Quantitative analysis of dendritic cell subsets in bone marrow of patients with acute myeloid leukemia / 中国实验血液学杂志

In order to study the quantity of dendritic cell (DC) subsets of bone marrow in patients with acute myeloid leukemia (AML), the bone marrow aspirate were collected from 77 newly diagnosed AML patients and from 30 healthy persons. The quantity of DC subsets (myeloid dendritic cells, mDC and plasmacytoid dendritic cells, pDC) were detected by flow cytometry and analysed by 3-color and 4-color cytometric gate. Based on the conventional 3-color panel, mDC were identified by Lin-HLA-DR+CD11c+ and pDC were identified by Lin-HLA-DR+CD123+. Based on the 4-color panel, mDC were identified by Lin-HLA-DR+CD11c+ BDCA-1+ and pDC were identified by Lin-HLA-DR+CD123+BDCA-2+. The results showed that a reduction of mDC was found in 74.0% (57/77) and 58.4% (45/77) patients, a reduction of pDC was found in 90.9% (70/77) and 46.8% (36/77) patients respectively by 3-color and 4-color cytometric analysis. Meanwhile an expansion of mDC was showed in 19.5% (15/77) and 22.1% (17/77) patients, an expansion of pDC was showed in 1.3% (1/77) and 27.3% (21/77) patients respectively by 3-color and 4-color cytometric analysis. In subtypes of AML-M2, AML-M3 or AML-M4/5, 81.4%, 100% and 42.1% patients showed mDC decrease and 88.4%, 100% and 89.5% patients showed pDC decrease respectively by 4-color cytometric analysis. It is concluded that the 4-color cytometric gate is better method for detection of mDC and pDC from bone marrow of newly diagnosed AML patients as compared with 3-color cytometric gate, the majority of AML patients showed reduction of mDC and pDC. The percentages of patients with mDC normal or mDC increase in AML-M4/5 subtypes are more than that in AML-M2/3 subtypes, while the pDC does not show difference between AML subtypes.

Identification and enumeration of dendritic cells in peripheral blood and bone marrow of healthy individuals / 中国实验血液学杂志

The study was aimed to analyse and enumerate the dendritic cells (DC) subsets in peripheral blood and bone marrow (BM) of healthy individuals in China by using 2 panel 4-color flow cytometry (FCM). The percentage and absolute number of Lin-HLA-DR+CD11chiBDCA1+ myeloid DC (mDC) and Lin-HLA-DR+CD123hiBDCA2+ plasmacytoid DC (pDC) were detected in 35 normal BM (NBM) and 29 normal peripheral blood (NPB), the results were compared with the Lin-HLA-DR+CD11chimDC and Lin-HLA-DR+CD123hi pDC obtained by 3-color FCM. The results indicated that both absolute count of DC subset and relative count of pDC in BM were decreased along with increase of age, the absolute count of DC subset in male BM was higher than that in femoral BM (p<0.05). The DC subsets in NBM and NPB were different whatever by 3 or 4-color cytometric analysis, there were more mDCs than pDCs in PB and the ratio of mDC to pDC was 2.70 and 2.31 respectively. In contrast, pDCs predominated in BM, the ratio of mDC to pDC in BM was 0.90 and 0.71 respectively. The quantity of DC subsets significantly correlated to both frequency (mDC r=0.86; pDC r=0.96, p<0.05) and absolute number (mDC r=0.95; pDC r=0.98, p<0.05) between 3 and 4-color cytometric analysis. The quantity of DC subsets in PB and BM were significantly different, counted by 3 and 4-color cytometric analysis except pDC in PB (p<0.001). The quantity of DC subsets were much higher by 3-color than that by 4-color analysis. Since some Lin-HLA-DR+CD11chimDC and Lin-HLA-DR+CD123hi pDC were BDCA1- and BDCA-2dim/- respectively, that more were in BM than in PB. It is concluded that the DC absolute enumeration is correlated with sample type, gender, age and total nucleated cells (p<0.05). 4-color antibody combination may help to identify the real DC subsets in BM. DC subsets in NBM and NPB are different, that more mDC are in PB whereas more pDC in BM.

Expression of 5 genes in CD7 positive acute myeloid leukemia stem/progenitor cells from bone marrow / 中国实验血液学杂志

This study was aimed to investigate abca5, mdr-1, kdr, dapk and irf-1 expressions in leukemia stem/progenitor cells (LSC) from CD7 positive acute myeloid leukemia, the expression of these 5 genes in mononuclear cells (MNC) from 15 normal bone marrow (NBM) and 16 AML patients bone marrow (AML BM) specimen were detected by real-time quantitative PCR (RQ-PCR). CD34(+)CD38(+) progenitor cells and CD34(+)CD38(-)Lin(-) stem cells were sorted by flow cytometry (FCM) from the MNCs of 10 NBM and 21 AML BM specimen. These 5 gene expressions in the sorted cells were detected by small amount cell RQ-PCR. The results showed that these 5 genes above mentioned all expressed in NBM-MNC, in which the expression levels of irf-1 and dapk were highest with the relative expression levels 4.08 and 3.86, the expression levels of abca 5 and mdr-1 were in the middle with the relative expression 0.49 and 0.84 respectively, the kdr expression was lowest with the relative expression level 0.02. In CD34(+)CD38(+) progenitor cells, the expression level of kdr increased dramatically (p < 0.05) while irf-1 and dapk dramatically decreased (p < 0.05). There was no obvious change of expression in the rest 2 genes. In CD34(+)CD38(-) stem cells the expression level of these 5 genes all increased nearly 2 times as much as that in CD34(+)CD38(+) progenitor cells, but kdr increased 3 times as much, and the increase of kdr and irf-1 expressions was of statistical significance (p < 0.05). Compared with the NBM, expression levels of 5 genes in AML-MNC decreased, and out of them abca 5, mdr-1, kdr and dapk were decreased most remarkably (p < 0.05). Comparison between AML CD34(+)CD38(+) cells and AML MNC showed that the expression level of irf-1 and dapk were decreased dramatically (p < 0.05) while the rest 3 genes increased their expression with statistical significance (p < 0.05). The expression levels of these 5 genes were higher in CD34(+)CD38(-) cells than those in CD34(+)CD38(+) stem cells, and the increase of kdr and irf-1 expressions showed statistical difference (p < 0.05). These 5 genes expression levels were all higher than those in CD34(+)CD38(+) cells whether in AML CD34(+)CD38(-)CD7(+) cells or CD34(+)CD38(-)CD7(-) cells. The increase of kdr expression in CD34(+)CD38(-)CD7(+) cells as well as kdr and irf-1 expressions in CD34(+)CD38(-)CD7(-) cells were all of statistical significance (p < 0.05). In conclusion the expression level of kdr in NBM was highest in stem cells while dapk and irf-1 were highest in differentiated cells. The expression levels of these 5 genes in CD34(+)CD38(-)Lin(-) stem cells were higher than those in CD34(+)CD38(+) progenitor cells. The gene expressions in AML CD34(+)CD38(-)CD7(+) cells and CD34(+)CD38(-)CD7(-) cells are in accordance with the characteristics of stem cells.