CAJ | 학술논문

目的建立八色流式细胞术(8c-FCM)检测急性B淋巴细胞白血病(B-ALL)微小残留病(MRD)的方法,并进行初步验证.方法采用CD19/侧向角散色光(SSC)、CD45/10和CD34(或cTdT)联合设门,两管八色抗体组合检测B-ALL的MRD.将B-ALL细胞株Nalm-6细胞掺入正常人骨髓细胞中,使Nalm-6细胞占有核细胞比例为10.00％、1.00％、0.10％、0.01％和0.005％,采用已建立的8c-FCM,进行回收试验和重复性试验,评价检测方法的准确度和精密度.检测抗体标记Nalm-6后保存不同时间的荧光强度变化,评价各荧光抗体的稳定性.检测并分析39份骨髓细胞免疫表型,包括对照者9例,初发或复发B-ALL患者9例,B-ALL化疗或骨髓移植(BMT)后缓解期病例21例,评价8c-FCM方法检测正常骨髓B淋巴系细胞群和B-ALL细胞的免疫表型结果,以及对B-ALL的MRD 检测结果,并探讨其与现用四色流式细胞术(4c-FCM)的可比性.结果建立了以CD19、CD45、CD10为主的两管八色抗体组合检测MRD的方法；当掺入的细胞株占有核细胞比例≥0.10％时,变异系数(CV)＜2.5％,平均回收率为95.81％；当获取106个细胞,Nalm-6细胞比例10.00％～0.01％时,检测到的Nalm-6细胞占骨髓有核细胞比例与实测值呈线性相关(r=0.99,P＜0.05),该法检测骨髓中Nalm-6细胞的灵敏度达到0.01％.Nalm-6细胞株标记抗体后随时间延长略有降低,但24h内平均荧光强度减低＜10％.应用该方法检测骨髓中CD19阳性的B淋巴系细胞,对照组呈现4个连续发育阶段,可分为4期:Ⅰ期CD45low/CD10stro/CD20 -/CD38stro/CD34+或cTdT+,Ⅱ期CD45 +/CD10+/CD20-/CD38stro/CD34+或cTdT+,Ⅲ期CD45 +/CD10 +/CD20-/CD38stro/CD34 -或cTdT-,Ⅳ期CD45stro/CD10 -/CD20 +/CD38low/CD34 -或cTdT -.初发和复发B-ALL患者骨髓中白血病细胞与对照组相比,均存在抗原表达异常；8c-FCM检测B-ALL缓解组,有5例存在MRD,主要抗原表达与4c-FCM检测结果一致,5例MRD细胞的范围为0.02％～5.42％.结论本研究所建立的两管8c-FCM新方法检测骨髓中白血病细胞的重复性好、准确度高,可鉴别骨髓正常B淋巴系细胞群、B-ALL治疗缓解后骨髓造血重建的B系前体细胞与MRD细胞群；和4c-FCM比较,两管8c-FCM新方法的标本用量少,检测速度快,能够有效诊断B-ALL的MRD. 目的建立八色流式細胞術(8c-FCM)檢測急性B淋巴細胞白血病(B-ALL)微小殘留病(MRD)的方法,併進行初步驗證.方法採用CD19/側嚮角散色光(SSC)、CD45/10和CD34(或cTdT)聯閤設門,兩管八色抗體組閤檢測B-ALL的MRD.將B-ALL細胞株Nalm-6細胞摻入正常人骨髓細胞中,使Nalm-6細胞佔有覈細胞比例為10.00％、1.00％、0.10％、0.01％和0.005％,採用已建立的8c-FCM,進行迴收試驗和重複性試驗,評價檢測方法的準確度和精密度.檢測抗體標記Nalm-6後保存不同時間的熒光彊度變化,評價各熒光抗體的穩定性.檢測併分析39份骨髓細胞免疫錶型,包括對照者9例,初髮或複髮B-ALL患者9例,B-ALL化療或骨髓移植(BMT)後緩解期病例21例,評價8c-FCM方法檢測正常骨髓B淋巴繫細胞群和B-ALL細胞的免疫錶型結果,以及對B-ALL的MRD 檢測結果,併探討其與現用四色流式細胞術(4c-FCM)的可比性.結果建立瞭以CD19、CD45、CD10為主的兩管八色抗體組閤檢測MRD的方法；噹摻入的細胞株佔有覈細胞比例≥0.10％時,變異繫數(CV)＜2.5％,平均迴收率為95.81％；噹穫取106箇細胞,Nalm-6細胞比例10.00％～0.01％時,檢測到的Nalm-6細胞佔骨髓有覈細胞比例與實測值呈線性相關(r=0.99,P＜0.05),該法檢測骨髓中Nalm-6細胞的靈敏度達到0.01％.Nalm-6細胞株標記抗體後隨時間延長略有降低,但24h內平均熒光彊度減低＜10％.應用該方法檢測骨髓中CD19暘性的B淋巴繫細胞,對照組呈現4箇連續髮育階段,可分為4期:Ⅰ期CD45low/CD10stro/CD20 -/CD38stro/CD34+或cTdT+,Ⅱ期CD45 +/CD10+/CD20-/CD38stro/CD34+或cTdT+,Ⅲ期CD45 +/CD10 +/CD20-/CD38stro/CD34 -或cTdT-,Ⅳ期CD45stro/CD10 -/CD20 +/CD38low/CD34 -或cTdT -.初髮和複髮B-ALL患者骨髓中白血病細胞與對照組相比,均存在抗原錶達異常；8c-FCM檢測B-ALL緩解組,有5例存在MRD,主要抗原錶達與4c-FCM檢測結果一緻,5例MRD細胞的範圍為0.02％～5.42％.結論本研究所建立的兩管8c-FCM新方法檢測骨髓中白血病細胞的重複性好、準確度高,可鑒彆骨髓正常B淋巴繫細胞群、B-ALL治療緩解後骨髓造血重建的B繫前體細胞與MRD細胞群；和4c-FCM比較,兩管8c-FCM新方法的標本用量少,檢測速度快,能夠有效診斷B-ALL的MRD.
목적 건립팔색류식세포술(8c-FCM)검측급성B림파세포백혈병(B-ALL)미소잔류병(MRD)적방법,병진행초보험증.방법 채용CD19/측향각산색광(SSC)、CD45/10화CD34(혹cTdT)연합설문,량관팔색항체조합검측B-ALL적MRD.장B-ALL세포주Nalm-6세포참입정상인골수세포중,사Nalm-6세포점유핵세포비례위10.00％、1.00％、0.10％、0.01％화0.005％,채용이건립적8c-FCM,진행회수시험화중복성시험,평개검측방법적준학도화정밀도.검측항체표기Nalm-6후보존불동시간적형광강도변화,평개각형광항체적은정성.검측병분석39빈골수세포면역표형,포괄대조자9례,초발혹복발B-ALL환자9례,B-ALL화료혹골수이식(BMT)후완해기병례21례,평개8c-FCM방법검측정상골수B림파계세포군화B-ALL세포적면역표형결과,이급대B-ALL적MRD 검측결과,병탐토기여현용사색류식세포술(4c-FCM)적가비성.결과 건립료이CD19、CD45、CD10위주적량관팔색항체조합검측MRD적방법；당참입적세포주점유핵세포비례≥0.10％시,변이계수(CV)＜2.5％,평균회수솔위95.81％；당획취106개세포,Nalm-6세포비례10.00％～0.01％시,검측도적Nalm-6세포점골수유핵세포비례여실측치정선성상관(r=0.99,P＜0.05),해법검측골수중Nalm-6세포적령민도체도0.01％.Nalm-6세포주표기항체후수시간연장략유강저,단24h내평균형광강도감저＜10％.응용해방법검측골수중CD19양성적B림파계세포,대조조정현4개련속발육계단,가분위4기:Ⅰ기CD45low/CD10stro/CD20 -/CD38stro/CD34+혹cTdT+,Ⅱ기CD45 +/CD10+/CD20-/CD38stro/CD34+혹cTdT+,Ⅲ기CD45 +/CD10 +/CD20-/CD38stro/CD34 -혹cTdT-,Ⅳ기CD45stro/CD10 -/CD20 +/CD38low/CD34 -혹cTdT -.초발화복발B-ALL환자골수중백혈병세포여대조조상비,균존재항원표체이상；8c-FCM검측B-ALL완해조,유5례존재MRD,주요항원표체여4c-FCM검측결과일치,5례MRD세포적범위위0.02％～5.42％.결론 본연구소건립적량관8c-FCM신방법검측골수중백혈병세포적중복성호、준학도고,가감별골수정상B림파계세포군、B-ALL치료완해후골수조혈중건적B계전체세포여MRD세포군；화4c-FCM비교,량관8c-FCM신방법적표본용량소,검측속도쾌,능구유효진단B-ALL적MRD.
Objective To establish and evaluate the new method of 8-color flow cytometry (8c-FCM) with two tube detecting bone marrow minimal residual disease (MRD) in B lineage acute lymphoblastic leukemia (B-ALL).Methods The MRD cells were analyzed by using two combinations of 8c-FCM antibody panels,gating with CD19/Side scatter(SSC),CD45/CD10 and CD34(or cTdT).Nalm-6 cell of B-ALL was mixed into normal marrow cells,with proportion of 10.00％,1.00％,0.10％,0.01％and 0.005％,and recovery test and reproducibility test were carried with 8c-FCM established to value its accuracy and precision of.Fluorescence intensity was detected on different time points after marked Nalm-6 by antibodies to evaluate the fluorescent stability of the antibodies.The immunophenotyping was analyzed in 39 bone marrow specimens,including 9 cases of normal control,9 cases of B-ALL primary or recurrent,21 cases of complete remission (CR) after chemotherapy or bone marrow transplantation ( BMT),to evaluate the detection of normal B lymphocyte lineage,leukemia cells of B-ALL,MRD cells of B-ALL using the 8c-FCM and compare it with 4c-FCM in being.Results The method of two tube 8c-FCM with main antibodies of CD19,CD45 and CD10 to detect MRD of B-ALL was founded; CV ＜ 2.5％,average recovery rate was 95.81％,when the actual percent of Nalm-6 mixed into normal bone marrow≥0.10％,and the percent of Nalm-6 detected and actual was linear dependent ( r =0.99,P ＜ 0.05 ) ranged from 10.00％ - 0.01 ％,when 106 cells were acquired ; the sensitivity of the method established could reach 0.01％.The fluorescent intensity decreased along with the time after Nalm-6 cell marked,but less than 10％ in 24 hours.Using the antibody combinations and analyze strategy,the immunophenotye of B lymphocyte in normal bone marrow presented four sequential stages:Stage Ⅰ CD45low/CD10stro/CD20 -/CD38stro/CD34 + or cTdT +,Stage ⅡCD45 +/CD10 + / CD20 -/CD38stro/CD34+ or cTdT +,Stage Ⅲ CD45 +/CD10 +/CD20 -/CD38stro/CD34 -or cTdT-,Stage Ⅳ CD45stro/CD10 -/CD20 +/CD38low/CD34 or cTdT.Antigen expressions of leukeamic cells of B-ALL primary or recurrent were different compared with control team; there were 5 cases with MRD positive in CR team,and the main antigen expression was consistent with the results from 4c-FCM.The range of the percent of MRD cells was 0.02％ - 5.42％ of the 5 cases of MRD positive.Conclusions The new method of two tube 8c-FCM established shows good reproducibility and high accuracy,and can identify normal B lymphocyte populations in bone marrow and regenerated B-precursors in CR cases with MRD cells;compared with 4c-FCM,the new method of two tube 8c-FCM with the fewer specimen is faster and efficient to diagnose MRD of B-ALL.