Development and Verification of an Automated Sample Processing Protocol for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

Abstract

We developed and verified an automated sample processing protocol for use with the AMPLICOR HIV-1 MONITOR test, version 1.5 (Roche Diagnostics, Indianapolis, Ind.). The automated method uses the MagNA Pure LC instrument and total nucleic acid reagents (Roche Applied Science, Indianapolis, Ind.) to extract human immunodeficiency virus type 1 (HIV-1) RNA from plasma specimens. We compared the HIV-1 load results for a dilution series (1 to 5 nominal log₁₀ copies/ml) and 175 clinical specimens processed by the automated method to those for the same samples processed by the manual methods specified by the manufacturer. The sensitivity, dynamic range, and precision of the viral load assay obtained by automated processing of specimens were similar to those obtained by an ultrasensitive manual processing method. The results were highly correlated (R², 0.95), and were in close agreement, with a mean difference of 0.09 log₁₀ (standard deviation, 0.292). The limits of agreement were ±0.58 log₁₀ for results for samples processed by both the manual and the automated methods. These performance characteristics were achieved with a smaller sample volume (200 versus 500 μl) and without a high-speed centrifugation step and required only 15 min of labor for a batch of 32 samples. In conclusion, the automated sample preparation protocol can replace both the standard and the ultrasensitive manual methods used with the AMPLICOR HIV-1 MONITOR test and can substantially reduce the labor associated with this test.

The AMPLICOR HIV-1 MONITOR test (Roche Diagnostics Corporation, Indianapolis, Ind.) is a PCR assay for the quantification of human immunodeficiency virus type 1 (HIV-1) that can be used with either of two manual sample preparation protocols, the standard protocol or the ultrasensitive protocol. By the standard manual protocol, HIV-1 RNA is isolated directly from 200 μl of plasma by lysis of the virus with a chaotropic agent followed by precipitation of the RNA with alcohol. By the ultrasensitive manual protocol, HIV-1 particles in 500 μl of plasma are concentrated by high-speed centrifugation, followed by lysis of the virus with a chaotropic agent and precipitation of the RNA with alcohol. These manual procedures are labor-intensive and prone to operator error.

We developed and verified an automated sample preparation protocol for use with the AMPLICOR HIV-1 MONITOR test on the MagNA Pure LC (MPLC) system (Roche Applied Science, Indianapolis, Ind.). The MPLC system uses robotics, precision pipettors, and magnetic glass particles to purify DNA, RNA, mRNA, or total nucleic acid from various sample types. The samples are dissolved and simultaneously stabilized by incubation with a buffer containing denaturing agents and proteinase K. Nucleic acids are bound to the surface of the magnetic glass particles, and several washing steps remove the unbound substances. The purified nucleic acids are then eluted in a low-salt buffer, with variable elution volumes ranging from 50 to 100 μl. The instrument can process up to 32 samples in 1.5 h. It can also automate the PCR setup and can transfer the purified nucleic acids directly into a wide variety of reaction vessels, including LightCycler capillary tubes (Roche Applied Science), 96-well microplates, standard PCR tubes, and COBAS AMPLICOR amplification rings (Roche Diagnostics).

Although originally developed for the research laboratory, the MPLC system is finding increased use in clinical laboratory settings where reproducible and cost-effective alternatives to manual methods for the processing of large numbers of specimens are needed (2-5). In this study the effects of the automated sample preparation protocol on the analytical sensitivity, precision, and dynamic range of the AMPLICOR HIV-1 MONITOR test, version 1.5, were investigated. The correlation and agreement of viral load results for plasma samples prepared in parallel by the manual and the automated methods were also assessed.

MATERIALS AND METHODS

Plasma samples.

Remaining aliquots of EDTA-anticoagulated plasma samples submitted to the Molecular Diagnostic Laboratory, Emory Medical Laboratories, for HIV-1 load determination were selected for use in this study. The plasma samples were collected, transported, and stored according to established guidelines for the AMPLICOR HIV-1 MONITOR test. A reference plasma sample, NAC-HIV-1, was purchased from Acrometrix, Benecia, Calif. Dilutions of some plasma samples were made in normal human plasma provided with the AMPLICOR HIV-1 MONITOR test kit to adjust the HIV-1 RNA concentrations.

Manual sample preparation.

Both the standard and the ultrasensitive manual sample preparation protocols for the AMPLICOR HIV-1 MONITOR test were performed as recommended by the manufacturer.

MPLC sample preparation.

The MPLC total nucleic acid isolation kit was used in conjunction with the MPLC instrument (software version 2.1) to process the plasma samples. The sample volume was 200 μl, and the total nucleic acid isolation reagents and instrument were used as recommended by the manufacturer, with the following exceptions. The HIV-1 quantitation standard provided with the AMPLICOR HIV-1 MONITOR test kit was added to the lysis buffer at a ratio of 1.7 μl per 300 μl of lysis buffer (0.0057) prior to its addition to the reagent tub. The nucleic acid was eluted from the magnetic glass particles in 65 μl of elution buffer. Fifty microliters of each processed sample was added to reaction tubes containing the working master mixture for reverse transcription and PCR amplification. The sample volume factor used to convert the number of copies per PCR mixture to the number of copies per milliliter of plasma for samples processed with the MPLC system was calculated to be 6.4. The high-positive control provided with the AMPLICOR HIV-1 MONITOR test kit diluted in water (12.5 μl in 200 μl) and a reference plasma sample, NAC-HIV-1, were processed with the samples on the MPLC instrument as positive controls. Two different lots of total nucleic acid kits were used in this study.

Viral load determinations.

Samples were processed in parallel by either the standard or the ultrasensitive manual method and the MPLC protocol for viral load determinations with the AMPLICOR HIV-1 MONITOR test, version 1.5. This test was performed according the instructions of the manufacturer, and two different lots of kits were used in this study. The dynamic range of the test with the standard manual sample processing protocol was from 400 (2.6 log₁₀) to 750,000 (5.87 log₁₀) HIV-1 RNA copies/ml, and that with the ultrasensitive manual sample processing protocol was 50 (1.7 log₁₀) to 100,000 (5 log₁₀) HIV-1 RNA copies/ml.

Statistics.

All statistics were calculated by using log₁₀-transformed viral load values. Descriptive statistics, Student's t tests and F tests for variances, and linear regression lines were calculated by using the data analysis tool pack that is part of Microsoft Excel 2000 software. Agreement between viral load results for samples processed by the different methods was assessed by the method of Bland and Altman (1).

RESULTS

Table 1 shows the essential parameters of the three sample processing methods used in this study. For each sample processing method, the number of HIV-1 copies per milliliter was calculated by the following equation: total HIV-1 OD₄₅₀/(total QS OD₄₅₀ × input number of QS copies/PCR mixture × sample volume factor), where OD₄₅₀ is the optical density at 450 nm and QS is the quantitation standard. Only the sample volume factor was changed when the number of HIV-1 copies per milliliter was calculated for samples extracted by different methods (Table 1).

TABLE 1.

Essential parameters for the sample preparation protocols used for AMPLICOR HIV-1 MONITOR Test

Parameter	Standard manual method	Ultrasensitive manual method	MPLC system
Plasma vol (μl)	200	500	200
Ultracentrifugation	No	Yes	No
QSa: lysis buffer (vol:vol)	0.011	0.0028	0.0057
Working lysis buffer vol (μl)	600	600	300
Diluent or eluant volume (μl)	400	100	65
Vol added to PCR mixture (μl)	50	50	50
Sample vol factor	40	4	6.4

^aQS, quantitation standard.

A dilution series was prepared from a pool of three high-titer positive plasma specimens diluted in normal human plasma. It spanned a 5-log₁₀ concentration range from approximately 10 to 10⁵ copies/ml. Table 2 shows the results of replicate viral load determinations of dilution series samples extracted by both the standard manual and the MPLC protocols. The viral load values for the samples extracted by the two protocols were highly correlated (R², 0.97) and in close agreement. The precision of the viral load determinations was similar for samples extracted by the standard manual and MPLC protocols, with average standard deviations of 0.124 and 0.151, respectively (P = 0.49, Student's t test).

TABLE 2.

Sensitivity and intra-assay variation of AMPLICOR HIV-1 MONITOR Test with dilution series samples processed in parallel by the standard manual and MPLC protocols

Nominal log10 HIV-1 copies/ml	Standard manual processing			MPLC processing
	No. detecteda/ no. tested	Mean log10 HIV-1 copies/ml	SD	No. detected/ no. tested	Mean log10 HIV-1 copies/ml	SD
5.0	12/12	5.09	0.098	12/12	5.07	0.129
4.7	8/8	4.87	0.070	8/8	4.73	0.076
3.7	8/8	3.98	0.036	8/8	4.03	0.175
3.0	8/8	3.21	0.091	8/8	3.26	0.150
2.7	8/8	3.20	0.206	7/8	3.06	0.151
2.0	5/8	2.24	0.245	8/8	2.42	0.096
1.7	4/8	2.29	0.126	8/8	2.09	0.282
1.0	1/8	2.16		5/8	1.70	0.374

^aNumber of replicates in which the HIV-1 RNA OD₄₅₀ was >0.2.

The MPLC sample processing protocol detected 100% of the eight replicates containing 1.7 nominal log₁₀ copies/ml and 50% of the replicates containing 1 nominal log₁₀ copy/ml. The standard manual protocol was approximately 1 log₁₀ less sensitive, detecting 100% of the replicates at a low concentration of 2.7 nominal log₁₀ copies/ml. A plot of the measured versus the expected log₁₀ number of HIV-1 copies per milliliter for samples processed by the MPLC protocol showed a linear response over the concentration range (R², 0.99) (Fig. 1).

FIG. 1.

Plot of measured against expected log₁₀ number of HIV-1 copies per milliliter for specimens processed by the MPLC protocol. Each point represents the mean for eight replicates. The equation for the linear regression line was y = 0.86x + 0.73 (R², 0.996).

We next processed a total of 140 plasma specimens in parallel by the manual and the MPLC protocols. The ultrasensitive manual method was used for 92 (66%) of the specimens, and the standard manual method was used for 48 (34%) of the specimens. Discrete values were obtained for both viral load determinations with 127 specimens. HIV-1 RNA was not detected (HIV-1 OD₄₅₀ < 0.2) in four specimens processed manually and six specimens processed by the MPLC protocol. The mean log₁₀ viral load determined for manually extracted specimens was 3.44 (median, 3.22), with values ranging from a low of 1.12 log₁₀ copies/ml to a high of 5.79 log₁₀ copies/ml.

The correlation of the viral load values obtained with specimens processed in parallel by the different protocols is shown in Fig. 2. The correlation was higher between values obtained with specimens processed by the manual and the MPLC protocols when the ultrasensitive (R², 0.91) rather than the standard manual (R², 0.86) protocol was used. In addition, the response of the AMPLICOR MONITOR test appeared to plateau for specimens processed by the MPLC protocol, with viral load values being >5 log₁₀.

FIG. 2.

Correlation of viral load results for all clinical specimens processed in parallel by the MPLC and the manual protocols. ▪, ultrasensitive manual method; ▴, standard manual method; solid line, trend line for values determined by the ultrasensitive manual and MPLC protocols (R², 0.913); dashed line, trend line for values determined by the standard manual and MPLC protocols (R², 0.863).

The agreement between viral load values for the samples processed by different methods was measured by determining the differences in log₁₀ viral loads for each sample (i.e., the loads obtained by the manual protocol minus the loads obtained by the MPLC protocol) and calculating the mean and standard deviation of the differences. The values were in good agreement, with a mean log₁₀ difference of 0.09 and a standard deviation of 0.291. The 95% confidence interval (±2 standard deviations) for the mean difference was −0.5 and 0.65 log₁₀. A plot of the difference versus the average log₁₀ viral load for each sample is shown in Fig. 3. As expected, the differences were larger at the extremes of the dynamic range of the assay, but the differences did not vary in a systematic way over the range of measurements.

FIG. 3.

Difference in log₁₀ number of HIV-1 copies per milliliter against average log₁₀ number of HIV-1 copies per milliliter for specimens processed manually and by the MPLC protocol. Solid line, mean difference (0.086 log₁₀); dashed lines, ±2 standard deviations (0.568 log₁₀); ▪, ultrasensitive manual method; ▴, standard manual method.

On the basis of the data presented above, the dynamic range of the assay with samples processed by the MPLC protocol was similar to that for samples processed by the ultrasensitive manual protocol (1.7 to 5 log₁₀). We reassessed the correlation and agreement of the viral load values for the 102 samples described above that had values between 1.7 and 5 log₁₀ copies/ml when they were processed by the standard or the ultrasensitive manual protocol. The correlation coefficient increased to 0.95 when only values in this range were considered (Fig. 4). However, the agreement was essentially the same as described for the larger data set, with a mean log₁₀ difference of 0.09 (standard deviation, 0.292).

FIG.4.

Correlation of viral load results for clinical specimens with viral load values between 1.7 and 5 log₁₀ copies/ml processed in parallel by the MPLC and the manual protocols. The equation for the linear regression line was y = 1.082x + 0.354 (R², 0.945).

An additional 35 specimens with viral loads between 5 and 5.87 log₁₀ copies/ml were diluted 10-fold in normal human plasma and processed by the MPLC protocol. These same specimens were also processed, undiluted, by the standard manual protocol. The viral load results were compared after correction for the additional dilution factor for the specimens processed by the MPLC protocol. The results were in good agreement, with a mean log₁₀ difference of −0.09 (standard deviation, 0.365).

The viral load results for two positive controls processed in parallel by the MPLC and ultrasensitive manual protocols are shown in Table 3. The data were pooled from four MPLC runs using two different lots of total nucleic acid kits. The NAC-HIV-1 positive control had a target concentration of 2 log₁₀ copies/ml. The mean viral load value was 2.37 with automated sample processing and 2.22 with manual sample processing (P = 0.08). There was a trend to increased variance (standard deviation squared) with automated sample processing (P = 0.06).

TABLE 3.

HIV-1 load results for positive controls processed in parallel by the MPLC and ultrasensitive manual protocols

Control	Processing protocol (vol)	No. of replicates	Mean log10 HIV-1 copies/ml	SD
NAC-HIV-1a	MPLC (200 μl)	11	2.37	0.267
	Ultrasensitive manual (500 μl)	8	2.22	0.147
AMPLICOR kit (high)	MPLC (12.5 μl/200 μl of H2O)	11	4.61	0.178
	Ultrasensitive manual (12.5 μl/500 μl of NHPb)	11	4.42	0.098

^aAcrometrix.

^bNHP, normal human plasma.

Both the mean viral load value and the variance were higher for the AMPLICOR kit high-positive control processed by the MPLC protocol (P = 0.005 and P = 0.04, respectively). However, only 1 (9%) of the 11 replicates processed by the MPLC protocol and none of the replicates processed manually were outside of the target range (4.04 to 4.99 log₁₀ copies/ml) given by the manufacturer. The kit high-positive control must be diluted in water rather than normal human plasma in the MPLC protocol to avoid degradation of the RNA transcript by RNases present in normal human plasma. Both the NAC-HIV-1 plasma sample and the AMPLICOR high-positive control, as described here, appear to be suitable controls for assay by the MPLC sample preparation protocol. However, our data indicate that the small RNA transcript in the high-positive control is not recovered as consistently by the MPLC protocol as it is by the manual method.

DISCUSSION

Although significant progress has been made in automating the amplification and detection steps of nucleic acid amplification tests, sample preparation is still performed manually in many laboratories. Nucleic acid isolation is technically demanding and labor-intensive. The response of the diagnostic industry to the growing needs of clinical laboratories for truly integrated systems for sample preparation, amplification, and product detection has been slow.

The COBAS AMPLIPREP instrument (Roche Molecular Systems, Pleasanton, Calif.), which was designed to automate sample preparation for COBAS AMPLICOR tests, has been in development for several years but is still not widely available in the United States. In a recent evaluation, this instrument was found to be a suitable replacement for the manual method used for sample preparation with the COBAS AMPLICOR HCV MONITOR test (7). However, its expense, lack of versatility, and large footprint may limit its use to all but a few laboratories performing large numbers of COBAS AMPLICOR tests.

The MPLC system is an open platform that allows the user to define protocols for a variety of different applications and formats. We previously developed and verified MPLC sample preparation protocols for the AMPLICOR HCV test, the AMPLICOR CMV MONITOR test, and a variety of PCR tests developed in-house (3). In this study, we describe the development and verification of a sample preparation protocol for use with the AMPLICOR HIV-1 MONITOR test. To our knowledge, this is the first report of an automated sample preparation protocol for this test.

We found that the AMPLICOR HIV-1 MONITOR test with the automated extraction protocol had a sensitivity and a dynamic range similar to those of the ultrasensitive test. In addition, we demonstrated that, on average, the viral load results for samples processed by the MPLC protocol had viral load values approximately 0.09 log₁₀ lower than those for samples processed manually. The limits of agreement, defined as the 95% confidence interval around the mean log₁₀ difference, for samples processed by the different methods were shown to be ±0.58 log₁₀ copies/ml. Since changes in viral load of <0.5 log₁₀ are generally not biologically significant (6), the MPLC protocol could replace the manual sample preparation protocols without the need to reestablish a patient's baseline HIV-1 load by the new method. Similar limits of agreement, ±0.5 log₁₀, were found between hepatitis C virus (HCV) load results determined for specimens processed manually and with the COBAS AMPLIPREP instrument (7).

Automated processing of samples did not improve the precision of the viral load values in our study. In fact, there appeared to be a trend to greater imprecision with the MPLC sample preparation protocol. However, the average standard deviation reported here, 0.15 log₁₀, is well within the range of values reported by others for AMPLICOR MONITOR tests. All of the sample processing was done by one medical technologist who was selected for participation in this study, in part, because of her skill in performing the manual sample processing associated with the AMPLICOR MONITOR test. The manual sample processing is technically demanding, and in practice, there is considerable variation among operators in the recovery of RNA by these methods. The operator-to-operator variation should be less with an automated protocol but was not assessed in our study.

The MPLC protocol has several advantages over the ultrasensitive manual protocol. The ultrasensitive manual protocol increases the sensitivity of the AMPLICOR MONITOR test by concentrating virus from 500 μl of plasma by high-speed centrifugation. The MPLC protocol achieves the same sensitivity with only 200 μl of plasma and no need for centrifugation to concentrate the virus. It also resulted in significant labor savings. An MPLC run of 32 specimens requires only about 15 min of hands-on time, whereas the manual method requires almost 2 h. It also offers the opportunity to unify the sample preparation protocols used for the assay. At present, laboratories use different manual sample preparation protocols for low- and high-titer specimens. The high-titer specimens (>5 log₁₀ copies/ml) can simply be diluted 1:10 in normal human plasma prior to processing on the MPLC instrument.

Automated sample processing adds additional costs to the AMPLICOR MONITOR test. The unit list cost for the reagents and disposables associated with the MPLC protocol is $3.77/sample, and the list cost of the instrument is approximately $85,000. The labor savings for the AMPLICOR HCV MONITOR test offset the modest incremental cost of the reagents. The ability to use the instrument for sample preparation for a variety of nucleic acid-based tests with different assay formats helps justify the substantial capital investment. Although the throughput of 32 samples in 1.5 h is a good fit for our laboratory, it may be a limitation for those laboratories with larger batch sizes. It is a compact, bench-top instrument measuring 40 in. wide, 26 in. deep, and 35 in. high and weighing 332 lbs. The software interface with the MPLC system is simple and uses graphical representations to guide the user through the setup and run of a batch.

In conclusion, the MPLC sample preparation protocol can replace both the standard and the ultrasensitive manual protocols described for the AMPLICOR HIV-1 MONITOR test without compromising the performance characteristics of the test. The automated protocol described here saved substantial labor costs, simplified the nucleic acid extraction, and proved reliable in a clinical laboratory setting.

ADDENDUM IN PROOF

While the manuscript was in press, Hölzl et al. reported the development and evaluation of a MagNA Pure LC protocol for use with the ultrasensitive COBAS AMPLICOR HIV-1 MONITOR test (G. Hölzl, M. Stöcher, V. Leb, H. Stekel, and Jörg Berg, J. Clin. Microbiol. 41:1248-1251, 2003).