Encapsulation of ribonucleic acid in human red blood cells for use as a reticulocyte quality control material for flow cytometric analysis.
The osmotic lysis procedure was employed to encapsulate ribonucleic acid (RNA) in human red blood cells in order to prepare a reticulocyte reference control. The procedure required the hypotonic dialysis of erythrocytes in the presence of RNA and cytosolic components of red blood cells followed by a short hypertonic dialysis to restore isotonicity and reseal the pores formed on the cell membrane during the hypotonic swelling. The procedure was monitored by a dedicated flow cytometer for reticulocyte counting and required 120 min. Approximately 20% of the erythrocytes undergoing the reversible osmotic lysis were encapsulated with various amounts of RNA. The morphology of the RNA-loaded erythrocytes were similar to those of normal erythrocytes and reticulocytes, however, their mean cell volume (MCV) was slightly smaller than normal cells. RNA-loaded erythrocytes prepared by this method were stable for several months as a reference control for identification and enumeration of reticulocytes using flow cytometric as well as manual analysis methods and resulted in a high correlation coefficient between these counting techniques.
Ebrahim
A
A
Streck Laboratories, Inc., Omaha, Nebraska, USA.
Ryan
W L
WL
D002469:Cell Separation; D004912:Erythrocytes; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D009995:Osmosis; D011786:Quality Control; D012313:RNA; D012015:Reference Standards; D017701:Reticulocyte Count / Q000379:methods*; D012156:Reticulocytes
D012313:RNA