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Policy # MI/MD/v07 Page 1 of 2
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Table of Contents
Issued by: LABORATORY MANAGER Original Date: June 08, 2007
Approved by: Laboratory Director Revision Date: May 31, 2011
Annual Review Date: May 31, 2011
MOLECULAR DIAGNOSTIC TESTS MANUAL
TABLE OF CONTENTS
Chlamydia trachomatis and Neisseria gonorrhoeae Molecular Testing .................................................... 3
Cepheid Xpert C. difficile Assay ................................................................................................................ 10
Hepatitis B (HBV) DNA Quantitative Testing ......................................................................................... 20
Hepatitis C RNA PCR .............................................................................................................................. 27
West Nile Virus rPCR................................................................................................................................ 34
Nucleic Acid Extraction – Manual Method .............................................................................................. 45
Sample Preparation ................................................................................................................................47
Lysis and Purification Steps...................................................................................................................47
Elution Steps ..........................................................................................................................................49
Clean-Up ................................................................................................................................................49

Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG ................................................................. 51
Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG................................... 59
BK Virus DNA Detection ......................................................................................................................... 67
Cytomegalovirus DNA Detection ............................................................................................................. 76
Epstein Barr Virus DNA Detection .......................................................................................................... 86
Enterovirus RNA Amplification and Detection Procedures ..................................................................... 95
Herpes simplex Virus/Varicella-Zoster Virus DNA Detection .............................................................. 105
Influenza A, B and H1N1 Virus rPCR ..................................................................................................... 116
Parvovirus B19 DNA PCR ..................................................................................................................... 127
Respiratory Virus Panel (RVP) Multiplex PCR (Luminex) ...................................................................... 137
Respiratory Synctial Virus (RSV) PCR .................................................................................................. 146
Procleix® Donor Ultrio® (HBV, HCV, HIV) Assay and West Nile Virus Assay ................................ 155
Procleix® Donor Discriminatory (HBV, HCV, HIV) Assay ................................................................. 168
APPENDIX I - Molecular Diagnostic Tests Schedule ........................................................................... 182
APPENDIX II - BDProbeTec ET Workflow.......................................................................................... 183
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 1
Policy # MI/MD/v07 Page 2 of 2
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Table of Contents

APPENDIX III - Autoverification for ProbeTec® ................................................................................... 184
APPENDIX IV - Wipes Test for ProbeTec® ........................................................................................... 186
APPENDIX V - General PCR Precautions and Decontamination Procedures .......................................... 187
General PCR Precautions .................................................................................................................187
Decontamination Procedure .............................................................................................................188
WNV Decontamination Procedure ...................................................................................................188

APPENDIX VI – Virology Specimen Accessioning Guide ..................................................................... 190
Record of Edited Revisions .................................................................................................................... 191

Chlamydia trachomatis and Neisseria gonorrhoeae Molecular Testing
I. 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.

II. 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 2oC to 27oC before testing.
Urines can be stored 24h at 2-4oC prior to being transfer to Urine Preservation Transport (UPT).
Urines should be transferred to UPT within 8h of collection if urine is stored at 2-30oC.

III. 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 330C.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 Tube

BD ProbeTec™ ET System supplies:
BD ProbeTec™ ET Instrument
Microcell plates
Lysing Heater and Lysing Rack
Priming and Amplification heater
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 3
Policy # MI/MD/01/v06 Page 2 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Molecular Testing - Chlamydia
trachomatis & Neisseria gonorrhoeae
Pipettor and Power Supply
Urine Preservation Transport Kit (UPT)
Pipette tips

Other Materials:
Centrifuge
Vortex mixer
Gloves (powder free)
Transfer Pipette
1% (V/V) Sodium hypochlorite
Timer
Absorbent Paper (roll)

Sample Processing:

1. 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.

Note: Specimens processed, but not yet lysed, maybe stored at room temperature for up to 6
hours or overnight at 2-8 o C

2. Urine:

a) Open Urine Preservation Transport Kit remove transfer pipette and label UPT tube with
patient specimen number.

b) Hold UPT upright and tap firmly on a flat surface to dislodge droplets from the cap.

c) Uncap UPT and using transfer pipette provided, transfer urine to the UPT until the fluid
level is between the two black lines on the ―Fill Window‖. Approximately 2.5-3.45 of
Urine. DO NOT OVERFILL. Cap UPT tightly and discard transfer pipette.

d) Invert the UPT tubes 3-4 times to mix. Urine in UPT can be stored 30 days at 2-30oC or
at -20o C for 2 months.

f) Insert the Lysing Rack into the Lysing Heater. Heat for 10 minutes.

g) Remove Lysing Rack from Lysing Heater and cool tubes at Room temperature for a
minimum of 15 minutes, or maximum 6 hours.

h) Centrifuge the tubes at 2000 x g for 30 minutes. (Centrifuge MICT12 program# 2.)

i) After centrifugation carefully remove tubes from the centrifuge. In a Biological Safety
Cabinet, uncap the first UPT and carefully decant the supernatant into liquid discard
container. End the decanting motion with a ―gentle flick‖ of the wrist to remove residual
liquid from the tube, and blot on a clean a separate piece of absorbent paper. Loose
replace cap on tube. Repeat this step for all spun UPT tubes.

j) Transfer the contents of one CT/GC (2mL) Diluent Tube into the each UPT tube. Tightly
recap the UPT tubes and vortex 5 seconds to resuspend the sediment in the diluent.
Processed urines may be stored at Room Temperature for 6 hours or overnight at 2-8oC.
Processed Urines samples are now ready for Sample Lysing Step.

Note: Specimens processed, but not yet lysed, maybe stored at room temperature for up to 6
hours or overnight at 2-8o C

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.

Sample Lysing:

a) Use plate layout template to place samples and controls in correct position in lysing rack.
b) Place rack in lysing heater for 30 minutes. Remove rack and let samples cool for at least
15 minutes.

Note: After lysing samples:
 They may be stored at 18-30 0C for up to 6 hr and may be tested without re-lysing.
 The may be stored up to 5 days at 2-8 0C. Samples must be vortexed and re-lysed
prior to testing.
 They may be stored up to 98 days at –20 0C. Samples must be thawed at room
temperature, vortexed and re-lysed prior to testing. Lysed samples may be frozen and
thawed twice.

Assay Procedure:
1. Allow samples to cool at room temperature for at least 15 minutes (may be held up to 6
hours) after lysing is completed.

2. Remove and discard caps from tubes.

3. Change gloves to avoid contamination

4. 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.

5. Using Program #2, expand pipettor by pulling the handle all the way out.

6. 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.

7. Gently collapse the pipettor over a discard container and dispense 150 ul into each of the
corresponding column of Priming Microwell Plate.

8. Continue transferring the remaining samples for the run.

Note: Recap lysed specimen tubes; hold these at room temperature until run is
completed.

9. 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.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 6
Policy # MI/MD/01/v06 Page 5 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Molecular Testing - Chlamydia
trachomatis & Neisseria gonorrhoeae

10. Prepare the Amplification Microwell Plate by Configuring the Amplification Microwell Plate
to match the Plate Layout Report.

11. Remove the cover from the Priming Microwell Plate.

12. Check the temperature of the Priming/Warming Heater:
Priming Heater: 72-73oC
Warming Heater: 53.5-54.5oC

13. Place the Priming Microwell Plate into the Priming Heater.

14. Immediately place the Amplification Microwell Plate into the Warming Heater

15. Set the timer for 10 minutes. (Note: This step is time critical.)
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.

17. Allow pipette tips to touch sides of microwells

18. 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.

19. When the last column has been transferred, seal the Amplification Microwell Plate using an
Amplification Plate Sealer.

20. 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.

21. After initiating the run, complete the following portion of the clean-up procedure:
a) Seal priming microwells and remove from Priming Heater. Place Microwells into a
disposal bag and seal. Discard bag in biohazard box.

b) Clean metal plate:
Rinse the plate with Eliminase, or DNA Away, or 1% sodium hypochlorite.
Rinse the plate with water. Allow plate to dry.
Wrap plate in a clean paper towel.

22. Test results will be printed out as soon as the testing is finished.

23. Remove plate form instrument. Seal microwells and place in disposal bag. Seal bag and
discard in biohazard box.

24. Clean metal plate as above.

25. After last run of the day, perform the following clean-up procedures:

a) Saturate paper towels with 1% sodium hypochlorite, or DNA Away, or Eliminase and
apply to bench top, exterior surfaces of the Lysing Heater, Priming and Warming Heater,
BD instrument and pipettor handle (only the handle). Leave solution on surfaces for 2-3
min. Saturate towels with water and remove cleaning solution.
b) Immerse the Lysing Rack base and Lysing Rack in 1% sodium hypochlorite or Eliminase
for 1-2 min. Rinse thoroughtly with water and allow to air dry.

26. See Appendix VIII for Workflow Overview.

IV. 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 organism in the
specimen.

MOTA Score for CT or GC:

CT/NG Positive Control : > or = 2000
CT/NG Negative Control: < 2000

If any of these two controls fails, the test is invalid. Should inform Senior/Charge
technologists and repeat testing.

VI. 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

Indeterminate report:
Indeterminate for Chlamydia trachomatis by Nucleic Acid Amplification
Indeterminate for Neisseria gonorhoeae by Nucleic Acid Amplification

VII. 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.

If any of these two controls fails, the test is invalid. Should inform Senior/Charge
technologists and repeat testing.

External proficiency testing is provided by CAP and QMP-LS.
Maintenance QC:
Enter temperature 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 Analyzer, Bench, Lysing rack, Pipette, Priming heater, Lysing heater are
done every two weeks (see appendix#).

VII. 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
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 9
Policy # MI/MD/15/v01 Page 1 of 10
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cepheid Xpert C. difficile Assay
Issued by: LABORATORY MANAGER Original Date: January 10, 2010
Approved by: Laboratory Director Revision Date:
Annual Review Date:

Cepheid Xpert C. difficile Assay

I. Introduction

The Cepheid Xpert C. difficile Assay, performed on the Cepheid GeneXpert® Dx System, is a
qualitative in vitro diagnostic test for rapid detection of toxin B gene sequences from unformed
(liquid or soft) stool specimens collected from patients suspected of having Clostridium difficile
infection (CDI). The test utilizes automated real-time polymerase chain reaction (PCR) to detect
toxin gene sequences associated with toxin producing C. difficile. The Xpert C. difficile Assay is
intended as an aid in the diagnosis of CDI. Concomitant culture is necessary only if further
typing or organism recovery is required.

II. Specimen Collection and Processing

Collect unformed stool specimen in a clean Starplex container, and send to the Virology
laboratory as soon as possible. Stool collected in Enteric Transport Medium, or in SAF is not
suitable for this assay. The specimen is stable for up to 5 days when stored at 2–8°C.
Alternatively, specimens can be kept at room temperature (20 – 30 °C) for up to 24 hours.

III. Materials & Equipment

 GeneXpert Dx System
 Vortex mixer
 Dry swab for transfer of the specimen, such as the swab found in the Cepheid Sample
Collection Device 900-0370 (Copan Venturi Transystem® Culture) or the Copan Dual Swab
and Transport System.
 Disposable transfer pipettes.
 Xpert C. difficile kit – each kit contains 10 or 120 Xpert C. difficile cartridges and buffer

Each Xpert C. difficile Assay Cartridge with integrated reaction tubes contain:
Bead 1 (freeze-dried)
 Polymerase
 dNTPs
 BSA (bovine serum albumin)
 Probe
Bead 2 (freeze-dried)
 Primers
 Probes
 BSA
Bead 3 (freeze-dried)
 Sample Processing Control (SPC) non-infectious sample preparation
control spores
Reagent 1 (Sodium Hydroxide)
Reagent 2 (Tris Buffer, EDTA, surfactants)

Xpert C. difficile reagent pouch
 Sample Reagent (Guanidinium thiocyanate, surfactants)

IV. Procedure

GeneXpert Dx System
i) GeneXpert Cartridge Preparation

1. The disposable single-use GeneXpert DX cartridge holds the samples and reagents
that you want to process in the GeneXpert DX System. Do not reuse spent cartridges.

2. Store new GeneXpert DX cartridge at 2 – 28 °C. The cartridge and reagents are stable
for up to 7 days after the package has been opened.

3. The test must be started within 30 minutes of adding reagents to the cartridge.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 11
Policy # MI/MD/15/v01 Page 3 of 10
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cepheid Xpert C. difficile Assay

4. Use one cartridge and one sample reagent vial for each stool sample that needs to be
tested. Label the cartridge with the corresponding sample ID barcode. Do not hold
the cartridge from the reaction tube.

5. Place a dry swab which came with the Cepheid Sample Collection Device into the
unformed stool sample. The swab does not need to be completely saturated.

6. Insert the swab into the elution vial containing the Sample Reagent.
Note: Use sterile gauze to minimize the risk of contamination.
7. Hold the swab by the stem near the rim of the vial, lift the swab a few millimeters
from the bottom of the tube and push the stem against the edge of the vial to break it.
Make sure the swab is short enough to allow the cap to close tightly.
8. Close the lid and vortex at high speed for 10 seconds.
9. Open the cartridge lid. Using a clean transfer pipette, transfer the entire contents of the
Sample Reagent into the ―S‖ chamber of the Xpert C. difficile Assay cartridge.
10. Close the cartridge lid.

Figure 1. Xpert C. difficile cartridge (top view)

ii) Assay Method:
1. Turn on the computer, and then turn on the GeneXpert Dx instrument. Log into
windows using the password ―cphd‖.
2. On the Windows® desktop, double-click the GeneXpert Dx shortcut icon.
3. Log on to the GeneXpert Dx System software using your user name and password,
for example (User Name: admin1, Password: admin1).

4. The Database Management dialog box appears on top of the GeneXpert Dx System
window once the system starts up. Click ―NO‖ in the Database Management dialog
box if you do not want to perform any database management tasks.
5. If a test archive is overdue, the Test Archive Reminder dialog box appears. If you do
not want to archive click ―No‖ and if you do want to archive click ―Yes‖.
6. In the GeneXpert Dx System window, click Create Test. The Scan Sample ID
Barcode dialog box appears. Scan the Sample ID barcode using the barcode scanner
or you can manually enter the sample ID by clicking ―Manual Entry‖. Type in the
Sample ID into the Manually Patient ID Barcode Entry dialog box that appears.
7. Scan the barcode on the Xpert C. difficile Assay cartridge. The Create Test window
appears. Using the barcode information, the software automatically fills in the boxes
for the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and Expiration
Date.
8. Click Start Test. In the Create Test dialog box that appears. In the Check Status
Window, the selected instrument module progress changes to Waiting.
9. Open the instrument module door with the blinking green light and load the cartridge.
10. Close the door. The test starts and the green light stops blinking.

11. When the test is complete the instrument module door unlocks and the green light
turns off. Wait until the system releases the door lock before opening the module door
and removing the cartridge.

12. Dispose of the used cartridge in an appropriate specimen waste container.

13. Once testing is complete the report is automatically printed. To view the result, in the
GeneXpert DX System window, click View Results on the menu bar. The View
Results window appears.

14. Click View Test. The Select Test To Be Viewed dialog box appears. Select the test
of interest and click OK. The results of the selected test appear in the View Results
window.

15. The View Results window contains three tabs: Results, Errors, and Support. The
Results tab displays the information for a test such as the Patient ID, Sample ID,
Assay name and the test Result. The Errors tab list the errors encountered during the
test process.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 13
Policy # MI/MD/15/v01 Page 5 of 10
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cepheid Xpert C. difficile Assay

16. Monthly Maintenance Procedure:
a. Disinfect the instrument surfaces
1. Dampen a paper towel with 10% bleach solution and wipe the
instrument surface thoroughly with the paper towel.
2. Wait 10 minutes
3. Dampen a paper towel with 70% alcohol solution and wipe the
instrument thoroughly with the paper towel.

b. Disinfect the cartridge bay interior
1. Dip a swab into 10% bleach solution. Press the swab against the
inside wall of the container to remove excess solution.
2. Open the instrument module door.
3. Wipe the surfaces inside the cartridge bay with the swab. Do not
touch the slit on the I-CORE module into which the cartridge reaction
tube is inserted.
4. Wait 10 minutes
5. Dip a new swab into 70% alcohol solution. Press the swab against the
wall of the container to remove excess solution.
6. Wipe the same surface with the new swab.
7. Repeat steps 5 and 6 two times.
8. Close the instrument module door.

c. Disinfecting the plunger rod
1. In the GeneXpert DX System window, click Maintenance on the
menu bar. The Maintenance window appears.
2. On the Maintenance menu, click Plunger Maintenance. The
Plunger Maintenance dialog box appears.
3. In the Module table, select the module you want to clean, and then
click Clean or select Clean All to clean all modules simultaneously.
The Plunger Cleaning dialog box then appears.
4. Follow the directions in the Plunger Cleaning dialog box, then click
OK. In the Plunger Maintenance dialog box, the Clean button changes
to Move Up (if you clicked Clean All button, it changes to Move Up
All). In the instrument, the plunger rod in the selected module (or all
modules if you clicked Clean All button) lowers into the cartridge bay.
5. Dip a number of swabs in the 10% bleach solution. Press the swabs
against the inside wall of the container to remove excess solution.

6. Wipe the plunger rods with the swabs. Use a fresh swab for each
plunger rod.
7. Wait 5 minutes
8. Dip a number of swabs into 70% alcohol solution. Press the swabs
against the inside wall of the container to remove excess solution.
9. Wipe the plunger rods with the swabs. Use a fresh swab for each
plunger rod.
10. Repeat steps 8 and 9 two times.
11. In the Plunger Maintenance dialog box, click Move Up (or Move Up
All). The plunger rod moves back into its resting position.
12. Click Close to dismiss the Plunger Maintenance dialog box.

d. Clean fan filters

17. The GeneXpert DX instrument needs to be recalibrated annually or after 2000 test per
instrument module, whichever comes first. The system monitors the number of tests
since last calibration. To check whether the system requires calibration:

1. In the Maintenance window, look at the ICORE Starts Since Cal
column. On the Maintenance menu, click Module Reports. The
Module Reports dialog box appears.

2. Check the calibration date. If calibration is required contact the
Cepheid Technical Support to schedule a calibration.

IV. Assay validation

Each test includes a Sample Processing Control (SPC) and Probe Check Control (PCC):

1. Sample Processing Control (SPC) — Ensures the sample was correctly processed.
The SPC contains spores of Bacillus globigii in the form of a dry spore cake that is
included in each cartridge to verify adequate processing of the sample bacteria. The
SPC verifies that lysis of C. difficile bacteria and a spore have occurred, if the
organism is present, and verifies that specimen processing is adequate. Additionally,
this control detects specimen-associated inhibition of the real-time PCR assay. The
SPC should be positive in a negative sample and can be negative or positive in a
positive sample. The SPC passes if it meets the validated acceptance criteria.

2. Probe Check Control (PCC) — Before the start of the PCR reaction, the GeneXpert
Dx System measures the fluorescence signal from the probes to monitor bead
rehydration, reaction-tube filling, probe integrity and dye stability. Probe Check
passes if it meets the assigned acceptance criteria.

V. Interpretation of Results

The results are interpolated by the GeneXpert Dx System from measured fluorescent
signals and embedded calculation algorithms and will be shown in the View Results
window. Possible results are:

1. Toxigenic C. difficile POSITIVE

 Toxin producing C. difficile target DNA sequences are detected.
 The toxin producing C. difficile target(s) have Cts within the valid range and
endpoint above the minimum setting.
 SPC — NA (not applicable); SPC is ignored since C. difficile target amplification
may compete with this control
 Probe Check – PASS; all probe check results pass.
2. Toxigenic C. difficile NEGATIVE with no Critical Threshold (ct) or Endpoint

 C. difficile target DNA sequences are not detected.
 Toxin producing C. difficile targets not detected.
 SPC — PASS; SPC has a Ct within the valid range and endpoint above the
endpoint minimum setting.
 Probe Check — PASS; all probe check results pass.

3. Toxigenic C. difficile NEGATIVE with Critical Threshold (ct) or Endpoint

 C. difficile target DNA sequences are detected but not sufficient to trigger a POS
result.
 The GeneXpert test report gives a non-zero numeric value under ct or >10 under
EndPt for Toxin B yet the Analyte Result is NEG
 Check the curve for amplification (exponential increase) with a senior/charge who
will review with a microbiologist to report out on a case by case basis until we
have seen enough cases to know how to best report them. All such stools should
be frozen (7 vials till further notice) and sent to PHL for toxigenic C. difficile
culture.
 If there is an amplification curve and reviews cannot be immediately carried out
(after hours), preliminary reports can be issued as Presumptive Positive, specimen
has been sent to PHL for toxigenic C. difficile culture…

4. INVALID
 Presence or absence of C. difficile target DNA cannot be determined
 SPC — FAIL; SPC target result is negative and the SPC Ct is not within valid
range and endpoint below minimum setting.
 Probe Check — PASS; all probe check results pass.
 The sample was not properly processed or PCR was inhibited.
 Inform Virology Charge/ Senior and repeat the test according to the following
retesting procedure below.

5. ERROR
 Presence or absence of C. difficile cannot be determined.
 Toxin producing C. difficile targets — NO RESULT
 Probe Check — FAIL*; one or more of the probe check results fail.
 If the probe check passed, possible causes for the error include: the reaction tube
was filled improperly; a reagent probe integrity problem was detected; or the
maximum pressure limits exceeded the acceptable range.
 Inform Virology Charge/ Senior and repeat the test according to the following
retesting procedure below.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 17
Policy # MI/MD/15/v01 Page 9 of 10
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cepheid Xpert C. difficile Assay
6. NO RESULT
 Indicated that insufficient data were collected, the presence or absence of C.difficile
cannot be determined.
 Possible causes include the operator stopping the test will it was in progress.
 Inform Virology Charge/ Senior and repeat the test according to the following
retesting procedure below.
Retesting Procedure
The test should be repeated if an ―INVALID‖, ―ERROR‖ or ―NO RESULT‖ result was
obtained.
To repeat a test within 3 hours of an indeterminate result:
1. Transfer the remaining contents from the S Chamber of the current Xpert C.
difficile Assay cartridge to a new Sample Reagent Vial using a disposal transfer
pipette.
2. Vortex and add the entire contents of the Sample Reagent to Chamber S of a new
Xpert C. difficile Assay cartridge.
3. Close the lid and start new test

To repeat a test after 3 hours of an “INVALID” result:
1. Repeat the test with a new swab sample.

VI. Reporting

Negative Report: ―Negative - No Clostridium difficile toxin B gene detected by Cepheid Xpert
C.difficile PCR Assay. A single negative test is sufficient to rule out
C.difficile.‖

Positive report: Report as ISOLATE code ―clodtb‖ – ―Clostridium difficile toxin B gene
DETECTED.‖ Add ISOLATE COMMENT ―by Cepheid Xpert C.difficile PCR
Assay. If your patient has recurrent C. difficile infection, please consider
enrollment in the Fecal Transplant Study. For further information, please
contact the study coordinator, Mary Jane Salpeter at 416-340-4800 ext 8353 or
mjsalpet@uhnresearch.ca.‖

Phone positive result to ward (in-patient) or physician (Out-patient), and also phone to Infection
Control Practitioner as per Isolate Notification and Freezing Table QPCMI15003.

VII. Quality Control

Each test includes a Sample Processing Control (SPC) and Probe Check Control (PCC). Refer to
a senior technologist if control results are outside of limits or for any other problems with
running or reporting the assay.
Run external control (C. difficile Toxin from Positive C. difficile culture) with each new lot, QC and
instrument problems. Result filed in External Control Binder. If result is negative, the run is
invalid. Inform Charge/senior technologist, and repeat testing.
CAP provides external proficiency testing.

VIII. Reference

Xpert C.difficile PCR Assay package insert

Hepatitis B (HBV) DNA Quantitative Testing
I. Introduction

The Roche COBAS AmpliPrep TaqMan HBV Test is an in vitro nucleic acid amplification test for
the quantitation of Hepatitis B virus DNA in human plasma on the COBAS TaqMan 48™
Analyzer. Viral DNA is extracted using the automated the AmpliPrep instrument. Amplification
and detection are performed with the automated TaqMan 48™ Analyser using real-time PCR
technology.

II. Specimen Collection and Processing

Collect approximately 10 mL of blood in Vaccutainer tubes with EDTA (two lavender top tubes)
and transport them to the lab as soon as possible. Whole blood can only be transported/stored at 2-
25oC for up to one day before processing.
In the lab, centrifuge samples at 3000 rpm for 10 minutes with the Centra GP8R centrifuge (800-
1600 g). After centrifugation, separate the plasma into two storage tubes and put into the ‗HBV
DNA‘ box in the –20oC freezer.

III. Procedure

AmpliPrep Start –up
1. Turn Computer ON.
2. Control-Alt-Delete
3. To log onto Windows
a. User name: aluser
b. Password : aluser
4. Click Amplilink icon
5. To log onto amplilink
a. User name: MSH (case sensitive)
b. Password: Msh12345 (case sensitive)
6. Check Wash Reagent Reservoir.
7. Empty Waste container.
8. To perform maintenance AmpliPrep must be OFF
9. Clean glass cover of instrument.
10. Clean all work surfaces of AmpliPrep
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 20
Policy # MI/MD/02/v05 Page 2 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HBV DNA Quantitative Testing
with TaqMan
11. Clean tube handler and SPU Gripper on both heads
12. Clean Reagent tips on both heads
13. Clean loading panel
14. Clean TaqMan
15. Turn AmpliPrep and TaqMan on
16. Load reagents as per instructions below
17. Go to instrument icon AmpliPrep
a. choose service due tab
b. choose daily maintenance
c. click done
d. click on prime
e. click perform
f. perform extended prime on Monday
1. choose service all tab
2. choose prime system extended
3. choose perform
18. Record maintenance on maintenance worksheet.

Load Reagents
1. Remove reagents from refrigerator
2. Load CS1 on one Rack and insert into position A
If doing multiple assays, load enough CS1 cassettes for all runs. Be careful not to smudge the
barcodes.
3. Load Cassettes CS2, CS3 and CS4 onto another rack and insert into positions B-E.

All reagents cassettes should be removed for the refrigerator and loaded immediately on to the
AmpliPrep and allowed to come to room temperature on the instrument for at least 30 min. before
the first specimen is processed. Do not let the cassettes come to room temperature outside the
instrument as condensation may form on the barcode labels. Do not wipe off condensation if it
appears on the barcode labels.

Load Consumables
One sample processing unit (SPU), one Input sample tube (s-tube), one K-tip and one K-tube is
needed for each specimen and control.
1. Load SPU‘s on positions J,K,L.
Check seating in rack; do not push on top of (S-tips)- only on sides
2. Load K-tips into positions M-P.
3. Place control Barcode clips on sample rack
1. HPC, 2. LPC, 3. Neg
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 21
Policy # MI/MD/02/v05 Page 3 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HBV DNA Quantitative Testing
with TaqMan

4. Place new Barcode clips on sample rack for each sample being tested.

Create Order
1. Click the orders button
2. Select the Sample-Rack tab and click NEW
3. Type the Sample rack ID in the Sample Rack ID box.
4. Put the worklist # in the Batch ID box.
5. Click the test button (HBMCAP48 for HBV).
6. To identify the controls highlight then click the ―S‖ in the sample type (T) column to select the
control types. 1. HPC, 2. LPC and 3. Neg
7. Highlight Sample ID on line 4
8. Scan or enter Sample ID
9. Repeat 7 and 8 for all samples
10. Click save when finished.
11. Print worksheet.

Prepare Specimens
1. Vortex (3-5 sec) thawed specimens and controls.
2. Transfer 1050 uL into an S-tube for each specimen and control. Close cap of S-tube finger tight.
Avoid contaminating the upper part of the S-tube with specimens or controls.
3. Place K-tubes on Sample rack for each specimen.
4. Check seating of Clips and S-tubes.
5. Load Sample rack onto positions F, G or H.

Load K-Carrier

1. Click the System button
2. Select the Cobas AmpliPrep instrument using the instrument Selection box.
3. Click the Samples tab to display the Samples folder.
4. Using the handheld barcode scanner, scan the K-carrier rack barcode.
5. Scan the K-carrier barcode.
Steps 4 and 5 must be completed within 5 seconds.
6. Load the K-carrier rack containing the K-carrier in position O and P.

Start AmpliPrep
1. Check Status of Cassettes, Specimens and System by clicking on each tab
2. Press start.
3. Note the time when the run is completed on the worksheet.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 22
Policy # MI/MD/02/v05 Page 4 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HBV DNA Quantitative Testing
with TaqMan

End of Run
1. Click the Messages button and examine for any error messages.
2. Click the Systems button
3. Examine the Samples folder – successfully processed samples are green
4. Open the load panel
5. Remove the K-carrier rack containing the processed samples.

Start COBAS Taqman 48 Analyzer
1. Select the COBAS Taqman 48 Analyzer using the instrument Selection box.
2. Select the System tab.
3. Click Open for the thermal cycler to be loaded.
4. Open the thermal cycler lid.
Balance the K-carrier if necessary.
5. Using the K-carrier transporter, place the K-carrier in the thermal cylcer.
6. Close the lid
7. Choose Start.
8. Store reagents for the AmpliPrep in the refrigerator.

Remove Instrument Waste

1. Remove used SPUs and S-tubes put in autoclave bag and seal it.
2. Remove empty K-tip racks
3. Remove Sample racks and dispose used bar-code clips
4. Discard used K-tubes

Results Interpretation
1. Click Results button
2. Select the Review tab
3. Select the desired worklist under the batch ID column
4. Examine the results
Double-click the results line to display detail
Check for flags and error messages
Click Measurement Details to view graph
Print graph of any _Q_QS_ INVALID, _S_Drift_High or any other unusual flag
5. Click Accept.
The K-carrier must be removed from the TaqMan before results can be accepted.
6. Print Results Report

Specimen Dilution Protocol:
The reportable range (up to 1.1 x E11 IU/mL) is extended above the upper linear range limit (1.1
x E8 IU/mL or _S_DRIFT_HIGH) of the assay by performing a set of serial dilutions.

Each specimen requiring a dilution (_S_DRIFT_HIGH) will be diluted 1:1000. See procedure
below.

NOTE: For a ―_ Q_QS_INVALID‖ result, check and print the graph.
(1) If both the QS and target are not amplified, repeat the specimen neat. If both are still
not amplified, then report: ―Indeterminate due to inhibitory substances, by Cobas
Ampliprep TaqMan HBV Assay‖.
(2) If the QS is not amplified but the target is strongly amplified, repeat the extraction
procedure using the 1:1000 dilution protocol. The printed graph should be initialled
and dated to indicate acceptance. Report the HBV DNA value in IU/mL after
multiplying the dilution factor of 1000 or +E3.

To perform the dilutions you will need 2 mL screw cap tubes and Basematrix (this is stored in
aliquots in freezer).

(1) Thaw the specimens to be diluted at room temperature and vortex for at least 10 seconds.
(2) Add 225 uL of Basematrix to the first row of tubes.
(3) Add 225 uL of Basematrix to the second row of tubes.
(4) Add 1350 uL of Basematrix to the third row of tubes.
(5) Add 25 uL of the undiluted specimens into the first row of Basematrix to make a 1:10
dilution. Mix up and down 10 times using the pipet.
(6) Remove 25 uL of the 1:10 dilution from first row and dispense into second row to make a
1:100 dilution. Mix up and down 10 times using the pipet.
(7) Remove 150 uL of the 1:100 dilution from second row and dispense into third row to
make a 1:1000 dilution. Mix up and down 10 times using the pipet.
(8) Use the 1:1000 dilution to process and run in the assay.
(9) Cap diluted samples and store at 2-8 oC for up to 3 days. 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 IU/mL
number by the appropriate dilution factor to determine the final result.

** If the results are from the previous day, Press „O‟ for ‗Open‘ and choose the previous day‘s date.
Then continue on from step 5.

Note: To check which orders have been verified, ‗F1‟ out of the Look option and then go back in. All
results that have already been verified will have a ‗V‟ beside the order number.

Posting calculated values after dilution:

Note: The policy is that if a dilution cannot be done within 3-4 days, a preliminary report must be sent
out when the results are a value > x. When the dilution is going to wait more than 4 days, post the initial
> value and then amend the patient record with the calculated value once the dilution is complete. When
the dilution will be completed within the 3-4 day framework, do not post the initial high (>) value but
run the dilution and post the calculated value to the patient record. The latter is preferred and will not
cause an amended result on the patient record as the amendment is made in the interface file itself.

i. Go to „lab‟.
2. „8‟- ‗Interface‘.
3. „I‟ – ‗Interface menu‘.
4. „6‟ – ‗AMPLI‘
5. The interface will be on today‘s date, press „L‟ – Look.
6. The display received results window will open, press „F12”.
7. Choose the order number(s) for the diluted sample(s) by using the down arrow and pressing
„Enter‟.
8. Press ‗Enter‘ to go to the result field. Change the result to the calculated value.
9. Press „[„ – Post. Press ‗F12‟.
10. Go to the next order number.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 25
Policy # MI/MD/02/v05 Page 7 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HBV DNA Quantitative Testing
with TaqMan
11. At the end, press „F1‟ to exit.
12. Report results as the numeric value posted from the COBAS TaqMan to the LIS.

All values over 1.1 xE8 IU/mL, _S_DRIFT_HIGH or QS INVALID should be diluted according to the
dilution protocol. Report the exact # of the IU/mL after multiplying the dilution factor.

Results will automatically be resulted with the numeric value or ―Not Detected‖ with the units IU/mL
and the phrase:
By Cobas AmpliPrep TaqMan HBV Assay
HBV DNA conversion: 1 copy/mL = 0.19 IU/mL
or 1 IU/mL = 5.26 copies/mL

V. Quality Controls
Roche QC:
One HBV TaqMan Negative control, one HBV TaqMan Low Positive control, and one HBV
TaqMan High Positive control are included with each run. Each result must be within the set
value for each Lot #, otherwise the run is invalid.
Record these results on-line in the T-Drive under ―Microbiology/Virology/HBV DNA QC…
Active‖. The results should be within mean ± 2.S.D., otherwise withhold reports and inform
charge/senior technologist for further review. Charge/senior will review QC values outside the
mean ± 2.S.D. range based on the Westgard multirules for rejection (also see T-Drive as above).

External QC:
Run external control with each new reagent lot, each new shipment and at least once a month.
May also need to run external QC after equipment / QC failures, consult charge/senior
technologist.
Record these results on-line in the T-Drive under ―Microbiology/Virology/HBV DNA QC…
Active‖. The results should be within ± 2.S.D., otherwise withhold reports and inform
charge/senior technologist for further review.

External proficiency testing:
Provided by College for American Pathologists (CAP).
Read and follow the accompanying instructions and process proficiency tests using standard
procedures but do not send to reference lab. Results are verified by Charge/Senior and submitted
to Microbiologist for review.

VI. Reference

Roche Diagnostics, AmpliPrep Software Version 3.2, Application Manual Version 1.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 26
Policy # MI/MD/03/v09 Page 1 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HCV RNA PCR
Issued by: LABORATORY MANAGER Original Date: March 14, 2001
Approved by: Laboratory Director Revision Date: June 23, 2008
Annual Review Date: July 31, 2010
Hepatitis C RNA PCR
I. Introduction
The Roche COBAS AmpliPrep TaqMan HCV Test is an in vitro nucleic acid amplification test
for the quantitation of Hepatitis C virus RNA in human plasma on the COBAS TaqMan 48™
Analyzer. Viral RNA is extracted using the automated the AmpliPrep instrument. Amplification
and detection are performed with the automated TaqMan 48™ Analyser using real-time PCR
technology.

II. Specimen Collection and Processing
Collect approximately 10 mL of blood in Vaccutainer tubes with EDTA (two lavender top tubes)
and transport them to the lab as soon as possible. Whole blood can only be transported/stored at
2-25oC for up to one day before processing.
In the lab, centrifuge samples at 3000 rpm for 10 minutes with the Centra GP8R centrifuge (800-
1600 g). After centrifugation, separate the plasma into two storage tubes and put into the ‗HCV-
RNA‘ box in the –20oC freezer.

III. Procedure
AmpliPrep Start –up
1. Turn Computer ON.
2. Control-Alt-Delete
3. To log onto Windows
a. User name: aluser
b. Password : aluser
4. Click Amplilink icon
5. To log onto amplilink
a. User name: MSH (case sensitive)
b. Password: Msh12345 (case sensitive)
6. Check Wash Reagent Reservoir.
7. Empty Waste container.
8. To perform maintenance AmpliPrep must be OFF
9. Clean glass cover of instrument.
10. Clean all work surfaces of AmpliPrep
11. Clean tube handler and SPU Gripper on both heads
12. Clean Reagent tips on both heads
13. Clean loading panel
14. Clean TaqMan

All reagents cassettes should be removed for the refrigerator and loaded immediately on to the
AmpliPrep and allowed to come to room temperature on the instrument for at least 30 min.
before the first specimen is processed. Do not let the cassettes come to room temperature outside
the instrument as condensation may form on the barcode labels. Do not wipe off condensation if
it appears on the barcode labels.

Create Order
1. Click the orders button
2. Select the Sample-Rack tab and click NEW
3. Type the Sample rack ID in the Sample Rack ID box.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 28
Policy # MI/MD/03/v09 Page 3 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HCV RNA PCR
4. Put the worklist # in the Batch ID box.
5. Click the test button (HCMCAP48 for HCV).
6. To identify the controls highlight then click the ―S‖ in the sample type (T) column to select
the control types. 1. HPC, 2. LPC and 3. Neg
7. Highlight Sample ID on line 4
8. Scan or enter Sample ID
9. Repeat 7 and 8 for all samples
10. Click ―save‖ when finished.
11. Print worksheet.

Prepare Specimens
1. Vortex (3-5 sec) thawed specimens and controls.
2. Transfer 1050 uL into an S-tube for each specimen and control. Close cap of S-tube finger
tight. Avoid contaminating the upper part of the S-tube with specimens or controls.
3. Place K-tubes on Sample rack for each specimen.
4. Check seating of Clips and S-tubes.
5. Load Sample rack onto positions F, G or H.

Load K-Carrier
1. Click the System button
2. Select the Cobas AmpliPrep instrument using the instrument Selection box.
3. Click the Samples tab to display the Samples folder.
4. Using the handheld barcode scanner, scan the K-carrier rack barcode.
5. Scan the K-carrier barcode.
Steps 4 and 5 must be completed within 5 seconds.
6. Load the K-carrier rack containing the K-carrier in position O and P.

Start AmpliPrep
1. Check Status of Cassettes, Specimens and System by clicking on each tab
2. Press start.
3. Note the time when the run is completed on the worksheet.

Start Cobas TaqMan 48 Analyzer
1. Select the Cobas TaqMan 48 Analyzer using the instrument Selection box.
2. Select the System tab.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 29
Policy # MI/MD/03/v09 Page 4 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HCV RNA PCR
3. Click Open for the thermal cycler to be loaded.
4. Open the thermal cycler lid.
Balance the K-carrier if necessary.
5. Using the K-carrier transporter, place the K-carrier in the thermal cylcer.
6. Close the lid
7. Choose Start.
8. Store reagents for the AmpliPrep in the refrigerator.

Remove Instrument Waste
1. Remove used SPUs and S-tubes put in autoclave bag and seal it.
2. Remove empty K-tip racks
3. Remove Sample racks and dispose used bar-code clips
4. Discard used K-tubes

Specimen Dilution Protocol:
The reportable range (up to 6.9 x E10 IU/mL) is extended above the upper linear range limit
(6.90 x E7 IU/mL or _S_DRIFT_HIGH) of the assay by performing a set of serial dilutions.

Each specimen requiring a dilution (_S_DRIFT_HIGH) will be diluted 1:1000. See procedure
below.
NOTE: For a ―_ Q_QS_INVALID‖ result, check and print the graph.
(1) If both the QS and target are not amplified, repeat the specimen neat. If both are still
not amplified, then report: ―Indeterminate due to inhibitory substances, by Cobas
Ampliprep TaqMan HCV Assay‖.
(2) If the QS is not amplified but the target is strongly amplified, repeat the extraction
procedure using the 1:1000 dilution protocol. The printed graph should be initialled
and dated to indicate acceptance. Report the HCV RNA value in IU/mL after
multiplying the dilution factor of 1000 or +E3.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 30
Policy # MI/MD/03/v09 Page 5 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HCV RNA PCR
To perform the dilutions you will need 2 mL screw cap tubes and Basematrix (this is stored in
aliquots in freezer).

NOTE: For specimens that have been pre-diluted, it will be necessary to multiply the IU/mL
number by the appropriate dilution factor to determine the final result.

V. Reporting

Posting of Amlilink Results
1. Go to „lab‟.
2. „8‟- ‗Interface‘.
3. „I‟ – ‗Interface menu‘.
4. „6‟ – ‗AMPLI‘ **
5. The interface will be on today‘s date, choose „L‟ – Look.
6. The display received results window will open, press „F12”.
7. Choose each order number in order by using the down arrow and pressing „Enter‟.
8. Review the result in the interface with the result sheet from the Amplilink. Press „[„ – Post. Press
‗F12‟.
9. Go to the next order number.
10. At the end, press „F1‟ to exit.

** If the results are from the previous day, Press „O‟ for ‗Open‘ and choose the previous day‘s date.
Then continue on from step 5.

Note: To check which orders have been verified, ‗F1‟ out of the Look option and then go back in. All
results that have already been verified will have a ‗V‟ beside the order number.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 31
Policy # MI/MD/03/v09 Page 6 of 7
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: HCV RNA PCR

Posting calculated values after dilution:
1. Go to „lab‟.
2. „8‟- ‗Interface‘.
3. „I‟ – ‗Interface menu‘.
4. „6‟ – ‗AMPLI‘
5. The interface will be on today‘s date, press „L‟ – Look.
6. The display received results window will open, press „F12”.
7. Choose the order number(s) for the diluted sample(s) by using the down arrow and pressing
„Enter‟.
8. Press ‗Enter‘ to go to the result field. Change the result to the calculated value.
9. Press „[„ – Post. Press ‗F12‟.
10. Go to the next order number.
11. At the end, press „F1‟ to exit.

Report results as the numeric value posted from the COBAS TaqMan to the LIS.

All values over 1.1 xE8 IU/mL, _S_DRIFT_HIGH or QS INVALID should be diluted according to the
dilution protocol. Report the exact # of the IU/mL after multiplying the dilution factor.

Results will automatically be resulted with the numeric value or ―Not Detected‖ with the units IU/mL
and the phrase :
By Cobas AmpliPrep TaqMan HBV Assay
HBV DNA conversion: 1 copy/mL = 0.19 IU/mL
or 1 IU/mL = 5.26 copies/mL

VI. Quality Control

Roche QC:
One HCV TaqMan Negative control, one HCV TaqMan Low Positive control, and one HCV
TaqMan High Positive control are included with each run. Each result must be within the set
value for each Lot #, otherwise the run is invalid.

Record these results on-line in the T-Drive under ―Microbiology/Virology/HCV RNA QC…. ‖.
The results should be within mean ± 2.S.D., otherwise withhold reports and inform charge/senior
technologist for further review. Charge/senior will review QC values outside the mean ± 2.S.D.
range based on the Westgard multirules for rejection (also see T-Drive as above).

External QC:
Run external control with each new reagent lot, each new shipment and at least once a month.
May also need to run external QC after equipment / QC failures, consult charge/senior
technologist.
Record these results on-line in the T-Drive under ―Microbiology/Virology/HCV RNA QC…
Active‖. The results should be within ± 2.S.D., otherwise withhold reports and inform
charge/senior technologist for further review.

External proficiency testing:
Provided by College for American Pathologists (CAP). Read and follow the accompanying
instructions and process proficiency tests using standard procedures but do not send to reference
lab. Results are verified by Charge/Senior and submitted to Microbiologist for review.
.

VII. Reference

Roche Diagnostics, AmpliPrep Software Version 3.2, Application Manual Version 1.

I. Introduction
West Nile virus (WNV) is a flavivirus belonging to the Japanese Encephalitis group. The
primary host is wild birds and the virus is usually transmitted to humans via mosquitoes.
Transplants and blood transfusions have also been implicated in its transmission.
This assay involves isolating the viral RNA using the Qiagen Viral RNA Kit and amplifying it
by RealArtTM WNV Reverse-Transcription (RT)-PCR Kit. The amplification and detection take
place simultaneously, continually and in real time in the Roche LightCyclerTM.
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 bp WNV amplicon; and the other for the Internal
Control (IC). The IC is added to each specimen in the initial step to both control the isolation
procedure and to check for PCR inhibition. The LightCyclerTM simultaneously monitors the two
different amplicons using two fluorimeter channels.

II. Collection and Transport
Blood samples for WNV PCR should be collected in EDTA (purple top vacutainer tube). CSF
specimens should be collected in a clean, sterile container and sent to the laboratory as soon as
possible. If specimens cannot be transported immediately, they should be kept at 4oC. If a delay
of more than 6 hours is anticipated, the CSF specimen should be frozen at –70oC. The EDTA
samples should be processed within 6 hours of collection (plasma separated and refrigerated;
frozen if delay is more than 6 hours). Allow only one freeze-thaw cycle.
NOTE: Repeated freezing-thawing will reduce test sensitivity and cryoprecipitates may
accumulate in the plasma.

I. Procedure
A. Worklist Preparation:

a. Log on to Softmic,
b. Select/type:
i. Result
ii. Worklist
iii. Pagedown to 85 (WNV PCR),
iv. Enter
v. F12
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy # MI/MD/04/v03 Page 2 of 11
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: West Nile Virus rPCR

c. Pending samples (plasma, serum or CSF) will appear on Worklist.
d. Mark received samples using the F5 key,
e. Type "`" to print Worklist. The numbers on the left column of the Worklist will be used
to label the 1.5 mL Eppendoff microcentrifuge tubes and the Qiagen Isolation Columns.

B. LIS Label Printing (if needed for the RNA eluate):
a. While still in the Worklist, press Enter to go to the Test Screen
b. Press F1 to move curser to Demographic field
c. Press: / (to Order/Entry)
d. Press: No (to confirm editing)
e. Press: F9
f. Press: \ (Print Labels)
g. Press: No (modify record)
h. Select label printer for the LIS label

C. 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.

b. CSF:
An aliquot (0.2-2 mL) of the CSF should be frozen unless PCR can be performed
immediately

After processing, an aliquot of the left-over specimens should be stored frozen at-70C.

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)
Microwave
Vortex

PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy # MI/MD/04/v03 Page 3 of 11
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: West Nile Virus rPCR
1.5 mL microcentrifuge tubes
Cooling Block with capillary adaptors pre-cooled to 4oC
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
From Qiagen Viral Isolation Kit:
 Collection Tubes
 Lysis Buffer AVL
 Carrier RNA (poly A)-add 310 uL Elution Buffer -AVE to lyophilized
carrier RNA (310 ug) dissolve thoroughly and aliquot into 5.6 uL and 30 uL
screw-cap microtubes, and store at -20C in the clean room. DO NOT
freeze-thaw the carrier RNA aliquots more than 3 times
 Spin Columns
 Wash Buffer 1 (Buffer AW1) – add 125 mL ethanol before use
 Wash Buffer 2 (Buffer AW2) – add 160 mL ethanol before use
 Elution Buffer (Buffer AVE)
From: RealArtTM WNV LC RT PCR Kit, Artus (stored at -20o C in clean room):
 Quantification Standards (QS). When a new kit is opened, the QS must be
moved to a freezer in the specimen preparation area.
 Internal Control (IC)
 Master Mix (12 tests)
 Water (PCR grade)
Ethanol, anhydrous, reagent grade, 96 - 100%
External Control: To be run when a new lot (Qiagen or RealArt) is used, during training
or if QC failure occurs.

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:
a. Clean room
b. Specimen preparation room
c. Amplification room
 Supplies and equipment must be dedicated to each PCR area and not used for
other activities or moved between areas.
 Change lab coats and gloves between work areas. Only blue gowns are to be
worn in the clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure

 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Prepare and store reagents (lysis buffer, RNA carrier, elution buffer, AW1,
AW2 and ethanol) in the clean room. Keep only working aliquots in the
specimen preparation area.
 Keep the Quantitation Standards and specimens in the specimen preparation
area.
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or Eliminase to disinfect equipment and
surfaces and then rinse with 70% alcohol or water.

Clean Room

In the Clean Room prepare the Working Lysis Buffer AVL; determine the number of tests to be
extracted and prepare the appropriate volume of Working Lysis Buffer AVL required according to
the chart. Mix gently, invert 10 times to mix. Do not vortex.

No. of Volume Lysis Buffer Volume Carrier RNA-in
samples -AVL (mL) Elution buffer -AVE (uL)
1 0.56 5.6
2 1.12 11.2
3 1.68 16.8
4 2.24 22.4
5 2.80 28.0
6 3.36 33.6
7 3.92 39.2
8 4.48 44.8
9 5.04 50.4
10 5.60 56.0
11 6.16 61.6
12 6.72 67.2

DO NOT freeze-thaw the carrier RNA aliquots more than 3 times.
Working Lysis Buffer AVL is stable for 48 hrs at 2-8C. Solution may precipitate at 2-8C; redisolve
by warming on the bench, DO NOT MICROWAVE.

Sample Preparation:
Use Specimen Preparation area; work in cabinet with gown and frequent glove change

Lysis and Purification Steps:

a. For each Specimen and External Control, prepare and label two 1.5 mL microcentrifuge
tubes (sterilized, RNase-free) with the corresponding number in the Worklist. Put the
specimens first on the worklist, followed by the external control (if using one). Label one of
them with the lab number (LIS label). This microcentrifuge tube will store the eluted RNA;
the other will be used for processing and will be discarded. Also, label one QIAamp Spin
Column (Qiagen) with the number on the Worklist. Arrange the 2 microcentrifuge tubes and
Columns into 3 rows.

b. Add 560 uL Working Lysis Buffer (AVL with RNA carrier) into each of the microcentrifuge
tubes in one row (these will contain the waste).

c. Add 140 uL Specimen to the Working Lysis Buffer (Buffer AVL with RNA carrier), vortex
for 15 seconds.

d. Incubate at Room Temperature (15-25oC) for 10 minutes.

e. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids.

f. Add 5 uL WNV Internal Control to each of the microcentrifuge tubes with Buffer AVL.

g. Add 560 uL ethanol (96-100%) to the samples. Vortex for 15 seconds. Centrifuge for 10
seconds at 3000rpm (2000 g) to remove drops from the lids.

h. Pipette 630 uL (half the volume) of the mixture (specimen-lysis buffer-ethanol) to the
QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000 rpm
(6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and
discard the collection tube containing the filtrate.

i. Repeat Step (h) by pipeting the remaining 630 uL of the mixture to the QIAamp spin column
(in a 2 mL collection tube), close the cap and centrifuge at 8000rpm (6000 g) for 1 minute.
Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection
tube containing the filtrate.

k. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection
tube containing the filtrate.

l. Open the spin column and add 500 uL of Buffer AW2. Close the cap and centrifuge at full
speed 14,000 rpm (20,000g) for 3 minutes.

m. Place the spin column into a clean 2 mL collection tube and discard the collection tube
containing the filtrate. Centrifuge at 14,000 rpm (20,000g) for 3 minutes.

Elution Steps:

a. Place the spin column in the second clean 1.5 mL microcentrifuge tube with the LIS
specimen label. Discard the collection tube containing the filtrate. Open the spin column
and add 50 uL of Elution Buffer (Buffer AVE). Close the caps and incubate at room
temperature for 1 minute.

b. Centrifuge at 8000rpm (6000 g) for 1 min. Discard the spin column. Store the eluate at -20oC
or –70oC if not able to proceed immediately.

PCR Amplification and Detection Procedures:

In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

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.

b. Place a capillary for each purified sample, QS, external QC and Negative control (H2O) into the
capillary adaptor of the Cooling Block (pre-cooled to 4oC).

c. Pipette 15 uL Master Mix into the reservoir of each capillary.

d. Pipette 0.5 uL of internal control into the capillaries designated for the QS and the Negative
control.

e. Pipette 5 uL water (PCR grade) into the negative control capillary and cap.

f. Changed out of dedicated clean room gown
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy # MI/MD/04/v03 Page 7 of 11
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: West Nile Virus rPCR

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, QS4, and
external QC (if using) directly into the capillary reservoir. Immediately close the capillary with a
lid.

b. Load capillary tubes into LightCycler (LC) adaptor and centrifuge at 3000 rpm for 15 seconds in
LC centrifuge. Do Not take the cooling block into the Amplification Room. Transfer the LC
adaptor to the LC instrument.

In the LightCycler room:
a. Turn LightCycler on first and then the computer.

b. Press Control-Alt-Delete to begin.

c. Put in your user name and password and click OK.

d. Click on the LightCycler icon and push ENTER.

e. Wait for the ―Self-test box‖ to appear.

f. Select "selftest", (skip if already done within 24 hr.).

g. Choose OK when the self-test passes.

h. Record that you did the self-test in the log book.

i. Select:
"Open Experimental File"
―WNV protocol"; choose ―open‖
―Run‖

j. Type in file name ―WNV-year-month-day- run number‖ e.g. WNV-1-04-02-01. Choose ―save‖.

k. Type in the number of samples to run including QS, ext. QC and Negative Control in the
―maximum position‖ field.

l. Click ―Edit Sample Later‖.

m. Click ―Edit Samples‖ (bottom right of screen).
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy # MI/MD/04/v03 Page 8 of 11
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: West Nile Virus rPCR

n. Type in the lab numbers; QS; ext. QC (if any) and Negative control.

o. On the QS row, click the arrow-down button to change its description from ―Unknown‖ to
―STD‖ and enter the number of copies/uL (eg. QS3=100)

p. Press ―done‖

Remember to freeze the end product and patient samples. (Use store program for patient samples)

To Print Results, go to:
―FLUORESCENCE‖ and click
 F1
 Quantification
 Print Window

To Print Internal Control, go to:
―FLUORESCENCE‖ and click:
 F3/BACK F1
 Print Window

At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed
by 70% alcohol:

Clean Room:
 Clean Biological Safety Cabinet and turn on UV light for a least ½ hour but not overnight.

Specimen Preparation Room:
 Clean Biological Safety Cabinet, pippettors, centrifuge, and bench top.
 Cap discard container.
 Wash racks.

Amplification Room:
 Discard capillaries into a sharps container and put the cap the back onto the sharps container
 If a capillary breaks in the carousel, use the small brush supplied to clean the holes with !%
hypochorite and then alcohol.

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 (>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 (<QS4). The curve will be rising at a high cycle number. The sample 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.
PROCEDURE MANUAL
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Page 42
Policy # MI/MD/04/v03 Page 10 of 11
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: West Nile Virus rPCR

Positive Report: POSITIVE by RT-PCR
This is a research test performed by using RealArt WNV assay, Artus
Biotech Inc.

Inform senior/charge technologist of Positive WNV PCR and call the ward/and or doctor. Record
isolate in LIS by F7 as ―77wnv‖.

V. Quality Control

Equipment QC:

 Colour Compensation (to compensate interference between different fluorochromes) should be
run to update the Colour Compensation File.

Reagent QCs:
Before every lot change of isolation and/or master mix kit:
 An External Control (external to Artus Biotech) is used to monitor the isolation, amplification
and detection procedures. The result must correspond to expected value supplied by the
manufacturer.
Before every lot change of master mix kit:
 All four Quantification Standards (QS 1, 2, 3, and 4) must be run.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor 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).
 A Positive Control (e.g. Quantification Standard #4) is included and shows a positive
reading in fluorimeter Channel F1 (WNV amplicon).
 A Negative Control (e.g. H2O 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

Failed QC:

Test is invalid without satisfactory QC results.

a. Do not release results pending resolution of QC failure.
b. Inform charge/senior technologist.
c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where
appropriate.
d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can be
released (positive QC material yielded positive result, negative yielded negative result).
e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate of
the run with its expected positive rate.
h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated
reagents and consumables and disinfect equipment and environment before repeating the run.
i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are often
non-repeatable positives), it is necessary to have a thorough environmental disinfection followed
by swabbing to monitor.
j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental swabs.

VI. References:

QIAamp Viral RNA Mini Kit Handbook 01/99, Qiagen
RealArtTM WNV LC RT PCR Kit User Manual March 2003, Artus
LightCycler, Roche Diagnostics

Nucleic Acid Extraction – Manual Method

I. Introduction

The QIAamp DNA Mini kit utilizes the QIAamp spin column for the manual purification
of total DNA for reliable PCR. Ideal for manual processing of multiple samples yielding
pure DNA ready for direct amplification in the Roche LightCycler, using Artus RealArt
LC PCR Kits.
Purified DNA using this method produces approximate fragments of 20-30 kb in size.
DNA of this length denatures completely during thermal cycling and can be amplified with
efficiency.

II. Specimens

The procedure is suitable for whole blood treated with citrate, or EDTA; buffy coat,
plasma, serum, and body fluids. Samples may be fresh or frozen.
Tissues for Viral DNA extraction should follow the methodology for Tissues.
After processing, an aliquot of the left-over specimens should be stored at-70C in
appropriate area.

III. Materials, Equipments and Facilities

From Qiagen QIAamp DNA Mini Kit:
 Collection Tubes
 Buffer AL (Lysis buffer) mix thoroughly before use, stable on year at room
temperature
 QIAGEN Protease K – Ready to use –stable for 1 year
at room temperature- to prolong lifetime of reagent store at 2-4 oC
 Buffer ALT (Lysis buffer for Tissue)
 Spin Columns- store at room temperature good for one year
 Buffer AW1(Wash Buffer 1) concentrate - add 125 mL ethanol( 100%) before
use, as indicatedon bottle- stable one year at room temperature
 Buffer AW2 (Wash Buffer 2) – add 160 mL ethanol(100%) before use, as
indicated on bottle- stable one year at room temperature
 Buffer AE (Elution Buffer)
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 45
Policy # MI/MD/05/v04 Page 2 of 6
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Manual Method
From: Amersham Biosciences Carrier RNA (RNA-Homopolymer Poly A)
 Lyophilized stock reconstitute with 10 mL Buffer AE
 Stock concentrate 10 ug/uL diluted 1:10 with Buffer AE
 Working Carrier 1 ug/uL aliquoted into 10uL and frozen at –200C
 Each aliquot should be thawed only once.
(For every 100 uL BufferAL (Lysis) / 1uL of working Carrier RNA)
Ethanol, anhydrous, reagent grade, 96 - 100%
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
Microcentrifuge (MICT14)
Microwave
Vortex
1.5 mL microcentrifuge tubes
Cooling Block with capillary adaptors pre-cooled to 4oC
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
External Control: To be run when a new lot (Qiagen or RealArt) is used, during training
or if QC failure occurs.

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:
1. Clean room
2. Specimen preparation room
3. Amplification room
 Supplies and equipment must be dedicated to each PCR area and not used
for other activities or moved between areas.
 Change lab coats and gloves between work areas. Only blue gowns are to
be worn in the clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free micro centrifuge tubes

 Use sterile, disposable polypropylene tubes throughout the procedure
 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 46
Policy # MI/MD/05/v04 Page 3 of 6
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Manual Method
 Prepare and store reagents not‖ in use‖ (lysis buffer, carrier DNA, elution
buffer, AW1, AW2 and ethanol) in the clean room. Keep only working
aliquots in the specimen preparation area.
 Keep the Quantitation Standards and specimens in the specimen
preparation area.
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 CAUTION: DO NOT ADD BLEACH OR ACIDIC SOLUTIONS
DIRECTLY TO SAMPLE-PREPATION WASTE. Buffers AL and
AW1 form highly reactive compounds when combined with bleach. If the
buffers are split clean-up with Virox-5 wipe and distilled water, then use
1% sodium hypochloride or Eliminase to disinfect equipment and surfaces
and then rinse with 70% alcohol or water.

IV. Procedure
Sample Preparation:

 Use Specimen Preparation area, work in cabinet with gown and frequent glove change
 Turn on heating block to 56 C.
 Equilibrate samples to room temperature
 Prepare Buffer AW1, Buffer AW2, QIAGEN Protease
 If precipitate has formed in Buffer AL, dissolve by microwaving for
5 second intervals
 All centrifugation should be at room temperature

Lysis and Purification Steps:
a. Prepare fresh required Working Buffer AL (Lysis Buffer) in Clean Room by adding
1000 uL Buffer AL (Lysis Buffer) to 10uL Working Carrier RNA (1ug/uL Working
Carrier RNA for every 100 uL Buffer AL required).

b. For each specimen and External Control, prepare and label two 1.5 mL micro centrifuge
tubes (sterilized, RNase-free) with the corresponding number in the Worklist. Put the
specimens first on the worklist, followed by the external control (if using one). Label one
of them with the lab number (LIS label). This micro centrifuge tube will store the eluted
DNA; 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 micro
centrifuge tubes and Columns into 3 rows.

c. Pipette 200 uL of specimen to micro centrifuge tube.

d. Add 20 uL of QIAGEN Protease K to each specimen or external control. Mix by pulse
vortexing 15s.

e. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids.

f. Add 200 uL Working Buffer AL (Lysis buffer + Carrier RNA) to each specimen or
external control.

g. Add 5 uL Internal Control (eg. HSV IC, EBV IC, CMV IC, ParvoB19 IC, VZV IC) to
each of the micro centrifuge tubes. Mix by pulse vortexing 15s. ONLY ADD ONE
INTERNAL CONTROL. Do not add Internal Control to sample material directly.

h. Incubate at 560C for 10 minutes.

i. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids.

j. Add 200 uL ethanol (96-100%) to the samples and external control, vortex for 15s.
Centrifuge for 10 seconds at 3000rpm (2000 g) to remove drops from the lids.

k. Pipette 650 uL of sample mixture to the QIAamp Spin Column and centrifuge at 8000
rpm (6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection
tube, and discard the collection tube containing the filtrate.

l. Carefully open the QIAamp Spin Column and add 500 uL Buffer AW1 without wetting
the rim. Close the cap and centrifuge at 8000 rpm (6000 g) for 1 minute. Place the
QIAamp spin column into a clean 2 mL collection tube, and discard the collection tube
containing the filtrate.

m. Carefully open the QIAamp Spin Column and add 500 uL Buffer AW2 without wetting
the rim. Close the cap and centrifuge at 14,000 rpm (20,000g) for 3 minute.

n. Place the QIAamp spin column into a clean 2 mL collection tube, and discard the
collection tube containing the filtrate. Centrifuge at 14,000 rpm (20,000 g) for 1 minute.

Elution Steps:
a. Place the spin column in the second clean 1.5 mL micro centrifuge tube with the
LIS specimen label. Discard the collection tube containing the filtrate. Open the
spin column and add 50 uL of Buffer AE (Elution Buffer). Close the caps and
incubate at room temperature for 5 minute.

b. Centrifuge at 8000 rpm (6000 g) for 1 min. Discard the spin column.
For long-term storage of eluted DNA in Buffer AE storing at –200C is
recommended.

PCR Amplification and Detection Procedures:
Go to specific Virus for LC Amplication and Detection Procedure

Clean-Up:
At the end of the day, follow the cleaning protocol below using 1% hypochlorite or
Eliminase followed by 70% alcohol.

CAUTION: DO NOT ADD BLEACH OR ACIDIC SOLUTIONS DIRECTLY TO
SAMPLE-PREPATION WASTE. Buffers AL and AW1 form highly reactive
compounds when combined with bleach. If the buffers are spilt clean-up with Virox-5
wipe and distilled water, then use 1% sodium hypochloride or Eliminase to disinfect
equipment and surfaces and then rinse with 70% alcohol or water.

Clean Room:
 Clean Biological Safety Cabinet and turn on UV light for a least ½ hour but not
overnight.

Specimen Preparation Room:
 Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.
 Cap discard container.
 Wash racks.

Amplification Room:
 Discard capillaries into a sharps container and put the cap the back onto the sharps
container
 If a capillary breaks in the carousel, use the small brush supplied to clean the
holes with 1% hypochorite and then alcohol.

V. References

QIAamp Viral DNA Mini Kit Handbook 02/2003, Qiagen
LightCycler, Roche Diagnostics

Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG
I. Introduction

The NucliSENS easyMAG is an automated platform for the isolation (purification and
concentration) of total nucleic acids (RNA/DNA) from biological specimens. Used for batch
processing of 1-24 samples.
The nucleic extraction method is based on Boom Chemistry using magnetic silica particles.
Briefly, under high salt conditions, nucleic acid will bind to the silica particles. These silica
particles act as solid phase and non-nucleic acid components are removed by several washing
steps performed in the NucliSENS easyMAG instrument. Next, nucleic acids are eluded from the
silica particles and the silica particles are removed from the extracted specimens by the
NucliSENS easyMAG instrument.the resulting eluate contains purified and concentrated total
nucleic acids.

II. Specimens Types

Plasma (minimum 200uL) collected in EDTA tube and separated by centrifugation; 3000rpm for
10 minutes. Do not test plasma collected in heparin tubes.
CSF, sterile body fluids (amniotic fluid, vitreous fluid), Urine, BAL
Viral transport media (VTM) from swabs collected in VTM and vortexed on high for 10 seconds.

III. Materials, Equipments

NucliSENS easyMAG
NucliSENS easyMAG extraction products:
 NucliSENS Lysis Buffer
 NucliSENS easyMAG Lysis Buffer
 NucliSENS easyMAG Magnetic Silica
 NucliSENS easyMAG Extraction Buffer 1
 NucliSENS easyMAG Extraction Buffer 2
 NucliSENS easyMAG Extraction Buffer 3
 NucliSENS easyMAG Disposables –vessels and pipettes

BioHit Multichannel pipettor
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Policy # MI/MD/13/v03 Page 2 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Biomerieux NucliSENS
easyMAG

Ranin 1000uL, 100ul, 20ul Pipettors
Aerosol Resistant Tips (ART) 1000ul, 200ul, 20ul
Powder-free gloves
Internal controls (for CMV, HSV, BKV, Parvovirus B19, WNV, VZ, and RSV)

GENERAL PRECAUTIONS:

Do not allow the NucliSENS easyMAG Lysis Buffer, NucliSENS easyMAG Extraction Buffer 1 or
waste from the instrument to come in contact with acidic materials.

Certain reagents contain guanidine thiocyanate.

Buffers containing guanidine thiocyanate MUST NOT be mixed with cleaning solutions containing
bleach. Liquid waste from extractions procedures contain guanidine thiocynate MUST NOT be
mixed with other laboratory waste. Proper waste disposal is required.

IV. Procedure

1. Turn ON easyMAG. Wait for orange light to turn GREEN, then switch ON the easyMAG
computer.

2. Perform and record easyMAG Daily Maintence: Inspect drip tray.
Inspect reagent cap filters.
Inspect carbon filter.
Empty & rinse waste bottle with water.

3. easyMAG Login name: bmx
Password: bmx

4. Click on ―reagent‖ icon

5. Scan reagent stand and scan reagent bottle for all four reagents (A, B, C, D).
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Policy # MI/MD/13/v03 Page 3 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Biomerieux NucliSENS easyMAG

6. Click on ―Sample‖ icon

For H1N1/RSV
Enter Protocol: Generic 2.0.1
Enter Matrix: Other
Enter Volume (mL): 0.500
Eluate (uL): 50

For BKV/CMV/HSV/VZV/PARVO/ENTERO/WNV
Enter Protocol: Generic 2.0.1
Enter Matrix: Plasma or ―other‖ for urine & BAL, CSF for CSF
Enter Volume (mL): 0.200
Eluate (uL): 50

7. Click on sample ID bar and scan specimen barcodes.

8. Click on ―organize run‖ icon

9. Click on ―create run‖ icon

10. A box will appear with today‘s date and run #. Click on OK.

11. Click on to transfer all scanned items onto the worklist.

12. Click on ―load run‖ icon
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Policy # MI/MD/13/v03 Page 4 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Biomerieux NucliSENS easyMAG
13. To identify vessels, click on vessel icon

14. Scan position A barcode and then vessel barcode for first 8 samples, continue with position B
and C depending on how many samples you have.

15. Click on the silica icon

16. Scan in silica barcode from top of silica box.

17. Highlight all samples by touching screen or with mouse.

18. To apply silica barcode to all samples click on ―downward arrow‖ icon

19. Click on the ―progress tab‖ >>>> on third menu bar from top.

20. Print worklist. Select printer icon on right hand vertical menu bar. Pop-up appears, click OK.

21. Click on the ―dispense lysis icon‖
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Policy # MI/MD/13/v03 Page 5 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Biomerieux NucliSENS easyMAG

22. Once the lysis buffer has been dispensed, remove vessels form easyMag and put them in rack
and take them to the BSC.

23. For H1N1 and RSV, add 500 uL of specimen to the lysis buffer.
For CMV/BKV/HSV/VZV/PARVO/ENTERO, add 200 uL to the lysis buffer.
Mix by pipetting up and down three times.

24. Vortex internal control (IC).
For H1N1, add 10uL IC to the lysis buffer/specimen mix.
For CMV/BKV/HSV/VZV/PARVO/ENTERO/WNV/RSV add 5 uL IC.
Mix up and down three times.

25. Incubate at room temperature for 10 minutes.

26. To make silica premix add 550 uL PCR grade water to silica (program 1 of pipette)

27. Vortex silica premix before dispensing.

28. Dispense 125 uL silica into Elisa strip (program 2 of pipette)

29. After the 10 incubation period add 100 uL silica premix to vessel (program 3 of pipette)

30. Reload vessels into the easyMAG.

31. Scan position barcode and then vessel barcode (make sure the vessel icon is highlighted).

32. Click on start icon on right hand vertical menu bar.
A pop-up box will appear asking if you have added the premix silica, click YES.

33. Once the samples have been eluted, transfer the eluate from the vessels to labeled microtube. Set
pipette at 55uL to remove all of the eluate.

34. If elutes are to be tested same day store eluates at 4C. If PCR testing is to be performed at a later
date, store eluate at -20oC as soon as possible.

2. Clean easyMAG according to maintenance worksheet and record maintenance on worksheet.

3. To shut down easyMAG computer:
Click on bmx key on the bottom menu bar.
Click on ―quit‖ in the pop-up box.

4. Wait 10 minutes after the easyMAG Computer is OFF before switching off the easyMAG.

TO EMPTY WASTE: as required maximum 1/3 full

1. Go to reagent icon.

(If reagent icon is not on the screen, then click on machine icon
and click on reagent icon.)

2. Click on icon with droplet picture
(top icon on right vertical bar)

3. Empty waste in media room fume hood.
The Waste container should be labeled with the ―Start date‖ and ―contains guanidine
thiocyanate‖. Empty any remaining buffer from easyMAG Lysis Buffer and easyMAG
Extraction Buffer1 into the fume hood waste container. Discard these empty plastic
containers (Lysis Buffer & Extraction Buffer 1) into the biohazard box.
Extraction buffer 2 and Extraction buffer 3 can be discarded in the sink and bottles can be
rinsed and recycled.

4. Rinse easyMAG waste container with water and return to easyMag.
PROCEDURE MANUAL
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Policy # MI/MD/13/v03 Page 7 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction –
Biomerieux NucliSENS easyMAG

5. Click ―I confirm that the waste container is empty. A check mark should appear in that box.

6. Press OK.

WEEKLY MAINTENANCE:

Clean Dispense Probe on the EasyMag

1. Click on maintenance icon .

2. Put in empty vessels and pipettes.

3. Choose start from vertical side bar menu

To file runs that has been completed.

 Choose the magnifying glass (second menu bar from the top)
 Highlight the runs you wish to file.
 Choose the filing cabinet (right menu bar).

Failed extraction runs, should be reviewed with senior and/or Charge technologist to establish causes
and corrective actions; repeating extraction run maybe necessary.

If the easyMag system becomes inoperable call Biomerieux technical support phone number, provided
on each easyMag.

External controls are extracted on the easyMag according to schedule in the table below.

Analyte/target External control Extraction schedule
BKV High positive and Low Each day when test is done
Positive (once/week)
CMV High positive and Low Each day when quant. test is
positive done (weekdays)
EBV High positive and Low Each day when quant. test is
Positive done
HSV1 & 2 Positive control Once a month and/or with
each new lot
VZV Positive control Once a month and/or with
each new lot
H1N1 Positive control Once a month and/or with
each new lot
Influenza A Positive control Once a month and/or with
each new lot
Influenza B Positive control Once a month and/or with
each new lot
RSV A Positive control Once a month and/or with
each new lot
RSV B Positive control Once a month and/or with
each new lot
Parvovirus B19 Positive control Once a month and/or with
each new lot
Enterovirus Positive control Once a month and/or with
each new lot

VI. References

NucliSENS easyMAG User Manual v2.0 ref: 280163
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 58
Policy # MI/MD/06/v03 Page 1 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction for
Whole Blood – Biomerieux
NucliSENS easyMAG
Issued by: LABORATORY MANAGER Original Date:
Approved by: Laboratory Director Revision Date: August 23, 2010
Annual Review Date:

Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG

I. Introduction
The NucliSENS easyMAG is an automated platform for the isolation (purification and
concentration) of total nucleic acids (RNA/DNA) from biological specimens. Used for batch
processing of 1-24 samples.
The nucleic extraction method is based on Boom Chemistry using magnetic silica particles.
Briefly, under high salt conditions, nucleic acid will bind to the silica particles. These silica
particles act as solid phase and non-nucleic acid components are removed by several washing steps
performed in the NucliSENS easyMAG instrument. Next, nucleic acids are eluded from the silica
particles and the silica particles are removed from the extracted specimens by the NucliSENS
easyMAG instrument.the resulting eluate contains purified and concentrated total nucleic acids.

II. Specimens Types:

EDTA Whole blood minimum volume 200uL

III. Materials, Equipments

BioHit Multichannel pipettor
Ranin 1000uL, 100ul, 20ul Pipettors
Aerosol Resistant Tips (ART) 1000ul, 200ul, 20ul
Powder-free gloves
Internal controls (EBV)
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 59
Policy # MI/MD/06/v03 Page 2 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction for
Whole Blood – Biomerieux
NucliSENS easyMAG

IV. GENERAL PRECAUTIONS:

Do not allow the NucliSENS easyMAG Lysis Buffer, NucliSENS easyMAG Extraction Buffer 1
or waste from the instrument to come in contact with acidic materials.

Certain reagents contain guanidine thiocyanate.

Buffers containing guanidine thiocyanate MUST NOT be mixed with cleaning solutions
containing bleach. Liquid waste from extractions procedures contain guanidine thiocynate MUST
NOT be mixed with other laboratory waste. Proper waste disposal is required.

V. PROCEDURE: Off-Board Preparation

1. Thaw whole blood samples at room temperature. DO NOT vortex samples while thawing.

2. When samples are completely thawed, mix by inverting sample tubes 5 times.

3. Prepare the NucliSENS easyMAG: see below.

4. Pipette 2mL Nuclisens Lysis Buffer into a labeled 5mL processing tube with a screw cap, one
5mL tube per sample.

5. Add 200uL of whole blood sample. Vortex immediately.

6. Add 5uL of Internal Control (IC) to Lysis-specimen mixture. Vortex.

7. Add 140uL UNDILUTED Magnetic Silica to the Lysis-specimen mixture. Pipette up and down
to mix. Vortex.

8. Transfer the entire contents of the processing tube (Lysis-sample-IC-silica) into the easyMAG
sample vessel, using a sterile transfer pipette. Avoid causing bubbles and do not touch the top
of the easyMAG sample vessel.

VI. ON the NucliSENS easyMAG

1. Turn ON easyMAG. Wait for orange light to turn GREEN, then switch ON the easyMAG
computer.

2. Perform and record easyMAG Daily Maintence: Inspect drip tray.
Inspect reagent cap filters.
Inspect carbon filter.
Empty & rinse waste bottle with water.

3. easyMAG Login name: bmx
Password: bmx

4. Click on ―reagent‖ icon

5. Scan reagent stand and scan reagent bottle for all four reagents (A, B, C, D).

6. Click on ―Sample‖ icon
Enter Protocol: Specific B Protocol
Enter Matrix: Whole Blood
Enter Volume (mL): 0.200
Eluate (uL): 50
Type: Lysed
Lot no: Nuclisens Lysis buffer lot used – type the lot number in using keyboard

7. Click on sample ID bar and scan specimen barcodes.

8. Click on ―organize run‖ icon

9. Click on ―create run‖ icon

10. A box will appear with today‘s date and run #. Click on OK.

11.

12. Click on to transfer all scanned items onto the worklist.

13. Click on ―load run‖ icon

14. Load easyMAG sample vessels and pipette tips.

15. To identify vessels, click on vessel icon

16. Scan position A barcode and then vessel barcode for first 8 samples, continue with position B
and C depending on how many samples you have.

17. Click on the silica icon

18. Scan in silica barcode from top of silica box.

19. Highlight all samples by touching screen or with mouse.

20. To apply silica barcode to all samples click on ―downward arrow‖ icon

21. Print worklist. Select printer icon on right hand vertical menu bar. Pop-up appears, click OK.

22. Click on start icon on right hand vertical menu bar.
A pop-up box will appear asking if you have added the premix silica, click YES.
Extraction of 24 samples will take approximately 60 minutes on the easyMAG.

23. Once the samples have been eluted, transfer the eluate from the vessels to labeled microtube. Set
pipette at 55uL to remove all of the eluate.

24. If elutes are to be tested same day store eluates at 4C. If PCR testing is to be performed at a later
date store at -20C as soon as possible.

25. Perform Clean Dispense Probe on the EasyMag after each whole blood extraction run. See
weekly maintenance below.

2. Clean easyMAG according to maintenance worksheet and record maintenance on worksheet.

3. To shut down easyMAG computer:
Click on bmx key on the bottom menu bar.
Click on ―quit‖ in the pop-up box.

4. Wait 10 minutes after the easyMAG Computer is OFF before switching off the easyMAG.

TO EMPTY WASTE: as required maximum 1/3 full

a. Go to reagent icon.

(If reagent icon is not on the screen, then click on machine icon
and click on reagent icon.)

b. Click on icon with droplet picture
(top icon on right vertical bar)

c. Empty waste in media room fume hood.
The Waste container should be labeled with the ―Start date‖ and ―contains guanidine
thiocyanate‖. Empty any remaining buffer from easyMAG Lysis Buffer and easyMAG
Extraction Buffer1 into the fume hood waste container. Discard these empty plastic
containers (Lysis Buffer & Extraction Buffer 1) into the biohazard box.
Extraction buffer 2 and Extraction buffer 3 can be discarded in the sink and bottles can be
rinsed and recycled.
5. Rinse easyMAG waste container with water and return to easyMag.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 64
Policy # MI/MD/06/v03 Page 7 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Nucleic Acid Extraction for
Whole Blood – Biomerieux
NucliSENS easyMAG

6. Click ―I confirm that the waste container is empty. A check mark should appear in that box.

7. Press OK

VIII. Quality Control

Failed extraction runs, should be reviewed with senior and/or Charge technologist to establish causes
and corrective actions; repeating extraction run maybe necessary.

If the easyMag system becomes inoperable call Biomerieux technical support phone number, provided
on each easyMag.

External controls should be extracted on the easyMag according to schedule in the table below.

Analyte/target External control Extraction schedule
EBV High positive and Low once a week/ per PCR run
positive

IX. WEEKLY MAINTENANCE:

Perform Clean Dispense after each whole blood extraction run.

Clean Dispense Probe on the EasyMag

4. Click on maintenance icon .

5. Put in empty vessels and pipettes.

6. Choose start from vertical side bar menu

To file runs that has been completed.

 Choose the magnifying glass (second menu bar from the top)
 Highlight the runs you wish to file.
 Choose the filing cabinet (right menu bar).

IX. References

NucliSENS easyMAG User Manual v2.0 ref. 280163

II. Specimen Types
Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

III. Materials, Equipments
Clean Room:
Dedicated Biosafety Cabinet (MIBCT4),
Designated clean room gowns and powder-free gloves
Designated Clean room Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
72 microtube Corbett Research block pre-cooled to 4oC
36 microtube Corbett Research block pre-cooled to 4oC
0.2mL microtubes/ or 0.1mL Strip Tubes and Caps
Aerosol Resistant Tips (ART)

Astra BKV PCR Kit 1.0, (stored at -20o C in clean room):
 Master A (12 tests per vial)
 Master B (12 tests per vial)
 Internal Control (IC)
 Water (PCR grade)

Specimen Preparation area:
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
Aerosol Resistant Tips (ART)
Astra BKV PCR Kit 1.0, (stored at -20o C in clean room):
 QS1 (10^4 Copies/µL)
 QS2 (10^3 Copies/µL)
 QS3 (10^2 Copies/µL)
 QS4 (10^1 Copies/µL)

External Control: To be run when a new lot is used, during training or if QC failure occurs.
PROCEDURE MANUAL
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Page 67
Policy # MI/MD/14/v03 Page 2 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: BK Virus Detection
Detection Area:
Rotorgene 6000 Multiplexing System programmed with ASTRA BKV PCR
36-Well Rotor with Locking Ring/ or 72-Well Rotor with Locking Ring

GENERAL PRECAUTIONS
There must be designated separate PCR work areas: 1. Clean Room
2. Specimen preparation area
3. Detection (Amplification) Area
Supplies and equipment must be dedicated to each PCR work area.
Use only aerosol resistant tips (ART).
Work with powder-free gloves; change gloves frequently.
Follow cleaning instructions outlined on daily maintenance schedules.

IV. Detection Set Up
Organize BKV eluates and store at 4oC until ready to load.

Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

a. Take out the necessary number of BKV Master Mix A and B vials (12 tests per vial) from
the -20C freezer, quickly and thoroughly thaw inside the Biosafety Cabinet. Prepare BKV
Master Mix (number of samples + controls + 1 extra test) required according to the
chart. Mix gently, do not vortex.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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should be checked against the document (titled as above) on the server prior to use
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Page 68
Policy # MI/MD/14/v03 Page 3 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: BK Virus Detection
b. Place proper number of microtubes for each sample, Controls (Standard and NTC-
Negative) into the Cooling Block (pre-cooled to 4oC).

c. Pipette 20 uL prepared BKV Master Mix (A+B) into each microtubes. Lightly cap
sample microtubes.

d. Pipette 1.0 uL of internal control into the microtubes designated for the Standard (BKV
QS3) and the NTC-Negative control.

e. Pipette 10uL water (PCR grade) into the negative control microtube and cap tightly.
Number microtube lids.

f. Changed out of dedicated clean room gown.

Specimen Processing Area:
a. Carefully pipette 10uL of each sample eluate, and Standard (BKV QS3), into designated
numbered microtube containing BKV Master Mix. Gently mix by pipetting up and down. Close
the lid tightly.

b. Visually check that each microtube is tightly closed and that all microtubes have the same liquid
level 30ul, before loading. Make sure there are no bubbles at the bottom of the microtubes.

Detection (RotorGene) Area:
a. Turn ON the RotorGene and computer.

i. Click the box ―Locking Ring Attached‖
j. Press “Next” button.
k. Enter initials in Operator space. Enter kit lot number and the Rotor-Gene Colour (ie.Red/Blue) in
the Notes box. Verify the Reaction Volume is 30ul.
l. Press “Next”
m. Temperature Profile window pops up; press ―Next”.
n. Summary window appears; press “Start Run”. The RotorGene will start.
o. Save as window appears; go to My documents -Open–select BKV result folder- Open.
p. Press Save -BKV yyyy-mm-dd (run no.)
q. Edit Samples window. Go to format select 1.23E+05. Under Units select copies/ul.
r. Enter sample LIS numbers using the barcode reader, enter BKV Standard (BKV QS3), and
NTC-water.

Name Type Given conc. Select
Sample Number Unknown Yes
QS3 CMV Standard 1.00E+02 Yes
Water NTC(no template control) Yes

s. Press ―Finish”. Click on “Name On”.

To analyze and print BKV report after a run has finished:
1. The run is done when Profile Program reads: Run has completed.

2. Analyze raw data by clicking each channel one at a time: Green channel (BKV) and Yellow
channel (IC).These tabs are found on the second menu bar from the top, on the left hand side.

3. In the Green Channel. Click “All Off” (bottom right). Select sample ―water (NTC)‖ and ―BKV
QS3 (Standard)‖. Click on Autoscale (bottom left).
Compare each specimen curve to the NTC-water curve and the BKV QS3 curve one at a time (by
clicking each sample on and off then proceeding to the next sample). A positive curve will have a
sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all
positives on the worklist.

4. Click on “Named On”.

5. Perform curve Analysis (Get report): Click “Analysis” on the top tool bar.

6. Analysis floating window pops up.

7. Click Quantitation Analysis.

8. Quantitation window is brought up to the front. There are should be two channels (Green/Yellow).

9. Highlight Cycling A. Green option and press Show.

10. Maximize the cycling green channel graph (or the channel you are working on).

11. Press “Import Curve” button on the right side of the screen.

12. Press “From other run” button.

13. Go to My Document -STD curves folder, press “Open”

14. Choose the latest BKV standard curve (Note: standard curves are changed every 6 months), -
“Open”

15. Highlight green channel, press “Import” button.

16. Adjust if only ONE Standard used (Note: Do NOT adjust if ALL Std used)
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 71
Policy # MI/MD/14/v03 Page 6 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: BK Virus Detection

17. Press button “Slope Correct” on the top.

18. Press button ―Ignore first‖ on the top, and type ―5‖, press OK.

19. Check curve quality, adjust the Threshold.

20. To Print Reports: Press ―Reports‖.

21. Choose Cycling A.Green (Page 1)

22. Highlight Quantitation full report, press ―Show‖
Maximize the report window, check that only the positives that you have identified from the raw
data are positive (i.e. have a crosspoint)

23. Print pages 1-4 for the green channel; pages 2-4 for the yellow channel.
Close the report window.

24. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab,
select Cycling A. Yellow -the internal control (IC), select Show.
Maximize the graph Quantitation Analysis –Cycling A. Yellow.
Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in
the first 5 cycles of the run).
Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph
screen. Move the threshold line to above the noise.
Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show.
All samples should have a positive IC curve, and a cycle number in the Ct column.
Print pages 2-4.
(For Cycling A. Yellow report, if there is message of ―NEG (Multi Ct)‖ for any specimen, you have
to go back to check Raw Data of cycling A.Yellow. If all specimens are amplified, adjust the
threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct.
Adjust the threshold line so that the threshold line crosses the curve at only one point.)

25. Close the outer most window.
―Save changes to BKV yyyy-mm-dd?‖ Select Yes.
Shut down the computer, and switch off the Rotor-Gene.

V. Calculations

Perform calculations on BKV PCR specimens (plasma/urines)

Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL)
Sample volume (mL)

Result (copies of DNA/mL) = Result (copies/uL) x 50(uL)
0.200 (mL)

These calculations can also be performed using PCR bench Math excel file.

VI. Reporting

BKV PCR negative (has no Ct value) report on the LIS test window, using the keypads:
Ctrl+D, Ctrl+D, Ctrl+D, select >BKJC, BK-

Negative for BK virus DNA.
This is a research test.
BKV PCR Kit v1.0, Astra Diagnostics Inc.

Under media ―PCBKV‖ F6 to enter the date tested.
Finalize the result (Ctrl+F)

BKV PCR positive, with value in copies of DNA/mL (ie.After calculation has been performed).
Report BKV as an isolate in the isolate window (F7).
Isolate #: 1 Org.ID. 62BKV
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖.
If >10 copies of DNA/mL select the keypad: \BK+
Type in the copies, in scientific format, in the space provided.
If result is <10 copies of DNA/mL select: \BK10
`Grave (`) the result.
Under media ―PCBKV‖ F6 to enter the date tested. If reporting <10 cp/mL enter the actual copy value.
Interim the result (Ctrl + L).

VII. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value
supplied by the manufacturer.
 BKV High Positive and BKV Low Positive External controls should be extracted on the
easyMag once a week and/or per BKV PCR run. Record external QC results in the T drive,
Virology folder, QC folder.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both extraction
and PCR inhibition.
 BKV QS3 Standard is included and shows a positive reading in Green Channel
 A Negative Control is included and shows a negative reading in Green Channel
Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.
a. Do not release results pending resolution of QC failure.
b. Inform charge/senior technologist.
c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where
appropriate.
d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can
be released (positive QC material yielded positive result, negative yielded negative result).
e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate
of the run with its expected positive rate.
h. If the contamination is extensive, it is necessary to quarantine/discard potentially
contaminated reagents and consumables and disinfect equipment and environment before
repeating the run.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 74
Policy # MI/MD/14/v03 Page 9 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: BK Virus Detection

i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are
often non-repeatable positives), it is necessary to have a thorough environmental disinfection
followed by swabbing to monitor.
j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental
swabs.

VIII. References

Astra BKV PCR Kit v1.0 Instructions, Astra Diagnostics

Cytomegalovirus DNA Detection

I. Introduction

Human cytomegalovirus (CMV) can cause severe, life threatening disease in
immunocompromised patients such as transplant patients, patients with HIV, and developing
fetus. Quantitative CMV can be used to monitor the course of CMV infection during and after
treatment.

II. Specimen Collection and Processing

Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

III. Materials, Equipments

Clean Room: dedicated Biosafety Cabinet (MIBCT4),
Designated clean room gowns and powder-free gloves
Designated Clean room Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
o
36 microtube Loading block pre-cooled to 4 C
72 microtube Loading block pre-cooled to 4oC
0.2mL microtubes/ or 0.1mL microtubes and Caps
Aersol Resistant Tips (ART)

Astra CMV PCR Kit 1.0, (stored at -20o C in clean room):
 Master A (12 tests per vial)
 Master B (12 tests per vial)
 Internal Control (IC)
 Water (PCR grade)

Specimen Preparation area: Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
Astra CMV PCR Kit 1.0, (stored at -20o C in clean room):
 QS1 (10^4 Copies/µL)
 QS2 (10^3 Copies/µL)
 QS3 (10^2 Copies/µL)
 QS4 (10^1 Copies/µL)

External Control: To be run when a new lot is used, during training or if QC failure
occurs.

Detection Area: Rotorgene 6000 Multiplexing System programmed with ASTRA CMV PCR
36-Well Rotor with Locking Ring/ or 72-Well Rotor with Locking Ring

GENERAL PRECAUTIONS:

There must be designated separate PCR work areas: 1. Clean Room
2. Specimen preparation area
3. Detection (Amplification) Area
Supplies and equipment must be dedicated to each PCR work area.
Use only aerosol resistant tips (ART).
Work with powder-free gloves; change gloves frequently.
Follow cleaning instructions outlined on daily maintenance schedules.

IV. DETECTION SET UP:

Organize CMV eluates and store at 4oC until ready to load.

Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

a. Take out the necessary number of CMV Master Mix A and B vials (12 tests per vial) from
the -20C freezer, quickly and thoroughly thaw inside the Biosafety Cabinet. Prepare
CMV Master Mix (number of samples + controls + 1 extra test) required according to
the chart. Mix gently, do not vortex.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 77
Policy # MI/MD/15/v03 Page 3 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: CMV DNA Detection

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180

b. Place proper number of microtubes for each sample, Controls (Positive and Negative) into
the Cooling Block (pre-cooled to 4oC).

c. Pipette 20 uL prepared CMV Master Mix (A+B) into each microtubes. Lightly cap
sample microtubes.

d. Pipette 1.0 uL of internal control into the microtubes designated for the Positive control
(CMV QS3) and the Negative control.

e. Pipette 10uL water (PCR grade) into the negative control microtube and cap tightly.
Number microtube lids.

f. Changed out of dedicated clean room gown.

Specimen Processing Area:

a. Carefully pipette 10uL of each sample eluate, and Positive Control (CMV QS3), into designated
numbered microtube containing CMV Master Mix. Gently mix by pipetting up and down. Close
the lid tightly.

Detection (RotorGene) Area:
a. Turn ON the RotorGene and computer.

b. FOR THE BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press ―→‖
button;
c. Double click (left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon. Wait for
―Initializing machine…..‖
d. Load microtubes into proper rotor (eg. 36 well/or 72-well). Load sample #1 into rotor position
#1, sample #2 into rotor position #2, and so on.
e. Fill the empty positions of the rotor using empty microtubes and snap in the locking ring. Load
the rotor in the RotorGene 6000.
f. Highlight CMV assay;
g. Press ―New‖ button;
h. Highlighting the correct rotor in use: Red 36-well Rotor
Blue 72-well Rotor

l. Press “Next”

m. Temperature Profile window pops up; press ―Next”.

n. Summary window appears; press “Start Run”. The RotorGene will start.

o. Save as window appears; go to My documents -Open–select CMV result folder- Open Press
Save -CMV yyyy-mm-dd (run no.)

p. Edit Samples window. Go to format select 1.23E+05. Under Units select copies/ul.

q. Enter sample LIS numbers using the barcode reader, enter CMV Positive (CMV QS3), and
water.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 79
Policy # MI/MD/15/v03 Page 5 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cytomegalovirus DNA Detection

Name Type Given conc. Select
Sample Number Unknown Yes
QS3 CMV Standard 1.00E+02 Yes
Water NTC(no template control) Yes

r. Press ―Finish”. Click on “Name On”

To analyze and print CMV report after a run has finished:
1. The run is done when Profile Program reads: Run has completed.

2. Analyze raw data by clicking each channel one at a time: Green channel (CMV) and Yellow
channel (IC).These tabs are found on the second menu bar from the top, on the left hand side.

3. In the Green Channel. Click “All Off” (bottom right). Select sample ―water (NTC)‖ and ―cmv
QS3 (Standard)‖. Click on Autoscale (bottom left).
Compare each specimen curve to the water curve and the cmv QS3 curve one at a time (by clicking
each sample on and off then proceeding to the next sample). A positive curve will have a sigmoidal
shape. A negative result will be a relatively flat line, like the water. Make a note of all positives on
the worklist.

4. Click on “Named On”.

5. Perform curve Analysis (Get report): Click “Analysis” on the top tool bar.

6. Analysis floating window pops up.

7. Click Quantitation Analysis.

8. Quantitation window is brought up to the front. There are should be two channels (Green/Yellow).

9. Highlight Cycling A. Green option and press Show.

10. Maximize the cycling green channel graph (or the channel you are working on).

11. Press “Import Curve” button on the right side of the screen.

12. Press “From other run” button.

13. Go to My Document -STD curves folder, press “Open”
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 80
Policy # MI/MD/15/v03 Page 6 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cytomegalovirus DNA Detection
14. Choose the latest CMV standard curve (Note: standard curves are changed every 6 months),
“Open”

15. Highlight green channel, press “Import” button.

16. Adjust if only ONE Standard used (Note: Do NOT adjust if ALL Std used)

17. Press button “Slope Correct” on the top.

18. Press button ―Ignore first‖ on the top, and type ―5‖, press OK.

19. Check curve quality, adjust the Threshold.

20. To Print Reports: Press ―Reports‖.

21. Choose Cycling A.Green (Page 1)

22. Highlight Quantitation full report, press ―Show‖ Maximize the report window, check that only
the positives that you have identified from the raw data are positive (i.e. have a crosspoint)

23. Print pages 1-4 for the green channel; pages 2-4 for the yellow channel.
Close the report window.

24. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab,
Select Cycling A. Yellow -the internal control (IC), select Show.
Maximize the graph Quantitation Analysis –Cycling A. Yellow.
Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in
the first 5 cycles of the run).
Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph
screen. Move the threshold line to above the noise.
Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show.
All samples should have a positive IC curve, and a cycle number in the Ct column.
Print pages 2-4.
(For Cycling A. Yellow report, if there is message of ―NEG (Multi Ct)‖ for any specimen, you have
to go back to check Raw Data of cycling A.Yellow. If all specimens are amplified, adjust the
threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct.
Adjust the threshold line so that the threshold line crosses the curve at only one point.)

25. Close the outer most window.
―Save changes to CMV yyyy-mm-dd?‖ Select Yes.
Shut down the computer, and switch off the Rotor-Gene.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 81
Policy # MI/MD/15/v03 Page 7 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cytomegalvirus DNA Detection
V. Calculations:
Perform calculations on CMV PCR blood (CMVB) specimens only. CMV PCR blood (CMVB)
specimens should be reported quantitatively; all CMV PCR non-blood (CMVNB) specimens are
reported qualitatively (CMV DNA detected/ No CMV DNA detected).

Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL)
Sample volume (mL)

Result (copies of DNA/mL) = Result (copies/uL) x 50(uL)
0.200 (mL)
These calculations can also be performed using PCR bench Math excel file.

VI. Reporting:
CMV PCR blood (CMVB) specimens should be reported quantitatively; all CMV PCR non-blood
(CMVNB) specimens are reported qualitatively (ie. CMV DNA detected/ No CMV DNA detected).

PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second
time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report.

CMV PCR Blood (CMVB) Specimens:
CMV PCR negative (has no Ct value) report on the LIS test window, use keypad:
No CMV DNA detected.
This is a research test.
CMV PCR Kit 1.0, Astra Diagnostics Inc.
Under media ―PCCMV‖ F6 to enter the date tested.
Finalize the result (Ctrl+F)

CMV PCR positive, with value in copies of DNA/mL (ie.After calculation has been performed). Report
CMV as an isolate in the isolate window (F7).
Isolate #: 1 Org.ID. 13cmv
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖.
From the keypad select: >CMV+ this will take you to CMV resulting keypads.
If result is <100 copies of DNA/mL select: <100\cmv1
If copies are to be reported select: \CMV+ Type in the copies, in scientific format, in the space
provided.
Grave (`) the result.
Under media ―PCCMV‖ F6 to enter the date tested. If reporting <100 cp/mL enter the actual copy
value.
Interim the result (Ctrl + L).
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 82
Policy # MI/MD/15/v03 Page 8 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cytomegalvirus DNA Detection

CMV PCR Non-Blood (CMVNB) Specimens: Report qualitatively only.
CMV PCR negative, no Ct value. Report on the front of LIS workcard, use keypad:

No CMV DNA detected.
This is a research test.
CMV PCR Kit 1.0, Astra Diagnostics Inc.

Under media ―PCCMV‖ F6 to enter the date tested.
Finalize the result (Ctrl+F)

CMV PCR positive (Ct value present), no calculations are performed. Report CMV as an isolate
window F7.
Isolate #: 1 Org.ID. 13cmv
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖.
Select: >CMV+
Select: \CMV+ Type: DETECTED and delete the ―copies of DNA/mL‖ phrase.
Isolate Comment window F8 should appear as:
DETECTED.
This is a research test.
CMV PCR Kit 1.0, Astra Diagnostics Inc.

Grave (`) the result.
Under media ―PCCMV‖ F6 to enter the date tested.
Interim the result (Ctrl+ L).

Calling Results:
BMT: Call all CMV PCR blood (CMVB) results to the Bone Marrow Transplant Clinic when tested;
CMV PCR positives and negative results.

PMH: Call Positive CMV PCR blood (CMVB) results

TGH: Call Positive CMV PCR blood (CMVB) results, if patients‘ have not had CMV detected in their
blood in more than 2 months. If patients‘ have had CMV detected in their blood within the last 2 months
no calling is necessary.

Outside clients: Call Positive CMV PCR blood (CMVB) and fax results.

Call all Positive CMV PCR Non-blood (CMVNB).

VII. Testing Schedule: CMV PCR blood (CMVB)

PMH patients will be tested as requested.
TGH patients: CMV PCR blood (CMVB) Positive no CMV PCR Blood (CMVB) will be tested for 5
days. Result as: Previous CMV PCR Positive, please resubmit a specimen for testing after 5 days.

VIII. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value
supplied by the manufacturer.
 CMV High Positive and CMV Low Positive External controls should be extracted on the
easyMag daily and run daily. Record external QC results in the T drive, Virology folder, QC
folder.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both extraction
and PCR inhibition.
 CMV QS3 Standard is included and shows a positive reading in Green Channel
 A Negative Control is included and shows a negative reading in Green Channel
 Report all failed QCs to senior/charge technologist.

Failed QC:
Test is invalid without satisfactory QC results.
a. Do not release results pending resolution of QC failure.
b. Inform charge/senior technologist.
c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where
appropriate.
d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can be
released (positive QC material yielded positive result, negative yielded negative result).
e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate of
the run with its expected positive rate.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 84
Policy # MI/MD/15/v03 Page 9 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Cytomegalvirus DNA Detection

h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated
reagents and consumables and disinfect equipment and environment before repeating the run.
i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are often
non-repeatable positives), it is necessary to have a thorough environmental disinfection followed
by swabbing to monitor.
j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental swabs.

IX. References

CMV PCR Kit 1.0, Astra Diagnostics

Epstein Barr Virus DNA Detection
I. Introduction

II. Specimen Collection and Processing:

Please refer to Nucleic Acid Extraction for Whole Blood – Biomerieux NucliSENS easyMAG

III. Materials, Equipments

Clean Room
dedicated Biosafety Cabinet (MIBCT4),
Designated clean room gowns and powder-free gloves
Designated Clean room Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
Cooling Block pre-cooled to 4oC: 72 microtube Corbett Research block
or 36 microtube Corbett Research block
0.1mL microtubes/ or 0.2mL Strip Tubes and Caps

Astra EBV PCR Kit v1.0, (stored at -20o C in clean room):
 Master A (12 tests per vial)
 Master B (12 tests per vial)
 EBV Internal Control (EBV IC)
 Water (PCR grade)

Specimen Preparation area
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
Astra EBV PCR Kit v1.0, (stored at -20o C in clean room):
 QS1 (10^4 Copies/µL)
 QS2 (10^3 Copies/µL)
 QS3 (10^2 Copies/µL)
 QS4 (10^1 Copies/µL)
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 86
Policy # MI/MD/07/v04 Page 2 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Epstein Barr Virus DNA Detection
External Control: To be run when a new lot is used, during training or if QC failure occurs.

Detection Area
Rotorgene 6000 Multiplexing System programmed with ASTRA EBV PCR template
72 Well (Blue) Rotor with Locking Ring/ or 36 Well (Red) Rotor with Locking Ring

GENERAL PRECAUTIONS:
There must be designated separate PCR work areas: 1. Clean Room
2. Specimen preparation area
3. Detection (Amplification) Area
Supplies and equipment must be dedicated to each PCR work area.
Use only aerosol resistant tips (ART).
Work with powder-free gloves; change gloves frequently.
Follow cleaning instructions outlined on daily maintenance schedules.

DETECTION SET UP:
Organize EBV eluates and store at 4oC until ready to load.

Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)
Take out the necessary number of EBV Master Mix A and B vials (12 tests per vial) from
the -20C freezer, quickly and thoroughly thaw inside the Biosafety Cabinet. Prepare EBV
Master Mix (number of samples + controls + 1 extra test) required according to the
chart. Mix gently, do not vortex.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 87
Policy # MI/MD/07/v04 Page 3 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Epstein Barr Virus DNA Detection

a. Place proper number of microtubes for each sample, Controls (Standard and NTC-
Negative) into the Cooling Block (pre-cooled to 4oC).

b. Pipette 20 uL prepared EBV Master Mix (A+B) into each microtube. Lightly cap sample
microtubes.

c. Pipette 1.0 uL of internal control into the microtubes designated for the Standard (EBV
QS3) and the NTC-Negative control.

d. Pipette 10uL water (PCR grade) into the negative control microtube and cap tightly.
Number microtube lids.

e. Changed out of dedicated clean room gown.

Specimen Processing Area:
a. Carefully pipette 10uL of each sample eluate, and Standard (EBV QS3), into designated
numbered microtube containing EBV Master Mix. Gently mix by pipetting up and down.
Close the lid tightly.

b. Visually check that each microtube is tightly closed and that all microtubes have the same
liquid level 30ul, before loading. Ensure that there are no bubbles at the bottom of the
microtubes.

Detection (RotorGene) Area:
a. Turn ON the RotorGene and computer.

b. FOR THE BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press
―→‖ button;
c. Double click (left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon
Wait for ―Initializing machine…..‖
d. Load microtubes into proper rotor (eg. 72 well/or 36-well). Load sample #1 into rotor
position #1, sample #2 into rotor position #2, and so on.
e. Fill the empty positions of the rotor using empty microtubes and snap in the locking ring.
Load the rotor in the RotorGene 6000.
f. Highlight EBV assay;
g. Press ―New‖ button;
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 88
Policy # MI/MD/07/v04 Page 4 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: EBV DNA Detection

h. Highlighting the correct rotor in use: Blue 72-well Rotor
Red 36-well Rotor

i. Click the box ―Locking Ring Attached‖
j. Press “Next” button.
k. Enter initials in Operator space. Enter kit lot number and the Rotor-Gene Colour
(ie.Red/Blue) in the Notes box. Verify the Reaction Volume is 30ul.
l. Press “Next”
m. Temperature Profile window pops up; press ―Next”.
n. Summary window appears; press “Start Run”. The RotorGene will start.
o. Save as window appears; go to My documents -Open–select EBV result folder- Open.
p. Press Save -EBV yyyy-mm-dd (run no.)
q. Edit Samples window. Go to format select 1.23E+05. Under Units select copies/ul.
r. Enter sample LIS numbers using the barcode reader, enter EBV Standard (EBV QS3), and
NTC-water.
Name Type Given conc. Select
Sample Number Unknown Yes
EBV QS3 Standard 1.00E+02 Yes
Water NTC(no template control) Yes

s. Press ―Finish”. Click on “Name On”

To analyze and print EBV report after a run has finished:

1. The run is done when Profile Program reads: Run has completed.

2. Analyze raw data by clicking each channel one at a time: Green channel (EBV) and Yellow
channel (IC).These tabs are found on the second menu bar from the top, on the left hand side.

3. In the Green Channel. Click “All Off” (bottom right). Select sample ―water (NTC)‖ and ―EBV
QS3 (Standard)‖. Click on Autoscale (bottom left).
Compare each specimen curve to the NTC-water curve and the EBV QS3 curve one at a time (by
clicking each sample on and off then proceeding to the next sample). A positive curve will have a
sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of all
positives on the worklist.

4. Click on “Named On”.

5. Perform curve Analysis (Get report): Click “Analysis” on the top tool bar.

6. Analysis floating window pops up.

7. Click Quantitation Analysis.

8. Quantitation window is brought up to the front. There are should be two channels (Green/Yellow).

9. Highlight Cycling A. Green option and press Show.

10. Maximize the cycling green channel graph (or the channel you are working on).

11. Press “Import Curve” button on the right side of the screen.

12. Press “From other run” button.

13. Go to My Document -STD curves folder, press “Open”

14. Choose the latest EBV Standard curve (Note: standard curves are changed every 6 months), -
“Open”

15. Highlight green channel, press “Import” button.

16. Adjust if only ONE Standard used (Note: Do NOT adjust if ALL Std used)

17. Press button “Slope Correct” on the top.

18. Press button ―Ignore first‖ on the top, and type ―5‖, press OK.

19. Check curve quality, adjust the Threshold.

20. To Print Reports: Press ―Reports‖.

21. Choose Cycling A.Green (Page 1)

22. Highlight Quantitation full report, press ―Show‖
Maximize the report window, check that only the positives that you have identified from the raw
data are positive (i.e. have a crosspoint)

23. Print pages 1-4(5) for the green channel; pages 2-4(5) for the yellow channel.
Close the report window.

25. Close the outer most window.
―Save changes to EBV yyyy-mm-dd?‖ Select Yes.
Shut down the computer, and switch off the Rotor-Gene.

Calculations:

Perform calculations on EBV PCR specimens (plasma/urines)

Result (copies of DNA/mL) = Result (copies/uL) x Elution volume (uL)
Sample volume (mL)

Result (copies of DNA/mL) = Result (copies/uL) x 50(uL)
0.200 (mL)

These calculations can also be performed using PCR bench Math excel file.

IV. Reporting

EBV PCR negative (has no Ct value) report on the LIS test window, using the keypads:
Ctrl+D, Ctrl+D, select }EBV-

Negative for EBV virus DNA.
This is a research test.
EBV PCR Kit v1.0, Astra Diagnostics Inc.

Under media ―PCEBV‖ F6 to enter the date tested.
Finalize the result (Ctrl+F)

PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second
time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report.

EBV PCR positive, with value in copies of DNA/mL (ie.After calculation has been performed).
Report EBV as an isolate in the isolate window (F7).
Isolate #: 1 Org.ID. 65ebv
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖.
If >600 copies of DNA/mL select the keypad: \EBV+
Type in the copies, in scientific format, in the space provided.
If result is <600 copies of DNA/mL select: \E600
`Grave (`) the result.
Under media ―PCEBV‖ F6 to enter the date tested. If reporting <600 copies/mL enter the actual copy
value.
Interim the result (Ctrl + L).

V. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value supplied
by the manufacturer.
 EBV High Positive and EBV Low Positive External controls should be extracted on the
easyMag once a week and/or per EBV PCR run. Record external QC results in the T drive,
Virology folder, QC folder.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both extraction
and PCR inhibition.
 EBV QS3 Standard is included and shows a positive reading in Green Channel
 A Negative Control is included and shows a negative reading in Green Channel
 Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.

a. Do not release results pending resolution of QC failure.
b. Inform charge/senior technologist.
c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where
appropriate.
d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can
be released (positive QC material yielded positive result, negative yielded negative result).
e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate of
the run with its expected positive rate.
h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated
reagents and consumables and disinfect equipment and environment before repeating the run.
i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are often
non-repeatable positives), it is necessary to have a thorough environmental disinfection
followed by swabbing to monitor.

j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental
swabs.

VI. References

Astra EBV PCR Kit v1.0 Instructions, Astra Diagnostics

Enterovirus RNA Amplification and Detection Procedures

I. Introduction
Enterovirus belongs to the family Picornaviridae including over 70 distinct serotypes(coxsackie
A and B, echoviruses, polioviruses, and enteroviruses 68-73). They infect a wide variety of
mammals and are associated with a board spectrum of diseases. There are 68 viruses within the
Enterovirus genus that are known to infect humans. Enteroviruses are transmitted primarily by
the fecal-oral route but respiratory spread is possible. Non-polio enteroviruses most commonly
cause rashes, upper respiratory infections. Enteroviruses infections account for a substantial
number of ascetic meningitis and encephalitis cases in summer and fall.

II. Collection and Transport
Blood samples for enterovirus PCR should be collected in EDTA (purple top vacutainer tube).
CSF specimens should be collected in a clean, sterile container and sent to the laboratory as soon
as possible. If specimens cannot be transported immediately, they should be kept at 4oC. If a
delay of more than 6 hours is anticipated, the CSF specimen should be frozen at –70oC. The
EDTA samples should be processed within 6 hours of collection (plasma separated and
refrigerated; frozen if delay is more than 6 hours). Allow only one freeze-thaw cycle.
NOTE: Repeated freezing-thawing will reduce test sensitivity and cryoprecipitates may
accumulate in the plasma.

III. Procedure

A. Worklist Preparation:
a. log on to Softmic,
b. Go to PCR Worklist
ii. Worklist : 1VPCR VIROL PCR
iii. Enter
iv. F12
c. Pending samples (plasma, serum or CSF) will appear on Worklist.
d. Mark received samples using the F5 key, that require Enterovirus PCR
e. Type "`" to print Worklist.

B. LIS Label Printing (if needed for the RNA eluate):
a. While still in the Worklist, press Enter to go to the Test Screen
b. Press F1 to move curser to Demographic field
c. Press: / (to Order/Entry)

D. Specimens Processing:

Specimens should be processed as soon as possible after arriving in virology laboratory.

CSF:
An aliquot (0.2-2 mL) of the CSF should be frozen unless PCR can be performed
immediately

After processing, an aliquot of the left-over specimens should be stored frozen at-70C.

E. Materials, Equipments and Facilities:

Clean Room with dedicated Biosafety Cabinet (MIBCT4), freezer (MIFTG), gowns and
gloves
Specimen Preparation area with Biosafety Cabinet and microcentrifuge
Detection Room for LightCycler
Roche LightCycler programmed for Artus Enterovirus RT-PCR
Microcentrifuge (MICT14)
Vortex
1.5 mL microcentrifuge tubes
Cooling Block with capillary adaptors pre-cooled to 4oC
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
ART-Aersol Resistant Tips for pipettes

From Qiagen QIAampR Viral RNA Mini Kit:
 Collection Tubes
 Lysis Buffer AVL
 Carrier RNA (poly A)-add 310 uL Elution Buffer -AVE to lyophilized carrier RNA
(310 ug) dissolve thoroughly and aliquot into 5.6 uL and 30 uL screw-cap microtubes,
and store at -20C in the clean room. DO NOT freeze-thaw the carrier RNA aliquots
more than 3 times
 Spin Columns
 Wash Buffer 1 (Buffer AW1) – add 125 mL ethanol before use
 Wash Buffer 2 (Buffer AW2) – add 160 mL ethanol before use
 Elution Buffer (Buffer AVE)

From: artusTM Enterovirus LC RT-PCR Kit (stored at -20o C in clean room):
 Quantification Standards (QS). When a new kit is opened, the QS must be
moved to a freezer in the specimen preparation area.
 Internal Control (IC)
 Master Mix (12 tests)
 Water (PCR grade)

Ethanol, anhydrous, reagent grade, 96 - 100%

External Control: To be run when a new lot (Qiagen or RealArt) is used, during training
or if QC failure occurs.

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:

1. Clean room
2. Specimen preparation room
3. Amplification room

 Supplies and equipment must be dedicated to each PCR area and not used for other
activities or moved between areas.
 Change gloves between work areas. Only blue gowns are to be worn in the clean
room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure
 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Prepare and store reagents (lysis buffer, RNA carrier, elution buffer, AW1, AW2 and
ethanol) in the clean room. Keep only working aliquots in the specimen preparation
area.
 Keep the Quantitation Standards and specimens in the specimen preparation area.
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces and
then rinse with 70% alcohol or water.

Clean Room

Volume Lysis Volume Carrier
No. of Buffer -AVL RNA-in Elution
samples (mL) buffer -AVE (uL)
1 0.56 5.6
2 1.12 11.2
3 1.68 16.8
4 2.24 22.4
5 2.80 28.0
6 3.36 33.6
7 3.92 39.2
8 4.48 44.8
9 5.04 50.4
10 5.60 56.0
11 6.16 61.6
12 6.72 67.2

Lysis and Purification:
a. Use Specimen Preparation area; work in cabinet with gown. Use only ART tips. Change
gloves frequently.

b. For each Specimen and External Control, prepare and label two 1.5 mL microcentrifuge
tubes (sterilized, RNase-free) with the corresponding number in the Worklist. Put the
specimens first on the worklist, followed by the external control (if using one). Label one of
them with the lab number (LIS label). This microcentrifuge tube will store the eluted RNA;
the other will be used for processing and will be discarded. Also, label one QIAamp Spin
Column (Qiagen) with the number on the Worklist. Arrange the 2 microcentrifuge tubes and
Columns into 3 rows.

c. Add 560 uL Working Lysis Buffer (AVL with RNA carrier) into each of the microcentrifuge
tubes in one row (these will contain the waste).

e. Incubate at Room Temperature (15-25oC) for 10 minutes.

f. Centrifuge for 10 seconds at 3000 rpm (2000 g) to remove drops from the lids.

g. Add 5 uL Enterovirus Internal Control to each of the microcentrifuge tubes with Buffer
AVL.

h. Add 560 uL ethanol (96-100%) to the samples, vortex for 15 seconds. Centrifuge for 10
seconds at 3000rpm (2000 g) to remove drops from the lids.

i. Pipette 630 uL (half the volume) of the mixture (specimen-lysis buffer-ethanol) to the
QIAamp spin column (in a 2 mL collection tube), close the cap and centrifuge at 8000 rpm
(6000 g) for 1 minute. Place the QIAamp spin column into a clean 2 mL collection tube and
discard the collection tube containing the filtrate.

j. Repeat Step (h) by pipeting the remaining 630 uL of the mixture to the QIAamp spin column
(in a 2 mL collection tube), close the cap and centrifuge at 8000rpm (6000 g) for 1 minute.
Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection
tube containing the filtrate.

k. Open the spin column and add 500 uL of Buffer AW1. Close the cap and centrifuge at 8,000
rpm (6,000 g) for 1 minute.

l. Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection
tube containing the filtrate.

m. Open the spin column and add 500 uL of Buffer AW2. Close the cap and centrifuge at full
speed 14,000 rpm (20,000g) for 3 minutes.

n. Place the spin column into a clean 2 mL collection tube and discard the collection tube
containing the filtrate. Centrifuge at 14,000 rpm (20,000g) for 3 minutes.

Elution Steps:
a. Place the spin column in the second clean 1.5 mL microcentrifuge tube with the LIS
specimen label. Discard the collection tube containing the filtrate. Open the spin column
and add 50 uL of Elution Buffer (Buffer AVE). Close the caps and incubate at room
temperature for 1 minute.

b. Centrifuge at 8000rpm (6000 g) for 1 min. Discard the spin column. Store the eluate at -20oC
or –70oC if not able to proceed immediately.

PCR Amplification and Detection Procedures:

In the Clean Room:
Change into dedicated clean room gown and gloves, work in Biosafety Cabinet. Use only ART tips.

a. Take out the necessary number of Enterovirus Master Mix vials (12 tests per vial) from the
freezer and thoroughly thaw inside the Biosafety Cabinet.

b. Place a capillary for each purified sample, QS, external QC and Negative control (H2O) into the
capillary adaptor of the Cooling Block (pre-cooled to 4oC).

a. Pipette 15 uL Master Mix into the reservoir of each capillary.
b. Pipette 0.5 uL of internal control into the capillaries designated for the QS and the Negative
control.

c. Pipette 5 uL PCR grade water into the negative control capillary and cap.
d. Changed out of dedicated clean room gown

In the Specimen Processing Room: Changed into specimen room gown.

a. Using ART tips, carefully pipette 5 uL each of purified sample, and QS4, (and external QC, if
using) directly into the capillary tube. Immediately close the capillary with a lid.

b. Load capillary tubes into LightCycler (LC) sample carousel and centrifuge at 3000 rpm for 15
seconds in LC centrifuge. Load the LC sample carousel into the LC instrument.

The LightCycler (LC) Instrument:

a. Turn LightCycler on first and then the computer.

b. Enter your user name and password and click OK.

c. Click on the LightCycler icon and push ENTER.

d. Wait for the ―Self-test box‖ to appear.

e. Perform "selftest". ―Skip selftest‖ if performed within 24 hr.

f. Choose OK when the self-test passes.

g. Record that you did the self-test in the log book.

h. Select: "Open Experimental File"
―Enterovirus protocol"; choose ―Open‖
―Run‖

i. Type in file name ―enterovir-year-month-day- run number‖ e.g. enterovir -2004-02-01. Choose
―save‖.

j. Type in the number of samples to run including QS, Negative Control, and External Control (if
using one) in the ―maximum position‖ field.

k. Select ―Enter Samples Later‖. LightCycler will begin protocol.

l. Select ―Edit Samples‖ (bottom right of screen).

m. Select ―Clear Sample List‖. OK

n. Type in the lab numbers; QS; ext. QC (if any) and Negative control.

o. On the QS row, click the arrow-down button to change its description from ―Unknown‖ to
―STD‖ and enter the number of copies/uL (eg. QS3=100)

p. Press ―Done‖

Remember to freeze the end product and patient samples.

To Print Results, go to:
―FLUORESCENCE‖ and click
 F1
 Quantification
 Print Window

To Print Internal Control, go to:
―FLUORESCENCE‖ and click:
 F3/BACK- F1
 Print Window

At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed
by 70% alcohol:

Clean Room:
 Clean Biological Safety Cabinet and turn on UV light for a least ½ hour but not overnight.
 Clean Pipettors

Specimen Preparation Room:
 Seal and discard waste bag
 Clean Biological Safety Cabinet thoroughly
 Wipe all pipettors, centrifuge, and bench top.
 Wash racks.

IV. Reporting:

Fluorimeter Fluorimeter
Channel F1 Channel F3 back- F1 Interpretations
(Target) (IC amplicon)
Possible PCR inhibition-Repeat assay.
- - If still -ve on both Fluorimeter Channels,
report: Indeterminate

- + Sample has no detectable Enterovirus RNA
Report: Negative

Review the F1 graph.
Sample contains Enterovirus RNA.
+ - May need to be repeated. Inform
charge/senior.
Report: POSITIVE
Review the F1 graph.
Sample contains Enterovirus RNA.
+ + May need to be repeated. Inform
charge/senior.
Report: POSITIVE

Positive results for the Enterovirus amplicon (Channel F1) will have the cycle number printed under
the Cross Point Column in the chart and the Specimen Cross point (Ct) will be less than the
Enterovirus QS4 (60 copies/uL).Review the curve, it will be rising exponentially and levels off to a
plateau. Internal Control amplicon need not be positive. Inform Charge of positive specimen, sample
may need to be repeated.

Indeterminate results may be those with a positive signal for the Enterovirus (F1 channel) cycle
number printed under the Cross Point Column, the Specimen Cross point (Ct) is less than the
Enterovirus QS4 (60 copies/uL). Review the curve. Inform Charge of Indeterminate specimen, sample
may need to be repeated.

Indeterminate results may also be those with no signal for the Enterovirus (F1) and also no signal for
the IC amplicon (Channel F3/Back-F1), indicating possible PCR inhibition. Report Indeterminate
specimens to Charge, repeat testing may be required.

Negative results for the Enterovirus amplicon (Channel F1) will be blank under the Cross Point
Column in the chart and the curve will be rather flat. The IC amplicon (Channel F3 back F1) must be
positive to be valid.

Negative Report: Negative by RT-PCR.
This is a research test performed by using artus Enterovirus LC RT-PCR
assay, Artus Biotech Inc.

Indeterminate Report: Indeterminate by RT-PCR
This is a research test performed by using artus Enterovirus LC RT-PCR
assay, Artus Biotech Inc.

Positive Report: POSITIVE by RT-PCR
This is a research test performed by using artus Enterovirus LC RT-PCR
assay, Artus Biotech Inc.

Inform Charge technologist of Positive and Indeterminate results. Record isolate in LIS in F7
Isolate window as 39ent Enterovirus, and call Positive results.

V. Quality Control

 Colour Compensation (to compensate interference between different fluorochromes) should be
run to update the Colour Compensation File.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor extraction
and PCR inhibition. The sample must show a positive reading in either fluorimeter
Channel F1 or fluorimeter Channel F3 back-F1 (IC amplicon) to be valid (no PCR
inhibition).
 A Positive Control (e.g.QS4) is included and shows a positive reading in fluorimeter
Channel F1
 A Negative Control (e.g. H2O spiked with IC) is include and shows a negative reading in
fluorimeter Channel F1 and a positive reading in fluorimeter Channel F3 back F1 (IC
amplicon).

Report all failed QCs to senior/charge technologist

VI. References:

QIAamp Viral RNA Mini Kit Handbook Dec. 2005, Qiagen
ArtusTM Enterovirus LC RT-PCR Kit User Manual March 2006, Artus
LightCycler, Roche Diagnostics

Herpes simplex Virus/Varicella-Zoster Virus DNA Detection

I. Collection and Transport

Sterile body fluids, swabs in viral transport media.
Store collected specimens at 4oC, process specimens as soon as possible.

II. Specimen Processing

Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

III. Materials, Equipments and Facilities:

Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and gloves
Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8)
and microcentrifuge(MICT17)
Vortex
Detection Area for Rotor-Gene 6000
Rotor-Gene 6000 Multiplexing System programmed for ASTRA HSV & VZV
36-Well Loading Block (pre-cooled to 4oC)
36-Well 6000 Series Rotor & Locking Ring
72-Well Loading Block (pre-cooled to 4oC)
72-Well 6000 Series Rotor & Locking Ring
0.1 mL Strip Tubes and Caps
0.2mL microtubes
Aerosol Resistant Tips (ART)
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL

RealStar alpha Herpes PCR Kit v1.0: stored at -20C until needed
Component Master A
Component Master B
HSV-1 Positive Control
HSV-2 Positive Control
VZV Positive Control
PCR Water
alpha Herpes Internal Control

External Control: To be run when a new lot is used, during training or if QC failure occurs.

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:
o Clean room
o Specimen preparation room
 Amplification room Supplies and equipment must be dedicated to each PCR
area and not used for other activities or moved between areas.
 Change lab coats and gloves between work areas. Only blue gowns are to be
worn in the clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure
 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or Eliminase to disinfect equipment and
surfaces and then rinse with 70% alcohol or water.

IV. Detection Set-up:

Organize eluates and controls according to worklist.

In the Clean Room:
(Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

Take out the necessary number of alpha Herpes Components Master A and Master B vials (12 tests per
vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet.

Prepare alpha Herpes Master Mix (number of test samples + controls + 1 extra test) required
according to the chart.
Work quickly. Protect Master A and Master B from light.
Mix gently, DO NOT VORTEX.
Avoid repeated thawing and freezing of Master A and Master B (maximum twice).
After Master A has been mixed to Master B it cannot be frozen again.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180

a. Place microtubes into the Cooling Block (pre-cooled to 4oC).

b. Pipette 20 uL alpha Herpes Master Mix (A+B) into each microtubes

g. Pipette 1.0 uL of internal control (IC) into the microtubes designated for the Positive controls
(HSV-1, HSV-2, VZV) and water (PCR grade).

h. Pipette 10uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and
cap tightly. Label each microtubes 1,2,3…. before leaving clean room.

i. Changed out of dedicated clean room gown.

Specimen Processing Area: (Changed into specimen area lab coat)

Carefully pipette 10 uL each of eluate sample, Positive controls (HSV-1, HSV-2, and VZV), and
external QC (if using) directly into the microtubes containg alpha Herpes Master Mix. Pipette up
and down three times (3X) into the Master Mix to mix sample with Master Mix. Close the cap
tightly.

Detection Area:

 Check each microtube before loading. Make sure the microtube is capped tightly and
there is no bubble at the bottom of the microtube.
 Load microtubes into rotor (e.g. 72 Rotor or 36 Rotor) and secure the locking ring.
 Fill the empty position of the rotor using empty microtubes.
 Place Rotor in to the Rotor-Gene 6000.

On the Rotor-Gene 6000:
a) Turn on computer, turn on Rotogene;
b) FOR THE BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press ―→‖
button;
c) Double click(left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon
d) Wait for ―Initializing machine…..‖
e) Highlight ―ASTRA HSV & VZV ” assay;
f) Press ―New‖ button;
g) Highlighting the correct Rotor type you are using
h) 72-well Rotor (Blue)
i) 36-well Rotor (Red)
j) Click the ―Locking Ring Attached‖ option, to confirm Locking Ring is in place.
k) Press ―Next‖ button
l) Enter initials in Operator space. Enter kit lot number and the Rotor-Gene Colour (ie.Red/Blue) in
the Notes box. Verify the Reaction Volume is 30ul.
m) Press ―Next‖ button.
n) Temperature Profile window pops up; press ―Next”.
o) Summary window appears; press “Start Run”. The RotorGene will start.
p) Save as window appears; go to My documents -Open–select hsv & vzv result folder- Open
Press Save –HSV VZV yyyy-mm-dd (run no.)
q) Edit Samples window. Enter sample LIS numbers using barcode scanner. Enter HSV-1, HSV-2,
VZV as Positive Controls, and water as negative Control.

Name Type Select

Sample Unknown Yes

Positive Controls HSV-1, HSV-2, VZV Positive Control Yes

Water Negative Control Yes

r) Once finishing all samples, press Finish button.
s) Once cycling screen comes on, click ―Named On‖ button.

To analyze and print HSV and VZV report after a run is finished:

The run is done when Profile Program reads: Run has completed.

Analyze raw data by clicking on the each of these channels (one at a time).
Green channel (VZV), Red channel (HSV-2), Orange channel (HSV-1) and Yellow channel (IC).
These tabs are found on the second menu bar from the top, on the left hand side.

Select the Green Channel (VZV). Click “All Off” (bottom right). Select sample ―water
Negative Control” and ―VZV Positive Control‖. Click on Autoscale (bottom left).
Compare each specimen curve to the water curve and the VZV Positive Control one at a time (by
clicking each sample on and off then proceeding to the next sample). A positive curve will have
a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a note of
all positives on the worklist.

Select the Orange Channel (HSV-1). Click “All Off”. Select sample ―water Negative
Control” and ―HSV-1 Positive Control‖. Click on Autoscale (bottom left).
Compare each specimen curve to the water curve and the HSV-1 Positive Control one at a time
(by clicking each sample on and off then proceeding to the next sample). A positive curve will
have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a
note of all positives on the worklist.

Select the Red Channel (HSV-2). Click “All Off”. Select sample ―water Negative Control”
and ―HSV-2 Positive Control‖. Click on Autoscale (bottom left).
Compare each specimen curve to the water curve and the HSV-2 Positive Control one at a time
(by clicking each sample on and off then proceeding to the next sample). A positive curve will
have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a
note of all positives on the worklist.

a. Click on ―Named On‖
b. Perform curve Analysis (Get report): Click ―Analysis‖ on the top tool bar.
c. Analysis floating window pops up;
d. Click Quantitation Analysis.
e. Quantitation window is brought up to the front. There should be four channels: Green (VZV),
Orange (HSV-1), Red (HSV-2) and Yellow (IC).

f. Highlight Cycling A. Green (VZV) option and press Show.
g. Maximize cycling green window (or the channel you are working on)
h. Press button ―Slope Correct‖ on the top.
i. Press button ―Ignore first‖ on the top, and type ―5‖, press OK.
j. Set threshold at 0.05. A threshold line will appear on the screen.
k. Move threshold line to above the noise (i.e. Negative specimens)
l. To Print reports: Press ―Reports‖.

m. Report Browser window pops up.
n. Choose Cycling A. Green (Page 1).
Highlight Quantitation full report, press ―Show‖.
Maximize the report window, check that only the positives that you have identified from the raw
data review are positive and have a crosspoint (ct value).
o. Print pages 1-4 for the green channel; pages 2-4 for all the other channels: orange, red.
p. Repeat the steps above 3-15 for Cycling A. Orange(HSV-1), Cycling A. Red (HSV-2).
q. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab, select Cycling
A. Yellow -the internal control (IC), select Show.
Maximize the graph Quantitation Analysis –Cycling A. Yellow.
Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the readings in
the first 5 cycles of the run).
Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph
screen. Move the threshold line to above the noise.
Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show.
All samples should have a positive IC curve, and a cycle number in the Ct column.
Print pages 2-4.
(For Cycling A. Yellow report, if there is message of ―NEG (Multi Ct)‖ for any specimen, you
have to go back to check Raw Data of cycling A.Yellow. If all specimens are amplified, adjust
the threshold in Quantitation Analysis for Cycling A.Yellow, so that all samples have one Ct.
Adjust the threshold line so that the threshold line crosses the curve at only one point.)
r. Close the outer most window.
s. ―Save changes to Astra HSV & VZV yyyy-mm-dd?‖ Select Yes.
t. Shut down the computer, and switch off the Rotor-Gene.

At the end of the day, follow the cleaning protocol below using 1% hypochorite followed by 70%
alcohol:
Clean Room:
 Clean Biological Safety Cabinet, pipettors, and bench tops

Specimen Preparation Area:
 Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.
 Cap discard container.
 Wash racks.

Green Orange Red Yellow
Channel Channel Channel Channel
Interpretations
(VZV) (HSV-1) (HSV-2) (IC)
-
+ - +/- Positive for VZV

- + -* +/- Positive for HSV-1

- - + +/- Positive for HSV-2

- - - + Negative for HSV and VZV

INVALID: internal control failed to
- amplify. Dilute sample 1:4 and re-extract
- - -
and test.

* Cross-talk: Samples and controls that are strong positives in the Orange HSV-1 Channel, can be
observed to exhibit a diminished weak curve in the Red HSV-2 Channel (y-axis fluorescence is very
weak); these samples and controls are not HSV-2 positive.

PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be repeated a second
time. Re-extract the sample on the easyMAG and repeat the PCR. Release a preliminary report,
see below “Reporting”.

VI. Reporting:

Report HSV and VZV results for sterile body fluids, tissues, eye swabs, and skin/lesion specimens only.
Report HSV results only on oral swabs/washings, and BALs.

Negative for HSV and VZV.
Under media on the back of the LIS workcard. PCHZ: F6 to add run date
Report on the front of the LIS workcard from the keypad select: }HVZ-

}HVZ- Negative for Herpes simplex virus.
Negative for Varicella zoster virus.
This is a research test.
Alpha Herpes Virus PCR Kit v1.0.
Astra Diagnostics Inc.

Ctrl-F to finalize the report.

Sterile sites: PRELIMINARY Positive for HSV-1, or HSV-2, or VZV.
Date the media on the back of the LIS workcard. PCHZ: F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Report the virus as an isolate in the F7 window.

Isolate #: 1
Org. ID: 15hsv1 for HSV-1
15hsv2 for HSV-2
18vzv for VZV
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖. Ctrl D. Select from the keypad: \HSV+ to add the comment
phrase:

\HSV+ DETECTED by PCR, confirmation to follow.
This is a research test.
Alpha Herpes Virus PCR Kit v1.0.
Astra Diagnostics Inc.

Grave (`) the result.
Ctrl-P to prelim the report. Call reports to ward/doctor.

Sterile sites: CONFIRMED Positive for HSV-1, or HSV-2, or VZV.
Date the media on the back of the LIS workcard. PCHZ: F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Report the virus as an isolate in the F7 window.

\HSV+ DETECTED by PCR, confirmed.
This is a research test.
Alpha Herpes Virus PCR Kit v1.0.
Astra Diagnostics Inc.

Grave (`) the result.
Ctrl-F to finalize the report.

Sterile sites: NOT CONFIRMED for HSV-1, or HSV-2, or VZV.
Date the media on the back of the LIS workcard. PCHZ: F6 to add run date

Isolate #: 1 must be suppressed, change to an alphabet
Front of the LIS workcard type the following:

UPDATED REPORT: Previously reported Herpes simplex virus type 1/ Herpes simplex
virus type 2/ or Varicella zoster virus not confirmed.

Report on the front of the LIS workcard (manually type):

This is a research test.
Alpha Herpes Virus PCR Kit v1.0.
Astra Diagnostics Inc.

Ctrl-F to finalize the report. Call Updated reports to ward/doctor.

Positive for HSV-1, or HSV-2, or VZV.
Date the media on the back of the LIS workcard. PCHZ: F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Report the virus as an isolate in the F7 window.

\HSV+ DETECTED by PCR.
This is a research test.
Alpha Herpes Virus PCR Kit v1.0.
Astra Diagnostics Inc.

Grave (`) the result.
Ctrl-F to finalize the report.

Calling Results:

HSV-1, HSV-2, and VZV DETECTED from sterile body fluids, tissues, and eye fluids/swabs should
be called to ward and/or doctor.
HSV-1, HSV-2, and VZV DETECTED from CSF must be faxed and phoned to the Medical Officer of
Health (MOH).
VZV DETECTED from skin/lesion specimens must be phoned to the ward and/or doctor and the
Infection Control Practitioner (ICP) must be notified.

VII. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value supplied
by the manufacturer.
 External control for VZV, HSV-1, and HSV-2 should be extracted on the on the easyMag and
run once a month and/or with each new lot.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both extraction
and PCR inhibition.
 A Positive Control is included and shows either a positive reading in Green Channel
(VZV) or a positive reading in Orange Channel (HSV-1), or a positive reading in Red
Channel (HSV-2)
 A Negative Control is included and shows a negative reading in Green Channel (VZV),
Orange Channel (HSV-1), and Red Channel (HSV-2)
Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.

e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate of
the run with its expected positive rate.
h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated
reagents and consumables and disinfect equipment and environment before repeating the run.
i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are often
non-repeatable positives), it is necessary to have a thorough environmental disinfection followed
by swabbing to monitor.
j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental swabs.

VIII. References:

Astra alpha Herpes Virus PCR Kit v1.0 Instructions, Astra Diagnostics

Influenza A, B and H1N1 Virus rPCR

I. Introduction

Influenza, commonly referred to as the flu, is and infectious disease caused by RNA viruses of
the family Orthomyxoviridae (the influenza viruses), that affects birds and mammals. The
influenza virus gets spread via aerosols. The major surface antigens are hemagglutinin (H) and
neuraminidase (N). The surface antigens change continuously (antigenic drift).
By using Astra Influenza Screen & Type rPCR Kit 1.2, this assay is designed to detect Influenza
B, all Influenza A strains in combination with a H1N1 (swine flu) specific primer probe system
for the detection of all swine influenza strains including the current pandemic strain. The
amplification and detection take place simultaneously, continually and in real time in the Rotor-
GeneTM.
In real-time PCR, fluorescent dyes are linked to oligonucleotide primer/probes, which bind
specifically to the amplified product. Monitoring the level of fluorescence allows the detection
of the amplicon. In the Astra Influenza Screen & Type rPCR assay version 1.2, four fluorescent
dyes are incorporated in the master mix to detect four amplified products: One for SwineFlu
specific amplicon; one for Influenza A specific (non-SwineFlu) amplicon; one for Influenza B
and another one for the Internal Control (IC). The IC is added to each specimen in the initial step
to both control the isolation procedure and to check for PCR inhibition.

The Rotor-GeneTM simultaneously monitors the four different amplicons using four fluorimeter
channels.

II. Collection and Transport

Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs
on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as
possible.

BAL specimens if mucoid treat with working sputolysin (see Appendix). Remove red blood cells
and large particulate matter by micro-centrifuging sample for 5minutes at 3000 rpm in the
Eppendroff 5415 C Micro-centrifuge. Aliquot supernatant and store at 4C, extract on the
easyMag as soon as possible.

III. Specimen Processing

Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

II. Procedure:

a. Worklist Preparation:

Take the Rotor Gene worklist from the virology planting along with the extra labels.

b. Specimens Processing:

 Specimens should be processed as soon as possible after arriving in virology
laboratory.

 Nasophargneal swabs, sputum and BAL‘s are used to detect H1N1 and
Influenza A & B.

 After processing, an aliquot of the left-over specimens should be stored frozen
at-70C.

c. Materials, Equipments and Facilities:

Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and
gloves.
Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8)
and microcentrifuge(MICT17)
Vortex
Detection Room for Rotor-Gene 6000
Rotor-Gene 6000 Multiplexing System programmed for ASTRA H1N1 virus RT PCR
96-Well Loading Block (pre-cooled to 4oC)
72-Well Loading Block (pre-cooled to 4oC)
36-Well High Profile Rotor
36-Well Rotor Locking Ring
72-Well 6000 Series Rotor
72-Well Rotor Locking Ring
0.2mL Tubes
0.1 mL Strip Tubes and Caps
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL

From: Astra Influenze Screen & Type rPCR Kit 1.0, (stored at -20o C in clean room):
 Swine Flu Positive Control
 Influenza A Positive Control
 Influenza B Positive Control
 Master A (12 tests per vial)
 Master B (12 tests per vial)
 Internal Control (IC)
 Water (PCR grade)

External Control: To be run when a new lot is used, during training or if QC failure
occurs.

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:
d. Clean room
e. Specimen preparation room
 Amplification room Supplies and equipment must be dedicated to each PCR
area and not used for other activities or moved between areas.
 Change lab coats and gloves between work areas. Only blue gowns are to be
worn in the clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure

 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or Eliminase to disinfect equipment and
surfaces and then rinse with 70% alcohol or water.

Sample Preparation:
Use Specimen Preparation area; work in cabinet with gown and frequent glove change
Use EasyMag to extract DNA and RNA

PCR Set-up:

In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

a. Take out the necessary number of H1N1 Master Mix A and B vials (24 tests per vial) from the
freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the number of tests and
prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do
not vortex. Make only enough master mix for the tests you are running. After vial A has
been added to Vial B it cannot be frozen again.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 10
2 10 20
3 15 30
4 20 40
5 25 50
6 30 60
7 35 70
8 40 80
9 45 90
10 50 100
11 55 110
12 60 120

b. Place proper micro tubes for each purified sample, Positive controls, external QC and Negative
control (H2O) into the Cooling Block (pre-cooled to 4oC).

c. Pipette 15 uL prepared Master Mix (A+B) into each micro tubes

j. Pipette 1.0 uL of internal control into the micro tubes designated for the Positive controls and
water.

k. Pipette 10uL water (PCR grade) into the negative control micro tube. Mix up and down 3X and
cap tightly. Label each microtubes 1,2,3…. before leaving clean room.

l. Place the cooling block in to a clean biohazard bag.

m. Changed out of dedicated clean room gown.

In the Specimen Processing Room: (Changed into specimen room gown)

a. Using a separate filter tip each time, carefully pipette 10 uL each of purified sample, Positive
controls, and external QC (if using) directly into the micro tubes. Mix by pipetting 3x up and
down into the mastermix. Immediately close the cap tightly.
b. Check each micro tube before loading. Make sure there is no bubble at the bottom of the tube.
c. Load micro tubes into a proper rotor (e.g. 36-well or 72-well) and snap close the locking ring.
Load the rotor in Rotor-Gene 6000.
d. Always fill up the empty position of the rotor using empty tubes.

In the Rotogene room:

a. Turn on computer, turn on Rotogene;
b. FOR THE BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press ―→‖
button;
c. Double click(left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon
d. Wait for ―Initializing machine…..‖
e. Highlight ―Influenza A,B & H1N1” assay;
f. Press ―New‖ button;
g. Highlighting the correct Rotor type you are using
h. 36-well Rotor (Red)
i. 72-well Rotor (Blue)
j. Tick the ―Locking Ring Attached‖ option
k. Press ―Next‖ button
l. Enter initials in operator space. Enter Master Mix lot. # in the Notes space. Make sure the
Reaction Volume is ―25µl‖;
m. Press ―Next‖ button
n. Temperature profile window pops up, press ―Next‖ button
o. Make sure your ring has been loaded in the Rotogene.
p. Summary window pops up, press ―Start Run‖ button;
q. Save As window pops up; the run is given a filename with a default template. Change it
according to the date and numerical runs the assay performed on the same day; e.g. H1N1 2009-
07-17-01 (assay name-year-month-date-numerical run#);
r. Note: Influenza results are stored in \desktop\My Doucument\Influenza A,B&H1N1
s. Press Save button;
t. Machine is starting and Edit Samples window pops up; Enter sample LIS# including positive
and negative controls.
u. Define the type of samples according to the chart:

Name Type Select
Sample Unknown Yes
Positive Controls H1N1, FluA, FluB Positive Control Yes
Water Negative Control Yes

v. Once finishing all samples, press Finish button.
w. Once cycling screen comes on, click ―Named On‖ button.

To analyze and print H1N1 report after a run is finished:

a. The run is done when Profile Program reads: Run has completed.
b. Analyze raw data by clicking on the each of these channels (one at a time): Green channel
(H1N1), Red channel (Flu A other than H1N1), Orange channel (Flu B) and Yellow channel (IC).
These tabs are found on the second menu bar from the top, on the left hand side.
c. Click on autoscale (bottom left). Click ―All Off‖ (bottom right). Click ―water‖ on. Compare the
specimen and control curves to the water one at a time (by clicking each sample on and then off
when proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative
result will be a relatively flat line, like the water. Make a note of the positives on the worklist.
d. Click on ―Named On‖
e. Perform curve analysis: Click Analysis on the top tool bar.
f. Analysis floating window pops up;
g. Choose ―Quantitation‖ tab.
h. Quantitation window is brought up to the front. There should be four channels: Green (H1N1),
Red (Flu A other than H1N1), Orange (Flu B) and Yellow (IC).
i. Highlight Cycling A. Green (Page 1) (H1N1) option and press Show button.
j. Maximize cycling green window (or the channel you are working on)
k. Highlight slope correct tab.
l. Click on Ignore first… tab. A window will pop-up, type in 5 (i.e. ignore 5 cycles).
m. Set threshold at 0.05. A threshold line will appear on the screen.
n. Move threshold line to above the noise (i.e. Negative specimens)

To Print reports: Press Reports button on the top toolbar.
a. Report Browser window pops up.
b. Choose Cycling A. Green (Page 1). Under templates box, choose ―Quantitation full report‖.
c. Press Show button
Maximize the report window, check that only the positives that you have identified from your raw
data analysis are positive (i.e. have a crosspoint)
d. Choose print report. (for the red, orange and yellow channels print only pages 2-4).
e. Once finishing the print, close the report window
f. Go to the floating Analysis window;

g. Repeat the step from ―e‖ to ―m‖ for Cycling A. Red (Influenza A), Cycling A. (Influenza B) and
Cycling A. Yellow (internal control)

Shut down computer:

a. Exit from software. A pop-up window will appear ―save changes‖. Click ―yes‖.
b. Click ―start‖ button. Click ―Turn off computer‖.
c. Turn off Rotogene

At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase
followed by 70% alcohol:

Clean Room:
 Clean Biological Safety Cabinet

Specimen Preparation Room:
 Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.
 Cap discard container.
 Wash racks.

Amplification Room:
 Discard micro tubes into a disposal bag and tight the bag.
 Discard the bag into biohazard waster container.
 Perform the cleaning procedure according the daily maintenance sheet.

IV. Interpretation and Reporting

Red
Green Yellow
Channel Orange
Channel Channel
(Influenza Channel
(H1N1 (IC Interpretations
A specific, (Influenza B)
specific) specific)
non-
swineflu)

+ - - +/- Positive for H1N1

- + - +/- Positive for Influenza A, NOT H1N1

Positive for H1N1 and Influenza A (ask
+ + - +/-
senior for confirmation)

- - + +/- Positive for Influenza B

- - - + Negative for both H1N1 and Influenza A

INVALID
- - - - Test needs to be repeated. Sample needs to
be diluted 1:4
Repeated Diluted 1:4 Sample tested:
- - - -
INDETERMINATE

Note: Check for each curve of the samples showing positive reactions either/or for the Red, Orange and
Green Channel in comparison with the positive and water (negative) controls. Do this step by clicking
the ALL OFF tab and then highlighting the samples showing positive reactions in the Quantitation result
channel.

Positive for H1N1, Influenza A, or Influenza B report as an isolate in the (F7) Isolate Window.
Date the media on the back of the LIS workcard. PCH1R: F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Report the virus as an isolate in the F7 window.
Isolate window (F7)
Isolate #: 1
Org. ID: 23ah1n1 Influenza A H1N1 (Swine) virus
23notsw Influenza A virus NOT H1N1 (Swine)
23infb Influenza B virus
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖. Ctrl D. Select from the keypad: \ANH1 to add the comment
phrase:
DETECTED by PCR.
This is a research test. Influenza Screen & Type RT-PCR Kit v1.2
Astra Diagnostics Inc.
This isolate has been forwarded to PHL (Public Health Laboratory) for subtyping.
Results to follow.

Note: Remove (delete) the sentences that refer to subtyping at PHL when reporting H1N1, and Influenza
B; subtyping is only required for Influenza A isolates.

Grave (`) the result.
Ctrl-F to finalize the report.

Call all positives to ward/doctor and Infection Control Practitioner (ICP). The Medical Officer of Health
(MOH) must be notified by fax and phone. Record all calls and faxes made on the LIS workcard; under
the CALL media and in the Call Window.

Negative for H1N1, Influenza A, and Influenza B.
Date the media on the back of the LIS workcard. PCH1R: F6 to add run date.
On the front of the LIS workcard. Select from the keypad: }H1-

}H1- Negative for Influenza A and B including H1N1 (Swine) virus.
This is a research test.
Influenza Screen and Type RT-PCR Kit v1.2
Astra Diagnostics Inc.

Ctrl-F to finalize the report.

Indeterminate Report. Date the media on the back of the LIS workcard. PCH1R: F6 to add run date.
Select from the keypad ―Failed single‖, type ―IC no amplification‖, select >FAIL
Select from keypad ^PRH1R. Under this media report the dilution test result.
On the front of the LIS workcard. Select from the keypad: }H1-
Change ―Negative‖ to INDETERMINATE.

INDETERMINATE for Influenza A and B including H1N1 (Swine) virus.
This is a research test.
Influenza Screen and Type RT-PCR Kit v1.2
Astra Diagnostics Inc.

Ctrl-F to finalize the report.

III. Quality Control

Reagent QCs:

 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value
supplied by the manufacturer.
 External control for H1N1, Influenza A (not H1N1), and Influenza B should be extracted on
the on the easyMag and run once a month and/or with each new lot.

Daily QCs:

Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both isolation
and PCR inhibition.
 A Positive Control is included and shows either a positive reading in Green Channel
(SwineFlu specific) or a positive reading in Red Channel (Infuenza A specific-non SwineFlu)
 A Negative Control is include and shows a negative reading in both Green Channel
(SwineFlu specific) and Red Channel (Infuenza A specific-non SwineFlu)
 Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.

IV. References:

Astra Influenza screen & Type RT-PCR Kit 1.2 User Manual, Astra

Parvovirus B19 DNA PCR

I. Introduction
Parvovirus B19 belongs to the DNA virus family Parvoviridae and the genus Erythrovirus.
Infection with B19 may result in an asymptomatic infection or produce a wide range of disease;
ranging from erythema infections in children (slapped cheek syndrome or fifth disease) to severe
anemia (the cause of aplastic crisis in hemolytic anemias and in sickle-cell disease), and systemic
disease involving the central nervous system, heart, liver depending on the immune competence
of the host. In pregnant women, Parvovirus B19 may cause hydrops fetalis, congenital anemia,
fetal death, or spontaneous abortion.

II. Collection and Transport
Amniotic fluid stored at 4C and extract on the BioMerieux EasyMag as soon as possible. EDTA
plasma; EDTA blood is spun at 3000 rpm for 10 minutes and the plasma is separated and extract
on the BioMerieux EasyMag as soon as possible.

III. Specimen Processing
Please refer to Nucleic Acid Extraction – Biomerieux NucliSENS easyMAG

GENERAL PRECAUTIONS:
 There must be separate PCR work areas:
 Clean room
 Specimen preparation room
 Amplification room
 Supplies and equipment must be dedicated to each PCR area and not used for other activities
or moved between areas.
 Change lab coats and gloves between work areas. Only blue gowns are to be worn in the
clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure
 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Keep the Quantitation Standards and specimens in the specimen preparation area.
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or RNAse Away to disinfect equipment and surfaces and then
rinse with 70% alcohol or water.

Materials, Equipments and Facilities:

Clean Room with dedicated Biosafety Cabinet (MIBCT4), freezer (MIFTG), gowns and gloves
Specimen Preparation area with Biosafety Cabinet and microcentrifuge
Detection Room for Rotor-Gene 6000
Rotor-Gene 6000 Multiplexing System programmed for ASTRA VZ PCR
72-Well Loading Block (pre-cooled to 4oC)
72-Well 6000 Series Rotor
72-Well Rotor Locking Ring
0.1 mL Strip Tubes and Caps
36-Well Loading Block (pre-cooled to 4oC)
36-Well Rotor Locking Ring
0.2mL Tubes
Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL

From: Astra Parvovirus B19 PCR Kit 1.0, (stored at -20o C in clean room):
 QS1 (10^4 Copies/µL)
 QS2 (10^3 Copies/µL)
 QS3 (10^2 Copies/µL)
 QS4 (10^1 Copies/µL)
 Master A (12 tests per vial)
 Master B (12 tests per vial)
 Internal Control (IC)
 Water (PCR grade)

External Control: To be run when a new lot is used, during training or if QC failure occurs.

IV. Procedure:

Prepare Parvovirus B19 Worklist and arrange eluates in order according to the worklist store at
4C until it is time to add sample to the master mix.
Remove the Parvo B19 QS3 from -20C freezer and place at 4C until ready to load.

In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety
Cabinet)

a. Take out the necessary number of Parvovirus B19 Master Mix A and B vials (12 tests per
vial) from the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the
number of tests and prepare the appropriate volume of Master Mix required according to the
chart. Mix gently, do not vortex.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180

b. Place proper micro tubes for each purified sample, Positive controls, external QC and
Negative control (H2O) into the Cooling Block (pre-cooled to 4oC).
c. Pipette 20 uL prepared Master Mix (A+B) into each micro tubes.
d. Pipette 1.0 uL of internal control into the micro tubes designated for the Positive control
(Parvo B19 QS3 100 cp/uL) and the Negative control.
e. Pipette 10uL water (PCR grade) into the negative control micro tube and cap tightly.
f. Place the cooling block in to a clean biohazard bag.
g. Changed out of dedicated clean room gown.

In the Specimen Processing Area: (Changed into specimen room gown)

a. Using a separate filter tip each time, carefully pipette 10 uL each of purified sample, Positive
controls (QS3), and external QC (if using) directly into the micro tubes. Mix by pipetting up
and down. Close the lid tightly.
b. Check that each micro tube is closed tightly before loading. Make sure there are no bubbles
at the bottom of the micro tubes.

In the RotorGene room:

a. Turn on computer, turn on rotorGene.
b. FOR THE BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press ―→”
button
c. Double click(left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon
d. Wait for ―Initializing machine…..‖
e. Load micro tubes into a proper rotor (eg. 36-well or 72-well).

f. Fill up the empty positions of the rotor using empty microtubes and snap close the locking
ring. Load the rotor in Rotor-Gene 6000.
g. Highlight Parvo B19 assay;
h. Press ―New‖ button;
i. Highlighting the correct Rotor type you are using
72-well Rotor (Blue)
36-well Rotor (Red)
j. Tick the ―Locking Ring Attached‖ option
k. Press ―Next‖ button
l. Enter initials at Operator space and make sure the Reaction Volume is ―30µl‖; enter lot no. in
the Notes section.
m. Press ―Next‖ button
n. Temperature profile window pops up, press ―Next‖ button.
o. Make sure your ring has been loaded in the Rotogene.
p. Summary window pops up, press ―Start Run‖ button. The RotorGene will start.
q. Save as window appears; go to My documents -Open–select Parvo result folder- Open
Press Save –Parvo yyyy-mm-dd (run no.)
r. Press Save button.
s. Machine is starting and Edit Samples window pops up
Enter sample LIS# including positive and negative controls. Define the type of samples
according to the chart:
Name Type Given conc. Select
Sample Number Unknown Yes

QS3 Parvo B19 Positive Control Yes

Water Negative Control Yes

t. Once finishing all samples, press Finish button.
u. Once cycling screen cmes on, click ―Named On‖ button.

To analyze and print Parvo B19 report after a run has finished:
The run is done when Profile Program reads: Run has completed.

Analyze raw data by clicking on the each of these channels (one at a time).
Green channel (Parvo B19), and Yellow channel (IC). These tabs are found on the second menu
bar from the top, on the left hand side.

Select the Green Channel (Parvo B19). Click “All Off” (bottom right). Select sample ―water
Negative Control” and ―QS3 Parvo B19 Positive Control‖. Click on Autoscale (bottom left).
Compare each specimen curve to the water curve and the QS3 Parvo B19 Positive Control one at
a time (by clicking each sample on and off then proceeding to the next sample). A positive curve
will have a sigmoidal shape. A negative result will be a relatively flat line, like the water. Make a
note of all positives on the worklist.

f. Highlight Cycling A. Green (Parvo B19) option and press Show.
g. Maximize cycling green window (or the channel you are working on)
h. Press button ―Slope Correct‖ on the top.
i. Press button ―Ignore first‖ on the top, and type ―5‖, press OK.
j. Set threshold at 0.05. A threshold line will appear on the screen.
k. Move threshold line to above the noise (i.e. Negative specimens)
l. To Print reports: Press ―Reports‖.
m. Report Browser window pops up.
n. Choose Cycling A. Green (Page 1).
Highlight Quantitation full report, press ―Show‖.
Maximize the report window, check that only the positives that you have identified from the
raw data review are positive and have a crosspoint (ct value).

o. Print pages 1-4 for the green channel; pages 2-4 for all the other channels.
p. Yellow channel Analysis. Go to the floating Analysis window: Quantitation tab, select
Cycling A. Yellow -the internal control (IC), select Show.
Maximize the graph Quantitation Analysis –Cycling A. Yellow.
Select Slope Correct. Select Ignore first and type “5” press OK (this will ignore the
readings in the first 5 cycles of the run).
Set the threshold (right-hand side middle) at 0.05. A threshold line will appear on your graph
screen. Move the threshold line to above the noise.
q. Press Reports. Select Cycling A.Yellow. Select Quantitation (Full report). Select Show.
All samples should have a positive IC curve, and a cycle number in the Ct column.
Print pages 2-4.
(For Cycling A. Yellow report, if there is message of ―NEG (Multi Ct)‖ for any specimen,
you have to go back to check Raw Data of cycling A.Yellow. If all specimens are
amplified, adjust the threshold in Quantitation Analysis for Cycling A.Yellow, so that all
samples have one Ct.
Adjust the threshold line so that the threshold line crosses the curve at only one point.)

r. Close the outer most window.
―Save changes to Astra Parvo B19 yyyy-mm-dd?‖ Select Yes.
Shut down the computer, and switch off the RotorGene.

At the end of the day, follow the cleaning protocol below using 1% hypochorite followed by 70%
alcohol:

Clean Room:
 Clean Biological Safety Cabinet, pipettors, and bench tops

Specimen Preparation Area:
 Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.
 Bag and securely close off bag and discard waste
 Wash racks.
 Clean the easyMag according to the daily maintenance sheet

Amplification Area:
 Discard microtubes into a disposal bag and tight the bag.
 Discard the bag into biohazard waster container.
 Perform the cleaning procedure according the daily maintenance sheet: clean the rotor and
cooling blocks with RNase Away, followed by double distilled water, and finally 70% alcohol

Interpretation:

Green Channel Yellow Channel
Parvo B19 target Internal Control Interpretation

- + Report: Negative for Parvo B19 DNA by PCR.

Preliminary report: Positive
Repeat extraction & Parvo B19 PCR to confirm.
+ + Parvovirus B19 DETECTED, confirmation to
follow.
Notify senior or Charge.
Preliminary report: Positive
Repeat extraction & Parvo B19 PCR to confirm.
+ - Parvovirus B19 DETECTED, confirmation to
follow.
Notify senior or Charge.
Possible PCR inhibition.
Repeat extraction & PCR.
- - If still negative on Yellow IC Channel, report:
INDETERMINATE for Parvo B19.

PCR Positives from sterile sites: CSF, eye specimens, biopsy, tissues; should be
repeated a second time. Re-extract the sample on the easyMAG and repeat the PCR.
Release a preliminary report, see below “Reporting”.

VII. Reporting:

Negative for Parvovirus B19.
Under media on the back of the LIS workcard. PCPAR: F6 to add run date
Report on the front of the LIS workcard from the keypad select: }PAR-

}PAR- Negative for Parovirus B19.
This is a research test.
Parvo B19 PCR Kit v1.0.
Astra Diagnostics Inc.
Ctrl-F to finalize the report.

Sterile sites: PRELIMINARY Positive for Parvovirus B19.
Date the media on the back of the LIS workcard. PCPAR: F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Order a repeat Parvo PCR test, on the back of the workcard. >FAIL, Ctrl D, select from keypad
^PRPAR Enter.
Report the virus as an isolate in the F7 window.

Isolate #: 1
Org. ID: 63parB Parvovirus B19
Open the Isolate Comment window F8.
Go to the Virology keypad, type ―V‖. Ctrl D. Select from the keypad: \B19+ to add the comment
phrase.

\B19+ DETECTED by PCR, confirmation to follow.
This is a research test.
PARVO B19 PCR Kit v1.0, Astra Diagnostics Inc.

Manually type the phrase ―confirmation to follow.‖
Grave (`) the result.
Ctrl-P to prelim the report. Call reports to ward/doctor.

Sterile sites: CONFIRMED Positive for Parvovirus B19.
Date the media on the back of the LIS workcard. ^PRPAR : F6 to add run date
Enter on the media line the cross point (ct) value: Ct=
Open the Isolate (F7) Comment window (F8)

Isolate #: 1
Org. ID: 63parB Parvovirus B19
Open the Isolate Comment window F8.
Delete the phrase ―confirmation to follow‖. Manually type: CONFIRMED
The final reporting phrase should appear as below:

\B19+ DETECTED by PCR, CONFIRMED.
This is a research test.
PARVO B19 PCR Kit v1.0, Astra Diagnostics Inc.

Grave (`) the result.
Ctrl-F to finalize the report.

Sterile sites: NOT CONFIRMED for Parvovirus B19.
Date the media on the back of the LIS workcard. ^PRPAR: F6 to add run date

Isolate #: 1 must be suppressed, change to an alphabet
Front of the LIS workcard type the following:

UPDATED REPORT: Previously reported Parvovirus B19 NOT CONFIRMED.

Report on the front of the LIS workcard (manually type):

This is a research test.
This is a research test.
PARVO B19 PCR Kit v1.0, Astra Diagnostics Inc.

Ctrl-F to finalize the report. Call Updated reports to ward/doctor.

V. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value supplied
by the manufacturer.
 External control for Parvo B19 should be extracted on the on the easyMag and run once a month
and/or with each new lot.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both extraction and
PCR inhibition.
 A Positive Control is included and shows either a positive reading in Green Channel (QS3 Parvo
B19)
 A Negative PCR Water Control is included and shows a negative reading in Green Channel
(QS3 Parvo B19)
 Report all failed QCs to senior/charge technologist.

Failed QC:

Test is invalid without satisfactory QC results.

a. Do not release results pending resolution of QC failure.
b. Inform charge/senior technologist.
PROCEDURE MANUAL
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Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Parvovirus B19 DNA PCR

c. Record in Reagent Log Chart, Instrument Maintenance Log or Incident Report where
appropriate.
d. If the QC failure was due to a simple matter of position reversal or misplacement, the run can be
released (positive QC material yielded positive result, negative yielded negative result).
e. If positive QC material yielded negative result, repeat the entire run.
f. If negative QC material yielded positive result, it may be due to cross-contamination from
adjacent positive sample within the run or carry-over contamination from previous runs via
equipment or the environment. Review procedure and equipment to establish and eliminate
potential sources of contamination.
g. The extent and nature of contamination can also be evaluated by comparing the positive rate of
the run with its expected positive rate.
h. If the contamination is extensive, it is necessary to quarantine/discard potentially contaminated
reagents and consumables and disinfect equipment and environment before repeating the run.
i. If a carry-over contamination is suspected (e.g. two or more runs with negative QC being
positive or patient samples have higher than expected positive rate and these samples are often
non-repeatable positives), it is necessary to have a thorough environmental disinfection followed
by swabbing to monitor.
j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental swabs.

Report all failed QCs to senior/charge technologist

VI. References:

Parvo B19 PCR kit v1.0, Astra Diagnostics Inc.

Respiratory Virus Panel (RVP) Multiplex PCR (Luminex)

I. Introduction

The Luminex ID-TagTM RVP is a qualitative PCR assay that simultaneously detects viral nucleic
acid from 18 common respiratory viruses and subtypes in nasopharyngeal and pulmonary
specimens. The RNA viruses detected are Influenza, RSV, Metapneumo, Rhino/Entero,
Parainfluenza and Corona viruses. Adenovirus is the DNA virus also detected by the ID-TagTM
RVP.

The ID-TagTM RVP uses multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR)
and multiplex Target Specific Primer Extension (TSPE) with Tm Bioscience‘s ID-TagTM sorting
system on the Luminex xMAP platform.

II. Collection and Transport

Nasopharyngeal specimens can be collected in Viral Transport Media (VTM) or Universal
Transport Media kits. Bronchoscopy specimens should be collected in a clean, sterile container
and sent to the laboratory as soon as possible. If specimens cannot be transported immediately,
they should be kept at 4oC.

III. Procedure

Sample Preparation

It is recommended that nucleic acid be extracted from nasopharyngeal aspirates or
nasal swabs. There are many commercially available viral nucleic acid extraction kits
available which will provide high quality nucleic acid. Methods which produce poor
quality RNA/DNA may produce sub-optimal results. We use NucliSENS easyMAG™ from
Biomerieux. The RNA/DNA eluates should be stored at -20C or -80C until ready for use.

It is suggested that the recommended extraction Internal Control, (E. coli phage MS 2,) be added to the
sample prior to extraction. MS 2 will be detected by ID-Tag™ RVP.
It is recommended 20 uL added to the extraction reaction.

Internal Control preparation

1 uL of MS2 RNA internal control stock solution + 300 uL of Viral Transport Media = working solution
(Store at -70C)

Multiplex RT-PCR
Preheat Thermal Cycler to 50°C using the preheat instructions:

50°C x 30min
Program RTRVP: 95°C x 15min
[95°C x 30sec / 55°C x 30sec / 72°C x 30sec] X35 cycles
72°C x 2min
4°C forever

Once the Sample reach 50°C, press Pause, then OK.

1. Thaw and bring to room temperature the RVP RT-PCR Primer Mix provided in ID-Tag™ RVP,
dNTPs and 5X Buffer (supplied with Qiagen One Step RT-PCR kit).

2. Label individual RT-PCR tubes and one for Master Mix.

3. Set up Master Mix as follows:

Reagent MM for 1 rxn MM for 8 rxns MM for 48 rxns MM for 96
RNase-free water 5.75 57.50 316.25 603.75
5X QIAGEN One Step RT-PCR Buffer 6.25 62.50 343.75 656.25
dNTP Mix QIAGEN One Step 1.00 10.00 55.00 105.00
RVP RT-PCR Primer Mix 5.00 50.00 275.00 525.00
QIAGEN OneStep RT-PCR Enzyme Mix 2.00 20.00 110.00 210.00
20.00 200.00 1100.00 2100.00
RNA 5.00
Total 25.00

4. Vortex the RT-PCR Master Mix for 2-5 seconds to mix reagents and centrifuge to bring reagents to
the bottom of the tube.
5. Aliquot 20 uL Master Mix into 1st (NC) and the last 2 tubes (NC & Lambda).

6. Add 5 uL of RNase-free water (NC) into 1st and last tube.

7. Get specimens, Lambda, and Internal Control from -70 ºC freezer.

8. (In main virology room) Add 5 uL of Lambda (Run Control) into the 2nd last tube.

9. Make Internal Control dilution by adding 300 uL VTM to the 1 uL IC centrifuge tube.

10. Add MS2 RNA Internal Control into the Master Mix (1uL X #sample + 1 extra). Vortex (Do not
centrifuge). Do not add MS2 into negative control and lambda DNA wells.

11. Aliquot 20 uL of Master Mix with MS2 internal control into the rest of the tubes.
PROCEDURE MANUAL
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Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Multiplex Luminex RVP

12. Add 5 uL of Sample into the remaining tubes.

13. Vortex and centrifuge.

14. Load the strips into Thermal Cycler, and press Resume, then OK.

15. Store RT-PCR tubes at 2-8 ºC until ready to use (for maximum of 48 hours) and DO NOT freeze.

Clean up
1. Make an enzyme mix as follows:

Enzyme MM for 1 rxn MM for 8 rxns MM for 48 rxns MM for 96
Shrimp alkaline phosphatase 2.50 25 137.5 275
Exonuclease 1 2 20 110 220
______
Total 4.50 45.00 247.50 495.00

2. Vortex Enzyme Mix for 2-5 seconds to mix reagents and centrifuge to bring reagents to the bottom
of tube.

3. Dispense 4.5uL enzyme mix into each RT-PCR tube, and pipette up and down to mix.

4. Vortex and centrifuge.

5. Load the strips into Thermal Cycler using the following program:

Program RVPCLEAN: 37°C x 30min
99°C x 30sec
4°C forever

Store treated RT-PCR tubes at 2-8 ºC until ready to use (for maximum of 48 hours)
And don‟t freeze.

Multiplex TSPE
1. Thaw and bring to room temperature the TSPE Primer Mix provided in ID-Tag™ RVP, and 10X
Buffer (supplied with TaKaRa HS).

2. Label TSPE tubes (new set) corresponding to RT-PCR reactions.

3. Set up Master Mix as follows:

Reagent MM for 1 rxn MM for 8 rxns MM for 48 rxns MM for 96
RNase-free Water 9.60 96 528 1008
10X buffer (small vial)3 30 165 315
RVP TSPE Primer Mix 2 20 110 210
TaKaRa (5U/uL) 0.4 4 22 42
15.00 150 825 1575
Treated PCR product 5
______
Total 20

4. Vortex the TSPE Master Mix for 2-5 seconds to mix reagents and centrifuge to bring reagents to the
bottom of the tube.

5. Aliquot 15 uL Master Mix into each TSPE tube.

6. Transfer 5 uL treated RT-PCR product to TSPE tubes with multichannel pipette, and pipette up
and down to mix.

7. Vortex and centrifuge.
8. .
9. Load the strips into Thermal Cycler using the following program:

Program RVPTSPE: 96°C x 2min
[95°C x 30sec / 54°C x 30sec / 72°C x 30sec] X35 cycles
4°C forever

Store TSPE tubes at 2-8 ºC until ready to use (for maximum of 48 hours)
And don‟t freeze.

Bead Hybridization

1. Prior to the hybridization reaction, turn on the Luminex xMAP system and warm up according to
Luminex manual.

2. Thaw and bring to room temperature the Bead Mix, 10X Wash buffer (big vial), and Cal 1, Cal 2,
Con 1, Con 2. Place in dark drawer.

Since the beads are light sensitive, limit their exposure to light at all times during the setup of the
hybridization reaction.

3. Cut Costar thermowells and sealer according to the number of strips.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Section: Molecular Diagnostic Tests Manual Subject Title: Multiplex Luminex RVP

4. Vortex and sonicate Bead Mix (for 10 seconds each) twice to re-suspend.

5. Turn off the light of hood

6. Aliquot 20 uL Bead Mix into Costar thermowells.

7. Transfer 3.5 uL TSPE products into wells with beads using multichannel pipette, and pipette up
and down to mix.

8. Place tubes in Thermal Cycler. Cover wells with PCR sealer and hybridize.

9. Load the strips into Thermal Cycler using the following program:

Program RVPHYB: 96°C x 2min
37 °C x 30min
37 °C forever

Detection
1. About 10 minutes before hybridization is complete, dilute 10X Wash Buffer to 1X Wash Buffer with
RNase-free water in a glass tube.
(Each strip = 100 uL of 10X Wash Buffer + 900uL of RNase-free water).
Cover tube with parafilm and vortex, protect from light until ready to use.

2. Warm up Streptavidin-R-Phycoerythrin reporter to room temperature.

3. Once bead hybridization is complete, transfer the strip to Luminex tray.

4. Make up 1:100 Streptavidin-R-Phycoerythrin Reporter Mix with 1X Wash Buffer.
(Each strip = 10 uL of Reporter + 1000 uL of 1X Wash Buffer).
Cover tube with parafilm and vortex.
Pour into plastic disposable V-shaped troughs (store in the drawer).

5. Add 100 uL Reporter Mix solutions to each well very slowly with multichannel pipette, and
pipette up and down 10X to mix.

6. Incubate for 20 minutes in the Luminex machine. The incubation should be carried out in a dark
place.

7. After 20 minutes incubation, click Start Plate to start the run.

8. Use "TDAS RVP-1.11" to analyze the data.

Luminex 100

1. Turn on the machine (3 switches)
2. Turn on the computer.
3. Select George, enter the password.
4. Select Warmup, press OK, wait about 30 min until Standby appear on Device Status field and
Warmed up appear on Laser Status field.
5. Select Prime, press OK, wait until Idle appear on Command State field.
6. Select Eject button to open the drawer, empty the cuvette and add 75% Alcohol. Click Eject
button to close the drawer.
7. Select Alcohol Flush, press OK, wait until Idle appear on Command State field.
8. Select Eject button to open the drawer, empty the cuvette and add ultraPure distilled H2O. Click
Eject button to close the drawer.
9. Select Wash, press OK, wait until Idle appear on Command State field. Repeat washing two
more times.
10. Select Run Batch, turn on Temp (green dot should appear 35 ºC). Select Home.
11. Cut a Costar thermowell strip with 4 wells only.
12. Select Eject button to open drawer, remove the tray from Luminex, and put Costar thermowell
into A B C D positions.
13. Vortex each bottle well, add 3 drops of each CAL 1, CAL 2, CON 1, CON 2 into A B C D
positions, reload tray into Luminex.
14. Select CAL 1, press OK to start the calibration, click Run Batch to see if calibration finished
with a green check mark. Select Home.
15. Select CAL 2, CON 1, and CON 2 as in step 14.
16. Click Maintenance, click Diagnostics to view if calibration success or not. (green-pass, red-
fail). Select Home.
17. Double click xTAG RVP T-B
18. Enter the number of wells, press Apply.
19. Enter the patient‘s laboratory number under Sample ID, and click Finish.
20. Press Eject button to bring out Luminex tray. Load strips. Press Eject button to close the
drawer.
21. Set timer and incubate for 20 minutes.
22. After 20 min incubation, click Start Plate to start the run.
23. Select Acq. Detail to view the detail result.
24. Select Run Batch to view if run succeeded or not.
25. Once the run is finished, select Home. Select Soak, press OK.
26. Minimize the current window.
27. Select TDAS RVP-I 1.11, press OK
28. Select File, click open
29. Select My Batches look for today‘s batch (month-day-year), click open.
30. Select Output, click open
31. Print the results. Close window (x).
32. Maximize Luminex 100 IS window.

33. Select Eject button to open the drawer, discard the strip. Click Eject button to close the drawer.
34. Turn off the machine (3 switches).
35. Turn off the computer.

Note: If Negative Control is not negative, then highlight another negative control, select Sample, and
mark as Negative Control.

C-1000 Thermocycler – Version 1
To pre-heat Thermocycler (50°C):
1. Turn on (switch at back).
2. Machine will do self testing and booting.
3. Home screen will appear-chose Files press F2.
4. Arrow down to USERS, press ENTER
5. Arrow down to George, press ENTER
6. Arrow down to RTRVP1, press ENTER
7. Press RUN
8. Select the Block or Blocks you are using A, B or both.
9. Press ENTER beside the block or blocks you want. A check mark will appear.
10. Press RUN and wait until current program screen appears.
11. Press RUN again.
12. Press STATUS button.
13. Wait until lid temperature goes to 96°C and the sample temperature reaches 50°C,
press PAUSE (F1), then OK.

After loading the strips:
Press RESUME (F1), then OK

After run finished:
1. Press SKIP STEP
2. Press OK
3. Press MAIN MENU

To set-up Thermocycler using program RVPCLEAN/RVPTSPE/RVPHYP:
1. Home screen will appear-chose Files press F2.
2. Arrow down to USERS, press ENTER
3. Arrow down to George, press ENTER
4. Arrow down to next program you are using CLEANUP/TSPE/RVP1HYB,
press ENTER.
5. Press RUN
6. Select the Block or Blocks you are using A, B or both.
7. Press ENTER beside the block or blocks you want. A check mark will appear.
8. Press RUN and wait until current program screen appears.
9. Press RUN again.
10. Press STATUS button.

After run finished:
11. Press SKIP STEP
12. Press OK
13. Press MAIN MENU

IV. Quality Controls

Run Controls:
Internal Control (IC) must be incorporated into each specimen before extraction to show proper
extraction, amplification and detection. IC is not added to the NC or Lambda Run Control.
Each run must have at least one Negative Control (NC) and a lambda Run Control.
It is recommended that a large run (>20 samples) should have two sets of NC and Lambda.

Expected QC results for each run:
Sample Run Control Internal Control
NC Blank (nothing detected) Blank (nothing detected)
Lambda ―PRES‖ ―ABS‖
Patient sample ―ABS‖ ―PRES‖

Unexpected QC results should be discussed with a senior/charge and results cannot be released until
issue is resolved. Repeating a single test or the run may be necessary.
- Detection of anything under NC suggests possible contamination. Negative patient sample may
be released. Positive patient results may require repeating.
- Patient sample with ABS (absent) under IC will have to be repeated from extraction, possibly
diluted 1:4 to reduce inhibition.
- A patient sample with PRES (present) under Run Control suggests contamination with RC only.
Negative results can still be reported but positive ones will have to be repeated.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 144
Policy # MI/MD/10/v03 Page 9 of 9
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Multiplex Luminex RVP

Statistical considerations:
Compare the positive rate of each run to a few previous runs. If the rate is unexpectedly changed, hold
the results and review with a senior/charge before release.

Reagent Lot Controls:
Each new reagent lot is tested with External QC (with known results) samples against targets in the
multiplex assay to assess assay performance. Due to the numerous targets, this can be achieved by
rotating targets (a different target each run), overlap testing with previous lots or assays and parallel
testing with other (reference) labs.

Proficiency Testing:
CAP
QMPLS

V. Interpretations

In addition to the QC requirement in the preceding section, results of reading <1000 and those with ―No
Call‖ should be withheld and reviewed. If patient was recently tested positive, or with positive results
from elsewhere, results may be released after consulting with a senior/charge.

Sample POS Run Internal _ Interpretations/REPORTING
Calls Control Control _virus
Virus ABS PRES POS __virus DETECTED by
name multiplex PCR
blank ABS PRES NEG Negative by multiplex PCR
No Review or repeat.
Call or
<1000

Specimens may be forwarded to PHL for subtyping, confirmation (low pos) or when equipment is not
operational.

VI. Reporting

Any positive respiratory virus patient results are phoned to the ward or doctor and Infection Control as
soon as possible. Document in LIS call log.
Influenza results are faxed to Public Health Departments.

VII. Reference

Luminex Insert ..\..\Inserts, package\Luminex RVP USA IUO package insert May 8 Clin and EAP2.pdf
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 145
Policy # MI/MD/17/v01 Page 1 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Respiratory Synctial Virus
(RSV) PCR
Issued by: LABORATORY MANAGER Original Date: May 31, 2011
Approved by: Laboratory Director Revision Date:
Annual Review Date:

Respiratory Synctial Virus (RSV) PCR

I. Introduction

Respiratory Synctial Virus (RSV) is a negative sense, single stranded RNA virus of the family
Paramyxoviridae. Based on antigenic properties and sequence analysis RSV are subdivided into
RSV A and RSV B. Different genotypes of both subgroups can co-circulate within one epidemic
with varying frequency. RSV causes respiratory tract infections. For most people, a RSV
infection produces only mild symptoms like common cold. But RSV is the most important cause
of severe respiratory illness in infants and young children and the major cause of infantile
bronchiolitis and pneumonia in industrialized countries. Nearly all children will have been
infected with the virus by 2-3 years of age. RSV also is a significant problem in the elderly, in
persons with cardiopulmonary diseases and in immunocompromised individuals. Repeated
reinfections occur throughout life. RSV is spread by infectious droplets or by contact with nasal
or oral secretions from infected people. Outbreaks of RSV infections are reported worldwide,
with striking annual epidemics in the winter and early spring in temperate climates, like Europe
and North America, and in the rainy season in the tropics.

II. Collection and Transport

Nasopharyngeal swabs collected in Viral Transport Media (VTM), store at 4C. Vortexed swabs
on high for 10 seconds; aliquot off the VTM and store at 4C, extract on the easyMag as soon as
possible.

III. Specimen Processing

Please refer to Nucleic Acid Extraction –Biomerieux NucliSENS easyMAG

GENERAL PRECAUTIONS:

 There must be separate PCR work areas:
f. Clean room
g. Specimen preparation room
 Amplification room Supplies and equipment must be dedicated to each PCR
area and not used for other activities or moved between areas.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
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Page 146
Policy # MI/MD/17/v01 Page 2 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Respiratory Synctial Virus
(RSV) PCR

 Change lab coats and gloves between work areas. Only blue gowns are to be
worn in the clean room.
 Use only filtered pipette tips
 Use only sterile RNase, Dnase-free microcentrifuge tubes
 Use sterile, disposable polypropylene tubes throughout the procedure
 Always wear powder-free gloves when handling reagents
 Change gloves frequently and keep tubes closed whenever possible
 Thaw components thoroughly at room temperature
 Mix components and centrifuge briefly
 Work quickly in the cooling block
 Use 1% sodium hypochloride or Eliminase to disinfect equipment and
surfaces and then rinse with 70% alcohol or water.

a. Materials, Equipments and Facilities:

Clean Room with dedicated Biosafety Cabinet (MIBCT3), freezer (MIFTG), gowns and
gloves
Specimen Preparation area with Biosafety Cabinet (MIBCT7 or MIBCT8)
and microcentrifuge(MICT17)
Vortex
Detection Room for Rotor-Gene 6000
Rotor-Gene 6000 Multiplexing System programmed for ASTRA RSV A & B RT PCR
72-Well Loading Block (pre-cooled to 4oC)
72-Well 6000 Series Rotor
72-Well Rotor Locking Ring
0.1 mL Strip Tubes and Caps
36-Well High Profile Rotor
36-Well Rotor Locking Ring
0.2mL Tubes

Variable volume pipettes: 1 to 20 uL
10 to 200 uL
100 to 1000 uL
From: Astra RSV A & B RT-PCR Kit 1.0, (stored at -20o C in clean room):
 RSV A Positive Control
 RSV B Positive Control
 RSV Master A (12 tests per vial)
 RSV Master B (12 tests per vial)
 RSV Internal Control (IC)
 Water (PCR grade)

External Control: To be run when a new lot is used, during training or if QC failure
occurs.

IV. Procedure:

Take RSV worklist from the virology prep bench and arrange eluates in order according to the
worklist, store the eluates at 4C until ready to add to the RSV Master mix.

PCR Set-up:

In the Clean Room: (Change into dedicated clean room gown and gloves, work in Biosafety Cabinet)

a. Take out the necessary number of RSV A & B Master Mix A and B vials (24 tests per vial) from
the freezer and thoroughly thaw inside the Biosafety Cabinet. Determine the number of tests and
prepare the appropriate volume of Master Mix required according to the chart. Mix gently, do
not vortex. Make only enough master mix for the tests you are running. After vial A has
been added to Vial B it cannot be frozen again.

Preparation of Master Mix
No. of samples
Master A (µl) Master B (µl)
1 5 15
2 10 30
3 15 45
4 20 60
5 25 75
6 30 90
7 35 105
8 40 120
9 45 135
10 50 150
11 55 165
12 60 180

In the Specimen Processing Area: (Change into specimen room gown)

a. Using a separate filter tip each time, carefully pipette 5 uL each of purified sample, Positive
controls, and external QC (if using) directly into the micro tubes. Mix by pipetting 3x up and
down into the mastermix. Immediately close the cap tightly.
b. Check each micro tube before loading. Make sure there is no bubble at the bottom of the tube.
c. Load micro tubes into a proper rotor (e.g. 36-well or 72-well) and snap close the locking ring.
Load the rotor in Rotor-Gene 6000.
d. Always fill up the empty position of the rotor using empty tubes.

In the Rotogene Detection Area:

a. Turn on computer, turn on Rotogene;
b. FOR BLUE ROTOGENE ONLY. Choose clinical icon; pass word ―msh‖; press ―→‖ button;
c. Double click(left on the mouse) ―Rotor-Gene 6000 series software 1.7‖ icon
d. Wait for ―Initializing machine…..‖
e. Highlight ―ASTRA RSV A&B” assay;
f. Press ―New‖ button;
g. Highlighting the correct Rotor type you are using
h. 72-well Rotor (Blue)
i. 36-well Rotor (Red)
j. Tick the ―Locking Ring Attached‖ option
k. Press ―Next‖ button
l. Enter initials in operator space. Enter Master Mix lot. # in the Notes space. Make sure the
Reaction Volume is ―25µl‖;
m. Press ―Next‖ button
Temperature profile window pops up, press ―Next‖ button
n. Make sure your ring has been loaded in the Rotogene.
o. Summary window pops up, press ―Start Run‖ button;
p. Save As window pops up; the run is given a filename with a default template. Change it
according to the date and numerical runs the assay performed on the same day; e.g. RSV A&B
2010.03.29 (e.g. assay name-year-month-date-numerical run#);
a. Note: RSV results are stored in \desktop\My Doucument\RSV A&B results.
q. Press Save button;
r. Machine starts and Edit Samples window pops up;
s. Enter sample LIS# including positive and negative controls. Define the type of samples
according to the chart:
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 149
Policy # MI/MD/17/v01 Page 5 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Respiratory Synctial Virus
(RSV) PCR

Name Type Select
Sample Unknown Yes
RSV A Positive Control Positive Control Yes
RSV B Positive Control Positive Control Yes
Water Negative Control Yes

t. Press Finish button.
u. Once cycling screen comes on, click ―Named On‖ button.

To analyze and print RSV A &B report after a run is finished:
a. The run is done when Profile Program reads: Run has completed.
b. Analyze raw data by clicking on the each of these channels (one at a time): Green channel
(RSV B), Red channel (RSV A) and Yellow channel (IC). These tabs are found on the second
menu bar from the top, on the left hand side.
c. Click on autoscale (bottom left). Click ―All Off‖ (bottom right). Click ―water‖ on. Compare the
specimen and control curves to the water one at a time (by clicking each sample on and then off
when proceeding to the next sample). A positive curve will have a sigmoidal shape. A negative
result will be a relatively flat line, like the water. Make a note of the positives on the RSV
worklist.
d. Click on ―Named On‖
e. Perform curve analysis: Click Analysis on the top tool bar.
f. Analysis floating window pops up
g. Choose ―Quantitation‖ tab.
h. Quantitation window is brought up to the front. There should be three channels: Green (RSV
B), Red (RSV A) and Yellow (IC).
i. Highlight Cycling A. Green (Page 1) (RSV B) option and press Show button.
j. Maximize cycling green window (or the channel you are working on)
k. Highlight slope correct tab.
l. Click on Ignore first… tab. A window will pop-up, type in 5 (i.e. ignore 5 cycles).
m. Set threshold at 0.05. A threshold line will appear on the screen.
n. Move threshold line to above the noise (i.e. Negative specimens)
o. To Print reports: Press Reports button on the top toolbar.
p. Report Browser window pops up.
q. Choose Cycling A. Green (Page 1). Under templates box, choose ―Quantitation full report‖.
r. Press Show button
s. Maximize the report window, check that only the positives that you have identified from your
raw data analysis are positive (i.e. have a crosspoint).
t. Choose print report. (for the red and yellow channels print only pages 2-4).
u. Once finished printing, close the report window
v. Go to the floating Analysis window;
w. Repeat the step from ―e‖ to ―t‖ for Cycling A. Red (RSV A) and Cycling A. Yellow (internal
control)
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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Page 150
Policy # MI/MD/17/v01 Page 6 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Respiratory Synctial Virus
(RSV) PCR

Shut down computer:

a. Exit from software. A pop-up window will appear ―save changes‖. Click ―yes‖.
b. Click ―start‖ button. Click ―Turn off computer‖.
c. Turn off Rotogene
At the end of the day, follow the cleaning protocol below using 1% hypochorite or Eliminase followed
by 70% alcohol:
Clean Room:
 Clean Biological Safety Cabinet

Specimen Preparation Room:
 Clean Biological Safety Cabinet, pipettors, centrifuge, and bench top.
 Cap discard container.
 Wash racks.

Amplification Room:
 Discard micro tubes into a disposal bag and close the bag.
 Discard the bag into biohazard waster container.
 Perform the cleaning procedure according the daily maintenance sheet.

IV. Interpretation and Reporting:

Green Red Yellow
Channel Channel Channel
Interpretations
(RSV B) (RSV A) (IC specific)

+ - +/- Positive for RSV B

- + +/- Positive for RSV A

Positive for RSV A & B (ask senior for
+ + +/-
confirmation)
- - + Negative for both RSV A & B
INVALID
- - - Test needs to be repeated. Sample needs
to be diluted 1:4 with lysis buffer
Note: Check for each curve of the samples showing positive reactions either/or for the Red, Orange
and Green Channel in comparison with the positive and water (negative) controls. Do this step by
clicking the ALL OFF tab and then highlighting the samples showing positive reactions in the
Quantitation result channel.

Positive for RSV A, or RSV B report as an isolate in the (F7) Isolate Window.
Date the media on the back of the LIS workcard PCRSV: F6 to add the date tested.
Enter on the PCRSV media line the crosspoint (ct) value: ct=
Report the virus as an isolate in the F7 window.
Isolate #: 1
Org. ID: 26rsvA Respiratory syncytial virus type A
26rsvB Respiratory syncytial virus type B
Open the Isolate Comment window F8
Go to the virology keypad, type ―V‖. Ctrl D. Select from the keypad: \RSV+ to add the comment
phrase:
\RSV+ DETECTED by PCR.
This is a research test.
RSV RT-PCR Kit v1.0 Astra Diagnostics Inc.

Grave (`) the result.
Ctrl-F to finalize the report.

Call all positives to ward/doctor and Infection Control Practitioner (ICP).

Negative for RSV A and RSV B.
Date the media on the back of the LIS workcard. PCRSV: F6 to add run date.
On the front of the LIS workcard, select from the keypad: }RAB-

}RAB- Negative for Respiratory Syncytial Virus A and B.
This is a research test.
RSV RT-PCR Kit v1.
Astra Diagnostics Inc.
Ctrl-F to finalize the report.

Indeterminate Report. (Internal control failed twice to amplify.) Date the media on the back of the LIS
workcard. PCRSV: F6 to add run date. Select from the keypad ―Failed single‖, type ―IC no
amplification‖, select >FAIL
Select from keypad ^PRRSV. Under this media ^PRRSV report the 1:4 dilution test result type ―IC
no amplification‖.
On the front of the LIS workcard. Select from the keypad: }RSV-
Change ―Negative‖ to INDETERMINATE; final report should appear as:

INDETERMINATE for Respiratory Syncytial Virus A and B.
This is a research test.
RSV RT-PCR Kit v1.
Astra Diagnostics Inc.
.
Ctrl-F to finalize the report.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
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Page 152
Policy # MI/MD/17/v01 Page 7 of 8
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: Respiratory Synctial Virus
(RSV) PCR

V. Quality Control

Reagent QCs:
 An External Control (external to Astra Diagnostics) is used to monitor the isolation,
amplification and detection procedures. The result must correspond to expected value supplied
by the manufacturer.
 External control for RSV A, and RSV B should be extracted on the on the easyMag and run once
a month and/or with each new lot.

Daily QCs:
Every run:
 Each patient specimen must have an Internal Control (IC) added to monitor both isolation
and PCR inhibition.
 A Positive Control is included and shows either a positive reading in Green Channel
(RSV B) or a positive reading in Red Channel (RSV A)
 A Negative Control is include and shows a negative reading in both Green Channel (RSV
B) and Red Channel (RSV A)
 Report all failed QCs to senior/charge technologist.

Failed QC:
Test is invalid without satisfactory QC results.

j. Successful ending to a carry-over contamination may be indicated by QC results and patient
positivity rate falling back to the expected normal range and three negative environmental swabs.

V. References:

RSV A & B RT-PCR Kit v1.0 User Manual, Astra

Procleix® Donor Ultrio® (HBV, HCV, HIV) Assay and West Nile Virus Assay

I. Introduction

The PROCLEIX® Ultrio® and WNV Assay is a qualitative, in vitro nucleic acid test that uses
three technologies-Target capture, Transcription Mediated Amplification (TMA), and
Hybridization Protection Assay (HPA)- to isolate, amplify, and detect HIV-1 RNA, HCV
RNA,HBV DNA, and WNV RNA in human/plasma.

II. Specimen Collection

Serum or plasma may be used. Specimen with visible precipitate or fibrous material should be
centrifuged for 10 minutes at 1000 to 3000 x g prior to testing. Cadeveric blood specimens may
be diluted to overcome potential inhibitory substances or specimen shortage. Plasma and/or
plasma may be diluted 1:5 in saline (0.9% sodium chloride), i.e. 100uL of specimen plus 400 uL
of saline. Diluted specimen should be mixed well by inverting several times before use.

III. Materials, Equipments and Facilities

Reagents:
a. Internal Control Reagent
b. Target Capture Reagent
c. Amplification Reagent
d. Enzyme Reagent
e. Probe Reagent
f. Selection Reagent
g. PROCLEIX® Negative Calibrator
h. PROCLEIX® HIV-1 Positive Calibrator
i. PROCLEIX® HCV Positive Calibrator
j. PROCLEIX® HBV Positive Calibrator
k. PROCLEIX® WNV Negative Calibrator
l. PROCLEIX® WNV Positive Calibrator

Equipments and Materials:
a. PROCLEIX® Target Capture System (TCS)
b. PROCLEIX®HC+ Luminometer and associated PC and printer
c. Worklist Editor ( CPU,Monitor and Printer )
d. PROCLEIX® System Software
e. Vacuum source
f. 3 dedicated circulating water baths: one at 41.5° ± 1°C, one at 60° ± 1°C and one at 62° ±
1°C
g. 3 dedicated water bath spacers to hold the tube racks approximately 1.5‖ from the bottom of
the water bath
h. 2 dedicated multi-tube vortexers
i. 3 dedicated Eppendorf repeat pipettors
j. TTU carriers
k. Ten tube units (TTUs)
l. Ten tip cassettes (TTCs)
m. Sealing Card
n. PROCLEIX® Assay Fluids
o. Wash solution
p. Oil
q. Buffer for Deactivation Fluid
r. Auto Detect 1 and 2
s. Eppendorf Combitips Plus repeat pipettor tips (10.0 ml, 5.0 mL, 2.5 mL) or equivalent
t. 5% sodium hypochlorite in water and 0.5% sodium hypochlorite in water
u. HIV-1 HCV ,HBV and WNV External Quality Controls

IV. Procedure

This assay requires a minimum of three dedicated circulating water baths and one room
temperature water bath. One, at 60° ± 1°C, is used during target capture and pre-amplification;
one at 62° ± 1°C and one at room temperature, are used during post amplification.

1st thing in the morning Turn on all water baths, Luminometer, and computer - (turn computer
on first, then the luminometer), type in User name and password in computer to log on.

Do daily Maintenance on water bath once the temperature has been reached:
i. Check water level
ii. Check for circulating water
iii. Record water bath actual temperature( 0 C)
iv. Record NIST Thermometer temperature( 0 C)
v. Decontaminate exterior at the end of the incubation.

Reagent preparation Area:

1. Thawed reagents, including Amplification Reagent, Enzyme Reagent, Probe Reagent,
working TCR(already prepared) in a 300C water bath .They may be kept at room temperature
up to 8 hours per 24 hour period while in use, not to exceed 80 hours at room temperature.
Do not refreeze these reagents.

2. If a new bottle of Wash Solution, Oil, Selection Reagent, Buffer for Deactivation Fluid, Auto
Detect 1, or Auto Detect 2 is needed, record the date the bottle is opened in the space
provided on the label. These reagents expire 30 days after they are opened, or when master
lot expired.

3. Prepare New working TCR:
a. Thaw one vial of Internal Control Reagent (IC) at room temperature or 2° to 8°C. Mix the
Internal Control Reagent thoroughly by inversion.
b. Remove the TCR from 2° to 8°C storage, and immediately mix vigorously (at least 10
inversions), but do not vortex. Warm the TCR at 22° to 30°C, shaking the bottle
approximately every ten minutes until the precipitate disappears.
c. If a gel forms, return the bottle to 2° to 8°C storage, and use a different bottle of TCR.
d. When both reagents have reached room temperature, mix the TCR by inversion, then
pour the entire vial of IC into the TCR bottle and mix thoroughly. This is now working
TCR.
e. The total time that each reagent left at room temperature is 80 hours.

f. Record the IC‘s lot number, the date it was added, and the expiration date on the bottle of
what is now working TCR.

4. Prepare Deactivation Fluid by mixing one part Buffer for Deactivation Fluid with one part
5% sodium hypochlorite. Deactivation Fluid is stable for 30 days at room temperature.

5. Label sterile, polypropylene conical tubes with sealing caps to use as reagent aliquot vials.

6. Use sterile serological pipettes to aliquot appropriate reagent volumes for the run size.

7. Change gloves.

8. Create tasklist in LIS for Ultrio and /or WNV tests

9. Prepare the samples.

10. Remove enough TTUs (Target Tube Unit) from storage bag carefully (hands do not touch
TTU) and sit them loosely on the TTU rack.

11. Prepare WorkList::
a. Double click on Procleix Worklist Editor
b. Click on ‗New‘
c. Scan in barcode(s) on the TTU.Click on ‘Edit‘, and ‗delete a TTU‘.
d. Scan in Negative and Positive Calibrators and specimens. Click on ‘Edit‘, and ‗delete
sample‘ if needed.We will use this function if less than 10 samples in the TTU.
e. Click on ‗File‘ and ‗Save‘. The file will then be saved on the C:\sneaker net.
f. Transfer the file from the sneaker net to a floppy disc.
g. Bring the disc with you when you go to the‘ Post Amplification Area‘, so the file will be
installed in the computer with the Luminometer.
h. Change Gloves.

12. Using repeater pipette, dispense 400 uL (10 mL Combitip Plus –I division=200 uL, setting
#2) working TCR to all tubes.

Add 500 uL of each Calibrator/Specimen to first TTU using a single used ART tip per
specimen:

For PROCLEIX® Ultrio®:

1. PROCLEIX® Negative Calibrator
2. PROCLEIX® Negative Calibrator
3. PROCLEIX® Negative Calibrator
4. PROCLEIX®HIV-1 Positive Calibrator
5. PROCLEIX®HIV-1 Positive Calibrator
6. PROCLEIX®HCV Positive Calibrator
7. PROCLEIX®HCV Positive Calibrator
8. PROCLEIX®HBV Positive Calibrator
9. PROCLEIX®HBV Positive Calibrator
10. Specimen

For PROCLEIX® WNV:

1. PROCLEIX® Negative Calibrator
2. PROCLEIX® Negative Calibrator
3. PROCLEIX® Negative Calibrator
4. PROCLEIX®WNV Positive Calibrator
5. PROCLEIX®WNV Positive Calibrator
6. PROCLEIX®WNV Positive Calibrator
7. Specimen
8. Specimen
9. Specimen
10. Specimen

Continue adding 500 ul of specimen each to the second TTU, according to the worklist.
Include external controls with each run.

13. Cover with sealing card.

Amplification Area:

Target Capture:

1. Vortex for at least 20 seconds at 1300 RPM (1240-1360) to resuspend particles. Tubes may
remain at room temperature for up to 75 minutes prior to proceeding to 60 ±10C incubation.

2. Incubate in water bath (60 ±10C for 20±1 minutes).

3. Perform Daily Startup on PROCLEIX® TCS(Target Capture System).

4. Incubate at room temperature (14- 20 minutes).

5. Incubate in TCS (9- 20 minutes).

6. Remove sealing card.

7. Change Glove.

8. Aspirate

9. Add 1.0±0.1 ml of Wash Solution.

10. Cover with sealing card.

11. Vortex for at least 20 seconds at 1300 RPM(1240-1360) to resuspend particles.

12. Incubate in TCS (4-10 minutes).

13. Remove sealing card.

14. Change Gloves.

15. Aspirate.

16. Add 1.0±0.1 ml of Wash Solution.

17. Cover with sealing card.

18. Vortex for at least 20 seconds at 1300 RPM (1240-1360) to resuspend particles.

19. Incubate in TCS (4-10 minutes).

20. Remove sealing card.

21. Change Gloves.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 160
Policy # MI/MD/11/v04 Page 7 of 13
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: PROCLEIX® Donor Ultrio®
Assay and West Nile Virus
Assay

22. Aspirate.

23. Cover with sealing card.

Amplification:

1. Remove sealing card.

2. Change Gloves.

3. Pipette 75 uL Amp Reagent (straight, 2.5 mL Combi-tip plus, 1 row) Color changes to
red/dark pink.

4. Pipette 200ulof Oil ( Angle, 10.0 mL Combi-tip plus, 3 rows)

5. Cover with sealing card.

6. Vortex for at least 20 seconds at 1800 RPM(1710-1800) to resuspend particles.

7. Incubate in water bath (60 ±10C for 10±1 minutes).

8. Do daily shut down on PROCLEIX® TCS(Target Capture System after TCS incubation:
a. Change Wash solution to DI water
b. Aspirate DI through manifold(3 ml)
c. Empty and clean Priming trough
d. Clean Workstation Base surface unit, Dispense and Aspiration manifold
e. Clean Ten tip Cassette(TTC) rack
f. Verify vacuum line is dry
g. Empty Waste Bottle and re-filled with 5% Bleach as needed

9. Incubate in water bath (41.5 ±10C for 9- 20 minutes).

10. Remove sealing card (rack remains in 41.5 ±10C water bath).

11. Change Gloves.

12. After the above incubation, while rack is in 41.5 ±10C water bath, pipette 25 uL Enzyme
Reagent.( straight, 2.5 ml Combi-tip plus, 10 row)

13. Cover with sealing card.

14. Manually shake to mix (DO NOT VORTEX).
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 161
Policy # MI/MD/11/v04 Page 8 of 13
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: PROCLEIX® Donor Ultrio®
Assay and West Nile Virus
Assay

15. Incubate in water bath (41.5 ±10C for 60±5minutes).

16. Decontaminate work surfaces and equipments with 0.5% sodium hypochlorite in water, leave
on for minimal of 15 minutes, and rinse with distilled water afterward. Decontamination can
be done during periods of incubation, or at the end of the run.
Soak all racks in 0.5% sodium hypochlorite in water for minimal of 15 minutes, and rinse
with distilled water, air dry.
Remove TTU rack from water bath and transfer to HPA area

Post Amplification (HPA-Hybridization Protective Assay) area:

Hybridization:
Tubes may remain at room temperature for up to 30 minutes prior to the addition of Probe
Reagent.

1. Remove sealing card

2. Change Gloves.

3. Pipette 100ulof Probe Reagent ( Angle, 5.0 ml Combi-tip plus, 3 rows)
Color changes to yellow.

4. Cover with sealing card.

5. Vortex for at least 20 seconds 1600 RPM (1520-1680) until well mixed.

6. Incubate in water bath (62 ±10C for 15±1 minutes).

Selection:
7. Remove the rack of TTU from 620C water bath.

8. Remove sealing card

9. Change Gloves.

10. Pipette 250ulof Selection Reagent ( Angle, 5 ml Combi-tip plus, 3 rows)
Color changes to pink.

11. Cover with sealing card.

12. Vortex for at least 20 seconds 1300 RPM (1240-1360) until well mixed.

13. Incubate in water bath (60 ±10C for 10±1 minutes).You have 75 minutes to complete the
luminometer reading after the Selection incubation.

14. Do daily maintenance on PROCLEIX® HC+ Luminometer:

a. Check detection area temperature and humidity.
b. Lab temperature: 21o to 27o C.
c. Verify that there is adequate amount of Auto Detect Reagents to complete the run.
d. Log on to PROCLEIX® System Software PC:
i. Double click on the icon on the desktop, click ‗OK‘.
ii. Enter the assigned user name and passwords, click ‗OK‘.
e. Press the red button; turn the latch handles to the center to open the lid of the
luminometer.
f. Initialize the instrument: Remove cassette position #1 and #14 from the luminometer.
Press ‗Ultility‘,‘4‘,‘Enter‘,‘0‘,‘Enter‘ and‘ Enter‘ using the keypad on the luminometer.
g. Barcode reader check: Double click on ‗HyperTerminal‘ on the desk top.
h. The ‗Connection description‘ dialog box is displayed.Type‘Test‘ at the ‗Name‘ prompt
and click ‗OK‘
i. Select the COM port 2 using the pull down arrow. Click ‗OK‘
j. The ‗Port Settings‘ screen will now be displayed. Enter the settings as:
 Bits per second: 9600
 Data bits: 8
 Parity: Odd
 Stop bits: 1
 Flow control: Xon/Xoff
Click ‗OK‘
h. Insert a empty TTU into cassette position #1, make sure that the barcode is facing the
front of the Luminometer.
i. Close and latch the luminometer lid.
j. From the computer keyboard, type a capital B using the ‗shift ‗key (make sure that Cap
lock and Num lock are off).
k. Verify that after a delay of no more than three sec, the screen display the character ‗!b‘
followed by the barcode of the TTU in position #1.Verify the barcode on the screen
matches the barcode on the TTU.
l. To exit, select ‗File.Exit‘. A dialogue box asks if you are sure you want to disconnect
now, click ‗YES‘. A dialogue box asks if you want to save the session, click ‗NO‘.

15. Load data from Disc to ‗Sneaker net‘ in computer.

16. Load an empty TTU into position # 1 of the luminometer.

17. Click On Ultrio Procleix System software:
a. Click ‗OK‘ to‘ ETF3‘ version.
b. Type in user name and password.
c. Click on ‗New Run‘.
d. Enter Lot # and expiration date twice.
e. Click ‗Next‘.
f. Start ‗PROCLEIX® HC+‘. Perform Wash Step, click ‗YES‘.
g. Each injector is primed five times into each of the three reaction tubes, for a total of 15
injectors from each injector. Luminometer will beep when wash is completed. Press ‗0‘
and ‗Enter‘. Remove the TTU from the luminometer and check the volume of 3rd tube.
The third tube should be at least ¾ full.

18. Change Gloves.

19. Incubate in water bath (23 ±10C for 10 minutes).

20. Remove TTU rack from container of water and place it on absorbent paper.

21. Remove sealing card.

22. Change Gloves.

23. Remove TTUs from rack and wipe sides and bottom of TTU with DI water dampened tissue.

24. Place TTUs in the luminometer with barcode facing forward and close the lid.

25. Click On ‗Start ‗PROCLEIX® HC+‘.
a. The reading of the TTU will start, can also see graphs(RLU) of each tube on the screen.
b. When finished, will ask to remove TTU from Luminometer,click ‗OK‘
c. Data will be transferred, and report will be printed.
If running WNV as well, TTU for WNV can be loaded the same time as the PROCLEIX®
Ultrio®, leave 1 rack space between the two assays.

26. Click on WNV Procleix System software, repeat Step 70, ii to ix. Do not do ‗Wash Step‘,
click on ‗NO‘ when prompted.

27. When prompted, remove TTUs from luminometer, click ‗OK‘.

28. Click on ‗Main Menu‘, ‗Are you sure you want to exit to the main menu‘, click ‗OK‘. Then
Click on‘ Exit Program‘. Turn off Luminometer. Do a complete ‗Shut Down‘ on the
computer.

29. Add at least 1ml Deactivation Fluid to each reaction tube to deactivate. Allow to sit for a
minimum of 30 minutes, or overnight. Empty all TTU into sink, put all TTU tubes into a
small plastic bag, tape bag tightly, and discard into Biohazard container. Soak the TTU rack
overnight in 0.5% sodium hypochlorite in water.. Rinse with distilled water, and air dry the
next day.

30. Decontaminate work surfaces and equipments with 0.5% sodium hypochlorite in water, leave
on for minimal of 15 minutes, and rinse with distilled water afterward.

V. Assay Validation

1. For the PROCLEIX® Ultrio® Assay At least seven of the nine calibrators must be valid.
Two out of the three replicates of each calibrator (Negative, HIV-1 Positive, HCV Positive,
and HBV Positive) must be valid.
2. For the PROCLEIX® WNV Assay At least four of the six calibrators must be valid.
3. For both PROCLEIX® Ultrio® and WNV Assay, no more than 10% of the calibrators and
specimens may be invalid.
4. The operator may invalidate a run or individual sample if specific technical, operator, or
instrument difficulties were observed and documented. If individual samples are invalidated,
then the 10% run validity must be manually recalculated.
5. Calibrator validity criteria:

For PROCLEIX® Ultrio® Assay:

1. The Negative Calibrator must have an analyte signal that is greater than or equal to 0 RLU
and less than or equal to 45,000 RLU. Its Internal Control (IC) signal must be between
75,000 RLU and 375,000 RLU.
2. The HIV-1 Positive Calibrator must have an analyte signal that is between 300,000 RLU and
1,800,000 RLU and an IC signal that is less than or equal to 475,000 RLU.
3. The HCV Positive Calibrator must have an analyte signal between 200,000 RLU and
1,000,000 RLU, with an IC signal less than or equal to 475,000 RLU.
4. The HBV Positive Calibrator must have an analyte signal between 300,000 RLU and
1,800,000 RLU, with an IC signal less than or equal to 475,000 RLU.
5. Cut-off value for internal control: 0.5 x Negative Calibrator Internal Control Mean RLU.
6. Cut-off for the each Analyte is:
HIV- NC Analyte Mean RLU + 0.02 x (HIV-1 PC Analyte Mean RLU)
HCV- NC Analyte Mean RLU + 0.04 x (HCV PC Analyte Mean RLU)
HBV- NC Analyte Mean RLU + 0.02 x (HBV PC Analyte Mean RLU)

7. If one of the Negative Calibrator replicate values in invalid due to an IC value or an analyte
value outside of these limit, the Negative Calibrator Mean will be recalculated based upon
the two acceptable values. The run is invalid and must be repeated if two or more of the three
Negative Calibrator replicate values have IC values or analyte values that are outside of these
limit.

For PROCLEIX® WNV Assay:

1. On the run report, each sample has a signal to cutoff ratio (S/CO).
2. Report the sample as Nonreactive if the Analyte signal < Analyte cutoff (S/CO <1) and
500,000 ≥ IC ≥ IC cutoff.
3. Report the sample as Invalid if the IC > 500,000 RLU or the Analyte signal < Analyte
cutoff (S/CO<1) and the IC < IC cutoff. For specimen with IC signal > 500,000 RLU, the
specimen will be invalidated by the software, and the reactive status cannot be assessed.
4. Report the sample as Reactive if the Analyte signal ≥ Analyte cutoff (S/CO ≥
the IC ≤ 500,000 RLU.

Reactive Non-Reactive Invalid
Analyte Signal ≥ Analyte Cutoff < Analyte Cutoff < Analyte Cutoff
S/CO ≥1 <1 <1
IC ≤ 500,000 RLU ≥ IC Cutoff and < IC Cutoff
≤ 500,000 RLU > 500,000 RLU

Specimens found to be reactive for PROCLEIX® Ultrio® must be run in individual HIV,
HCV and HBV Discriminatory Assay to determine if they are reactive for HIV-1, HCV,
HBV, or any combination of the three.

VI. Reporting
:
Positive: Reactive result
Negative: Non-Reactive result
Invalid result: inform supervisor for further instruction

VII. Quality Control :

Run External HIV+, HCV+, HBV+ and Negative control with each run.

VIII. Other Maintenances:

Water Bath:
Monthly Maintenance: Empty and clean water bath.

PROCLEIX® TCS (Target Capture System):
Weekly Maintenance: Flush Aspiration Manifold with bleach and rinse with Deionzed water.

PROCLEIX® HC+ Luminometer
Weekly Maintenance: a. Cassette Cleaning:
i. Remove all the Cassettes form the instrument.
ii. Soak the Cassettes in 0.5% sodium hypochlorite in water for 15
minutes.
iii. Rinse thoroughly with distilled water.
b. Interior Chamber Cleaning:
i. Using lint-free tissue, wipe the interior of the instrument
including the barcode scanner with Distilled water.
ii. Dry with lint-free tissue.
iii. Re-install all cassettes. Make sure the cassettes are completely
dry before returning them to the Luminometer chamber.
c. Shut Down:
 When shutting down, always turn the luminometer off first
then the PC.
 When starting up, always turn the PC on first, then the
i. Luminometer
ii. Click the ‗Log off ‗button from the PROCLEIX® System
Software Main Menu screen.
iii. Click the ‗Exit program‘ button from the log on dialog box.
iv. Wait for the PROCLEIX® system Software to exit.
v. Turn off the PROCLEIX® HC+ luminometer.
vi. Exit the operating system by selection Start, Shut Down.
vii. Select Shut Down, and then click OK.

Bi-weekly Maintenance:
Refer to PROCLEIX® System Training Manual Book 2
a. Warm Deionized Water Flush
b. Pump Volume Verification-Protocol 10 and 11.
c. SysCheck

IX. Reference

PROCLEIX® Ultrio Assay Package Insert, 500810, Rev. A.
PROCLEIX® WNV Assay Package Insert, 500630, Rev. A

Procleix® Donor Discriminatory (HBV, HCV, HIV) Assay

I. Introduction:

The PROCLEIX® Donor Discriminatory Assay is a qualitative, in vitro nucleic acid test that
uses three technologies-Target capture, Transcription Mediated Amplification (TMA), and
Hybridization Protection Assay (HPA) - to isolate, amplify, and detect HIV-1 RNA, HCV RNA,
or HBV DNA in human/plasma.
Specimens found to be reactive for PROCLEIX® Ultrio® must be run in individual HIV, HCV,
and HBV Discriminatory Assay to determine if they are reactive for HIV-1, HCV, HBV, or any
combination of the three.

II. Specimens

Specimens found to be reactive for PROCLEIX® Ultrio®.

III. Reagents

1. Internal Control Reagent
2. Target Capture Reagent
3. Amplification Reagent
4. Enzyme Reagent
5. HIV Discriminatory Probe Reagent
6. HCV Discriminatory Probe Reagent
7. HBV Discriminatory Probe Reagent
8. Selection Reagent
9. PROCLEIX® Negative Calibrator
10. PROCLEIX® HIV-1 Positive Calibrator
11. PROCLEIX® HCV Positive Calibrator
12. PROCLEIX® HBV Positive Calibrator

IV. Equipments and Materials

1. PROCLEIX® Target Capture System (TCS)
2. PROCLEIX®HC+ Luminometer and associated PC and printer
3. Worklist Editor ( CPU,Monitor and Printer )
4. PROCLEIX® System Software
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 168
Policy # MI/MD/12/v03 Page 2 of 14
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: PROCLEIX® Donor
Discriminatory Assay

5. Vacuum source
6. 3 dedicated circulating water baths: one at 41.5° ± 1°C, one at 60° ± 1°C and one at 62° ±
1°C
7. 3 dedicated water bath spacers to hold the tube racks approximately 1.5‖ from the bottom of
the water bath
8. 2 dedicated multi-tube vortexers
9. 3 dedicated Eppendorf repeat pipettors
10. TTU carriers
11. Ten tube units (TTUs)
12. Ten tip cassettes (TTCs)
13. Sealing Card
14. PROCLEIX® Assay Fluids
a. Wash solution
b. Oil
c. Buffer for Deactivation Fluid
15. Auto Detect 1 and 2
16. Eppendorf Combitips Plus repeat pipettor tips (10.0 ml, 5.0 mL, 2.5 mL) or equivalent
17. 5% sodium hypochlorite in water and 0.5% sodium hypochlorite in water
18. HIV-1 HCV ,HBV and WNV External Quality Controls

V. Procedure

Perform the assay using a unidirectional workflow in order to minimize the potential for
contamination. Do not return samples, equipment, or reagents to areas where previous steps were
performed. Do not move into previous work areas without taking anti-contamination precautions.
A minimum of three pipettors must be dedicated for use with this assay, including one for target
capture, one for use in amplification, and one for use in post amplification.
This assay requires a minimum of three dedicated circulating water baths and one room
temperature water bath. One, at 60° ± 1°C, is used during target capture and pre-amplification;
one at 62° ± 1°C and one at room temperature, are used during post amplification.

1st thing in the morning Turn on all water baths, Luminometer, and computer-(turn computer on
first, then the luminometer), type in User name and password in computer to log on.

Do daily Maintenance on water bath once the temperature has been reached:
a. Check water level
b. Check for circulating water
c. Record water bath actual temperature( oC)
d. Record NIST Thermometer temperature( oC)
e. Decontaminate exterior at the end of the incubation.

Reagent preparation Area:

1. Thawed reagents, including Amplification Reagent, Enzyme Reagent, Probe Reagent, working
TCR(already prepared) in a 30oC water bath .They may be kept at room temperature up to 8
hours per 24 hour period while in use, not to exceed 80 hours at room temperature. Do not
refreeze these reagents.

2. If a new bottle of Wash Solution, Oil, Selection Reagent, Buffer for Deactivation Fluid, Auto
Detect 1, or Auto Detect 2 is needed, record the date the bottle is opened in the space provided
on the label. These reagents expire 30 days after they are opened, or when master lot expired.

3. Prepare New working TCR, if required:
i. Thaw one vial of Internal Control Reagent (IC) at room temperature or 2° to
8°C. Mix the Internal Control Reagent thoroughly by inversion.
ii. Remove the TCR from 2° to 8°C storage, and immediately mix vigorously (at
least 10 inversions), but do not vortex. Warm the TCR at 22° to 30°C, shaking
the bottle approximately every ten minutes until the precipitate disappears.
iii. If a gel forms, return the bottle to 2° to 8°C storage, and use a different bottle of
TCR.
iv. When both reagents have reached room temperature, mix the TCR by
inversion, then pour the entire vial of IC into the TCR bottle and mix
thoroughly. This is now working TCR.
v. The total time that each reagent left at room temperature is 80 hours.
vi. Record the IC‘s lot number, the date it was added, and the expiration date on
the bottle of what is now working TCR.

4. Prepare Deactivation Fluid by mixing one part Buffer for Deactivation Fluid with one part 5%
sodium hypochlorite. Deactivation Fluid is stable for 30 days at room temperature.

5. Label sterile, polypropylene conical tubes with sealing caps to use as reagent aliquot vials.

6. Use sterile serological pipettes to aliquot appropriate reagent volumes for the run size.

7. Change gloves.

8. Prepare the samples.

9. Remove enough TTUs (Target Tube Unit) from storage bag carefully (hands do not touch
TTU) and sit them loosely on the TTU rack.

10. Prepare WorkList::
i. Double click on Procleix Worklist Editor
ii. Click on ‗New‘
iii. Scan in barcode(s) on the TTU. Use 1 TTU for each assay. Click on ‘Edit‘, and
‗delete a TTU‘.
iv. Scan in Negative and Positive Calibrators and specimens. Click on ‘Edit‘, and
‗delete sample‘ if needed.We will use this function if less than 10 samples in
the TTU.
v. Click on ‗File‘ and ‗Save‘. Click on ‗Worlist pathway‘, click ‗OK‘. The file
will then be saved on the C:\sneaker net.
vi. Transfer the file from the sneaker net to a floppy disc.
vii. Bring the disc with you when you go to the‘ Post Amplification Area‘, so the
file will be installed in the computer with the Luminometer.
viii. Change Gloves.

11. Using repeater pipette, dispense 400 uL (10mL combitip – I division=200 uL, setting #2)
working TCR to all tubes.

For PROCLEIX® Ultrio®: Using a single ART tip per calibrator/specimen

Add 500 uL of each Calibrator/Specimen to the 1st and 2nd TTU:
1st TTU:
1. PROCLEIX® Negative Calibrator
2. PROCLEIX® Negative Calibrator
3. PROCLEIX® Negative Calibrator
4. PROCLEIX®HIV-1 Positive Calibrator
5. PROCLEIX®HIV-1 Positive Calibrator
6. PROCLEIX®HIV-1 Positive Calibrator
7. PROCLEIX®HCV Positive Calibrator
8. PROCLEIX®HCV Positive Calibrator
9. PROCLEIX®HBC Positive Calibrator
10. PROCLEIX®HBC Positive Calibrator

2nd TTU:
1. Specimen
2. Negative External Control
3. Positive HIV External Control

Add 500 uL of each Calibrator/Specimen to the 3rd and 4th TTU:
3rd TTU
1. PROCLEIX® Negative Calibrator
2. PROCLEIX® Negative Calibrator
3. PROCLEIX® Negative Calibrator
4. PROCLEIX® HIV Positive Calibrator
5. PROCLEIX® HIV Positive Calibrator
6. PROCLEIX® HCV Positive Calibrator
7. PROCLEIX® HCV Positive Calibrator
8. PROCLEIX® HCV Positive Calibrator
9. PROCLEIX® HBV Positive Calibrator
10. PROCLEIX® HBV Positive Calibrator

4th TTU:
1. Specimen
2. Negative External Control
3. Positive HCV External Control

Add 500 uL of each Calibrator/Specimen to the 5th and 6th TTU:
5rh TTU
1. PROCLEIX® Negative Calibrator
2. PROCLEIX® Negative Calibrator
3. PROCLEIX® Negative Calibrator
4. PROCLEIX® HIV Positive Calibrator
5. PROCLEIX® HIV Positive Calibrator
6. PROCLEIX® HCV Positive Calibrator
7. PROCLEIX® HCV Positive Calibrator
8. PROCLEIX® HBV Positive Calibrator
9. PROCLEIX® HBV Positive Calibrator
10. PROCLEIX® HBV Positive Calibrator

6th TTU:
1. Specimen
2. Negative External Control
3. Positive HBV External Control

12. Cover with sealing card.

Amplification Area:

Target Capture:

1. Vortex for at least 20 seconds at 1300 RPM (1240-1360) to resuspend particles.
Tubes may remain at room temperature for up to 75 minutes prior to proceeding to 60 ±1oC
incubation.

2. Incubate in water bath (60 ±1oC for 20 ±1 minutes).

3. Perform Daily Startup on PROCLEIX® TCS(Target Capture System).
a. Record room temperature and humidity
b. Change H2O bottle to Wash bottle.
c. Check Wash solution level and Prime x3.
d. Check tubing for kinks.
e. Check dispenser volumes (1.0 ± 0.1 ml) using TTU marked with level for 0.9 and 1.1
mL.
f. Record vacuum pressure( Range 11-36 cm Hg)
g. Check aspiration efficiency.

4. Incubate at room temperature (14- 20 minutes).

5. Incubate in TCS (9- 20 minutes).

6. Remove sealing card.

7. Change Glove.

8. Aspirate

9. Add 1.0 ± 0.1 mL of Wash Solution.

10. Cover with sealing card.

11. Vortex for at least 20 seconds at 1300 RPM (1240-1360) to resuspend particles.

12. Incubate in TCS (4-10 minutes).

13. Remove sealing card.

14. Change Gloves.

15. Aspirate.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 173
Policy # MI/MD/12/v03 Page 7 of 14
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: PROCLEIX® Donor
Discriminatory Assay

16. Add 1.0±0.1 ml of Wash Solution.

17. Cover with sealing card.

18. Vortex for at least 20 seconds at 1300 RPM (1240-1360) to resuspend particles.

19. Incubate in TCS (4-10 minutes).

20. Do daily shut down on PROCLEIX® TCS(Target Capture System after TCS incubation:
i. Change Wash solution to DI water
ii. Aspirate DI through manifold(3 ml)
iii. Empty and clean Priming trough
iv. Clean Workstation Base surface unit, Dispense and Aspiration manifold
v. Clean Ten tip Cassette(TTC) rack
vi. Verify vacuum line is dry
vii. Empty Waste Bottle and re-filled with 5% Bleach as needed.

21. Remove sealing card.

22. Change Gloves.

23. Aspirate.

24. Cover with sealing card.

Amplification:

1. Remove sealing card.

2. Change Gloves.

3. Pipette 75 uL Amp Reagent (straight, 2.5 mL Combi-tip plus, 1 row). Color changes to
red/dark pink.

4. Pipette 200uL of Oil ( Angle, 10.0 mL Comb-tip plus, 3 rows)

5. Cover with sealing card.

6. Vortex for at least 20 seconds at at 1800 RPM (1710-1800) to resuspend particles.

7. Incubate in water bath (60 ±10C for 10±1 minutes).

8. Do daily shut down on PROCLEIX® TCS(Target Capture System after TCS incubation:
i. Change Wash solution to DI water
ii. Aspirate DI through manifold(3 ml)
iii. Empty and clean Priming trough
iv. Clean Workstation Base surface unit, Dispense and Aspiration manifold
v. Clean Ten tip Cassette(TTC) rack
vi. Verify vacuum line is dry
vii. Empty Waste Bottle and re-filled with 5% Bleach as needed

9. Incubate in water bath (41.5 ±10C for 9- 20 minutes).

10. Remove sealing card (rack remains in 41.5 ±10C water bath).

11. Change Gloves.

12. After the above incubation, while rack is in 41.5 ±10C water bath, pipette 25 uL Enzyme
Reagent.( straight, 2.5 ml Combi-tip plus, 10 row)

13. Cover with sealing card.

14. Manually shake to mix (DO NOT VORTEX).

15. Incubate in water bath (41.5 ±10C for 60±5minutes).

17. Remove TTU rack from water bath and transfer to HPA area

Post Amplification (HPA-Hybridization Protective Assay) area:

Hybridization:

Tubes may remain at room temperature for up to 30 minutes prior to the addition of Probe Reagent.

1. Remove sealing card

2. Change Gloves.

3. Pipette 100ulof Probe Reagent ( Angle, 5.0 ml Combi-tip plus, 3 rows). Color changes to
yellow.

4. Cover with sealing card.

5. Vortex for at least 20 seconds 1600 RPM (1520-1680) until well mixed.

6. Incubate in water bath (62 ±10C for 15±1 minutes).

Selection:

1. Remove the rack of TTU from 620C water bath.

2. Remove sealing card

3. Change Gloves.

4. Pipette 200ulof Selection Reagent ( Angle, 12.5 ml Combi-tip plus, 3 rows). Color changes to
pink.

5. Cover with sealing card.

6. Vortex for at least 20 seconds 1300 RPM (1240-1360) until well mixed.

7. Incubate in water bath (60 ±1oC for 10±1 minutes). You have 75 minutes to complete the
luminometer reading after the Selection incubation.

8. Do daily maintenance on PROCLEIX® HC+ Luminometer:
a. Check detection area temperature and humidity. (Lab temperature: 21o to 27o C).
b. Verify that there is adequate amount of Auto Detect Reagents to complete the run.

c. Log on to PROCLEIX® System Software PC:
i. Double click on the icon on the desktop, click ‗OK‘.
ii. Enter the assigned user name and passwords, click ‗OK‘.
d. Press the red button; turn the latch handles to the center to open the lid of the
luminometer.
e. Initialize the instrument: Remove cassette position #1 and #14 from the luminometer.
Press ‗Ultility‘,‘4‘,‘Enter‘,‘0‘,‘Enter‘, and‘ Stop‘.
f. Barcode reader check: Double click on ‗HyperTerminal‘ on the desk top.
g. The ‗Connection description‘ dialog box is displayed.Type‘Procleix HC+‘ at the
‗Name‘ prompt and click ‗OK‘
h. Select the COM port 2 using the pull down arrow. Click ‗OK‘
i. The ‗Port Settings‘ screen will now be displayed. Enter the settings as:
 Bits per second: 9600
 Data bits: 8
 Parity : Odd
 Stop bits: 1
 Flow control: On/off
Click ‗OK‘
j. Insert a TTU into cassette position #1, make sure that the barcode is facing the front of
the Luminometer.
k. Close and latch the luminometer lid.
l. From the computer keyboard, type a capital B using the ‗shift ‗key (make sure that Cap
lock and Num lock are off).
m. Verify that after a delay of no more three sec, the screen display the character ‗!b‘
followed by the barcode of the TTU in position #1.Verify the the barcode on the screen
matches the barcode on the TTU.
n. To exit, select ‗File.Exit‘.A dialogue box asks if you are sure you want to disconnect
now, click ‗YES‘. A dialogue box asks if you want to save the session, click ‗NO‘.

9. Load data from Disc to ‗Sneaker net‘ in computer.

10. Load an empty TTU into position # 1 of the luminometer.

11. Click On Ultrio Procleix System software:
i. Click ‗OK‘ to‘ ETF3‘ version.
ii. Type in user name and password.
iii. Click on ‗New Run‘.
iv. Enter Lot # and expiration date twice.
v. Click ‗HIV, HCV or HBV.
vi. Click ‗Next‘.
vii. Start ‗PROCLEIX® HC+‘. Perform Wash Step; click ‗YES‘.

viii. Each injector is primed five times into each of the three reaction tubes, for a
total of 15 injectors from each injector. Luminometer will beep when wash is
completed. Press ‗0‘ and ‗Enter‘. Remove the TTU from the luminometer and
check the volume of 3rd tube. The third tube should be at least ¾ full.

12. Change Gloves.

13. Incubate in water bath (23 ±10C for 10 minutes).

14. Remove TTU rack from container of water and place it on absorbent paper.

15. Remove sealing card.

16. Change Gloves.

17. Remove TTUs from rack and wipe sides and bottom of TTU with DI water dampened tissue.

18. Place TTUs in the luminometer with barcode facing forward and close the lid. Load all TTUs,
leave 1 rack space between different assays.

19. Click On ‗Start ‗PROCLEIX® HC+‘.
i. The reading of the TTU will start, can also see graphs(RLU) of each tube on
the screen.
ii. When finished, will ask to remove TTU from Luminometer, click ‗OK‘
iii. Data will be transferred, and report will be printed.

20. Do the same for HCV and HBV.Repeat step 70- i to iv, say ‗No‘ to Wash Step.

21. Repeat step 78.

22. When prompted, remove TTUs from luminometer.

23. Add at least 1ml Deactivation Fluid to each reaction tube to deactivate. Allow to sit for a
minimum of 30 minutes, or overnight. Empty all TTU into sink, put all TTU tubes into a small
plastic bag, tape bag tightly, and discard into Biohazard container. Soak the TTU rack
overnight in 0.5% sodium hypochlorite in water.. Rinse with distilled water, and air dry the
next day.

24. Decontaminate work surfaces and equipments with 0.5% sodium hypochlorite in water, leave
on for minimal of 15 minutes, and rinse with distilled water afterward.

VI. Assay Validation

Calibrator validity criteria. For each PROCLEIX® Donor Discriminatory Assay:
1. The Negative Calibrator must be run in triplicates. The Negative Calibrator must have an
analyte signal that is greater than or equal to 0 RLU and less than or equal to 45,000 RLU. Its
Internal Control (IC) signal must be between 75,000 RLU and 375,000 RLU.
2. The HIV-1 Positive Calibrator must have an analyte signal that is between 300,000 RLU and
1,800,000 RLU and an IC signal that is less than or equal to 475,000 RLU.
3. The HCV Positive Calibrator must have an analyte signal between 400,000 RLU and
2,700,000 RLU, with an IC signal less than or equal to 475,000 RLU.
4. The HBV Positive Calibrator must have an analyte signal between 300,000 RLU and
1,800,000 RLU, with an IC signal less than or equal to 475,000 RLU.
5. Cut-off value for internal control: 0.5 x Negative Calibrator Internal Control Mean RLU.
6. Cut-off for the each Analyte is:
HIV- NC Analyte Mean RLU + 0.04 x (HIV-1 PC Analyte Mean RLU)
HCV- NC Analyte Mean RLU + 0.04 x (HCV PC Analyte Mean RLU)
HBV- NC Analyte Mean RLU + 0.04 x (HBV PC Analyte Mean RLU)
7. If one of the Negative Calibrator replicate values in invalid due to an IC value or an analyte
value outside of these limit, the Negative Calibrator Mean will be recalculated based upon
the two acceptable values. The run is invalid and must be repeated if two or more of the three
Negative Calibrator replicate values have IC values or analyte values that are outside of these
limit.

VII. Interpretation of results

For Each PROCLEIX® Donor Discriminatory Assay:
1. On the run report, each sample has a signal to cutoff ratio (S/CO).
2. Report the sample as Nonreactive if the Analyte signal < Analyte cutoff (S/CO < 1) and
475,000  IC  IC cutoff.
3. Report the sample as Invalid if the IC > 475, 000 RLU or the Analyte signal < Analyte cutoff
(S/CO<1) and the IC < IC cutoff.
4. Report the sample as Reactive if the Analyte signal Analyte cutoff (S/CO 1) and the IC
475, 000 RLU.

Reactive Non-Reactive Invalid
Analyte Signal  Analyte Cutoff < Analyte Cutoff < Analyte Cutoff
S/CO 1 <1 <1
IC 475,000 RLU IC Cutoff and < IC Cutoff
 475,000 RLU >475,000 RLU

VIII. Reporting

Positive: Reactive result
Negative: Non-Reactive result

IX. Quality Control

Run External HIV+, HCV+, HBV+ and Negative control with each run.
Perform proficiency testing by CAP for HBV DNA and HCV RNA.
Perform proficiency testing by NML for WNV RNA PCR.
Perform proficiency testing by QMPLS for HIV RNA viral load.

X. Other Maintenances

Water Bath:
Monthly Maintenance: Empty and clean water bath.

PROCLEIX® TCS (Target Capture System):
Weekly Maintenance: Flush Aspiration Manifold with bleach and rinse with Deionzed
water.

PROCLEIX® HC+ Luminnometer
Weekly Maintenance: a. Cassette Cleaning:
i. Remove all the Cassettes form the instrument.
ii. Soak the Cassettes in 0.5% sodium hypochlorite in water for 15
minutes.
iii. Rinse thoroughly with distilled water.
b. Interior Chamber Cleaning:
i. Using lint-free tissue, wipe the interior of the instrument
including the barcode scanner with Distilled water.
ii. Dry with lint-free tissue.
iii. Re-install all cassettes. Make sure the cassettes are completely
dry before returning them to the Luminometer chamber.
c. Shut Down:
 When shutting down, always turn the luminometer off first
then the PC.
 When starting up, always turn the PC on first, then the
Luminometer
i. Click the ‗Log off ‗button from the PROCLEIX® System
Software Main Menu screen.
ii. Click the ‗Exit program‘ button from the log on dialog box.
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 180
Policy # MI/MD/12/v03 Page 14 of 14
Microbiology Department
Policy & Procedure Manual
Section: Molecular Diagnostic Tests Manual Subject Title: PROCLEIX® Donor
Discriminatory Assay

iii. Wait for the PROCLEIX® system Software to exit.
iv. Turn off the PROCLEIX® HC+ luminometer.
v. Exit the operating system by selection Start, Shut Down.
vi. Select Shut Down, and then click OK.
Two week Maintenance: Refer to PROCLEIX® System Training Manual Book 2
a. Warm Deionized Water Flush
b. Pump Volume Verification-Protocol 10 and 11.
c. SysCheck

XI. Reference

PROCLEIX® Ultrio Assay Package Insert, 500810, Rev. A.

APPENDIX I - Molecular Diagnostic Tests Schedule

Chlamydia PCR ............................................................................................................................. daily
C. difficile GeneXpert PCR ......................................................................................................... hourly
HCV RNA PCR......................................................................................... twice per week (as needed)
HBV DNA ............................................................................................................. daily (if needed)
West Nile PCR........................................................................................................................................

* Stat testing may be required where clinically justified.

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

Good for 6 hrs at RT

APPENDIX III - Autoverification for ProbeTec®

POLICY:

ProbTtec 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 ?NEGATIVE Autoverifies/Autofi Canned
gonorrhoeae nalizes Message:
}GCNE

Canned Messages:

}CHLP - POSITIVE for Chlamydia 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.

APPENDIX IV - 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) Bench
b) Lysing rack
c) Pipette
d) Priming heater
e) Lysing heater

4. Run all 6 swabs with specimen run.
5. Record results in BD ProbeTec® ET Binder.

APPENDIX V - General PCR Precautions and Decontamination Procedures

General PCR Precautions:

 There must be separate PCR work areas:

 Clean room
 Specimen preparation room
 Amplification room

 Workflow must proceed in a uni-directional manner beginning in the clean room area and
moving to the amplification area.

 Supplies and equipment must be dedicated to each area and not used for other activities or
moved between areas.

 Change laboratory coat and gloves between work areas.

 Wear powder-free gloves at all times. Hands and dust particles may carry bacteria and molds
and are the most common sources of Rnase contamination.

 All reagents and master mixes must be prepared and stored in the clean room.

 Use sterile aerosol resistant pipette tips.

 Use 1% sodium hypochloride or Eliminase to disinfect equipment and surfaces
and then rinse with 70% alcohol or water.

 Racks, surfaces and equipment that can be cleaned are cleaned daily.

Decontamination Procedure:

When contamination occurs the following steps must be followed:

 Use 1% sodium hypochlorite followed by alcohol (water) to decontaminate.
 Change into clean lab coat
 Begin cleaning in the clean room and end in the amplification room.
 Discard all reagents and disposable items (eg. pipette tips, capillary caps)
 Clean biological safety cabinet, equipment and surfaces.
 For WNV PCR decontamination, see checklist below.

WNV Decontamination Procedure:

Clean room:

 Discard pipette tips, capillaries, caps, pens and eppendorf tubes
 Clean biological safety cabinet, pipettors, racks and capper
 Turn UV light on in the safety cabinet

Specimen preparation area:

 Discard pipette tips, caps, pens, and reagents (AW1, AW2, alcohol, elution buffer and lysing
buffer)
 Clean: Biological safety cabinet
Pipettors and holder
Racks
Vortex
Centrifuge
Timer
Bench
Capillary capper

Amplification area:

 Clean: Microwave
Capillary centrifuge
Capillary carousel
Light Cycler

Perform wipe tests on all 3 areas:

 Clean room: biological safety cabinet, pipettors, racks and capper

 Specimen preparation area: Biological safety cabinet
Pipettors and holder
Racks
Vortex
Centrifuge
Timer
Bench
Capillary capper

 Clean room:
Microwave
Capillary centrifuge
Capillary carousel
Light Cycler

If any wipe test is positive, repeat cleaning procedure for that area.

APPENDIX VI – Virology Specimen Accessioning Guide

Virology Specimen Accessioning Guide.xls

Manual Section Name: Molecular Diagnostic Tests

Page Number / Item Date of Revision Signature of
Approval
Molecular Testing - HBV DNA-Reporting Added Para. October 10, 2003 Dr. T. Mazzulli
Page 67 - Molecular Testing - Chlamydia Trachomatis & May 20, 2005 Dr. T. Mazzulli
Neisseria gonorrhoeae
Page 67 – urine but not more than 60 mL added May 20, 2005 Dr. T. Mazzulli
Page 68 – specimen not lysed…added May 20, 2005 Dr. T. Mazzulli
Page 68 – tubes may be blotted May 20, 2005 Dr. T. Mazzulli
Page 70 – handling samples after lysing May 20, 2005 Dr. T. Mazzulli
Page 71,72 Procedure change May 20, 2005 Dr. T. Mazzulli
Page 72, interpretation of result - change MOTA score May 20, 2005 Dr. T. Mazzulli
Page 73, reporting – indeterminate May 20, 2005 Dr. T. Mazzulli
Page 76 – do not vortex working master mix May 20, 2005 Dr. T. Mazzulli
Page 76 – specimen dilution protocol May 20, 2005 Dr. T. Mazzulli
Page 77 – 80 procedure and reporting changed May 20, 2005 Dr. T. Mazzulli
Page 81 – QC procedure changed May 20, 2005 Dr. T. Mazzulli
Page 93 – procedure changed May 20, 2005 Dr. T. Mazzulli
Page 86 – 88, instrument instructions May 20, 2005 Dr. T. Mazzulli
Page 90 – WNV RT-PCR May 20, 2005 Dr. T. Mazzulli
Page 92 – lysis buffer May 20, 2005 Dr. T. Mazzulli
Page 93 – General Precaution section revised May 20, 2005 Dr. T. Mazzulli
Page 94-100 – procedure revised May 20, 2005 Dr. T. Mazzulli
Page 128 – Updated decontamination procedure June 2, 2005 Dr. T. Mazzulli
Added links to TGLN Procedures March 21, 2007 Dr. T. Mazzulli
Revised HBV-DNA and HCV-RNA procedures with May 11, 2009 Dr. T. Mazzulli
Ampliprep instructions
Procleix Ultrio and WNV added July 21, 2008 Dr. T. Mazzulli
Procleix Discriminatory Assay July 21, 2008 Dr. T. Mazzulli
Annual Review July 21, 2008 Dr. T. Mazzulli
CMV DNA Amplification and Detection Procedures November 01, 2008 Dr. T. Mazzulli
updated
Molecular Testing – updated November 01, 2008 Dr. T. Mazzulli
EBV DNA Amplification and Detection Procedures November 01, 2008 Dr. T. Mazzulli
updated
HSV DNA Amplification and Detection Procedures November 01, 2008 Dr. T. Mazzulli
updated
Parvovirus B19 DNA PCR Amplification and Detection November 01, 2008 Dr. T. Mazzulli
Procedures updated
Enterovirus RNA Amplification and Detection November 01, 2008 Dr. T. Mazzulli
Procdeures added
West Nile Virus RT PCR - Failed QC added July 10, 2009 Dr. T. Mazzulli
Annual Review July 31, 2009 Dr. T. Mazzulli
Annual Review July 31, 2010 Dr. T. Mazzulli
Added revised easyMag August 23, 2010 Dr. T. Mazzulli
PROCEDURE MANUAL
UNIVERSITY HEALTH NETWORK / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and
should be checked against the document (titled as above) on the server prior to use
C:\Docstoc\Working\pdf\96ce2aa2-d35b-4791-bc6b-7058999b5767.doc
Page 191
Page Number / Item Date of Revision Signature of
Approval
Added revised CMV PCR August 23, 2010 Dr. T. Mazzulli
Added revised BKV PCR October 31, 2010 Dr. T. Mazzulli
GeneXpert C. difficile added January 10, 2011 Dr. T. Mazzulli
Updated Procleix Ultrio April 14, 2011 Dr. T. Mazzulli
Updated QC section of easyMag extraction May 31, 2011 Dr. T. Mazzulli
Updated Influenza procedure May 31, 2011 Dr. T. Mazzulli
Added Virology specimen accessioning guide link May 31, 2011 Dr. T. Mazzulli
Deleted CMV DNA Amplification and Detection May 31, 2011 Dr. T. Mazzulli
Procedures by Lightcycler
Deleted VZV Amplification and Detection Procedures by May 31, 2011 Dr. T. Mazzulli
Lightcycler
Deleted HSV Amplification and Detection Procedures by May 31, 2011 Dr. T. Mazzulli
Lightcycler
Added HSV/VZV Detection (Astra Alpha herpes) May 31, 2011 Dr. T. Mazzulli
Added Multiplex Respiratory virus detection May 31, 2011 Dr. T. Mazzulli
Annual Review May 31, 2011 Dr. T. Mazzulli
Parvovirus PCR revised May 31, 2011 Dr. T. Mazzulli
RSV PCR added May 31, 2011 Dr. T. Mazzulli