Objective To analyze the immunophenotyping characteristics of myeloid leukemia in transgenic mouse.Methods According to differential antigen expression profile on various hematopoietic lineages,flow cytometric analysis of bone marrow sample was performed on 5 myeloid leukemia mice and 10 healthy BL6 mice.Cell cycle analysis was further performed to assess cell proliferation.Results Expressions of Mac-1+ Gr-1+ and c-Kit+ in bone marrow cells in transgenic leukemia mice were(72.6±6.5)% and (20.5±4.8)%,and it were significantly higher than those in normal mice[(52.8±4.8)% and(2.1±0.3)%](t=6.66,12.66,P＜0.01).And expressions of B220+,CD3+,CD41+ and Ter119+ in leukemia mice were(2.7±1.1)%,(1.2±0.3)%,(1.2±0.6)% and(2.8±1.1)%,respectively.It were significantly lower than those in normal mice[(20.2±2.1)%.(6.6±1.3)%,(4.7±1.1)% and(10.6±1.2)%](t=-17.63,-8.69,-6.30,-12.28,P＜0.01).The percentages of S phase and G2/M phase in leukemia mice were(25.7±4.2)% and(21.1±4.2)%,respectively.It were significantly increased as compared with normal mice[(11.8±2.1)% and(8.9±1.8)%](t=8.59,7.98,P＜0.01).Conclusions Immunophenotyping of myeloid leukemia in transgenic mouse was characterized by hish expression of myeloid specific marker(Mac-1 and Gr-1)and hematopoietic stem/progenitors cells specific marker(c-Kit),and by low expression of B-lymphoid specific marker(B220),T-lymphoid(CD3),megakaryocyte(CD41)and Erythroid(Ter119).

Objective To inhibit the expression level of SALI4 in AML cell line THP-1 and investigate its potential effects on pathogenesis of leukemia. Methods AML cell line THP-1 was transfected with plasmids that expressed small interfering RNA targeting SALL4. The samples were divided into 4 groups:(1) blank group: samples with not any treatments; (2) control group: cells with empty pRS vector alone;(3) test1 group:cells with SALL4-shRNA-pRS-1 plasmid transfection complex; (4) test2 group:cells with SALL4-shRNA-pRS-2 plasmid transfection complex. The expression levels of SALL4 mRNA and protein were measured by real time fluorescence quantitative PCR and WB. C-myc, Cyclin D1 and β-catenin were important components of Wnt/β-catenin signaling pathway and their expression levels in SALL4 knockdown THP-1 cells were detected by real-time fluorescence PCR. Furthermore, THP-1 apoptosis was analyzed by flow cytometry after Annexin V-PI staining. Results Real time fluorescent quantitative PCR illustrated that the expression of SALL4 in testl group, test2 group, control group and blank group were ( 36. 0 ± 4. 3 ) %,(32. 0 ± 2. 4) %, ( 102. 0 ± 6.5 ) % and ( 100. 0 ± 2. 6 ) % respectively. There was statistical significance ( F = 226. 3, P < 0. 05 ). The expression of SALL4 in testl and test2 group respectively were significant lower than that in blank group (t = 19.7,19. 1, P<0. 05). The expression of SALL4 had no significant difference between blank group and control group (t = 1.1, P ＞0. 05). Western blot analysis revealed SALL4 protein in testl and test2 group were significantly decreased compared with those of control and blank group. All above data indicated the high efficiency of RNA interference targeting SALL4. Comparing with the blank group, the relative expression of C-myc, Cyclin D1 and β-catenin mRNA in test1, test2 and control group were(44.0 ±6.2)%,(44.0 ±5.1)% and (107.0±13.6)%;(22.0±4.5)%,(25.0±3.5)% and (48.0 ± 7. 6 ) %; ( 42.0 ± 3.5 ) %, ( 59. 0 ± 3.7 ) % and ( 79. 0 ± 5.6 ) %. The expression of C-myc,β-catenin and Cyclin D1 mRNA in testl and test2 group were significant lower than that in blank group (t = 10. 1,9. 5, 23. 3, 22. 9; 17.4, 12. 4; P < 0. 05). The percentage of apoptotic cells in group of test1,test2,control, blank were (57.2 ±9.1)%, (34.4 ±8.6)%, (14.4 ±3.6)% and (14.8 ±4.8)%respectively. There was statistical significance ( F = 42. 5, P < 0. 05 ). After the inhibition of SALL4, the percentages of apoptotic cell in testl and test2 group were significantly increased( t =9. 7, 4. 5 ;P <0. 05).Conclusion The inhibition of SALL4 in leukemia cell line THP-1 downregulates the expression of cell proliferation related genes such as C-myc, Cyclin D1,β-catenin and promoted apoptosis.

Objective To study the clinical significance of a novel marker of platelet clumps count provided by hematology analyzer in differentiating true thrombocytopenia from EDTA-dependent pseudothrombocytopenia (EDTA-PTCP). Methods Samples from 65 cases of thrombocytopenia (including 15 EDTA-PCTP samples and 50 random samples of true thrombocytopenia) and 50 healthy controls were analyzed using hematology analyzers, and samples with low platelet counts were checked by replacing citric acid and using manual microscope observation to identify true thrombocytopenia from EDTA-PTCP. A novel marker of platelet clumps count was used to differentiate the two diseases for samples anficoagulated with EDTA or citric acid. Results In 65 patients with thrombocytopenia, platelet counts were (48±11)×109/L detected by automatic hematology analyzers. Fifty of 65 cases were true thrombocytopenia which showed low platelet counts [(48±10)×109/L by automated analyzer and (46±11)×109/L by manual assay]. No significance was observed between them (t=-1.26, P0.05). Platelet clumps counts were 86±15. No platelet clamps were detected under microscope. The other 15 cases were EDTA-PTCP [platelet counts were (48±12)×109/L and platelet clumps counts (840±184) were increased significantly by automated analyzer and using EDTA anticoagulant] which showed obviously platelet clumps and no less platelet counts under microscope. After replacing citric acid, platelet counts [(141±13)×109/L by automated analyzer and (134±17)×109/L by manual microscope assay] were increased significantly. No significance was observed between them (t=-1.29, P0.05). Platelet clumps counts (75±12) were decreased obviously compared with EDTA anticoagulant method (t=-6.82, P<0.001). No platelet clumps were detected under microscope. Conclusion Platelet clumps counts may be a useful clinical indicator for monitoring of platelet aggregates, especially for EDTA-PTCP caused by platelet clumping.

Objective To detect the expression of SALL4 in patients with acute myeloid leukemia (AML) and analyze its potential clinical significance. Methods Reverse transcription polymerase chain reaction and Real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) was used to examine SALLA expression in peripheral blood mononuclear cells (PBMCs) of 68 cases of AML including 36 cases in acute phase and 32 cases in remission phase, 30 healthy controls, Kasumi-1 cells and THP-1 cells. Then, flow cytometry, bone marrow smear and automated hematology analyzer were used to analyze the relationship between the SALL4 expression and blast cell counts in the bone marrow, peripheral white blood cell (WBC) counts, peripheral large unstained cell (LUC), CD34 in blast cells. Further, the change of SALL4 level during pre-chemotherapy, chemotherapy (2nd w to 3rd w) and remission were investigated in 5 AML cases. Results The level of SALL4 expression in patients with AML in acute phase [69.01 (17.20-120.28)] was 26-fold and 61-fold high compared with that in remission phase [2.64(1.35-5.41)] and in healthy control [1.14(0.50-1.62)] (Z=-6.48,-6.83,P<0.01). The level of SALL4 expression in remission phase was 2.3-fold high compared with that in healthy control (Z=-3.61 ,P<0.01). The expression level of SALL4 was decreased along with efficient chemotherapy in 5 AML cases in which SALL4 expression level was 79.74 (33.76-89.09), 7.19 (5.97-20.21) and 3.40 (1.44-15.53) during pre-chemotherapy, chemotherapy (2nd w to 3rd w) and remission, respectively. In groups of abnormal increased counts of blast cell, peripheral LUC% and CD34%, expression of SALL4 [33.82 (16.00-144.01), 30.70(23.75-72.50) and 56.25(23.79-153.81), respectively] were higher than that in groups of normal counts [2.74 (1.59-5.13), 5.71 (2.52-22.40) and 20.82 (14.03-55.12), respectively ] (Z=-4.64,-2.18,-3.66,P<0.01 or P<0.05). The expression of SALL4 in the group of increased WBC counts [89.26(23.75-154.34)] was higher than that in the group of normal WBC counts [3.86(2.03-6.01)] and the group of decreased WBC counts [6.66(2.51-17.06)] (Z=-4.91,-4.21,P<0.01). The level of SALL4 expression was positively correlated with blast cell counts in bone marrow and peripheral WBC counts (r=0.45,0.40,P<0.01). Conclusions FQ-RT-PCR method can be used successfully to detect the expression of SALL4,and the expression of SALLA may be useful to predict disease progression of AML.

Objective To investigate the appropriate setting up of normal reference ranges of lymphocyte subsets in some flow cytometry laboratories and to study the effects of different flow cytometers and various reagents by different manufacturers on the analysis of peripheral blood lymphocyte subsets. Methods Three FCM labs (named A, B and C) in Beijing region were selected representing 3 commonly used flow cytometers (Beckman Coulter Epics XL, Beckman Coulter Cytomics FC500, BD FACS Calibur). 50 samples from healthy donors were distributed to 3 labs and tested according to individual lab's standard operating procedure to verify whether the normal reference ranges of peripheral blood lymphocyte subsets established were appropriate. The application of internal quality control was also investigated. Commercial blood quality control reagents were given to the 3 FCM labs and tested within 20 working days paralleled with routine samples. In addition, 20 patients' samples were prepared using 4 different combinations of reagents ( a , b , c and d). The results from combination a, which used the Beckman Coulter reagents and instrument, were compared to the results from combination b, c and d, which used reagents from different manufacturers. Then the prepared samples were tested on Beckman Coulter Epics XL to evaluate the effects of different combinations of reagents on the results of peripheral blood lymphocyte subsets analyzed by the same instrument. Furthermore, 24 patients' samples prepared by same reagents from Beckman Coulter company were tested on both Beckman Coulter Epics XL and BD FACS Calibur respectively to assess the effects of different instruments on peripheral blood lymphocyte subsets. 20 patients' samples prepared by same reagents and instruments were analyzed by Beckman Coulter Epics XL analytic system and BD FACS Calibur analytic system respectively to assess the effects of the two analytic systems on the lymphocyte subsets. Results Over 10% of the results for NK and T4/T8 in lab A as well as T4 in labs B and C fell outside of their normal reference ranges. The probabilities exceeding corresponding normal reference ranges were 16% ( 9/50 ), 24% ( 12/50 ), 22% (11/50) and 12% ( 6/50 ), respectively. The results using internal blood quality control in 3 FCM labs within 20 working days were all within the reference ranges of the quality control provided by the kit. The biases from b and c reagent combinations were substantial compared with that of reagent a combination. Among the biases from b and c reagent combinations, the lowest probability of bias exceeding 10% was T8 of combination b, which had probability of 70% (14/20). The highest probabilities of hias exceeding 10% were T3 and T4 of b and c reagent combinations, which reached 100% (20/20) . Furthermore, the biases of T3, T8 and B of d reagent combination compared with that of reagent a combination were also substantial. The probabilities of bias exceeding 10% were 35% (7/20) ,85% (17/20) and 75% (15/20), respectively. Comparing the results of samples prepared and analyzed by reagents and instruments from different manufacturers to that of samples prepared and analyzed by the same company's reagents and instruments showed that there were great discrepancies in T3, T4 , T8 , B and NK. The probabilities of bias exceeding 10% were 71% ( 17/24), 80% (19/24) ,38% (9/24), 33% (8/24) and 92% (22/24), respectively. The biases of T8, NK and B were substantial when compared the results from Beckman Coulter Epics XL analytic systems and BD FACS Calibur analytic systems. The probabilities of bias exceeding 10% were 55% (11/20 ), 70% ( 14/20 ) and 55% (11/20), respectively. Conclusions FCM labs should set up their own normal reference range for peripheral blood lymphocyte subsets. The normal reference range should be verified periodically. It is important to apply internal blood quality control regularly and accumulate the quality control results. The reagents and instrument for preparing peripheral blood samples should be from the same manufacturers.

Objective To evaluate the biological variations of 8 lymphocyte subsets using flow cytometric double-platform method.Methods Twenty healthy adults were recruited from Peking Union Medical College Hospital in September 2013.At 8:00 AM,12:00 PM,and 4:00 PM on days 1,3,and 5,venous blood was collected from the volunteers.The percent and absolute lymphocyte subset counts were measured using duel-platform method.The sample collection and handling techniques were standardized.Before each batch analysis,the instrument quality controls were performed using the same lots of reagents.The intra-individual coefficient of variation (CVI) and inter-individual coefficient of variation (CVG) were calculated by nested ANOVA with SPSS 13.0 software.The analytical coefficient of variation (CVA),index of individuality (Ⅱ) and reference change value (RCV) were calculated by Excel2003.A mean pairwise comparison was determined by one-way ANOVA and variance analysis.The values between groups were analyzed by independent sample t test.Results For T cells (CD3 +),helper T cells (CD3 + CD4 + CD8-),suppressor T cells (CD3 + CD4-CD8 +) and B cells (CD3-CD19 +),the intra-individual coefficient of variation (CVI) and inter-individual coefficient of variation (CVG) were 0.03,0.06,0.05,0.14 and 0.12,0.16,0.23,0.31 respectively,which were all similar to those in previous studies.However,the Ⅱ and RCV of the four lymphocyte subsets were very different from those in previous studies,which were 0.26,0.40,0.22,0.44 and 8.77,16.86,14.93,39.69,respectively.Moreover,variations in absolute count,CVI,CVG,and analysis coefficient of variation (CVA) of all 8 lymphocyte subsets were greater than those of relative count.Variations in the percent and absolute counts for the CD3 + CD4-CD8-,CD3 + CD4 + CD8 +,and CD3+ CD16+ CD56+ cell subsets were relatively high.The CVI,CVG and CVA for the cells of CD3 + CD4-CD8-were 0.12,0.49 and 0.16.The CVI,CVG and CVA for the cells of CD3+ CD4+ CD8+ were 0.40,0.93 and 0.55.The CVI,CVG and CVA for the cells of CD3 + CD16 + CD56 + were 0.28,1.11 and 0.16.Conclusions Investigation on the CVI,CVG and CVA may allow us to obtain Ⅱ and RCV,by which we can determine the utility of traditional population based reference ranges.Documentation of the RCV indices may be used as objective delta-check values in quality management and decide whether clinical significance existed in the continuously detected results.

Objective To investigate the correlation between RDW and disease activity in patients with IBD and evaluate clinical significance of RDW as a potential indicator to monitor IBD activity. Methods 256 patients with IBD were divided into two groups. One was UC group including 136 patients with 80 active period cases and 56 emission period cases. Another was CD group including 120 patients with 75 active period cases and 45 emission period cases. 60 bacillary dysentery diseases and 80 healthy controls were selected as bacillary dysentery group and healthy control group. RDW, Hb, WBC, PLT, CRP, ESR, MCV were tested and monitored with development of disease at different stages. We compared RDW with CRP,ESR, PLT, Hb, MCV parameters. By ROC curve analysis, the sensitivity and specificity of RDW was estimated in identifying the IBD's activity. Results The mean values of RDW in active UC group, remission UC group, bacillary dysentery group and control group were ( 16. 1 ± 2. 7), ( 13.5 ± 2. 1 ), ( 13.0 ± 2. 0)and ( 12. 8 ± 1.8), respectively. There was significant difference among four groups ( F = 51.9, P < 0. 01 ).RDW in active UC group was significant higher than that in remission UC group, bacillary dysentery group and healthy control group ( t = 8. 12, 9. 67, 11.85, P < 0.05) and RDW in remission UC group was significant higher than that in bacillary dysentery group and healthy control group as well ( t = 2. 45, 2. 67,P <0. 05). The mean values of RDW in active CD group, remissive CD group,bacillary dysentery group and control group were ( 16. 9 ± 2. 2 ), ( 13. 8 ± 1.1 ), ( 13.0 ± 2. 0), ( 12. 8 ± 1.8 ). There was significant difference among four groups ( F = 113.9, P < 0. 01 ). RDW in the active CD group was significant higher than that in remission CD group, bacillary dysentery group and healthy control group (t = 11.47,18.63,18. 72, P < 0. 05 ) and RDW in remission CD group was also significant higher than that in bacillary dysentery group and healthy control group ( t = 3.60, 3. 72, P < 0. 05 ). RDW in UC and CD groups demonstrated a positive correlation with CRP and ESR (r=0. 484, 0. 525, 0. 286, 0. 358 and P<0. 01, <0. 01, < 0. 05, < 0. 01, respectively) but an inverse correlation with Hb and MCV (r = -0. 378, -0. 271,- 0. 329, - 0. 298 and P < 0. 01, < 0. 01, < 0. 05, < 0. 01, respectively). In UC groups RDW represented a larger area under ROC curve (0. 8.54) compared with CRP, ESR, PLT, Fib and MCV. When the cut-off value of RDW was 14. 0, the sensitivity and specificity for identifying active UC were 82% (65/80) and 72% (40/56) respectively. In CD groups, the area of RDW under ROC curve was the largest (0. 925 )among all indicators. When the cut-off of RDW was 14.5, the sensitivity and specificity for identifying active CD was 88% (66/75) and 82% (37/45) respectively. Conclusion RDW in patients with IBD is a useful indicator to estimate the IBD activity and predict disease progression.

Objective To evaluate the clinical performance of INNOVANCE D-Dimer,and provide information for clinical application.Methods 402 cases of sodium citrate anticoagulant blood were tested with INNOVANCE assay and PLUS assay on CA7000 analyzer to measure plasma D-Dimer levels.VIDAS-30 immunology analyzer was also used to validate the two assays.4 patients with elevated D-Dimer were monitored continuously during 5 days using INNOVANCE assay and PLUS assay respectively,then the consistency of trend between 2 assays was analyzed.Plasma specimens added with hemoglobin,bilimbin and triglyceride were used to verify the anti-interference capability of INNOVANCE D-Dimer assay.Results In 402 specimens,the result ranges of INNOVANCE D-Dimer and PLUS D-Dimer were [2.15 (0.33,8.63)]mg/L FEU and [325.50 (123.75,974.00)] μ,g/L DDU,respectively.The consistency between two assays was poor (Z =-17.375,P =0.000),especially the results in the range of PLUS D-Dimer (201-300) μg/L DDU and (301-400) μg/L DDU,the coincidence rates were only 25％ and 15％,respectively; the coincidence rate was up to 85％ during PLUS D-Dimer (500-600) μg/L DDU; the coincidence rate was close to 100％ when PLUS D-Dimer over 700 μg/L DDU.Totally 47 of 402 cases were unmatched between two assays.Verified by VIDAS 30,83.0％ (39/47) was false negative for PLUS assay,4.3％ (2/47) was false negative for INNOVANCE assay,12.7％ (6/47) was false positive for PLUS assay.There were 5 false positives and 39 false negative for PLUS assay,totally 45 cases; Two false negative for INNOVANCE assay.Four patients with elevated D-Dimer were monitored and the results showed similar trend between 2 assays.For INNOVANCE assay,the capacity of anti-interference to free bilirubin,unconjugated bilirubin,hemoglobin,and triglyceride was up to 217 μmol/L,337 μmol/L,41.04 g/L,18.35 mmol/L,respectively.Conclusions INNOVANCE assay can markedly reduce false negative results of D-Dimer compared with PLUS assay.INNOVANCE D-Dimer has good performance on anti-interference to jaundice,hemolysis and lipemia samples.

Objective:To explore the effect of heparanase (HPSE) on the cell adhesion and invasion ability of hepatoma carcino-ma (HC) cell. Methods:HPSE expressions in human HC cell lines (BEL-7402, HepG2, and HCCLM3) were measured by real-time re-verse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Four recombinant miRNA vectors, pcD-NATM6.2-GW/EmGFP-miR-HPSE (pcDNA-miR-HPSE), were constructed and transfected into HCCLM3 cells. Full-length cDNA of HPSE gene was cloned into pIRES2-EGFP vector and transfected into HepG2 cells. Transfection efficiency was observed with fluores-cence microscope. HPSE expressions in transfected cells were measured by real-time RT-PCR and Western blot analysis. Adher-ence ability was determined with microplate reader, and invasion and migration abilities were detected with Transwell chambers. Re-sults:Both HPSE mRNA and protein relative expression levels were higher in the three types of HC cells than those in normal hepato-cyte (P<0.05). HPSE had the highest expression level in HCCLM3 cells and the lowest expression level in HepG2 cells (P<0.05). All five recombinant vectors met the experimental requirements. The transfection efficiencies were 75%-85%. The four miRNA vectors, pcDNA-miR-HPSE, significantly decreased HPSE expression in transfected HCCLM3 cells (P<0.05), and pcDNA-miR-HPSE-1 showed the best interference effect (P<0.05). Plasmid pIRES2-EGFP-HPSE increased HPSE expression in transfected HepG2 cells (P<0.05). After pcDNA-miR-HPSE-1 was transfected, the HCCLM3 cell adherence rate and the cell invasion and migration numbers dropped by almost 50%(P<0.01). After transfection of pIRES2-EGFP-HPSE, the HepG2 cell adherence rate and the cell invasion and migration numbers increased by nearly 40%(P<0.05). Conclusion:Different HPSE vectors could regulate bi-directionally the adher-ence, invasion, and migration abilities of transfected HC cells. HPSE may be related with adherence aside from invasion of HC cell.

Objective: To investigate the effect of taurine on learning and memory impairment, cytokines secretion in rats intrahippocampally injected with ?-amyloid (A?) 1-40. Methods: SD rats were randomly divided into control group, A? injected group, taurine (0.3g/kg?d, 0.6g/kg?d) groups. The rats were fed with taurine for 7 days, and then subjected to bilateral intrahippocampus injection of A?1-40 or vehicle. Two weeks later, all rats performed Morris water maze test. The contents of IL-6, TNF-? were checked by way of radio-immunity assay for hippocampus samples. Results: Compared with A?model group, the escape latency and distance were significantly reduced in taurine (0.6g/kg?d) group; the ratio of swimming distance in the target quadrant to that in the whole pool of the probe trial; the content of cytokines of IL-6 and TNF-?in hippocampus were reduced significantly. Conclusion: Taurine can effectively attenuate the cognitive dysfunction caused by A?1-40 in rats. The reduced cytokines content in hippocampus might contribute to this effect.