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Objective: To analyze the practicality of current diagnostic criteria of invasive fungal infection (IFI) in patients with hematologic diseases/malignant tumors, so as to enhance the recognition of characteristics of pulmonary IFI after allogeneic hematopoietic stem cell transplantation (allo-HSCT)., Methods: The clinical features of 51 cases with IFI after allo-HSCT were analyzed retrospectively., Results: Pulmonary IFI accounted for 42.1% (51/121) of the whole infectious pneumonia diagnosed among the patients admitted during the study. One (2.0%) case was proven diagnosis; 24 (47.1%) were probable diagnosis and 26 (51.0%) were possible diagnosis. The using of immuno-suppressors and corticosteroids, and the presence of graft-versus-host disease (GVHD) were the main host factors. The patients with two or more host factors simultaneously accounted for 66.7% (34/51) of all pulmonary IFI patients. Totally 94.1% (48/51) of the patients with pulmonary IFI presented nodules and/or patches as the main features in high resolution computed tomography (HRCT) scanning. The positive rates of fungal antigen detection were 58.6% for G test and 33.3% for GM test, which were relatively high. Twenty patients (39.2%) showed decrease of arterial partial pressure of oxygen and hypoxia in blood-gas analysis., Conclusions: For the diagnosis of pulmonary IFI post allo-HSCT, the administration of immuno-suppressors and corticosteroids, and the presence of GVHD were the main host factors. Nodules and/or patches were the main features in HRCT image. Fungus antigen detection is the main tool to support clinical diagnosis.

Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.

The objective of this study was to evaluate the value of morphologic diagnosis for acute leukemia (AL), to explore the relation of morphologic diagnosis with immunology, cytogenetics and molecular biology diagnosis of AL and to analyze the onset characteristics of AL in 10 years. The samples of bone marrow and peripheral blood from 233 newly diagnosed cases of AL were collected during 2001-2011 years; the morphologic examination and immunologic, cytogenetic and molecular biologic examination (ICM) were carried out, the consistency of morphologic diagnosis with ICM diagnosis was compared, the onset characteristics of AL was analyzed. The results showed that: (1) the consistent rate of immunology, cytogenetics, molecular biology diagnosis with morphologic diagnosis was 84.3%. The order of consistent rat was AUL, M0 < M1 < HAL < M4 < M2 < M3 < M5 < ALL < M6, M7, AP; (2) Misdiagnosis always occurred among AUL, M0, M1, ALL and HAL or among M2a, M3v, M4 and M5. (3) In 233 cases, the highest ratio of blast was observed in M1 (92.5%), while the lowest ratio of blast was observed in M2 (49.5%). (4) AL occurred more frequently in males than that in female (147:86). (5) AL occurred in patients aged from 1 to 88 years. The median age was 41.5 for AUL, 40.8 for M0, 43.4 for M1, 46.3 for M2, 33.8 for M3, 42.6 for M4, 48.8 for M5, 77.3 for M6, 2.5 for M7, 65.0 for AP, 29.1 for ALL and 40.3 for HAL. (6) The number of patients in the later five years (139 cases) was significantly greater than that in the first five years (94 cases), especially the patients with M1, M2, M3, M4, and M5. It is concluded that morphologic diagnosis has important clinical value in the MICM diagnosis of AL. Attaching importance to the confusing cell morphology and onset characteristics of AL can improve the diagnostic accuracy.

Objective: In order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs)., Methods: Improved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process., Results: TPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration., Conclusion: These findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.

The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.

This study was aimed to compare the influences of bone marrow mesenchymal stem cells (BMMSCs) from patients with acute myeloid leukemia (AML), AML patients with complete remission (CR) and non-leukemia patients on HL-60 cells. The HL-60 cells were divided into three groups: group of co-cultivation with BMMSCs of AML patients, group of co-cultivation with BMMSCs of AML patients with CR and group of co-cultivation with BMMSCs of non-leukemia patients. The count of HL-60 cells, the CD11b and survivin expression of HL-60 cells, the cell cycle distribution of the HL-60 cells in 3 groups were compared by flow cytometry, the morphology and differentiation rate of HL-60 cells in 3 groups were observed and compared by microscopy. The results showed that there were no differences in HL-60 cell count at five and seven days, in HL-60 distribution at the G(0)/G(1) phase, in survivin and CD 11b expressions in 3 groups. All cells of 3 groups began to mature, and the differentiation rates in 3 groups were 18.0 +/- 3, 17.0 +/- 1.3 and 19.0 +/- 2.0 respectively, therefore there were no significant differences between the 3 groups (p = 0.23). It is concluded that there is no influence of BMMSCs in 3 groups on the proliferation and differentiation of HL-60 cells.

This study was aimed to compare partial biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) in AML patients, AML patients with complete remission (CR) and non-leukemia patients. The bone marrow (BM) MSCs were divided into 3 groups: group of MSCs from AML patients, group of MSCs from AML patients with CR, group of MSCs from non-leukemia patients. The morphologic features of MSCs were observed by light microscopy; CFU-F numbers of MSCs were counted after Wright-Giemsa staining in situ; the fusion times of MSCs were determined; the growth curves of MSCs were drawn by counting cell numbers; the immunophenotypes and cell cycle of MSCs were detected by flow cytometry; the DI values of MSCs were calculated. The results showed that the morphologic features of MSCs in 3 groups did not display difference; there was significant difference (p < 0.01) of CFU-F numbers in 3 groups, while CFU-F number of MSCs in AML group was minimal; there was significant difference of MSC fusion time in 3 groups, while fusion time of MSCs in AML group was most long; the growth curves of MSCs in 3 groups were similar; MSCs in 3 groups highly expressed CD105 and CD106, but not expressed CD45; the cell distribution ratios at phase of G(0)/G(1) for MSCs in 3 groups were 89.9 +/- 4.0%, 90.2 +/- 3.0% and 91.0 +/- 3.0% respectively; the DI values of MSCs in 3 groups were between 0.9 and 1.1. It is concluded that no significant difference of biological characteristics of the second generations of MSCs is found between those in leukemia and non-leukemia patients.

This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.

Aim: To explore proper cryopreservative systems for hematopoietic stem cells., Methods: Peripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation., Results: The recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group., Conclusion: 5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

This study was aimed to investigate the influence of cryopreservation on biological properties and function of leukemic dendritic cells (L-DCs) derived from patients with acute or chronic leukemia. Some fresh leukemic cells were detected immediately; some were cultured immediately; some were cryopreserved in -80 degrees C with 5% DMSO-6% HES as cryopreservor. After being thawed, they were cultured. The combination of rhGM-CSF, rhIL-4, rhTNF-alpha and other cytokines were added into the culture system. 12 days later, L-DCs were assayed for morphology, immunophenotype, mixed lymphocytic reaction (MLR) and CTL cytotoxicity on autologous leukemic cells. The results showed that both fresh and cryopreserved leukemic cells obtained from patients with acute or chronic leukemia revealed typical DC morphologically by means of using combinations of cytokines in culture, but there was no significant difference between pre-or post cryopreservations. L-DCs also upregulated the expression of CD80, CD54, HLA-DR, CD1a, CD83 and CD86, and downregulated the expression of CD14, but there was also no difference as compared with L-DCs befor cryopreservation. L-DCs derived from leukemic cells were also capable of stimulating MLR and inducing CTL which could kill autologous leukemic cells obviously. It is concluded that leukemic cells, regardless of fresh or frozen, can induce L-DCs after culture with cytokine combination. The L-DCs can induce CTL targeting autologous leukemic cells, and may be used to treat MRD as immunotherapy. The induction and biological properties of L-DCs are not influenced by cryopreservation.