![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Clinical Microbiology, April 1998, p. 1070-1073, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Optimization of Specimen-Handling Procedures for Accurate
Quantitation of Levels of Human Immunodeficiency Virus RNA in
Plasma by Reverse Transcriptase PCR
Ruth E.
Dickover,1
Steven A.
Herman,2
Khaliq
Saddiq,1
Deborah
Wafer,1
Maryanne
Dillon,1 and
Yvonne J.
Bryson1,*
Department of Pediatrics, UCLA School of
Medicine, Los Angeles, California,1 and
Roche Molecular Systems, Somerville, New
Jersey2
Received 19 June 1997/Returned for modification 27 August
1997/Accepted 20 January 1998
 |
ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma
are currently widely used clinically for prognostication and in
monitoring antiretroviral therapy. Accurate and reproducible results
are critical for patient management. To determine the effects of
specimen collection and handling procedures on quantitative measurement
of HIV-1 RNA, we compared anticoagulants and sample processing times.
Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid
citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at
room temperature. Plasma was separated from whole-blood aliquots
prepared at 
1, 3, 6, 24, and 48 h postcollection and
then stored at 
70°C until use. HIV-1 RNA levels were determined by
the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest
baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels
decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes,
respectively. From 6 to 48 h postcollection, HIV-1 RNA levels
decreased in all anticoagulants, albeit at a slower, more consistent
rate. Our results indicate that EDTA should be the anticoagulant of
choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR,
but ACD tubes are acceptable if the plasma is separated within 6 h
of blood collection. Caution must be applied in the interpretation of
absolute HIV-1 RNA copy number values obtained with suboptimal specimen
collection and processing procedures.
| |
INTRODUCTION |
The level of human immunodeficiency
virus type 1 (HIV-1) RNA in plasma of infected individuals serves as an
indicator of total viral load and can be used to infer viral
replication rates (10, 17). Quantitation of plasma HIV-1 RNA
levels has been shown to be an important marker of disease progression,
antiretroviral therapy efficacy, and risk of perinatal transmission
(6-9, 12). A variety of commercial assays for quantitation
of HIV-1 RNA in patient samples have recently become available (4,
16, 20). The availability of standardized laboratory assays for
HIV-1 RNA quantitation should help to decrease both intra- and
interlaboratory variability in results, which is essential both in
clinical trials and for patient management. A better understanding of
the factors influencing the outcome of quantitative HIV-1 RNA assays is
needed to compare results from studies conducted with the various
methodologies in real time or with frozen, batched specimens and to
provide clinicians with the most accurate and reproducible results for use in patient management.
The AMPLICOR HIV-1 MONITOR assay uses reverse
transcriptase (RT) PCR amplification to achieve high levels
of sensitivity (
400 HIV-1 RNA copies/ml) in the quantitation of
HIV-1 RNA levels (16). PCR has limitations as a quantitative
assay, however, including decreased efficiency at higher initial copy
numbers (5) and inhibition by heparin present in blood
samples (1). The accuracy of quantitative HIV-1 RNA
measurements in plasma specimens can be affected by both the choice of
anticoagulant and specimen-handling procedures (13, 19, 21).
In order to determine the effects of specimen collection and handling
procedures on quantitation of plasma HIV-1 RNA, we compared
anticoagulants and sample processing times with quantitative RT-PCR. We
also analyzed possible patient variables influencing HIV-1 RNA loss,
including age, gender, pregnancy status, antiretroviral treatment
status, initial HIV-1 RNA copy numbers, and CD4 cell counts.
(This work was presented in part at the 36th Interscience Conference on
Antimicrobial Agents and Chemotherapy, New Orleans, La., September
1996.)
| |
MATERIALS AND METHODS |
Study population.
Twenty HIV-1-seropositive patients
monitored at the University of California, Los Angeles, MCIC and CARE
clinics were studied, with informed consent obtained under the
guidelines of the UCLA Human Subjects Protection Committee. The study
population consisted of 12 adults ranging from 22 to 38 years old and 8 children ranging from 2 to 16 years old. The current antiretroviral
treatment history, CD4 cell count, and Centers for Disease Control and
Prevention (CDC) classification (2, 3) were obtained for
each patient at the time of sample collection.
Serological assays.
HIV-1 antibodies were detected by enzyme
immunoassay (Abbott Laboratories, North Chicago, Ill.) and confirmed by
Western blot assay (Du Pont, Wilmington, Del.).
Sample preparation.
Whole blood was collected into EDTA,
acid citrate dextrose (ACD), and heparinized VACUTAINER tubes (Becton
Dickinson, Franklin Lakes, N.J.), aliquoted, and stored at room
temperature. Plasma was separated from the whole-blood aliquots within
1 h of collection (baseline) and then at 3, 6, 24, and 48 h
postcollection. Blood was centrifuged at 400 × g for
10 min in a Sorvall RT 6000B swinging bucket centrifuge (Du Pont)
at room temperature. Plasma was transferred to a new tube, and
platelets were removed by a second spin at 800 × g for
10 min. The clarified plasma aliquots were stored at 70°C until
use.
Quantitative RNA PCR.
Quantitative HIV-1 RNA PCR was
performed on batched plasma samples with the AMPLICOR HIV-1 MONITOR
assay according to the manufacturer's instructions (Roche Molecular
Systems, Somerville, N.J.). RNA was prepared from EDTA- and ACD-treated
plasma according to the MONITOR assay protocol. Briefly, plasma samples
(200 µl) were added to lysis buffer (600 µl) containing guanidinium
thiocyanate and an internal quantitation standard. After a 10-min
incubation, RNA was precipitated by the addition of 800 µl of
isopropanol. RNA was pelleted by centrifugation at 16,000 × g for 15 min in a microcentrifuge (Brinkman Instruments,
Westbury, N.Y.), washed once with 70% ethanol, and pelleted again at
16,000 × g for 5 min. RNA was resuspended in a
low-ionic-strength diluent (400 µl). Heparinized samples were
pretreated for 1 h at room temperature with 7.5 units of
heparinase I (Sigma Chemical Company, St. Louis, Mo.) in 1× heparinase
buffer (15 mM Tris base, 3 mM CaCl2, 45 mM NaCl, 0.02%
bovine serum albumin [pH 7.5]). The heparinase I reaction was stopped
by the addition of EDTA to a final concentration of 10 mM prior to
extraction by the MONITOR assay protocol.
Fifty microliters of each prepared RNA sample was used for PCR.
Following amplification and detection of the PCR product, the starting
HIV-1 RNA copy number in each sample was calculated by comparison with
the internal quantitation standard, and results were expressed as the
numbers of HIV-1 RNA copies per milliliter of plasma. Baseline (1 h
postcollection) HIV-1 RNA levels were determined in duplicate and the
results were averaged.
Statistics.
Descriptive statistics are provided as
means ± standard deviations (SD). Correlations between patient
variables were determined with the Spearman rank correlation
coefficient. Virologic variables between groups were compared by the
Mann-Whitney and Kruskal-Wallis tests. Statistical significance was
defined as a P value of less than 0.05.
| |
RESULTS |
Study population.
The average CD4 cell count for the 12 adults
enrolled in this study was 275 ± 185/mm3 (range, 22 to 522/mm3). Among the 12 adults in the study there were 4 men, 4 nonpregnant women, and 4 pregnant women. Nine of these 12 adults
were receiving zidovudine (ZDV) monotherapy at the time of sample
collection. All 12 adults had symptomatic HIV-1 disease (CDC classes B2
through C3). Among the eight children in our study, the average CD4
count was 527 ± 478/mm3 (range, 14 to
1,429/mm3). Six of the eight children had symptomatic
disease (CDC classes A2 through C3), and five were being treated with
ZDV monotherapy at the time of sample collection. A total of 360 quantitative HIV-1 RNA PCRs were run on the samples from the 20 patients enrolled in this study.
Effects of anticoagulants on baseline plasma HIV-1 RNA
levels.
The HIV-1 RNA copy numbers measured at baseline
varied over a wide range for all three anticoagulants, as would be
predicted from the range in CD4 cell counts among study
participants (Fig. 1A). Baseline (within
1 h postcollection) HIV-1 RNA levels were highest in plasma
collected in VACUTAINER tubes with EDTA as the anticoagulant.
HIV-1 RNA levels in plasma collected in EDTA VACUTAINER tubes were,
on average, 14% higher than those measured in ACD tubes and 31%
higher than those measured in heparinized tubes (P < 0.0001) (Fig. 1B) at baseline.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
(A) Baseline (1 h) HIV-1 RNA copy numbers in plasma
collected from infected individuals with three anticoagulants. HIV-1
RNA levels are expressed as the numbers of HIV-1 RNA copies per
milliliter of plasma. Symbols:  , patients who were being treated
with ZDV at the time of specimen collection;  , patients who were not
receiving ZDV at the time of specimen collection. Horizontal bars
indicate the means of the measured variables. (B) Comparison of HIV-1
RNA copy numbers, as determined by RT-PCR, of EDTA-, ACD-, and
heparin-treated plasma at baseline. Results are expressed as
percentages of the mean EDTA baseline HIV-1 RNA copy number. Horizontal
bars indicate SD. P values were determined by the
Kruskal-Wallis test.
|
|
Stability of HIV-1 RNA in whole blood.
Whole-blood aliquots
were made within 1 h of blood collection, and the aliquots
were held at room temperature for either 3, 6, 24, or 48 h before
plasma processing. Plasma HIV-1 RNA levels decreased over time in
all anticoagulant tubes tested but were most stable when collected in
EDTA tubes (Fig. 2A). At 24 h
postcollection, HIV-1 RNA levels had decreased by 20, 29, and 40% in
EDTA, ACD, and heparinized plasma, respectively (P 0.0001) (Table 1). In comparison to
baseline EDTA plasma, ACD and heparinized plasma samples had 39 and
60% less HIV-1 RNA, respectively, when measured at 24 h
postcollection (P 0.0001) (Fig. 2B; Table 1).
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 2.
(A) Stability of HIV-1 RNA in whole blood stored at room
temperature. Results are expressed as mean percentages of baseline copy
numbers for EDTA (top curve), ACD (middle curve), and heparinized
(bottom curve) plasma. Horizontal bars represent SD. (B) Stability of
HIV-1 RNA in whole blood stored at room temperature in comparison to
mean baseline (1 h) EDTA-treated specimens. Results are expressed as
mean percentages of EDTA baseline copy numbers for EDTA (top curve),
ACD (middle curve), and heparinized (bottom curve) plasma. Horizontal
bars represent SD.
|
|
The average rate of HIV-1 RNA loss in whole blood was 0.8%/h for EDTA,
1.2%/h for ACD, and 1.7%/h for heparinized blood samples held at room
temperature for 24 h (P < 0.0001). However, as
shown in Fig. 2, the rate of HIV-1 RNA loss was greater during the
first 6 h postcollection for all three anticoagulants tested. The
decay rate during the first 6 h of room temperature storage was
1.8%/h for EDTA samples, 3.3%/h for ACD samples, and 5.0%/h for
heparinized samples (P 0.0001) (Fig. 2). Between 6 and 48 h postcollection, the HIV-1 RNA decay rate decreased
dramatically and remained consistent for all three anticoagulants. This
suggests the presence of a population of highly labile HIV-1 virions in
the blood of infected patients which is degraded rapidly in the first
few hours postcollection.
Correlation between patient variables and HIV-1 RNA loss.
We
also correlated a variety of patient parameters with the average
rate of HIV-1 RNA loss over time. The stability of HIV-1 RNA in plasma was not influenced by the status of the patient as
adult versus child (RNA loss, 0.849%/h versus 0.761%/h
[P = 0.643]) or male versus female (0.761%/h versus
0.617%/h [P = 0.671]). Among the eight adult women
in our study, four were pregnant and four were not pregnant at the time
of sample collection; however, this did not influence the stability of
HIV-1 RNA levels (RNA loss, 0.795%/h versus 0.880%/h
[P = 0.797]). No correlation was seen between the use
or nonuse of antiretroviral therapy and the rate of HIV-1 RNA loss over
time (0.772%/h versus 0.912%/h [P = 0.322]).
Neither patient CD4 count (r = 0.085; P = 0.716) nor initial HIV-1 RNA copy number (r = 0.196;
P = 0.395) correlated with the stability of HIV-1 RNA
in plasma stored at room temperature.
| |
DISCUSSION |
Determination of HIV-1 RNA levels in plasma is now used widely in
laboratory evaluations of HIV disease status and prognosis (15,
18). Caution must be applied in the interpretation of absolute HIV-1 RNA values determined in real time for measurement of antiretroviral effects and patient management, as a variety of
factors can influence the outcome of quantitative HIV-1 RNA measurements, including the method used to make the
determination, the genotype of the virus, and specimen-handling
procedures (13). We compared different anticoagulants and
various specimen processing times in order to determine optimal
handling procedures for plasma HIV-1 RNA quantitation by RT-PCR.
The differences between baseline HIV-1 RNA levels measured in
EDTA and ACD plasma can be accounted for by the increased volume of
anticoagulant present in ACD VACUTAINER tubes, resulting in dilutions of plasma HIV-1 RNA levels in these tubes. However, it is not
clear why heparinized plasma showed the lowest baseline HIV-1 RNA
levels. Because heparin has been shown to inhibit PCR (11), we attempted to inactivate heparin by pretreating the plasma samples with heparinase. The significantly lower levels of HIV-1
RNA measured in heparinized plasma at baseline may, therefore, reflect
incomplete inactivation by heparinase.
The fact that plasma HIV-1 RNA levels decreased over time in blood
stored in all three anticoagulants but were most stable in EDTA-treated
whole blood is of major importance both for in-house assays and in
clinical trials which may involve shipment of whole-blood specimens
overnight at ambient temperatures to control laboratories. HIV-1 RNA
levels showed a significant decline from baseline in heparin-treated
samples after as little as 24 h of storage, indicating that
heparinized blood is not suitable for studies involving shipment of
blood at ambient temperatures. Interestingly, plasma HIV-1 RNA levels
decreased more rapidly in the first 6 h postcollection than in
hours 6 to 48 with all three anticoagulants tested. This may reflect
the presence of defective virions in which RNA may be degraded by RNase
present in plasma soon after blood draw.
In this study, we found that the stability of HIV-1 RNA in whole blood
was not influenced by a variety of individual patient parameters,
including gender, age, and pregnancy status. We also saw no correlation
between antiretroviral treatment status, initial CD4 count, or initial
HIV-1 RNA copy number and the rate of HIV-1 RNA loss over time. These
data strongly suggest that anticoagulants and specimen processing times
can influence RNA stability, while host factors seem to be less
important.
Proper specimen-handling procedures are of major importance in the
design of new clinical trials and the interpretation of previous trials
involving quantitative HIV-1 RNA analysis. Samples collected during
such studies may not have been processed within optimal time limits or
with EDTA used as an anticoagulant. Direct comparison of previous
reports shows that HIV-1 RNA levels measured in patients with similar
profiles by different methods can give rise to severalfold-different
absolute copy numbers (14, 17, 20). Our results indicate
strongly that valid comparisons of study results will occur only if
all studies involved use the same specimen-handling procedures and
quantitate HIV-1 RNA levels with consistent methodologies.
In summary, samples collected in EDTA VACUTAINER tubes provided the
highest baseline HIV-1 RNA levels and showed the best stability of
HIV-1 RNA levels in whole blood stored up to 48 h at room
temperature. These results indicate that EDTA should be the
anticoagulant of choice for quantitative HIV-1 RNA PCR performed either
in-house or following overnight shipment to a testing facility. Samples
collected in ACD tubes are acceptable for quantitation of HIV-1 RNA
levels if plasma is separated within 6 h of blood draw. In
contrast, heparinized plasma is poorly suited to HIV-1 RNA quantitation
by RT-PCR due both to difficulties in specimen processing and to the
poor quality of results generated regardless of the length of storage.
Our results indicate clearly that tube type and sample processing time
should be consistent in order to ensure optimal and reproducible
results with quantitative HIV-1 RNA PCR.
| |
ACKNOWLEDGMENTS |
This research was supported in part by grants HD30629 and
HD26621 from the National Institute of Child Health and Human
Development, by grants ACTG AI27550, AI30629, and AI32440 from the
National Institute of Allergy and Infectious Diseases, by
Universitywide AIDS Research Program grant R91-LA-152, and by Clinical
Research Center grant RR-00-865.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Pediatrics, UCLA Medical Center, 10833 LeConte Ave., Los
Angeles, CA 90095. Phone: (310) 825-9161. Fax: (310) 206-4764. E-mail: YBryson{at}Pediatrics.medsch.UCLA.edu.
| |
REFERENCES |
| 1.
|
Beutler, E.,
T. Gelbart, and W. Kuhl.
1990.
Interference of heparin with polymerase chain reaction.
BioTechniques
90:166-167.
|
| 2.
|
Centers for Disease Control and Prevention.
1994.
1994 revised classification system for human immunodeficiency virus infection in children less than 13 years of age.
Morbid. Mortal. Weekly Rep.
43:1-9.
|
| 3.
|
Centers for Disease Control.
1989.
Guidelines for prophylaxis against Pneumocystis carinii pneumonia for persons infected with human immunodeficiency virus.
Morbid. Mortal. Weekly Rep.
5:1-9.
|
| 4.
|
Dewar, R. L.,
H. C. Highbarger,
M. D. Sarmiento,
J. A. Todd,
M. B. Vasudevachari,
R. T. Davey, Jr.,
J. A. Kovacs,
N. P. Salzman,
H. C. Lane, and M. S. Urdea.
1994.
Application of branched DNA signal amplification to monitor human immunodeficiency virus type 1 burden in human plasma.
J. Infect. Dis.
170:1172-1179[Medline].
|
| 5.
|
Dickover, R. E.,
R. M. Donovan,
E. Goldstein,
S. Dandekar,
C. E. Bush, and J. R. Carlson.
1990.
Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction.
J. Clin. Microbiol.
28:2130-2133[Abstract/Free Full Text].
|
| 6.
|
Dickover, R. E.,
E. M. Garratty,
S. A. Herman,
M. S. Sim,
S. Plaeger,
P. J. Boyer,
M. Keller,
A. Deveikis,
E. R. Stiehm, and Y. J. Bryson.
1996.
Identification of levels of maternal HIV-1 RNA associated with risk of perinatal transmission. Effect of maternal zidovudine treatment on viral load.
JAMA
275:599-605[Abstract].
|
| 7.
|
Fang, G.,
H. Burger,
R. Grimson,
P. Tropper,
S. Nachman,
D. Mayers,
O. Weislow,
R. Moore,
C. Reyelt,
N. Hutcheon,
D. Baker, and B. Weiser.
1995.
Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission.
Proc. Natl. Acad. Sci. USA
92:12100-12104[Abstract/Free Full Text].
|
| 8.
|
Galetto-Lacour, A.,
S. Yerly,
T. V. Perneger,
C. Baumberger,
B. Hirschel,
L. Perrin, and the Swiss HIV Cohort Study Group.
1996.
Prognostic value of viremia in patients with long standing human immunodeficiency virus infection.
J. Infect. Dis.
173:1388-1393[Medline].
|
| 9.
|
Henrard, D. R.,
J. F. Phillips,
L. R. Muenz,
W. A. Blattner,
D. Wiesner,
M. E. Eyster, and J. J. Goedert.
1995.
Natural history of HIV-1 cell-free viremia.
JAMA
274:554-558[Abstract].
|
| 10.
|
Ho, D. D.,
A. U. Neumann,
A. S. Perelson,
W. Chen,
J. M. Leonard, and M. Markowitz.
1995.
Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection.
Nature
373:123-126[Medline].
|
| 11.
|
Holodniy, M.,
S. Kim,
D. A. Katzenstein,
M. Konrad,
E. Groves, and T. C. Merigan.
1991.
Inhibition of human immunodeficiency virus gene amplification by heparin.
J. Clin. Microbiol.
29:676-679[Abstract/Free Full Text].
|
| 12.
|
Holodniy, M.,
L. Mole,
D. Margolis,
J. Moss,
H. Dong,
E. Boyer,
M. Urdea,
J. Kolberg, and S. Eastman.
1995.
Determination of human immunodeficiency virus RNA in plasma and cellular viral DNA genotypic zidovudine resistance and viral load during zidovudine-didanosine combination therapy.
J. Virol.
69:3510-3516[Abstract].
|
| 13.
|
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].
|
| 14.
|
Mellors, J. W.,
L. A. Kingsley,
C. R. Rinaldo, Jr.,
J. A. Todd,
B. S. Hoo,
R. P. Kokka, and P. Gupta.
1995.
Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion.
Ann. Intern. Med.
122:573-579[Abstract/Free Full Text].
|
| 15.
|
Mellors, J. W.,
C. R. Rinaldo,
P. Gupta,
R. M. White,
J. A. Todd, and L. A. Kingsley.
1996.
Prognosis in HIV-1 infection predicted by the quantity of virus in the plasma.
Science
272:1167-1170[Abstract].
|
| 16.
|
Mulder, J.,
N. McKinney,
C. Christopherson,
J. Sninsky,
L. Greenfield, and S. Kwok.
1994.
Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection.
J. Clin. Microbiol.
32:292-300[Abstract/Free Full Text].
|
| 17.
|
Piatak, M., Jr.,
M. S. Saag,
L. C. Yang,
S. J. Clark,
J. C. Kappes,
K. C. Luk,
B. H. Hahn,
G. M. Shaw, and J. D. Lifson.
1993.
High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR.
Science
259:1749-1754.
|
| 18.
|
Shearer, W. T.,
T. C. Quinn,
P. LaRussa,
J. F. Lew,
M. L. S. Almy,
K. Rich,
E. Handelsman,
C. Diaz,
M. Pagano,
V. Smeriglio, and L. A. Kalish.
1997.
Viral load and disease progression in infants infected with human immunodeficiency virus type 1.
N. Engl. J. Med.
336:1337-1342[Abstract/Free Full Text].
|
| 19.
|
van Bueren, J.,
R. A. Simpson,
P. Jacobs, and B. D. Cookson.
1994.
Survival of human immunodeficiency virus in suspension and dried onto surfaces.
J. Clin. Microbiol.
32:571-574[Abstract/Free Full Text].
|
| 20.
|
van Gemen, B.,
R. van Beuningen,
A. Nabbe,
D. van Strijp,
S. Jurriaans,
P. Lens, and T. Kievits.
1994.
A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes.
J. Virol. Methods
49:157-167[Medline].
|
| 21.
|
Winters, M. A.,
L. B. Tan,
D. A. Katzenstein, and T. C. Merigan.
1993.
Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction.
J. Clin. Microbiol.
31:2960-2966[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, April 1998, p. 1070-1073, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Niedrig, M., Meyer, H., Panning, M., Drosten, C.
(2006). Follow-Up on Diagnostic Proficiency of Laboratories Equipped To Perform Orthopoxvirus Detection and Quantification by PCR: the Second International External Quality Assurance Study. J. Clin. Microbiol.
44: 1283-1287
[Abstract]
[Full Text]
-
Stewart, M., Desport, M., Hartaningsih, N., Wilcox, G.
(2005). TaqMan Real-Time Reverse Transcription-PCR and JDVp26 Antigen Capture Enzyme-Linked Immunosorbent Assay To Quantify Jembrana Disease Virus Load during the Acute Phase of In Vivo Infection. J. Clin. Microbiol.
43: 5574-5580
[Abstract]
[Full Text]
-
Marteau, J.-B., Mohr, S., Pfister, M., Visvikis-Siest, S.
(2005). Collection and Storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study. Clin. Chem.
51: 1250-1252
[Full Text]
-
Ghosh, M. K., Kuhn, L., West, J., Semrau, K., Decker, D., Thea, D. M., Aldrovandi, G. M.
(2003). Quantitation of Human Immunodeficiency Virus Type 1 in Breast Milk. J. Clin. Microbiol.
41: 2465-2470
[Abstract]
[Full Text]
-
Kuritzkes, D. R., Grant, R. M., Feorino, P., Griswold, M., Hoover, M., Young, R., Day, S., Lloyd, R. M. Jr., Reid, C., Morgan, G. F., Winslow, D. L.
(2003). Performance Characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System. J. Clin. Microbiol.
41: 1594-1599
[Abstract]
[Full Text]
-
Singer, E. J., Aronow, H. A., Lee, S.-Y., Hinkin, C. H., Lazarus, T.
(2002). Stability of Human Immunodeficiency Virus Type 1 RNA in Cerebrospinal Fluid Determined with the AMPLICOR HIV-1 MONITOR Test, Version 1.5 (Ultrasensitive). J. Clin. Microbiol.
40: 3863-3864
[Abstract]
[Full Text]
-
Garcia, M. E., Blanco, J. L., Caballero, J., Gargallo-Viola, D.
(2002). Anticoagulants Interfere with PCR Used To Diagnose Invasive Aspergillosis. J. Clin. Microbiol.
40: 1567-1568
[Full Text]
-
Kellogg, J. A., Atria, P. V., Sanders, J. C., Eyster, M. E.
(2001). Intra- and Interlaboratory Variabilities of Results Obtained with the Quantiplex Human Immunodeficiency Virus Type 1 RNA bDNA Assay, Version 3.0. CVI
8: 560-563
[Abstract]
[Full Text]
-
Jennings, C., Bremer, J. W., Brambilla, D. J.
(2000). Investigation of Effects of Acid Citrate Dextrose and EDTA on Ability To Quantitatively Culture Human Immunodeficiency Virus. J. Clin. Microbiol.
38: 3522-3522
[Full Text]
-
Fiscus, S. A., Chakraborty, H., Shepard, R., Goodman, M.
(2000). Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures. J. Clin. Microbiol.
38: 858-860
[Abstract]
[Full Text]
-
Holodniy, M., Rainen, L., Herman, S., Yen-Lieberman, B.
(2000). Stability of Plasma Human Immunodeficiency Virus Load in VACUTAINER PPT Plasma Preparation Tubes during Overnight Shipment. J. Clin. Microbiol.
38: 323-326
[Abstract]
[Full Text]
-
Kirstein, L. M., Mellors, J. W., Rinaldo, C. R. Jr., Margolick, J. B., Giorgi, J. V., Phair, J. P., Dietz, E., Gupta, P., Sherlock, C. H., Hogg, R., Montaner, J. S. G., Muñoz, A.
(1999). Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma. J. Clin. Microbiol.
37: 2428-2433
[Abstract]
[Full Text]
Top
Abstract
Introduction
