Policy # MI_MD_MPXNAT Department of Microbiology. Page Quality Manual

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1 Department of Microbiology Version: 1.0 CURRENT 1 of 20 Prepared by QA Committee Issued by: Laboratory Manager Revision Date: 9/11/2017 Approved by Laboratory Director: Annual Review Date: 11/1/2018 Microbiologist-in-Chief Uncontrolled When Printed TABLE OF CONTENTS Introduction... 2 Specimen Collection, Transport & Storage... 2 Materials, Equipments and Facilities... 3 Procedure... 4 Synchronize Hamilton with Ampliprep...12 Assay Validation...12 Interpretation of Results...12 Reporting Trouble Shooting Data Archiving Best Practice Recommendations Recommendations to Avoid Contamination...17 Quality Control Related Documents Reference Record of Edited Revisions (titled as above) on the server prior to use

2 Department of Microbiology Version: 1.0 CURRENT 2 of 20 Prepared by QA Committee Issued by: Laboratory Manager Revision Date: 9/11/2017 Approved by Laboratory Director: Annual Review Date: 11/1/2018 Microbiologist-in-Chief Uncontrolled When Printed Introduction A major concern surrounding tissue and blood donations from live or cadaveric donors involves the potential of viral infection transmission including that of Human Immunodeficiency Virus (HIV-1) and Type 2 (HIV-2), Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV). These agents are primarily transmitted by exposure to contaminated blood or blood products, exposure to certain body tissues or fluids, by sexual contact or by an infected mother to the newborn child. The cobas TaqScreen MPX Test, v2.0 is a qualitative multiplex test that enables the simultaneous detection and discrimination of HIV RNA, HCV RNA and HBV DNA and the internal Control in a single test of an infected pool or individual plasma donation. The cobas TaqScreen MPX Test, v2.0 uses a generic nucleic acid preparation technique on the cobas Ampliprep Instrument. HIV RNA (HIV- 1 Group M and O RNA,, HIV-2 RNA). HCV RNA and HBV DNA are amplified, detected and discriminated using automated, real time PCR on the cobas TaqMan Analyzer. The test does not discriminate between HIV-1 Group M, HIV-1 Group O and HIV-2. The test also incorporates an Internal Control for monitoring test performance in each individual test as well as the AmpErase enzyme to reduce potential contamination by previously amplified material (amlicon). Discriminatory tests for HIV-1 Group O and HIV-2 are not available from Roche Specimen Collection, Transport & Storage Living donor specimen: Blood collected in EDTA tube(s) may be stored for up to 48 hours at 2-25 o C and up to 72 hours at 2-8 o C Following separation from cells, plasma may be stored at 2-8 o C for up to 7 days, and up to 30 days at <-18 o C with 3 freeze/thaw cycles. Cadaveric blood specimen-edta plasma or serum may be used Specimens from cadaveric donors displaying a straw to pink color are classified as Moderately Hemolyzed and specimens displaying a red to dard-red or brown color are classified as Highly Hemolyzed specimens. Cadaveric blood collected in EDTA or serum tube(s) may be stored for up to 24 hours at 2-30 o C followed by up to 24 hours at 2-25 o C prior to plasma or serum separation from red blood cells. For storage longer than 48 hours, separate the plasma or serum from the red (titled as above) on the server prior to use

3 Version: 1.0 CURRENT 3 of 20 blood cells by centrifugation and remove the separated plasma or serum from the red blood cells prior to storage. Following removal, plasma or serum may be stored at 2-8 o C for an additional 8 days. Alternatively, plasma or serum may be stored at -18 o C for up to 30 days after removal from red blood cells with 3 freeze/thaw cycles. Cadaveric plasma or serum is tested at 1:5 dilution. Do not freeze whole blood. Heparin Plasma is NOT acceptable Materials, Equipments and Facilities Reagents: 1. Cobas TaqScreen MPX Test (CS1-CS4) stored at 2-8 o C magnetic glass particles reagent cassette lysis reagent cassette Multi-reagent cassette Test-specific reagent cassette 2. Cobas TaqScreen MPX/WNV Control Kit stored at 2-8 o C HIV-1M positive control HIV-1 O positive control HIV-2 positive control HCV positive control HBV positive control TaqScreen negative control 3. Cobas TaqScreen Wash Reagent stored at o C 4. Cobas TaqScreen Cadaveric Specimen Diluents Unopened CADV SPEC DIL is stable until the expiration date. Once opened, this reagent is stable for 30 days at 2-8 o C or until the expiration date, whichever comes first. Equipment: Hamilton MICROLAB STAR/STARlet IVD Pipettor

4 Version: 1.0 CURRENT 4 of 20 Cobas Ampliprep Instrument Cobas Taqman Analyzer Pooling, AMPLILINK and Data Management Workstation, Data Station Pipette tips (high volume CO-RE tips (1000mL), filter Waste bags Sample Input tubes (S-tubes) and barcode clips 24 and 32 test tube sample carrier Plate carrier Tip carrier Sample Racks (SK24) SK24 Rack Carrier SPUs SPU racks Reagent Racks Racks of K-tips K-tubes BD Vacutainer Procedure 1. Take out MPX reagent kits and controls from the fridge, and leave at room temperature. 2. If Hamilton STAR was off during the weekend, turn on Hamilton STAR at least 1 minute before turn on the computer! 3. Re-start computers for Pooling Manager, and Data Manager every week day. 4. Perform daily maintenance procedures for HAMILTON and AMPLIPREP. 5. Use Pooling Manager Terminal for the maintenance of Hamilton: a. Click Wizard icon b. Click Maintenance Tab c. Click Daily Maintenance d. Click Start e. Follow instructions to finish daily maintenance procedure

5 Version: 1.0 CURRENT 5 of 20 Perform weekly maintenance every Tuesday. Push Back Up tape into driver every Tuesday morning after the weekly maintenance Turn off Hamilton STAR and computer at the end of the Friday! * Do not touch the red scanner * Clean inside of HAMILTON with de-ionized water and 70%alcohol * Clean the pipette ejector ledge with alcohol 6. Loading of reagents and disposables in AmpliPrep a. Loading of Reagent Cassettes in AmpliPrep Place all CS1 for 8MPX onto a same reagent cassette Place CS2, CS3, CS4 for MPX onto to a separate reagent cassettes Load the reagent cassette containing all CS1 onto position A of the AmpliPrep Load the reagent cassettes containing CS2, CS3, CS4 onto position B,C,D or E of the AMPLIPREP b. Loading of Disposables in AmpliPrep Load enough SPUs in the SPU rack(s) and load the rack(s) onto position J, K or L of the AmpliPrep. Load enough K-tubes onto position M-N of the AmpliPrep. Load enough K-tips onto position O-P of the AmpliPrep. Do NOT unload any partial rack of K-tube or K-tips even the LEDs are green Position M-N are designated for K-tubes, position O-P are designated for K-tips. 7. Prepare Sample rack and S-tube rack a. Using the Work list, match labels of sample tube with the 6mL BD Vacutainer tube b. Add 1200ul plasma of Living Donor samples to the empty 6mL BD Vacutainer tubes c. Add 300ul plasma/serum Cadaveric samples to the 6mL BD Vacutainer tube with 1200 ul Cadaveric diluent d. Centrifuge at 3000 rpm for 5-10 minutes Make sure the sample tube is free of bubbles, clots and flocculates. e. De-cap and place 6mL BD Vacutainer tubes onto 32 test tube sample carrier f. Take out enough clips and S-tubes (4 controls plus the sample number) g. Slightly bend the clip before place it onto the S-tubes rack starting position#1 h. Place S-tubes into clips i. Place S-tube rack onto SK24 Rack Carrier j. De-cap S-tubes 8. Set up MPX NEGATIVE CONTROL and POSITIVE CONTROL racks

6 Version: 1.0 CURRENT 6 of 20 a. Mix controls by inversion at least 3 times, avoiding the creation of bubbles, as specified below: b. Place clean Kleenex paper on Control Working Area c. Remove positive control lids and leave on Kleenex paper. d. Load positive controls in Positive Control Rack Position #1, 2, and 3 with barcode labels visible. Yellow cap positive control is in position#1 Orange cap positive control is in position#2 Red cap positive control is in position#3 CHANGE GLOVES AFTER SETTING UP POSITIVE CONTROL RACK e. Load negative control in negative Control Rack Position #1 with barcode label visible. Pop bubbles from control tubes by lightly tapping with control tube cover Return Control boxes back to fridge after finish loading.

7 Version: 1.0 CURRENT 7 of Pooling Manager Terminal a. Click Wizard Icon b. Make sure Batch Run is selected c. Click Assay/Start b. c. d. Choose MPX v2.0 e. Click Next

8 Version: 1.0 CURRENT 8 of 20 f. Click Pools of 1 g. Follow the instruction to load sample rack h. Click Done button after all samples are loaded f. h.

9 Version: 1.0 CURRENT 9 of 20 i. All required quantities of S-Tubes,SK24 racks,tips, RMEC (roche manufactured external controls) are calculated j. Follow instructions to load S-tube rack, control racks, and pipette tip rack.

10 Version: 1.0 CURRENT 10 of 20 i. k. After tips are loaded, pipetting is automatically started l. All racks are automatically unloaded after the pooling procedure is finished m. Re-cap the S-tubes.Double check that caps are not so tight and properly placed n. Press the ctrl on the keyboard + the logoff button on the lower right corner of the pooling wizard to exit the pooling wizard o. To start a new pooling run, click pooling wizard and click tab Batch Run p. If window is freeze, press Ctrl key and Logoff icon at the same time to active the windows

11 Version: 1.0 CURRENT 11 of Extraction/Amplification/Detection AMPLIPREP USER ID: MSH PASSWORD: Msh12345 Load in AMPLIPREP AMPLIPREP Terminal NO order needs to be manually created. The order is automatically transmitted from Pooling manager to Ampliprep a. In the Amplilink computer, click order icon to check if the order is send to Amliprep. If the order is not sent over, proceed the procedure of Synchronize Hamilton with Amliprep b. Remove S-tube rack from carrier. Load S- tube rack in AMPLIPREP, select start. Check the End time (Time to transfer k-tubes to Taqman) c. Load only FULL K-tube / K-tip rack when required. d. When the run is complete, the indicator light on the Cobas Ampliprep will be orange, and SK24 racks may be removed. 11. Load the SK24 racks directly to the Cobas TaqMan. 12. If the Amplification and detection run does not start automatically, click Start 13. Check finished time. 14. When testing is finished in Cobas TaqMan, the results are automatically transmitted to Data Manager. 15. Print Results from Data Manager (follow instructions) Print Donor Result Report only a. DMS b. Query Tab c. Today d. Search e. Highlight batch, Next f. Confirm any information in Alarm Review g. Donor Review Tab, h. Print 16. Report results in LIS manually. If all sample tests are negative, only check Donor Result Report If there is positive result for patient s sample, check both PCR Batch Report and Donor Result Report 17. Clean Up and Shutdown Procedures a. Check number of reagents left and write in CS1 then cover with parafilm and store in fridge.

12 Version: 1.0 CURRENT 12 of 20 b. Discard used SPU tubes and save clean ones. Synchronize Hamilton with Ampliprep Click Pooling icon Click Amplilink Status tab Under Amplilink workstation, click PCR-LAB-W1-220 Under Current Batches, click the Rack ID Click Synchronize button Assay Validation All controls have to be valid before specimen results are released. All result data are stored in main frame computer HPR 300XR in Rm Interpretation of Results If all controls are valid; Non Reactive: reported as Negative Reactive: reported as Positive. Invalid: repeat once. If still invalid, reported as Indeterminate

13 Version: 1.0 CURRENT 13 of 20 Reporting For Negative Result Result CT Values HIV HBV HCV I C Internal Control Report Comment Non- Reactive ### Valid 8MPX:Negative@RMPx Verify 8MPX Non- Reactive Invalid If there is enough sample for repeating, repeat the test Order 8COM to record initial invalid result After repeating, if the IC is still invalid, result as following: 8MPX: Indeterminate@RMPx If there is not enough sample for repeating, result as following: 8MPX: Indeterminate@RMPx In the comment for 8MPX, type: Not Sufficient Quantity for repeating Verify 8MPX In the comment for 8COM, record the 2nd result Verify 8COM Verify 8MPX Verify 8COM

14 Version: 1.0 CURRENT 14 of 20 For Positive Result Result HBV Reactive HIV Reactive HCV Reactive CT Values Internal Serology Result HIV HBV HCV IC Control ### ### Valid 8HAGX:Negative 8HBC:Positive Or 8HAGX:Positive 8HBC:Positive Or 8HAGX:Positive 8HBC:Negative ### ### Valid ### ### Valid 8TSC: Positive 8HCA: Positive Report 1. 8MPX: HBV DNA Detected 2. Order 8COM in LIS and record HBV CT 1. 8MPX: HIV DNA Detected 2. Order 8COM in LIS and record HIV CT 1. 8MPX: HCV DNA Detected 2. Order 8COM in LIS and record HCV CT Note: If an MPX target is positive and the corresponding serology is negative: a. Review run with senior/charge if feasible 1, report positive MPX if satisfied. b. Repeat only when necessary 2. 1 Evaluate the probability of contamination, specimen mix up, expected positive rate and Ct values. 2 Check the original tube is labelled correctly. If repeat is necessary, then repeat from the original tube if possible.

15 Version: 1.0 CURRENT 15 of 20 Trouble Shooting Problem Internal Control fails on ONE specimen Internal Control fails on a specimen for the second time Pooling manager computer is frozen while loading specimen Any Equipment failures / malfunctions Repeat specimen Solutions Result specimen as: Invalid In result comment: hit F5 to add Likely due to specimen integrity, unable to obtain valid test result after repeated attempts). Verify result. 1. Press Ctrl key and Logoff icon at the same time to active the windows 2. If computer is still frozen, press Ctrl+Alt+Del to restart the pooling manager computer 3. If the computer is still frozen, turned off the Hamilton STAR and computer 4. Manually unload the specimens from the Hamilton STAR 5. Wait for couple of minutes, turn back the Hamilton STAR at least 1 minute before turn on the computer 6. Wait for the log on screen before pressing Ctrl+Alt+Del to log in 7. Restart the pooling procedure Immediately: 1. Inform senior technologist immediately 2. Troubleshoot equipment 3. Notify clients if results will be delayed Ensure to: 4. Document in Analyzer history log: Find error message on Run log in equipment computer. Click on Add note. You can document here steps taken to address error. Print note, initial and file in equipment binder. Roche Hot Line Tel# michael.salem@roche.com

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17 Version: 1.0 CURRENT 17 of 20 Data Archiving The Data Manager must be archived monthly. 1. Retrieve a DVD disk: Verbatim discs 4x DVD+RW (Verbatim cat# ) from a senior technologist. 2. Insert the formatted DVD into the Data manager computer. 3. Right click the Archiving Management icon (B) in the Data Management workstation taskbar. 4. Select Perform Archiving Now (A) from the displayed popup menu. 5. The Archiving Management icon updates to show archiving is in progress. 6. Multiple discs may be needed to complete archiving. 7. Data records that have resided in the PDM database for more than the specified number of daysare automatically archived and purged from the system. Best Practice Recommendations Recommendations to Avoid Contamination 1. Change gloves frequently. This is particularly important for : Loading SPUs and other consumables onto the Ampliprep Opening S-tubes before loading them onto the Hamilton STAR Immediately after handling/opening positive controls After unloading controls from the Hamilton STAR When closing the S-tube caps 2. Use the plastic SPU covers upside down for temporary storage of S-tube caps while pipetting in the Hamilton STAR. Use a new cover on a daily basis. The covers should be used shortly after removing them from the packaging as they are considered contamination free when in packaging. 3. Handle and uncap the positive controls only AFTER the negative control has been loaded. Controls should have a separate and devoted handling area.

18 Version: 1.0 CURRENT 18 of After pipetting is complete: Inspect pools to ensure volumes are consistant Cap with S-tubes in the following order: i. Cap Negative Control ii. Cap all Specimens iii. Cap Positive Control Transfer the Specimens to the Ampliprep first Remove donor tubes Lastly remove and discard controls tubes (change gloves) Quality Control Run External Positive and negative control (previously tested CAP proficiency samples) monthly. CAP and NML provide external proficiency testing. Related Documents PCR QC and Reagent Inventory Log T:\Microbiology\Virology\QC statistics\external QC and INVENTORY Logs\PCR Reagent QC & Inventory Current.xls Reference Roche cobas s 201 System Hardware and Software reference Manual, P/N PDM Software Software Guide,P/N Operator s Manual for Use with small Pools(Pools of 1 and 6) P/N

19 Version: 1.0 CURRENT 19 of 20 Record of Edited Revisions Manual Section Name: Hepatitis B/Hepatitis C/HIV Donor Ampliprep/TaqMan Number / Item Date of Revision Signature of Approval Manual Transferred from Molecular Diagnostics Manual December 2, 2015 Dr. T. Mazzulli Policy # MI/MD/v51 archived Any Equipment failures / malfunctions to March 31, 2016 Dr. T. Mazzulli troubleshooting table. added with monthly archiving procedures. practice recommendations added with monthly archiving procedures. Reporting section updated: -removed automatic repeating when mpx positive, serology negative. Added caveat for these specimens. April 25, 2016 Dr. T. Mazzulli

20 Version: 1.0 CURRENT 20 of 20 Number / Item Date of Revision Signature of Approval