Erythocytapheresis is a highly effective procedure used in the management of both acute and chronic complications of sickle cell disease (SCD). Indeed, exchange transfusion is recommended as a part of the initial treatment of vaso-occlusive stroke in children. Red cells exchange can also be proposed in primary and secondary stroke prevention in order to maintain a low level of HbS. Microparticles (MPs) are small vesicles released from cells after activation or apoptosis. Microparticles number is increased in SCD both at the steady state and during crisis. The majority of these microparticles are originated from erythrocytes and platelets. In SCD, erythrocytes MPs are strongly correlated with hemolysis and activation of coagulation.
In this study, we aimed to measure the level of total, platelets-derived and erythrocytes-derived MPs in patients with SCD undergoing erythrocytapheresis; we compared the level of MPs before and after red cells exchange in order to evaluate if the reduction of stroke observed in patients undergoing erythrocytapheresis could be due to a specific decrease in erythrocytes-derived MPs known to be implicated in coagulation activation in this disease.
After informed consent, 24 samples were harvested on a tube containing buffered sodium citrate from 22 patients with SCD undergoing erythrocytapheresis. For each patient, a sample was collected before and just after the exchange. 20 samples from donor with neither anemia nor coagulation disorders were used as controls. Each sample was centrifugated at 1500g during 15 mn, the supernatant was collected and centrifugated 2mn at 13000g at 20°C and then frozen at -80°C until analysis. For MPs detection, 38mL of the thawed supernatant was labelled with 5mL of 10X Annexine V buffer, 1mL of Annexine V-FITC, 1 mL of anti-GPA-PE and 5 mL of anti-CD41-APC. 5mL of calibrated beads at known concentration were added to 345 mL of 1X Annexine buffer just before analysis on the MoFlow cytometer (Beckman Coulter). The Megamix beads system was used in order to delimitate the FSC/SSC gate of MPs.
Total number of plasma MPs (1755/mL vs 273), erythrocytes-derived MPs (174/mL vs 8) and platelets-derived MPs (1520/mL vs 236) were significantly increased in SCD patients in comparison with controls. For SCD patients undergoing erythrocytapheresis, the mean HbS level was respectively 52% and 24% before and after red cells exchange. No significant difference in the total hemoglobin concentration was noted. The mean number of MPs did not differ before and after the exchange (1755/mL versus 1940/mL respectively, student test p>0.05). The mean number of platelets-derived MPs remained stable before and after the exchange (1520/mL versus 1250mL, student test, p>0.05). In sharp contrast, we observed a significant clearance of the erythrocytes-derived MPs in the plasma of SCD patients after red cells exchange (mean number of GPA+-MPs: 174/mL before exchange versus 77mL after, student test, p<0.05). However, we could also observe a great variation of the number of erythrocytes and platelets-derived MPs between the different patients.
The number of erythrocytes-derived MPs decreased specifically after red cells exchange, in contrast with the total and the platelets-derived MPs which remained stable; this selective decrease in the GPA+-MPs of SCD patients could be due to a reduction in the total number of red cells containing a high HbS content in the circulation, because of their replacement by normal erythrocytes that have a far lower capacity to produce MPs. It has been recently shown that in SCD, GPA+-MPs are specifically correlated with coagulation and fibrinolysis activation, underlying the fact that a direct link exists between hemolysis, erythrocytes- derived MPs liberation and procoagulant state. The specific clearance of the GPA+-MPs after red cells exchange could partially explain the positive effects of this procedure in primary and secondary prevention of stroke in SCD. However, that GPA+-MPs remained statistically increased after red cells exchange in comparison with controls; whether a low number of post-exchange MPs could be correlated with a better outcome is actually under investigation.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.