Policy # MI_MD_ADEBR Department of Microbiology. Page Quality Manual

Transcription

1 Department of Microbiology Version: 1.0 CURRENT 1 of 25 Prepared by QA Committee Issued by: Laboratory Manager Revision Date: 10/2/2017 Approved by Laboratory Director: Annual Review Date: 11/1/2018 Microbiologist-in-Chief Uncontrolled When Printed ADENOVIRUS QUANTITATIVE PCR BIO-RAD Introduction:... 2 Specimen Collection, Transport & Storage... 2 Materials, Equipments and Facilities:... 2 Specimen Processing... 3 Procedure:... 4 General Precautions:...4 Build Worklist...4 PCR Set-up:...5 To be loaded by epmotion...7 To be loaded by manual pipetting;...7 BIORAD Detection Area:...7 Import worklist...10 Analysis...15 Interpretation: Reporting Cleaning Quality Control Related Documents References: Record of Edited Revisions (titled as above) on the server prior to use

2 Department of Microbiology Version: 1.0 CURRENT 2 of 25 Prepared by QA Committee Issued by: Laboratory Manager Revision Date: 10/2/2017 Approved by Laboratory Director: Annual Review Date: 11/1/2018 Microbiologist-in-Chief Uncontrolled When Printed Introduction: Adenovirus is a double stranded non-enveloped DNA virus in the family Adenoviridae first isolated in 1950 from adenoid tissue. Adenoviruses is classified into 7 species A to G with several serotypes; causing a wide range of illnesses from colds, diarrhea, eye infections to neurological diseases. Recently, Adenovirus has been seen to cause severe infections in transplant recipients resulting in graft loss. Adenovirus PCR is a qualitative real-time PCR, used for the detection of Adenovirus in sterile body fluids, including plasma. Specimen Collection, Transport & Storage Fluids collected in a sterile container, store at C after collection, if processed within 24 hours; store at -20 o C if processing >24 hours. EDTA blood: plasma should be removed from red cells 4-6 hours after collection. Centrifuge EDTA blood at 10,000 RCF (Relative Centrifugal Force), and remove plasma 4-6 hours after collection. Store plasma at C if processed within 24 hours; store at -20 o C if processing >24 hours. Materials, Equipments and Facilities: Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) BIO-RAD Deep Well TM Real-Time System BIO-RAD Hard-Shel PCR Plates 96-Well WHT/CLR BIO-RAD Microseal B seal Seals BIO-RAD Optical Flat 8-Cap Strips for 0.2ml tube strips BIO-RAD Low-Profile PCR Tubes 8-tube strip, white 96-Well Loading Block (pre-cooled to -20 C) Variable volume Rainin pipettes: 1 to 20 ul, 10 to 200 ul, 100 to 1000 ul Reagents: Altona Adenovirus PCR Kit 1.0: MasterA, MasterB, DNA Internal Control, Adenovirus QS3 as positive control and PCR grade water as a negative control. External Controls: Adenovirus Externa control and a known negative external control to be run in every new shipment. (titled as above) on the server prior to use

3 Version: 1.0 CURRENT 3 of 25 Specimen Processing Please refer to Nucleic Acid Extraction Biomerieux NucliSENS easymag

4 Version: 1.0 CURRENT 4 of 25 Procedure: General Precautions: There must be separate PCR work areas: Clean room Specimen preparation room Powder-free Gloves should only be in use in PCR areas. Change gloves frequently and keep tubes closed whenever possible. Prepare Working 1% sodium hypochloride daily. Specimen Preparation Supplies and equipment must be dedicated to Specimen Prep Area and not used for other activities and never used in Clean Room. Change lab coats and gloves between work areas. Use only Aerosol Resistant Tips (ART) Use only sterile RNase-free, DNAse-free microtubes Thaw components thoroughly at room temperature. PCR work areas (Clean Room and Specimen Preparation Area) benchtops and equipment after each shift. Build Worklist Open the worklist file according to the following path: T:Microbiology>Virology>Bio Rad PCR>Worklist Mastcopy>HZ_CMV_PARVO_ADENO Worklist Password window pops up, click Read only button Scan the samples information

5 Version: 1.0 CURRENT 5 of 25 File > Save as CSV at T:Microbiology>Virology>Bio Rad PCR>Import Worklist File name: Adeno yyyy.mm.dd.run No. eg.adeno_ Save as type: CSV (Comma delimited) Note:Worklist can only be imported as CSV type Click Save button Click OK The following window pops up, click Yes button Close current excel file without any savings. PCR Set-up: Prepare eluate samples including controls in order according to the Adenovirus worksheet.

6 Version: 1.0 CURRENT 6 of 25 In the Clean Room: Change into dedicated clean room gown and gloves, work in Biological Safety Cabinet. Remove the required vials from -20 o C freezer to thaw at room temperature the required number of vials of Adenovirus Master A, and Adenovirus Master B (12 reactions/vial) for your Adenovirus PCR run. Prepare Adenovirus Master Mix in 1.5mL conical (sarstedt) microtube; Number of Test Samples + 2 Controls (Adenovirus QS3&NC) + one extra Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added together to Master B it cannot be frozen again.. Number of Reactions 1 Adenovirus Master A 5 Adenovirus Master B 15 Volume of Master Mix 20 Sample/Control Volume 10 No. of Test reactions Adenovirus Master Mix Adenovirus Master A (µl) Adenovirus Master B (µl)

7 Version: 1.0 CURRENT 7 of 25 To be loaded by epmotion See Pipetting by epmotion Manualfor further loading and programming instructions. To be loaded by manual pipetting; Place 96-well plate onto pre-cooled block Pipette 20 ul prepared Adenovirus Master Mix into each reaction well; Pipette 1.0 ul of internal control into the wells designated for the positive and negative controls Pipette 10 ul PCR grade Water into the well designated for negative control. In the Specimen Processing Area: Pipette 10uL of each sample, QS, Negative control into reaction wells according to the plate map. Mix by pipetting up and down 3 times into the Master Mix. Seal the plate after pipetting is completed. Load plate into the centrifuge carrier and spin at until speed hits 3000 rpm, then hit stop Check reaction wells before loading into Deep Well Real-Time System: ensuring the liquid levels are at the same height and there are no bubbles at the bottom of each reaction well. BIORAD Detection Area: Double click Bio-Rad CFX Manager icon Startup Wizard window pops up

8 Version: 1.0 CURRENT 8 of 25 Make sure Deep Well is selected Under Select run type, click User-defined button Run Set up window pops up, including three tabs-protocol, Plate, and Start Run Click Open Lid button to open the lid Load sealed plate to the block Click Close Lid button to close the lid WARNING! Do NOT manually close the motorized lid Under Protocol tab, select Altona DNA PCR.prcl from Express Load pull-down menu Express Load pulldown menu Open and Close Lid button

9 Version: 1.0 CURRENT 9 of 25 Click Plate tab on the top or click Next button on the bottom right side to load the plate profile Select hz_cmv_parvo_adeno.pltd plate profile from the Express Load pull-down menu Click Start Run tab on the top or click Next button on the bottom right side

10 Version: 1.0 CURRENT 10 of 25 Confirm the following information: Protocol: Altona DNA PCR.prcl Plate: Altona hz_cmv_parvo_adeno.pltd Scan Mode: All Channels If any information above needs to be edited, click Prev button If all information is correct, click Start Run button Save Optical Data File [CT014845] window pops up Change admin to Adeno Save the file in the designated folder: T:Microbiology>Virology>Bio Rad PCR>Save Run Click Save button Import worklist Click Realtime Status tab Select View/Edit Plate from Plate Setup pull-down menu Plate Setup pull-down menu Plate Editor window pops up

11 Version: 1.0 CURRENT 11 of 25 Click Spreadsheet View/Importer button Plate Spreadsheet View window pops up Select HEX from Fluors List pull-down menu

12 Version: 1.0 CURRENT 12 of 25 Click Import button Select Import File window pops up T:Microbiology>Virology>Bio Rad PCR>Import Worklist Select appropriate worklist and click Open, all samples and controls information are imported Click OK button Select all wells by clicking top left side corner

13 Version: 1.0 CURRENT 13 of 25 Type IC as target Name for HEX channel Hit Enter key on the key board Click Well groups button Click top left side corner to select all Type IC and hit Enter key Well Groups Manager window pops up Click Add button Select wells including control wells for Adeno detection (selected wells should be highlighted)

14 Version: 1.0 CURRENT 14 of 25 Change the name Group 1 to Adeno Click OK button Click Ok button from Plate Editor window

15 Version: 1.0 CURRENT 15 of 25 Click Yes button from the popping up window Click Time Status tab from the Run Details window Analysis Choose Adeno from Well Groups pull-down menu Change the channel names from Fluorophore to Target

16 Version: 1.0 CURRENT 16 of 25 Set the threshold line for each target channel (Adeno & IC). Click off the ic leaving adeno on. Drag the threshold over all the flat (negative) reaction. Repeat on the ic channel. Check and mark on the worksheet the result of each sample and controls. A positive PCR is observed if there is a rise or amplification in the Target channel e.g. FAM (Adeno) and HEX (IC). The graph should be exponential and sigmoidal in shape. Conversely a negative PCR is a flat line or no signal. Click Export Select Custom Export Click Export button

17 Version: 1.0 CURRENT 17 of 25 Save as Window pops up Change the File name according to the target and save in the following folder: T:\microbiology\Virology\Bio Rad PCR\Exported Excel Results\ Click Save button The final results with Ct values are exported in Excel sheet Change Starting Quantity to Scientific Notation Print the excel sheet and attach it to the Adeno worksheet Close excel Click OK button

18 Version: 1.0 CURRENT 18 of 25 Click Close button Close Deep Well Real-Time Systim Shut down computer Shut down Biorad CFX 96 Thermocycler.

19 Version: 1.0 CURRENT 19 of 25 Interpretation: Sample ID FAM (Adenovirus) HEX (IC) Interpretation A Positive Negative POSITIVE for Adenovirus* B Negative Positive NEGATIVE for Adenovirus C Negative Negative PCR Inhibition: Re-extract and repeat PCR from extraction * Negative Internal Control readings in HEX (IC) may occur in strongly positive FAM channels. High Adenovirus viral loads in the sample often deplete the amplification/detection materials (eg. nucleotides), leading to reduced or absent Internal Control signals. Reporting NEGATIVE RESULTS: Negative for Adenovirus. Under media on the back of the LIS workcard. PCADE: F6 to add run date Report on the front of the LIS workcard from the keypad select: }ADE- Ctrl-F to finalize the report. POSITIVE RESULTS:. A. Non-Sterile sites: Negative for Adenovirus. This is a research test. Adenovirus PCR Kit, Altona Diagnostics GmbH. Positive for Adenovirus Date the media on the back of the LIS workcard. PCADE: F6 to add run date Enter on the media line the cross point (ct) value: Ct= Report the virus as an isolate in the F7 window. Isolate #: 1 Org.ID. 21ade Open the Isolate Comment window F8.

20 Version: 1.0 CURRENT 20 of 25 Go to the Virology keypad, type V. Select from the keypad: \ADE+ DETECTED by PCR, This is a research test. Adenovirus PCR Kit, Altona Diagnostics Verify the result. Ctrl-F to finalize the report. B. Sterile sites: PRELIMINARY Positive for Adenovirus Date the media on the back of the LIS workcard. PCADE: F6 to add run date Enter on the media line the cross point (ct) value: Ct= Report the virus as an isolate in the F7 window. Isolate #: 1 Org.ID. 21ade Open the Isolate Comment window F8. Go to the Virology keypad, type V. Select from the keypad: \ADE+ DETECTED by PCR, confirmation to follow This is a research test. Adenovirus PCR Kit, Altona Diagnostics Verify the result. Ctrl-P to prelim the report. Call reports to ward/doctor. Order media PRADE for repeat and repeat the sample from extraction to amplification. If Repeat is positive: CONFIRMED Positive Adenovirus. Date the media on the back of the LIS workcard. PRADE: F6 to add run date Enter on the media line PRADE the cross point of the repeat (ct) value: Ct= Open the Isolate Comment window F8, change the previous report confirmation to follow to Confirmed e.g. \ADE+ Verify the result. Ctrl-F to finalize the report. If repeat is negative : DETECTED by PCR, confirmed. This is a research test. Adenovirus PCR Kit, Altona Diagnostics

21 Version: 1.0 CURRENT 21 of 25 NOT CONFIRMED for Adenovirus. Date the media PRADE on the back of the LIS workcard. PRADE: F6 to add run date Isolate #: 1 must be suppressed, change to the corresponding alphabet letter eg. 1(A), 2(B) In front of the LIS workcard type the following: UPDATED REPORT: Previously reported Adenovirus not confirmed. Report in front of the LIS workcard: This is a research test. Adenovirus PCR Kit, Altona Diagnostics GmbH Ctrl-F to finalize the report. Call Updated reports to ward/doctor. Calling Results: Adenovirus DETECTED from sterile body fluids, tissues, and eye fluids/swabs should be called to ward and/or doctor. Cleaning Clean Room: Wipe down with RNAse Away or NucleoClean on paper towel, followed by distilled water, and then 70% alcohol Biological Safety Cabinet Pipettes Bench tops Specimen Preparation Area: Wipe down with Working 1% hypochloride (made daily), followed by distilled water, and then 70% alcohol Biological Safety Cabinet (BSC), pipettes, centrifuge, and bench top. Seal and discard BSC waste Wash racks. Amplification Area: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe, followed by UltraPure water, and then 70% alcohol Wipe Seal & discard reaction microtubes into biohazard waste after each run. Perform the cleaning procedure according the daily maintenance sheet.

22 Version: 1.0 CURRENT 22 of 25 Quality Control Reagent QCs: An External Control (external to Altona Diagnostics) is used to monitor the isolation, amplification and detection procedures. The result must correspond to expected value supplied by the manufacturer. External control positive and negative for Adenovirus should be extracted on easymag and run at with each new lot or shipment of reagent. Daily QCs: Every Run Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition. A Positive Control (QS3) is included and should show a positive signal (amplification) in FAM Channel (Adenovirus). A Negative Control (water) is included and shows a negative reading in FAM Channel (VZV), Texas Red Channel (HSV-1), and Cy5 (HSV-2). Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. a. Do not release results pending resolution of QC failure. b. Inform charge/senior technologist. c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can be released (positive QC material yielded positive result, negative yielded negative result).

23 Version: 1.0 CURRENT 23 of 25 Related Documents Virology Accessioning Manual Biorad Worksheet Nucleic Acid Extraction Biomerieux NucliSENS easymag Pipetting by epmotion Manual Qualitative PCR External Control Log T:\microbiology\Virology\QC statistics\external QC and INVENTORY Logs\Qualitative PCR EXTERNAL QC.xls References: Altona Adenovirus PCR Kit v1.0 Instructions, Altona Diagnostics

24 Version: 1.0 CURRENT 24 of 25 Record of Edited Revisions Manual Section Name: Adenovirus PCR Bio-Rad 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 Added Interpretation Section March 17, 2016 Dr. T. Mazzulli Annual Review August 24, 2016 Dr. T. Mazzulli Updated MSH logo in header. Updated External QC and Daily QC section: Added on each new lot or shipment Updated Threshold Instructions: Set the threshold line for each target channel (Adeno & IC). Click off the ic leaving adeno on. Drag the threshold over all the flat (negative) reaction. Repeat on the ic channel. Updated file paths to acces throughout manual Update related link to new Qualitative PCR excel for QC and inventory logging Modifed centrifugation time of spinning from 2 minutes to when centrifuge reaches 3000rpm. Moved build worklist section to beginning of procedure. Updated reporting of sterile fluids and non-sterile specimens Added note for positive samples with negative internal control in interpretation table. December 9, 2016 Dr. T. Mazzulli

25 Version: 1.0 CURRENT 25 of 25 Number / Item Date of Revision Signature of Approval