探讨流式细胞术检测胞浆抗原的方法及其在白血病免疫分型中的意义

Detection of Cytoplasmic Antigens by Flow Cytometry and It′s Implication for Leukemia Immunophenotyping

为探讨用流式细胞术检测胞浆抗原的方法及其在免疫分型中的意义 ,采用三色或四色直接免疫荧光标记法检测 5 6例患者 17种膜抗原表达 ,并采用 4种透膜剂处理细胞后用流式细胞仪测定胞浆 (c)CD3,CD2 2 ,CD79a ,CD13,CD33及髓过氧化物酶 (MPO)的表达。结果显示 ,用FACS透膜液处理细胞后 ,胞浆抗原的阳性百分比最高 ,而细胞的光散射 (SSC)及CD4 5的荧光强度不变。 18例急性髓细胞白血病 (AML)患者 ,16例MPO阳性 ;4例T ALL患者 ,虽然膜cCD3阴性 ,而胞浆cCD3均为阳性 ;13例B ALL患者中 ,9例cCD2 2阳性。对 5例B ALL检测了cCD79a ,结果全部阳性。 38例急性白血病 (AL)患者中 ,18例表达 1个系列以上的膜标记 ;在 2 1例急性非淋巴细胞白血病 (ANLL)患者中 ,8例表达CD7;17例ALL中 ,7例CD13阳性。经胞浆抗原检测后 ,只有 2例分别符合双表型和混和型AL特点 ,提示cCD3,cCD2 2 ,cCD79a和cMPO为系列特异性标志 。

The purpose of this study was to optimize a fixation procedure for detection of cytoplasmic antigens by flow cytometry(FCM) and to evaluate the effect of intracellular CD3, CD22, CD79a and myeloperoxidase(MPO) in lineage assignment. Four kinds of fixation procedure and three or four color direct immunofluorescence staining were used to permeate cell membrane and label cell surface and intracellular antigens by means of FCM. Results showed that percentage of cytoplasmic antigens positive cells was the highest and cell scatter and fluorescence intensity of CD45 were not changed after using of FACS Permeabilization Solution. MPO protein was positive in 16/18 acute myeloid leukemia(AML) patients. 4 cases of T cell acute lymphoblastic leukemia(T ALL) cases were positive for cytoplasmic CD3(cCD3) but surface CD3 was negative. cCD22 was only detected in 9/13 of B ALL and cCD79a was positive in 5/5 B ALL. 18/38 cases of acute leukemia were expressed in more than one lineage marker, 8/21 cases of acute non lymphocytic leukemia(ANLL) were CD7 positive. 7/17 cases of acute lymphocytic leukemia (ALL) expressed CD13. After further cytoplasmic antigens detection, one was considered to be a T/myeliod biphenotypic leukemia, another one was diagnosed a biclonal or mixed leukemia. The results suggest that intracellular CD3,CD22,CD79a and MPO are lineage specific markers, they are very important for biphenotypic and biclonal/mixed acute leukemia identification.