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Analytical performance of an automated assay quantifying HIV-1 from dried blood spots
Journal of Clinical Virology 57 (2013) 271–273
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Analytical performance of an automated assay quantifying HIV-1 from dried blood spots Felix Kleshik, Jesse Brooks, Carlo Cosenza, Thomas R. Battersby ∗ Siemens Healthcare Diagnostics, Berkeley, CA, USA
a r t i c l e
i n f o
Article history: Received 9 January 2013 Received in revised form 2 March 2013 Accepted 2 March 2013 Keywords: Infectious disease Quantitative PCR Whole blood
a b s t r a c t Background: Performance of an automated sample preparation and viral load quantification for HIVpositive dried blood spots (DBS) on the Siemens VERSANT® kPCR Molecular System has been previously demonstrated with clinical samples. Objectives: Evaluation of the analytical performance of the automated assay using HIV-positive DBS prepared from a dilution series. Study design: Over 300 DBS of HIV-1 nucleic acids from a dilution series in lysed whole blood (322 copies/mL to over 1.6 × 107 copies/mL) were spotted onto Whatman 903 cards and analyzed to evaluate analytical performance. Cross contamination was examined with a checkerboard pattern of 82 alternating negative and 1 × 106 copies/mL samples. Results: Analytical sensitivity evaluation with a single 50 L spot demonstrated a limit of detection (LoD) of 866 copies/mL. Above the LoD, linearity (difference between linearized and observed mean values) was within ±0.10 log, and accuracy (difference between expected and observed mean values) was within ±0.18 log. Imprecision for dilution series levels more than two-fold above the LoD was measured as 20% to 27% CV of quantification. No cross contamination was observed. Conclusions: The HIV-1 DBS Assay performed similarly to the VERSANT HIV-1 RNA 1.0 Assay (kPCR) in assay linearity, accuracy, and imprecision. The assay was sensitive enough to run single 50 L spots and used an unmodified VERSANT® SP Module with only a 30 min incubation prior to automated sample preparation. DBS-specific assay calibrators and controls, expressly formulated for easy frozen storage and identical processing to samples, were employed. © 2013 Published by Elsevier B.V.
1. Background Dried blood spot (DBS) specimens in diagnostic testing present potential advantages of lower cost, greater simplicity, and increased safety over other blood-derived sample types like frozen plasma. Small aliquots (typically 50–100 l) of whole blood are soaked into filter paper and dried. The spots can then be shipped to a central lab, with the collection and subsequent storage process taking place at ambient temperature. The simple collection procedure of DBS is ideal for limited-resource settings, circumventing the need for trained phlebotomists and material resources for the collection, processing, and shipping of samples.
An automated sample preparation and viral load quantification for HIV-positive DBS specimens is under development at Siemens Healthcare Diagnostics. Performance of this assay, automated on the Versant kPCR Molecular System, has already been examined by Pirillo et al.1 Pirillo et al. found that quantification of HIVpositive DBS clinical samples with the DBS assay strongly correlated with quantification of paired plasma samples with the VERSANT® HIV-1 RNA 1.0 Assay (kPCR), and that the HIV-1 DBS assay had a 100% detection rate among the samples examined with viral load >1000 copies/mL. They concluded that the Siemens assay was useful in detecting virological failure in treatment of HIV-patients. 2. Objectives
Abbreviations: CI, confidence interval; DBS, dried blood spot; HPC, high positive control; LoD, limit of detection; LPC, low positive control. ∗ Corresponding author at: Siemens Healthcare Diagnostics, 725 Potter St., Berkeley, CA 94710, USA. Tel.: +1 510 982 4279; fax: +1 510 982 4238. E-mail address: [email protected] (T.R. Battersby). 1386-6532/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jcv.2013.03.001
To complement the clinical sample study of Pirillo et al., we present here a brief evaluation of analytical performance of the assay using controlled target material containing only extracellular HIV-1 RNA. We also provide a detailed description of the HIV-1 DBS viral load assay procedure.
272
F. Kleshik et al. / Journal of Clinical Virology 57 (2013) 271–273
Fig. 1. Analytical performance plots. (a) Percent detection versus sample concentration was plotted with confidence intervals. (b) A combined linearity log plot of observed versus expected values for a dilution series with perfect linearity and accuracy indicated.
3. Study design
3.4. Assay procedure
3.1. Instrumentation and software
50 L of hemolysate (test sample, calibrator, or control) was spotted individually and dried on Whatman 903 filter paper. After drying, a single spot was punched from the paper with a ½inch punch. The punch was carefully decontaminated4 between processing of sample levels. DBS Lysis Buffer (1.25 mL) was added and the spot incubated (30 min) at room temperature with shaking (300 rpm). The paper was drawn onto the side of the tube with a pipette tip and removed. The solution was loaded onto the VERSANT kPCR Sample Preparation Module for automated nucleic acid extraction. The prior manual incubation in DBS Lysis Buffer allowed a specific DBS protocol with room temperature lysis incubation conditions to be used. Extracted nucleic acid and all required amplification reagents were automatically loaded onto a 96-well plate, ready for amplification.
The DBS procedure was run using a standard VERSANT kPCR Molecular System (Siemens Healthcare Diagnostics, Tarrytown, US; CE-Marked; not available in US) without hardware modifications. A DBS-specific extraction protocol was used. Amplification software and settings of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) assay were used without modification. 3.2. Reagents VERSANT Sample Preparation 1.0 Reagents – except for Lysis Buffer – were used in extraction. A DBS Lysis Buffer (not a Siemens product – not available for sale), similar to the guanidine thiocyanate Lysis Buffer used with VERSANT Reagents except for the neutral pH of the reagent, was substituted. VERSANT HIV-1 RNA 1.0 Assay (kPCR) Primer/Probe mix, Enzyme mix, and Internal Control were all used without modification. 3.3. Calibrators, controls, and test samples Assay calibrators and controls, as well as a dilution series for analytical testing (10 levels from 322 to 16,115,732 copies/mL) were prepared by gravimetrically diluting a stock of 8E5/LAV noninfectious virus2 (Tables S1 and S2, respectively). The viral stock, previously quantified by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) against RNA calibrators traceable to a US NIST phosphate standard,3 was diluted in lysed, human whole blood (lysis by freeze/thaw). Lysed blood behaved physically like whole blood in spotting and drying, but allowed frozen storage. VERSANT HIV-1 RNA 1.0 Assay (kPCR) Calibrators, Positive Controls, and Negative Controls were replaced by dedicated calibrators and controls in the HIV-1 DBS assay. Each run had two replicates each of Calibrator A and Calibrator B, and single high (HPC), low (LPC), and negative controls. Calibrators and controls were stored frozen and thawed, spotted, and dried on filter paper prior to use, identically to samples.
3.5. Data analysis For run validity the slope and intercept of Ct versus log10 copies/mL for calibrators, the across-plate mean Ct of the Internal Control, and HPC and LPC quantifications must be within specification. Negative controls must not be positive. Individual well validity was established by an Internal Control signal within specification. Imprecision was evaluated with a linear mixed-effect model to estimate the variance components for each level. Linearity of quantification was evaluated via the log difference from perfect linearity. This was done by taking the difference between mean log quantification and the linearized value for each concentration, where the linearized value is the mean quantification in the log scale adjusted by the dilution factor of the serial dilution. The log difference is reported for each level, along with the observed mean quantification and its linearized value. Accuracy, based on log recovery (difference between observed mean log quantification and log-assigned concentration), was also calculated for each level. The LoD is the lowest concentration where the percent detected is estimated to be at least 95%.
F. Kleshik et al. / Journal of Clinical Virology 57 (2013) 271–273
273
Table 1 Analytical performance: accuracy, linearity, and imprecision.
a
Level
Assigned concentration (copies/mL)
Log assigned concentration (copies/mL)
N
Observed mean log quant
Log recovery (accuracy)
Linearized log quant
Log difference from linearity
Total %CV
LP1 LP2 LP3 LP4 LP5 LP6a LP7a LP8a LP9a LP10a
16,115,732 1,611,499 161,149 16,113 1612 860 645 537 430 322
7.2073 6.2072 5.2072 4.2072 3.2074 2.9345 2.8096 2.7300 2.6335 2.5079
20 20 20 20 23 40 34 35 30 27
7.1347 6.1992 5.0590 4.1041 3.0300 2.8247 2.7429 2.6985 2.5291 2.5499
−0.0725 −0.0080 −0.1482 −0.1031 −0.1773 −0.1096 −0.0664 −0.0316 −0.1041 0.0416
7.1308 6.1307 5.1307 4.1307 3.1308 2.8578 2.7329 2.6537 2.5568 2.4318
0.0040 0.0685 −0.0717 −0.0266 −0.1008 −0.0331 0.0101 0.0448 −0.0277 0.1181
27.08 20.53 20.18 25.87 47.51 50.67 55.88 45.15 63.44 43.16
Below assay LoD.
4. Results
Funding
Four 96-well plates were run in an analytical performance study (Tables S2 and S3). An additional brief cross contamination study consisted of a single 96-well plate with alternating HIV-positive (1 × 106 copies/mL) and HIV-negative samples. This “checkerboard” pattern presented an extreme contamination challenge. All runs and all individual wells were valid. No outlier analysis was performed. Our evaluation of analytical sensitivity demonstrated a limit of detection (LoD) of 866 copies/mL (95% CI from 676 to 1633 copies/mL, Fig. 1a and Table S4). Our LoD estimate is different in one regard from LoD estimates of DBS assays utilizing paired DBS and plasma specimens. Intracellular nucleic acid, such as proviral DNA, complicates interpretation of assay sensitivity in paired DBS and plasma clinical specimens because at low concentrations of plasma RNA, intracellular HIV-1 nucleic acid contributions to assay signal are relatively significant. Our LoD result is not complicated by contributions from unknown amounts of intracellular nucleic acid. The LoD determined here may be related to an equivalent plasma viral load, after correcting for hematocrit volume.5 Thus, the assay minimally detects clinical specimens at the plasma equivalent LoD 95% of the time. In practice, the assay may detect clinical specimens below the equivalent plasma viral load ≥95% of the time because of additional signal contribution from intracellular nucleic acid. The HIV DBS assay was linear above the LoD, with assay linearity (difference between linearized and observed mean values) within ±0.10 log (Table 1 and Fig. 1b). Assay accuracy (difference between expected and observed mean values) was within ±0.18 log above the LoD (Table 1 and Fig. 1b). Despite increased complexity in processing DBS versus plasma samples, imprecision for DBS dilution series levels (Table 1) from 1.6 × 104 to 1.6 × 107 copies/mL was 20% to 27% CV of linear quantification, only slightly worse than imprecision observed with clinical plasma samples6 or a dilution series of a clinical sample in plasma7 in the VERSANT HIV-1 RNA 1.0 Assay (kPCR). No detectable signal from cross contamination was observed in any of the 41 HIV-negative samples in the checkerboard experiment.
No external funding, apart from the support of the authors’ institution, was available for this study.
5. Conclusions Additional processing required by the DBS sample type had only minor effects on analytical performance versus the plasmabased VERSANT HIV-1 RNA 1.0 Assay (kPCR). An assay LoD of 866 copies/mL was achieved despite the 50 L sample input volume. DBS-specific calibrators and controls were conveniently stored frozen until use and processed identically to samples. Manual processing of DBS was minimal, with an additional 30 min incubation prior to automated sample preparation on an unmodified VERSANT kPCR Molecular System.
Competing interests None declared. Ethical approval Not required. Authors’ contributions FK and TB conceived of and designed the assay and experiments, and wrote the manuscript. FK and JB performed the experiments. TB and CC analyzed the data. Acknowledgements The authors would like to thank Suzanne Lain for assistance in editing the manuscript and Dr. Johan Surtihadi for helpful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2013.03.001. References 1. Pirillo MF, Recordon-Pinson P, Andreotti M, Mancini MG, Amici R, Giuliano M. Quantification of HIV-RNA from dried blood spots using the Siemens VERSANT® HIV-1 RNA (kPCR) assay. J Antimicrob Chemother 2011;66(12): 2823–86. 2. Folks TM, Powell D, Lightfoote M, Koenig S, Fauci AS, Benn S, et al. Biological and biochemical characterization of a cloned Leu-3-cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med 1986;164(1): 280–90. 3. Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, et al. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem 1995;226(1):120–9. 4. Bonne N, Clark P, Shearer P, Raidal SR. Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination. J Vet Diagn Invest 2008;20(1):60–3. 5. Leelawiwat W, Young NL, Chaownachan T, OuCY, Culnane M, Vanprapa N, et al. Dried blood spots for the diagnosis and quantitation of HIV-1: stability studies and evaluation of sensitivity and specificity for the diagnosis of infant HIV-1 infection in Thailand. J Virol Methods 2009;155(2):109–17. 6. Troppan KT, Stelzl E, Violan D, Winkler M, Kessler HH. Evaluation of the new VERSANT HIV-1 RNA 1.0 assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA. J Clin Virol 2009;46(1):69–74. 7. Ruelle J, Jnaoui K, Lefèvre I, Lamarti N, Goubau P. Comparative evaluation of the VERSANT® HIV-1 RNA 1.0 kinetic PCR molecular system (kPCR) for the quantification of HIV-1 plasma viral load. J Clin Virol 2009;44(4):297–301.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Analytical performance of an automated assay quantifying HIV-1 from dried blood spots Felix Kleshik, Jesse Brooks, Carlo Cosenza, Thomas R. Battersby ∗ Siemens Healthcare Diagnostics, Berkeley, CA, USA
a r t i c l e
i n f o
Article history: Received 9 January 2013 Received in revised form 2 March 2013 Accepted 2 March 2013 Keywords: Infectious disease Quantitative PCR Whole blood
a b s t r a c t Background: Performance of an automated sample preparation and viral load quantification for HIVpositive dried blood spots (DBS) on the Siemens VERSANT® kPCR Molecular System has been previously demonstrated with clinical samples. Objectives: Evaluation of the analytical performance of the automated assay using HIV-positive DBS prepared from a dilution series. Study design: Over 300 DBS of HIV-1 nucleic acids from a dilution series in lysed whole blood (322 copies/mL to over 1.6 × 107 copies/mL) were spotted onto Whatman 903 cards and analyzed to evaluate analytical performance. Cross contamination was examined with a checkerboard pattern of 82 alternating negative and 1 × 106 copies/mL samples. Results: Analytical sensitivity evaluation with a single 50 L spot demonstrated a limit of detection (LoD) of 866 copies/mL. Above the LoD, linearity (difference between linearized and observed mean values) was within ±0.10 log, and accuracy (difference between expected and observed mean values) was within ±0.18 log. Imprecision for dilution series levels more than two-fold above the LoD was measured as 20% to 27% CV of quantification. No cross contamination was observed. Conclusions: The HIV-1 DBS Assay performed similarly to the VERSANT HIV-1 RNA 1.0 Assay (kPCR) in assay linearity, accuracy, and imprecision. The assay was sensitive enough to run single 50 L spots and used an unmodified VERSANT® SP Module with only a 30 min incubation prior to automated sample preparation. DBS-specific assay calibrators and controls, expressly formulated for easy frozen storage and identical processing to samples, were employed. © 2013 Published by Elsevier B.V.
1. Background Dried blood spot (DBS) specimens in diagnostic testing present potential advantages of lower cost, greater simplicity, and increased safety over other blood-derived sample types like frozen plasma. Small aliquots (typically 50–100 l) of whole blood are soaked into filter paper and dried. The spots can then be shipped to a central lab, with the collection and subsequent storage process taking place at ambient temperature. The simple collection procedure of DBS is ideal for limited-resource settings, circumventing the need for trained phlebotomists and material resources for the collection, processing, and shipping of samples.
An automated sample preparation and viral load quantification for HIV-positive DBS specimens is under development at Siemens Healthcare Diagnostics. Performance of this assay, automated on the Versant kPCR Molecular System, has already been examined by Pirillo et al.1 Pirillo et al. found that quantification of HIVpositive DBS clinical samples with the DBS assay strongly correlated with quantification of paired plasma samples with the VERSANT® HIV-1 RNA 1.0 Assay (kPCR), and that the HIV-1 DBS assay had a 100% detection rate among the samples examined with viral load >1000 copies/mL. They concluded that the Siemens assay was useful in detecting virological failure in treatment of HIV-patients. 2. Objectives
Abbreviations: CI, confidence interval; DBS, dried blood spot; HPC, high positive control; LoD, limit of detection; LPC, low positive control. ∗ Corresponding author at: Siemens Healthcare Diagnostics, 725 Potter St., Berkeley, CA 94710, USA. Tel.: +1 510 982 4279; fax: +1 510 982 4238. E-mail address: [email protected] (T.R. Battersby). 1386-6532/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jcv.2013.03.001
To complement the clinical sample study of Pirillo et al., we present here a brief evaluation of analytical performance of the assay using controlled target material containing only extracellular HIV-1 RNA. We also provide a detailed description of the HIV-1 DBS viral load assay procedure.
272
F. Kleshik et al. / Journal of Clinical Virology 57 (2013) 271–273
Fig. 1. Analytical performance plots. (a) Percent detection versus sample concentration was plotted with confidence intervals. (b) A combined linearity log plot of observed versus expected values for a dilution series with perfect linearity and accuracy indicated.
3. Study design
3.4. Assay procedure
3.1. Instrumentation and software
50 L of hemolysate (test sample, calibrator, or control) was spotted individually and dried on Whatman 903 filter paper. After drying, a single spot was punched from the paper with a ½inch punch. The punch was carefully decontaminated4 between processing of sample levels. DBS Lysis Buffer (1.25 mL) was added and the spot incubated (30 min) at room temperature with shaking (300 rpm). The paper was drawn onto the side of the tube with a pipette tip and removed. The solution was loaded onto the VERSANT kPCR Sample Preparation Module for automated nucleic acid extraction. The prior manual incubation in DBS Lysis Buffer allowed a specific DBS protocol with room temperature lysis incubation conditions to be used. Extracted nucleic acid and all required amplification reagents were automatically loaded onto a 96-well plate, ready for amplification.
The DBS procedure was run using a standard VERSANT kPCR Molecular System (Siemens Healthcare Diagnostics, Tarrytown, US; CE-Marked; not available in US) without hardware modifications. A DBS-specific extraction protocol was used. Amplification software and settings of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) assay were used without modification. 3.2. Reagents VERSANT Sample Preparation 1.0 Reagents – except for Lysis Buffer – were used in extraction. A DBS Lysis Buffer (not a Siemens product – not available for sale), similar to the guanidine thiocyanate Lysis Buffer used with VERSANT Reagents except for the neutral pH of the reagent, was substituted. VERSANT HIV-1 RNA 1.0 Assay (kPCR) Primer/Probe mix, Enzyme mix, and Internal Control were all used without modification. 3.3. Calibrators, controls, and test samples Assay calibrators and controls, as well as a dilution series for analytical testing (10 levels from 322 to 16,115,732 copies/mL) were prepared by gravimetrically diluting a stock of 8E5/LAV noninfectious virus2 (Tables S1 and S2, respectively). The viral stock, previously quantified by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) against RNA calibrators traceable to a US NIST phosphate standard,3 was diluted in lysed, human whole blood (lysis by freeze/thaw). Lysed blood behaved physically like whole blood in spotting and drying, but allowed frozen storage. VERSANT HIV-1 RNA 1.0 Assay (kPCR) Calibrators, Positive Controls, and Negative Controls were replaced by dedicated calibrators and controls in the HIV-1 DBS assay. Each run had two replicates each of Calibrator A and Calibrator B, and single high (HPC), low (LPC), and negative controls. Calibrators and controls were stored frozen and thawed, spotted, and dried on filter paper prior to use, identically to samples.
3.5. Data analysis For run validity the slope and intercept of Ct versus log10 copies/mL for calibrators, the across-plate mean Ct of the Internal Control, and HPC and LPC quantifications must be within specification. Negative controls must not be positive. Individual well validity was established by an Internal Control signal within specification. Imprecision was evaluated with a linear mixed-effect model to estimate the variance components for each level. Linearity of quantification was evaluated via the log difference from perfect linearity. This was done by taking the difference between mean log quantification and the linearized value for each concentration, where the linearized value is the mean quantification in the log scale adjusted by the dilution factor of the serial dilution. The log difference is reported for each level, along with the observed mean quantification and its linearized value. Accuracy, based on log recovery (difference between observed mean log quantification and log-assigned concentration), was also calculated for each level. The LoD is the lowest concentration where the percent detected is estimated to be at least 95%.
F. Kleshik et al. / Journal of Clinical Virology 57 (2013) 271–273
273
Table 1 Analytical performance: accuracy, linearity, and imprecision.
a
Level
Assigned concentration (copies/mL)
Log assigned concentration (copies/mL)
N
Observed mean log quant
Log recovery (accuracy)
Linearized log quant
Log difference from linearity
Total %CV
LP1 LP2 LP3 LP4 LP5 LP6a LP7a LP8a LP9a LP10a
16,115,732 1,611,499 161,149 16,113 1612 860 645 537 430 322
7.2073 6.2072 5.2072 4.2072 3.2074 2.9345 2.8096 2.7300 2.6335 2.5079
20 20 20 20 23 40 34 35 30 27
7.1347 6.1992 5.0590 4.1041 3.0300 2.8247 2.7429 2.6985 2.5291 2.5499
−0.0725 −0.0080 −0.1482 −0.1031 −0.1773 −0.1096 −0.0664 −0.0316 −0.1041 0.0416
7.1308 6.1307 5.1307 4.1307 3.1308 2.8578 2.7329 2.6537 2.5568 2.4318
0.0040 0.0685 −0.0717 −0.0266 −0.1008 −0.0331 0.0101 0.0448 −0.0277 0.1181
27.08 20.53 20.18 25.87 47.51 50.67 55.88 45.15 63.44 43.16
Below assay LoD.
4. Results
Funding
Four 96-well plates were run in an analytical performance study (Tables S2 and S3). An additional brief cross contamination study consisted of a single 96-well plate with alternating HIV-positive (1 × 106 copies/mL) and HIV-negative samples. This “checkerboard” pattern presented an extreme contamination challenge. All runs and all individual wells were valid. No outlier analysis was performed. Our evaluation of analytical sensitivity demonstrated a limit of detection (LoD) of 866 copies/mL (95% CI from 676 to 1633 copies/mL, Fig. 1a and Table S4). Our LoD estimate is different in one regard from LoD estimates of DBS assays utilizing paired DBS and plasma specimens. Intracellular nucleic acid, such as proviral DNA, complicates interpretation of assay sensitivity in paired DBS and plasma clinical specimens because at low concentrations of plasma RNA, intracellular HIV-1 nucleic acid contributions to assay signal are relatively significant. Our LoD result is not complicated by contributions from unknown amounts of intracellular nucleic acid. The LoD determined here may be related to an equivalent plasma viral load, after correcting for hematocrit volume.5 Thus, the assay minimally detects clinical specimens at the plasma equivalent LoD 95% of the time. In practice, the assay may detect clinical specimens below the equivalent plasma viral load ≥95% of the time because of additional signal contribution from intracellular nucleic acid. The HIV DBS assay was linear above the LoD, with assay linearity (difference between linearized and observed mean values) within ±0.10 log (Table 1 and Fig. 1b). Assay accuracy (difference between expected and observed mean values) was within ±0.18 log above the LoD (Table 1 and Fig. 1b). Despite increased complexity in processing DBS versus plasma samples, imprecision for DBS dilution series levels (Table 1) from 1.6 × 104 to 1.6 × 107 copies/mL was 20% to 27% CV of linear quantification, only slightly worse than imprecision observed with clinical plasma samples6 or a dilution series of a clinical sample in plasma7 in the VERSANT HIV-1 RNA 1.0 Assay (kPCR). No detectable signal from cross contamination was observed in any of the 41 HIV-negative samples in the checkerboard experiment.
No external funding, apart from the support of the authors’ institution, was available for this study.
5. Conclusions Additional processing required by the DBS sample type had only minor effects on analytical performance versus the plasmabased VERSANT HIV-1 RNA 1.0 Assay (kPCR). An assay LoD of 866 copies/mL was achieved despite the 50 L sample input volume. DBS-specific calibrators and controls were conveniently stored frozen until use and processed identically to samples. Manual processing of DBS was minimal, with an additional 30 min incubation prior to automated sample preparation on an unmodified VERSANT kPCR Molecular System.
Competing interests None declared. Ethical approval Not required. Authors’ contributions FK and TB conceived of and designed the assay and experiments, and wrote the manuscript. FK and JB performed the experiments. TB and CC analyzed the data. Acknowledgements The authors would like to thank Suzanne Lain for assistance in editing the manuscript and Dr. Johan Surtihadi for helpful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2013.03.001. References 1. Pirillo MF, Recordon-Pinson P, Andreotti M, Mancini MG, Amici R, Giuliano M. Quantification of HIV-RNA from dried blood spots using the Siemens VERSANT® HIV-1 RNA (kPCR) assay. J Antimicrob Chemother 2011;66(12): 2823–86. 2. Folks TM, Powell D, Lightfoote M, Koenig S, Fauci AS, Benn S, et al. Biological and biochemical characterization of a cloned Leu-3-cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med 1986;164(1): 280–90. 3. Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, et al. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem 1995;226(1):120–9. 4. Bonne N, Clark P, Shearer P, Raidal SR. Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination. J Vet Diagn Invest 2008;20(1):60–3. 5. Leelawiwat W, Young NL, Chaownachan T, OuCY, Culnane M, Vanprapa N, et al. Dried blood spots for the diagnosis and quantitation of HIV-1: stability studies and evaluation of sensitivity and specificity for the diagnosis of infant HIV-1 infection in Thailand. J Virol Methods 2009;155(2):109–17. 6. Troppan KT, Stelzl E, Violan D, Winkler M, Kessler HH. Evaluation of the new VERSANT HIV-1 RNA 1.0 assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA. J Clin Virol 2009;46(1):69–74. 7. Ruelle J, Jnaoui K, Lefèvre I, Lamarti N, Goubau P. Comparative evaluation of the VERSANT® HIV-1 RNA 1.0 kinetic PCR molecular system (kPCR) for the quantification of HIV-1 plasma viral load. J Clin Virol 2009;44(4):297–301.