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Intracranial hemorrhage (ICH) is a rare and life-threatening hemorrhagic event in patients with immune thrombocytopenia (ITP). However, its mortality and related risk factors remain unclear. Herein, we conducted a nationwide multicenter real-world study of ICH in adult ITP patients. According to data from 27 centers in China from 2005 to 2020, the mortality rate from ICH was 33.80% (48/142) in ITP adults. We identified risk factors by logistic univariate and multivariate logistic regression for 30-day mortality in a training cohort of 107 patients as follows: intraparenchymal hemorrhage (IPH), platelet count ≤10 × 109/L at ICH, a combination of serious infections, grade of preceding bleeding events, and Glasgow coma scale (GCS) level on admission. Accordingly, a prognostic model of 30-day mortality was developed based on the regression equation. Then, we evaluated the performance of the prognostic model through a bootstrap procedure for internal validation. Furthermore, an external validation with data from a test cohort with 35 patients from 11 other centers was conducted. The areas under the receiver operating characteristic (ROC) curves for the internal and external validation were 0.954 (95% confidence interval [CI], 0.910-0.998) and 0.942 (95% CI, 0.871-1.014), respectively. Both calibration plots illustrated a high degree of consistency in the estimated and observed risk. In addition, the decision curve analysis showed a considerable net benefit for patients. Thus, an application (47.94.162.105:8080/ich/) was established for users to predict 30-day mortality when ICH occurred in adult patients with ITP.

To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators.The percentage of CD8In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.PD-1、ICOS在ITP患者中的表达及意义.检测原发免疫性血小板减少症（ITP）患者外周血CD8选取2020年9月至12月于天津医科大学总医院血液科就诊的ITP患者28例，包括 13例新诊断患者，15例慢性患者，同时选取健康志愿者22名作为对照组。采用流式细胞术检测外周血CD8慢性组ITP患者CD8在ITP患者中，PD-1和ICOS的不平衡表达可能干扰机体的免疫稳态，可以作为ITP患者的治疗靶标。.

To investigate the inhibitory effect of ascorbic acid single or combination of decitabine on tumor cells of myelodysplastic syndrome (MDS) and explore its related mechanism.The human MDS cell lines SKM-1 and MUTZ-1 were treated with different concentrations of ascorbic acid, and the cell proliferation activity was detected by the CCK-8 assay. The reactive oxygen species (ROS) level, labile iron pool (LIP), cell cycle, and apoptosis of SKM-1 and MUTZ-1 cells were detected by flow cytometry. The control group, ascorbic acid monotherapy group, decitabine monotherapy group, and combination group of ascorbic acid and decitabine were set up, the cell proliferation activity and apoptosis were detected in each group.High-dose ascorbic acid could reduce the cell proliferation activity of SKM-1 (R=0.886, p=0.000) and MUTZ-1 (R=0.880, p=0.000). With the increase of ascorbic acid concentration, the ROS level in SKM-1 and MUTZ-1 cells increased (r=0.816, r=0.942), the proportion of cells stagnation in GHigh-dose ascorbic acid shows a cytotoxic effect on MDS tumor cells, inhibiting cell proliferation and increasing apoptosis. Ascorbic acid combined decitabine have a synergistic effect of anti-MDS tumor cells.抗坏血酸对骨髓增生异常综合征细胞的抑制作用及相关机制.观察抗坏血酸单药或与地西他滨联合用药对抑制骨髓增生异常综合征（MDS）肿瘤细胞的抑制作用，并探讨其相关机制。.用不同浓度抗坏血酸干预人MDS细胞系SKM-1和MUTZ-1，CCK-8法检测细胞增殖活性，流式细胞术检测细胞内活性氧和不稳定铁池水平、细胞周期及凋亡程度。分别设立对照组、抗坏血酸组、地西他滨单药组及二者联合用药组，检测细胞增殖活性和凋亡程度。.高剂量抗坏血酸可降低SKM-1和MUTZ-1细胞的增殖活力，且这种抑制增殖作用呈剂量依赖性（复相关系数R=0.886，p=0.000和复相关系数R=0.880，p=0.000）；随着抗坏血酸浓度升高，SKM-1和MUTZ-1细胞内活性氧水平增高（r=0.816，r=0.942），停滞在G高剂量抗坏血酸对MDS肿瘤细胞存在细胞毒作用，抑制细胞增殖并增加其凋亡；抗坏血酸与地西他滨有协同抗MDS肿瘤细胞的作用。.

Congenital pure red cell aplasia, also known as Diamond-Blackfan anemia (DBA), is a hereditary disease characterized by pure red cell aplasia and congenital malformation. Its main clinical features are anemia, dysplasia, and tumor susceptibility. Ribosomal protein (RP) gene mutation is the main pathogenesis of DBA. The most common type of gene mutation is RPS19 gene mutation. Heterozygous mutations in as many as 19 RP genes and other non-RP genes mutations have been identified in DBA. This review summarized briedfly the latest research advances in the pathogenesis of DBA.先天性纯红细胞再生障碍性贫血发病机制的研究进展.先天性纯红细胞再生障碍又名Diamond-Blackfan贫血（DBA），是以单纯红系再生障碍和先天性畸形为特征的遗传性疾病，以贫血、身体发育异常和肿瘤易感性为主要临床特征。核糖体蛋白质（RP）基因突变是DBA的主要发病机制，最常见的基因突变类型为RPS19基因突变，目前已在DBA患者中发现了19个RP基因的杂合突变及其他非RP基因的突变。本文对基因突变引起DBA的最新研究进展进行综述。.

To analyze the subtype and functional biomarker expression changes of natural kill cells(NK) in peripheral blood of patients with myelodysplastic syndrome(MDS) and normal people, so as to evaluate the relationships between these changes and hematopoietic functions and to explore the role of NK cells in the pathogenesis of MDS.The quantity of NK cells and the expression of biomarkers(NKp30,NKp46,NKG2A) on NK cells were detected by flow cytometry in 35 MDS patients from 2015 to 2016 in our hospital and 34 normal controls. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil(ANC), hemoglobin in peripheral blood and the hematopoietic function in bone marrow(CD34The percentage and quantity of NK cells and CD56The decrease of NK number and function may cause the immune surveillance and lead to disease progression.

To explore the single nucleotide polymorphism(SNP) of mitochondrial DNA (mtDNA) D-LOOP region in peripheral blood lymphocytes of immuno-related pancytopenia (IRP) patients and its correlation with immune parameters.The D-LOOP region in mitochondrial DNA of lymphocytes in peripheral blood mononuclear cells from 43 patients with untreated IRP was detected by polymerase chain reaction(PCR). The PCR products were sequenced by the pros and cons direct sequencing methods. The sequencing results were compared with the revised Cambridge reference sequence (rCRS) and the Polymorphic Sites of Human Mitochondrial Genome Database.Among total of 110 variant positions of D-LOOP region in 43 patients, 62 was SNP sites and 48 was mutation sites, of which 14 were the new mutation sites not yet registered in the database, 516 base variations were observed at 110 positions, the most common variations were base substitutions, among them T/C and A/G was 184/410 and 113/410 respectively. In the 110 variant positions, the high frequency variation sites were 73 and 263 for 43/43,311 for 32/43,310 and 16 224 for 27/43,16 519 for 25/43, 489 and 16 362 for 24/43. By the analysis of mitochondrial DNA D-LOOP polymorphism and related clinical immunology indicators of the patient's lymphocytes, it was found that D-loop in adult patients (age≥ 18 years old) significantly correlated with CD15 IgM, GLYCoAThe high frequency of polymorphism exists in mitochondrial DNA D-loop region of lymphocytes in IRPA patients, and was significantly correlates with the autoantibodies in bone marrow mononuclear cells in adult patients, which may be associated with the IRP occurrence.

To investigate the expression pattern of HOXA9 in myelodysplastic syndrome (MDS) patients and its relation with clinical characteristics and treatment response.The mRNA and protein expression levels of HOXA9 in bone marrow cells from 33 cases of MDS, 12 cases of AML, 20 cases of ITP and 18 normal controls were detected by real-time guautitative PCR(RT-PCR) and flow cytometry, respectively.The percentage of HOXA9(+)/CD34(+) and HOXA9(+)/CD34(+)CD38(-) in MDS patients were significantly higher than that in control group (P0.05), and the mRNA and protein expression of HOXA9 in MDS patients had a similar trend. The percentages of HOXA9(+)/CD34(+) and HOXA9(+)/CD34(+)CD38(-) before decitabine treatment were (50.64±27.59)% and (55.67±28.57)% respectively, which were both higher than those in control group (P0.05). After decitabine treatment, expression of HOXA9 significantly decreased (P0.05).HOXA9 is overexpressed in MDS patients and associated with several clinical characteristics. The detection of HOXA9 expression may have guide roles for diagnosis and treatment of MDS patients.

To explore the effect of costimulatory signaling molecules (CD80, CD86) expression on the quantity and function of B lymphocytes in peripheral blood (PB) of the patients with immune thrombocytopenia (ITP).A total of 55 ITP patients (30 cases were newly diagnosed and 25 cases were in remission), 25 healthy volunteers as controls were enrolled in this study. The levels of CD19(+)CD5(+), CD19(+)CD80(+), CD19(+)CD86(+), CD41a(+)IgG, CD41a(+)IgM and IgG, IgM in CD19(+)B cells were measured by flow cytometry (FCM). The correlation of CD19(+)B cells with clinical parameters of ITP patients was analyzed.The level of B1 (CD19(+)CD5(+)) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). The level of CD19(+)CD80(+) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). And the expression of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). The levels of IgG and IgM in remitted ITP patients after treatment were significantly lower than that before treatment (P0.05). The level differences of IgG and IgM before and after treatment in refractory ITP patients were not statistically significant (P0.05). The expression of CD19(+)CD80(+) in newly diagnosed ITP patients positively correlated with the level of Th1 and Th1/Th2 (r=0.502, r=0.471, P0.05). The expression of CD19(+)CD80(+) of newly diagnosed ITP patients positively correlated with the level of IgG and IgM in CD19(+)B cells (r=0.552, r=0.467, P0.05), and negatively correlated with PB platelet count (r= -0.424, P0.05). The levels of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients negati- vely correlated with PB platelet count (r=-0.658, r=-0.526, P0.05).The enhacement of costimulatory signaling pathway of CD19(+)B cells in ITP patients results in the abnormal activation of B lymphocytes, thereby mediates the dysfunction of immune system and involves in the pathogenesis of ITP.

Myelodysplastic syndromes (MDS) are a group of bone marrow failure diseases. The bone marrow microenvironment consists of bone marrow stromal cells (BMSC), growth factors and cytokines. The BMSC supporting haemopoiesis include mesenchymal stem cells (MSC), osteoblasts, endothelial cells and macrophages, but the adipocytes play a role in the suppression of hematopoiesis. Recently more and more researches indicate that the abnormality of bone marrow microenvironment involves in the pathogenesis and progression of MDS. In this review the abnormality of MDS bone marrow microenvironment is summarized briefly.

To investigate the complete blood count, morphological changes, follicular T helper (Tfh) cells and expression of PD-1 in bone marrow and spleen of mice with myelodysplastic syndrome(MDS) and to explore their significance in pathogenesis of MDS.The 10 male NUP98-HOXD13 transgenic mice and 10 male homologous wild-type C57BL/6J mice were used for experments. The complete blood count, morphological change of NUP98-HOXD13 transgenic mice and wild-type C57BL/6J were detected by routine methods. The level of Tfh cells and expression of PD-1 in bone marrow and spleen were measured by flow cytometry. The PD-1 mRNA of bone marrow mononuclear cells and spleen cells were analyzed by real-time PCR method.The counts of RBC, neutrophile and platelet in above- mentioned transgenic mice were less than that in wild type C57BL/6J mice. As compared with wild type C57BL/6J mice, the morphology of RBC and platelet in transgenic mice was some abnormal, including bi-nucleated erythrocytes, ringed mucleated neutrophil and erythroblastic islands. The count of Tfh cells in transgenic mice was less than that in wild type mice, but the expression of PD-1 was higher. The expression of BMMNC PD-1 mRNA was obviously higher than that in wild type mice.The pancytopenia and dysplasia, decrease of Tfh cells and increase of PD-1 expression have been observed in NUP98-HOXD13 transgenic mice, which may be one of important reasons for promoting malignant clone and leading to impair anti immune respones.

This study was purposed to investigate the role of regulatory T cells (Treg) in the immune unbalance for patients with acquired severe aplastic anemia (SAA). The flow cytometry was used to detect the quantity of CD4(+) CD25(+) CD127(dim) Tregs, T cell subset (CD4(+)/CD8(+) ratio), dendritic cell(DC) subset(mDC/pDC ratio) in 44 SAA patients(25 untreated patients and 19 recovery patients) and 23 normal controls. The correlation between Tregs and T cell subset, DC subset and hemogram were analyzed. The results showed that the percentage of CD4(+) CD25(+) CD127(dim) Tregs in peripheral blood lymphocyte(PBL) of untreated patients was (0.83 ± 0.44) %, which was obviously lower than that in recovery patients (2.91 ± 1.24)% and normal controls (2.18 ± 0.55)% (P0.05), but the difference was not statistically significant between latter two groups. The ratio of CD4(+)/CD8(+) was (0.5 ± 0.3) in untreated patients, which was obviously lower than that in recovery patients (1.2 ± 0.4) and normal controls (1.11 ± 0.24) (P0.05). The ratio of mDC/pDC was (3.08 ± 0.72) in untreated patients, which was significantly higher than that in recovery patients(1.61 ± 0.49) and normal controls (1.39 ± 0.36) (P0.05). The percentage of CD4(+) CD25(+)CD127(dim) Tregs in PBL positively correlated with CD4(+)/CD8(+) ratio (r = 0.695, P0.01), and that negatively correlated with mDC/pDC ratio (r = -0.796, P0.01). There were significant positive correlations between CD4(+)CD25(+)CD127(dim) Tregs/PBL and WBC, Ret% (r = 0.761, 0.749 respectively, P0.01). It is concluded that the decrease of CD4(+)CD25(+)CD127(dim) Tregs quantity in SAA may be one of mechanisms underlying bone marrow failure resulting from the deterioration of immune tolerance and hyperfunction of T-cells.

This study was purposed to detect the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of patients with immune thrombocytopenia, and to explore the role of Tfh cells in the pathogenesis of ITP. Twenty-one newly diagnosed ITP patients, twenty ITP patients in recovery stage and eighteen normal controls were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 in BM were detected by flow cytometry (FCM), and the mRNA expression of BCL-6 in BMMNC was determined by semi-quantitive RT-PCR. Correlation of Tfh cell level with the disease severity of ITP patients was analysed. The results showed that the ratio of CD4(+)CXCR5(+)/CD4(+) cells in newly diagnosed ITP patients [(5.532 ± 2.599)%] was significantly higher than that in ITP patients with recovery stage [(4.064 ± 2.026)%] and controls [(4.048 ± 1.413)%] (P0.05). The ratio of CD4(+)CXCR5(+)ICOS(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(14.586 ± 8.561)%] was higher than that in recovery stage ITP patients [(12.884 ± 10.161)%] and controls [(7.487 ± 5.176)%]. The differences be-tween newly diagnosed ITP patients and controls were statistically significant (P0.05). The ratio of CD4(+)CXCR5(+) CD40L(+)/CD4(+) CXCR5(+) cells in newly diagnosed ITP patients [(15.309 ± 10.756)%] and in ITP patients with recovery stage [(18.242 ± 12.243)%] were significantly higher than that in controls [(8.618 ± 5.719) %] (P0.05). The ratio of intracytoplasm CD4(+) CXCR5(+) IL-21(+)/CD4(+)CXCR5(+) cells in newly diagnosed ITP patients [(58.560 ± 26.285)%] and in ITP patients with recovery stage [(57.035 ± 30.936)%] were significantly higher than that in controls [(36.289 ± 24.868)%] (P0.05). The relative expression levels of BCL-6 mRNA in BMMNC of three groups were (1.407 ± 0.264), (1.149 ± 0.217) and (0.846 ± 0.157), respectively. The differences between 3 groups were significant(P0.05). It is concluded that the quantity and function of Tfh cells in ITP patients increase, which may play an important role in the pathogenesis of ITP.

This study was aimed to investigate the expression of microRNA-21 and its correlation with PTEN in diffuse large B cell lymphoma (DLBCL) paraffin-embedded tissues, and evaluate its potential relevance with clinical characteristics. The expression levels of miR-21 in 26 primary DLBCL and 10 normal lymph node tissue specimens were examined by real-time polymerase chain reaction. The expression of PTEN was detected by immunohistochemical staining. The results indicated that the expression of miR-21 was significantly higher in tumor tissues [6.586(1.10,38.22)] than that in normal tissues [0.791 (0.35,2.87)] (P0.05). Among 26 patients with DLBCL the expression of PTEN protein was positive in 6 patients (23%), and was negative in 20 patients (77%). In patients with DLBCL, the expression level of miR-21 was negatively correlated with the level of PTEN protein. The high expression of miR-21 was positively correlated with the level of serum LDH. The expression level of miR-21 in patients with Ann Arbor III-IV stage was obviously higher than that of patients with Ann Arbor I-II stage, but did not correlate with the subtype of patients in clinic (P0.05). It is concluded that the expression of miR-21 is high in DLBCL and its overexpression may be related with poor prognosis of DLCBL. These findings suggest that PTEN is possibly one of the targets of miR-21 in DLBCL.

Leukocyte interleukin-3 receptor α (CD123) is regarded as a marker of leukemia stem cells. We previously found that CD123 was also highly expressed on CD34(+)CD38(-) cells in myelodysplastic syndrome (MDS) patients, but it is unclear whether the level and the characteristics of CD34(+)CD38(-)CD123(+) cells in MDS are similar to those in acute myeloid leukemia (AML). Based on previous research by our team, we further enlarged the specimens and found that the mean proportion and the mean MFI of CD34(+)CD38(-)CD123(+) cells in low-grade MDS were lower than that in AML, and those in high-grade MDS were similar to those in AML. CD34(+)CD38(-)CD123(+) cells expressed lower granulocyte stimulating factor receptor, CD11b, and apoptosis molecule (Annexin V), meanwhile, these cells showed upregulation of transcription factors (GATA-1, GATA-2) and transferrin receptor (CD71) in MDS and AML. Furthermore, an increase in CD34(+)CD38(-)CD123(+) cells was closely related to the number of cytopenias involving hematopoietic lineages, anemia, blast count in bone marrow smear, fluorescence in situ hybridization analysis and WHO prognostic scoring system score. Thus, increases in CD34(+)CD38(-)CD123(+) cells may reflect malignant clonal cells with aberrant differentiation, overproliferation, and decreased apoptosis in MDS, which were similar to AML. CD123 may thus be a promising indicator for identifying malignant clonal cells in MDS and a candidate for targeted therapy.

To explore the relationship between the dendritic cell (DC) subsets and abnormal expression of transcription factors Gata-3 and T-bet in patients with immune thrombocytopenia (ITP).The plasmacytoid DC (pDC) and myeloid DC (mDC) of 33 ITP (16 untreated, 17 remitted) patients and 12 healthy controls were analyzed by flow cytometry (FCM) . The expressions of Gata-3 mRNA and T-bet mRNA in peripheral blood mononuclear cell (PBMNC) were detected by reverse transcription-polymerase chain reaction (RT-PCR) .The levels of interleukin-4 (IL-4) and interferon-gamma (IFN-γ) were measured by FCM in 33 ITP patients and 12 healthy controls.The percentage of pDC in PBMNC was 0.49% ± 0.18% in untreated and it was higher than that in remitted ITP patients (0.27% ± 0.17%) and in controls (0.32% ± 0.13%) (both P0.05). The percentage of mDC in PBMNC was 0.23% ± 0.17% in untreated, which was lower than that in remitted ITP patients (0.33% ± 0.18)% and in controls (0.31% ± 0.11%), but no statistic difference in mDC expression existed among 3 groups (P0.05). pDC/mDC ratios was (3.15 ± 2.01) in untreated ITP patients and it was higher than that in remitted ITP patients (0.81 ± 0.32) and in controls (1.07 ± 0.44) (both P0.05). The relative mRNA expression levels of Gata-3 were 2775 ± 489, 1357 ± 307 and 652 ± 165 respectively. And the expression of Gata-3mRNA in untreated group was higher than that in remission group or healthy controls (both P0.05). The relative mRNA expression levels of T-bet were 782 ± 394, 583 ± 176 and 576 ± 120. No statistic difference in T-bet expression existed among 3 groups (P0.05). Gata-3mRNA/T-bet mRNA ratio was (4.13 ± 1.69 ) in untreated group and it was higher than that of remission group (2.45 ± 0.69) or controls (1.15 ± 0.27) (both P0.05). The level of IL-4 in the untreated group was 9.14% ± 4.34% and it was higher than that of remission group (4.78% ± 1.69%) or controls (4.86% ± 1.41%). The level of IFN-γ in the untreated group was lower than that of controls (P0.05). Significant positive correlations existed between Gata-3 and pDC/mDC ratio (r = 0.585, P0.01). Significant positive correlations existed between Gata-3 and IL-4 ( r = 0.463, P0.05).The mechanism of ITP may be due to a disorder of DC subsets and a high expression of Gata-3.

To explore the inhibitory effects of tacrolimus (FK506) on effector T cells in vitro and examine the relationship between effector T cells and clinical features in patients with severe aplastic anemia (SAA) to elucidate its immune mechanism.The CD8(+) HLA-DR(+) cells, sorted by immunomagnetic separation from bone marrow mononuclear cells (BMMNC) of 16 SAA patients, were cultured in different concentrations of interleukin-2 (IL-2) alone or with FK506 for 72 hours. The proliferation effect was measured with methyl thiazolyl tetrazolium (MTT) method. The T lymphocytes were sorted from the SAA patients by lymphocyte separation medium and cultured alone or with IL-2 or with FK506 or FK506 plus cyclosporin A (CsA) for 18 hours. The expression of tumor necrosis factor-β (TNF-β) in CD8(+) HLA-DR(+) T cells was analyzed by flow cytometry. The relationship between the expression of TNF-β and the clinical data, including percentages of reticulocyte and lymphocytes in peripheral blood cell count and ratio of CD4(+) T cells and CD8(+)T cells, was also analyzed.At the concentration of IL-2 greater than or equal to 20 U/ml, the cell proliferation (A values, 0.538 ± 0.142) were significantly higher than that in the blank culture hole (0.505 ± 0.153) (P0.05). The A values significantly decreased (0.386 ± 0.124) after the addition of FK506 (P0.05). Compared with control group, the expression of TNF-β was significantly higher in IL-2 group (73.36% ± 16.73% vs 66.61% ± 16.20%, P0.05), significantly lower in FK506 and FK506 plus CsA groups (P0.05). No significant differences existed between the FK506 and FK506 plus CsA groups (47.78% ± 20.09% and 42.23% ± 21.35%, P0.05). The expression of TNF-β in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4(+) T cell and CD8(+) T cell, positively correlated with the percentage of lymphocyte in peripheral blood count (r = -0.86, -0.90, 0.77, all P0.05).IL-2 can enhance the proliferation and expression of TNF-β of CD8(+)HLA-DR(+)T cells from SAA patients. Such an effect is inhibited by FK506. And FK506 and FK506 plus CsA have similar effects.

To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA).Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR).Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P0.05).Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

To study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP.Forty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR.The ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P0.05).There exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.

This study was aimed to investigate the expression level and mechanism of microRNA-223 and LMO2 in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) cells and the mechanism. MicroRNA-223 mimics was transfected to increase the expression of MicroRNA-223 in the lymphocytes sorted by ficoll separation from the bone marrow mononuclear cells (BMMNC) of ALL and CLL patients. MicroRNA-223 inhibitor was transfected to decrease the expression of the MicroRNA-223 in the lymphocytes of normal controls. Then the expression of the MicroRNA-223 and LMO2 in transfected lymphocytes before and after cultivating for 72 hours were detected by RT-PCR, the apoptosis and cell cycle of these cells were measured by flow cytometery. The results indicated that before the transfection, the expression of MicroRNA-223 in ALL and CLL cells was (433.11 ± 144.88), which was significantly lower than that in the normal lymphocyte (949.59 ± 267.39); the expression of LMO2 was (807.10 ± 238.41), which was significantly higher than that in the normal lymphocytes (455.32 ± 176.83) (P0.05); after the transfection, the expression of MicroRNA-223 was (571.86 ± 142.00) in ALL and CLL cells, which was significantly higher than that before transfection (P0.05), but the expression of LMO2 was significantly lower than that before transfection (651.97 ± 230.12) (P0.05); in the normal control the expression of MicroRNA-223 obviously decreased (646.32 ± 172.93) (P0.05), the expression of LMO2 was significantly increased (541.27 ± 158.86.2) (P0.05). After transfection, the cell cycle G1/G2 phase and apoptosis changed in ALL and CLL cells. Before transfection the cell ratio in cell cycle G1/G2 phase was (94.75 ± 3.15)%, the cell ratio in S phase was (5.14 ± 3.12)%; after transfection the cell ratio in cell cycle G1/G2 phase was (97.03 ± 2.08)% and obviously increased (P0.05), the cell ratio in S phase was (2.97 ± 2.08)% and significantly decreased (P0.05). Before transfection the apoptosis rate was (54.47 ± 8.72)%, and obviously was higher than that after transfection (60.48 ± 8.81)%. And in the normal control, the cell ratio in G1/G2 phase was significantly higher than that after transfection [(96.73 ± 2.26)%, (94.55 ± 2.77)%, P0.05)], and the cell ratio in S phase was significantly increased [(3.25 ± 2.26)%, (5.45 ± 2.77)% (P0.05)]. The apoptotic rate in the ALL and CLL patients was significantly higher than that after the transfection [(54.47 ± 8.72)% vs (60.48 ± 8.81)%, respectively (P0.05)]. The apoptotic rate in the normal control was significantly lower than that after the transfection [(59.02 ± 10.20)%, (51.96 ± 10.20)%, respectively (P0.05)]. It is concluded that the expression of MicroRNA-223 decreases, and the expression of LMO2 increases in lymphocytic leukemia cells which leads to the lymphocytes over-proliferation and abnormal apoptosis, thus may be one of pathogenesis in lymphocytic leukemia.

To investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP).Twenty-one iron overloading, 26 non-iron overloading IRP patients and 10 normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3 and apoptosis of BMMNC were analyzed by flow cytometry (FCM). Antioxidants were added to iron overloading IRP BMMNC, and then the changes of indices above were detected by FCM. The number and apoptosis of T lymphocytes of IRP patients were also detected.ROS and apoptosis of BMMNC, myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones and normal controls (P0.05). The expressions of Bcl-2 on BMMNC, erythrocytes and stem cells of iron overloading IRP patients were significantly lower than those of non-iron overloading IRP ones (P0.05). The levels of Caspase-3 on myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones and normal controls (P0.05). After treatment with antioxidants, the expressions of ROS, Caspase-3 and apoptosis of iron overloading IRP BMMNC significantly decreased, but opposite for Bcl-2. The percentages of CD4(+) lymphocytes [ ( 40.86 ± 8.74）%] and CD4(+)/CD8(+) (1.44 ± 0.36) in PB of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones [(35.96 ± 7.03)% and 1.14 ± 0.37] and normal controls [(28.00 ± 6.73)% and 0.79 ± 0.21], respectively (P0.05), as opposite for CD8(+) lymphocytes (P0.05). The apoptosis of CD8(+) lymphocytes [(27.35 ± 10.76)%] and the ratio of CD8(+) apoptosis/CD4(+) apoptosis (2.51 ± 0.81) in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones [(15.47 ± 8.99)%] and normal controls (1.39 ± 0.47), respectively (P0.05). The apoptosis of erythrocytes and stem cells coated with auto-antibodies in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP and normal controls.Mechanisms underlying bone marrow damage by iron overload might be through the follows: ①The increased ROS induced by excessive iron deposition affected the expressions of Caspase-3 and Bcl-2, which caused more BMMNC apoptosis; ②The abnormal number and ratio of T lymphocytes caused by iron overload aggravated the abnormality of immunity of IRP; ③Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.目的 探究铁过载对骨髓单个核细胞（BMMNC）Coombs试验阳性全血细胞减少症（又称免疫相关性全血细胞减少症，IRP）患者骨髓造血损伤的机制。方法 采用流式细胞术检测21例铁过载IRP患者、26例非铁过载IRP患者及10名正常对照者骨髓造血细胞活性氧自由基（ROS）、Bcl-2、Caspase-3水平及凋亡情况；并检测铁过载组BMMNC经N-乙酰半胱氨酸处理后上述指标的变化；同时检测IRP患者T细胞数量、比例及凋亡率。结果 铁过载组BMMNC、粒系、红系和干细胞ROS水平及凋亡率均明显高于非铁过载组及正常对照组（P0.05）；铁过载组BMMNC、红系和干细胞Bcl-2水平均明显低于非铁过载组（P0.05）；铁过载组粒系、红系和干细胞Caspase-3水平均明显高于非铁过载组及正常对照组（P0.05）。铁过载组经N-乙酰半胱氨酸处理后，BMMNC ROS、Caspase-3水平及凋亡率均较前明显下降，Bcl-2水平较前明显升高。铁过载组外周血CD4+细胞、CD4+/CD8+细胞比值分别为（40.86±8.74）%及1.44±0.36，均明显高于非铁过载组[（35.96±7.03）%及1.14±0.37]和正常对照组[（28.00±6.73）%及0.79±0.21]（P0.05），铁过载组CD8+细胞百分率明显低于非铁过载组和正常对照组（P0.05）。铁过载组骨髓CD8+细胞凋亡率、CD8+细胞凋亡率/CD4+细胞凋亡率比值分别为（27.35±10.76）%及2.52±0.81，均明显高于非铁过载组[（15.47±8.99）%及1.39±0.47]（P0.05）。在骨髓有核红细胞膜抗体或干细胞膜抗体阳性IRP患者中，铁过载组骨髓红系或干细胞凋亡率均高于非铁过载组（P0.05）。结论 铁过载可能通过以下机制影响IRP患者骨髓造血功能：①过量铁沉积可诱导骨髓造血细胞ROS生成增加，影响Bcl-2、Caspase-3凋亡信号的表达，引起IRP患者骨髓造血细胞凋亡增加；②铁过载可通过影响T细胞数量及比例加重IRP患者免疫异常；③铁过载可增加IRP患者被覆自身抗体的骨髓有核红细胞和干细胞的破坏。

To investigate the function of dendritic cells (DC) of patients with immune related pancytopenia (IRP) and explore the role of DC in IRP.The expression of CD80 and CD86 on myeloid DC (mDC, Lin-HLA-DR(+) CD11c(+) cells) and plasmacytoid DC (pDC, Lin-HLA-DR(+) CD123(+) cells) of 65 IRP (37 untreated and 28 remitted) patients and 17 healthy controls were analyzed by flow cytometry.The expression of CD86 on pDC was (82.47 ± 13.17)% in untreated group and (60.08 ± 14.29)% in remission group, which were significantly higher than that of controls (47.95 ± 18.59)% (P0.05), while the expression in untreated group was higher than that of remission group (P0.05). The expression of CD80 on pDC was (6.31 ± 4.49)% in untreated group, which was significantly higher than that of remitted patients (3.09 ± 2.93)% and controls (2.33 ± 2.25)% (P0.05). The expression of CD86 on mDC was (97.06 ± 4.82)% in untreated group and (91.35 ± 12.20)% in control group, while the expression in untreated group was higher than that of control group (P0.05). The expression of CD80 on mDC was (6.20 ± 5.44)% in untreated group and (3.97 ± 3.24)% in remission group, which were significantly higher than that of controls (1.86 ± 1.73)% (P0.05). The expression of CD86 on pDC was negatively correlated to Th1/Th2 (r = -0.733, P0.05), it was positively correlated to the antibody on membrane of BMMNC (r = 0.283, P0.05) and the quantity of CD5(+)B cells (r = 0.436, P0.05), while it was negatively correlated to the level of hemoglobin, platelets and white blood cells (r = -0.539, P0.05; r = -0.519, P0.05; r = -0.567, P0.05, respectively). The expression of CD80 on pDC was negatively correlated to the level of hemoglobin and platelets (r = -0.431, P0.05; r = -0.464, P0.05).The function of pDC in PB of IRP were strengthened, which was relevant to the immunopathogenesis of IRP.

To investigate the expressions of STAT5 phosphorylation in CD34(+)CD38(-)CD123(+) bone marrow cells of the patients with myelodysplastic syndromes (MDS), and then evaluate the level of activation of STAT5 associated with cell proliferation in MDS clone cells.The bone marrow mononuclear cells (BMMNC) were extracted from 36 MDS patients and 14 normal controls. The mean fluorescence intensities (MFI) of phosphorylated STAT5(P-STAT5) in CD34(+)CD38(-)CD123(+) and CD34(+)CD38(-)CD123(-)cells, with or without the stimulation of 10 U/ml EPO, were examined by flow cytometry (FCM).Without stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 113.71 ± 67.22/173.05 ± 102.78, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (58.84 ± 27.51/68.99 ± 50.42, P0.01, P0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (63.06 ± 21.06, P0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; With the EPO stimulation, the P-STAT5 MFI in CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients was 144.04 ± 58.11/239.45 ± 152.05, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (68.41 ± 25, 10/64.21 ± 23.43, P0.01) and the normal controls CD34(+)CD38(-)CD123(-) cells (75.21 ± 27.02, P0.01), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal control CD34(+)CD38(-)CD123(-) cells; The P-STAT5 MFI in the CD34(+)CD38(-)CD123(+) cells of low/high risk MDS patients with or without EPO stimulation were 21.80/28.86, which was significantly higher than that of CD34(+)CD38(-)CD123(-) cells (7.42/5.50, P0.01, P0.05) and the normal controls CD34(+)CD38(-)CD123(-) cells (6.39, P0.05), there was no significant difference between the CD34(+)CD38(-)CD123(-) cells of MDS patients and the normal controls CD34(+)CD38(-)CD123(-) cells; There was no significant difference of P-STAT5 MFI with or without EPO stimulation and the increased P-STAT5 MFI between the CD34(+)CD38(-)CD123(+) cells of low and high risk MDS.STAT5 associated with cell proliferation was activated in CD34(+)CD38(-)CD123(+) bone marrow cells in MDS, which had more significant reactions to EPO than CD34(+)CD38(-)CD123(-) cells, indicating that CD34(+)CD38(-)CD123(+) bone marrow cells might be the real malignant MDS clone cells in MDS.

To assess the efficacy and safety of monoclonal antibody rituximab combined with cyclophosphamide (CTX) in the treatment of refractory and recurrent autoimmune hemolytic anemia.Seven cases with refractory and recurrent autoimmune hemolytic anemia (including 1 case of Evans syndrome) were recruited during January, 2007 to December, 2010. Treatment regimens were as follows: rituximab: 375 mg/m², 1 time/week, 2-6 courses; CTX:1 g, 1/10 d, 2-7 courses; combined with intravenous immunoglobulin (IVIG) 5 g, 1 time/week, given 1 day after rituximab administration. The efficacy and safety of this regimen were assessed during follow-up.All the patients showed good responses (7/7). Six patients achieved complete remission (6/7) and one achieved partial remission (1/7). Average follow-up time for the patients was 27 months. All patients remained in remission during the 12-month follow-up visits. Two patients showed elevated indirect bilirubin and increased reticulocyte counts within 24 months. One patient achieved complete remission after additional rituximab therapy, and another patient remained partial remission after cyclosporine therapy. At the time of 36-month follow-up visit, the patient relapsed and was retreated with 3 courses of rituximab combined with CTX and eventually achieved partial remission. All patients tolerated the treatment well with few mild side effects.Rituximab combined with CTX is effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia. Additional treatment to relapse patients about 12 - 24 months after drug withdrawal continues to be effective.

To explore the regulative factors on CD8(+)HLA-DR(+) T cells in the patients with severe aplastic anemia (SAA) and examine the roles of these cells in the immunopathogenesis of SAA.CD8(+)HLA-DR(+) T cells were sorted from bone marrow mononuclear cells of 13 SAA patients from July 2011 to March 2012 by magnetic activated cell sorting system and were divided into 3 groups: interleukin 2 (IL-2) group (0, 0.1, 1, 10, 100 and 1000 U/ml), cyclosporine A (CsA) group (addition of 400 ng/ml CsA in each IL-2-containing well),receptor antagonist group (addition of IL-2 receptor antagonist 8 µg/ml in each IL-2-containing well). Then cell proliferation rate was evaluated by MTT assay after a 72-hour culturing. Bone marrow mononuclear cells of the SAA patients were divided into CsA group, IL-2 group and control group and cultured for 18 hours and another 4 hours following the dosing of phorbol ester. The expression of tumor necrosis factor β (TNF-β) in CD8(+)HLA-DR(+) T cells was analyzed by flow cytometry.The cell proliferations of IL-2 wells at the concentrations of 10, 100 and 1000 U/L (0.36 ± 0.12, 0.41 ± 0.12, 0.46 ± 0.14) were significantly higher than those of the control wells (0.23 ± 0.11), CsA group (0.18 ± 0.05, 0.19 ± 0.00, 0.20 ± 0.04) and receptor antagonist group (0.18 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, all P0.05). No statistic difference existed between CsA and receptor antagonist groups (P0.05). The expressions of TNF-β of CD8(+)HLA-DR(+)T cells of the IL-2 group were higher than those of the control group (64% ± 25% vs 46% ± 22%) whereas the CsA group (27% ± 20%) were lower than those of the control group (both P0.05).IL-2 can significantly stimulate the proliferation of CD8(+)HLA-DR(+) T cells and accelerate the in vitro secretion of TNF-β in SAA patients. The proliferation may be inhibited by CsA and receptor antagonist. And the expression of TNF-β is suppressed significantly by CsA.

To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity.CD(3)(+) T cells were sorted by magnetic activated cell-sorting system. The expressions of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR.The expression of TET2 mRNA in CD(3)(+) T cells was down-regulated in the MDS patients by (0.16 ± 0.15) fold compared with the controls (P0.05). The expression of TET2 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with serum complement C(3) (r = 0.404, P0.05). The expression of DLK1 mRNA in CD(3)(+) T cells was up-regulated in the MDS patients by (1.61 ± 0.88) folds compared with the controls (P0.05). Grouped by the chromosomes, the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [(1.45 ± 0.44) folds, P0.05]. The expression of DLK1 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with the proportion of bone marrow blasts (r = 0.343, P0.05).The mRNA expression of TET2 in CD(3)(+) T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased, which might decline the immune surveillance function. The findings would be useful for exploring the mechanism of immune tolerance.

To examine the expression of lymphokines damaging hematopoietic cells by CD8(+)HLA-DR(+) effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia (SAA) and explore further the heterogeneous immunopathogenesis of SAA.The CD8(+)HLA-DR(+) cells were sorted by immunomagnetic separation from the PB of 24 untreated SAA patients and 23 normal controls. The mRNA expressions of perforin, granzyme B, FasL and tumor necrosis factor β (TNF-β) of sorted cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR).The mRNA levels of perforin, granzyme B of CD8(+)HLA-DR(+)T cells in untreated group were higher than those of the controls (0.66 ± 0.25, 0.56 ± 0.26 vs 0.53 ± 0.14, 0.40 ± 0.13, P = 0.042, 0.012). The mRNA level of FasL in CD8(+)HLA-DR(+) T cells of untreated SAA patients was higher than that of the controls (0.77 ± 0.24 vs 0.61 ± 0.16, P = 0.011). The mRNA of TNF-β in CD8(+)HLA-DR(+) T cells of untreated SAA patients was also higher than that of the controls (0.58 ± 0.16 vs 0.46 ± 0.15, P = 0.011).CD8(+)HLA-DR(+) T cells may damage hematopoiesis through the actions of perforin, granzyme B, TNF-β and FasL. And it thus contributes to the immunopathogenesis of SAA.

To investigate the expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) plasma cells in the patients with multiple myeloma (MM), and explore the importance of Notch signaling pathway in the formation and progression of MM.Thirty three MM patients and 15 healthy controls were enrolled in this study. The expression of Notch1 on the membrane of bone marrow CD38(+)CD138(+) and CD38(+)CD138(-) plasma cells were analyzed by flow cytometry. The clinical data of MM patients were also analyzed.The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was (60.21 ± 25.06)% which was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients (39.84 ± 18.94)% (P = 0.000) and controls (38.34 ± 19.39)% (P = 0.004). There was no statistical difference between the two latter groups (P0.05). The expression of Notch1 on CD38(+)CD138(+)plasma cells from 24 newly diagnosed MM patients was correlated to the level of malignant plasma cells in there bone marrow (r = 0.914, P = 0.000), serum level of lactate dehydrogenase (LDH) (r = 0.754, P = 0.007), and β(2)-MG(r = 0.716, P = 0.013). The ratio of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients who had renal dysfunction was correlated to their abnormal serum creatinine levels. The expression of Notch1 on CD38(+)CD138(+) plasma cells from 17 MM patients who received VD (bortezamib and dexamethasone) chemotherapy was correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r = 0.842, P = 0.000).The expression of Notch1 on the membrane of CD38(+)CD138(+) plasma cells of MM patients was significantly higher than those of CD38(+)CD138(-) plasma cells of MM patients and controls. Notch1 overexpressed plasma cells were sensitive to the early VD therapy, and correlated to the progression and long term outcome of MM.

This study was aimed to investigate the quantity and subtypes of dendritic cells (DC) in patients with immune related pancytopenia (IRP) and to explore the role of DC in pathogenesis of IRP. The quantity of plasmacytoid dendritic cells (pDC, Lin(-)HLA-DR(+) CD123(+) cells) and myeloid dendritic cells (mDC, Lin(-)HLA-DR(+) CD11c(+)cells) in peripheral blood of 65 patients with IRP (37 new diagnosed and 28 remitted) and 17 healthy controls were analyzed by flow cytometry. The results indicated that the ratio of pDC in peripheral blood mononuclear cells (PBMNC) was (0.91 ± 064)% in new diagnosed group, which was significantly higher than that in remission group (0.39 ± 0.11)% and control group (0.29 ± 0.13)% (P0.01), while this ratio of pDC in remission group was higher than that in control group (P0.05). The ratio of mDC in PBMNC was (0.21 ± 0.20)% in new diagnosed group and (0.34 ± 0.21)% in remission group respectively, there was no statistical difference as compared with control group (0.29 ± 0.09)% (P0.05). The ratio of pDC to mDC in new diagnosed group was 6.75 ± 7.11, which was significantly higher than that in remission group (1.55 ± 0.93) and control group (1.07 ± 0.43, P0.01), there was no statistical difference between the ratio of remission group and control group (P0.05). The ratio of pDC in PBMNC of IRP group negatively correlated to ratio of Th1/Th2 (r = -0.347, P0.05), and positively correlated to the ratio of auto-antibody on membrane of BMMNC (r = 0.606, P0.05) and to the quantity of CD5(+)B cells (r = 0.709, P0.05), while it negatively correlated to the levels of hemoglobin (r = -0.381, P0.01) and platelets (r = -0.343, P0.01). The ratio of mDC in PBMNC positively correlated to the ratio of Th1/Th2 (r = 0.595, P0.05) and the level of hemoglobin (r = 0.292, P0.05). The ratio of pDC/mDC negatively correlated to ratio of Th1/Th2 (r = -0.395, P0.05), it positively correlated to the level of antibody on membrane of BMMNC (r = 0.421, P0.05) and the quantity of CD5(+)B cells (r = 0.423, P0.05), while it negatively correlated to the levels of hemoglobin (r = -0.304, P0.05) and platelets (r = -0.287, P0.05). It is concluded that the quantity of pDC in peripheral blood of IRP patients increases, which may be related to the immunopathogenesis of IRP.

This study was purposed to applicate the bone marrow indirect Coombs test and investigate its clinical significancies in diagnosis of immuno-related pancytopenia (IRP). 30 patients with pancytopenia including 22 cases of IRP and 8 cases of idiopathic cytopenia of undetermined significance (ICUS), and 15 patients with iron-deficiency anemia as controls were enrolled in this study. After incubation of the bone marrow supernatant of IRP patients and bone marrow nucleated cell (BMNC) of controls was used as experiment group, while the incubation of BMNC and bone marrow supernatant of controls was used as control group. After incubation for 45 min, the positive rate of membrane antibodies in bone marrow hematopoietic cells (CD15(+), GlyCoA(+) and CD34(+)cells) was detected by flow cytometry, and correlation analysis of positive rate with clinical data of patients were analyzed. The results showed that among 30 patients with pancytopenia (16 positive and 14 negative for bone marrow direct Coombs test) 16 cases showed positive for bone marrow indirect Coombs test, with positive rate 53.33. In the experiment group, the median positive rate of CD15(+)IgM was 0.34, which was significantly higher than that in control group (0.20, P0.05); the median positive rates of CD34(+) IgG and IgM were 0.64 and 0.21 respectively, which were significantly higher than those in control group (0.00, P0.05) and (0.00, P0.05); the positive rates of GlyCoA(+)IgG and IgM were (0.83 ± 0.75) and (2.12 ± 1.98) respectively, which were significantly higher than those in control group [(0.47 ± 0.43), P0.05, (0.68 ± 0.64), P0.01]; the positive rates of CD15(+) IgG and IgM were positively correlated with the ratio of CD5(+)B cells. The positive rates of GlyCoA(+) IgG and IgM negatively correlated with the Hb level, percentage of reticulocytes, the ratio of bone marrow erythroid lineage and DC1/DC2 positively correlated with the ratio of CD5(+)B cells and indirect bilirubin level. It is concluded that antibodies (IgG or IgM) aiming at the bone marrow hematopoietic cells exist in the supernatant of some IRP and ICUS patients, and may act on the membrane protein of the normal BMNC. These antibodies correlate with the prognosis of IRP.

This study was purposed to investigate the expressions of miR-21, miR-155 and miR-210 in plasma of patients with lymphoma, and explore their role played in diagnosis, evaluation of chemotherapy effect and prognosis of lymphoma. The expressions of miR-21, miR-155 and miR-210 were assayed by RT-PCR in plasma of 54 cases of lymphoma, 10 cases of lymphonode inflammation and 27 cases of normal controls. The results indicated that the expressions of miR-21, miR-155 and miR-210 in plasma of lymphoma patients were higher than those of control group and lymphonode inflammation group (P0.05). The expressions of miR-21 and miR-210 in plasma of control group and lymphonode inflammation group had no significant differences (P0.05). The expression of miR-21 in plasma of lymphoma patient group significantly correlated with their serum LDH level. The expressions of miR-21 and miR-210 in plasma of previously untreated lymphoma patient group were higher than those of the patients treated for 6 or more courses (P0.05). The diagnostic accuracy of miR-21, miR-155 and miR-210 used for lymphoma patients was 56, 65, 48 respectively, and reached to 83 when combined three of them. It is concluded that the expressions of miR-21, miR-155 and miR-210 in plasma of lymphoma patients were significantly higher. Detection of these 3 miRNA in plasma of patients can contribute to the clinical diagnosis, treatment and prognosis evaluation of lymphoma.

To investigate the quantity and the pathway to damage hematopoietic cells of CD8+CD25+ and CD8+ HLA-DR+ effector T cells in peripheral blood (PB) of severe aplastic anemia(SAA) patients and explore the immunopathogenesis of SAA.The quantity of CD8+ CD25+ and CD8+ HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-beta (TNF-beta) and FasL in 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry.The fraction of CD8+ CD25+ T cells in CD8+ T cells was (3.67 +/- 2.58)% in untreated SAA patients, (5.19 +/- 4. 29)% in recovered patients and (4.84 +/- 2.31)% in normal controls, and that of CD8+ CD25+ T cells in CD3+ cells in the three groups was (2.25 +/- 1.35)%, (2.98 +/- 1.35)% and (2.11 +/- 1.88)%, respectively. They had no statistic difference among the 3 groups (P0.05). The fraction of CD8+ HLA-DR+ T cells in CD8+ T cells was (39.30 +/- 8.13)% in untreated patients, which was significantly higher than that in recovered patients [(20.65 +/- 5.38)%] and controls [(18.34 +/- 6.68)%] (P0.001), while there was no statistic difference between the latter two groups (P0.05). CD8+ HLA-DR+ T cells in CD3+ cells was (27.81 +/- 7.10)% in untreated group, which was significantly higher than that of recovered group [(12.02 +/- 3.03)%] and controls [(8.50 +/-2.33)%] (P0.01). And that in recovered group was higher than that in control group (P0.05). The expressions of perforin, granzyme B, TNF-beta and FasL of CD8+ HLA-DR+ T cells in untreated group were 8.51%, 96.08%, 72.11% and 94.25% respectively, which were higher than those in recovered group (1.78%, 85.20%, 34.38% and 51.20%) and controls (1.86%, 82.09% ,17.92% and 32.91%). There was no statistic difference between recovered patients and controls (P0.05).There were elevated quantity of CD8+ HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-beta and FasL in SAA, which might contribute to the bone marrow failure.

To study the STAT5 phosphorylation levels of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH).The bone marrow mononuclear cells (BMMNC) were extracted from 23 PNH patients treated at our department from April 2010 to February 2011 and 11 normal controls. The mean fluorescence intensity (MFI) of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(+) cells and CD34(+)CD59(-) cells with or without the stimulation of 10 U/ml EPO and 50 U/ml TPO were examined by flow cytometry.(1) Without stimulation, the P-STAT5 MFI in CD34(+)CD59(-) cells of PNH patients was significantly lower than that of CD34(+)CD59(+) cells (31 ± 15 vs 74 ± 47, P0.01). And it was 59 ± 23 in normal control CD34(+)CD59(+) cells (P0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. (2) Under the stimulations of EPO and TPO, the P-STAT5 MFI was significantly lower in CD34(+)CD59(-) cells of PNH patients than that of CD34(+)CD59(+) cells (49 ± 24 and 51 ± 41 vs 120 ± 82 and 124 ± 87, both P0.01). For the normal control CD34(+)CD59(+) cells, they were 79 ± 47 and 98 ± 53 respectively (P0.05). No statistic difference existed between the CD34(+)CD59(+) cells of PNH patients and the normal control CD34(+)CD59(+) cells. P-STAT5 MFI was elevated after the stimulations of EPO and TPO. The increments of CD34(+)CD59(+) cells in PNH patients were significantly higher than those of CD34(+)CD59(-) cells (49 ± 11 and 54 ± 43 vs 17 ± 4 and 16 ± 6, both P0.01).Under the in vitro stimulations of EPO and TPO, the STAT5 phosphorylation levels of EPO and TPO receptors in normally cloned hematopoietic stem cells in PNH patients are obviously superior to those in abnormally cloned counterparts.

To explore the effects of CD8(+)CXCR3(+)T cells on autoimmune hemolytic anemia (AIHA).Twenty-two AIHA patients, including 11 untreated and 11 recovered ones, and 23 normal controls were recruited from July 2010 to November 2010. The percentage of CD8(+)CXCR3(+)/CD8(+)T cells in peripheral blood and the expression of interleukin-10 (IL-10) in CD8(+)CXCR3(+)T cells were detected by flow cytometry. Their correlations with the count of CD3(+)CD4(+)cells and the percentage of CD5(+)CD19(+) in CD19(+) B cells were analyzed. The expression level of CXCR3 mRNA in PBMC was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).The percentage of CD8(+)CXCR3(+)/CD8(+) of untreated AIHA patients was (39.80 ± 19.96)%. And it was lower than that of recovered patients [(58.76 ± 14.22)%, P0.05] and normal controls [(59.66 ± 12.62)%, P0.01]. The percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells of untreated patients was (22.98 ± 14.96)% and it was lower than that of normal controls [(38.15 ± 17.03)%, P0.05]. The expression level of CXCR3 mRNA for untreated AIHA patients was (0.51 ± 0.19) and it was lower than that of normal controls (1.67 ± 1.17, P0.01). The percentage of CD8(+)CXCR3(+)/CD8(+)T cells had a negative correlation with the count of CD3(+)CD4(+) cells and the percentage of CD5(+)CD19(+)/CD19(+) B cells (r = -0.571, -0.583, both P0.05). So did the percentage of IL-10(+) T cells in CD8(+)CXCR3(+)T cells (r = -0.524, -0.523, both P0.05).The decreased count of CD8(+)CXCR3(+)T cells and the lowered level of IL-10 may disturb the immune tolerance and lead to the occurrence of AIHA.

To analyze the percentage and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cells in the pathogenesis of SAA.By flow cytometry, the percentages of NK cells (CD3(-)CD56(+)CD16(+)) and its subsets [CD3(-)CD56(bright)CD16(-)(CD56(bright)), CD3(-)CD56(dim) CD16(+)(CD56(dim)), CD3(-)CD56(-)CD16(+)] in peripheral blood lymphocytes were detected in 12 untreated patients, 30 recovered patients and 13 normal controls respectively from April 2010 to December 2010 in our hospital. NK cells activating receptors (NKG2D and NKp46), perforin and granzyme-β of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte and reticulocyte absolute value in peripheral blood, and hyperplasia degree, percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes absolute value in bone marrow were evaluated.(1) The percentages of NK cells (10.30% ± 6.08%) and CD56 bright cells (0.11%) in untreated patients were significantly lower than those of recovered patients (16.47% ± 8.29%, 0.68%, both P0.05) or normal controls (19.45% ± 6.88%, 0.53%, both P0.05). The percentage of CD56(dim) cells in untreated patients was significantly lower than that of normal controls (9.62% ± 6.04% vs 18.21% ± 7.16%, P0.05). The percentage of CD3(-)CD56(-)CD16(+) cells was significantly higher in recovered patients than that of untreated patients or normal controls (0.79% vs 0.37%, 0.41%, both P0.05). (2) The expression of NKp46 and perforin of NK cells in untreated (88.23%, 64.97% ± 21.61%) and recovered patients (82.97%, 66.14% ± 20.73%) were significantly higher than those of healthy controls (40.99%, 42.11% ± 27.25%, all P0.05). (3) The percentage of NK CD56(bright) and CD3(-)CD56(-)CD16(+) cells of patients was positively correlated with ANC% (r = 0.423, 0.609, 0.468 respectively, all P0.05) and the percentage of granulocytes in bone marrow (r = 0.357, 0.517, 0.434 respectively, all P0.05). The percentages of NK, CD56(bright), CD56(dim) and CD3(-)CD56(-)CD16(+) cells were positively correlated with the hyperplasic degree of bone marrow (r = 0.455, 0.412, 0.404, 0.451 respectively, all P0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow (r = -0.522, -0.435, -0.411, -0.547 respectively, all P0.05). The expression of NKG2D, NKp46, perforin and granzyme-β of NK cells had no correlation with hematopoiesis (all P0.05).The lowered percentage of NK CD56(bright), CD56(dim) cells and a higher expression of perforin may cause the over-function of T lymphocytes and thus lead to hematopoietic failure in SAA.

This study was purposed to investigate the BCL-2/IgH gene rearrangement in major break point region (MBR) and IgH gene rearrangements of patients with non-Hodgkin's lymphoma (NHL), and explore their significance for improving early diagnosis and accurately evaluating chemotherapy effect. DNA for BCL-2/IgH and IgH gene assays was extracted from bone marrow mononuclear cells in 70 cases of lymphoma (60 cases of B-NHL and 10 cases of T-NHL), 7 cases of lymph node inflammatory and 20 healthy controls. The BCL-2/IgH, IgH gene rearrangements were assayed by polymerase chain reaction (PCR), the assayed results were compared with results of pathological biopsy; the factors related with occurrence of these 2 kinds of gene rearrangement were analyzed and the dynamic changes of BCL-2/IgH and IgH gene rearrangements after chemotherapy were compared, the chemotherapy effect was evaluated. The results indicated that (1) BCL-2/IgH gene rearrangement in bone marrow mononuclear cells was observed in 10 cases out of 30 DLBCL cases (33.3%), and was more frequent than that in 30 other B-NHL cases (6.7%), 10 T-NHL cases (0%), 7 lymph nodes inflammatory cases (0%) and 20 healthy controls (5%) (p0.05). (2) the quantity of rearranged BCL-2/IgH gene of 8 DLBCL cases reduced from 0.59 to 0.16 (p0.05) after 2 courses of R-CHOP chemotherapy and completely disappeared after 6 courses of R-CHOP chemotherapy. (3) 81.8% patients with BCL-2/IgH gene rearrangement showed high serum LDH level, while it was observed in 28.6% patients without this gene rearrangement (p0.05). Lymphoma staging, systemic symptoms, β(2)-MG level, bone marrow involvement, infiltration of liver and spleen were not significantly correlated with BCL-2/IgH gene rearrangement. (4) IgH gene rearrangement was found in 9 cases out of 20 DLBCL patients (all newly diagnosed patients) (45%), IgH rearrangement was observed in 14 cases out of 30 other B-NHL (all newly diagnosed or relapsed patients, except patients with DLBCL) (46.7%) and there was no statistical difference between these 2 groups, however IgH rearrangement all were not observed in 20 healthy persons, 10 T-NHL cases and 7 lymph nodes inflammatory cases. (5) the quantity of rearranged IgH gene in 7 DLBCL cases was reduced from 0.42 to 0.13 after one course of R-CHOP chemotherapy (p0.05) and completely disappeared after 2 courses of R-CHOP chemotherapy. (6) 90% patients with IgH gene rearrangement had high serum LDH level, while it was found in 30% patients without this gene rearrangement (p0.05). Lymphoma staging, systemic symptoms, β(2)-MG levels, bone marrow involvement, infiltrations liver and spleen all were not significantly correlated with IgH gene rearrangement. It is concluded that the BCL-2/IgH and IgH gene rearrangements may be used as specific indicators in early diagnosis and accurate evaluation of therapy efficacy in B-NHL, these 2 kind of rearrangement correlate with LDH level. The BCL-2/IgH gene rearrangement is more specific for in DLBCL.

To detect the abnormalities of differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes (MDS).Forty-five newly diagnosed MDS cases from July 2008 to March 2010 in our hospital and 30 normal controls were enrolled. There were 17 low-risk and 28 high-risk patients. The CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor (SCF-R), erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptors (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry.The mean percentage of CD34+ in karyocyte of MDS cases in high-risk patients [0.53% (0.10%-1.68%)] was significantly higher than that of control group [0.13% (0.08%-0.32%), P0.01]. The mean percentages of CD34+CD38+ cells were significantly lower in low and high-risk groups (86.3%±8.5% and 82.6%±11.1%) than those in control group (92.3%±3.4%). And the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group (13.7%±8.5% and 17.4%±11.0%) than that in control group (7.7%±3.4%, both P0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells than that in CD34+CD38- cells (18.7%±18.3% vs 63.6%±20.0%, P0.01). The expressions of SCF-R, G-CSFR and TpoR were not significantly different between two cell populations. The expressions of EpoR on CD34+CD38+ cells of low and high-risk MDS groups [9.0% (1.4%-12.7%), 5.2% (1.1%-14.1%)] were significantly lower than those of control group [9.6% (5.1%-30.1%), both P0.05]. The expressions of G-CSFR on CD34+CD38+ cells of low and high-risk MDS groups (29.8%±19.1%, 28.7%±21.1%) were significantly lower than those of control group (44.4%±23.4%, both P0.05). The quantities of EpoR on CD34+CD38- cells of low and high-risk MDS groups (42.2%±21.9%, 25.7%±15.6%) were significantly lower than those of control group (63.6%±20.0%, both P0.01). The expressions of TpoR on CD34+CD38- cells of low and high-risk MDS groups (5.4%±4.7%, 4.1%±4.0%) were significantly lower than those of control group (10.1%±8.3%, both P0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells was higher than that of MDS with high expression rates.The abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS are associated with MDS cytopenia and may be useful for the diagnosis of MDS.

To detect the abnormalities of CD(34)(+) cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS).Fifty newly diagnosed MDS (17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD(34)(+)CD(38)(+), CD(34)(+)CD(38)(-) bone marrow cells and bone marrow cell cycle were measured with flow cytometry.The mean percentages of CD(34)(+) cells in bone marrow karyocyte of high risk [(2.29 ± 2.17)%] and MDS-AML groups [(18.69 ± 17.47)%] were significantly higher than that of control group [(0.36 ± 0.49)%, P0.05]. The mean percentages of CD(34)(+)CD(38)(+) cells were significantly lower in low risk, high risk and MDS-AML groups [(86.09 ± 7.79)%, (81.68 ± 11.82)% and (82.88 ± 2.60)%, respectively] than that in control group [(92.21 ± 3.85)%, P0.05], thus the percentages of CD(34)(+)CD(38)(-) cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [(13.91 ± 7.79)%, (18.32 ± 11.82)% or (17.13 ± 2.60)%, respectively] than that in control group [(7.79 ± 3.85)%, P0.05]. The percentages of CD(34)(+)CD(38)(-) cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r = 0.493, P = 0.001) and WHO Adapted Prognostic Scoring System (WPSS) (r = 0.586, P = 0.000) scores. The percentages of bone marrow mononuclear cells (BMMNCs) in G(0)/G(1) phase of in low risk, high risk and MDS-AML groups [(94.52 ± 4.32)%, (96.07 ± 3.88)% and (94.65 ± 4.55)%, respectively] were significantly higher than that in control group [(88.94 ± 7.30)%, P0.01], thus the percentages of BMMNCs in S and G(2)/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P0.05). MDS patients with low percentages of CD(34)(+)CD(38)(-) cells presented higher therapeutic efficacy than those with high percentages of CD(34)(+)CD(38)(-) cells, while without significant differences (P0.05).There are abnormalities of differentiation of CD(34)(+) bone marrow cells and high proportion of G(0)/G(1) cells which indicates a G(1) phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD(34)(+) bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.

To observe the efficacy and safety of linezolid for the treatment of gram positive coccus infections in hematological disease patients.One hundred and two hematological disease patients with suspected or proven gram positive coccus bacteria infection were enrolled in this study. Linezolid was given at a dosage of 600 mg, iv, q12h. The mean treatment period was (10.82 ± 5.12) days (1 to 51 days) with 74.5% over 7 d and 51.0% over 10 d.Among 102 patients, 57 were male, 45 female aged 11 to 81 years, with a mean of (45.26 ± 19.15) years. Ninety four cases were nosocomial infection (92.2%) and 8 community infection (7.8%); There were pneumonia in 80 (78.4%), septicemia in 11 (10.8%), and infection of other organsin 11 (10.8%); Forty five cases were proven gram positive coccus bacteria infection, and 57 were suspected infection; Fifty one bacteria strains were isolated from cultivated samples of proven patients, in which 22 were staphylococcus aureus with 19 methicillin resistant 13 hemolytic streptococcus, 9 staphylococcus epidermidis with 7 methicillin resistant 6 enterococcus faecom, and 1 enterococcus hirae. Seven cases were mixed with one kind gram negative bacillus infection, 4 mixed with two kinds of gram negative bacillus infection, and 12 mixed with fungal infection; Total clinical response rates by ITT (intention to treatment) analysis was 69.6%, in which 40 (39.2%) were curative and 31 (30.4%) obviously effective; PP (per-protocol) analysis was 70.9%, in which 39 (41.9%) were curative and 27 (29.0%) obviously effective. Bacteria clearance rate was 70.6%, and in this group the clinical effective rate was 88.9%; Adverse effect rate was 2.9%, being transient thrombocytopenia and increased transaminase.Linezolid is a safe and effective antibiotic used in hematological disease patients complicated with infections of gram positive coccus.

To investigate the effects and related factors of itraconazole in the treatment of invasive fungal infection (IFI) in the patients with blood diseases (BD).A total of 156 BD patients with IFI treated with itraconazole in General Hospital, Tianjin Medical University from 2005 to 2008, were retrospectively analyzed.Of these patients, 92 were with underlying malignant BD, and 64 with non-malignant BD; 77 possible IFI, and others proven IFI. A total of 94 (63.5%) patients were responded to itraconazole successfully, while 54 (36.5%) failed. The underlying malignant BD, post-chemotherapy, neutrophil count less than 0.5×10(9)/L, positive fungus culture, and bacteria infection were related with the response to itraconazole significantly, while patient's age, application of other antibiotics, positive G test, IFI localization, haemoglobin level and platelet counts were not. Five patients was changed other anti-IFI therapy because of side effects, including gastrointestinal ill (3 cases with nausea or vomiting) and tachycardia (2 cases).Itraconazole was effective and safe in the treatment of IFI in the patients with BD. Underlying malignant BD, agranulocytosis, bacteria infection, and delayed anti-IFI therapy might reduce itraconazole therapeutic effects.

Recent studies have shown that interleukin-3 receptor alpha (CD123) is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis. The expression of CD123 may also be high in patients with myelodysplastic syndrome (MDS). In this study, the expression and clinical significance of CD123 and granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of patients with MDS were investigated to explore the molecular marker of the malignant clone of MDS.Forty-two patients with MDS, who were diagnosed in the Hematological Department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study. Fluorescence activiated cell sorter (FACS) was used to measure the expression of CD123 on CD34(+)CD38(-) cells and CD114 on CD34(+) cells of the bone marrow of these patients and controls and the clinical significance was analyzed. The expression of CD114 on CD123(+)CD34(+)CD38(-) cells was further measured to explore the molecular marker of the malignant clone in MDS.MDS patients displayed significantly higher proportion of CD34(+)CD38(-)/CD34(+) ((14.03 +/- 5.27)%) than normal controls ((7.70 +/- 4.36)%, P0.05). The expression rate of CD123(+)CD34(+)CD38(-)/CD34(+)CD38(-) was significantly higher in MDS patients ((48.39 +/- 28.15)%) than that in normal controls ((8.75 +/- 11.71)%, P0.01). The expression level of CD123 was significantly correlated with the proportion of bone marrow blasts (r = 0.457, P0.05). The expression rate of CD114(+)CD34(+)/CD34(+) was lower in MDS patients ((33.05 +/- 21.71)%) than that in normal controls ((38.99 +/- 19.07)%) but was not statistically significant (P0.05). The expression of CD114 on CD123(+)CD34(+)CD38(-) cells ((34.82 +/- 29.58)%) was significantly lower than that on CD123(-)CD34(+)CD38(-) cells ((53.48 +/- 27.41)%) of MDS patients (P0.05).MDS patients displayed higher proportion of CD34(+)CD38(-)/CD34(+) than normal controls. CD123 was highly expressed in the bone marrow of the patients with MDS, significantly correlated with the proportion of bone marrow blasts, and thus might be the marker of MDS malignant clone. CD123(+)CD34(+)CD38(-) cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responds worse to G-CSF in vitro and in vivo.

This study was purposed to investigate the immune state of T cells, the quantity and function of GPI(+) T cells and GPI(-) T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH). 22 cases of PNH and 18 normal controls were enrolled in this study. Their T lymphocyte subsets, Th lymphocyte subsets were assayed by flow cytometry with the monoclonal antibodies concerned. The proportion of GPI(+) T cells or GPI(-) T cells in CD3(+) T cells, CD4(+) T cells, CD8(+) T cells and the expressions of CD69 on these T cells were also respectively assayed. The results showed that the proportion of CD4(+) T cells in CD3(+) T cells in PNH [(47.7670 +/- 13.91139)%] was lower than that in controls [(54.9592 +/- 7.11678)%] (p0.05). CD8(+) T cells in CD3(+) T cells of PNH cases [(52.2767 +/- 13.90395)%] were higher than that of controls [(45.2418 +/- 6.75306)%] (p0.05). The ratio of CD4(+) T cells to CD8(+) T cells was reverse in PNH. Those were more significantly in PNH-AA (0.77763 +/- 0.409153) (p0.05). The proportion of Th1 cells in PNH [(16.9136 +/- 6.78899)%], especially in PNH-AA [(22.8000 +/- 5.45244)%], was significantly higher than that in controls [(4.4600 +/- 1.81879)%] (p0.05). The proportion of Th2 cells in PNH [(4.7582 +/- 1.98441)%] had no difference from controls [(3.7960 +/- 1.13810)%]. The number of GPI(-) T cells in CD8(+) T cells and CD4(+) T cells were (14.6797 +/- 11.96718)% and (3.9241 +/- 2.46263)% respectively. The expression of CD69 on GPI(+) T cells or GPI(-) T cells in PNH [CD8(+) GPI(+) T cells (17.67881 +/- 8.562493)%, CD8(+) GPI(-) T cells (15.86575 +/- 7.279743)%, CD4(+) GPI(+) T cells (4.65431 +/- 1.984378)%, CD4(+) GPI(-) T cells (4.93181 +/- 1.730001)%]was significantly higher than that in normal controls [CD8(+) GPI(+) T cells (4.68038 +/- 1.216645)%, CD4(+) GPI(-) T cells (1.77339 +/- 0.645259)%] (p0.05), but the expression of CD69 on GPI(+) T cells was not different from that on GPI(-) T cells in PNH. It is concluded that high function of cytoimmunity in PNH may be responsible for bone marrow failure but not relates to the existence of PNH clone in T cell population.

To detect the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients.Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality and the prognosis of MDS cases were analyzed.The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+ (0.39 +/- 0.34) and CD16-/CD16+ (1.33 +/- 0.77) were significantly higher in high-risk MDS group than those of control group (0.07 +/- 0.05 and 0.39 +/- 0.31 respectively) (P0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ 3.88% +/- 3.07%, CD11b-HLA-DR- 16.23% +/- 15.59%, CD16-HLA-DR- 41.12% +/- 24.53%, CD11b+CD16- 33.53% +/- 17.26% and CD13+CD16- 44.51% +/- 21.99% granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r = 0.690, P = 0.000) and WPSS (WHO adapted prognostic scoring system) (r = 0.651, P = 0.000) scores.There is an abnormal expression of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity is correlated with the prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

To explore the expression of antigen activated of macrophages (MPhi) of bone marrow and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells (BMMNC)-Coombs test (immunorelated pancytopenia, IRP).Sixty-one IRP patients, 10 severe aplastic anemia (SAA) patients and 13 healthy controls were enrolled in this study. The categories of auto-antibodies (IgG, IgM) on BMMNC (CD(34)(+)/CD(15)(+)/GlycoA(+) hematocytes), the quantity (CD(68)(+)/CD(45)(+))% and expression of antigen activated (CD(69)) of MPhi (CD(68)(+)CD(69)(+)/CD(+)(68))% in bone marrow of all cases and controls were measured by fluorescence activated cell sorting (FACS).The quantity and expression ratio of activated antigen of bone marrow (BM) MPhi in IRP patients [(0.57 +/- 0.30)% and (40.30 +/- 18.49)%] were respectively significantly higher than those in SAA [(0.46 +/- 0.08)% and (32.44 +/- 19.37)%] and healthy controls [(0.44 +/- 0.69)% and (29.71 +/- 11.67)%] (both P0.05). The quantity presented high-positive correlation with the expression ratio of activated antigen of BM MPhi (r = 0.89, P0.01). Patients with IRP were classified into two subgroups according to the quantity of MPhi: Group A (MPhi/= 0.5%, 34 cases) and Group B (MPhi0.5%, 27 cases). Thirty-two cases (94.12%) were with auto antibody (IgG) in Group A, while only 2 (7.41%) with auto antibody (IgG) in Group B. There was significant difference in expression ratio of activated antigen of BM MPhi between Group A (49.19 +/- 16.63)% and Group B (29.11 +/- 14.30)% (P0.05), but no difference was found between Group B and the control group (P0.05). Total curative rates at 3 and 6 month (47.06% and 79.41%) of Group A were better than those of Group B (22.22% and 51.85%). Thirty-four IRP patients with autoantibody (IgG)(+) were divided into two subgroups according to the quantity of MPhi: high level group (/= 0.75%, 9 cases) and low level group (0.75%, 25 cases), 24 cases (96%) in MPhi low level group were found auto-antibody (IgG) on one hemotopoietic cell lineage, 1 on two lineages, while 8 (88.89%) in MPhi high level group were detected auto-antibody (IgG) on two cell lineages, and 1 on three cell lineages. Expression ratio of activated antigen (56.12 +/- 15.11)% was much higher in MPhi high level group than that in MPhi low level group (44.58 +/- 18.16)% (P0.05). The count of red blood cell concentration of hemoglobin and platelet in peripheral blood in MPhi high level group were respectively lower than those in MPhi low level group, while the percentage of Ret, the level of total bilirubin and indirect bilirubin, the ratio of erythroid of sternal bone marrow in MPhi high level group were higher than those in MPhi low level group.The expression of activated antigen of BM MPhi was enhanced in IRP especially with auto-antibody (IgG), which might be involved in damage process of hemotopoietic cell.