Extent of agreement between the body fluid model of Sysmex XN-20 and the manual microscopy method.

BACKGROUND: Although the correlations concerning cellular component analysis between the Sysmex XN-20 body fluid (BF) model and manual microscopy have been investigated by several studies, the extent of agreement between these two methods has not been investigated. METHODS: A total of 90 BF samples were prospectively collected and analyzed using the Sysmex XN-20 BF model and microscopy. The extent of agreement between these two methods was evaluated using the Bland-Altman approach. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic accuracy of high-fluorescence (HF) BF cells for malignant diseases. RESULTS: The agreements of white blood cell (WBC), red blood cell (RBC), and percentages of neutrophils, lymphocytes, and monocytes between the Sysmex XN-20 BF model and manual microscopy were imperfect. The areas under the ROC curves for absolute and relative HF cells were 0.67 (95% confidence interval [CI]: 0.56-0.78) and 0.60 (95% CI: 0.48-0.72), respectively. CONCLUSION: Due to the Sysmex XN-20 BF model's imperfect agreement with manual microscopy and its weak diagnostic accuracy for malignant diseases, the current evidence does not support replacing manual microscopy with this model in clinical practice.

[Pathogen Identification for An Imported Case with African Trypanosomiasis].

Objective: To perform laboratory diagnosis for an imported case of human African trypanosomiasis and identify the pathogen. Methods: Clinical and epidemiological information was collected. Blood and cerebrospinal fluid samples were collected, stained with Wright-Giemsa, and microscopically examined. Genomic DNA from the blood samples was amplified with primers specific for Trypanosoma sp. expression site-associated gene (ESAG), Trypanosoma brucei gambiense specific glycoprotein (TgsGP) and 18S rRNA（M18S-Ⅱ-Tb） gene, and Trypanosoma brucei rhodesiense specific serum resistance associated （SRA） gene. Complete blood count, blood chemistry, and CSF examination were also conducted. Results: The patient had a 4-month history of lower extremity weakness and swelling of surface lymph nodes. Physical examination showed somnolence, and occasional emotional abnormalities, accompanied by anemia (hemoglobin 85 g/L), electrolyte disturbance (sodium 124 mmol/L; chlorine 87 mmol/L) and significantly increased nonspecific immune globulin protein （globulin 63 g/L）. Epidemiological survey showed that the patient suffered insect bites and stings for several times during his work in the Republic of Gabon in Africa. Microscopic examination revealed flagella of trypanosome in peripheral blood. PCR amplification produced bands of 286, 308, and 150 bp with primers specific for ESAG, TgsGP and M18S-Ⅱ-Tb, respectively. Conclusion: The patient was diagnosed with Trypanosoma brucei gambiense infection from the clinical information, epidemiological history, etiology and PCR results.

[Preliminary effect of VEGF promoter-driven recombinant adenovirus containing double suicide genes on apoptosis of human gastric carcinoma cells].

OBJECTIVE: To study the effect of the adenovirus containing CD/TK fusion gene controlled by the human vascular endothelial growth factor (VEGF) promoter on apoptosis of human gastric carcinoma cells SGC-7901. METHODS: VEGF-expressing SGC-7901 cells were infected by the recombinant adenovirus Ad-VEGFP-CD/TK, and the infection efficiencies were observed with fluorescence microscopy. The toxic effect and intracellular calcium concentration induced by 5-fluorocytosine (5-FC) and ganciclovic (GCV) were determined by light microscopy, electron microscopy and flow cytometry. RESULTS: The transfection efficiency of the recombinant adenovirus in SGC-7901 cells increased with the viral titer. At the multiplicity of infection (MOI) of 100, 5-FC and GCV could induce apoptosis of SGC-7901 cells within a given dose range in a dose- and time-dependent manner, and apoptotic changes of the cells were observed with electron microscopy. Apoptotic peak was also detected by flow cytometry. Cell cycle analysis revealed increased cell percentage in G(0)-G(1) phase and decreased percentage of cells in G(2)-M and S phases in response to treatment with the pro-drugs, which also induced marked elevation of intracellular calcium concentration in the infected cells. CONCLUSIONS: CD/TK fusion gene system driven by VECF promoter selectively induces apoptosis of VEGF-expressing SGC-7901 cells, the action of which is probably mediated by intracellular calcium variation.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells.

AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector. METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001). CONCLUSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells.

AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR-CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured. RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.