中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2014年
8期
623-627
,共5页
张欢欢%张春燕%杨晋%郑萍%赵绍林%吴惠毅
張歡歡%張春燕%楊晉%鄭萍%趙紹林%吳惠毅
장환환%장춘연%양진%정평%조소림%오혜의
中性白细胞%细胞膜%碱性磷酸酶%流式细胞术%感染
中性白細胞%細胞膜%堿性燐痠酶%流式細胞術%感染
중성백세포%세포막%감성린산매%류식세포술%감염
Neutrophils%Cell membrane%Alkaline phosphatase%Flow cytometry%Infection
目的 建立流式细胞术检测中性粒细胞膜碱性磷酸酶(mNAP)表达.方法 方法学建立.采集研究对象乙二胺四乙酸二钾(EDTA-K2)抗凝静脉血,应用PE标记抗碱性磷酸酶单克隆抗体,建立流式细胞术检测外周血mNAP表达方法.运用BD公司QuantiBRITETM PE于流式细胞术评估每个细胞结合的抗体数(AB/c),间接反映待测细胞mNAP的表达量.观察肝素、乙二胺四乙酸二钾、柠檬酸钠3种抗凝剂、标本放置温度、时间以及血浆中碱性磷酸酶对结果的影响,评价方法精密度.用所建方法检测481名健康人群外周血mNAP表达水平,对数据进行正态性检验,确立临床参考区间.检测84例重症感染和局部感染患者和39例病毒感染患者外周血mNAP表达水平.结果 标本检测前,应用磷酸盐缓冲液洗涤可以有效消除血浆中碱性磷酸酶(ALP)干扰.3种不同抗凝剂采集的标本对mNAP检测结果无影响.标本放置室温或4℃12 h内对检测结果无影响.精密度实验结果,批内变异系数(CV) 2.01％ ～3.33％,平均CV 2.67％;批间CV 5.80％ ～6.00％,平均CV5.90％.健康成年人无论男女,mNAP表达水平均呈非正态分布.男、女中位数(P25,P75)分别为1 758(1 378 ～2 310) AB/c、1 897(1 369 ～3 249) AB/c,性别之间差异无统计学意义(U=27 140,P=0.243 8).以2.5％ ～97.5％位数作为临床参考区间,结果为920.5 ～3 493.0 AB/c.细菌感染患者外周血中mNAP表达水平13 532(9 756 ～ 16 869) AB/c高于病毒感染患者1 143(536～2 012)AB/c以及健康对照人群1 879(1 399 ～2 497) AB/c(H=221.5,P＜0.01).结论 应用QuantiBRITETM PE试剂盒来标准化流式细胞仪的设置,可定量分析待测样品荧光强度,进而获得细胞所含荧光素分子数,并计算出每个细胞表达的抗原位点或抗原分子数目,从而达到定量检测的目的.应用流式细胞术检测外周血中mNAP,操作简便、快速、结果准确,mNAP表达对于鉴别细菌、病毒感染具有重要的临床价值.
目的 建立流式細胞術檢測中性粒細胞膜堿性燐痠酶(mNAP)錶達.方法 方法學建立.採集研究對象乙二胺四乙痠二鉀(EDTA-K2)抗凝靜脈血,應用PE標記抗堿性燐痠酶單剋隆抗體,建立流式細胞術檢測外週血mNAP錶達方法.運用BD公司QuantiBRITETM PE于流式細胞術評估每箇細胞結閤的抗體數(AB/c),間接反映待測細胞mNAP的錶達量.觀察肝素、乙二胺四乙痠二鉀、檸檬痠鈉3種抗凝劑、標本放置溫度、時間以及血漿中堿性燐痠酶對結果的影響,評價方法精密度.用所建方法檢測481名健康人群外週血mNAP錶達水平,對數據進行正態性檢驗,確立臨床參攷區間.檢測84例重癥感染和跼部感染患者和39例病毒感染患者外週血mNAP錶達水平.結果 標本檢測前,應用燐痠鹽緩遲液洗滌可以有效消除血漿中堿性燐痠酶(ALP)榦擾.3種不同抗凝劑採集的標本對mNAP檢測結果無影響.標本放置室溫或4℃12 h內對檢測結果無影響.精密度實驗結果,批內變異繫數(CV) 2.01％ ～3.33％,平均CV 2.67％;批間CV 5.80％ ～6.00％,平均CV5.90％.健康成年人無論男女,mNAP錶達水平均呈非正態分佈.男、女中位數(P25,P75)分彆為1 758(1 378 ～2 310) AB/c、1 897(1 369 ～3 249) AB/c,性彆之間差異無統計學意義(U=27 140,P=0.243 8).以2.5％ ～97.5％位數作為臨床參攷區間,結果為920.5 ～3 493.0 AB/c.細菌感染患者外週血中mNAP錶達水平13 532(9 756 ～ 16 869) AB/c高于病毒感染患者1 143(536～2 012)AB/c以及健康對照人群1 879(1 399 ～2 497) AB/c(H=221.5,P＜0.01).結論 應用QuantiBRITETM PE試劑盒來標準化流式細胞儀的設置,可定量分析待測樣品熒光彊度,進而穫得細胞所含熒光素分子數,併計算齣每箇細胞錶達的抗原位點或抗原分子數目,從而達到定量檢測的目的.應用流式細胞術檢測外週血中mNAP,操作簡便、快速、結果準確,mNAP錶達對于鑒彆細菌、病毒感染具有重要的臨床價值.
목적 건립류식세포술검측중성립세포막감성린산매(mNAP)표체.방법 방법학건립.채집연구대상을이알사을산이갑(EDTA-K2)항응정맥혈,응용PE표기항감성린산매단극륭항체,건립류식세포술검측외주혈mNAP표체방법.운용BD공사QuantiBRITETM PE우류식세포술평고매개세포결합적항체수(AB/c),간접반영대측세포mNAP적표체량.관찰간소、을이알사을산이갑、저몽산납3충항응제、표본방치온도、시간이급혈장중감성린산매대결과적영향,평개방법정밀도.용소건방법검측481명건강인군외주혈mNAP표체수평,대수거진행정태성검험,학립림상삼고구간.검측84례중증감염화국부감염환자화39례병독감염환자외주혈mNAP표체수평.결과 표본검측전,응용린산염완충액세조가이유효소제혈장중감성린산매(ALP)간우.3충불동항응제채집적표본대mNAP검측결과무영향.표본방치실온혹4℃12 h내대검측결과무영향.정밀도실험결과,비내변이계수(CV) 2.01％ ～3.33％,평균CV 2.67％;비간CV 5.80％ ～6.00％,평균CV5.90％.건강성년인무론남녀,mNAP표체수평균정비정태분포.남、녀중위수(P25,P75)분별위1 758(1 378 ～2 310) AB/c、1 897(1 369 ～3 249) AB/c,성별지간차이무통계학의의(U=27 140,P=0.243 8).이2.5％ ～97.5％위수작위림상삼고구간,결과위920.5 ～3 493.0 AB/c.세균감염환자외주혈중mNAP표체수평13 532(9 756 ～ 16 869) AB/c고우병독감염환자1 143(536～2 012)AB/c이급건강대조인군1 879(1 399 ～2 497) AB/c(H=221.5,P＜0.01).결론 응용QuantiBRITETM PE시제합래표준화류식세포의적설치,가정량분석대측양품형광강도,진이획득세포소함형광소분자수,병계산출매개세포표체적항원위점혹항원분자수목,종이체도정량검측적목적.응용류식세포술검측외주혈중mNAP,조작간편、쾌속、결과준학,mNAP표체대우감별세균、병독감염구유중요적림상개치.
Objective To establish a flow cytometric method for the detection of alkaline phosphatase (ALP) expression on the membrane of neutrophils (mNAP).Methods EDTA-K2 anticoagulant venous bloods were collected.Expression of mNAP in peripheral blood was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-ALP monoclonal antibody.BD QuantiBRITE PE was used to generate a calibration curve for PE fluorescence and ratios of PE to anti-ALP antibody and detect the bound ALP antibodies per cell (antibodies bound per cell,AB/c).Preanalytical handling including anticoagulants (EDTA-K2,citrate,and heparin),storage temperature,storage time,and plasma ALP were optimized and measured the precision.The expression levels of mNAP from 481 healthy controls were measured to establish a clinical reference range.The mNAP levels of 84 patients with severe infection and local infection,39 patients with virus infection were determined by this method.Results For preanalytical handling,application of PBS washing can effectively eliminate the interference of plasma ALP.The mNAP levels were not influenced by different anticoagulants and storage conditions (stored for 12 h either at room temperature or 4 ℃).This method had preferable reproducibility (CV in batch were 2.01％-3.33％,average 2.67％ ; CV between batch were 5.80％-6.00％,average 5.90％).The median (quartiles) of mNAP in health controls were 1 758 (1 378-2 310) AB / c for men and 1 897 (1 369-3 249) AB / c for women.There was no significant difference between genders (U =27 140,P =0.243 8).The clinical reference ranges (2.5 percentile to 97.5 percentile) of mALP was 920.5-3 493.0AB / c.The expression levels of mNAP of patients with bacterial infections (13 532,9 756-16 869 AB / c) were significantly higher than those of patients with virus infection(1 143,536-2 012 AB / c) and healthy controls (1879,1399-2497 AB / c) (H=221.5,P＜0.01).Conclusion BD QuantiBRITE PE kit can be used to standardize flow cytometer settings and quantitatively detect molecules per cell.The flow cytometric method for detection of mNAP has important clinical application for differentiating bacterial and virus infection.
