Biologicals 33 (2005) 9e16 www.elsevier.

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Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage
*, Rodrigo Gajardo, Juan I. Jorquera Marta Jose
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 amplication 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 eect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after storage at dierent temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years at 20  C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also been studied. At 20  C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decay in 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 donations made during the serological window period. In addition to the routinely performed serological tests, the introduction of nucleic acid amplication techniques (NAT) has allowed the window period to be reduced to 6e10 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

* Corresponding author. Tel.: C34 935710593; fax: C34 935710855. ). E-mail address: marta.jose@grifols.com (M. Jose

handling and/or storage of samples. Many authors have reported that the storage conditions of samples may aect the stability and, hence, the detectability of nucleic acid of viruses, although apparently discrepant conclusions have been drawn [4e14]. Dierent facts might have an eect on the nucleic acid stability: shipping conditions, 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) are of 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

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

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M. Jose et al. / Biologicals 33 (2005) 9e16

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 dierent HCV RNA concentrations at 70  C [21]. We found absence of decay in HCV RNA attributable to the storage at 20  C during the period studied (approximately 2.6e2.7 years) in samples with high HCV RNA titre. We also demonstrated the absence of signicant titre decay during storage at 20  C for approximately 1 year of study in samples with intermediate concentrations. 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, the half-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 dierent temperatures (5  C, 25  C, %20  C, %70  C).

2.2. Study design for HIV-1 RNA and HBV DNA Samples containing dierent 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 and 26  C) and at %70  C (range between 70  C and 80  C). The RNA/DNA concentrations were chosen according 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 reagent (PWS-1) for nucleic acid-based techniques, code 99/634 (NIBSC, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK), stored at %70  C, was thawed and adjusted to yield expected nal titres of approximately 103 IU/ml [22]. The dilutions were prepared in an HIV-1 RNA-negative plasma pool in a laminar ow hood. The diluted material was aliquoted in 2 ml vials and stored at %20  C and %70  C. After dierent storage periods, the samples were thawed in a water bath at 30  C, and dierent 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]. Briey, 200 ml 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 amplied by 35 cycles of a qualitative PCR in a single tube using gene-specic primers for the gag region of HIV-1. The amplied products were detected by means of a specic biotinlabelled 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-o 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 and 70  C corresponding to a specic storage period were analysed in the same run of tests, to avoid the variability that dierent test runs might cause.

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 nal titres of approximately 100 IU/ml. The sample was aliquoted and stored at %20  C and %70  C. After dierent storage periods, the samples were analysed in triplicate using a qualitative PCR technique as described above. Briey, the extracted RNA was reverse-transcribed and amplied by nested PCR in a single tube using gene-specic 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 validation, the 95% cut-o point of the method was determined 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).

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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, Potters Bar, 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 log10 IU/ml [22]. The diluted material was aliquoted in 1.5 ml vials and stored at 5 G 3  C and 25 G 2  C. Dilutions and aliquots were prepared in a laminar ow hood. After dierent storage periods, the samples were quantied 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, respectively, expressed in c/ml or log10 c/ml), according to the manufacturers instructions, at an external laboratory (General Lab, Barcelona, Spain). Both techniques, routinely employed in clinical laboratories, use the same specic primers and probes for the gag region of HIV-1.

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 signicant change of titre during storage. The half-life decay of each sample at dierent storage conditions, t1/2, dened as the time needed to reduce the initial titre by half (50% of the initial titre, on an arithmetical scale), equivalent to 0.3 log10 titre loss, was also calculated (Eq. (2)) log titreZ k !tClog initial titre 2:303 1

t1=2 Z

2:303 !log 2 k

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 after dilutions 1/2, 1/4 and 1/8, at the above mentioned storage periods. After 5 years at 20  C, 80 positive results 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 the neat 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, no evaluable 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 and followed up for 3 years (36 months). Table 2 shows the results obtained by a qualitative RT-PCR technique as described above, testing the sample neat and after

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 amplication 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 approximately 104 IU/ml and 103 IU/ml, using an HBV DNAnegative plasma pool. The diluted material was aliquoted in 1.5 ml vials and stored at 5 G 3  C and 25 G 2  C. Dilutions and aliquots were prepared in a laminar ow hood. After dierent storage periods, the samples were quantied by PCR (Roches Amplicor HBV-1 HIV-1 Monitor, quantitation limit 200 c/ml, expressed in c/ml or log10 c/ml) according to the manufacturers instructions, at an external laboratory (General Lab, Barcelona, Spain). The technique, routinely employed in clinical laboratories, uses specic primers and probes against the core/precore region of human HBV genome.

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M. Jose et al. / Biologicals 33 (2005) 9e16

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) Neat 0 7 14 21 28 35 42 49 56 85 108 140 168 224 280 337 366 457 562 639 731 909 1095 1284 1462 1649 1829 Total
a

Dilution series (%70  C) 1/4 3 2 3 2 3 2 1 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0 72/81 1/8 3 2 3 3 3 3 2 2 1 2 2 2 2 3 3 3 3 0 2 2 1 2 1 2 2 3 0 57/81 Neat 3 3 3 3 3 3 2 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2a 3 3 3 76/80 1/2 3 3 3 3 3 2 2 3 2 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 2 76/81 1/4 3 3 3 3 3 2 1 3 3 2 2 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 1 73/81 1/8 3 2 3 3 3 3 1 2 0 2 2 2 1 3 2 3 3 1 3 3 3 2 3 3 2 3 2 63/81

1/2 3 3 3 3 2 2 3 2 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 2 76/81

3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 80/81

period at 70  C, 20 positives were found in the neat sample, 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 signicant decrease in HIV-1 RNA detectability has been observed at 20  C, neither in absolute terms nor when compared to the samples stored at 70  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 log10 c/ml) were stored at the same temperature during 28 days (Fig. 1). A linear regression analysis was performed in order to determine the eect 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 dierences from a zero value slope were observed in both cases (slope of 0.00038 days1 and of 0.00057 days1, respectively, non-signicant (n.s.)). Consequently, at 5  C, no statistically signicant 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 of 0.00433 days1, p Z 0.051, 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 log10 c/ml, had a titre of 2.46 log10 c/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 (equivalent to 3.56 log10 c/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 eect of the storage temperature on the HBV DNA titre (Table 4). After 28 days, the results obtained at both temperatures indicate no statistical dierences from a zero value slope (sample containing 104 IU/ml: slope of 0.0009 days1 and 0.0048 days1, at 5  C and 25  C, respectively,

Two positives from a total of 2 (1 test failed).

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 neat sample, 16 positives at 1/2 dilution and 6 positives at 1/20 dilution out of 20 tests per dilution. After the same
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) Neat 0 3 6 9 12 15 18 24 30 36 Total 2 2 2 2 2 2 2 2 2 2 20/20 1/2 2 2 0 2 1 2 2 1 2 2 16/20 1/20 1 1 1 1 0 0 0 0 2 0 6/20 Dilution series (%70  C) Neat 2 2 2 2 2 2 2 2 2 2 20/20 1/2 2 2 2 2 2 2 2 1 2 2 19/20 1/20 1 1 0 0 0 0 0 1 1 1 5/20

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A
4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00
0 5 10 15 20 25 30

B
4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 0 5 10 15 20 25 30

log c/ml

DAYS

log c/ml

DAYS

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 log10 c/ml) (closed symbols) and 103 IU/ml (equivalent to 2.46 log10 c/ml) (open symbols). The gure shows the HIV-1 RNA titre of samples stored at 5  C (A) and at 25  C (B), at dierent storage periods, expressed in log10 c/ml.

p O 0.05; sample containing 103 IU/ml: slope of 0.0290 days1 and 0.0048 days1, at 5  C and 25  C, respectively, p O 0.05).

4. Discussion It is important to know the nucleic acid stability of dierent viruses, in terms of PCR reactivity, during handling in the clinical and transfusional settings in order to avoid any false negative. Dierent studies show that many parameters aect the capacity of a virus to survive in the environment, including the concentration of virus, the temperature or the nature of the surrounding medium. The storage conditions are also important in 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 eect of antiviral treatments [11,17e19,24,25]. Likewise, it is important that blood banks can detect, by nucleic acid amplication technology (NAT), a unit positive for one of these important viruses in order to identify and discard the
Table 3 Stability of HIV-1 RNA (log c/ml, Monitor HIV-1 Amplicor) in plasma samples under cold-room and room-temperature storage conditions (regression analysis (log titre versus time)) 5 C Sample Number of assays Time, days Slope, days1 p-value to test signicance of decay t1/2, daysa 104 IU/ml 4 28 0.00038 0.542 n.a. 103 IU/ml 3 14 0.00057 0.806 n.a. 25  C 104 IU/ml 4 28 0.00433 0.051 6.9

n.a., Not applicable. a Half-life expressed in arithmetical scale (i.e. 50% or 0.3 log titre reduction).

stored units corresponding to a specic window-period donation. Results have been published whose discrepancies might arise from dierent ways of handling samples. Many authors have reported several studies on the stability of HCV RNA in plasma, serum or blood cellcontaining samples. Most of them focused their eorts on the stability of HCV RNA in terms of RT-PCR reactivity during handling in the clinical and transfusional settings. In addition, the stability of plasma samples at 70  C is well established and this is the temperature 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 dierent analytical methods (PCR, bDNA, qualitative and quantitative assays) [21]. We did not nd dierences between the titre of HCV RNA samples stored at 70  C and those stored at 20  C. Using quantitative techniques, we showed the absence of decay in HCV RNA attributable to storage at 20  C during the period studied (approximately 2.6e2.7 years) in samples with high HCV RNA titre. We also demonstrated the absence of signicant titre decay during storage at 20  C for approximately 1 year of study in samples with intermediate concentrations. 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 concentration 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 Evaluation Agency (EMEA) stipulated, as of July 1st, 1999,

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M. Jose et al. / Biologicals 33 (2005) 9e16

A
5,00 4,50 4,00 3,50

B
5,00 4,50 4,00 3,50

log c/ml

2,50 2,00 1,50 1,00 0,50 0,00 0 5 10 15 20 25 30

log c/ml

3,00

3,00 2,50 2,00 1,50 1,00 0,50 0,00 0 5 10 15 20 25 30

DAYS

DAYS

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 log10 c/ml) (closed symbols) and 103 IU/ml (equivalent to 3.56 log10 c/ml) (open symbols). The gure shows the HBV DNA titre of samples stored at 5  C (A) and at 25  C (B), at dierent storage periods, expressed in log10 c/ml.

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 date show 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 knowledge of the nucleic acid stability of other important transfusion-transmitted viruses, the stability of HIV-1 RNA and HBV DNA at dierent temperatures was also studied 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 dierent origins (plasma, blood, and serum), in the presence of dierent preservatives (citrate, EDTA, etc.), at dierent storage temperatures (70  C, 15  C, 4  C, room temperature, etc.) and using dierent analytical methods. These studies were not carried out under standardised conditions and discrepant conclusions were sometimes drawn.

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 Sample Number of assays Time, days Slope, days1 p-value to test signicance of decay 10 IU/ml 4 28 0.0009 0.791
4

25  C 10 IU/ml 4 28 0.0290 0.377

104 IU/ml 4 28 0.0048 0.093

103 IU/ml 4 28 0.0007 0.865

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] observed long-term stability (6 months) at 70  C with no signicant 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 signicant loss of RNA titres in whole blood stored for no longer than 72 h below 10  C, no signicant 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 the period 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 dierent 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 all concentrations analysed, no decrease in HIV-1 RNA detectability at 20  C was observed, neither in absolute terms nor when compared to samples stored at 70  C. These results are in agreement with the stability 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 demonstrated 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 log10 of titre reduction) of nearly

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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 nonrelevant. In the present work, we also studied the stability at 5  C and 25  C of HBV DNA-positive plasma samples at dierent 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 dierences, attributable to the DNA concentration, were found. Several factors can aect the nucleic acid inactivation in liquid-frozen preparations. These factors, like natural hydrolysis or nuclease content, among others, can aect the nucleic acids to a dierent extent depending on the RNA/DNA being naked or protected by protein-coated virus. Although the HBV DNA should be more resistant to the eect 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 dierent titres of HCV, HIV-1 and HBV. This study was carried out under specic 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) and DNA (HBV) of viruses studied in samples stored at 5  C do not show signicant dierences. 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 dierent virus titres, under the described conditions of handling, at dierent temperatures and for dierent 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 n y Ciencia, Spain) and FEDER. (Ministerio de Educacio

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