Positive Coincidence Research Articles

Development and evaluation of a C-ELISA for rapid detection of antibody to AIV- H7

Since 2013, the H7 subtype avian influenza virus (AIV-H7) has seriously endangered human life and health, and has had a serious impact on the poultry industry in China. A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects the antibody for AIV-H7 was developed, basing on a monoclonal antibody (mAb) against the neutralizing epitopes on hemagglutinin (HA)gene. Twelve hybridoma cell lines were screened by cell fusion. Hemagglutination inhibition (HI) assay and indirect ELISA were used to identify the competitive effect of the mAbs. High-affinity mAb 1H11 was selected as a competitive antibody. The reaction conditions for the C-ELISA were optimized for AIV-H7 antibody detection. The cross-reactivity of the C-ELISA was determined by AIV-(H1H15), NDV, IBV and IBDV positive serum. A total of 1294 field samples (chicken (462), duck (318), goose (219), quail (203) and pigeon (92) were simultaneously detected by C-ELISA and HI assay. The C-ELISA was found to have a high specificity of 93.23% and a sensitivity of 96.24%. These results reveal a positive coincidence between C-ELISA and HI assay at a coincidence rate of 97.52%. In addition, It confirmed that this method can be used for the diagnosis of AIV-H7 antibodies from chicken, ducks, goose, quail and pigeons.

The Clinical Performance of a New Chemiluminescent Immunoassay in Measuring Anti-β2 Glycoprotein 1 and Anti-Cardiolipin Antibodies.

BackgroundLaboratory criterion is needed for the classification of antiphospholipid syndrome (APS), which contain anticardiolipin antibodies (aCL) and anti-β2-glycoprotein 1 antibodies (aβ2GP1). They are commonly identified by enzyme-linked immunosorbent assay (ELISA), but lack standardized kits, resulting in substantial variations in the antibody positivity between different laboratories. The emergence of chemiluminescence automated BIO-FLASH may improve the situation.Material/MethodsWe selected 185 patients with APS, systemic lupus erythematosus (SLE), infertility, connective tissue disease (CTD), and other conditions in Peking University Third Hospital. We tested the aCL and aβ2GP1 levels by EUROIMMUN ELISA and 105 patients had at least one positive result for aCL and aβ2GP1, while the others had negative results. We retested them by chemiluminescence assay (CIA) and analyzed the result and compared the coincidence rate. The IgM levels were retested by AESKU ELISA. Data were analyzed using SPSS.ResultsOur result suggested that CIA had good performance for IgG isotype of aCL and aβ2GP1 in the coincidence rate. The positive coincidence rate of aCL IgM between CIA and EUROIMMUN ELISA was only 41.67%, but two ELISA kits showed good coincidence, CIA and AESKU ELISA had an obviously higher positive rate. CIA and AESKU had a higher coincidence than that of AESKU and EUROIMMUN in aβ2GP1-IgM.ConclusionsThe new automated CIA BIO-FLASH is suitable for detecting aCL and aβ2GP1 antibodies, especially IgG isotype, which may provide an alternative to time-consuming conventional ELISA method.

Relationship between PD-L1 protein expression and gene amplification in gastric cancer tissues

Objective: To investigate the relationship of PD-L1 protein expression and gene amplification in gastric cancer and their correlation with clinicopathologic factors. Methods: The cohort included 247 gastric cancer specimens with follow-up data and clinicopathologic data obtained from Shanxi Cancer Hospital in 2011. PD-L1 expression was detected by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results: PD-L1 protein was expressed in 25.9% (64/247) of the tumor cells and 26.7% (66/247) of the tumor infiltrating immune cells (IC). There was a correlation between the two (P<0.01). The expression of PD-L1 in tumor cells correlated with the degree of differentiation and tumor diameter(P<0.05). The PD-L1 expression in IC correlated with vascular tumor thrombi(P<0.05). The amplification rate of PD-L1 gene detected by FISH was 19.0% (47/247), and was associated with age, large/small curvature of the stomach, tumor location, tumor diameter, and lymph node metastasis(P<0.05). The positive coincidence rate of the two methods was 25.0% (16/64), negative coincidence rate was 83.0% (152/183), and total coincidence rate was 68.0% (168/247), suggesting that the coincidence of IHC and FISH was poor (P=0.157). There was a negative correlation between PD-L1 protein expression on tumor cells and prognosis in gastric cancer. There was no significant correlation between PD-L1 protein expression on IC and PD-L1 gene amplification with prognosis. Vascular tumor thrombi, tumor diameter, depth of invasion, and lymph node metastasis were all poor prognostic factors of gastric cancer(P<0.05). Multivariate Cox regression analysis showed that PD-L1 protein expression, depth of invasion and lymph node metastasis were all independent prognostic risk factors for gastric cancer. Conclusions: Concordance between PD-L1 protein expression and gene amplification is poor. PD-L1 protein expression may signify poor prognosis. There is no significant correlation between PD-L1 gene amplification and prognosis of patients with gastric cancer.

Comparison of two different combined test strips with fluorescent microspheres or colored microspheres as tracers for rotavirus and adenovirus detection

BackgroundRotavirus (RV) and enteric adenovirus (AdV) mainly cause infantile infectious gastroenteritis. Several separate test methods for the detection of RV or AdV are currently available, but few tests are able to simultaneously detect both RV and AdV viruses, especially in primary medical institutions.MethodsThe present study was mainly designed to compare the performance of two combined test strips for the detection of RV and AdV: a rotavirus–adenovirus strip with fluorescent microspheres for tracers (FMT); and the CerTest rotavirus–adenovirus blister strip with colored microspheres for tracers (CMT). To test the strips cultures of RV, AdV and from other enteric pathogens were used, in addition to 350 stool specimens from 45 symptomatic patients with gastrointestinal infections.ResultsDetection thresholds for RV and AdV cultures using serial dilutions showed that the sensitivity of FMT was significantly higher than that of CMT (both P < 0.05). Specificity evaluation demonstrated that with culture mixtures of Coxsackie (A16), ECHO (type30), and entero- (EV71) viruses there was no detection of cross reaction using the two test strips, i.e., all the results were negative. With regard to the detection of RV in 350 clinical specimens, the total coincidence rate was 92.9%, the positive coincidence rate was 98.2%, and the negative coincidence rate was 90.8%. With regard to AdV detection, the total coincidence rate was 95.4%, the positive coincidence rate was 95.2%, and the negative coincidence rate was 95.5%.ConclusionsFMT performed better than CMT with regard to the combined detection of RV and AdV.

Analysis on the value of cytological and histological noninvasive methods in endometrial cancer screening

Objective To explore the value of cytological and histological noninvasive methods in the screening of endometrial cancer (EC). Methods Seventy-six cases of suspected EC high risk patients diagnosed in the gynecological outpatient department of the People Hospital of Longhua from January 2015 to May 2017 were selected as the research object, the endometrial cells and tissues were collected for cytological and histological examination, the diagnosis of curettage or hysteroscopy performed as agold standardto determine the effect of cytology, histology and combined examination on the ensitivity, specificity, positive coincidence rate of EC diagnosis. Results 76 patients with suspected EC high risk factors were diagnosed as EC 48 cases (63.16%) viagold standard. The sensitivity, specificity and positive coincidence rate of cytology were (75.00%, 89.29%, 80.26%, χ2=1.0105, P=0.6033), histology were(79.17%, 92.86%, 84.21%, χ2=0.3117, P=0.8557) and combined examination in EC were(83.33%, 92.86%, 86.84%, χ2=1.2260, P=0.5417). Conclusion Cytological and histological noninvasive screening has high sensitivity, specificity and positive coincidence rate in EC and has a high diagnostic value for individual or combined exams and can be used as a routine screening for EC in clinical practice. Key words: Endometrial Cancer; Cytology; Histology

Accuracy analysis and comparison of different serological detection methods in syphilis

Objective: To compare the accuracy of different methods by reviewing syphilis serologic screening external quality assessment (EQA) data of Chinese primary health care institutions. Methods: The EQA was based on one or two distinct shipments from 2011 to 2016. Each shipment consisted of five serum samples. The laboratories performed the EQA and sent back the results by the deadline. The results were analyzed using the standard procedures and methods. Results: Six different methods were used for syphilis serologic screening: enzyme linked immunosorbent assay, colloidal gold method, Treponema pallidum particle agglutination (TPPA) test, rapid plasma reagin card test, toluidine red unheated serum reagin test and chemiluminescence immunoassay. From 2011 to 2016, the qualified rate of the colloidal gold method and TPPA had improved from 67.78% to 98.89% and 27.27% to 100%, respectively. The positive coincidence rate of TPPA had changed from 54.55% to 100%. The negative coincidence rate of TRUST had changed from 78.95% to 100%. Conclusions: There were differences between different methods of syphilis serological detection. With the implementation of the EQA performance, the qualified rate, positive coincidence rate, and negative coincidence rate were improved in the primary maternal and child health care institutions.

Comparison of three different assays for measuring thyroglobulin and thyroglobulin antibodies in patients with chronic lymphocytic thyroiditis

ObjectiveTo evaluate how thyroglobulin (Tg) and thyroglobulin antibody (TgAb) results compare among different chemiluminescence methods. MethodsSerum specimens from 153 patients with thyroiditis and 127 apparently healthy individuals were collected at the Peking Union Medical College Hospital from September 2014 to December 2014. TgAb and Tg levels were analyzed by using three different platforms manufactured by Roche, Beckman, and Siemens. The χ2 test was performed to analyze the TgAb results from the different methods. Passing-Bablok regression equations and Bland-Altman plots were used to estimate the relationship and bias among the Tg results obtained with the different analyses. ResultsThe overall coincidence rates for the TgAb results were 82.9% between the Beckman and Roche methods, 78.6% between the Siemens and Roche methods, and 81.1% between the Siemens and Beckman methods. The borderline positive coincidence rate among the different TgAb methods was very poor (9.1–24.2%). Of the 280 samples, the TgAb results for 26.4% of the samples differed among the three CLIA platforms. A good linear relationship among the Tg levels was shown in TgAb-negative subjects analyzed with the different CLIA systems. However, for TgAb-positive subjects, the linear relationship of the Tg levels measured with the Siemens system, compared with the other two systems, was poor. Bland-Altman plots demonstrated significant differences among the different Tg assays. ConclusionsCurrent Tg and TgAb methods are highly variable and cannot be used interchangeably.

Evaluation of preliminary screening strategies for human immunodeficiency virus: a single center experience

Background: To compare and evaluate the available screening tests for human immunodeficiency virus (HIV) antibody screening test. Methods: A retrospective analysis of 331,968 screening specimens for HIV antibody test from the First Affiliated Hospital of Nanjing Medical University between January 2010 and April 2014 was performed. Three hundred and thirty-two cases were screened positive with the third-generation enzyme-linked immunosorbent assay (3G ELISA) and colloidal gold-immunochromatography assay (GICA) and these samples were repeat-tested by the fourth-generation ELISA (4G ELISA). Repeat tested positive samples were again confirmed by western blot (WB) analysis. Comparative study was used to analyze the effectiveness of the 3G ELISA, 4G ELISA and/or combined with GICA, respectively for screening of presence of HIV antibody. Results: Of the 332 positive 3G ELISA screening samples, 189 samples were confirmed to be positive for HIV by WB. The confirmed positive coincidence rate of 3G ELISA with WB was 57.27% and with 4G ELISA was 89.15%. The difference between the 3G and 4G ELISA in HIV antibody detection was statistically significant (P Conclusions: The 4G ELISA is a better HIV antibody screening test than 3G ELISA, especially when combined with GICA. The 4G ELISA combined with GICA appears to be a more suitable strategy for HIV antibody screening test in clinical practice.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

JCES01.22 Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Patients with Non-Small Cell Lung Carcinoma

This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC). Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet DigitalTM PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen). If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%. The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

P3.02b-011 Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Patients with Non-Small Cell Lung Carcinoma: Topic: EGFR Biomarkers

This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC). Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet DigitalTM PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen). If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx ,cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%. The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

Clinical utility of real-time fluorescent PCR for combined detection of anaplastic lymphoma kinase and c-ros oncogene 1 receptor tyrosine kinase in non-small cell lung cancer

Objective: To investigate the clinical application value of combined detection of ALK fusion gene and c-ros oncogene 1 receptor tyrosine kinase (ROS1) fusion gene in non-small cell lung cancer (NSCLC) using real-time fluorescent PCR. Methods: A kit for combined detection of ALK fusion gene and ROS1 fusion gene based on fluorescent PCR was used to simultaneously detect the two fusion genes in 302 cases of NSCLC specimens. The results were validated through Sanger sequencing. The consistency of the two detection methods was analyzed. Results: All 302 cases of NSCLC specimens were successfully analyzed through fluorescent PCR (302/302). 12 cases (4.0%) were found to contain ALK fusion gene, including 3 cases with ALK-M1, 3 with ALK-M2, 3 with ALK-M3, 1 with ALK-M4, and 2 with ALK-M6 fusion gene.12 cases (4.0%) were found to contain ROS1 fusion gene, including 1 case with ROS1-M7, 8 cases with ROS1-M8, 1 case with ROS1-M12, 1 case with ROS1-M14, and 1 case with double-positive ROS1-M3 and ROS1-M8 fusion genes. The total detection rate of ALK fusion gene and ROS1 fusion gene was 7.9% (24/302) and 278 cases showed to be negative for ALK fusion gene and ROS1 fusion gene. The successful detection rates for Sanger DNA sequencing were also 100%. The positive, negative and total coincidence rates obtained by real-time fluorescent PCR and by Sanger DNA sequencing were all 100%. Conclusions: The results of Sanger DNA sequencing demonstrate that the real-time fluorescent PCR assay is equally effective in detecting ALK and ROS1 fusion genes in NSCLC tissues. Furthermore, real-time fluorescent PCR assay can be used to detect trace ALK and ROS1 fusion gene simultaneously in tiny samples, and can save time and avoid repeated sampling. It is worthy of recommendation as a rapid and reliable detection technique.

Integrating immunomagnetic bead enrichment with immunochromatography for the detection of influenza A virus

Objective To establish a novel method by integrating immunomagnetic bead enrichment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influenza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was further combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influenza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensitivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold (Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochromatography was successfully established and suitable for the rapid and on-site detection of influenza A virus. Key words: Influenza A virus; Immunomagnetic beads; Immunochromatography; Rapid detection

Development of a novel protein chip for the detection of bluetongue virus in China

Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.

Value of serum HE4, CYFRA21-1, CA199 in the diagnosis of ovarian benign and malignant tumor

Objective To investigate the diagnosis significance of human epididymis (HE4), cytokeratin-19-fragment (CYFRA21-1), carbohydrate antigen 199 (CA199) on the malignant ovarian tumor, and to evaluate the value of individual and combined detection in judging high risk ovarian cancer. Methods The serum HE4, CYFRA21-1, CA199 levels of 45 patients with primary ovarian cancer, 56 patients with benign ovarian tumor and 50 healthy check-up women in our hospital were detected by electrochemiluminescence immunoassay (ECL). The positive rates and coincidence rates of them were analyzed and the diagnostic value of them were compared. Results HE4 and CYFRA21-1 levels of ovarian cancer group were significantly higher compared with the benign tumor group and healthy control group and the differences were statistically significant (Z=-8.61, P<0.001; Z=-8.39, P<0.001; Z=-8.60, P<0.001; Z=-8.39, P<0.001), however there is no difference between benign tumor group andcontrol group(Z=-1.31, P=0.189; Z=-1.29, P=0.191). The difference of CA199 between benign tumor group and control group was statistically significant (Z=-8.79, P<0.001). For the malignant tumors, the specificity and positive predictive value of HE4 (99.8%, 99.7%), CYFRA21-1 (99.0%, 96.7%) were very high and the diagnostic accordance rates were relatively high (93.4%, 88.7%), but the sensibilities of them were lower compared with CA199.CA199 was increased both in benign and malignant ovarian tumors with different degrees, but the specificity was not very high (85.8%), and the positive predictive value and the diagnosis accordance rate were low (70.6%, 84.1%). The combined detection of HE4, CYFRA 21-1 and CA199 could improve the sensitivity and of the diagnosis of ovarian cancer (100%) and accuracy (89.4%). Conclusions HE4 and CYFRA21-1 only increas in malignant ovarian tumors, and they are mainly used for the diagnosis of ovarian cancer; CA199 level is increased both in benign and malignant ovarian tumors, but the extent is different and it can be used for distinguishing and diagnosing of benign and malignant ovarian tumors. Combining the three indexes can improve the sensitivity and accuracy of the diagnosis of ovarian cancer, and has an important significance for the early diagnosis and treatment of ovarian cancer. Key words: Ovarian neoplasms; Keratins; Antigens, tumor-associated, carbohydrate; human epididymis protein 4

Size-controlled preparation of monodisperse gold nanoparticles for detection of cardiac troponin I by immunochromatography assay

Objective To prepare the monodisperse, colloidal gold nanoparticles (AuNPs) with controllable sizes (50 nm, 65 nm, 79 nm and 102 nm) for the qualitative detection of cardiac troponin I (cTnI) by immunochromatography assay, and to evaluate the effectiveness of the detection. Methods Four kinds of monodisperse citrate-stabilized AuNPs were prepared using small AuNPs as growth centers (seeds) by a seeded growth-thermal aging protocol. As controls, two conventional AuNPs (20 nm, 40 nm) were prepared by the traditional citrate-reduction method. The mouse monoclonal antibody against cTnI -labeled AuNPs were dropped on polyester mat to make AuNPs conjugate pad. The detection line and quality control line of immunochromatography assay kits for detection of cTnI were coated by mouse anti-human cTnI monoclonal antibody paired with antibody in AuNPs and goat anti-mouse polyclonal antiboy respectively. The six kinds of AuNPs were employed as color-labels in immunochromatography assay kits for detecting cTnI, and the corresponding detection effects were evaluated in signal intensity, sensitivity, specificity and stability. The assay kit with the best performance was chosen and compared with the commercialized kits for the detection of cTnI in clinical samples. Results Four kinds of monodisperse AuNPs with large sizes of 50 nm, 65 nm, 79 nm, 102 nm respectively were successfully synthesized by the seeded growth-thermal aging method. The signal strength of four kinds of kits produced by the four large-sized AuNPs was superior to the kits produced by 20 nm AuNPs in detecting cTnI(all P<0.01). The signal strength of the kits produced by 65nm AuNPs showed the best performance among the six kinds of AuNPs(all P<0.01). The lowest detectable limit was 0.50 ng/ml. To compare the agreement of results from chemiluminescent immunoassay versus the results from kits produced by 65nm AuNPs, 100 serum samples have been used for detecting cTnI.Their positive coincidence rate was 97.30% and negative coincidence rate was 100%, the sensitivity and signal strength of the kits produced by 65nm AuNPs was superior to similar products which produced by ABON and Bottests company(all P<0.01). Conclusions Monodisperse, large-sized, citrate-stabilized AuNPs are controllably prepared by a seeded growth-thermal aging method. The development of large-size AuNPs-based immunochromatography assay kits is feasible. 65 nm AuNPs can be a suitable candidate for cTnI immunochromatography assay kit. Our findings provides a new idea for the current immunochromatography assay kits which still adopt small-sized AuNPs as color labels. Key words: Gold; Nanoparticles; Particle size; Immunoassay; Troponin I

Optimization of candidate proteins for serological screening of Chlamydia trachomatis infection.

The aim of this study was to optimize candidate antigen proteins for serological screening of Chlamydia trachomatis infection. C. trachomatis positive serum and swabs of genital secretions were collected from 50 patients in the Tianjin Medical University General Hospital, as well as from 30 patients negative for C. trachomatis. Samples were assessed by colloidal gold assay in a sexually transmitted disease clinic as follows: serum antibodies for eight kinds of C. trachomatis immunodominant proteins (Pgp3, CPAF, CT143, CT101, CT694, CT875, CT813, and IncA) were detected, and two traditional gold standards, immunofluorescence and C. trachomatis cell culture of genital secretions, were used for comparison in order to determine the antigen protein combinations with the highest sensitivity and specificity. Of the 50 samples that tested positive for C. trachomatis infection by colloidal gold assay, 44 tested positive by micro-immunofluorescence, whereas 6 tested negative. In contrast, 14 samples tested positive by cell culture, whereas 36 tested negative. Serological results of the immunodominant protein combination of Pgp3, CT694, and CT875 shared positive coincidence rates of 97.73 and 92.86% with C. trachomatis micro-immunofluorescence and cell culture, respectively. No antibodies of the three proteins were detected in the 30 C. trachomatis samples that tested negative by colloidal gold assay; these samples also tested negative in C. trachomatis genital secretion culture. Overall, the combination of the three immunodominant proteins Pgp3, CT694, and CT875 had good sensitivity and specificity for serological screening of C. trachomatis infection, and the process was simple and easy to apply.

Analysis of Human Immunodeficiency Virus Infections Among Voluntary Blood Donors in Foshan from 2001 to 2013

Objective To investigate the human immunodeficiency virus(HIV) infections among voluntary unpaid blood donors in Foshan,in order to provide references for improvement of appropriate strategies to ensure blood safety in Foshan.Methods From January 2001 to December 2013,a total of 538 107 voluntary unpaid blood donors in Foshan Blood Stations were collected into this study.The blood samples of voluntary blood donors were tested for HIV.The variation trend of HIV infection rates,the dectetion of HIV infection and the epidemiological data on HIV infector were analyzed.Blood samples were screened by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blotting (WB) method.Results ① The HIV infection rates of voluntary unpaid blood donors in Foshan from 2001 to 2013 were 7.02,3.23,6.17,6.83,3.94,17.86,16.38,17.51,5.11,14.39,18.27,12.56,21.74 per 100 000.Total HIV infectors were 70 cases,and total HIV infection rate was 13.01 per 100 000.As a whole,HIV infection rate was increasing year by year (x2 trend=8.03,P＜0.05).HIV infection rate was increased from 7.02 per 100 000 in 2001 to 21.74 per 100 000 in 2013,and the amplitude was 209.69％.HIV infection rate in Foshan was significantly higher than those in Zhejiang and Hainan (P＜0.05),and was close to that in Zhengzhou (P＞0.05),but significantly lower than those in Guangzhou and Shenzhen (P＜0.05).② The screened positive blood samples were 1 346 in 538 107,and the screened positive rate was 0.25％.The confirmed positive samples were 70 cases,and the confirmed positive rate was 13.01 per 100 000.The confirmed positive coincidence rate was 5.20％.The confirmed uncertainty samples were 14 cases,and 6 cases didn't turn positive in follow-up.③ Most of the bands in WB were gp160,gp120,gp41,p66,p51,p31,p24,and the frequency of occurrence were above 90％.The frequency of occurrence of p17 and p55 bands were lower,p39 band was the rarest.Most blood samples occurred more than 8 bands in WB,accounted for 84.3％ (59/70).④ Above 80％ of comfirmed positive blood samples had high S/CO value (S/CO value≥10.00) in ELISA.Less than 10％ of the blood samples had medium or low S/CO value (1.00≤S/CO value＜6.00,6.00≤S/CO value＜10.00).⑤ 85.71％ (60/70) of the HIV infectors were male.75.71％ (53/70) were between ages of 21 to 41.72.86％ (51/70) occupations were migrant workers,company employee and service industry practitioners.82.86％ (58/70) had low-levels of education under college.55.71％ (39/70) were unmarried.84.29％ (59/70) were non-indigenous.82.86％ (58/70) risk experience history were sexual contact,and 81.43％ (57/70) infections were sexual transmitted.Most HIV infectors were infected by hepatitis C virus (HCV) and syphilis,the infection rates were both 12.86％,and significantly higher than the combined infection rate of hepatitis B virus (HBV) (1.43％,x2=6.89,P＜0.05) and the average (HCV:0.38％,x2 =286.59,P＜0.05; syphilis:0.42％,x2=257.88,P＜0.05).78.57％ (55/70) were primary blood donors.Conclusions Under the new situation of HIV infection among voluntary blood donors,the risk of HIV infection by blood transfusion in Foshan was increasing year by year.In order to control HIV transmission via blood tranafusion and ensure blood safety,health education for voluntary blood donation should be strengthened,stable low-risk blood donation team should be established,and more accurate and effective screening technology should be used to improve the sensitivity of HIV tests. Key words: Blood donors; Volunteers; Human immunodeficiency virus; Infection

Comparative study of placental α‐microglobulin‐1, insulin‐like growth factor binding protein‐1 and nitrazine test to diagnose premature rupture of membranes: A randomized controlled trial

The aim of this study was to compare the accuracy of placental α-microglobulin-1 (PAMG-1), insulin-like growth factor binding protein-1 (IGFBP-1) and nitrazine test to diagnose premature rupture of membranes. A total of 120 pregnant women between 11 and 42 weeks with signs/symptoms of membrane rupture were eligible for our study. These women were evaluated with the PAMG-1, IGFBP-1, and nitrazine tests. In the 120 women, the sensitivity, specificity, positive predictive value, and negative predictive value of PAMG-1, IGFBP-1 and nitrazine test were 100%, 100%, 100%, and 100%, 93.33%, 98.89%, 96.55% and 97.80%, and 93.33%, 94.44%, 84.85%, and 97.7%, respectively. In a comparison of the PAMG-1 test and the nitrazine test, positive coincidence rate was 84.85%, negative coincidence rate was 97.70%, total coincidence rate was 94.17%, and kappa value was 0.85. In a comparison of the PAMG-1 test and the IGFBP-1 test, the positive coincidence rate, negative coincidence rate and total coincidence rate were 96.55%, 97.80%, and 97.50%, and kappa value was 0.93. PAMG-1 assay was the most accurate method to diagnose premature rupture of membranes with the highest sensitivity, specificity, positive predictive value and negative predictive value.

A multi-center comparison of smooth muscle autoantibodies and mitochondrial autoantibodies in 154 laboratories of China

Objective Survey and analyses the current situation of autoantibodies testing of ASMA and AMA in China for evidence of further improvement of detecting quality. Methods Survey study.154 laboratories (at least participated in one item) participated voluntarily in the study of multi-center comparison of autoantibodies testing of autoimmune liver diseases.The samples (serum liquid) were prepared by the group of Clinical promotion and translational application research of key technology of diagnosis and treatment of autoimmune diseases of the Research Special Fund for Public Welfare Industry of Health (“the group” for short), including 5 ASMA samples and 5 AMA samples (for AMA-M2, too), which were mailed to laboratories under normal temperature on 15th Dec 2012.The laboratories were required to detect the samples using their routine methods before 31th Dec 2012, and mail the results and methods in a special form back to our group before 14th Jan 2013. The group preformed analysis of the results by Excel, including coincidence rate and titer report rate. Results The reference results of the samples were ASMA-1 positive, others negative, AMA-3 and -4 positive for both AMA and M2, others negative for both AMA and M2.The laboratories that eventually participated in ASMA, AMA and AMA-M2 were 103, 103 and 138, and the positive coincidence rate were 57.3%, 66.5% and 93.5%，meanwhile the negative coincidence rate were 96.8%、99.0%和97.8%, respectively.Indirect immunofluorescence (IIF) was the most common method for detection of ASMA and AMA, while low positive coincidence rate was found in the testing of ASMA and AMA, 57.6% and 65.2%, respectively.In addition, only 39.1% and 43.5% labs reported the titer results of ASMA and AMA.About 57.0% of the labs reported results of AMA and AMA-M2 at the same time. Conclusions In 2012, the positive coincidence rate of ASMA and AMA are not satisfying in clinical labs of China, while the negative coincidence rate of them and the coincidence rate of AMA-M2 are good.The titer report rate of the detection of ASMA and AMA are low by IIF.Standardization and elevation of detecting quality of autoimmune liver disease related autoantibodies are in urgent need of in China.(Chin J Lab Med, 2014,37：127-131) Key words: Hepatitis, autoimmune; Liver cirrhosis, biliary; Muscle, smooth; Mitochondria; Autoantibodies