Objective To explore a quick and feasible method of screening malaria parasite by using a Sysmex XE-2100 hematology analyzer though alarm information on high eosinophil count and atypical eosinophil distributions in the WBC scattergram. Methods Sysmex XE-2100 hematology analyzer was used for complete blood cell analysis. Microscopic review was used when high eosinophil count and atypicaleosinophil distributions in the WBC scattergram were found. If the review showed normal eosinophil cells, wecontinued to focus on red cell for searching malaria parasite. Results Among 1 501 specimens showing higheosinophil counts and atypical eosinophil distributions, nine cases with normal eosinophil cells were indisaccordance with the hematology analyzer, six of them showed high eosinophil count in the Sysmex XE-2100 hematology analyzer, whose distribution was located close to neutrophil clusters in scattergram. The otherthree had an abnormal WBC scattergram. There was no gap between eosinophil clusters and neutrophilclusters, which brought no classified results. But all the nine specimens had been found the trophozoite,schizont and merozoite in blood smears. Conclusions There were great possibility of the existence of themalaria parasite in specimens when hematology analysis showed high eosinophil count and atypical eosinophildistributions in the WBC scattergram in a Sysmex XE-2100 hematology analyzer, although these alarm wasnot comfirmed by microscopic review. This method provides not only a simple and reliable way for quickscreening malaria parasite but also has a great value in preventing the undetected ratio on malaria parasite.

Objective To investigate the changes of CD4+ CD25+ CD127low regulatory T cells (Treg) in peripheral blood samples collected from the patients with cervical cancer and its clinical signifi -cance , and to evaluate the correlations between Treg cells and the infection of high-risk human papillomavir-us ( HR-HPV) .Methods Flow cytometry analysis was performed to measure the percentages of CD4+ CD25+ CD127low Treg cells among CD4+T cells in peripheral blood samples collected from 249 patients with cervical cancer , 30 patients with cervix intraepithelial neoplasia ( CIN) and 60 healthy subjects .The corre-lations between the levels of Treg cells and the clinicopathological features of cervical cancer were analyzed . Flow-through hybridization and gene chip technology ( HybriMax) were used to analyze the genotypes of HPV strains isolated from the 339 subjects.The correlations between Treg cells and HR-HPV infection were ana-lyzed.Results Compared with the healthy subjects , the patients with cervical cancer or CIN showed higher percentages of CD4+ CD25+ CD127low Treg cells and rates of HR-HPV infection (P<0.01).The percentages of CD4+ CD25+ CD127low Treg cells in patients with cervical cancer were significantly correlated with the sta-ges of cervical cancer , which was staged by the Federation International of Gynecology and Obstetrics (FIGO) staging system,and the degrees of differentiation (P<0.05).The percentages of CD4+ CD25+ CD127low Treg cells in the peripheral blood of patients with positive HR-HPV were significantly higher than those in patients without HR-HPV infection (P<0.01).Conclusion The CD4+ CD25+ CD127low Treg cells in peripheral blood might play an important role in the oncogenesis and development of cervical carcinoma , thus it could be used as a potential marker for the evaluation of disease progression .Moreover , the CD4+ CD25+ CD127low Treg cells were closely related to the HR-HPV infection.

Objective To investigate the significance of the combined detection of several serum tumor markers with high-risk human papillomavirus (HR-HPV) in the diagnosis of cervical cancer.Methods Chemiluminescent microparticle immunoassay(CMIA) was employed to measure the levels of serum CA125,CA19-9,SCCA and CEA in peripheral blood samples collected from 249 patients with cervical cancer,30 patients with cervix intraepithelial neoplasia(CIN),and 60 healthy controls.The levels of serum CA72-4,HE4 were measured by electrochemiluminescence immunoassay(ECLI).Flow-through hybridization and gene chip technology (HybriMax) was employed to measure the HPV genotypes in all the 339 cases of women.Results Serum CA72-4 level in the cervical cancer group [3.13 (2.25,7.63) ng/mL] was significantly higher than that in the C IN group [2.37 (1.98,6.25) ng/mL] and the control group [2.69 (2.35,3.35) ng/mL] (P =0.028;P =0.003).Serum CEA level in the cervical cancer group 3.08 (2.28,4.75) ng/mL was significantly higher than that in the CIN group [1.45 (1.00,1.83) ng/mL] and the control group [2.13 (1.45,2.67) ng/mL] (P =0.000;P =0.000).Serum CA 125 level in the cervical cancer group [24.4(20.30,44.15) U/mL] was significantly higher than that in the CIN group[12.85(7.90,16.23)U/mL] and the control group[12.33 (11.26,17.11) U/mL] (P =0.000;P =0.000).Serum CA19-9 level in the cervical cancer group[27.05(18.48,38.01) U/mL] was significantly higher than that in the CIN group[13.01 (8.79,17.12) U/mL] and the control group[12.83 (10.89,17.93) U/mL] (P =0.000;P =0.000).Serum SCCA level in the cervical cancer group [1.7 (0.90,4.75) ng/mL] was significantly higher than that in CIN group [0.8 (0.60,1.10) ng/mL] and the control group [0.6 (0.50,0.70) ng/mL] (P =0.000;P =0.000).However,there was no significant difference of HE-4 among three groups (P ＞ 0.05).The HR-HPV infection rates in the cervical cancer (92.4％) and CIN patients (90.0％) were significantly higher than that in controls (15.0％)(x2 =46.875,P =0.000;x2 =165.178,P =0.000).In the cervical cancer patients,the levels of serum tumor marker was associated with Federation International of Gynecology and Obstetrics (FIGO) stage (P ＜ 0.05).ROC curves showed HR-HPV was the most sensitive in the diagnosis of cervical cancer.The sensitivity of the diagnosis of cervical cancer by serum tumor markers combined with HR-HPV(97.99％) was significantly higher than that of single marker,CA724 25.30％,CEA 20.89％,CA125 27.71％,CA199 24.90％,SCCA 52.61％,HPV 92.37％ (x2 =278.237,P =0.000;x2 =307.036,P =0.000;x2 =263.348,P =0.000;x2 =280.769,P =0.000;x2 =137.864,P =0.000;x2 =8.580,P =0.003).Conclusion Serum tumor markers combined with HR-HPV check can improve the detection rate of cervical cancer and has important clinical value.

Objective To study the pharmacokinetics and detection window of clozapine and its metabolites in human blood, so as to provide experimental basis for forensic cases of identification of clozapine poisoning. Methods 29 Taiyuan Han people's elbow venous blood was collected after given oral administration of 12.5mg clozapine at different time point, in which clozapine and its metabolites were extracted with solid phase extraction (SPE) and determined by HPLC-MS-MS. The qualitative analysis was based on retention time and MRM ions. The quantitative analysis was based on an internal standard method and calibration curve. Using the 3p97 pharmacokinetic software, pharmacokinetic equation of clozapine in the blood were imitated from the C-T data, and pharmacokinetic parameters were calculated. Results The pharmacokinetics of clozapine met a two compartment open model with a first kinetics absorption. The Tmax of clozapine(CLP), demethylclozapine(DMCLP), N-oxidation-clozapine(NO-CLP) respectively were 2.96±1.32h, 8.65±3.00h, 9.31±26.38h; The Cmax of CLP, DMCLP, NO-CLP respectively were 34.68±9.32ng/mL, 11.16±4.15ng/mL, 9.62±13.88ng/mL;The t1/2 of CLP, DMCLP, NO-CLP respectively were 17.02±23.63h, 27.06±12.58h, 41.27±29.75h; The detection window of CLP, DMCLP, NO-CLP respectively were 81.72±26.19h, 93.21±29.40h and 19.93±14.62h. Conclusion The pharmacokinetics of clozapine in blood of Han people is consistent with two compartment open model with a first kinetics absorption. The pharmacokinetics model and parameters of clozapine can provide expirimental basis for forensic identification of clozapine poisoning cases.

Objective To study the safety and efficacy of transurethral resection of bladder tumor(TURBt)under block anesthesia of bilateral obturator nerves.Methods Seventy-seven patients were chronologically divided into two groups.Forty-six of patients with lateral,bilateral or multiple tumors in the bladder,which underwent transurethral resection of bladder tumor under epidural anesthesia from April 2003 to October 2004,were chosen as the Control Group.Thirty-one patients whom were administrated with epidural anesthesia plus bilateral block of the obturator nerve from October 2004 to July 2005 served as the Study Group.Incidences of bladder perforation and obturator nerve reflex were compared between the two groups.Results In the Control Group,obturator nerve reflex occurred in 25 patients(including intense reflex in 11 patients),giving an incidence of 54.3%(25/46),and bladder perforation resulted from the reflex was observed in 8 patients,with an incidence of 17.3%(8/46).In the Study Group,slight obturator nerve reflex happened in 3 patients(9.9%,3/31)and bladder perforation was found in 1 patient(3.2%,1/31).A significant higher rate of obturator nerve reflex was noted in the Control Group than in the Study Group(?2=15.970,P=0.000),but no statistical difference was seen in bladder perforation rate between the two groups(?2=2.359,P=0.125).Conclusions Bilateral block of the obturator nerve can improve the safety remarkably during transurethral resection of bladder tumor,especially when the tumor was located in the lateral bladder wall.

Objective To investigate the changes of CD 4+CD25+CD127 low regulatory T ( Treg ) cells in peripheral blood samples from patients with lung cancer and their correlation with clinicopathologic features of lung cancer .Methods Flow cytometry was used to measure the percentages of CD 4, CD8, nat-ural killer ( NK) and Treg cells in peripheral blood samples collected from 160 patients with lung cancer and 60 healthy subjects .The correlations between the levels of Treg cells and clinicopathologic features of lung cancer were analyzed .The percentages of Treg cells in 60 patients with lung cancer before and after surgery were compared .Results The percentages of CD 4+and NK cells and the ratios of CD 4+/CD8+cells in pa-tients with lung cancer were significantly lower than those in healthy control , while the percentages of Treg cells in patients with lung cancer were decreased as compared with those in healthy subjects .The percenta-ges of Treg cells in patients with advanced cancer were significantly higher than those in patients at early stage (P<0.05), which dropped significantly after surgery (P<0.01).Conclusion The results of this study indicated that Treg cells in patients with lung cancer might inhibit antitumor immune responses and correlate with the progression of cancer .It would be worthwhile to check Treg cells in patients with lung cancer for monitoring the prognosis and treatment effects .

Objective To establish the blood Septin9 methylation detection system for early screening of colorectal cancer based on fluorescence PCR technology .Methods The PCR primer of Septin9 was designed by searching the CpG island site of Septin9 methylation in the NCBI database .The high methylation site of Septin9 gene promoter region was confirmed by PCR amplification and sequencing after extracting DNA from colorectal cancer and para-carcinoma tissues .The fluorescence PCR and TaqMan probe detection technique was designed by aiming at high methylation site for constructing the plasma sample methylation detection sys-tem .Then its accuracy ,specificity ,repeatability and minimum detectable amount were performed the assess-ment and analysis .The SETP9 methylation detection was performed in plasma samples from 57 cases of color-ectal cancer and 30 healthy persons .Results The high methylation site of Septin9 gene in tissue samples was confirmed by sequencing .This site served as the target for designing fluorescence PCR detection system .After assessment ,the accuracy ,specificity and repeatability of this detection system were 100％ ,the lowest detection amount reached 0 .1 ng/μL .Among the plasma samples in 57 cases of colorectal cancer ,the positive rate of Septin9 methylation site detection was 71 .92％ (41/57) and the positive rate of in the patients with pathologi-cal stage Ⅰ and Ⅱ of colorectal cancer reached 64 .2％ .But Septin9 gene had no methylation in plasma of healthy population .Conclusion The plasma fluorescence PCR detection system with Septin9 gene methylation as the target has the characteristics of high sensitivity ,high specificity and high accuracy ,which is the reliable detection technique for the early screening of colorectal cancer and has good clinical application prospect .

OBJECTIVE:To establish the method for simultaneous determination of berberine hydrochloride and baicalin in Jianpi zhixiening granules. METHODS:HPLC switching walvelength method was adopted. The determination was performed on Hypersil BDS C18 column with mobile phase consisted of methanol-0.45％ phosphoric acid solution-triethylamine(50:49:1,V/V/V) at the flow rate of 1.0 mL/min. The detection wavelength was set at 265 nm(berberine hydrochloride)and 280 nm(baicalin). The column temperature was set at 30 ℃,and sample size was 10 μL. RESULTS:The linear range of berberine hydrochloride and baicalin were 60.3-312.8 ng(r=0.9997)and 81.5-368.9 ng(r=0.9999). The limits of quantitation were 0.6668,0.7740 ng,andthe limits of detection were 0.2226,0.2580 ng,respectively. RSDs of intermediate precision,stability and repeatability tests were all lower than 1.0％. The recoveries were 96.48％-99.30％(RSD=1.06％,n=6) and 95.20％-99.39％(RSD=1.66％,n=6), respectively. RSDs of durability test were all lower than 2.0％. CONCLUSIONS:The method is simple, precise, stable, reproducible,accurate and durable. It can be used for simultaneous determination of berberine hydrochloride and baicalin in Jianpi zhixiening granules.

BACKGROUND:Gold nanoparticles are of great significance in the development of multifunctional transdermal drug delivery systems.Smaller gold nanoparticles can penetrate the dermis through the intercellular pathway,but are limited to their easy agglomeration and colloidal morphology,which makes it difficult to exert effects on low delivery efficiency. OBJECTIVE:To develop an ultrasound-optimized hydrogel delivery system by combining phase change nanodroplets with bio-adhesive hydrogel for percutaneous delivery of gold nanoparticles. METHODS:The ultrasound-responsive nanodroplets loaded with gold nanoparticles were prepared by the emulsion solvent evaporation method and loaded into the polydopamine-modified methylacryloyl gelatin hydrogel to prepare a composite hydrogel scaffold.The structure and chemical composition of the ultrasound-responsive nanogold carrier were characterized.The microstructure,porosity,permeability,rheology,in vitro hemostasis,and antibacterial properties of the composite hydrogel were characterized.The cell compatibility of the hydrogel scaffold was evaluated by live/dead staining,and the optimization effects of low-intensity pulsed ultrasound on the permeability,porosity,and mechanical properties of hydrogel were evaluated. RESULTS AND CONCLUSION:(1)Transmission electron microscopy and ultraviolet-visible spectroscopy proved the successful construction of nanogold carriers.The particle size and potential results demonstrated that the synthesized nanoscaled ultrasonic responsive carrier had good stability.(2)Live/dead cell staining proved that the prepared composite hydrogel scaffold had certain biocompatibility.(3)Scanning electron microscopy exhibited that the prepared composite hydrogel scaffold had a porous network structure,and numerous pores of about 2 μm appeared inside the macropores after the addition of nanodroplets and ultrasonic irradiation.The permeability experiment displayed that low-intensity pulsed ultrasound could optimize the porosity and permeability of hydrogel materials.The hemostatic performance of the composite hydrogel scaffold was better than that of the hemostatic sponge and polydopamine@methylacrylylated gelatin hydrogel scaffold.Under the irradiation of low-intensity pulsed ultrasound,the composite hydrogel scaffolds had good antioxidant effects and antibacterial properties.(4)Thermal imaging results manifested that gold nanoparticles were encapsulated in ultrasound-responsive nanobubbles,and more uniform dispersion could be obtained under ultrasonic excitation.(5)The results of the mechanical property test demonstrated that the storage modulus of the hydrogel increased before and after loading gold nanoparticles-nanodroplets,which showed stronger mechanical properties.The elongation at break was 122%,and the ductility was better than that without gold nanoparticles-nanodroplets(P<0.05).(6)These findings indicate that the composite hydrogel scaffold has good biocompatibility,antibacterial property,oxidation resistance,and hemostatic effect.

Objective:To explore the potential of thrombus-targeted nanoprobes for ultrasound/near-infrared bimodal imaging and their synergistic therapeutic effects on thrombosis in vitro.Methods:Nanoprobes loaded with arginine-glycine-aspartate peptide (RGD), perfluoropentane (PFP) and indocyanine green (ICG) were prepared by ultrasonic vibration and carbodiimide method with mesoporous silica nanoparticle (MSN) as the carrier. The probe morphology was observed by scanning and transmission electron microscopy. The loading of RGD and ICG was detected by Bicinchoninic Acid Assay (BCA) and UV-Visible-NIR spectroscopy respectively. The imaging performance and photothermal response of the nanoprobe under near infrared light (NIR) irradiation were studied in vitro. Its biological safety was tested by cytotoxicity test and hemolysis test. The phase transformation was studied under ultrasound and NIR irradiation. The nanoprobe was incubated with fresh arterial thrombus, and its target-seeking ability was observed by frozen section. Ultrasound and NIR irradiation were used to evaluate its thrombolytic ability by the weight changes of thrombus before and after irradiation.Results:The prepared nanoprobe had regular morphology and uniform size. The particle diameter was (156.83±5.05)nm, and the surface potential was (11.47±0.25)mV. The RGD coupling rate was (77.67±4.50)%, which could mediate the targeting of nanoprobe to fresh extracorporeal arterial thrombus. UV-Visible-NIR spectroscopy confirmed the successful loading of ICG, and its encapsulation rate was (80.47±0.05)%. After ultrasound and NIR irradiation, the nanoprobe could undergo acoustically induced phase transition, thermally induced phase transition and enhance the ultrasonic development effect. With the increase of the concentration of the nanoprobe solution, the NIR signal gradually increased, and the temperature rose in a concentration-dependent and intensity-dependent manner after NIR irradiation. The cytotoxicity test and hemolysis test showed that the nanoprobe had good biological safety, and it could play a thrombolytic role under the combined irradiation of ultrasound and NIR, and the weight of thrombus was significantly reduced after the treatment ( P<0.01). Conclusions:In this study, the nanoprobe (RGD/ICG/PFP@MSN) were successfully prepared possesses excellent dual mode imaging capabilities of ultrasound and NIR, excellent phase transition ability and photothermal conversion efficiency, as well as efficient targeted penetration and therapeutic effects against thrombosis. This study provides strong in vitro experimental evidence and new strategies for the integration of diagnosis and treatment of thrombotic diseases under the cooperation of ultrasound and NIR.