2009 College of American Pathologists. The College does not permit reproduction of any substantial portion of the material in this Report without its written authorization. The College hereby authorizes participants in the program to use the material in this Report solely for educational purposes within their own institutions. The College prohibits use of the material in the Report - and any unauthorized use of the College s name or logo - in connection with promotional efforts by marketers of laboratory equipment, reagents, materials, or services. Data from this program do not necessarily indicate the superiority or inferiority of instruments, reagents, or other materials used by participating laboratories. Use of these data to suggest such superiority or inferiority may be deceptive and misleading. The College will take all steps open to it under the law to prevent unauthorized reproduction of substantial portions of the material in this Report, deceptive use of any such material, and any unauthorized use of the College s name or logo in connection with promotional efforts by marketers of laboratory equipment, reagents, materials, or services.
Results of the College of American Pathologists (CAP) Proficiency Program for CMV, EBV, and BKV Viral Load Testing Christine C. Ginocchio, PhD, MT (ASCP) North Shore-LIJ Health System Laboratories Angela Caliendo, MD, PhD Emory University Medical Center SoGAT Clinical Diagnostics II Istanbul Oct 1, 2009
CAP Microbiology Resource Mike Mitchell Gary Procop Angie Caliendo Sheldon Campbell Karen Carroll Dierdre Church Chris Ginocchio Gerri Hall Randy Hayden Gordon Love Cathy Petti Bobbi Pritt Julie Ribes Diane Roscoe Robert Schlaberg John Steele Elizabeth Wagar Christina Wojewoda Megan Wick
CAP Proficiency Testing Must follow CLIA guidelines Regulated analytes Specified by CMS 5 samples/3 times yr at regular intervals Grading based on a 80% consensus Results reported to CMS Testing can not be designed to fail users Repetitive failures Loss of licensure Loss of CMS reimbursement Educational or challenge specimens Unregulated analytes New or novel targets Ungraded, not reported to CMS but PT is still required with resolution of incorrect results Allow for comparison across other labs, methods
Molecular Viral Panel Requirements Must be inactivated (matrix specific samples) Requirement for select countries outside US Requirement for all military labs outside US Can result in a 2-3 log reduction in viral load Can not access the integrity of the virion Problems Obtaining viral loads across the dynamic range Especially for viruses with high VLs (BKV, B19) Compatibility with multiple test platforms Compatibility with multiple targets RNA vs DNA Target regions (whole genomes or specific regions) Preparations in high quantities
CAP Viral Load Survey (VLS) 2007: CMV, EBV 2008: CMV, EBV, BKV 2 challenges per target, twice yearly Varying and replicate concentrations Reported as integers and as log 10 transformed values Results are compared within peer groups Not graded Methods vary greatly
Nucleic Acid Extraction Methods Magnetic bead Column Liquid Phase Platforms biomerieux: NucliSENS easymag Qiagen: Mini Kits, BioRobot, EZ-1, QIACube Roche: COBAS, MagNA Pure, MagNA Pure Compact, High Pure
Assay Characteristics Detection Standard PCR: EIA Real-Time PCR: Probes Specific probe hybridization: Taqman, dual-probe FRET, molecular beacons SYBR green dye with melt curve analysis Quantification Method Full standard curve periodically with external controls Full standard curve with every run
CMV Methods Targets Abbott ASR DNA polymerase Qiagen Artus ASR Immediate Early Nanogen ASR Glycoprotein B Roche ASR Other COBAS Amplicor RUO LDT
CMV Survey Data Expressed as log 10 transformed values/ml Year No Labs Mean SD Median Low Value High Value Variance LV/HV^ 07-1 69 3.561 0.618 3.72 1.03 5.51 4.48 07-2 58 4.807 0.787 5.03 1.04 6.99 5.95 07-3 69 2.918 0.665 2.78 <LDL* 4.40 4.40 08-1 95 4.904 0.496 4.94 3.20 5.80 2.60 08-2 86 3.338 0.471 3.26 2.30 5.20 2.90 08-3 94 5.096 0.638 5.19 2.30 6.50 4.20 09-1 139 4.729 0.616 4.84 2.23 5.84 3.61 09-2 154 4.748 0.609 4.87 2.32 6.15 3.83 * 18 labs (850 cps) ^LV: Low value; HV: High value
EBV Methods Abbott ASR Qiagen Artus ASR Nanogen ASR Roche ASR LDT Targets DNA polymerase EBER EBNA Other
EBV Survey Data Expressed as log 10 transformed values/ml Year No Labs Mean SD Median Low Value High Value Variance LV/HV^ 07-1 40 5.850 0.734 5.89 1.70 7.56 5.86 07-2 41 5.804 0.721 5.67 4.07 7.41 3.34 07-3 31 3.312 0.951 3.36 1.85 7.08 5.23 08-1 61 5.189 0.705 5.07 3.60 6.76 3.16 08-2 47 2.569 0.811 2.70 <LDL* 3.89 3.89 08-3 60 3.966 0.570 3.90 2.74 5.10 2.36 09-1 78 4.164 0.586 4.21 1.58 5.23 3.65 09-2 77 4.172 0.578 4.23 1.58 5.15 3.57 * 22 labs (400 cps) ^LV: Low value; HV: High value
BKV Methods Abbott ASR Qiagen Artus ASR Nanogen ASR Roche ASR LDT Targets T Ag L1 Other
BKV Survey Data Expressed as log 10 transformed values/ml Year No Labs Mean SD Median Low Value High Value Variance LV/HV^ 08-1 61 5.152 0.565 5.28 4.10 5.94 1.84 08-2 48 3.753 0.755 3.26 <LDL* 5.44 2.84 09-1 52 4.831 0.741 4.94 3.14 6.53 3.39 09-2 53 4.748 0.609 4.87 2.32 6.15 3.83 * 14 labs (5,750 cps) ^LV: Low value; HV: High value
Comparison of Performance to Assays with International Standards HIV FDA-cleared HCV-both FDA-cleared and non-fda cleared assays
HCV VLS Example
Comparison of SDs from Mean for Assays without and with International Standards Expressed as log 10 transformed values/ml Year CMV EBV BK HCV HIV 07-1 0.618 0.734 0.162 0.360 07-2 0.787 0.721 0.233 0.227 07-3 0.665 0.951 0.212 0.173 08-1 0.496 0.705 0.565 0.163 0.138 08-2 0.471 0.811 0.755 0.182 0.159 08-3 0.638 0.570 0.159 0.130 09-1 0.616 0.586 0.741 0.248 0.132 09-2 0.609 0.578 0.609 0.281 0.120 Mean 0.613 0.707 0.668 0.205 0.180
Comparison of Variances for Assays without and with International Standards Expressed as log 10 transformed values/ml Year CMV EBV BK HCV HIV 07-1 4.48 5.86 0.66 0.88 07-2 5.95 3.34 0.77 0.73 07-3 4.40 5.23 1.05 0.68 08-1 2.60 3.16 1.84 0.92 0.66 08-2 2.90 3.89 2.84 0.92 0.73 08-3 4.20 2.36 0.80 0.66 09-1 3.61 3.65 3.39 0.68 0.73 09-2 3.83 3.57 3.83 0.96 0.57 Mean 4.00 3.88 2.98 0.85 0.71 Variance is difference between lowest and highest value reported
Summary Lack of method standardization Extraction, amplification and detection methods Quantification method Controls/calibrators from various manufacturers/in house developed Large variances in results as compared to assays calibrated against international standards Poor inter-laboratory correlations Patient monitoring by same assay/same lab Mobility of patients Treatment decision points based on VL will vary between institutions
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