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Reina Karunaratne, Mona Shahbazian, Jerry Boonyaratanakornkit, Lorenzo Sapinoso, and Ralf Schönbrunner. AcroMetrix, part of Life Technologies, Benicia, California, 94510 USA.
RESULTS
Table 2. Linearity and Precision
Summary of slope, coefficients of determination (R2), and number of replicates (n=) from each lab. Slope is a linear regression analysis of Expected Titers (log10cp/ml) versus Observed Titers (log10 cp/ml).
Figure 1. Overall Evaluation
The OptiQuant® BKV Quantification panel consists of BKV serially diluted to 5E2, 5E3, 5E4, 5E5, and 5E6 copies/ml. The panel was tested in 1-4 replicates by 10 independent labs on various combinations of extraction systems, amplification platforms, and assays being routinely run in their laboratory . The results were reported in titers (cp/ml). The mean observed titer (log10 cp/ml) for each panel member was calculated per site and plotted against the expected titer (log10 cp/ml). The graphs for all 10 sites were combined. Theoretical line is the line if Observed Titers (log10 cp/ml) were exactly equal to the Expected Titers (log10 cp/ml).
Table 1. Test Methods-Panel
Summary of extraction systems, amplification platforms, and assay used in each lab. LDT = Lab Developed Test.
Figure 2. Lab A: Run to Run Consistency-BKV Low Control
Table 6. Lab A: Run to Run Consistency
Two lots of OptiQual® BKV Low and High Controls were tested in 3-5 replicates on 2 separate runs in Lab B using the Roche Magna Pure® extraction, ABI 7500 instrument, and their lab developed assay. Each run was performed on separate days. The mean titer (log10 cp/ml) was calculated for each lot.
Table 7. Lab B: Lot to Lot Consistency
*These labs tested two different lots of OptiQual BKV Low and High Controls; # of replicates tested is the combined number of replicates from both lots for each the Low and High Controls.
ABSTRACT Introduction: Quantitative viral load assays monitor BK virus (BKV) associated diseases which are common in many immuno-compromised patients. There is currently no FDA approved NAT assay for the in-vitro diagnosis of BKV. The majority of these assays are either laboratory-developed tests or analyte specific reagents (ASR/RUO). To standardize and compare different testing methods and verify operator proficiency, quality control materials that span the linear range of these assays are necessary.
Objective: To evaluate the linearity, precision, and consistency of the AcroMetrix OptiQuant® BKV Quantification Panel AND OptiQuant® BKV Controls across multiple laboratories using different extraction systems, assays, and amplification platforms.
Methods: The OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls consist of intact whole BKV diluted in normal human EDTA plasma. The panel consists of BKV serially diluted to 5E2, 5E3, 5E4, 5E5, and 5E6 copies/ml, and the BKV controls are targeted to 5E3 and 1E5 copies/ml for the low and high controls respectively. The OptiQuant® BKV Quantification Panel and/or OptiQual®BKV Controls were tested across 15 different laboratories in replicates of 3-20 using different combinations of extraction systems, assays, and amplification platforms that represent the BKV molecular diagnostic market.
Results: The OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls performed well across the 15 laboratories and different test methods. For the panel, when the nominal titers and observed titers were log transformed and plotted, a good degree of linearity was observed; the slopes ranged from 0.86 to 1.17, indicating consistent agreement between the expected and observed values. The coefficients of determination for the panel were greater than 0.99 at all sites, supporting a high degree of accuracy and precision. There was 100% detection at all sites for both the low and high controls. The mean observed values for the low and high controls were 3.61 log copies/ml (3.70 log expected) and 4.95 log copies/ml (5.00 log expected) respectively. The mean log difference observed between the high and low controls was 1.34 log which corresponds to a 23-fold difference (20-fold expected).
Conclusion: The OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls provides a wide range of titers for the detection of BKV in viral monitoring NAT assays. These quality control materials are useful for assay validation and verification, operator qualification, daily run controls and verification of extraction and detection efficiency across the linear range of multiple test methods.
INTRODUCTIONBK Virus (BKV) is a member of the Polyomaviridae family and closely resembles JC virus (JCV) and simian virus (SV40). BKV primary infection typically occurs in childhood and is rarely associated with clinical disease. The virus persists in circulating leukocytes and renal uroepithelium. Reactivation can occur in immuno-suppressed patients resulting in BKV nephropathy, ureteral stenosis, asymptomatic hematuria or hemorrhagic cystitis. The use of quantitative real-time PCR for BKV can provide a useful tool for the clinician in diagnosing BKV associated diseases, as well as, monitoring the patients response to treatment. There is currently no FDA approved real-time PCR assay for the in-vitro diagnosis of BKV. The majority of these assays are either homebrew or analyte specific reagents (ASR/RUO). To standardize and compare different testing methods and verify operator proficiency, quality control materials that span the linear range of these assays are necessary.
MATERIALS AND METHODS The OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls consist of intact whole BKV diluted in normal human EDTA plasma. The panel consists of BKV serially diluted to 5E2, 5E3, 5E4, 5E5, and 5E6 copies/ml, and the BKV controls are targeted to 5E3 and 1E5 copies/ml for the low and high controls respectively. Two different lots of the OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls were manufactured according to ISO 17511 and QC release tested by AcroMetrix to meet internal acceptance criteria. Extraction was performed using the QIAamp ® MinElute ® Virus Spin Kit on the QIAcube ® and real-time PCR was completed using the Eragen MultiCode ® RTx BKV RUO kit on the ABI 7300. The BKV materials were evaluated independently across 15different laboratories. Labs tested either lot 1, lot 2, or both lots in replicates of 3-20 using different combinations of extraction systems, assays, and amplification platforms that represent the BKV molecular diagnostic market. Results from all labs were compiled by AcroMetrix and analyzed in MS Excel.
CONCLUSIONSThe OptiQuant® BKV Quantification Panel and OptiQual® BKV Controls provides a wide range of titers for the detection of BKV in viral monitoring NAT assays. The OptiQuant® BKV Quantification Panel was shown to have good accuracy and linearity. Good consistency (run-to-run and lot-to-lot) and precision were demonstrated by the OptiQual® BKV Controls. The data from this study displays that these quality control materials performed well in a broad range of extraction and detection methods, demonstrating their usefulness for assay validation and verification, operator qualification, daily run controls, and verification of extraction and detection efficiency across the linear range of multiple test methods.
ACKNOWLEDGEMENTS 1. Charles P. Cartwright and Kalpana Ramachandran. ViroMed (LabCorp)
Laboratories.2. Olivo C. and Chiaramello S. Nanogen Advanced Diagnostics s.r.l3. Jill Gerriets, Dr. Cathy Gebhart, and Sharleen Rapp. The Nebraska Medical
Center.4. Heather Bentley, Joel Lefferts, PhD., and Gregory J. Tsongalis, Ph.D.
Dartmouth-Hitchcock Medical Center.5. Ray Hein Ph. D. Clinical Reference Laboratory.6. Pritt BS, Grys TE, and Espy MJ. Mayo Clinic, Rochester, MN7. Robert L. Barker, Ph.D. and James W. Snyder, Ph.D. Dept. of Pathology & Lab
Medicine, Univ. of Louisville School of Medicine 8. Zi-Xuan Wang, Ph.D. Thomas Jefferson University Hospital, Clinical
Microbiology/Molecular Lab9. John Sipley and Hanna Rennert. New York Presbyterian Hospital-Weill Cornell
Medical Center.10. Paula J. Romano, PhD, Robert Scott, CHS, MT, and Ken Bogdanovich, CHS,
MT. Central PA Transplant Assoc. HLA Lab.11. Michael Ho and Maria Foster. EraGen Biosciences.12. Linda Cook, PhD, and Shannon Nesbitt. University of Washington and the
Fred Hutchinson Cancer Research Center.13. Hayley Webber, PhD and Gabrielle Royal, MS. NorDx.14. Dr. Holly Alexander. Via Christi Regional Medical Center15. David Ferguson and Robin Garner. Yale-New Haven Hospital, Clinical Virology.16. Dr. Bobby Boyanton and Anne Prada. William Beaumont Hospital. 17. Dr. Manfred Brigl, Linda Cotugno, and Andrea M. DuBois. Brigham and
Women's Hospital, Department of Pathology Clinical Laboratories.
TRADEMARKS/LICENSINGThe trademarks mentioned herein are the property of Life TechnologiesCorporation unless otherwise noted.
Magna Pure® and LightCycler® are registered trademarks of Roche Diagnostics
ABI 7500 and ABI 7900 are registered trademarks of Life Technologies
NucliSENS® easyMAG® is a registered trademark of bioMérieux
QIAamp®, EZ1®, BioRobot®, QIAcube®, artus®, MinElute®, andRotorGene ® are registered trademarks of Qiagen Group.
Nanogen ® /Epoch are registered trademarks of the Elitech Group
SmartCycler® is a registered trademark of Cepheid
MultiCode®-RTx is a registered trademark of Eragen BioScicences
Bio Rad CFX96TM is a trademark of Bio Rad labortories.
Excel is a trademark of Microsoft Corporation.
ExtraGen is a trademark of Tosoh Corporation.
QIAxtractor is a trademark of Corbett Research.
Nanogen is a trademark of Nanogen, Inc.
The OPTIQUANT® BKV QUANTIFICATION PANEL is for Research Use Only.Not for use in diagnostic procedures.
A MULTI-SITE EVALUATION OF THE OPTIQUANT® BKV QUANTIFICATION PANEL AND OPTIQUAL® BKV CONTROLS
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
OptiQuant® BKV Quantification Panel
OptiQuant® BKV Quantification PanelEvaluation
1.002.003.004.005.006.007.008.00
2.00 3.00 4.00 5.00 6.00 7.00Expected Titer (log10 cp/ml)
Obs
erve
d Ti
ter
(log 1
0 cp
/ml)
Lab 1Lab 2Lab 3Lab 4Theoretical LineLab 5Lab 6Lab 7Lab 8Lab 9Lab 10
OptiQual® BKV ControlsTable 3. Test methods-Controls
Lab Low High ∆ Log Fold1 3.77 5.29 1.52 332 3.48 4.89 1.41 263 3.19 4.58 1.39 254 3.09 4.33 1.24 175 3.99 5.19 1.20 166 3.10 4.55 1.45 287 3.00 4.48 1.48 308 3.81 5.05 1.24 179 3.83 5.20 1.37 23
10 3.67 4.95 1.28 1911 4.08 5.50 1.42 2612 4.10 5.26 1.16 1413 3.66 4.97 1.31 2014 3.71 4.51 0.80 615 3.79 5.05 1.26 18
AVG 3.62 4.92 1.30 21Target 3.70 5.00 1.30 20Bias -0.08 -0.08 0.00 n/a
Table 4. Overall Results
Lab Slope R2 n=1 1.10 0.998 32 1.08 0.999 33 0.86 0.999 34 1.04 0.997 35 0.96 0.995 36 1.11 0.996 37 0.93 0.987 48 0.96 0.999 19 1.00 0.999 110 1.13 0.999 3
AVG 1.02 0.997 3
Summary of extraction systems, amplification platforms, and assay used in each lab. LDT = Lab Developed Test.
The results were reported in titers (cp/ml). The mean observed titer (log10 cp/ml) was calculated for both the low and high controls. The ΔLog (High (log10 cp/ml) – Low (log10cp/ml) was calculated for each lab. The fold difference is the 10^x where x is the ΔLog.
Sample Run AVG TiterLow 1 3.78Low 2 3.89Low 3 3.87Low 4 3.77High 1 5.19High 2 5.21High 3 5.28High 4 5.10
OptiQual® BKV Low Control
0.00
1.00
2.00
3.00
4.00
5.00
1 2 3 4
Run
Obs
erve
d Ti
ter
(Log
10
cp/m
l)
Figure 3. Lab A: Run to Run Consistency-BKV High Control
OptiQual® BKV High Control
0.001.00
2.003.004.00
5.006.00
1 2 3 4
Run
Obs
erve
d Ti
ter
(log
10
cp/m
l)
Lot n= Sample Titer1 3 Low 3.462 5 Low 3.511 3 High 4.912 5 High 4.87
OptiQual® BKV High and Low Controls
01
234
56
Low High
Controls
Obs
erve
d Lo
g Ti
ter
(cp/
ml) Lot 1
Lot 2
∆log=0.05
∆log=0.04
Figure 4. Lab B: Lot to Lot Consistency
One lot of OptiQual® BKV Low and High Controls were tested in 2-3 replicates on 4 separate runs in Lab A using the bioMérieuxeasyMag® extraction system, ABI 7500 amplification platform, and a lab developed assay. Each run was performed on separate days. The mean titer (log10 cp/ml) was calculated for each run.
Δlog= absolute value of mean titer (log10 cp/ml) lot 2 -mean titer (log10 cp/ml) of lot 1.
Table 5 . Detection and Precision
# of ReplicatesLab Low High %CV Low %CV High Tested
1 100% 100% 47 2 3*2 100% 100% 25 14 8*3 100% 100% 20 12 264 100% 100% 5 11 3*5 100% 100% 14 3 156 100% 100% n/a n/a 1*7 100% 100% 123 86 348 100% 100% 17 11 59 100% 100% 22 18 1010 100% 100% 26 23 511 100% 100% 54 18 512 100% 100% 80 40 1213 100% 100% 49 48 1014 100% 100% 5 19 1515 100% 100% 40 23 6
AVG 100% 100% 38 23
Detection Precision
Lab Extraction Systems Amplification Platform Assay1 Magna Pure® LightCycler® 2.0 LDT2 Magna Pure® ABI 7500 LDT3 BioRobot® M48 ABI 7500 LDT4 Magna Pure® LightCycler® 480 LDT5 easyMag® ABI 7500 LDT6 ExtraGen® RotorGene® 6000 Nanogen®/Epoch7 Magna Pure® LightCycler® 2.0 Nanogen®/Epoch8 QIAamp® MinElute® Bio-Rad CFX96TM Nanogen®/Epoch9 easyMag® ABI 7500 LDT
10 easyMag® RotorGene® 6000 Nanogen®/Epoch11 EZ1® SmartCycler® Nanogen®/Epoch12 EZ1® ABI 7500 LDT13 easyMag® SmartCycler® Nanogen®/Epoch14 QIAamp® MinElute® SmartCycler® Nanogen®/Epoch15 QIAamp®/QIAxtractor® ABI 7900 MultiCode®-RTx
Lab Extraction Systems Amplifcation Platforms Assay1 Magna Pure® LightCycler® 2.0 LDT2 Magna Pure® ABI 7500 LDT3 easyMag® ABI 7500 LDT4 Magna Pure® LightCycler® 480 LDT5 Magna Pure® LightCycler® 2.0 Nanogen®/Epoch6 BioRobot® M48 ABI 7500 LDT7 ExtraGen® RotorGene® 6000 Nanogen®/Epoch8 EZ1® SmartCycler® Nanogen®/Epoch9 easyMag® RotorGene® 6000 Nanogen®/Epoch10 QiaCube® RotorGene® artus®
