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SSP typing results from diluted DNA samples
Abstracts
S87
7.3 #101
TRANSFORMATION EFFICIENCY OF B–LCL IN HEALTHY SUBJECTS AND PATIENTS WITH DIFFERENT DISEASES A. Vazquez,1 R. Cruz,1 M. Rodriguez,2 H. Flores,1 J. M. Mejia,3 C. Gorodezky.1 1 Dept. of Immunogenetics, InDRE, SSA; 2Centro Dermatol. Pascua; 3IMSS CMN SXXI, Mexico City, Mexico B cell lines (LCL) are obtained using EBV supernatants to infect B cells isolated from peripheral blood samples. The EBV is obtained from B–LCL (B95– 8 and MCuV5) from the Marmoset monkey, or from seropositive EBV individuals. The aim of this study was to compare the ability and efficiency to transform B– cells obtained from healthy subjects and from patients with several diseases, and to assess differences in the transformation between fresh and frozen cells. We included cells from: 66 healthy subjects (47 fresh, 19 frozen); 26 lepromatous leprosy (LL) (6 fresh, 20 frozen); 34 Down syndrome (DS); 30 acute leukemia (AL) and 3 DS⫹AL patients. The EBV supernatant of B95– 8 cell line was harvested, filtered, and frozen at ⫺70°C. After titration it was used to transform the B cells. Mononuclear cells were isolated from heparinized blood and were cultured (2 ⫻ 106 cel/mL) with 1mL EBV and supplemented medium. Cyclosporine–A was used to eliminate cytotoxic T cells. Cells of healthy individuals were stored in liquid N2 for 2 years and LL patient cells from 7 to 11 years. They were cultured in the same conditions as fresh cells and they were monitored daily to determine when clones first started and when the LCL was established. T–Student and X2Y test were done. Fresh cells of healthy controls showed a greater transformation and cloning efficiency as compared to the patients. AL and DS⫹AL cells demonstrated a lower efficiency. The transformation speed of LL and DS cells was similar to the found in control cells. However cells of AL and DS showed a lower transformation speed (p ⬍ 0.05; p ⬍ 0.05). Speed of cloning was lower in cells of all patient groups but LA (X ⫽ 9.9 days), probably because of the intrinsic ability of tumoral cells to grow. Freezing time was critical for transformation time and efficiency since even in controls this was 11 times less efficient. Viability was very poor in frozen LL cells (p ⬍ 0.05) (50 times less than in frozen controls).In conclusion, the differences shown herein indicate that changes and lack of uniformity in the transformation process are probably due to cell changes and the nature of the cells used.
7.4 #102
SSP TYPING RESULTS FROM DILUTED DNA SAMPLES Cynthia S. Turino,1 Sarah Jones,1 Nancy Murphy.1 1Research and Development, GenoVision, Inc., West Chester, PA High quality DNA is required for labs performing HLA typing assays. For Sequence Specific Priming (SSP) assays, which are highly specific and sensitive, concentration and quality of DNA is one of the most critical variables. Recommended concentrations of DNA range from 20 –50 ng/l. Often clinical samples from very ill individuals or historic samples do not provide adequate DNA. If intact, pure DNA is used, amounts less than the recommended range should amplify successfully. The purpose of this study was to test the amplification ability of diluted DNA isolated and purified from various samples using the GenoPrepDNA™ from Blood – Higher Yield kit in combination with the GenoM™– 48 Robotic Workstation, when using an SSP kit. Serial dilutions were performed from 20 ng/l of DNA. High resolution Class I and Class II Olerup SSP kits were used to amplify the DNA. Successful typings were achieved from all diluted samples, including 0.625 ng/l DNA. These results show that SSP can work with DNA concentrations significantly less than recommended if the DNA is intact and of high quality.
S87
7.3 #101
TRANSFORMATION EFFICIENCY OF B–LCL IN HEALTHY SUBJECTS AND PATIENTS WITH DIFFERENT DISEASES A. Vazquez,1 R. Cruz,1 M. Rodriguez,2 H. Flores,1 J. M. Mejia,3 C. Gorodezky.1 1 Dept. of Immunogenetics, InDRE, SSA; 2Centro Dermatol. Pascua; 3IMSS CMN SXXI, Mexico City, Mexico B cell lines (LCL) are obtained using EBV supernatants to infect B cells isolated from peripheral blood samples. The EBV is obtained from B–LCL (B95– 8 and MCuV5) from the Marmoset monkey, or from seropositive EBV individuals. The aim of this study was to compare the ability and efficiency to transform B– cells obtained from healthy subjects and from patients with several diseases, and to assess differences in the transformation between fresh and frozen cells. We included cells from: 66 healthy subjects (47 fresh, 19 frozen); 26 lepromatous leprosy (LL) (6 fresh, 20 frozen); 34 Down syndrome (DS); 30 acute leukemia (AL) and 3 DS⫹AL patients. The EBV supernatant of B95– 8 cell line was harvested, filtered, and frozen at ⫺70°C. After titration it was used to transform the B cells. Mononuclear cells were isolated from heparinized blood and were cultured (2 ⫻ 106 cel/mL) with 1mL EBV and supplemented medium. Cyclosporine–A was used to eliminate cytotoxic T cells. Cells of healthy individuals were stored in liquid N2 for 2 years and LL patient cells from 7 to 11 years. They were cultured in the same conditions as fresh cells and they were monitored daily to determine when clones first started and when the LCL was established. T–Student and X2Y test were done. Fresh cells of healthy controls showed a greater transformation and cloning efficiency as compared to the patients. AL and DS⫹AL cells demonstrated a lower efficiency. The transformation speed of LL and DS cells was similar to the found in control cells. However cells of AL and DS showed a lower transformation speed (p ⬍ 0.05; p ⬍ 0.05). Speed of cloning was lower in cells of all patient groups but LA (X ⫽ 9.9 days), probably because of the intrinsic ability of tumoral cells to grow. Freezing time was critical for transformation time and efficiency since even in controls this was 11 times less efficient. Viability was very poor in frozen LL cells (p ⬍ 0.05) (50 times less than in frozen controls).In conclusion, the differences shown herein indicate that changes and lack of uniformity in the transformation process are probably due to cell changes and the nature of the cells used.
7.4 #102
SSP TYPING RESULTS FROM DILUTED DNA SAMPLES Cynthia S. Turino,1 Sarah Jones,1 Nancy Murphy.1 1Research and Development, GenoVision, Inc., West Chester, PA High quality DNA is required for labs performing HLA typing assays. For Sequence Specific Priming (SSP) assays, which are highly specific and sensitive, concentration and quality of DNA is one of the most critical variables. Recommended concentrations of DNA range from 20 –50 ng/l. Often clinical samples from very ill individuals or historic samples do not provide adequate DNA. If intact, pure DNA is used, amounts less than the recommended range should amplify successfully. The purpose of this study was to test the amplification ability of diluted DNA isolated and purified from various samples using the GenoPrepDNA™ from Blood – Higher Yield kit in combination with the GenoM™– 48 Robotic Workstation, when using an SSP kit. Serial dilutions were performed from 20 ng/l of DNA. High resolution Class I and Class II Olerup SSP kits were used to amplify the DNA. Successful typings were achieved from all diluted samples, including 0.625 ng/l DNA. These results show that SSP can work with DNA concentrations significantly less than recommended if the DNA is intact and of high quality.