不同流式细胞分析系统对外周血淋巴细胞亚群分析结果的影响

目的 探讨部分临床实验室FCM淋巴细胞亚群分析参考范围应用的合理性及不同生产厂家的流式细胞仪和试剂组合对淋巴细胞亚群分析结果的影响.方法 根据国内临床实验室常用的3个流式细胞仪型号(Beckman Coulter Epics XL、Beckman Coulter Cytomics FC500、BD FACS Calibur),分别选取3家北京地区临床流式细胞室(A、B、c室),按照各室的实际检测方案分别测定50份健康人静脉血标本,以验证各室淋巴细胞亚群分析参考范围是否合理.调查3家实验室室内全血质控品使用情况,并将商品化全血质控品分发各室,按照各自的实际实验方案在20个工作日内与常规标本平行处理、检测和分析.针对不同生产厂家的试剂,在同一流式细胞仪(型号为Beckman Coulter Epics XL)上用a、b、c、d 4种不同的试剂组合对20份患者标本进行检测,其中试剂组合a为美国Beckman Coulter公司同厂配套试剂和仪器,试剂组合b、c、d的检测结果分别与试剂组合a比较,计算b、c、d试剂组合偏倚>10%的概率.采用相同试剂和溶血素(美国Beckman Coulter公司)对24份患者标本进行前处理,分别在2台不同厂家和型号的流式细胞仪(型号为Beckman Coulter Epics XL和BD FACS Calibur)上检测,比较相同试剂处理标本后不同仪器对淋巴细胞亚群分析结果的影响.采用同厂配套试剂和仪器,比较Beckman Coulter Epics XL和BD FACS Calibur两个流式细胞检测系统对20份患者标本检测结果的影响.结果 A室的自然杀伤(NK)细胞及CD+4 T淋巴细胞/CD+8 T淋巴细胞(T4/T8),B、c两室的T4均有大于10%的结果落在相应的参考范围之外,超出相应参考范围的概率分别为16%(9/50)、24%(12/50)、22%(11/50)、12%(6/50).3家实验室20个工作日内的室内质控均在参考范围内.与试剂组合a比较,试剂组合b、c的所有项目偏倚均较大,其中偏倚>10%的概率最低为试剂组合b的T8,为70%(14/20);最高为试剂组合b、c的T淋巴细胞(T3)、T4,均达到100%(20/20).试剂组合d的T3、T8和B淋巴细胞(B)偏倚较大,偏倚>10%的概率分别为35%(7/20)、85%(17/20)、75%(15/20).不同生产厂家的试剂、仪器处理和分析标本的结果,与采用同一生产厂家的试剂、仪器处理和分析的结果相比,T3、T4、T8、B、NK均存在较大偏倚,偏倚>10%的概率分别为71%(17/24)、80%(19/24)、38%(9/24)、33%(8/24)、92%(22/24).Beckman Coulter Epics XL和BD FACS Calibur两个流式细胞检测系统相比较,T8、NK和B的偏倚均较大,偏倚>10%的概率分别为55%(11/20)、70%(14/20)、55%(11/20).结论 流式细胞实验室需要建立自己的参考范围并定期验证,以便合理进行调整.建议定期采用全血质控品,并累计质控数据.各实验室应选择同厂配套试剂处理标本.

Abstract：

Objective To investigate the appropriate setting up of normal reference ranges of lymphocyte subsets in some flow cytometry laboratories and to study the effects of different flow cytometers and various reagents by different manufacturers on the analysis of peripheral blood lymphocyte subsets. Methods Three FCM labs (named A, B and C) in Beijing region were selected representing 3 commonly used flow cytometers (Beckman Coulter Epics XL, Beckman Coulter Cytomics FC500, BD FACS Calibur). 50 samples from healthy donors were distributed to 3 labs and tested according to individual lab's standard operating procedure to verify whether the normal reference ranges of peripheral blood lymphocyte subsets established were appropriate. The application of internal quality control was also investigated. Commercial blood quality control reagents were given to the 3 FCM labs and tested within 20 working days paralleled with routine samples. In addition, 20 patients' samples were prepared using 4 different combinations of reagents ( a , b , c and d). The results from combination a, which used the Beckman Coulter reagents and instrument, were compared to the results from combination b, c and d, which used reagents from different manufacturers. Then the prepared samples were tested on Beckman Coulter Epics XL to evaluate the effects of different combinations of reagents on the results of peripheral blood lymphocyte subsets analyzed by the same instrument. Furthermore, 24 patients' samples prepared by same reagents from Beckman Coulter company were tested on both Beckman Coulter Epics XL and BD FACS Calibur respectively to assess the effects of different instruments on peripheral blood lymphocyte subsets. 20 patients' samples prepared by same reagents and instruments were analyzed by Beckman Coulter Epics XL analytic system and BD FACS Calibur analytic system respectively to assess the effects of the two analytic systems on the lymphocyte subsets. Results Over 10% of the results for NK and T4/T8 in lab A as well as T4 in labs B and C fell outside of their normal reference ranges. The probabilities exceeding corresponding normal reference ranges were 16% ( 9/50 ), 24% ( 12/50 ), 22% (11/50) and 12% ( 6/50 ), respectively. The results using internal blood quality control in 3 FCM labs within 20 working days were all within the reference ranges of the quality control provided by the kit. The biases from b and c reagent combinations were substantial compared with that of reagent a combination. Among the biases from b and c reagent combinations, the lowest probability of bias exceeding 10% was T8 of combination b, which had probability of 70% (14/20). The highest probabilities of hias exceeding 10% were T3 and T4 of b and c reagent combinations, which reached 100% (20/20) . Furthermore, the biases of T3, T8 and B of d reagent combination compared with that of reagent a combination were also substantial. The probabilities of bias exceeding 10% were 35% (7/20) ,85% (17/20) and 75% (15/20), respectively. Comparing the results of samples prepared and analyzed by reagents and instruments from different manufacturers to that of samples prepared and analyzed by the same company's reagents and instruments showed that there were great discrepancies in T3, T4 , T8 , B and NK. The probabilities of bias exceeding 10% were 71% ( 17/24), 80% (19/24) ,38% (9/24), 33% (8/24) and 92% (22/24), respectively. The biases of T8, NK and B were substantial when compared the results from Beckman Coulter Epics XL analytic systems and BD FACS Calibur analytic systems. The probabilities of bias exceeding 10% were 55% (11/20 ), 70% ( 14/20 ) and 55% (11/20), respectively. Conclusions FCM labs should set up their own normal reference range for peripheral blood lymphocyte subsets. The normal reference range should be verified periodically. It is important to apply internal blood quality control regularly and accumulate the quality control results. The reagents and instrument for preparing peripheral blood samples should be from the same manufacturers.     

透析患者血清HBsAg假阳性伴HIV可疑一例分析

HBsAg和HIV抗体的检测分别在HBV感染和HIV感染的诊疗中具有重要意义.现阶段HBsAg和HIV抗体主要通过血清免疫学方法检测,其中ELISA法检测HBsAg或HIV抗体的假阳性病例时有报道,而全自动化学发光免疫分析仪的假阳性病例较为少见.该文即报道了一例用全自动化学发光免疫分析仪检测透析患者血清HBsAg假阳性伴HIV抗体可疑的病例,并对可能的原因进行了深入探讨.     

糖化血红蛋白浓度的短期生物学变异

  目的  研究糖化血红蛋白(glycated hemoglobin-A1c, HbA_1c)浓度短期内的生物学变异。  方法  30名健康志愿者在第1天、第3天和第5天的上午8:00、中午12:00和下午4:00分别采集静脉血, 进行HbA_1c浓度的测定。分析计算HbA_1c浓度的个体内变异(within-subject coefficient of variations, CV_I)、个体间变异(between-subject coefficient of variations, CV_G)、分析变异(analytical coefficient of variations, CV_A)、个体指数(index of individuality, II)和参考变化值(reference change value, RCV)。  结果  总体、女性及男性CV_I分别为1.39%、1.49%和1.32%;CV_G分别为2.65%、2.33%和2.90%;CV_A均为0.6%;II分别为0.5、0.6和0.5;95% RCV分别为4.22%、4.45%和4.02%;99% RCV分别为5.19%、5.86%和5.29%。HbA_1c上午8:00、中午12:00和下午4:00的日内变异差异无统计学意义(P=0.28);第1天、第3天和第5天的周内日间变异差异无统计学意义(P=0.31);男女性别间差异亦无统计学意义(P=0.18)。  结论  HbA_1c浓度的日内和周内日间生物学变异很小, 结果相对稳定, 男性和女性之间亦无明显差异。     

造血发育调控基因在白血病中的研究进展

正常造血干细胞(HSC)具有自我更新和多向分化的潜能,能在体内重建造血系统.而在病理状态下,当HSC不断地增殖但分化受阻时,就会形成白血病干细胞(LSC),从而促进和维持了白血病的发生[1-2].     

信号转导淋巴细胞激活分子家族的研究进展

人造血干细胞(HSC)和肿瘤干细胞标志物的探寻一直是造血干细胞移植监测、治疗及临床研究关注的焦点,而最近报道的新标志物--信号转导淋巴细胞激活分子(SLAM)家族和侧群(side population,SP)细胞极大地推进了干细胞临床和基础研究.     

胆囊炎证治

胆囊炎早期:可见右胁下时常作痛,伴见舌苔糙垢根厚、质红且干,脉多弦滑而数,大便干结,小溲赤少。此为胆热郁滞,气机不畅,治宜清泻胆热,疏调气机,以缓疼痛。处方:柴胡6克,黄芩10     

RNA干扰技术抑制急性白血病细胞THP-1中SALL4基因表达的研究

目的 抑制SALL4基因在急性白血病细胞THP-1中的表达,探讨其在白血病发病机制中的作用.方法 将表达SALL4干扰序列的质粒载体转染至急性白血病细胞THP-1中,分为4组:(1)空白对照组:不加处理;(2)转染对照组:加入空pRS质粒载体;(3)转染一组:加入SALL4-shRNA-pRS-1干扰质粒的转染复合体;(4)转染二组:加入SALL4-shRNA-pRS-2干扰质粒的转染复合体.采用实时荧光定量PCR和WB技术观察THP-1细胞中SALL4基因mRNA及蛋白表达的变化,建立抑制SALL4表达的体系.实时荧光定量PCR检测SALL4受抑后Wnt/β-catenin信号通路中C-myc、Cyclin D1、β-catenin基因的表达改变,采用流式细胞术分析THP-1细胞凋亡情况.结果 实时荧光定量PCR结果 显示,转染一组、转染二组、转染对照组、空白对照组SALL4基因的表达水平分别为(36.0±4.3)%、(32.0±2.4)%、(102.0±6.5)%、(100.0±2.6)%,差异有统计学意义(F=226.3,P<0.05);转染一组、转染二组的SALL4基因表达水平显著低于空白对照组,差异有统计学意义(t值分别为19.7、19.1,P<0.05);转染对照组SALL4基因表达水平与空白对照组比较差异无统计学意义(t=1.1,P＞0.05).WB检测结果 显示,转染一组、转染二组SALL4蛋白表达与空白对照组和转染对照组相比明显下降.以上基因和蛋白表达水平均提示SALL4干扰效率良好.SALL4基因抑制表达后,转染一组C-myc基因、β-catenin基因、Cyclin D1基因的表达分别为(44.0±6.2)%、(44.0±5.1)%和(107.0±13.6)%,转染二组为(22.0±4.5)%、(25.0±3.5)%和(48.0±7.6)%,转染对照组为(42.0±3.5)%、(59.0±3.7)%及(79.0±5.6)%.转染一组、转染二组C-myc基因表达水平显著低于空白对照组的(103.0±7.5)%,差异有统计学意义(t值分别为10.1、9.5,P均<0.05);转染一组、转染二组β-catenin基因的表达显著低于空白对照组的(100.0±4.1)%,差异有统计学意义(t值分别为23.3、22.9,P均<0.05);转染一组、转染二组Cyclin D1基因的表达显著低于空白对照组的(102.0±6.0)%,差异有统计学意义(t值分别为17.4、12.4,P均<0.05).SALL4基因被抑制后,转染一组、转染二组、转染对照组及空白对照组的细胞凋亡率分别为(57.2±9.1)%、(34.4±8.6)%、(14.4±3.6)%及(14.8±4.8)%,差异有统计学意义(F=42.5,P<0.05).转染一组、转染二组细胞凋亡率显著高于空白对照组(t值分别为9.7、4.5,P均<0.05).结论 抑制急性白血病细胞THP-1中SALL4基因表达后,Wnt/β-catenin信号通路的C-myc、Cyclin D1及β-catenin基因的表达随之下调,并促进了细胞凋亡.     

全自动尿沉渣分析仪的临床应用评价

  目的  对一种全自动尿沉渣分析仪的临床应用进行评价, 并建立其对尿液有形成分分析的正常参考范围。  方法  从仪器精密度、携带污染率、分析测量范围以及与人工镜检的可比性4个方面进行评价, 同时建立仪器检测红细胞(red blood cell, RBC)、白细胞(white blood cell, WBC)、鳞状上皮细胞(squamous epithelial cell, EC)及管型的正常参考范围。  结果  RBC低值、中值和高值精密度的变异系数分别为129.10%、20.59%和7.80%;WBC低值、中值和高值精密度的变异系数分别为65.73%、14.30%和13.00%;EC中值和高值精密度的变异系数分别为27.01%和21.46%。RBC、WBC和EC的携带污染率均为0;RBC分析测量范围为0~83 608个/μl, WBC分析测量范围为0~15 624个/μl, 细菌分析测量范围根据菌种不同而结果有明显差异。仪器与人工镜检相比, RBC、WBC和EC的一致率分别为82.80%、73.82%和57.56%。男性尿液有形成分的参考范围显示, RBC为0~8个/μl, WBC为0~12个/μl, EC为0~13个/μl, 管型为0~4个/μl, 结晶为0~34个/μl; 女性尿液有形成分的参考范围显示, RBC为0~12个/μl, WBC为0~21个/μl, EC为0~42个/μl, 管型为0~4个/μl, 结晶为0~2个/μl。  结论  该全自动尿沉渣分析仪对尿中的RBC和WBC有较好的识别性能, 对结晶、管型、真菌、异形细胞等难以自动识别的成分, 可利用仪器的审核功能进行人工识别, 必要时进行人工复检, 为临床提供可靠的检验报告。