Study on the elimination of leukocyte interference with apheresis platelet RNA assay

Abstract

Objective To investigate the effect of a small amount of leukocyte mixed in platelets on platelet RNA related study, and to further propose a method to remove leukocyte contamination. Methods The collected platelets were divided into groups and then processed. 1) Control group: Cells were collected by direct centrifugation; 2) Centrifuge purification group: Centrifuge with different centrifugal forces to collect precipitated cells and recover suspended cells; 3) Magnetic bead purification group: Remove CD45 positive leukocyte from platelets using magnetic bead method, and recover CD45 negative platelet cells. Total RNA was extracted from the cells, and cDNA was reverse transcribed from the extracted RNA. The fragments of housekeeping gene β-actin, platelet marker ITGA2B and leukocyte marker PTPRC were PCR amplified using cDNA as a template. The results of the PCR amplification were used to determine the influence of leukocyte contamination in platelet mRNA study and the effect of centrifugation purification and magnetic bead purification. Results PTPRC gene could be detected in cDNA from platelet cells obtained by direct centrifugation. PTPRC gene was also detected in cell segments collected from both precipitated and suspended cells after centrifugation under different conditions. But PTPRC was not detected in platelet after removal of CD45 positive cells by magnetic bead. Conclusion A small amount of leukocyte contamination in platelets can interfere with the mRNA study of platelets. The simple centrifugation could not remove the leukocyte contamination in platelets, but the magnetic bead had a better purification effect.

Keywords

• apheresis platelets purification
• leukocyte contamination
• platelet rna