Dual Assessment of the RBC Fraction Abnormally Retaining Mitochondria and of the RBC Mitochondrial Load Provides a New Clinical Parameter for Sickle Cell Disease Patients.

BACKGROUND

Mitochondria and other organelles are normally eliminated in a process called mitophagy during the maturation of hematopoietic precursors, leading to the release of enucleated red blood cells (RBCs) in circulation. In sickle cell disease (SCD), a significant fraction of the RBCs of patients abnormally retain mitochondria. This process increases the oxygen consumption rate and formation of reactive oxygen species, augmenting known pathways of hemolysis and playing a significant role in SCD pathophysiology. The retention of mitochondria in RBC is detectable by flow cytometry analysis of whole blood, but this approach does not quantify the number of mitochondria in individual cells.

METHODS

Mitochondrial DNA was isolated from sorted RBC (10 cells) of a cohort of pediatric sickle patients (HbSS, = 19; HbSC/HbSE, = 8) and quantified by qPCR with a standard curve.

RESULTS

The methodology is suitable for the clinical quantification of the severity of mitochondrial retention in RBC of individuals with SCD. The number of mitochondria in RBCs are obtained from quantification of the copy numbers of mtDNA = 21 copies/total RBC) and the mitochondria positive RBCs fraction is determined by flow cytometry ( = 11%), which provides the number of mitochondria present in the population of RBCs retaining mitochondria ( = 400 copies/positive RBC).

CONCLUSION

With this approach, future therapies that circumvent mitophagy defects could be assessed for their efficacy in reducing both the size of the RBC fraction retaining mitochondria and the mitochondrial load in each cell.