stability of nat samples

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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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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.

10 M. Joseet al. / Biologicals 33 (2005) 9e16


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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


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

11M. Joseet al. / Biologicals 33 (2005) 9e16


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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


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


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



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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).



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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



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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.

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