Objective
To determine the number and intensity of phosphatidylserine (PS) expression of the red blood cells (RBCs), fragmented RBCs, and RBC-derived microparticles (RMPs) in patients with β-thalassemia/hemoglobin (Hb)E.
Methods
We used flow cytometry to determine the number and levels of PS expression.
Results
The number of PS-exposing RBCs was statistically significantly higher (P <.001) than that of PS-exposing fragmented RBCs or RMPs. In contrast, the intensity of PS expression was significantly higher (P <.001) in RMPs than in RBCs or fragmented RBCs. Our study showed a trend of association between RBC distribution width (RDW) and both the number of fragmented RBCs and RMPs and their intensity of PS expression.
Conclusion
In β-thalassemia/HbE, PS-exposing RBCs, fragmented RBCs, and RMPs all differed in their numbers and their intensity of PS expression. The effects of these differences among PS-exposing populations on the pathophysiology of the disease require further investigation.
Relatively recent investigations1,2 have shown the clinical importance of phosphatidylserine (PS)–exposing red blood cells (RBCs). In vitro and in vivo studies have documented the coagulation activation properties of PS-exposing RBCs.3 Another study4 has reported increased adhesion to the vascular endothelium by PS-exposing RBCs. Therefore, to understand the clinical significance of PS in the pathophysiology of β-thalassemia, it is important to examine PS-exposing populations in β-thalassemia disease.
Recently, fragmented RBCs or RBC vesicles have been found in patients with β-thalassemia; the size of these fragmented RBCs was similar to that of the platelets, according to their forward scatter (FSC) and side scatter (SSC) profiles.5,6 Studies have also suggested that the number of people in a PS-exposing population are inversely correlated with the degree of anemia. RBC-derived microparticles (RMPs) have also been shown to express PS on their surfaces. RMPs are small vesicles, from 0.1 to 1.0 μm in diameter, that are released from the RBCs and can be characterized using flow cytometry in combination with standard-size microbeads. A study7 has shown that these PS-exposing RMPs can activate thrombin and coagulation protein. To date, no study, to our knowledge, has fully elucidated the characteristics of these 3 PS-exposing populations in thalassemia/hemoglobin (Hb)E disease.
In the present study, we compared the numbers of PS-exposing RBCs, fragmented RBCs, and RMPs and their intensity of PS expression in patients with β-thalassemia/HbE. The association between each of the 3 populations and the severity of the illness was also addressed.
In this study, we used allophycocyanin-conjugated cluster of differentiation 41a (CD41a-APC), fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC), and 10× annexin V binding buffer (Becton, Dickinson and Company). We also used phycoerythrin-conjugated cluster of differentiation (CD)235a (CD235a-PE; Dako); CountBright counting beads Thermo Fisher Scientific, Inc); and blank calibration particles, size 1.09 μm (Spherotech, Inc.).
This study was approved by the Institutional Review Board of Siriraj Hospital, Mahidol University School of Medicine, Bangkok, Thailand (COA no. Si341/2013). We collected blood specimens from patients with β-thalassemia/HbE in the Department of Pediatrics, Faculty of Medicine, Siriraj Hospital. After informed written consent was obtained, 3 mL of venous blood was collected from each patient and preserved in K2EDTA (dipotassium ethylenediaminetetraacetic acid). Then, the blood specimens were divided into groups to be analyzed by flow cytometry and automated cell counters. For all patients, the diagnosis of thalassemia had been made using standard hematological techniques and Hb analysis. Also, none of the patients in the cohort had received a blood transfusion for at least 3 months.
We used an AcT 5-part differential (5 diff; Beckman Coulter, Inc.) to analyze complete blood counts according to manufacturer protocol. Before the procedure, internal quality control was conducted to check the accuracy and precision of the instrument.
Whole blood specimens were diluted 1:100 with phosphate-buffered saline (PBS). Then, 5 μL of diluted specimen material was incubated with 2 μL of annexin V–fluorescein isothiocyanate (FITC), 3 μL of CD235a–phycoerythrin (PE), 5 μL of CD41a–allophycocyanin (APC), and 20 μL of 1× annexin V binding buffer in the dark at room temperature for 15 minutes. Next, we added 300 μL of 1× annexin V binding buffer and 25 μL of counting beads to the stained specimens, which we then analyzed using FACSDiva software, version 6.0, on a FACSCanto flow cytometer (Becton, Dickinson and Company).
For flow cytometry analysis, the FSC, SSC, and fluorescence (FL) parameters were set at logarithmic scale. The threshold was set at FSC to eliminate debris and noise signals. Figure 1 summarizes the flow-cytometry gating strategies. A CD41a vs SSC dot plot was used to define the counting beads (R-1) and total cell population (R-2). The gated populations were analyzed on an FSC vs SSC dot plot to define the gate for MPs (R-3), fragmented RBCs (R-4), and RBCs (R-5). The MP gate was defined using standard-size beads. Then, gated populations were analyzed on a CD235a vs annexin V dot plot to determine RMPs (R-6), PS-exposing fragmented RBCs (R-7), and PS-exposing RBCs (R-8). Data were collected for at least 1300 events of counting beads at medium speed. Then, the numbers per microliter were calculated according to the instructions for the counting beads. Before the analysis, the performance of the instrument was verified using Cytometer Setup & Tracking (CS&T) beads (Becton, Dickinson and Company).
Figure 1
Open in new tabDownload slideFlow cytometry gating strategy for enumerating phosphatidylserine (PS)–bearing populations. A cluster of differentiation (CD)41a vs side scatter (SSC) dot plot represents the counting beads in gate R-1 and the red blood cells (RBCs) and platelets in gate R-2. A front scatter (FSC) vs SSC dot plot shows the microparticles (MPs), fragmented RBCs, and RBCs in the R-3, R-4, and R-5 gates, respectively. A CD235a vs annexin V dot plot shows the PS-exposing RBC-derived microparticles (RMPs) (R-6), fragmented RBCs (R-7), and RBCs (R-8) from the previous gated population.
Data analysis and graphing were performed using GraphPad Prism software, version 5.0 (GraphPad Software Inc.). The results were expressed as mean, standard error (SE), and range. The Mann-Whitney U test was used to determine the mean differences in the levels of PS-exposing RBCs and RMPs among the groups. Linear regression was used to determine the relationship between the 2 variables. P ≤.05 was considered statistically significant.
The current study analyzed 149 patients with β-thalassemia/HbE. The average patient age and male/female ratio were 13 years (range, 1‒30 years) and 1:1.2 (68:81), respectively. The mean (SE) RBC, WBC, and platelet counts were 3.8 (0.8) × 106per μL (range, 2.2–6.8 × 106/μL), 15.7 (26.3) × 103 per μL (3.2–194 × 103/μL), and 402 (228) × 103 per μL (range, 100–1167 × 103/μL), respectively. The hemoglobin, hematocrit, mean corpuscular volume, mean cell hemoglobin, and mean corpuscular hemoglobin concentration were 9.1 (1.3) g per dL (range, 5.4–13.9 g/dL), 28.1% (4.3%) (16.7%–46.2%), 73.4 (8.5) fL (49.9–105.5 fL), 23.9 (3.5) pg (14.3–37.8 pg), and 32.5 (2.0) g per L (26.6–36 g/L), respectively.
First, we determined the numbers of PS-exposing RBCs, fragmented RBCs, and RMPs and their levels of PS expression. The results showed that the number of PS-exposing RBCs (410,943 [27,368]/μL [range, 6322–1.300 × 106/μL]) was significantly higher (P <.001) than that of fragmented RBCs (40,088 [5325]/μL [2537–536,113/μL]) and RMPs (13,527 [1751]/μL [1687–201,230/μL]) (Figure 2A). In contrast, the highest intensity of PS expression was documented in RMPs (376.7 [12.8] arbitrary units [AU] [range, 124–956 AU]), compared with fragmented RBCs (261.5 [9.1] AU [96.4–632.1 AU]) and RBCs (180.5 [3.6] AU [93.9–319.1 AU]) (Figure 2B).
Figure 2
Open in new tabDownload slideBar plots show the numbers per microliter (A) and phosphatidylserine (PS) intensity (B) of PS-exposing red blood cells (RBCs), fragmented RBCs, and RMPs in patients with β-thalassemia/hemoglobin (Hb)E. The asterisk indicates the statistically significant differences among the groups (P <.001).
Next, we investigated the relationship between the number and intensity of PS expression in each PS-exposing population and the severity of β-thalassemia/HbE among the patients. Our data revealed no association between the number of PS-exposing RBCs, fragmented RBCs, and RMPs and the Hb level (Figure 3A, Figure 3B, and Figure 3C). Also, there was no relationship between the number of PS-exposing RBCs and the RBC distribution width (RDW) (Figure 3D). However, the scattergram showed a trend of positive association between the RDW and the number of fragmented RBCs (Figure 3E) and RMPs (Figure 3F). Similarly, there was no association between the intensity of PS expression on RBCs and the Hb level (Figure 4A, Figure 4B, and Figure 4C). The data also showed no relationship between the intensity of PS expression on RBCs and the RDW (Figure 4D). However, a trend of negative association was observed between the RDW and the intensity of PS expression on fragmented RBCs (Figure 4E) and RMPs (Figure 4F).
Figure 3
Open in new tabDownload slideLinear regression analysis of hemoglobin level vs the number of phosphatidylserine (PS)–exposing red blood cells (RBCs) (A), PS-exposing fragmented RBCs (B), RBC-derived microparticles (RMPs) (C), and RBC distribution width (RDW) vs the number of PS-exposing RBCs (D), PS-exposing fragmented RBCs (E), and RMPs (F).
Figure 4
Open in new tabDownload slideLinear regression analysis of hemoglobin level vs intensity of phosphatidylserine (PS) expression on red blood cells (RBCs) (A), fragmented RBCs (B), RBC-derived microparticles (RMPs) (C), and RBC distribution width (RDW) vs the intensity of PS expression on RBCs (D), fragmented RBCs (E), and RMPs (F).
Expression of PS has been linked to the pathophysiology of β-thalassemia disease.8–10 Kuypers et al1 demonstrated that in a subpopulation of thalassemic RBCs, the number of PS-exposing RBCs and the intensity of PS expression varied among patients. Other studies have identified 3 distinct populations in patients with β-thalassemia that exhibit PS expression on their surface membranes.5–7 These populations can be classified using flow cytometry analysis. To understand the pathophysiology associated with PS in patients with β-thalassemia, it is important to examine the PS expression of these populations. The current study quantitated PS-exposing RBCs, fragmented RBCs, and RMPs in patients with β-thalassemia/HbE and addressed the association of each of these populations with the severity of the disease. Our results demonstrated for the first time the differences in the numbers of PS-exposing RBCs, fragmented RBCs, and RMPs and the differences in their intensity of PS expression.
The numbers of PS-exposing cells and the intensity of their PS expression are both important factors contributing to the procoagulant properties of PS-exposing cells.7,11 The present study demonstrated numbers of PS-exposing RBCs that were 10 times and 30 times higher, respectively, than those of PS-exposing fragmented RBCs and RMPs. In contrast, the intensity of PS expression by RMPs was 1.4-fold higher than that by fragmented RBCs and twice as high as that by RMPs.
This observation may be explained by the clearance mechanism of PS-exposing cells. Accumulated evidence12–14 has suggested that PS-exposing cells are removed from the blood circulation by the reticuloendothelial (RE) system in the spleen. The PS on the outer membranes of cells is recognized by tissue macrophages as a signal for phagocytosis. On recognition, PS-bearing RBCs, fragmented RBCs, and RMPs are engulfed by macrophages. Considering the high PS expression on the surface of RMPs, RMPs might be the first target for removal by the RE system, resulting in a lower number of RMPs.
In contrast, the RBCs and fragmented RBCs, which have lower PS expression, might survive longer than the RMPs. A study15 demonstrated that exposed PS on the surface membranes can potentiate adhesion between the PS-exposing cells and the vascular endothelial cells. In the case of high PS expression, interaction between the PS-exposing RMPs and the endothelial cells may occur more frequently than interaction between PS-exposing RBCs or fragmented RBCs and endothelial cells. Also, patients with β-thalassemia are regularly monitored for Hb levels. To avoid anemia, blood transfusion is recommended when the Hb level is low. Considering the short lifespan of PS-exposing RBCs,16 to normalize their numbers, patients with higher numbers might require more frequent blood transfusions than patients with low numbers.
Two recent studies documented a negative correlation between the number of PS-exposing RBC vesicles and the hemoglobin concentration in patients with β-thalassemia.5,17 However, our data showed no correlation, negative or positive, between the number of PS-exposing RBCs, fragmented RBCs, and RMPs and the level of Hb. The discrepancy between our results and those of previous studies can be explained by the differences in the severity of the β-thalassemia/HbE in the patients involved in each study. This hypothesis is supported by the results of a previous study that documented a significant increase in PS-bearing fragmented RBCs in a patient with β-thalassemia/HbE who had average Hb levels of 6 g per dL.5
In the present study, the patients had an average Hb level of 9 g per dL, suggesting mild anemia. Also, our data showed an average of 40,088 fragmented RBCs per μL, which was similar to the number of fragmented RBCs in healthy volunteers found by another study, namely, 30,000 per μL.5 Further, in the present study, the average number of RMPs was lower than in another previous study.18 However, despite the poor correlation between the RDW and the number of PS-exposing fragmented RBCs and RMPs, our scatterplot showed a trend of positive association between these parameters. Also, our results documented a trend of negative association between the RDW and the intensity of PS expression on fragmented RBCs and RMPs. Considering this observation, further investigation is required to confirm the roles of various PS-exposing populations in the severity of illness in patients with β-thalassemia.
The present study has several limitations. Previous studies3,4 have demonstrated a possible association between MP levels and thrombosis complications in patients with β-thalassemia. However, the present cross-sectional study examined only the relationship between PS-exposing RBCs, fragmented RBCs, and RMPs and their hematological data.
Also, further study should be conducted to address whether the number of PS-exposing RBCs, fragmented RBCs, and RMP is associated with the degree of severity in severe, moderate, and mild anemia and lack of anemia in patients with β-thalassemia/HbE. Such a study would provide new insight into the role of different PS-exposing populations in pathophysiology of β-thalassemia. In addition, the present study investigated only PS-exposing RBCs and RMPs originating from RBCs. Several lines of evidence have shown that increased MPs originate from platelets and endothelial and white blood cells.6,18,19 The heterogeneity of these MPs and their association with pathophysiological manifestations warrant further study.
In conclusion, we have demonstrated the differences among 3 PS-exposing populations in terms of the number of cells and their intensity of PS expression in patients with β-thalassemia/HbE. The findings of the present study warrant further research into the heterogeneity of these PS-bearing populations and the pathophysiological characteristics of patients with β-thalassemia. LM
PS
phosphatidylserine
RBC
red blood cell
FSC
forward scatter
SSC
side scatter
RMPs
red blood cell–derived microparticles
Hb
hemoglobin
CD41a-APC
allophycocyanin-conjugated cluster of differentiation 41a
annexin V–FITC
fluorescein isothiocyanate-conjugated annexin V
CD
cluster of differentiation
K2EDTA
dipotassium ethylenediaminetetraacetic acid
5 diff
5-part differential
PBS
phosphate-buffered saline
FITC
fluorescein isothiocyanate
PE
phycoerythrin
APC
antigen-presenting cell
FL
fluorescence
CS&T
Cytometer Setup &Tracking
SE
standard error
AU
arbitrary unit
RDW
red blood cell distribution width
RE
reticuloendothelial
We thank the Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand for supporting this research project. This project was also supported by Thailand Research Fund (TRF) to KP.
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