Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples stored at different temperatures

1
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
2
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.

SoGAT XVIII, Washington May 2005
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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 RNA
• To evaluate the RNA and DNA stability of HIV-1
  and HBV, stored at different temperatures

SoGAT XVIII, Washington May 2005
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Stability of low level HCV RNA in samples under
freezing conditions
Results I
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).

José et al, Biologicals 2005 33 9-16
SoGAT XVIII, Washington May 2005
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Stability of low level HCV RNA in samples under
freezing conditions Results II
TIME, years Positive results for all dilutions Positive results for all dilutions
TIME, years -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)
Study design

• 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).

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) Results II
TIME, years Positive results for all dilutions Positive results for all dilutions
TIME, years -20 ºC -70 ºC
3 40/54 44/54
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 53 ºC
  and 252 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.

José et al, Biologicals 2005 33 9-16
SoGAT XVIII, Washington May 2005
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Stability of HIV-1 RNA in samples stored at5 3
C and RT Results
Regression Analysis (log titer versus time) Regression Analysis (log titer versus time) Regression Analysis (log titer versus time) Regression Analysis (log titer versus time)
5 ºC 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
n.a., not applicable (no decay) a, Half-life
expressed to arithmetical scale (i.e. 50 or
0.3 log titer reduction).
José et al, Biologicals 2005 33 9-16
SoGAT XVIII, Washington May 2005
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Stability of HBV DNA in samples stored at5 3
C and RT Results
Regression Analysis (log titer versus time) Regression Analysis (log titer versus time) Regression Analysis (log titer versus time) Regression Analysis (log titer versus time) Regression Analysis (log titer versus time)
5 ºC 5 ºC 25 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
SoGAT XVIII, Washington May 2005
José 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 2005
José 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 2005
José et al, Biologicals 2005 33 9-16