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

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

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.

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

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:

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

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

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

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

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)

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

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

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