Isolation of Peripheral Blood Nucleated Red Blood Cells from β-Thalassaemia Patients Using CD71 Magnetic Activated Cell Sorting

Abstract

The molecular biology knowledge in β-thalassaemia is limited due to the involvement of various erythropoeitic processes where the genetic information is lack due to nucleus ejection throughout the maturation of red blood cell activities concurrence with the accumulation of haemoglobin. Nucleated red blood cells (NRBCs) are typically found in peripheral blood (PB) of β-thalassaemia transfusion dependent patients and abundant in post splenectomy (Fig. 1A) [1]. The presence of NRBCs will provide further understanding on the molecular aspect of ineffective erythropoiesis in β-thalassaemia patients. Therefore, the objectives of this study were to isolate the NRBCs using CD71 magnetic beads from PB of β-thalassaemia patients and to compare the quantity of NRBCs enriched between non-splenectomised transfusion dependent and post-splenectomised transfusion dependent β-thalassaemia patients

NRBCs were isolated from 6mL PB of both groups after the isolation of mononuclear cells (MNCs) based on density gradient and Magnetic activated cell sorting (MACs) with anti-CD71 was used to enrich isolated NRBCs (Figure 1B). Cell count for NRBCs positive for CD71 was determined by trypan blue using haemocytometer (Weber,UK) and flow cytometry analysis (Becton-Dickson,USA) was performed for method validation. NRBCs were successfully isolated from PB of 15 non-splenectomised transfusion dependent and 7 post-splenectomised β-thalassaemia patients with >90% specificity (Fig. 2). The median number of NRBCs (×10⁴) enriched were 58.5 (283) in non-splenectomised transfusion dependent and 340 (338) in post-splenectomised transfusion β-thalassaemia patients (Table 1). Higher NRBCs were isolated from the post-splenectomised compared with non-splenectomised patients (P = 0.012). 

This finding supports the idea of isolating the NRBCs from β-thalassaemia patients by using specific antibodies, CD71. This was consistent with the literature in which CD71 antibodies have been reported as the surface marker of NRBCs in cord blood [2]. It was important to isolate NRBCs from other haematopoietic cells as heterotopic interaction between cells could impact the molecular studies of this erythropoeitic cell especially in term of epigenetic activity [3]. Epigenetic mechanisms such as methylation was reported to play role in regulating and maintaining the globin gene expression as the cells proliferate which starts in the preproerythroblast and achieved high levels of transcription after the proerythroblast stage of development. This finding may provides the basis in understanding β-thalassaemia  in term of epigenetics involvement which required a specific cells. In conclusions, MACs method is convenient, simple, and can be efficiently applied in isolating NRBCs from both non-splenectomised and post-splenectomised β-thalassaemia patients.