Study on the elimination of leukocyte interference with apheresis platelet RNA assay
10.13303/j.cjbt.issn.1004-549x.2024.10.013
- VernacularTitle:滤白机采血小板消除白细胞干扰血小板RNA实验的研究
- Author:
Fengqiu LIN
1
;
Xiaofeng LI
;
Jianping LI
Author Information
1. 辽宁省血液中心辽宁省血液安全研究重点实验室沈阳市血液安全研究重点实验室,辽宁沈阳 110044
- Keywords:
apheresis platelets purification;
leukocyte contamination;
platelet RNA
- From:
Chinese Journal of Blood Transfusion
2024;37(10):1164-1168
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of a small amount of leukocyte mixed in platelets on platelet RNA relat-ed study,and to further propose a method to remove leukocyte contamination.Methods The collected platelets were divid-ed into groups and then processed.1)Control group:Cells were collected by direct centrifugation;2)Centrifuge purifica-tion 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 u-sing cDNA as a template.The results of the PCR amplification were used to determine the influence of leukocyte contamina-tion 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.
