热变性对血细胞流式细胞术分析结果的影响

目的 初步探讨热变性对外周血细胞在流式细胞术分析结果中的影响,从而提高流式-荧光原位杂交（Flow-FISH）技术的应用.方法 收集5例非骨髓造血干细胞疾病患者肝素抗凝外周血标本,以CD45-Alexa Fluor（R）647标记细胞表面抗原,高温变性后,采用流式细胞术分析热变性前后外周血有核细胞的散射光信号和荧光信号变化.结果 热变性后,外周血粒细胞的侧向散射光明显缩小;单核细胞不易通过散射光被区分和设门.所有细胞CD45表达强度均减弱,以淋巴细胞为著;通过侧向散射光和CD45设门虽能大致区分各群细胞,但不及未热变性细胞清晰.结论 热变性后,外周血细胞在流式细胞术中的散射光信号和荧光信号均发生了变化,按照常规FSC/SSC和CD45/SSC方法设门进行细胞亚群分析的结果不精确,利用系列特异性荧光抗体标记是一种有前景的方法.

Effect of Heat Denaturation on Blood Cells Analyzed by Flow Cytometry

Objective To investigate the effect of heat denaturation on peripheral blood cells analyzed by flow cytometry and to improve the application of Flow cytometry-Fluorescence in situ hybridization （Flow-FISH）.Methods Heparinized peripheral blood were sampled from 5 patients without hematopoietic stem cell disease,isolated nucleated cells were labeled with CD45-Alexa Fluor （R） 647,then denatured by high temperature.The scatter signals and fluorescence signals were analyzed by flow cytometry,on cells with and without heat denaturation separately.Results After heat denaturation,the sideward light scatter of granulocytes became markedly smaller,and the monocytes were difficult to be gated.CD45 expression of all cells became weaker,especially the lymphocytes; and cell subgroups could be differentiated by CD45 and sideward light scatter,while it was not as clear as cells without heat denaturation.Conclusion Heat denaturation could change both light scatter signal and fluorescence intensity by flow cytometry,and the routine method to gate cell groups might be not precise.Therefore,fluorescence labeled lineage specific antibody may be useful method to apply in Flow-FISH.