Molecular Diagnostic Tests Manual

Policy # MI/MD/v50 Microbiology Page 1 of 291 Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Issued by: LABORATORY MANAGER Approved by: Laboratory Director Subject Title: Table of Contents Original Date: June 08, 2007 Revision Date: October 26, 2015 Annual Review Date: June 15, 2015 MOLECULAR DIAGNOSTIC TESTS MANUAL TABLE OF CONTENTS Virology Accessioning Manual .................................................................................................................. 3 PCR Order Entry Chart .......................................................................................................................5 Adenovirus Qualitative Real-time PCR by Rotorgene ............................................................................. 14 BK Virus Quantitative PCR Bio-Rad CFX96 .......................................................................................... 23 BK Virus Quantitative PCR by Rotorgene ............................................................................................... 41 Chlamydia trachomatis and Neisseria gonorrhoeae PCR by Cobas® ..................................................... 53 Clostridium difficile toxin B gene PCR by Cepheid GeneXpert .............................................................. 64 Testing for C. difficile toxin from eSwab for Infection Control Purpose .......................................77 Cytomegalovirus Quantitative PCR by Cobas® Ampliprep/TaqMan ...................................................... 78 Cytomegalovirus Qualitative PCR by Rotorgene ..................................................................................... 84 Epstein Barr Virus Quantitative PCR by Rotorgene................................................................................. 94 Enterovirus RNA PCR by Lightcycler ................................................................................................... 106 Hepatitis B/Hepatitis C/HIV Donor Screen NAT PCR by Cobas® s201 Ampliprep/TaqMan ............. 116 Herpes simplex Virus/Varicella-Zoster Virus PCR by Rotorgene ......................................................... 127 Human Metapneumovirus RT-PCR by Rotorgene ................................................................................. 138 Human Metapneumovirus and Parainfluenza RT-PCR Bio-Rad CFX96............................................... 146 Human Papilloma Virus (HPV) PCR by Cobas® 4800 ........................................................................... 171 Influenza A/B & RSV PCR by Focus Diagnostics Simplexa™ ............................................................. 181 Parainfluenza RT-PCR by Rotorgene ..................................................................................................... 189 Parvovirus B19 PCR by Rotorgene ........................................................................................................ 197 West Nile Virus PCR by Rotorgene ......................................................................................................... 207 West Nile Virus Donor Screen NAT PCR by Cobas® s201 Ampliprep/TaqMan .................................... 217 APPENDIX I - Molecular Diagnostic Tests Schedule ........................................................................... 225 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 2 of 291 APPENDIX II - Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG .................................... 226 APPENDIX III - Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG .... 234 APPENDIX IV – Influenza A, B and H1N1 Virus rPCR back up testing by RotorGene ...................... 242 APPENDIX V - Respiratory Synctial Virus (RSV) PCR backup testing by RotorGene ....................... 253 APPENDIX VI - General PCR Precautions and Decontamination Procedures......................................... 262 General PCR Precautions .................................................................................................................262 Decontamination Procedure .............................................................................................................263 WNV Decontamination Procedure ...................................................................................................263 Appendix VII: Creating Standard Curve for Quantitative PCRs............................................................... 265 Appendix VIII - Instructions for Pipetting by epMOTION ...................................................................... 267 Instructions for Pipetting One Test on epMOTION ......................................................................267 Instruction For Pipetting Two Or More Tests On Epmotion: ......................................................272 Appendix IX – RotorGene Worksheet ..................................................................................................... 280 Appendix X – RotorGene Calculations for Reports.................................................................................. 281 Appendix XI – Rotorgene Optical Temperature Verification (OTV) Procedure ....................................... 282 Appendix XII – WNV PCR Procedure Abbreviated * ............................................................................. 286 Record of Edited Revisions .................................................................................................................... 287 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 3 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Virology Accessioning Manual Respiratory specimen accessioning guide: DF A Qiagen/ Light Cycler PCR PCP Entero EasyMag – Altona Rotorgene PCR Targets Specimens Resp Baby WNV Adeno Alpha HSV +VZ BKV CMV EBV Parvo Meta/ Para Flu A/B RSV ‘Virology’ If requested Resp (NP) Adult Simplexa Requests/ Comment If requested If requested Yes ‘Virology’ Yes Resp (NP) Adult BAL BAL BAL/Spt LIS CODE PMH – 14A/14B/ 14C/15A/ 15B (Regardless of comment) UHN Lung Tx / Non-tx Routine tx “CMV” PCP Yes If requested If requested Yes Yes Other *Call ward if suspect virus not indicated -Add Multiplex testing: order “resp virus detection referred out” -Label ‘NICU’ if sent to PHL -For NP MSH ICU add Multiplex testing: order “resp virus detection referred out” -MSH NP: 2 extra aliquots for study -Do not send NPs to PHL for “multiplex” despite comments. Meta/Para done on PMH wards NP only. - Add media in Flu test - multiplex/luminex or other virus requests; order “resp virus detection ref out” Yes Yes PCENT PCWNV PCADE PCHZ PCBKV PCCMV PCEBV PCPAR PCMET/ PCPIN PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 4 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Virology specimen accessioning guide: Qiagen/ Light Cycler PCR EasyMag – Altona Rotorgene PCR Targets Entero Specimens Oral/Mouth Urine Skin Genital Eye swab/Corneal scraping Vitreous/BX Fetal specimen Stool EDTA plasma WNV Adeno ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ ‘Virology’ CMV/EBV/BK CSF ‘Virology’ Peritoneal, Pleural, Pericardial fluids Esoph, colon Bx Non-fetal Tissue/ Fluid/bile LIS CODE ‘Virology’ Simplexa Requests/ Comment Alpha HSV +VZ BKV CMV If requested If requested EBV Parvo Flu A/B RSV Other *Call ward if suspect virus not indicated Yes PHL for EM Yes Yes Yes Yes If requested Yes Yes Yes Yes PHL TGLN (seasonal) Yes If requested Tx pt only WNV (Seasonal) Baby or Tx Yes Yes Yes Yes Yes PHL for EM NML for CMV resistance, HHV Whole Blood Yes Require Approval Require Approval PCEBV PCPAR NML for HHV-6,7 or JC Yes ‘Virology’ ‘Virology’ Yes Yes PCENT Yes PCWNV PCADE PCHZ Yes PCBKV PCCMV For processing instrcutions on specimens being sent out for testing see Send out Manual PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 5 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual PCR Order Entry Chart REQUSITIONS FROM Bay and Algoma Health Centre-Patients blood drawn at LIFEA Bluewater Health LaboratoryNorman Site CML Healthcare Inc (Canadian Medical Laboratories) Credit Valley Hospital (Gamma) Dynacare Laboratories London with UHN MRN (Gamma) Dynacare Laboratories London WITHOUT UHN MRN USE UHN MRN VISIT/WARD Yes, if have UHN MRN use UHN visit/ward NO, if no UHN MRN. Use Last name first name Yes, if have UHN MRN LIFEA use UHN visit/ward REPORTS TO FIELD IN ORDER ENTRY SORT REQ TO FILE HOLDER WHO GETS REPORT LIFEA OTHER PCR UHN (EPR) and LIFEA N BHNS OTHER PCR UHN (EPR) and BHNS N OTHER PCR UHN (EPR) and CMLL Y OTHER PCR UHN (EPR) and CVH Y NO, if no UHN MRN. Use Last name first name BHNS Yes, use UHN MRN use UHN visit CMLL Yes, if have UHN MRN use UHN visit/ward CVH NON UHN/MSH AUTOFAX BUILT Y/N NO, if no UHN MRN. Use Last name first name CVH Yes, use UHN MRN use UHN visit GDMLL OTHER PCR UHN (EPR) and GDMLL Y NO, Last name first name MRN GDMLL Leave Blank OTHER PCR GDMLL Y Leave Blank PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 6 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM (Gamma) Dynacare Laboratories Main Lab Brampton with UHN MRN (Including Huronia Medical Centre Midland, LaSalle Community Health Care in Windsor, 240 Old Penetanguishene Rd. (Tremble) and Highlands Family Health or Dufferin Area Family Health in Orangeville (140 Rollinghills Dr.)) (Gamma) Dynacare Laboratories Main Lab Brampton WITHOUT UHN MRN Georgian Bay General Hospital Midland Site Grand River Hospital Grey Bruce Health ServicesMeaford Site USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY Yes, use UHN MRN use UHN visit DYNAC OTHER PCR UHN (EPR) and DYNAC Y NO, Last name first name MRN DYNAC Leave Blank OTHER PCR DYNAC Y Yes, use UHN MRN use UHN visit GBGHM OTHER PCR UHN (EPR) and GBGHM N KWHC Leave Blank OTHER PCR KWHC N use UHN visit GBHSM OTHER PCR UHN (EPR) and GBHSM Y NO, Last name first name MRN Yes, use UHN MRN Put their lab# (found under barcode) in site NO, Use their MRN top left of label XXXX XX XX No spaces in LIS SORT REQ TO FILE HOLDER WHO GETS REPORT NON UHN/MSH AUTOFAX BUILT Y/N PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 7 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY SORT REQ TO FILE HOLDER WHO GETS REPORT use UHN visit GBRHC OTHER PCR UHN (EPR) and GBRHC Y OTMH Leave Blank OTHER PCR OTMH Y NON UHN/MSH AUTOFAX BUILT Y/N Halton Healthcare ServicesOakville Yes, use UHN MRN Put their lab# (found under barcode) in site NO, Use their MRN top left of label XXXX XX XX No spaces in LIS NO, Last name first name MRN Horizon Health Network Yes, use UHN MRN use UHN visit DECRH OTHER PCR UHN (EPR) and DECRH H Hospital in Common Lab NO, Last name first name MRN HICLM Leave Blank OTHER PCR HICLM Y Humber River Regional Hospital (Church St Site) NO, Last name first name MRN HRRH Leave Blank OTHER PCR HRRH Y Humber River Regional Hospital (Finch Ave Site) NO, Last name first name MRN HRRHF Leave Blank OTHER PCR HRRHF N Lakeridge Hospital NO, Last name first name MRN LHC1 Leave Blank OTHER PCR LHC1 Y NO, Use UNIT# as MRN RTCH LIFEA LifeLabs box LIFEA and RTCH Y NO, Last name first name MRN LIFEA HRRH LifeLabs box LIFEA and HRRH Y Grey Bruce Regional Health Services-Owen Sound Site {GBHS-Owen Sound on req) LifeLabs BUT 50 Charlton Ave Hamilton: May say Renal Transplant Clinic or Nephrology Clinic or just address LifeLabs BUT Humber River Regional Hospital at top. (May also have Dr's name here) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 8 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY LifeLabs NON-TGH doctor NO, Last name first name MRN LIFEA Leave Blank LifeLabs box LIFEA N LifeLabs Orillia NON-TGH doctor NO, Last name first name MRN LIFEO Leave Blank LifeLabs box LIFEO N LifeLabs Orillia requesting Tx doctor (TGH doctor) Yes, use UHN MRN use UHN visit LIFEO LifeLabs box UHN (EPR) and LIFEO N LifeLabs requesting Tx doctor (TGH doctor) Yes, use UHN MRN use UHN visit LIFEA LifeLabs box UHN (EPR) and LIFEA Y LHSCM Leave Blank OTHER PCR LHSCM Y MSH Leave Blank OTHER PCR MSH N Yes, use UHN MRN use UHN visit MLW OTHER PCR UHN (EPR) and MLW N Yes, if have UHN MRN use UHN visit/ward NFLDP OTHER PCR UHN (EPR) and NFLDP London Health Science Centre Microbiology Laboratory Markham Stouffville Hospital Medical Laboratories of Windsor Newfoundland Public Health Laboratory North Bay General Hospital. If together with NBRHC requisition use NBRHC in Report to field. Use their MRN top left of label XXXX XX XX No spaces in LIS Dr Code: JOHNM Put their lab# (top right) in site NO, Last name first name MRN NO, if no UHN MRN. Use Last name first name Yes, if have UHN MRN NO, if no UHN MRN. Use Last name first name NFLDP use UHN visit/ward NBRHC NBRHC SORT REQ TO FILE HOLDER WHO GETS REPORT OTHER PCR NON UHN/MSH AUTOFAX BUILT Y/N N UHN (EPR) and NBCH N NBCH PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 9 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM North Bay Regional Health Centre North York General Hospital USE UHN MRN VISIT/WARD Yes, if have UHN MRN use UHN visit/ward NO, if no UHN MRN. Use Last name first name Yes, if have UHN MRN NBRHC use UHN visit/ward REPORTS TO FIELD IN ORDER ENTRY NBRHC SORT REQ TO FILE HOLDER OTHER PCR NYGCL OTHER PCR WHO GETS REPORT NON UHN/MSH AUTOFAX BUILT Y/N UHN (EPR) and NBRHC N NBRHC UHN (EPR) and NYGCL NO, if no UHN MRN. Use Last name first name NYGCL Notre Dame Hospital Yes, use UHN MRN use UHN visit NDAM OTHER PCR Orillia Soldier's Memorial Hospital Main Laborarory Yes, if have UHN MRN NO, if no UHN MRN. Use Last name first name use UHN visit/wardOSMHO OSMHOLeave Blank OTHER PCR UHN (EPR) and OSMHOOSMHO N Parry Sound (West Parry Sound Health Centre) Laboratory Yes, use UHN MRN use UHN visit WPSHL OTHER PCR UHN (EPR) and WPSHL N Pembroke Regional Hospital Yes, use UHN MRN use UHN visit PRH -Pembroke OTHER PCR UHN (EPR) and PRH N Peterborough Regional Healthcare Yes, use UHN MRN use UHN visit PRHC OTHER PCR UHN (EPR) and PRHC N Queen Elizabeth Hospital, PEI NO, Last name first name MRN QEPEI Leave Blank OTHER PCR QEPEI Y Leave Blank Y NYGCL UHN (EPR) and NDAM N PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 10 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY Queen Elizabeth II Health Science Centre, Halifax NS NO, Last name first name MRN QE2HS Leave Blank OTHER PCR QE2HS Y RVHB OTHER PCR UHN (EPR) and RVHB Y SJRH Leave Blank OTHER PCR SJRH Y SGHGC-CMV PCR SGHGC SGHGC Y TSHBC Y Yes, if have UHN MRN Royal Victoria Hospital Barrie Saint John Regional Hospital, Saint John NB Scarborough General Hospital-Birchmount Campus (Grace) The Scarborough Hospital Birchmount Campus Scarborough General Hospital-General CampusThe Scarborough Hospital Main Campus (x6075) South Bruce Grey Health Center WALKERTON Laboratory South West Health NO, if no UHN MRN. Use Last name first name NO, Last name first name MRN Use Last name first name Use Last name first name Use their MRN WKXXXXXX Dr code: NEWK1 Put their lab# (under barcode) in site Yes, if have UHN MRN NO, if no UHN MRN. Use Last name first name use UHN visit/ward SORT REQ TO FILE HOLDER WHO GETS REPORT NON UHN/MSH AUTOFAX BUILT Y/N RVHB OTHER PCR TSHBC-VRESP TSHBC Leave Blank SGH-CMV PCRTSH-VRESP SGHTSHLeave Blank OTHER PCR SGHTSH YY SBGHW Leave Blank OTHER PCR SBGWH Y SWH OTHER PCR UHN (EPR) and SWH Y use UHN visit/ward SWH PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 11 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY Southlake Regional Health Centre NO, Last name first name MRN YCHN Leave Blank use UHN visit/ward SJRE Yes, if have UHN MRN St John's Rehab Hospital Toronto (Part of Sunnybrook) NO, if no UHN MRN. Use Last name first name Yes, if have UHN MRN St Joseph's Hospital GuelphRehab St Mary's General Hospital Kitchener St. Joseph's General Hospital, Lab, Elliot Lake Sunnybrook Health Science Centre Study Dr Anita Rachlis Sunnybrook Health Science Centre Microbiology NO, if no UHN MRN. Use Last name first name Yes, use UHN MRN Yes, use UHN MRN SJRE use UHN visit/ward SJHG use UHN visit/ward use UHN visit/ward SORT REQ TO FILE HOLDER WHO GETS REPORT OTHER PCR YCHN N OTHER PCR UHN (EPR) and SJRE N OTHER PCR UHN (EPR) and SJHG N Leave Blank SJHG Leave Blank STMHK OTHER PCR SJGEL OTHER PCR UHN (EPR) and STMHK UHN (EPR) and SJGEL NON UHN/MSH AUTOFAX BUILT Y/N N N NO, Last name first name MRN SHSCR Leave Blank OTHER PCR SHSCR N NO, Last name first name MRN SHSC Leave Blank OTHER PCR SHSC Y Sunnybrook Health Science Centre/ ACCAPELA Study NO, use last name: ACCAPELA SRCAC Leave Blank OTHER PCR SRCAC Y Thunder Bay Regional Hospital Yes, use UHN MRN TBRHS OTHER PCR UHN (EPR) and TBRHS Y Toronto Rehab Institute Yes, use UHN MRN Leave Blank OTHER PCR TRI (EPR) N (EPR) use UHN visit/ward use UHN visit/ward PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 12 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual REQUSITIONS FROM USE UHN MRN VISIT/WARD REPORTS TO FIELD IN ORDER ENTRY Trillium Health Centre NO, Last name first name MRN TRHC Leave Blank OTHER PCR TRHC Y Yes, use UHN MRN use UHN visit/ward WMRHN OTHER PCR UHN (EPR) and WMRHN N WOHCB Leave Blank OTHER PCR WOHCB Y WOHCE Leave Blank OTHER PCR WOHCB Y WOHCB use UHN visit/ward WRH OTHER PCR UHN (EPR) and WRH Western Memorial Regional Hospital (Western Health) William Osler Health CentreBrampton William Osler Health CentreEtobicoke NO, Last name first name MRN NO, Last name first name MRN Yes, if have UHN MRN Windsor Regional Hospital Laboratory NO, if no UHN MRN. Use Last name first name WRH Leave Blank SORT REQ TO FILE HOLDER WHO GETS REPORT NON UHN/MSH AUTOFAX BUILT Y/N N PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 13 of 291 Stool Preperation: Stool specimens being processed in house for virology PCR should be processed in the following manner: 1. Fill a clear small tube with 1.5ml VTM 2. Touch the tip of a wooden stick 3mm into the stool sample and inoculate in the VTM prepared. 3. Vortex the sample until well emulsified. 4. Centrifuge the sample in the Eppindorf 5417C at 10 000 rcf for 3 minutes. 5. Transfer the supernatant into a sample cryovial for extraction by easymag. Sputolysin Procedure: Sputolysin reagent is a concentrate of dithiothreitol (DTT) in phosphate buffer used in the isolation of pathogens from mucoid sputum, BAL and other fluids. 1. Dilute 1ml of Sputolysin Reagent in a conical tube with 9ml of sterile dH20. This solution is stable for up to 48hrs at 2-8°C 2. Suspend specimen with an equal volume of Sputolysin in a microcentrifuge tube by vortexing for 30 seconds. 3. Incubate at room temperature for 15 minutes. 4. Centrifuge at 5000rpm for 5 minutes and remove supernatant for use. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 14 of 291 Adenovirus Qualitative Real-time PCR by Rotorgene I. Introduction Adenovirus is a double stranded nonenveloped DNA virus in the family Adenoviridae first isolated in 1950 from adenoid tissue. Adenoviruses is classified into 7 species A to G with many 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 thedetection of Adenovirus in sterile body fluids, including plasma. II. Specimen Collection and Transport Fluids collected in a sterile container, store at C after collection, if processed within 24 hours; store at -20oC 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 with 24 hours; store at -20oC if processing >24 hours. III. Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG GENERAL PRECAUTIONS: There must be separate PCR work areas:Clean room and Specimen preparation area  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 Preparation 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 15 of 291 Materials, Equipments and Facilities: Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System with experiment template: Adenovirus 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor with Locking Ring 0.1 mL reaction microtubes Tubes and Caps Variable volume Rainin pipettes: 1 to 20 uL, 10 to 200 uL, 100 to 1000 uL (assigned to designated areas) Reagent: Altona RealStar Adenovirus-PCR Kit 1.0: MasterA, MasterB, Internal Control, Adenovirus Quantitative Standards 1, 2, 3, 4 (QS1, QS2, QS3, and QS4), PCR Grade Water External Control: Adenovirus Positive, Adenovirus Negative, to be extracted and run in this order on once amonth, or if QC failure occurs. IV. Procedure: Prepare eluate samples in order according to the Adenovirus worksheet. Add the following Adenovirus Quantitative Standard and NC Water to the worksheet in the following order: Adenovirus QS3, Negative-Water. PCR Set-up: In the Clean Room: Change into dedicated clean room gown and gloves, work in Biological Safety Cabinet. Remove from -20oC 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; to load on the Eppendorf epMotion. Prepare the Master Mix reactions required: Number of Test Samples + 1 Standard (Adenovirus QS3&NC) + one 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 Adenovirus Master A Adenovirus Master B Volume of Master Mix Sample/Control Volume 1 5 15 20 10 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Number of Test reactions 1 2 3 4 5 6 7 8 9 10 11 12 Page 16 of 291 Adenovirus Master Mix Adenovirus Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Adenovirus Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 See Eppendorf epMotion Manual for further loading and programming instructions. Manual Loading: Load one reaction microtube (0.1mL) for each sample/control. Pipette 20uL prepared Adenovirus Master Mix into each reaction microtube. Pipette 1.0uL of internal control into each reaction microtube designated for the 1 Standards (Adenovirus QS3 and NC water). Specimen Processing Area: Pipette 10uL of Sample/Standard into reaction microtube. Mix by pipetting 3x up and down into the master mix. Cap each segment after pipetting is completed. Check reaction microtube segments before loading into the rotor, ensure the liquid levels are at the same height, there are no bubbles at the bottom of the microtubes, and the lid is tightly in place. Load reaction microtubes in 72-Well blue rotor, fill empty rotor spaces with blank reaction microtubes. Attach the Locking Ring. Load the rotor in the Rotor-gene 6000. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 17 of 291 RotorGene Detection Area: Switch ON computer, switch ON RotorGene. FOR THE BLUE ROTORGENE ONLY. Select clinical icon; password “msh”. Enter. Open “Rotor-Gene 6000 series software 1.7” icon Wait for “Initializing machine…..”  New Run Window  Select Adenovirus PCR (or CMV this is the same pcr experiment template)  Select New    Select 72 Well Rotor (blue) Check the box for “Locking Ring Attached” Select “Next” button. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual            Page 18 of 291 Enter operator intials. In Notes enter RotorGene colour (i.e. Red or Blue) Reagent Lot Number. Make sure the reaction volume is 30uL. Select Next. Temperature Profile window .Select Next. Rotor must be loaded. New Run Wizard window: Select Start Run. The RotorGene will start. Save As window appears; the run filename is given with the default template. Save in: My Documents in Adenovirus folder for the current year: e.g. Adenovirus 20150326(1) (assay name yyyymmdd run#) Select Save. RotorGene will start running. Edit Samples window appears. Enter sample numbers including Adenovirus QS3. Define the sample types according to the chart below. Name Sample Type Samples Adenovirus QS3 NC Water Unknown Positive Negative “Finish”. Click on “Name On”. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc  P r e s s Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 19 of 291 Analysis  Profile Program reads: Run has completed.  Analyze raw data by clicking each channel one at a time: Green channel (Adenovirus) and Yellow channel (IC). Target Channel Report Dye Adenovirus Internal Control Green Yellow FAM JOE  Perform Raw Green (AdenovirusTarget) Channel. Select All Off.  Select sample water (NC) and Adenovirus QS3. Select Autoscale.  Compare each sample curve to the NC-water curve and the Adenovirus QS3 one at a time, then proceeding to the next sample. A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worksheet.  Select Named On.      Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Green. Select Show. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Select Reports. Select Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Quantitation (Full Report)  Maximize report window. Check that only the positives that you have identified, from the raw data analysis, are positive (i.e. have a crossing threshold ct value).  Print all pages for first channel. Print pages 2-5(or 4) for the second channel.  Close Report window.  Perform Raw Yellow (Internal Control) Channel. Select Name On. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 20 of 291  All samples, including Adenovirus QS3, and NC water, should have a good amplification curve; all should be positive in the Yellow IC Channel; with the exception of samples with high ct values (ct <20.00) in the Adenovirus Target Green Channel.      Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Yellow. Select Show. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Select Reports. Select Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Quantitation (Full Report)  Print all pages for first channel. Print pages 2-5(or 4) for the second channel.  Close Report window.  Note: Cycling A. Yellow report: if there is message of “NEG (Multi Ct)” for any specimen, you must adjust the threshold. This indicates the threshold has crossed the curve at multiple points. The threshold can only cross a curve at one point.  Save changes to Adenovirus yyyy-mm-dd. Select Yes.  Shut down the computer, and switch off the Rotor-Gene. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual V. Page 21 of 291 Reporting Adenovirus Not detected: no ct value. Report in LIS using the keypads: Virology Worklist > Viral PCR worklist > Select from the reporting keypads: ADENegative for Adenovirus. This is a research test. RealStar Adenovirus Kit v1.0. Altona Diagnostics Inc. Under media PCADE. F6 to enter the date tested. Finalize the result (Ctrl+F) Adenovirus Detected: with ct value. Report Adenovirus as an isolate in the isolate window (F7). Isolate #: 1 Org.ID. 21ade Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Adenovirus Detected. This is a research test. RealStar Adenovirus Kit v1.0. Altona Diagnostics Inc. Verify all in the Isolate window Under media “PCADE” F6 to enter the date tested. And the ct value. VI. Quality Control Reagent QCs: An External Control (external to Altona Diagnostics) is used to monitor the isolation, amplification and detection procedures beginning of every month. The result must correspond to expected value supplied by the manufacturer. Adenovirus Positive and Adenovirus Negative External controls should be extracted on the easyMag once a month. Record external QC results in the T drive, Virology folder, QC folder. Daily QCs: Every run: Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition. Adenovirus QS3 Standard is included and shows a positive reading in Green Channel. Quantitative Standards must meet valid quantitative parameters. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 22 of 291 A Negative Water Control is included and shows a negative reading in Green Channel Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in: Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs VII References Realstar Adenovirus PCR Kit v1.0 Instructions, Altona Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 23 of 291 BK Virus Quantitative PCR Bio-Rad CFX96 I. Introduction: BK virus is a Polyomavirus, first isolated in 1970 from kidney transplant receipient. The virus is commonly reactivated in immunocompromised transplant patients. BK virus in renal transplant recipients is associated with nephropathy, in bone marrow transplant with haemorrhagic cystitis. Monitoring and treatment of BK virus reactivation in transplant patients is vital graft survival II. Collection and Transport Urine collected in a sterile container, store at C after collection, if processed within 24 hours; store at -20oC 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 with 24 hours; store at -20oC if processing >24 hours. III. Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG 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. Materials, Equipments and Facilities:  Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG)  Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8)  BIO-RAD CFX96 Deep WellTM Real-Time System  BIO-RAD Hard-Shel®PCR Plates 96-Well WHT/CLR  BIO-RAD Microseal ®‘B’ seal Seals PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual      Page 24 of 291 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 Reagent: Altona RealStar BKV-PCR Kit 1.0: MasterA, MasterB, Internal Control, BKV Quantitative Standards 1, 2, 3, 4 (QS1, QS2, QS3, and QS4), PCR Grade Water External Control: BKV High Positive, BKV Negative, BKV Low Positive, to be extracted and run in this order on once a week, or if QC failure occurs. IV. Procedure: Prepare samples in the order according BKV worksheet. Add the following controls to the Worksheet: BKV Quantitative Standards 1, 2, 3, 4 (QS1, QS2, QS3, and QS4), PCR Grade Water PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) Remove the required vials of BKV Master A and Master B (12 reactions/ vial) from the freezer and thaw them at room temperature A. To be loaded by epMotion   Prepare BKV Master Mix in Sarstedt 1.5 mL conical microtube; Prepare the number of Master Mix reactions required: Number of Test samples + 5 Controls + extra one Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added to Master B it cannot be frozen again. Number of Reactions BKV Master A BKV Master B Volume of Master Mix Sample/Control Volume . No. of Test reactions 1 2 3 4 5 1 5 15 20 10 BKV Master Mix BKV Master A (µl) 5 10 15 20 25 BKV Master B (µl) 15 30 45 60 75 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 6 7 8 9 10 11 12 30 35 40 45 50 55 60 Page 25 of 291 90 105 120 135 150 165 180 See Eppendorf epMotion Manual for further loading and programming instructions. B. To be loaded by manual:     Place 96-well plate onto pre-cooled block Pipette 20 uL prepared BKV Master Mix into each reaction well; Pipette 1.0 uL of internal control into the wells designated for the five BKV Controls (BKV QS1,QS2,QS3,QS4), PCR Water Control NTC; Pipette 10 uL PCR Water into the well designated for NTC. In the Specimen Processing Area:  Pipette 10uL of each sample, QS, NTC into reaction wells. Mix by pipetting 3 times up and down into the Master Mix.  Seal the plate after pipetting is completed.  Check reaction wells before loading into CFX96 Deep Well Real-Time System: ensure the liquid levels are at the same height, and there are no bubble at the bottom of each reaction well.  Double click Bio-Rad CFX Manager icon  Startup Wizard window pops up PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Make sure CFX96 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  Load sealed plate to the block  Click Close Lid button  WARNING! Do NOT manually close the motorized lid Page 26 of 291 to open the lid to close the lid PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 27 of 291 Under Protocol tab, select Altona DNA PCR.prcl from Express Load pull-down menu Express Load pulldown menu Open and Close Lid button  Click Plate tab on the top or click Next button side to load the plate profile  Select BKV.pltd plate profile from the Express Load pull-down menu on the bottom right PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Start Run tab bottom right side  Confirm the following information:  Protocol: Altona DNA PCR.prcl Plate: Altona BKV.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 “BKV”  Save the file in the designated folder: on the top or click Next button Page 28 of 291 on the T:Microbiology>Virology>Bio Rad CFX96 PCR>Save Run>DNA PCR 2015  Click Save button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 29 of 291 Build Worklist  Open the worklist file according to the following path: T:Microbiology>Virology>Bio Rad CFX96 PCR>Worklist Mastcopy>BKV Worklist  Password window pops up, click Read only button  Scan the samples’ information  File > Save as CSV at T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist>DNA PCR Worklist 2015>BKV File name: BKV yyyy.mm.dd.run No. eg. Metapara2015.04.07.1 Save as type: CSV (Comma delimited) Note:Worklist can only be imported as CSV type  Click Save button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 30 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click OK  The following window pops up, click Yes button  Close current excel file without any savings. Import wordlist  Click Realtime Status tab  Select View/Edit Plate… from Plate Setup pull-down menu Plate Setup pull-down menu  Plate Editor window pops up  Click Spreadsheet View/Importer button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Plate Spreadsheet View window pops up  Select HEX from Fluors List pull-down menu  Click Import button Page 31 of 291 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 32 of 291 Select Import File window pops up T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist>DNA PCR Worklist 2015 Click top left side corner to select all wells  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  Type IC as target Name for HEX channel  Hit Enter key on the key board  Click Well groups button Type IC and hit Enter key PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 33 of 291  Well Groups Manager window pops up  Click Add button  Select wells including control wells for BKV detection (selected wells should be highlight)  Change the name Group 1 to BKV  Click OK button  Click Ok button from Plate Editor window PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Yes button from the popping up window  Click Time Status tab Page 34 of 291 from the Run Details window PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 35 of 291 Analyze results after the PCR is completed  Choose BKV from Well Groups pull-down menu  Change the channel names from Fluorophore to Target  Set the threshold line for each target channel  Check if the QC parameters meet requirement Control Parameter M Slope PCR Efficiency R square (R2) Valid Values -3.00 to -3.74 85% to 115% >0.98 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Export  Select Custom Export…  Select Starting Quantity  Click Export button  Save as Window pops up  Change the File name according to the target and save in the following folder:  Click Save button Page 36 of 291 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  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 BK worksheet  Close excel  Click OK button  Click Close button  Close CFX96 Deep Well Real-Time Systim  Shut down computer Page 37 of 291 End of Shift Cleaning: Perform cleaning protocol as outlined below. 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. EasyMAG , Lysis Rack, and BioHit Multi-channel Pipette: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe, followed by UltraPure water, and then 70% alcohol Wipe 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 38 of 291 V. BKV Reporting: BKV Not detected: Specimen has no ct value. Report in LIS using the keypads: Virology Worklist > BK PCR worklist > Select from the reporting keypads: >BKJC }BKNegative for BK virus DNA. This is a research test. BKV PCR Kit v1.0. Astra Diagnostics Inc. Under media PCBKV. F6 to enter the date tested. Finalize the result (Ctrl+F) BKV Detected: Specimen with ct value. Report quantitation “copies of DNA/mL” Report BKV as an isolate in the isolate window (F7). Isolate #: 1 Org.ID. 62BKV Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Greater than 10 copies (>10) of DNA/mL. Select the keypad: \BK+ Turn off the reporting keypad. Ctrl + A or Ctrl + K Enter the copies, in scientific number format, in the space provided, remove extra spacing. Example: 3.20E+04 copies of DNA/mL. This is a research test. BKV PCR Kit v1.0, Astra Diagnostics Inc. Verify all in the Isolate window Less than 10 copies (<10) of DNA/mL. Select: \BK10 Under media “PCBKV” F6 to enter the date tested. If reporting Less than 10 copies (<10) of DNA/mL, enter the actual copy value. Interim (Ctrl + L) the report. Second technologist will check transcription in LIS and Finalize Ctrl + F the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 39 of 291 VI. 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 for hMPV A, hMPV B, PIV1/3, and PIV 2/4 should be extracted on the on the easyMag and run once a month and/or with each new lot.     a. b. c. d. e. f. g. h. i. j. Daily QCs: Every Run Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition. A Positive Control is included and shows a positive reading in FAM channels (hMPV B and PIV 1/3) Channel or a positive reading in Cy5 Channels ( hMPV A and PIV 2/4) A Negative Control is include and shows a negative reading in both FAM channels (hMPV B and PIV 1/3) Channel and Cy5 Channels ( hMPV A and PIV 2/4) Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 40 of 291 References: RealStar hMPV RT-PCR Kit v1.0, altona Diagnostics RealStar PIV RT-PCR Kit v1.0, altona Diagnostics cdc.gov/Human Parainfluenza Viruses (HPIVs) (/parainfluenza/index.html Emerging Infectious Disease Journal Volume 14, Number1-January 2008 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 41 of 291 BK Virus Quantitative PCR by Rotorgene I. Introduction BK virus is a Polyomavirus, first isolated in 1970 from kidney transplant receipient. The virus is commonly reactivated in immunocompromised transplant patients. BK virus in renal transplant recipients is associated with nephropathy, in bone marrow transplant with haemorrhagic cystitis. Monitoring and treatment of BK virus reactivation in transplant patients is vital graft survival II. Specimen Types Urine collected in a sterile container, store at C after collection, if processed within 24 hours; store at -20oC 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 with 24 hours; store at -20oC if processing >24 hours. III. Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG GENERAL PRECAUTIONS: There must be separate PCR work areas:Clean room and Specimen preparation area  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 Preparation 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. IV. Materials, Equipments and Facilities: Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System with experiment template: BK Virus PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 42 of 291 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor with Locking Ring 0.1 mL reaction microtubes Tubes and Caps Variable volume Rainin pipettes: 1 to 20 uL, 10 to 200 uL, 100 to 1000 uL (assigned to designated areas) Reagent: Altona RealStar BKV-PCR Kit 1.0: MasterA, MasterB, Internal Control, BKV Quantitative Standards 1, 2, 3, 4 (QS1, QS2, QS3, and QS4), PCR Grade Water External Control: BKV High Positive, BKV Negative, BKV Low Positive, to be extracted and run in this order on once a week, or if QC failure occurs. V. Procedure: Prepare eluate samples in order according to the BKV worksheet. Add the following BKV Quantitative Standards and NTC Water to the worksheet in the following order: BKV QS4, BKV QS3, BKV QS2, BKV QS1, NTC (Non-Template Control) Water. PCR Set-up: In the Clean Room: Change into dedicated clean room gown and gloves, work in Biological Safety Cabinet. Remove from -20oC freezer to thaw at room temperature the required number of vials’ of BKV Master A, and BKV Master B (12 reactions/vial) for your BKV PCR run. Prepare BKV Master Mix in 1.5mL conical (sarstedt) microtube; to load on the Eppendorf epMotion. Prepare the Master Mix reactions required: Number of Test Samples + 5 Standards (4QS&NTC) + one 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 BKV Master A BKV Master B Volume of Master Mix Sample/Control Volume 1 5 15 20 10 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Number of Test reactions 1 2 3 4 5 6 7 8 9 10 11 12 Page 43 of 291 BKV Master Mix BKV Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 BKV Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 See Eppendorf epMotion Manual for further loading and programming instructions. Manual Loading: Load one reaction microtube (0.1mL) for each sample/control. Pipette 20uL prepared BKV Master Mix into each reaction microtube. Pipette 1.0uL of internal control into each reaction microtube designated for the 5 Standards (BKV QS4, QS3, QS2, QS1, and NTC). Specimen Processing Area: Pipette 10uL of Sample/Standard into reaction microtube. Mix by pipetting 3x up and down into the master mix. Cap each segment after pipetting is completed. Check reaction microtube segments before loading into the rotor, ensure the liquid levels are at the same height, there are no bubbles at the bottom of the microtubes, and the lid is tightly in place. Load reaction microtubes in 72-Well blue rotor, fill empty rotor spaces with blank reaction microtubes. Attach the Locking Ring. Load the rotor in the Rotor-gene 6000. RotorGene Detection Area: Switch ON computer, switch ON RotorGene. FOR THE BLUE ROTOGENE ONLY. Select clinical icon; password “msh”. Enter. Open “Rotor-Gene 6000 series software 1.7” icon Wait for “Initializing machine…..” PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 44 of 291  New Run Window  Select BKV PCR  Select New    Select 72 Well Rotor (blue) Check the box for “Locking Ring Attached” Select “Next” button. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual            Page 45 of 291 Enter operator intials. In Notes enter RotorGene colour (i.e. Red or Blue) Reagent Lot Number. Make sure the reaction volume is 30uL. Select Next. Temperature Profile window .Select Next. Rotor must be loaded. New Run Wizard window: Select Start Run. The RotorGene will start. Save As window appears; the run filename is given with the default template. Save in: My Documents in BK Virus folder for the current year: e.g. BK Virus 20150326(1) (assay name yyyymmdd run#) Select Save. RotorGene will start running. Edit Samples window appears. Enter sample numbers including Quantitative Standards (BKV QS4, QS3, QS2, QS1, NTC) and concentrations. Define the sample types according to the chart below. Name Samples BKV QS4 BKV QS3 BKV QS2 BKV QS1 NC Water Sample Type Given concentration Copies/uL *Conversion factor x250 Enter Concentration 1.00E+01 1.00E+02 1.00E+03 1.00E+04 x250 x250 x250 x250 2.50E+03 2.50E+04 2.50E+05 2.50E+06 Copies/mL Unknown Standard Standard Standard Standard NTC (No template control) *conversion factor is based on the extraction sample volume and eluate volume *Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL) Sample volume (mL) *Result (copies of DNA/mL) = Result (copies/uL) x 50(uL) 0.200 (mL)   Under Given Conc. Format: Select 1.23E+05 (2 decimal points expressed as an exponent). Units: Select copies/mL  Press “Finish”. Click on “Name On”. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 46 of 291 Analysis  Profile Program reads: Run has completed.  Analyze raw data by clicking each channel one at a time: Green channel (BKV) and Yellow channel (IC). Target Channel Report Dye BKV Internal Control Green Yellow FAM JOE  Perform Raw Green (BKV Target) Channel. Select All Off.  Select sample water (NTC) and BKV QS4, QS3, QS2, QS1. Select Autoscale.  Compare each sample curve to the NTC-water curve and the BKV QSs’ one at a time, then proceeding to the next sample. A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worksheet.  Select Named On.       Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Green. Select Show. Calculate Automatic Threshold. Select OK. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Verify the Quantitative Standard Curve on the Green (FAM) Channel. The following parameters must be met, this information can be found on page one of the Green Channel Quantitation –Full Report or on the screen in the Green Channel Graph. Control Parameter M Slope PCR Efficiency R square (R2) Valid Values -3.00 to -3.74 85% to 115% >0.98 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 47 of 291 If the quantitative parameters are not valid, it must be brought to the attention of the senior/and or charge. External Controls must be valid, if not valid, it must be brought to the attention of the senior/and or charge. Patients results must not be released. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 48 of 291  Select Reports. Select Cycling A Green (Target) or Quantitation Analysis Cycling A Yellow (Internal Control) depending on what channel you are analyzing.  Select Quantitation (Full Report)  Maximize report window. Check that only the positives that you have identified, from the raw data analysis, are positive (i.e. have a crossing threshold ct value).  Print all pages for Green (Target-FAM) channel. Print pages 2-5(or 4) for the Yellow (Internal Control-JOE) channel.  Close Report window.  Perform Raw Yellow (Internal Control) Channel. Select Name On.  All samples, including BKV QS1-4, and NTC water, should have a good amplification curve; all should be positive in the Yellow IC Channel; with the exception of samples with high ct values (ct <20.00) in the BKV Target Green Channel.       Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Yellow. Select Show. Calculate Automatic Threshold. Select OK. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Select Reports. Select Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Quantitation (Full Report)  Print all pages for first channel. Print pages 2-5(or 4) for the Yellow IC channel.  Close Report window.  Note: Cycling A. Yellow report: if there is message of “NEG (Multi Ct)” for any specimen, you must adjust the threshold. This indicates the threshold has crossed the curve at multiple points. The threshold can only cross a curve at one point.  Save changes to BKV yyyy-mm-dd. Select Yes.  Shut down the computer, and switch off the Rotor-Gene. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VI. Page 49 of 291 Reporting BKV Not detected: Specimen has no ct value. Report in LIS using the keypads: Virology Worklist > BK PCR worklist > Select from the reporting keypads: >BKJC }BKNegative for BK virus DNA. This is a research test. BKV PCR Kit v1.0. Astra Diagnostics Inc. Under media PCBKV. F6 to enter the date tested. Finalize the result (Ctrl+F) BKV Detected: Specimen with ct value. Report quantitation “copies of DNA/mL” Report BKV as an isolate in the isolate window (F7). Isolate #: 1 Org.ID. 62BKV Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Greater than 10 copies (>10) of DNA/mL. Select the keypad: \BK+ Turn off the reporting keypad. Ctrl + A or Ctrl + K Enter the copies, in scientific number format, in the space provided, remove extra spacing. Example: 3.20E+04 copies of DNA/mL. This is a research test. BKV PCR Kit v1.0, Astra Diagnostics Inc. Verify all in the Isolate window Less than 10 copies (<10) of DNA/mL. Select: \BK10 Under media “PCBKV” F6 to enter the date tested. If reporting Less than 10 copies (<10) of DNA/mL, enter the actual copy value. Interim (Ctrl + L) the report. Second technologist will check transcription in LIS and Finalize Ctrl + F the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 50 of 291 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. BKV High Positive and BKV Low Positive External controls should be extracted on the easyMag once a week. Record external QC results in the T drive, Virology folder, QC folder. Daily QCs: Every run: Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition. BKV QS1, QS2, QS3, QS4 Standard is included and shows a positive reading in Green Channel. Quantitative Standards must meet valid quantitative parameters. A Negative NTC (No template control) Control is included and shows a negative reading in Green Channel Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in: Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 51 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Summary Information: BKV PCR Number of Reactions BKV Master A BKV Master B Volume of Master Mix Sample/Control Volume Name Sample Type 1 5 15 20 10 Given concentration Copies/uL *Conversion factor x250 Enter Concentration Copies/mL Samples Unknown BKV QS4 Standard 1.00E+01 x250 2.50E+03 BKV QS3 Standard 1.00E+02 x250 2.50E+04 BKV QS2 Standard 1.00E+03 x250 2.50E+05 BKV QS1 Standard 1.00E+04 x250 2.50E+06 NC Water NTC (No template control) Control Parameter M Slope PCR Efficiency R square (R2) Valid Values -3.00 to -3.74 85% to 115% >0.98 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 52 of 291 VIII. References RealStar BKV PCR Kit v1.0 Instructions, altona Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 53 of 291 Chlamydia trachomatis and Neisseria gonorrhoeae PCR by Cobas® I. Introduction Chlamydia are gram-negative, non-motile bacteria consisting of four reported species. Chlamydia trachomatis (CT) is the most frequently reported bacterial sexually transmitted disease, and the second most leading cause of sexually transmitted disease worldwide. CT can cause a variety of diseases in men and has severe consequences for women if left untreated. It also can cause severe complications to pregnant women and their child. Neisseria gonorrhoeae are gram-negative diplococci and the causative agent of gonorrhoeae. In women the primary site of infection is the endocervix, and can cause conjunctivitis in infants with infected mothers. The Cobas® 4800 CT/NG is an in vitro nucleic acid amplification test for the qualitative detection of Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) in patient specimens. The Cobas® 4800 CT/NG assay includes targets for all fifteen of the major Chlamydia trachomatis serotypes, and both wild type and variant sequences of Neisseria gonorrhoreae. The test utilizes amplification of target DNA be the Polymerase Chain Reaction (PCR) followed by nucleic acid hybridization for the detection of CT and NG DNA in a variety of specimens including, endocervical swabs, vaginal swabs, and urine samples collected in Cobas® PCR media. II. Specimen Collection and Processing a. Cobas® PCR Female Swab Kit  For endocervical, cervical and vaginal swab specimens  Transport at 2-30ºC. Endocervical specimens can be stored at 2-30ºC for up to 12 months, and cerivcal/vaginal swabs can be stored at 2-30ºC for 90 days once samples have been stabilized in Cobas® PCR media.  Endocervical swab and vaginal swab specimens containing a SINGLE swab in the cobas® PCR Media tube can be directly processed on the cobas® 4800 System.  A properly collected endocervical swab and vaginal swab specimen should have a single swab with the shaft broken at the scoreline. Swab shafts which are broken above the scoreline will appear longer than normal and may also be bent over to fit into the cobas® PCR Media tube. This can produce an obstruction to the system which may cause the loss of test results. In the event that a swab specimen has an improperly broken shaft, remove the swab prior to sample processing on the cobas x 480 instrument.  Do not process endocervical and vaginal swab specimens that appear bloody or have a dark brown color. Compare the swab against 5% (v/v) whole blood standard. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 54 of 291 b. Cobas® PCR Urine Sample Kit  Transport at 2-30ºC. Urine specimens can be stored at 2-30ºC for up to 12 months once the specimens have been stabilized in Cobas® PCR media.  Do not process urine specimens that appear bloody or have a dark brown color. Compare the urine against 0.35% (v/v) whole blood standard. *Note: Specimens in Cobas® PCR media must be brought to room temperature for at least 30 minutes before loading on to the cobas x 480 instrument. III. Procedure Reagents: a) Cobas® 4800 System Sample Preparation Kit - 240/960 test kits  Magnetic glass particles (MGP)  Elution buffer (EB) b) Cobas® 4800 System Wash Buffer Kit – 240/960 test kits  Wash Buffer c) Cobas® 4800 CT/NG Amplification/Detection Kit – 240/960 test kits  Master Mix (CT/NG MMX)  Manganese Solution (CT/NG Mn) d) Cobas® 4800 System Control Diluent Kit e) Cobas® 4800 CT/NG Controls Kit  Positive Control  Negative Control  Internal Control f) Reagent Reservoirs – 50mL and 200 mL containers g) CO-RE pipette tips h) Plate carrier i) Tip carrier j) Sample and reagent racks *Note: All reagents except CT/NG Master Mix and CT/NG Manganese solution must be brought to room temperature before loading on the cobas x 480 instrument. CT/NG Master Mix and CT/NG Manganese solutions must be removed from 2-8ºC just prior to loading on the cobas x 480 instrument. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 55 of 291 Sample Processing: 1. Turn on the system as the following sequence: cobas z 480; Heater/Shaker unit ; cobas x 480 instrument 2. When the left orange light on the z 480 turns to solid green, turn on the computer workstation. 3. Remove Cobas® Sample Preparation Kit and System Preparation Kit reagents from fridge, and allow reagents to come to room temperature 4. Logon to the cobas 4800 software User ID: LABOPERATOR Password: 00labOPR 5. Perform instrument maintenance: a. Select Overview tab and check maintenance status of the cobas x 480 instrument.  If weekly maintenance is due, click Run Weekly Maintenance.  If daily maintenance is due, click Run Daily Maintenance. b. Follow the online instructions. 6. Start new run a. After the completion of the maintenance, in the overview tab, click New Run Tab on the right hand vertical bar b. The following window pops up. Select test type: CT/NG workflow. c. d. Type run name (optional).ie: CTNG 2012.05.29-1 Click OK. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 56 of 291 7. Refer to the following figure while you are loading the deck: E-plate Holder Tip Rack Holder Dynamic Loading AD-plate Holder Plate Carrier Track 1-6 Tip Rack Holder Tip Rack Carrier Track 11-16 Sample Loading Area Track 17-34 Tip Rack Carrier Track 35-40 Reservoirs Reagent Track Track 48-50 51 8. Load samples a. Decap samples. b. Place samples on corresponding carrier. c. Insert sample carriers on autoload tray when the LEDs for the designated track (1734) are flashing. d. Click Load Samples PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 57 of 291 9. Load consumables a) Place listed consumables on appropriate carriers.  Both tip carriers are fully filled and barcodes facing the right hand side  Ensure deepwell plate is loaded flatly and properly –barcode facing the right hand side  Ensure microwell plate is loaded flat and properly (barcode facing away from you) b) Insert carriers on autoload tray. c) Click Load Consumables. 10. Load reagents a) 200 mL reagent reservoir carrier  Load Wash Buffer on 200 mL reagent reservoir carrier as indicated in the wizard (scan-scan-pour).  Insert carrier on autoload tray.  Click Load Reagents. b) 50 mL reagent reservoir carrier PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 58 of 291  Load reagents on 50 mL reagent reservoir carrier as indicated in the wizard.(scan-scanpour).  vortex MGP for 10s prior to scan  Insert carrier on autoload tray.  Click Load Reagents. c) Reagent carrier  Take out CT/NG MMX and CT/NG Mn from the clean room  Open reagent vials and load them on reagent carrier as indicated in the wizard.  Change gloves after uncapping positive control  Insert carrier on autoload tray.  Click Load Reagents. 11. Start sample preparation run a) Click Start Run. b) The sample preparation starts. c) Check the timer in the wizard PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 59 of 291 12. Unload and seal micro plate. a) Click Sample Prep Results to review the results of the sample preparation. b) Click Unload. c) Seal the micrwell oplate sealing foil applicator as indicated on screen.  Be sure not to touch the cover with your fingers as it may affect detection. Hold the sides of the plate and use the smoothing applicator provided to properly seal.  The plate must be transferred from the cobas x 480 to the cobas z 480 within 90 minutes of completion d) Click NEXT 13. Load mirowell plate on cobas z 480 analyzer. a) Press load button on the cobas z 480 analyzer. b) Place the sealed microwell plate into the extended loader. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 60 of 291 c) Press load button again. d) The amplification and detection run starts automatically. e) Check the timer in the wizard. 14. Remove used reagents, samples, and deepwell plate from the cobas x 480 instrument. 15. Review and accept results. a) Click Show Results. b) Review and accept results in Results work area.  Do NOT accept FAILED results c) Select results and click  to print the results report. 16. Unload the cobas z 480 analyzer. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 61 of 291 IV. Interpretation of Results All assay and run validation is determined by the Cobas® 4800 software. Controls must be run with each sample run and valid results must be obtained for both the positive and negative control in order for sample results to be displayed. Cobas® 4800 CT/NG Test Interpretation CT POS, NG POS Specimen is positive for both CT and NG DNA CT NEG, NG NEG Neither CT or NG DNA could be detected CT POS, NG NEG or CT NEG, NG POS Specimen is positive for CT DNA Invalid or Failed Results V. Specimen is positive for NG DNA Do not accept the result from cobasX480. Refer to the trouble shooting. Troubleshooting X3: Error Clot Detected 1. Check the sample for clots. 2. If swab is present, votex 30 seconds and remove swab from the sample. 3. Rerun the sample X4: Error Processing of the sample was aborted due to one or more pipetting error(s) 1. Make sure that the sample must have a minimum volume of 1.0mL. 2. If swab is present, remove it. Internal Control fails on ONE specimen Repeat specimen Internal Control fails on a specimen for the second time Result specimen as: Invalid In result comment: hit F5 to add comment “@SPIN” “Likely due to specimen integrity, unable to obtain valid test result after repeated attempts)”. Verify result. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VI. Page 62 of 291 Reporting Positive report: * Chlamydia Detection – Urine by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Urine by Cobas 4800 CT/NG Assay Positive * Chlamydia Detection – Urine by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Urine by Cobas 4800 CT/NG Assay Negative * Chlamydia Detection – Cervical by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Cervical by Cobas 4800 CT/NG Assay Positive Chlamydia Detection – Cervical by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Cervical by Cobas 4800 CT/NG Assay Negative Chlamydia Detection – Semen by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Semen by Cobas 4800 CT/NG Assay Positive Chlamydia Detection – Semen by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Semen by Cobas 4800 CT/NG Assay Negative Negative report: * Chlamydia Detection – Urine by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Urine by Cobas 4800 CT/NG Assay * Chlamydia Detection – Cervical by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Cervical by Cobas 4800 CT/NG Assay Negative Positive Negative Positive Negative Positive Negative Negative Negative Negative PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual * Chlamydia Detection – Semen by Cobas 4800 CT/NG Assay * N. gonorrhoeae Detection – Semen by Cobas 4800 CT/NG Assay VI. Page 63 of 291 Negative Negative Quality Control One Positive Control and one Negative Control must be included in each run. Run external control (Accurun 341)) first week of each month and before a new reagent is put in service and after the instrument is calibrated or services. Result filed in Reagent Lot Binder. If any of these two controls fails, the test is invalid. Should inform Senior/Charge technologists and repeat testing. External proficiency testing is provided by CAP and QMP-LS. Maintenance QC: VII. Reference Package insert for Roche 4800 CT/NG Assay and Operators Manual PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 64 of 291 Clostridium difficile toxin B gene PCR by Cepheid GeneXpert I. Introduction The Cepheid Xpert C. difficile Assay, performed on the Cepheid GeneXpert® Dx System, is a qualitative in vitro diagnostic test for rapid detection of toxin B gene sequences from unformed (liquid or soft) stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). The test utilizes automated real-time polymerase chain reaction (PCR) to detect toxin gene sequences associated with toxin producing C. difficile. The Xpert C. difficile Assay is intended as an aid in the diagnosis of CDI. Concomitant culture is necessary only if further typing or organism recovery is required. II. Specimen Collection and Processing Collect unformed stool specimen in a clean Starplex container, and send to the Virology laboratory as soon as possible. Stool collected in Enteric Transport Medium, or in SAF is not suitable for this assay. The specimen is stable for up to 5 days when stored at 2–8°C. Alternatively, specimens can be kept at room temperature (20 – 30 °C) for up to 24 hours. Eswab rectal samples may be used for testing for Infection Control purposes upon request by Infection Control Practioners. III. Materials & Equipment  GeneXpert Dx System  Vortex mixer  Dry swab for transfer of the specimen, such as the swab found in the Cepheid Sample Collection Device 900-0370 (Copan Venturi Transystem® Culture) or the Copan Dual Swab and Transport System.  Disposable transfer pipettes.  Xpert C. difficile kit – each kit contains 10 or 120 Xpert C. difficile cartridges and buffer PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 65 of 291 Each Xpert C. difficile Assay Cartridge with integrated reaction tubes contain: Bead 1 (freeze-dried)  Polymerase  dNTPs  BSA (bovine serum albumin)  Probe Bead 2 (freeze-dried)  Primers  Probes  BSA Bead 3 (freeze-dried)  Sample Processing Control (SPC) non-infectious sample preparation control spores Reagent 1 (Sodium Hydroxide) Reagent 2 (Tris Buffer, EDTA, surfactants) Xpert C. difficile reagent pouch  Sample Reagent (Guanidinium thiocyanate, surfactants) IV. Procedure GeneXpert Dx System i) GeneXpert Cartridge Preparation 1. The disposable single-use GeneXpert DX cartridge holds the samples and reagents that you want to process in the GeneXpert DX System. Do not reuse spent cartridges. 2. Store new GeneXpert DX cartridge at 2 – 28 °C. The cartridge and reagents are stable for up to 7 days after the package has been opened. 3. The test must be started within 30 minutes of adding reagents to the cartridge. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 66 of 291 4. Use one cartridge and one sample reagent vial for each stool sample that needs to be tested. Label the cartridge with the corresponding sample ID barcode. Do not hold the cartridge from the reaction tube. 5. Place a dry swab which came with the Cepheid Sample Collection Device into the unformed stool sample. The swab does not need to be completely saturated. 6. Insert the swab into the elution vial containing the Sample Reagent. Note: Use sterile gauze to minimize the risk of contamination. 7. Hold the swab by the stem near the rim of the vial, lift the swab a few millimeters from the bottom of the tube and push the stem against the edge of the vial to break it. Make sure the swab is short enough to allow the cap to close tightly. 8. Close the lid and vortex at high speed for 10 seconds. 9. Open the cartridge lid. Using a clean transfer pipette, transfer the entire contents of the Sample Reagent into the “S” chamber of the Xpert C. difficile Assay cartridge. 10. Close the cartridge lid. Figure 1. Xpert C. difficile cartridge (top view) ii) Assay Method: 1. Turn on the computer, and then turn on the GeneXpert Dx instrument. Log into windows using the password “cphd”. 2. On the Windows® desktop, double-click the GeneXpert Dx shortcut icon. 3. Log on to the GeneXpert Dx System software using your user name and password, for example (User Name: admin1, Password: admin1). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 67 of 291 4. The Database Management dialog box appears on top of the GeneXpert Dx System window once the system starts up. Click “NO” in the Database Management dialog box if you do not want to perform any database management tasks. 5. If a test archive is overdue, the Test Archive Reminder dialog box appears. If you do not want to archive click “No” and if you do want to archive click “Yes”. 6. In the GeneXpert Dx System window, click Create Test. The Scan Sample ID Barcode dialog box appears. Scan the Sample ID barcode using the barcode scanner or you can manually enter the sample ID by clicking “Manual Entry”. Type in the Sample ID into the Manually Patient ID Barcode Entry dialog box that appears. 7. Scan the barcode on the Xpert C. difficile Assay cartridge. The Create Test window appears. Using the barcode information, the software automatically fills in the boxes for the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and Expiration Date. 8. Click Start Test. In the Create Test dialog box that appears. In the Check Status Window, the selected instrument module progress changes to Waiting. 9. Open the instrument module door with the blinking green light and load the cartridge. 10. Close the door. The test starts and the green light stops blinking. 11. When the test is complete the instrument module door unlocks and the green light turns off. Wait until the system releases the door lock before opening the module door and removing the cartridge. 12. Dispose of the used cartridge in an appropriate specimen waste container. 13. Once testing is complete the report is automatically printed. To view the result, in the GeneXpert DX System window, click View Results on the menu bar. The View Results window appears. 14. Click View Test. The Select Test To Be Viewed dialog box appears. Select the test of interest and click OK. The results of the selected test appear in the View Results window. 15. The View Results window contains three tabs: Results, Errors, and Support. The Results tab displays the information for a test such as the Patient ID, Sample ID, Assay name and the test Result. The Errors tab list the errors encountered during the test process. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 68 of 291 16. Monthly Maintenance Procedure: a. Disinfect the instrument surfaces 1. Dampen a paper towel with 10% bleach solution and wipe the instrument surface thoroughly with the paper towel. 2. Wait 10 minutes 3. Dampen a paper towel with 70% alcohol solution and wipe the instrument thoroughly with the paper towel. b. Disinfect the cartridge bay interior 1. Dip a swab into 10% bleach solution. Press the swab against the inside wall of the container to remove excess solution. 2. Open the instrument module door. 3. Wipe the surfaces inside the cartridge bay with the swab. Do not touch the slit on the I-CORE module into which the cartridge reaction tube is inserted. 4. Wait 10 minutes 5. Dip a new swab into 70% alcohol solution. Press the swab against the wall of the container to remove excess solution. 6. Wipe the same surface with the new swab. 7. Repeat steps 5 and 6 two times. 8. Close the instrument module door. c. Disinfecting the plunger rod 1. In the GeneXpert DX System window, click Maintenance on the menu bar. The Maintenance window appears. 2. On the Maintenance menu, click Plunger Maintenance. The Plunger Maintenance dialog box appears. 3. In the Module table, select the module you want to clean, and then click Clean or select Clean All to clean all modules simultaneously. The Plunger Cleaning dialog box then appears. 4. Follow the directions in the Plunger Cleaning dialog box, then click OK. In the Plunger Maintenance dialog box, the Clean button changes to Move Up (if you clicked Clean All button, it changes to Move Up All). In the instrument, the plunger rod in the selected module (or all modules if you clicked Clean All button) lowers into the cartridge bay. 5. Dip a number of swabs in the 10% bleach solution. Press the swabs against the inside wall of the container to remove excess solution. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 69 of 291 6. Wipe the plunger rods with the swabs. Use a fresh swab for each plunger rod. 7. Wait 5 minutes 8. Dip a number of swabs into 70% alcohol solution. Press the swabs against the inside wall of the container to remove excess solution. 9. Wipe the plunger rods with the swabs. Use a fresh swab for each plunger rod. 10. Repeat steps 8 and 9 two times. 11. In the Plunger Maintenance dialog box, click Move Up (or Move Up All). The plunger rod moves back into its resting position. 12. Click Close to dismiss the Plunger Maintenance dialog box. d. Clean fan filters 17. The GeneXpert DX instrument needs to be recalibrated annually or after 2000 test per instrument module, whichever comes first. The system monitors the number of tests since last calibration. To check whether the system requires calibration: 1. In the Maintenance window, look at the ICORE Starts Since Cal column. On the Maintenance menu, click Module Reports. The Module Reports dialog box appears. 2. Check the calibration date. If calibration is required contact the Cepheid Technical Support to schedule a calibration. IV. Assay validation Each test includes a Sample Processing Control (SPC) and Probe Check Control (PCC): 1. Sample Processing Control (SPC) — ensures the sample was correctly processed. The SPC contains spores of Bacillus globigii in the form of a dry spore cake that is included in each cartridge to verify adequate processing of the sample bacteria. The SPC verifies that lysis of C. difficile bacteria and a spore have occurred, if the organism is present, and verifies that specimen processing is adequate. Additionally, this control detects specimenassociated inhibition of the real-time PCR assay. The SPC should be positive in a negative sample and can be negative or positive in a positive sample. The SPC passes if it meets the validated acceptance criteria. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 70 of 291 2. Probe Check Control (PCC) — Before the start of the PCR reaction, the GeneXpert Dx System measures the fluorescence signal from the probes to monitor bead rehydration, reaction-tube filling, probe integrity and dye stability. Probe Check passes if it meets the assigned acceptance criteria. V. Interpretation of Results The results are interpolated by the GeneXpert Dx System from measured fluorescent signals and embedded calculation algorithms and will be shown in the View Results window. Possible results are: 1. Toxigenic C. difficile POSITIVE   Toxin producing C. difficile target DNA sequences are detected. The toxin producing C. difficile target(s) have Cts within the valid range and endpoint above the minimum setting.  SPC — NA (not applicable); SPC is ignored since C. difficile target amplification may compete with this control  Probe Check – PASS; all probe check results pass. 2. Toxigenic C. difficile NEGATIVE with no Critical Threshold (ct) or Endpoint   C. difficile target DNA sequences are not detected. Toxin producing C. difficile targets not detected.  SPC — PASS; SPC has a Ct within the valid range and endpoint above the endpoint minimum setting.  Probe Check — PASS; all probe check results pass. 3. Toxigenic C. difficile NEGATIVE with Critical Threshold (ct) or Endpoint (INDETERMINATE)     C. difficile target DNA sequences are detected but not sufficient to trigger a POS result. The GeneXpert test report gives a non-zero numeric value under ct or >10 under EndPt for Toxin B yet the Analyte Result is NEG LIS will trigger a ?END: SIG although test is “NEG” in the test result window to notify of an indeterminate result. All such stools should be frozen (7 vials) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 71 of 291 4. INVALID  Presence or absence of C. difficile target DNA cannot be determined  SPC — FAIL; SPC target result is negative and the SPC Ct is not within valid range and endpoint below minimum setting.  Probe Check — PASS; all probe check results pass.  The sample was not properly processed or PCR was inhibited.  Inform Virology Charge/ Senior and repeat the test according to the following retesting procedure below. 5. ERROR  Presence or absence of C. difficile cannot be determined.  Toxin producing C. difficile targets — NO RESULT  Probe Check — FAIL*; one or more of the probe check results fail.  If the probe check passed, possible causes for the error include: the reaction tube was filled improperly; a reagent probe integrity problem was detected; or the maximum pressure limits exceeded the acceptable range.  Inform Virology Charge/ Senior and repeat the test according to the following retesting procedure below. 6. NO RESULT  Indicated that insufficient data were collected, the presence or absence of C.difficile cannot be determined.  Possible causes include the operator stopping the test will it was in progress.  Inform Virology Charge/ Senior and repeat the test according to the following procedure below. retesting PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 72 of 291 Retesting Procedure The test should be repeated if an “INVALID”, “ERROR” or “NO RESULT” result was obtained. To repeat a test within 3 hours of an “INVALID/ERROR/NO RESULTS” result: 1. Transfer the remaining contents from the S Chamber of the current Xpert C. difficile Assay cartridge to a new Sample Reagent Vial using a disposal transfer pipette. 2. Vortex and add the entire contents of the Sample Reagent to Chamber S of a new Xpert C. difficile Assay cartridge. 3. Close the lid and start new test 4. Follow LIS Instructions below VI. Reporting for “INVALID”, “ERROR” or “NO RESULT” To repeat a test after 3 hours of an “INVALID” result: 1. Repeat the test with a new swab sample. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VI. Page 73 of 291 Reporting From Xpert C. difficile Assay Result LIS Codes Result Comments Negative ?END: NSG ?NAP1: NEGATIVE ?CLD: NEG Positive ?END: SIG ?NAP1: SIG or NEG ?CLD: ?DETECTED Auto resulting TEST Comment: }NCTB Negative “Negative - No Clostridium difficile toxin B gene detected by Cepheid Xpert C. difficile/Epi assay. This assay has a negative likelihood ratio between 0.03-0.06. A second test does NOT significantly increase the sensitivity or change the negative likelihood ratio. (Ref: Eur J Clin Microbiol Infect Dis. 2013 Jan;32(1):97-9) ISOLATE Comment: “Clostridium difficile toxin B gene DETECTED by PCR using Cepheid Xpert C.difficile/Epi assay. Note: Strain designation is provided for Infection control reasons only. 1. In test window, pick ‘P’ from keypad 2. Chose ‘1’ or ‘D’ from keypad - ‘1’ will generate: ~^clodn-; (C.diff pos, nap-1 neg) - ‘D’ will generate: ~^clodn+; (C.diff pos, nap-1 pos) 3. Verify Isolate, Accept “YES” Pop Up Comment Clodn+/- - ~\GXPT Clodn+/- - ~\Cdif 4. Verify, Finalize, View & Save 5. Notify ward/ICP, 6. Add media FRZ, freeze 4 vials PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Indeterminate These will print out as NEG but have a endpoint >10. ?END: SIG ?NAP1: NEGATIVE ?CLD: NEG 1. In test window, pick ‘P’ from keypad. 2. Chose ‘I’ from keypad Page 74 of 291 ISOLATE Comment: “INDETERMINATE for C.difficile toxin B gene by PCR using Cepheid Xpert C.difficile/Epi Assay. This result indicates either C. difficile toxin B gene at low concentrations or a false positive result. Clinical correlation is required. If you have any questions, please contact the Medical Microbiologist on call. 3. Accept “YES” Pop Up Comment cloptb - ~\Ceph cloptb - ~\Cptb cloptb - ~\Cdif “INVALID”, “ERROR” or “NO RESULT” 4. Verify, Finalize all tests, View & Save 5. Notify ward/ICP, freeze 7 vials ?END: ERROR ?NAP1: ERROR ?CLD: ERROR 1. To add repeat test media, under the CLD test, click in the CLDT media and press “R” in the keypad. This will drop “CLDRP” repeat media. 2 .DO NOT remove any significant flags in Test Tab OR any results until you receive the repeat result. 3. Once the second result is available , Report accordingly 4. Should the repeat test give another Errot/Invalid/No result, DO NOT SET UP AGAIN; Report as: M- Not processed }Due to interfering Upon repeat, if the second result is: NEGATIVE 1.Remove the significant flag so it will not be posted as abnormal. 2.Under all tests, delete “ERROR”. In ?CLD Use keypad 1 }NCDB Negative. 3.Finalize all test ?END,?NAP1 and CLD. POSITIVE 1.DO NOT remove the significant flag. 2.Follow POSITIVE reporting instructions. 3.Finalize all test ?END,?NAP1 and CLD. INDETERMINATE 1.DO NOT remove the significant flag 2.Follow INDETERMINATE reporting instructions 3.Finalize all test ?END,?NAP1 and CLD. INVALID, ERROR, NO RESULT 1. Remove the significant flag. 2. Delete “ERROR” and use keypad M Not Processed} DUE to interfering 3.Finalize all test ?END,?NAP1 and CLD. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 75 of 291 *Any ?ERROR comment in the CLD test window will trigger a “significant” flag (even after saving and finalizing result). Delete any ?ERROR left in the CLD test window before finalizing result. Phone positive result to ward (in-patient) or physician (Out-patient), and also phone to Infection Control Practitioner as per Isolate Notification and Freezing Table QPCMI15003. Referred Result Previous Result Negative within 7 days LIS Code }PNEG PrevNeg TEST Comment “This patient has tested negative for C. difficile toxin B gene within the past week. This assay has a negative likelihood ratio between 0.03-0.06. A second test does NOT significantly increase the sensitivity or change the negative likelihood ratio. (Ref: Eur J Clin Microbiol Infect Dis. 2013 Jan;32(1):97-9) If your patient has NEW, ONGOING or WORSENING signs/symptoms since the last test that suggest C difficile infection, please contact the Microbiologist-on-call to arrange repeat testing.” Positive within 15 days }CLD+ PrevPos Indeterminate within 7 days }IND+ PrevIND+ “This patient has tested positive for C. difficile toxin B gene within the past 15 days. Do not repeat C. difficile toxin testing unless the patient has received a full course of treatment (14 days) and has developed recurrent signs/symptoms of C. difficile. Repeat C. difficile toxin testing is not indicated in patients who have clinically responded to treatment.” “This patient has tested indeterminate for C.difficile toxin B gene within the past week. Indeterminate results indicate either detection of low levels of C.difficile toxin B gene or a false positive result. If your patient has signs/symptoms consistent with C.difficile infection, an indeterminate result most likely reflects a low level positive result and the test does not need to be repeated.If you would like repeat testing for another reason, please contact the Microbiologist-on-call.” PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 76 of 291 Rejected Samples VII. Rejection Criteria Formed stool received LIS Code Formed}FSTL TEST Comment “Not Processed. Formed stool received. Test cancelled.” Rectal swab received Rectal}RECT Duplicate Orders received in the same day Specimen received in SAF transport media }DUPL ICATE Charcoal Swab received }CHAR coal received Specimen received in enteric pathogen transport medium Patient’s Age is less than 1 year old }CDTX ETT received “Not Processed. Rectal swab received; unsuitable for testing.Please resubmit a stool sample.” “This is a duplicate order. This test has been cancelled.” “Not Processed. Specimen received in SAF (fixative for parasite) and is unsuitable for Clostridium difficile toxin testing. If you have any question, please call the Microbiology lab.” “Not Processed. Charcoal swab received is unsuitable for this test.If you have any question, please call the Microbiology lab.” “Not Processed. Specimen received in enteric pathogen transport medium.Unsuitable for C.difficile testing. Please repeat.” “Test not performed on patients less than one year of age.” }SAF received }<1y Age Quality Control Each test includes a Sample Processing Control (SPC) and Probe Check Control (PCC). Refer to a senior technologist if control results are outside of limits or for any other problems with running or reporting the assay. Run external control (C. difficile Toxin from Positive C. difficile culture) with each new lot, QC and instrument problems. Result filed in External Control Binder. If result is negative, the run is invalid. Inform Charge/senior technologist, and repeat testing. CAP provides external proficiency testing. VIII. Reference Xpert C.difficile PCR Assay package insert PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 77 of 291 Testing for C. difficile toxin from eSwab for Infection Control Purpose Infection Control Practioners may request C. difficile on specimens collected in eSwab during admission screening to rule out C. difficile infection prior to admission. 1. From a previously collected eSwab culture, place a new order in the LIS and order the C. difficile toxin test 2. use the Cepheid instrument by the VRE bench so that automated results through the interface do not occur 3. use 100 uL of eSwab liquid transport media for testing and follow the Procedure above 4. Report results as above with additional Comment: “Note: This specimen type is not appropriate for C. difficile testing but has been tested for infection control purposes only.” PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 78 of 291 Cytomegalovirus Quantitative PCR by Cobas® Ampliprep/TaqMan I. Introduction Human cytomegalovirus (CMV) can cause severe, life threatening disease in immunocompromised patients such as transplant patients, patients with HIV, and developing fetus. In conjunction with clinical presentation and other lab markers, the quantitative CMV test can indicate clinical prognosis, be used to monitor anti-viral treatment and manage CMV infection in patients at risk. The quantitative CMV test is not intended for use as a screening test for CMV in blood, as a diagnostic test to confirm the presence of CMV infection or in specimens other than human plasma. ® ® The COBAS AmpliPrep/TaqMan CMV test is an in vitro nucleic acid amplification test for the quantitation of CMV DNA in EDTA plasma using the AmpliPrep instrument for automated DNA ® extraction and the TaqMan analyzer for automated amplification and detection. The test can quantitate CMV DNA over the range of 150 - 10,000,000 copies/mL. One copy of CMV DNA (as ® defined by TaqMan ) is equivalent to 0.91 International Unit (IU) [1.1 cp/IU] on the WHO Standard). Below the lower limit of quantitation of 150 cp/mL, CMV DNA can be detected but its quantitation cannot be accurately calculated. The CMV QS, a non-infectious DNA construct, is added to each specimen at a known copy number to compensate effects of inhibition, allowing a more accurate quantitation of CMV DNA in each specimen The TaqMan Analyzer calculates the CMV DNA concentration in the test specimens by comparing the CMV signal to the CMV QS signal for each specimen. II. Specimen Collection and Processing Five to 10 mL of blood is collected in a sterile lavender top (EDTA) tube and is transported at 2-25oC to the lab within 6 hours of collection. In the lab, samples are centrifuged at 800-1600 g (3000 rpm in centrifuge MICT36) for 20 minutes. Plasma is separated within 6 hours of collection into two storage tubes and put in CMV DNA’ box in – 20oC freezer. Plasma specimens may be stored at 2-8°C for up to 7 days. Plasma specimens are stable for 6 weeks if frozen at ≤-20°C. Plasma specimens may be frozen and thawed up to 3 times without a significant loss of CMV DNA. III. Materials and Instruementation: Instruments:  Cobas Ampliprep Instruement  Cobas Taqman 48 Analyzer Reagent Kit: Cobas Ampliprep/Cobas TaqMan CMV Test PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 79 of 291 Disposables:  Sample processing units: SPUs  Sample input tubes (S-tubes) with barcode clips  Racks of K-tips  Racks of K-tubes Other materials:  Sample Rack (SK 24 rack)  Reagent Rack  SPU Rack  K-Carrier  K-Carrier Transporter  K-Carrier rack IV. Procedure: Start –up 1. Turn Compter ON. 2. Control-Alt-Delete 3. To log onto Windows a. User name: aluser b. Password : aluser 4. Click Amplilink icon 5. To log onto amplilink a. User name: MSH (case sensitive) b. Password: Msh12345 (case sensitive) 6. Check Wash Reagent Reservoir. 7. Empty Waste container. 8. Perform all maintenance in the “Service Due” tab and as listed below 9. Clean glass cover of instrument. 10. Clean as per figure 1: Work surfaces of Ampliprep 11. Clean figure 2 and 3 on both heads 12. Clean Reagent tips on both heads 13. Clean loading panel 14. Clean Taqman 15. Taqman on 16. Record maintenance on maintenance worksheet. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 80 of 291 Load Reagents 1. Remove reagents from refrigerator 2. Load CS1 on one Rack and insert into position A If doing multiple assays, load enough CS1 cassettes for all runs. Be careful not to smudge the barcodes. 3. Load Cassettes CS2, CS3 and CS4 onto another rack and insert into positions B-E. All reagents cassettes should be removed for the refrigerator and loaded immediately on to the Ampliprep and allowed to come to room temperature on the instrument for at least 30 min. before the first specimen is processed. Do not let the cassettes come to room temperature outside the instrument as condensation may form on the barcode labels. Do not wipe off condensation if it appears on the barcode labels. Load Consumables One sample processing unit (SPU), one Input sample tube (s-tube), one K-tip and one K-tube is needed for each specimen and control. 1. 2. 3. 4. Load SPU’s on positions J,K,L. Check seating in rack; do not push on top of (S-tips)- only on sides Load K-tips into positions M-P. Place control Barcode clips on sample rack 1. HPC , 2. LPC ,3. Neg Place new Barcode clips on sample rack for each sample being tested. Create Order 1. Click the orders button 2. Select the Sample-Rack tab and click NEW 3. Type the Sample rack ID in the Sample Rack ID box. 4. Put the worklist # in the Batch ID box. 5. Click the test button (CMVCAP48 for CMV). 6. To identify the controls highlight then click the “S” in the sample type (T) column to select the control types: CMV (-)C, CMV L(+)C and CMV H(+)C 7. Highlight Sample ID on line 4 8. Scan or enter Sample ID 9. Repeat 7 and 8 for all samples 10. Click save when finished. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 81 of 291 Prepare Specimens 1. Transfer 500 uL into an S-tube for each specimen and control. Close cap of S-tube finger tight. Avoid contaminating the upper part of the S-tube with specimens or controls. 2. Place K-tubes on Sample rack for each specimen. 3. Check seating of Clips and S-tubes. 4. Load Sample rack onto positions F, G or H. Load K-Carrier 1. Click the System button 2. Select the Cobas Ampliprep instrument using the instrument Selection box. 3. Click the Samples tab to display the Samples folder. *Steps 4 and 5 must be completed within 5 seconds. 4. Using the handheld barcode scanner, scan the K-carrier rack barcode. 5. Scan the K-carrier barcode. 6. Load the K-carrier rack containing the K-carrier in position O and P. Start Ampliprep 1. Check Status of Cassettes, Specimens and System by clicking on each tab 2. Press start. 3. Note the time when the run is completed on the worksheet. End of Run 1. Click the Messages button and examine for any error messages. 2. Click the Systems button 3. Examine the Samples folder – successfully processed samples are green 4. Open the load panel 5. Remove the K-carrier rack containing the processed samples. Note: All processed specimens and controls should not be exposed to light. Note: The K-carrier must be loaded into the Taqman within 2 hours of processing on the Ampliprep Start Cobas TaqMan 48 Analyzer 1. Select the Cobas TaqMan 48 Analyzer using the instrument Selection box. 2. Select the System tab. 3. Click Open for the thermal cycler to be loaded. 4. Open the thermal cycler lid. Balance the K-carrier if necessary. 5. Using the K-carrier transporter, place the K-carrier in the thermal cylcer. 6. Close the lid 7. Choose Start. 8. Store reagents for the Ampliprep in the refrigerator. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 82 of 291 Remove instrument waste 1. Remove used SPUs and S-tubes put in autoclave bag and seal it. 2. Remove empty K-tip racks 3. Remove Sample racks and dispose used bar-code clips 4. Discard used K-tubes Results interpretation 1. Click Results button 2. Select the Review tab 3. Select the desired worklist under the batch ID column 4. Examine the results Double-click the results line to display detail Check for flags and error messages Click Measurement Details to view graph Print graph of any _Q_QS_ INVALID, _S_Drift_High or any other unusual flag and consult Senior Tech or Charge Tech. 5. Click Accept. The K-carrier must be removed from the Taqman before results can be accepted. 6. Print Results Report V. Reporting Titre Result Target Not Detected <1.37E+02 IU/mL Interpretation No CMV DNA detected CMV DETECTED, <137 IU/mL >=1.37E+02 IU/mL and <=9.10E+06 IU/mL >9.10E+06 IU/mL CMV, (# value) IU/mL CMV, >9.10E+06 IU/mL The conversion factor between CMV DNA copies/mL (as defined by COBAS® Ampliprep/ COBAS® Taqman ® CMV test) is 1.1cp/IU [0.91 IU/cp]. Note: Specimens that produce an invalid result with a flag “QS_INVALID” review the graph. Repeat testing. Once the results have been finalized a report is faxed to the BMT clinic using the LIS : -Lab-Reports-Verified ResultsReports; (remove checks from all items under print flags) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 83 of 291 -put in date in Verification Date field -in Ward field put “BMTCL” -in the Test ID field put in “8CMVP” -fax to BMT VI. Quality Controls One CMV (-)C, CMV L(+)C and CMV H(+)C is included with each run. Each result must be within the set value for each Lot #, otherwise the run is invalid. Record these results on-line in the T-Drive under “Microbiology/Virology/CMV QC… Active”. The results should be within mean ± 2.S.D., otherwise withhold reports and inform charge/senior technologist for further review. External QC: Run external control with each new reagent lot and at least once a month. May also need to run external QC after equipment / QC failures, consult charge/senior technologist. Record these results on-line in the T-Drive under “Microbiology/Virology/CM QC… Active”. The results should be within ± 2.S.D., otherwise withhold reports and inform charge/senior technologist for further review. External proficiency testing : Provided by National Microbiology Lab (NML). Alternatively, send 2 samples to PHL for parallel testing (January and July). Reference Roche Diagnostics, Ampliprep Software Version 3.2, Application Manual Version 1. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 84 of 291 Cytomegalovirus Qualitative PCR by Rotorgene I. Introduction Human cytomegalovirus (CMV) can cause severe, life threatening disease in immunocompromised patients such as transplant patients, patients with HIV, and developing fetus. Quantitative CMV can be used to monitor the course of CMV infection during and after treatment. II. Specimen Collection and Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG III. Materials, Equipments Clean Room: dedicated Biosafety Cabinet (MIBCT4), Designated clean room gowns and powder-free gloves Designated Clean room Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL o 36 microtube Loading block pre-cooled to 4 C 72 microtube Loading block pre-cooled to 4oC 0.2mL microtubes/ or 0.1mL microtubes and Caps Aersol Resistant Tips (ART) Altona CMV PCR Kit 1.0, (stored at -20o C in clean room):  Master A (12 tests per vial)  Master B (12 tests per vial)  Internal Control (IC)  Water (PCR grade) Specimen Preparation area: Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL o Altona CMV PCR Kit 1.0, (stored at -20 C in clean room):  QS1 (10^4 Copies/µL)  QS2 (10^3 Copies/µL)  QS3 (10^2 Copies/µL)  QS4 (10^1 Copies/µL) External Control: To be run when a new lot is used, during training or if QC failure occurs. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 85 of 291 Detection Area: Rotorgene 6000 Multiplexing System programmed with ALTONA CMV PCR 36-Well Rotor with Locking Ring/ or 72-Well Rotor with Locking Ring GENERAL PRECAUTIONS: There must be designated separate PCR work areas: 1. Clean Room 2. Specimen preparation area 3. Detection (Amplification) Area Supplies and equipment must be dedicated to each PCR work area. Use only aerosol resistant tips (ART). Work with powder-free gloves; change gloves frequently. Follow cleaning instructions outlined on daily maintenance schedules. IV. DETECTION SET UP: Organize CMV eluates and store at 4oC until ready to load. Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) a. Take out the necessary number of CMV Master Mix A and B vials (12 tests per vial) from the -20C freezer, quickly and thoroughly thaw inside the Biosafety Cabinet. Prepare CMV Master Mix (number of samples + controls + 1 extra test) required according to the chart. Mix gently, do not vortex. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Page 86 of 291 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 b. Place proper number of microtubes for each sample, Controls (Positive and Negative) into the Cooling Block (pre-cooled to 4oC). c. Pipette 20 uL prepared CMV Master Mix (A+B) into each microtubes. Lightly cap sample microtubes. d. Pipette 1.0 uL of internal control into the microtubes designated for the Positive control (CMV QS3) and the Negative control. e. Pipette 10uL water (PCR grade) into the negative control microtube and cap tightly. Number microtube lids. f. Changed out of dedicated clean room gown. Specimen Processing Area: a. Carefully pipette 10uL of each sample eluate, and Positive Control (CMV QS3), into designated numbered microtube containing CMV Master Mix. Gently mix by pipetting up and down. Close the lid tightly. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 87 of 291 b. Visually check that each microtube is tightly closed and that all microtubes have the same liquid level 30ul, before loading. Make sure there are no bubbles at the bottom of the microtubes. Detection (RotorGene) Area: a. Turn ON the RotorGene and computer. b. FOR THE BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; c. Double click (left on the mouse) “Rotor-Gene 6000 series software 1.7” icon. Wait for “Initializing machine…..” d. Load microtubes into proper rotor (eg. 36 well/or 72-well). Load sample #1 into rotor position #1, sample #2 into rotor position #2, and so on. e. Fill the empty positions of the rotor using empty microtubes and snap in the locking ring. Load the rotor in the RotorGene 6000. f. Highlight CMV assay; g. Press “New” button; h. Highlighting the correct rotor in use: Red 36-well Rotor Blue 72-well Rotor i. Click the box “Locking Ring Attached” j. Press “Next” button. k. Enter initials in Operator space. Enter kit lot number and the Rotor-Gene Colour (ie.Red/Blue) in the Notes box. Verify the Reaction Volume is 30ul. l. Press “Next” m. Temperature Profile window pops up; press “Next”. n. Summary window appears; press “Start Run”. The RotorGene will start. o. Save as window appears; go to My documents -Open–select CMV result folder- Open Press Save -CMV yyyy-mm-dd (run no.) p. Edit Samples window. Go to format select 1.23E+05. Under Units select copies/ul. q. Enter sample LIS numbers using the barcode reader, enter CMV Positive (CMV QS3), and water. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Name Sample Number QS3 CMV Water Type Unknown Standard NTC(no template control) Given conc. 1.00E+02 Page 88 of 291 Select Yes Yes Yes r. Press “Finish”. Click on “Name On” To analyze and print CMV report after a run has finished: 1. The run is done when Profile Program reads: Run has completed. 2. Analyze raw data by clicking each channel one at a time: Green channel (CMV) and Yellow channel (IC).These tabs are found on the second menu bar from the top, on the left hand side. 3. In the Green Channel. Click “All Off” (bottom right). Select sample “water (NTC)” and “cmv QS3 (Standard)”. Click on Autoscale (bottom left). Compare each specimen curve to the water curve and the cmv QS3 curve one at a time (by clicking each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worklist. 4. Click on “Named On”. 5. Perform curve Analysis (Get report): Click “Analysis” on the top tool bar. 6. Analysis floating window pops up. 7. Click Quantitation Analysis. 8. Quantitation window is brought up to the front. There are should be two channels (Green/Yellow). 9. Highlight Cycling A. Green option and press Show. 10. Maximize the cycling green channel graph (or the channel you are working on). 11. Press “Import Curve” button on the right side of the screen. 12. Press “From other run” button. 13. Go to My Document -STD curves folder, press “Open” PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 89 of 291 14. Choose the latest CMV standard curve (Note: standard curves should changed every 6 months unless instructed by Charge/Microbiologist), “Open” 15. Highlight green channel, press “Import” button. 16. Adjust if only ONE Standard used (Note: Do NOT adjust if ALL Std used) 17. Press button “Slope Correct” on the top. 18. Press button “Ignore first” on the top, and type “5”, press OK. 19. Check curve quality, adjust the Threshold. 20. To Print Reports: Press “Reports”. 21. Choose Cycling A.Green (Page 1) 22. Highlight Quantitation full report, press “Show” Maximize the report window, check that only the positives that you have identified from the raw data are positive (i.e. have a crosspoint) 23. Print pages 1-4 for the green channel; pages 2-4 for the yellow channel. Close the report window. 24. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab, Select Cycling A. Yellow -the internal control (IC), select Show. Maximize the graph Quantitation Analysis –Cycling A. Yellow. Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in the first 5 cycles of the run). Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph screen. Move the threshold line to above the noise. Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show. All samples should have a positive IC curve, and a cycle number in the Ct column. Print pages 2-4. (For Cycling A. Yellow report, if there is message of “NEG (Multi Ct)” for any specimen, you have to go back to check Raw Data of cycling A.Yellow. If all specimens are amplified, adjust the threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct. Adjust the threshold line so that the threshold line crosses the curve at only one point.) 25. Close the outer most window. “Save changes to CMV yyyy-mm-dd?” Select Yes. Shut down the computer, and switch off the Rotor-Gene. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 90 of 291 V. Calculations: Perform calculations on CMV PCR blood (CMVB) specimens only. CMV PCR blood (CMVB) specimens should be reported quantitatively; all CMV PCR non-blood (CMVNB) specimens are reported qualitatively (CMV DNA detected/ No CMV DNA detected). Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL) Sample volume (mL) Result (copies of DNA/mL) = Result (copies/uL) x 50(uL) 0.200 (mL) These calculations can also be performed using PCR bench Math excel file. VI. Reporting: CMV PCR blood (CMVB) specimens should be reported quantitatively; all CMV PCR non-blood (CMVNB) specimens are reported qualitatively (ie. CMV DNA detected/ No CMV DNA detected). PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report. CMV PCR Blood (CMVB) Specimens: CMV PCR negative (has no Ct value) report on the LIS test window, use keypad: No CMV DNA detected. This is a research test. CMV PCR Kit 1.0, Altona Diagnostics Inc. Under media “PCCMV” F6 to enter the date tested. Finalize the result (Ctrl+F) CMV PCR positive, with value in copies of DNA/mL (ie.After calculation has been performed). Report CMV as an isolate in the isolate window (F7). Isolate #: 1 Org.ID. 13cmv Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. From the keypad select: >CMV+ this will take you to CMV resulting keypads. If result is <100 copies of DNA/mL select: <100\cmv1 If copies are to be reported select: \CMV+ Type in the copies, in scientific format, in the space provided. Grave (`) the result. Under media “PCCMV” F6 to enter the date tested. If reporting <100 cp/mL enter the actual copy value. Interim the result (Ctrl + L). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 91 of 291 CMV PCR Non-Blood (CMVNB) Specimens: Report qualitatively only. CMV PCR negative, no Ct value. Report on the front of LIS workcard, use keypad: No CMV DNA detected. This is a research test. CMV PCR Kit 1.0, Altona Diagnostics Inc. Under media “PCCMV” F6 to enter the date tested. Finalize the result (Ctrl+F) CMV PCR positive (Ct value present), no calculations are performed. Report CMV as an isolate window F7. Isolate #: 1 Org.ID. 13cmv Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Select: >CMV+ Select: \CMV+ Type: DETECTED and delete the “copies of DNA/mL” phrase. Isolate Comment window F8 should appear as: DETECTED. This is a research test. CMV PCR Kit 1.0, Altona Diagnostics Inc. Grave (`) the result. Under media “PCCMV” F6 to enter the date tested. Interim the result (Ctrl+ L). Calling Results: BMT: Call all CMV PCR blood (CMVB) results to the Bone Marrow Transplant Clinic when tested; CMV PCR positives and negative results. PMH: Call Positive CMV PCR blood (CMVB) results TGH: Call Positive CMV PCR blood (CMVB) results, if patients’ have not had CMV detected in their blood in more than 2 months. If patients’ have had CMV detected in their blood within the last 2 months no calling is necessary. Outside clients: Call Positive CMV PCR blood (CMVB) and fax results. Call all Positive CMV PCR Non-blood (CMVNB). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 92 of 291 Testing Schedule: CMV PCR blood (CMVB) PMH patients will be tested as requested. TGH patients: CMV PCR blood (CMVB) Positive no CMV PCR Blood (CMVB) will be tested for 5 days. Result as: Previous CMV PCR Positive, please resubmit a specimen for testing after 5 days. VIII. 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.  CMV High Positive and CMV Low Positive External controls should be extracted on the easyMag daily and run daily. Record external QC results in the T drive, Virology folder, QC folder. Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition.  CMV QS3 Standard is included and shows a positive reading in Green Channel  A Negative Control is included and shows a negative reading in Green Channel  Report all failed QCs to senior/charge technologist. a. b. c. d. e. f. g. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 93 of 291 h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. IX. References CMV PCR Kit 1.0, Altona Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 94 of 291 Epstein Barr Virus Quantitative PCR by Rotorgene I. Introduction Epstein-Barr Virus is a double stranded DNA virus, in the family Herpesviridae. An asymptomatic infection most commonly during childhood; EBV has been associated with some cancers, including Burkett’s lymphoma and nasopharyngeal carcinoma. EBV PCR is most commonly used for transplant recipients for monitoring post-transplant. EBV PCR is a real time PCR, for the detection and quantitation of EBV from whole blood. II. Specimen Collection and Transport EDTA whole blood stored at C if processed with 24 hours; processing >24 hours aliquot and freeze whole blood at -20oC. Sterile body fluids (CSF, plasma) for EBV PCR testing require Medical Microbiologist consent before testing. III. Specimen Processing: Whole blood processing refer to: Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG Sterile body processing refer to: Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG GENERAL PRECAUTIONS: There must be separate PCR work areas:Clean room and Specimen preparation area  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 Preparation 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual IV. Page 95 of 291 Materials, Equipments Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System with experiment template: EBV 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor with Locking Ring 0.1 mL reaction microtubes Tubes and Caps Variable volume Rainin pipettes: 1 to 20 uL, 10 to 200 uL, 100 to 1000 uL (assigned to designated areas) Reagent: Altona RealStar EBV-PCR Kit 1.0: MasterA, MasterB, Internal Control, EBV Quantitative Standards 1, 2, 3, 4 (QS1, QS2, QS3, and QS4), PCR Grade Water External Control: EBV High Positive, EBV Negative, EBV Low Positive, to be extracted and run in this order on once a week, or if QC failure occurs. V. Procedure: Prepare eluate samples in order according to the EBV worksheet. Add the following EBV Quantitative Standards and NTC Water to the worksheet in the following order: EBV QS4, EBV QS3, EBV QS2, EBV QS1, NTC (Non-Template Control) Water. PCR Set-up: In the Clean Room: Change into dedicated clean room gown and gloves, work in Biological Safety Cabinet. Remove from -20oC freezer to thaw at room temperature the required number of vials’ of EBV Master A, and EBV Master B (12 reactions/vial) for your EBV PCR run. Prepare EBV Master Mix in 1.5mL conical (sarstedt) microtube; to load on the Eppendorf epMotion. Prepare the Master Mix reactions required: Number of Test Samples + 5 Standards (4QS&NTC) + one 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 EBV Master A EBV Master B Volume of Master Mix Sample/Control Volume 1 5 15 20 10 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Page 96 of 291 EBV Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 See Eppendorf epMotion Manual for further loading and programming instructions. Manual Loading: Load one reaction microtube (0.1mL) for each sample/control. Pipette 20uL prepared EBV Master Mix into each reaction microtube. Pipette 1.0uL of internal control into each reaction microtube designated for the 5 Standards (EBV QS4, QS3, QS2, QS1, and NTC). Specimen Processing Area: Pipette 10uL of Sample/Standard into reaction microtube. Mix by pipetting 3x up and down into the master mix. Cap each segment after pipetting is completed. Check reaction microtube segments before loading into the rotor, ensure the liquid levels are at the same height, there are no bubbles at the bottom of the microtubes, and the lid is tightly in place. Load reaction microtubes in 72-Well blue rotor, fill empty rotor spaces with blank reaction microtubes. Attach the Locking Ring. Load the rotor in the Rotor-gene 6000. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 97 of 291 Detection (RotorGene) Area: Switch ON computer, switch ON RotorGene. FOR THE BLUE ROTOGENE ONLY. Select clinical icon; password “msh”. Enter. Open “Rotor-Gene 6000 series software 1.7” icon Wait for “Initializing machine…..”       New Run Window Select EBV PCR Select New Select 72 Well Rotor (blue) Check the box for “Locking Ring Attached” Select “Next” button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 98 of 291  Enter operator intials.  In Notes enter RotorGene colour (i.e. Red or Blue) Reagent Lot Number. Make sure the reaction volume is 30uL.  Select Next.  Temperature Profile window .Select Next.  Rotor must be loaded.  New Run Wizard window: Select Start Run. The RotorGene will start.  Save As window appears; the run filename is given with the default template. Save in: My Documents in EBV Virus folder for the current year: e.g. EBV Virus 20150326(1) (assay name yyyymmdd run#) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 99 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual     Select Save. RotorGene will start running. Edit Samples window appears. Enter sample numbers including Quantitative Standards (EBV QS4, QS3, QS2, QS1, NTC) and concentrations. Define the sample types according to the chart below. Name Sample Type Samples EBV QS4 EBV QS3 EBV QS2 EBV QS1 NC Water Unknown Given concentration IU/uL *Conversion factor x250 Enter Concentration IU/mL 1.00E+01 1.00E+02 1.00E+03 1.00E+04 x250 x250 x250 x250 2.50E+03 2.50E+04 2.50E+05 2.50E+06 Standard Standard Standard Standard NTC (No template control) *conversion factor is based on the extraction sample volume and eluate volume *Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL) Sample volume (mL) *Result (copies of DNA/mL) = Result (copies/uL) x 50(uL) 0.200 (mL)  Under Given Conc. Format: Select 1.23E+05 (2 decimal points expressed as an exponent).  Units: Select copies/mL  Press “Finish”. Click on “Name On”. Analysis  Profile Program reads: Run has completed.  Analyze raw data by clicking each channel one at a time: Green channel (EBV) and Yellow channel (IC). Target Channel Report Dye EBV Internal Control Green Yellow FAM JOE PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 100 of 291  Perform Raw Green (EBV Target) Channel. Select All Off.  Select sample water (NTC) and EBV QS4, QS3, QS2, QS1. Select Autoscale.  Compare each sample curve to the NTC-water curve and the EBV QSs’ one at a time, then proceeding to the next sample. A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worksheet.  Select Named On.       Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Green. Select Show. Calculate Automatic Threshold. Select OK. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Verify the Quantitative Standard Curve on the Green (FAM) Channel. The following parameters must be met, this information can be found on page one of the Green Channel Quantitation –Full Report or on the screen in the Green Channel Graph. Control Parameter M Slope PCR Efficiency R square (R2) Valid Values -3.00 to -3.74 85% to 115% >0.98 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 101 of 291 If the quantitative parameters are not valid, it must be brought to the attention of the senior/and or charge. External Controls must be valid, if not valid, it must be brought to the attention of the senior/and or charge. Patients results must not be released.  Select Reports. Select Cycling A Green (Target) or Quantitation Analysis Cycling A Yellow (Internal Control) depending on what channel you are analyzing.  Select Quantitation (Full Report)  Maximize report window. Check that only the positives that you have identified, from the raw data analysis, are positive (i.e. have a crossing threshold ct value).  Print all pages for Green (Target-FAM) channel. Print pages 2-5(or 4) for the Yellow (Internal Control-JOE) channel. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 102 of 291  Close Report window.  Perform Raw Yellow (Internal Control) Channel. Select Name On.  All samples, including EBV QS1-4, and NTC water, should have a good amplification curve; all should be positive in the Yellow IC Channel; with the exception of samples with high ct values (ct <20.00) in the EBV Target Green Channel.       Select Analysis Icon In Analysis window. Select Quantitation Tab. (This should be the default). Two channel option: Cycling A Green and Cycling A Yellow Select Cycling A. Yellow. Select Show. Calculate Automatic Threshold. Select OK. Maximize the window: Quantitation Analysis Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Slope Correct.  Select Ignore first: 5 Cycles. OK  Select Threshold Icon and adjust the threshold. The threshold can only cross a curve at one point.  Select Reports. Select Cycling A Green or Quantitation Analysis Cycling A Yellow depending on what channel you are analyzing.  Select Quantitation (Full Report)  Print all pages for first channel. Print pages 2-5(or 4) for the Yellow IC channel.  Close Report window.  Note: Cycling A. Yellow report: if there is message of “NEG (Multi Ct)” for any specimen, you must adjust the threshold. This indicates the threshold has crossed the curve at multiple points. The threshold can only cross a curve at one point.  Save changes to EBV yyyy-mm-dd. Select Yes.  Shut down the computer, and switch off the Rotor-Gene. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VI. Page 103 of 291 Reporting EBV Not detected: Specimen has no ct value. Report in LIS using the keypads: Virology Worklist > EBV PCR worklist > Select from the reporting keypads: }EBVNegative for EBV virus DNA. This is a research test. EBV PCR Kit v1.0. Astra Diagnostics Inc. Under media PCEBV. F6 to enter the date tested. Finalize the result (Ctrl+F) EBV Detected: Specimen with ct value. Report quantitation “IU/mL” Report EBV as an isolate in the isolate window (F7). Isolate #: 1 Org.ID. 65ebv Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Greater than 600 IU/mL. Select the keypad: \EBV+ Turn off the reporting keypad. Ctrl + A or Ctrl + K Enter the IU/mL, in scientific number format, in the space provided, remove extra spacing. Example: 3.20E+04 IU/mL. This is a research test. EBV PCR Kit v1.0, Astra Diagnostics Inc. Verify all in the Isolate window Less than 600 (<600) of IU/mL. Select: \E600 Under media “PCEBV” F6 to enter the date tested. If reporting Less than 600 IU/mL, enter the actual IU/mL. Interim (Ctrl + L) the report. Second technologist will check transcription in LIS and Finalize (Ctrl + F) the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 104 of 291 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. EBV High Positive and EBV Low Positive External controls should be extracted on the easyMag once a week. Record external QC results in the T drive, Virology folder, QC folder. Daily QCs: Every run: Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition. EBV QS1, QS2, QS3, QS4 Standard is included and shows a positive reading in Green Channel. Quantitative Standards must meet valid quantitative parameters. A Negative NTC (No template control) Control is included and shows a negative reading in Green Channel Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in: Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 105 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Summary Information: EBV PCR Number of Reactions EBV Master A EBV Master B Volume of Master Mix Sample/Control Volume Name Sample Type 1 5 15 20 10 Given concentration IU/uL *Conversion factor x250 Enter Concentration IU/mL Samples Unknown EBV QS4 Standard 1.00E+01 x250 2.50E+03 EBV QS3 Standard 1.00E+02 x250 2.50E+04 EBV QS2 Standard 1.00E+03 x250 2.50E+05 EBV QS1 Standard 1.00E+04 x250 2.50E+06 NC Water NTC (No template control) Control Parameter M Slope PCR Efficiency R square (R2) Valid Values -3.00 to -3.74 85% to 115% >0.98 VIII. References Altona EBV PCR Kit v1.0 Instructions, Altona Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 106 of 291 Enterovirus RNA PCR by Lightcycler I. Introduction Enterovirus belongs to the family Picornaviridae including over 70 distinct serotypes(coxsackie A and B, echoviruses, polioviruses, and enteroviruses 68-73). They infect a wide variety of mammals and are associated with a board spectrum of diseases. There are 68 viruses within the Enterovirus genus that are known to infect humans. Enteroviruses are transmitted primarily by the fecal-oral route but respiratory spread is possible. Non-polio enteroviruses most commonly cause rashes, upper respiratory infections. Enteroviruses infections account for a substantial number of ascetic meningitis and encephalitis cases in summer and fall. II. Collection and Transport Blood samples for enterovirus PCR should be collected in EDTA (purple top vacutainer tube). CSF specimens should be collected in a clean, sterile container and sent to the laboratory as soon as possible. If specimens cannot be transported immediately, they should be kept at 4oC. If a delay of more than 6 hours is anticipated, the CSF specimen should be frozen at –70oC. The EDTA samples should be processed within 6 hours of collection (plasma separated and refrigerated; frozen if delay is more than 6 hours). Allow only one freeze-thaw cycle. NOTE: Repeated freezing-thawing will reduce test sensitivity and cryoprecipitates may accumulate in the plasma. III. Procedure A. Worklist Preparation: a. log on to Softmic, b. Go to PCR Worklist i. Worklist : 1VPCR VIROL PCR ii. Enter iii. F12 c. Pending samples (plasma, serum or CSF) will appear on Worklist. d. Mark received samples using the F5 key, that require Enterovirus PCR e. Type "`" to print Worklist. B. LIS Label Printing (if needed for the RNA eluate): a. While still in the Worklist, press Enter to go to the Test Screen b. Press F1 to move curser to Demographic field c. Press: / (to Order/Entry) d. Press: No (to confirm editing) e. Press: F9 f. Press: \ (Print Labels) g. Press: No (modify record) h. Select label printer for the LIS label PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 107 of 291 C. Specimens Processing: Specimens should be processed as soon as possible after arriving in virology laboratory. CSF: An aliquot (0.2-2 mL) of the CSF should be frozen unless PCR can be performed immediately After processing, an aliquot of the left-over specimens should be stored frozen at-70C. E. Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT4), freezer (MIFTG), gowns and gloves Specimen Preparation area with Biosafety Cabinet and microcentrifuge Detection Room for LightCycler Roche LightCycler programmed for Artus Enterovirus RT-PCR Microcentrifuge (MICT14) Vortex 1.5 mL microcentrifuge tubes Cooling Block with capillary adaptors pre-cooled to 4oC Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL ART-Aersol Resistant Tips for pipettes From Qiagen QIAampR Viral RNA Mini Kit:  Collection Tubes  Lysis Buffer AVL  Carrier RNA (poly A)-add 310 uL Elution Buffer -AVE to lyophilized carrier RNA (310 ug) dissolve thoroughly and aliquot into 5.6 uL and 30 uL screw-cap microtubes, and store at -20C in the clean room. DO NOT freeze-thaw the carrier RNA aliquots more than 3 times  Spin Columns  Wash Buffer 1 (Buffer AW1) – add 125 mL ethanol before use  Wash Buffer 2 (Buffer AW2) – add 160 mL ethanol before use  Elution Buffer (Buffer AVE) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 108 of 291 From: artusTM Enterovirus LC RT-PCR Kit (stored at -20o C in clean room):  Quantification Standards (QS). When a new kit is opened, the QS must be moved to a freezer in the specimen preparation area.  Internal Control (IC)  Master Mix (12 tests)  Water (PCR grade) Ethanol, anhydrous, reagent grade, 96 - 100% External Control: To be run when a new lot (Qiagen or RealArt) is used, during training or if QC failure occurs. GENERAL PRECAUTIONS:  There must be separate PCR work areas: 1. Clean room 2. Specimen preparation room 3. Amplification room              Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas. Change gloves between work areas. Only blue gowns are to be worn in the clean room. Use only filtered pipette tips Use only sterile RNase, Dnase-free microcentrifuge tubes Use sterile, disposable polypropylene tubes throughout the procedure Always wear powder-free gloves when handling reagents Change gloves frequently and keep tubes closed whenever possible Prepare and store reagents (lysis buffer, RNA carrier, elution buffer, AW1, AW2 and ethanol) in the clean room. Keep only working aliquots in the specimen preparation area. Keep the Quantitation Standards and specimens in the specimen preparation area. Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 109 of 291 Clean Room In the Clean Room prepare the Working Lysis Buffer AVL; determine the number of tests to be extracted and prepare the appropriate volume of Working Lysis Buffer AVL required according to the chart. Mix gently, invert 10 times to mix. Do not vortex. Volume Lysis Buffer No. of AVL samples (mL) 1 0.56 2 1.12 3 1.68 4 2.24 5 2.80 6 3.36 7 3.92 8 4.48 9 5.04 10 5.60 11 6.16 12 6.72 Volume Carrier RNA-in Elution buffer AVE (uL) 5.6 11.2 16.8 22.4 28.0 33.6 39.2 44.8 50.4 56.0 61.6 67.2 DO NOT freeze-thaw the carrier RNA aliquots more than 3 times. Working Lysis Buffer AVL is stable for 48 hrs at 2-8C. Solution may precipitate at 2-8C; redisolve by warming on the bench, DO NOT MICROWAVE. Lysis and Purification: a. Use Specimen Preparation area; work in cabinet with gown. Use only ART tips. Change gloves frequently. b. For each Specimen and External Control, prepare and label two 1.5 mL microcentrifuge tubes (sterilized, RNase-free) with the corresponding number in the Worklist. Put the specimens first on the worklist, followed by the external control (if using one). Label one of them with the lab number (LIS label). This microcentrifuge tube will store the eluted RNA; the other will be used for processing and will be discarded. Also, label one QIAamp Spin Column (Qiagen) with the number on the Worklist. Arrange the 2 microcentrifuge tubes and Columns into 3 rows. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 110 of 291 c. Add 560 uL Working Lysis Buffer (AVL with RNA carrier) into each of the microcentrifuge tubes in one row (these will contain the waste). d. Add 140 uL Specimen to the Working Lysis Buffer (Buffer AVL with RNA carrier), vortex for 15 seconds. e. Incubate at Room Temperature (15-25oC) for 10 minutes. f. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids. g. Add 5 uL Enterovirus Internal Control to each of the microcentrifuge tubes with Buffer AVL. h. Add 560 uL ethanol (96-100%) to the samples, vortex for 15 seconds. Centrifuge for 10 seconds at 3000rpm (2000 g) to remove drops from the lids. i. Pipette 630 uL (half the volume) of the mixture (specimen-lysis buffer-ethanol) to the QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000 rpm (6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. j. Repeat Step (h) by pipeting the remaining 630 uL of the mixture to the QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000rpm (6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. k. Open the spin column and add 500 uL of Buffer AW1. Close the cap and centrifuge at 8,000 rpm (6,000 g) for 1 minute. l. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. m. Open the spin column and add 500 uL of Buffer AW2. Close the cap and centrifuge at full speed 14,000 rpm (20,000g) for 3 minutes. b. Place the spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. Centrifuge at 14,000 rpm (20,000g) for 3 minutes. Elution Steps: a. Place the spin column in the second clean 1.5 mL microcentrifuge tube with the LIS specimen label. Discard the collection tube containing the filtrate. Open the spin column and add 50 uL of Elution Buffer (Buffer AVE). Close the caps and incubate at room temperature for 1 minute. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 111 of 291 b. Centrifuge at 8000rpm (6000 g) for 1 min. Discard the spin column. Store the eluate at -20oC or –70oC if not able to proceed immediately. PCR Amplification and Detection Procedures: In the Clean Room: Change into dedicated clean room gown and gloves, work in Biosafety Cabinet. Use only ART tips. a. Take out the necessary number of Enterovirus Master Mix vials (12 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. b. Place a capillary for each purified sample, QS, external QC and Negative control (H2O) into the capillary adaptor of the Cooling Block (pre-cooled to 4oC). a. Pipette 15 uL Master Mix into the reservoir of each capillary. b. Pipette 0.5 uL of internal control into the capillaries designated for the QS and the Negative control. c. Pipette 5 uL PCR grade water into the negative control capillary and cap. d. Changed out of dedicated clean room gown In the Specimen Processing Room: Changed into specimen room gown. a. Using ART tips, carefully pipette 5 uL each of purified sample, and QS4, (and external QC, if using) directly into the capillary tube. Immediately close the capillary with a lid. b. Load capillary tubes into LightCycler (LC) sample carousel and centrifuge at 3000 rpm for 15 seconds in LC centrifuge. Load the LC sample carousel into the LC instrument. The LightCycler (LC) Instrument: a. Turn LightCycler on first and then the computer. b. Enter your user name and password and click OK. c. Click on the LightCycler icon and push ENTER. d. Wait for the “Self-test box” to appear. e. Perform "selftest". “Skip selftest” if performed within 24 hr. f. Choose OK when the self-test passes. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 112 of 291 g. Record that you did the self-test in the log book. h. Select: "Open Experimental File" “Enterovirus protocol"; choose “Open” “Run” i. Type in file name “enterovir-year-month-day- run number” e.g. enterovir -2004-02-01. Choose “save”. j. Type in the number of samples to run including QS, Negative Control, and External Control (if using one) in the “maximum position” field. k. Select “Enter Samples Later”. LightCycler will begin protocol. l. Select “Edit Samples” (bottom right of screen). m. Select “Clear Sample List”. OK n. Type in the lab numbers; QS; ext. QC (if any) and Negative control. o. On the QS row, click the arrow-down button to change its description from “Unknown” to “STD” and enter the number of copies/uL (eg. QS3=100) p. Press “Done” Remember to freeze the end product and patient samples. To Print Results, go to: “FLUORESCENCE” and click  F1  Quantification  Print Window To Print Internal Control, go to: “FLUORESCENCE” and click:  F3/BACK- F1  Print Window PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 113 of 291 At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet and turn on UV light for a least ½ hour but not overnight.  Clean Pipettors Specimen Preparation Room:  Seal and discard waste bag  Clean Biological Safety Cabinet thoroughly  Wipe all pipettors, centrifuge, and bench top.  Wash racks. Amplification Room:  Discard capillaries into a sharps container and put the cap the back onto the sharps container  If a capillary breaks in the carousel, use the small brush supplied to clean the holes with 1% hypochorite and then alcohol. IV. Reporting: Fluorimeter Channel F1 (Target) Fluorimeter Channel F3 back- F1 (IC amplicon) Interpretations - - Possible PCR inhibition-Repeat assay. If still -ve on both Fluorimeter Channels, report: Indeterminate - + + - + + Sample has no detectable Enterovirus RNA Report: Negative Review the F1 graph. Sample contains Enterovirus RNA. May need to be repeated. Inform charge/senior. Report: POSITIVE Review the F1 graph. Sample contains Enterovirus RNA. May need to be repeated. Inform charge/senior. Report: POSITIVE PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 114 of 291 Positive results for the Enterovirus amplicon (Channel F1) will have the cycle number printed under the Cross Point Column in the chart and the Specimen Cross point (Ct) will be less than the Enterovirus QS4 (60 copies/uL).Review the curve, it will be rising exponentially and levels off to a plateau. Internal Control amplicon need not be positive. Inform Charge of positive specimen, sample may need to be repeated. Indeterminate results may be those with a positive signal for the Enterovirus (F1 channel) cycle number printed under the Cross Point Column, the Specimen Cross point (Ct) is less than the Enterovirus QS4 (60 copies/uL). Review the curve. Inform Charge of Indeterminate specimen, sample may need to be repeated. Indeterminate results may also be those with no signal for the Enterovirus (F1) and also no signal for the IC amplicon (Channel F3/Back-F1), indicating possible PCR inhibition. Report Indeterminate specimens to Charge, repeat testing may be required. Negative results for the Enterovirus amplicon (Channel F1) will be blank under the Cross Point Column in the chart and the curve will be rather flat. The IC amplicon (Channel F3 back F1) must be positive to be valid. Negative Report: Negative by RT-PCR. This is a research test performed by using artus Enterovirus LC RT-PCR assay, Artus Biotech Inc. Indeterminate Report: Indeterminate by RT-PCR This is a research test performed by using artus Enterovirus LC RT-PCR assay, Artus Biotech Inc. Positive Report: POSITIVE by RT-PCR This is a research test performed by using artus Enterovirus LC RT-PCR assay, Artus Biotech Inc. Inform Charge technologist of Positive and Indeterminate results. Record isolate in LIS in F7 Isolate window as 39ent Enterovirus, and call Positive results. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual V. Page 115 of 291 Quality Control    Colour Compensation (to compensate interference between different fluorochromes) should be run to update the Colour Compensation File. Reagent QCs: Before every lot change of isolation and/or master mix kit: An External Control (external to Artus Biotech) is used to monitor the isolation, amplification and detection procedures. The result must correspond to expected value supplied by the manufacturer. Before every lot change of master mix kit: All four Quantification Standards (QS 1, 2, 3, and 4) must be run. Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor extraction and PCR inhibition. The sample must show a positive reading in either fluorimeter Channel F1 or fluorimeter Channel F3 back-F1 (IC amplicon) to be valid (no PCR inhibition).  A Positive Control (e.g.QS4) is included and shows a positive reading in fluorimeter Channel F1  A Negative Control (e.g. H2O spiked with IC) is include and shows a negative reading in fluorimeter Channel F1 and a positive reading in fluorimeter Channel F3 back F1 (IC amplicon). Report all failed QCs to senior/charge technologist VI. References: QIAamp Viral RNA Mini Kit Handbook Dec. 2005, Qiagen ArtusTM Enterovirus LC RT-PCR Kit User Manual March 2006, Artus LightCycler, Roche Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 116 of 291 Hepatitis B/Hepatitis C/HIV Donor Screen NAT PCR by Cobas® s201 Ampliprep/TaqMan I. 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 II. Specimen Collection and Storage Living donor specimen:  Blood collected in EDTA tube(s) may be stored for up to 48 hours at 2-25 oC and up to 72 hours at 2-8oC  Following separation from cells, plasma may be stored at 2-8 oC for up to 7 days, and up to 30 days at <-18 oC 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 oC followed by up to 24 hours at 2-25 oC prior to plasma or serum separation from red blood cells. For storage longer than 48 hours, separate the plasma or serum from the red 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 2-8oC for an additional 8 days. Alternatively, plasma or serum may be stored at ≤-18 oC 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 for each MPX and/or WNV PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 117 of 291 Do NOT freeze whole blood. Heparin Plasma is NOT acceptable Donor Serology and Molecular Testing Specimen Storage III. Materials, Equipments and Facilities Reagents: 1. Cobas® TaqScreen MPX Test (CS1-CS4) stored at 2-8 oC  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 oC  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 15-30 oC 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 oC or until the expiration date, whichever comes first. Equipment:  Hamilton MICROLAB STAR/STARlet IVD Pipettor  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 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual      IV. Page 118 of 291 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 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 5. 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 119 of 291 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. 6. 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 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 7. Set up MPX NEGATIVE CONTROL and POSITIVE CONTROL racks !!!Mix controls by inversion at least 3 times, avoiding the creation of bubbles, as specified below:  Inversion each time is defined as turning the control upside down and right side up again  Within inversion each time, hold the control for at least 2 seconds in each orientation (i.e. turn the control upside down, and hold for at least 2 seconds. Then, turn the control right side up again, and hold for at least 2 seconds a. Place clean Kleenex paper on Control Working Area b. Remove positive control lids and leave on Kleenex paper. c. 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 d. 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. 8. Pooling Manager Terminal a. Click Wizard Icon PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 120 of 291 b. Make sure Batch Run is selected c. Click Assay/Start b. c. d. e. f. g. h. i. Choose MPX v2.0 Click Next Click Pools of 1 Follow the instruction to load sample rack Click Done button after all samples are loaded All required quantities of S-Tubes,SK24 racks,tips, RMEC (roche manufactured external controls) are calculated PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 121 of 291 f. h. j. Follow instructions to load S-tube rack, control racks, and pipette tip rack. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 122 of 291 i. k. l. m. n. After tips are loaded, pipetting is automatically started All racks are automatically unloaded after the pooling procedure is finished Re-cap the S-tubes .Double check that caps are not so tight and properly placed 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 9. Extraction/Amplification/Detection AMPLIPREP USER ID: MSH PASSWORD: Msh12345 Load in AMPLIPREP – AMPLIPREP Terminal PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 123 of 291 !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 (Start with Track F), 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. 10. Load the SK24 racks directly to the Cobas® TaqMan. 11. If the Amplification and detection run does not start automatically, click Start 12. Check finished time. 13. When testing is finished in Cobas® TaqMan , the results are automatically transmitted to Data Manager. !DO NOT NEED TO MANULLY ACCEPT RESULTS FROM Cobas® TaqMan 14. 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  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 15. Report results in LIS manually. 16. Clean Up and Shutdown Procedures a. Check number of reagents left and write in CS1 then cover with parafilm and store in fridge. b. Discard used SPU tubes and save clean ones. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 124 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual V. Synchronize Hamilton with Ampliprep      VI. 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 1438. VII. Interpretatiion 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 VIII. Reporting For Negative Result Result NonReactive NonReactive CT Values HIV│HBV│HCV│IC ─│─│ ─ │### ─│─│ ─ │─ Internal Control Valid Invalid Report Comment  8MPX:Negative@RMPx Verify 8MPX  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  Verify 8MPX  In the comment for 8COM, record the 2nd result  Verify 8COM  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  Verify 8COM PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 125 of 291 For Positive Result Result HBV Reactive HIV Reactive HCV Reactive CT Values HIV│HBV│HCV│IC ─│###│ ─ │### ###│─│ ─ │### Internal Control Valid Valid Valid ─│─│ ###│### Architect Result 8HAGX:Negative 8HBC:Positive Or 8HAGX:Positive 8HBC:Positive Or 8HAGX:Positive 8HBC:Negative Repeat in Duplicate No 8HAGX:Negative 8HBC:Negative Or No serology results available Yes 8TSC:@MOH+ 8TSC: Positive No Report 1. 8MPX: Positive@RMPx HBV DNA Detected 2. Order 8COM in LIS and record HBV CT a. 8MPX: Positive@RMPx HBV DNA Detected b. Order 8COM in LIS and record HBV CT Enough Volume for repeating: a. 2/3 Positive8MPX :Positive@RMPx HBV DNA Repeatedly Detected  Order 8COM in LIS and record all HBV CT b. 2/3 Negative8MPX: Negative@RMPx  Order 8COM in LIS and record all results NSQ for repeating 8MPX :Positive@RMPx HBV DNA Detected, not sufficient sample for repeating a. 8MPX:Positive@RMPx HIV RNA Detected b. Order 8COM in LIS and record HIV CT 8TSC:Negative Or No serology result availalbe Yes Enough Volume for repeating: a. 2/3 Positive8MPX:Positive@RMPx HIV RNA Repeatedly Detected  Order 8COM in LIS and record all HIV CT b. 2/3 Negative8MPX: Negative@RMPx  Order 8COM in LIS and record all results 8HCA: Positive Or 8HCA:@MOH+ No NSQ for repeating 8MPX :Positive@RMPx HIV RNA Detected, not sufficient sample for repeating 8MPX: Positive@RMPx HCV RNA Detected 8HCA:Negative Or No serology result available Yes Enough Volume for repeating: a. 2/3 Positive8MPX:Positive@RMPx HCV RNA Repeatedly Detected  Order 8COM in LIS and record all HCV CT b. 2/3 Negative8MPX: Negative@RMPx  Order 8COM in LIS and record all results NSQ for repeating 8MPX :Positive@RMPx HCV RNA Detected, not sufficient sample for repeating PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual IX. 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 X. Page 126 of 291 Solutions Repeat specimen Result specimen as: Invalid In result comment: hit F5 to add comment “@SPIN” “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 Quality Control : Run External Positive and negative control (previously tested CAP proficiency samples) monthly. XI. CAP and NML provide external proficiency testing. Reference: Roche cobas s 201 System Hardware and Software reference Manual, P/N 05575982190-01. PDM Software Software Guide,P/N 05575974190-01. Operator’s Manual for Use with small Pools(Pools of 1 and 6) P/N 05576008190-01 Roche Hot Line Tel# 877-273-3433 michael.salem@roche.com xinyu.song@roche.com PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 127 of 291 Herpes simplex Virus/Varicella-Zoster Virus PCR by Rotorgene I. Introduction Herpes simplex virus (HSV) and Varicella zoster virus (VZV) are doube stranded DNA virus in the family Herpesviridae. HSV and VZV remain in the host as a latent lifelong infection in the peripheral nervous system, and can reactivate in the host, causing serious complications in immunocompromised patients. The RealStar alpha Herpesvirus PCR Kit is a real-time PCR, for the qualitative detection and differentiation of Herpes simlex virus type 1, type 2, and Varicella zoster virus in a single PCR reaction. II. Collection and Transport Sterile body fluids, swabs in viral transport media. Store collected specimens at 4oC, process specimens as soon as possible. III. Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG IV. Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and gloves Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8) and microcentrifuge(MICT17) Vortex Detection Area for Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System programmed for ALTONA HSV & VZV 36-Well Loading Block (pre-cooled to 4oC) 36-Well 6000 Series Rotor & Locking Ring 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor & Locking Ring 0.1 mL Strip Tubes and Caps 0.2mL microtubes Aerosol Resistant Tips (ART) Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL RealStar alpha Herpes PCR Kit v1.0: stored at -20C until needed PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 128 of 291 Component Master A Component Master B HSV-1 Positive Control HSV-2 Positive Control VZV Positive Control PCR Water alpha Herpes Internal Control External Control: To be run when a new lot is used, during training or if QC failure occurs. GENERAL PRECAUTIONS:             V. There must be separate PCR work areas: o Clean room o Specimen preparation room Amplification room Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas. Change lab coats and gloves between work areas. Only blue gowns are to be worn in the clean room. Use only filtered pipette tips Use only sterile RNase, Dnase-free microcentrifuge tubes Use sterile, disposable polypropylene tubes throughout the procedure Always wear powder-free gloves when handling reagents Change gloves frequently and keep tubes closed whenever possible Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water. Detection Set-up: Organize eluates and controls according to worklist. In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) Take out the necessary number of alpha Herpes Components Master A and Master B vials (12 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Prepare alpha Herpes Master Mix (number of test samples + controls + 1 extra test) required according to the chart. Work quickly. Protect Master A and Master B from light. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 129 of 291 Mix gently, DO NOT VORTEX. Avoid repeated thawing and freezing of Master A and Master B (maximum twice). After Master A has been mixed to Master B it cannot be frozen again. No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 a. Place microtubes into the Cooling Block (pre-cooled to 4oC). b. Pipette 20 uL alpha Herpes Master Mix (A+B) into each microtubes a. Pipette 1.0 uL of internal control (IC) into the microtubes designated for the Positive controls (HSV-1, HSV-2, VZV) and water (PCR grade). b. Pipette 10uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and cap tightly. Label each microtubes 1,2,3…. before leaving clean room. c. Changed out of dedicated clean room gown. Specimen Processing Area: (Changed into specimen area lab coat) Carefully pipette 10 uL each of eluate sample, Positive controls (HSV-1, HSV-2, and VZV), and external QC (if using) directly into the microtubes containg alpha Herpes Master Mix. Pipette up and down three times (3X) into the Master Mix to mix sample with Master Mix. Close the cap tightly. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 130 of 291 Detection Area:     Check each microtube before loading. Make sure the microtube is capped tightly and there is no bubble at the bottom of the microtube. Load microtubes into rotor (e.g. 72 Rotor or 36 Rotor) and secure the locking ring. Fill the empty position of the rotor using empty microtubes. Place Rotor in to the Rotor-Gene 6000. On the Rotor-Gene 6000: a. Turn on computer, turn on Rotorgene; b. FOR THE BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; c. Double click(left on the mouse) “Rotor-Gene 6000 series software 1.7” icon d. Wait for “Initializing machine…..” e. Highlight “ALTONA HSV & VZV ” assay; f. Press “New” button; g. Highlighting the correct Rotor type you are using h. 72-well Rotor (Blue) i. 36-well Rotor (Red) j. Click the “Locking Ring Attached” option, to confirm Locking Ring is in place. k. Press “Next” button l. Enter initials in Operator space. Enter kit lot number and the Rotor-Gene Colour (ie.Red/Blue) in the Notes box. Verify the Reaction Volume is 30ul. m. Press “Next” button. n. Temperature Profile window pops up; press “Next”. o. Summary window appears; press “Start Run”. The RotorGene will start. p. Save as window appears; go to My documents -Open–select hsv & vzv result folder- Open Press Save –HSV VZV yyyy-mm-dd (run no.) q. Edit Samples window. Enter sample LIS numbers using barcode scanner. Enter HSV-1, HSV-2, VZV as Positive Controls, and water as negative Control. Name Type Select Sample Unknown Yes Positive Controls HSV-1, HSV-2, VZV Positive Control Yes Water Negative Control Yes r. Once finishing all samples, press Finish button. s. Once cycling screen comes on, click “Named On” button. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 131 of 291 To analyze and print HSV and VZV report after a run is finished: The run is done when Profile Program reads: Run has completed. Analyze raw data by clicking on the each of these channels (one at a time). Green channel (VZV), Red channel (HSV-2), Orange channel (HSV-1) and Yellow channel (IC). These tabs are found on the second menu bar from the top, on the left hand side. Select the Green Channel (VZV). Click “All Off” (bottom right). Select sample “water Negative Control” and “VZV Positive Control”. Click on Autoscale (bottom left). Compare each specimen curve to the water curve and the VZV Positive Control one at a time (by clicking each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worklist. Select the Orange Channel (HSV-1). Click “All Off”. Select sample “water Negative Control” and “HSV-1 Positive Control”. Click on Autoscale (bottom left). Compare each specimen curve to the water curve and the HSV-1 Positive Control one at a time (by clicking each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worklist. Select the Red Channel (HSV-2). Click “All Off”. Select sample “water Negative Control” and “HSV-2 Positive Control”. Click on Autoscale (bottom left). Compare each specimen curve to the water curve and the HSV-2 Positive Control one at a time (by clicking each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worklist. a. b. c. d. e. Click on “Named On” Perform curve Analysis (Get report): Click “Analysis” on the top tool bar. Analysis floating window pops up; Click Quantitation Analysis. Quantitation window is brought up to the front. There should be four channels: Green (VZV), Orange (HSV-1), Red (HSV-2) and Yellow (IC). f. g. h. i. j. k. l. Highlight Cycling A. Green (VZV) option and press Show. Maximize cycling green window (or the channel you are working on) Press button “Slope Correct” on the top. Press button “Ignore first” on the top, and type “5”, press OK. Set threshold at 0.05. A threshold line will appear on the screen. Move threshold line to above the noise (i.e. Negative specimens) To Print reports: Press “Reports”. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual m. n. o. p. q. r. s. t. Page 132 of 291 Report Browser window pops up. Choose Cycling A. Green (Page 1). Highlight Quantitation full report, press “Show”. Maximize the report window, check that only the positives that you have identified from the raw data review are positive and have a crosspoint (ct value). Print pages 1-4 for the green channel; pages 2-4 for all the other channels: orange, red. Repeat the steps above 3-15 for Cycling A. Orange(HSV-1), Cycling A. Red (HSV-2). Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab, select Cycling A. Yellow -the internal control (IC), select Show. Maximize the graph Quantitation Analysis –Cycling A. Yellow. Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in the first 5 cycles of the run). Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph screen. Move the threshold line to above the noise. Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show. All samples should have a positive IC curve, and a cycle number in the Ct column. Print pages 2-4. (For Cycling A. Yellow report, if there is message of “NEG (Multi Ct)” for any specimen, you have to go back to check Raw Data of cycling A.Yellow. If all specimens are amplified, adjust the threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct. Adjust the threshold line so that the threshold line crosses the curve at only one point.) Close the outer most window. “Save changes to Altona HSV & VZV yyyy-mm-dd?” Select Yes. Shut down the computer, and switch off the Rotor-Gene. At the end of the day, follow the cleaning protocol below using 1% hypochorite followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet, pipettors, and bench tops Specimen Preparation Area:  Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.  Cap discard container.  Wash racks. Amplification Area:  Discard microtubes into a disposal bag and tight the bag.  Discard the bag into biohazard waster container.  Perform the cleaning procedure according the daily maintenance sheet. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VI. Page 133 of 291 Interpretation: Green Channel (VZV) Orange Channel (HSV-1) + - - + - - Red Channel (HSV-2) - Yellow Channel (IC) Interpretations +/- Positive for VZV -* +/- Positive for HSV-1 - + +/- Positive for HSV-2 - - + - - - Negative for HSV and VZV INVALID: internal control failed to amplify. Dilute sample 1:4 and re-extract and test. * Cross-talk: Samples and controls that are strong positives in the Orange HSV-1 Channel, can be observed to exhibit a diminished weak curve in the Red HSV-2 Channel (y-axis fluorescence is very weak); these samples and controls are not HSV-2 positive. PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report, see below “Reporting”. VI. Reporting: Report HSV and VZV results for sterile body fluids, tissues, eye swabs, and skin/lesion specimens only. Report HSV results only on oral swabs/washings, and BALs. Negative for HSV and VZV. Under media on the back of the LIS workcard. PCHZ: F6 to add run date Report on the front of the LIS workcard from the keypad select: }HVZ}HVZ- Negative for Herpes simplex virus. Negative for Varicella zoster virus. This is a research test. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 134 of 291 Alpha Herpes Virus PCR Kit v1.0. Altona Diagnostics Inc. Ctrl-F to finalize the report. Sterile sites: PRELIMINARY Positive for HSV-1, or HSV-2, or VZV. Date the media on the back of the LIS workcard. PCHZ: 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: 15hsv1 for HSV-1 15hsv2 for HSV-2 18vzv for VZV Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Ctrl D. Select from the keypad: \HSV+ to add the comment phrase: \HSV+ DETECTED by PCR, confirmation to follow. This is a research test. Alpha Herpes Virus PCR Kit v1.0. Altona Diagnostics Inc. Grave (`) the result. Ctrl-P to prelim the report. Call reports to ward/doctor. Sterile sites: CONFIRMED Positive for HSV-1, or HSV-2, or VZV. Date the media on the back of the LIS workcard. PCHZ: 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: 15hsv1 for HSV-1 15hsv2 for HSV-2 18vzv for VZV Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Ctrl D. Select from the keypad: \HSV+ to add the comment phrase: \HSV+ DETECTED by PCR, confirmed. This is a research test. Alpha Herpes Virus PCR Kit v1.0. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 135 of 291 Altona Diagnostics Inc. Grave (`) the result. Ctrl-F to finalize the report. Sterile sites: NOT CONFIRMED for HSV-1, or HSV-2, or VZV. Date the media on the back of the LIS workcard. PCHZ: F6 to add run date Isolate #: 1 must be suppressed, change to an alphabet Front of the LIS workcard type the following: UPDATED REPORT: Previously reported Herpes simplex virus type 1/ Herpes simplex virus type 2/ or Varicella zoster virus not confirmed. Report on the front of the LIS workcard (manually type): This is a research test. Alpha Herpes Virus PCR Kit v1.0. Altona Diagnostics Inc. Ctrl-F to finalize the report. Call Updated reports to ward/doctor. Positive for HSV-1, or HSV-2, or VZV. Date the media on the back of the LIS workcard. PCHZ: 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: 15hsv1 for HSV-1 15hsv2 for HSV-2 18vzv for VZV Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Ctrl D. Select from the keypad: \HSV+ to add the comment phrase: \HSV+ DETECTED by PCR. This is a research test. Alpha Herpes Virus PCR Kit v1.0. Altona Diagnostics Inc. Grave (`) the result. Ctrl-F to finalize the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 136 of 291 Calling Results: HSV-1, HSV-2, and VZV DETECTED from sterile body fluids, tissues, and eye fluids/swabs should be called to ward and/or doctor. HSV-1, HSV-2, and VZV DETECTED from CSF must be faxed and phoned to the Medical Officer of Health (MOH). VZV DETECTED from skin/lesion specimens must be phoned to the ward and/or doctor and the Infection Control Practitioner (ICP) must be notified. IX. 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 for VZV, HSV-1, and HSV-2 should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition.  A Positive Control is included and shows either a positive reading in Green Channel (VZV) or a positive reading in Orange Channel (HSV-1), or a positive reading in Red Channel (HSV-2)  A Negative Control is included and shows a negative reading in Green Channel (VZV), Orange Channel (HSV-1), and Red Channel (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). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 137 of 291 e. If positive QC material yielded negative result, repeat the entire run. f. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. g. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. X. References: Altona alpha Herpes Virus PCR Kit v1.0 Instructions, Altona Diagnostics PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 138 of 291 Human Metapneumovirus RT-PCR by Rotorgene I. Introduction: Human metapneumovirus (hMPV) is a Paramyxovirus first isolated 2001. hMPV infections may resemble RSV, causing mild upper respiratory tract infections to severe lower respiratory tract infections. hMPV is most frequently seen during winter months. All ages may be affected, but children and people with compromised immune systems are most at risk. RealStar hMPV RT-PCR Kit 1.0 is a real time PCR test for the qualitative detection of hMPV RNA; differentiating between hMPVsubtype A and hMPV subtypeB. II. Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as possible. BAL specimens if mucoid treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the Eppendroff 5415 C Microcentrifuge. Aliquot supernatant and store at 4C, extract on the easyMag as soon as possible. III. Specimen Processing Please refer to Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG 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. Materials, Equipments and Facilities: Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) Rotor-Gene 6000 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 139 of 291 Rotor-Gene 6000 Multiplexing System with experiment template: Altona RealStar PIV & hMeta 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor 72-Well Rotor Locking Ring 0.1 mL reaction microtubes Tubes and Caps Variable volume Rainin pipettes: 1 to 20 uL, 10 to 200 uL, 100 to 1000 uL Reagent: Altona RealStar hMPV RT-PCR Kit 1.0: MasterA, MasterB, Internal Control, hMPV A Positive Control, hMPV B Positive Control, PCR Grade Water External Control: To be run first week of the month/ or with every new lot; run during training or if QC failure occurs. IV. Procedure: Prepare samples in order according to hMetapneumovirus (hMPV) worksheet. Add the following controls to the hMetapneumovirus Worksheet: hMPV A Positive control, hMPV B Positive Control, Negative Control (PCR grade Water). PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) Remove to thaw at room temperature the required vial’s hMPV Master A and Master B (12 reactions/ vial) from the freezer. Prepare hMPV Master Mix in Sarstedt 1.5 mL conical microtube; to load on the Eppendorf epMotion. Prepare the number of Master Mix reactions required: Number of Test samples + 3 Controls + one Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added to Master B it cannot be frozen again. Number of Reactions hMPV Master A hMPV Master B Volume of Master Mix Sample/Control Volume 1 5 10 15 10 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual No. of Test reactions 1 2 3 4 5 6 7 8 9 10 11 12 Page 140 of 291 hMPV Master Mix hMPV Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 hMPV Master B (µl) 10 20 30 40 50 60 70 80 90 100 110 120 See Eppendorf epMotion Manual for further loading and programming instructions. Manual Loading: Load one reaction microtube (0.1 mL) for each sample/control. Pipette 15 uL prepared hMPV Master Mix into each reaction microtube. Pipette 1.0 uL of internal control into the reaction microtubes designated for the 3 hMPV Controls (hMPV A Positive Control, hMPV B Positive Control, Negative PCR Water Control . In the Specimen Processing Area: Pipette 10uL each of Sample/Control into reaction microtubes. Mix by pipetting 3x up and down into the Master Mix. Cap each segment, after pipetting is completed. Check reaction microtubes before loading into the rotor ensure the liquid levels are at the same height, and there are no bubble at the bottom of the reaction microtube. Load reaction microtubes into 72-Well Blue rotor, fill empty rotor spaces with blank reaction microtubes, attach the Locking Ring. Load the rotor in Rotor-Gene 6000. In the Rotorgene Detection Area: a. Switch ON computer, switch ON Rotorgene; b. FOR BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; c. Open “Rotor-Gene 6000 series software 1.7” icon d. Wait for “Initializing machine…..” e. Highlight “Altona RealStar PIV & hMeta” experiment f. Select “New” button; g. Highlighting the correct Rotor type you are using h. 72-well Rotor (Blue) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 141 of 291 i. Check the box for “Locking Ring Attached” option j. Select “Next” button k. Enter operator initials. In Notes enter RotorGene Colour (i.e. RED or BLUE) Reagent Lot number. Make sure the Reaction Volume is “25µl”; l. Select “Next” button Temperature profile window appears, select “Next” button m. Rotor must be loaded. n. Summary window appears, select “Start Run” button; o. Save As window appears; the run is given a filename with a default template. Save in My documents in PIV & hMeta folder for the current year: e.g. Altona Realstar PIV & hMeta 20150101(1) (e.g. assay name-year-month-date-numerical run#); a. Note: PIV & hMeta results are stored in \desktop\My Document\Altona RealStar PIV & hMeta p. Select Save button; q. Machine starts and Edit Samples window appears. r. Enter sample numbers including positive and negative controls. Define the type of samples according to the chart: Name Sample hMPV A Positive Control hMPV B Positive Control PCR Grade Water Type Unknown Select Yes Positive Control Yes Positive Control Yes Negative Control Yes s. Select Finish button. t. Once cycling screen appears select “Named On” button. To analyze and print PIV report after a run is finished: a. The run is done when Profile Program reads: Run has completed. b. Analyze Raw data by selecting each channel, one at a time: Green hMPV B Channel, Red hMPV A Channel and Yellow channel Internal Control (IC). Target Channel hMPV B RNA Green hMPV A RNA Red Internal Control Yellow Report Dye FAM Cy5 JOE c. Select Autoscale. Select “All Off”. Select Negative (water) Control. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s. t. u. v. Page 142 of 291 Compare the specimen and control curves to the water one at a time. A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of the positives on the hMPV Worksheet. Select on “Named On” Select Analysis. Analysis floating window pops up Select “Quantitation” Tab. Quantitation window is brought up to the front. There should be three channels: Green hMPV B, Red hMPV A, and Yellow (IC). Highlight Cycling A. Green hMPV B. Select Show. Maximize Target (Green/Red/Yellow) graph window Select slope correct Select Ignore first… 5 cycles Select Threshold icon, and set threshold. Move threshold to remove noise.(i.e. Negative specimens) Print reports: Select Reports. Report Browser window pops up. Choose Cycling A. Green hMPV B. Under templates box, choose “Quantitation full report”. Select Show button Maximize the report window, check that only the positives that you have identified from your raw data analysis are positive (i.e. have a crosspoint). Choose print report. (red and yellow channels print only pages 2-4). Once finished printing, close the report window Analysis, perform analysis on remaining channels: Red hMPV A Channel, Yellow IC Channel. Shut down computer: Exit from software. “Save changes”. Select “yes”. Select “start” button. Select “Turn off computer”. Switch off Rotorgene End of Shift Cleaning: Perform cleaning protocol as outlined below. 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 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 143 of 291 Wash racks. EasyMAG , Lysis Rack, and BioHit Multi-channel Pipette: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe, followed by UltraPure water, and then 70% alcohol Wipe 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. IV. Interpretation and Reporting: Red Channel hMPV A Yellow Channel (IC specific) Interpretations + - +/- hMPV B DETECTED - + +/- hMPV A DETECTED + + +/- hMPV B DETECTED. hMPV A DETECTED. (ask charge/senior for confirmation) - - + hMPV NOT DETECTED - - - INVALID Respiratory sample needs to be diluted 1:4 with VTM and re-extracted and RT-PCR repeated. Green Channel hMPV B VTM=Viral Transport Media hMPV B DETECTED. or hMPV A DETECTED Report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard PCMET: F6 to add the date tested. In the media PCMET enter crosspoint (ct) value : ct= Report the virus as an isolate in the F7 window. Isolate #: 1, or next available isolate number. Org. ID: 25mpvA Metapneumovirus A DETECTED 25mpvB Metapneumovirus B DETECTED PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 144 of 291 The reporting isolate comment is attached to the Organism ID. Verify All in the isolate window. Review report entry: media, and isolates. Finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). Document calls and emails in the media CALL. hMPV NOT DETECTED Date the media on the back of the workcard. PCMET: F6 to add run date. On the front of the workcard, select from the keypad: }MET}MET- Negative for metapneumovirus by RealStar hMPV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. hMPV INDETERMINATE Internal control has failed twice to amplify in a sample. Date the media on the back of the LIS workcard. PCMET: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRMET Under this media ^PRMET report the 1:4 dilution test result type “IC no amplification”. Report: select from the keypad: }METChange “Negative” to INDETERMINATE; final report should appear as: INDETERMINATE for metapneumovirus by RealStar hMPV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. V.  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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual       a. b. c. d. e. f. g. h. i. j. Page 145 of 291 External control for hMPV A, and hMPV B should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every Run Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition. A Positive Control is included and shows a positive reading in Green hMPV B Channel or a positive reading in Red hMPV A Channel A Negative Control is include and shows a negative reading in both Green hMPV B Channel or a negative reading in Red hMPV A Channel Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. V. References: RealStar hMPV RT-PCR Kit v1.0, altona Diagnostics Emerging Infectious Disease Journal Volume 14, Number1-January 2008 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 146 of 291 Human Metapneumovirus and Parainfluenza RT-PCR Bio-Rad CFX96 VIII. Introduction: Human metapneumovirus (hMPV) is a Paramyxovirus first isolated 2001. hMPV infections may resemble RSV, causing mild upper respiratory tract infections to severe lower respiratory tract infections. hMPV is most frequently seen during winter months. All ages may be affected, but children and people with compromised immune systems are most at risk. RealStar hMPV RT-PCR Kit 1.0 is a real time PCR test for the qualitative detection of hMPV RNA; differentiating between hMPVsubtype A and hMPV subtypeB. Parainfluenza viruses (PIV) can cause upper and lower respiratory tract infections. Commonly seen in children, but may occur at any age group causing severe disease in older adults, and people with compromised immune systems. PIV-1 and PIV -2 often causes cold-like symptoms, and croup in children. PIV-3 is often associated with bronchiolitis, bronchitis, and pneumonia. PIV-4 may cause mild to severe respiratory tract infections. RealStar PIV RT-PCR is a real time PCR for the qualitative detection of human PIV RNA; differentiating PIV-1and 3, and PIV-2 and 4. IX. Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as possible. BAL specimens if mucoid treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the Eppendroff 5415 C Microcentrifuge. Aliquot supernatant and store at 4C, extract on the easyMag as soon as possible. X. Specimen Processing Please refer to Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual     Page 147 of 291 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. XI. Materials, Equipments and Facilities:  Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG)  Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8)  BIO-RAD CFX96 Deep WellTM 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  Reagent: Altona RealStar hMPV RT-PCR Kit 1.0: MasterA, MasterB, Internal Control; Altona RealStar PIV RT-PCR Kit 1.0: MasterA, MasterB, Internal Control; hMPV A Positive Control, hMPV B Positive Control PIV-1 Positive Control, PIV-2 Positive Control, PIV-3 Positive Control, PIV-4 Positive Control PCR Grade Water  External Control: To be run first week of the month/ or with every new lot; run during training or if QC failure occurs. XII. Procedure: Prepare samples in the order according to hMetapneumovirus and Parainfluenza viruses (hMPV and PIV) worksheet. Add the following controls to the Worksheet: hMPV A Positive control, hMPV B Positive Control, Negative Control (PCR grade Water); PIV1/3 Positive control, PIV2/4 Positive control, Negative Control(PCR grade Water). PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) Remove the required vials of hMPV Master A and Master B (12 reactions/ vial) from the freezer and thaw them at room temperature C. To be loaded by epMotion   Prepare hMPV Master Mix in Sarstedt 1.5 mL conical microtube; Prepare the number of Master Mix reactions required: Number of Test samples + 3 Controls + extra one PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 148 of 291 Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added to Master B it cannot be frozen again. Number of Reactions hMPV Master A hMPV Master B Volume of Master Mix Sample/Control Volume No. of Test reactions 1 2 3 4 5 6 7 8 9 10 11 12 1 5 10 15 10 hMPV Master Mix hMPV Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 hMPV Master B (µl) 10 20 30 40 50 60 70 80 90 100 110 120 See Eppendorf epMotion Manual for further loading and programming instructions. D. To be loaded by manual:     Place 96-well plate onto pre-cooled block Pipette 15 uL prepared hMPV Master Mix into each reaction well; Pipette 1.0 uL of internal control into the wells designated for the 3 hMPV Controls (hMPV A Positive Control, hMPV B Positive Control, Negative PCR Water Control; Pipette 10 uL PCR Water into the well designated for Negative Control. In the Specimen Processing Area: Prepare working P1/P3 and P2/P4 positive controls:  Prepare Working aliquot P1/P3 Positive Control and P2/P4 Positive. Prepare as needed, use aliquot until finished, and then prepare a new aliquot.  In a 1.5mL conical microtube pipette 180 uL PCR grade water add 20uL PIV-1 Positive Control and add 20uL PIV-3 Positive Control; label as P1/P3 Positive Control with date. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 149 of 291 In a 1.5mL conical microtube pipette 180 uL PCR grade water add 20uL PIV-2 Positive Control and add 20uL PIV-4 Positive Control; label as P2/P4 Positive Control with date. Working Positive Control PCR Grade Water PIV Control 180uL 20uL PIV-1 Positive Control P1/P3 Positive Control 20uL PIV-3 Positive Control 180uL 20uL PIV-2 Positive Control P2/P4 Positive Control 20uL PIV-4a Positive Control 20uL PIV-4b Positive Control  Pipette 10uL each of Sample/Control into reaction wells. Mix by pipetting 3 times up and down into the Master Mix.  Seal the plate after pipetting is completed.  Check reaction wells before loading into CFX96 Deep Well Real-Time System: ensure the liquid levels are at the same height, and there are no bubble at the bottom of each reaction well.  Double click Bio-Rad CFX Manager icon  Startup Wizard window pops up  Make sure CFX96 Deep Well is selected  Under Select run type, click User-defined button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 150 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Run Set up window pops up, including three tabs-Protocol, Plate, and Start Run  Click Open Lid button  Load sealed plate to the block  Click Close Lid button  WARNING! Do NOT manually close the motorized lid  Under Protocol tab, select Altona PIV RT-PCR.prcl from Express Load pull-down menu to open the lid to close the lid Express Load pulldown menu Open and Close Lid button  Click Plate tab on the top or click Next button side to load the plate profile on the bottom right PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Select hMPV and PIV.pltd plate profile from the Express Load pull-down menu  Click Start Run tab bottom right side on the top or click Next button Page 151 of 291 on the PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Confirm the following information:  Protocol: Altona PIV RT-PCR.prcl  Plate: hMPV and PIV.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  Name and save the file in the designated folder: Page 152 of 291 T:Microbiology>Virology>Bio Rad CFX96 PCR>Save Run>Respiratory PCR 2015  Click Save button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 153 of 291 Build Worklist  Open the worklist file according to the following path: T:Microbiology>Virology>Bio Rad CFX96 PCR>Worklist Mastcopy>Metapara Worklist-excel  Password window pops up, click Read only button  Scan the samples’ information  Row A to Row D are designated for hMeta detection  Row E to Row H are designated for PIV detection  File>Save as PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 154 of 291 Save the worklist at T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist>Respiratory Worklist2015 File name: Metapara yyyy.mm.dd.run No. eg. Metapara2015.04.07.1 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 155 of 291 Import wordlist  Click Realtime Status tab  Select View/Edit Plate… from Plate Setup pull-down menu Plate Setup pull-down menu  Plate Editor window pops up PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Spreadsheet View/Importer button  Plate Spreadsheet View window pops up  Select HEX from Fluors List pull-down menu  Click Import button Page 156 of 291 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 157 of 291  Select Import File window pops up  T:Microbiology>Virology>Bio Rad CFX96 PCR>Import Worklist>Respiratory Worklist2015  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  Type IC as target Name for HEX channel  Hit Enter key on the key board  Click Well groups button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Click top left side corner to select all wells Page 158 of 291 Type IC and hit Enter key  Well Groups Manager window pops up  Click Add button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 159 of 291  Select wells including control wells for Meta detection (selected wells should be highlight)  Change the name Group 1 to meta  Click Add button  Group 2 shows up  Select wells including control wells for Para detection (selected wells should be highlight)  Change group 2 to para PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click OK button  Click Ok button from Plate Editor window  Click Yes button from the popping up window Page 160 of 291 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Time Status tab Page 161 of 291 from the Run Details window PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 162 of 291 Analyze results after the PCR is completed Export and Print Hmpv result  Choose meta from Well Groups pull-down menu  Change the channel names from Fluorophore to Target  Set the threshold line for each target channel  Click Export PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Select Custom Export…  Click Export button  Save as Window pops up  Change the File name according to the target and save in the following folder: Page 163 of 291 T:\Microbiology\Virology\Bio Rad CFX96 PCR\Exported Excel Results  Click Save button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Once Export completed, Click OK button  The final results with Ct values are exported in Excel sheet  Print the excel sheet and attach it to the hMPV worksheet  Close excel Page 164 of 291 Export and Print PIV result  Choose para from Well Groups pull-down menu  Change the channel names from Fluorophore to Target  Set the threshold line for each target channel  Click Export  Save as Window pops up  Change the File name according to the target and save in the following folder: T:\Microbiology\Virology\Bio Rad CFX96 PCR\Exported Excel Results  Click OK button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Click Close button  Print the excel sheet and attach it to the hMPV worksheet  Close excel  Close CFX96 Deep Well Real-Time Systim  Shut down computer Page 165 of 291 End of Shift 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. EasyMAG , Lysis Rack, and BioHit Multi-channel Pipette: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe, followed by UltraPure water, and then 70% alcohol Wipe 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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 166 of 291 XIII. hMPV Interpretation and Reporting: Cy5 Channel hMPV A HEX Channel (IC specific) Interpretations + - +/- hMPV B DETECTED - + +/- hMPV A DETECTED FAM Channel hMPV B + + +/- hMPV B DETECTED. hMPV A DETECTED. (ask charge/senior for confirmation) - - + hMPV NOT DETECTED - INVALID Respiratory sample needs to be diluted 1:4 with VTM and re-extracted and RT-PCR repeated. - - VTM=Viral Transport Media hMPV B DETECTED or hMPV A DETECTED Report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard PCMET: F6 to add the date tested. In the media PCMET enter crosspoint (ct) value : ct= Report the virus as an isolate in the F7 window. Isolate #: 1, or next available isolate number. Org. ID: 25mpvA Metapneumovirus A DETECTED 25mpvB Metapneumovirus B DETECTED The reporting isolate comment is attached to the Organism ID. Verify All in the isolate window. Review report entry: media, and isolates. Finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). Document calls and emails in the media CALL. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 167 of 291 hMPV NOT DETECTED Date the media on the back of the workcard. PCMET: F6 to add run date. On the front of the workcard, select from the keypad: }MET}MET- Negative for metapneumovirus by RealStar hMPV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. hMPV INDETERMINATE Internal control has failed twice to amplify in a sample. Date the media on the back of the LIS workcard. PCMET: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRMET Under this media ^PRMET report the 1:4 dilution test result type “IC no amplification”. Report: select from the keypad: }METChange “Negative” to INDETERMINATE; final report should appear as: INDETERMINATE for metapneumovirus by RealStar hMPV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 168 of 291 XIV. PIV Interpretation and Reporting: FAM Channel PIV1/3 Cy5 Channel PIV2/4 HEX Channel (IC specific) Interpretations + - +/- PIV-1/3 DETECTED - + +/- PIV-2/4 DETECTED + + +/- PIV-1/3 DETECTED. PIV-2/4 DETECTED. (ask charge/senior for confirmation) - - + PIV NOT DETECTED - INVALID Respiratory sample needs to be diluted 1:4 with VTM and re-extracted and RT-PCR repeated. - - VTM=Viral Transport Media PIV-1/3 DETECTED. or PIV-2/4 DETECTED Report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard PCPIN: F6 to add the date tested. In the media PCPIN enter crosspoint (ct) value : ct= Report the virus as an isolate in the F7 window. Isolate #: 1, or next available isolate number. Org. ID: 24par13 Parainfluenza virus 1 and/or 3 24par24 Parainfluenza virus 2 and/or 4 The reporting isolate comment is attached to the Organism ID. Verify All in the isolate window. Review report entry: media, isolate. Finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). Document calls and emails in the media CALL. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 169 of 291 PIV-1/3 NOT DETECTED. or PIV-2/4 NOT DETECTED Date the media on the back of the workcard. PCPIN: F6 to add run date. On the front of the workcard, select from the keypad: }PI}PI- Negative for parainfluenza virus by RealStar PIV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. PIV INDETERMINATE Internal control has failed twice to amplify in a sample. Date the media on the back of the LIS workcard. PCPIN: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRPIN Under this media ^PRPIN report the 1:4 dilution test result type “IC no amplification”. Report: select from the keypad: }PIChange “Negative” to INDETERMINATE; final report should appear as: INDETERMINATE for parainfluenza virus by RealStar PIV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. XV.       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 for hMPV A, hMPV B, PIV1/3, and PIV 2/4 should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every Run Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition. A Positive Control is included and shows a positive reading in FAM channels (hMPV B and PIV 1/3) Channel or a positive reading in Cy5 Channels ( hMPV A and PIV 2/4) A Negative Control is include and shows a negative reading in both FAM channels (hMPV B and PIV 1/3) Channel and Cy5 Channels ( hMPV A and PIV 2/4) Report all failed QCs to senior/charge technologist. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual a. b. c. d. e. f. g. h. i. j. Page 170 of 291 Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. XVI. References: RealStar hMPV RT-PCR Kit v1.0, altona Diagnostics RealStar PIV RT-PCR Kit v1.0, altona Diagnostics cdc.gov/Human Parainfluenza Viruses (HPIVs) (/parainfluenza/index.html Emerging Infectious Disease Journal Volume 14, Number1-January 2008 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 171 of 291 Human Papilloma Virus (HPV) PCR by Cobas® 4800 I. Introduction The cobas® 4800 HPV Test is for the qualitative in vitro detection of Human Paillomavirus in patients specimens using PCR. There are 118 types of HPV, and approximately 40 different HPVs that can infect the human anogenital mucosa. The cobas® 4800 HPV test specifically identifies types HPV 16 and HPV 18, and also detects high risk types (31, 33, 39, 45, 51, 52, 56, 58, 59, 66, and 68). HPV is a small, non-enveloped, double stranded DNA virus with a genome of approximately 8000 nucleotides. The presence of HPV has been found in 99% of cervical cancers, with types 13 to 18 is considered high-risk for the development of cervical cancer, and its precursor lesions. II. Specimen Collection/Transport/Storage Cervical specimens collected in primary container: PreservCyt Solution, cobas® PCR Cell Collection Media, and SurePath Preservative Fluid. Minimum volume in primary container should be 3.0 mL. Primary containers for PreservCyt Solution, and cobas® PCR Cell Collection Media should be vortex prior to loading on the cobas® x480. Cervical specimens in PreservCyt Solution, cobas® PCR Cell Collection Media, and SurePath Preservative Fluid can be transported at 2-30°C. Cervical specimens collected in SurePath Preservative Fluid should be refrigerated 24 hour after collection. Cervical specimens collected in PreservCyt Solution, cobas® PCR Cell Collection Media, may be stored at 2-30°C for up to 6 months after collection date. Cervical samples collected in SurePath Preservative Fluid may be stored at 2-8°C for up to 4 weeks after collection. III. Reagents and Materials a) cobas® 4800 System Liquid Cytology Preparation Kit – 240 tests (stored at 2-8°C) PK- Proteinase K SDS- 4800 System SDS Reagent LYS- 4800 Lysis Buffer b) cobas® 4800 System Sample Preparation Kit – 240 test (stored at 2-8°C) MGP-Magnetic glass particles EB-Elution buffer c) cobas® 4800 System Wash Buffer Kit – 240 test kits (stored at 15-25°C) WB-Wash Buffer d) cobas® 4800 HPV Amplification/Detection Kit – 240 test (stored in Clean Room at 2-8°C) HPV MMX-Master Mix HPV Mg/Mn-Magnesium/Magnesium e) cobas® 4800 HPV Controls Kit (stored at 2-8°C) HPV (+) C - Positive Control PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual f) g) h) i) j) k) l) Page 172 of 291 HPV (-) C - Negative Control Reagent Reservoirs – 50mL and 200 mL containers cobas® 4800 System Extraction (deep well) Plate cobas® 4800 AD (microwell) Plate CO-RE pipette tips (1000 uL) rack of 96 Plate carrier Tip carrier Sample and reagent racks IV. Instrumentation and Software cobas® x 480 Instrument cobas® z 480 Analyzer Heater/Shaker Unit cobas® 4800 System Control Unit V. Procedure NOTE: All reagents must be at except HPV Amplification/Detection Kit should be at room temperature before loading on the cobas® x480. HPV MMX and HPV Mg/Mn can be loaded directly on cobas® x480 from 2-8°C. Specimens in PreservCyt Solution, cobas® PCR Cell Collection Media, and SurePath Preservative Fluid must be at room temperature before loading on the cobas® x480. 1. Turn on the system as the following sequence: 1. cobas® z 480 2. Heater/Shaker unit 3. cobas® x 480 instrument 2. When the left orange light on the cobas® z 480 turns to solid green, turn on the computer workstation. 3. Remove one set of reagents from cobas® 4800 System Liquid Cytology Preparation Kit, one set of reagents from cobas® System Sample Preparation Kit from fridge, and allow reagents to come to room temperature 4. Logon to the cobas 4800 software User ID: LABOPERATOR Password: 00labOPR 5. Perform instrument maintenance: follow the online window dialogue boxes 1) Select Overview tab and check maintenance status of the cobas x 480 instrument.  If weekly maintenance is due, click Run Weekly Maintenance.  If daily maintenance is due, click Run Daily Maintenance. 2) Follow the online instructions. 6. Start New Run a. After the completion of the maintenance, in the overview tab, select New Run icon. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 173 of 291 on the right hand vertical bar b. The following window pops up. Select test type: HPV workflow. c. Type run name ie: HPV 2012.05.29-1 d. Click OK. 7. Refer to the following figure while you are loading the deck: E-plate Holder Tip Rack Holder Dynamic Loading AD-plate Holder Plate Carrier Track 1-6 Tip Rack Holder Tip Rack Carrier Track 11-16 Sample Loading Area Track 17-34 Tip Rack Carrier Track 35-40 Reservoirs Reagent Track Track 48-50 51 8. Load samples Vortex the primary PreservCyt Solution samples prior to loading on the cobas® x480. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 174 of 291 Un-cap samples. Place samples on corresponding carrier. Insert sample carriers on autoload tray when the LEDs for the designated track(17-34) are flashing. Select Load Samples 9. Load consumables Place listed consumables on appropriate carriers.  Both tip carriers are fully filled and barcodes facing the right hand side  Ensure deepwell plate is loaded flatly and properly –barcode facing the right hand side  Ensure microwell plate is loaded flat and properly (barcode facing away from you) Insert carriers on autoload tray. Select Load Consumables. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 175 of 291 10. Load reagents 200 mL reagent reservoir carrier  Load Wash Buffer (WB) on 200 mL reagent reservoir carrier as indicated in the wizard (scan-scan-pour).  Insert carrier on autoload tray.  Click Load Reagents. Note: Once the Wash Buffer is scanned you have one hour to complete the loading process and Press Start button. 50 mL reagent reservoir carrier  Load reagents on 50 mL reagent reservoir carrier as indicated in the wizard.(scan-scan-pour).  Elution Buffer (EB), SDS Reagent (SDS), Lysis Buffer (LYS)  vortex MGP for 10s prior to scanning and loading  Insert carrier on autoload tray.  Click Load Reagents. Reagent carrier  Take out HPV MMX and HPV Mg/Mn from the clean room  Open reagent vials and load them on reagent carrier as indicated in the wizard.  Change gloves after uncapping positive control  Insert carrier on autoload tray.  Click Load Reagents. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 11. Page 176 of 291 Start sample preparation run Select Start Run. The sample preparation starts. Check the timer in the wizard; timer should count down. 12. Unload AD (microwell) Plate. Seal AD plate. Select Sample Prep Results to review the results of the sample preparation. Select Unload. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 177 of 291 Seal AD (microwell) plate with Sealing Film and applicator as indicated on screen. Be sure not to touch the seal with your fingers as it may affect detection. Hold the sides of the plate and use the smoothing applicator provided to properly seal. The plate must be transferred from the cobas x 480 to the cobas z 480 within 90 minutes of completion Select NEXT 13. Load mirowell plate on cobas® z 480 analyzer. Press load button on the cobas z 480 analyzer. Place the sealed AD (microwell) plate into the extended loader. Select load button again. The amplification and detection run starts automatically. Check the timer in the wizard; timer should be counting down. 14. Remove used reagents, reservoirs, samples, and deepwell plate from the cobas x 480 instrument. Discard waste appropriately, used reagents should be disposed of properly. (Some reagents may contain Guanidine HCl and should be disposed of properly.) PCR waste should put in sealed waste bags into the biohazard boxes. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 15. Page 178 of 291 Detection is completed in the cobas® z 480. Run is finished. Select Show Results. Review and accept results in Results. ●DO NOT ACCEPT FAILED or INVALID results● Select results and select printer icon  ; to print the results report. Select results to Export. 16. Unload the AD (microwell) plate from the cobas® z 480. Discard the AD plate in a sealed waste bag into the biohazard box. Log-off and Exit from the cobas® 4800 program. Shutdown the PC. Switch-off the cobas® z 480, switch-off the cobas® x 480, and switch-off the heater. VI. Interpretation of Results All assay and run validation is determined by the Cobas® 4800 software. Controls must be run with each sample run and valid results must be obtained for both the positive and negative control in order for sample results to be displayed. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 179 of 291 Result Reporting and Interpretation Cobas® 4800 HPV Test Interpretation Other HR HPV NEG, HPV16 NEG, HPV18 NEG Specimen is negative for Other High Risk HPV, Negative for HPV16, Negative for HPV18 Other HR HPV POS, HPV16 NEG, HPV18 NEG Specimen is POSITIVE for Other High Risk HPV, Negative for HPV16, Negative for HPV18 Other HR HPV POS, HPV16 POS, HPV18 NEG Specimen is Negative for Other High Risk HPV, POSITIVE for HPV16, Negative for HPV18 Other HR HPV NEG, HPV16 NEG, HPV18 POS Specimen is Negative for Other High Risk HPV, Negative for HPV16, POSITIVE for HPV18 INVALID or FAILED SAMPLE RESULTS NO NOT ACCEPT the results in the results field. These results should not be exported. Review Details. Refer to trouble shooting guidelines. Trouble Shooting INVALID X3: Error Clot Detected Re-test the original specimen. Vortex 30s to break-up excessive mucous. Re-test. X4: Check for excessive mucous. Vortex 30s to break-up Error Processing of the sample was aborted excessive mucous. Re-test. due to one or more pipetting error(s) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VIII. Page 180 of 291 Quality Control Each HPV run includes a set of HPV Positive and Negative Controls. The HPV controls must be valid, before specimens results are displayed, in the cobas 4800 HPV result field. Each sample has an internal control, ß-globin, to monitor cell adequacy, extraction and amplification; internal control must be valid for specimen test to be valid. CAP provides external proficiency testing. All invalid failed runs should be brought to the attention of Charge/seniors for trouble shooting, and resolution. IX. Reference cobas® 4800 HPV Test 2010, Roche Molecular Systems, Inc. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 181 of 291 Influenza A/B & RSV PCR by Focus Diagnostics Simplexa™ I. Introduction The Simplexa™ Flu A/B & RSV Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of human influenza A virus RNA, human influenza B virus RNA and respiratory syncytial virus (RSV) RNA from unprocessed nasopharyngeal swabs (NPS) that have not undergone an extraction process. The system consists of the Simplexa™ Flu A/B & RSV Direct assay, the 3M Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories In the Simplexa™ Flu A/B & RSV Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The assay provides three results; conserved regions of influenza A viruses (matrix gene), influenza B viruses (matrix gene) and RSV (M gene) are targeted to identify these viruses in the specimen. An RNA internal control is used to detect RTPCR failure and/or inhibition. II. Specimen Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM) are stored at 4oC. Vortex swabs on high for 10 seconds, aliquot the VTM into freezer vials and store at 4oC. If BAL specimens are mucoid, treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5 minutes at 3000 rpm in the Eppendorf 5415 C Micro-centrifuge. Aliquot supernatant and store at 4oC. III. Materials and Equipments       3M Integrated Cycler (with Integrated Cycler Studio Software v4.1 or higher) Simplexa™ Flu A/B & RSV Direct assay (MOL2650) 24 tests i) Direct Amplification Discs-24 tests stored at room temperature ii) Simplexa ™ Flu A/B & RSV Reaction Mix (RM) – 24 tests stored in Clean Room freezer (-10 to -30°C) Yse within 30 minutes after thawing Variable volume Ranin Pipitte: 10 to 200 uL Aerosol Resistant Tips (ART) Powder free gloves Biological Safety Cabinet PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual IV. Page 182 of 291 Procedure: NOTE: Do not wear gloves when using the computer and Cycler. Perform daily maintenance. Slightly dampen KimWipe with 70% alcohol, wipe external surfaces of the 3M Cyclers, and laptop keyboards. Wipe benchtop with 70% alcohol. Cautionary notes: Avoid creating aerosols. Use only ART tips. Change gloves frequently. Dispose of waste properly, in sealed discard bags. Do not open foil wedges or pierce foil of post amplified tests. Avoid cross contamination. 1. Switch ON 3M Cycler -first Then switch on lap top computer –second. Windows… User name: MSH Password: MSH12345 Enter 2. Select and open: Integrated Cycler Studio Icon on desktop. User name: MSH Password: MSH12345 LogOn 3. In the Clean Room freezer remove Simplexa™ Flu A/B & RSV Reacion Mix (RM) vials. Please check lot numbers; ensure you are using the current “in use Reagent lot”. Only one Reagent Mix (RM) Lot can be used in specimen testing batch. DO NOT MIX LOTS. One Reagent Mix (RM) is required for every specimen. Thaw thoroughly before use, 3 minutes. DO NOT VORTEX. Spin down reagent mix. Reagent Mix must be used within 30 minutes. 4. New Reagent Lot ONLY. Menu bar at the top Select: Tools Select: Scan Assay Barcode Dialogue window appears: Please scan the Assay barcode(s) now. Scan the Simplexa Reagent Lot card (provided with each kit), the square barcode. See diagram below. Dialogue window appears: “Import assay parameters… Select: OK PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 183 of 291 <<<Scan Large Data Barcode for NEW Reagent lots Only. 5. Remove Direct Amplification Disc (8 tests per disc) from the foil package. Note: Partially used discs are remembered in the machine they were first run on. Ensure the right disc is used and the available spaces on the disc corresponds to what is shown in the computer 6. Load specimens in Specimen processing area Biological Safety Cabinet. Load one test at a time. Opening one foil wedge at a time. Lift the foil from the wedge to be loaded. DO NOT CREATE AEROSOLS. Pipette 50 uL of the Reagent Mix (RM) into the well marked R. Pipette 50 uL of the specimen into the well marked SAMPLE. Re-seal the wedge with foil firmly and tear off the perforated tab edge. Open the lid of the 3M Cycler with glove-free hand, load the Amplification Disc into 3M Cycler. Close the lid. NOTE: Different Reagent Mix (RM) lots cannot be mixed on a disc. Only one Reagent Mix (RM) Lot can be used with each specimen testing batch on a disc. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 184 of 291 7. Left-hand toolbar Configure Runs. Select: Simplexa FluA_B & RSV Direct 5. Running window appears. A run is automatically generated under Run name, add your initials to the end of the run file; example: Run 11-11-2012 At 0900 lws. This will identify you as operator. 8. In Reagent Lot: Scan reagent barcode on Reagent Mix RM) Vial used to load your batch of samples. If your reagent lot is new, the program will prompt you and require you to scan the data bar code. See New Reagent Lot (above). NOTE: Different Reagent Mix (RM) lots cannot be mixed on a disc. Only one Reagent Mix (RM) Lot can be used with each specimen testing batch on a disc. In Disc ID: Scan the barcode, located on the side of the Direct Amplication Disc. 9. Under Sample ID: Scan the sample(s) barecodes(s). Ensure specimens are loaded in the correct wedge identified. Create VRESP Worklist Select: Save 10. Select: Run. Select Run. Run time is 64 minutes. Run status window allows you to monitor the whole run. 11. Run complete. Select Print. Print report window appears Select: include CT values and print with graph. 12. If Direct Amplification disc can still be used; test wedges still available, leave on 3M Cycler. If the Direct Amplification disc is finished, no test wedges available, seal the used disc in a bag, and discard into biohazard waste. 13. Shutdown lap top computer –first. Then Switch-off the 3M Cycler. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 185 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual V. Interpretation of Results Upon completion of the run, the software automatically interprets and displays results. For each Specimen entered, software displays a result (“Detected”, “Not Detected” or “Invalid”) for Flu A, Flu B and RSV. Flu A (FAM) Flu B (JOE) RSV (CFR610) Not detected Not detected Not detected Internal Control –IC (Q670) Detected Detected Not Detected Not Detected Detected Not Detected Detected Not Detected Detected Not Detected Not Detected Detected Detected Invalid Invalid Invalid Invalid Interpretation Flu A/B RNA and RSV RNA not detected. Influenza A RNA Detected Influenza B RNA Detected RSV RNA Detected Invalid. Review trouble shooting guidelines. Invalid Results Troubleshooting: Internal Control (IC) failure: Dilute sample 1: 10 with Viral Transport Media (VTM); pipette 50uL specimen to 450ul VTM for a 1:10 dilution, and retest the specimen. In LIS order media PRABR, when repeating Simplexa testing. If repeat test is still Invalid, report as INDETERMINATE. EC500 error code result points to a data quality error for the particular viral analyte(s). The software was unable to determine a valid amplification for that analyte(s). Dilute sample 1: 10 with Viral Transport Media (VTM); pipette 50uL specimen to 450ul VTM for a 1:10 dilution, and retest the specimen. In LIS order media PRABR, when repeating Simplexa testing. If repeat test is still Invalid, report as INDETERMINATE. Insufficient Specimen Volume; may be due sample integrity, air bubble was introduce during pipetting, pipetting to quickly, or R reagent was not thawed completely. Dilute sample 1: 10 with Viral Transport Media (VTM); pipette 50uL specimen to 450ul VTM for a 1:10 dilution, and retest the specimen. In LIS order media PRABR, when repeating Simplexa testing. If repeat test is still Invalid, report as INDETERMINATE. Unresolved problems should be brought to the attention of senior/charge technologist and/or contact Focus Diagnostics Technical Services department. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 186 of 291 VI. Reporting Result influenza samples using the Virology Worklist “Respiratory PCR” Negative Report: In the Respiratory Viral Detection test comment: Select: }SIMNegative for Influenza A , Influenza B, and Respiratory syncytial virus by Simplexa™ Flu A/B & RSV Direct kit. Focus Diagnostics. In the media PCABR add date tested (F6). Finalize all negative reports. EXCEPTION: Interim reports originating from NP swasb from PMH wards 14A/14B/14C/15A/15B waiting for results on parainfluenza and metapneumovirus. MULTIPLEX REQUESTS: For negative samples requesting multiplex testing, under test “Respiratory viral detection referred out” select keypad option }PHLV. Enter “multiplex testing” in the comment. Enter a date in the media as the day sent to PHL and interim test. Print a PHL requisition using instant report MIC10. Affix a “Respiratory Multiplex testing” and “Negative for FluA, FluB and RSV by Focus Simplexa” label under test requested on the requisition. Package 0.5mL of samples with the requisition to be sent to PHOL. Positive Report: Record date tested (F6) and positive ct value in media PCABR Report as a numbered Isolate in the isolate window. Influenza A Positive. Select: \SIMA for send DETECTED. By Simplexa™ Flu A/B & RSV Direct kit. Focus Diagnostics Inc.” This isolate has been forwarded to PHL (Public Health Laboratory) for subtyping. Results are to follow. Add date to media PHL when sample is sent to PHL. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 187 of 291 Phone all positive results to ward / physician, and notify Infection Control Practitioner as per Isolate Notification and Freezing Table QPCMI15003 Notify Medical Officer of Health (MOH) for Influenza A, and Influenza B. *All Influenza A isolates should be forwarded to Toronto Public Health (PHL) for subtyping. Use instant report MIC10 to print a PHL requisition. Affix a TIBDN Flu A subtyping label under test requested. Make a 0.5mL aliquot to send out. Keep samples and PHL requisition in order and package for send out at the end of the shift. MULTIPLEX REQUESTS: Samples testing positive in house for a respiratory virus will not be sent out for multiplex testing at PHOL. For positive samples requesting multiplex testing, under test “Respiratory viral detection referred out” select keypad option }REFV and finalize test. Influenza B and RSV Positive. Select: \SIM+ DETECTED. By Simplexa™ Flu A/B & RSV Direct kit. Focus Diagnostics Inc.” Phone all positive results to ward/physician, and notify Infection Control Practitioner as per Isolate Notification and Freezing Table QPCMI15003 Notify Medical Officer of Health (MOH) for Influenza A, and Influenza B. All positive Reports should be finalized by reporting technologist. EXCEPTION: Interim reports originating from NP swasb from PMH wards 14A/14B/14C/15A/15B waiting for results on parainfluenza and metapneumovirus. MULTIPLEX REQUESTS: Samples testing positive in house for a respiratory virus will not be sent out for multiplex testing at PHOL. For positive samples requesting multiplex testing, under test “Respiratory viral detection referred out” select keypad option }REFV and finalize test. Indeterminate Report: Select: }SIMi Indeterminate for Influenza A, Influenza B, and Respiratory syncytial virus by Simplexa™ Flu A/B & RSV Direct kit. Focus Diagnostics. Finalize all Indeterminate reports. EXCEPTION: Interim reports originating from NP swasb from PMH wards 14A/14B/14C/15A/15B waiting for results on parainfluenza and metapneumovirus. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 188 of 291 Quality Control a. All In-house External control materials: FluA, FluB, and RSV should be run first week of each month on both 3M Cyclers for non flu season. VIII. Troubleshooting Follow the instructions in “Troubleshooting” folder on desktop. IX. Clean Up & Maintenance Start-up. Perform daily maintenance. Slightly dampen KimWipe with 70% alcohol, wipe external surfaces of the 3M Cyclers, and laptop keyboards. Wipe benchtop with 70% alcohol. End of day. Clean Biological Safety Cabinet (BSC) by wiping surfaces with a 1% working solution hypo chloride (made daily), followed by wiping surfaces with 70% alcohol. Clean pipette with slightly dampened Kimwipe with a 1% working solution hypo chloride, followed by slightly dampened KimWipe with 70% alcohol. Dispose of PCR waste in sealed discard waste bags into the biohazard boxes. Once a week, every Monday, perform Database Backup on both Analyzers. At the Menu bar, Select Tools > Database Tools > Backup Database > Create backup X. Reference 1. Simplexa Flu A/B & RSV Assay package insert. 2. On line: http://www.cdc.gov/rsv/clinical/description.html and http://www.cdc.gov/Features/dsRSV/ PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 189 of 291 Parainfluenza RT-PCR by Rotorgene I. Introduction: Parainfluenza viruses (PIV) can cause upper and lower respiratory tract infections. Commonly seen in children, but may occur at any age group causing severe disease in older adults, and people with compromised immune systems. PIV-1 and PIV -2 often causes cold-like symptoms, and croup in children. PIV-3 is often associated with bronchiolitis, bronchitis, and pneumonia. PIV-4 may cause mild to severe respiratory tract infections. RealStar PIV RT-PCR is a real time PCR for the qualitative detection of human PIV RNA; differentiating PIV1and 3, and PIV-2 and 4. II. Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as possible. BAL specimens if mucoid treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the Eppendroff 5415 C Microcentrifuge. Aliquot supernatant and store at 4C, extract on the easyMag as soon as possible. III. Specimen Processing Please refer to Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG 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. Materials, Equipments and Facilities: Clean Room: Biosafety Cabinet (MIBCT3), freezer (MIFTG) Specimen Preparation area: Biosafety Cabinet (MIBCT7 or MIBCT8) Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System with experiment template: Altona RealStar PIV & hMeta PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 190 of 291 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor 72-Well Rotor Locking Ring 0.1 mL reaction microtubes Tubes and Caps Variable volume Rainin pipettes: 1 to 20 uL, 10 to 200 uL, 100 to 1000 uL Reagent: Altona RealStar PIV RT-PCR Kit 1.0: MasterA, MasterB, Internal Control, PIV-1 Positive Control, PIV-2 Positive Control, PIV-3 Positive Control, PIV-4 Positive Control, PCR Grade Water External Control: To be run first week of the month/ or with every new lot; run during training or if QC failure occurs. IV. Procedure: Prepare samples in order according to PIV worksheet. Add the following controls to the PIV Worksheet: P1/P3 Positive control, P2/P4 Positive Control, Negative Control (PCR grade Water). PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) Remove to thaw at room temperature the required vial’s PIV Master A and Master B (12 reactions/ vial) from the freezer. Prepare PIV Master Mix in Sarstedt 1.5 mL conical microtube; to load on the Eppendorf epMotion. Prepare the number of Master Mix reactions required: Number of Test samples + 3 Controls + one Mix gently, do not vortex. Make only enough master mix for the tests you are running. After Master A has been added to Master B it cannot be frozen again. Number of Reactions PIV Master A PIV Master B Volume of Master Mix Sample/Control Volume No. of Test reactions 1 2 3 4 5 6 1 5 10 15 10 PIV Master Mix PIV Master A (µl) 5 10 15 20 25 30 PIV Master B (µl) 10 20 30 40 50 60 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 191 of 291 7 35 70 8 40 80 9 45 90 10 50 100 11 55 110 12 60 120 See Eppendorf epMotion Manual for further loading and programming instructions. Manual Loading: Load one reaction microtube (0.1 mL) for each sample/control. Pipette 15 uL prepared PIV Master Mix into each reaction microtube. Pipette 1.0 uL of internal control into the reaction microtubes designated for the 3 PIV Controls (P1/P3 Positive Control, P2/P4 Positive Control, Negative PCR Water Control . In the Specimen Processing Area: Prepare Working aliquot P1/P3 Positive Control and P2/P4 Positive. Prepare as needed, use aliquot until finished, and then prepare a new aliquot. In a 1.5mL conical microtube pipette 180 uL PCR grade water add 20uL PIV-1 Positive Control and add 20uL PIV-3 Positive Control; label as P1/P3 Positive Control with date. In a 1.5mL conical microtube pipette 180 uL PCR grade water add 20uL PIV-2 Positive Control and add 20uL PIV-4 Positive Control; label as P2/P4 Positive Control with date. Working Positive Control PCR Grade Water PIV Control 180uL 20uL PIV-1 Positive Control P1/P3 Positive Control 20uL PIV-3 Positive Control 180uL 20uL PIV-2 Positive Control P2/P4 Positive Control 20uL PIV-4a Positive Control 20uL PIV-4b Positive Control Pipette 10 uL each of Sample/Control into reaction microtubes. Mix by pipetting 3x up and down into the Master Mix. Cap each segment, after pipetting is completed. Check reaction microtubes before loading into the rotor ensure the liquid levels are at the same height, and there are no bubble at the bottom of the reaction microtube. Load reaction microtubes into 72-Well Blue rotor, fill empty rotor spaces with blank reaction microtubes, attach the Locking Ring. Load the rotor in Rotor-Gene 6000. In the Rotorgene Detection Area: u. Switch ON computer, switch ON Rotorgene; v. FOR BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; w. Open “Rotor-Gene 6000 series software 1.7” icon x. Wait for “Initializing machine…..” y. Highlight “Altona RealStar PIV & hMeta” experiment PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 192 of 291 z. Select “New” button; aa. Highlighting the correct Rotor type you are using bb. 72-well Rotor (Blue) cc. Check the box for “Locking Ring Attached” option dd. Select “Next” button ee. Enter operator initials. In Notes enter RotorGene Colour (i.e. RED or BLUE) Reagent Lot number. Make sure the Reaction Volume is “25µl”; ff. Select “Next” button Temperature profile window appears, select “Next” button gg. Rotor must be loaded. hh. Summary window appears, select “Start Run” button; ii. Save As window appears; the run is given a filename with a default template. Save in My documents in PIV &hMeta folder for the current year: e.g. Altona Realstar PIV & hMeta 20150101(1) (e.g. assay name-year-month-date-numerical run#); a. Note: PIV & hMeta results are stored in \desktop\My Doucument\Altona RealStar PIV & hMeta jj. Select Save button; kk. Machine starts and Edit Samples window appears. ll. Enter sample numbers including positive and negative controls. Define the type of samples according to the chart: Name Sample P1/P3 Positive Control P2/P4 Positive Control PCR Grade Water Type Unknown Positive Control Positive Control Negative Control Select Yes Yes Yes Yes mm. Select Finish button. nn. Once cycling screen appears, select “Named On” button. To analyze and print PIV report after a run is finished: w. The run is done when Profile Program reads: Run has completed. x. Analyze raw data by selecting each channel, one at a time: Green channel PIV-1 and PIV3, Red channel PIV-2 and PIV-4 and Yellow channel Internal Control (IC). Target Channel PIV-1 and PIV-3 RNA Green PIV-2 and PIV-4a/b Red Internal Control Yellow Report Dye FAM Cy5 JOE y. Select autoscale (bottom left). Click “All Off” (bottom right). Click “water” on. Compare the specimen and control curves to the water one at a time (by selecting each sample on and then off, PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 193 of 291 then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of the positives on the PIV Worksheet. z. Select on “Named On” aa. Perform curve analysis: Select Analysis on the top tool bar. bb. Analysis floating window pops up cc. Select “Quantitation” tab. dd. Quantitation window is brought up to the front. There should be three channels: Green PIV-1/3, Red PIV-2/4 and Yellow (IC). ee. Highlight Cycling A. Green PIV-1/3 option and select Show button. ff. Maximize Target (Green/Red/Yellow)window gg. Select slope correct hh. Select Ignore first… 5 cycles ii. Set threshold at 0.05. A threshold line will appear on the screen. jj. Move threshold line to above the noise (i.e. Negative specimens) kk. To Print reports: Select Reports button on the top toolbar. ll. Report Browser window pops up. mm. Choose Cycling A. Green PIV-1/3 . Under templates box, choose “Quantitation full report”. nn. Select Show button oo. Maximize the report window, check that only the positives that you have identified from your raw data analysis are positive (i.e. have a crosspoint). pp. Choose print report. (red and yellow channels print only pages 2-4). qq. Once finished printing, close the report window rr. Go to the floating Analysis window; ss. Repeat the step from “e” to “t” for Cycling A. Red PIV-2/4 and Cycling A. Yellow Internal control Shut down computer: Exit from software. “save changes”. Select “yes”. Select“start” button. Select “Turn off computer”. Turn off Rotorgene End of Shift Cleaning: Perform cleaning protocol as outlined below. 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 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 194 of 291 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. EasyMAG , Lysis Rack, and BioHit Multi-channel Pipette: Wipe down surfaces with RNAse Away or NucleoClean on KimWipe, followed by UltraPure water, and then 70% alcohol Wipe 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. IV. Interpretation and Reporting: Green Channel PIV1/3 Red Channel PIV2/4 Yellow Channel (IC specific) Interpretations + - +/- PIV-1/3 DETECTED - + +/- PIV-2/4 DETECTED + + +/- PIV-1/3 DETECTED. PIV-2/4 DETECTED. (ask charge/senior for confirmation) - - + PIV NOT DETECTED - - - INVALID Respiratory sample needs to be diluted 1:4 with VTM and re-extracted and RT-PCR repeated. VTM=Viral Transport Media PIV-1/3 DETECTED. or PIV-2/4 DETECTED Report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard PCPIN: F6 to add the date tested. In the media PCPIN enter crosspoint (ct) value : ct= Report the virus as an isolate in the F7 window. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 195 of 291 Isolate #: 1, or next available isolate number. Org. ID: 24par13 Parainfluenza virus 1 and/or 3 24par24 Parainfluenza virus 2 and/or 4 The reporting isolate comment is attached to the Organism ID. Verify All in the isolate window. Review report entry: media, isolate. Finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). Document calls and emails in the media CALL. PIV-1/3 NOT DETECTED. or PIV-2/4 NOT DETECTED Date the media on the back of the workcard. PCPIN: F6 to add run date. On the front of the workcard, select from the keypad: }PI}PI- Negative for parainfluenza virus by RealStar PIV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. PIV INDETERMINATE Internal control has failed twice to amplify in a sample. Date the media on the back of the LIS workcard. PCPIN: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRPIN Under this media ^PRPIN report the 1:4 dilution test result type “IC no amplification”. Report: select from the keypad: }PIChange “Negative” to INDETERMINATE; final report should appear as: INDETERMINATE for parainfluenza virus by RealStar PIV RT-PCR, Altona Diagnostics. Review report entry. Finalize the report. V.  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. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual       a. b. c. d. e. f. g. h. i. j. Page 196 of 291 External control for PIV-1/3, and PIV-2/4 should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every Run Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition. A Positive Control is included and shows a positive reading in Green PIV-1/3 Channel or a positive reading in Red PIV-2/4 Channel A Negative Control is include and shows a negative reading in both Green PIV-1/3 Channel or a negative reading in Red PIV-2/4 Channel Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. Do not release results pending resolution of QC failure. Inform charge/senior technologist. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where appropriate. 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). If positive QC material yielded negative result, repeat the entire run. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. V. References: RealStar PIV RT-PCR Kit v1.0, altona Diagnostics cdc.gov/Human Parainfluenza Viruses (HPIVs) (/parainfluenza/index.html PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 197 of 291 Parvovirus B19 PCR by Rotorgene I. Introduction Parvovirus B19 belongs to the DNA virus family Parvoviridae and the genus Erythrovirus. Infection with B19 may result in an asymptomatic infection or produce a wide range of disease; ranging from erythema infections in children (slapped cheek syndrome or fifth disease) to severe anemia (the cause of aplastic crisis in hemolytic anemias and in sickle-cell disease), and systemic disease involving the central nervous system, heart, liver depending on the immune competence of the host. In pregnant women, Parvovirus B19 may cause hydrops fetalis, congenital anemia, fetal death, or spontaneous abortion. II. Collection and Transport Amniotic fluid stored at 4C and extract on the BioMerieux EasyMag as soon as possible. EDTA plasma; EDTA blood is spun at 3000 rpm for 10 minutes and the plasma is separated and extract on the BioMerieux EasyMag as soon as possible. III. Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG GENERAL PRECAUTIONS:  There must be separate PCR work areas:  Clean room  Specimen preparation room  Amplification room  Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas.  Change lab coats and gloves between work areas. Only blue gowns are to be worn in the clean room.  Use only filtered pipette tips  Use only sterile RNase, Dnase-free microcentrifuge tubes  Use sterile, disposable polypropylene tubes throughout the procedure  Always wear powder-free gloves when handling reagents  Change gloves frequently and keep tubes closed whenever possible  Keep the Quantitation Standards and specimens in the specimen preparation area.  Thaw components thoroughly at room temperature  Mix components and centrifuge briefly  Work quickly in the cooling block  Use 1% sodium hypochloride or RNAse Away to disinfect equipment and surfaces and then rinse with 70% alcohol or water. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 198 of 291 Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT4), freezer (MIFTG), gowns and gloves Specimen Preparation area with Biosafety Cabinet and microcentrifuge Detection Room for Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System programmed for ALTONA VZ PCR 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor 72-Well Rotor Locking Ring 0.1 mL Strip Tubes and Caps 36-Well Loading Block (pre-cooled to 4oC) 36-Well Rotor Locking Ring 0.2mL Tubes Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL From: Altona Parvovirus B19 PCR Kit 1.0, (stored at -20o C in clean room):  QS1 (10^4 Copies/µL)  QS2 (10^3 Copies/µL)  QS3 (10^2 Copies/µL)  QS4 (10^1 Copies/µL)  Master A (12 tests per vial)  Master B (12 tests per vial)  Internal Control (IC)  Water (PCR grade) External Control: To be run when a new lot is used, during training or if QC failure occurs. IV. Procedure: Prepare Parvovirus B19 Worklist and arrange eluates in order according to the worklist store at 4C until it is time to add sample to the master mix. Remove the Parvo B19 QS3 from -20C freezer and place at 4C until ready to load. In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) a. Take out the necessary number of Parvovirus B19 Master Mix A and B vials (12 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 199 of 291 number of tests and prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do not vortex. No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 b. Place proper micro tubes for each purified sample, Positive controls, external QC and Negative control (H2O) into the Cooling Block (pre-cooled to 4oC). c. Pipette 20 uL prepared Master Mix (A+B) into each micro tubes. d. Pipette 1.0 uL of internal control into the micro tubes designated for the Positive control (Parvo B19 QS3 100 cp/uL) and the Negative control. e. Pipette 10uL water (PCR grade) into the negative control micro tube and cap tightly. f. Place the cooling block in to a clean biohazard bag. g. Changed out of dedicated clean room gown. In the Specimen Processing Area: (Changed into specimen room gown) a. Using a separate filter tip each time, carefully pipette 10 uL each of purified sample, Positive controls (QS3), and external QC (if using) directly into the micro tubes. Mix by pipetting up and down. Close the lid tightly. b. Check that each micro tube is closed tightly before loading. Make sure there are no bubbles at the bottom of the micro tubes. In the RotorGene room: a. Turn on computer, turn on rotorGene. b. FOR THE BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s. Page 200 of 291 Double click(left on the mouse) “Rotor-Gene 6000 series software 1.7” icon Wait for “Initializing machine…..” Load micro tubes into a proper rotor (eg. 36-well or 72-well). Fill up the empty positions of the rotor using empty microtubes and snap close the locking ring. Load the rotor in Rotor-Gene 6000. Highlight Parvo B19 assay; Press “New” button; Highlighting the correct Rotor type you are using 72-well Rotor (Blue) 36-well Rotor (Red) Tick the “Locking Ring Attached” option Press “Next” button Enter initials at Operator space and make sure the Reaction Volume is “30µl”; enter lot no. in the Notes section. Press “Next” button Temperature profile window pops up, press “Next” button. Make sure your ring has been loaded in the Rotorgene. Summary window pops up, press “Start Run” button. The RotorGene will start. Save as window appears; go to My documents -Open–select Parvo result folder- Open Press Save –Parvo yyyy-mm-dd (run no.) Press Save button. Machine is starting and Edit Samples window pops up Enter sample LIS# including positive and negative controls. Define the type of samples according to the chart: Name Sample Number QS3 Parvo B19 Water Type Given conc. Select Unknown Yes Positive Control Yes Negative Control Yes t. Once finishing all samples, press Finish button. u. Once cycling screen cmes on, click “Named On” button. To analyze and print Parvo B19 report after a run has finished: The run is done when Profile Program reads: Run has completed. Analyze raw data by clicking on the each of these channels (one at a time). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 201 of 291 Green channel (Parvo B19), and Yellow channel (IC). These tabs are found on the second menu bar from the top, on the left hand side. Select the Green Channel (Parvo B19). Click “All Off” (bottom right). Select sample “water Negative Control” and “QS3 Parvo B19 Positive Control”. Click on Autoscale (bottom left). Compare each specimen curve to the water curve and the QS3 Parvo B19 Positive Control one at a time (by clicking each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on the worklist. a. b. c. d. e. Click on “Named On” Perform curve Analysis (Get report): Click “Analysis” on the top tool bar. Analysis floating window pops up; Click Quantitation Analysis. Quantitation window is brought up to the front. There should be four channels: Green (Parvo B19), and Yellow (IC). f. g. h. i. j. k. l. m. n. Highlight Cycling A. Green (Parvo B19) option and press Show. Maximize cycling green window (or the channel you are working on) Press button “Slope Correct” on the top. Press button “Ignore first” on the top, and type “5”, press OK. Set threshold at 0.05. A threshold line will appear on the screen. Move threshold line to above the noise (i.e. Negative specimens) To Print reports: Press “Reports”. Report Browser window pops up. Choose Cycling A. Green (Page 1). Highlight Quantitation full report, press “Show”. Maximize the report window, check that only the positives that you have identified from the raw data review are positive and have a crosspoint (ct value). o. Print pages 1-4 for the green channel; pages 2-4 for all the other channels. p. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab, select Cycling A. Yellow -the internal control (IC), select Show. Maximize the graph Quantitation Analysis –Cycling A. Yellow. Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in the first 5 cycles of the run). Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph screen. Move the threshold line to above the noise. q. Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show. All samples should have a positive IC curve, and a cycle number in the Ct column. Print pages 2-4. (For Cycling A. Yellow report, if there is message of “NEG (Multi Ct)” for any specimen, you have to go back to check Raw Data of cycling A.Yellow. If all specimens are PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 202 of 291 amplified, adjust the threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct. Adjust the threshold line so that the threshold line crosses the curve at only one point.) r. Close the outer most window. “Save changes to Altona Parvo B19 yyyy-mm-dd?” Select Yes. Shut down the computer, and switch off the RotorGene. At the end of the day, follow the cleaning protocol below using 1% hypochorite followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet, pipettors, and bench tops Specimen Preparation Area:  Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.  Bag and securely close off bag and discard waste  Wash racks.  Clean the easyMag according to the daily maintenance sheet Amplification Area:  Discard microtubes into a disposal bag and tight the bag.  Discard the bag into biohazard waster container.  Perform the cleaning procedure according the daily maintenance sheet: clean the rotor and cooling blocks with RNase Away, followed by double distilled water, and finally 70% alcohol PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 203 of 291 Interpretation: Green Channel Parvo B19 target Yellow Channel Internal Control Interpretation - + Report: Negative for Parvo B19 DNA by PCR. + + + - - - Preliminary report: Positive Repeat extraction & Parvo B19 PCR to confirm. Parvovirus B19 DETECTED, confirmation to follow. Notify senior or Charge. Preliminary report: Positive Repeat extraction & Parvo B19 PCR to confirm. Parvovirus B19 DETECTED, confirmation to follow. Notify senior or Charge. Possible PCR inhibition. Repeat extraction & PCR. If still negative on Yellow IC Channel, report: INDETERMINATE for Parvo B19. PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report, see below “Reporting”. VII. Reporting: Negative for Parvovirus B19. Under media on the back of the LIS workcard. PCPAR: F6 to add run date Report on the front of the LIS workcard from the keypad select: }PAR}PAR- Negative for Parovirus B19. This is a research test. Parvo B19 PCR Kit v1.0. Altona Diagnostics Inc. Ctrl-F to finalize the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 204 of 291 Sterile sites: PRELIMINARY Positive for Parvovirus B19. Date the media on the back of the LIS workcard. PCPAR: F6 to add run date Enter on the media line the cross point (ct) value: Ct= Order a repeat Parvo PCR test, on the back of the workcard. >FAIL, Ctrl D, select from keypad ^PRPAR Enter. Report the virus as an isolate in the F7 window. Isolate #: 1 Org. ID: 63parB Parvovirus B19 Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Ctrl D. Select from the keypad: \B19+ to add the comment phrase. \B19+ DETECTED by PCR, confirmation to follow. This is a research test. PARVO B19 PCR Kit v1.0, Altona Diagnostics Inc. Manually type the phrase “confirmation to follow.” Grave (`) the result. Ctrl-P to prelim the report. Call reports to ward/doctor. Sterile sites: CONFIRMED Positive for Parvovirus B19. Date the media on the back of the LIS workcard. ^PRPAR : F6 to add run date Enter on the media line the cross point (ct) value: Ct= Open the Isolate (F7) Comment window (F8) Isolate #: 1 Org. ID: 63parB Parvovirus B19 Open the Isolate Comment window F8. Delete the phrase “confirmation to follow”. Manually type: CONFIRMED The final reporting phrase should appear as below: \B19+ DETECTED by PCR, CONFIRMED. This is a research test. PARVO B19 PCR Kit v1.0, Altona Diagnostics Inc. Grave (`) the result. Ctrl-F to finalize the report. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 205 of 291 Sterile sites: NOT CONFIRMED for Parvovirus B19. Date the media on the back of the LIS workcard. ^PRPAR: F6 to add run date Isolate #: 1 must be suppressed, change to an alphabet Front of the LIS workcard type the following: UPDATED REPORT: Previously reported Parvovirus B19 NOT CONFIRMED. Report on the front of the LIS workcard (manually type): This is a research test. This is a research test. PARVO B19 PCR Kit v1.0, Altona Diagnostics Inc. Ctrl-F to finalize the report. Call Updated reports to ward/doctor. V. 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 for Parvo B19 should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every run: Each patient specimen must have an Internal Control (IC) added to monitor both extraction and PCR inhibition. A Positive Control is included and shows either a positive reading in Green Channel (QS3 Parvo B19) A Negative PCR Water Control is included and shows a negative reading in Green Channel (QS3 Parvo B19) Report all failed QCs to senior/charge technologist. Failed QC: Test is invalid without satisfactory QC results. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 206 of 291 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). e. If positive QC material yielded negative result, repeat the entire run. f. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. g. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. Report all failed QCs to senior/charge technologist VI. References: Parvo B19 PCR kit v1.0, Altona Diagnostics Inc. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 207 of 291 West Nile Virus PCR by Rotorgene I. Introduction West Nile virus (WNV) is a flavivirus belonging to the Japanese Encephalitis group. The primary host is wild birds and the virus is usually transmitted to humans via mosquitoes. Transplants and blood transfusions have also been implicated in its transmission. This assay involves isolating the viral RNA using the Qiagen Viral RNA Kit and amplifying it by Altona (previously known as Altona) RealStarTM WNV Reverse-Transcription (RT)-PCR Kit. The amplification and detection take place simultaneously, continually and in real time in the Rotor-GeneTM. In real-time PCR, fluorescent dyes are linked to oligonucleotide probes, which bind specifically to the amplified product. Monitoring the level of fluorescence allows the detection of the amplicon. In the RealStarTM WNV assay, two fluorescent dyes are incorporated in the master mix to detect two amplified products: One for the WNV amplicon; and the other for the Internal Control (IC). The IC is added to each specimen in the initial step to both control the isolation procedure and to check for PCR inhibition. The Rotor-Gene TM simultaneously monitors the two different amplicons using two fluorimeter channels. II. Collection and Transport Blood samples for WNV PCR should be collected in EDTA (purple top vacutainer tube). CSF specimens should be collected in a clean, sterile container and sent to the laboratory as soon as possible. If specimens cannot be transported immediately, they should be kept at 4oC. If a delay of more than 6 hours is anticipated, the CSF specimen should be frozen at –70oC. The EDTA samples should be processed within 6 hours of collection (plasma separated and refrigerated; frozen if delay is more than 6 hours). Allow only one freeze-thaw cycle. NOTE: Repeated freezing-thawing will reduce test sensitivity and cryoprecipitates may accumulate in the plasma. III. Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and gloves Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8) and microcentrifuge (MICT17) Vortex Detection Room for Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System programmed for ALTONA WNV RT PCR PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 208 of 291 36-Well High Profile Rotor 36-Well Rotor Locking Ring 0.2 mL Tubes 1.5 mL microcentrifuge tubes Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL From Qiagen Viral Isolation Kit:  Collection Tubes  Dissolve the lyophilized carrier RNA with 320uL of Elution Buffer. Individually pipette 5.6 uL into microcentrifuge tubes, freeze at – 20oC.  Spin Columns  Wash Buffer 1 (Buffer AW1) – add 125 mL ethanol before use  Wash Buffer 2 (Buffer AW2) – add 160 mL ethanol before use  Elution Buffer (Buffer AVE) From: Altona WNV RT-PCR Kit 1.0, (stored at -20o C in clean room):  WNV Positive Control  Master A (12 tests per vial)  Master B (12 tests per vial)  Internal Control (IC)  Water (PCR grade) External Control: To be run when a new lot is used, during training or if QC failure occurs. IV. General Precautions:          There must be separate PCR work areas: a. Clean room b. Specimen preparation room Amplification room Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas. Change lab coats and gloves between work areas. Only blue gowns are to be worn in the clean room. Use only filtered pipette tips Use only sterile RNase, Dnase-free microcentrifuge tubes Use sterile, disposable polypropylene tubes throughout the procedure Always wear powder-free gloves when handling reagents Change gloves frequently and keep tubes closed whenever possible Thaw components thoroughly at room temperature PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual    V. Page 209 of 291 Mix components and centrifuge briefly Work quickly in the cooling block Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water. Procedure Sample Preparation: Lysis and Purification Steps: a. For each specimen and External Control, prepare and label one 1.5 mL microcentrifuge tubes (sterilized, RNase-free) with the corresponding number in the Worklist. Put the specimens first on the worklist, followed by the external control (if using one). Label one of them with the lab number (LIS label). This microcentrifuge tube will store the eluted RNA, the other will be used for processing and will be discarded. Also, label one QIAamp Spin Column (Qiagen) with the number on the Worklist. Arrange the microcentrifuge tube and Columns into 3 rows. b. Add 560 uL Lysis Buffer into 5.6 uL cRNA microcentrifuge tubes. c. Add 140 uL specimen to the Lysis Buffer (Buffer AVL with RNA carrier), vortex for 15 seconds. d. Incubate at Room Temperature (15-25oC) for 10 minutes. e. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids. f. Add 6 uL WNV Internal Control to each of the microcentrifuge tubes with Buffer AVL. g. Add 560 uL ethanol (96-100%) to the samples, vortex for 15 seconds. Centrifuge for 10 seconds at 3000rpm (2000 g) to remove drops from the lids. h. Pipette 630 uL (half the volume) of the mixture (specimen-lysis buffer-ethanol) to the QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000 rpm (6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. i. Repeat Step (h) by pipeting the remaining 630 uL of the mixture to the QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000rpm (6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 210 of 291 j. Open the spin column and add 500 uL of Buffer AW1. Close the cap and centrifuge at 8,000 rpm (6,000 g) for 1 minute. k. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. l. Open the spin column and add 500 uL of Buffer AW2. Close the cap and centrifuge at full speed 14,000 rpm (20,000g) for 3 minutes. m. Place the spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate. Centrifuge at 14,000 rpm (20,000g) for 3 min. Elution Steps: a. Place the spin column in the clean 1.5 mL microcentrifuge tube with the LIS specimen label. Discard the collection tube containing the filtrate. Open the spin column and add 60 uL of Elution Buffer (Buffer AVE). Close the caps and incubate at room temperature for 1 minute. b. Centrifuge at 8000rpm (6000 g) for 1 min. Discard the spin column. Store the eluate at -20oC or –70oC if not able to proceed immediately. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 211 of 291 PCR Amplification and Detection Procedures: PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) a. Take out the necessary number of WNV Master Mix A and B vials (24 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the number of tests and prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do not vortex. Make only enough master mix for the tests you are running. After vial A has been added to Vial B it cannot be frozen again. No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 20 40 60 80 100 120 140 160 180 200 220 240 b. Place a 1.5ml microcentrifuge tube for each purified sample, Positive control, external QC and Negative control (H2O) into the Cooling Block (pre-cooled to 4oC). c. Pipette 25 uL prepared Master Mix (A+B) into each micro tubes d. Pipette 2.5 uL of internal control into the micro tubes designated for the Positive control and water. e. Pipette 25 uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and cap tightly. Label each microtube 1, 2, 3…. before leaving clean room. f. Place the cooling block into a clean biohazard bag. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 212 of 291 g. Change out of dedicated clean room gown. In the Specimen Processing Room: (Change into specimen room gown) a. Using a separate filter tip each time, carefully pipette 25 uL each of purified sample, Positive controls, and external QC (if using) directly into the micro tubes. Mix by pipetting 3x up and down into the mastermix. Immediately close the cap tightly. b. Check each micro tube before loading. Make sure there is no bubble at the bottom of the tube. c. Load micro tubes into a proper rotor (36-well) and snap close the locking ring. Load the rotor in Rotor-Gene 6000. d. Always fill up the empty position of the rotor using empty tubes. In the Rotorgene room: a. Turn on computer, turn on Rotorgene; b. FOR BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; c. Double click(left on the mouse) “Rotor-Gene 6000 series software 1.7” icon d. Wait for “Initializing machine…..” e. Highlight “ALTONA WNV” assay; f. Press “New” button; g. Highlighting the correct Rotor type you are using h. 36-well (Red) or 72-well Rotor (Blue) i. Tick the “Locking Ring Attached” option j. Press “Next” button k. Enter initials in operator space. Enter Master Mix lot. # In the Notes space and Rotogene used (Blue or Red). Make sure the Reaction Volume is “50µl”; l. Press “Next” button m. Temperature profile window pops up, press “Next” button. n. Make sure your ring has been loaded in the Rotorgene. o. Summary window pops up, press “Start Run” button; p. Save As window pops up; the run is given a filename with a default template. Change it according to the date and numerical runs the assay performed on the same day; e.g. WNV 2010.03.29 (e.g. assay name-year-month-date-numerical run#); q. Note:WNV results are stored in \desktop\My Document\WNV results. r. Press Save button; s. Machine starts and Edit Samples window pops up; t. Enter sample LIS# including positive and negative controls. Define the type of samples according to the chart: PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Name Sample Type Unknown Select Yes Positive Control WNV Positive Control Yes Water NTC Yes Page 213 of 291 u. Press Finish button. v. Once cycling screen comes on, click “Named On” button. To analyze and print WNV report after a run is finished: a. The run is done when Profile Program reads: Run has completed. b. Analyze raw data by clicking on the each of these channels (one at a time): Green channel (WNV) and Yellow channel (IC). These tabs are found on the second menu bar from the top, on the left hand side. c. Click on autoscale (bottom left). Click “All Off” (bottom right). Click “water” on. Compare the specimen and control curves to the water one at a time (by clicking each sample on and then off when proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of the positives on the worklist. d. Click on “Named On” e. Perform curve analysis: Click Analysis on the top tool bar. f. Analysis floating window pops up; g. Choose “Quantitation” tab. h. Quantitation window is brought up to the front. There should be two channels: Green (WNV) and Yellow (IC). i. Highlight Cycling A. Green (Page 1) (WNV) option and press Show button. j. Maximize cycling green window (or the channel you are working on) k. Highlight slope correct tab. l. Click on Ignore first… tab. A window will pop-up, type in 5 (i.e. ignore 5 cycles). m. Set threshold at 0.05. A threshold line will appear on the screen. n. Move threshold line to above the noise (i.e. Negative specimens) o. To Print reports: Press Reports button on the top toolbar.  Report Browser window pops up.  Choose Cycling A. Green (Page 1). Under templates box, choose “Quantitation full report”.  Press Show button  Maximize the report window, check that only the positives that you have identified from your raw data analysis are positive (i.e. have a crosspoint)  Choose print report. (for the red and yellow channels print only pages 2-4).  Once finished printing, close the report window PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 214 of 291  Go to the floating Analysis window; p. Repeat the step from “e” to “m” for Cycling A. Yellow (internal control) VI. Reporting: Green Channel (WNV) Yellow Channel (IC specific) Interpretations + + or - Preliminary report: Positive for WNV: Repeat assay to confirm* (Donor samples should be repeated in duplicates) If repeat is positive in the green channel, report: Positive If repeat is negative, consult Senior/Charge on final report - + Negative for WNV Indeterminate Test needs to be repeated. Sample may be diluted 1:4 with lysis buffer *Repeated specimen should be re-extracted from the original clot Negative Report: Negative for WNV This is a research test. WNV RT-PCR Kit v1.0. Altona Diagnostics Inc. and RotorGene, ?Corbett Life Sciences Indeterminate Report: Indeterminate by RT-PCR This is a research test. WNV RT-PCR Kit v1.0. Altona Diagnostics Inc. and RotorGene, Corbett Life Sciences Positive Report: POSITIVE for WNV This is a research test. WNV RT-PCR Kit v1.0. Altona Diagnostics Inc. and RotorGene, Corbett Life Sciences Record CT value in media PCWNV Notify Senior/Charge Technologist/Microbiologist on positive WNV results Notify Medical Officer of Health on positive WNV results PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 215 of 291 Shut down computer: a. Exit from software. A pop-up window will appear “save changes”. Click “yes”. b. Click “start” button. Click “Turn off computer”. c. Turn off Rotorgene At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet Specimen Preparation Room:  Clean Biological Safety Cabinet, pippettors, centrifuge, and bench top.  Cap discard container.  Wash racks. Amplification Room:  Discard micro tubes into a disposal bag and close the bag.  Discard the bag into biohazard waster container.  Perform the cleaning procedure according the daily maintenance sheet. VII. Quality Control Reagent QCs: Before every lot change of isolation and/or master mix kit:  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. Before every lot change of master mix kit: Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition.  A Positive Control is included and shows a positive reading in Green Channel (WNV)  A Negative Control is include and shows a negative reading in Green Channel (WNV)  Report all failed QCs to senior/charge technologist. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 216 of 291 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). e. If positive QC material yielded negative result, repeat the entire run. f. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. g. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. ROTORGENE QC: Semi-Annual:  Perform optical temperature verification (See Appendix - Rotorgene Optical Temperature Verification (OTV) Procedure) VI. References: QIAamp Viral RNA Mini Kit Handbook 01/99, Qiagen WNV RT-PCR Kit 1.2 User Manual, Altona Diagnostics, 22767 Hamburg, Germany PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 217 of 291 West Nile Virus Donor Screen NAT PCR by Cobas® s201 Ampliprep/TaqMan I. Introduction West Nile Virus is a member of the Flaviviridiae family, the genus Flavivirus and the Japanese encephalitis virus serocomplex. Viruses in this complex are arboviruses with the potential to cause meningitis, encephalitis and meningoencephalitis. The Cobas® TaqScreen West Nile Virus Test is a qualitative test that enables the screening and detection of WNV RNA in infected pooled and individual specimen donations. The Cobas® TaqScreen West Nile Virus Test uses a generic nucleic acid preparation technique on the Cobas® Am;liPrep Instument. WNV test 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 (amplicon). II. Specimen Collection and Storage Living donor specimen:  Blood collected in EDTA may be stored in one of two conditions: For condition 1, blood may be stored for up to 24 hours at 2-30 oC, followed by 24 hours at 2-25 oC, followed by 72 hours at 2-8 oC, prior to plasma separation. For storage longer than five days, separate the plasma from the red blood cells by centrifugation at 8001,600 x g for 20 minutes. Following removal, plasma my be stored at 2-8 for seven days, followed by up to 30 days at ≤-20 oC For condition 2, blood may be stored for up to 72 hours at 2-25 oC, followed by 48 hours at 2-8 oC, prior to plasma separation. For storage longer than five days, separate the plasma from the red blood cells by centrifugation at 800-1,600 x g for 20 minutes. Following removal, plasma my be stored at 2-8 for seven days, followed by up to 30 days at ≤-20 oC  EDTA plasma can be frozen and thawed a maximum of three(3) times Cadaveric blood specimen-EDTA plasma or serum may be used  Cadaveric blood specimens collected in EDTA may be stored for up to 24 hours at 225oC, prior to plasma separation. For storage long than one day, separate the plasma from the red blood cells by centrifugation at 800-1,600 x g for 20 minutes. Following removal, plasma may be stored at 2-8oC for an additional seven days. Alternatively, plasma my be stored at ≤18 oC for up 30 days. Cadaveric EDTA plasma can be frozen and thawed a miximum of three (3) times.  Cadaveric blood collected as serum may be stored for up to 24 hours at 2-8 oC, prior to separation. For storage longer than one day, separate the serum from the clot by centrifugation at 800-1,600 x g for 20 minutes. Following removal, serum specimens may PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual  Page 218 of 291 be stored for up to three days at 2-8 oC. Alternatively, in the case of serum removed from the clot, moderately hemolyzed cadaveric serum specimens may be stored at ≤18 oC for up 15 days and highly hemolyzed cadaveric serum specimens may be stored at at ≤18 oC for up 8 days.Cadaveric serum specimens can be frozen and thawed a mximum of three (3) times. Cadaveric plasma or serum is tested at 1:5 dilution. !Do not freeze whole blood !Heparin Plasma is NOT acceptable Donor Serology and Molecular Testing Specimen Storage III. Materials, Equipments and Facilities Reagents: 1. Cobas® TaqScreen WNV Test (CS1-CS4) stored at 2-8 oC  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 oC  WNV negative control  WNV positive control 3. Cobas® TaqScreen Wash Reagent stored at 15-30 oC 4. Cobas® TaqScreen Cadaveric Specimen Diluents:  Unopened CADV SPEC DIL is table until the expiration date.  Once opened, this reagent is stable for 30 days at 2-8 oC or until the expiration date, whichever comes first. Equipment:  Hamilton MICROLAB STAR/STARlet IVD Pipettor  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 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual          IV. Page 219 of 291 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 WNV 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 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 WNV onto a same reagent cassette Place CS2, CS3, CS4 for WNV onto to a separate reagent cassettes Load the reagent cassette containing all CS1 onto position A of the AmpliPrep PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 220 of 291 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 8. Using the Work list, match labels of sample tube with the 6mL BD Vacutainer tube a. Add 1200ul plasma of Living Donor samples to the empty 6mL BD Vacutainer tubes b. Add 300ul plasma/serum Cadaveric samples to the 6mL BD Vacutainer tube with 1200 uL Cadaveric diluent c. Centrifuge at 3000 rpm for 5-10 minutes ! Make sure the sample tube is free of bubbles, clots and flocculates. d. De-cap and place 6mL BD Vacutainer tubes onto 32 test tube sample carrier e. Take out enough clips and S-tubes (4 controls plus the sample number) f. Slightly bend the clip before place it onto the S-tubes rack starting position#1 g. Place S-tubes into clips h. Place S-tube rack onto SK24 Rack Carrier i. De-cap S-tubes 9. Set up MPX NEGATIVE CONTROL and POSITIVE CONTROL racks !!!Mix controls by inversion at least 3 times, avoiding the creation of bubbles, as specified below:  Inversion each time is defined as turning the control upside down and right side up again  Within inversion each time, hold the control for at least 2 seconds in each orientation (i.e. turn the control upside down, and hold for at least 2 seconds. Then, turn the control right side up again, and hold for at least 2 seconds a. Place clean Kleenex paper on Control Working Area b. Remove positive control lids and leave on Kleenex paper. c. 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 d. Load negative control in negative Control Rack Position #1 with barcode label visible. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 221 of 291 ! Pop bubbles from control tubes by lightly tapping with control tube cover ! Return Control boxes back to fridge after finish loading. 10. Pooling Manager Terminal a. Click Wizard Icon b. Make sure Batch Run is selected c. Click Assay/Start d. Choose WNV e. Click Next f. Click Pools of 1 g. Follow the instruction to load sample rack h. Click Done button after all samples are loaded 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. 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. 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 (Start with Track F), 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. 12. Load the SK24 racks directly to the Cobas® TaqMan. 13. If the Amplification and detection run does not start automatically, click Start 14. Check finished time. 15. When testing is finished in Cobas® TaqMan , the results are automatically transmitted to Data Manager. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 222 of 291 !DO NOT NEED TO MANULLY ACCEPT RESULTS FROM Cobas® TaqMan 16. 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  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. Report results in LIS manually. 18. Clean Up and Shutdown Procedures a. Check number of reagents left and write in CS1 then cover with parafilm and store in fridge. b. Discard used SPU tubes and save clean ones. V. Synchronize Hamilton with Ampliprep      VI. 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 1438. VII. Interpretatiion of Results  If all controls are valid: Non Reactive: reported as Negative Reactive: reported as Positive.  Invalid Result: repeat test once. If still invalid, reported as Indeterminate PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 223 of 291 VIII. Reporting For Negative Result Result NonReactive NonReactive CT Values WNV│IC ─ │### ─ │─ Internal Control Valid Report Comment  8WNVR:Negative@RWNv Verify 8WNVR  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: 8WNVR: Indeterminate@RWNv  Verify 8WNVR  In the comment for 8COM, record the 2nd result  Verify 8COM  If there is not enough sample for repeating, result as following: 8WNVR: Indeterminate@ RWNv  In the comment for 8WNVR, type: Not Sufficient Quantity for repeating  Verify 8WNVR  Verify 8COM Invalid For Positive Result Result CT Values Internal Control WNV│IC Reactive ─ │### Valid Repeat in Duplicate YES Report Living Donor?? Cadaveric Donor: a. 2/3 Positive8WNVR:Positive@ RWNv  Order 8COM in LIS and record all CT b. 2/3 Negative8WNVR:Negative@ RWNv Order 8COM in LIS and record all results PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual IX. 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 X. Page 224 of 291 Solutions Repeat specimen Result specimen as: Invalid In result comment: hit F5 to add comment “@SPIN” “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 Quality Control : Run External Positive and negative control (previously tested CAP proficiency samples) monthly. XI. CAP and NML provide external proficiency testing. Reference: Roche cobas s 201 System Hardware and Software reference Manual, P/N 05575982190-01. PDM Software Software Guide,P/N 05575974190-01. Operator’s Manual for Use with small Pools(Pools of 1 and 6) P/N 05576008190-01 Roche Hot Line Tel# 877-273-3433 michael.salem@roche.com xinyu.song@roche.com PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 225 of 291 APPENDIX I - Molecular Diagnostic Tests Schedule BK Virus PCR………………………………………………………………………………...…….weekly Chlamydia trachomatis and Neisseria gonorrhoeae PCR …………………………………………weekly Clostridium difficile toxin B gene PCR ……………………………………………………………..hourly Cytomegalovirus Quantitative PCR ………………………………………………….……………….daily Cytomegalovirus Qualitative PCR ………………………………………..………….……………….daily Epstein Barr Virus PCR. ……………………………………………...……………………………weekly Enterovirus RNA PCR …………………………………………………………………………....weekly* Hepatitis B/Hepatitis C/HIV Donor Screen NAT PCR………………………………………..…….daily* Herpes simplex Virus/Varicella-Zoster Virus PCR …………………………………………...…….daily* Human Papilloma Virus PCR …………………………..………………………………………..biweekly Influenza A/B & RSV PCR …………………………………………………………………...…….hourly Parvovirus B19 PCR …………………………………………………………………...…………..weekly West Nile Virus PCR ……………………………………………………………………………as needed West Nile Virus Donor Screen NAT PCR ……………………………………………………….….daily* * Stat testing may be required where clinically justified. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 226 of 291 APPENDIX II - Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG I. Introduction The NucliSENS easyMAG is an automated platform for the isolation (purification and concentration) of total nucleic acids (RNA/DNA) from biological specimens. Used for batch processing of 1-24 samples. The nucleic extraction method is based on Boom Chemistry using magnetic silica particles. Briefly, under high salt conditions, nucleic acid will bind to the silica particles. These silica particles act as solid phase and non-nucleic acid components are removed by several washing steps performed in the NucliSENS easyMAG instrument. Next, nucleic acids are eluded from the silica particles and the silica particles are removed from the extracted specimens by the NucliSENS easyMAG instrument.the resulting eluate contains purified and concentrated total nucleic acids. II. Specimens Types Plasma (minimum 200uL) collected in EDTA tube and separated by centrifugation; 3000rpm for 10 minutes. Do not test plasma collected in heparin tubes. CSF, sterile body fluids (amniotic fluid, vitreous fluid), Urine, BAL Viral transport media (VTM) from swabs collected in VTM and vortexed on high for 10 seconds. III. Materials, Equipments NucliSENS easyMAG NucliSENS easyMAG extraction products:  NucliSENS Lysis Buffer  NucliSENS easyMAG Lysis Buffer  NucliSENS easyMAG Magnetic Silica  NucliSENS easyMAG Extraction Buffer 1  NucliSENS easyMAG Extraction Buffer 2  NucliSENS easyMAG Extraction Buffer 3  NucliSENS easyMAG Disposables –vessels and pipettes BioHit Multichannel pipettor Ranin 1000uL, 100ul, 20ul Pipettors Aerosol Resistant Tips (ART) 1000ul, 200ul, 20ul Powder-free gloves Internal controls (for CMV, HSV, BKV, Parvovirus B19, VZ, Enterovirus) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 227 of 291 GENERAL PRECAUTIONS: Do not allow the NucliSENS easyMAG Lysis Buffer, NucliSENS easyMAG Extraction Buffer 1 or waste from the instrument to come in contact with acidic materials. Certain reagents contain guanidine thiocyanate. Buffers containing guanidine thiocyanate MUST NOT be mixed with cleaning solutions containing bleach. Liquid waste from extractions procedures contain guanidine thiocynate MUST NOT be mixed with other laboratory waste. Proper waste disposal is required. IV. Procedure 1. Turn ON easyMAG. Wait for orange light to turn GREEN, then switch ON the easyMAG computer. 2. Perform and record easyMAG Daily Maintence: Inspect drip tray. Inspect reagent cap filters. Inspect carbon filter. Empty & rinse waste bottle with water. 3. easyMAG 4. Click on “reagent” icon 5. Scan reagent stand and scan reagent bottle for all four reagents (A, B, C, D). 6. Click on “Sample” icon Login name: bmx Password: bmx For BKV/CMV/HSV/VZV/PARVO/ENTERO/ Enter Protocol: Generic 2.0.1 Enter Matrix: Plasma or “other” for urine & BAL, CSF for CSF Enter Volume (mL): 0.200 Eluate (uL): 50 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 7. Click on sample ID bar and scan specimen barcodes. 8. Click on “organize run” icon 9. Click on “create run” icon 10. A box will appear with today’s date and run #. Click on OK. 11. Click on 12. Click on “load run” icon Page 228 of 291 to transfer all scanned items onto the worklist. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 13. 14. Page 229 of 291 To identify vessels, click on vessel icon Scan position A barcode and then vessel barcode for first 8 samples, continue with position B and C depending on how many samples you have. 15. Click on the silica icon 16. Scan in silica barcode from top of silica box. 17. Highlight all samples by touching screen or with mouse. 18. To apply silica barcode to all samples click on “downward arrow” icon 19. Click on the “progress tab” 20. Print worklist. Select printer icon on right hand vertical menu bar. Pop-up appears, click OK. Sign your initials beside “Operater”. 21. Click on the “dispense lysis icon” >>>> on third menu bar from top. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 230 of 291 22. Once the lysis buffer has been dispensed, remove vessels form easyMag and put them in rack and take them to the BSC. 23. For CMV/BKV/HSV/VZV/PARVO/ENTERO, add 200 uL to the lysis buffer. Mix by pipetting up and down three times. 24. Vortex internal control (IC). For CMV/BKV/HSV/VZV/PARVO/ENTERO add 5 uL IC. Mix up and down three times. 25. Incubate at room temperature for 10 minutes. 26. To make silica premix add 550 uL PCR grade water to silica (program 1 of pipette) 27. Vortex silica premix before dispensing. 28. Dispense 125 uL silica into Elisa strip (program 2 of pipette) 29. After the 10 incubation period add 100 uL silica premix to vessel (program 3 of pipette) 30. Reload vessels into the easyMAG. 31. Scan position barcode and then vessel barcode (make sure the vessel icon is highlighted). 32. Click on start icon on right hand vertical menu bar. A pop-up box will appear asking if you have added the premix silica, click YES. 33. Once the samples have been eluted, transfer the eluate from the vessels to labeled microtube. Set pipette at 55uL to remove all of the eluate. 34. If elutes are to be tested same day store eluates at 4C. If PCR testing is to be performed at a later date, store eluate at -20oC as soon as possible. 35. A printout with reagent lot numbers must be printed at the end of the run. - When the extraction is complete, click on the magnifying glass icon. - Select your run from the list on the left. Make sure it is highlighted. - Click on the printer icon, print pages 1 of 1 only. - A pop up will appear “The run results are being printed. Mark this run ASSESSED?”. Click YES to this and the document will print. - File this with the corresponding tasklist printed at the beginning of the procedure. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 231 of 291 DAILY CLEAN UP: 1. Remove consumables from easyMAG, bag them and discard in biohazard waste box. 2. Clean easyMAG according to maintenance worksheet and record maintenance on worksheet. 3. To shut down easyMAG computer: Click on bmx key on the bottom menu bar. Click on “quit” in the pop-up box. 4. Wait 10 minutes after the easyMAG Computer is OFF before switching off the easyMAG. TO EMPTY WASTE: as required maximum 1/3 full 1. Go to reagent icon. (If reagent icon is not on the screen, then click on machine icon and click on reagent icon.) 2. Click on icon with droplet picture (top icon on right vertical bar) 3. Empty waste in media room fume hood. The Waste container should be labeled with the “Start date” and “contains guanidine thiocyanate”. Empty any remaining buffer from easyMAG Lysis Buffer and easyMAG Extraction Buffer1 into the fume hood waste container. Move the waste container to the 6th floor outside the Autopsy Suite for disposal every Tuesday afternoon. Discard these empty plastic containers (Lysis Buffer & Extraction Buffer 1) into the biohazard box. Extraction buffer 2 and Extraction buffer 3 can be discarded in the sink and bottles can be rinsed and recycled. 4. Rinse easyMAG waste container with water and return to easyMag. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 232 of 291 5. Click “I confirm that the waste container is empty. A check mark should appear in that box. 6. Press OK. WEEKLY MAINTENANCE: Clean Dispense Probe on the EasyMag 1. Click on maintenance icon . 2. Put in empty vessels and pipettes. 3. Choose start from vertical side bar menu To file runs that has been completed.    V. Choose the magnifying glass (second menu bar from the top) Highlight the runs you wish to file. Choose the filing cabinet (right menu bar). Quality Control Failed extraction runs, should be reviewed with senior and/or Charge technologist to establish causes and corrective actions; repeating extraction run maybe necessary. If the easyMag system becomes inoperable call Biomerieux technical support phone number, provided on each easyMag. External controls are extracted on the easyMag according to schedule in the table below. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Analyte/target BKV HSV1 & 2 External control High positive and Low Positive High positive and Low positive High positive and Low Positive Positive control VZV Positive control H1N1 Positive control Influenza A Positive control Influenza B Positive control RSV A Positive control RSV B Positive control Parvovirus B19 Positive control Enterovirus Positive control CMV EBV VI. Page 233 of 291 Extraction schedule Each day when test is done (once/week) Each day when quant. test is done (weekdays) Each day when quant. test is done Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot Once a month and/or with each new lot References NucliSENS easyMAG User Manual v2.0 ref: 280163 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 234 of 291 APPENDIX III - Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG I. Introduction The NucliSENS easyMAG is an automated platform for the isolation (purification and concentration) of total nucleic acids (RNA/DNA) from biological specimens. Used for batch processing of 1-24 samples. The nucleic extraction method is based on Boom Chemistry using magnetic silica particles. Briefly, under high salt conditions, nucleic acid will bind to the silica particles. These silica particles act as solid phase and non-nucleic acid components are removed by several washing steps performed in the NucliSENS easyMAG instrument. Next, nucleic acids are eluded from the silica particles and the silica particles are removed from the extracted specimens by the NucliSENS easyMAG instrument.the resulting eluate contains purified and concentrated total nucleic acids. II. Specimens Types: EDTA Whole blood minimum volume 200uL III. Materials, Equipments NucliSENS easyMAG NucliSENS easyMAG extraction products:  NucliSENS Lysis Buffer  NucliSENS easyMAG Lysis Buffer  NucliSENS easyMAG Magnetic Silica  NucliSENS easyMAG Extraction Buffer 1  NucliSENS easyMAG Extraction Buffer 2  NucliSENS easyMAG Extraction Buffer 3  NucliSENS easyMAG Disposables –vessels and pipettes BioHit Multichannel pipettor Ranin 1000uL, 100ul, 20ul Pipettors Aerosol Resistant Tips (ART) 1000ul, 200ul, 20ul Powder-free gloves Internal controls (EBV) IV. GENERAL PRECAUTIONS: Do not allow the NucliSENS easyMAG Lysis Buffer, NucliSENS easyMAG Extraction Buffer 1 or waste from the instrument to come in contact with acidic materials. Certain reagents contain guanidine thiocyanate. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 235 of 291 Buffers containing guanidine thiocyanate MUST NOT be mixed with cleaning solutions containing bleach. Liquid waste from extractions procedures contain guanidine thiocynate MUST NOT be mixed with other laboratory waste. Proper waste disposal is required. Only Aerosol Resistant Tips (ART) should be used in this procedure; single use tips, one tip per sample. V. PROCEDURE: Off-Board Preparation I. II. III. Thaw whole blood samples at room temperature. DO NOT vortex samples while thawing. When samples are completely thawed, mix by inverting sample tubes 5 times. Prepare the Premix. Invert 10 times, vortex 10 seconds, and invert x10 again to mix. Avoid creating bubbles. Number of extractions One 8 extractions/ 24 extractions/ extraction one vessel 3 vessels 600 uL 5.1 mL 15 mL Lysis Buffer volume 5 uL 44 uL 125 uL EBV Internal Control (IC) 140 uL *1190 (595 x2) **3.5 mL Silica uL *4 silica aliquots needed **6 silica aliquots needed IV. Prepare the NucliSENS easyMAG. See Section VI VI. ON Board the NucliSENS easyMAG 1. Turn ON easyMAG. Wait for orange light to turn GREEN, then switch ON the easyMAG computer. 2. Perform and record easyMAG Daily Maintence: Inspect drip tray. Inspect reagent cap filters. Inspect carbon filter. Empty & rinse waste bottle with water. 3. easyMAG Login name: bmx Password: bmx PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 236 of 291 4. Click on “reagent” icon Scan reagent stand and scan reagent bottle for all four reagents (A, B, C, D). 5. Click on “Sample” icon Enter Protocol: Specific B Protocol 2.0.1 Enter Matrix: Whole Blood Enter Volume (mL): 0.200 Eluate (uL): 50 Type: Primary 6. Click on sample ID bar and scan specimen barcodes. 7. Click on “organize run” icon 8. Click on “create run” icon 9. A box will appear with today’s date and run #. Click on OK. 10. Click on to transfer all scanned items onto the worklist. 11. Click on “load vessel” icon 12. Load easyMAG sample vessels and pipette tips. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 237 of 291 13. To identify vessels, click on vessel icon 14. Scan position A barcode, then scan vessel barcode; continue with position B and C, depending on how many samples are being extracted. 15. Click on the silica icon 16. Scan silica barcode from top of silica box in use. 17. Highlight all samples by touching screen or with mouse. 18. To apply silica barcode to all samples click on “downward arrow” icon 19. Print worklist. Select printer icon on right hand vertical menu bar. Pop-up printer window appears, click OK. 20. On the right side menu bar. Select vessels. to dispense lysis buffer into the extraction PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 238 of 291 21. Once the lysis buffer has been dispensed, remove vessels from the easyMag . Place vessels into rack and take them to the Biological Safety Cabinet (BSC). 22. Carefully pipette 200uL of each EBV whole blood sample into each well of the extraction vessels; pipette up and down three times to mix. 23. Invert and vortex the Premix. Using one ART tip per sample. Pipette 740uL of the Premix into each well of the extraction vessels. 24. Using the BioHit Multichannel Pipette on Program 3; mix the sample, silica, and lysis buffer in the vessel wells. Using one ART tip per sample. 25. Load the vessels back on-board the easyMag. Scan position A barcode, then scan vessel barcode; continue with position B and C. 26. Click on start icon on right hand vertical menu bar. A pop-up window will appear asking if you have added the premix silica, select YES. Extraction of 24 samples will take approximately 60 minutes on the easyMAG. 27. Once extraction is completed, transfer the eluate from the vessel well to labeled microtube. Set pipette at 55uL to remove all of the eluate. 28. If elutes are to be tested same day store eluates at 4C. If PCR testing is to be performed at a later date store at -20C as soon as possible. 29. Perform Clean Dispense Probe on the EasyMag after each whole blood extraction run. See weekly maintenance below. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual VII. Page 239 of 291 DAILY CLEAN UP: 1. Remove consumables from easyMAG, bag them and discard in biohazard waste box. 2. Clean easyMAG according to maintenance worksheet and record maintenance on worksheet. 3. EMPTY WASTE: Maximum 1/3 full performed as required. a. Select Buffer icon. (If the Buffer icon is not on the screen, then select the Main easyMag to access the Buffer icon.) icon b. Select Waste icon with droplet picture (top icon on right vertical bar) c. Empty waste in media room fume hood. The Waste container should be labeled with the “Start date” and “contains guanidine thiocyanate”. Empty any remaining buffer from easyMAG Lysis Buffer and easyMAG Extraction Buffer1 into the fume hood waste container. Discard these empty plastic containers (Lysis Buffer & Extraction Buffer 1) into the biohazard box. Extraction buffer 2 and Extraction buffer 3 can be discarded in the sink and bottles can be rinsed and recycled. Rinse easyMAG waste container with water and return to easyMag. d. Place the Waste Bottle back into the stand and attach the waste line to the Waste Bottle. Select the box “I confirm that the waste container is empty. A check mark should appear in that box. Select OK. 4. To shut down easyMAG computer: Select bmx key on the bottom menu bar. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 240 of 291 Select “Quit” in the pop-up window. Select Shutdown. 5. Wait 10 minutes after the easyMAG Computer is OFF before switching off the easyMAG. VIII. Quality Control Failed extraction runs, should be reviewed with senior and/or Charge technologist to establish causes and corrective actions; repeating extraction run maybe necessary. If the easyMag system becomes inoperable call Biomerieux technical support phone number, provided on each easyMag. External controls should be extracted on the easyMag according to schedule in the table below. Analyte/target External control EBV High Positive (HP), Negative, and Low Positive (LP) Extraction schedule once a week/ per PCR run IX. WEEKLY MAINTENANCE: Perform Clean Dispense after each whole blood extraction run. Clean Dispense Probe on the EasyMag 4. Click on maintenance icon . 5. Put in empty vessels and pipettes. 6. Choose start from vertical side bar menu To file runs that has been completed.  Choose the magnifying glass (second menu bar from the top)  Highlight the runs you wish to file.  Choose the filing cabinet (right menu bar). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual IX. Page 241 of 291 References NucliSENS easyMAG User Manual v2.0 ref. 280163 Vignoles, M. and Bertrand, M. et al, CVS May 2013, “Improvement of Nuclisens EasyMAG Whole Blood Extraction Protocol For Virus” PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 242 of 291 APPENDIX IV – Influenza A, B and H1N1 Virus rPCR back up testing by RotorGene I. Introduction Influenza, commonly referred to as the flu, is and infectious disease caused by RNA viruses of the family Orthomyxoviridae (the influenza viruses), that affects birds and mammals. The influenza virus gets spread via aerosols. The major surface antigens are hemagglutinin (H) and neuraminidase (N). The surface antigens change continuously (antigenic drift). By using Altona Influenza Screen & Type rPCR Kit 1.2, this assay is designed to detect Influenza B, all Influenza A strains in combination with a H1N1 (swine flu) specific primer probe system for the detection of all swine influenza strains including the current pandemic strain. The amplification and detection take place simultaneously, continually and in real time in the Rotor-GeneTM. In real-time PCR, fluorescent dyes are linked to oligonucleotide primer/probes, which bind specifically to the amplified product. Monitoring the level of fluorescence allows the detection of the amplicon. In the Altona Influenza Screen & Type rPCR assay version 1.2, four fluorescent dyes are incorporated in the master mix to detect four amplified products: One for SwineFlu specific amplicon; one for Influenza A specific (non-SwineFlu) amplicon; one for Influenza B and another one for the Internal Control (IC). The IC is added to each specimen in the initial step to both control the isolation procedure and to check for PCR inhibition. The Rotor-GeneTM simultaneously monitors the four different amplicons using four fluorimeter channels. II. Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as possible. BAL specimens if mucoid treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the Eppendroff 5415 C Micro-centrifuge. Aliquot supernatant and store at 4C, extract on the easyMag as soon as possible. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual III. Page 243 of 291 Specimen Processing Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG I. Procedure: a. Worklist Preparation: Take the Rotor Gene worklist from the virology planting along with the extra labels. b. Specimens Processing:  Specimens should be processed as soon as possible after arriving in virology laboratory.  Nasophargneal swabs, sputum and BAL’s are used to detect H1N1 and Influenza A & B.  After processing, an aliquot of the left-over specimens should be stored frozen at-70C. c. Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and gloves. Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8) and microcentrifuge(MICT17) Vortex Detection Room for Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System programmed for ALTONA H1N1 virus RT PCR 96-Well Loading Block (pre-cooled to 4oC) 72-Well Loading Block (pre-cooled to 4oC) 36-Well High Profile Rotor 36-Well Rotor Locking Ring 72-Well 6000 Series Rotor 72-Well Rotor Locking Ring 0.2mL Tubes 0.1 mL Strip Tubes and Caps Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 244 of 291 From: Altona Influenze Screen & Type rPCR Kit 1.0, (stored at -20o C in clean room):  Swine Flu Positive Control  Influenza A Positive Control  Influenza B Positive Control  Master A (12 tests per vial)  Master B (12 tests per vial)  Internal Control (IC)  Water (PCR grade) External Control: To be run when a new lot is used, during training or if QC failure occurs. GENERAL PRECAUTIONS:             There must be separate PCR work areas: c. Clean room d. Specimen preparation room Amplification room Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas. Change lab coats and gloves between work areas. Only blue gowns are to be worn in the clean room. Use only filtered pipette tips Use only sterile RNase, Dnase-free microcentrifuge tubes Use sterile, disposable polypropylene tubes throughout the procedure Always wear powder-free gloves when handling reagents Change gloves frequently and keep tubes closed whenever possible Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water. Sample Preparation: Use Specimen Preparation area; work in cabinet with gown and frequent glove change Use EasyMag to extract DNA and RNA PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 245 of 291 PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) d. Take out the necessary number of H1N1 Master Mix A and B vials (24 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the number of tests and prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do not vortex. Make only enough master mix for the tests you are running. After vial A has been added to Vial B it cannot be frozen again. No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 10 20 30 40 50 60 70 80 90 100 110 120 b. Place proper micro tubes for each purified sample, Positive controls, external QC and Negative control (H2O) into the Cooling Block (pre-cooled to 4oC). c. Pipette 15 uL prepared Master Mix (A+B) into each micro tubes h. Pipette 1.0 uL of internal control into the micro tubes designated for the Positive controls and water. i. Pipette 10uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and cap tightly. Label each microtubes 1,2,3…. before leaving clean room. j. Place the cooling block in to a clean biohazard bag. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual k. Page 246 of 291 Changed out of dedicated clean room gown. In the Specimen Processing Room: (Changed into specimen room gown) e. f. g. h. Using a separate filter tip each time, carefully pipette 10 uL each of purified sample, Positive controls, and external QC (if using) directly into the micro tubes. Mix by pipetting 3x up and down into the mastermix. Immediately close the cap tightly. Check each micro tube before loading. Make sure there is no bubble at the bottom of the tube. Load micro tubes into a proper rotor (e.g. 36-well or 72-well) and snap close the locking ring. Load the rotor in Rotor-Gene 6000. Always fill up the empty position of the rotor using empty tubes. In the Rotorgene room: a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s. t. u. Turn on computer, turn on Rotorgene; FOR THE BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; Double click(left on the mouse) “Rotor-Gene 6000 series software 1.7” icon Wait for “Initializing machine…..” Highlight “Influenza A,B & H1N1” assay; Press “New” button; Highlighting the correct Rotor type you are using 36-well Rotor (Red) 72-well Rotor (Blue) Tick the “Locking Ring Attached” option Press “Next” button Enter initials in operator space. Enter Master Mix lot. # in the Notes space. Make sure the Reaction Volume is “25µl”; Press “Next” button Temperature profile window pops up, press “Next” button Make sure your ring has been loaded in the Rotorgene. Summary window pops up, press “Start Run” button; Save As window pops up; the run is given a filename with a default template. Change it according to the date and numerical runs the assay performed on the same day; e.g. H1N1 200907-17-01 (assay name-year-month-date-numerical run#); Note: Influenza results are stored in \desktop\My Doucument\Influenza A,B&H1N1 Press Save button; Machine is starting and Edit Samples window pops up; Enter sample LIS# including positive and negative controls. Define the type of samples according to the chart: PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 247 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual v. w. Name Type Select Sample Unknown Yes Positive Controls H1N1, FluA, FluB Positive Control Yes Water Negative Control Yes Once finishing all samples, press Finish button. Once cycling screen comes on, click “Named On” button. To analyze and print H1N1 report after a run is finished: a. b. c. d. e. f. g. h. i. j. k. l. m. n. The run is done when Profile Program reads: Run has completed. Analyze raw data by clicking on the each of these channels (one at a time): Green channel (H1N1), Red channel (Flu A other than H1N1), Orange channel (Flu B) and Yellow channel (IC). These tabs are found on the second menu bar from the top, on the left hand side. Click on autoscale (bottom left). Click “All Off” (bottom right). Click “water” on. Compare the specimen and control curves to the water one at a time (by clicking each sample on and then off when proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of the positives on the worklist. Click on “Named On” Perform curve analysis: Click Analysis on the top tool bar. Analysis floating window pops up; Choose “Quantitation” tab. Quantitation window is brought up to the front. There should be four channels: Green (H1N1), Red (Flu A other than H1N1), Orange (Flu B) and Yellow (IC). Highlight Cycling A. Green (Page 1) (H1N1) option and press Show button. Maximize cycling green window (or the channel you are working on) Highlight slope correct tab. Click on Ignore first… tab. A window will pop-up, type in 5 (i.e. ignore 5 cycles). Set threshold at 0.05. A threshold line will appear on the screen. Move threshold line to above the noise (i.e. Negative specimens) To Print reports: Press Reports button on the top toolbar. a. b. c. d. e. f. Report Browser window pops up. Choose Cycling A. Green (Page 1). Under templates box, choose “Quantitation full report”. Press Show button Maximize the report window, check that only the positives that you have identified from your raw data analysis are positive (i.e. have a crosspoint) Choose print report. (for the red, orange and yellow channels print only pages 2-4). Once finishing the print, close the report window Go to the floating Analysis window; PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual g. Page 248 of 291 Repeat the step from “e” to “m” for Cycling A. Red (Influenza A), Cycling A. (Influenza B) and Cycling A. Yellow (internal control) Shut down computer: d. Exit from software. A pop-up window will appear “save changes”. Click “yes”. e. Click “start” button. Click “Turn off computer”. f. Turn off Rotorgene At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet Specimen Preparation Room:  Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.  Cap discard container.  Wash racks. Amplification Room:  Discard micro tubes into a disposal bag and tight the bag.  Discard the bag into biohazard waster container.  Perform the cleaning procedure according the daily maintenance sheet. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 249 of 291 IV. Interpretation and Reporting Green Channel (H1N1 specific) Red Channel (Influenza A specific, nonswineflu) Orange Channel (Influenza B) Yellow Channel (IC specific) Interpretations + - - +/- Positive for H1N1 - + - +/- Positive for Influenza A, NOT H1N1 + + - +/- Positive for H1N1 and Influenza A (ask senior for confirmation) - - + +/- Positive for Influenza B - - - + Negative for both H1N1 and Influenza A - - - - INVALID Test needs to be repeated. Sample needs to be diluted 1:4 - - - - Repeated Diluted 1:4 Sample tested: INDETERMINATE Note: Check for each curve of the samples showing positive reactions either/or for the Red, Orange and Green Channel in comparison with the positive and water (negative) controls. Do this step by clicking the ALL OFF tab and then highlighting the samples showing positive reactions in the Quantitation result channel. Positive for H1N1, Influenza A, or Influenza B report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard. PCH1R: 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 window (F7) Isolate #: 1 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 250 of 291 Org. ID: 23ah1n1 Influenza A H1N1 (Swine) virus 23notsw Influenza A virus NOT H1N1 (Swine) 23infb Influenza B virus Open the Isolate Comment window F8. Go to the Virology keypad, type “V”. Ctrl D. Select from the keypad: \ANH1 to add the comment phrase: DETECTED by PCR. This is a research test. Influenza Screen & Type RT-PCR Kit v1.2 Altona Diagnostics Inc. This isolate has been forwarded to PHL (Public Health Laboratory) for subtyping. Results to follow. Note: Remove (delete) the sentences that refer to subtyping at PHL when reporting H1N1, and Influenza B; subtyping is only required for Influenza A isolates. Grave (`) the result. Ctrl-F to finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). The Medical Officer of Health (MOH) must be notified by fax and phone. Record all calls and faxes made on the LIS workcard; under the CALL media and in the Call Window. Negative for H1N1, Influenza A, and Influenza B. Date the media on the back of the LIS workcard. PCH1R: F6 to add run date. On the front of the LIS workcard. Select from the keypad: }H1}H1- Negative for Influenza A and B including H1N1 (Swine) virus. This is a research test. Influenza Screen and Type RT-PCR Kit v1.2 Altona Diagnostics Inc. Ctrl-F to finalize the report. Indeterminate Report. Date the media on the back of the LIS workcard. PCH1R: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRH1R. Under this media report the dilution test result. On the front of the LIS workcard. Select from the keypad: }H1Change “Negative” to INDETERMINATE. INDETERMINATE for Influenza A and B including H1N1 (Swine) virus. This is a research test. Influenza Screen and Type RT-PCR Kit v1.2 Altona Diagnostics Inc. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 251 of 291 Ctrl-F to finalize the report. II. 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 for H1N1, Influenza A (not H1N1), and Influenza B should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition.  A Positive Control is included and shows either a positive reading in Green Channel (SwineFlu specific) or a positive reading in Red Channel (Infuenza A specific-non SwineFlu)  A Negative Control is include and shows a negative reading in both Green Channel (SwineFlu specific) and Red Channel (Infuenza A specific-non SwineFlu)  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). e. If positive QC material yielded negative result, repeat the entire run. f. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. g. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 252 of 291 h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. III. References: Altona Influenza screen & Type RT-PCR Kit 1.2 User Manual, Altona PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 253 of 291 APPENDIX V - Respiratory Synctial Virus (RSV) PCR backup testing by RotorGene VI. Introduction Respiratory Synctial Virus (RSV) is a negative sense, single stranded RNA virus of the family Paramyxoviridae. Based on antigenic properties and sequence analysis RSV are subdivided into RSV A and RSV B. Different genotypes of both subgroups can co-circulate within one epidemic with varying frequency. RSV causes respiratory tract infections. For most people, a RSV infection produces only mild symptoms like common cold. But RSV is the most important cause of severe respiratory illness in infants and young children and the major cause of infantile bronchiolitis and pneumonia in industrialized countries. Nearly all children will have been infected with the virus by 2-3 years of age. RSV also is a significant problem in the elderly, in persons with cardiopulmonary diseases and in immunocompromised individuals. Repeated reinfections occur throughout life. RSV is spread by infectious droplets or by contact with nasal or oral secretions from infected people. Outbreaks of RSV infections are reported worldwide, with striking annual epidemics in the winter and early spring in temperate climates, like Europe and North America, and in the rainy season in the tropics. VII. Collection and Transport Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as possible. BAL specimens if mucoid treat with working sputolysin. Remove red blood cells and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the Eppendroff 5415 C Micro-centrifuge. Aliquot supernatant and store at 4C, extract on the easyMag as soon as possible. VIII. Specimen Processing Please refer to Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG GENERAL PRECAUTIONS:  There must be separate PCR work areas: e. Clean room f. Specimen preparation room  Amplification room Supplies and equipment must be dedicated to each PCR area and not used for other activities or moved between areas. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual           Page 254 of 291 Change lab coats and gloves between work areas. Only blue gowns are to be worn in the clean room. Use only filtered pipette tips Use only sterile RNase, Dnase-free microcentrifuge tubes Use sterile, disposable polypropylene tubes throughout the procedure Always wear powder-free gloves when handling reagents Change gloves frequently and keep tubes closed whenever possible Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water. a. Materials, Equipments and Facilities: Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and gloves Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8) and microcentrifuge(MICT17) Vortex Detection Room for Rotor-Gene 6000 Rotor-Gene 6000 Multiplexing System programmed for ALTONA RSV A & B RT PCR 72-Well Loading Block (pre-cooled to 4oC) 72-Well 6000 Series Rotor 72-Well Rotor Locking Ring 0.1 mL Strip Tubes and Caps 36-Well High Profile Rotor 36-Well Rotor Locking Ring 0.2mL Tubes Variable volume pipettes: 1 to 20 uL 10 to 200 uL 100 to 1000 uL From: Altona RSV A & B RT-PCR Kit 1.0, (stored at -20o C in clean room):  RSV A Positive Control  RSV B Positive Control  RSV Master A (12 tests per vial)  RSV Master B (12 tests per vial)  RSV Internal Control (IC)  Water (PCR grade) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 255 of 291 External Control: To be run when a new lot is used, during training or if QC failure occurs. IX. Procedure: Take RSV worklist from the virology prep bench and arrange eluates in order according to the worklist, store the eluates at 4C until ready to add to the RSV Master mix. PCR Set-up: In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet) a. Take out the necessary number of RSV A & B Master Mix A and B vials (24 tests per vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the number of tests and prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do not vortex. Make only enough master mix for the tests you are running. After vial A has been added to Vial B it cannot be frozen again. No. of samples 1 2 3 4 5 6 7 8 9 10 11 12 Preparation of Master Mix Master A (µl) 5 10 15 20 25 30 35 40 45 50 55 60 Master B (µl) 15 30 45 60 75 90 105 120 135 150 165 180 b. Place proper micro tubes for each purified sample, Positive controls, external QC and Negative control (H2O) into the Cooling Block (pre-cooled to 4oC). c. Pipette 20 uL prepared Master Mix (A+B) into each micro tubes d. Pipette 0.5 uL of internal control into the micro tubes designated for the RSV A Positive Control, RSV B Positive Control and PCR grade Water- Negative Control. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 256 of 291 e. Pipette 5uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and cap tightly. Label each microtubes 1,2,3…. before leaving clean room. f. Place the cooling block into a clean biohazard bag. g. Changed out of dedicated clean room gown. In the Specimen Processing Area: (Change into specimen room gown) a. Using a separate filter tip each time, carefully pipette 5 uL each of purified sample, Positive controls, and external QC (if using) directly into the micro tubes. Mix by pipetting 3x up and down into the mastermix. Immediately close the cap tightly. b. Check each micro tube before loading. Make sure there is no bubble at the bottom of the tube. c. Load micro tubes into a proper rotor (e.g. 36-well or 72-well) and snap close the locking ring. Load the rotor in Rotor-Gene 6000. d. Always fill up the empty position of the rotor using empty tubes. In the Rotorgene Detection Area: oo. Turn on computer, turn on Rotorgene; pp. FOR BLUE ROTORGENE ONLY. Choose clinical icon; pass word “msh”; press “→” button; qq. Double click(left on the mouse) “Rotor-Gene 6000 series software 1.7” icon rr. Wait for “Initializing machine…..” ss. Highlight “ALTONA RSV A&B” assay; tt. Press “New” button; uu. Highlighting the correct Rotor type you are using vv. 72-well Rotor (Blue) ww. 36-well Rotor (Red) xx. Tick the “Locking Ring Attached” option yy. Press “Next” button zz. Enter initials in operator space. Enter Master Mix lot. # in the Notes space. Make sure the Reaction Volume is “25µl”; aaa. Press “Next” button Temperature profile window pops up, press “Next” button bbb. Make sure your ring has been loaded in the Rotorgene. ccc. Summary window pops up, press “Start Run” button; ddd. Save As window pops up; the run is given a filename with a default template. Change it according to the date and numerical runs the assay performed on the same day; e.g. RSV A&B 2010.03.29 (e.g. assay name-year-month-date-numerical run#); a. Note: RSV results are stored in \desktop\My Doucument\RSV A&B results. eee. Press Save button; fff. Machine starts and Edit Samples window pops up; PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 257 of 291 ggg. Enter sample LIS# including positive and negative controls. Define the type of samples according to the chart: Name Sample RSV A Positive Control RSV B Positive Control Water Type Unknown Positive Control Positive Control Negative Control Select Yes Yes Yes Yes hhh. Press Finish button. iii. Once cycling screen comes on, click “Named On” button. To analyze and print RSV A &B report after a run is finished: tt. The run is done when Profile Program reads: Run has completed. uu. Analyze raw data by clicking on the each of these channels (one at a time): Green channel (RSV B), Red channel (RSV A) and Yellow channel (IC). These tabs are found on the second menu bar from the top, on the left hand side. vv. Click on autoscale (bottom left). Click “All Off” (bottom right). Click “water” on. Compare the specimen and control curves to the water one at a time (by clicking each sample on and then off when proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of the positives on the RSV worklist. ww. Click on “Named On” xx. Perform curve analysis: Click Analysis on the top tool bar. yy. Analysis floating window pops up zz. Choose “Quantitation” tab. aaa. Quantitation window is brought up to the front. There should be three channels: Green (RSV B), Red (RSV A) and Yellow (IC). bbb. Highlight Cycling A. Green (Page 1) (RSV B) option and press Show button. ccc. Maximize cycling green window (or the channel you are working on) ddd. Highlight slope correct tab. eee. Click on Ignore first… tab. A window will pop-up, type in 5 (i.e. ignore 5 cycles). fff. Set threshold at 0.05. A threshold line will appear on the screen. ggg. Move threshold line to above the noise (i.e. Negative specimens) hhh. To Print reports: Press Reports button on the top toolbar. iii. Report Browser window pops up. jjj. Choose Cycling A. Green (Page 1). Under templates box, choose “Quantitation full report”. kkk. Press Show button lll. Maximize the report window, check that only the positives that you have identified from your raw data analysis are positive (i.e. have a crosspoint). PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 258 of 291 mmm. Choose print report. (for the red and yellow channels print only pages 24). nnn. Once finished printing, close the report window ooo. Go to the floating Analysis window; ppp. Repeat the step from “e” to “t” for Cycling A. Red (RSV A) and Cycling A. Yellow (internal control) Shut down computer: a. Exit from software. A pop-up window will appear “save changes”. Click “yes”. b. Click “start” button. Click “Turn off computer”. c. Turn off Rotorgene At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed by 70% alcohol: Clean Room:  Clean Biological Safety Cabinet Specimen Preparation Room:  Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.  Cap discard container.  Wash racks. Amplification Room:  Discard micro tubes into a disposal bag and close the bag.  Discard the bag into biohazard waster container.  Perform the cleaning procedure according the daily maintenance sheet. IV. Interpretation and Reporting: Green Channel (RSV B) Red Channel (RSV A) Yellow Channel (IC specific) + - +/- Positive for RSV B - + +/- Positive for RSV A + + +/- Positive for RSV A & B (ask senior for confirmation) - - + Negative for both RSV A & B Interpretations PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 259 of 291 INVALID Test needs to be repeated. Sample needs to be diluted 1:4 with lysis buffer Note: Check for each curve of the samples showing positive reactions either/or for the Red, Orange and Green Channel in comparison with the positive and water (negative) controls. Do this step by clicking the ALL OFF tab and then highlighting the samples showing positive reactions in the Quantitation result channel. Positive for RSV A, or RSV B report as an isolate in the (F7) Isolate Window. Date the media on the back of the LIS workcard PCRSV: F6 to add the date tested. Enter on the PCRSV media line the crosspoint (ct) value: ct= Report the virus as an isolate in the F7 window. Isolate #: 1 Org. ID: 26rsvA Respiratory syncytial virus type A 26rsvB Respiratory syncytial virus type B Open the Isolate Comment window F8 Go to the virology keypad, type “V”. Ctrl D. Select from the keypad: \RSV+ to add the comment phrase: \RSV+ DETECTED by PCR. This is a research test. RSV RT-PCR Kit v1.0 Altona Diagnostics Inc. Grave (`) the result. Ctrl-F to finalize the report. Call all positives to ward/doctor and Infection Control Practitioner (ICP). Negative for RSV A and RSV B. Date the media on the back of the LIS workcard. PCRSV: F6 to add run date. On the front of the LIS workcard, select from the keypad: }RAB}RAB- Negative for Respiratory Syncytial Virus A and B. This is a research test. RSV RT-PCR Kit v1. Altona Diagnostics Inc. Ctrl-F to finalize the report. Indeterminate Report. (Internal control failed twice to amplify.) Date the media on the back of the LIS workcard. PCRSV: F6 to add run date. Select from the keypad “Failed single”, type “IC no amplification”, select >FAIL Select from keypad ^PRRSV. Under this media ^PRRSV report the 1:4 dilution test result type “IC no amplification”. On the front of the LIS workcard. Select from the keypad: }RSVChange “Negative” to INDETERMINATE; final report should appear as: PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 260 of 291 INDETERMINATE for Respiratory Syncytial Virus A and B. This is a research test. RSV RT-PCR Kit v1. Altona Diagnostics Inc. . Ctrl-F to finalize the report. V. 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 for RSV A, and RSV B should be extracted on the on the easyMag and run once a month and/or with each new lot. Daily QCs: Every run:  Each patient specimen must have an Internal Control (IC) added to monitor both isolation and PCR inhibition.  A Positive Control is included and shows either a positive reading in Green Channel (RSV B) or a positive reading in Red Channel (RSV A)  A Negative Control is include and shows a negative reading in both Green Channel (RSV B) and Red Channel (RSV A)  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). e. If positive QC material yielded negative result, repeat the entire run. f. If negative QC material yielded positive result, it may be due to cross-contamination from adjacent positive sample within the run or carry-over contamination from previous runs via equipment or the environment. Review procedure and equipment to establish and eliminate potential sources of contamination. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 261 of 291 g. The extent and nature of contamination can also be evaluated by comparing the positive rate of the run with its expected positive rate. h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated reagents and consumables and disinfect equipment and environment before repeating the run. i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being positive or patient samples have higher than expected positive rate and these samples are often non-repeatable positives), it is necessary to have a thorough environmental disinfection followed by swabbing to monitor. j. Successful ending to a carry-over contamination may be indicated by QC results and patient positivity rate falling back to the expected normal range and three negative environmental swabs. X. References: RSV A & B RT-PCR Kit v1.0 User Manual, Altona PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 262 of 291 APPENDIX VI - General PCR Precautions and Decontamination Procedures General PCR Precautions:  There must be separate PCR work areas:    Clean room Specimen preparation room Amplification room  Workflow must proceed in a uni-directional manner beginning in the clean room area and moving to the amplification area.  Supplies and equipment must be dedicated to each area and not used for other activities or moved between areas.  Change laboratory coat and gloves between work areas.  Wear powder-free gloves at all times. Hands and dust particles may carry bacteria and molds and are the most common sources of Rnase contamination.  All reagents and master mixes must be prepared and stored in the clean room.  Use sterile aerosol resistant pipette tips.  Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and then rinse with 70% alcohol or water.  Racks, surfaces and equipment that can be cleaned are cleaned daily. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 263 of 291 Decontamination Procedure: When contamination occurs the following steps must be followed:       Use 1% sodium hypochlorite followed by alcohol (water) to decontaminate. Change into clean lab coat Begin cleaning in the clean room and end in the amplification room. Discard all reagents and disposable items (eg. pipette tips, capillary caps) Clean biological safety cabinet, equipment and surfaces. For WNV PCR decontamination, see checklist below. WNV Decontamination Procedure: Clean room:    Discard pipette tips, capillaries, caps, pens and eppendorf tubes Clean biological safety cabinet, pipettors, racks and capper Turn UV light on in the safety cabinet Specimen preparation area:   Discard pipette tips, caps, pens, and reagents (AW1, AW2, alcohol, elution buffer and lysing buffer) Clean: Biological safety cabinet Pipettors and holder Racks Vortex Centrifuge Timer Bench Capillary capper Amplification area:  Clean: Microwave Capillary centrifuge Capillary carousel Light Cycler PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 264 of 291 Perform wipe tests on all 3 areas:  Clean room: biological safety cabinet, pipettors, racks and capper  Specimen preparation area: Biological safety cabinet Pipettors and holder Racks Vortex Centrifuge Timer Bench Capillary capper  Clean room: Microwave Capillary centrifuge Capillary carousel Light Cycler If any wipe test is positive, repeat cleaning procedure for that area. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 265 of 291 Appendix VII: Creating Standard Curve for Quantitative PCRs Create standard curve for PCR: CMV, BKV, and EBV Standard curves are created every 6 months, or as needed; recalibration of a standard curve may be required. Materials CMV PCR Kit, Altona Diagnostics BKV PCR Kit, Altona Diagnostics EBV PCR Kit, Altona Diagnostics Variable pipettes: 1-20uL Aerosol resistant tips (ART) 20uL 0.01 microtubes and caps eppendroff epMotion 5075 (benchtop robotic pipettor) Procedure: 1. In the Clean Room prepare master mix for the standard curve PCR reaction; 12 reactions are required to create a standard curve. Make 13 reactions (12 + 1), to load on the epMotion 5075. 2. In the processing area thaw the Quantitative Standards: QS1, QS2, QS3, QS4. 3. Use the epMotion 5075 to pipette the master mix into the PCR reaction microtubes. See Appendix I. 4. Once the master mix is pipetted. Manually pipette 10uL of the Quantitative Standard (QS) into the master mix in the following order: Position 1 2 3 4 5 6 7 8 9 10 11 12 QS4 QS4 QS4 QS3 QS3 QS3 QS2 QS2 QS2 QS1 QS1 QS1 Do not add internal control (IC) to the reaction microtubes. Only the target Green channel will be analyzed. Do not load a negative/PCR water control. 5. After the QSs’ has been added to the master mix cap the PCR reaction microtubes. Load the Rotorgene. Identify each quantitative standard (QS) as sample type Standard and remember to enter the concentration of the QSs’ under “Given Conc.” in the Edit Samples window. The QS concentrations are on the QS sample vials. 6. Name the file as Standard curve and save into the Standard curve (STD) folder. For example: CMV STD yyyymmdd. My documents… STD folder. 7. When the PCR reaction is complete. Select Analysis, each quantitative standard (QS) will have three curves. Make sure “Name on” has been selected. Select Slope correct, and Ignore first 5 Cycles; create a threshold. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 266 of 291 8. Observe the Quant. Results chart window, paying attention to the Given Conc. Column and the Calc. Conc. Column (calculate concentration column). Look on the Analysis Green Channel Graph deselect QS curves that cause the calculated concentration of the standard to be far from the given concentration of the standard. The objective is to have the calculated concentration of the standards be as close to the given concentration of the standards on the quantitative results chart. When satisfied save the standard curve run. 9. Clean Up: 10. Place PCR reaction microtubes in a bag, seal shut, and discard into the biohazard waste containers. 11. Clean Rotorgene rotors and locking rings with Kimwipe dampen with RNAseAway, followed with Kimwipe dampened with distilled water, and then a Kimwipe dampen with 70% alcohol. 12. Complete daily equipment maintenance, as outlined in RotorGene Maintenance schedule. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 267 of 291 Appendix VIII - Instructions for Pipetting by epMOTION Instructions for Pipetting One Test on epMOTION NOTE: It takes about 30 minutes (15 samples), from putting the gloves on, removing specimens from the freezer, making up the Master Mix, loading the epMotion, epMotion at work, loading the Rotorgene and scanning specimens numbers onto the Rotorgene. It takes the epMotion 20 minutes to pipette Master Mix only for 30 samples. 40 Minutes, excluding setup, to pipette both Master Mix and samples for about 70 samples. You can prepare the Master Mix slightly ahead of time. Load the Master Mix, and samples and start pipetting before the first extraction is finished. Pause (by clicking the Red Button on the left side) the epMotion either right after pipetting the Master Mix or after it pipetted the last sample, to load the rest of the specimens after the extraction is finished and finish the pipetting to save time. Press the Green Arrow without the line, (the lower one) to CONTINUE the pipetting. REMEMBER TO SPIKE THE QS, POSITIVE CONTROL AND WATER WITH IC 1. Turn on switches in the sequence for computer/epMotion/monitor, wait until the short cut for epBlue Server appears on the bottom left side of the screen. 2. Double click the short cut for “epBlue Client” in the center of the screen 3. Type “msh” in both Account and password, click log in 4. Click “Home” tab 5. Click “Open/run Applications” from the bottom Task Card 6. “Open/Run applications” window pops up, make sure the following items are selected: i. “msh” under user ii. “(msh)” under Folder iii. Select and right click PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 268 of 291 a) “OtherVirus Rotorgene.dws” under Applications for CMV, BKV, HSV/VSV, EBV, PARVO and ADENO; b) “Flu Rotorgene.dws” for H1N1, RSV A/B and ParaInfluenza. iv. Click “Copy” v. Click “Daily Run” vi. Click the scissors icon in the Application pane and click “Paste” vii. Click “Edit Properties” viii. Change the “Name” from “Other Virus Rotorgene.dws” to Virus type and todays date (run #) e.g. CMV 2011-08-26 (1) 7. Click “Save” button 8. Double click on the file just created in (6 viii) in the Application pane or click Open Application with cursor on the file e.g. CMV 2011-08-26 (1).dws 9. If the following window does not pops up, click the “Run” tab. Make sure “Filter Devicelist” is checked. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 269 of 291 10. Load the microtubes into the cooling block according to diagram: 11. Prepare the mastermix in the 1.5mL snapcap conical microtubes and placed in first position of PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 270 of 291 12. the Reagents Rack. 13. Press the microtube down firmly so that it sits flushed with the top of rack 14. Place samples column-wise in Sample Racks labeled “Sample 1-24” according to the worklist including necessary controls. Subsequent samples may be placed in “Sample 25-28” and “Sample 49-72” for a maximum of 72 reactions. 15. Close the epMotion hood firmly. You should hear a click. 16. Click “Run>>” button. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 271 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 17. The Process Control window pops up 18. Enter the total number of reactions including controls over the highlighted ”1”. 19. Click “OK” 20. Cancel the level sensing for Levels and Location. Keep the level sensing for Tips. 21. Keep clicking the “RUN>>” button until the “MasterMix A+B” window shows up * 22. At the “Labware information” menu, enter the number “*” shown in the “Minimum Volume” column in the ““Volume” column highlighted with 23. Click “Run>>” 24. Close the run by highlighting the run, right click and click “Close”. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 272 of 291 25. Wipe down the blocks and the epMotion with RNase AWAY, distilled water, alcohol and distilled water in the above sequence. 26.Turn off the Computer/computer switch/epMotion/Monitor Instruction For Pipetting Two Or More Tests On Epmotion: 1. Turn on switches in the sequence for computer/epMotion/monitor, wait until the short cut for epBlue Server appears on the bottom left side of the screen. 2. Double click the short cut for “epBlue Client” in the center of the screen 3. Type “msh” in both Account and password, click log in 4. Click “Home” tab 5. Click “Open/run Applications” from the bottom Task Card 6. “Open/Run applications” window pops up, make sure the following items are selected: i. “msh” under user ii. “(msh)” under Folder iii. Select and right click iv. Select and right click a) “OtherVirus Rotorgene.dws” under Applications for CMV, BKV, HSV/VSV, EBV, PARVO and ADENO; b) “Flu Rotorgene.dws” for H1N1, RSV A/B and ParaInfluenza. v. Click “Copy” vi. Click “Daily Run” vii. Click the scissors icon in the Application pane and click “Paste” viii. Click “Edit Properties” ix. Change the “Name” from “Other Virus Rotorgene.dws” to Virus type and todays date (run #) e.g. CMV 2011-08-26 (1) PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 273 of 291 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 274 of 291 7. Click “Save” button. 8. i) RSV can be pipetted right after the FLU; ii) PARVO, ADENO can be pipetted after HSV/VZV. 9. Create the second or third test as above, edit the name and save with the corresponding “TEST NAME date” 10. Double click on the file (the new file i.e. the second or third file) just created in (6 viii) in the Application pane. e.g. RSV 2011-08-26 (1).dws 11. Click the “Procedure” Tab on the left side. 12. Click on the “Master Mix Dispense” –#3 i) Click “New Pattern” on the bottom left corner. a) Click the highlighted circle, the placement of the second Master Mix. b) Click 2 consecutive circles on the samples “Cooling block” to indicate the direction the Master mix are to be dispensed PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 275 of 291 13. Click on the “Sample dispense” #5 i) Click “New Pattern” on the bottom left corner. a) Click the highlighted circle, the placement of the second Master Mix. b) Click 2 consecutive circles on the samples “Cooling block” to indicate the direction the Master mix are to be dispensed 14. Click “File” then “Save”. 15. Click the first test to be dispensed. 16. Run the first test until it is completed. 17. Close the window by right click and close the first run. 18. Start the second run by clicking on the run. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 276 of 291 19. If the following window does not pops up, click the “Run” tab. Make sure “Filter Devicelist” is checked. 20. Load the microtubes into the cooling block according to diagram: PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 277 of 291 21. Prepare the mastermix in the 1.5mL snapcap microtubes and placed in first position of the Reagents Rack. 22. Press the microtube down firmly so that it sits flushed with the top of rack 23. Place samples column-wise in Sample Racks labeled “Sample 1-24” according to the worklist including necessary controls. Subsequent samples may be placed in “Sample 25-28” and “Sample 49-72” for a maximum of 72 reaction 24. Close the epMotion hood firmly. You should hear a click. 25. Click “Run>>” button. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Page 278 of 291 Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual 26. The Process Control window pops up 27. Enter the total number of reactions including controls over the highlighted ”1”. 28. Click “OK” 29. Cancel the level sensing for Levels and Location. Keep the level sensing for Tips. 30. Keep clicking the “RUN>>” button until the “MasterMix A+B” window shows up * 31. At the “Labware information” menu, enter the number “*” shown in the “Minimum Volume” column in the ““Volume” column highlighted with 32. Click “Run>>”. 33. Close the run by highlighting the run, right click and click “Close”. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 279 of 291 34. Wipe down the blocks and the epMotion with RNase AWAY, distilled water, alcohol and distilled water in the above sequence. 35. Turn off the Computer/computer switch/epMotion/Monitor PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 280 of 291 Appendix IX – RotorGene Worksheet Forms_Worksheets\RotorGene Worksheet MI_MD_06_09 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 281 of 291 Appendix X – RotorGene Calculations for Reports RotorGene Calculations for Reports MI_MD_06_10 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 282 of 291 Appendix XI – Rotorgene Optical Temperature Verification (OTV) Procedure I. II. III. Introduction: Optical Temperature Verification (OTV) Kit uses 3 thermochromic liquid crystals (TLCs) optical properties to verify the temperature of the RotorGene real-time cyclers. When heated the thermochromic liquid crystals change from opaque to transparent at very precise temperatures (50oC, 70oC, and 90oC). The thermochromic liquid crystals do not fluoresce, and it is necessary to cover the excitation source with a fluorescence scatter plate allowing the liquid crystal temperature transition points to be detected. The temperature transition reported by the RotorGene cycler is compared to the temperature encoded in the OTV Rotor-Disc serial number. Every batch of thermochromic liquid crystals has specific melting temperatures encoded in the OTV Rotor-Disc serial number. If the instrument is not within temperature specifications, automatic recalibration of the RotorGene can be performed by selecting “Apply Adjustment” in the OTV Temperature Verification Results window. Optical Temperature Verification (OTV) should be performed every 6 months. Equipment: RotorGene 6000 Rotor-Disc 72 Rotor and Rotor-Disc 72 Locking Ring Rotor-Disc OTV Kit (Qiagen catalog no. 981400) Store at Room Temperature (15-25oC). Keep OTV Rotor-Disc away from light. Can be used a maximum of 30 times until expiration kit date. Procedure: Place the large green fluorescent insert over the excitation lens –bottom right of the RotorGene cycler. Place the OTV Rotor-Disc into the Rotor-Disc 72 Rotor. Ensure that the disc is properly aligned in the rotor; the locator tab on the disc must be seated into the slot on the rotor. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 283 of 291 Attach the locking ring. Load the Rotor with OTV disc into the RotorGene 6000. Select the RotorGene 6000 icon to open the RotorGene 6000 application. New Run Window. Select Advanced (this menu tab should be the default). Select Instrument Maintenance. PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 284 of 291 Select OTV. The program will prompt for the OTV Rotor-Disc serial number. Insert the CD provided with each OTV Kit and select the Browse. Select the OTV calibration file provided on the CD (*.otv file). The serial number and be imported from the OTV calibration file on the CD. (Alternatively the serial number can be read from the label on the OTV Rotor-Disc.) Ensure the check box for “Fluorescent insert placed correctly” is marked off; and the insert is in place. Save the run file in My Documents > OTV folder > OTV-lot.number _yyyy-mm-dd PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 285 of 291 The run begins. The OTV run performs a series of melts that determine the thermal characteristics of the RotorGene Cycler. The total run time is approximately 45 minutes. When the run is completed. OTV Temperature Verification Results window will appear indicating if “No Adjustments are required.” Select Report and print for filing in Equipment binder. Record, with inital, that Optical Temperature Verification OTV was performed in the Analyzer History Log RotorGene. Or “Adjustments Required”. Select Apply Adjustments. This will prompt for a verification run; after the verification run no further adjustments should be required. Select Report and print. Record, with inital, that Optical Temperature Verification OTV was performed in the Analyzer History Log RotorGene. If further adjustment is required inform seniors/and Charge. Contact: Montreal BioTech Inc. 75 Guthrie, Suite202 Dorval, QC H9P 2P1 PH: 514-697-2312 References: Qiagen Rotor-Disc OTV Handbook, January 2011 PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 286 of 291 Appendix XII – WNV PCR Procedure Abbreviated * (In clean room) 1. Add 560uL Lysis buffer into 5.6 uL cRNA microcentrifuge tubes (In hood) 1. Add 140 uL specimen to Lysis buffer, vortex 15 seconds 2. Incubate for 10 minutes at room temperature 3. Centrifuge for 10 seconds at 3000 rpm to remove drops from lid 4. Change gloves 5. Add 6uL WNV internal control 6. Add 560uL ethanol (96-100%), vortex 15 seconds 7. Centrifuge for 10 seconds at 3000 rpm to remove drops from lid 8. Change gloves 9. Add 630 uL to the spin column, centrifuge for 1 minute at 8000 rpm 10. Discard filtrate in collection tube and transfer spin column to a new collection tube 11. Add remaining 630 uL to spin column, centrifuge for 1 minute at 8000 rpm 12. Discard filtrate in collection tube and transfer spin column to a new collection tube 13. Change gloves 14. Add 500 uL of Buffer AW1, centrifuge for 1 minute at 8000 rpm 15. Discard filtrate in collection tube and transfer spin column to a new collection tube 16. Add 500 uL of Buffer AW2, centrifuge for 3 minutes at 14,000 rpm 17. Discard filtrate in collection tube and transfer spin column to a new collection tube 18. Dry spin for 3 minutes at 14,000 rpm 19. Discard filtrate in collection tube and place spin column in clean microcentrifuge tube 20. Add 60 uL of Elution Buffer. Incubate for 1 minute at room temp 21. Centrifuge for 1 minute at 8000 rpm. 22. Store the eluate at -20C if not using immediately * Abbreviated version for bench quick reference only. For complete manual, please refer to WNV PCR by ALTONA KIT v. 1.0 and ROTORGENE PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page 287 of 291 Record of Edited Revisions Manual Section Name: Molecular Diagnostic Tests Page Number / Item Date of Revision Signature of Approval Molecular Testing - HBV DNA-Reporting Added Para. October 10, 2003 Annual Review Page 67 - Molecular Testing - Chlamydia Trachomatis & Neisseria gonorrhoeae Page 67 – urine but not more than 60 mL added Page 68 – specimen not lysed…added Page 68 – tubes may be blotted Page 70 – handling samples after lysing Page 71,72 Procedure change Page 72, interpretation of result - change MOTA score Page 73, reporting – indeterminate Page 76 – do not vortex working master mix Page 76 – specimen dilution protocol Page 77 – 80 procedure and reporting changed Page 81 – QC procedure changed Page 93 – procedure changed Page 86 – 88, instrument instructions Page 90 – WNV RT-PCR Page 92 – lysis buffer Page 93 – General Precaution section revised Page 94-100 – procedure revised Page 128 – Updated decontamination procedure Annual Review Added links to TGLN Procedures Revised HBV-DNA and HCV-RNA procedures with Ampliprep instructions Procleix Ultrio and WNV added Procleix Discriminatory Assay Annual Review CMV DNA Amplification and Detection Procedures updated Molecular Testing – updated EBV DNA Amplification and Detection Procedures updated HSV DNA Amplification and Detection Procedures updated May 30, 2004 May 20, 2005 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 May 20, 2005 June 2, 2005 May 30, 2006 March 21, 2007 May 11, 2008 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli July 21, 2008 July 21, 2008 July 21, 2008 November 01, 2008 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli November 01, 2008 November 01, 2008 Dr. T. Mazzulli Dr. T. Mazzulli November 01, 2008 Dr. T. Mazzulli PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page Number / Item Parvovirus B19 DNA PCR Amplification and Detection Procedures updated Enterovirus RNA Amplification and Detection Procdeures added West Nile Virus RT PCR - Failed QC added Annual Review Annual Review Added revised easyMag Added revised CMV PCR Added revised BKV PCR GeneXpert C. difficile added Updated Procleix Ultrio Updated QC section of easyMag extraction Updated Influenza procedure Added Virology specimen accessioning guide link Deleted CMV DNA Amplification and Detection Procedures by Lightcycler Deleted VZV Amplification and Detection Procedures by Lightcycler Deleted HSV Amplification and Detection Procedures by Lightcycler Added HSV/VZV Detection (Altona Alpha herpes) Added Multiplex Respiratory virus detection Annual Review Parvovirus PCR revised RSV PCR added Replace Procleix with MPX Updated WNV PCR with Altona kits Annual Review Added Appendix VIII - epMotion procedure Added Appendix VII - Creating Standard Curve for Quantitative PCRs Added Links to RotorGene Worksheets and calculations and reports Chlamydia/Neisseria gonorrhoeae detection method changed to Roche COBAS 4800 Appendix II to IV BD ProbeTec related documents removed Added HPV testing Added Focus Simplexa Flu A/B and RSV testing Date of Revision Page 288 of 291 Signature of Approval November 01, 2008 Dr. T. Mazzulli November 01, 2008 Dr. T. Mazzulli July 10, 2009 July 31, 2009 July 31, 2010 August 23, 2010 August 23, 2010 October 31, 2010 January 10, 2011 April 14, 2011 May 31, 2011 May 31, 2011 May 31, 2011 May 31, 2011 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli May 31, 2011 Dr. T. Mazzulli May 31, 2011 Dr. T. Mazzulli May 31, 2011 May 31, 2011 May 31, 2011 May 31, 2011 May 31, 2011 September 11, 2011 June 13, 2012 June 13, 2012 June 13, 2012 June 13, 2012 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli June 13, 2012 Dr. T. Mazzulli October 17, 2012 Dr. T. Mazzulli October 17, 2012 Dr. T. Mazzulli December 12, 2012 December 12, 2012 Dr. T. Mazzulli Dr. T. Mazzulli PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page Number / Item Moved RotorGene Flu and RSV tests to Appendix II and III for backup use Renumbered procedures Revised Positive C. diff resulting message Espanded C. diff resulting messages Realigned bullets and numbering format Added Appendix XI WNV PCR Procedure abbreviated Update QC & Maintenance for Focus Simplexa Flu A/B and RSV testing Annual Review MPX procedure updates Simplexa Flu A/B and RSV reporting procedure updated Simplexa Flu A/B and RSV testing procedure updated Addition of Appendix IV: Simplexa A/B/RSV worksheet Manual Creation: Cobas Ampliprep/TaqMan CMV PCR MI/MD/19/v01 Reporting C.diff GeneExpert Updating C.diff reporting update, WNV PCR manual and WNV PCR appreviated appendix dry spin time update. Annual Review Removed Luminex, HBV DNA, HCV RNA procedure Updated MPX NAT to new version and in its own manual as well as WNV NAT Updated Virology accessioning chart to it’s own manual Move Easymag manuals to Appendix Updated APPENDIX I - Molecular Diagnostic Tests Date of Revision Page 289 of 291 Signature of Approval December 12, 2012 Dr. T. Mazzulli December 12, 2012 February 05, 2013 April 18, 2013 July 31, 2013 July 31, 2013 July 31, 2013 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli July 31, 2013 December 9th, 2013 December 18,2013 January 15, 2014 January 24, 2014 February 21, 2014 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli April 14, 2014 May 28, 2014 Dr. T. Mazzulli Dr. T. Mazzulli November 3, 2014 Dr. T. Mazzulli November 12, 2014 January 28, 2015 Dr. T. Mazzulli Dr. T. Mazzulli February 18, 2015 Dr. T. Mazzulli February 26, 2015 March 15, 2015 Dr. T. Mazzulli May 6, 2015 May 6, 2015 May 6, 2015 Dr. T. Mazzulli Dr. T. Mazzulli Dr. T. Mazzulli Schedule C. diff reporting update for results Addition of : Sputolysin Procedure and Stool preparation in Virology Accessioning tables split and updated Simplexa manual resulting updated. Parainfluenza and human Metapneumovirus procedure added Update C.diff comments Parainfluenza and human Metapneumovirus BIORAD procedure added Added C. diff with eSwab section Added links for BK/EBV to calculation excel worksheet p.8 BK general precautions modified PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page Number / Item Added introduction and specimen collection/transport to BKV section and updated materials section Adenovirus PCR by Rotorgene Section Added in Added: ROTORGENE QC Semi-Annual: Perform optical temperature verification Annual Review Added Appendix XI – Rotorgene Optical Temperature Verification (OTV) Procedure Added link in WNV PCR to OTV Procedure Added PCR ordering chart to accessioning section Updated BK Virus procedure Updated BK Manual section, Updated full EBV manual BK and EBV manual addition of summary information South West Health Ordering information added to PCR accessing chart. Virology specimen accessioning guide changes: -add tx pt to edta for hz, -add pleural to peritoneal/pericardial fluids (entero) -removed “if requested” Entero from BALs -removed adeno to PHL from eye specimens (done in house) Influenza Simplexa section: - added report using virology worklist Respiratory PCR - Error EC500 retest 1:10 - Add media PRABR if retesting simplexa sample - Add dates in media PCABR WNV Rotorgene: - Removed use of 72-well rotor parts - Document which rotorgene was used (blue or red) - Removed Red Channel RSV A (typo) p.172 - Reporting section: note for positives to re-extract from original clot and re run, for donors repeat in duplicates & record CT values in PCWNV media. -Updated troubleshooting section Influenza Simplexa manual & added instructions for insufficient sample error. -Reorganize manuals alphabetically Date of Revision Page 290 of 291 Signature of Approval May 6, 2015 Dr. T. Mazzulli May 6, 2015 June 15, 2015 Dr. T. Mazzulli Dr. T. Mazzulli June 15, 2015 Dr. T. Mazzulli June 25, 2015 July 16, 2015 Dr. T. Mazzulli Dr. T. Mazzulli August 12, 2015 Dr. T. Mazzulli September 1, 2015 Dr. T. Mazzulli September 11, 2015 Dr. T. Mazzulli September 25, 2015 Dr. T. Mazzulli PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc Policy # MI/MD/v50 Microbiology Department Policy & Procedure Manual Section: Molecular Diagnostic Tests Manual Page Number / Item Date of Revision Page 291 of 291 Signature of Approval Under trouble shooting section of manuals: MPX NAT, WNV NAT, Chlamydia PCR added to the table if IC fails: First time: repeat Second time: result as invalid with result comment @SPIN “Likely due to specimen integrity, unable to obtain valid test result after repeated attempts.” Added BK BIO RAD section Virology Accessioning Chart changes: Adeno request for stool send to PHL, parvo request on plasma: requires microbiologist approval Under Virology ordering chart added link to Send out Manual for processing specimens to be send for external testing. October 7, 2015 Dr. T. Mazzulli October 15, 2015 Dr. T. Mazzulli October 20, 2015 Dr. T. Mazzulli Updated MPX NAT and WNV NAT to include storage temperature of reagents Updated WNV storage conditions of specimen October 26, 2015 Dr. T. Mazzulli PROCEDURE MANUAL UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use D:\106748206.doc

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