Identification of subpopulations of multipotent progenitor cells in hematopoietic stem-cell transplant patients using flow cytometry

Abstract

Background The implemented flow-cytometric protocol for enumeration of CD34+ cells in bone marrow, peripheral, or cord blood addresses only CD34+ cells, irrespective of their distinct subsets. However, the definition of CD34+ different subsets has gained importance concerning the engraftment kinetics and immune reconstitution, after hematopoietic stem-cell transplantation. Objectives This study aimed to describe and enumerate CD34 subpopulations, using a multicolor flow-cytometric protocol, based on the expression of CD133, SSEA-4, CD38, and CD10, in an attempt to explore the impact of the different CD34 subsets on engraftment kinetics, patient, and graft outcomes. Methods A total of 33 bone marrow transplant (25 autologous and 8 allogenic) Egyptian patients were included. Detailed demographic, clinical, and laboratory data, as well as echocardiography and pulmonary-function tests, were collected from all participants enrolled in the study, before transplantation. All patients were monitored up to 1 year post transplantation, for the development of complications. Discrimination of the distinct stem-cell subsets in the harvest was performed on NAVIOS flow cytometer, using multicolor FCM phenotyping. Results Seven CD34+ hematopoietic stem cell subpopulations were identified in the harvest blood by flow cytometry: the multipotent progenitor (MPP), erythromyeloid progenitor, B-lymphoid progenitor (BLP), multilymphoid progenitor (MLP), lymphomyeloid progenitor (LMPP), granulocyte and macrophage progenitor (GMP), and the late GMP. The MPP was the most frequently encountered subpopulation, whereas the BLP was the least-encountered one. In addition, the % population and absolute count of the late GMP were significantly higher after autologous transplantation (P=0.049 and 0.048, respectively). The absolute count of the MLP was significantly higher after G-CSG + chemotherapy-mobilization technique (P=0.039). Higher absolute count of the MLP in the harvest was associated with longer post-transplant 1-year survival of patients (P=0.025). The % population of the GMP in the harvest was significantly correlated with faster engraftment (P=0.039). However, a higher proportion of the late GMP (P=0.041and 0.028, respectively), along with higher absolute count of the LMPP, has been significantly encountered in patients who developed post-transplant disease relapse (P=0.050). By drawing a receiver-operating characteristic curve, only the size of the MPP population at cutoffs of ≤18.85% and ≤165312, respectively, could be significantly used to predict the persistence of cytopenia at 3 months after transplantation. Conclusion This study found that discrimination and quantification of the different CD34 stem-cell subsets might play a pivotal role for better understanding of engraftment kinetics and prediction of post-transplant graft and patient outcomes.