VQA HIV DNA Control SOP Version 5.0 HIV DNA Testing 13 March 2012
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1 1. PRINCIPLE 1.1. The Virology Quality Assurance (VQA) Laboratory provides external cell pellet controls for use in the validation of assays that detect HIV proviral DNA HIV seronegative peripheral blood mononuclear cells (PBMC) are generated from source leukocytes purchased from the Ohio Region American Red Cross. The cells are spiked with 8E5/LAV cells that are propagated fresh before each production from a frozen cell stock. 8E5/LAV cells are used because each cell contains one provirus DNA copy per cell. A known number of infected cells can be calculated per cell pellet to assist in evaluating assay sensitivity and performance. The use of blinded controls, which contain either no HIV provirus or 160 copies of provirus per pellet, provides an extraction control that evaluates sample handling techniques This SOP describes the proper use, interpretation, and template setup for the use of VQA copy controls to validate assays performed using the Roche Amplicor HIV-1 DNA Test, version 1.5, the Abbott RealTime HIV-1 DNA assay, or the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test. VQA controls are required for the validation of assays containing VQA-related data (i.e. proficiency testing data) and clinical trial data as outlined by your Clinical Trial Network. The use of Roche Amplicor HIV-1 DNA kit controls must also be included if required by your laboratory s SOP or required by the manufacturer. 2. VQA CONTROLS 2.1. VQA DNA controls can be ordered from the VQA Laboratory using the Reagent Order Form located on the HANC website at Samples are shipped on dry ice and should be maintained at -70 C until extracted. VQA DNA cell pellets are washed cell pellets that are ready for extraction and should be handled in the same manner as patient samples VQA DNA copy controls contain 0, 20, 40 or 80 HIV-1 provirus copies per cell pellet. One-fourth of the extracted pellet is added to the amplification mixture for the Roche Amplicor HIV-1 DNA Test, version 1.5, which equates to a total of 0, 5, 10 or 20 copies of HIV-1 provirus per amplification input which corresponds to the nominal values used for these controls VQA Real-time Blinded Control cell pellets contain 0 or 160 provirus copies per cell pellet. One-fourth of the extracted pellet is added to the amplification mixture for the Roche Amplicor HIV-1 DNA Test, version 1.5, which equates to a total of 0 or 40 copies of HIV-1 provirus per amplification input VQA cell pellets must be thawed prior to use and microfuged for 1 minute to ensure the cell pellet is present and visible. Discard the control if a cell pellet is not visible prior to extraction. Do not freeze/thaw the pellets. Pellets are intended for single use only. Do not stockpile reagents domestic laboratories should not order more than 3 months supply of controls and international laboratories should not order more than 6 months 1
2 supply of controls to permit accurate inventory tracking by the VQA laboratory. 3. PROCEDURE: Roche Amplicor HIV-1 DNA Test, v One set of DNA copy controls must be included in each assay The 0 copy control must be extracted in singleton, amplified in duplicate, and each replicate is detected in singleton, to yield 2 HIV-1 OD values per control (NOTE: internal control (IC) results must also be obtained on all VQA DNA controls, and the interpretation of the IC result will be handled in the same manner as the package insert for sample results) The 5 copy control must be extracted in singleton amplified in duplicate and then each replicate is detected in singleton to yield 2 HIV-1 OD values per control The 10 copy control must be extracted, amplified and detected in singleton to yield 1 HIV-1 OD per control The 20 copy control must be extracted, amplified and detected in singleton to yield 1 HIV-1 OD per control Two Real-time Blinded control pellets must be extracted, amplified and detected in singleton to yield 1 HIV-1 OD per control per pellet. The first 2 blinded control pellets are included for the first 10 patients added to the template. A single additional blinded pellet control must be added after every 10 patients are added to the template Sample Templates: VQA Reagent Setup Roche Amplicor HIV-1 Test, v1.5 (Singleton Testing - No Kit Controls) A VQA 0 copy sample 1 sample 9 sample 16 sample 23 blinded #5 sample 38 sample 45 sample 52 sample 60 sample 67 sample 74 B VQA 0 copy sample 2 sample 10 sample 17 sample 24 sample 31 sample 39 sample 46 sample 53 blinded #8 sample 68 sample 75 C VQA 5 copy sample 3 blinded #3 sample 18 sample 25 sample 32 sample 40 sample 47 sample 54 sample 61 sample 69 sample 76 D VQA 5 copy sample 4 sample 11 sample 19 sample 26 sample 33 blinded #6 sample 48 sample 55 sample 62 sample 70 sample 77 E VQA 10 copy sample 5 sample 12 sample 20 sample 27 sample 34 sample 41 sample 49 sample 56 sample 63 blinded #9 sample 78 F VQA 20 copy sample 6 sample 13 blinded #4 sample 28 sample 35 sample 42 sample 50 sample 57 sample 64 sample 71 sample 79 G blinded #1 sample 7 sample 14 sample 21 sample 29 sample 36 sample 43 blinded #7 sample 58 sample 65 sample 72 sample 80 H blinded #2 sample 8 sample 15 sample 22 sample 30 sample 37 sample 44 sample 51 sample 59 sample 66 sample 73 blinded #10 VQA Reagent Setup Roche Amplicor HIV-1 Test, v1.5 (Singleton Testing - With Kit Controls) A VQA 0 copy sample 1 sample 9 sample 16 sample 23 blinded #5 sample 38 sample 45 sample 52 sample 60 sample 67 sample 74 B VQA 0 copy sample 2 sample 10 sample 17 sample 24 sample 31 sample 39 sample 46 sample 53 blinded #8 sample 68 sample 75 C VQA 5 copy sample 3 blinded #3 sample 18 sample 25 sample 32 sample 40 sample 47 sample 54 sample 61 sample 69 sample 76 D VQA 5 copy sample 4 sample 11 sample 19 sample 26 sample 33 blinded #6 sample 48 sample 55 sample 62 sample 70 sample 77 E VQA 10 copy sample 5 sample 12 sample 20 sample 27 sample 34 sample 41 sample 49 sample 56 sample 63 blinded #9 Kit Pos F VQA 20 copy sample 6 sample 13 blinded #4 sample 28 sample 35 sample 42 sample 50 sample 57 sample 64 sample 71 Kit Neg G blinded #1 sample 7 sample 14 sample 21 sample 29 sample 36 sample 43 blinded #7 sample 58 sample 65 sample 72 Kit Neg H blinded #2 sample 8 sample 15 sample 22 sample 30 sample 37 sample 44 sample 51 sample 59 sample 66 sample 73 Kit Neg 2
3 VQA Reagent Setup Roche Amplicor HIV-1 Test, v1.5 (Duplicate Testing - No Kit Controls) A VQA 0 copy sample 1 sample 5 sample 9 sample 12 sample 16 sample 20 sample 24 sample 28 sample 31 sample 35 sample 39 B VQA 0 copy sample 1 sample 5 sample 9 sample 13 sample 17 blinded #4 sample 24 sample 28 sample 32 sample 36 sample 40 C VQA 5 copy sample 2 sample 6 sample 10 sample 13 sample 17 sample 21 sample 25 sample 29 sample 32 sample 36 sample 40 D VQA 5 copy sample 2 sample 6 sample 10 sample 14 sample 18 sample 21 sample 25 sample 29 sample 33 sample 37 blinded #6 E VQA 10 copy sample 3 sample 7 blinded #3 sample 14 sample 18 sample 22 sample 26 sample 30 sample 33 sample 37 sample 41 F VQA 20 copy sample 3 sample 7 sample 11 sample 15 sample 19 sample 22 sample 26 sample 30 sample 34 sample 38 sample 41 G blinded #1 sample 4 sample 8 sample 11 sample 15 sample 19 sample 23 sample 27 blinded #5 sample 34 sample 38 sample 42 H blinded #2 sample 4 sample 8 sample 12 sample 16 sample 20 sample 23 sample 27 sample 31 sample 35 sample 39 sample 42 VQA Reagent Setup Roche Amplicor HIV-1 Test, v1.5 (Duplicate Testing - With Kit Controls) A VQA 0 copy sample 1 sample 5 sample 9 sample 12 sample 16 sample 20 sample 24 sample 28 sample 31 sample 35 sample 39 B VQA 0 copy sample 1 sample 5 sample 9 sample 13 sample 17 blinded #4 sample 24 sample 28 sample 32 sample 36 sample 40 C VQA 5 copy sample 2 sample 6 sample 10 sample 13 sample 17 sample 21 sample 25 sample 29 sample 32 sample 36 sample 40 D VQA 5 copy sample 2 sample 6 sample 10 sample 14 sample 18 sample 21 sample 25 sample 29 sample 33 sample 37 blinded #6 E VQA 10 copy sample 3 sample 7 blinded #3 sample 14 sample 18 sample 22 sample 26 sample 30 sample 33 sample 37 Kit Pos F VQA 20 copy sample 3 sample 7 sample 11 sample 15 sample 19 sample 22 sample 26 sample 30 sample 34 sample 38 Kit Neg G blinded #1 sample 4 sample 8 sample 11 sample 15 sample 19 sample 23 sample 27 blinded #5 sample 34 sample 38 Kit Neg H blinded #2 sample 4 sample 8 sample 12 sample 16 sample 20 sample 23 sample 27 sample 31 sample 35 sample 39 Kit Neg 4. PROCEDURE: Roche COBAS AmpliPrep / COBAS TaqMan HIV-1 Qual Test, v Kit controls must be included in every run per the manufacturer s requirement One set of DNA copy controls must be included for each batch of 24 samples. A single set of DNA copy controls may be split between two rings of 24 samples if extracted and amplified simultaneously The 0 copy control must be extracted, amplified and detected in singleton The 5 copy control must be extracted, amplified and detected in singleton The 10 copy control must be extracted, amplified and detected in singleton The 20 copy control must be extracted, amplified and detected in singleton A single real-time blinded control pellet must be added after every 8 patients are added to the template. 3
4 5. Sample Templates: HIV DNA ASSAY TEMPLATE FOR THE ROCHE COBAS AMPLIPREP / COBAS TAQMAN HIV-1 QUAL ASSAY VQA HIV DNA CONTROL REQUIREMENTS FOR 24-SAMPLE AND 48-SAMPLE BATCH SIZES 24-Sample Template 48-Sample Template 1 Kit PC 1 Kit PC 25 Kit PC 2 Kit NC 2 Kit NC 26 Kit NC 3 VQA 0 copy 3 VQA 0 copy 27 VQA 5 copy 4 VQA 5 copy 4 VQA 10 copy 28 VQA 20 copy 5 VQA 10 copy 5 Sample 1 29 Sample 19 6 VQA 20 copy 6 Sample 2 30 Sample 20 7 Sample 1 7 Sample 3 31 Sample 21 8 Sample 2 8 Sample 4 32 Sample 22 9 Sample 3 9 Sample 5 33 Sample Sample 4 10 Sample 6 34 Sample Sample 5 11 Sample 7 35 Blinded #3 12 Sample 6 12 Sample 8 36 Sample Sample 7 13 Blinded #1 37 Sample Sample 8 14 Sample 9 38 Sample Blinded #1 15 Sample Sample Sample 9 16 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample blinded #4 21 Sample Sample Sample Sample blinded #2 46 Sample Sample Sample Sample Blinded #2 24 Sample Sample PROCEDURE: Abbott RealTime HIV-1 Qual Assay 6.1. Kit controls must be included in every run per the manufacturer s requirement One set of DNA copy controls must be included for each batch of 24 samples. A single set of DNA copy controls may be split between two rings of 24 samples if extracted and amplified simultaneously The 0 copy control must be extracted, amplified and detected in singleton The 5 copy control must be extracted, amplified and detected in singleton The 10 copy control must be extracted, amplified and detected in singleton The 20 copy control must be extracted, amplified and detected in singleton A single real-time blinded control pellet must be added after every 8 patients are added to the template. 4
5 7. Sample Templates: HIV DNA ASSAY TEMPLATE FOR THE ABBOTT REALTIME HIV-1 QUAL ASSAY VQA HIV DNA CONTROL REQUIREMENTS FOR 24-SAMPLE AND 48-SAMPLE BATCH SIZES 24-Sample Template 48-Sample Template 1 Kit PC 1 Kit PC 25 VQA 5 copy 2 Kit NC 2 Kit NC 26 VQA 20 copy 3 VQA 0 copy 3 VQA 0 copy 27 Sample 19 4 VQA 5 copy 4 VQA 10 copy 28 Sample 20 5 VQA 10 copy 5 Sample 1 29 Sample 21 6 VQA 20 copy 6 Sample 2 30 Sample 22 7 Sample 1 7 Sample 3 31 Sample 23 8 Sample 2 8 Sample 4 32 Sample 24 9 Sample 3 9 Sample 5 33 Blinded #3 10 Sample 4 10 Sample 6 34 Sample Sample 5 11 Sample 7 35 Sample Sample 6 12 Sample 8 36 Sample Sample 7 13 Blinded #1 37 Sample Sample 8 14 Sample 9 38 Sample Blinded #1 15 Sample Sample Sample 9 16 Sample Sample Sample Sample Sample Sample Sample blinded #4 19 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample blinded #2 46 Sample Sample Sample Sample Blinded #2 24 Sample Sample INTERPRETATIONS 8.1. Whenever kit controls are included for testing, they must be deemed valid per the package insert for that kit For all assays, the VQA 0 copy control must be undetectable. For the RA kit, the HIV-1 OD for both replicates must also be <0.2; the IC OD result also must be >=0.2; and for TQ and AQ, the controls must be valid per the package insert For all assays, the VQA 5 copy control is not used for assay validation, but the expectation is that the control results will be detectable >95% of the time. Even though the control result does not invalidate the run, the control result must be valid for the run to be deemed valid For all assays, the VQA 10 copy control must be 100% detectable. For the RA kit, the HIV-1 OD for the control must also be >= 0.8 (the IC OD result can be anything and the control will be considered valid, per the package insert); and for TQ and AQ, the controls must be valid per the package insert. 5
6 8.5. For all assays, the VQA 20 copy control must be 100% detectable. For the RA kit, the HIV-1 OD for the control must also be >=2.000 (the IC OD result can be anything and the control will be considered valid, per the package insert); and for TQ and AQ, the controls must be valid per the package insert VQA real-time control pellets must yield the correct result as defined by the key for that production lot number. For all kits, a positive control result must be detectable; for RA, the HIV-1 OD must also be >= 0.8 (the IC OD result can be anything and the control will be considered valid, per the package insert); and for TQ and AQ, the control must be valid per the package insert. For all kits, a negative control result must be undetectable; for RA, the HIV-1 OD for both replicates must also be <0.2 and the IC OD result must also be >=0.2; and for TQ and AQ the control must be valid per the package insert If any of the VQA control results are invalid, then the run is invalid and the assay must be repeated (For RA only, if the user suspects a detection problem, then an assay may be redetected and deemed valid upon redetection if all the criteria for a valid run are met). For all other repeats due to invalid control results, all samples and controls must be re-extracted, amplified and detected. A new set of real-time blinded controls and DNA copy controls must be used. Contact the VQA if you have any questions regarding how VQA HIV DNA control performance should be interpreted. 9. REAL-TIME BLINDED CONTROL RESULT VERIFICATION 9.1. VQA blinded controls may be validated by use of the Laboratory Data Management System (see LDMS user manual, The blinded pellet code should be logged into the LDMS exactly as it appears on the sample label Failed runs will be tracked by the LDMS. Two consecutive failures or three failures out of 10 runs will result in the user being locked out of the DNA assay module. Users will need to contact the VQA lab (vqa@rush.edu) and LDMS User Support (ldmshelp@fstrf.org) if they are locked out of their assay module Users who do not have access to an LDMS may verify their blinded pellet control results by ing the control results to dna.pcr.pellet@fstrf.org. The user should send their data using the following template included in the text of the 6
7 Dear FSTRF Staff: Please verify the following results for VQA HIV DNA real-time blinded control pellets. The results are as follows: Lab Number: 078 Requested By: Cheryl Jennings Method/Assay: Roche Amplicor HIV-1 DNA Test, v1.5 Date of Request: 10 March 2012 Blinded Pellet ID HIV OD IC OD Result 022d Reactive 022d Reactive 022d Non-Reactive In addition to my blinded pellet results, the results for our VQA HIV DNA Copy Controls are as follows: DNA Copy Control HIV OD IC OD Result 0 copy 0.079, , Non-Reactive, Non-Reactive 5 copy 2.835, , Reactive, Reactive 10 copy Reactive 20 copy Reactive Dear FSTRF Staff: Please verify the following results for VQA HIV DNA real-time blinded control pellets. The results are as follows: Lab Number: 078 Requested By: Cheryl Jennings Method/Assay: Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test Date of Request: 10 March 2012 Blinded Pellet ID HIV CT IC CT Result 022d Reactive 022d Reactive 022d Non-Reactive In addition to my blinded pellet results, the results for our VQA HIV DNA Copy Controls are as follows: DNA Copy Control HIV OD IC OD Result 0 copy Non-Reactive 5 copy Reactive 10 copy Reactive 20 copy Reactive 10. REFERENCES Jackson, J.B., J. Drew, H.J. Lin, P. Otto, J.W. Bremer, F.B. Hollinger, S.M. Wolinsky, The ACTG PCR Working Group, and The ACTG PCR Virology laboratories Establishment of a Quality Assurance Program for Human Immunodeficiency Virus Type 1 DNA Polymerase Chain Reaction Assays by the AIDS Clinical Trials Group. J. Clin. Microbiol. 31: Roche Amplicor HIV-1 DNA Test, version 1.5 package insert. 3/2005. Roche Molecular Systems, Inc. Branchburg, NJ, USA. 7
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