin receptor, a transmembrane cellular protein, primarily expressed in cells that require iron. Serum sTfR concentration reflects the receptor density on cells and the number of cells expressing receptors; therefore, it is closely related to cellular iron demands and to erythroid proliferation rate.
The aim of this study was to investigate the importance of NRBCs counts and the concentrations of serum transferrin receptor as markers of ineffective erythropoiesis and adequate transfusion therapy in pediatric patients with beta thalassemia major. Also, we aimed to evaluate the performance of automated Sysmex XN-1000 hematology analyzer and flow cytometry in enumerating NRBCs in comparison with manual microscopy.
The present study included 61 pediatric patients with beta thalassemia major who were selected from patients admitted to Pediatric Department, Menoufia University for transfusion therapy in the duration between September 2018 and December 2018.
All patients were subjected to laboratory investigations included: Complete blood count, peripheral blood smear, reticulocyte count and reticulocyte production index estimation, NRBCs count by manual microscopic counting, Sysmex XN-1000 and flow cytometry; and serum soluble transferrin receptors assay.
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The results of the present study showed excellent correlation and good agreement between the reference MC method and both XN-1000 analyzer and flow cytometric method in counting NRBCs.
Comparison analysis between different methods indicated that the NRBCs% by XN-1000 analyzer was underestimated when compared with the reference MC method. However, NRBCs% by FC was mildly overestimated when compared with the reference MC method.
Analysis demonstrated that NRBCs%, reticulocyte % and reticulocyte production index were significantly increased in splenectomized group in comparison with non-splenectomized group while no significant differences were found between both groups regarding Hb level and sTFR.
Further analysis revealed that pre-transfusion Hb was significantly increased while reticulocyte %, RPI and sTFR were significantly decreased in thalassemia patients with NRBCs less than 5 % in comparison with patients with NRBCs equal to or more than 5%.
In all patients, pre-transfusion Hb levels showed significant negative correlation with NRBCs%. Moreover, reticulocyte % and RPI demonstrated significant positive correlation with NRBCs%.
Statistically significant negative correction was observed between sTfR and pre-transfusion Hb while significant positive correlation was found between sTfR and reticulocyte %.
Positive correlation was found between NRBCs% and sTfR regarding all patients, splenectomized and non-splenectomized group.
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Conclusion
The results of this study indicated that Sysmex XN-1000 analyzer and flow cytometry showed excellent correlation and good agreement with manual microscopic NRBCs counting.
The automated NRBCs counting using Sysmex XN-1000 analyzer offered many advantages in comparison with manual counting and flow cytometry, including labor savings, faster turnaround time and cost effectiveness.
In addition to Hb, reticulocytes count, RPI and sTfR, NRBCs could be used with cutoff value of 5% as a marker to differentiate between effective and ineffective erythropoiesis.
sTfR negatively correlated with Hb concentration and positively correlated with both NRBCs and reticulocytes. No statistically significant differences were found between splenectomized and non-splenectomized groups regarding sTFR levels.