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Stability of HCV, HIV-1 and HBV nucleic acids in
plasma samples under long-term storage
Marta Jose *, Rodrigo Gajardo, Juan I. Jorquera
Research and Development Area, Instituto Grifols, S.A., Poligon Llevant, C/Can Guasch, 2,
08150-Parets del Valle`s, Barcelona, Spain
Received 22 July 2004; accepted 24 October 2004
Abstract
The implementation of nucleic acid amplification technology (NAT) for detection of HCV, HIV-1 and HBV has undoubtedly
contributed to the viral safety of blood, reducing the window period. One important matter related to the stability of RNA/DNA is
the effect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after
storage at different temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years
at20 C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also beenstudied. At20 C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decayin HIV-1 RNA detectability. Regardless of the HIV-1 RNA concentration, samples stored at 5 C maintain their titre for at least 14
days. At 25 C, the HIV-1 RNA half-life was determined at nearly 7 days. The HBV DNA, at 5 C and 25 C, is stable for at least
28 days, regardless of the initial titre.
2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
1. Introduction
HCV, HIV-1 and HBV are the most important
pathogenic viruses potentially transmissible via blood
transfusion. The vast majority of residual cases of
transfusion-transmitted infections occur through dona-
tions made during the serological window period. In
addition to the routinely performed serological tests, the
introduction of nucleic acid amplification techniques
(NAT) has allowed the window period to be reduced to6e10 days for HCV[1,2], to 11 days for HIV-1[2]and
to 34 days for HBV[3].
A major point relative to the screening for important
viruses is the stability of their RNA or DNA during
handling and/or storage of samples. Many authors have
reported that the storage conditions of samples may
affect the stability and, hence, the detectability of nucleic
acid of viruses, although apparently discrepant conclu-
sions have been drawn[4e14]. Different facts might have
an effect on the nucleic acid stability: shipping con-
ditions, screening in blood banks, handling of samples,
among others. The storage conditions of samples might
be important before testing an infected sample to avoid
any false negative, especially in low-titre samples.Furthermore, long-term storage conditions (samples
stored at 70 C versus samples stored at 20 C) areof great importance to minimise logistics problems,
especially at reference testing laboratories (retained
samples, shipping conditions, etc.).
The screening for HCV, HIV-1 or HBV by NAT
plays a major role in the area of blood safety[15,16]but
also, monitoring the viral loads by quantitative assays is
very valuable in the performance of antiviral therapy in* Corresponding author. Tel.: C34 935710593; fax: C34 935710855.
E-mail address: [email protected](M. Jose ).
1045-1056/04/$30.00 2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.biologicals.2004.10.003
Biologicals 33 (2005) 9e16
www.elsevier.com/locate/biologicals
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8/12/2019 stability of NAT samples
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patients with advanced infection[11,17e19], as well as
in the evolution of the infection. Furthermore, low viral
loads cannot always be detected by serological tests,
particularly in long-term survivors [20].
In a previous work, we demonstrated that no
advantage was derived from storing samples containing
different HCV RNA concentrations at70
C[21]. Wefound absence of decay in HCV RNA attributable to the
storage at 20 C during the period studied (approxi-mately 2.6e2.7 years) in samples with high HCV RNA
titre. We also demonstrated the absence of significant
titre decay during storage at 20 C for approximately1 year of study in samples with intermediate concen-
trations. The HCV RNA of these samples showed
a half-life between 231 and 261 days. In samples
containing low levels of HCV RNA (100 IU/ml) no loss
of HCV RNA reactivity was detected during storage at
20 C for approximately 3.5 years. Furthermore, thehalf-life of an HCV RNA sample diluted to 104 IU/ml
and 105 IU/ml and stored at 5 C and 25 C was nearly
3 months and 14 days, respectively [21].
The aim of this study was, on the one hand, to update
the stability study results of samples containing low
levels of HCV RNA and, on the other hand, to evaluate
the RNA and DNA stability of other important
transfusion-transmitted viruses, such as HIV-1 and
HBV, stored at different temperatures (5 C, 25 C,
%20 C, %70 C).
2. Materials and methods
2.1. Stability of HCV RNA in frozen samples
with a concentration of 100 IU/ml
An HCV RNA-positive sample was diluted in an
HCV RNA-negative fractionation plasma pool sample
(cryoprecipitate supernatant) and adjusted to yield
expected final titres of approximately 100 IU/ml. The
sample was aliquoted and stored at %20 C and%70 C. After different storage periods, the sampleswere analysed in triplicate using a qualitative PCR
technique as described above. Briefly, the extracted
RNA was reverse-transcribed and amplified by nested
PCR in a single tube using gene-specific primers for the
5#-UTP region of human HCV. The PCR method was
validated according to the European guideline for
validation of NAT techniques [27]. During the valida-
tion, the 95% cut-off point of the method was de-
termined by Probit test at 20.7 IU/ml (CI 95%:
13.8e58.8 IU/ml) [21]. We have previously published
results of these samples up to 1284 days of storage [21].
In this paper we present follow-up data up to 1829 days
(approximately 5 years).
2.2. Study design for HIV-1 RNA and HBV DNA
Samples containing different concentrations of HIV-1
RNA and HBV DNA were aliquoted and stored at 5 C
(range between 2 C and 8 C) and at 25 C (range
between 23 C and 27 C). The HIV-1 samples were
also stored at %20
C (range between 20
C and26 C) and at %70 C (range between 70 C and80 C). The RNA/DNA concentrations were chosenaccording to both the detection or quantitation limit of
each method and the storage temperature, in order to
detect the least variation in the viral titre.
2.3. Stability of HIV-1 RNA in a diluted positive
sample under freezing conditions
The original HIV-1 RNA provisional working re-
agent (PWS-1) for nucleic acid-based techniques, code
99/634 (NIBSC, South Mimms, Potters Bar, Hertford-
shire EN6 3QG, UK), stored at %70 C, was thawedand adjusted to yield expected final titres of approxi-
mately 103 IU/ml[22]. The dilutions were prepared in an
HIV-1 RNA-negative plasma pool in a laminar flow
hood. The diluted material was aliquoted in 2 ml vials
and stored at %20 C and %70 C. After differentstorage periods, the samples were thawed in a water
bath at 30 C, and different dilutions (from neat to 1 in
20) were analysed in duplicate, as follows.The viral RNA was extracted by a variation of the
method described by Chomcznski and Sacchi [23].
Briefly, 200ml of plasma sample was denatured using
a guanidine thiocyanate solution. The viral RNA was
extracted with phenolechloroform in an acid medium
and concentrated by precipitation with isopropanol.
Fifteen microlitre of the HIV-1 RNA extracted was
reverse-transcribed and amplified by 35 cycles of
a qualitative PCR in a single tube using gene-specific
primers for the gag region of HIV-1. The amplified
products were detected by means of a specific biotin-
labelled capture probe and subsequent colorimetric
reaction (ELISA-DIG Detection, Roche).
The PCR method was validated according to the
European guideline for validation of NAT techniques
[27]. During the validation, the 95% cut-off point of the
method was determined by Probit test at 237 IU/ml (CI
95%: 188e339 IU/ml). Positive controls of 595, 188 and
59 IU/ml were included in each run. The control of
595 IU/ml had to be positive in a valid run.
In all experiments, the samples stored at 20 C and70 C corresponding to a specific storage period wereanalysed in the same run of tests, to avoid the variability
that different test runs might cause.
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2.4. Stability of HIV-1 RNA in positive plasma
samples under cold-room and room-temperature
conditions
An aliquot of the original HIV-1 RNA provisional
working standard 2 (PWS-2) for nucleic acid-based
techniques, code 97/632 (NIBSC, South Mimms, PottersBar, Hertfordshire EN6 3QG, UK), stored at %70 C,was thawed and diluted in an HIV-1 RNA-negative
plasma pool sample. Two dilutions were prepared with
titres of approximately 104 IU/ml and 103 IU/ml. The
PWS-2 working reagent contains 10-fold more virus
than the HIV-1 RNA provisional working standard 1
(PWS-1, NIBSC code 99/634) and consequently, it
might have an approximate titre of 4.6 log10IU/ml[22].
The diluted material was aliquoted in 1.5 ml vials
and stored at 5G 3 C and 25G 2 C. Dilutions and
aliquots were prepared in a laminar flow hood. After
different storage periods, the samples were quantified by
PCR, using the Amplicor HIV-1 Monitor and/or the
ultrasensitive Amplicor HIV-1 Monitor, both from
Roche (quantitation limit, 500 c/ml and 50 c/ml, re-
spectively, expressed in c/ml or log10c/ml), according to
the manufacturers instructions, at an external labora-
tory (General Lab, Barcelona, Spain). Both techniques,
routinely employed in clinical laboratories, use the same
specific primers and probes for the gag region of HIV-1.
2.5. Stability of HBV DNA in positive plasma
samples under cold-room and room-temperature
conditions
One aliquot of the WHO International Standard for
Hepatitis B virus DNA for nucleic acid amplification
technology (NAT) assays, code 97/746 (NIBSC, South
Mimms, Potters Bar, Hertfordshire EN6 3QG, UK) was
reconstituted according to NIBSCs instructions. In an
international study, it was assigned a concentration of
5! 105 IU/vial. Consequently, after reconstitution with
0.5 ml of sterile nuclease-free water, the International
Standard had a titre of 106 IU/ml.
Two dilutions were prepared with titres of approx-
imately 104 IU/ml and 103 IU/ml, using an HBV DNA-
negative plasma pool. The diluted material was
aliquoted in 1.5 ml vials and stored at 5G 3 C and
25G 2 C. Dilutions and aliquots were prepared in
a laminar flow hood. After different storage periods, the
samples were quantified by PCR (Roches Amplicor
HBV-1 HIV-1 Monitor, quantitation limit 200 c/ml,
expressed in c/ml or log10c/ml) according to the
manufacturers instructions, at an external laboratory
(General Lab, Barcelona, Spain). The technique, rou-
tinely employed in clinical laboratories, uses specific
primers and probes against the core/precore region of
human HBV genome.
2.6. Statistical analysis
The HIV-1 RNA and HBV DNA logarithmic titre
decay was analysed by linear regression against time
(Eq. (1)) to determine if there was any statistically
significant change of titre during storage. The half-life
decay of each sample at different storage conditions, t1/2,defined as the time needed to reduce the initial titre by
half (50% of the initial titre, on an arithmetical scale),
equivalent to 0.3 log10titre loss, was also calculated (Eq.
(2))
log titreZ k
2:303!tClog initial titre 1
t1=2Z2:303
k !log 2 2
3. Results
3.1. Stability of HCV RNA in frozen samples
with a concentration of 100 IU/ml
The stability study on samples containing 100 IU/ml
HCV RNA had been followed up for 1829 days (5 years)
at the time this manuscript was submitted. Table 1
shows the results obtained by a qualitative RT-PCR
technique expressed in number of positives out of three
replicates (new results correspond to 909e1829 days of
storage). The samples were tested neat and afterdilutions 1/2, 1/4 and 1/8, at the above mentioned
storage periods. After 5 years at 20 C, 80 positiveresults were found in the neat sample, 76 positives at 1/2
dilution, 72 positives at 1/4 dilution and 57 positives at
1/8 dilution out of 81 tests per dilution. After the same
period at 70 C, 76 positive results were found in theneat sample out of 80 tests, 76 positives at 1/2 dilution,
73 positives at 1/4 dilution and 63 positives at 1/8
dilution out of 81 tests. For all dilutions, the following
results were obtained: 285 positives out of 324 tests and
288 positives out of 233 tests, at 20 C and 70 C,respectively. After 5 years of storage at 20 C, noevaluable decrease in HCV RNA detectability has been
observed, neither in absolute terms nor when compared
to the samples stored at 70 C.
3.2. Stability of HIV-1 RNA in diluted positive
samples under freezing conditions
These samples, containing approximately 1000 IU/ml
HIV-1 RNA, were stored at 20 C and 70 C andfollowed up for 3 years (36 months). Table 2shows the
results obtained by a qualitative RT-PCR technique as
described above, testing the sample neat and after
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dilutions 1/2 and 1/20. The results are expressed in
number of positives out of two replicates. After 3 years
at 20 C, 20 positive results were found in the neatsample, 16 positives at 1/2 dilution and 6 positives at
1/20 dilution out of 20 tests per dilution. After the same
period at 70 C, 20 positives were found in the neatsample, 19 positives at 1/2 dilution and 5 positives at
1/20 dilution out of 20 tests per dilution. For all dilutions,
the following results were obtained: 40 positive results at
20 C and 44 positives at 70 C out of 54 tests.Consequently, no significant decrease in HIV-1 RNA
detectability has been observed at 20
C, neither inabsolute terms nor when compared to the samples stored
at70 C.
3.3. Stability of HIV-1 RNA in positive plasma
samples under cold-room and room-temperature
conditions
Samples containing 103 IU/ml (equivalent to
2.72 log10 c/ml) were stored at 5 C and 25 C for 14
days and 7 days, respectively. Samples of 104 IU/ml
(equivalent to 3.99 log10c/ml) were stored at the same
temperature during 28 days (Fig. 1).
A linear regression analysis was performed in order
to determine the effect of the storage temperature on the
HIV-1 RNA titre (Table 3). After 28 days of storage at
5 C of the sample with a titre of 104 IU/ml and 14 days
of samples containing 103 IU/ml, no statistical differ-
ences from a zero value slope were observed in both
cases (slope of 0.00038 days1 and of 0.00057days1, respectively, non-significant (n.s.)). Consequ-
ently, at 5 C, no statistically significant decay in HIV-1
RNA titre was observed for the periods studied (28 days
or 14 days).
For the sample containing 104 IU/ml HIV-1 RNA,
stored at 25 C, a slope of0.00433 days1
, p Z0.051,was obtained. The estimated half-life (t1/2) was nearly 7
days (6.9 days).
Since only two results were available for the sample
containing 103 IU/ml HIV-1 RNA stored at 25 C, the
linear regression analysis was not performed. After 7
days of storage the initial titre, determined at
2.72 log10c/ml, had a titre of 2.46 log10c/ml, equivalent
to 0.26 log10 of titre reduction.
3.4. Stability of HBV DNA in positive plasma
samples under cold-room and room-temperature
conditions
Samples containing 103 IU/ml HBV DNA (equiva-
lent to 3.56 log10c/ml) and 104 IU/ml HBV DNA
(equivalent to 4.51 log10 c/ml) were followed up for 28
days at 5 C(Fig. 2A) and at 25 C (Fig. 2B).
For these samples, a linear regression analysis was
also performed in order to determine the effect of the
storage temperature on the HBV DNA titre (Table 4).
After 28 days, the results obtained at both temperatures
indicate no statistical differences from a zero value slope
(sample containing 104 IU/ml: slope of0.0009 days1
and 0.0048 days1, a t 5 C and 25 C, respectively,
Table 1
Stability of HCV RNA (diluted in a fractionation plasma pool) at
approximately 100 IU/ml
Time,
days
Positive results out of three replicates
Dilution series
(%20 C)Dilution series
(%70 C)
Neat 1/2 1/4 1/8 Neat 1/2 1/4 1/80 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 2337 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 2a 3 3 3
1462 3 3 3 2 3 3 3 2
1649 3 3 3 3 3 3 3 3
1829 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
a
Two positives from a total of 2 (1 test failed).
Table 2
Stability of HIV-1 RNA (diluted in a plasma pool) at approximately
1000 IU/ml
Time,
months
Positive results out of two replicates
Dilution series (%20 C) Dilution series (%70 C)
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
12 M. Joseet al. / Biologicals 33 (2005) 9e16
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pO0.05; sample containing 103 IU/ml: slope of0.0290days1 and 0.0048 days1, a t 5 C and 25 C, re-
spectively, pO0.05).
4. Discussion
It is important to know the nucleic acid stability of
different viruses, in terms of PCR reactivity, during
handling in the clinical and transfusional settings in
order to avoid any false negative. Different studies show
that many parameters affect the capacity of a virus to
survive in the environment, including the concentration
of virus, the temperature or the nature of the surround-
ing medium. The storage conditions are also importantin order to minimise logistics problems relative to
central laboratories.
The measurement of HCV RNA, HIV-1 RNA or
HBV DNA in plasma allows an accurate follow-up of
patients, as well as monitoring the effect of antiviral
treatments [11,17e19,24,25]. Likewise, it is important
that blood banks can detect, by nucleic acid amplifica-
tion technology (NAT), a unit positive for one of these
important viruses in order to identify and discard the
stored units corresponding to a specific window-period
donation.
Results have been published whose discrepancies
might arise from different ways of handling samples.
Many authors have reported several studies on the
stability of HCV RNA in plasma, serum or blood cell-
containing samples. Most of them focused their efforts
on the stability of HCV RNA in terms of RT-PCR
reactivity during handling in the clinical and trans-
fusional settings. In addition, the stability of plasma
samples at 70 C is well established and this is thetemperature recommended to store the reference liquid
preparations or NAT working reagents. In our previous
work, we gave a comprehensive view on the stability of
HCV RNA in plasma at a wide range of temperatures(70 C, 20 C, 5 C and 25 C) using differentanalytical methods (PCR, bDNA, qualitative and
quantitative assays) [21]. We did not find differences
between the titre of HCV RNA samples stored at
70 C and those stored at 20 C.Using quantitative techniques, we showed the ab-
sence of decay in HCV RNA attributable to storage at
20 C during the period studied (approximately2.6e2.7 years) in samples with high HCV RNA titre.
We also demonstrated the absence of significant titre
decay during storage at 20 C for approximately 1year of study in samples with intermediate concentra-
tions. We demonstrated no loss of HCV RNA reactivity
during storage at 20 C for approximately 3.5 years,using a qualitative method, in samples with a concentra-
tion close to 100 IU/ml.
In the same work, HCV RNA samples stored at 5 C
and 25 C showed high stability, the half-life being
nearly 3 months and 14 days, respectively, regardless of
the RNA concentration tested.
Taking into account the viral removal capacity of the
production process, the Committee for Proprietary
Medicinal Products of the European Medicines Evalu-
ation Agency (EMEA) stipulated, as of July 1st, 1999,
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
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 0 5 10 15 20 25 30
DAYS
logc/ml
DAYS
logc/ml
A B
Fig. 1. Stability of HIV-1 RNA in samples stored under cold-room and room-temperature conditions. Plasma samples were diluted to 104 IU/ml
(equivalent to 2.72 log10c/ml) (closed symbols) and 103 IU/ml (equivalent to 2.46 log10c/ml) (open symbols). The figure shows the HIV-1 RNA titre
of samples stored at 5 C (A) and at 25 C (B), at different storage periods, expressed in log10c/ml.
Table 3
Stability of HIV-1 RNA (log c/ml, Monitor HIV-1 Amplicor) inplasma samples under cold-room and room-temperature storage
conditions (regression analysis (log titre versus time))
5 C 25 C
Sample 104 IU/ml 103 IU/ml 104 IU/ml
Number of assays 4 3 4
Time, days 28 14 28
Slope, days1 0.00038 0.00057 0.00433p-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.a Half-life expressed in arithmetical scale (i.e. 50% or 0.3 log titre
reduction).
13M. Joseet al. / Biologicals 33 (2005) 9e16
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that only batches derived from plasma pools tested
negative for HCV RNA by NAT can be released by
plasma product manufacturers (CPMP/BWP/390/97).
Also, the method used must be able to detect a run
control with an HCV RNA content equivalent to
100 IU/ml [26]. Due to the relevance of the sample
containing 100 IU/ml HCV RNA, we decided to
continue the stability study of this sample stored at
70 C and 20 C. The results obtained up to dateshow that it will remain reactive after at least 5 years of
storage, either at 20 C or at 70 C.Due to the results obtained in our previous work with
samples containing HCV RNA and the limited knowl-
edge of the nucleic acid stability of other important
transfusion-transmitted viruses, the stability of HIV-1
RNA and HBV DNA at different temperatures was alsostudied in the present work.
Not many studies have been conducted about the
stability of HIV-1 RNA. These few reports also studied
the stability of HIV-1 RNA [9e13] in samples from
different origins (plasma, blood, and serum), in the
presence of different preservatives (citrate, EDTA, etc.),
at different storage temperatures (70 C, 15 C,4 C, room temperature, etc.) and using different
analytical methods. These studies were not carried out
under standardised conditions and discrepant conclu-
sions were sometimes drawn.
With regard to frozen plasma samples, Smith and
Heldebrant[9]showed that HIV-1 was stable for up to
45 days at 15 C and other authors [12,13] observedlong-term stability (6 months) at 70 C with nosignificant titre decrease. The same authors showed that
HIV-1 RNA is stable for up to 7 days at 5 C or 8 C,up to 10 h at 24 C[9] or up to 30 h at 4 C and 23 C
[12] or up to 14 days at 4 C [13]. The American Red
Cross (ARC) [10] also performed stability studies of
RNA in the presence of EDTA. Their data indicated no
significant loss of RNA titres in whole blood stored for
no longer than 72 h below 10 C, no significant loss in
the separated plasma after 7 days of storage below
10 C, and stability at room temperature for no longer
than 24 h. Although the conclusions of the studies might
seem discrepant, most of these works were limited by theperiod of time studied. Consequently, after these storage
periods, not always was the decay in virus titre made
evident.
In the present work, we studied the stability of
plasma samples containing HIV-1 RNA, stored at
different temperatures under standardised conditions.
Using a qualitative technique, a sample containing
approximately 1000 IU/ml HIV-1 RNA was followed
up for 3 years at 20 C and 70 C. For allconcentrations analysed, no decrease in HIV-1 RNA
detectability at 20 C was observed, neither inabsolute terms nor when compared to samples stored
at 70 C. These results are in agreement with thestability of HCV RNA in a sample containing 100 IU/
ml, which we found to be reactive after 5 years of
storage at 20 C or 70 C.Another HIV-1 RNA sample was diluted in plasma
to 103 IU/ml and 104 IU/ml and stored at 5 C and
25 C. Using a quantitative technique, we have demon-
strated the absence of decay in HIV-1 RNA caused by
storage at 5 C during the period studied (28 days for
the sample of 104 IU/ml and 7 days for the sample of
103 IU/ml). The sample of 104 IU/ml, stored at 25 C,
showed a half-life (0.3 log10of titre reduction) of nearly
A
DAYS
logc/ml
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
B
DAYS
logc/ml
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
Fig. 2. Stability of HBV DNA in samples stored under cold-room and room-temperature conditions. Plasma samples were diluted to 104 IU/ml
(equivalent to 4.51 log10c/ml) (closed symbols) and 103 IU/ml (equivalent to 3.56 log10c/ml) (open symbols). The figure shows the HBV DNA titre
of samples stored at 5 C (A) and at 25 C (B), at different storage periods, expressed in log10c/ml.
Table 4
Stability of HBV DNA (log c/ml, Monitor HBV Amplicor) in plasma
samples under cold-room and room-temperature storage conditions
(regression analysis (log titre versus time))
5 C 25 C
Sample 104 IU/ml 103 IU/ml 104 IU/ml 103 IU/ml
Number of assays 4 4 4 4
Time, days 28 28 28 28
Slope, days1 0.0009 0.0290 0.0048 0.0007p-value to test
significance of decay
0.791 0.377 0.093 0.865
14 M. Joseet al. / Biologicals 33 (2005) 9e16
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14 days, similar to the one previously found for HCV
RNA[21]. After 7 days of storage of the sample with
103 IU/ml at 25 C, the titre reduction was lower than
0.3 log10 (0.26 log10), which can be considered non-
relevant.
In the present work, we also studied the stability at
5
C and 25
C of HBV DNA-positive plasma samplesat different concentrations (103 IU/ml and 104 IU/ml).
The results obtained showed high stability, since after 28
days of storage at both temperatures, no decrease in
HBV DNA titre was detected. No relevant differences,
attributable to the DNA concentration, were found.
Several factors can affect the nucleic acid inactivation
in liquid-frozen preparations. These factors, like natural
hydrolysis or nuclease content, among others, can affect
the nucleic acids to a different extent depending on the
RNA/DNA being naked or protected by protein-coated
virus. Although the HBV DNA should be more resistant
to the effect of handling in the clinical and transfusional
settings and to storage conditions (since DNA is, in
principle, more stable than RNA) [28], not many
published studies (to the best of our knowledge) have
been carried out to study the stability of HBV DNA. A
recent work studied the stability of samples containing
different titres of HCV, HIV-1 and HBV. This study was
carried out under specific storage conditions and
without distinction of samples based on their titre.
Under these conditions, at 4 C, while the HCV and
HIV-1 RNA can be stored until 72 h, the HBV DNA
can be stored until 168 h without lowering the viral titre
[14].
The stability results of RNA (HCV and HIV-1) andDNA (HBV) of viruses studied in samples stored at 5 C
do not show significant differences. Although a 3-month
half-life could be established for HCV RNA [21], the
half-life for HIV-1 RNA and HBV DNA could not be
calculated because no titre decrease was detected during
the period studied (28 days or 14 days). On the other
hand, the HBV DNA at 25 C seems more stable than
the RNA of the viruses studied. These results suggest
that, under these conditions, the viral DNA is more
stable than the viral RNA.
The studies described in this paper complete our
previous report about the stability of HCV RNA in
plasma samples. The new stability data of 100 IU/ml
HCV RNA, HIV-1 DNA and HBV DNA show
a comprehensive view of the stability of HCV RNA,
HIV-1 RNA and HBV DNA in plasma at a wide range
of temperatures (70 C,20 C, 5 C and 25 C).This work also shows the stability of samples
containing different virus titres, under the described
conditions of handling, at different temperatures and for
different storage periods, regardless of the NAT
technique employed. It is therefore demonstrated that
the nucleic acid of viruses, in terms of NAT reactivity, is
stable under a wide range of storage conditions.
Acknowledgements
The authors thank Ms Curtu S., Ms Maya A., Ms
Prat M. and Ms Morales E. for technical assistance. This
work was supported in part by grants from MEC
(Ministerio de Educacio n y Ciencia, Spain) and FEDER.
References
[1] Busch MP, Rawal BD, Nowicki M, Operskalski EA, Mosley JW.
HCV RNA and ALT patterns in seroconverting transfusion
recipients, and correlation with donor ALT and RNA. Trans-
fusion huuib 1997;37:S443.
[2] Busch MP, Kleinman SH, Nemo GJ. Current and emerging
infectious risks of blood transfusions. JAMA 2003;289:95962.
[3] Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of
transfusion-transmitted viral infections. The retrovirus epidemi-
ology donor study. N Engl J Med 1996;334:168590.[4] Halfon P, Khiri H, Gerolami V, Bourliere M, Feryn JM,
Reynier P, et al. Impact of various handling and storage
conditions on quantitative detection of hepatitis C virus RNA.
J Hepatol 1996;25:30711.
[5] Quan CM, Krajden M, Zhao J, Chan AW. High-performance
liquid chromatography to assess the effect of serum storage
conditions on the detection of hepatitis C virus by the polymerase
chain reaction. J Virol Methods 1992;43:299308.
[6] Damen M, Sillekens P, Sjerps, Melsert R, Frantzen I,
Reesink HW, et al. Stability of hepatitis C virus RNA during
specimen handling and storage prior to NASBA amplification.
J Virol Methods 1998;72:17584.
[7] Wang JT, Wang TH, Sheu JC, Lin SM, Lin JT, Chen DS. Effects
of anticoagulants and storage of blood samples on efficacy of the
polymerase chain reaction assay for hepatitis C virus. J ClinMicrobiol 1992;30:7503.
[8] da Silva Cardoso M, Koerner K, Kubanek B. PCR screening in
the routine of blood banking of the German Red Cross blood
transfusion service of BadeneWu rttemberg. Infusionsther Trans-
fusionsmed 1998;25:11620.
[9] Smith R, Heldebrant C. Large scale PCR screening of pooled
plasma samples for HIV-1 and HCV. Advances in transfusion
safety. Dev Biol Stand 2000;102:10911.
[10] Dodd RY, Stramer SL, Aberle-Grasse J, Notari E. Risk of
hepatitis and retroviral infections among blood donors and
introduction of nucleic acid testing (NAT). Dev Biol Stand
2000;102:1927.
[11] Mellors JW, Munoz A, Giorgi JV, Margolick JB, Tassoni CJ,
Gupta P, et al. Plasma viral load and CD4C lymphocytes as pro-
gnostic markers of HIV-1 infection. Ann Intern Med 1997;126:94654.
[12] Ginocchio CC, Wang XP, Kaplan MH, Mulligan G, Witt D,
Romano JW, et al. Effects of specimen collection, processing, and
storage conditions on stability of human immunodeficiency virus
type 1 RNA levels in plasma. J Clin Microbiol 1997;35:288693.
[13] Bruisten SM, Oudshoorn P, Vanswieten P, Boesernunnink B,
Vanaarle P, Tondreau SP, et al. Stability of HIV-1 RNA in blood
during specimen handling and storage prior to amplification by
NASBA-QT. J Virol Methods 1997;67:199207.
[14] Gessoni G, Barin P, Valverde S, Giacomini A, Di Natale C,
Orlandini E, et al. Biological qualification of blood units;
considerations about the effects of samples handling and storage
on stability of nucleic acids. Transfus Apheresis Sci 2004;30:
197203.
15M. Joseet al. / Biologicals 33 (2005) 9e16
-
8/12/2019 stability of NAT samples
8/8
[15] Roth WK, Buhr S, Drosten C, Seifried E. NAT and viral safety in
blood transfusion. Vox Sang 2000;78(Suppl. 2):2579.
[16] Willkommen H, Schmidt I, Lower J. Safety issues for plasma
derivatives and benefit from NAT testing. Biologicals 1999;27:
32531.
[17] Chew CB, Zheng F, Byth K, Van Asten M, Workman C,
Dwyer DE. Comparison of three commercial assays for the
quantification of plasma HIV-1 RNA from individuals with low
viral loads. AIDS 1999;13:19778.
[18] Giachetti C, Linnen JM, Kolk DP, Dockter J, Gillotte-Taylor K,
Park M, et al. Highly sensitive multiplex assay for detection of
human immunodeficiency virus type 1 and hepatitis C virus RNA.
J Clin Microbiol 2002;40:240819.
[19] Paraskevis D, Haida C, Tassopoulos N, Raptopoulou M,
Tsantoulas D, Papachristou H, et al. Development and assess-
ment of a novel real-time PCR assay for quantitation of HBV
DNA. J Virol Methods 2002;103:20112.
[20] Candotti D, Richetin A, Cant B, Temple J, Sims C, Reeves I, et al.
Evaluation of a transcription-mediated amplification-based HCV
and HIV-1 RNA duplex assay for screening individual blood
donations: a comparison with a minipool testing system. Trans-
fusion 2003;43:21525.
[21] Jose M, Curtu S, Gajardo R, Jorquera JI. The effect of storage at
different temperatures on the stability of Hepatitis C virus RNA
in plasma samples. Biologicals 2003;31:18.
[22] Davis C, Heath A, Best S, Hewlett I, Lelie N, Schuurman R, et al.
Calibration of HIV-1 working reagents for nucleic acid amplifi-
cation techniques against the 1st international standard for HIV-1
RNA. J Virol Methods 2003;107:3744.
[23] Chomcznski P, Sacchi N. Single step method of RNA isolation by
acid guanidinium thiocyanateephenolechloroform extraction.
Anal Biochem 1987;162:1569.
[24] Roth WK, Weber M, Petersen D, Drosten C, Buhr S, Sireis W,
et al. NAT for HBV and anti-HBc testing increase blood safety.
Transfusion 2002;42:86975.
[25] Brechtbuehl K, Whalley SA, Dusheiko GM, Saunders NA. A
rapid real-time quantitative polymerase chain reaction for
hepatitis B virus. J Virol Methods 2001;93:10513.
[26] Committee for Proprietary Medicinal Products. The introduction
of nucleic acid amplification technology (NAT) for the detection
of hepatitis C virus RNA in plasma pools (CPMP/BWP/390/97).
Addendum to note for guidance on plasma-derived medicinal
products (CPMP/BWP/269/95) 1998.
[27] European Network of Official Medicines Control Laboratories,
Council of Europe. Guideline for validation of Nucleic Acid
Amplification Technology (NAT) for the detection of Hepatitis C
Virus (HCV) RNA in plasma pools (PA/PH/OMLC(98)22, DEF)
1999.
[28] Ehrlich GD, Greenberg SJ. In: PCR-based diagnostics in infectious
disease, vol. 2. Blackwell Scientific Publications; 1994. p. 38.
16 M. Joseet al. / Biologicals 33 (2005) 9e16