Roche Cobas Amplicor HIV-1 MonitorTM Test
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Title/Subject
Document Number:
PRO610-03
Effective (or Post) Date:
10 Feb 09
Document Origin : Site Laboratory
Company:
CAPRISA
SMILE Approved by:
Peggy Coulter
Review by
Heidi Hanes
Review date
8 Feb-2012
SMILE Comments: This document is provided as an example only. It must be revised to accurately
reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to
countercheck facts when considering their use in other applications. If you have any questions contact
SMILE.
Author: K. Coetzee
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CAPRISA
PREPARED BY
K. Coetzee
REVIEW DATE
July 2008
CENTRE FOR THE AIDS PROGRAMME OF
RESEARCH IN SOUTH AFRICA
DATE IMPLEMENTED
30 July 2007
REVISION DATE
CAPRISA is a UNAIDS
Collaborating Centre for
HIV Prevention Research
SUPERCEDES SOP #
Version 004
NAME
SIGNATURE
APPROVAL OF STANDARD OPERATING PROCEDURE
Requires the signatures of the following persons :
Principle investigator or designate
Signature : …………………………………
Date
Author
Signature : ………………………………..
Date
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: …………………………………
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: ………………………………..
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I.
PURPOSE
CAPRISA
Use of the Roche Amplicor HIV 1 Monitor test, version 1.5 for quantitative determination of HIV.
II.
SCOPE
This procedure applies to all specimens collected for CAPRISA projects/ studies and
processed in the CAPRISA laboratory by the laboratory staff.
III.
RESPONSIBILITY
The laboratory staff are responsible for compliance with this SOP.
The Laboratory Manager and/ or designate is responsible for training and compliance of all
staff on this SOP.
The Laboratory Manager and/ or designate is responsible for reviewing and revision of this
SOP annually or if an operational change warrants it.
IV.
DEFINITIONS
PCR
PID
SID
QS
NHP
TCA
TCB
EQA
–
-
V.
REFERENCES
Polymerase Chain Reaction
Patient Identification number
Sample Identification number, also known as the Laboratory number
Quantitation Standard
Negative Human Plasma
ThermoCycler A
ThermoCycler B
External Quality Assurance (programme)
Package insert
Amplicor Operator’s Manual
Ampliprep Operator’s Manual
SOP SPPE003
SOP SHAZ006
SOP SSHP007
SOP QEQA021
SOP QRES022
SOP LP-CRL-017
VI.
Safety Personal Protective Equipment
Safety Handling of Biohazardous Waste
Safety Use of Sharpsafes
External Quality Assurance (Proficiency Testing)
Results and Reporting
COBAS TAQMAN HIV-1 TEST
INSTRUCTIONS AND PROCEDURES
Principle
The test is a quantitative PCR procedure including 5 stages: RNA extraction; reverse
transcription of target RNA to cDNA; PCR amplification of a 155 base pair sequence in the gag
region at position 789-2290 defined by primers SK145 and SKCC1B (The nucleotide sequence
of the primers has been optimised to yield equivalent amplification of Group M subtypes of
HIV-1); hybridisation of the denatured amplified products to oligonucleotide probes, specific to
the target sequence; and detection of the probe–bound amplified products by colourimetry.
Safety Precautions
 Standard safety operating procedures are to be followed at all times
 Treat all material as potentially infectious
 Refer to SOP SPPE003 for the use of personal protective equipment
 Refer to SOP SHAZ006 for the handling and disposal of biohazardous waste
 Refer to SOP SSHP007 for the handling and disposal of sharps
Suitable Specimens
1. Plasma collected in ACD or EDTA tubes only.
2. Other body fluids are suitable, but their viral load is generally low compared to plasma.
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3. Blood should be stored for no longer than 6 hours before plasma is separated and
stored at –70oC.
4. If a specimen is delayed before the plasma can be separated it may be stored between
2 and 8C for no more than 18 hours before separation. A comment must be added to
the result report.
5. ACD specimens will result in viral load measurements approximately 15% lower than
EDTA specimens refer to XI. Method Limitations.
Unsuitable Specimens
1. Specimens older than 24 hours
2. Grossly haemolysed specimens
3. Test requires
a. 200μL of plasma for the Standard test
b. 500µL of plasma for the Ultrasensitive test.
Procedure
1. Refer to the package insert.
2. Two specimen preparation procedures are illustrated.
o In the Standard specimen preparation procedure, HIV-1 RNA is isolated directly
from plasma by lysis of virus particles with a chaotropic agent, followed by
precipitation of RNA with alcohol.
 The reportable range is 400 to 750 000 copies/mL
o With the UltaSensitive specimen preparation procedure, HIV-1 viral particles in
body fluids are concentrated by a high speed centrifugation, followed by lysis
of the virus particles with a chaotropic agent and precipitation of the HIV-1 RNA
with alcohol.
 The reportable range is 50 to 100 000 copies/mL
3. Use the standard method for patients not on treatment (especially in the acute phase of
HIV infection) where results are expected to be very high
4. Use the UltraSensitive procedure for body fluids with low viral loads and for samples
from patients on treatment when the viral load is expected to be undetectable. This
ultrasensitive preparation utilises a high speed centrifugation of the plasma to
concentrate the virus before extraction.
Preliminary
1. Take out specimens from the freezer and allow to thaw on the bench for 0.5 hour.
2. If using the UltraSensitive procedure, pre-cool the Ultracentrifuge and rotor to 2-8°C
(Refer to the Operator’s Manual; Instruction and Service Manual).
3. Make up 70% Ethanol (14ml absolute EtOH and 6ml Baxter water/ 12 specimen and
controls).
4. Take out kit reagents.
5. Vortex the specimens for 5 seconds (Virus settles to the bottom in standing
specimens).
6. Warm Lysis reagent at 37oC to dissolve guanidinium crystals in the waterbath, refer to
SOP QBAT012.
Reagent Preparation (In the Master Mix Room):
1. Vortex the Mn to mix thoroughly before use.
2. Add 100 μl Manganese (HIM-1 Mn2+, v1.5) into one vial of Master Mix provided (HIM-1
MMX, 1.5)
3. Mix by inversion 10-15 times, do not vortex.
4. Aliquot 50 μl MMR into each of the 12 MicroAmp reaction tubes. Do not cap the reaction
tubes.
5. Replace the A-rings into the sterile plastic bags and transport to the BioSafety cabinet
in the Amplification room.
6. Store tubes in clean fridge (2-8°C) and use within 4 hours of preparation.
7. For 1 A-ring (12 samples) you need 1 Mn + 1 MM.
8. For 2 A-rings (24 samples) you need 2 Mn + 2 MM.
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Manual Method
HIV RNA Extraction (In the Extraction Room).
Standard Procedure
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27.
Always change tips between sampling to prevent cross-contamination.
Vortex the QS for 10 seconds.
Make sure crystals are dissolved in Lysis reagent bottle.
Add 100 μl QS RNA into Lysis buffer – to make the working buffer.
Vortex for 10 seconds to mix.
Dispense 600 μl of the Working Lysis reagent into labelled 2.0ml Sarstedt tubes
(Label with date, specimen number and “RNA”).
Add 200 μl of patient specimen into appropriate tubes.
To each control tube add 200μL negative human plasma (NHP) and 50μL of the
appropriate control (low positive, high positive, negative).
Vortex gently for 10 seconds
Centrifuge for 2 minutes to remove liquid from lid.
Incubate at room temperature for 10 min – this activates Guanidium.
Add 800 μl 100% isopropanol and vortex for 10 seconds to mix the layers.
Put an orientation mark near the top of the tubes.
Centrifuge for 15 minutes at max speed (14000 RPM) – place the tube with the
orientation mark to the outside of rotor to allow easier detection of pellet when you
retrieve the tube.
Use a new transfer pipette for each tube and carefully remove and discard the
supernatant from each tube without disrupting the pellet. Remove as much liquid as
possible without disturbing the pellet
If the pellet is not visible it may have floated off the side of the tube. Before
aspirating, centrifuge again for 1 minute. Often the pellet dislodges if one is doing a
batch of specimens by the time one has reached the last few specimens.
Add 1ml 70% ethanol and vortex for 10 seconds.
Shake tube slowly and check for pellet, which detaches from side of the tube.
Vortex for 10 seconds.
Centrifuge for 15 minutes at max speed (14000 RPM) – place the tube with the
orientation mark to the outside of rotor to allow easier detection of pellet when you
retrieve the tube.
Use a new transfer pipette for each tube and carefully remove and discard the
supernatant from each tube without disrupting the pellet. The pellet should be
clearly visible at this step.
Centrifuge for 20 seconds at 14 000 RPM (The small A14 ultracentrifuges may be
used) and remove residual ethanol (as it inhibits PCR) with a pipette or pastette
without disrupting the pellet.
Add 400 μl Specimen Diluent reagent to each tube.
VORTEX for 10 seconds! Note that a white precipitate often remains.
Label the A-rings with their respective PIDs. i.e. ensure that the sample ID on the
tube matches that on the worksheet (in the correct position) and label the A-ring to
match. Check afterwards by going through each one again. Add 50 μl RNA samples
to corresponding PCR tube (A-ring) containing MMX.
o If precipitate is visible, allow it to settle and take the liquid from the top as
PCR input RNA.
Record the positions of the controls and specimens. Reverse Transcription and
Amplification must be started within 45 minutes (no longer than 2 hours) of the time
that the processed specimens and controls are added to the reaction tubes.
Processed specimens and controls can be stored frozen at -20°C or colder for up to
one week with no more than 1 freeze-thaw.
UltraSensitive Procedure
1. Precool the centrifuge by switching it on approximately 0.5 hours before you use it.
It needs to be between 2 and 8C.
2. Always change tips between sampling to prevent cross-contamination.
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3. Vortex the QS for 10 seconds.
4. Make sure crystals are dissolved in HIV-1 Lysis reagent bottle.
5.
µL HIV-1 QS v1.5 to one bottle HIV-1 Lyse.
6. Vortex for 10 seconds to mix.
7. UltraSensitive Working Lysis Reagent is stable for 4 hours at room temperature.
8. Add 500µL of each patient specimen to the appropriately labelled 1,5ml Sarstedt
tubes (date, specimen number).
9.
µL NHP to each of the appropriately labelled control 1,5ml Sarstedt tubes
(Negative, Low Positive, High Positive).
10. Put an orientation mark near the top of the each sample tube and each control tube.
11. Centrifuge for 60 min at maximum speed (20 000 RPM) at 2 – 8oC.
o Place the tube with the orientation mark to the outside of rotor to allow easier
detection of pellet when you retrieve the tube
12. Remove the plasma using a sterile pastette. Do not use vacuum aspiration.
13. Add 600 µL UltraSensitive Working Lysis Reagent into each specimen and control
tubes. Vortex each tube for 3-5 seconds.
14. Add 12.5µLof the appropriate control tube into the labelled control 1,5ml Sarstedt
tube (negative, low positive, high positive). – vortex for 3-5 seconds all control
tubes before and after adding into the appropriate tubes.
15. Incubate at room temperature for 10 min - activates Guanidium.
16. Add 600µL 100% isopropanol and vortex for 10 seconds to mix layers.
17. Centrifuge for 15 min at max speed- place the tube with the orientation mark to the
outside of rotor to allow easier detection of pellet when you retrieve the tube
18. Use a new transfer pipette for each tube and carefully remove and discard the
supernatant from each tube without disrupting the pellet. If the pellet is not visible
it may have floated off the side of the tube.
o Before aspirating, centrifuge again for 1 min. Often the pellet dislodges if one is
doing a batch of specimens by the time one has reached the last few
specimens.
19. Add 1ml 70% ethanol.
20. Shake tube slowly and check for pellet, which detaches from side of the tube.
21. Vortex for 10 seconds. Centrifuge for 15 min at max speed (14000 RPM) – place the
tube with the orientation mark to the outside of rotor to allow easier detection of
pellet when you retrieve the tube
22. Remove all supernatant with a sterile pastette.
23. Quick centrifuge and remove residual ethanol (inhibits PCR) with pipette or pastette
without disrupting the pellet.
24. Add 100µL Specimen Diluent reagent to each tube.
25. VORTEX 10 sec! Note that a white precipitate often remains.
26. Add 50µ
tubes. i.e. ensure that the sample ID on the tube matches that on the worksheet (in
the correct position) and label the A-ring to match. Check afterwards by going
through each one again.
o If the precipitate is visible, allow it to settle and take the liquid from the top as
PCR input RNA.
27. Record the positions of the controls and specimens. Reverse Transcription and
Amplification must be started within 45 minutes of the time that the processed
specimens and controls are added to the reaction tubes.
o Processed specimens and controls can be stored frozen at –20oC or colder for
up to one week with no more than one freeze –thaw.
28. Take prepared samples to the Amplification room.
Amplification (On the Roche Cobas Amplicor)
 Refer to Operator’s Manual for additional information and/ or diagrams.
1. Perform Daily maintenance before performing any testing – enter in Maintenance
Log (LFORM016)
a. Perform Normal Prime (or Extended Prime if new buffer has been added and
there are bubbles in the line)
b. Remove old A-Rings and used D-cups from the instrument
2. Prepare reagents
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a. I.Q. (Quantitation Standard probe suspension )
i. Gently swirl the IQ4 cassette on the bench to mix and vortex the IQ PSI
for 10 seconds.
ii. Pipette 2.5mL of the IQ PSI into the IQ4 cassette.
iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
b. I.M. (Probe suspension)
i. Gently swirl the IM4 cassette on the bench to mix and vortex the IM PSI
for 10 seconds.
ii. Pipette 2.5mL of the IM PSI into the IM4 cassette.
iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
c. A.D. (Amplicor Dilution Reagent)
i. Ready for use.
d. Conjugate
i. Ready for use.
e. Denaturation solution
i. Ready for use.
f. Substrate
i. Gently swirl the SB3 cassette on the bench to mix and vortex the SB for
10 seconds.
ii. Pipette 5mL SB into SB3 cassette.
iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
3. Load D-cups if required
4. Load Reagent cassettes – press “Escape” to save
5. Load Reagents on the platform – press “Escape” to save
6. Load A-Rings – press “Escape” to save
a. Press “Edit” – select TCA (for the first A-Ring)
b. Enter the A-Ring PID number press “Enter”
c. (Repeat process for TCB)
7. Order A-Ring worklist (Refer to the Roche Cobas Amplicor System Quick Reference
Guide (Pages 9 and 10).
a. Press “Order” and select “Edit”
b. Enter the A-Ring ID Nr (with scanner)
c. Press “Enter”
d. Enter the Profile number (e.g. 1 and 2 are for the Ampliprep, whilst 3 and 4 are
manual extraction).
e. Press “Enter”
f. The first A-ring will allocate positions 1 to 3 for the controls, followed by the
sample positions up to position 12.
i. In position 1 scan in the control ID found on the cards provided in each
kit (e.g. G08650). To do this scan the alphanumeric (G) from the
laminated card provided with the instrument and type in the numeric
(08650).
ii. Do this for the Negative, Low Positive and High Positive.
iii. To get to the next position (sample) press “ ⊳ ”.
iv. For each sample use the keypad to enter the PIDs.
g. Remember to enter the QS Data which are the cards provided with the kits –
these have the control ranges, scan the barcodes for each of the following:
1. QS#
2. L Lo
3. H Lo
4. L Hi
5. H Hi
h. Press “Escape” to save.
i. Scan in the second A-ring in the same manner – it only has samples (no QC).
j. After the second A-ring you have the option to enter the QS data again – check
to see that it is correct (you don’t have to enter again) and press “Escape”.
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k. Press “Start”, instrument performs a “Load Check”. If “Failed” press “Stop”
and then “Shift” and “Enter” simultaneously and take corrective action.
l. If the “Load Check” has passed the run will commence.
Automated Extraction using the Roche Ampliprep
HIV RNA extraction may be performed on the Roche Ampliprep in the extraction laboratory.
1. Refer to Operator’s Manual for additional information and/ or diagrams.
2. On the Ampliprep computer select “Ampliprep” using the mouse.
3. Using the mouse select the icon for Service.
a. Select “Extended Prime”
b. Select “Perform” (At the bottom of the screen).
c. The timer will indicate the end of the prime.
4. In the S-tube rack replace the S-tubes and ensure that the caps are loose by
checking that the seal is broken.
5. Replace the K-tips.
6. Replace the SPUs (24) onto the rack. Line up the word “Roche” with the arrow on
the rack.
7. Ensure that the SPUs are placed firmly on the rack and that the tips are loose.
Loading the Reagents
1. The Roche Ampliprep kit (CAP/CA Amplicor HIV Monitor) is different to the manual
extraction kit (Cobas Amplicor HIV-1 Monitor Test) see Vendor information below.
2. Load the HIV Beads onto the Shaking rack “A” on the Roche Ampliprep.
3. Load the Lysis reagent onto rack “B” or “C” (Remove the plastic cover).
4. Load the Probe dilution onto rack “B” or “C”.
5. Ensure the barcode on the reagent cassette is on the same side as the rack’s
barcode (On the right).
a. When loading these listen for a click and push with a gentle but continuous
motion.
b. The light on the panel above should turn green.
i. If the light does not turn green remove and repeat until it does.
Loading the Samples
1. Slot barcodes onto the sample input rack (Transparent) and insert 24 sample input
tubes (Transparent screw caps) into the places on the rack.
2. The first 3 positions are for the controls:
a. Standard (PHM):
i. Negative control
– white
ii. Low positive control – Orange
iii. High positive control – red
b. Ultrasensitive (PHS):
i. Negative control
– white
ii. Sensitive low positive control – yellow
iii. High positive control
- Red
3. Vortex the negative control for 10 seconds and pipette 300µLinto the tube in
position 1 of the sample input rack.
4. Vortex the low positive control for 10 seconds and pipette 300µLinto the tube in
position 2 of the sample input rack.
5. Vortex the high positive control for 10 seconds and pipette 300µLinto the tube in
position 3 of the sample input rack.
6. Vortex the samples for 10 seconds and pipette 300µL into the tubes in the sample
rack which correspond with their entries onto the Worklist. i.e. make sure that the
PID of the sample in position 1 is the same as the PID on the Worklist for that same
position. Check after you have completed all the pipetting to ensure that you have
placed everything in the correct order.
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Worklist Setup.
1. On the Roche Ampliprep computer, using the mouse select the “Order” icon.
2. Using the mouse select “Sample”.
3. Using the mouse select “New” (this will initiate a new date and Worklist. If a second
run is performed for the day it will continue sequentially.
4. For each sample enter the PID plus Visit Code (if there is no Visit Code enter the
collection date) and select “New”.
5. Using the mouse, from the top of the Sample screen select “Quality Control”.
6. Using the mouse select “New”.
7. From the drop down menu select the desired control and:
a. Enter the Lot numbers of each control by scanning in the barcodes at the
bottom of the cards provided in the kit:
i. The controls are listed on the left-hand panel on the screen.
ii. Using the mouse select –PHM and scan the Lot No. barcode for –PHM
when the entry screen pops up. Ensure you select the correct barcode
i.e. Either for the Standard or Ultrasensitive control.
iii. Using the mouse select the correct expiry date
iv. Repeat this procedure for each of the +PHM and the #PHM.
v. For the positive controls select “Ranges” and scan the correct ranges.
Note: the negative control does not have a range.
vi. Check all your entries.
8. Load the Sample Input Rack (from 6 above) onto the Roche Ampliprep in one of
“F”,”G” or “H” channels.
9. On the computer screen sample numbers 1 – 24 will appear on the screen.
10. Using the mouse select –PHM from the Q.C. screen and drag and drop it into
position 1 (ID). Similarly drop the +PHM in position 2 and the #PHM in position 3.
11. Using the mouse select the “Sample” tab and select and drag and drop the sample
IDs into their corresponding positions with respect to their load position on the
rack.
12. Using the mouse select “System” tab.
13. Using the mouse select “Start”.
14. “End time” will appear on the screen; if there is a problem an error code will appear
on the screen and an audible alarm.
a. If any appear refer to the Operator’s Manual for troubleshooting and call the
Roche Applications Specialist if you are unable to clear an error.
15. About 15 minutes before the “Endtime” on the AmpliPrep make up the Master Mix
reagents as per “Reagent Preparation” above.
16. Make up the Ampliprep-specific reagents:
a. S.Q. (Quantitation Standard probe suspension )
i. Gently swirl the SQ4 cassette on the bench to mix and vortex the SQ PSI
for 10 seconds.
ii. Pipette 2.5mL of the SQPSI into the SQ4 cassette.
iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
b. I.M. (Probe suspension)
i. Gently swirl the SM4 cassette on the bench to mix and vortex the SM
PSI for 10 seconds.
ii. Pipette 2.5mL of the SM PSI into the SM4 cassette.
iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
c. A.D. (Amplicor Dilution Reagent)
i. Ready for use.
d. Conjugate
i. Ready for use.
e. Denaturation solution
i. Ready for use.
f. Substrate
i. Gently swirl the SB3 cassette on the bench to mix and vortex the SB for
10 seconds.
ii. Pipette 5mL SB into SB3 cassette.
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iii. Do not invert but mix by gently swirling the cassette on the bench.
iv. Ready for use.
17. Remove the samples from the AmpliPrep ensuring that all of them (Including the
Q.C.s) have a grey top – this indicates that they have been extracted.
18. Vortex each for 10 seconds.
19. Add 50 μL RNA samples to corresponding PCR tube (A-ring) containing MMX.
(Remember: the first 3 are the Q.C. samples).
20. Using the mouse select the “Order” icon on the AmpliPrep computer screen.
21. Using the mouse select the “A-ring” tab.
22. The Worklist compiled previously appears onscreen and each sample should have
a blue tick next to it indicating extraction.
23. The A-ring Worklist appears empty below – this is for the Worklist (from the
AmpliPrep extraction) to be loaded onto the Amplicor now.
24. Using the mouse select “New”.
25. Scan in barcode on the A-ring.
26. Drag and drop the controls into their respective positions on the Worklist.
27. Using the mouse select “Save”.
28. Using the mouse select the “Amplicor” icon.
29. Using the mouse select “System” tab and double-click on TCA icon.
30. In the pop-up screen scan the A-ring barcode available, click on this number and
select “OK”.
31. Repeat this process for a second A-ring if necessary into TCB.
32. Using the mouse select “Cassette” tab and check for reagents which may be low or
depleted.
33. Replace any low ones or else load new specific reagents onto the shaking rack (SM,
SQ, AD3).
34. Load the generic reagents onto a separate (not shaking) rack (CN, DN, SB3).
35. Check the D-cups and replace as necessary.
36. Check the Wash Buffer reservoir and fill with a 1 / 10 solution if necessary.
a. 5400mL DH2O + 600mL W.B. concentrate.
37. Empty Waste as per SOP SHAZ006.
38. Switch the system off (Amplicor) and open the blue catch on the TCB lid.
39. Switch the system on and press the system button 4 times until “System Check”.
40. Select 1: Prime – step to “Extended”.
41. Press “Escape” to save, this will perform an extended prime.
42. Close the blue lid of the TCB.
43. After the prime has completed load the reagents.
a. Press “Load” until “L/Cass”.
b. Press Edit.
c. Scan the rack number and position barcode.
d. Scan the cassette barcode.
e. Press “Escape” to save.
f. Save into specific rack position and the generic rack positions on the system.
44. Ensure A-rings are correct and select “Escape” to save.
45. Press “Start” – system performs a Load-check.
46. If the check fails follow the troubleshooting guide from the Operator’s Manual.
Results
1. The results can be printed from the Amplicor or from the AmpliLink computer.
2. On the AmpliLink select the “Results” icon.
a. Select the correct A-ring (Check the date).
b. Select “Print”.
c. Attach the result printout to the Extraction Worklist.
3. Check that the controls are within tolerance limits and if so accept. If the controls
are not within their ranges reject the run as invalid and repeat the entire testing
process.
4. Enter results as per SOP RES022.
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NOTE:
 Standard: Test results greater than 750,000 copies/ml should be reported as “Greater
than 750,000 copies/ml”.
o These must then be repeated after dilution with NHP.
o Use a 1 / 10 dilution (30 µL patient plasma + 270 µLNHP)
o If result is still high repeat again using a 1 / 100 dilution (3µL patient plasma +
297µL NHP).
 Ultrasensitive: Test results greater than 100,000 should be reported as “Greater than
100,000 copies/ml”. If quantitative results are desired for such specimens, the original
plasma should be retested using the Standard Specimen Preparation Procedure.
Interpretation of tests with out-of range or out-of-sequence OD values refer to package insert,
section 8.
Amplification and Detection using the Roche Cobas TaqMan
Refer to SOP LP-CRL-017 COBAS TAQMAN HIV-1 TEST
QUALITY CONTROL
1. Run the Amplicor HIV-1 negative control, the Cobas Amplicor HIV-1 Monitor Test
low positive control and the Amplicor HIV-1 Monitor high positive control each time
the test is performed.
2. The requirement for the positions is that to the first three wells we should add the
negative, the low positive and the high positive control respectively.
3. All controls should yield Quantitation Standard OD (QS OD) values that meet the
criteria described in Step 2 of the Results section of the Operator’s Manual,
demonstrating that the specimen processing, reverse transcription, amplification
and detection steps were performed correctly.
a. Refer to the Package Insert “Results” section. The Negative Control should
yield a Not Detected result, i.e., all HIV-1 OD values less than 0.20. If the HIV-1
Negative Control does not meet these criteria, the entire run is invalid, the
results rejected and the entire process must be repeated.
b. If the absorbance of the Negative Control is consistently above 0.099A660
contact the Roche Response Centre (see last page of the package insert) for
technical assistance.
c. The assigned range for the Cobas Amplicor HIV-1 Monitor Test Low Positive
and High Positive Controls in this procedure is specific for each lot of control
and is provided on the Cobas Amplicor HIV-1 Monitor Test v1.5 Data Card
supplied with the kit.
d. The HIV-1 RNA copy number/mL for both Low positive and High Positive
controls should fall within the range indicated on the Data Card.
e. If one or both HIV-1 Monitor Positive Controls does not meet this criteria, the
entire run is invalid. Repeat the entire process
f. If the HIV-1 RNA copy number/mL of one or both Cobas Amplicor HIV-1 Monitor
Test Positive Controls is consistently outside the assigned range, contact the
Roche Response Centre (see last page of the package insert) for technical
assistance.
g. QC must be reviewed by the Q.A./Q.C. Officer and/ or Laboratory Manager and
signed off before result release
h. Results must be within the parameters listed; otherwise the run (all test results)
are to be rejected and repeated. Refer to Table 1 for flags on the report which
indicate an “Invalid” run.
Table 1.
FLAG
LAMP011
COMMENT
INTERPRETATION
_Q_
QS_INVALID
_N_
NC_INVALID
HIV-1 QS A660 value for any control above or below the
acceptable range
HIV-1 (-) C A660 value above the acceptable limit (>0.099)
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Revision Nr 005
_H_
_L_
CAPRISA
HPC_INVALID
LPC_INVALID
OD_SEQUENCE
QS_SEQUENCE
HIV-1 H (+)C copy number above or below the assigned range
HIV-1 L (+)C copy number above or below the assigned range
Indicates an out-of-sequence target A660 value. No results will
be calculated. Repeat the entire test procedure including
specimen and control preparation, amplification and detection.
Indicates an out-of-sequence QS A660 value. No results will be
calculated. Repeat the entire test procedure including
specimen and control preparation, amplification and detection.
If the run is invalid repeat the entire test with all samples.
The absorbance values (O.D.) must follow a pattern of decreasing
values as the dilution factor increases i.e. the OD should decrease from
tubes 1 to 2 to 3 to 4.
i.
Once a week one specimen must be duplicated in each batch of samples tested
to give an indication of intra-run reproducibility.
1. The results of the 2 samples should be within 10% of each other
on comparison.
2. The results will be filed and a report compiled and reviewed by
the Q.A./Q.C. Officer.
j. Once a month a sample must be retested between different batches of samples
to give an indication of inter-run reproducibility.
1. A sample from the last batch of samples processed must be
retained and included for testing in the new batch.
2. The results of this sample from each of the 2 runs should be
within 10% of each other on comparison.
3. The results will be filed and a report compiled and reviewed by
the Q.A./Q.C. Officer.
k. EQA control panels will be tested at regular intervals and the results
transmitted for comparative reviews, refer to SOP QEQA021.
REPORTING (Refer to SOP QRES022).
VII.
REAGENTS, EQUIPMENT, SUPPLIES
Water bath
Powderless, disposable gloves
BioSafety Cabinet
Roche Cobas Amplicor
Roche Cobas AmpliPrep
Ultracentrifuge
Cryofuge
Vortex mixer
Automatic pipette 0 - 200µL
Automatic pipette 0 - 1000µL
Pipette tips (RNAse-free)
Sterile pastettes
70% Alcohol
Cobas Amplicor HIV-1 Monitor Test - specimen preparation reagents HIM PREP
Cobas Amplicor HIV-1 Monitor Test - control reagents
HIM CTL
Cobas Amplicor HIV-1 Monitor Test – amplification reagents
HIM AMP
Cobas Amplicor HIV-1 Monitor Test – detection reagents
HIM MWP DK
NHP - [Negative Plasma (Human)]
COBAS AmpliScreen HIV-1 Amplification Reagents, version 1.5
COBAS AMPLICOR A-rings
COBAS AMPLICOR D-cups
Sarstedt 1.5-mL tubes
VIII.
INTERPRETATION / RESULTS
Refer to the package insert.
LAMP011
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IX.
CAPRISA
CALCULATIONS
Refer to the Package Insert.
X.
EXPECTED VALUES
Refer to the Package Insert.
XI.
METHOD LIMITATIONS
1. Body fluids other than blood will yield lower viral loads but may be requested.
2. Viral loads may be performed on ACD specimens at the Project Director’s discretion if
no other valid samples are available. Include a comment on the result report “ACD
sample used which has a lower yield than EDTA samples”.
3. Heparin inhibits PCR – DO NOT use heparin specimens for the Cobas Amplicor HIV-1
Monitor Test.
XII.
PROCEDURAL NOTES
1. The 2 options for the kit are the Standard and UltraSensitive methods.
2. The Standard kit would generally be used for baseline viral load measurements; whilst
the UltraSensitive method would be useful for detecting the lowest limits for patients
on treatment.
3. Elevated levels of lipids, icterus and haemoglobin do not interfere with the test.
4. Workflow in the laboratory must be unidirectional moving from the Pre-Amplification
(Master Mix) then to Extraction to the Post-Amplification room (Amplification/
Detection).
a. Do not move between these rooms in any other sequence otherwise
contamination of the samples/ results may occur.
b. Each room has its own equipment which must be left in place – including
pipettes and laboratory coats.
5. When the results are transferred to the AmpliLink (pc attached to the Ampliprep) and
are viewed, please select “Accept” before you print them. This will ensure that they are
saved in the correct format and place on the system.
6. In order for the Ampliprep to perform its own backup it is necessary that it be switched
off. Please ensure that the Ampliprep is switched off every week to enable this function.
7. Please make a backup copy on disc from the Ampliprep each Month. Label “Ampliprep
Backup September 2006” etc.
XIII.
CLINICAL APPLICATION
HIV RNA is not present in healthy individuals and presence of virus indicates infection.
Infected patients have varying viral loads depending on the progression of the disease state.
i.e. acutely infected individuals develop a high viral load initially which drops very low as setpoint is reached.
Patients on successful treatment should exhibit an undetectable viral load. If a patient on
treatment has detectable virus it may be indicative of treatment failure.
XIV.


QUALITY CHECKS
Maintenance Log
Quality control results
XV.
ESSENTIAL DOCUMENTATION
Package insert
LFORM016
Cobas Amplicor Maintenance Log
XVI.
AMPLICOR OPERATION AND MAINTENANCE
Operation
I.
Only staff who have undergone instrument specific training may operate this
instrument
LAMP011
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CAPRISA
Revision Nr 005
II.
Refer to Operator’s Manual; COBAS AMPLICOR General Instrument Document 8078882
and the System Quick Reference Guide
1. refer to Operator’s Manual; COBAS AMPLICOR General Instrument Document 8078882
2. Refer to Amplicor Maintenance log for daily, weekly, monthly and 6-monthly
procedures
3. to be logged in Maintenance/Service Log
4. Indicate corrective action taken
o Date
o name
o sign
Servicing
1. Six services are scheduled each year and performed by Roche service technicians
2. Decontaminate the instrument by wiping all surfaces and the probe thoroughly with a
swab soaked in 70% alcohol; soak and clean the racks.
3. Provide a Decontamination log to record this; sign and date it; place in instrument file.
4. to be logged in Maintenance/Service Log
5. Indicate corrective action taken
i. Date
ii. name
iii. sign
XVII.
VENDOR INFORMATION
Supplier
Roche Diagnostics
+27 11 504 4600
Roche Ampliprep
LAMP011
Item Description
Cobas Amplicor HIV-1 Monitor v1.5
Roche Cobas Amplicor A-Rings
Roche COBAS Amplicor wash buffer
Roche COBAS Amplicor D-cups
Roche COBAS Amplicor Maintenance kit
Roche CAP/CA Amplicor HIV Monitor 1.5
Roche COBAS Ampliprep S tubes-output
Roche COBAS Ampliprep S tubes (input)
Roche COBAS Ampliprep K tips
Roche COBAS Ampliprep SPU rack
Roche COBAS Ampliprep system wash
reagent
Page 14 of 14
Catalogue
Number
21118390123
21045636001
20759899123
21045644001
28166072001
3309398123
3137058001
3137040001
3287343001
3137066001
Unit
48 tests
Box 24
2 X 250 tests
Box 12
Kit
48 tests
12 X 36
12 X 24
13 X 36
12 X 24
3309380123
100 tests
CONTROLLED