摘要
目的建立荧光显微镜自动化检测血液残留白细胞的方法。方法用不同浓度的残留白细胞[(1—150)个/μL]对LEICA-DM6000B荧光显微镜做放大倍数、荧光试剂配方、样品与试剂比例等参数优化及精密度试验,以ADAM白细胞计数仪做参比配对验证准确度试验。结果 LEICA-DM6000B荧光显微镜放大倍数选100倍形成88个图谱保存电脑;荧光试剂配方:碘化丙啶(PI)含量4 mg%(W/V),吐温含量0.60%(V/V);红细胞样品与荧光试剂比例为1∶14,曝光时间选择591 ms;血小板样品与荧光试剂比例为1∶4,曝光时间选择329 ms;红细胞样品与荧光试剂混合后取样量100μL;加入Nageotte计数板上静置时间选择20 min;Z轴调节上下范围(97±40)μm;避光保存2年的试剂检测残留白细胞值无明显变化(P>0.05);红细胞和血小板样品均为残留白细胞精密度<15%;LEICA-DM6000B荧光显微镜与ADAM计数仪配对检测残留白细胞相关性:红细胞样品为y=0.978 9x+0.381 6,r=0.9951(P>0.05),血小板样品为Y=0.985 3X+0.494 2,r=0.996 9(P>0.05)。结论采用荧光显微镜自动检测白细胞计数,准确、省工省时、检测费用低,克服了人为差错,并实现数据图像存档,有助于减少临床输血不良反应,保证临床输血的有效性及安全性。
Objective To establish an automatic method for the determination of residual WBCs in blood by fluorescence microscope.Methods LEICA-DM6000 B fluorescence microscope was optimized by microscope magnification, fluorescence reagent formula, sample-to-reagent ratio, and precision test with residual WBCs at different concentrations(a range of 1-150 WBC/μL). ADAM WBC counter was used as a reference test to verify the accuracy in paralle. Results The image was magnified 100 diameters by LEICA-DM6000 B fluorescence microscope to form 88 chromatograms for computer storage. Fluorescence reagent formula was as follows: propidium iodide(PI) content of 4 mg%(W/V), Triton content of 0.60%(V/V). RBC sample was diluted 1∶14 with fluorescent reagent, and exposed for 591 ms;platelet sample was diluted 1∶4 with fluorescent reagent, and exposed for 329 ms.The mixture was sampled at 100μL, then transferred to the Nageotte chamber and allowed to settle for 20 min.When the vision was magnified 100 diameters, the entire range of Z-axis adjustment was(97±40)μm. As to the stability of reagent, no difference in the determination values of residual WBC was observed between the one-and two-year reagent kept in dark(P > 0.05).The accuracy of residual WBC in RBC and platelets samples was of less than 15%. The comparison of LEICA-DM6000 B fluorescence microscope and ADAM counter for determination of residual WBC in RBCs and platelets demonstrated a correlation of r=0.995 1 and 0.996 9,respectilvely, between these two methods. Conclusion Residual WBC can be accurately counted using LEICA fluorescence microscopy. It represents a objetive, effificient and cost-effective method and achieves data and image archiving, and it is helpful to decrease the risk of adverse reactions after blood transfusion and further ensure the efficacy and safety of clinical blood transfusion.
作者
王珺
王兆福
程四国
刘玉振
王媛
WANG Jun;WANG Zhaofu;CHENG Siguo;LIU Yuzhen;WANG Yuan(Henan Province Red Cross Blood Center,Zhengzhou 450012,China)
出处
《中国输血杂志》
CAS
2019年第2期158-162,共5页
Chinese Journal of Blood Transfusion
基金
河南省医学科技攻关计划项目(201403228)
关键词
残留白细胞
荧光显微镜
碘化丙啶
吐温
自动化检测
residual white blood cells
fluorescence
microscope
propidine iodide
Trion
automatic detection
