[Detection of cytoplasmic antigens by flow cytometry and its implication for leukemia immunophenotyping]

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(c CD3) but surface CD3 was negative. c CD22 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 antigen detection, one was considered to be a T/myeloid biphenotypic leukemia, another one was diagnosed as 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