Red cells exchanges in sickle cells disease lead to a selective reduction of erythrocytes‐derived blood microparticles (2011)

Red cell exchange (RCE) is a highly effective procedure in the management of complications of sickle cell disease (SCD) (Swerdlow, 2006). During acute vaso-occlusive complications, RCE permits both the input of normal red cells and the removal of sickle cells without increasing blood viscosity and is a recommended part of the initial treatment of vaso-occlusive stroke in children (National Institutes of Health, National Heart, Lung and Blood Institute, 2002). RCE can also be used as a long-term therapy to maintain a low level of HbS in primary and secondary stroke prevention (Platt, 2006), with the major advantage of avoiding the risk of iron overload observed with simple transfusions (Kim et al, 1994). Microparticles (MPs) are small vesicles released from cells after activation or apoptosis (Piccin et al, 2007). In SCD, their total number is robustly increased at steady state or during crisis (Shet et al, 2003; van Beers et al, 2009; van Tits et al, 2009). Of interest, the number of erythrocyte-derived MPs is particularly high in SCD. These MPs seem to play a specific role in the activation of coagulation and thrombin generation and, consequently, could participate in the global pro-coagulant state observed in this pathology (Horne et al, 2006; van Beers et al, 2009). This study aimed to compare the level of total, platelets-derived and erythrocytes-derived MPs before and after RCE in SCD patients. After informed consent, 37 blood samples were collected from 27 patients with SS genotype undergoing a long-term RCE program using apheresis. Two patients underwent RCE before surgery; all the others were included in a RCE program for pregnancy (n = 2), recurrent vaso occlusive complications (n = 7) or chronic complications, such as leg ulcers or vasculopathy (n = 16). For each patient, a blood sample was drawn before and just after the exchange. Samples from donors without neither anaemia nor coagulation disorders were used as controls (n = 19). MPs were isolated with a protocol with no pre-analytic artifacts related to any ultracentrifugation or filtration step (Robert et al, 2009). MPs were isolated from blood after two centrifugation steps (1500 g during 15 mn and 13000 g during 2 mn). Samples were stored as 200 μl aliquots and immediately frozen at −80°C. A method of standardization has been published for the detection and quantification of platelets-derived MPs, based on the use of calibrated beads that clearly identify MP forward and sideways scatter (FSC/SSC) gate within a diameter of 0·5–1 μm (Robert et al, 2009). As we wished to quantify both platelets-derived and erythrocytes-derived MPs in the same sample, we applied this method using triple-labelling, combining Annexin V staining in the FL1 channel with GPA-phycoerythrin (PE) and a CD41-allophycocyanin (APC) staining. Briefly, 38 μl of platelet-free plasma (PFP) was labelled with 5 μl 10 × Annexin V binding buffer, 1 μl Annexin V-fluorescein isothiocyanate (FITC; Beckman Coulter, Brea, CA, USA), 1 μl anti-Glycophorin A-PE (CalTag, Burlingame, CA, USA) and 5 μl anti-CD41-APC (BD Biosciences, San Jose, CA, USA). For MP quantification, 5 μl of APC-calibrated beads (BD Biosciences) at known concentration (Cc beads) were diluted in 345 μl of 1 × Annexin buffer and added to samples just before acquisition of 1000 events on a MoFlo Legacy cell sorter (Beckman Coulter). The concentration (Cc) of MPs was estimated using the following formula: Cc MPs = (absolute number of events in the MPs gate * Cc beads)/(38* 1000). The analysis gate was first defined on a SSC/FSC scattergram using the Megamix beads system (ByoCytex, Marseille, France) as previously described (Robert et al, 2009); MPs were defined as all Annexin-V positive events inside that gate (Fig 1). The mean number of total MPs was clearly higher in SCD patients than in controls, confirming the significant increase in the total number of circulating MPs described previously (Shet et al, 2003; van Beers et al, 2009; van Tits et al, 2009). This was true both before (2208 vs. 273 MPs/μl; Student test: P = 0·0002) and after the exchange (2313 vs. 273 MPs/μl; Student test: P = 0·0008) (Fig 2A). Of note, the steady state MP number in our patients was probably underestimated as the majority of them still had some exogenous circulating red cells from a former exchange at the time of testing. In agreement with a recent study (van Beers et al, 2009), most of the circulating MPs in SCD were of platelet origin and both the CD41+ (1843 vs. 256 MPs/μl; Student test: P = 0·001) and the GPA+-MPs (281 vs. 8 MPs/μl; Student test: P = 0·003) were increased in comparison with controls (Fig 2B, C). There were no significant differences between MP numbers before and after RCE in either the number of total MPs (2208 vs. 2314 MPs/μl; Student test, P = 0·89, Fig 2A), or the number of platelets-derived MPs (1843 vs. 1807 CD41+-MPs/μl; Student test, P = 0·9, Fig 2B). In contrast, there was a statistically significant reduction in the number of erythrocytes-derived MPs after RCE (101 vs. 282 GPA+-MPs/μl; Student test: P = 0·003, Fig 2C). The mechanism of this selective decrease is not clear, but more probably reflects the clearance from the circulation of red cells with a high HbS content, which may release more MPs than normal erythrocytes, rather than a consequence of the centrifugation step, which is too slow to discard MPs from the plasma. Whether this selective reduction of GPA+-MPs could contribute to the efficiency of RCE in primary or secondary prevention of stroke remains uncertain, but a recent study focused on the specific pro-coagulant role of the erythrocytes-derived MPs in SCD (van Beers et al, 2009). In that study, the number of GPA+-MPs correlated with in vivo markers of coagulation, in contrast to the total and platelets-derived MPs. However, this correlation was not confirmed in the present study, using a thrombin generation assay (data not shown). This may be due to the fact that the activation of coagulation and thrombin generation may require a certain ‘threshold’ of phopshatidylserine exposure, which may be reached only during acute crisis and not in patients undergoing RCE as a preventive therapy. Flow cytometric detection and quantification of MPs: Representative dot plots obtained on the MoFlow cytometer used for the detection and quantification of MPs: The SSC/FSC MP gate was determined using the calibrated Megamix beads (upper left). The samples were analysed in that predefined gate, and a known number of APC-stained calibrated beads with a diameter of 3 μm (size larger than the MPs) was added in each sample for quantification (upper right). MPs were defined as the Annexin V+-events in that FSC/SSC gate (lower right). Origin of the MPs was defined according to the GPA and the CD41 expression in that Annexin V+ gate (lower left). Quantification of circulating MPs before and after RCE in SCD patients: Histograms representing the number of total (2a), platelets-derived (2b) and erythrocytes-derived MPs (2c) in controls and SCD patients just before and after RCE. In conclusion, using a triple Annexin V/CD41/GPA staining that enabled us to detect and quantify both platelets-derived and erythrocytes-derived MPs in the same sample, we confirmed that the MPs from both origins are clearly increased in SCD patients. Moreover, we observed a selective reduction in the number of erythrocytes-derived MPs after RCE, while the number of platelets-derived MPs and total MPs remained unchanged. This selective reduction of erythrocytes-derived MPs could contribute to the benefit of this procedure as a prevention of stroke in SCD patients. This work was supported by a grant from the Institut National de la Santé et de la Recherche Médicale (INSERM, France) and from the Direction Générale de la Recherche Scientifique (DGRS, Tunisia). EM, YL and CF performed the research. SA read and corrected the paper. FD was in charge of the patients and performed RCE. LG designed the study and wrote the paper. We are grateful to Shwetha Manjunath for correcting the English language. Competing interests: the authors have no competing interests.