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

Abstract

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, −0.12 log₁₀ copies/ml; RT-PCR, −0.05 log₁₀ copies/ml) and after 18 h (bDNA assay, −0.27 log₁₀ copies/ml; RT-PCR, −0.15 log₁₀ copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log₁₀ copies/ml; RT-PCR, −0.02 log₁₀ copies/ml), but it was after 18 h (bDNA assay, −0.09 log₁₀ copies/ml; RT-PCR, −0.09 log₁₀ copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log₁₀ copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4⁺-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log₁₀ copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4⁺-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

Many studies of the natural history of human immunodeficiency virus (HIV) infection have shown that the quantity of HIV type 1 (HIV-1) RNA in plasma is strongly associated with the rate of CD4⁺ T-lymphocyte decline, the time to AIDS, and the time to death from AIDS (5, 18, 22). In addition, the magnitude of plasma HIV-1 RNA suppression with antiretroviral therapy is associated with the degree of clinical benefit and the durability of the treatment effect (16, 17, 25). Given the essential role that quantification of HIV-1 RNA plays in the clinical care of the HIV-infected individual, it is important to characterize the variability in HIV-1 RNA levels that arise from different processing conditions and laboratory procedures.

Several technical issues must be considered when quantifying plasma HIV-1 RNA, including the assay method, the anticoagulant used for blood collection, and plasma processing procedures. Three different assays that measure plasma HIV-1 RNA accurately and reproducibly are commercially available (21, 24, 28). These include the branched-DNA (bDNA) signal amplification assay (Chiron, Emeryville, Calif.), reverse transcriptase-initiated PCR (RT-PCR) target amplification (Roche Diagnostic Corporation, Indianapolis, Ind.), and nucleic acid sequence-based amplification (Organon Teknika, Durham, N.C.). Currently, four anticoagulants are used to collect plasma: EDTA, heparin, acid citrate dextrose (ACD), and sodium citrate.

Previous studies have indicated that the stability of HIV-1 RNA in plasma samples depends on the anticoagulant used during blood collection (7, 9, 13, 14), blood processing time (7, 14, 20), the length of time that plasma samples are in frozen storage (9), type of clinical sample processed (9, 11), and assay method (8, 18, 27). Published recommendations and current manufacturer instructions in kits suggest that plasma viral load assays are best performed with EDTA- or ACD-anticoagulated plasma that has been processed within 2 h of collection. However, in the Multicenter AIDS Cohort Study (MACS), blood samples were historically collected in heparin-containing tubes and were separated into plasma after a delay of approximately 6 h. These differences could affect the plasma viral load results that have been reported from the MACS and on which treatment guidelines have been based (3).

To examine the impact of assay, anticoagulant, and processing time on the decay of plasma HIV-1 RNA, we conducted two analyses. First, we nested a controlled experiment (hereafter referred to as experimental decay study) within MACS in which fresh samples collected from MACS participants were subjected to different anticoagulants and processing times and were measured by different assays. Second, to quantify variables that could affect interpretation of HIV-1 RNA levels at the cohort level, we compared HIV-1 RNA levels in plasma samples from participants in MACS and the British Columbia Drug Treatment Program (BCDTP), with a different protocol used for the measurement of HIV-1 RNA levels in each cohort (hereafter referred to as observational cohort studies).

MATERIALS AND METHODS

Experimental decay study. (i) Study population.

Beginning in December 1996, a subset of homosexual and bisexual men participating in the MACS (see the Observational Cohort Studies section below for a description of the MACS) was enrolled in the experimental plasma HIV-1 RNA decay study. Seropositive participants in the MACS were eligible for this study if they attended a semiannual clinic visit between October 1996 and March 1997. At this visit, individuals had to be able to donate six tubes of blood, each containing at least 7 ml and a maximum of 10 ml. No restrictions were placed on the stage of disease or on reported therapy. Men were enrolled in this study by the order of their scheduled visits at each of the four MACS clinical sites.

(ii) Study design.

The decay study was designed to directly assess the effects of different assays, anticoagulants, and processing times on the rate of HIV-1 RNA decay in plasma specimens. Specifically, we analyzed whether the decrease in HIV-1 RNA levels in heparin-anticoagulated plasma samples was greater over time than that in EDTA-anticoagulated plasma. Whole blood was collected from participants in 10-ml Vacutainer (Becton Dickinson Vacutainer Systems, Franklin Lakes, N.J.) tubes containing potassium EDTA (lavender top) and into 10-ml Vacutainer tubes containing sodium heparin (green top). Blood from each participant was collected in three EDTA-containing tubes and three heparin-containing tubes, for a total of six tubes per individual. One heparin-containing tube and one EDTA-containing tube were then processed as close to the following times as possible: (i) 2 h after collection, (ii) 6 h after collection, and (iii) 18 h after collection. We categorized the observed processing times into the following three time groups: (i) 0 to 3 h postcollection, (ii) 3.01 to 9 h postcollection, and (iii) 15 to 27 h postcollection. For identification purposes, these three categories of processing times will be referred to as 2, 6, and 18 h postcollection, respectively. All tubes were held at room temperature (23°C) until processing, which involved separation of the plasma by centrifugation at 300 × g for 15 min in a swinging-bucket rotor. Aliquots (4 ml) of plasma were then stored in cryovials at −70°C until they were assayed for HIV-1 RNA. For each tube, clinic personnel recorded the date and time that the sample was drawn, and laboratory personnel recorded the times at which the processing began and was completed (placement of plasma at −70°C).

HIV-1 RNA for each tube was quantified by both bDNA (version 2.0) and RT-PCR (Amplicor version 1.0) assays. The bDNA assay measures levels of particle-associated genomic RNA, while the RT-PCR assay quantifies HIV-1 RNA extracted from plasma (19). The lower limit of quantification for the bDNA assay is 500 copies/ml, and that for the Amplicor RT-PCR assay is 400 copies/ml. Heparin-anticoagulated plasma samples were treated with heparinase prior to testing by RT-PCR, as described previously (13), to eliminate the inhibitory effect of heparin on the RT-PCR. Additional details and performance characteristics of both assays are described elsewhere (21, 24, 26). Each measurement of HIV-1 RNA was classified into one of the following four anticoagulant-assay categories: (i) heparin-bDNA assay, (ii) heparin–RT-PCR, (iii) EDTA-bDNA assay, or (iv) EDTA–RT-PCR. By design, each participant should have contributed a plasma HIV-1 RNA level to each of the four anticoagulant-assay categories at 2, 6, and 18 h postcollection, for a total of 12 measures per individual.

(iii) Statistical analysis.

For all analyses, plasma HIV-1 RNA measures were log₁₀ transformed. Additionally, all data from individuals with HIV-1 RNA levels below an assay’s detection limit for any of their samples processed at 2 h were excluded from the decay analysis because decay could not be quantified. Mean HIV-1 RNA decay was defined as the mean of the differences between HIV-1 RNA levels in samples processed 2 h after collection and the levels in corresponding samples processed 6 and 18 h after collection. The paired t test was used to evaluate the statistical significance of differences between HIV-1 RNA levels at 6 and 18 h with respect to the HIV-1 RNA levels at 2 h.

Observational cohort studies. (i) Study population.

To compare HIV-1 RNA levels across two cohort studies, viral load measurements from participants in the MACS and in the BCDTP were used. MACS was initiated in 1983 to study the natural history of HIV infection among homosexual and bisexual men in the United States. MACS comprises four centers located in Baltimore, Md.; Chicago, Ill.; Los Angeles, Calif.; and Pittsburgh, Pa. MACS enrolled a total of 4,954 men between March 1984 and April 1985. Semiannually, participants in the MACS cohort return to one of these four centers for a follow-up visit. For this study, HIV-1 RNA measurements from the third or fourth MACS follow-up visit (approximately 1 to 1.5 years after the MACS enrollment period) were used (18). The participants eligible for this study consisted of men who were both HIV-1 seroprevalent and free of AIDS at this visit, who had a CD4⁺ lymphocyte count measure and an available plasma sample, and who returned for at least one further follow-up visit. Since antiretroviral therapy was not available when these samples were collected, all men were antiretroviral therapy naive. Further details of the study and characteristics of the MACS cohort are reported elsewhere (15).

The parallel cohort from British Columbia, Canada, comprised seropositive individuals requesting antiretroviral therapy or plasma HIV-1 RNA testing from the BCDTP between June 1996 and March 1997 (12). BCDTP was the only free source of antiretroviral medications and HIV-1 RNA testing in British Columbia. Program participants were eligible for this study if they were antiretroviral therapy naive and age 18 years or over.

(ii) Study design.

The comparison of the two cohorts for HIV-1 seroprevalence was designed to see if variables of interest affected or could account for differences between cohorts. The laboratory variables of interest were assay method, assay detection limit, anticoagulant, processing time, and length of time in frozen storage. Pertinent laboratory methods for HIV-1 RNA quantification for samples from both the MACS and the BCDTP cohorts are detailed in Table 1. For the MACS cohort, whole blood for the collection of plasma was drawn into Vacutainer tubes containing sodium heparin. Specimens remained at room temperature from the time of blood collection to the time of processing to plasma. It is estimated that the average time between blood collection and freezing of plasma was approximately 6 h, although times were not always recorded. Plasma was then frozen at −70°C for approximately 10 years. In 1995, the samples were thawed and the bDNA assay (version 2.0) was used to quantify the HIV-1 RNA in the plasma samples. CD4⁺ T-cell numbers were obtained by standardized methods that have been described previously (10).

TABLE 1.

Comparison of methods used for measurement of plasma HIV-1 RNA levels in samples from the MACS and the BCDTP cohorts

Cohort	Assay	Limit of quantification (no. of RNA copies/ml)	Anticoagulant	Avg time from blood collection to processing of plasma (h)	Length of time in frozen storage (yr)
MACS	bDNA, version 2.0 (Chiron)	500	Heparin	∼6	∼10a
BCDTP	RT-PCR, version 1.0 (Roche-AMPLICOR)	400	EDTA or ACD	≤2	0b

^aStored samples. 

^bReal-time testing. 

At the British Columbia Centre for Excellence, whole blood from BCDTP participants was drawn into Vacutainer tubes containing either EDTA or ACD. All blood samples were processed to plasma within 2 h of collection. Specimens were then immediately assayed by the Amplicor RT-PCR assay (version 1.0). CD4⁺ T-cell numbers were obtained by standardized methods described elsewhere (1).

(iii) Statistical analysis.

The main purpose of this analysis was to compare HIV-1 RNA levels in individuals with similar CD4⁺-cell counts between two cohorts. After adjusting for assay method, putative effects of anticoagulant or length of time in frozen storage would result in differences in the log₁₀ transformed HIV-1 RNA levels in individuals in the two cohorts with similar CD4⁺-cell counts. For this comparison, the MACS HIV-1 RNA levels obtained by the bDNA assay were adjusted to those which would have been expected had the samples been quantified by the RT-PCR assay. The formula used to perform this conversion was as follows: number of HIV-1 RNA copies per milliliter by RT-PCR = 5.13 × (number of copies of HIV-1 RNA per milliliter by bDNA assay)^0.9 (18). We then compared the log₁₀ transformed levels in strata defined by CD4⁺-cell count. HIV-1 RNA levels were stratified by CD4⁺-cell count and cohort, and box plots were drawn for each of the strata. The nonparametric Wilcoxon rank sum test was used to assess the statistical significance of differences in HIV-1 RNA levels between the groups.

RESULTS

Experimental decay study. (i) Descriptive statistics.

A total of 101 seropositive men were selected and enrolled in the decay study at the four MACS sites. Of these, 84.3% were free of clinical AIDS according to the 1993 Centers for Disease Control and Prevention definition of AIDS based on clinical symptoms but not according to CD4⁺ lymphocyte counts. The mean ± standard deviation CD4⁺ lymphocyte count for the study population was 408 ± 258 cells/mm³. Twenty-eight of the subjects (31.5%) reported being on potent antiretroviral therapy (6) within the 6 months prior to blood collection. The 101 men contributed a total of 302 bDNA assay measurements and 302 RT-PCR measurements of plasma HIV-1 RNA levels. Processing times for these 604 measurements ranged from a minimum of 20 min to a maximum of 27 h and 32 min postcollection. Since the primary purpose of the study was to measure the loss (decay) of HIV-1 RNA over time, we restricted our study population to the 69 individuals who had detectable HIV-1 RNA, as determined with the sample processed at 2 h postcollection across all four anticoagulant-assay categories.

(ii) Effect of anticoagulant and processing time.

Table 2 shows the loss of HIV-1 RNA according to type of anticoagulant, assay method, and processing interval. Decay of HIV-1 RNA in heparinized blood was significant after 6 h, but the magnitude was small: 24% loss (−0.12 log₁₀ copies/ml) and 11% loss (−0.05 log₁₀ copies/ml) for the bDNA and RT-PCR assays, respectively. After 18 h, the decay in heparinized samples increased to 46% (−0.27 log₁₀ copies/ml) and 29% (−0.15 log₁₀ copies/ml) for the bDNA and RT-PCR assays, respectively. Decay of HIV-1 RNA in EDTA-anticoagulated blood was not significant after 6 h but was significant after 18 h. However, even after 18 h the magnitude of the decay was only 19% (−0.09 log₁₀ copies/ml) for both the bDNA and the RT-PCR assays. Across all four anticoagulant-assay combinations, the average decay at 6 h was −0.04 log₁₀ copies/ml, or 9%.

TABLE 2.

Loss of plasma HIV-1 RNA over time by type of anticoagulant, assay method, and processing interval

Anticoagulant	Assay	Plasma HIV-1 RNA levels (log10 copies/ml) at the following times postcollectiona:
		2 h (n = 69)	6 hb(n = 65)	18 hb(n = 66)
Heparin	bDNA	3.83 ± 0.72	−0.12 ± 0.11c	−0.27 ± 0.15c
	RT-PCR	4.41 ± 0.65	−0.05 ± 0.15c	−0.15 ± 0.16c
EDTA	bDNA	3.93 ± 0.73	−0.00 ± 0.15	−0.09 ± 0.19c
	RT-PCR	4.44 ± 0.69	−0.02 ± 0.14	−0.09 ± 0.17c

^aValues are means ± standard deviations. 

^bLevels are differences with respect to the levels at 2 h. 

^cStatistically significant difference (P < 0.05; paired t test). 

Figure 1 depicts the distribution of all the differences in plasma HIV-1 RNA levels measured in the samples at 6 h relative to the levels in the samples at 2 h for each of the four anticoagulant-assay groups. Of the 260 samples processed 6 h after collection, only 11 (4.2%) of the samples lost greater than 50% (0.3 log₁₀ copies/ml) of the HIV-1 RNA measured at 2 h postcollection. These 11 samples were spread across all four anticoagulant-assay groups. Mean differences (depicted by horizontal lines in Fig. 1) were clearly influenced by the skewness of the data towards low values. Excluding all values away from zero by more than 0.3 log₁₀ copies/ml (n = 12), the mean differences shrunk to 21% (−0.10 log₁₀ copies/ml), 5% (−0.02 log₁₀ copies/ml), 0% (−0.00 log₁₀ copies/ml), and 5% (−0.02 log₁₀ copies/ml) for the four anticoagulant-assay groups, respectively.

FIG. 1.

Distribution of differences between HIV-1 RNA levels in samples processed 6 h after collection relative to the levels in the same samples processed 2 h after collection. Horizontal lines indicate mean decay rates for each of the four anticoagulant-assay groups.

Observational cohort studies. (i) Descriptive statistics.

Of the 4,954 men enrolled in the MACS, 1,604 were eligible for inclusion in the cohort comparison study (18). As indicated in the eligibility criteria, all participants were antiretroviral therapy naive and free of clinical AIDS at the time of specimen collection. From the BCDTP cohort, 1,304 HIV-1-seropositive and antiretroviral therapy-naive individuals, including 817 men, 129 women, and 358 of unreported gender were selected for the comparison study. Eighty-nine of the 1,304 individuals had AIDS at the time of entry into the BCDTP. Further characteristics of the two cohorts are described in Table 3, including the median CD4⁺-cell count and viral load for each cohort.

TABLE 3.

Comparison of plasma HIV-1 RNA levels and CD4⁺-cell counts for subjects in the MACS and BCDTP cohorts

Cohort	Median HIV-1 RNA level (copies/ml [IQRa])	Median CD4+ cell count (IQR)	Spearman correlationc
MACS (n = 1,604)	23,766b (8,329–65,107)	527 (376–716)	−0.40
BCDTP (n = 1,304)	45,000  (10,500–130,000)	390 (240–560)	−0.47

^aThe interquartile range (IQR) is the range between the 75th and 25th percentiles. 

^bOriginal HIV-1 RNA levels were obtained with the Chiron version 2.0 bDNA assay; they were converted to the values expected by the Roche AMPLICOR RT-PCR assay with the formula given in Materials and Methods (18). 

^cCorrelation between CD4⁺ cell count and plasma HIV-1 RNA level. 

(ii) Effects of anticoagulant and processing times.

Table 3 shows that the CD4⁺-cell counts differed between the two cohorts. Therefore, to compare HIV-1 RNA levels between the two cohorts, we stratified individuals in the two cohorts by CD4⁺-cell count. Figure 2 depicts the distributions of viral load in the two cohorts stratified by six categories of CD4⁺-cell count, using 100, 200, 350, 500, and 750 cells/mm³ as cutoff points. When individuals were stratified by CD4⁺-cell count, the log₁₀ viral load distributions of the two cohorts were very similar. Across all strata, there were no significant differences (P > 0.05) between the log₁₀ HIV-1 RNA distributions in the MACS and the BCDTP cohorts.

FIG. 2.

Box plots of distributions of log₁₀ plasma HIV-1 RNA levels in the MACS and BCDTP cohorts stratified by CD4⁺ category. Box plot whiskers are drawn for the minimum and maximum HIV-1 RNA levels for each group.

DISCUSSION

The development of accurate and reproducible assays for quantification of HIV-1 RNA in plasma provided essential insights into the natural history of HIV-1 infection and the optimal goals of antiretroviral therapy (18, 22). Measures of HIV-1 RNA are now routinely used in clinical practice to guide decisions about initiation and changes in antiretroviral therapy. Several commercial assays that use different methodologies for quantification of HIV-1 RNA are available. Additionally, several different anticoagulants for the preservation of blood plasmas between collection and testing are available. While the availability of different assays and anticoagulants provides many options for research studies and clinical laboratories, this creates the potential for disparities between viral load measurements obtained by different blood collection, processing, and measurement technologies.

Previous studies have focused on this issue of disparity in the framework of small laboratory studies. Studies by Ginocchio et al. (9), Holodniy et al. (14), Dickover et al. (7), and Coombs et al. (4) looked at the effects of various specimen collection, processing, and storage conditions on the stability of HIV-1 RNA in plasma. Results varied, with Ginocchio et al. (9) showing that EDTA-, ACD-, and heparin-treated samples processed to plasma within 2 h of collection had comparable levels of HIV-1 RNA, while Holodniy et al. (14) and Dickover et al. (7) reported that for the same time interval, plasma HIV-1 RNA levels in samples collected in EDTA-containing tubes were significantly higher than those in samples collected in ACD- or heparin-containing tubes. Coombs et al. (4) reported no significant difference between HIV-1 RNA levels measured in heparin- or citrate-treated samples. In terms of the effect of processing delays, Coombs et al. (4) reported that storage at room temperature for 6 h before processing had no effect on the HIV-1 RNA level in EDTA-anticoagulated samples. Likewise, Ginocchio et al. (9) found that plasma HIV-1 RNA levels obtained from EDTA-treated plasma were stable for samples stored at room temperature for up to 30 h before processing. Additional studies have supported these findings (23, 27). In contrast, Holodniy et al. (14) reported a significant drop in HIV-1 RNA levels when citrate-treated whole blood was allowed to remain at room temperature for 8 h before processing to plasma, and Dickover et al. (7) estimated the rate of decay during the first 6 h of room temperature storage to be 1.8%/h for EDTA-treated samples, 3.3%/h for ACD-treated samples, and 5.0%/h for heparin-treated samples. Finally, with respect to samples in frozen storage, Ginocchio et al. (9) reported that plasma HIV-1 RNA levels in all samples decreased over a 6-month period of frozen storage but that only the heparin-treated samples showed a loss of 50% or greater (≥−0.30 log₁₀ copies/ml).

Results from the experimental study reported here indicate that plasma HIV-1 RNA decayed in whole blood if the blood was not processed to plasma within 2 h of collection. This decay increased as the time between collection and processing lengthened and was greater if whole blood was collected in tubes containing heparin rather than in tubes containing EDTA. While anticoagulant and processing times affected HIV-1 RNA levels, this effect was small relative to the inherent variability of the methods used to measure viral load. Across all samples in the experimental decay study, regardless of the anticoagulant or assay, the average decay in HIV-1 RNA level measured in samples processed to plasma 6 h after blood collection relative to the level in the respective sample processed within 2 h was 9% (−0.04 log₁₀ copies/ml). Of the 260 samples that were processed 6 h after collection, only 11 (4.2%) of the samples showed decays of greater than 50% (−0.3 log₁₀ copies/ml). Although the magnitude of the effect of anticoagulant and processing times on HIV-1 RNA was small, it was statistically significant; therefore, small studies that do not account for different blood collection and processing protocols run the risk of drawing improper conclusions from the data.

In contrast, large cohort studies may not be heavily influenced by differences in blood collection and processing protocols. Indeed, to explore the magnitude of this problem on the population level (i.e., in the setting of the larger cohort study), we conducted the comparison of observational cohort studies. Smaller laboratory studies have suggested a need to adjust for heparin usage, a longer average processing time, and the length of time in frozen storage. We did not apply corrections to the values for these factors for the MACS cohort; however, the plasma HIV-1 RNA measurements in the MACS and the BCDTP cohorts were remarkably similar after accounting only for CD4⁺-cell count and assay type. Thus, the importance of anticoagulant and processing time in cohort studies appears to be small.

Both the experimental decay study and the comparison of observational cohort studies indicate that the loss of HIV-1 RNA in archived samples treated with heparin and processed by the bDNA assay within 6 h of collection has likely been overestimated in past reports. In particular, HIV-1 RNA levels in frozen samples from the MACS cohort were comparable to the HIV-1 RNA levels obtained by current processing standards. Thus, our data suggest that a correction factor is not needed for samples collected in heparin and stored within 6 h. However, it should be noted that viral loads measured by the RT-PCR assay are still approximately 2 times higher than those viral loads obtained by the bDNA assay. As derived by Mellors et al. (18), bDNA assay values should still be converted to RT-PCR-equivalent values by using the conversion formula that was used in this study.

There are still many difficulties in comparisons of longitudinal viral load measurements collected under distinct blood collection protocols. Currently, the recommendation is to use the same protocol to measure the HIV-1 RNA load in the same person over time (3). However, technological advances and refinements of the assays may make it difficult or impossible to adhere to the same method over long periods of time. Indeed, the MACS itself chose to take advantage of the advances in technologies in that, starting on 1 April 1997, heparin-bDNA assay was replaced by EDTA–RT-PCR as the preferred anticoagulant-assay. In these cases, it is essential that substudies be properly designed so that calibration formulas can be developed to convert values from one assay so that they are equivalent to those from another assay (8, 18, 27). With each new decrease in assay detection limit, researchers will need to consider sampling from the previously “undetectable” region to describe the distribution of HIV-1 RNA levels by the new assay. Thus, all viral load measures will be on the scale of the most current threshold of detection.

With the exception of the National Institute of Allergy and Infectious Diseases Virology Quality Assurance Program (2, 29), we are not aware of large-scale studies designed to address the standardization of viral load measurements across various assay and blood collection and processing protocols. It is critical to perform such research and to institute standard equations to provide the translation between various viral load measurements as well as to identify the aspects of blood collection and processing that do not significantly affect viral load measurements. This research will ultimately enable informed recommendations to be made regarding the laboratory techniques that produce the most valid and reliable quantitative viral load results as well as concerning the optimal conditions for blood collection, processing, and storage.