ObjectiveTo facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay,a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads.Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process,for the method development,screening and selecting the magnetic beads based on magnetism,suspension power and extraction effects of HBV DNA,the optimization of the selected key components of extraction reagents were performed,which including the quantity of magnetic beads( 1 - 1.75 μl/ml),the buffer pH of lysis (4 -9),washing conditions with or without alcohol,elution for proliferation with or without magnetic beads,and etc.The evaluation for the extraction method was carried out including linearity,precision,sensitivity,specificity,anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples,the magnetic beads method was also compared with the interntional Gold standard Roche COBAS Tagman test method ( Roche method) in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression.ResultsOptimization experiment result showed superparamagnetic beads A was the best among the candidates ( C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml,buffer pH 7 - 8 of lysis,ethanol was eliminated from washing buffer,proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4 × 101 -4 × 108 IU/ml and that of boiling method was 4 × 102 -4 × 108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0％ and boiling method was 11.9％.The false positive rate was 0 for beads method vs 3.1％ for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However,sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86％ vs boiling method 74％.The correlation between the two method was R2 =0.948,P ＜ 0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95％ (49/50) vs Roche method was 100％ (50/50).The correlation was R2 =0.963,P ＜ 0.05.ConclusionsThe established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction,broader testing range,accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.( Chin J Lab Med,2012,35:843 -850 )

Objectives To explore the humoral immunologic mechanisms of the susceptibility to invasive pneumococcal diseases (IPD) in asthmatic children. Methods Plasma samples were collected from 43 asthmatic and 20 non-asthmatic chil-dren. Anit-pneumococcal capsular polysaccharide (PPS)-IgG concentrations were measured by enzyme-linked immunosorbent assays. Results The mean concentrations of anti-PPS 14, 19A and 23F-IgG were signiifcantly higher in asthmatic children than in non-asthmatic children (P<0.05). The ratios of the asthmatic children who had anti-PPS 14, 19A and 23F-IgG concentrations higher than the protective antibody level (≥0.2 μg/ml ) were 100%for all the serotypes. Conclusions The immune responses of producing anti-PPS IgG to defense IPD were normal in asthmatic children. Asthmatic children may be more susceptive to pneumococcal infection or colonization than non-asthmatic children.

Objective To investigate the relationship between noninvasive brachial artery blood pressure (B) and radial artery blood pressure (R) of the right arm.Methods Two hundred and ninetyfive patients with 149 males and age of (47 ± 16) years were studied.The height of patients was 163 ± 8 cm,and weight of patients was (61.2 ± 7.8) kg.The patients with peripheral vascular disease,wounds of arm skin or subcutaneous tissue infection were all excluded.Their B (with adult cuffs) and R (with infant cuffs) of the right arm were measured and analyzed after the patients under general anesthesia and stable hemodynamics.The relationships between B and R were analyzed by linear regression,the differences between B and R of each interval were compared using one-way ANOVA and then followed by SNK procedure.Results Right brachial artery blood pressure was significantly lower than radial artery blood pressure.The differences between the two varied from 13 to 18 mmHg in systolic BP (SBP),diastolic BP (DBP) and mean blood pressure (MAP).And linear regression was most applicable to describe their correlation [r=0.841 (SBP),0.808 (DBP),0.833 (MAP),all P＜0.01].Conclusions Radial artery blood pressure measured with infant cuffs can well reflect the variation of brachial artery blood pressure.

Objective To investigate the clinical features of non-steroidal anti-inflammatory drugs (NSAIDs) caused gastrointestinal bleeding in the elderly.Methods We performed a retrospective analysis ofNSAIDs caused gastrointestinal bleeding with 308 cases admitted to Department of Gastroenterology,The Second Hospital of Tianjin Medical University.Patients were divided into elderly group(≥65 years,n =208) and young group (＜ 60 years,n =100) to make a comparative analysis of two group differences in clinical characteristics.Results The NSAIDs in the elderly taking were mainly Aspirin (55.8 ％ (116/208)),which was significantly more frequent than that of young group (37.0％ (37/100),x2 =9.517,P ＜ 0.05).There was no significant difference on the blood routine and coagulation between the two groups (P ＞ 0.05).The recent bleeding rate of the elderly group (21.2％ (44/208)) was lower than the young group (63.0％ (63/100))(x2 =52.161,P ＜0.05).The two groups had no significant difference on the history of uleer and Hp infection (P ＞ 0.05).Conclusion The clinical characteristics of NSAIDs caused gastrointestinal bleeding on the elderly were different with young or middle-aged.This infers that elderly patients need individualized treatment.

OBJECTIVE To evaluate the distribution of pathogens causing infection and current status of antimicrobial resistance in order to offer experimental data for clinical administration.METHODS The resistance tests were performed using K-B method.WHONET5.2 and EXCEL were used to analyze bacteria distribution and resistance.RESULTS From 119 cases of patients were earned the routine bacteria culture results,the tested rate was 96.75%,the positive specimens were 379 and the postitive rate was 83.85%.The strains of Pseudomonas aeruginosa were the most frequent organisms isolated then were the strains of Streptococcus pyogenes,coagulase-negative Staphylococcus,Nesseria spp,and Stenotrophomonas maltophilia.CONCLUSIONS It is important to strengthen antibiotics administration,bacteria isolation and resistance surveillance.

Objective To investigate the role of TLR9 in the glomerulonephritis through observing the changes of Toll-like receptor 9 in the glomerulo nephritis kidney tissue with or without CpG-ODN stimulation .Methods Wistar male rats were randomly divided into normal group(N),nephritis model group(M),CpG-ODN group(CpG) and GpC-ODN group(GpC).The urine samples were collected at 2,4,6 and 8 weeks after treatment,respectively.Blood samples were collected at the end of the last urine sample was collected,and the kidney tissue was collected,then the rats were killed.24 h urine protein was measured by Coomassie light blue technique.Serum album and renal function were determined by serological method.The pathologic changes of kidney were observed by light microscope and NF-?B p65 expression was detected using immunohistochemystry,RT-PCR was performed to detect the expressions of TLR9,INF-? and IL-6 mRNA.Results The expression of TLR9 was lighter in group M,and significantly increased after CpG-ODN stimulation compared with group M.Furthermore,24 h urine protein excretion was markedly increased,serum album was markedly decreased.The histopathologic changes of kidney were more severe.The mesangial cells(MCs) proliferated diffusifully in midrange and wide range,some of the glomeruli formatted cellularity crescent,micrangium loop was limitted,mononuclear macrophile cells were seen in the mesangial region.Conclusion Inflammatory factors mediated by TLR9 can deteriorate the biochemical and histopathologic changes.The immunologic reaction mediated by TLR9 is one of the mechanisms for the glomerulonephtitis' progression.

Objective:To explore the role of receptor-interaction protein 3 (RIP3) in regulating the infiltration of monocytes/macrophages into the liver in autoimmune hepatitis (AIH).Methods:From January to June in 2018, at Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, 10 AIH patients who underwent liver biopsy were enrolled, and at the same time, 5 age and gender matched individuals with normal liver function and hepatic cyst were selected as control. The infiltration of monocytes/macrophages in the liver tissues was observed by immunofluorescence detection in the patients with AIH and controls. Raw264.7 macrophages were divided into control group, lipopolysaccharide group and lipopolysaccharide+ RIP3 inhibitor GSK872 (GSK872) group. The expression of RIP3, mixed lineage kinase domain like pseudokinase ( MLKL), tumor necrosis factor ( TNF)- α, interleukin ( IL)-6, IL-1 β, nod-like receptor protein 3 ( NLRP3), CC motif chemokine ligand ( CCL)2 and CCL5 at mRNA levels were detected by quantitative polymerase chain reaction (qPCR). Raw264.7 macrophages were also divided into control group, lipopolysaccharide group and lipopolysaccharide + dexamethasone group. The relative expression of TNF- α, NLRP3, RIP3 and MLKL at mRNA level in macrophage were detected by qPCR. Twenty-four 6-week-old female C57BL/6 mice were chosen to establish AIH mice model and were randomly divided into control group, concanavalin A (ConA) group, ConA+ dexamethasone group and ConA+ GSK872 group (6 mice in each group). After the mice were executed, the peripheral blood and liver tissues were collected. The histopathology of mice liver were observed and the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. The expression of CCL2 and CC motif chemokine receptor 2 ( CCR2) at mRNA level were detected by qPCR. The proportion of macrophages in mice livers were analyzed by flow cytometry. The independent sample t test and one-way analysis of variance were performed for statistical analysis. Results:The percentages of CD68 positive macrophages and MAC387 positive infiltrated mononuclear macrophages in livers of AIH patients were both higher than those of controls ((0.84±0.21)% vs. (0.09±0.03)%, (0.79±0.13)% vs. (0.03±0.01)%), and the differences were statistically significant ( t=3.00 and 4.84; all P<0.05). The expression of RIP3, MLKL, TNF- α, IL-6, IL-1 β, NLRP3, CCL2 and CCL5 at mRNA level of lipopolysaccharide group were all higher than those of control group and lipopolysaccharide+ GSK872 group (1.64±0.16 vs. 1.07±0.07 and 0.63±0.11; 10.45±1.37 vs. 1.10±0.33 and 1.51±0.63; 5.43±0.59 vs. 0.94±0.06 and 2.59±0.45; 204.20±30.73 vs. 1.26 ±0.19 and 111.40±11.62; 20 848.00±362.00 vs. 1.09 ±0.26 and 10 940.00±566.60; 7.47±1.17 vs. 1.09±0.09 and 3.79±0.89; 68.03±5.15 vs. 1.14±0.19 and 14.09±2.62; 5 935.12±96.20 vs. 1.43±0.46 and 673.50±49.10), and the differences were all statistically significant ( t=3.11, 5.21, 6.65, 6.55, 7.57, 3.96, 6.60, 3.06, 8.83, 4.08, 5.46, 2.56, 12.97, 10.16, 25.34 and 14.99; all P<0.05). The expression of TNF- α, NLRP3, RIP3 and MLKL at mRNA level of lipopolysaccharide group were all higher than those of control group and lipopolysaccharide+ dexamethasone group (8.85±1.43 vs. 1.44±0.43 and 3.63±0.63; 6.42±0.86 vs. 0.99±0.12 and 2.07±0.17; 1.72±0.21 vs. 0.93±0.09 and 0.43±0.07; 6.87±0.85 vs. 1.62±0.31 and 1.41±0.29), and the differences were all statistically significant ( t=4.95, 3.33, 6.24, 4.95, 3.04, 5.11, 5.77 and 6.07, all P<0.05). The mice liver of ConA group showed obviously inflammatory cells infiltration and hepatocytes necrosis. The serum ALT and AST levels of ConA group were both higher than those of control group, ConA+ dexamethasone group and ConA+ GSK872 group ((2 569.00±45.44) U/L vs. (49.38±9.07), (103.00±14.07) and (759.30±34.99) U/L; (3 335.00±88.79) U/L vs. (108.50±18.10), (460.00±97.40) and (1 573.85±36.06) U/L), the serum ALT and AST levels of ConA+ dexamethasone group were both lower than those of ConA+ GSK872 group, and the differences were all statistically significant ( t=5.54, 5.42, 3.90, 4.63, 4.16, 3.79, 6.70 and 2.71; all P<0.05). The expression of CCL2 and CCR2 at mRNA levels in mice liver of ConA group were both higher than those of control group, ConA+ dexamethasone group and ConA+ GSK872 group (92.64±10.57 vs. 0.78±0.15, 5.64±1.00 and 9.47±2.06; 5.73±0.39 vs. 0.98±0.22, 2.18±0.22 and 2.98±0.33), and the differences were all statistically significant ( t=7.66, 7.24, 5.87, 8.71, 8.58 and 5.45; all P <0.01). The proportion of CD45 + CD11b + F4/80 + total macrophages and CD45 + CD11b hiF4/80 lo infiltrated macrophages in mice livers of ConA group were both higher than those of control group, ConA+ dexamethasone group and ConA+ GSK872 group (0.86±0.02 vs. 0.73±0.03, 0.68±0.02 and 0.72±0.03; 0.56±0.02 vs. 0.08±0.02, 0.11±0.01 and 0.08±0.01), however the proportion of CD45 + CD11b loF4/80 hi liver macrophages (Kupffer cells) was lower than those that of control group, ConA+ dexamethasone group and ConA+ GSK872 group (0.24±0.03 vs. 0.58±0.04, 0.52±0.07 and 0.56±0.07), and the differences were all statistically significant ( t=4.27, 5.90, 3.89, 18.70, 19.87, 20.52, 7.35, 3.82 and 3.87, all P<0.05). Conclusions:The number of macrophages incread in the livers of AIH patients. RIP3 signaling mediates the migration of monocytes/macrophages infiltration in immune hepatitis, which may be a potential therapeutic target for AIH.

Objective:To compare the effect of fluorescence staining and periodic acid-Schiff staining in the diagnosis of fungal keratitis (FK).Methods:A total of 147 corneal specimens from 147 FK patients treated in Eye Hospital of Shandong First Medical University from January 2017 to May 2019 with positive corneal scraping or fungal culture were collected.Among them, there were 84 cases with penetrating keratoplasty (PKP), 42 cases with lamellar keratoplasty (LKP) and 21 cases with lesion resection.Another 11 cases with herpes simplex virus keratitis served as negative control.The corneal tissue specimens were performed with fungal fluorescence staining and periodic acid-Schiff staining, respectively.The stained sections were placed under fluorescence microscope and optical microscope to observe fungal hyphae or spores, respectively.The positive rates of the two staining methods were compared, and the positive cases of the diagnosis of FK in corneal tissue samples obtained by different surgical methods and corneal infection caused by different strains of the two staining methods were compared.Written informed consent was obtained from each patient.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY-2016012).Results:The positive rate of periodic acid-Schiff staining and fungal fluorescence staining was 60.5% (89/147) and 79.6% (117/147), respectively.The positive rate of fluorescence staining in the diagnosis of fungal keratitis was significantly higher than that of periodic acid-Schiff staining ( χ2=28.00, P<0.01), and both the specificity of the two staining methods was 100%.The positive rate of specimens obtained by PKP with fluorescent staining was 85.7% (72/84), and the positive rate with periodic acid-Schiff staining was 65.5% (55/84), and the difference was statistically significant ( χ2=17.00, P<0.01). The positive rate of specimens obtained by LKP with fluorescent staining was 71.4% (30/42), and the positive rate with periodic acid-Schiff staining was 52.4% (22/42), and the difference was statistically significant ( χ2=8.00, P<0.01). The positive rate of resected foci specimens with fluorescent staining was 71.4% (15/21), and the positive rate with periodic acid-Schiff staining was 57.1% (12/21), and the difference was not statistically significant ( χ2=1.30, P=0.25). The positive cases of two kinds of staining were different among different fungal strains.Among them, the positive cases of Fusarium solani complex, Pythium insidiosum, Aspergillus fumigatus complex, Candida guilliermondii, Trichoderma and Nigrograna mackinnonii with fluorescence staining were 19, 5, 5, 1, 1 and 1, and the positive cases of periodic acid-Schiff staining were 11, 0, 3, 0, 0 and 0, respectively.The staining results of the 11 negative controls were negative. Conclusions:Fluorescence staining is more sensitive than periodic acid-Schiff staining in the detection of fungal components in paraffin-embedded corneal tissues, and it can significantly improve the fungal detection rates.

ObjectiveTo detect the value of ultrasound-guided steroids injection for the treatment of plantar fasciitis.MethodsThirty-eight physical therapy ineffective plantar fasciitis patients were enrolled in this study, and randomly divided into ultrasound-guided and palpation-guided groups. Pain intensity was measured using a visual analog scale (VAS) and plantar fasciitis were evaluated by high frequency ultrasound including assessment of the thickness before injection and at 4 weeks, 12 weeks post injection.ResultsThirty-eight patients who received either ultrasound guided or palpation-guided injection had significantly lower visual analog scale scores and lower plantar fascia thickness (bothP<0.01) at 4 weeks, 12 weeks post injection. The differences in plantar fascia thickness, VAS score between the two groups before, and at the 4 weeks follow-up were not statistically significant [2.52±0.77vs 2.68±0.82,P>0.05; (4.56±0.25) mmvs (4.72±0.38) mm, P>0.05]. However, the ultrasound guided group had lower mean visual analog scale score (1.47±0.77vs 2.37±0.68,P<0.01) and lower mean plantar fascia thickness [(4.02±0.24) mmvs (4.53±0.35) mm,P<0.01] than the palpation-guided group at 12 weeks post injection. The calcification of the plantar fascia at 12 weeks post injection in ultrasound guided group was completely disappeared or significantly decreased.ConclusionUltrasound-guided injection for treating plantar fasciitis is more accurate and effective than palpation-guided injection, and is of great clinical value for both patients and doctors.