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Objective: To analyze the prognostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, absolute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations., Methods: According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophenotype CLL, 16 with score 4 or 3 were atypical immunophenotype CLL. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, sIg, CD20, CD79b expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations., Results: There was no significant difference between the two groups in sex, age, ZAP-70 and IgVH mutation status (P=0.398, P=0.189, P=0.268 and P=0.131, respectively). The incidence of ALC> or =50 x 10(9)/L, Binet B + C, CD38> or =30% in atypical CLL patients (43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P=0.026, P<0.01 and P=0.026, respectively). The proportion of typical patients (26.8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P=0.022)., Conclusion: There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.

This study was aimed to investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL). CD45/Side Scatter (SSC) gating strategy and multiparametric flow cytometry were used to determine immunophenotype of 143 patients with APL. The immunophenotypic features were compared between newly diagnosed APL patients and relapsed APL patients. 42 patients with HLA-DR(-) (non-APL AML, DR(-)AML) were randomly selected as controls. 31 out of 42 AML patients were CD34 negative, and their immunophenotypes were compared with those in newly diagnosed APL patients. The results showed that (1) CD34 and HLA-DR were both negative in 91.9% of newly diagnosed APL, while the positive rate of CD34 and HLA-DR elevated in relapsed cases (3.0% vs 37.5%, 3.9% vs 37.5%). The positive rate of CD34 in HLA-DR(-) AML group was higher than that in newly diagnosed APL group (23.4% vs 3.0%). The positive level of CD34 in newly diagnosed APL group was lower than that in HLA-DR(-) AML group; (2) the positive rate of CD33 in newly diagnosed APL group was higher than that in other groups (97.0% vs 75.0%, 83.3%, 83.9%), as well as the the positive level of CD33 (p < 0.05). (3) no lymphoid antigen other than CD2 was expressed in newly diagnosed APL group. The positive rate of CD7 was 9.5% in DR(-) AML group and 6.5% in CD34(-)/DR(-) AML group, both were higher than those of newly diagnosed APL group (p < 0.05). It is concluded that the immunophenotyping can provide proof to the rapid diagnosis of APL. For those patients with DR(-) AML, it may be helpful to identify APL depending on following features: low or negative CD34 expression, homogeneous and bright expression of CD33, no lymphoid antigens other than CD2, higher SSC.

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western countries, however, infrequent in the Eastern. It shows a remarkable heterogeneity, with some patients having an almost normal lifespan, others surviving only several years after diagnosis despite intensive therapy. To explore the prognostic significance of CD38 expression in Chinese patients with CLL, multi-parameter flow cytometry was used to detect the expression of CD38 on CD5(+)CD19(+) cells of 147 patients. CD38 expression and its association with some other prognostic factors such as Binet stage, lymphocyte count in peripheral blood, serum lactate dehydrogenase (LDH), beta2-microglobulin (beta2-MG), ZAP-70 expression and cytogenetic abnormalities were analyzed. The Kaplan-Meier method was used to construct survival curves, and results were compared using the log-rank test. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. Out of the 147 CLL patients, positive expression of CD38 was found in 45 (30.6%) cases. CD38-positivity identified a subgroup of CLL patients with aggressive disease of Binet stage at the time of the test (P=0.036). Furthermore, the presence of higher serum LDH and beta2-MG levels at diagnosis was strongly correlated with CD38-positive (P=0.016 and 0.025, respectively). Prognostically unfavorable cytogenetic abnormalities, including 17p13 and 11q22 deletions, were significantly more frequent in CD38-positive patients than in CD38-negative ones (P=0.047 and 0.001, respectively). There was no significant difference between CD38-positive and CD38-negative groups in molecular cytogenetic aberrations of del(6q23), del(13q14), 14q32 translocation, or trisomy 12. In addition, in CD38-positive patients, the percentage of leukemic cells expressing ZAP-70 protein was not significantly higher than in CD38-negative ones (P=0.120). CD38 expression was associated with poor outcome. Patients with positive expression of CD38 had significantly shorter overall survival (mean, 81 months) than patients without CD38 expression (mean, 179 months) (P=0.015). Univariate analysis showed that serum levels of LDH and beta2-MG, del(17p13) and CD38 expression were the significant factors in determining overall survival (OS). Del(17p13) and CD38 expression were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that the patients with high level of CD38 expression had poorer outcome; CD38 was a good predictor of OS in Chinese patients with CLL.

This study was aimed to compare K562 cell proliferation, cell cycle and apoptosis before and after adhesive culture with MSCs of the same and different counts, so as to investigate the effect of human bone marrow mesenchymal stem cells (MSCs) on the growth of K562 cells. Culture system of bone marrow MSCs and co-culture system of K562 cells and BMSCs in vitro were established. And K562 cell proliferation curves were drawn by co-cultured of K562 cells with different counts of BMSCs. Cell cycle and apoptosis were determined by flow cytometry. The results showed that compared with the control, the proliferation of K562 cells cultured with the same amounts of MSCs was inhibited. After co-culture with the different amounts of MSCs, MSCs exhibited a dose-dependent anti-proliferation effect on K562 cells. The percentages of K562 cells in G(1) phase and G(2) phase were higher than those of the control. It is concluded that the normal bone marrow mesenchymal stem cells can inhibit the proliferation, the progress of cell cycle and the rate of apoptosis of K562 cells. As the number of mesenchymal stem cells increased, their anti-proliferation effect on the K562 cells were enhanced in a certain range.

Objective: To investigate lipoprotein lipase (LPL) and serum thymidine kinase (TK) levels in chronic lymphocytic leukemia (CLL) and their correlations with other prognostic factors., Methods: LPL expression level in peripheral blood samples of 58 CLL patients was detected by semi-quantitative reverse transcription PCR (RT-PCR). Serum TK1 level in 39 CLL patients was detected by enhanced chemiluminescence (ECL) and TK monoclonal antibody (Anti-TK mAb). IgVH mutation status was detected by multiplex PCR and sequencing of purified PCR products. The expression of ZAP-70 protein and CD38 were determined by flow cytometry . Panel probes and fluorescence in situ hybridization (FISH) were used to detect cytogenetic aberrations., Results: The median LPL expression level in CLL was 0.26 (0 -6.29), while undetectable in normal controls. LPL expression level was significantly correlated with IgVH mutation status, Binet stages, CD38 and cytogenetic aberrations. Patients with unmutated IgVH genes had higher LPL than those with IgVH mutations (P = 0.010). Patients in Binet stage B and C had higher LPL than those in stage A (P = 0.011). LPL level was higher in patients with CD38 > or = 30% (P = 0.001). Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) than those with favorable cytogenetics (deletion in 13q as the sole abnormality) (P = 0.002). LPL level was not significantly correlated with sex, age, and ZAP-70 protein (P > 0.05). The level of TK1 was higher in CLL patients than that in normal control (P < 0.05). Patients with higher level of absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), unmutated IgVH genes and ZAP-70 had higher levels of TK1 than those with lower level of ALC, LDH, mutated IgVH genes and ZAP-70 (P = 0.018, P = 0.018, P = 0.030 and P = 0.038, respectively). TK1 level had no correlation with sex, age, Binet stages, CD38, and cytogenetic aberrations (P > 0.05)., Conclusions: LPL expression and serum TK1 levels significantly correlate with other CLL prognostic factors and could be predictive markers for IgVH mutation status. LPL and serum TK1 might be applied to the assessment of prognosis in CLL patients.

To investigate the clinical, cellular morphology, immunophenotype, and cytogenetic characteristics of acute myeloid leukemia (AML) which are very similar to the morphological characteristics of prolymphocytic leukemia (PLL), the morphological features of bone marrow cells from patient were observed by light microscope, the immunophenotypes were detected by flow cytometry, the karyotypes were analyzed by conventional cytogenetic method, the hybridization signals were determined by fluorescence in situ hybridization. The results indicated that the clinical features were in accordance with acute leukemia and the immunophenotyping results showed malignant cells originated from myeloid lineage, while the cytomorphology analysis showed that the blastic cells were more like the lymphoid lineage. Trisomy 8 was found in the patient by cytogenetic study, the patient did not show good response to chemotherapy. In conclusion, acute leukemia has high heterogenicity, which could be defined as AML, but more like lymphocytic origination by morphological study. Immunophenotyping analysis could contribute to the final diagnosis of malignant cells.

In order to evaluate the efficiency of rituximab combined with fludarabine, cyclophosphamide and rituximab (FCR) regimen for chronic lymphocytic leukemia (CLL). Five patients with CLL were treated with FCR regimen for 2 - 6 courses. FCR regimen included fludarabine 25 mg/m(2) via intravenous drip at day 2 - 4, cyclophosphamide 250 mg/m(2) via intravenous drip at day 2 - 4 and rituximab 375 mg/m(2) via intravenous drip at day 1. Courses were repeated every 4 weeks. Minimal residual disease (MRD) was determined by multiparametic flow cytometry. The results showed that three patients achieved complete remission, 2 patients achieved partial remission. MRD was negative in two patients. In conclusion, FCR is an effective therapeutic regimen for treating CLL patients and is worth to be used in clinic.

The objective of study was to evaluate the efficiency and safety of bortezomib for the treatment of multiple myeloma. Bortezomib in combination with dexamethasone was administered as first-line treatment in all 7 newly diagnosed patients with multiple myeloma. The patients with refractory myeloma were treated with bortezomib in combination with dexamethasone or with other traditional agents such as mitoxantrone and thalidomide. The results showed that according to the EMBT criteria, out of 7 patients one achieved complete response (CR), five achived partial response (PR) and one achived minor response (MR). The 3 patients with refractory/relapsed myeloma achieved PR (2/3) and MR (1/3). The overall response rate (CR + PR) was 80%. The most frequent adverse events observed were thrombocytopenia in three patients, diarrhea and peripheral neuropathy in one respectively. In conclusion, bortezomib demonstrates efficiency in the treatment of new-diagnosed and refractory/relapsed multiple myeloma, and the side effects from treatment are acceptable and manageable.

Objective: To explore the prognostic significance of p53 and ATM gene deletion in patients with chronic lymphocytic leukemia (CLL)., Methods: Interphase fluorescence in situ hybridization (FISH) and probes of LSI p53 and LSI ATM were used to detect p53 and ATM gene deletion in 80 patients with CLL. p53 and ATM gene deletion and their association with some prognostic factors were analyzed. The Kaplan-Meier method was used to calculate survivals, and results were compared using the Log-rank test. Multivariate COX regression analysis was used to assess associations between survival and potential risk factors., Results: Out of the 80 CLL patients, p53 gene deletion was found in 14 (17.5%) cases, ATM gene deletion in 9 (11.3% ) cases, and both the two genes deletion in 3 (3.8%) cases. There was no significant differences of p53 and ATM gene deletion rates in sex, age, Binet stages, peripheral lymphocyte count, and the levels of LDH, beta2-MG, and ZAP-70. The p53 and ATM gene deletion rates were higher in the group of CD38 high expression than that in the group of low expression (P=0.025 and P=0.001). Among 41 patients who received fludarabine containing protocol, none of the nine cases with p53 and/or ATM gene deletion achieved complete response (CR), while 12 of 32 (37.5%) cases without the gene deletion achieved CR. The survival time was shorter in patients with p53 gene deletion (P<0.01). There was no association between outcome and ATM gene deletion (P=0.556). On multivariate analysis by COX regression, p53 gene deletion and CD38 expression (P=0.014 and P=0.017, respectively) were found to be independent factors in predicting survival., Conclusion: CLL patients with p53 and/or ATM gene deletion had poor therapeutic effect, and hence poor prognosis.

Chronic lymphocytic leukemia (CLL) is the most common adult form of leukemia in the Western world, however, infrequent in the Eastern. It shows a remarkable heterogeneity, with some patients having an almost normal lifespan, others surviving only several years after diagnosis despite intensive therapy. To prospectively explore the prognostic significance of ATM and TP53 deletions in Chinese patients with CLL, interphase fluorescence in situ hybridization (FISH) and probes of LSI ATM and LSI p53 were used to detect ATM and TP53 deletions in 95 patients with CLL. ATM and TP53 deletions and their association with some other prognostic factors such as Binet stage, lymphocyte count in peripheral blood, serum lactate dehydrogenase (LDH), beta2-microglobulin (beta2-MG), CD38 and ZAP-70 expressions were analyzed. The Kaplan-Meier method was used to construct survival curves, and results were compared using the log-rank test. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. Out of the 95 patients with CLL, ATM gene deletion was found in 9 (9.5%) patients, TP53 gene deletion in 16 (16.8%) cases. There were no significant differences between ATM or TP53 deletion and clinical parameters of sex, age, Binet stage, lymphocyte count, LDH, beta2-MG or ZAP-70 expression. However, the frequency of ATM and TP53 deletions were obviously higher in CD38-positive group than in CD38-negative group (P=0.001 and P=0.047, respectively). Among 41 patients received treatment with fludarabine and cyclophosphamide, there were nine patients with TP53 or ATM deletion, and no patient with these cytogenetic abnormalities achieved complete response (CR). Survival analysis showed that the patients with TP53 deletion had significantly shorter survival times than the patients without TP53 deletion. There was no evidence of important association between outcome and ATM gene deletion. Serum levels of LDH and beta2-MG, CD38 expression, and TP53 deletion were the significant factors in determining overall survival (OS). TP53 deletion and CD38 expression were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that ATM or TP53 deletion is associated with high expression level of CD38 and TP53 deletion as a possible prognostic factor in Chinese patients with CLL.

We used two molecular cytogenetic techniques, multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH), to analyze three established lung adenocarcinoma cell lines (A549, H1650, and SPC-A-1) and primary lung adenocarcinoma samples, to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q, and 22q to be commonly overrepresented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be underrepresented. The most common gains were found in 16p13 (in 50% of samples), and 16p13 amplification was associated with relatively poor differentiation and late stage. M-FISH and CGH can be a powerful tool in identification of genomic alterations in lung cancer, as well as in diagnosis. The overrepresented regions may harbor potential candidate genes involved in lung adenocarcinoma pathogenesis.

Objective: To evaluate the frequency and mutation status of IgVH gene expression in patients with chronic lymphocytic leukemia (CLL) in China., Methods: IgVH mutation was detected by multiplex PCR and directly sequencing in 29 CLL patients. IgH somatic hypermutation and mutation site were analysed by IMGT/V-QUEST., Results: Of 29 CLL patients, 21 had IgVH mutation (72%). The most frequently expressed VH gene family was found to be VH3 (55%) followed by VH4 (38%), VH2 (3.5%) and VH7 (3.5%), with no expression of VH1, VH5 and VH6., Conclusions: The expression frequency of IgVH gene families in Chinese CLL patients is significantly different from that in Western CLL patients, suggesting the involvement of ethnic and/or environmental factors in CLL development, which might partly explain the different incidence of CLL between China and Western countries.

Objective: To explore the potential of human bone marrow stromal cells (MSCs) as the feeding-layer to promote ex vivo expansion of cord blood CD34+ cells and engraftment of the expanded cells in NOD/SCID mice., Methods: Human MSCs were routinely isolated and cultured. MSCs at passage 3 were used as feeding-layer for the expansion of cord blood CD34+ cell in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF). The engraftment potential between unexpanded and expanded cord blood cells transplanted into NOD/SCID mice was compared., Results: The total nucleated cells (TNC), CD34 cells and colony forming units (CFUs) in the MSC feeding culture were increased by 111.6-, 19.3- and 58-fold after 1 week expansion and 532.8-, 41.3- and 563.5- fold increased after 2 weeks expansion respectively as compared with that in non MSC feeding culture. In transplant experiment, the percentage of human CD45+ cells (45.3% -59.1%) in bone marrow of recipient mice transplanted with the MSC feeding expanded cells was the highest in all the groups at six weeks after transplantation., Conclusion: Human MSCs enhance CB CD34+ cells in vitro expansion and their capacity of short-term engraftment in NOD/SCID mice.

To investigate the effects of interaction between human bone marrow mesenchymal stem cells (MSCs) and K562 cells on the expression of proangiogenic factor IL-8, the K562 cells were co-cultured in direct contact with MSCs or cultured in MSCs conditioned medium while the controlled K562 cells were cultured alone. The IL-8 gene expression of K562 cells and MSCs was determined by RT-PCR. The results indicated that the expression of IL-8 in K562 cells when co-cultured in direct contact with MSCs or cultured in MSCs conditioned medium was obviously higher than that of K562 cells cultured alone (p<0.01). MSCs co-cultured with K562 cells also had an increased level of IL-8 compared with MSCs cultured alone (p<0.01). It is concluded that the interaction between MSCs and K562 cells via direct contact and the cytokine network promotes expression of IL-8.

To investigate the influence of As2O3, dexamethasone (Dex) and thalidomide (Thal) on apoptosis-induced myeloma cell line U266 cytoplasmic calcium concentrations ([Ca2+]i), U266 cells were incubated in the culture of RPMI 1640 with 15% FBS in 24-well plate and exposed to different concentrations of As2O3, Dex and Thal for 8 hours, respectively, then cell apoptosis was analyzed by fluorescence microscopy and flow cytometry (FCM) with Annexin V-FITC/PI double staining, and cytoplasmic free calcium were detected on FCM through Fluo-3/AM loading. The results indicated that (1) apoptotic cells were gradually increased with enhancement of As2O3, Dex and Thal concentrations; (2) apoptotic cell rates increased from 0.56% in control to 31.54%, 28.35% and 21.97% respectively after treatment with As2O3, Dex and Thal; (3) As2O3, Dex induced U266 cell apoptosis accompanied with raise of [Ca2+]i; (4) [Ca2+]i had no statistically significant changes in Thal-induced apoptotic U266 cells. It is concluded that the raise of [Ca2+]i is one of the mechanisms for As2O3 and Dex-induced U266 cells apoptosis, whereas Thal-induced U266 apoptosis has no significant relation to [Ca2+]i changes.

Objective: To investigate CLLU1 expression and its relationship with clinical stage, expression of CD38 and ZAP-70, and chromosome abnormalities in patients with chronic lymphoid leukemia (CLL)., Methods: Fifty CLL patients were studied. Semiquantitative RT-PCR was performed to detect CLLU1 expression levels; three-color flow cytometry to detect the ZAP-70 and CD38 expression, interphase fluorescence in situ hybridization (FISH) to detect chromosomal aberrations., Results: The expression of CLLU1 mRNA was detected in 26 of the 50 cases (52%), of them 7 cases (26.92%) in Binet A and 19 (73. 08%) in Binet B + C. The expression levels of CLLU1 were significantly higher in Binet stage B + C than in Binet stage A (P < 0. 01). Among 24 CD38+ CLL cases, 17 (70. 83%) expressed high CLLU1 mRNA, whereas in 26 CD38- CLL patients only 9 (34.62%) were CLLU1 positive. The expression of CLLU1 in CD38+ CLL was significantly higher than that in CD38- CLL (P < 0.05). However, no significant difference of CLLU1 expression was found between ZAP-70+ and ZAP-70- patients (P > 0. 05), and between chromosomal aberrations (P > 0. 05)., Conclusions: CLLU1 expression was significantly associated with clinical stage and CD38 expression, and might be an important prognostic factor in CLL patients.

Mantle cell lymphoma (MCL) is a rare group of non-Hodgkin's lymphoma (NHL), which is difficult to discriminate from other subtype of small lymphocytic lymphoma in morphologic appearance. In order to enhance the understanding of MCL, a case of MCL first diagnosed as follicular lymphoma (FL) was reported. The clinical and laboratory characteristics of MCL were analyzed and summarized. The results showed that the findings of flow cytometry (FCM) and immunohistochemical staining technique were compatible with the diagnosis of MCL. Cytogenetic analysis can detect multiple types of chromosomal abnormalities, including t (11; 14). In conclusion, MCL is a disease which diagnosis is difficulty confirmed. Interphase fluorescence in situ hybridization, multiplex fluorescence in situ hybridization, FCM and immunohistochemical staining technique play important roles in the diagnosis of MCL.

The objective of this study was to investigate the immunophenotypic characteristics of T-cell acute lymphoblastic leukemia (T-ALL). Immunophenotyping was performed in 140 T-ALL patients by flow cytometry using a panel of monoclonal antibodies and CD45/SSC gating. The results showed that the T-lineage-associated antigen expressions were CD7 > CD2 > CD3 > CD5 successively. The positive rate of CD10 was 19.42% in patients. Among 140 cases of T-ALL, 12 (8.57%) was accompanied by B-lineage associated antigen expression. Myeloid antigen expression was identified in 31 out of 136 cases (22.79%). None of them expressed CD14 antigen. The positive rate of CD34 was 31.06%. The positive rate of myeloid antigen expression in CD34(+) T-ALL (36.58%) was significantly higher than that in CD34(-) T-ALL (15.38%) (p < 0.01). The expression of CD3 in child T-ALL was higher than that in adult T-ALL, whereas the expression of CD33 in children was lower than that in adults. It is concluded that immunophenotyping is an important tool for diagnosis of T-ALL. Immunophenotypic characteristics of T-ALL is heterogeneous.

IgVH mutation status is one of the most important independent prognostic factor of chronic lymphocytic leukemia (CLL). In order to evaluate IgVH mutation status in patients with CLL, IgVH mutation was detected by multiplex PCR in 9 CLL patients and purified PCR amplification products were directly sequenced, IgH somatic hypermutation and mutation site were analysed by IMGT/V-QUEST. The results showed that 5 patients had mutated IgVH, and IgVHs were IGHV3-11*03, IGHV3-9*01, IGHV3-23*01, IGHV4-59*01, IGHV4-34*02, respectively; whereas 4 others had unmutated IgVH, these IgVHs were IGHV3-53*01, IGHV3-23*03, IGHV3-33*05 and IGHV3-7*01. It is concluded that multiplex PCR is a rapid and easy method to detect IgVH mutation status, and it solves the limitations and pitfalls of routine PCR, and it is worth being extensively used both in clinic and scientific researches.

Background & Objective: The expression of some antigens has been involved in cytogenetic abnormalities in leukemia. This study was to explore the immunophenotypes of acute myelomonocytic leukemia (M4), analyze the correlation of M4 to cytogenetic abnormalities, and provide evidences for the diagnosis and therapy., Methods: Immunophenotypic analysis was performed using a panel of monoclonal antibodies and three-color immunofluorescent staining methods of flow cytometry in 81 patients with M4 leukemia. Cytogenetic records of 73 patients were available, among which 35 were further investigated by dual-color interphase fluorescent in situ hybridization (FISH) and the immunophenotype of inv(16)-positive patients were analyzed., Results: Among the 81 M4 leukemia patients, CD33 (84.0%) was the most commonly expressed antigen, followed by CD13 (81.5%) and CD14 (24.7%). CD34 was detected in 48 (59.3%) patients. T lymphoid-and B lymphoid-associated antigens were expressed in 23 (28.4%) and 10 (12.3%) patients, respectively. All the 11 patients with M4Eo had CD13 expression. CD33, CD34, and CD2 were expressed in 10, 7, and 5 patients, respectively. Of the 14 patients with inv(16), 13 expressed CD13, 11 expressed CD33, 8 expressed CD34, 5 expressed CD14, 3 expressed CD7, and 3 expressed CD2. Of the 73 patients with cytogenetic records, 30 (41.1%) had clonal chromosomal abnormalities. The expression of CD2 and CD34 was associated with karyotypic abnormalities. The expression of CD2 was higher in M4Eo patients, with no correlation to inv(16)., Conclusions: Acute myelomonocytic leukemia mainly expresses myeloid lineage antigens. The expression of CD2 and CD34 is correlated to karyotypic abnormalities. The expression of CD2 is higher in M4Eo. Inv(16) exists in 40% of M4 patients and the expression of all the antigens has no correlation to inv(16).

The study was aimed to investigate the different prognosis of acute myeloid leukemia (AML) with inv (16). A 13-year-old patient diagnosed as M4Eo presenting with bulky lymphadenopathy was reported, the curative process of patients was presented and the related issues were discussed. The karyotype and inv (16) were detected by conventional cytogeneties and fluorescence in situ hybridization (FISH), respectively, the immunophenotype was detected by flow cytometry. The results showed that conventional cytogenetics and FISH analysis revealed inv (16). Induction therapy included idarubicin and cytarabine. After complete remission, patient received consolidation theray containing high-dose cytarabine (HDAC). FISH analysis revealed poor response of patient to HDAC. It is concluded that bulky lymphadenopathy in AML with inv (16) may be a negative prognostic sign. FISH for inv (16) is specific and constitutes an reliable tool to be used for diagnosis and minimal residual disease (MRD).

This study was aimed to investigate the correlation between circulating myeloma cells (CMC) and bone marrow myeloma cells (MMC) in patients with multiple myeloma (MM) and its clinical significance. Four-color flow cytometry was used to detect the percentage of CMC and MMC in 55 patients with MM. Other prognosis-associated factors such as beta(2) microglobulin (beta(2)-MG), serum albumin (Alb), chromosomal abnormalities and renal function were simultaneously analyzed. The patients were divided into four groups: group A, in which CMC and MMC were simultaneously detected; group B, in which only MMC were detected; group C, in which only CMC were detected; group D, in which no myeloma cells were detected in peripheral blood or bone marrow. The results showed that the concentrations of beta(2)-MG and creatinine were significantly increased and Alb markedly decreased in group A as compared with other groups. Statistical differences existed in the above-mentioned factors between patients with myeloma cells detected and not detected. The percentages of CMC or MMC in newly diagnosed, refractory and relapsed patients were apparently higher than those in patients with partial and complete remission, respectively. CMC were strikingly correlated with MMC. It is concluded that the percentages of CMC and MMC not only imply tumor load in MM patients, but also predict the progression of MM, respectively for patients with MM, in those patients CMC and MMC were simultaneously detected.

To evaluate the expressions of proliferative antigen Ki-67 and apoptosis-antagonizing protein Bcl-2 as well as their clinical significance, immunohistochemistry staining with SAP was used to detect Ki-67 antigen and Bcl-2 protein in 18 cases of children with acute lymphoblastic leukemia (ALL) and 43 cases of adults with ALL. The results showed that the levels of Ki-67 and Bcl-2 expression in children with ALL were lower than that in adults, but only Bcl-2 expression had significant difference. Both in children and in adults, the levels of Ki-67 expression in T-ALL and My(+) ALL were higher than that in B-ALL and null-ALL. The highest complete remission rate (CR) was seen in the group with lower expression of both indexes (Ki-67 and Bcl-2). The lowest CR rate was seen in the group with higher expression of both indexes. It is concluded that the levels of Ki-67 and Bcl-2 expression in children and adults with ALL were closely related with the subtype of ALL and chemotherapeutic effects.

Many diseases cause bone marrow necrosis (BMN), especially lymphocytic leukemia. To explore the complexity of the pathogenesis and pathology of BMN and understand the multiplicity of clinical features, a case of Philadelphia chromosome positive (Ph+) B acute lymphoblastic leukemia (ALL) expressing myeloid antigens was reported. The results indicated that the clinical features of this case were complicated and multiplex, the diagnosis was confirmed by using bone marrow smear and biopsy, immunophenotype analysis, conventional cytogenetics and fluorescence in situ hybridization (FISH), the prognosis of patients improved by active treatment for primary disease. In conclusion, the Ph+ B ALL expressing myeloid antigen with BMN is very rare, its diagnosis should be confirmed by using multiple methods, and the active treatments should be performed.

Objective: To explore the diagnostic value of intracellular antibody combination in acute leukemia (AL) expressing cross-lineage cell-surface antigens., Methods: Flow cytometric immunophenotyping using intracellular antibody combination (cMPO/cCD79alpha/cCD3/CD45) was performed additionally in 60 patients who expressed cross-lineage antigens from 269 previously untreated adult AL., Results: Fifty-four of 269 previously untreated adult AL patients who expressed only one kind of intracellular antigen were diagnosed as cross-lineage AL, the percentage of cross-lineage AL in T cell acute lymphoblastic leukemia (T-ALL), B-ALL and acute myeloid leukemia (AML) was 28.6%, 43.6% and 13.4%, respectively. The positive rate of CD7, CD19, CD5 and CD20 in cross-lineage AML was 65.4%, 15.4%, 11.5%, and 7.7%, respectively. The positive rate of CD13, CD33 and CD15 in cross-lineage ALL was 89.3%, 21.4% and 3.6%, respectively. Six (2.3%) patients expressed two-lineage intracellular antigens were diagnosed as biphenotypic AL: 2 of T/B type and 4 B/M (B/myeloid) type., Conclusion: Intracellular antibodies possess lineage specificity and four-color combination flow cytometric immunophenotyping can provide fast and multi-parameter data. To ensure accuracy of the results, CD45/SSC gating and normal cells as internal reference should be used in the immunophenotyping of abnormal cells.

To investigate the expressions of proliferative antigen Ki-67 and apoptosis-antagonizing protein Bcl-2 in patients with chronic lymphocytic leukemia (CLL) and their clinical significance, immunohistochemistry method was used for detection of Ki-67 and Bcl-2 in bone marrow or peripheral blood of 29 patients with CLL. The results showed that the level of Ki-67 expression in advanced stage of CLL was higher than that in early stage of CLL and there was significant difference between these stages of CLL, but there was no significant difference between expression levels of Bcl-2 in two stages. The survival time in the group with Ki-67 expression < or = 8% was longer than that in the group of Ki-67 > 8%, and there was no significant difference of survival time between high and low groups in terms of Bcl-2 expression. It is concluded that detection of Ki-67 antigen and Bcl-2 protein for CLL patients can reflect the status of proliferation activity and apoptosis suppression of leukemia cells in patients; the level of Ki-67 expression closely correlate with the Binet stage and prognosis of CLL.

To explore the ZAP-70 expression in chronic lymphocytic leukaemia (CLL) and its relationship with other prognostic factors, the expressions of ZAP-70 protein and CD38 in bone marrow or peripheral blood of 24 patients with B-CLL were determined by four-color flow cytometry. The results showed that ZAP-70 was positively expressed in 37.5% of all B-CLL patients, 20% (3/15) patients in Binet A stage, and 66.7% in Binet B + C. The expression of ZAP-70 had significant difference between Binet A and Binet B + C (P < 0.05). CD38 was expressed in 29.1% of B-CLL patients and 3 out of these cases were in stage A, 2 out of 3 cases in stage A co-expressed CD38 and ZAP-70. The expression of CD38 had no significant difference between Binet A and Binet B + C stage (P > 0.05). ZAP-70+ and CD38+ were both expressed in 83.3% of B-CLL patients (P < 0.05). It is concluded that ZAP-70 protein can be routinely measured by flow cytometry in the laboratory. High expression of ZAP-70 is correlated with other prognostic factors such as clinic stage, chromosome abnormalities and CD38 in B-CLL.

To explore the efficacy and safety of high-dose of etoposide with granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells, 10 patients with hematologic malignancies including 6 patients with multiple myeloma and 4 with non Hodgkin' s lymphoma received an etoposide dose of 1.6 g/m2. The total dose of undiluted etoposide was given on day 1 as a continuous intravenous infusion via a central vein for 10 hours. G-CSF 5 microg/kg was used on day 3 and given daily subcutaneously until leukopheresis was completed. The results showed that leukopheresis was started at days 11 (range 9-13 days) following etoposide therapy, the mean number of CD34+ cells collected in all 10 patients was 9.4 x 10(6)/kg (range 4.2 - 17.3 x 10(6)/kg), by an average of 2.6 leukophereses (range 1-4) times. Mobilization procedure that produced yields of greater than 4.0 x 10(6)/kg were achieved in every patient. Toxicity showed oropharyngeal mucositis, faucitis and urethritis respectively in 3 patients. It is concluded that high-dose etoposide with G-CSF is an effective and safe mobilizing regimen for autologous peripheral blood stem progenitor cells in patients with hematologic malignancies.

This study was aimed to investigate the cytogenetic changes of MDS cell line (MUTZ-1) with chromosome 5q deletion. R-banding analysis was used to identify chromosome aberrations in MDS cell line and Vysis Spectra Vysion M-FISH was used to further characterize chromosomal complex karyotype. The results indicated that M-FISH exhibited obvious chromosomal aberrations with high frequency including translocation, insertion, breakage and rearrangement, deletion and increasement of chromosome number, the complex karyotype of MUTZ-1 was shown as 50, xx, der (1) t (1;2), ins (1;14), +der (2) t(2;19), der (3) t (3;5), der (3) (3::5::22), 5q-, der (6) t (3;6), der (7) (18::7::17), +8, +der (9) t (1;9), der (10) t (1;10), +11, +12, der (?13) (10::13::5::8), der (14) t (8;14), der (14) t (14, 15), der (15) t (15;21) x 2, +17, +18, -21, -22. It is concluded that M-FISH analysis revealed obvious changes in complex karyotype of MDS cell line MUTZ-1, and the M-FISH technique can increase accuracy of detection for chromosomal complex karyotype, and help diagnosis and prognostic evaluation of MDS.

To explore the effects of proteasome inhibitor PS-341 on the cytokine expressions of mesenchymal stem cells (MSC) in patients with multiple myeloma (MM), MSCs of 11 patients were cultured in medium of RPMI 1640 containing 10% FBS. When cells grew to 5 x 10(5) - 1 x 10(6), cells were exposed to 50 nmol/L PS-341 for 4 hours, then harvested. The expressions of IL-6, IL-1beta and SCF were detected by RT-PCR. The results indicated that after treatment with PS-341 the expressions of IL-6, IL-1beta and SCF of MSCs decreased markedly, especially that of IL-1beta, compared with control (P < 0.05, P < 0.01, P < 0.05, respectively). There were obviously differences of IL-1beta expression between refractory/relapsed group and complete remission (CR) group and IL-1beta expression was inhibited more seriously in CR group, whereas there were no significant differences of IL-6 and SCF expression between two groups; IL-1beta expression of patients treated with PS-341 was not detected; there were not effects of IL-1beta expression on expressions of IL-6 and SCF. It is concluded that proteasome inhibitor PS-341 downregulated the expressions of IL-6, IL-1beta and SCF of MSCs in patients with MM.

This study was aimed at exploring the immunophenotypic features of blasts in patients with myelodysplastic syndromes (MDS). Four-color flow cytometry using conventional and secondary gating strategies was used to immunophenotype blasts and the CD34 positive cells in bone marrow nucleated cells of 29 patients with MDS. The results showed: (1) with progression of MDS from RA/RAS, RAEB to RAEB-T, the proportion of CD34(+) cells were gradually increased from 8.0%, 46.4% to 76.8% (P < 0.05); (2) using CD45 vs SSC gating strategy, with the transformation of RA/RAS, RAEB to RAEB-T, the expression of HLA-DR, CD13, CD33, CD117 were also gradually increased (P < 0.05), and the expression of CD15 was gradually decreased (P < 0.05); (3) using CD45 vs CD34 gating strategy, the expression of HLA-DR, CD13, CD33, CD117 on blasts were higher by secondary gating method than those by conventional gating (P < 0.05). However, there were no significant difference (P > 0.05) in the expression of above-mentioned antigens on CD34(+) cells among different MDS subtypes. It is concluded that conventional gating method can reflect MDS progression from RA/RAS, RAEB to RAEB-T, and secondary gating strategy may accurately reflect the biological features of blasts in MDS. Abnormal expression of CD34 is related to the immaturity level and heterogeneity of blast cells, which is beneficial to the diagnosis of clinically suspected MDS incapable of diagnosing with morphology and cytogenetics.