Clinical Significance of Peripheral Blood EBV-DNA Determination and Genotyping in Lymphoma Patients / 中国实验血液学杂志

OBJECTIVE@#To explore the clinical significance of Epstein-Barr virus（EBV） detection and classification in peripheral blood of lymphoma patients.@*METHODS@#101 lymphoma patients were enrolled, the clinical characteristics of the patients were collected, including ages, sex, types of lymphoma, Ann Arbor stages, extranodal infiltration and lactate dehyhrogenase. Fluorescent quantitative PCR technology was used to detect the EBV-DNA. Polymerase chain reaction and Agarose gel electrophoresis was used for determination of EB genotyping. The difference between curative effect in EBV-DNA+ and EBV-DNA－ patients, the correlation of adverse factors and EBV infection of the patients were analyzed.@*RESULTS@#68.3% (69/101) of the patients showed EBV-DNA positive. EBV-positive lymphoma patients showed more adverse prognostic factors than the patients with EBV-negative, which may lead to poorer disease outcome. Among the 46 B-cell non-Hodgkin's lymphoma patients, the overall response rate of EBV-positive patients (60.7%) was lower than EBV-negative patients(88.9%) (P<0.05); For 19 patients with Hodgkin's lymphoma, the overall response rate of EBV-positive patients (46.2%) was lower than EBV-negative patients (100%), the differences were statistically significant (P<0.05). Among 69 patients with EBV-infected lymphoma, 98.6% (68/69) showed type-2 EB virus, and 1.4% (1/69) were type-1 and type-2 mixed infections.@*CONCLUSION@#Most of EBV-positive in lymphoma patients were EBV type 2, patients with EBV-DNA+ shows poorer efficacy than EBV-DNA－ patients.

Detection of JAK2V617F and CALR Gene Mutations by Multiplex of Patients with Myeloproliferative Neoplasms PCR-Capillary Electrophoresis / 中国实验血液学杂志

OBJECTIVE@#To evaluate the proformance of multiplex PCR and capillary electrophoresis(MPCE) in the detection of JAK2V617F and CALR mutation in myeloproliferative neoplasms(MPN).@*METHODS@#The specificity primers of JAK2617F gene mutation and the primers of CALR gene were designed at the same time. The JAK2V617F and CALR gene primers were labeled with Cy5 fluorescence, all the primers were mixed in one tube for multiplex PCR and the PCR prodcuts were analysised by capillary electrophoresis. Then detection limit and sensitivity of MPCE were evaluated, and compared with comercial diagnostic kit.@*RESULTS@#JAK2V617F and CALR gene mutations could be detect by MPCE in one PCR test. JAK2V617F mutation could be detected at 0.01 ng genomic DNA, double positive JAK2V617F and CLAR gene mutations could be detected at 0.1 ng genomic DNA, at least 0.1% JAK2V617F positive mutation could be detected. The consistency between MPCE and commercial diagnostic gene mutation kit was 100%.@*CONCLUSION@#It is developed that a new gene mutation detection method of JAK2 V617F and CLAR gene based on MPCE in our experiment and it can be used as a new reagent for molecular diagnosis of MPN patients.

Expression and Clinical Significance of Serum LRG1 in Patients with Diffuse Large B-Cell Lymphoma before Treatment / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of serum LRG1, LDH and β2-MG in the recurrence of diffuse large B-cell lymphoma(DLBCL) after chemotherapy.@*METHODS@#The serum levels of LRG1, LDH and β2-MG of 80 patients with DLBCL were detected before treatment and followed up for these patients was performed. The cut-off value of non-recurrent survival was determined by ROC analysis. The correlation of three serum markers for predicting recurrence with prognostic factors of diffuse large B-cell lymphoma patients after treatment was analyzed by ROC curve.@*RESULTS@#The serum levels of LDH, LRG1 and β2-MG were higher in the groups with high tumor stageing, extranodal invasion and bone marrow involvement, respectively(P＜0.05). The optimal cut-off values for predicting recurrence risk determined by ROC analysis: LDH 402.37 U/L, LRG1 1.81 mg/L and β2-MG 168.3 ng/L, respectively.COX multivariate regression analysis showed that serum LRG1 was an independent factor affecting the recurrence of diffuse large B-cell lymphoma(P＜0.05).@*CONCLUSION@#The serum level of LRG1 may become a new biological marker to predict the recurrence risk of diffuse large B-cell lymphoma.

Efficacy of Quercetin-sensitized Adriamycin for Treatment of Refractory Acute Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the chemotherapeutic efficency of quercetin sensitized adriamycin.@*METHOD@#CCK-8 was used to detect the inhibitory effect of different doses of adriamycin, quercetin and quercetin combined with adriamycin on the proliferation of primary leukemia cells from patients with clinically refractory acute leukemia. Quercetin, adriamycin and their combination were used to treat non-irradiated T-ALL leukemia mice to observe the changes of survival curve and myocardial injury.@*RESULT@#There was no significant difference in the inhibition rate of primary leukemia cell proliferation between the adriamycin concentration group (6, 0.6 and 0.06 μg/ml) and the adriamycin half-dose (3, 0.3 and 0.03 μg/ml) plus quercetin (0.25 mmol/L) group at three different time points (24, 48 and 72 hours). There was a significant difference in the inhibition rate of primary leukemia cell proliferation among the drug concentration groups, and the inhibition rate of primary leukemia cell proliferation was time-and concentration-dependent (r=0.995、r=1.000、r=0.984、r=0.993、r=0.999、r=0.960). In vivo experiments showed that the survival time of non-irradiated T-ALL leukemia mice treated with low-dose adriamycin combined with quercetin was not significantly prolonged compared with the high-dose adriamycin treatment group. The survival time of non-irradiated T-ALL leukemia mice treated with high dose of adriamycin and quercetin was significantly prolonged (P＜0.05). Compared with adriamycin group, the SOD activity in adriamycin combined with quercetin group increased significantly and the MDA content decreased. The results of transcriptome sequencing analysis showed that the expression of Ighv1-84 and Igkv6-14 in adriamycin combined quercetin group and quercetin group was lower than that in adriamycin group. The Ms4a1, Podx1, Mecom, Sh3bgr12, Bex4 and Tdrp expression in adriamycin combined quercetin group and adriamycin group were higher than that in quercetin group, while Crabp1 expression was lower.@*CONCLUSION@#Quercetin can inhibit the proliferation of primary leukemia cells in a time-dependent manner. Quercetin combined with adriamycin inhibit the proliferation of primary leukemia cells significantly, and had synergistic and additive effects on the proliferation of primary leukemia cells, and the inhibiting effect of quercetin combined with adriamycin is concentration-and time-dependent. Quercetin combined with high-dose adriamycin can significantly prolong the survival time of non-irradiated T-ALL leukemia mice and reduce the myocardial damage caused by adriamycin.

External Quality Analysis of Quality Indicators on Specimen Acceptability / 现代检验医学杂志

Objective To analyze the status of quality indicators(QI) on specimen acceptability and establish preliminary qual ity specification.Methods Web based External Quality Assessment system was used to collect data of laboratories partici pated in "Medical quality control indicators in clinical laboratory" from 2015 to 2017,including once in 2015 and 2017 and twice in 2016.Rate and sigma scales were used to evaluate incorrect sample type,incorrect sample container,incorrect fill level and anticoagulant sample clotted.The 25th percentile (P25) and 75th percentile (P75) of the distribution of each QI were employed to establish the high,medium and low specification.Results 5 346,7 593,5 950 and 6 874 laboratories sub mitted the survey results respectively.The P50 of biochemistry (except incorrect fill level),immunology and microbiology reach to 6σ.The P50 of clinical laboratory is 4 to 6σ except for incorrect sample container.There is no significant change of the continuous survey results.Based on results in 2017 to establish the quality specification,the P25 and P75 of the four QIs is 0 and 0.084 4 ％,0 and 0.047 6 ％,0 and 0.114 2 ％,0 and 0.078 4 ％,respectively.Conclusion According to the results of the survey,most laboratories had a faire performance in biochemistry,immunology and microbiology,and clinical laboratory needs to be strengthened.Laboratories should strengthen the laboratory information system construction to ensure the actual and reliable data collection,and make a long time monitoring to achieve a better quality.

Influence of Mouse Spleen Microenviroment on T-ALL Progression / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the influence of spleen on disease status of mouse T-ALL.</p><p><b>METHODS</b>The leukemia cells were transplanted into the mice, then the development levels of leukemia cells in different organs of transplanted mice were monitored at different time points after transplantation; the transplanted leukemia cell level in different organs was detected by flow cytometry at different time points after transplantation; the survival of transplanted mice was analyzed by means of splenectomy.</p><p><b>RESULTS</b>The spleen change displayed most severely in process of T-ALL, the number of T-ALL cells in the spleen obviously increased at initial period. The detection of organs showed that along with the progression of leukemia, spleen weight change was the most significant, following by the lever change. The splenectomy test showed that the spleen played a promotive role in progession of T-ALL, and the spleneetomy could difinitely postpone the progression of T-ALL in mice, there was significant difference between splenectomy and non-splenectomy.</p><p><b>CONCLUSIONS</b>In early stage after transplantation of T-ALL cells, the spleen has the promotive effect on function of T-ALL cells, which suggests that the spleen may be a important microenvironment for T-ALL cell migrating into body.</p>

Quercetin enhances the anti-leukemic effect of adriamycin / 中国实验血液学杂志

This study was purposed to explore the anti-leukemic mechanism of quercetin (Que) in vivo and it enhancing chemotherapeutic effect of adriamycin (ADR) by establishing the quercetin-treated P388 transplanted nude mouse model. The P388 leukemic cells in logarithmic growth phase were taken and injected subcutaneously into BALB/c nude mice so as to establish the leukemia-transplanted nude mouse model. The model mice were treated by quercetin, ADR and their combination, and the survival changes of model mice were observed, the hemogram and peripheral blood cell count examination were performed regularly; the cell cycle was detected and the influence of quercetin on cell proliferation was analyzed by flow cytometry; the caspase-3 protein expression level was detected by ELISA; the mRNA and protein changes of NF-κB, BCL-2, BAX were measured by real-time quantitative flourascence PCR and Western blot respectively. The results indicated that the quercetin and adriamycin could significantly prolong the survival of P388 leukemia nude mice, and their combination displayed significantly prolonged effect. Quercetin and adriamycin alone or in their combination could reduce the ratio of G0/G1 phase in mice, the cell ratio in S phase and G2/M phase increased, and the effects of the combination group were more significant than that of the single agent groups. Quercetin could activate caspase-3 and promote leukemic cell apoptosis. Meanwhile, quercetin could down-regulate the expression of BCL-2 and NF-κB gene, and up-regulate the expression of BAX gene. It is concluded that through modulating the expression of apoptosis-related genes, the quercetin can inhibit leukemia cell proliferation, promote apoptosis, and enhance the chemotherapeutic effects of adriamycin. These results provide some valuable data for further research and development of quercetin as a new and effective anti-leukemic drug.

Effects of quercetin on multidrug resistance and expression of related genes in human erythroleukemic K562/a cells / 中国实验血液学杂志

The study was aimed to investigate the effect of quercetin, flavonoid molecules on reversing leukemia multidrug resistance and its mechanism. K562/A cells were cultured in vitro with different concentrations of quercetin. Cell growth inhibition and adriamycin (ADR) sensitivity were detected by MTT method. Intracellular ADR concentration was determined by flow cytometry. Cell apoptosis was assayed by Annexin V/PI staining method. The expressions of drug transporter and apoptosis related genes were measured by real-time PCR array. The results indicated that quercetin inhibited the proliferation of K562 and K562/A in 5-160 µmol/L and with dose-dependent manner. Quercetin increased the sensitivity of K562/A cells to ADR in a low toxicity concentration. Flow cytometry showed that the quercetin increased the accumulation of ADR in K562/A cells when cells were co-cultured with 5 µmol/L ADR for 2 hours. Quercetin could induce the apoptosis of K562 and K562/A cells with dose dependent manner. Furthermore, some drug transport related genes such as ATP-binding cassette (ABC) and solute carrier (SLC) and some apoptosis-related genes such as BCL-2, tumor necrosis factor (TNF), tumor necrosis factor receptor (TNFR) families were down-regulated by quercetin. It is concluded that quercetin reverses MDR of leukemic cells by multiple mechanisms and the reversing effect is positively related to drug concentration.

Apoptosis-inducing effect of quercetin and kaempferol on human HL-60 cells and its mechanism / 中国实验血液学杂志

The purpose of this study was to explore the anti-leukemia effect of quercetin and kaempferol and its mechanism. The HL-60 cells were used as the leukemia models. The inhibitory effects of quercetin and kaempferol on growth of HL-60 cells was assessed by MTT assay. The effect of quercetin and kaempferol on cell cycle in HL-60 cells was detected by flow cytometry. The cytotoxic effect of these 2 drugs was analysed by single cell electrophoresis assay. Western blot analysis was used to study the apoptotic mechanism of HL-60 cells. The results showed that the quercetin and kaempferol had a significant anti-leukemia effect in vitro. The proliferation of HL-60 cells was significantly inhibited in dose-and time-dependent manners after treating with quercetin (r = 0.77) and kaempferol (r = 0.76) respectively, and the cytotoxicity of quercetin was superior to that of kaempferol. The quercetin and kaempferol induced G(2)/M arrest and apoptosis of HL-60 cells. The quercetin and kaempferol could down-regulate the survivin expression. It is concluded that the quercetin and kaempferol have significant anti-leukemia effect in vitro. Furthermore the apoptosis-inducing effect of quercetin is stronger than that of kaempferol, both of which induce apoptosis of HL-60 cells through depressing cell growth, arresting cell cycle and inhibiting expression of survivin.

Reversal effect of berbamine on multidrug resistance of K562/A02 cells and its mechanism / 中国实验血液学杂志

This preject is to explore the reversal efficacy of calmodulin antagonist berbamine (BBM) on multidrug resistance (MDR) and its mechanism. Human erythroleukemic cell line K562 and its adriamycin-resistant counterpart K562/A02 were used in the study. The cells were co-cultured with ADR and BBM in different concentrations. MTT assay was used to analyze the effect of BBM on cell growth inhibition. According to the MTT assay, the 50% inhibitory concentration (IC(50)), the multiples of drug resistance and increased sensitivity of ADR were calculated. The concentration of intracellular ADR and expression level of P-gp were detected by flow cytometry (FCM). The expression level of mdr1 mRNA and survivin mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with beta-actin as internal reference. The results showed that IC(50) of ADR in K562 and K562/A02 cells was 1.16 +/- 0.09 micro mol/L and 37.47 +/- 1.76 micro mol/L, respectively. The resistant multiple of K562/A02 cells to ADR was 32.30 higher than that of K562 cells. BBM increased the chemo-sensitivity of ADR in K562/A02 cells with dose-dependent relationship, i.e. when 5, 10 and 20 micro mol/L BBM was added in the culture the chemo-sensitivity of ADR was increased to 2.01-, 9.68-, and 41.18-fold (P < 0.01), respectively. After treating K562/A02 cells by 5 or 10 micro mol/L BBM for 2 hours the accumulation of intracellular ADR was increased to 1.41- and 1.52-fold (P < 0.01), respectively. Treating by BBM for 72 hours decreased 4.12% (P < 0.05) and 27.09% (P < 0.01) of P-gp expression, respectively, meanwhile down-regulated expression of mdr1 mRNA and survivin mRNA was found. In conclusion, BBM could increase intracellular concentration of ADR in K562/A02 that down-regulated expression level of mdr1 mRNA and P-gp and survivin so that the sensitivity of K562/A02 to ADR was increased significantly.