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1. TEST NAME SOFT RESULT TRANSLATION ACTION REFLEX TEST TESTS CODE Hep B Surface 8HAG NEGATIVE Negative Autoposted Ag Autoverified Hep B Surface 8HAG REACTIVE Review NOT 8HBC Ag Autoposted Hep B NOT Core Ab Autoverified Hep B Surface 8HAB REACTIVE POSITIVE Autoposted Ab Autoverified Hep B Surface 8HAB NEGATIVE Negative Autoposted Ab Autoverified Hep B Surface 8HAB GZ NEGATIVE Neg Not Ab Autoposted Not Autoverified Hep B Core Ab 8HBC REACTIVE REACTIVE Not 8HBC2 Autoposted Not Autoverified Hep B Core Ab 8HBC NEGATIVE Negative Autoposted Autoverified Hep A IgM Ab 8HAV REACTIVE REACTIVE Not Autoposted Not Autoverified PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 110 TML MSH Microbiology Department Policy MI VIR 17 07 v02 Page 3 of 3 Policy amp Procedure Manual Serology Manual Hep B Core 8HBCM REACTIVE POSITIVE Not 8HBC2 IgM Ab Autoposted Not Autoverified Hep Be 8HBEB NONREACTIVE NONREACTIVE Autoposted Antibody Autoverified Hep Be 8HBEB REACTIVE REACTIVE Autoposted Antibody Autoverified Hep Be 8HBEG NONREACTIVE NONREACTIVE Autoposted Antigen Autoverified Hep Be 8HBEG REACTIVE REACTIVE Autoposted Antigen Autoverified Rubella 8RUB NEGATIVE Negative Autoposted Autoverified Rubella 8RUB GRAYZONE Negative Autoposted Autoverified PROCEDURE MANUAL TORONTO MEDICAL LA
2. TML MSH Microbiology Department Policy MI SER 15 v04 Page 4 of 7 Policy amp Procedure Manual Serology Manual 17 Check the 6 status bars at right side of screen Cover should be closed cover lock should be locked lock bar should be locked heat unit should have pass cool units 1 amp 2 should have pass An OK box will appear at bottom right hand corner of screen which was not there before once status of all are correct Click on this OK and machine will start If OK box doesn t appear check that everything on the screen is in place and that each item has been clicked 18 Prepare COBAS as per HCV protocol 19 After the MagNA Pure has finished the extraction the Results screen will appear Check to see if all passed Cap the A rings and transfer them to the COBAS Continue as per HCV package insert 20 Discard disposables trays and tip stands from the MagNA Pure into biohazard box Wash buffers 1 2 3 and the elution buffers can be saved for one week Cover with tub lid seals date and put whole rack in fridge Proteinase K and MGP tubs must be discarded 21 Choose File and Exit then file and save When saving enter current date 22 Exit to main screen 23 Click the Decontamination button Set decontamination time for 8 hours On Friday set timer until end of workday Click on Actions and choose Start Decontamination When the decontamination
3. 2 Working Conjugate Solution 0 25 ml of Anti Human IgG into 1 bottle of 12 5 ml Conjugate Buffer 3 Anti EBV Reference P N From Supplementary Reagent Kit OUVP 17 4 Wash Solution 100 ml of Wash Solution 1900 ml of distilled H20 Store at 4 C Fill up Wash Solution in Behring 2000 when needed 5 Working Chromogen Solution 1 ml of chromogen TMB 10 ml of Buffer Substrate TMB Rinse and keep container when test is done 6 Stop Solution PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 37 TML MSH Microbiology Department Policy MI SER 06 v01 Page 2 of 5 Policy amp Procedure Manual Serology Manual ii Method 1 Turn hard drive and Behring 2000 Analyser ON 2 Double click on Short Cut to BEP 2000 3 Enter Technologist s initials click on Don t know Password 4 The machine will start Initialization and check all instrument functions The results of this check are then displayed on the screen Nn Check DH20 and Buffer level by opening the bottom part of the Analyzer 6 Perform Washer Assay every morning Sa mono op jat j k Click on New Worklist Click on Add Plate Click on Add Assay Highlight DB Washer asy click on Open click OK Lot specific values for plate 1 click OK Click on Green arrow to start LOAD cl
4. 6 Press F6 ADD 7 Press Abbott logo a if no other assay calibration is required 8 Print the displayed orderlist using the PRINT KEY 9 Mix the calibrator and place appropriate volume in the sample cup 10 Check for bubbles in the sample and place the segments in the sample carousel 11 Load the appropriate assay reagents in reagent carousel Remember to open reagent bottle 4 on the reagent pack if present 12 Press RUN MASTER CALIBRATION HBsAb CMV IgG Ab Rubella IgG Ab Master calibration requires two calibrators in separate S P locations Check required volume on printed orderlist 1 From the MAIN MENU select STORED RESULTS 2 Select calibration review 3 Select F4 NEW LOT 4 Enter the master curve information by selecting F4 SCAN CURVE 5 Scan the bar code label top and bottom 6 Select F6 SAVE 7 Master Cal is performed through the calibration orders and calibration type screen PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 10 TML MSH Microbiology Department Policy MI SER 02 v03 Page 4 of 9 Policy amp Procedure Manual Serology Manual 8 9 10 11 12 13 14 15 16 17 18 19 20 E 1 2 F 1 3 4 6 7 9 IG 1 Go to the MAIN MENU select ORDERLIST Select F4 Cal Select the assay to be calibrated by using the touchscreen The S P fo
5. Cleaning Processing Wash Station Remove the Wash Station Rinse the Wash Station with Deionized H20 Rinse the Wash Station with 95 Ethanol Rinse the Wash Station with Deionized HO Clean the inside and outside of the Wash Station with a cotton swab moistened with Deionized H20 to remove any salt buildup Reinstall the Wash Station Home Probes Cleaning Dispenser Nozzles Remove the Bulk Solution 1 Dispenser by pulling olive green lever toward you Lift the Dispenser to inspect the nozzle for buffer deposits Moisten a cotton swab with Deionized H20 and clean the outside of the nozzle and the surrounding collar Inspect the nozzle for obstruction Reinstall the Dispenser by lifting the black clip to vertical position should hear click Check the Dispenser position Make sure that the Dispenser sits properly not on a raised position Remove the Bulk Solution 3 Dispenser by Dispenser by pulling olive green lever toward you J Lift the Dispenser to inspect the nozzle for buffer deposits Moisten a cotton swab with Deionized H20 and clean the outside of the nozzle and the surrounding collar Inspect the nozzle for obstruction Reinstall the Dispenser by sliding the metal hinge pin on the right end of the Dispenser into the openings on the base Lower the left end into the circular opening of the base plate Check the Dispenser position Make sure that the Dispenser sits properly not on a raised position
6. Serology Manual IV Results Results should be sorted and viewed by S P to ensure that all tests were completed then resorted and printed 1 2 3 4 5 6 7 8 9 10 Note From the MAIN MENU select RESULTS Select F5 SORT Select S P by touchscreen OK Scroll down the list checking that every S P with a sample has a result Select F5 SORT Select SID OK Once sorted select F2 Select All and PRINT Select Print a listing of all selected results OK using touchscreen to activate printing After successful printing of results select F3 Release Results Are you sure Yes Check print out for low level positive CMV Rubella and enter the low level message in the LIS Check for reactive HBsAg HBcAb HCVAb HIV 1 2 for which repeat testing or reflex testing may be necessary Initial and give print out to senior technologist for verifying HB Ab Repeat all reactive results for HB Ab After repeat testing complete Select the appropriate result for 8HBC test from keypad in LIS Exception HB Ab reflex testing 83 HBC2 performed on HB Ag reactive sample for which HB Ab SHBC was not originally ordered as outlined below HB Ag Perform reflex confirmatory testing and HB Ab on all reactive HB Ag samples except for patients with previously confirmed positive HB Ag Preliminary report of stat requests will be phoned as presumptive positive PROCEDURE MANUAL TORONTO MED
7. TML MSH Microbiology Department Policy MI SER v11 Page 1 of 2 Policy amp Procedure Manual Section Serology Manual Subject Title Table of Contents Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date April 02 2004 Review Date May 26 2004 SEROLOGY MANUAL TABLE OF CONTENTS SEROLOGY TESTS B TTI ay AEE 3 AXSYM System for HBsAg HBsAb HBeAg HBeAb HBcAb IgM HBcAb Total HAV IgM HCVAb CMV IgG HIV 1 2 Rubella 128G oe ecesescnccseecsceeeeecneesesceceeeetseneeees 8 AXSYM HEPATITIS B SURFACE ANTIGEN CONFIRMATORY ASSAY uence 17 REPATITIS FIRUS SEROL OG veces eee E REE E EE AA EA TEA 21 HBSAG a HIN P2 eresiaren iaa E NAE E EE EEEa EE EEEE E AAEE SEENE ENAERE E ETENEE 30 BR e E E E EE E E E TN EA 37 HUMAN T LYMPHOTROPIC VIRUS TYPE 1 HTLV 1 EIA ssseeessesesssssessessseesessererssesee 42 INFECTIOUS MONONUCLEOSIS HETEROPHILE ANTIBODIES 0 cccssssssscoceeeceneees 47 ov PE SO RERNING cs ccchssnicseswonniersnctcas shia ies SE E EE EEE AEEA EENES 49 VARICELLA ZOSTER VIRUS JG ccritiiisisissisiesiinisresrisiisessiariseskta rinasa ise na E EA TEASA SA EEE 52 WEST NILE FPLAVYIVIRUS PO accionista EEE aTi Ea a EERE Ra 56 WEST NILE PLAYIVIRUS IEM een ne ene ee ee eae mee renner nee EEA EEA 61 MOLECULAR TESTS CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAE ccescceeseeeeeeesees 66 HEPATITIS BBY DNA oera eiiie e i EE E EAEE EE ETE TERRA 72 HEPA
8. AMPLICOR Hepatitis C Virus Test Specimen Collection and Processing Blood should be collected in sterile red topped tube or in sterile tube using EDTA Lavender topped and spun at 2000 rpm for 15 minutes Serum or plasma is separated and stored in three serum tubes and placed in HCV RNA box in 20 C freezer Separated specimen may be frozen and thawed up to two times without a loss in copy number Specimen collected in green topped Heparin tube is unsuitable for this test Procedure Pre Amplification Area Reagents Preparation Using the MagNA Pure Make worklist in LIS lab using 8HCAP Label as follows 1 1A 13 2E 2 1B 14 2F 3 1C 15 2G 4 1D 16 2H 5 1E 17 3A 6 1F 18 3B 7 1G 19 3C 8 1H 20 3D 9 2A 21 3E 10 2B 22 3F 11 2C 23 3G 12 2D 24 3H PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 77 Subject Title Molecular Testing HCV RNA TML MSH Microbiology Department Policy MI SER 15 v04 Page 2 of 7 Policy amp Procedure Manual Serology Manual 2 Turn on MagNA Pure instrument Now turn computer on Leave password blank 3 Make 2 vials of Master Mix 24 samples in clean room as per HCV protocol Load Master Mix vials onto the MM position in the MagNa Pure Put 2 new A rings onto MagNaA Pure into the correct A or B position Remember to take off Master Mix caps later Master Mix
9. Control well when needed Cover plate with sealing tape and incubate for 60 minutes at Room Temperature Wash the wells three times using the Automatic Washer under Program 10 Select Program Press memory F1 press Number F 1 For washing and soaking use Program 10 lt Program No press 10 enter Program 10 Name IgG WN should appear Press Set at this point press Change F2 then press Enter until number of strips to wash appears check that of strip appears is what is needed If it is the same presses enter If is not the same enter the right of strip and press enter until 11 Name IgG WN appears press Enter Press Run F1 and press No F1 under Prime Will wash strip for 3 times each time with working buffer End washing will appear when washing cycle is finished Repeat Washing will appear press No F2 Remove plate from Plate Washer Add 100 ul of Conjugate into each well using an 8 channel pipette cover plate with sealing tape and incubate for 30 minutes at room temperature Repeat wash steps 9a g Add 100 ul of Substrate into each well using an 8 channel pipette cover and incubate for 10 minutes at room temperature Add 100 ul of Stop Reagent into each well using an 8 channel pipette In antibody positive wells color should change from blue to yellow Read the plate in spectrophotometer at dual wavelength 45
10. Interpretation of Index Values Positive gt 1 10 Negative lt 0 9 Equivocal gt 0 9 and lt 1 1 All positive and equivocal specimens should be repeated the next day Validation The mean value for the Cut Off Calibrator must be within 0 100 to 0 700 O D units minus the blank well The Positive Control Index value should be between 1 5 and 3 5 The Negative Control Index value should be less than 0 8 Reporting Positive result To PHL Report PHL result in LIS and inform physician if result is confirmed Positive Negative result West Nile Virus Flavivirus IgM by EIA Negative This is a research test Equivocal result repeat testing If result remains equivocal becomes Positive Send to PHL Inform physician if result from PHL is Positive If result becomes negative West Nile Virus Flavivirus IgM by EIA Negative PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 64 Policy MI SER 12 v03 Page 4 of 5 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 12 v03 Serology Manual Page 5 of 5 VI uality Control Each plate must include 1 Positive Control 1 Negative Control and 3 Cut off Calibrators An External Positive Control Accurun 165 West Nile virus IgM IgG Positive Control Series 5000 must be included with each new lot Result filed in Reagent Lot Binder Inform charge senior techn
11. MI SER 11 v03 Serology Manual Page 5 of 5 VII Quality Control Each plate must include 1 Positive Control 1 Negative Control and 3 Cut off Calibrators An External Positive Control Accurun 165 West Nile virus IgM IgG Positive Control Series 5000 must be included with each new lot Result filed in Reagent Lot Binder Reference Manufacture s insert Focus Technologies Cypress California 90630 U S A 2003 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 60 TML MSH Microbiology Department Policy MI SER 12 v03 Page 1 of 5 Policy amp Procedure Manual Section Serology Manual Subject Title West Nile IgM Capture ELISA Issued by LABORATORY MANAGER Original Date September 30 2003 Approved by Laboratory Director Revision Date October 27 2003 Il HI WEST NILE FLAVIVIRUS IgM Introduction The Focus Technologies Flavivirus West Nile IgM capture ELISA is intended for qualitatively detecting IgM antibodies to flaviviruses West Nile in human serum In conjunction with the Focus Technologies Flavivirus West Nile ELISA IgG the test is indicated for testing persons having symptoms of arbovirus infection as an aid in the presumptive diagnosis of flavivirus infection Specimen Collection and Processing 5 ml of blood is collected in a serum separator tube and separated by centrifugation The serum is remo
12. Two negative specimens are sent to PHL every 6 months for verification The PHL reports are filed in Reagent Lot binder vii Reference Package insert from DETECT HTLV EIA Test Kit for the Detection of Antibodies to Human T Lymphotropic Viruses HTLV Types I and II Adaltis rev 06 02 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 46 TML MSH Microbiology Department Policy MI SER 08 v01 Page 1 of 2 Policy amp Procedure Manual Section Serology Manual Subject Title Epstein Barr Virus Serology Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date May 27 2003 Il HI INFECTIOUS MONONUCLEOSIS HETEROPHILE ANTIBODIES Introduction The MONOSPOT LATEX slide test is a latex particle agglutination test for in vitro qualitative detection of infectious mononucleosis heterophile antibodies IgM in serum or plasma These antibodies appear in the sera of 85 to 90 of patients with infectious mononucleosis within 2 to 3 weeks after onset of illness Specimen Collection and Processing 5 mL of blood is collected in a serum separator tube and separated by centrifugation The tube is refrigerated until testing Specimens are stored at 70 C after testing and discarded after 3 months Procedure i Reagents MONOSPOT LATEX Kit Store refrigerated Allow the reagent to warm up to RT Mix well befo
13. date performed e g Biorad 040528 BioRad tested May 28 04 Note session e g 31 Click on writer Name enter initial Click OR The screen will show e g BioRad0400528 31 ssn highlighted click OK Click on 5 th square from the left Profile Include click on BioRad HIV no Dilution 2 prf click OK Click on 7th square from the left Sample programming First click on Clear sample rack then click on sample code and enter lab and load specimens onto specimen rack at the same time Also PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 31 TML MSH Microbiology Department Policy MI SER 05 v01 Page 3 of 7 Policy amp Procedure Manual Serology Manual 10 11 load second half of the rack with dilution tubes only for HIV only Once finished double click on Small Rack icon in top left corner X s should appear next to all lab s click done The screen will show plate format of wells needed Take out the required of strips and wells Load HBsAg plate onto the left side in the P Lab HIV plate onto the right side in the P Lab Click on done Click on 8 th square from the left Setup Entry Click on HBsAg then Click on pulldown arrow under Lot If it is a new lot Click on Lot type in new lot Then click on Change Date and specify kit expiration date
14. least 2 hours which will remove inhibitors of SDA Urine can be stored for 2 days at 15 27 C or 4 to 6 days at 2 8 C PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 66 TML MSH Microbiology Department Policy MI SER 13 v02 Page 2 of 6 Policy amp Procedure Manual Serology Manual HI Procedure Reagents BD ProbeTec ET CT GC reagent pouches containing CT priming and Amplification microwells GC priming and Amplification microwells Reagent pouches may be stored at 2 to 33 C Once a pouch is opened the microwells are stable for 4 weeks if sealed properly or until expiry date whichever comes first BD ProbeTec ET control set CT GC Positive Controls CT GC Negative Controls BD ProbeTec ET Sample Dilution Tubes BD ProbeTec ET System supplies BD ProbeTec ET Instrument Microcell plates Lysing Heater and Lysing Rack Primary and warming rack Pipettor and Power Supply Urine Processing Kit UPP Sample tubes Pipette tips ii Other Materials Centrifuge Vortex mixer Gloves powder free Transfer Pipette 1 V V Sodium hypochlorite Timer Absorbent Paper roll PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 67 TML MSH Microbiology Department Policy MI SER 13 v02 Page 3 of 6 Policy amp Procedure Manual Serology Manual iii S
15. work in cabinet with gown and frequent glove change Lysis and Purification Steps a For each specimen Quantification Standard QS External Control and Negative Control H20 prepare and label two 1 5 mL microcentrifuge tubes sterilized RNase free with the corresponding number in the Worklist Label one of them with the lab number LIS label also 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 If the Lysis Buffer is stored in the refrigerator and crystals are present heat to dissolve the crystals Add 560 uL Lysis Buffer AVL with RNA carrier into each of the microcentrifuge tubes in one row these will contain the waste Add 5 uL WNV Internal Control to each of the microcentrifuge tubes with Buffer AVL Add 140 uL specimen to the Lysis Buffer Buffer AVL with RNA carrier vortex for 15 seconds Incubate at Room Temperature 15 25 C for 10 minutes Centrifuge for 10 seconds at 2000 g to remove drops from the lids Add 560 uL ethanol 96 100 to the samples vortex for 15 seconds Centrifuge for 10 seconds at 2000 g to remove drops from the lids 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 6000g
16. 275 275 9 Patient Sample 275 275 10 Patient Sample 275 270 uL 8 drops 250 uL 7 drops PROCEDURE MANUAL Page 18 TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT TML MSH Microbiology Department Policy MI SER 03 v01 Page 3 of 4 Policy amp Procedure Manual Serology Manual Index Calibrators 2 3 4 5 6 Select F4 CAL HBs Ag CNF by touch screen input Assign S P H4 Enter lot number and expiry date found on confirmatory kit used Select F6 ADD Positive Control 7 8 9 10 11 12 13 Select F6 PATIENT Type Pos Control at SID Assign S P H5 Select HBs Ag CNF by touch screen Select F4 DIL REPS Order Reagent B 1 Reagent A 1 Reagent B dil 0 Reagent A dil 0 Select F6 NEXT Select F6 ADD Patient Samples 14 15 16 17 18 19 20 21 22 23 24 25 Select F6 PATIENT Barcode in SID type in patient s last name Select HBsAg CNF by touch screen Assign S P H6 Select F4 DILS REPS Order Reagent B 1 Reagent A 1 Reagent B dil 1 Reagent A dil 1 Select F6 NEXT Select F6 ADD for more patient entry Select Abbott Logo to MAIN MENU Review the displayed order list Print the orderlist using the PRINT KEY Load Segment H containing the required confirmatory reagents and patient sample Load HBsAg reagent in reagent carousel PRESS RUN PROCEDURE MANUAL TORONTO M
17. Allow samples to cool at room temperature for at least 15 minutes may be held up to 6 hours after lysing is completed Remove and discard caps from tubes Change gloves to avoid contamination Using the Plate Layout Report for the run preparing the Priming Microwell Plate Microwells must be in vertical correct order with CT and follow by GC Using Program 3 expand pipettor by pulling the handle all the way out Aspirate samples from the first vertical column of tubes Note It is important to dispense liquid against the inside of the microwells to insure complete specimen addition and to avoid cross contamination Gently collapse the pipettor and dispense 150 ul into each of the corresponding column of Priming Microwell Plate Note Recap lysed specimen tubes hold these until run is completed Cover the Priming Microwell Plate with the priming cover and let the plate sit at room temperature for at least 20 minutes Note The plate may sit up to 6 hours before proceeding with the assay Prepare the Amplification Microwell Plate by Configuring the Amplification Microwell Plate to match the Plate Layout Report Remove the cover from the Priming Microwell Plate Check the temperature of the Priming Warming Heater Priming Heater 72 73 C Warming Heater 53 5 54 5 C PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 69 TML MSH Microbiology Department Policy
18. Director Revision Date BDProbeTec ET Workflow Overview Prepare for test run Enter sample date Assemble Priming Microwells according L te piate Fayout mpor fararey TRE Heat Samples 30 min ool 15 min Tranafer samples rimming Microwelks Incubate Priming eae Sat Up Microwell plats _ Amplification 20 min RT G Mierowelle Incubate Primi To pem NEL ng tiaisia Microwell plata 40 KENE A 416 min PAN Haater Transfer to Amplification Microwells SEAL Transfer Plate to BDProbaTec ET Start Aun RESULTS Printout PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 107 lt Good for 6 hrs at RT gt PROCEDURE MANUAL Processed swabs good for 6 hrs at RT Overnight at 4 C Cooled samples good for 6 hrs at RT 5 days at 4 C then re lysed 98 days frozen 20 C then re lysed TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 108 TML MSH Microbiology Department Policy MI VIR 17 07 v02 Page 1 of 3 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix VII Autoverification Process Issued by LABORATORY MANAGER Original Date June 25 2002 Approved by Laboratory Director Revision Date APPENDIX VII AUTOVERIFICATION PROCESS I Introduction To document the au
19. Flushing Pumps and Syringes Select Prime and Flush Enter 1 under sampling syringe flush and Processing syringe flush Select F2 Perform Maint Check for leaks or bubbles in the tubings Exit to Main Menu PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 27 TML MSH Microbiology Department Policy MI SER 04 v01 Page 8 of 9 Policy amp Procedure Manual Serology Manual 10 Cleaning Processing Carousel Remove the Processing Carousel Cover by unscrewing the two thumbscrews Grasp Processing Carousel Motor Handle push back and hold open Remove by tilting Carousel and pulling up careful not to hit the probe Clean the carousel and the surrounding area with lint free tissue moistened with water Inspect the clips on the under side of the carousel for broken pieces loose connections Dry the Carousel with Lint free tissue Reinstall the Carousel by grasping the Processing Carousel Motor Handle push back and hold open Place the rim of the Carousel in the V wheel located under the Carousel Motor Housing Tilt the Carousel downward and fit the rim into the two front V wheels Careful not to hit the probe Reinstall the Processing Carousel Cover by tightening the two thumbscrews Cleaning the Matrix Carousel Remove the Matrix Cell Carousel Access Panel Remove the Air Deflector Select F2 Shutdown from the Main Menu Verify that Shutdown was selected
20. If there is a next rack of specimens the next page of specimens will appear shortly Repeat step e Lot specific vales for plate 1 click OK unless a new lot If it is a new lot just highlight the lot type in new lot Do the same for expired date Plate format will also show the total of specimens to be tested and of wells needed Load C difficile wells onto the plate including controls Fill in unused space with blanks Load plate onto plate carrier Click OK Click on the green Initialization will start immediately LOAD Purple squares and circles represent Reagents controls If you hover the mouse around the squares or circles it will tell you what it is Click on the Square and drag it over to the correct position on the Rack Make sure that the arrow is pointed at the center of the square circle Chromogen Substrate is in the Rack 5 Conjugate amp Stop Solution are in Rack I and Controls are in Rack R Ifa Grey Green Pipette tip box appears it is to load a new set of tips into that space Noted the size of tip required Yellow for 300 ul Grey for 1100 ul Load tips Once finished with loading click OK Are you sure that The Resources have been loaded in the correct positions Click Yes The analyzer will check the levels of the reagents LOAD PLATEF appears Under Plate ID enter cd year month date cd030106 Load plate carrier into Analyser Click OK Are you sure plate l
21. LOAD Load a Dilution Plate into top left hand corner If you hover the mouse around the circle it will tell you that this is the Anti EBV Reference P N Click on the circle and drag it over to the correct position on the Rack R Make sure that the arrow is pointed at the center of the square circle If a Grey Pipette tip box appears it is to load a new set of tips into that space Noted the size of tip required Yellow for 300ul Grey for 1100 ul Load tips Once finished with loading click OK p Are you sure that The Resources have been loaded in the correct positions click Yes q The analyzer will check the levels of the reagents r LOAD PLATE appears Under Plate ID enter EBVyear month date EBV030106 s Load plate carrier into Analyser Click OK t Are you sure plate layout is correct click OK u Downloading Progress will appear and then the Orange square will also appear the testing will start v To see what stage the test is at close the inside window on the screen Click on beside Work 2 w Click on Schedule You can see the red line move across the screen with each step x When finished Remove plate amp Carrier from the system remove plate and click OK y Click on Print Report z Re capped all reagents and place the whole kit in the refrigerator PROCEDURE MANUAL TORONTO MEDIC
22. MI SER 13 v02 Page 5 of 6 Policy amp Procedure Manual Serology Manual II 12 Place the Priming Microwell Plate into the Priming Heater 13 Immediately place the Amplification Microwell Plate into the Warming Heater 14 Set the timer for 10 minutes 15 At the end of the 10 minutes incubation select Program 5 on the pipettor 16 Pick up new tips and transfer 100 ul from the column 1 of the Priming Microwell Plate to column 1 of the Amplification Microwell Plate Allow pipette tips to touch sides of microwells After dispensing the liquid allow pipettor to automatically mix the liquid in the wells and discard tips Continue transferring from column to column using new tips each time 17 When the last column has been transferred seal the Amplification Microwell Plate using an Amplification Plate Sealer 18 Place the Amplification Microwell Plate into the BD ProbeTec ET Instrument for analysis The Amplification Microwell Plate must be loaded into the instrument within 30 seconds of transferring the last column of samples Timing is extremely critical Do Not Delay entry of plate into the instrument 19 Test results will be printed out as soon as the testing is finished 20 See Appendix VIII for Workflow Overview Validation MOTA Score is a metric used to assess the magnitude of signal generated as a result of the reaction The magnitude of the MOTA Score is not indicative of the level of orga
23. MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 105 TML MSH Microbiology Department Policy MI SER 17 05 v01 Page 3 of 3 Policy amp Procedure Manual Serology Manual Move cursor to next blank test area A turn off keypad enter 9 F2 F12 to get a list of all send out tests highlight the test to be ordered ENTER repeat until all tests have been ordered F12 Confirm modification Yes q F8 to enter refer out results r Highlight the appropriate test and enter the result from the keypad If no keypad appears type in SEE BELOW s After the last result is entered highlight the last result free text F5 Canned message select 7 9rep t Space enter date that PHL reported the result Arrow up wand PHL bar code or enter PHL lab number F12 F12 Confirm modification Y u Record the Clinic Dr MRN and order number on the reference lab report if not already recorded v Give reports to senior charge technologist to verify W Forward reports to Report Controllers for photocopying and mailing PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 106 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 17 06 v01 Page of 1 Section Serology Manual Subject Title Appendix VI BDProbeTec ET Workflow Issued by LABORATORY MANAGER Original Date May 26 2003 Approved by Laboratory
24. Manual Page 7 of 7 vii Reference Package insert from Bio Rad Human Immunodeficiency Virus Types 1 and 2 synthetic Pepide Revised September 2000 Package insert from Bio Rad Human Immunodeficiency Virus Types 1 and 2 synthetic Pepide Revised September 2000 Bio Rad Laboratories blood Virus division Redmond WA 98052 U S A U S License No 1109 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 36 TML MSH Microbiology Department Policy MI SER 06 v01 Page 1 of 5 Policy amp Procedure Manual Section Serology Manual Subject Title Epstein Barr Virus Serology Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date June 6 2003 EBV VCA IgG I Introduction This test is used to detect the presence of EBV Viral Capsid Antigen VCA IgG antibodies in human serum using an ELISA system In general antibodies to EBV VCA appear within the first week after infection The presence of EBV VCA IgG antibodies may indicate recent or past infection with EBV II Specimen Collection and Processing a Approximately 10 ml of blood is collected in a red topped tube b Centrifuge the tube at 2000 rpm for 10 minutes c Transfer serum into 2 ml storage tube and store in designated rack in 4 C fridge HI Procedure Behring 2000 Analyzer i Reagents From EBV Kit OWIS 15 1 Sample Buffer
25. Mix the carbon antigen reagent well Squeeze the dropper bottle and withdraw sufficient reagent into the bottle Discard the first few drops into the reagent stock bottle and then dispense 1 drop into each circle in a vertical position Do not mix the sample and the antigen Rotate the card at 100 rpm for 8 minutes Observe for agglutination by two technologists independently Interpretation of Results Positive Result Any agglutination Repeat test see senior technologist for any discrepant results Send all positive sera to PHL for VDRL and confirmatory tests Write on PHL requisition RPR positive please do confirmatory test DO NOT mark prenatal box for serum from prenatal patients Negative Result No agglutination PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 50 TML MSH Microbiology Department Policy MI SER 09 v02 Page 3 of 3 Policy amp Procedure Manual Serology Manual IV VI Reporting Positive Result Enter in LIS as TO PHL VDRL send out test is ordered reflexively Send to PHL next day Negative Result Negative Quality Control Strongly reactive weakly reactive and non reactive control sera are included in each run If controls are not working or the antigen is not falling cleanly from the needle perform a needle check as outlined in the method Record control results and kit lot number on the task list Run ext
26. all fluid is drained off and bubbles appear Empty waste Click OK Empty tips click OK Don t forget to shut off P Lab click OK 18 Click on left top corner File and Exit will ask exit from P lab Processor click Yes 19 Click on 4 box from the top Open Results Click on New Result Then click on your session and OK PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 33 TML MSH Microbiology Department Policy MI SER 05 v01 Page 5 of 7 Policy amp Procedure Manual Serology Manual 20 Click on 3 box from the left report click on HBsAg and also the 21 square with the printer Click on HIV 4 and also the square with the printer Report will be printed out Close all windows by clicking on X at top right corner three times 22 Turn off hard drive monitor P lab and printer iv Validation HBsAg l Calculate the Mean Absorbance of the Negative Control NCX Sum of the absorbance values of the three negative controls divided by three Each individual absorbance must be gt 0 000 AU and lt or to 0 100 AU One Negative Control value may be discarded if it is outside this range The NCX may be calculated from the two remaining absorbance values Calculate the Mean Absorbance of the Positive Control PCX Sum of the absor
27. enzyme linked immunoreacting substance antibody antigen or hapten adsorbed onto microparticles to detect the primary interaction of antibody or antigen The reaction mixture is transferred to an inert glass fibre matrix to which the microparticles bind irreversibly The immune complex on the glass fibre matrix is detected by alkaline phosphatase labelled conjugate II Specimen Collection and Processing Blood is collected 5 mL for adult and 1 mL for neonates in a serum separator tube Sodium heparin tube Sodium citrate tube ACD tube CPDA 1 tube or EDTA tube Sample is centrifuged 3 000 g x 10 minutes and the serum is stored refrigerated until testing After testing is completed specimens are stored at 70 C for 10 years for bone marrow and bone bank patients and 3 months for other patients IHI Procedure Reagents Reagents calibrator and control packs for the assays are stored refrigerated All other reagents are stored at room temperature except MUP Solution 1 which is stored refrigerated Once MUP solution is opened it is only good for 2 weeks AxSYM Operation A Daily Maintenance 1 Check inventory and waste containers Flush tubings 2 Scan reagent packs Add additional reagent packs and calibrate if necessary sufficient for the days work 3 Probe clean to be done at the end of the shift 4 Initial the maintenance log book PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEP
28. is complete next day or end of day turn off instrument Click on Start at bottom left corner of screen and choose shut done Post Amplification Area Amplification and Detection Perform Daily Instrument Maintenance as outline in the Operator s Manual of the COBAS AMLPIOCR Analyzer including e Wipe initialization post with a lint free moist cloth and dry e Wipe D cup handler tip with a lint free moist cloth and dry e Check Wash Buffer Reservoir and fill if necessary e Prepare Working Wash Buffer 1x by adding 1 volume of WB 10x to 9 volume of Deionized water Mix well Keep a minimum of 5 6 liters of Wash Buffer 1x in the Wash Buffer Reservoir at all times e Empty waste container e Prime the system 1 Normal prime 2 Extended prime use if wash reservoir was refilled or been idle for a long time PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 80 TML MSH Microbiology Department Policy MI SER 15 v04 Page 5 of 7 Policy amp Procedure Manual Serology Manual Instrument Loading and System Operation using AmpliLink 1 Maintenance Click on Maintenance window 5 from the top at the side application bar Click on Service actions tab to get into Service Action folder Performing prime a Click on tools from menu bar Highlight prime c Choose either Normal of Extended prime Record daily and weekly
29. maintenance by clicking on action to highlight it and click Update and OK 2 Reagent Check Click on Instrument Window 2 from the top at the side application bar Click on Cassette tab To alter rack double click on Rack and change click OK To add cassette double click on any of the cassette icons and scan in barcode on new cassette A single click on any cassette will show the of tests left in that cassette Color coding green reagent on rack and has gt 10 test left Yellow reagent on rack expired and or has lt 10 test left Red zero test left or mismatched volumes 3 Creating orders Click on Order 3 window from the top at the side application bar Click on at top right corner to create a new order Type in A ring at top left corner Double click on Profile HCV 11 12 specimens HCV 12 10 specimens negative control and Positive control Click on first space to enter specimen either type in or use barcode wand Use J to move down to the next position until finished Click on save to save order Click on again to create another order repeat the above steps 4 Loading the A rings Click on Analyzer tab Double clicks on thermocycler A click on arrow select A ring click OR Do the same for thermocycler B PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 81 T
30. means of specific antibody neutralization II Specimen Collection and Processing A minimum of 300 uL of preliminary positive HBs Ag patient sample in a sample cup is required Ill Procedure i Reagents AxSYM Confirmatory Kit AxSYM HBs Ag Kit ii Method Use the assigned H segment for the confirmatory test Using the following chart for the number of samples to be confirmed add appropriate volume of reagents to the corresponding sample cups at position 1 to position 5 Patient sample starts at position 6 onward Segment H S P 1 S P 2 S P 3 S P 4 Reagent A Reagent B Dilution Reagent Index Calibrator 9use cal Received with reagent kit PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 17 TML MSH Microbiology Department Policy MI SER 03 v01 Page 2 of 4 Policy amp Procedure Manual Serology Manual S P 5 HBs Ag Positive Control S P 6 Patient sample Sample Reagent For Cal Minimum Volume Required uL Segment or Reagent Control No of Patient Samples Position Sample Color Only 1 2 3 4 5 1 Reagent A Violet 100 150 200 250 300 350 2 Reagent B Yellow 150 200 250 300 350 400 3 Dilution Natural 0 500 875 1250 1625 2000 Reagent 4 Index Green 270 270 270 270 270 270 Calibrator 5 Positive Blue 250 250 250 250 250 250 Control 6 Patient 275 275 275 275 275 Sample 7 Patient 275 275 275 275 Sample 8 Patient Sample 275
31. press No F2 under Prime The Washer will dispense the 350 ml of buffer into each well and soak for 5 minutes 6 After 5 minutes Aspirate Wash Buffer from wells For aspiration of Wash Buffer use Program 1 lt a Select Program Press memory F1 press Number F1 b Program No press I enter c 001 Name 01 should appear d Press Set at this point press Change F2 then press Enter until Number of strips to wash appears check that of strip appears is what is needed If it is the same press enter If is not the same enter the right of strip and press enter until 001 Name 01 appears press Enter Press Run F1 and press No F2 under Prime The Washer will aspirate the buffer End washing will appear when washing cycle is finished remove plate Repeat Washing will appear press No F2 Ba rh PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 57 TML MSH Microbiology Department Policy MI SER 11 v03 Page 3 of 5 Policy amp Procedure Manual Serology Manual 7 Add 100 ul of Blk to well 1 100 ul of Pos to well 2 100 ul of Neg to well PaO Tie 0 90 10 11 12 13 14 15 3 100 ul each of Cal to well 4 5 and 6 Add 100 ul of specimen to each well starting from well 7 Use last well as external
32. run 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 Bartels lot G 666 C difficile Toxin Control with each new lot 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 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 6 TML MSH Microbiology Department Policy MI SER 01 v01 Page 5 of 5 Policy amp Procedure Manual Serology Manual VIII References 1 Package insert from TechLab an ELISA for the detection of Clostridium difficile Toxin A and B 2 BEP 2000 Quick Reference Guide Dade Behring Marburg GMBH PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 7 TML MSH Microbiology Department Policy MI SER 02 v03 Page 1 of 9 Policy amp Procedure Manual Section Serology Manual Subject Title AXSYM System Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date October 28 2003 AXSYM SYSTEM FOR HBsAg HBsAb HBeAg HBeAb HBcAb IgM HBcAbTotal HAV IgM HCVAb CMV IgG HIV 1 2 Rubella IgG I Introduction The Abbott AxSYM System is a fully automated random access analyser that utilizes an
33. select OK Turn the power switch to OFF Once power is off make sure that it is off for at least 5 minutes before turning AXSYM back ON Rotate the Carousel so that the red dot is facing you Remove the Carousel by pushing back on the Carousel tilting down and pulling out Clean the carousel and the surrounding area with lint free tissue moistened with water Inspect the clips on the under side of the carousel for broken pieces loose connections Dry the Carousel with Lint free tissue Reinstall the Carousel by holding the carousel with the red dot facing you and fit the rim of the carousel into the rear V wheel and push in towards the spring loaded V wheel Tilt up and fit the rim of the carousel into the front V wheels Manually rotate Carousel to ensure proper positioning Reinstall Air Deflector Reinstall the Matrix Cell Carousel Access Panel Close the Processing Center Cover Re install Matrix Cell Hopper Turn the power ON Select Startup from the Main Menu If the power is off for less than 15 minutes there is a 15 minute warm up If the power is off for more than 15 minutes there is an hour warm up PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 28 TML MSH Microbiology Department Policy MI SER 04 v01 Page 9 of 9 Policy amp Procedure Manual Serology Manual 11 12 13 MEIA Verification Select MAINTENANCE from the Main Me
34. the Waste and Supply Center Doors ii Update inventory 1 Select Inventory from Main Menu 2 Select F5 Bulk Solutions 3 Select F2 Replace Solution 1 F4 Replace Solution 3 F5 Replace Solution 4 4 F6 Save Exit to Main Menu 4 Flushing Pumps and Syringes Select Maintenance from the Main Menu Select Prime and Flush Enter 1 under sampling syringe flush and Processing syringe flush Select F2 Perform Maint Check for leaks or bubbles in the tubings Exit to Main Menu 5 After loading all reagent packs press F5 Scan pack Press Inventory Press F3 Reagent Load List Check to see how many tests are left in each reagent pack To print a copy Press Alt and PRINT at the same time PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 24 TML MSH Microbiology Department Policy MI SER 04 v01 Page 5 of 9 Policy amp Procedure Manual Serology Manual 6 7 8 Scan reagent packs at the end of the day a Remove all Reagent Packs b Select F5 scan pack from Main Menu Probe Clean at the end of the day a Select Maintenance from the Main Menu b Select Probe Clean Follow instructions on the screen c Load two Reaction Cells at the designated locations on the Reaction Vessel Carousel d Pipette 1 ml of TEAH Probe Clean Solution into both the buffer and predilution wells of both Reaction Vessel
35. with no more than 1 freeze thaw More than one freeze thaw cycle may result in loss of copy number PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 73 TML MSH Microbiology Department Policy MI SER 14 v02 Page 3 of 5 Policy amp Procedure Manual Serology Manual If processed specimens and controls were stored frozen prior to amplification thaw at room temperature vortex for 5 seconds centrifuge for 15 minutes before proceeding to step 17 17 Preparation of Working Master Mix to be done in clean room a Vortex HBM Mg 2 for 5 seconds Prepare Working Master Mix by adding 100uL HBM Mg 2 to one vial HBM MMX It is not necessary to measure the volume of Master Mix Mix well by inverting the tube 10 15 times Discard remaining HBM Mg 2 The pink dye in HBM Mg 2 is used for visual confirmation that HBM Mg 2 has been added to HBM MMX b Pipette 50 uL Working Master Mix into each A tube using a pipette with an aerosol barrier tip Do not close covers of the A tubes at this time Discard unused Working Master Mix c Place A rings containing Working Master Mix into a resealable plastic bag and seal plastic bag securely Move A rings into specimen preparation area and store at 2 8 C until ready for use Working Master Mix must be used within 4 hours of preparation 18 Add 50 ul of each processed specimen or control to the appropriate A tubes containing worki
36. 0 C or 70 C if not able to proceed immediately PCR Amplification and Detection Procedures In the Clean Room Changed to dedicated clean room gown and gloves work in Cabinet a Take out the necessary number of WNV Master Mix vials 12 tests per vial from the freezer and thoroughly thaw inside the Biosafety Cabinet Place a capillary for each purified sample QS external QC and H20 into the capillary adaptor of the Cooling Block pre cooled to 4 C Pipette 15 uL Master Mix into the reservoir of each capillary Changed out of dedicated clean room gown PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 88 TML MSH Microbiology Department Policy MI SER 16 v01 Page 6 of 10 Policy amp Procedure Manual Serology Manual In the Specimen Processing Room Changed into specimen room gown a Using a separate filter tip each time carefully pipette 5 uL each of purified sample QS3 external QC and H20 already spiked with 0 1 mL internal control per 1 mL H20 directly into the capillary reservoir Immediately close the capillary with a lid Load capillary tubes into LightCycler LC adaptor and centrifuge at 3000 rpm for 15 seconds in LC centrifuge Transfer the LC adaptor to the LC instrument Alternatively if the LC centrifuge is not available centrifuge the capillary in the adaptors for 10 seconds at 2000 rpm 400g to transfer the mixture fr
37. 0 Index Reactive POSITIVE Negative Negative HBcAb Total Assay Cut off lt 1 0 S CO Reactive POSITIVE Negative Negative Non repeatable reactive NEGATIVE HAV IgM Assay Cut off gt 1 20 index Reactive POSITIVE Grey zone Reactive 0 8 1 2 Index Inconclusive Please send repeat specimen Non reactive Negative HCV Ab Assay Cut off gt 1 0 S CO 1 If S CO gt 10 reflex to 8SHCA2 Report positive if repeatably reactive If S CO lt 10 enter in the LIS as To PHL MOHC Send to PHL next day Preliminary report of STAT testing will be phoned as Presumptive Positive Grey zone 1 6 2 0 S N NON REACTIVE Non reactive NON REACTIVE PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 14 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 02 v03 Page 8 of 9 x CMV IgG Antibody Cut off gt 15 AU mL Positive gt 25 AU ml POSITIVE Positive 15 25 AU mL POSITIVE Low Level Negative lt 15 AU ml Negative xi Rubella IgG Antibody Cut off gt 10 0 IU mL Positive gt 15 IU mL POSITIVE Positive 10 15 IU mL POSITIVE Low Level Greyzone 5 9 9 IU mL Negative Negative Negative xii HIV 1 2 Assay Reactive Enter in the LIS as TO PHL Send to PHL next day for confirmation Negative Negative Review with Charge Technologist before reporting Quality Control Abbott
38. 0 nm 630 nm within 1 hour of stopping the assay PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 58 TML MSH Microbiology Department Policy MI SER 11 v03 Page 4 of 5 Policy amp Procedure Manual Serology Manual IV Calculation Index value Optical Density of Sample the mean Optical Density of the Cut Off Calibrators Interpretation of Index Values Positive gt 1 10 Negative lt 0 9 Equivocal gt 0 9 and lt 1 1 All positive and equivocal specimens should be repeated the next day V Validation The mean value for the Cut Off Calibrator must be within 0 100 to 0 700 O D units minus the blank well The Positive Control Index value should be between 1 5 and 3 5 The Negative Control Index value should be less than 0 8 VI Reporting Positive result Sent to PHL for confirmation Report PHL result in LIS Negative result West Nile Virus Flavivirus IgG by EIA Negative This is a research test Equivocal result repeat testing the next working day If result is negative report as West Nile Virus Flavivirus IgG by EIA Negative This is a research test If result is still equivocal positive Send to PHL for confirmation Enter PHL for result in LIS PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 59 TML MSH Microbiology Department Policy amp Procedure Manual Policy
39. 16 v01 Page 8 of 10 Policy amp Procedure Manual Serology Manual IV Reporting Fluorimeter Channel F1 Fluorimeter Channel F3 WNV amplicon back F1 IC amplicon Interpretations Possible PCR inhibition Repeat assay If still ve on both Fluorimeter Channels report Indeterminate Report Negative Preliminary report Positive Repeat assay to confirm If still ve on fluorimeter Channel F1 Report POSITIVE Preliminary report Positive Repeat assay to confirm If still ve on fluorimeter Channel F1 Report POSITIVE Positive results for the WNV amplicon Channel F1 will have the cycle number printed under the Cross Point Column in the chart and the Calculated values will be at least 10 copies uL gt QS4 The curve will be rising exponentially and levels off to a plateau The sample must be repeated for confirmation Internal Control amplicon need not be positive Indeterminate results may be those with a positive signal for the WNV amplicon Channel F1 cycle number printed under the Cross Point Column in the chart but the Calculated values are less than 10 copies uL lt QS4 The curve will be rising at a high cycle number The sample must be repeated PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 91 TML MSH Microbiology Department Policy MI SER 16 v01 Page 9 of 10 Policy amp Procedure Ma
40. 20 TML MSH Microbiology Department Policy MI SER 04 v01 Page of 9 Policy amp Procedure Manual Section Serology Manual Subject Title Hepatitis Virus Serology Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date September 20 2001 HEPATITIS VIRUS SEROLOGY Although there are many infectious causes of hepatitis the majority are caused by hepatitis A B and C viruses Acute Hepatitis A is diagnosed by detection of IgM antibodies Anti HAV IgM becomes positive just before the development of clinical hepatitis and remains positive for at least 4 months after infection There is no chronic carrier state for Hepatitis A Detection of total antibodies to Hepatitis A indicates immunity due to either past infection or immunization Hepatitis B is diagnosed by either detecting hepatitis B surface antigen HBsAg which indicates the presence of infectious virus HBcAb IgM or Anti hepatitis B surface antibodies HBsAb which indicate immunity due to either past infection or immunization Anti hepatitis B core IgM antibodies indicate acute infection and HBcAb Total indicates previous or current infection HBsAg should be cleared within 6 months of acute infection Persistence of HBsAg beyond 6 months is consistent with chronic hepatitis B infection Some of these tests may occasionally be performed on a STAT basis because of concern regarding transmission of the virus to
41. 8000 rpm 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 TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 87 TML MSH Microbiology Department Policy MI SER 16 v01 Page 5 of 10 Policy amp Procedure Manual Serology Manual b i Repeat Step 7 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 6000g 8000 rpm for 1 minute Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate j Open the spin column and add 500 uL of Buffer AW1 Close the cap and centrifuge at 6 000g 8 000 rpm for 1 minute k Open the spin column and add 500 uL of Buffer AW2 Close the cap and centrifuge at full speed 20 000g 14 000 rpm for 3 minutes l Place the spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate Centrifuge at 20 000g for 1 min Elution Steps 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 Centrifuge at 6000g 8000 rpm for 1 min Store the eluate at 2
42. AL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 40 TML MSH Microbiology Department Policy MI SER 06 v01 Page 5 of 5 Policy amp Procedure Manual Serology Manual iii iv v vi vii Validation Each pair of Reference P N wells must reach or exceed a value of 0 5 A A A A Reference P N gt 0 5 Interpretation of Results Positive AA sample gt 0 200 Negative AA sample lt 0 100 Equivocal 0 100 lt AA sample lt 0 200 Reporting Positive by EIA Negative by EIA Equivocal by EIA Quality Control A reference P N control must be run at the start and at the end of the each run Run External Control Lot 20030509 pooled positive sera with each new lot If external control result is negative the test is invalid Inform Charge senior technologist and repeat testing Results filed in Reagent Lot Binder CAP provides external proficiency testing Reference Package insert from Dade Behring Enzygnost Anti EBV IgG May 1999 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 41 TML MSH Microbiology Department Policy MI SER 07 v01 Page 1 of 5 Policy amp Procedure Manual Section Serology Manual Subject Title Human T Lymphotropic Virus Type 1 HTLV 1 EIA Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date May 26 2003 I HI H
43. ARTMENT Page 8 TML MSH Microbiology Department Policy MI SER 02 v03 Page 2 of 9 Policy amp Procedure Manual Serology Manual B Assay Procedure for Patient Sample Primary tube 1 2 3 4 C 1 2 4 5 6 7 8 9 10 D 1 2 3 5 6 7 8 9 Check that label on tube contains ordered tests as per the requisition and the name on the label matched the name on the tube The bar code label is centered and vertical on the tube Remove the cap Check for and remove any bubbles in the serum Ensure that the level of serum is adequate Load the samples and place the segments in the sample carousel Load the appropriate assay reagents in the reagent carousel Remember to open reagent bottle 4 on the reagent pack if present Assay Procedure for Patient Sample Sample cup From the MAIN MENU select ORDERLIST select F6 Patient Scan LIS type in Name and then press TAB Type S P location of the sample cup Select F6 ADD continue the above steps for the next sample cup Select F1 EXIT once all the patients have been entered Print the displayed orderlist using the PRINT KEY Verify sample cups are in correct location Load the samples Check for bubbles in the sample and place the segments in the sample carousel Load the appropriate assay reagents in reagent carousel Remember to open reagent bottle 4 on the reagent pack if pre
44. Appendix X Posting of AxSYM results Issued by LABORATORY MANAGER Original Date Dec 20 2003 Approved by Laboratory Director Revision Date APPENDIX X 10 POSTING OF AxSYM RESULTS Go to lab 8 Inerface T Interface Menu 9 AXSYM Open will be highlighted Enter Choose today s day and Enter P Posting How to Post change to By Order using space bar starting From enter LIS to be posted F12 Do you want to verify results with Posting Y N Press N Reflex window will appear F12 Press to post result Verify result with posting Press N Under ST column O will change to P 11 F12 to go to the next reflex repeat steps 10 12 At the end press F1 to exit Test to be posted Report as HBsAgt HBC HBsAg Positive HBC HBC 2 x HBsAg HBC HBC2 HBC Positive HBC2 HCV S CO gt 10 00 HCV 2 HCV Positive HCV 2 S CO gt 10 00 HCV S CO lt 10 00 HCV2 Send to PHL HCV 2 Not tested PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 115 TML MSH Microbiology Department Policy MI VIR 17 11 v02 Page 1 of 1 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix XI Looking Up Previous Hepatitis Result
45. At VIDAS instrument start at Main Menu a Enter 1 Create a Work List ENTER b Do you want to reserve sections now y ENTER c Enter technologist name _ enter initials here d Enter assay code enter VZG here e Enter number of VZG tests enter number here Include standards and controls f Enter sample ID Enter s two positions automatically used C1 C2 5 6 etc g When finished hit ENTER PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 53 TML MSH Microbiology Department Policy MI SER 10 v02 Page 3 of 4 Policy amp Procedure Manual Serology Manual h Recheck worklist entries Hit ENTER to accept worklist d to delete any entries OR m to modify any entries i Do you want a worklist fill report hit n ENTER j Load VZG strips according to assigned module positions on the worklist k Load one VZG SPR for each VZG strip 1 Run worklist now hit y ENTER m Assay is complete when results are printed and module lights are blinking n If instrument is beeping exit program 11 to the vid prompt Type message To re enter to the main menu type vid enter o If computer is not responding log in vid password enter blank Interpretation of Results Specimens with test values greater than 0 9 MEA VZ are considered positive Test values ranging from 0 6 0 9 are equivocal Samples wi
46. BIOLOGY DEPARTMENT Page 30 TML MSH Microbiology Department Policy MI SER 05 v01 Page 2 of 7 Policy amp Procedure Manual Serology Manual HI Procedure 0S P Lab Analyzer i ii Reagents 1 HBsAg Conjugate Concentrate 11 HIV 2 Peptide coated 2 HBsAg Cojugate Diluent Microwell Plates 3 AntiHBsAg Microwell Strip Plates 12 HIV 4 Peptide Specimen 4 HBsAg Negative Control Diluent 5 HBsAg Positive Control 13 HIV1 2 Peptide Conjugate 6 HBsAg Low Positive Control Concentrate 7 EIA Wash Solution 14 HIV 4 Peptide Conjugate 8 EIA Chromogen Reagent Diluent 9 EIA Chromogen diluent 15 HIV 1 Positive Control 10 EIA Stopping Reagent 16 HIV 2 Positive Control 17 HIV Negative Control Method HBsAg amp HIV 2 can be run simultaneously in the P Lab analyzer Before beginning EIA assay Allow all components to reach room temperature Prepare wash solution Add 100 ml of 25x wash solution to 2400 ml of distilled water Dilute each of HIV 1 Positive Control HIV2 Positive Control and Negative Control 1 10 in a test tube 50 ul control 450 ul of HIV 2 specimen diluent Fill up P Lab wash bottle Place bottle in Buffer 1 position in P Lab Turn hard drive and monitor Wait for software to be fully loaded before turning the P Lab ON Type in Qw as username and password Enter Click on third square from the left open session Click on New Type in assay amp
47. BORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 111 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI VIR 17 08 v02 Page of 2 Section Serology Manual Subject Title Appendix VIII Autoverification for the Probetec Issued by LABORATORY MANAGER Original Date June 25 2002 Approved by Laboratory Director Revision Date POLICY APPENDIX VIII AUTOVERIFICATION FOR THE PROBETEC The Probetec performs the following tests for the Virology department 1 Chlamydia trachomatis detection 2 Neisseria gonorrhoeae detection Autoverification Autofinalizing will occur as follows TEST SOFT RESULT TRANSLATION ACTION FINAL NAME TEST RESULT CODE Chlamydia CHLA POSITIVE POSITIVE No Autoverification Canned Detection Message CHLP Chlamydia CHLA NEGATIVE Autoverifies Autofi Canned Detection nalizes Message CHLN Neisseria NGON POSITIVE POSITIVE No Autoverification Canned gonorrhoeae Message GCPO Neisseria NGON NEGATIVE 7 NEGATIVE Autoverifies Autofi Canned gonorrhoeae nalizes Message GCNE PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 112 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI VIR 17 08 v02 Page 2 of 2 Section Serology Manual Canned Messages CHLP POSITIVE for Ch
48. Clinical Category HBsAg HBsAb HAV IgM HCVAb Hepatitis B Screen x Hepatitis A Screen x Hepatitis B Immune Status X Needlestick Patient source X Staff exposed X Pre postnatal Mother Hepatitis Screen X X X X X X For additional requests within the above categories consult with the charge technologist or medical microbiologist In the absence of one of the above clinical categories do the tests as requested Table 2 Guidelines for STAT Testing Clinical Serum Time frame Days of Call setting tested from exposure week back to report Neonate Mother lt 12 hours All No Prenatal Mother lt 12 hours All No Needlestick See below lt 72 hours All No mucosal exposure Renal dialysis Patient lt 12 hours All No 1 May require call back on weekends if time from exposure to reporting exceeds 12 hours Must be approved by microbiologist on call 2 Perform HBsAg and HCVAb on the source Test HBsAb and HCVAb on the employee Whichever of these arrives first is to be tested STAT If both arrive at the same time test both simultaneously If the source HBsAg is positive test the employee STAT If the source is negative test employee in the next routine run PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 22 TML MSH Microbiology Department Policy MI SER 04 v01 Page 3 of 9 Policy amp Procedure Manual Serology Manual HUMAN IMMUNODE
49. Controls Positive and negative controls must be run for the following assays once per 8 hour shift HCV Ab CMV Ab and Rubella IgG Positive and negative controls must be run for the following assays once per 24 hour shift HBsAg HBsAb HAV IgM HBeAg HBeAb and HIV 4 Results are verified by a senior technologist and filed in AXSYM printout log External Controls Run Positive External Control with each new reagent lot Accurun 5100 is for HBsAg HIV 2 HBcore Ab HCV Ab and CMV IgG Accurun 51 is for HBeAg HbcIgM and HAVIgM Accurun 52 is for AUSAB and HBeAb Accurun 140 for Rubella IgG Viroclear is used as negative control for all except Rubella IgG PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 15 TML MSH Microbiology Department Policy MI SER 02 v03 Page 9 of 9 Policy amp Procedure Manual Serology Manual VIL All stock Accuruns are stored at 70 C freezer MIFTJ External Controls are verified by a senior technologist and filed in External QC binder If any external control result is wrong the test is invalid Inform Charge senior technologist and repeat testing CAP QMP LS and LCDC provide external proficiency testings Calibrations Run calibration for each new reagent lot or when control s fail Failed QC Test is invalid without satisfactory QC results Do not release reagent lot for use pending resolution of QC error
50. EDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 19 TML MSH Microbiology Department Policy MI SER 03 v01 Page 4 of 4 Policy amp Procedure Manual Serology Manual IV VI VIL Results 1 From the MENU select RESULTS 2 Highlight all control and patient results associated with the confirmatory run 3 Select PRINT choose Print a formatted result for each displayed test 4 Select OK by touch screen Reporting Results HBsAg Confirmatory 8CON testing results are entered into the LIS using the keypads but do not print on the report The repeat HBcAb 8HBC2 results also do not print but are automatically posted to the LIS These confirmatory and repeat HBcAb 8HBC2 results are used to edit if necessary the HBsAg SHAG and HBcAb 8HBC results which will print on the report HBsAg Confirmatory Assay Neut or Neut Dil Positive Confirmed Positive Neut AND Neut Dil Negative Not Confirmed HBcAb 8HBC2 Assay Reactive Reactive Negative Negative Refer to AXSYM portion of manual for guidelines on reporting HBsAg and HBcAb based on reflex testing Quality Control A positive control confirmation should be included on each run Reference AXSYM operational manual and manufacturer s package insert Abbott Laboratories Abbort Park IL 60064 U S A PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page
51. FICIENCY VIRUS HIV SEROLOGY HIV testing will be done on patient source involved in Needle Stick Incident as STAT so appropriate prophylaxis can be given in case it is positive It is also done for transplant patients donor amp recipient Two tubes of blood should be received If the result is positive send the unopened tube to PHL The positive result will be reported as sent to PHL for confirmation Specimen will be sent out as STAT the next working day to PHL Put specimen with PHL HIV form in a separate brown bag clearly marked as STAT Call 416 340 6022 to inform PHL HIV lab that this specimen is coming as STAT and ask to call result to Lab when it is available Daily Maintenance 1 Empty Waste Containers first thing in the morning or at the end of the day Open the Waste and Supply Center Doors DO NOT OPEN THIS DOOR WHEN THE AXSYM IS RUNNING then open the Interior Waste Door a Remove Consumable waste i Remove biohazard bag from the consumable waste container and dispose it into Biohazard waste box ii Place a new biohazard bag in the container b Remove Liquid Waste i Hold down the tubing to the Liquid Waste Container while pressing the metal tab on the side of the clip ii Remove the Liquid Waste Container iii Transfer liquid waste into decontamination containers near the sinks Fill container with 4 5 full of waste and 1 5 full of Javex Label date on the lid iv Rin
52. ICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 12 TML MSH Microbiology Department Policy MI SER 02 v03 Page 6 of 9 Policy amp Procedure Manual Serology Manual 2 Reporting Results i HB Ag Assay Cut off gt 2 0 S N Reactive and confirmed HB Ab reactive POSITIVE Reactive and previously confirmed positive POSITIVE Reactive and non confirmed Negative HBsAg HBcAb Action Required S N Result lt 2 or Report Negative 2 5 T Send to PHL gt 5 10 F Perform Confirmatory Testing 2 10 Send to PHL gt 10 Perform Confirmatory Testing gt 10 S CO lt 0 2 Report Positive gt 10 S CO gt 0 2 Perform Confirmatory Testing ii HB Ab Assay Cut off gt 10 0 IU mL Reactive POSITIVE Negative Negative iii HB Ab Assay Cut off lt 1 0 S CO Repeatable reactive POSITIVE Non reactive Negative Non repeatable reactive NEGATIVE iv HBeAb Assay Cut off gt 1 0 S CO Reactive POSITIVE Negative Negative PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 13 TML MSH Microbiology Department Policy MI SER 02 v03 Page 7 of 9 Policy amp Procedure Manual Serology Manual v vi vii viii ix HBeAg Assay Cut off gt 1 0 S N Reactive POSITIVE repeat specimen with value of gt 1 0 and lt 10 Negative Negative HBcAb IgM Assay Cut off gt 0 8
53. Inform charge senior technologist Record in Reagent Log Chart Instrument Maintenance Log if microscope incubator is involved in the failure and Incident Report if necessary Re run failed control materials in parallel to fresh controls to evaluate the QC material itself If the re run shows the old QC material still fails and fresh QC is satisfactory the error may be attributed to the old QC material itself and the reagent is satisfactory If the re run shows both the old and fresh QC materials fail or other QCs not satisfactory the error may be attributed to the reagent then the reagent cannot be released for use Reference AXSYM operational manual and manufacturer s package insert for the appropriate assay Abbott Laboratories Diagnostics Division Abbott Park IL 60064 USA PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 16 TML MSH Microbiology Department Policy MI SER 03 v01 Page of 4 Policy amp Procedure Manual Section Serology Manual Subject Title AxSym HBsAg Surface Antigen Confirmatory Assay Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date September 20 2001 AXSYM HEPATITIS B SURFACE ANTIGEN CONFIRMATORY ASSAY I Introduction AXSYM HBsAg Confirmatory assay is a Microparticle Enzyme Immunoassay MEIA used for the detection of HBsAg in human serum or plasma by
54. ML MSH Microbiology Department Policy MI SER 15 v04 Page 6 of 7 Policy amp Procedure Manual Serology Manual 5 Start the Run Click Start to start the run a Load check will be performed Select Run Mode basic Wait for Load check to pass see status at top right corner print a copy by clicking on Print Icon If Load Check failed click on Stop which will stop the transferring step but amplification will continue Find out the reason for failing e g not enough reagent in cassette wrong rack Ratify problem s and click on Start again Wait for Load check to pass see status at top right corner print a copy by clicking on Print icon 6 Printing the results IV When the run is finished click on Result 4 window from the top at the side application bar Scroll down to find A ring on the left side Double click on A ring check results on screen click on Accept All at the bottom of the screen Click on View List by tube and print Validation Internal control for each negative specimen must be positive to make sure the specimen does not contain inhibitory substance If internal control is negative or result is equivocal repeat using another tube of the same specimen in next run If internal control is still negative after repeat testing send out report in as No result available du
55. MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 29 TML MSH Microbiology Department Policy MI SER 05 v01 Page 1 of 7 Policy amp Procedure Manual Section Serology Manual Subject Title BIO RAD HBsAg Issued by LABORATORY MANAGER Original Date January 5 2004 Approved by Laboratory Director Revision Date HBsAG amp HIV 1 2 I Introduction Genetic Systems HBsAg EIA 2 0 is a qualitative enzyme immunoassay for the detection of Hepatitis B Surface Antigen HBsAg in human serum and plasma and also in cadaveric serum specimens Genetic Systems HIV 1 HIV 2 Peptide EIA 2 0 is the Genetic Systems Corporation qualitative enzyme immunoassay for the detection of Human Immunodeficiency Virus type 1 and or Human Immunodeficiency Virus type 2 in human serum and plasma and also in cadaveric serum specimens These kits are only used for postmortem specimens forwarded from the following Eye Banks a Eye Bank of Canada Ontario Division b New Brunswick Eye bank c Lions Eye Bank Manitoba amp Northwest Onatrio and Tissue Lab Hospital For Sick Children II Specimen Collection and Processing a Blood or serum collected by Eye Banks b If blood tube received centrifuge at 3000 rpm for 10 minutes Transfer approximately 1 1 5 ml serum into 2 ml storage tube give blood tube serum to AXSYM bench for HCV Ab testing PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICRO
56. PR test card 0 05 mL disposable stirrer pipettes Serological rotator at 100 rpm dH20 iii Precaution Refrigerate reagents until required Warm to RT and mix well before use To ensure stability return the antigen suspension to the original glass bottle after testing The dispenser and needle assembly must be thoroughly washed in dH2O and air dried after use PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 49 TML MSH Microbiology Department Policy MI SER 09 v02 Page 2 of 3 Policy amp Procedure Manual Serology Manual iv Needle Accuracy Check When New Kit Opened v vi This procedure is performed to check the needle delivering antigen each time before testing Using a pipette deliver 0 5 mL antigen to the dropper bottle Attach the needle and holding in a vertical position count the number of drops delivered in 0 5 mL The needle is considered satisfactory if 30 1 drops are obtained Ifthe needle is unsatisfactory repeat the check Record the lot number of the newly opened kit in the reagent lot number binder Method Using the stirrer pipette held vertically dispense one drop 50 uL of serum onto a circle on the test card Use a fresh stirrer pipette for each sample Repeat with the control sera Using the flat end of the stirrer pipette spread the sample over the entire area of the test circle Attach the needle to the dropper bottle
57. SH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 07 v01 Page 4 of 5 13 Click on Fill lung screen will ask you to open lid Open lid and click on OK Water level should fill up to just under the lower black line Click 1 2 times to Add 250ul of water each time to raise the level to just above the lower black line Close lid and click on OK Click on Buffer 1 prime listen to the noise to make sure that the vacuum is working 14 15 Click on 3 rd square Tips if need to replace pipette box Click on 6th square from the left to execute the run will ask to put sample rack in click OK The P lab will start processing Click on Time chart to see what stages the testing is at After assay is finished Session terminated will appear on screen click OK Click on Maintenance and click on Endwork Will ask to fill in buffer 2 with DH 0 open lid make sure enough DH20 in the bottle also change buffer 1 bottle to DH20 bottle Click OK The machine is rinsing the tubing with DH 0O Next will ask to empty waste press on Black Button on the side of the machine and hold it until almost all fluid is drained off and bubbles appear Empty waste Click OK Empty tips click OK Don t forget to shut off P Lab click OK 16 17 18 Click on left top corner File an
58. TITIS CENA POR enra a een a a Ra 71 WEST NEE yIRUS RI PER cererea rE R E a E a aa Ta heen 84 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 1 TML MSH Microbiology Department Policy MI SER v11 Page 2 of 2 Policy amp Procedure Manual Section Serology Manual Subject Title Table of Contents APPENDICES Appendix T FP Test NS sa cssrixsuncsenvvansacrvnnrexinestusasinnencvaniaciaanvatievaiaeeesasinennsiesiiacaeatons 94 Appendix U ten La 6 ele ie ee eee eee eee eee area ee 96 Appendix II Shipment of Parvovirus PCR Sante vc csccscvcessunesdsactiavsaanversevenseatceaivonesuavieciarsvess 98 Appendix IV The Thermo Cycler Gene Amp PCR System 9600 cc ccssecsseceseeseeeeneeeseeees 99 Appendix V Entering Serology Refer Out Results scsccsssssseresesescessscesereesseecensscssensessencenes 104 Appendix VI BD Probe Tec ET Wy Orb hi ssc isccisaitesissanscsisiaisadveniGeiasanvocsiadimaniiienaans 107 Appendix VI Antoyerihicaton Pas ee eee 109 Appendix VIII Autoverification for the Probetec i isscis css cesassnisescedusadustseediasasnasvsouesassnsseseeaneteuiars 112 Appendix IX Shipment of Parvovirus PCR to HSC qo cccscnsssicessaicsssnsssssnenssncssecssanennnosncavensecens 114 Appendix X Posting of AXSYM Pests aoc cssuscasviasssasssovaindsiasidersscorsassdanscaivassevadedadisenssinnveantace 115 Appendix XI Looking Up Previous Hepatitis Results in Vista ciccscssc
59. UMAN T LYMPHOTROPIC VIRUS TYPE 1 HTLV 1 EIA Introduction The Adaltis DETECT HTLV EIA Test Kit is a solid phase enzyme immunoassay utilizing a mixture of synthetic peptides for the detection of antibodies to HTLV I and II in human serum or plasma Specimen Collection and Processing Blood is collected 5 mL for adults and mL for neonates in a serum separator tube and separated by centrifugation The serum is stored at 4 C Procedure i Reagents Positive Control Negative control Anti Human IgG Peroxidase Conjugate Conjugate Diluent Tetramethylbenzidine TMB Reagent Peroxide Reagent Stop Solution 2x 96 well Microplate coated with HTLV I amp HTLVII peptide ii Other Materials P Lab Analyser System 5 ml graduated Pipettes 10 ml graduated Pipettes 1 ml graduated Pipettes 10 100 ul pipettor Pipette tips 55x12 3 5 ml tubes Automatic pipettor PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 42 TML MSH Microbiology Department Policy MI SER 07 v01 Page 2 of 5 Policy amp Procedure Manual Serology Manual iii Method A Using P Lab Personal Lab Before beginning EIA assay 1 Allow all components to reach room temperature Prepare wash solution Add 100 ml of 25x wash solution to 2400 ml of distilled water Fill up P Lab wash bottle Place bottle in Buffer 1 position in P Lab Turn hard drive and monitor Wait for sof
60. Wells 96 wells Sample Diluent PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 56 TML MSH Microbiology Department Policy MI SER 11 v03 Page 2 of 5 Policy amp Procedure Manual Serology Manual ili Method l h Pe moagcgp M Label a set of 12x75 mm glass tubes as follows Blk Pos Neg Cal 7 8 9 10 and External Control when required e g a new lot Add 1 ml of Sample Diluent to all tubes Leave 1 tube blank add 10 ul of Controls Calibrator and patient samples to corresponding tubes Take out the required of strips Open bag containing strips when strips reaches room temperature 10 15 minutes Return the unused strips to the bag and refrigerate immediately Change bottle from D H20 to Wash Buffer Turn Washer ON Press button under Prime F2 Press button under washing F1 Select Program Press memory F1 press Number F1 For dispensing 350 ul of buffer use Program 2 Program No press 2 enter 002 Name 1 should appear Press Set at this point press Change F2 then press Enter until Number of strips to wash appears check that of strip appears is what is needed If it is the same press Escape If is not the same enter the right of strip and press enter until 002 Name 01 appears press Enter g Press Run F1 and
61. Y MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date September 20 2001 APPENDIX III SHIPMENT OF JC BK PCR SAMPLES PHL requires that these samples be packaged in a standardized way before they will forward them to Hamilton for us l Put sample tube inside plastic bag and place together with requisition into a plastic cylinder 2 Place plastic cylinder into a cardboard box padded with paper towels 3 Seal the box with packing tape and label the box as follows Public Health Laboratory 81 Resources Rd DO NOT OPEN Please forward to JC BK PCR Dr James Mahoney REGIONAL VIROLOGY LABORATORY ST JOSEPH S HOSPITAL 50 CHARLTON AVE E HAMILTON ONTARIO L8N2X6 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 98 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 17 04 v01 Page 1 of 5 Section Serology Manual Subject Title Appendix IV The Thermo Cycler Gene Amp PCR System 9600 Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date September 20 2001 APPENDIX IV THE THERMO CYCLER USED IN AMPLIFICATION IS GENE AMP PCR SYSTEM 9600 To use this machine for amplification create Creating a Method the Method and the program for the experiment A method is a series of up to 17 programs linked together Fo
62. a susceptible individual following exposure i e needlestick newborn to infected blood body fluids and the need to prevent the disease by administering vaccine and or immunoglobulin Hepatitis due to the delta virus is rare in Canada It only occurs in association with patients who are positive for hepatitis B surface antigen Requests for this virus should be forwarded to the Public Health Laboratory Hepatitis C HCV is a blood borne virus closely associated with blood transfusion and intravenous drug use The presence of antibodies to HCV indicates that an individual may have been infected with HCV may harbour infectious HCV and or may be capable of transmitting HCV infection The following requests will be handled in our laboratory Hepatitis B surface antigen HBsAg Hepatitis B surface antibody HBsAb Hepatitis B core antibody HBcAb Hepatitis B e antigen HBeAg Hepatitis B e antibody HBeAb Hepatitis A IgM antibody HAV IgM Hepatitis A Total antibody HAV IgG PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 21 TML MSH Microbiology Department Policy MI SER 04 v01 Page 2 of 9 Policy amp Procedure Manual Serology Manual Note 1 These tests will be performed on request only if the patient is HBsAg positive 2 Perform only HAV IgM if type of Hepatitis A test is not specified Table 1 Tests performed as per designated categories Test Performed
63. al Serology Manual i RS aie oa Lot specific vales for plate 1 click OK unless a new lot If this is a new lot just highlight the area that needs changes and type in new Value for lot upper margin lower margin a and B and save Click on Green arrow to start Load click OR Are you sure that the resources have been loaded in the correct position click Yes Load Plate load a NEW microtiter plate clear one with strips into a metal carrier Open the door on the far right and load the whole tray into Behring 2000 the two pins on the right side of the carrier should fit into the two rings in the Behring 2000 Under Plate 1 enter validyear month date e g valid030106 Click OK Are you sure plate layout correct click Yes When finished remove plate and carrier from system appears click OK Remove plate and visually check strips 9 10 for equally distributed liquid strips 11 12 for complete aspiration Print report and checked that V C Passed printed on right top corner of the report File report in Behring 2000 Validation Assay binder 8 Perform testing a b Remove Reagent Rack 5 L load Sample Buffer Working Chromogen Solution and Stop Solution into rack and re load into Analyzer Remove Reagent Rack I Load Working Conjugate Solution into rack and reload into Analy
64. ample Processing l Swabs a Label and remove cap from sample diluent tube b Insert swab and swirl in diluent for 5 10 seconds c Express liquid from swab and discard d Tightly recap the tube and vortex for 5 seconds 2 Urine a UPP must be in contact with specimen for a minimum of two hours b Mix urine by swirling and pipetting 4 ml into empty sample tube c Cap tube and centrifuge at 2000xg for 30 minutes d Decant tubes End decanting motion with a gentle flick of the wrist to remove the residual fluid from the tube e Add 2 ml of sample diluent f Recap tubes and vortex 5 seconds 3 Controls a Use 1 Positive Control and 1 Negative control for each run b Add 2 ml of sample diluent to each tube c Recap tubes and vortex 5 seconds Note It is important to dispense liquid against the inside wall of the microwells to insure complete specimen addition and to avoid cross contamination iv Sample Lysing d Use plate layout template to place samples and controls in correct position in lysing rack e Place rack in lysing heater for 30 minutes Remove rack and let samples cool for at least 15 minutes PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 68 TML MSH Microbiology Department Policy MI SER 13 v02 Page 4 of 6 Policy amp Procedure Manual Serology Manual iv Assay Procedure l 0S 10 11
65. ayout is correct click OK Downloading Progress will appear and then the Orange square will also appear the testing will start To see what stage the test is at close the inside window on the screen Click on beside Work 2 Click on Schedule You can see the red line move across the screen with each step PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 5 TML MSH Microbiology Department Policy MI SER 01 v01 Page 4 of 5 Policy amp Procedure Manual Serology Manual IV VI VII s When finished Remove plate amp Carrier from the system remove plate and click OK t Click on Print Report u Repeat any specimen that has Clot detected the next working day v Re capped all reagents and place the whole kit in the refrigerator Assay validation Positive Control should read gt 0 500 at 450 620nm Negative Control should read lt 0 080 at 450 620 nm Interpretation of Results Spectrophotometric Dual Wavelength 450 620 nm Negative OD lt 0 08 Positive OD gt 0 08 Reporting Positive Clostridium difficile toxin detected Negative Clostridium difficile toxin not detected If patient is positive for C difficile toxin phone result to ward and also phone INFECTION CONTROL NURSE at UHN TG TW amp PMH or CHC in patient only Quality Control Negative and positive controls must be included with each
66. bance values of the two Positive controls divided by two The PCX must be gt or to 1 00 AU Each individual absorbance must be within the range of 0 65 to 1 35 times the PCX No Positive Control absorbance value may be discarded Calculate the Mean Absorbance of the Low Positive Control LPCX Sum of the absorbance values of the two Low Positive controls divided by two The LPCX must be Positive gt or to the assay Cut off value Each individual absorbance must be within the range of 0 65 to 1 35 times the PCX No Positive Control absorbance value may be discarded 4 Cutoffvalue NCx negative control mean 0 70 HIV 5 Calculate the Mean Absorbance of the Negative Control NCX Sum of the absorbance values of the three negative controls divided by three Each individual absorbance must be gt 0 020 AU and lt or to 0 140 AU One Negative Control value may be discarded if it is outside this range The NCX may be calculated from the two remaining absorbance values Calculate the Mean Absorbance of the HIV 1 Positive Control HIV 1 PCX Sum of the absorbance values of the two HIV 1 Positive controls divided by two The HIV 1PCX must be gt or to 0 900 AU Each individual absorbance must be within the range of 0 65 to 1 35 times the PCX No Positive Control absorbance value may be discarded PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 34 TML MSH M
67. d Exit will ask exit from P lab Processor click Yes 19 Click on 4 box from the top Open Results Click on New Result Then click on you session and OK 20 Click on 3 box from the left report click on DetectHTLV and also the square with the printer Report will be printed out 21 Close all windows by clicking on X at top right corner three times 22 Turn off hard drive monitor P lab and printer iv Validation 9 Absorbance of the blank must be lt or 0 100 10 The Positive Control mean must be gt or 0 800 If the value is less than 0 80 the run must be repeated 11 Cut off NCx negative control mean 0 150 If the positive control fails the test is invalid Should inform Senior Charge technologists and repeat testing PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 45 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 07 v01 Serology Manual Page 5 of 5 v Interpretation of Results 3 Absorbance value is lt cut off reported as Negative 4 Absorbance value is gt or cut off send to PHL for confirmation vi Quality Control Two positive and three negative controls must be included with each run Run Virotrol I as external control Do not dilute with each new lot All positive specimens are sent to PHL for confirmation
68. dule When the reports come back from a reference lab the reporting date reference lab number and result are entered A senior charge technologist verifies the results in the LIS and the reference lab reports are photocopied and then forwarded to the appropriate clinic or doctor a b Log on lab Press 1 Order Entry Press F3 scan barcode on reference lab requisition Press F12 Check that it is the correct patient Press F8 for result field Check that referred out tests have been ordered for all results coming back from the reference lab If not follow steps i to vi for free text referred out tests or proceed to step p for specific non free text refer out tests i F1 ii Move cursor to next blank test field iii On the Microbiology Order Entry screen press W for Serology tests iv Press S for Referred out Test 8REF for MSH and 9MIS for UHN patients v Press F8 for result field vi Press 1 To PHL Skip steps vii xi if send out tests results are complete go to step g vii Press 4 MOHR on the 8REF or 9MIS result field viii Press to see the canned message This sample has been referred to the Public Health Lab PHL 81 Resources Rd Etobicoke Ont M9P 3T1 for the tests listed below will appear PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 104 TML MSH Microbiology Department Policy MI SER 17 05 v01 Page 2 of 3 Policy amp Pr
69. e to inhibitory substance present in specimen please send another specimen Internal control for each positive specimen can be positive or negative Because specimen has already amplified therefore giving a positive result Positive gt 0 15 at A660 Negative lt 0 15 at A660 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 82 TML MSH Microbiology Department Policy MI SER 15 v04 Page 7 of 7 Policy amp Procedure Manual Serology Manual vV Reporting VI VIL Positive All positive patients should be checked if they belong to the enlisted doctors If not the patients should be highlighted on the tasklist before summitting it to the charge technogist for verification Negative No result available due to inhibitory substance present in specimen please send another specimen Quality Control One AMPLICOR HCV control and one AMPLICOR HCV control are processed with each run The absorbance of HCV should be less than 0 1 at 660 nm The absorbance of HC V should be greater than or equal to 1 0 at 660 nm Run External Control Accurun 305 monthly If result is invalid the run has to be repeated Inform charge senior and repeat testing Result filed in Reagent Lot Binder External proficiency testing is provided by LCDC Reference COBAS Amplicor Operator s Manual and Insert for Hepatitis C Virus Test vers
70. eisesscsseseasveacssscenceosseve 116 Appendix XII Printing of Pending ist iia seisincdcecdestev iia cotedvindesaealaa vada een 117 Appendix XII Wipes Test for Pre 1 C0 sas csezscacccacvecasesasacesiadeuetsasmauwaareridiacdeienencs 118 Record of Edited Reyko ericeira ieaie E EERE beaded EEEE E AEREAS 119 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 2 TML MSH Microbiology Department Policy MI SER 01 v01 Page 1 of 5 Policy amp Procedure Manual Section Serology Manual Subject Title C difficile Toxin A B EIA Test Issued by LABORATORY MANAGER Original Date April 25 2001 Approved by Laboratory Director Revision Date May 27 2003 I HI C difficile TOXIN A B Introduction C difficile Tox A B is an enzyme immunoassay for the detection of toxins A and B produced by toxigenic strains of C difficile It can be used to detect toxins Aand B in fecal specimens from persons suspected of having C difficile disease Specimen Collection and Processing Collect stool specimen in 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 a Dispense 600 ul of diluent into 2 ml microtube b Add 150 ul of liquid stool mix stool well first or approx 6 mm diameter of solid stool c Vortex for 10 seconds spin for 5 minutes at 14 in Ep
71. entrifuge tubes Cooling Block with capillary adaptors pre cooled to 4 C Variable volume pipettes 1 to 20 uL 10 to 200 uL 100 to 1000 uL From Qiagen Viral Isolation Kit Collection Tubes Lysis Buffer AVL with RNA carrier thawed or heated to dissolve crystals formed during refrigeration Spin Columns Wash Buffer 1 Buffer AW1 add 125 mL ethanol before use e Wash Buffer 2 Buffer AW2 add 160 mL ethanol before use Elution Buffer Buffer AVE From RealArt WNV LC RT PCR Kit Artus e Quantification Standard no 3 10 copies uL e Internal Control Ethanol anhydrous reagent grade 96 100 GENERAL PRECAUTIONS e Use only filtered pipette tips e Store positive materials specimens controls and amplicons separately from all other reagents and add to reaction mixes in a spatially separated facility clean room e Clean and specimen pipettes and tips stay separated in their designated rooms Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Put on fresh gloves and clean gown colour coded blue when entering the clean room PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 86 TML MSH Microbiology Department Policy MI SER 16 v01 Page 4 of 10 Policy amp Procedure Manual Serology Manual E Sample Preparation Use Specimen Preparation area
72. er until 11 Name IgM WN appears press Enter Press Run F1 and press No F1 under Prime Will wash strip for 3 times each time with working buffer n End washing will appear when washing cycle is finished soak strip for 5 minutes o Aspirate Wash Buffer using Program 1 steps 6 a i 10 11 12 13 14 15 16 17 Remove plate from Plate Washer Add 100 ul of Antigen into each well using an 8 channel pipette cover plate with sealing tape and incubate for 2 hours 120 minutes at room temperature Repeat wash steps 9a h Add 100 ul of Conjugate into each well using an 8 channel pipette cover plate with sealing tape and incubate for 30 minutes at room temperature Repeat wash steps Add 100 ul of Substrate into each well using an 8 channel pipette cover and incubate for 10 minutes at room temperature Add 100 ul of Stop Reagent into each well using an 8 channel pipette In antibody positive wells color should change from blue to yellow Read the plate in spectrophotometer at dual wavelength 450 nm 630 nm within 1 hour of stopping the assay PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 63 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual IV iv Calculation Index value Optical Density of Sample the mean Optical Density of the Cut Off Calibrators v
73. er Technologist s initials click on Don t know Password 4 The machine will start Initialization and check all instrument functions The results of this check are then displayed on the screen 5 Check DH20 and Buffer level by opening the bottom part of the Analyzer 6 Perform Washer Assay every morning see EBV testing page 21 step 6 7 Perform Validation Assay every Friday see EBV testing page 21 step7 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 4 TML MSH Microbiology Department Policy MI SER 01 v01 Page 3 of 5 Policy amp Procedure Manual Serology Manual 8 Performing testing a b c d OBTA Remove Reagent Rack 5 and re load into analyzer Remove Reagent Rack I and re load Reagent Rack I into Analyzer Remove Control Rack R and re load into Analyzer Load 1 rack of specimens 1 20 into Analyzer with bar codes facing the Bar code reader Do the same for the subsequent rack s 21 40 41 60 Patient Editor will appear with the specimen s Check that specimen s are correct If there is a blank space click on it and enter the manually use TAB to move to another row Click on the column header highlight C diff toxin Click on the first space and hold then drag the mouse down the whole column A V check mark will appear beside each specimen Click Close to save
74. erature checks on lysing heater priming heater warming heater and instrument in System Maintenance Log every time the test is performed Clean replace Air Filter and verify BD ProbeTec ET temperature monthly Wipe checks for Analyser Bench Lysing rack Pipette Priming heater Lysing heater are done every two weeks see appendix Reference Package insert for BD ProbeTec ET CT GC Amplified DNA Assay Becton Dickinson and Company 7 Loveton Circle Sparks MD 21152 USA PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 71 TML MSH Microbiology Department Policy MI SER 14 v02 Page of 5 Policy amp Procedure Manual Section Serology Manual Subject Title Molecular Testing HBV DNA Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date October 10 2003 Il HI HEPATITIS B HBV DNA Introduction The COBAS Amplicor HBV Monitor Test is an in vitro nucleic acid amplification test for the quantitation of Hepatitis B virus DNA in human serum or plasma on the COBAS AMPLICOR Analyser Specimen Collection and Processing 10 ml of blood is collected in a sterile red topped or lavender topped EDTA tube and spun at 3000 rpm for 15 minutes Serum plasma is separated into two storage tubes and put in HBV DNA box in 20 C freezer Procedure Pre PCR Specimen Preparation Use eit
75. ernal control Accurun 156 with each new lot When opening a new kit record the lot number needle check and external control results in the reagent lot binder Refer to a senior technologist if control results are outside of limits or for any other problems with running or reporting the assay CAP provides external proficiency testing Reference Manufacturer s package insert RPR Card Test NCS Diagnostics Inc 130 Matheson Blvd E Mississauga Ontario L4X 1Y6 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 51 TML MSH Microbiology Department Policy MI SER 10 v02 Page 1 of 4 Policy amp Procedure Manual Section Serology Manual Subject Title Varicella Zoster Virus IgG VIDAS System Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date May 27 2003 II II VARICELLA ZOSTER VIRUS IgG Introduction Varicella Zoster IgG assays is used for evaluating patient s immune status to Varicella Zoster viruses infection The VIDAS is an enzyme linked fluorescent immunoassay ELFA utilizing a virus coated Solid Phase Receptacle SPR to which antibody in serum binds Anti Human IgG conjugated with Alkaline Phosphatase reacts with substrate 4 Methylumbelliferyl Phosphate to form a fluorescent product All necessary reagents and test serum are contained in the VZG reagent strips Specimen Collecti
76. est Schedule Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date March 19 2002 APPENDIX I SEROLOGY TEST SCHEDULE BE WSU yas aac cace den Sues tees ceded ee e E cca dace He Foca ea STG A Thursday EBS th aes Saosin eset sere Gs tah ada Sag Senet aac ae lat Sotoae acon al anced tetas E daily JE UB yA WeMTel0 0NSIBONT 1NL0 0 epee te Manat ter an nan E OD tree inn Sh Ne rien Seren ere en npn when needed FAIS SAND is s35 ances tees tha delice saa hee a Maal yaaa sian sayaweh ta Saale are Saale tha sban a aa daily PUBCA B Total sais ah ssacexssasaneassususansse ai nE a E a VEEE K ANEETA ENTA ss E aOR Enana Nar aeaaea Sanaa daily HBCAb eM oessicurinnaeunui g aas when needed HAV M a A A R a ae lag da E R a daily HBAS H BEA Dreonia e AE eet E E ENA RRR daily Monospot IM heterophile Ab gssascuiel ec ccesaeasaumaieedatidenasaeaaligaces Macey laleces eeteotae A aan tedaee same day RA oye a eee ne arr arc Oe Ia ana RPEN Pee a PRESS ONE A VEN CTE MRT ER SUE RITES PPE daily PV ALCS VNR Z OUT PD sh eee ela cal ge care aa ea a a fo te crate aot STAT or Thursday 454 22 FA for Syphilis soss me eN ire nS et a oe nO Rn NOE eT eT Oe Wednesday HEVA bindas anaa ilies ula ical Atlas a iat cists ht ted et fait daily CMV IGAD Jace ceaaseuceat gate etss te aa a aE E ENEE T E O ETE a ASENA EES aS e daily VA a A E aae E R EAA daily HTEV A o ere ee ene eS O rE T A O Thursday Chlamydia PER ase eters eee a caged a we ga
77. he following procedure to store the program Storing the program 1 Press the OPTION key to move the cursor beneath STORE then press ENTER The following display appears Store Enter program x The number x is the next available number If this is the first file being stored 66 99 x would be a 1 on the display Up to 150 programs can be stored PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 101 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 17 04 v01 Page 4 of 5 2 Press ENTER to store the method under the displayed number OR To use a different number type the number 1 150 then press ENTER The following display appears Store Protect program NO The above display gives you the option of protecting the file under a user I D By doing so the file cannot be overwritten or deleted unless the user number is entered Select YES or NO by pressing the OPTION key then press ENTER Always selects NO unless if you want to keep it forever and will be under your I D number CYCLE The Cycle Program A CYCL program contains the thermal ramp and hold segment patterns for PCR cycling It contains up to nine set points Each set point comprises a ramp segment and a hold segment for the specific target temperature The pattern can be repeated up to 99 t
78. he required of strips Open bag containing strips when strips reaches room temperature 10 15 minutes Return the unused strips to the bag and refrigerate immediately Change bottle from D H20 to Wash Buffer Turn Washer ON Press button under Prime F2 Press button under washing F1 Select Program Press memory F1 press Number F1 For dispensing 350 ul of buffer use Program 2 Program No press 2 enter 002 Name 1 should appear Press Set at this point press Change F2 then press Enter until Number of strips to wash appears check that of strip appears is What is needed If it is the same press Escape If is not the same enter the right of strip and press enter until 002 Name 1 appears press Enter i Press Run F1 and press No F2 under Prime j The Washer will dispense the 350 ml of buffer into each well and soak for 5 minutes After 5 minutes Aspirate Wash Buffer from wells For aspiration of Wash Buffer use Program 1 lt a Select Program Press memory F1 press Number F1 a Program No press l enter b 001 Name 01 should appear c Press Set at this point press Change F2 then press Enter until Number of strips to wash appears check that of strip appears is what is needed If it is the same press enter If is not the same enter the
79. her pre diluted specimens See Specimen Dilution Protocol to follow or if using frozen serum or plasma specimens thaw the specimens at room temperature and vortex for at least 5 seconds Label one 1 5 ml screw cap tube for each specimen to be processed including one tube for the Negative Control one tube for the Low Control and one tube for the High Control Place a pellet orientation mark on each tube Vortex HBM LYS 1 for 5 seconds Add 50ul of HBM LYS 1 to each of the tubes using a Repeater Pipette and 1 25 ml Combitip Reservoir or a micropipette with a plugged tip Vortex NHP for 5 seconds To each of the three control tubes containing HBM LYS 1 add 100ul of NHP Vortex for at least 10 seconds To each of the patient tubes containing HBM LYS 1 add 100uL of patient serum or plasma and vortex for at least 5 10 seconds PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 72 TML MSH Microbiology Department Policy MI SER 14 v02 Page 2 of 5 Policy amp Procedure Manual Serology Manual 6 10 11 12 13 14 15 16 Place tubes in the microcentrifuge with the orientation mark facing outward Centrifuge tubes for 5 minutes at 12 500 16 000 x g at room temperature Remove and discard the supernatant using a new fine tip disposable transfer pipette or an aerosol resistant tip for each tube Remove as much liquid a
80. ick OK Are you sure that the resources have been loaded in the correct positions Click Yes Load Plate load a clean microtiter plate into a metal tray carrier Open the door on then far right and load the carrier into Behring 2000 the two pins on the right side of the carrier should fit into the two rings in the Behring 2000 Close door Click OK Are you sure plate layout correct click Yes Result already exist Replace click Yes When finished Remove plate and carrier from system remove plate and visually check strips 1 6 for equally distributed liquid Strips 7 12 for complete aspiration click OK Discard buffer in sink and rinse plate wit DH20O air dried 7 Perform Validation Assay every Friday a b moe mo Ao Remove Reagent Rack 5 and re load into analyzer Remove Reagent Rack I Open lids and stoppers from Reference solution A B and C and Load into Rack 1 Load Reagent Rack I into Analyser Remove Control Rack R and re load into Analyser Click on New Worklist Click on Add Plate Click on Add Assay Highlight DB Validation asy click on Open Set Up Panel click OK PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 38 TML MSH Microbiology Department Policy MI SER 06 v01 Page 3 of 5 Policy amp Procedure Manu
81. icles TO BE MADE AFTER ALL OTHER REAGENTS AND DISPOSABLES ARE ON THE MAGNA PURE this reagent needs to be mixed well by inversion then pipet exact amount into medium 20 tub letting it drain well into tub put in reagent tray click on image on screen e Elution buffer invert bottle to mix pour amount noted on screen into medium 20 tub put lid onto tub with points facing down put into metal tray in correct position colour coded click on image on screen e Buffer 2 as wash buffer 1 e Place reagent tray on MagNA Pure in designated position left side in machine lock bar needs to be opened in order to place reagent tray and tips in next step Load tips processing cartridges tip stands and elution and storage cartridges as per screen and click on each image as you complete task Check and change liquid waste bottle Click on image on screen Check and change waste bag if needed Click on image on screen 13 Click on image of drop catcher this is changed weekly see weekly maintenance for procedure Click on sample cartridge image and confirm barcode MGP magnetic glass particles TO BE MADE NOW AND PLACED IN REAGENT TRAY ALREADY ON MAGNA PURE NOTE LAST STEPS ARE TO PEEL OFF PLASTIC COVER FROM SAMPLE CARTRIDGE CLOSE LOCKING BAR OVER REAGENT TRAY AND TIPS AND TAKE OFF LIDS TO BOTH MASTER MIX VIALS PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 79
82. icrobiology Department Policy MI SER 05 v01 Page 6 of 7 Policy amp Procedure Manual Serology Manual 7 Calculate the Mean Absorbance of the HIV 2 Positive Control HIV 2 PCX Sum of the absorbance values of the two HIV 2 Positive controls divided by two TheHIV 2 PCX must be Positive gt or to 0 700 Each individual absorbance must be within the range of 0 65 to 1 35 times the HIV 2 PCX No Positive Control absorbance value may be discarded 8 Cut off value NCx negative control mean 0 240 If any control fails the test is invalid Should inform Senior Charge technologists and repeat testing v vi Interpretation of Results Absorbance value is lt cut off reported as Negative Absorbance value is gt or cut off send to PHL for confirmation Quality Control Two Positive Controls two Low Positive Controls and three Negative Controls must be included with each HBsAg run Two HIV 1 Positive Controls two HIV 2 Positive controls and three Negative Controls must be included with each HIV1 2 Run Run Accurun 5100 as external control for both HBsAg and HIV with each new lot Result entered in Reagent Lot Binder All positive specimens are sent to PHL for confirmation PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 35 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 05 v01 Serology
83. if needed always change year first Then do the same for HIV Click OK Click on last square Start Session Save document using the new file will appear click OK Profile vial locations for controls or standards and reagents will appear Right click on each colored position will show where to load each diluent controls conjugate substrate and stop solution Click continue and wait for instrument initialization Position Reagent amp Controls R3 HIV Sample Diluent R4 HIV Working Conjugate R5 HBsAg Working Conjugate R6 Working Chromogen R7 Stop Solution 2 Diluted HIV 1 Positive Control 4 Diluted HIV 2 Positive Control 6 Diluted HIV Negative Control 7 HBsAg high Positive Control 8 HBsAg Low Positive Control 9 HBsAg Negative Control Prepare Fresh same Day Working Conjugate as follows always make 1 extra strip Number of Strips 40X conjugate ul Conjugate Diluent ml 1 10 1 0 2 20 2 0 3 30 3 0 4 40 4 0 5 50 5 0 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 32 TML MSH Microbiology Department Policy MI SER 05 v01 Page 4 of 7 Policy amp Procedure Manual Serology Manual Prepare Fresh Same Day Working Chromogen as follows always make extra strip Number of Strips Chromogen Reagent ul Chromogen Diluent ml 1 10 1 0 2 20 2 0 3 30 3 0 4 40 4 0 5 50 5 0 11 Click on 2 nd square from the lef
84. imes Typically there are two or three set points ina CYCL program A complete cycle typically required less than two to four minutes The Displaying in a CYCL Program example for displays CYCL program 3 Temperature PCR Setpt 1 Ramp 0 00 94 0C Hold 0 30 Setpt 2 Ramp 0 00 55 0C Hold 0 30 Setpt 3 Ramp 0 00 72 0C Hold 0 30 Total cycles 35 Pause during run NO PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 102 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 17 04 v01 Serology Manual Page 5 of 5 Adjust your program temperature and number of cycles according your experiment protocol by following the table above Store your programs as usual under the same number you create PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 103 TML MSH Microbiology Department Policy MI SER 17 05 v01 Page of 3 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix V Entering Serology Refer Out Results Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date January 15 2004 APPENDIX V ENTERING SEROLOGY REFER OUT RESULTS Refer out test results for blood specimens can only be recorded in the LIS using the SCC lab mo
85. initial step to both control the isolation procedure and to check for PCR inhibition The LightCycler simultaneously monitors the two different amplicons using two fluorimeter channels 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 4 C If a delay of more than 6 hours is anticipated the CSF specimen should be frozen at 70 C 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 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 84 TML MSH Microbiology Department Policy MI SER 16 v01 Page 2 of 10 Policy amp Procedure Manual Serology Manual HI Procedure A Worklist Preparation a b p C ro mPoaRaogTp Log on to Softmic Select type i Result ii Worklist iii Pagedown to 86 WNV PCR iv Enter v F12 Pending samples plasma serum or CSF will appear on Worklist Mark received samples using the F5 key Type to print Worklist The numbers on the left column
86. ion 2 0 by Roche Diagnostics PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 83 TML MSH Microbiology Department Policy MI SER 16 v01 Page of 10 Policy amp Procedure Manual Section Serology Manual Subject Title Molecular Testing West Nile Virus RT PCR Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date October 10 2003 WEST NILE VIRUS RT PCR I Introduction I 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 RealArt WNV Reverse Transcription RT PCR Kit The amplification and detection take place simultaneously continually and in real time in the Roche LightCycler 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 Artus WNV assay two fluorescent dyes are incorporated in the master mix to detect two amplified products One for the 80 bb WNV amplicon and the other for the Internal Control IC The IC is added to each specimen in the
87. is only stable for 4 hours once made and prepared A ring s must be placed in Cobas before this time expired 4 Make working lysis buffer in a 15 ml conical centrifuge tube Vortex the HCV IC from HCV specimen prep kit for 5 10 s For 24 samples including controls add 162 ul HCV IC to 7800 ul Lysis Binding Buffer vial 4 green cap from MagNA Pure kit don t touch sides of tube when adding the HCV IC Invert gently to mix 5 For each sample and control pipet 300 ul of working lysis reagent with repeat pipettor into 24 vials of the sample cartridge Mark last two vials as negative and positive controls respectively 6 Preparation of controls a Prior to use vortex the NHP HCV control and HCV control for 5 10 s b Add 200 ul NHP to each of the two corresponding control vials of the sample cartridge containing the working lysis reagent and mix up and down with pipette c Add 20 ul HCV control to the vial marked with HCV and mix up and down with pipette d Add 20 ul HCV control to the vial marked with HCV and mix up and down with pipette 7 For each sample to be extracted pipet 200 ul of sample material into the corresponding vial in the sample cartridge containing the working lysis reagent and mix up and down with pipette Seal plate and place onto MagNA Pure Remember to take off seal later 8 At MagNA Pure computer screen e pick A ring ordering Barcode order numbers or enter them
88. lamydia trachomatis by Nucleic Acid Amplification CHLN Negative for Chlamydia trachomatis by Nucleic Acid Amplification GCPO POSITIVE for Neisseria gonorrhoeae by Nucleic Acid Amplification GCNE Negative for Neisseria gonorrhoeae by Nucleic Acid Amplification TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT PROCEDURE MANUAL Page 113 TML MSH Microbiology Department Policy MI VIR 17 09 v02 Page 1 of 1 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix IX Shipment of Parvovirus PCR to HSC Issued by LABORATORY MANAGER Original Date Dec 20 2003 Approved by Laboratory Director Revision Date APPENDIX IX SHIPMENT OF PARVOVIRUS PCR TO HSC Fill up a HSC requisition 2 Put specimen and requisition in biohazard bag and then in a brown paper bag 3 Put Hospital for Sick Children Microbiology Receiving 3 Floor Atrium Rm3676 sticker on brown bag 4 Put labeled brown bag into blue specimen container and send to TG specimen management 5 Enter in LIS as specimen send to HSC and finalize 6 Also enter in serology send out binder PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 114 TML MSH Microbiology Department Policy MI VIR 17 10 v02 Page 1 of 1 Policy amp Procedure Manual Section Serology Manual Subject Title
89. ls ate a a a r aR daily HEV RNA PCER sateen tale seit ea et a eee anc castles a once or twice per week EBV DIAG cn casei a T EE E A E avanas Ranind sides Gee ETE Daily PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 94 TML MSH Microbiology Department Policy MI SER 17 01 v02_ Page 2 of 2 Policy amp Procedure Manual Serology Manual Stat testing may be required where clinically justified All routine pre Bone Marrow Transplant serology screening will be completed by Friday afternoon weekly except RPR for Syphilis for samples received Thurs pm or Fri Friday afternoon print an LIS report for 8TSC template For all BMT patients donors appearing on the 8TSC printout check that they were faxed successfully To maintain confidentiality results for HIV testing will be reported using Transplant Screen These results will be printed in lab and sent to the PMH BMT office daily by hospital mail in an envelope marked CONFIDENTIAL FAX number 926 6585 Occasionally a pre BMT serology screen will be STAT The microbiologist will inform the laboratory in addition to the requisition being clearly marked STAT Such specimens will be processed within 24 hours of receipt and results will be FAXed back to the BMT office as soon as they are available PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 95 TML MSH Micr
90. manually Select protocol and choose total NA and then total NA external lysis blk Select post elution protocol and choose HCV Cobas setup pep Change lysed sample volume to 350ul Change elution volume to 65ul Click on Liquid Waste Discard square this will remove liquid at end of cycle before we discard them from cartridges e Click on stage setup PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 78 TML MSH Microbiology Department Policy MI SER 15 v04 Page 3 of 7 Policy amp Procedure Manual Serology Manual 9 10 11 12 14 15 16 Load reagents according to MagNA Pure screen e Wash buffer 1 invert bottle to mix pour amount noted on screen into large tub put lid onto tub with points facing down put into metal tray in correct position color coded click image on MagNA Pure screen e Wash buffer 3 as wash buffer 1 e Proteinase K reconstitute by first adding 3 ml elution buffer mix by inversion then add another 2 ml of elution buffer mix by inversion pour amount noted on screen into medium 20 tub put lid onto tub with points facing down put into metal tray in correct position color coded click on image on screen refrigerate unused portion immediately put date on lid fresh aliquot must be used each day when test is run e MGP magnetic glass part
91. n the dirty filter under running tap water in the opposite direction of the airflow arrows Shake off excess water and air dried Select Maintenance from Main Menu Select PRIME AND FLUSH Select F6 Raise probes Open the sampling Pipette Cover Cleaning Sampling Probe Moisten a cotton swab with Deionized H20 Wipe the Sampling probe several times Moisten a cotton swab with 95 Ethanol Wipe the Sampling probe several times Moisten a cotton swab with Deionized H20 Wipe the Sampling probe several times Visually check the probe for damage Cleaning Processing Probe Moisten a cotton swab with Deionized H20 Wipe the Processing probe several times Moisten a cotton swab with 95 Ethanol Wipe the Processing probe several times Moisten a cotton swab with Deionized H20 Wipe the Processing probe several times Visually check the probe for damage Cleaning Sampling Wash Station Remove the Wash Station Rinse the Wash Station with Deionized HO Rinse the Wash Station with 95 Ethanol Rinse the Wash Station with Deionized H20 Clean the inside and outside of the Wash Station with a cotton swab moistened with Deionized H20 to remove any salt buildup Reinstall the Wash Station PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 26 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 04 v01 Page 7 of 9
92. ng Master Mix using a micropipettor with a plugged tip Do not transfer any precipitated material Cap the tubes after each sample addition 19 Transfer A rings to COBAS AMPLICOR Amplification on the COBAS AMPLICOR analyzer must be started within 1 hour of the time processed specimens and controls are added to the A tubes containing Working Master Mix The remainder of the processed specimens may be frozen at 20 to 80 C for up to one month Specimen Dilution Protocol If a quantitation value is required for specimens containing high levels of HBV DNA gt 200 000 copies ml then a set of serial dilutions should be performed For this dilution you will need a new disposable microwell plate and Basematrix this is stored in aliquots in freezer If using frozen serum or plasma specimens thaw the specimens to be diluted at room temperature and vortex for at least 10 seconds PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 74 TML MSH Microbiology Department Policy MI SER 14 v02 Page 4 of 5 Policy amp Procedure Manual Serology Manual a IV Add 150 ul of each specimen to a well in the first row of a new disposable microwell plate Row A Add 225 ul of Basematrix to the next 6 wells below each specimen to be diluted Rows B through G Using a 25 ul pipet remove 25 ul of the undiluted specimens located in Row A and dispense into the first
93. nism in the specimen MOTA Score for CT or GC CT NG Positive Control gt or 2000 CT NG Negative Control lt 2000 If any of these two controls fails the test is invalid Should inform Senior Charge technologists and repeat testing PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 70 TML MSH Microbiology Department Policy MI SER 13 v02 Page 6 of 6 Policy amp Procedure Manual Serology Manual V VI VIL VIII Interpretation of Results MOTA score for CT NG is gt or 2000 is considered as Positive reported as Positive for Chlamydia trachomatis Neisseria gonorhoeae by Nucleic Acid Amplification MOTA Score for CT NG is lt 2000 is considered as Negative reported as Negative for Chlamydia trachomatis Neisseria gonorhoeae by Nucleic Acid Amplification Reporting Positive report Positive for Chlamydia trachomatis by Nucleic Acid Amplification Positive for Neisseria gonorhoeae by Nucleic Acid Amplification Negative report Negative for Chlamydia trachomatis by Nucleic Acid Amplification Negative for Neisseria gonorhoeae by Nucleic Acid Amplification Quality Control One Positive Control and one Negative Control must be included in each run Run external control Accurun 341 every second week Result filed in Reagent Lot Binder External proficiency testing is provided by CAP and QMP LS Maintenance QC Enter temp
94. nu Select CALS AND CHECKS Select OPTICS Select MEIA VERIFICATION Remove Matrix Cell Hopper Select CONTINUE Follow instructions on screen Place the Standard Hopper into position Insert the Standard into the Standard Hopper Select CONTINUE to begin the MEIA Verification This will take 10 minutes When finished select OK to acknowledge completion of the verification Press PRINT on the keyboard Retrieve the Standard by pressing the white MEIA Standard Release lever Open the MEIA Optical Retrieval Door and remove the Standard Store it upside down in the black box Reinstall Matrix Cell Hopper Exit to the MAIN MENU MAINTENANCE OPTICS FPIA Verification Select FPIA VERIFICATION Follow instruction on the screen select Continue Press down the back of the clip on top of the FPIA Optical Standard Load the Standard into the Reaction Vessel Carousel at the Load Station Press on the front of the clip to provide a flat surface Select Continue to begin the procedure This will take 20 minutes When finished select OK to acknowledge completion of the verification Press PRINT on the keyboard Press down on the back of the clip on top of the FPIA Optical Standard Lift and remove the Standard store in assigned place in the black box Exit to Main Menu Cleaning Segments and Sample Cup Adapters Use disinfectant wipes to clean segment and Sample Cup Adapters PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES
95. nual Serology Manual Indeterminate results may also be those with no signal for the WNV amplicon Channel F1 and also no signal for the IC amplicon Channel F3 back F1 indicating possible PCR inhibition The test must be repeated Negative results for the WNV 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 RealArt WNV assay Artus Biotech Inc Indeterminate Report Indeterminate by RT PCR This is a research test performed by using RealArt WNV assay Artus Biotech Inc Positive Report POSITIVE by RT PCR This is a research test performed by using RealArt WNV assay Artus Biotech Inc Phone all Report positive results and inform senior charge technologist Record isolate in LIS by F7 as 77wnv Quality Control Equipment QC Every six months 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 fou
96. obiology Department Policy amp Procedure Manual Policy MI SER 17 02 v01 Page 1 of 2 Section Serology Manual Subject Title Appendix IT Send Outs Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date September 20 2001 APPENDIX II List of Tests Referred Out to Other Laboratories TEST 1 Aspergillus precipitins 2 Chlamydial culture 3 Mycoplasma culture 4 Mycoplasma pneumoniae Ab 5 Genital Mycoplasma Ureaplasma culture 6 Viral antibodies 7 Legionella tissue Legionella urine antigen 8 Lyme disease Ab 9 Fungal antibodies 10 Diphtheria antitoxin toxin 11 Tetanus antitoxin titre 12 Electron microscopy 13 VDRL serum or CSF 14 Amoebic serology REQUISITION PHL General PHL General PHL General PHL General PHL General PHL General PHL General PHL General PHL General PHL General Reference Bacteriology Reference Bacteriology PHL General PHL General PHL form 97 44 08 99 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 96 DESTINATION Serology PHL Virology PHL Bacteriology PHL Virology PHL Mycoplasma PHL Virology PHL Bacteriology PHL Serology Serology PHL Serology PHL Special Bacteriology PHL Special Bact PHL Virology PHL Serology PHL Serology PHL TML MSH Microbiolog
97. obiology Department Policy MI SER 11 v03 Page 1 of 5 Policy amp Procedure Manual Section Serology Manual Subject Title West Nile Flavivirus IgG Issued by LABORATORY MANAGER Original Date April 25 2001 Approved by Laboratory Director Revision Date May 27 2003 Il HI i a9 ii WEST NILE FLAVIVIRUS IgG Introduction The Focus Technologies Flavivirus West Nile ELISA IgG is intended for qualitatively detecting IgG antibodies to flaviviruses West Nile in human serum In conjunction with the Focus Technologies Flavivirus West Nile ELISA IgM Capture ELISA the test is indicated for testing persons having symptoms of arbovirus infection as an aid in the presumptive diagnosis of flavivirus infection Specimen Collection and Processing 5 ml of blood is collected in a serum separator tube and separated by centrifugation The serum is removed from the clot and refrigerated until testing Specimens are stored at 70 C after testing Procedure Reagent Preparation Wash Buffer 100 ml 10X Prepare working buffer 1X by adding 100 ml of 10X Wash Buffer to 900 ml of Distilled H2O Mix well Labeled and dated Controls 1 vial of Positive Control 1 vial of Negative Control 1 vial of Cut off Calibrator IgG Conjugate Substrate Reagent 2 vials of Tetramethylbenzidine TMB and Hydrogen Peroxide in buffer Stop Reagent 1 vial of 1M sulfuric acid Other Materials IgG Antigen
98. ocedure Manual Serology Manual ix Type in the referred out test s x Press F12 F12 xi Yes to confirm Continue from steps g o below if referred out tests are already ordered and results are ready to be entered Press F8 to enter refer out results Select the appropriate test and enter the result from the keypad If no keypad appears type in TO PHL or TO HSC as appropriate in the result field only 9 characters allowed Delete the canned message MOHR This sample has been referred to but leave the referred out tests names Free text the results by opening the text window by pressing in the result field and type in the results after each referred out test name After the last result enter the reporting date reference lab number either by free text or by upressing F5 Canned message and select 31 9rep Space enter date that PHL reported the result Arrow up wand PHL bar code or enter PHL lab number F12 F12 Confirm modification Y Record the Clinic Dr MRN and order number on the reference lab report if not already recorded n Give reports to senior charge technologist to verify oO Forward reports to Report Controllers for photocopying and mailing Alternate Method for non free text specific refer out tests continued from steps a f above p Specific non free text refer out tests as can be ordered as follows F1 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES
99. of the Worklist will be used to label the 1 5 mL Eppendoff microcentrifuge tubes and the Qiagen Isolation Columns LIS Label Printing if needed for the RNA eluate While still in the Worklist press Enter to go to the Test Screen Press F1 to move curser to Demographic field Press to Order Entry Press No to confirm editing Press F9 Press Print Labels Press No modify record Select label printer for the LIS label Specimens Processing Specimens should be processed as soon as possible after arriving in virology laboratory a EDTA blood The blood should be centrifuged and an aliquot of about 2 mL plasma frozen unless PCR can be performed immediately CSF An aliquot 0 2 2 mL of the CSF should be frozen unless PCR can be performed immediately PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 85 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 16 v01 Page 3 of 10 Serology Manual After processing an aliquot of the left over specimens should be stored frozen at 70 C D 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 WNV LC PCR Microcentrifuge MICT14 1 5 mL microc
100. ologist of any QC failure VII Reference Manufacture s insert Focus Technologies Cypress California 90630 U S A 2003 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 65 TML MSH Microbiology Department Policy MI SER 13 v02 Page of 6 Policy amp Procedure Manual Section Serology Manual Subject Title Molecular Testing Chlamydia Trachomatis amp Neisseria gonorrhoeae Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date May 26 2003 I CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAE Introduction Chlamydia trachomatis CT and Neisseria gonorrhoeae GC testing are performed using BD ProbeTec ET System The assay uses a Strand Displacement Amplification SDA technology for the direct quantitative detection of CT and GC DNA in endocervical swabs male urethral swabs and urine specimens Specimen Collection and Processing Use Culturette Direct Swab for collecting endocervical specimen and Mini Tip Culturette for urethral specimen The swabs can be stored between 4 to 6 days at 2 C to 27 C before testing 15 20 ml of the first voided urine can be collected in a preservative free sterile container The specimen should be forwarded to the Virology laboratory as soon as possible Upon arrival in the laboratory a UPP Urine Processing pouch is added to the specimen for at
101. om the reservoir to the capillary making sure that the engraved numbers on the adaptors correspond to the correct sample Place the adaptors and capillaries back in the Cooling Block and proceed to the LightCycler In the LightCycler room Turn LightCycler and the computer on Click on the LightCycler icon and click run Select selftest skip if already done within 24 hr or Press OK Select Run Experimental File WNYV protocol Type in file name WNV run number year month date Select run Type in the number of samples to run including QS ext QC and H20 Click Edit Sample Later Type in the lab numbers QS ext QC if any and H2O PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 89 TML MSH Microbiology Department Policy MI SER 16 v01 Page 7 of 10 Policy amp Procedure Manual Serology Manual k 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 l Press done m To Print Results go to FLUORESCENCE and click e Fl e Quantification e Print Window m To Print Internal Control go to FLUORESCENCE and click e F3 BACK FI e Print Window PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 90 TML MSH Microbiology Department Policy MI SER
102. on and Processing Blood 5 mL is collected in a serum separator tube and separated by centrifugation The serum is removed to a vial and refrigerated until testing Specimens are stored at 20 C after testing Procedure Reagents VIDAS Varicella Zoster IgG test kit VZG Reagent Strips VZG SPRs Standard Positive Control Negative Control Other materials Pipettor 100 uL Method 1 Bring test kit and serum samples to room temperature Check that lot number of kit matches lot currently in use as posted on VIDAS instrument If lot number does not match ensure that all kits test strips for the posted lot number have been used PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 52 TML MSH Microbiology Department Policy MI SER 10 v02 Page 2 of 4 Policy amp Procedure Manual Serology Manual Enter new lot data by inserting bar code card found in kit into instrument using 7 Calibration Menu 2 Read Master Lot Entry Card from Instrument Enter section to read data from enter section letter Post card of new lot currently in use on VIDAS along with the date 2 Label MSG VZG strips as follows s Standard s Standard C1 Positive Control C2 Negative Control 5 6 etc up to 30 3 Pipette 100 uL of Standard Control or serum into the specimen well of the corresponding VZG strips Check for adequate sample level and remove any bubbles 4
103. pendorf microcentrifuge Load onto Behring 2000 Analyser sample rack according to worklist for testing Procedure Behring 2000 Analyzer i Reagent Preparation 1 Prepare Behring Wash Solution as needed e g 100 ml of Wash Solution 1900 ml of distilled HO Store at 4 C Fill up Wash Solution in Behring 2000 when needed 2 Prepare C difficle working buffer 50 ml of 20X Wash Buffer Concentrate 950 ml of D H20 Store at 4 C Fill up C difficile working buffer in Behring 2000 Analyser when needed PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 3 TML MSH Microbiology Department Policy MI SER 01 v01 Page 2 of 5 Policy amp Procedure Manual Serology Manual 3 Pour Conjugate into Behring medium sized container and labeled as Conjugate Pour Substrate into Behring large sized container and labeled as Substrate Pour Stop Solution into Behring medium sized container and labeled as Stop Solution All these reagents can be stored in the respective containers in 4 C refrigerator Use new containers for each new Lot Bring microwell pouch and the above mentioned containers to room temperature before use 5 Empty waste change D H20 and clean control bottles replace with new controls every Friday ii Assay Method l Turn hard drive and Behring 2000 Analyser ON 2 Double click on Short Cut to BEP 2000 3 Ent
104. r Quantification Standards QS 1 2 3 and 4 must be run PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 92 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 16 v01 Serology Manual Page 10 of 10 Daily QCs Every run e Fach patient specimen must have an Internal Control IC added to monitor both isolation and PCR inhibition The sample must show a positive reading in either fluorimeter Channel F1 WNV amplicon or fluorimeter Channel F3 back F1 IC amplicon to be valid no PCR inhibition e A Positive Control eg Quantification Standard 3 is included and shows a positive reading in fluorimeter Channel F1 WNV amplicon e A Negative Control eg H20 spiked with IC is include and shows a negative reading in fluorimeter Channel F1 WNV amplicon 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 01 99 Qiagen RealArt WNV LC RT PCR Kit User Manual March 2003 Artus LightCycler Roche Diagnostics PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 93 TML MSH Microbiology Department Policy MI SER 17 01 v02 Page of 2 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix I Serology T
105. r example you can link a HOLD program to a CYCL program and conclude you experiment Access the main menu Select option RUN CREATE EDIT UTIL 9600 l Press the OPTION key to select CREATE then press ENTER the following display appear Create program HOLD CYCL AUTO METH 2 Press the OPTION key to select METH appears then press ENTER The following display Link progs 3 Type the program numbers of the programs you want linked After you type each number press ENTER For example to link programs 1 3 and 5 your display would look like this Link progs Dede Ors ee PROCE DURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 99 TML MSH Microbiology Department Policy MI SER 17 04 v01 Page 2 of 5 Policy amp Procedure Manual Serology Manual 4 Press STEP to move to the next display If you are linking seven or more programs in a method the following display appears This display let you link up to ten additional programs in the method NOTE You cannot link a method 5 Type the program numbers of the programs you want linked After you type each number press ENTER 6 Press STEP to move to the next display Meth 25 6 C RUN STORE PRINT HOME This display allows you to run store or print the method 7 Press OPTION key to move the c
106. r two master cal samples is automatically assigned Change the S P location if necessary Enter master cal lot number and expiry date number on box Make sure calibration type is Master Cal if not enter MASTER CAL by touchscreen Select F6 ADD Select F1 ABBOTT LOGO if no further request is needed Print the displayed orderlist using the PRINT KEY Dispense master calibrators into two sample cups AUSAB calibrators must be microcentrifuged for 10 minutes on maximum speed before using Check for bubbles in the samples and place the segments in the sample carousel Load the appropriate assay reagent in the reagent carousel Remember to open reagent bottle 4 on the reagent pack if present Press RUN Daily Clean up Procedure Ensure that all reagents have been removed and scanned Clean probe Weekly Maintenance MEIA amp FPIA Verification Clean sample segments and sample cup adaptors Clean matrix cell amp processing carousels Clean air filters Clean outside of probes Clean wash cups waste stations Clean dispenser nozzles and baseplate Flush pumps and syringes Weekly Archive patient results must be performed before 1 500 records Monthly Maintenance Tubing decontamination PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 11 TML MSH Microbiology Department Policy MI SER 02 v03 Page 5 of 9 Policy amp Procedure Manual
107. re use ii Other Materials Supplied with kit Test slides Paddle pipettes iii Method 1 Dispense 1 drop of the latex reagent onto a labelled oval ring of the test card 2 Add 1 drop 50 uL of patients s serum or control to the same ring PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 47 TML MSH Microbiology Department Policy MI SER 08 v01 Page 2 of 2 Policy amp Procedure Manual Serology Manual IV VI 3 Mix the latex reagent and serum together and spread to cover the entire area of the ring with the blade end of the paddle pipette 4 Immediately rotate the card on the serologic rotator at 100 rpm for 3 minutes 5 Observe for agglutination using a light source to aid in visualization iv Interpretation of Results Negative No agglutination Positive Any degree of agglutination Reporting Positive Report Infectious mononucleosis heterophile antibody POSITIVE Negative Report Infectious mononucleosis heterophile antibody NEGATIVE Quality Control Negative and positive controls must be included with each run and results and kit lot number recorded on the tasklist When opening a new kit record the lot number in the reagent lot number binder 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 Accurrun 31 with each new lo
108. right of strip and press enter until 001 Name 01 appears press Enter d Press Run F1 and press No F2 under Prime e The Washer will aspirate the buffer PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 62 TML MSH Microbiology Department Policy MI SER 12 v03 Page 3 of 5 Policy amp Proc edure Manual Serology Manual L pe ne pe m f End washing will appear when washing cycle is finished remove plate g Repeat Washing will appear press No F2 Add 100 ul of Blk to well 1 100 ul of Pos to well 2 100 ul of Neg to well 3 100 ul each of Cal to well 4 5 and 6 Add 100 ul of specimen to each well starting from well 7 Use last well as external Control well when needed Cover plate with sealing tape and incubate for 60 minutes at Room Temperature Wash the wells three times using the Automatic Washer under Program I Select Program Press memory F1 press Number F1 For washing and soaking use Program 11 Program No press 11 enter Program 11 Name IgM WN should appear Press Set at this point press Change F2 then press Enter until number of strips to wash appears check that of strip appears is what is needed If it is the same presses enter If is not the same enter the right of strip and press ent
109. row of Basematrix Row B to make a 1 10 dilution Mix up and down 10 times using the pipet Remove 25 ul of the 1 10 dilution from row B and dispense into Row C to make a 1 100 dilution Mix up and down 10 times using the pipet Repeat step 4 a total of four more times to make 1 1 000 1 10 000 1 100 000 and 1 1 000 000 dilutions using Rows D E F and G respectively Use the 1 1 000 000 Row G dilution to process and run in the assay Date and seal used rows with a plastic seal and place lid over the plate place in a zip lock bag and store at 2 8 C The remaining specimen dilutions may be used if necessary on a subsequent run if the initial processed specimen falls outside of the reportable range NOTE For specimens that have been pre diluted it will be necessary to multiply the copy number by the appropriate dilution factor to determine the final result Reporting Report results as the numeric value generated by the COBAS of copies ml by Roche Assay HBD DNA is reported for research purposes All Target OD LO and Result LO results should be reported as lt 200 copies ml by Roche Assay HBD DNA is reported for research purposes All values over 200 000 copies ml i e Result HI or QS INVALID should Be diluted once according the dilution protocol check with charge senior regarding appropriate dilution to repeat testing Then report the exact of copies ml plus dilution factor unless it is Resul
110. s Select OK f When finished exit to Main Menu m Archive new results of the week on Friday Weekly Maintenance O O ONEAN SA A Replace and cleaning Air Heater Inlet Filter Replace and cleaning Card Cage Air Filter Cleaning Sampling Probe Cleaning Processing Probe Cleaning Sampling Wash Station Cleaning Processing Wash Station Cleaning Dispenser Nozzles Flushing Pumps and Syringes Cleaning Processing Carousel Cleaning Matrix Cell Carousel MEIA Verification FPIA Verification Cleaning Segments and Sample Cup Adapters Remove Cell Hopper and lift Processing Center Cover 1 Replace and Cleaning Air Heater Inlet Filter The Air Heater Inlet Air Filter is located on the left hand side and near the back of the AXSYM Lift filter up and replace with a clean filter in the filter slot Clean the dirty filter under running tap water in the opposite direction of the airflow arrows Shake off excess water and air dried PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 25 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 04 v01 Page 6 of 9 Replace and Cleaning Card Cage Air Filter Open the Card Cage Access Panel located on the left side of the system Remove the Card Cage Filter Place the spare filter in the filter slot Install the Card Cage Access Panel Clea
111. s in Vista Issued by LABORATORY MANAGER Original Date Dec 20 2003 Approved by Laboratory Director Revision Date APPENDIX XI LOOKING UP PREVIOUS HEPATITIS RESULTS IN VISTA 1 Log on Vista 2 Enter user ID amp Password 3 Click on All UHN Patients 4 Enter MRN in Patient ID press Enter 5 Click on any visit then click on Goto Selected visit 6 Click on Chart Review Continue Click on y yes 7 Click on Hepatitis Profile click on OK 8 All previous Hepatitis test results will be displayed e g HBsAg HBsAb HBcAbIgM HBeAg HBeAb HC Ab and HAAbIgM PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 116 TML MSH Microbiology Department Policy MI VIR 17 12 v02 Page of 1 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix XII Printing of Pending List Issued by LABORATORY MANAGER Original Date Dec 20 2003 Approved by Laboratory Director Revision Date APPENDIX XII PRINTING OF PENDING LIST 1 Log on Lab 2 Enter ID amp Password 3 3 Results 4 View Enter Results by Sel Tests 5 Select tests by Template Enter 6 Enter 8SERO under Template Status pend nonver 7 From order enter last month s lab to leave it blank F12 8 F9 to prin
112. s possible without disturbing the pellet Withdraw the supernatant slowly allowing the liquid to drain off the sides of the tube Do not use vacuum aspiration Mix the HBV C by vortexing for 5 10 seconds Add 25uL of HBV C to the tube labeled HBV C Add 25uL of HBV C to each of the patient tubes Mix the HBV L C by vortexing for 5 10 seconds Add 25uL of HBV L C to the tube labeled HBV L C Mix the HBV H C by vortexing for 5 10 seconds Add 25uL of HBV H C to the tube labeled HBV H C Prepare Working Lysis 2 Reagent as follows vortex the HBM QS for 10 seconds Add 50uL of HBM QS to one tube HBM LYS 2 and mix well by vortexing for at least 15 seconds Add 100uL of Working Lysis 2 Reagent to all the tubes and mix well by vortexing 15 seconds The pellet does not always dissolve completely but this will not interfere with assay performance Incubate all tubes for 60 minutes at 60 C 2 C Pulse spin tubes for 5 seconds to remove condensate from cap Add 100uL of HBM LYS 3 to each tube and mix well by vortexing for at least 10 seconds Incubate all tubes for 10 minutes at 100 C 2 C Place tubes in the microcentrifuge with the orientation mark facing outward Centrifuge tubes for 15 minutes at 12 500 16 000 x g at room temperature to collect insoluble particles Amplify the processed specimens and controls within 1 hour of preparation or store frozen at 20 to 80 C for up to 1 month
113. se container with a little bit of Javex and then lots of H2O Wipe dry outside of container v Reattach the tubing by pressing the tubing assembly until it locks into place Close the Interior Waste Door and close the Waste and Supply Center Doors 2 Update Waste Levels Select Inventory from the Main Menu Select F4 Waste Select F3 Empty Waste Select F6 Save Exit to Main Menu aa a e Ff PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 23 TML MSH Microbiology Department Policy MI SER 04 v01 Page 4 of 9 Policy amp Procedure Manual Serology Manual 3 Update Matrix cells and Bulk Solutions a Matrix Cells i Visually check inventory level ii If level is around or less than 100 add a box by removing the plastic cover and the paper tabs in the middle of the box Place the box between the plastic walls at the top of the Matrix Cell Hopper Rest the box ends firmly on the roll bars and apply pressure to the center of the box The Matrix cells pour into the Hopper Make sure that no Matrix Cells lie sideways iii Update inventory Select F6 Save a oe b Bulk Solutions Exit to Main Menu Select Inventory in Main Menu Select F2 Matrix cell Select F3 Add a box i Open Waste and Supply Center Doors 1 Remove the bulk solution that you are replacing 2 Load the replacement solution bottle and screw on the cap 3 Close
114. sent PRESS RUN Assay Procedure for Control Sample From the MAIN MENU select ORDERLIST Select F5 control Select the assay for the control by touchscreen Type in the location of the control sample S P Select positive or negative control by touchscreen Select F6 ADD Continue for next control follow Select F1 EXIT Repeat control for next assay or Press ABBOTT LOGO TO MAIN MENU Review the displayed orderlist To print the orderlist select the PRINT KEY Load the control samples PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 9 TML MSH Microbiology Department Policy MI SER 02 v03 Page 3 of 9 Policy amp Procedure Manual Serology Manual 10 Check for bubbles in the sample and place the segments in the sample carousel 11 Load the appropriate assay reagents in reagent carousel Remember to open reagent bottle 4 on the reagent packs if present 12 Press RUN INDEX CALIBRATION HBsAg HBeAg HBeAb HBcAb IgM HBcAb Total HAV IgM Ab HCV Ab HIV 1 2 Index Calibration procedure requires only one sample cup Check required volume on printed orderlist 1 Order Cal from the MAIN MENU select ORDERLIST 2 Select F4 CAL 3 Select the assay to be calibrated using the touch screen 4 The S P is automatically assigned Change the S P location if necessary 5 Enter reagent lot number and expired date found on bottle
115. ser Remove Controls Rack R Load Anti EBV Reference P N into rack and re load into Analyser Load 1 rack of specimens 1 20 into Analyser with bar codes facing the Bar code reader Do the same for the subsequent rack s 21 40 41 60 Patient Editor will appear with the specimen s Check that specimen s are correct If there is a blank space click on it and enter the manually use TAB to move to another row Click on the column header highlight DBA EBV G Click on the first space and hold then drag the mouse down the whole column A check mark will appear beside each specimen Click Close to save If there is a next rack of specimens the next page of specimens will appear shortly Repeat step e PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 39 TML MSH Microbiology Department Policy MI SER 06 v01 Page 4 of 5 Policy amp Procedure Manual Serology Manual h Click on New Worklist i Click on beside Plate 1 EBV IgG appears j Click on folder beside EBV IgG k Plate format will also show the total of specimens to be tested and of strips needed Load EBV stripes onto the plate Load plate onto plate carrier l Click OK m Lot Specific Values For Plate 1 Check the Lot Click OK n Click on the green Initialization will start immediately o
116. t Maintenance Click on Start to start up Self Test Machine will do all checks then asks Print Self Test report click on Yes Staple printout with day list 12 Click on Daily Maintenance Tab click on Fill syringes check for bubbles in syringes and if no bubbles present click gt No 13 Click on Fill lung screen will ask you to open lid Open lid and click on OK Water level should fill up to just under the lower black line Click 1 2 times to Add 250ul of water each time to raise the level to just above the lower black line Close lid and click on OK 14 Click on Buffer 1 prime listen to the noise to make sure that the vacuum is working 15 Click on 3 rd square Tips if need to replace pipette box Click on 6th square from the left to execute the run will ask to put sample rack in click OK The P lab will start processing Click on Time chart to see what stages the testing is at 16 After assay is finished Session terminated will appear on screen click OK 17 Click on Maintenance and click on Endwork Will ask to fill in buffer 2 with DH20 open lid make sure there is enough DH20O in the bottle also change buffer 1 bottle to DH20 bottle Click OK The machine is rinsing the tubing with DH20 Next will ask to empty waste press on RED Button on the side of the machine and hold it until almost
117. t Choose either TC2RVIR or TC3R MIC printer to print 9 Look up each record and find out why the results are still pending PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 117 TML MSH Microbiology Department Policy MI VIR 17 13 v02 Page of 1 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix XIII Wipes Test for ProbeTec Issued by LABORATORY MANAGER Original Date Dec 20 2003 Approved by Laboratory Director Revision Date APPENDIX XII WIPES TEST FOR ProbeTec Wipes Test is done every two weeks 1 Dip swab in a tube of specimen diluent labeled as Analyser and swab surfaces of Analyser including the screen keyboard and all around the surface 2 Put swab back in specimen diluent tube and twirl around and remove swab Vortex well and treat this as a specimen 3 Do the same for a b c d e Bench Lysing rack Pipette Priming heater Lysing heater 4 Run all 6 swabs with specimen run 5 Record results in BDProbeTec ET Binder PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 118
118. t Result filed in Reagent Lot Binder If result is negative the run is invalid Inform Charge senior technologist and repeat testing CAP provides external proficiency testing References Manufacturer s package insert Meridian Diagnostics Inc 3471 River Hills Dr Cincinnati Ohio 45244 U S A 1 513 271 3700 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 48 TML MSH Microbiology Department Policy MI SER 09 v02 Page 1 of 3 Policy amp Procedure Manual Section Serology Manual Subject Title Rapid Plasma Reagin Test for Syphilis Screening Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date May 27 2003 Il HI SYPHILIS SCREENING Introduction The NCS Rapid Plasma Reagin RPR Card test is a macroscopic non treponemal flocculation test used to detect reagin antibodies Specimen Collection and Processing Blood is collected 5 mL for adult and 1 mL for neonate in a serum separator tube and separated by centrifugation The serum is removed to a tube and refridgerated until testing Specimens are stored in the refrigerator for 2 weeks after testing A request for VDRL on spinal fluid CSF or neonate blood will be sent to PHL for testing Procedure i Reagents RPR reagent kit NCS Diagnostics Inc ii Other Materials 3 mL dropper bottle Dispensing needle 17 uL drop R
119. t click on each colored position will show where to load each diluent controls conjugate substrate and stop solution Click continue and wait for instrument initialization PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 43 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 07 v01 Page 3 of 5 Serology Manual Position Reagent amp Controls R3 Sample Diluent R4 Conjugate RS Substrate R6 Stop Solution 1 Negative Control 2 Positive Control Prepare Fresh same Day Conjugate as follows always make 1 extra strip Number of Strips 40X conjugate ul Conjugate Diluent ml 1 30 LZ 2 50 2 0 3 80 2 9 4 100 3 9 5 130 4 9 6 150 5 9 7 180 6 8 Prepare Fresh Same Day Substrate as follows always make 1 extra strip Number of Strips TMB Reagent ml Peroxide Reagent ml 1 0 2 1 0 2 0 4 2 0 3 0 6 3 0 4 0 8 4 0 5 1 0 5 0 6 1 2 6 0 7 1 4 7 0 11 Click on 2 nd square from the left Maintenance Click on Start to start up Self Test Machine will do all checks then asks Print Self Test report click on Yes Staple printout with day list 12 Click on Daily Maintenance click on Fill syringes check for bubbles in syringes and if no bubbles present click No PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 44 TML M
120. t Hi again The Result Hi after dilution will be reported as gt 2xE10 copies ml by Roche Assay HBD DNA is reported for research purposes PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 75 TML MSH Microbiology Department Policy MI SER 14 v02 Page 5 of 5 Policy amp Procedure Manual Serology Manual vV Quality Control One HBV Monitor Negative control One HBV Monitor Low Positive control and one HBV Monitor High Positive control are included with each run Each result must be within the set value for each Lot otherwise the run is invalid Run Accurun HBV DNA 300 and 600 on each run Plot results in chart provided results has to be within 3 S D otherwise the run is invalid Inform charge senior technologist External proficiency testing is provided by LCDC VI Reference Package insert from COBAS AMPLICOR HBV MONITOR version 2 0 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 76 TML MSH Microbiology Department Policy MI SER 15 v04 Page 1 of 7 Policy amp Procedure Manual Section Serology Manual PCR Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date October 10 2003 Il HI HEPATITIS C RNA PCR Introduction To test for the presence of HCV RNA in serum or plasma using The COBAS
121. th test values less than 0 6 MEA VZ are considered negative IV Reporting Positive Varicella Zoster antibody Positive Equivocal Varicella Zoster antibody Equivocal Negative Varicella Zoster antibody Negative PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 54 TML MSH Microbiology Department Policy MI SER 10 v02 Page 4 of 4 Policy amp Procedure Manual Serology Manual V Quality Control Standard RFV must be greater than or equal to RFV range posted on VIDAS instrument Positive and Negative Control Test Values must be within ranges posted on VIDAS Quality Control ranges posted on VIDAS may change from lot to lot therefore it is essential that the lot number of the test kit in use corresponds to that of the posted values 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 pooled positive sera with each new lot Results filed in Reagent Lot Binder If result is negative the run is invalid Inform Charge senior technologist and repeat testing CAP provides external proficiency testing Reference Manufacturer s Package Insert VIDAS System Operational Manual bioMerieux Vitek Inc Missouri USA 64042 2395 PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 55 TML MSH Micr
122. toverification process Autoverification is the process by which the computer performs the initial verification of test results Any value that falls outside of the defined criteria must be assessed by a technologist before release of results II Procedure l The laboratory has a policy signed by the laboratory director approving the autoverification procedure a The results of autoverification is thoroughly tested appropriately documented and signed by the section head designee before implementation 3 If changes are made to the autoverification rules initially chosen and documented the process is reverified as to its accuracy 1 The autoverification process is checked yearly AUTOVERIFICATION FOR THE AXSYM INSTRUMENT POLICY The Axsym instrument performs the following tests for the Serology department 1 Hepatitis B Surface Antigen 2 Hepatitis B Surface Antibody 3 Hepatitis B Core Antibody PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 109 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI VIR 17 07 v02 Page 2 of 3 Hepatitis A IgM Antibody Hepatitis B Core IgM Antibody Hepatitis Be Antigen Hepatitis Be Antibody Hepatitis C Antibody 9 Rubella Antibody 10 Cytomegalovirus IgG Antibody 11 Transplant Screen Autoverification will occur as follows Oy ae
123. tware to be fully loaded before turning the P Lab ON 2 Click once on third square from the left open session Click on New Type in assay amp date performed e g HTLV 280499 HTLYV tested April 28 99 Note session e g 31 Click on writer Name enter initial Click OK 3 The screen will show e g HTLV280499 31 ssn highlighted click OK 4 Click on 5 th square from the left Profile Include click on DetectHTLV 0 prf click OK 5 Click on 7th square from the left Sample programming First click on Clear sample rack then click on sample code and enter lab and load specimens onto specimen rack at the same time Also load second half of the rack with dilution tubes Once finished double click on Small Rack icon in top left corner X s should appear next to all lab s click done 6 The screen will show plate format of wells needed Take out the required of strips and wells Load plate onto the left side in the P Lab Click on done 7 Click on 8 th square from the left Setup Entry Click on pulldown arrow under Lot If it is a new lot Click on Change Date and specify kit expiration date if needed Click OK 8 Click on last square Start Session Save document using the new file will appear click OK 9 Profile vial locations for controls or standards and reagents will appear Righ
124. ursor beneath STORE then press ENTER The following display appears Store Enter program x X is the next available number 8 Press ENTER to store the method under the displayed number 1 150 the following display appears Store Protect program NO Now your method stored so you need to program your programs which linked in your method To do this follow the steps as following PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 100 TML MSH Microbiology Department Policy amp Procedure Manual Serology Manual Policy MI SER 17 04 v01 Page 3 of 5 Creating a program 1 Access the main menu Select option 9600 RUN CREATE EDIT UTIL 2 Press the OPTION key to select CREATES then press ENTER The following display appears Create program HOLD CYCL AUTO METH Press the OPTION key to select the type of program HOLD CYCL or AUTO to create program then press ENTER 3 Specify the thermal cycling parameters for the program The last display to appear allows you to run store or print the program For example this Display appears at the end of a CYCL program Program Number not yet assigned Program pec XTE Ypa RUN STORE PRINTHOME xx XC 4 In the most cases you will want to store the program and assign it a number so that it can be run at any time Use t
125. ved from the clot and refrigerated until testing Specimens are stored at 70 C after testing Procedure i Reagent Preparation Wash Buffer 100 ml 10X Prepare working buffer 1X by adding 100 ml of 10X Wash Buffer to 900 ml of Distilled H2O Mix well Labeled and dated West Nile Antigen 2 vials Add 8 ml of the Sample Diluent to 1 vial of Antigen Do not use Distilled H20 Keep re constituted Antigen in fridge until use and return it immediately to fridge after use Each re constituted Antigen can perform aproximately 80 tests and expired in 14 days Controls 1 vial of Positive Control 1 vial of Negative Control 1 vial of Cut off Calibrator IgM Conjugate Substrate Reagent 2 vials of Tetramethylbenzidine TMB and Hydrogen Peroxide in buffer Stop Reagent 1 vial of 1M sulfuric acid PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 61 TML MSH Microbiology Department Policy MI SER 12 v03 Page 2 of 5 Policy amp Procedure Manual Serology Manual ii Other Materials IgM Capture Wells 96 wells Sample Diluent iii Method l Poe mo Ao Te Y Label a set of 12x75 mm glass tubes as follows Blk Pos Neg Cal 7 8 9 10 and External Control when required e g a new lot Add 1 ml of Sample Diluent to all tubes Leave 1 tube blank add 10 ul of Controls Calibrator and patient samples to corresponding tubes Take out t
126. y Department Policy MI SER 17 02 v01 Page 2 of 2 Policy amp Procedure Manual Serology Manual TEST REQUISITION DESTINATION 15 Toxoplasma IgG Total PHL form 97 44 08 99 Serology PHL Toxoplasma IgM 16 Cystercosis CDC 50 34 Rev 09 2002 Center for Disease Echinococcus must be completed by Control and prevention Leishmania ref physician 1600 Clifton Road N E Schistosoma Atlanta Georgia Strongyloides U S A 30333 Toxocara Trichinella Trypanosoma Miscellaneous Filaria NIH Filaria form Laboratory of ParasiticDiseases must be completed by National Institute of Allergy amp ref physician Infectious Diseases National Institutes of Health Building 4 Rm 126 Bethesda Maryland U S A 20892 17 Avian precipitins HICL label requisition Hospital In Common Lab Specify bird in question 1 William Morgan Dr Toronto Ontario M4H 1N6 Tel 416 422 3000 18 Parvovirus Ab IgG IgM PHL General Serology PHL 19 BK Virus Urine PHL General Virology PHL EM 20 Parvovirus PCR Sick Kids Virology HSC 3 Floor Elm Wing 21 JC BK PCR MSH Miscellaneous St Joseph s Hospital PROCEDURE MANUAL Virology Lab Hamilton Via PHL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 97 TML MSH Microbiology Department Policy MI SER 17 03 v01 Page 1 of 1 Policy amp Procedure Manual Section Serology Manual Subject Title Appendix ITI Shipment of JC BK Parvovirus PCR Samples Issued by LABORATOR

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