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GENERAL Human immunodeficiency virus type 1 (HIV-1) has been identified as the primary cause of Acquired Immunodeficiency Syndrome

(AIDS). This lethal retrovirus is spread by sexual contact, exposure to infected blood or blood products, and perinatal transmission. Acute HIV-1 syndrome develops approximately 3 to 6 weeks after initial infection and is associated with high levels of viremia. Following seroconversion, viremia declines dramatically and most patients enter a phase of apparent latency that may continue for years. Depletion of peripheral blood CD4+ T-cells, the primary target for the virus occurs during this clinically latent phase, sometimes without large increases in plasma concentrations of virus. Clinically apparent disease or an AIDS-defining illness, coupled with severe immunosuppression, eventually results. The ability to measure viral load is a valuable tool in the management of HIV-1 infected patients. An increase in viral load has been shown to correlate with progression of HIV-1 disease, as characterized by decreasing CD4+ cell counts and increasing symptoms. Studies have shown that viral load testing can be useful in predicting outcome or survival time, in determining the need to initiate, change or assess response to an antiretroviral regimen, and in determining the likelihood of transplacental viral transmission. PRINCIPLE The VERSANTTTM, HIV-1 RNA 3.0 Assay (bDNA) is a signal amplification nucleic acid probe assay for the direct quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in specimens from HIV-1 infected individuals using the Bayer System 340 bDNA Analyzer. Viral load markers, such as serum HIV-1 p24 antigen and quantitative HIV-1 micro culture of plasma or peripheral blood mononuclear cells have been used to monitor the progression of HIV1 infection. Although useful, these markers have limitations in sensitivity, range, reproducibility, cost and labour intensiveness. Other methods for measuring viral load include target amplification (e.g. Polymerase Chain Reaction or PCR) and signal amplification (e.g. branched DNA or bDNA) technologies. The bDNA technology amplifies the non-isotopic signal of a direct hybridisation to the target sequence and the signal amplification is linear. The bDNA technology is quantitative, sensitive, and easy to perform for routine laboratory use. The VERSANT HIV-1 RNA 3.0 Assay (bDNA) is a sandwich nucleic acid hybridisation procedure for the direct quantitation of HIV-1 RNA in human plasma. HIV-1 is first concentrated from plasma by centrifugation. After HIV-1 genomic RNA is released from the virions, the RNA is captured to a microwell by a set of specific, synthetic oligonucleotide capture probes. A set of target probes hybridise to both the viral RNA and the pre-amplifier probes. The capture probes, comprised of 17 individual capture extenders, and the target probes, comprised of 81 individual target extenders, bind to different regions of the pol gene of the viral RNA. The amplifier probe hybridises to the pre-amplifier forming a branched DNA (bDNA) complex. Multiple copies of an alkaline phosphatase (AP) labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the complex with a chemiluminescent substrate. Light emission is directly related to the amount of HIV-1 RNA present in each sample, and results are recorded as relative light units (RLU's) by the analyzer. A standard curve is

defined by light emission from standards containing known concentrations of beta propiolactone (BPL)-treated virus. Concentrations of HIV-1 RNA in specimens are determined from this standard curve. Overnight incubation can be done in the S2340, Versant 440 or in a Quantiplex bDNA Heater. Use of the S2340 requires that the user make up and add reagents to plates on the second day of the assay. The Versant 440 has a fully automated second day, with the user making up the detection reagents on day one of the assay. Using the Quantiplex bDNA heaters is a manual method used if there are irreparable errors on the 340 or 440 during an assay, use of these heaters requires user to make up reagents and manually cool and wash the assay plates.

SAMPLE REQUIREMENTS - move somewhere else? Specimen Collection: Collect blood observing universal precautions for venipuncture Collect blood in sterile tubes containing an anticoagulant K3 EDTA. Store whole blood at room temperature (18-30C) for up to 4 hours. Remove plasma from cells within 4 hours of collection. Separate by centrifugation at 1000 x g for 10 to 15 minutes. Do not clarify plasma by filtration or further centrifugation. Specimens with levels of triglycerides up to 2000 mg/dL, bilirubin up to 20 mg/dL or hemoglobin up to 200 mg/dL showed no clinically significant interference with HIV-1 RNA quantitation by this assay. Store plasma at -60 to -80 C in sterile, screw-capped tubes if viral pellets are not prepared within 30 minutes of separation. EDTA plasma may be stored at 2 to 8 C for up to 48 hours before freezing at -60 to -80 C. Up to 3 feeze/thaw cycles of samples are acceptable. 1. Label 2 microtubes with patient ID. 2. Beads before adding plasma 3. Use a forward pipette to put 1 ml in each microtube, one for processing and one for reserve. 4. Reserve tube put in freezer at -60 to -80 C 5. Processing tube add 50 l of beads. 6. Regrigerated centrifuge for 1 hour at 4 C. 7. Aspirate Do we need info about specimens other than blood? MATERIALS PROVIDED BOX 1 Box 1 contains sufficent reagents and materials to perform partial plates on four assay runs. Box 2 contains sufficient reagents and materials to perform partial plates on two assay runs. Component Quantity Description Storage
Bead Suspension Lysis Diluent 1 x 6.0 mL 1 x 15 mL Inert polystyrene beads in buffered solution with sodium azide (<0.1%) and other preservatives Buffered protein solution with sodium azide (<0.1%) 2 to 8 C 2 to 8 C

Lysis Reagent Capture Probes Target Probes Wash A Wash B Capture Wells Barrier Film Pre-Amplifier/Amplifier Diluent Label Diluent Dextran Sulfate Pre-Amplifier Probes Amplifier Probe Substrate Substrate Enhancer

1 x 2.2 mL 1 x 120 L 1 x 120 L 2 x 440 mL 1 x 320 mL 8 strips/12 wells each 1 x 6 sheets 1 x 28 mL 1 x 15 mL 1 x 14 mL 1 x 130 L 1 x 130 L 1 x 12 mL 1 x 1.2 mL

and other preservatives Proteinase K solution with sodium azide (<0.1%) and other preservatives Synthetc oligonucleotides in buffered solution with sodium azide (<0.1%) and other preservatives Synthetic oligonucleotides in buffered solution with sodium azide (<0.1%) and other preservatives Buffered solution with sodium azide (<0.1%) and other preservatives Buffered solution with sodium azide (<0.1%) and other preservatives Polysterene microwells coated with synthetic oligonucleotides High density polyethylene sheets Buffered protein solution with sodium azide (<0.1%) and other perservatives Buffered protein solution with sodium azide (<0.1%) and other perservatives 40% dextran sulfate solution with sodium azide (<0.1%) and other perservatives Synthetic oligonucleotides in buffered solution with sodium azide (<0.1%) and other perservatives Synthetic oligonucleotides in buffered solution with sodium azide (<0.1%) and other perservatives Chemiluminescent substrate (Lumi-Phos Plus) aqueous solution with sodium azide (<0.1%) and other perservatives

2 to 8 C 2 to 8 C 2 to 8 C 2 to 30 C 2 to 30 C 2 to 8 C 2 to 30 C 2 to 8 C 2 to 8 C 2 to 8 C 2 to 8 C 2 to 8 C 2 to 8 C 2 to 8 C

BOX 2 Component
Label Probe High Positive Control Low Positive Control Negative Control Standards A, E, F Standards B, C, D

Quantity
1 x 140 L 1 x 2.25 mL 1 x 2.25 mL 1 x 2.25 mL 1 x 4.25 mL/level 1 x 2.25 mL/level

Description
Enzyme-labeled synthetic oligonucleotides in buffered solution with sodium azide (<0.1%) and other preservatives Human plasma containing HIV-18E5/LAV virus with sodium azide (<0.1%) and other preservatives Human plasma containing HIV-18E5/LAV virus with sodium azide (<0.1%) and other preservatives Human plasma nonreactive for HIV-1with sodium azide (<0.1%) and other preservatives Human plasma containing HIV-18E5/LAV virus with sodium azide (<0.1%) and other preservatives Human plasma containing HIV-18E5/LAV virus with sodium azide (<0.1%) and other preservatives

Storage
-60 to -80 C -60 to -80 C -60 to -80 C -60 to -80 C -60 to -80 C -60 to -80 C

MATERIALS REQUIRED BUT NOT PROVIDED 1 mL plastic transfer pipettes 2 and 10 mL sterile-packaged serological pipettes 12-channel pipette capable of dispensing 50 to 200 L and 50 mL sterile-packaged, disposable reservoirs Positive displacement pipette capable of dispensing 1 to 7 mL Precision pipettes capable of dispensing 25 to 1000 L Sterile-packaged, aerosol-resistant, disposable tips Sterile-packaged, disposable 200 L tips without filters - for aspiration

1.5 mL sterile-packaged screw-capped, conical polypropylene microcentrifuge tubes with O-rings capable of withstanding 23,500 x g for 1 hour 15 and 50 mL sterile-packaged, disposable polypropylene tubes Biological Safety Cabinet, Class II Blank microwells, black Bleach, unscented (5.25% sodium hypochlorite) Calibrated thermometer, traceable to standards provided by NIST, with an immersion depth of 35mm and accurate to +/- 1.0C Dry heat block incubator (63 +/- 5C) with a capacity for > 96 x 1.5 mL tubes (Labnet Vortemp 56) Refrigerated centrifuge with RCF of 23,5000 x g and 45 fixed angle rotor with capacity for > 24x 1.5 mL tubes (Eppendorf Centrifuge 5417R) Vacuum (source supply > 10 inches Hg) system with 0.2m filter (HEPA or equivalent) and 2 to 4 L primary and secondary vacuum flasks Vortex mixer (Heidolph) Water bath (37 +/- 2.5C) 70% Ethanol One four-channel timer or four timers

WARNINGS AND PRECAUTIONS Reagents labeled with "X": Harmful! Irritant! May cause sensitization by inhalation and skin contact. Irritating to eyes, respiratory system and skin. IN case of contact with eyes rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing. Do no breathe spray. Contains: Proteinase K; Lysis Reagent. Irritant! May cause sensitization by skin contact. Avoid contact with skin. Wear suitable gloves. Contains: 5-chloro-2-methyl-2H-isothiazol-3-one and 2-methyl-2H-isothiazol-3-one; Lysis Reagent, Bead Suspension, Lysis Diluent, Capture Probes, Target Probes, Wash A, Wash B, PreAmplifier/Amplifier Diluent, Label Diluent, Dextran Sulphate, Pre-Amplifier Probes, Amplifier Probe, Substrate Enhancer, and Label Probe. Reagents labeled by "4 circles": CAUTION! POTENTIAL BIOHAZARD: This product contains human plasma or other human source material or biological source material of non-human origin. Handle this product according to established good laboratory practices and universal precautions. CAUTION! POTENTIAL BIOHAZARD: The positive controls and standards contain human plasma and HIV-18E5/LAV viral material, which has a frame shift mutation in the viral genome

that prevents production of the reverse transcriptase enzyme necessary to cause infection. The infectivity of the HIV-18E5/LAV viral material has been reduced to a level that is not detected by the Tissue Culture Infectious Dose50 (TCID50) assay

NOTE: Sodium azide in the reagents can react with copper and lead plumbing to form explosive metal azides. On disposal, flush reagents with a large volume of water to prevent the buildup of metal azides. NOTE: This test is run in singlet. Be careful during pellet preparation. Perform steps 2 to 5 of Preparing the Viral Pellet and step 6 of Lysing the Pellets in a Biological Safety Class II cabinet. The rest of the procedure may be performed using universal precautions.

1. Wear protective disposable gloves and laboratory coats when handling specimens and kit reagents. Specimens should be handled as if infectious using safe laboratory procedures.

2. Lysis Reagent contains a harmful irritant, which may cause sensitization by inhalation and skin contact. In the case of contact with eyes, rinse immediately with plenty of water and seek medical advice. 3. This kit contains human plasma or other human source material or biological source material of non-human origin. Handle according to established good laboratory practices. 4. Some reagents contain sodium azide. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. While disposing of sodium azide containing solutions down laboratory sinks, flush the drains with a large volume of water to prevent azide build up.

System 340 bDNA Analyzer Maintenance Checklist (cleaning Day 1)

Dispense Test 1. Fill the Rinse bottle with rinse solution (Type I water) 2. Install Rinse bottle 3. Fill two assay plates with spacer wells. Remove the tabs at the end of each strip. 4. From the Main Menu of the System 340 bDNA Analyzer, select System Management. 5. Select Performance Checks. 6. Select Dispense. 7. Load the two plates without sealing pads onto the heater tray. 8. Close the access door and press Start. 9. At the end of the performance check, remove the plates and visually inspect that the wells are evenly filled. 10. Close the access door and press Start. The heater tray will return to the home position. Aspirate Test 1. From the Main Menu of the System 340 bDNA Analyzer, select System Management. 2. Select Performance Checks. 3. Select Aspirate. 4. Load the two plates (which contain 350u1 of rinse solution) without sealing pads onto the heater tray. 5. Close the access door and press Start. 6. At the end of the performance check, remove the plates and visually inspect that the remaining volume in each well is less than 15l. 7. Close the access door and press Start. The heater tray will return to the home position. Checking and Cleaning the Read Head Compression Pad 1. From the Main Menu of the Systm 340 bDNA Analyzer, select System Management. 2. Select Maintenance. 3. Select Service Read/Wash. 4. When prompted open the access door. 5. Unscrew the Read Head Compression Pad, using caution when touching the Read Head since the fiber optic cable is fragile and can be easily damaged. 6. Check for deformities, damage and buildup of buffer salts. 7. Wipe the compression pad with a lint-free tissue soaked with type 1 water. Allow the pad to air dry completely.

Hand-tighten the Read Head compression pad onto the Read Head. 9. Close the access door and press Start.
8.

Background Check A. Setting up the DMS 1. At the DMS Workspace, select Read from the toolbar. 2. At the Read Data- Left Tray window, select Background in Select Assay Type field. 3. Enter in an Operator Name. 4. In the Kit Lot Number field, enter System 340 bDNA Analyzer Serial Number. 5. In the Expiration Date field, enter today's date. 6. Enter a file name. 7. Select OK. A window listing standards and controls will appear. 8. Select Yes to accept the values in the Standards and Controls fields 9. Select OK 10. The Read Data- Right Window Tray opens. 11. Enter the file name "bkgR". 12. Select OK. "Waiting for data...." Appears. B. Setting up the System 340 bDNA Analyzer 1. From the Main Menu, select System Management. 2. Select Performance Checks. 3. Select Background. 4. Fill two plates with white spacer wells. 5. Load the plates without sealing pads onto the heater tray. 6. Close the door and press Start. 7. At the end of the performance check, check the luminescence value for each wellon both the left and right plates. Ensure that the luminescence value for each well is less than 0.050. 8. When prompted, remove the plates. 9. Close the access door and press Start. Cleaning the Wash and Rinse Bottles 1. From the Main Menu of the System 340 bDNA Analyzer, select System Pause. 2. Remove the bottles out of the module, and fill the Wash and Rinse bottles with cleaning solution. 3. Load the filled bottles onto the System 340 bDNA Analyzer and lower the lids. 4. Allow the bottles to soak with cleaning solution for 15 minutes. 5. Discard cleaning solution and rinse out the bottles with type 1 water. 6. Continue with Cleaning the Fluid System Cleaning the Fluid System 1. From the Main Menu of the System 340 bDNA Analyzer, select System Pause. 2. Fill the Wash, Rinse and Cleaning bottles halfway with cleaning solution. 3. Load the filled bottles onto the System 340 bDNA Analyzer and lower the lids. 4. Press the Previous to return to the Main Menu. 5. Select System Management. 6. Select Maintenance.

7. Select Clean System and press Start. The cleaning solution cleans the fluid lines and the wash head probes. Soaking will last for 10 minutes. 8. At the end of the soak period, the instrument will prompt you to fill Wash A, Wash B, and the Rinse bottles with rinse solution. 9. Rinse the Rinse, Wash A and Wash B bottles a minimum of five times with rinse solution (type 1 water) to ensure that no cleaning solution residue remains. 10. Fill the Wash A, Wash B and Rinse bottles with rinse solution. 11. Load the bottles onto the System 340 bDNA Analyzer and press Start. The analyzer rinses the fluid lines and wash head probes with rinse solution. 12. At the end of the rinse cycle, discard the rinse solution in the Wash A, Wash B and the Rinse bottles. 13. Fill the Rinse bottle with fresh rinse solution and load it back onto the System 340. Close the lid and press Start to return to the Main Menu. 14. Invert the Wash A and Wash B bottle on absorbent paper and allow them to air dry. Cleaning the Waste Bottle 1. From the Main Menu of the System 340 bDNA Analyzer, select System Pause. 2. Remove the waste bottle out of the module and plug the waste bottle tube fitting. 3. Take lid off and empty waste. 4. Pour 500 mL of cleaning solution into the waste bottle. 5. Place the cap on the bottle and close tightly 6. Swirl the solution completely around the inside of the bottle a few times. 7. Allow the bottle containing cleaning solution to sit for approximately 15 minutes. 8. Empty the solution. 9. Repeat cleaning. 10. Rinse the inside of the bottle with fresh reagent water a minimum of five times, inverting the bottle frequently to rinse out the bleach residue from all interior surfaces. 11. Add six drops of antifoam reagent into waste bottle. 12. Install waste bottle back into module. 13. Press Previous to return to main menu Cleaning the Exterior Surface and Display 1. Turn the analyzer off and disconnect the power cord. 2. Wipe the display with an anti-static cleaning agent. 3. Wipe all exterior surfaces of your analyzer with a cloth or gauze moistened with warm, soapy water. 4. Wipe all soapy surfaces with a cloth or gauze moistened with water. 5. Dry all surfaces with a dry cloth or gauze. 6. Plug in the analyzer and turn on the power. Updating the Maintenance Log 1. From the Main Menu of the System 340 bDNA Analyzer, Select System Management. 2. Select Maintenance. 3. Select Maintenance Log. 4. Select Scheduled or As Needed. 5. Scroll to the task you want to update. The current date reflects when the task was last performed. The absence of a checkmark indicates the task is due to be performed. 6. Press the Options key once to update the task. A checkmark will appear to the left

of the task and the date will change, reflecting the new date when the task was performed. *NOTE: If you performed a maintenance task ahead of schedule, press the Option key twice. A new date will appear next to the task reflecting when it was performed. 8. Press Previous to exit from the log screen. 9. Press Enter to save the updates.
7.

CALIBRATION QUALITY CONTROL METHOD (PROCEDURE) DAY 2 Preparation of Pellets from Standards and Controls: 1. Pre-cool the Eppendorf 5417R centrifuges to 2 to 8C. 2. Label four 1.5ml Sarstedt tubes for each of Standards A, E and F and two 1.5m1 Sarstedt tubes for Standards B, C and D, as well as Negative, Low Positive and High Positive Controls. 3. Add 50l of Bead Suspension to each 1.5m1 tube. Completely thaw specimens, standards and controls. Invert to mix completely. Add 1.0ml sample to the appropriately labeled centrifuge tube. Invert the tube to mix the sample with the beads. 4. Place the Standards and controls into the pre-cooled rotor in the Eppendorf centrifuge and centrifuge at 23,500 x g for 1 hour at 2 to 8C. 5. Remove the samples from the rotor and immediately vacuum aspirate the supernatant using a new sterile yellow tip for each sample. Leave approximately 20l liquid on the pellet. Avoid aspirating the lipid layer if possible. If no aspirate apparatus is available the supernatant can be aspirated using pastettes. Follow the meniscus while aspirating carefully. Take care not to aspirate the pellet and leave 20l liquid on the pellet. 6. Immediately freeze the pellets at -60 to -85C for at least 30 minutes before proceeding with the assay. Preparation of the Viral Pellets from Specimens (to be done within 30 minutes of whole blood centrifugation) 1. Clean all pipettes and the Safety Cabinets with 70% ethanol before starting. 2. Pre-cool the Eppendorf 5417R centrifuges to 2 to 8C. 3. Separate the plasma from cells in whole blood by centrifuging at 2000 rpm for 10 minutes at room temperature. Do not clarify plasma by any means. NOTE: Perform steps 3 to 5 in a Biological Safety Class II cabinet 4. Label one 1.5 ml Sarstedt tube for each specimen on the top and side of the tube with the laboratory number. Label one 2ml Sarstedt tube for each specimen the same way, for storage of unused specimen. The storage tube must be dated. 5. Add 50l of Bead Suspension to each 1.5ml tube. 6. If specimens have been frozen, completely thaw in cold water. Invert or vortex to mix comletely. Keep on ice or at 2 to 8 C. 7. Add 1.0ml sample to the appropriately labeled centrifuge tube. Invert the tube to mix the specimen with the beads. Pipette the remainder of the specimen into the 2ml storage box. The aliquot in the 2ml tube must be stored. 8. If there is less than 1ml of specimen, make up to 1ml with Phosphate Buffered Saline (PBS) and make a note of the dilution factor on the bDNA Dilutions Chart.

9. If the specimen was a serum sample, it must be noted as such on the bDNA Dilutions Chart. 10. If the specimen is haemolysed, use personal judgement as to whether plasma can be separated from the cells. Siemens has shown that specimens that are haemolysed show no changes up to 200 mg/dl of haemoglobin. Note on the dilutions chart that the sample is haemolysed. 11. Place the specimens into the pre-cooled rotor in the Eppendorf centrifuge and centrifuge at 23,500 x g for 1 hour at 2 to 8C. Refer to LE-STC-010. 12. Aspiration of liquid. Remove the specimens from the rotor and immediately vacuum aspirate the supernatant using a new sterile yellow tip for each specimen. Leave approximately 20l liquid on the pellet. Avoid aspirating the lipid layer if possible. If no aspirate apparatus is available the supernatant can be aspirated using pastettes. Follow the meniscus while aspirating carefully. Take care not to aspirate the pellet and leave 20l liquid on the pellet. 13. Immediately freeze the pellets at -60 to -85C for at least 30 minutes before proceeding with processing the samples. NOTE: At this point, pellets can remain frozen for up to four weeks before performing the assay. PREPARATION AND START OF HIV VIRAL LOAD ASSAY How to prepare the lysinq solution (Day 1) Switch on dry heat incubators 1. Turn the dry heat block incubator on and ensure the temperature is at 63C 0.5C. 2. Turn Thermostar on. Use the following settings to incubate the specimens. TEMP = 63C ACCEL = 3 RPM = 1300 TIME = 120 min. 3. Turn the VorTemp 56 on. 4. Place the Lysis Diluent in a 37C water bath for 10 to 20 minutes or until any visible crystals dissolve. 5. Place the Lysis Reagent, Capture Probes and Target Probes on the bench top and bring to room temperature. 6. Prepare the Lysis Working Reagent in a 50ml polypropylene tube. Add the volume appropriate to the number of strips used in the assay:
Capture wellstrips Specimens Lysis Diluent (ml) Lysis Reagent (ml) CaptureProbes (l) Target Probes (l)

8 6 5 4 3

84 60 48 36 24

14.1 10.6 8.8 7.1 5.3

1.8 1.4 1.1 0.9 0.7

100 75 63 50 38

100 75 63 50 38

12

3.5

0.5

25

25

7. Cap, then invert tube 10 times, vortex for 5 seconds, and invert for another 10 times to mix. Store at room temperature and let foam settle. Discard if not used within 2.5 hours. 8. Thawing of pellets: Thaw frozen pellets at room temperature for 5 minutes in batches of up to 48. Stagger processing of pellet sets by 10 minutes. Add 120l of Lysis Working Reagent to each tube. Cap tightly and vortex for 20 seconds. CAUTION: Process each pellet set within 15 minutes (5 minutes to thaw and 10 minutes to add reagent). 9. Place tubes in a 63C heating block for 2 hours. NOTE: Perform step 10 in a Biological Safety Class II Cabinet 10. Pellets for Thermostar and Vortemp incubation; add 120l Lysis Working Reagent to each pellet. Place tubes in Thermostar or Vortemp and press start button. 11. Bring the pouch containing the Capture Wells to room temperature before opening. 12. Wearing clean gloves, place capture well strips in a plate. Press the capture well strips firmly into the plate until the tops of the strips are level. Remove the tabs. Immediately return unused strips to the pouch, seal, date and store at 2 to 8C. 13. Create a sample identification (ID) file using the DMS. Print a plate map. Click on New ID List Choose HIV RNA 3.0 Enter the sample ID's Save the file as the date, the instrument name and if it is the left or right tray (e.g. 020508 T003L) How to prepare the analyser 1. Ensure that all maintenance is performed. If required, update the maintenance log. 2. Check the levels of rinse and cleaning solutions on the analyzer. Add solution as necessary. 3. Wearing clean gloves, place a new barrier film on the frame. Place a silicone layer over the barrier film to complete the Sealing Pad. 4. Select TEST from the Main Menu. Select AUTOMATED and follow the instructions to select HIV RNA 3.0 Assay. 5. Select the number of strips per plate. If only the left plate has samples, select 0 strips for the right plate. Hybridizing the Capture and Target Probes 1. Remove the tubes from the heating block/ Thermostar/Vortemp56. Cool tubes at room temperature for 10 minutes. As the tubes cool, condensation collects in the caps. To recover the condensation, centrifuge tube briefly or while holding firmly, snap tube in a downward motion, then vortex tubes for 10 seconds. 2. Ensure that the plate notches are on the left. Using a 200l manual pipette and a new filter tip for each sample, transfer 100l from each tube and place in the appropriate capture well according to the plate map:

1 A B C D E F G H Std A

2 Std A

3 Std B

4 Std C

5 Std D

6 Std E

7 Std E

8 Std F

9 Std F

10 N-Ct1

11 L-PCt1

12 H-PCt1

Std is Standard A through F, N-Ctl is Negative Control, L-Pct1 are low and High Positive Controls respectively. 3. Ensure that the samples are loaded on the plate in exactly the same order as they appear on the plate map. Format of this procedure is slightly different the package insert. Incubation of plates Plates can be incubated using the bDNA System 340 or Versant 440 or in the bDNA Heaters at 52C. Incubation using the system 340 bDNA analyzer 4. When prompted, place the plated on the loading tray of the analyzer. If 8 strips or less are run, use a right plate filled with blank, black micro wells. If less than 8 strips are used on the left plate, fill the remainder of the plate with blank, black micro wells. 5. Place the assembled Sealing Pad on each plate. Close the analyzer access door and press START. 6. Incubate 16 to 18 hours (overnight). After 16 hours, the analyzer beeps at regular intervals to indicate that 16 hours of incubation is complete. Hybridizing the Pre-Amplifier and Amplifier Probes (Day 2) NOTE: Each cool / wash cycle takes approximately 15 minutes depending on the room temperature. The instrument washes and aspirates reagent from the wells, beeps at the end of each cycle, then ejects the plates. Leave the plates on the analyzer heater tray while adding the next reagent. CAUTION: Do not let the plate stand dry. Prepare the next reagent before the plates are ejected. Add the next reagent to the plate and press START within 4 minutes of ejection. If more than 4 minutes elapse, then the assay performance may be compromised and the System 340 enters a warning message into the event log.

If Wash A and Wash B are stored at 2 to 8C, allow 2 hours to bring them to room temperature, and mix by inverting each bottle several times before adding to the analyzer bottles. Ensure Wash A and wash B bottles have enough solution. Add solution as necessary. (In package insert, there is a table here). 2. Place the Pre-Amplifier / Amplifier Diluent, Dextran Sulfate (and label diluent?) in a 37C water bath for at least 10 minutes. Swirl the bottle with Dextran Sulfate and invert each bottle containing diluent several times to mix until homogeneous. Keep at 37C. 3. Prepare the Pre-Amplifier / Amplifier Working Diluent in 50ml polypropylene tube. Add the volume appropriate to the number of strips used in the assays. Add Dextran Sulfate using a stepper tip. (or positive displacement pipette?)
1.

Capture Well Strips 8 6 5 4 3 2

Specimens 84 60 48 36 24 12

Pre-Amplifier /Amplifier Diluent (ml) 21.0 16.2 14.1 12.3 8.8 6.6

Dextran Sulfate (ml) 7.0 5.4 4.7 4.1 2.9 2.2

4. Cap tube, then invert 10 times and vortex 20 seconds to mix. Repeat until diluent is thoroughly mixed. Working diluent will be very foamy. Place in 37C water bath and let foam settle. Discard if not used within 2 hours. 5. Place the Pre-Amplifier Probes on the bench top and bring to room temperature. 6. Prepare the Pre-Amplifier Working Reagent in a 15ml or 50ml polypropylene tube. Add the volume appropriate to the number of strips used in the assay:

Capture Well Strips 8 6 5 4 3 2


7.

Specimens 84 60 48 36 24 12

Pre-Amplifier / Amplifier Working Diluent (ml) 13.0 9.8 8.4 7.2 5.1 3.9

Pre-Amplifier Probes (l) 100 75 65 55 39 30

Cap tube, then invert 10 times and vortex for 20 seconds to mix. Repeat once to ensure reagents are thoroughly mixed. (Or Repeat until reagent is thoroughly mixed.) Place in 37C water bath and let foam settle. Discard if not used within 30 minutes. 8. When the overnight incubation is completed, press START on the analyzer to cool and wash the plates. 9. The cool/wash cycle take about 15 minutes. When the plates are ejected, immediately add 100l of Pre-Amplifier Working Reagent to each well. Use a twelve-channel pipette and fill the plate from row A to row H. 10. Press START. 11. The incubation takes 30 minutes and is followed by a cool/wash cycle. Prepare the Amplifier Working Reagent when the cool/wash cycle starts. 12. Place the Amplifier Probe on the bench top and bring to room temperature. 13. Prepare the Amplifier Working Reagent in a 15ml or 50ml polypropylene tube. Add the volume appropriate to the number of strips used in the assay:
Capture Well Strips 8 6 5 4 3 2 Specimens 84 60 48 36 24 12 Pre-Amplifier/Amplifier Working Diluent (ml) 13.0 9.8 8.4 7.2 5.1 3.9 Amplifier Probes (l) 100 75 65 55 39 30

14. Cap the tube, then invert 10 times and vortex for 20 seconds to mix. Repeat once to ensure reagent is thoroughly mixed. Place in 37C water bath and let foam settle. 15. Discard if not used within 30 minutes. 16. When the plates are ejected, immediately add 100l of Amplifier Working Reagent to each well. Use a twelve-channel pipette and fill the plate from row A to row H.

17. Press START. 18. The incubation takes 30 minutes and is followed by a cool/wash cycle. Hybridizing the Label Probe 1. Place the Label Diluent in a 37C water bath for 10-15 minutes. Mix by inverting the bottle several times. 2. Thaw the Label Probe under running tap (cold) water just before using. Vortex to mix. NOTE: Immediately return an unused Label Probe to -60C to -80 C freezer. 3. When the cool/wash cycle starts, prepare the Label Working Reagent in a 15m1 or 50m1 polypropylene tube. Add the volume appropriate to the number of strips used in the assay:
Capture Well Strips 8 6 5 4 3 2 Specimen Samples 84 60 48 36 24 12 Label Diluent (ml) 12.0 9.0 7.5 6.0 5.0 3.6 Label Probe (l) 100 75 65 55 39 30

4. Cap tube, and then invert 10 times. Store at room temperature and let foam settle. Discard if not used within 30 minutes. 5. When the plates are ejected, immediately add 100l of Label Working Reagent to each well. Use a twelve-channel pipette and fill the plate from row A to row H. 6. Press START. 7. The incubation takes 45 minutes and is followed by a cool/wash cycle. 8. Place the Substrate and Substrate Enhancer on the bench top and bring to room temperature. Measuring the Light Output 1. When the wash cycle starts, prepare the Substrate Working Reagent in a 15m1 or 50ml polypropylene tube. 2. Add the volume appropriate to the number of strips used in the assay:
Capture Well Strips 8 6 5 4 Specimens 84 60 48 36 Substrate (ml) 11.0 8.3 6.9 5.5 Substrate Enhancer (ml) 1.0 0.75 0.63 0.50

3 2

24 12

4.1 2.8

0.38 0.25

3. Cap tube and invert 10 times and vortex to mix. Store at room temperature in the dark until ready to use. Discard if not used within 30 minutes. 4. When the plates are ejected, immediately add 80l of Substrate Working Reagent to each well. 5. Use a twelve-channel pipette and fill the plate from row A to row H. 6. Press START. The incubation takes 30 minutes. 7. During the substrate incubation, prepare the Bayer Data Management Software (DMS) to receive the data from the analyzer. 8. The analyzer automatically reads the light units in each of the wells of the plates and then transfers the data to the DMS for analysis. VALIDATION 1. The data output from the System 340 are reported as relative light units (RLUs). The light emitted is directly related to the number of HIV-1 RNA copies/ml. A 4-parameter logistic curve is used to model the logarithm of the RLUs as a function of HIV-1 RNA concentration. 2. The DMS uses the observed RLUs and the concentrations of the six standard curve points to determine the best fitting curve to plot the standard curve and calculate the concentration of HIV-1 RNA for each control and patient sample. The standard is calculated from nine wells of standards (one well each of Standards B, C and D, two wells each of Standards A, E and F). 3. To monitor the assay performance, three levels of control material must be included with each assay i.e. Negative, Low Positive and High Positive Controls. 4. The assay is considered valid if all the following conditions occur: a. The relative light units (RLUs) for the standards are mean RLU Std A> RLU Std B> RLU Std C> RLU Std D> mean RLU Std E. b. The values determined for at least one of the HIV-1 Positive controls are within the specified range. c. The HIV-1 Negative Control has a value <50 copies/ml. d. A senior member of staff reviews the standard curve and finds it acceptable. 5. If mean RLU Std F> mean RLU Std E. the RLU's of the entire assay must be carefully analysed before validating or invalidating the assay. There are several conditions to check for: a. If there appears to be no elevation of RLUs, the assay can be called valid by a senior member of the laboratory staff. b. If the lower end of the curve is invalid as a result of overall elevation of RLUs, the assay can be called valid at the top end of the curve. If the high positive is within range and the IQC is also within range, the curve can be validated to approximately the RLU of Std C and above. An RLU between 5 and 10 can be used as the cutoff for validation of viral loads of <50 copies/ml, this cutoff will each time be dependent on the background level of the particular assay in question. 6. If one of the HIV-1 Positive controls fails, the RLUs of the entire assay must be carefully analyzed before validating the assay. If there is an overall dampening or elevation of

RLUs resulting in the curve being invalid, the assay must be called invalid and the entire assay repeated. Conversely, if the dampening or elevation of the RLUs does not invalidate the curve, the assay can be validated by confirming that the other positive control result and the IQC result are within range. If, however, there appears to be a problem only with the Positive control, with no associated dampening or elevation of RLUs over the entire plate, the assay can be called valid by a senior staff member. RESULTS 1. The results of each run are printed on reports. 2. Use the following criteria to evaluate the results: a. The lowest value reported by the assay is 50 copies/ml. b. Samples with values less than 50 copies/ml are below the reporting threshold of the assay, and are reported as <50 copies/ml. c. Samples with values equal to or greater than 50 copies/ml contain HIV-1 RNA in the quantity reported. d. Samples with values greater than 500,000 copies/ml are above the upper limit of quantitation and can be diluted to obtain an accurate quantitative value. If required, dilute the specimen in Negative Human Plasma. Retest the diluted specimen following the assay procedure and multiply the result by the dilution factor to determine the HIV-1 RNA copies/ml in the original specimen. If a more accurate results is not required, report the results as >500,000 copies/ml. e. If the original specimen was less than 1 ml, multiply the result by the dilution factor to determine the HIV-1 RNA copies/ml in the original specimen, except if the diluted specimen gives a result of below 50 copies/ml. In this case the result reported is <50 copies/ml. LIMITATIONS The assay can only be used for samples from patients infected with Group M HIV-1. REPORTING 1. The raw data along with the plate map should be handed over to the senior staff member to validate the raw data and review the results. Including in the validation is the verification that raw data numbers and plate map numbers correspond for specific data file. 2. The results are then signed off and reported POTENTIALLY HAZARDOUS PROCEDURES, SAFETY AND PRECAUTIONS 5. Wear protective disposable gloves and laboratory coats when handling specimens and kit reagents. Specimens should be handled as if infectious using safe laboratory procedures. 6. Lysis Reagent contains a harmful irritant, which may cause sensitization by inhalation and skin contact. In the case of contact with eyes, rinse immediately with plenty of water and seek medical advice. 7. This kit contains human plasma or other human source material or biological source material of non-human origin. Handle according to established good laboratory practices. 8. Some reagents contain sodium azide. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. While disposing of sodium azide containing solutions down laboratory sinks, flush the drains with a large volume of water to prevent azide build up.

Error Code Recovery Error Code Probable Cause Corrective Action CL0004 Prior to the overnight 1. Open the access door. Frame pickup incubation one of the 2. Remove both the left and right frames failure frames is misplaced on and place them back on the plates the plate. 3. Close the door. 4. Press START. 5. Confirm that the frames have been picked up and that the system proceeds into the overnight incubation. After the overnight 1. Press the ENTER key to silence the incubation one of the 2. Open the access door and frames comes loose observe which frame has fallen. from the upper assembly 3. Carefully reach into the System 340, as the upper and lower being careful not to bump into the aspirate/ heater trays are wash manifold, and retrieve only the fallen separating. Frame. 4. Change the barrier film on this frame. Possible source of contamination 5. Properly position the frame on the appropriate plate and close the door. 6. Press START. 7. Confirm that the frames have been picked up and that the system proceeds to the next step.

Page 1 of the Results Report 1. Header: Contains information on the name of the assay, software version, name of operator, kit lot number, kit lot expiration date, data file name, ID list file name, date/time the data was collected; and position of the plate (left or right) 2. Relative Luminescence Table: Provides the raw data in Relative Luminescence Units (RLU) and the corresponding capture well locations on the plate from which the data was generated 3. Standards and Controls Table: Lists the capture well locations and the average RLU values of the standards and controls. Comments generated by the data management software (DMS) regarding standards and controls are also listed. The DMS uses the RLUs to plot the standard curve and to quantitate the controls. The values for controls above the quantitation limit for a specific assay are provided in the Control Results boxes [6] 4. Standard Curve Graph: 5. Provides a visual plot of the standards and controls. 6. Diagnostics Section: Provides statistical information regarding the linear equation and goodness of fit of the standard curve data o Four parameter logistic curve: LOg10 (RLU) = b Q + (1 + e The four parameters are a, b, c and d and are estimated from the standard curve data on each plate. The letter e denotes the exponential function. PROCEDURAL NOTES 1. Use aerosol-resistant pipette tips and use a new tip every time a volume is dispensed. 2. Do not use reagents if they appear turbid or couldy after bringing to specified temperature. 3. Use all kit components within 4 weeks after opening any component.

4. For stability of working reagents, refer to the Assay Procedure Section 5. Do not mix reagents from different lots 6. Do not interchange vial caps as cross-contamination may occur