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Study of CMV immunoassays for use with cadaveric samples
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
Abstract No: 1449
Abstract No: 1451
Presentation at ESCV 2015: Poster 2 Study of CMV immunoassays for use with cadaveric samples
Presentation at ESCV 2015: Poster 2 Demonstration of human papillomavirus type 16 positivity by real time-PCR assay in patients with colorectal carcinoma
E. Piccin ∗ , F. Capuano, M. Calleri, L. Pallavicini Product Development Dept, DiaSorin, Italy Background: current CE-marked assays are usually not validated for the use with cadaveric specimens. A validation study is required and based on international guidelines (Proposal for the validation of anti-HIV-1/2 or HIV Ag/Ab combination assays, antiHCV assays, HBsAg and anti-HBc assays for use with cadaveric samples”, PEI 08/05/2014) in order to use LIAISON® CMV IgG and LIAISON® CMV IgM assays for the release of tissues for transplantation. The aim is to determine if post-mortem changes in serum may influence test results for LIAISON® CMV specific assays. Methods: the LIAISON® immunoassays for the determination of IgG and IgM against CMV are used in the study. Collection and preparation of cadaveric specimens have to strictly follow procedures and limitations, since hemolysis and other changes occur in blood specimens after death, which might lead to false results either positive or negative in serological testing. In order to determine if changes interfere with the assays, accuracy is evaluated by assessing cadaveric samples, unspiked and spiked at 2 different levels of reactivity (low and medium/high positive), compared to same number of human sera from living donors. Specimens are collected within 24 h after last heartbeat. The same procedure is used for sera from living donors, then tested in parallel as reference. The results are calculated as the ratio between mean of doses for living donors and for post-mortem subjects, at each reactivity level. Accuracy testing is considered successful if the results are comparable within the range of precision and do not differ by more than 25%. Precision is evaluated by testing a cadaveric specimen with reactivity close to the cutoff, in 6 replicates. Precision testing is considered successful if the CV is <15%. Results: In the accuracy testing, a ratio of below 5% between mean of living donors and post-mortem is obtained, for both kits at all evaluated reactivity levels. Precision testing shows CV below 6% for both kits. Conclusions: No significant differences are observed when testing cadaveric specimens versus living donors. LIAISON® assays ensure reliable data, showing suitable performance in the determination of specific CMV antibodies in serum or plasma cadaveric specimens. This option improves the potential of the LIAISON® fully automated solution for the detection of CMV antibodies and of all other DiaSorin markers for infectious disease panel including Hepatitis and Retroviruses which are under evaluation. http://dx.doi.org/10.1016/j.jcv.2015.07.176
S75
Ayca Ozer Durmuslu 1,∗ , Guumllendam Bozdayi 1 , Aylin Altay 1 , Ozgur Ekinci 1 , Ayse Dursun 1 , Erdal Birol Bostanci 2 1
Gazi University Faculty of Medicine Department of Microbiology, Ankara, Turkey 2 Ministry of Health Turkey, Yuksek Ihtisas Hospital, Department of General Surgery, Ankara, Turkey Background: The purpose of our study is to indicate the HPV type 16 positivity by real time-PCR in patients with colorectal carcinoma and to determine whether an association of HPV with age, gender, location of tumor, grade and histological type exists. Methods: One hundred and twenty one paraffin embedded colorectal carcinoma tissues, 90 fresh carcinoma tissues and 107 normal tissue samples as control group were included in the study. After deparaffinisation by xylene, HPV DNA extraction was performed by phenol chloroform isoamylalcohol method. HPV DNA and HPV type 16 were detected by real time PCR using the L1 region. Real-time nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV and HPV 16 DNA specific probe. Real time PCR product analysis was done by melting curve analysis on LightCycler Software version 3.5.3 (LC 2.0 Roche Diagnostics, Germany). Melting peaks of 82–83 ◦ C showed the detection of HPV DNA in the sample. Probe melting peaks of positive samples has been analyzed in the same run and HPV16 positive samples gave peaks around 69.5–78.5 ◦ C. Results: Nineteen percent of patients were HPV positive, 34.1% of HPV positive patients were detected as HPV type 16. 18.6% of HPV positive samples were under 50 years old, 23.3% of them were between the age of 50–59 and 5.8% of them were over 60. Thirteen percent of HPV type 16 positive patients were under 50, 26% of them were between the age of 50–59 and 60% of them were over 60. 31.8% of HPV positive patients were female and 68.2% of them were male. 44.2% of HPV positive and 9.1% of HPV type 16 positive tumors were located in rectum and 55.8% and 5.8% of them were located in colon respectively. 12.3% of well-differentiated tumors were HPV positive, 24.3% of moderately tumors were HPV positive and 21.4% of poorly differentiated tumors were HPV positive. 6.7% of HPV type 16 samples were well-differentiated tumors, 60% of HPV type 16 samples were moderately differentiated tumors, 26.7% of HPV type 16 samples were poorly differentiated tumors. Conclusion: A statistically significant difference was not detected between HPV positivity in colorectal tumors and patients’ age, gender, location of tumor, grade and histological type. However, it was seen that HPV positivity increases with age. From the perspective of gender, HPV positivity of male was more than HPV positivity of female patients. However, as a result, colon cancer and HPV positivity could not be associated statistically. http://dx.doi.org/10.1016/j.jcv.2015.07.177
Abstract No: 1449
Abstract No: 1451
Presentation at ESCV 2015: Poster 2 Study of CMV immunoassays for use with cadaveric samples
Presentation at ESCV 2015: Poster 2 Demonstration of human papillomavirus type 16 positivity by real time-PCR assay in patients with colorectal carcinoma
E. Piccin ∗ , F. Capuano, M. Calleri, L. Pallavicini Product Development Dept, DiaSorin, Italy Background: current CE-marked assays are usually not validated for the use with cadaveric specimens. A validation study is required and based on international guidelines (Proposal for the validation of anti-HIV-1/2 or HIV Ag/Ab combination assays, antiHCV assays, HBsAg and anti-HBc assays for use with cadaveric samples”, PEI 08/05/2014) in order to use LIAISON® CMV IgG and LIAISON® CMV IgM assays for the release of tissues for transplantation. The aim is to determine if post-mortem changes in serum may influence test results for LIAISON® CMV specific assays. Methods: the LIAISON® immunoassays for the determination of IgG and IgM against CMV are used in the study. Collection and preparation of cadaveric specimens have to strictly follow procedures and limitations, since hemolysis and other changes occur in blood specimens after death, which might lead to false results either positive or negative in serological testing. In order to determine if changes interfere with the assays, accuracy is evaluated by assessing cadaveric samples, unspiked and spiked at 2 different levels of reactivity (low and medium/high positive), compared to same number of human sera from living donors. Specimens are collected within 24 h after last heartbeat. The same procedure is used for sera from living donors, then tested in parallel as reference. The results are calculated as the ratio between mean of doses for living donors and for post-mortem subjects, at each reactivity level. Accuracy testing is considered successful if the results are comparable within the range of precision and do not differ by more than 25%. Precision is evaluated by testing a cadaveric specimen with reactivity close to the cutoff, in 6 replicates. Precision testing is considered successful if the CV is <15%. Results: In the accuracy testing, a ratio of below 5% between mean of living donors and post-mortem is obtained, for both kits at all evaluated reactivity levels. Precision testing shows CV below 6% for both kits. Conclusions: No significant differences are observed when testing cadaveric specimens versus living donors. LIAISON® assays ensure reliable data, showing suitable performance in the determination of specific CMV antibodies in serum or plasma cadaveric specimens. This option improves the potential of the LIAISON® fully automated solution for the detection of CMV antibodies and of all other DiaSorin markers for infectious disease panel including Hepatitis and Retroviruses which are under evaluation. http://dx.doi.org/10.1016/j.jcv.2015.07.176
S75
Ayca Ozer Durmuslu 1,∗ , Guumllendam Bozdayi 1 , Aylin Altay 1 , Ozgur Ekinci 1 , Ayse Dursun 1 , Erdal Birol Bostanci 2 1
Gazi University Faculty of Medicine Department of Microbiology, Ankara, Turkey 2 Ministry of Health Turkey, Yuksek Ihtisas Hospital, Department of General Surgery, Ankara, Turkey Background: The purpose of our study is to indicate the HPV type 16 positivity by real time-PCR in patients with colorectal carcinoma and to determine whether an association of HPV with age, gender, location of tumor, grade and histological type exists. Methods: One hundred and twenty one paraffin embedded colorectal carcinoma tissues, 90 fresh carcinoma tissues and 107 normal tissue samples as control group were included in the study. After deparaffinisation by xylene, HPV DNA extraction was performed by phenol chloroform isoamylalcohol method. HPV DNA and HPV type 16 were detected by real time PCR using the L1 region. Real-time nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV and HPV 16 DNA specific probe. Real time PCR product analysis was done by melting curve analysis on LightCycler Software version 3.5.3 (LC 2.0 Roche Diagnostics, Germany). Melting peaks of 82–83 ◦ C showed the detection of HPV DNA in the sample. Probe melting peaks of positive samples has been analyzed in the same run and HPV16 positive samples gave peaks around 69.5–78.5 ◦ C. Results: Nineteen percent of patients were HPV positive, 34.1% of HPV positive patients were detected as HPV type 16. 18.6% of HPV positive samples were under 50 years old, 23.3% of them were between the age of 50–59 and 5.8% of them were over 60. Thirteen percent of HPV type 16 positive patients were under 50, 26% of them were between the age of 50–59 and 60% of them were over 60. 31.8% of HPV positive patients were female and 68.2% of them were male. 44.2% of HPV positive and 9.1% of HPV type 16 positive tumors were located in rectum and 55.8% and 5.8% of them were located in colon respectively. 12.3% of well-differentiated tumors were HPV positive, 24.3% of moderately tumors were HPV positive and 21.4% of poorly differentiated tumors were HPV positive. 6.7% of HPV type 16 samples were well-differentiated tumors, 60% of HPV type 16 samples were moderately differentiated tumors, 26.7% of HPV type 16 samples were poorly differentiated tumors. Conclusion: A statistically significant difference was not detected between HPV positivity in colorectal tumors and patients’ age, gender, location of tumor, grade and histological type. However, it was seen that HPV positivity increases with age. From the perspective of gender, HPV positivity of male was more than HPV positivity of female patients. However, as a result, colon cancer and HPV positivity could not be associated statistically. http://dx.doi.org/10.1016/j.jcv.2015.07.177