两步和三步法分离样本资源库抗凝血单个核细胞和血浆效果比较
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摘要
目的比较两步和三步法分离外周血单个核细胞(PBMC)和血浆的效果。方法对送至样本资源库的16例抗凝血标本分别采用两步法和三步法分离PBMC及血浆,并对2种不同处理方法的分离效果进行比较。两步法为将抗凝血加入淋巴细胞分离液,离心后分别分离PBMC和血浆;三步法则先离心分离血浆后再加入淋巴细胞分离液,离心后分离PBMC。结果 2种处理方法处理后检测的活细胞数、总细胞数及活细胞比例结果相近,差异均无统计学意义(P>0.05)。两步法血浆中细胞因子水平均低于三步法,其中25种细胞因子比较,差异均有统计学意义(P<0.05)。结论两步法省时,操作少,在分离PBMC方面效果与三步法类似;但两步法在分离血浆方面会影响血浆细胞因子水平。
Objective The quality of the specimens affects the subsequent scientific research,and the different separate methods significantly influence the specimen quality.We compared the effects of two different separate methods on the peripheral blood mononuclear cells(PBMC)and plasma.Methods 16 specimens of Biobank were separated respectively by two-step method and three-step method,and then compared the effect of the two different methods.Two-step method:add the anticoagulant blood specimen into the lymphocyte separation medium,then separate the peripheral blood mononuclear cells and plasma after centrifugation.Three-step method:first,separate plasma after centrifugation and dilute the rest specimen;then add the rest specimen into the lymphocyte separation medium;finally,separate PBMC by the second centrifugation.Results To separate PBMC:live cells,total cells and cell viability were similar between two-step method and three-step method,there were no statistical differences.To separate plasma:27 plasma cytokines were analysed,the level of plasma cytokines with two-step method were lower than that of three-step method,25 cytokines were statistically significant difference(P<0.05).Conclusion Compared with three-step method,twostep method has similar effect of separating PBMC with less time and less operation,but the two-step method could reduce the level of plasma cytokines.
Objective The quality of the specimens affects the subsequent scientific research,and the different separate methods significantly influence the specimen quality.We compared the effects of two different separate methods on the peripheral blood mononuclear cells(PBMC)and plasma.Methods 16 specimens of Biobank were separated respectively by two-step method and three-step method,and then compared the effect of the two different methods.Two-step method:add the anticoagulant blood specimen into the lymphocyte separation medium,then separate the peripheral blood mononuclear cells and plasma after centrifugation.Three-step method:first,separate plasma after centrifugation and dilute the rest specimen;then add the rest specimen into the lymphocyte separation medium;finally,separate PBMC by the second centrifugation.Results To separate PBMC:live cells,total cells and cell viability were similar between two-step method and three-step method,there were no statistical differences.To separate plasma:27 plasma cytokines were analysed,the level of plasma cytokines with two-step method were lower than that of three-step method,25 cytokines were statistically significant difference(P<0.05).Conclusion Compared with three-step method,twostep method has similar effect of separating PBMC with less time and less operation,but the two-step method could reduce the level of plasma cytokines.
引文
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[2] BUTLER D.Translational research:crossing the valley of death[J].Nature,2008,453(7197):840-842.
[3]李海燕,张雷,张雪娇,等.北京重大疾病临床数据和样本资源库建设成果初探[J].中华医学管理杂志,2013,29(11):863-865.
[4]李海燕,张雪娇,邵雪梅,等.疾病资源库建设的重要性及现状分析[J].中华医院管理杂志,2010,26(11):801-804.
[5]孙焕芹,刘梦佳,戴北宸,等.用转化医学理念指导样本资源库建设[J].北京医学,2012,34(6):495-496.
[6]孙坚萍,孙焕芹,刘宁,等.传染病样本资源库质量控制体系建设经验[J].中华医院管理杂志,2016,32(9):698-699.
[7] BOLEN C R,UDUMAN M,KLEINSTEIN S H.Cell subset prediction for blood genomic studies[J].BMC Bioinformatics,2011,12(6):258.
[8] WIECZOREK L,KREBS S J,KALYANARAMAN V,et al.Comparable antigenicity and immunogenicity of oligomeric forms of a novel,acute HIV-1subtype C gp145envelope for use in preclinical and clinical vaccine research[J].J Virol,2015,89(15):7478-7493.
[9] WETZEL K S,YI Y,ELLIOTT S T,et al.CXCR6-Mediated simian immunodeficiency virus SIVagmSab entry into sabaeus African green monkey lymphocytes implicates widespread use of Non-CCR5pathways in natural host infections[J].J Virol,2017,91(4):91.
[10]DONAHUE R N,LEPONE L M,GRENGA I,et al.Analyses of the peripheral immunome following multiple administrations of avelumab,a human IgG1 anti-PD-L1monoclonal antibody[J].J Immunother Cancer,2017,5(1):20.
[11]CHRET A,NEMBOT G,MLARD A,et al.Intensive five-drug antiretroviral therapy regimen versus standard triple-drug therapy during primary HIV-1infection(OPTIPRIM-ANRS 147):a randomised,open-label,phase 3trial[J].Lancet Infect Dis,2015,15(4):387-396.
[12]SIMEON-DUBACH D,PERREN A.Better provenance for biobank samples[J].Nature,2011,475(7357):454-455.
[13]郜恒骏.中国生物样本库向标准化迈进[J].实用器官移植电子杂志,2017,5(6):453.
[14]MALM L,TYBRING G,MORITZ T,et al.Metabolomic quality assessment of EDTA plasma and serum samples[J].Biopreserv Biobank,2016,14(5):416-423.
[15]GRIEVINK H W,LUISMAN T,KLUFT C,et al.Comparison of three isolation techniques for human peripheral blood mononuclear cells:cell recovery and viability,population composition,and cell functionality[J].Biopreserv Biobank,2016,14(5):410-415.