Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples stored at different temperatures Marta José, Rodrigo Gajardo and Juan I. Jorquera Instituto Grifols S. A. , Barcelona, SPAIN So. GAT XVIII, Washington May 2005
Importance of the stability of nucleic acids in stored plasma samples • To avoid any false negative before testing a contaminated sample, especially in low-titer samples. • To minimise logistic problems during long term storage (-70 ºC vs -20 ºC) of retained samples. • To minimise logistic problems due to the shipping conditions. • To monitor the viral loads by quantitative assays in the performance of antiviral therapy, as well as in the evolution of the infection. So. GAT XVIII, Washington May 2005
Stability of nucleic acids in stored plasma samples: previous data (José et al, Biologicals 2003; 31: 1 -8) • We demonstrated that no advantage was derived from storing samples containing different HCV RNA concentrations at -70 ºC vs -20 ºC: ü Absence of decay attributable to the storage at -20 ºC during the period studied (2. 6 -2. 7 years) in samples with high HCV RNA titer. ü Absence of significant titer decay at -20 ºC for approximately 1 year of study at intermediate concentrations (half-life between 231 and 261 days). ü In samples containing low levels of HCV RNA (100 IU/ml) no loss of reactivity was detected during the storage at -20 ºC for approximately 3. 5 years. • The half-life of a HCV sample diluted to 104 IU/ml and 105 IU/ml and stored at 5 ºC and 25 ºC was nearly 3 months and 14 days, respectively. The aim of the present study was: §To update the stability study results of samples containing low levels of HCV RNA §To evaluate the RNA and DNA stability of HIV-1 and HBV, stored at different temperatures So. GAT XVIII, Washington May 2005
Stability of low level HCV RNA in samples under freezing conditions Results I: Time, Days Study design: • A HCV RNA-positive sample was diluted in cryosupernatant to approximately 100 IU/ml. • The sample was aliquoted and stored at ≤ -20 ºC and ≤ -70 ºC. • After different storage periods, different dilutions of samples were analysed by RT-PCR in triplicate. • The samples were analysed using an in-house qualitative RT-PCR (95 % detection limit was established at 21 IU/ml by Probit analysis). 0 1/8 3 7 14 21 28 35 42 49 56 85 108 140 168 224 280 337 366 457 562 639 731 909 1095 1284 3 3 3 2 3 3 3 3 3 2 2 3 3 3 2 3 3 2 3 2 1 3 2 3 3 3 3 2 3 3 2 2 1 2 2 3 3 0 2 2 1 2 3 3 3 3 3 3 3 2* 3 3 2 2 3 3 3 3 3 3 3 2 1 3 3 2 2 3 3 3 2 3 3 1 2 0 2 2 2 1 3 2 3 3 1 3 3 3 2 3 3 1462 1649 1829 3 3 3 2 3 3 2 0 0 3 2 1 2 (5 years ) TOTAL José et al, Biologicals 2005; 33: 9 -16 Positive results out of 3 replicates £ - 20 ºC £ - 70 ºC Dilution Series Neat 1/2 1/4 1/8 Neat 1/2 1/4 3 3 3 3 80/81 76/81 72/81 57/81 76/80 76/81 73/81 63/81 *2 positives from a total of 2 (1 test failed) So. GAT XVIII, Washington May 2005
Stability of low level HCV RNA in samples under freezing conditions: Results II TIME, years Positive results for all dilutions ≤ -20 ºC 5 ≤ -70 ºC 285/324 288/323 José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HIV-1 RNA in samples under freezing conditions (-70 ºC vs -20 ºC) Study design: • • Results: The NIBSC HIV-1 RNA W. R. PWS-1 (code 99/634) was diluted in a negative plasma pool at approximately 1000 IU/ml. Positive results out of 2 replicates Months The sample was aliquoted and stored at ≤ -20 ºC and ≤ -70 ºC. • £ - 70 ºC Dilution Series Neat 1/20 Neat 1/2 0 3 6 9 12 15 18 24 30 After different storage periods, different dilutions of samples were analysed by RT-PCR in duplicate. • £ - 20 ºC Time, The samples were analysed using an in-house qualitative RT-PCR (95 % detection limit was established at 237 IU/ml by Probit analysis). 36 2 2 2 0 2 1 2 2 1 1 0 0 2 0 2 2 2 2 2 1 2 2 1/20 1 1 0 0 0 1 1 1 TOTAL 20/20 16/20 20/20 19/20 5/20 José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HIV-1 RNA in samples under freezing conditions (-70 ºC vs -20 ºC): Results II TIME, years 3 Positive results for all dilutions ≤ -20 ºC ≤ -70 ºC 40/54 44/54 José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HIV-1 RNA and HBV DNA in samples stored at 5 ± 3 °C and RT: Study Design • The NIBSC HIV-1 RNA W. R. PWS-2 (code 97/632) and the WHO HBV DNA I. S. (code 97/746) were diluted in a negative plasma pool to approximately 103 IU/ml and 104 IU/ml. • The samples were aliquoted and stored at 5± 3 ºC and 25± 2 ºC. • After different storage periods (between 0 and 28 days), the samples were quantified by PCR using: ü Amplicor HIV-1 Monitor and/or the ultra sensitive Amplicor HIV-1 Monitor from Roche (quantitation limit, 500 c/ml and 50 c/ml, respectively). ü Amplicor HBV Monitor from Roche (quantitation limit 200 c/ml). • The HIV-1 RNA and HBV DNA titer decay was analysed by: ü Linear regression against time. ü The half-life (t 1/2) decay of each sample under different storage conditions. José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HIV-1 RNA in samples stored at 5 ± 3 °C and RT: Results Cold-room (5 ºC) RT (25 ºC) 104 IU/ml (3. 99 log 10 c/ml) 103 IU/ml (2. 72 log 10 c/ml) Regression Analysis (log titer versus time) 5 ºC 25 ºC SAMPLE 104 IU/ml 103 IU/ml 104 IU/ml Slope, days-1 -0. 00038 -0. 00057 -0. 00433 p-value to test significance of decay 0. 542 0. 806 0. 051 t 1/2, daysa n. a. 6. 9 José et al, Biologicals 2005; 33: 9 -16 n. a. , not applicable (no decay) a, Half-life expressed to arithmetical scale (i. e. : 50 % or 0. 3 log titer reduction). So. GAT XVIII, Washington May 2005
Stability of HBV DNA in samples stored at 5 ± 3 °C and RT: Results Cold-room (5 ºC) RT (25 ºC) 104 IU/ml (4. 51 log 10 c/ml) 103 IU/ml (3. 56 log 10 c/ml) Regression Analysis (log titer versus time) 5 ºC 25 ºC SAMPLE 104 IU/ml 103 IU/ml Slope, days-1 -0. 0009 -0. 0290 0. 0048 0. 0007 p-value to test significance of decay 0. 791 0. 377 0. 093 0. 865 José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HCV, HIV-1 and HBV in stored plasma samples: Conclusions I • A sample containing 100 IU/ml HCV RNA will remain RT-PCR reactive after at least 5 years of storage, either at -20 ºC or at -70 ºC. No differences between -70 ºC and -20 ºC are evidenced up to date. José et al, Biologicals 2005; 33: 9 -16 So. GAT XVIII, Washington May 2005
Stability of HCV, HIV-1 and HBV in stored plasma samples: Conclusions II • For HIV-RNA: ü A sample containing 1000 IU/ml HIV-1 RNA will remain RT-PCR reactive after at least 3 years, either at -20 ºC or at -70 ºC. No differences between -70 ºC and -20 ºC are evidenced up to date. ü Absence of decay in HIV-1 RNA caused by storage at 5 ºC during the period studied (28 days for the sample of 104 IU/ml and 14 days for the sample of 103 IU/ml). ü The HIV-1 RNA sample of 104 IU/ml, stored at 25 ºC, showed a half-life (0. 3 log 10 of titer reduction) of nearly 7 days. ü After 7 days of storage of the sample with 103 IU/ml of HIV-1 RNA, at 25 ºC, the titer reduction was lower than 0. 3 log 10 (0. 26 log 10), which can be considered non-relevant. • After 28 days of storage at 5 ºC or at 25 ºC, no decay of HBV observed, neither at 104 IU/ml, nor at 103 IU/ml. • The nucleic acids of viruses, in terms of NAT reactivity, appear to be very stable under a wide range of storage conditions. José et al, Biologicals 2005; 33: 9 -16 DNA titer was So. GAT XVIII, Washington May 2005
Скачать презентацию Stability of HCV HIV-1 and HBV nucleic acids
897ea9d26d22c4d800cddcf5eb3bfba6.ppt