stability of hcv, hiv-1 and hbv nucleic acids in plasma samples stored at different temperatures
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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. SoGAT XVIII, Washington May 2005. Importance of the stability of nucleic acids in stored plasma samples. - PowerPoint PPT PresentationTRANSCRIPT
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Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples stored at different temperatures
Marta José, Rodrigo Gajardo and Juan I. JorqueraInstituto Grifols S.A., Barcelona, SPAIN
SoGAT XVIII, Washington May 2005
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SoGAT 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.
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SoGAT 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:
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.
Absence of significant titer decay at -20 ºC for approximately 1 year of study at intermediate concentrations (half-life between 231 and 261 days).
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 RNATo evaluate the RNA and DNA stability of HIV-1 and HBV, stored at different temperatures
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Stability of low level HCV RNA in samples under freezing conditions
• 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).
SoGAT XVIII, Washington May 2005
Study design:
José et al, Biologicals 2005; 33: 9-16*2 positives from a total of 2 (1 test failed)
Positive results out of 3 replicates
- 20 ºC - 70 ºC Dilution Series Dilution Series
Time, Days
Neat 1/2 1/4 1/8 Neat 1/2 1/4 1/8 0 3 3 3 3 3 3 3 3 7 3 3 2 2 3 3 3 2
14 3 3 3 3 3 3 3 3 21 3 3 2 3 3 3 3 3 28 3 2 3 3 3 3 3 3 35 3 2 2 3 3 2 2 3 42 3 3 1 2 2 2 1 1 49 2 2 3 2 1 3 3 2 56 3 3 2 1 2 2 3 0 85 3 3 3 2 3 3 2 2
108 3 3 3 2 3 3 2 2 140 3 3 3 2 3 3 3 2 168 3 3 3 2 3 3 3 1 224 3 3 3 3 3 2 3 3 280 3 3 3 3 3 3 3 2 337 3 3 3 3 3 3 3 3 366 3 3 3 3 3 3 3 3 457 3 3 3 0 3 3 3 1 562 3 3 3 2 3 3 3 3 639 3 2 3 2 3 3 3 3 731 3 3 3 1 3 3 3 3 909 3 3 3 2 3 3 3 2
1095 3 3 3 1 3 3 2 3 1284 3 3 3 2 2* 3 3 3
1462 3 3 3 2 3 3 3 2
1649 3 3 3 3 3 3 3 3
1829 (5 years )
3 2 0 0 3 2 1 2
TOTAL 80/81 76/81 72/81 57/81 76/80 76/81 73/81 63/81
Results I:
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Stability of low level HCV RNA in samples under freezing conditions: Results II
TIME, years
Positive results for all dilutions
≤ -20 ºC ≤ -70 ºC
5 285/324 288/323
José et al, Biologicals 2005; 33: 9-16
SoGAT XVIII, Washington May 2005
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Stability of HIV-1 RNA in samples under freezing conditions (-70 ºC vs -20 ºC)
SoGAT XVIII, Washington May 2005
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.
• 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 duplicate.
• The samples were analysed using an in-house qualitative RT-PCR (95 % detection limit was established at 237 IU/ml by Probit analysis).
Study design:
José et al, Biologicals 2005; 33: 9-16
Positive results out of 2 replicates - 20 ºC - 70 ºC
Dilution Series Dilution Series Time,
Months
Neat 1/2 1/20 Neat 1/2 1/20 0 2 2 1 2 2 1 3 2 2 1 2 2 1 6 2 0 1 2 2 0 9 2 2 1 2 2 0 12 2 1 0 2 2 0 15 2 2 0 2 2 0 18 2 2 0 2 2 0 24 2 1 0 2 1 1 30 2 2 2 2 2 1 36 2 2 0 2 2 1
TOTAL 20/20 16/20 6/20 20/20 19/20 5/20
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TIME, years
Positive results for all dilutions
≤ -20 ºC ≤ -70 ºC
3 40/54 44/54
Stability of HIV-1 RNA in samples under freezing conditions (-70 ºC vs -20 ºC):
Results II
José et al, Biologicals 2005; 33: 9-16 SoGAT XVIII, Washington May 2005
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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:
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).
Amplicor HBV Monitor from Roche (quantitation limit 200 c/ml).
• The HIV-1 RNA and HBV DNA titer decay was analysed by:
Linear regression against time. The half-life (t1/2) decay of each sample under different storage
conditions.
SoGAT XVIII, Washington May 2005José et al, Biologicals 2005; 33: 9-16
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Stability of HIV-1 RNA in samples stored at5 ± 3 °C and RT: Results
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
t1/2, daysa n.a. n.a. 6.9
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
0 5 10 15 20 25 30
DAYS
log
c/m
l
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
0 5 10 15 20 25 30
DAYS
log
c/m
l
103 IU/ml (2.72 log10 c/ml)
104 IU/ml (3.99 log10 c/ml)
RT (25 ºC)
SoGAT XVIII, Washington May 2005José 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).
103 IU/ml (2.72 log10 c/ml)
104 IU/ml (3.99 log10 c/ml)
Cold-room (5 ºC)
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Stability of HBV DNA in samples stored at5 ± 3 °C and RT: Results
Regression Analysis (log titer versus time)
5 ºC 25 ºC
SAMPLE 104 IU/ml 103 IU/ml 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
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
5,00
0 5 10 15 20 25 30
DAYS
log
c/m
l
103 IU/ml (3.56 log10 c/ml)
104 IU/ml (4.51 log10 c/ml)
RT (25 ºC)
103 IU/ml (3.56 log10 c/ml)
104 IU/ml (4.51 log10 c/ml)
Cold-room (5 ºC)
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
5,00
0 5 10 15 20 25 30
DAYS
log
c/m
l
SoGAT XVIII, Washington May 2005José et al, Biologicals 2005; 33: 9-16
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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.
SoGAT XVIII, Washington May 2005José et al, Biologicals 2005; 33: 9-16
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Stability of HCV, HIV-1 and HBV in stored plasma samples: Conclusions II
• For HIV-RNA:
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.
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).
The HIV-1 RNA sample of 104 IU/ml, stored at 25 ºC, showed a half-life (0.3 log10 of titer reduction) of nearly 7 days.
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 log10 (0.26 log10), which can be considered non-relevant.
• After 28 days of storage at 5 ºC or at 25 ºC, no decay of HBV DNA titer was 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.
SoGAT XVIII, Washington May 2005José et al, Biologicals 2005; 33: 9-16