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Rush Medical College, 1653 W. Congress Parkway, Chicago, Illinois 60612
New England Research Institutes, Inc., 9 Galen Street, Watertown, Massachusetts 02472
ABSTRACT
The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25°C).
TEXT
The Roche Amplicor HIV-1 Test is a "research use only" kit that is commonly used to detect human immunodeficiency virus type 1 (HIV-1) proviral DNA in whole blood (1). The package insert of the kit states that blood should be maintained at 2 to 25°C and should be processed within 4 days of collection (3). This limitation on processing time poses a potential problem for testing in rural settings where blood must be sent to laboratories distant from the site of collection. Furthermore, ambient conditions that occur en route to both national and international testing sites may exceed the recommended storage conditions, and since multiple anticoagulants are suitable for this assay, it's not clear if one anticoagulant may provide better stability under adverse storage conditions. As a result, the Virology Quality Assurance Laboratory (2) designed a study to evaluate the effects of anticoagulant, time, and storage temperature on the stability of proviral HIV DNA in whole blood by using the Roche Amplicor test.
(These data were presented as a poster at the 43rd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 14 to 17 September 2003 [control no. 2493].)
EDTA- and acid citrate dextrose (ACD)-anticoagulated whole blood was obtained from six HIV-infected donors and aliquoted for storage at room temperature (RT) (25°C), 4°C, or 37°C. Whole-blood pellets (WBP) were created by using the Amplicor whole-blood sample preparation kit according to the manufacturer's instructions, on the day of blood collection and after 2, 4, 6, 8, and 10 days of storage, from samples under each combination of anticoagulant and storage temperature. Day 4 pellets from two donors were not created under any storage conditions because day 4 fell on a weekend. Day 10 pellets from ACD-treated samples were not created for two donors because of fungal contamination. All WBPs were frozen at –70°C as soon as they were created and held for batch testing at the end of each experiment.
WBPs were extracted according to the manufacturer's instructions. Briefly, the pellets were lysed in the extraction reagent (0.2 ml), digested for 30 min at 60°C, and inactivated at 100°C for 30 min. Fifty microliters of extracted material was amplified and detected according to the Roche Amplicor package insert.
Extracted samples from day 0 and day 10 were also serially diluted (1:10, 1:100, and 1:1,000) in inactivated extraction buffer and then amplified and detected according to the package insert to determine if the DNA titer declined under any of the storage conditions. Serial dilutions of extracted material for the two samples with fungal contamination were performed from the day 8 pellets.
Positive results were obtained from all undiluted pellets derived from samples stored at 4°C and RT through day 10 (Table 1). Negative or indeterminate results were observed for pellets derived from both EDTA- and ACD-treated blood stored at 37°C for at least 4 days. One of 8 results was indeterminate on day 4, 1 of 12 was negative on day 6, 8 of 12 were negative on day 8, and 7 of the 10 were either negative (n = 3) or indeterminate (n = 4) on day 10.
These data show that HIV proviral DNA could be detected in ACD- or EDTA-treated whole blood obtained from HIV-infected donors for up to 10 days after collection as long as the blood was maintained between 2 and 25°C. HIV proviral DNA could be detected in blood that was stored at 37°C, but the results became unreliable if the storage exceeded 2 days. Therefore, if a blood specimen is going to be shipped to a laboratory where storage conditions may exceed 37°C for longer than 2 days, cold packs should be used to maintain ambient conditions.
The purpose of diluting extracts before amplification was twofold: first, to get a sense of the differences in proviral titers that may have existed between the donors used for this study, and second, to see if there was any noticeable loss of DNA over time and to determine if anticoagulant or storage condition had any affect on this loss. Dilution testing of the day 0 extracts yielded 12/12 (100%) positive results at the 1:10 dilution, 11/12 (92%) positive results at the 1:100 dilution, and 4/12 (33%) positive results at the 1:1,000 dilution. No differences between the two anticoagulants were observed.
Results from the diluted extracts indicated some loss of HIV DNA over 10 days of storage at all combinations of temperature and anticoagulant. For ACD-treated blood held at RT and 4°C, only 2/6 (33%) and 3/6 (50%) of the pellets were detectable for HIV DNA at the 1:100 dilution, respectively. In EDTA-treated blood held at RT and 4°C, 5/6 (83%) and 4/6 (67%) of the pellets were detectable for HIV DNA at the 1:100 dilution, respectively. For ACD- and EDTA-treated blood held at 37°C for 10 days, 4/6 (67%) and 1/6 (17%) of the pellets were detectable for HIV DNA at the 1:100 dilution, respectively. Interestingly, 11/12 (92%) extracts of the pellets from blood stored at 37°C and diluted 1:10 were positive for HIV DNA, which is a higher percentage of positive results than that observed in undiluted extracts. This may indicate that the reduced detection rate in blood held at 37°C was attributed to an increase in inhibitors rather than an accelerated loss of DNA and diluting the extract helped to remove this inhibition.
In conclusion, it is possible to qualitatively detect HIV proviral DNA in whole blood that has been maintained at 4 to 25°C for up to 10 days, but in order to achieve maximum sensitivity, the specimens should be processed as soon as possible after collection.
ACKNOWLEDGMENTS
This work was supported by National Institute of Allergy and Infectious Diseases contract NO-AI-85354.
REFERENCES
Bremer, J. W., J. F. Lew, K. P. Malobisky, E. Cooper, G. V. Hillyer, J. Pitt, E. Handelsman, D. Brambilla, J. Moye, and R. Hoff. 1996. Early diagnosis of HIV-1 by the Roche Amplicor HIV-1 DNA PCR assay in infants enrolled in the Women and Infants Transmission Study (WITS). J. Pediatr. 129:198-207.
Jackson, J. B., J. Drew, H. J. Lin, P. Otto, J. W. Bremer, F. B. Hollinger, S. Wolinsky, the ACTG PCR Working Group, and the ACTG PCR Virology Laboratories. 1993. Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays in the AIDS Clinical Trials Group. J. Clin. Microbiol. 31:3123-3128.
Roche Molecular Systems. 2002. Roche Amplicor HIV-1 Test manual. Roche Molecular Systems, Inc., Pleasanton, Calif.