![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Clinical Microbiology, February 1998, p. 382-386, Vol. 36, No. 2
Department of Microbiology,
Received 4 August 1997/Returned for modification 26 September
1997/Accepted 30 October 1997
Quantification of hepatitis B virus (HBV) DNA in serum is used to
establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the
conditions that preserve sample integrity is of paramount importance.
We therefore evaluated the stability of HBV DNA in 26 previously frozen
(PF) and 5 fresh, never previously frozen serum specimens. PF
specimens, covering a 3-log10 HBV DNA dynamic range, were
thawed and stored at Quantification of hepatitis B virus
(HBV) DNA in serum is used to determine eligibility for antiviral
therapy and to monitor treatment response. With the trend toward
centralized testing of HBV DNA, it is important to define the shipping
and storage conditions that preserve specimen integrity. This
information can minimize the risk of HBV DNA degradation, can ensure
that specimens are properly handled within laboratories, and may reduce handling costs. We therefore evaluated the stability of HBV DNA in
serum specimens stored at different temperatures for various lengths of
time.
Assessment of the effect of serum storage conditions on the ability to
detect an analyte such as HBV DNA quantitatively requires the
following: (i) an assay capable of generating a reproducible relationship between the quantity of the analyte and its output signal;
(ii) the measurement of the quantity of HBV DNA in sufficient numbers
of specimens to ensure that the study has sufficient statistical power
to demonstrate that changes in the quantity of HBV DNA reflect the
effect of specimen storage and/or handling and not inter- or
intra-assay variability, (iii) a descriptive endpoint that reflects a
clinically relevant change in the quantity of the analyte which can be
reliably measured, and (iv) appropriate statistical analysis of the
data.
HBV DNA levels were measured by the Chiron Quantiplex HBV DNA assay,
which is based on branched-DNA (bDNA) technology, because it has the
widest dynamic range of the commercially available HBV DNA assays and
it can reliably detect small (twofold) changes in HBV DNA concentration
(2, 7). We used several statistical methods to examine
the stability of HBV DNA in serum and evaluated sufficient numbers of
specimens to ensure that the statistical power of our analysis could
reliably detect changes in HBV DNA stability. First, linear
regression was used to estimate the change in the quantity of HBV DNA
stored at the various temperatures over time. Subsequently, three
different statistical methods (Kaplan-Meier, two-way
probability table, and logistic regression analyses) were used to
estimate the probability of specimen failure, defined as a loss of 20%
or more of HBV DNA and/or coagulation of serum.
(This study was presented in part at the 97th General Meeting of the
American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [9a].)
Specimens.
HBV DNA-positive sera were obtained from routine
clinical samples or from patients involved in clinical trials. All sera
were separated from the clot within 4 h of collection. Both
previously frozen (PF; n = 26) and fresh, never
previously frozen (n = 5) specimens were evaluated.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Assessment of Hepatitis B Virus DNA Stability in
Serum by the Chiron Quantiplex Branched-DNA Assay
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at 70°C prior to
testing. Five fresh specimens were split into two groups. Both group
FG1 and group FG2 specimens were handled as described above; however,
group FG1 specimens were subsequently maintained at 4°C and were
never frozen prior to testing. Linear regression analysis of PF
specimens demonstrated no significant HBV DNA degradation at 
4°C
over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20%
per day at 23, 37, and 45°C, respectively. Three independent
statistical methods confirmed that the probability of specimen failure,
defined as a loss of 20% or more of HBV DNA and/or coagulation of
serum, was lowest at 4°C and increased with temperature. Because
only 10 to 20% of individual patient specimens demonstrated losses of
HBV DNA of 
20% at 23 or 37°C, sufficient numbers of serum
specimens must be evaluated to determine overall statistical trends. In
conclusion, HBV DNA integrity in separated serum specimens is preserved
for at least 5 days when the specimens are stored at 70 or 4°C.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and
120 h (±2 h). Following incubation, the specimens were centrifuged at 14,000 rpm for 1 min in an Eppendorf Microfuge (catalog
no. 5415C) to collect the condensate and were frozen at 70°C until
assayed. PF specimens were independently tested by two laboratories.
The Toronto Hospital (n = 20) and Covance Laboratories
(n = 6; specimens from Covance Laboratories were not
incubated at 45°C). All specimens and controls underwent the same
number of freeze-thaw cycles prior to being assayed.
70°C prior to undergoing testing for HBV DNA. FG2 samples underwent
only one freeze-thaw cycle. Both FG1 and FG2 specimens were assayed
within 10 days of collection.
HBV DNA quantification. The Quantiplex HBV DNA assay (Chiron Corporation), based on bDNA technology, was used according to the manufacturer's instructions. The Chiron bDNA assay has been shown to be sensitive, specific, and linear over a nearly 4-log10 quantification range (2, 7). The Chiron HBV bDNA assay demonstrates inter- and intrarun coefficients of variation of 10 to 15% and has been shown to reproducibly detect twofold changes in HBV DNA levels (7). All specimens were tested in duplicate, time zero controls were tested in quadruplicate, and the quantity of HBV DNA in each specimen was determined from a standard curve of HBV DNA run in parallel for each assay. To further enhance reproducibility, all of the specimens derived from an individual patient were assayed in the same assay run. Results were expressed as megaequivalents of HBV DNA per milliliter, with 1 Meq defined as the amount of HBV DNA which generates a level of light emission equivalent to that of 106 copies of the HBV DNA standard (7).
Statistical methods. The raw data were evaluated by examining the daily mean HBV DNA level at each temperature. Changes in the HBV DNA level at each temperature over time were evaluated by linear regression (18). The data from each incubation temperature were grouped into one category and analyzed as a specific or a fixed effect; i.e., any inferences from the data were considered specific to that particular temperature. In contrast, site- and patient-related differences in HBV DNA levels were analyzed as random effects, i.e., as inferences which are nonspecific to the particular patient or site in the study but which are applicable to any selection from the distribution of all possible sites and patients.
The probability of specimen failure was estimated by three methods: Kaplan-Meier, two-way probability table, and logistic regression analyses for PF specimens and by logistic regression analysis for FG1 and FG2 specimens. For PF, FG1, and FG2 samples, specimen failure was arbitrarily defined as the loss of 20% or more of the amount of HBV DNA quantified from the corresponding control specimen (either immediately frozen or stored at 4°C after collection) and/or the coagulation of serum. To facilitate comparison of the estimates obtained by Kaplan-Meier analysis with those obtained by linear regression analysis, the data were categorized by temperature, and a simple moving time average over three adjacent values was obtained to minimize the impact of an isolated false specimen failure on the results obtained by Kaplan-Meier analysis. In time series signal processing, a moving average serves as a filter for high-frequency patterns, which are assumed to be noise. The moving average serially processes data for each time point, weighting the data for the current time point more heavily than those for adjacent time points; the sum of the weights typically are normalized to 1. One-way table analysis is typically used to show the effects of different levels of a single factor. Similarly, two-way table analysis show the effect of combinations of levels for two factors and are frequently analyzed by using a chi-square statistic. Two-way probabilities were created by dividing the number of measured specimen failures by the total number of measurements in each temperature category. Logistic regression analysis was performed in two stages (10). Each temperature level was considered to be a specific category or fixed effect, and both site- and patient-related variations in assay levels were considered to be random or nonspecific effects. For the fresh specimens, the probability of specimen failure was evaluated only by logistic regression because the sample size was too small for Kaplan-Meier and two-way table analyses. In all cases, a sufficient number of specimens was assessed to achieve at least a 95% confidence interval (CI) or its equivalent. All the data were natural log transformed prior to analysis. Such transformation was necessary to ensure that the error was constant, because regression analysis assumes that error properties are the same over the dynamic range, whereas intrinsic assay error tends to be proportional to the viral load. Another advantage of natural log transformation is that the standard error in natural log units is directly related to the coefficient of variance of the raw units when the coefficient of variance is less than 50%.| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
PF specimens. Twenty-six PF specimens were assessed. The quantity of HBV DNA in these specimens ranged from 1.57 to 5,029 Meq/ml, with a mean of 641 Meq/ml. The scatter in quantitation for each day is shown for each temperature category in Fig. 1A to D by plotting the quantity of HBV DNA (in megaequivalents per milliliter) on the y axis and time (in days) on the x axis. In order to illustrate the overall trends in the daily means, the y axis has been cropped such that four data points above 3,000 Meq are not shown. Day 0 has the highest number of datum points because the quantity of HBV DNA in the control samples was measured in quadruplicate and specimens that coagulated (which occurred only in some specimens incubated at 45°C) were not assessed. For Fig. 1D (45°C), only 20 of 26 PF specimens were tested (specimens tested at Covance Laboratories were not tested at 45°C). The specimens for this temperature category demonstrated a lower baseline mean HBV DNA level.
|
20% HBV DNA loss at these temperatures. Of note,
the rates of HBV DNA loss at 23 and 37°C did not differ significantly. The highest rate of HBV DNA loss was observed in samples
incubated at 45°C, which showed a decrease in HBV DNA levels of 0.246 ln Meq per day, and this was the only temperature at which specimen
coagulation occurred.
|
|
Fresh specimens.
Five fresh, never previously frozen specimens
containing between 0.70 and 73.58 Meq of HBV DNA per ml, with a mean of
42.79 Meq/ml, were also assessed. Specimens from FG1 were divided into aliquots and incubated at 4, 23, 37, or 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h). Following the allotted incubation times, each specimen was either stored at 4°C for up to 10 days (FG1) or
frozen at 70°C (FG2) prior to testing.
70°C. The greatest DNA losses
were observed in specimens incubated at the higher temperatures (23, 37, and 45°C) for 120 h prior to being frozen at 70°C. HBV
DNA levels decreased by 0.062, 0.070, and 0.124 ln Meq per day at the
respective temperatures. There were also HBV DNA losses, albeit to a
lesser extent, for the aliquots incubated at 23, 37, and 45°C
followed by storage at 4°C, with HBV DNA levels decreasing by 0.033, 0.064, and 0.099 ln Meq per day, respectively.
The probability of specimen failure for stored fresh specimens was
evaluated by logistic regression (Table 1). The probability of specimen
failure did not reach significance for specimens maintained at 4°C
for 120 h. However, the probability of specimen failure increased
with increasing temperature, such that at the highest temperature,
45°C, there was a 50% probability of specimen failure for samples
that had never been frozen. Of interest, the probability of specimen
failure was higher for all fresh samples that were frozen at 70°C
(FG2) than for specimens that had never been frozen (FG1).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Improvements in molecular biology-based technologies have dramatically enhanced the accuracy and reproducibility of quantitative viral nucleic acid detection in serum and plasma (5, 7, 20). Accurate viral load determinations are important because the viral load provides an indirect measure of the amount of viral replication in vivo and is used to help predict an individual's response to therapy and/or clinical outcome (12, 15). In order to properly validate the relationship between HBV viral load and clinical disease and to monitor the effects of antiviral therapy, it is crucial to maintain specimen integrity during shipping and handling. To date, we are unaware of an adequate stability study that has evaluated the stability of HBV DNA in serum under various temperature and storage conditions.
Most published studies of nucleic acid stability in serum or plasma have focused on hepatitis C virus and human immunodeficiency virus. In many cases those studies were limited because they involved the use of nonstandardized assays, tested a limited number of samples, used different and sometimes poorly defined endpoints, and did not involve rigorous statistical analysis (1, 3, 16). While PCR-based assays are generally more sensitive than hybridization assays for nucleic acid quantification, in-house PCR assays tend to be very poorly standardized (9, 17, 21).
When investigators have used standardized, commercially available hybridization or PCR-based assays such as the Chiron bDNA or the Amplicor HCV Monitor assay (4, 6, 8, 13, 14), several factors have been shown to affect either specimen integrity or the ability of a given assay to accurately quantify the analyte. These factors include (i) the time between specimen collection and the separation of the plasma or serum, with short intervals of 4 to 6 h maximizing the amount of nucleic acid that can be detected in a given sample (4, 6, 13); (ii) the nature of the sample collection tube and/or the anticoagulant used (8, 14, 19); and (iii) the temperature and duration of storage. For example, once a specimen has been separated, storage at 4°C for a few days does not generally lead to a loss of the viral nucleic acid (3, 6, 8, 13).
Although this study did not assess the impact of the time between separation of the serum from the clot, because all specimens were separated within 4 h, we have defined the critical elements required for accurate assessment of analyte stability in clinical specimens. These elements include (i) the use of a standardized and reproducible assay, (ii) assessment of sufficient numbers of specimens to ensure statistical power, (iii) the use of a clinically relevant descriptive endpoint, and (iv) the use of appropriate statistical analysis.
Our data demonstrated that in individual specimens there was a marked
variability in HBV DNA stability and that this variability was a
function of the storage temperature and the viral load. We found that
examination of the raw data alone could obscure the overall degradation
trends, in part, because only 10 to 20% of individual patient
specimens lose 20% of their HBV DNA when they are stored at
temperatures above 4°C. Thus, it is critical that sufficient numbers
of specimens be evaluated. We also found that logarithmic
transformation of the raw data, which has the effect of linearizing the
decay rate, enhanced the ability to assess the overall statistical
trend.
Although the percentage of HBV DNA lost could be projected from the estimated daily rates of change at each temperature, a more meaningful estimation of the potential clinical implications was provided by the probability of specimen failure, which we arbitrarily defined as a 20% or greater loss of HBV DNA and/or coagulation of the specimen. Although it could be debated whether a 20% loss of HBV DNA is clinically relevant, we did test sufficient numbers of specimens to document a 20% loss of HBV DNA, and it is common laboratory practice to discard coagulated specimens (which occurred for only some of the specimens stored at 45°C). We then used three different statistical methods to assess the probability of specimen failure (i.e., Kaplan-Meier, two-way probability table, and logistic regression analyses), and all three statistical methods yielded concordant results, such that the lowest probability of specimen failure was observed at the lowest temperature and the probability of specimen failure increased with increasing temperature. Kaplan-Meier analysis produced the highest estimates for the probability of specimen failure. Results of the two-way table analysis, which indicated a lower probability of specimen failure than Kaplan-Meier analysis, provided a lower limit of HBV DNA loss. Logistic regression analysis provided intermediate and probably the most realistic estimates of specimen failure, primarily because it is a model-based analysis that uses intrinsic trends in the data (11).
A substantial number of patient specimens had to be tested in order to
achieve adequate statistical power at the 95% CI assuming that
measurements are taken at three to four time points for each temperature. Of the three methods, linear regression analysis required
the least number of samples to achieve statistical power. In order to
approximate the number of uncorrelated samples required to achieve a
95% CI, the following formula was used: N 
8 (
/
)2, where N equals the number of
patients, is the inherent error for each quantitation, and is
the smallest change that is detected with a 95% CI. Typically, a
relative error of 80% would require the inclusion of five patients.
The power of two-way probability tables is based on the equation
n 4p(1 p)/(p)2, where n is the
total number of time points multiplied by the total number of patients,
p is the estimated probability of specimen failure, and
p represents the acceptable uncertainty of p.
When the probability of specimen failure is 70%, which occurred at 45°C, the number of patients required to achieve adequate statistical power at the 95% CI is 24. The requirements of logistic regression fall between those of linear regression and two-way tables. For instance, if a 10% error rate in the probability of specimen failure is considered acceptable, a cohort of 8 to 10 patients would satisfy the requirements for adequate statistical power (95% CI) for all three
statistical methods. The numbers of specimens in the cohort of frozen
specimens far exceeded these criteria because a total of 26 specimens
were used.
We selected PF specimens with quantities of HBV DNA over the entire range of detectability by the bDNA assay to assess HBV DNA stability but found no correlation between viral load at time zero and specimen failure. It is possible that specimens containing less than 0.7 Meq of HBV DNA per ml, the lower limit of quantitation of the Chiron bDNA assay, may not have the same stability profile. Such specimens will need to be evaluated when newer, more sensitive standardized assays become available.
We also evaluated five fresh serum specimens that had never been frozen
prior to testing. From this limited data set, it appears that freezing
somewhat alters the stability of HBV DNA in specimens once they have
been maintained at 4°C for any length of time compared to the
stability of HBV DNA in a matched set of unfrozen specimens. Further
study of fresh specimens is warranted to confirm this observation. It
is possible that freezing may cause immune complex and/or viral
precipitation, which may affect the amount of HBV DNA that is
subsequently detected in the clinical specimen after it has been
frozen. This may be especially important when using as controls
specimens which have never been frozen (6).
In summary, we have evaluated the stability of HBV DNA in PF and in
fresh serum specimens that were incubated at various temperatures for
discrete lengths of time and which were then stored at 4°C or frozen
at 70°C prior to testing by the Chiron bDNA assay. The data support
the fact that HBV DNA in separated serum stored at 70 or 4°C is
stable for at least 5 days.
| |
ACKNOWLEDGMENTS |
|---|
This study was supported in part by Toronto Medical Laboratories and The Toronto Hospital and by Chiron Diagnostics, Emeryville, Calif.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: The Toronto Hospital, Department of Microbiology, 13-NU-102, 200 Elizabeth St., Toronto, Ontario, Canada M5G 2C4. Phone: (416) 340-3336. Fax: (416) 971-6362. E-mail: mkrajden{at}torhosp.toronto.on.ca.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Busch, M. P., J. C. Wilber, P. Johnson, L. Tobler, and C. S. Evans. 1992. Impact of specimen handling and storage on detection of hepatitis C virus RNA. Transfusion 32:420-425[Medline]. |
| 2. | Butterworth, L.-A., S. L. Prior, P. J. Buda, J. L. Faoagalo, and W. G. E. Cooksley. 1996. Comparison of four methods for quantitative measurement of hepatitis B viral DNA. J. Hepatol. 24:686-691[Medline]. |
| 3. |
Cuypers, H. T. M.,
D. Bresters,
I. N. Winkel,
H. W. Reesink,
A. J. Weiner,
M. Houghton,
C. L. van der Poel, and P. N. Lelie.
1992.
Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.
J. Clin. Microbiol.
30:3220-3224 |
| 4. | Davis, G. L., J. Y.-N. Lau, M. S. Urdea, P. D. Neuwald, J. C. Wilber, K. Lindsay, R. P. Perrillo, and J. Albrecht. 1994. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology 19:1337-1341[Medline]. |
| 5. | Detmer, J., R. Lagier, J. Flynn, C. Zayati, J. Kolberg, M. Collins, M. Urdea, and R. Sánchez-Pescador. 1996. Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology. J. Clin. Microbiol. 34:901-907[Abstract]. |
| 6. | Halfon, P., H. Khiri, V. Gerolami, M. Bourliere, J. Feryn, M., P. Reynier, A. Gauthier, and G. Cartouzou. 1996. Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA. J. Hepatol. 25:307-311[Medline]. |
| 7. | Hendricks, D. A., B. J. Stowe, B. S. Hoo, J. Kolberg, B. D. Irvine, P. D. Neuwald, M. S. Urdea, and R. P. Perrillo. 1995. Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay. Am. J. Clin. Pathol. 104:537-546[Medline]. |
| 8. | Holodniy, M., L. Mole, B. Yen-Lieberman, D. Margolis, C. Starkey, R. Carroll, T. Spahlinger, J. Todd, and J. B. Jackson. 1995. Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes. J. Clin. Microbiol. 33:1562-1566[Abstract]. |
| 9. |
Jackson, B. J.,
J. Drew,
H. J. Lin,
P. Otto,
J. W. Bremer,
F. B. Hollinger,
S. M. 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 by the AIDS clinical trials group.
J. Clin. Microbiol.
31:3123-3128 |
| 9a. | Krajden, L., Comanor, J. Minor, O. Rifkin, A. Grigoriew, and G. Kapke. 1997. Effect of storage on hepatitis B virus (HBV) stability in serum as measured by the Chiron Quantiplex + assay (bDNA), abstr. S-11, p. 533. In Abstracts of the 97th General Meeting of the American Society for Microbiology 1997. American Society for Microbiology, Washington, D.C. |
| 10. | Lau, D., L. Comanor, J. M. Minor, G. Watson, and J. H. Hoofnagle. 1996. Statistical models for predicting a response to alpha interferon in patients with chronic hepatitis B. Hepatology 24(Suppl.):279A (abstr. 611). |
| 11. | Lee, E., T. 1992. Statistical methods for survival data analysis, 2nd ed. John Wiley & Sons, Inc., New York, N.Y. |
| 12. |
Mellors, J. W.,
A. Munoz,
J. V. Giorgi,
J. B. Margolick,
C. J. Tassoni,
P. Gupta,
L. A. Kingsley,
J. A. Todd,
A. J. Saah,
R. Detels,
J. P. Phair, and C. R. Rinaldo.
1997.
Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection.
Ann. Intern. Med.
126:946-954 |
| 13. | Miskovsky, E. P., A. V. Carrella, K. Gutekunst, C.-A. Sun, T. C. Quinn, and D. L. Thomas. 1996. Clinical characterization of a competitive PCR assay for quantitative testing of hepatitis C virus. J. Clin. Microbiol. 34:1975-1979[Abstract]. |
| 14. |
Mole, L.,
D. Margolis,
R. Carroll,
J. Todd, and M. Holodniy.
1994.
Stabilities of quantitative plasma culture for human immunodeficiency virus, RNA, and p24 antigen from samples collected in VACUTAINER CPT and standard VACUTAINER tubes.
J. Clin. Microbiol.
32:2212-2215 |
| 15. |
Nowak, M. A.,
S. Bonhoeffer,
A. M. Hill,
R. Boehme,
H. C. Thomas, and H. McDade.
1996.
Viral dynamics in hepatitis B virus infection.
Proc. Natl. Acad. Sci. USA
93:4398-4402 |
| 16. | Quan, C. M., M. Krajden, J. Zhao, and A. W. Chan. 1993. 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 43:299-307[Medline]. |
| 17. | Quint, W. G. V., R. A. Heijtink, J. Schirm, W. H. Gerlich, and H. G. M. Niesters. 1995. Reliability of methods for hepatitis B virus DNA detection. J. Clin. Microbiol. 33:225-228[Abstract]. |
| 18. | Sall, J., and A. Lehman. 1993. JMP start statistics. A guide to statistics and data analysis using JMP and JMP IN® software. SAS Institute, Duxbury Press, Hockessin, Del. |
| 19. |
Wang, J.-T.,
T.-H. Wang,
J.-C. Sheu,
S.-M. Lin,
J.-T. Lin, and D.-S. Chen.
1992.
Effects of anticoagulants and storage of blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus.
J. Clin. Microbiol.
30:750-753 |
| 20. | Yen-Lieberman, B., D. Brambilla, B. Jackson, J. Bremer, R. Coombs, M. Cronin, S. Herman, D. Katzenstein, S. Leung, H. Ju Lin, P. Palumbo, S. Rasheed, J. Todd, M. Vahey, and P. Reichelderfer. 1996. Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group Virology Laboratories. J. Clin. Microbiol. 34:2695-2701[Abstract]. |
| 21. | Zaaijer, H. L., H. T. M. Cuypers, H. W. Reesink, I. N. Winkel, G. Gerken, and P. N. Lelie. 1993. Reliability of polymerase chain reaction for detection of hepatitis C virus. Lancet 341:722-724[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
