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ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 4 | Page : 214-226
Identification of subpopulations of multipotent progenitor cells in hematopoietic stem-cell transplant patients using flow cytometry
Amera H Elsayed1, Soha R Youssef PhD 1, Mohamed M Moussa2, Yasmine N Elsakhawy1, Dalia D Salem1, Mariam K Youssef PhD 1
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine/Hematology and Bone Marrow Transplantation, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Correspondence Address:
Mariam K Youssef
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Abassia, Cairo, 11566
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_69_21
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.
Keywords: bone marrow transplantation, CD, CD34 HSPCs, complications, engraftment, flow cytometry
Abbreviation: AA, aplastic anemia; AML, acute myeloid leukemia; APC, allophycocyanin; AUC, area under the curve; BM, bone marrow; BMT, bone marrow transplantation; B-NHL, B-cell non-Hodgkin lymphoma; CMV, cytomegalovirus; ECD, phycoerythrin Texas red conjugate; EMP, erythromyeloid progenitor; FITC, fluorescein isothiocyanate; FL, Florida; GMP, granulocyte and macrophage progenitors; HSC, hematopoietic stem cell; HSCT, hematopoietic stem-cell transplant; HSPCs, hematopoietic stem and progenitor cells; ISHAGE, International Society of Haemato-therapy and Graft Engineering; K2-EDTA, ethylenediamine tetraacetic acid; LMPP, lymphomyeloid progenitor; MLP, multilymphoid progenitor; MM, multiple myeloma; MoAbs, monoclonal antibodies; MPP, multipotent progenitor; PB, peripheral blood; PBS, phosphate-buffer saline; PC 5.5, phycoerythrin cyanin 5.5; PC7, phycoerythrin cyanine 7; PE, phycoerythrin; PoGF, poor graft functions; Pre-B ALL, pre-B-cell acute lymphoblastic leukemia; SSEA-4, stage-specific embryonic antigen 4; T-ALL, T-cell acute lymphoblastic leukemia; TBV, total blood volume.
ORCID: https://orcid.org/0000-0003-2075-3889
Introduction
In the current clinical setting for hematopoietic stem cell (HSC), the implemented flow-cytometric protocol for enumeration of CD34+ cells in bone marrow (BM), peripheral or cord blood, focuses only on CD45 and CD34, irrespective of their different subsets. However, the definition of CD34+ distinct subsets has gained importance concerning the engraftment kinetics and immune reconstitution post transplantation [1].
A classical model of the hematopoietic lineage tree has been presented by studying the expression of CD34 with CD38, CD10, and CD45, which led to the discovery of functional CD34+ subpopulations [2]. Stage-specific embryonic antigen-4 (SSEA-4) has also been used, along with CD133 (prominin 1), to isolate primitive stem cells [3]. In context with these findings, a composite model of human hematopoiesis has been suggested by Görgens et al., to define the different hematopoietic stem and progenitor cell (HSPC) subsets [4]. According to the proposed model, the multipotent progenitors (MPP) represent an early CD34 developmental stage, characterized by being CD45−CD133+CD38lowCD10−. These MPP divide asymmetrically, creating pairs of daughter cells: the lymphoid-primed multipotent progenitors (LMPP) (CD45+CD133+CD38lowCD10−), and the erythromyeloid progenitors (EMP) (CD45−CD133−/lowCD38+CD10−). Division of LMPP creates granulocyte and macrophage progenitors (GMP), as well as multilymphoid precursors (MLP), the latter upregulate CD10, and create CD19+ B-lymphoid progenitors (BLP) (CD45+CD133−CD38++CD10+) [5]. The more the EMP and the GMP in the harvest, the faster the engraftment of neutrophils and platelets, and the less the tendency for infections and bleeding after HSC transplantation (HSCT) [6]. Also, when present in sufficient quantity in the harvest, BLP could prevent the development of chronic graft-versus-host disease (GVHD) [7]. Based on the definition of these distinct HSC subsets, we established a multicolor flow-cytometric protocol, to describe and enumerate CD34 subpopulations, 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.
MethodologyStudy settings and participants
This cross-sectional study was conducted on a convenience sample of BM-transplant patients. The sample included 33 Egyptian patients who have met the eligibility criteria [8] for autologous (25 patients) or allogenic (8 patients) HSC transplantation. The patients were recruited from the Hematology and Bone Marrow Transplantation Unit, Ain-Shams University Hospitals. Harvest samples were collected and analyzed, during the period from December 2019 to November 2020. 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. The demographic data included age, sex, blood group, and body mass index (BMI). The clinical data included information about the original diagnosis and the disease status pretransplant, the presence of comorbidities, the type of transplant and the stem-cell source, sex mismatch in allogenic transplantation, the conditioning regimen, and the mobilization technique. Laboratory data included complete blood picture with differential counts, coagulation, and chemistry profiles and viral markers. After completion of transplantation procedure, all patients were monitored up to 1 year post transplantation for the development of complications, and the results of complete blood-picture analysis were followed till the first day of engraftment, as well as at 1 and 3 months after engraftment.
Flow-cytometric immunophenotyping
Discrimination of the distinct stem-cell subsets in the harvest was performed on NAVIOS flow cytometer (Beckman Coulter, Electronics, Hialeah, FL, USA). Six fluorescent-coupled monoclonal antibodies (MoAbs) were used: phycoerythrin Texas Red conjugate (ECD)-labeled anti-CD45, phycoerythrin cyanin 5.5 (PC 5.5)-labeled anti-CD34, fluorescein isothiocyanate (FITC)-labeled anti-CD38, allophycocyanin (APC)-labeled anti-CD133, phycoerythrin (PE)-labeled anti-SSEA-4, and phycoerythrin cyanin 7 (PC7)-labeled anti-CD10. All MoAbs were supplied by Beckman Coulter and Thermo-Fisher Scientific, USA.
Procedure
One-to-two-ml blood samples were immediately collected from the harvest bag on ethylenediamine tetraacetic acid (K2-EDTA) anticoagulated vacutainer tubes, and transported to the laboratory, while being maintained in a vertical steady state. Before analysis on the flow cytometer, sample dilution (1/10) and adjustment of the total leukocytic count, using phosphate-buffered saline (PBS) (8.5 g of NaCl, 1.07 g of Na2-HPO4, and 0.39 g of NaH2PO4–2H2O, commercially available from Sigma, St Louis, MO), was first done. Five ul of each MoAb was added to 50 ul of each sample in a test tube, vortexed, and incubated for 15 min in the dark at room temperature. The cells were washed with 2 ml of PBS, centrifuged at 3000 rpm for 10 min, and then the supernatant was discarded. Tubes were vortexed and incubated for 5–10 min in the dark, after addition of 1–2 ml of ammonium chloride-based erythrocyte lysing solution to each tube [8.29 g (0.15 mM) NH4Cl, 1 g (10 mM) KHCO3, 0.037 g (0.1 mM) EDTA, and 1 l of distilled water, adjusted to pH 7.3]. The tubes were then centrifuged for 5 min at 3000 rpm, and the supernatant was discarded. The cell pellet was suspended in 500 ul of PBS and data were then analyzed on the flow cytometer.
Gating strategies
Initially, (Forward scatter/side scatter) FS/SS gating was done on all cellular populations, then singlet cellular population was gated using FS log/linear histogram. Gating was then done on HSC according to the International Society of Haemato-therapy and Graft Engineering (ISHAGE) guidelines [9]. Viable and true HSPCs were determined by their positivity for CD34, and their typical position in the lymphomonocytic area of the (Forward scatter/Side scatter) FSC/SSc dot plot. To define subpopulations, CD34+ cells were first divided into an earlier CD45− and a more committed CD45+ cell fractions. Each fraction was then separately depicted in CD38 vs. CD10 and CD133 vs. SSEA-4 dot plots, where each of the resulting subpopulation was examined for CD38, CD10, CD133, and SSEA-4 expression.
Interpretation of the results
Subpopulations were identified according to markers they expressed as follows: MPP, MLP, LMPP, EMP, BLP, GMP, and late GMP. The size of each population was quantified in %, whereas expression of individual markers on the designated population was quantified in mean channel intensity. The absolute count of CD34 was calculated as follows:
The absolute count of the other markers was calculated according to the CD34 cell dose (considered as the total CD34 count in millions) quantified in the harvest.
Data management and analysis
Recorded data were analyzed using the Statistical Package for Social Sciences, version 23.0 (SPSS Inc., Chicago, IL). The mean, median, percentiles, numbers, and percentages were used to represent the data. In the comparisons, the Mann–Whitney U and the Kruskal–Wallis tests were utilized. Spearman rank coefficient was used for the correlation studies. At <0.05, the probability of error is considerable. Receiver-operating characteristic (ROC) curve was constructed to obtain the most sensitive and specific cutoffs for each subpopulation, area under the curve (AUC) was also calculated.
ResultsCharacteristics of the study participants
This study included 33 Egyptian BM-transplant patients, of whom 25 underwent autologous transplantation and 8 underwent allogenic transplantation. As regards the original diagnosis of the included patients, 10 patients (30.3%) had Hodgkin disease10 (30.3%) had multiple myeloma, 4 (12.1%) had B-cell non-Hodgkin lymphoma, 2 (6.1%) had acute myeloid leukemia, 2 (6.1%) had aplastic anemia, 2 (6.1%) had pre-B-cell acute lymphoblastic leukemia, 2 (6.1%) had T-cell acute lymphoblastic leukemia, and only 1 patient (3%) had Langerhans histiocytosis ([Figure 1]). [Table 1] describes the clinical data of the included participants.
Seven CD34+ HSC subpopulations were identified in the harvest blood by flow cytometry: the MPP, EMP, BLP, MLP, LMPP, GMP, and the late GMP. The MPP was the most frequently encountered subpopulation in our studied participants (range: 0.36%–53.82%), whereas the BLP was the least-encountered subpopulation (range: 0%–0.39%) ([Table 2]).
Table 2 Descriptive flow-cytometric identification of different HSPC subpopulations in the harvest (n=33)[Table 3] reveals the complications and the outcome of the included patients, up to 1 year post transplantation.
Table 3 Descriptive post-transplant clinical data of the studied patients (n=33)After transplantation, 3 patients (9.1%) had not engrafted successfully (died before engraftment), 20 patients (60.6%) developed general complications, 4 patients (12.1%) developed GVHD, 12 patients (36.4%) developed infections, 1 patient (3%) had veno-occlusive disease, 6 patients (18.2%) relapsed, and 9 patients (27.3%) died. One month after engraftment, 24 patients (82.8%) were still cytopenic, whereas 5 patients (17.2%) had normal (complete blood count) CBC. Also, 3 months after engraftment, 12 patients (44.4%) were still cytopenic, whereas 15 patients (55.6 %) had normal CBC.
Comparison of the different stem-cell subpopulations regarding the characteristics of the study participants ([Table 4],[Table 5],[Table 6],[Table 7],[Table 8],[Table 9],[Table 10],[Table 11],[Table 12])
Table 4 Comparison of different HSPC subpopulations regarding age of harvest donors
Table 5 Comparison of different HSPC subpopulations regarding sex of harvest donors
Table 6 Comparison of different HSPC subpopulations regarding BMI of harvest donors
Table 7 Comparison of different HSPC subpopulations regarding type of transplant of the studied patients pretransplant (n=33)
Table 8 Comparison of different HSPC subpopulations regarding type of mobilization of the studied patients pretransplant (n=33)
Table 9 Comparison of different HSPC subpopulations regarding graft-related complications of the studied patients post transplant (n=33)
Table 10 Comparison of different HSPC subpopulations regarding patient- (especially infections) related complications of the studied patients post transplant (n=33)
Table 11 Comparison of different HSPC subpopulations regarding disease-related complications of the studied patients post transplant (n=33)
Table 12 Comparison of different HSPC subpopulations regarding complications in general of the studied patients post transplant (n=33)In our study, no significant difference was found between the different stem-cell subsets regarding age. However, the % population of the EMP was significantly higher in males (P=0.027), whereas the % population and absolute count of the GMP were significantly higher in females (P=0.01 and P=0.018, respectively). In addition, the size of population of the late GMP was significantly higher after autologous transplantation (P=0.049 and P=0.048, respectively). Also, the absolute count of the MLP was significantly higher after G-CSG + chemotherapy-mobilization technique (P=0.039).
Patients who developed post-transplant graft- and patient- (especially infections) related complications, received grafts with significantly smaller size of late GMP population (% and absolute count) (P=0.023, P=0.023 and P=0.031, P=0.035, respectively). However, higher % population and absolute count of the late GMP, along with higher absolute count of the LMPP, have been significantly encountered in patients who developed post-transplant disease relapse (P=0.05, P=0.041, and P=0.028, respectively). Moreover, higher absolute count of the LMPP, along with higher % population and absolute count of the GMP, has been significantly found in those who developed post-transplant complications in general (P=0.02, P=0.016, and P=0.021, respectively). The % population, as well as the absolute count of the different subpopulations, did not differ significantly between cytopenic and normal CBC patients, 1 and 3 months post transplantation.
Correlation between the different stem-cell subpopulations and the post-transplantation first day of engraftment, the post-transplantation CBC, and the post-transplantation 1-year survival
In the present study, the % population of the GMP in the harvest was significantly correlated with faster engraftment (P=0.039) ([Figure 2]). The % population and absolute count of the GMP were significantly positively correlated with the leukocytic count at engraftment (P=0.013 and P=0.019, respectively) ([Figure 3] and [Figure 4]). However, the absolute count of the GMP, as well as the % population and absolute count of the late GMP, was negatively correlated with the leukocytic count at 1 and 3 months after engraftment (P=0.05, P=0.023, and P=0.022, respectively). In addition, the % population of the EMP was negatively correlated with Hb level at engraftment (P=0.018). Also, the absolute count of the MPP was positively correlated with the leukocytic count at 1 and 3 months after engraftment (P=0.043 and P=0.028, respectively). Higher absolute count of the MLP in the harvest was associated with longer post-transplant 1-year survival of patients (P=0.025).
Figure 2 Statistical negative correlation between % population of the granulocyte and macrophage progenitor and the first day of engraftment.
Figure 3 Significant positive correlation between engraftment TLC and % of granulocyte and macrophage progenitor subpopulation in the harvest.
Figure 4 Significant positive correlation between engraftment TLC and absolute count of granulocyte and macrophage progenitor subpopulation in the harvest.Receiver-operating characteristic (ROC) curve to show the performance of the different stem-cell subpopulations for prediction of disappearance/persistence of cytopenia at 1 and 3 months post transplantation
Using an ROC curve analysis in an attempt to calculate a cutoff for the different stem-cell subpopulations, which can predict the disappearance or persistence of cytopenia at 1 and 3 months after BM transplantation, only the % population and absolute count of the MPP, at cutoffs of ≤18.85% and ≤165312, respectively (AUC: 0.706, 95% confidence interval [CI]: 0.5–0.864; AUC: 0.722, 95% CI: 0.518–0.878, respectively), can be significantly used to predict the persistence of cytopenia 3 months after transplantation. The % population and absolute count of the rest of the stem-cell subpopulations fail to show statistical significance for prediction of post-transplantation cytopenia ([Table 13]).
Table 13 Diagnostic validity test to show the performance of the different subpopulations in predicting post-transplant cytopenias Discussion In the present study, which included 33 Egyptian BM-transplant patients (25 autologous and 8 allogenic), we established a multicolor flow-cytometric protocol, based on the expression of CD133, SSEA-4, CD38, and CD10, to describe and enumerate CD34 subpopulations in harvest blood, in an attempt to explore the impact of the different CD34 subsets on engraftment kinetics, patient, and graft outcomes.
Seven CD34+ HSC subpopulations were identified in the peripheral harvest blood by flow cytometry: the MPP, EMP, BLP, MLP, LMPP, GMP, and the late GMP. The MPP was by far the most frequently encountered subpopulation in our studied participants, whereas the BLP was the least-encountered subpopulation. In accordance to our study, Dmytrus et al. found that PB stem cells contain significantly higher proportions of earlier and lower proportions of more committed CD34 subsets than BM stem cells, this could be the reason for the more rapid and sustained engraftment, after infusion of PB rather than BM stem cells [5].The more abundant earlier rather than more committed precursors during mobilization can be attributed to the fact that the earlier CD34 subsets are less adherent to the BM niche, and show a better stimulation response to myeloid growth factors than the late progenitors [10].
Mohammed [11] reported that the number of HSCs significantly declines with advancing age. That was supported by Ings et al., who found in their study that donors aged ≥55 years, significantly collected less CD34+ cells than younger donors, and that donors in the 38–54-year age group, collected similar to those aged <38 years [12]. In contrast, both a murine cell model, along with a study in humans, suggested that stem-cell marrow reserve increases with age [13],[14].However, comparison between different HSPC subpopulations in relation to age, revealed nonsignificant results in our studied participants.
Mohammed [11] reported that BM donors’ sex did not have any impact on the HSC yield. However, previous studies reported that sex hormones, mainly androgens, regulate development of murine, as well as human HSPCs [15]. That was supported by our study, where % population of the EMP was found to be statistically higher in males. On the other hand, it has been proposed that estrogens and progesterone indirectly regulate human erythropoiesis. Estrogen, in physiologic concentrations, led to decline in the amount of glycophorin-expressing erythroid cells [16]. In contrast, based on murine studies, increased oxygen consumption during pregnancy can be overcome by high estrogen level, which stimulates the HSPCs to produce excess erythrocytes [17]. In a detailed comparison of stem cells collected from male and female donors, male donors had significantly greater numbers of CD34+ cells and granulocyte–macrophage colony-forming cells. This could be attributed to the greater total blood volume and body weight of male donors [12]. In contrast, our study found that the % population and absolute count of the GMP were higher in female donors, this could be biased by the lower number of female-harvest donors enrolled in the study (13 donors).
In a study by Chen et al. [18], a greater BMI of donors was associated with a greater CD34+ stem-cell yield. In addition, previous animal studies reported that adipose tissue contains significant numbers of HSCs, and studies in humans must be undertaken to elucidate whether adipose tissue could be a potential source of HSCs [19]. In our study, the proportions of the different HSPC subpopulations did not significantly differ as regards to the BMI of donors. This may be due to the fact that the majority of the enrolled participants in our study were autologous donors, who had underlying conditions and/or had received chemotherapy before marrow mobilization, which both can adversely affect HSC yield, resulting in patients becoming ‘poor mobilizers’.
In our study, absolute count and % population of the late GMP were found to be significantly higher after autologous transplantation.
Moreover, absolute count of the MLP was found to be significantly higher after mobilization with G-CSG + chemotherapy, more than the regimen that added Mozobil. Mozobil was only used in patients who failed to produce the least-sufficient CD34 cell dose in response to the routine mobilization regimens. Disruption of the SDF-1/CXCR4 axis by (Granulocyte colony stimulating factor) G-CSF administration results in mobilization of myeloid cells to the peripheral circulation, especially, the late GMP, as it is attached the least to BM niche, and thus enhancing engraftment kinetics [20]. It is noteworthy that only cytokine-mobilization regimens used in healthy donors for allogenic transplantation, are well tolerated, but their utility is limited by suboptimal yields. In autologous transplantation, addition of a myelosuppressive chemotherapeutic agent to a cytokine-mobilization regimen, is associated with 2–5-fold greater stem-cells’ yield [20].
In our study, higher % population of the late GMP was significantly correlated with faster engraftment. In line with our findings, Singh et al. [21] found that patients who received G-CSF, had more GMP cells, earlier neutrophil engraftment (median days 11 vs. 14), and shorter post-transplant hospital stay.
Lower % population and absolute count of the late GMP in our participants, were associated with higher incidence of post-transplant GVHD. In contrast to our findings, Hülsdünker et al. [22] described a negative impact of GMP and neutrophils on graft outcome. The authors found that antibody-mediated depletion of GMP and neutrophils in transplant recipients on the day before transplantation, significantly decreased the severity of acute GVHD. This could be linked to the small number of allogenic donors in our study (only eight donors), in which four recipients experienced GVHD. Another reason is that all allogenic recipients underwent post-transplant immunosuppressive regimens, suppressing neutrophils, and hence decreasing the incidence of GVHD.
Not surprisingly that the reduction in the GMP, with subsequent decrease in the absolute number and phagocytic actions of mature neutrophils, was found to be significantly correlated with higher incidence of post-transplant infections in our studied participants. Tang et al. [23] assessed post-transplant absolute monocyte count on the clinical outcomes of 59 patients with acute myeloid leukemia who had undergone myeloablative-conditioning allogeneic HSCT. The authors found that patients with a post-transplant high monocytic count (≥0.57×109/l) had a significantly worse overall survival and post-relapse survival. In context with these findings, higher monocyte progenitors in the harvest were associated with higher incidence of post-transplantation disease relapse in our studied participants.
After a conditioning regimen based on high-dose chemotherapy, resolution of the aplastic phase, together with progressive normalization of blood counts, is expected to occur after engraftment of HSCs. Usually, neutrophil engraftment (defined as absolute neutrophil count (ANC) >500/μl) and platelet engraftment (defined as platelet count >20 000/μl), are expected within 3–4 weeks from HSCT [24]. Our study demonstrated a significant positive correlation between % population of the GMP with engraftment (total leucocytic count) TLC, whereas a significant negative correlation with TLC at 1- and 3 months post transplant. This could be attributed to the fact that transplanted patients mostly receive post-transplant prophylactic chemotherapy as well as immunosuppressive drugs to prevent GVHD, and so masking the effect of harvest myeloid progenitors. The absolute count of the MPP was positively correlated with the leukocytic count at 1 and 3 months after engraftment. Furthermore, % population of the EMP in the harvest was negatively correlated with engraftment hemoglobin. The explanation could be that the (peripheral blood stem cell) PBSC harvest had more early immature forms of erythroid progenitors, which indeed need time for hemoglobin-level elevation [25]. Higher absolute count of the MLP in the harvest was associated with longer post-transplant 1-year survival of patients.Although lack of engraftment (i.e., primary graft failure) could happen only in <2–3% of patients, persistent cytopenias, mostly thrombocytopenia, referred to as poor graft function (PoGF), could be noticed in up to 20% of patients [24]. Different causes may contribute to PoGF, including grafted HSC dose (and possibly also the T-cell dose), (Human leucocyte antigen) HLA disparity between donor and recipient, intensity of the conditioning regimen, and intensity of post-HSCT immunosuppression [26]. Furthermore, some post-transplantation conditions may contribute to the quality of the engraftment, such as GVHD, cytomegalovirus reactivation, and other viral or bacterial infections [27]. PoGF can be diagnosed according to the following criteria: the presence of persistent thrombocytopenia up to 35 days after transplantation, with or without other cytopenias, persistent hyporegenerative anemia (hemoglobin <10.0 g/dl, and absolute reticulocyte count <60 000/ul), and persistent ANC <1000/ul [28]. In the current study, 1 month after engraftment, 24 patients (82.8%) were still cytopenic, whereas 5 patients (17.2%) had normal CBC. Also, 3 months after engraftment, 12 patients (44.4%) were still cytopenic, whereas 15 patients (55.6%) had normal CBC. This could be caused by the post-transplant immunosuppressive and prophylactic chemotherapy taken by patients to prevent GVHD and disease relapse. Another cause for post-transplant cytopenia could be the presence of antithrombopoietin-receptor antibodies, which have been described after allogeneic HSCT [29].
Finally, by drawing an ROC curve, in an attempt to calculate a cutoff for the different stem-cell subpopulations, which can predict the disappearance or persistence of cytopenia at 1 and 3 months after BM transplantation, only the % population and absolute count of the MPP, at cutoffs of ≤18.85% and ≤165 312, respectively (AUC: 0.706, 95% CI: 0.5–0.864; AUC: 0.722, 95% CI: 0.518–0.878, respectively), can be significantly used to predict the persistence of cytopenia 3 months after transplantation.
The small sample size, as well as the relatively small percent population of the different stem-cell subsets, has been a major limitation in our study. Also, harvest blood was collected from a single stem-cell source, that is, peripheral blood stem cells. Neither BM nor umbilical cord were analyzed in our study. Studies that include larger samples, with comparison of the different stem-cell sources regarding the proportion and effect of the different stem-cell subsets, are still required for better understanding of the impact of the different stem-cell subpopulations on engraftment kinetics and graft outcome.
ConclusionsOur study supposed that discrimination and categorization of the various CD34+ stem-cell subsets in HSC-transplant patients by flow cytometry, could play an important role in prediction of post-transplant engraftment kinetics, cytopenias, graft, and patient outcomes.
AcknowledgementsAvailability of data and materials: All the data needed to support the findings will be provided upon request.
Ethical approval and consent to participate: The current study protocol was approved by the Research Ethics Committee (REC) of Ain Shams University Faculty of Medicine (FWA 000017585) on the basis of ethical considerations. All procedures were explained to all participants, and their informed consent was obtained before their enrollment in the study. This study adheres to the Declaration of Helsinki.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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