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 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 construct a recombinant lentivirus containing human beta defensins -3 ( hBD3 ) , connective tissue growth factor gene (CTGF) and enhanced green fluorescent protein (EGFP), and to detect its translation in rabbit bone marrow mesenchymal stem cells (BMSC).Methods The lentivirus containing hBD3, CTGF and EGFP genes was constructed in vitro.The titer of lentivirus was tested with end-paint dilution assay .Rabbit BMSCs were transfected with recombinant virus.The best value of multiplicity of infection (MOI) was tested.The expression condition, transfection efficacy and genetic stability of the target genes were evaluated by using fluorescence microscopy and flow cytometry . Western blotting was used to detect the expression of the target protein .Results Recombinant lentivirus vectors: Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained . The titer of the recombinant lentiviruses was 3.21 ×108, 5.80 ×108, and 1.16 ×109, respectively.The best MOI value to transfect BMSCs was 150. The transfection efficacy of these lentivirus vectors was high , reaching 79.72%as assessed by flow cytometry , and it could be stably inherited .Western blotting displayed that target protein expression was successful .Conclusion The construction of recombinant lentiviruses carrying hBD3 and CTGF genes is successful and can be effectively transfected into BMSCs .

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.

ObjectiveIn an attempt to clarify the usefulness of combined nerve and muscle biopsy in the diagnosis of neuromuscular disease when compared with traditional sural nerve biopsy.Methods Fifteen biopsies of superficial peroneal nerve (SPN) and peroneus brevis muscle ( PBM ) by one incision performed within one neurological clinic were reviewed.All patients had peripheral neuropathy while 3 of them had myopathy clinically.The diagnostic significance of SPN and PBM biopsies were classified into 3 grade: essential,helpful,no value.ResultsOf 15 SPN and PBM biopsies,7 showed essential pathological findings whichreachedthe etiologicaldiagnosis, including 5definitevasculitis, 1inflammatory demyelinating polyneuropathy and 1 amyloid neuropathy.Five biopsies are helpful for etiological diagnosis,including demyelinating neuropathy,mild inflammation,and microvascular lesion,et al.Three biopsies are of no value for etiological diagnosis which only have nonspecific change such as type 2 fiber atrophy,neurogenic atrophy and axonal degeneration et al. Finally,SPN and PBM biopsies made the definite etiological diagnosis possible in 12 patients.ConclusionsSPN and PBM biopsy improved the yield of specific pathological and etiological diagnosis of neuropathy and myopathy such as vasculitis and amyloidosis with minor trauma and side effect.Further clinical and pathological studies will be necessary for a better practice of combined nerve and muscle biopsy.

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 explore the relationship of depth of trophoblastic invasion with trophoblast cell activity and serum β-hCG according to the expression of proliferation antigen Ki-67 which viewed as an indicator of cell proliferation activity.Methods Fallopian tube specimens collected from 108 patients who underwent operation treatment for fallopian tubal pregnancy in our hospital were investigated by light microscopic examination.They were divided into three groups according to the depth of trophoblastic infiltration: Ⅰ group (stage): trophoblastic invasion of tubal mucosa,Ⅱ group(stage): trophoblastic invasion of the muscularis,Ⅲ group(stage): trophoblast invasion of serosa layer(muscularis penetration).The expression of Ki-67 was detected by SP method and blood β-hCG was detected within 2 hours of preoperative.The level of β-hCG,the expression of Ki-67 and the depth of trophoblast invasion were analyzed.Results Mean level of serumβ-hCG in Group Ⅰ,Ⅱ and Ⅲ were(1416.64 ± 859.94)U/L,(3380.33 ± 2392.36)U/L and(6999.33 ± 4949.90)U/L respectively.Positive expression rate of cell proliferation antigen Ki-67 in Group Ⅰ,Ⅱ and Ⅲ were 21.95％,53.66％ and 6.40％ respectively.There were significant difference on the expression of Ki-67 between group Ⅰ and group Ⅱ,group Ⅱ and Ⅲ group,group Ⅰ and group Ⅲ(x2 =3.94,4.07,4.35,respectively,P ＜ 0.05).The serumβ-hCG level also displayed statistics difference in the three groups(F =9.914,P ＜ 0.01).The positive expression of Ki-67 and serum β-hCG level were positively correlated with each other(r =0.678,P ＜ 0.05)Conclusion The high level of the serum β-hCG indicates high expression of Ki-67 and deeper trophoblast invasion of tubal wall.

Objective To investigate the environmental impact of the Japanese Fukushima No.1 nuclear power plant accident on radiation levels in some areas of Liaoning province.Methods The emergent monitoring was performed by detecting atmosphere aerosols,precipitations,drinking water,vegetables,milk and seafood by gamma spectrometry analysis and gross activity measurements.Results The fission radionuclides of 131I,134Cs and 137Cs were detected in atmosphere aerosols 20 d after nuclear accident.The rad ionuclides of 131I,134Cs and 137Cs were found existing in atmosphere for 25,4 and 6 d,respectively,with the highest concentrations of 4.6 × 10 3,2.9 × 10-4 and 4.2 × 10-4 Bq/m3,respectively.The man-made fission radionuclides could not be detected in vegetablcs,drinking water and milk from Shenyang city and seafood from Dandong city.Conclusions The atmosphere might be slightly contaminated in Liaoning province due to the nuclear accident,whereas the vegetables,milk and drinking water not contaminated.

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.