1.Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium.
Wei WANG ; Yi QIN ; Yaru WANG ; Jiejie ZOU ; Jing CHEN ; Jinwu CHEN ; Yan ZHANG ; Ming GENG ; Zhongdong XU ; Min DAI ; Lilong PAN
Chinese Journal of Biotechnology 2020;36(7):1431-1439
The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 μg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.
Animals
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Bone Marrow Cells
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physiology
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Cells, Cultured
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Culture Media, Conditioned
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Lipopolysaccharides
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metabolism
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Macrophages
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classification
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physiology
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Mice
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Phagocytosis
2.The clinic and pathologic significance of plasma cell myeloma with CCND1.
Qi SUN ; Gang AN ; Enbin LIU ; Zhanqi LI ; Hongju ZHANG ; Qingying YANG ; Fujun SUN ; Yue MA ; Mu XIAN ; Peihong ZHANG ; Kun RU
Chinese Journal of Hematology 2015;36(9):775-779
OBJECTIVETo study the clinical and pathologic features of multiple myeloma(MM) with CCND1.
METHODSRetrospectively analyzed the clinical and pathologic profiles of 158 patients with MM from 2010 to 2013. The clinical and morphologic features of bone marrow aspiration, biopsy and immunophenotypic analysis which was carried out by flow cytometry and immunohistochemistry were analyzed in all patients with MM respectively. CCND1 translocation was studied by FISH method in all cases. Classical cytogenetic studies of bone marrow were performed in 24 cases whose CCND1 was positive.
RESULTSIn the 158 patients with MM, CCND1 was detected in 31 patients (19.6%). In 31 patients, type IgA, IgD, IgG, IgM, light-chain only and nonsecretory MM were 4 cases,4 cases,11 cases,1 case, 6 cases and 5 cases respectively. A high incidence of CCND1 was observed in IgD and nonsecretory MM comparied with IgA and IgG respectively (P<0.05). but no statistical significance was reached between κ and λ type patients (P=0.627). The morphology of plasma cell in bone marrow biopsies were small Lymphocyte- Like 24 cases,mature plasma cell 6 cases and immature plasma cell 1 case. Immunophenotype of all 31 cases was CD38⁺CD138⁺CD19⁻CD45⁻, (CD56⁺ in 11 cases, CD20⁺ in 9 cases, CD117⁺ in 3 cases. MM with CCND1 showed a strong association with CD20 expression, the lack of CD56 expression. Immunohistochemistry showed positive for cyclinD1 in 22 cases.
CONCLUSIONA high incidence of CCND1 was detected in the IgD and nonsecretory MM, and correlated with Small Lymphocyte- Like, higher positive rate of CD20, cyclinD1 and the lack of CD56 expression. MM with CCND1 must be distinguished from LPL and other mature B cell lymphomas which have plasmacytoid differentiation.
Biopsy ; Bone Marrow ; Cyclin D1 ; metabolism ; Flow Cytometry ; Humans ; Immunohistochemistry ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Multiple Myeloma ; classification ; metabolism ; Plasma Cells ; Retrospective Studies ; Translocation, Genetic
3.Ultrastructural analysis of 5' nucleotides distribution in acute myeloid leukemia subtypes.
Yong-Xin RU ; Shi-Xuan ZHAO ; Jin-Hua LIU ; Yin-Chang MI ; Xiao-Fan ZHU ; Tian-Xiang PANG
Journal of Experimental Hematology 2008;16(3):484-487
5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.
5'-Nucleotidase
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metabolism
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ultrastructure
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Adolescent
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Adult
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Aged
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Bone Marrow Cells
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cytology
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enzymology
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Child
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Female
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Granulocytes
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enzymology
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Humans
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Leukemia, Myeloid, Acute
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classification
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enzymology
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Male
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Middle Aged
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Young Adult
4.Category, quantity and clinical significance of autoantibodies on bone marrow hematopoietic cells in patients with immunorelated cytopenia.
Rong FU ; Zong-hong SHAO ; Hong LIU ; Hong HE ; Hai-rong JIA ; Juan SUN ; Ming-feng ZHAO ; Guang-sheng HE ; Jun SHI ; Jie BAI ; Yu-lin CHU ; Tian-ying YANG
Chinese Journal of Hematology 2003;24(4):177-180
OBJECTIVESTo explore the category, quantity and clinical significance of autoantibodies on bone marrow hematopoietic cells in patients with immunorelated cytopenia and evaluate the sensitivity of direct antiglobulin reaction (Coombs test ) of bone marrow mononuclear cells (BMMNC).
METHODSThe category and the positive rate of autoantibodies on bone marrow hematopoietic stem cells, nucleated erythrocytes, granulocytes in 32 patients with uncertain immunorelated cytopenia were investigated by using BMMNC-Coombs test and double immunofluorescence flow cytometry.
RESULTSThe positive rate of autoantibodies on bone marrow hematopoietic cells tested by flow cytometry was 90.63% which was higher than that by BMMNC-Coombs test (50.0%) (p < 0.05). In 29 positive cases, IgG autoantibody accounted for 6.90%, IgM13.8%, IgG+IgA 3.4%, IgG+IgM 31.0%, and IgG+IgM+IgA 44.8%. Of the 29 Patients, 25 (86.2%) with IgG autoantibody, 26 (89.7%) with IgM and 14 (48.3%) with IgA. The patients with IgG autoantibody alone had the lowest hemoglobin levels, and those with IgM autoantibody might have intravascular hemolytic findings. The response time of patients with IgG and IgG+IgM was shorter than that of the other patients. 91.3% of the patients had autoantibodies on bone marrow hematopoietic stem cells and showed pancytopenia, and 50% of the patients had autoantibodies on nucleated erythrocytes and granulocytes. Eleven of 13 patients with negative BMMNC-Coombs tests had autoantibodies on bone marrow hematopoietic stem cells detected by FACS. There was no significant difference of the quantities of the three categories of autoantibodies of nucleated erythrocytes and stem cells. The quantities of IgA on granulocytes were lower than that of IgG and IgM. There was no significant difference between IgG and IgM on granulocytes. The quantity of IgA on hematopoietic stem cells was significantly higher than that on nucleated erythrocytes or granulocytes.
CONCLUSIONSThe sensitivity of double immunofluorescence flow cytometry assay was higher than that of BMMNC-Coombs test for detecting autoantibodies. In immunorelated cytopenia patients, the predominant autoantibody was IgM which could cause intravascular hemolysis, and the second one was IgG which could cause severe anemia. Most immunorelated cytopenia patients had autoantibodies on hematopoietic stem cells and showed pancytopenia. IgA was more easily seen on the hematopoietic stem cells.
Adolescent ; Adult ; Aged ; Autoantibodies ; classification ; metabolism ; Autoimmune Diseases ; immunology ; Bone Marrow Cells ; immunology ; Child ; Coombs Test ; Female ; Flow Cytometry ; Humans ; Immunoglobulin A ; metabolism ; Immunoglobulin G ; metabolism ; Immunoglobulin M ; metabolism ; Male ; Middle Aged ; Pancytopenia ; immunology
5.Distribution of Antigenic Aberration in the Bone Marrow of Acute Leukemia in Complete Remission.
Soyoung SHIN ; Jimin KAHNG ; Myungshin KIM ; Jihyang LIM ; Younggoo KIM ; Kyungja HAN
The Korean Journal of Laboratory Medicine 2008;28(1):1-7
BACKGROUND: The aberrant, leukemia-associated antigen expression patterns allow us to discriminate leukemic blasts from normal precursor cells. Our major goal was to determine a guideline for the detection of minimal residual disease using CD20+/CD34+ and myeloid Ag+/CD19+ combination in the bone marrow of acute leukemia in complete remission (CR) after chemotherapy. METHODS: Bone marrow samples from 117 patients with acute leukemia in complete remission after chemotherapy and from 22 healthy controls were immunophenotyped by triple staining and measured by flow cytometry. RESULTS: The CD20+/CD34+ cells in the large lymphocyte gate (R1) ranged from 0% to 3.24% (0.8+/-0.82%, P=0.000) in CD20+/CD34+ B-lineage ALL CR (N=31), from 0.03% to 4.2% (0.7+/-0.83%, P=0.000) in CD20-/CD34- B-lineage ALL CR (N=66), from 0.1% to 0.96% (0.45+/-0.32%, P=0.016) in T-ALL CR (N=10), and from 0.02% to 0.48% (0.18+/-0.15%, P=0.776) in AML CR (N=10). The CD13,33+/CD19+ cells in R1 gate ranged from 0% to 2.69% (0.37+/-0.48%, P<0.001) in CD13,33+/CD19+ B-lineage ALL CR (N=31), from 0% to 1.8% (0.31+/-0.28%, P<0.001) in CD13,33-/CD19+B-lineage ALL CR (N=65), from 0.02% to 0.64% (0.29+/-0.22%, P=0.071) in T-ALL CR (N=9), and from 0% to 0.17% (0.07+/-0.09%, P=0.341) in AML CR (N=3). CONCLUSIONS: Using an immunophenotypic method for the detection of early relapse or minimal residual disease of B-lineage ALL bone marrow in CR after chemotherapy, different cutoff values should be applied according to antigen combination and gating. When the proportion of aberrant antigen combination was less than 5% in large lymphocyte gate, the results should be interpreted with caution.
Acute Disease
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Antigens, CD/*metabolism
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Antigens, CD19/metabolism
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Antigens, CD20/metabolism
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Antigens, CD34/metabolism
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Antigens, Differentiation, Myelomonocytic/analysis/metabolism
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Bone Marrow Cells/*classification/metabolism
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Flow Cytometry
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Hematopoietic Stem Cells/classification/metabolism
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Humans
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Immunophenotyping
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Leukemia/*diagnosis/drug therapy
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Leukemia, Myeloid, Acute/diagnosis/drug therapy
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Neoplasm, Residual
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Remission Induction
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Tumor Markers, Biological/immunology
6.Immunophenotypic Features of Granulocytes, Monocytes, and Blasts in Myelodysplastic Syndromes.
Hee Won MOON ; Jung Won HUH ; Miae LEE ; Ki Sook HONG ; Wha Soon CHUNG
The Korean Journal of Laboratory Medicine 2010;30(2):97-104
BACKGROUND: Despite the diagnostic utility of immunophenotyping for myelodysplastic syndromes (MDS), it has not been widely performed, and reports on this are absent in Korea. We aimed to evaluate the immunophenotypic features of non-blastic granulocytes, monocytes, and blasts in patients with MDS and non-clonal disorders using routine flow cytometry (FCM). Moreover, we evaluated the phenotypic abnormalities of mature cells in leukemic patients. METHODS: Marrow aspirates from 60 patients, including 18 with MDS, 18 with leukemia, and 24 with non-clonal disorders (control group), were analyzed using FCM. Blasts, non-blast myeloid cells, and monocytes were gated based on CD45 expression and side scatter (SSC). The phenotypes were then compared among the 3 groups. RESULTS: Compared to non-clonal disorders, the granulocytic lineages of MDS showed decreased SSC (P=0.005), increased CD45 intensity (P=0.020), decreased CD10-positive granulocytes (P= 0.030), and a higher CD56-positive rate (P=0.005). It is noteworthy that similar results were obtained in the leukemia group, and these findings were not related to the phenotypes of the leukemic cells. Using blast and monocytic gating, useful parameters for generating a differential diagnosis were not found. CONCLUSIONS: Gating the granulocytic region is a relatively easy method for MDS immunophenotyping. Among the parameters studied, SSC, CD10, and CD56 were the most useful for differentiating MDS from non-clonal disorders. While immunophenotypic changes in MDS appear to be useful for differentiating MDS from non-clonal disorders, these changes were also noted in the mature cells of leukemic patients.
Adolescent
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Adult
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Aged
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Aged, 80 and over
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Antigens, CD45/metabolism
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Antigens, CD56/metabolism
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Bone Marrow Cells/cytology
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Cell Lineage
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Child
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Child, Preschool
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Diagnosis, Differential
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Female
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Flow Cytometry
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Granulocytes/*classification
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Humans
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*Immunophenotyping
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Leukemia/diagnosis/pathology
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Male
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Middle Aged
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Monocytes/*classification
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Myelodysplastic Syndromes/*diagnosis
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Neprilysin/metabolism
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Phenotype
7.Expression of cyclin-dependent kinase CDC2 and its significance in malignant progression of gliomas.
De-zhong ZHAI ; Qiang HUANG ; Qing ZHU ; Hong-mei HUO ; Jun DONG ; Zhi-yuan QIAN ; Ai-dong WANG ; Qing LAN
Chinese Journal of Pathology 2007;36(3):196-197
Adolescent
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Adult
;
Aged
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Aged, 80 and over
;
Animals
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Bone Marrow
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metabolism
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Brain
;
metabolism
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Brain Neoplasms
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metabolism
;
pathology
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CDC2 Protein Kinase
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metabolism
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Cell Line, Tumor
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Child
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Child, Preschool
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Female
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Gene Expression Regulation, Neoplastic
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Glioma
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classification
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metabolism
;
pathology
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Humans
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Male
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Mice
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Mice, Nude
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Middle Aged
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Neoplasm Transplantation
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Neoplastic Stem Cells
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metabolism
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Young Adult