How to Run HIV Viral Load Using the Gene Expert Machine

Assay Technical Training Xpert® HIV-1 Viral Load For CE-IVD Use Only Cepheid Training Center 1 © Cepheid 301-4648 Rev. D April 2019 Training Agenda • Xpert HIV-1 Viral Load ‒ Clinical utility ‒ Reagents ‒ Sample collection ‒ Kit storage and handling ‒ Preparing the cartridge ‒ Quality Controls ‒ Results Analysis • Discussion 2 © Cepheid Training Objectives • At the end of the training, user will be able to: ‒ Properly store and handle the Xpert® HIV-1 Viral Load cartridge kit and sample collection kits ‒ Follow proper laboratory safety precautions ‒ Collect appropriate specimen types and transport specimen ‒ Perform the cartridge set up and run the assay ‒ Understand the assay control strategy 3 ‒ Report the various software generated results © Cepheid The Cepheid Solution • Detects and quantifies HIV-1 target ‒ Reliable results with a linear range from 40 10,000,000 HIV-1 RNA copies/mL • On-board controls for each individual sample ‒ Sample Volume Adequacy (SVA) ‒ Probe Check Control (PCC) ‒ Internal Quantitative Standards (IQS) High (H) and Low (L) • Results in approximately 90 minutes • Closed cartridge system minimizes risk of contamination • On-demand results 24/7 • Random access 4 © Cepheid Intended Use • The Xpert HIV-1 Viral Load In vitro diagnostic test for the rapid quantitation of Human Immunodeficiency Virus type 1 (HIV-1) RNA from HIV-1 infected individuals, assay is based on reverse transcriptase polymerase chain reaction (RT-PCR) technology • The assay can quantify HIV-1 RNA over the range of 40 to 10,000,000 copies/mL • The Xpert HIV-1 Viral Load assay is validated for quantification of RNA from HIV-1 Group M (subtypes A, B, C, D, F, G, H, J, K, CRF01_AE, CRF02_AG, and CRF03_AB), Group N, and Group O 5 • Xpert HIV-1 Viral Load is intended for use in conjunction with clinical presentation and other laboratory markers for disease prognosis and for use as an aid in assessing viral response to antiretroviral treatment as measured by changes in plasma concentration of HIV-1 RNA • The Xpert HIV-1 Viral Load Assay is not intended to be used as a donor screening test for HIV-1 infection © Cepheid Specimen, Targets and Probes • Specimen ‒ Human Plasma collected with EDTA or EDTA (PPT) or ACD tubes • Target ‒ HIV-1 • Probes – 1 probe binds to IQS-H – 1 probe binds to IQS-L ‒ 2 probes bind to 3’ end of 5’LTR region of HIV-1 RNA (do not bind to HIV-2) 6 ‒ © Cepheid Depending on the HIV group, one of the probes will bind. http://www.mdpi.com/2227-9059/2/1/14/htm Assay Requirements GeneXpert Systems • Modules • GeneXpert Dx Software v4.7b or higher / Infinity Dx Software v6.5 or higher Test Kits (CE-IVD) • GXHIV-VL-CE-10 Sample Collection • EDTA / EDTA-PPT or ACD tubes Other materials • • • • • Personal Protective Equipment (PPE) 1:10 Bleach 70% ethanol or denatured ethanol Vortex Centrifuge for plasma preparation Optional • Uninterruptible Power Supply /Surge Protector • Printer 7 © Cepheid Good Laboratory Practice Personnel Protective Equipment (PPE) Lab Bench area Specimens, Samples, and Kits Storage • Wear clean lab coats and gloves • Change gloves between processing samples • Clean work surfaces routinely with: ✓ 1:10 dilution of household bleach ✓ 70% Ethanol Solution • After cleaning, ensure work surfaces are dry • Store specimens and sample away from kit to prevent contamination • Use filtered tips when recommended Equipment(s) 8 © Cepheid • Follow the manufacturer’s requirements for calibration and maintenance of equipment(s) Kit Handling 9 © Cepheid Xpert HIV-1 Viral Load Kit Contents Catalog Number GXHIV-Viral Load-CE-10 Tests Per Kit 10 Assay Definition File (ADF) Kit CD Assay Import Instructions Package Insert (PDF) Transfer Pipettes 10 (1ml) Storage 2-28 °C Cartridges contain chemically hazardous substances-please see Package Insert and Safety Data Sheet for more detailed information. 10 © Cepheid Warnings and Precautions ‒ Store the Xpert HIV-1 Viral Load Assay cartridges and reagents at 2–28°C ‒ Follow your institution’s safety procedures for working with chemicals and handling biological samples ‒ Do not use Collection Reagent tubes that have not been validated by Cepheid ‒ Open the Assay cartridge lid only when adding the Sample, close the lid and proceed with processing 11 © Cepheid Warnings and Precautions • Do not shake the cartridge • Do not use a cartridge that… : ‒ appears wet, has leaked or if the lid seal appears to have been broken ‒ appears damaged ‒ has been dropped after removing it from packaging ‒ has been dropped or shaken after adding the sample to it ‒ has a damaged reaction tube ‒ has been used: each cartridge is single-use to process one test ‒ is expired • Do not reuse spent disposable pipettes 12 © Cepheid Dispose Xpert HIV-1 Viral Load Assay cartridges and reagents according to your institution’s and country's guidelines for disposal of hazardous materials Warnings and Precautions • Biological specimens, transfer devices, and used cartridges should be considered capable of transmitting infectious agents requiring standard precautions. • Follow your institution’s environmental waste procedures for proper disposal of used cartridges and unused reagents. These materials may exhibit characteristics of chemical hazardous waste requiring specific national or regional disposal procedures. • If national or regional regulations do not provide clear direction on proper disposal, biological specimens and used cartridges should be disposed per WHO [World Health Organization] medical waste handling and disposal guidelines. 13 © Cepheid Xpert HIV-1 Viral Load Assay Limitations • The performance of Xpert HIV-1 Viral Load has not been demonstrated from specimens other than the that has been validated, i.e. plasma. • The performance of the Xpert HIV-1 Viral Load test has not been evaluated with specimens processed by methods other than those described in the package insert. • Failure to follow assay procedures may lead to false negative results. • Inhibitors present in the samples may lead to invalid results. For detailed information, refer to the current Package Insert 14 © Cepheid Specimen Collection, Storage and Transport 15 © Cepheid Plasma Sample Collection • Whole blood ‒ Collect whole blood specimens in EDTA, EDTA-PPT or ACD tubes as per manufacturer’s instructions EDTA tube ACD Tube • Plasma ‒ Centrifuge to separate the plasma and red blood cells per the manufacturer's instructions ‒ Prepare minimum of 1.2mL of plasma 16 © Cepheid Heparin tube Specimen Collection, Transport and Storage Prior to testing Temperature (°C) Storage Time 24 hours EDTA anticoagulated whole blood 72 hours Plasma specimens are stable up to three freeze/thaw cycles. Prior to testing Temperature (°C) Storage Time 24 hours Plasma 6 days 6 weeks 17 © Cepheid Cartridge Preparation 18 © Cepheid Cartridge Preparation - Plasma 2 1 3 Label the side of the cartridge with the sample ID as the Vortex the equilibrated sample for 15 seconds. If specimen is cloudy, centrifuge it for a few seconds If using frozen or refrigerated samples, place at room temperature until completely thawed and equilibrated to room temperature 4 6 5 Open the cartridge lid 7 Bulb Start the test on your GeneXpert System time frame specified in the package insert Fill above the Mark *HIV-1 VL Assay Transfer pipette 19 © Cepheid Adapted from 301-4647 Pipette a minimum of 1 mL of the sample using the provided pipette* Slowly empty the pipette into the sample chamber of the cartridge Close the lid firmly Run a Test 1 Create Test 2 Scan barcode messages: 3 Scan the cartridge Cartridge/ Patient and/or Sample ID GeneXpert Start the test within 4 hours after adding the sample to the cartridge By default, do not click on Manual Entry or Cancel GeneXpert Infinity Place the cartridge on the conveyor within 30 minutes of adding the sample. "For complete details on how to run a test, refer to the Package Insert and the GeneXpert Dx or Xpertise Dx Operator Manuals. 20 © Cepheid Create a Test on GeneXpert Dx Software 4 Complete the fields as required 5 The Assay Protocol is selected automatically 6 The module is selected automatically DO NOT CHANGE IT!!! 7 Click on Start Test 8 A green light will flash on the module Load the cartridge into module and close the door 21 © Cepheid Xpert HIV-1 Viral Load Create a Test on Xpertise Dx Software 4 Complete the fields as required 5 The Assay Protocol is selected automatically 22 6 Click on SUBMIT 7 Place the cartridge into the conveyor belt © Cepheid Xpert HIV-1 Viral Load Automated Xpert Protocol 3 2 1 23 © Cepheid The cartridge is loaded into the instrument Sample is added to the cartridge Nucleic acids are purified Eluted DNA Purified mixes Nucleicwith acids dried-down mix with the reagent PCR reagents beads 4 Simultaneous amplification and detection occurs 5 Results are ready to view 6 Quality Controls 24 © Cepheid Cepheid Control Strategy • Assay Quality Controls • Each Xpert cartridge is a self-contained test device • Cepheid designed specific molecular methods to include internal controls that enable the system to detect specific failure modes within each cartridge ‒ Sample Volume Adequacy (SVA) ‒ Probe Check Controls (PCC) ‒ Internal Quantitative Standard IQS 25 © Cepheid Internal Quality Controls • Sample Volume Adequacy (SVA) ‒ Verifies that the correct sample volume is added to the cartridge • Probe Check Controls (PCC) • Before the PCR step, the fluorescence signal is measured from all probes and compared with default settings to monitor ‒ bead rehydration ‒ probe integrity ‒ reaction tube filling ‒ dye stability • Internal Quantitative Standard IQS ‒ 2 non infectious armored RNAs® non specific to HIV in the form of a dry bead ‒ Called IQS Low (103 copies /ml) and IQS High (106 copies/ml) calibrated against WHO 3rd IS ‒ Verifies that sample was correctly processed ‒ Verifies lysis, presence of the organism and detects inhibition ‒ IQS Low and IQS High Ct and fluorescence values must always be within the valid range 26 © Cepheid Commercially Available External Controls Thermofisher - https://www.thermofisher.com/order/catalog/product/964001 Part Number Description Configuration Storage 964003 HIV-1 High control 1.2 mL x 5 vials ≤ -20ºC 964002 HIV-1 Mid control 1.2 mL x 5 vials ≤ -70ºC 964001 HIV-1 Low control 1.2 mL x 5 vials ≤ -70ºC 1. 2. 3. 4. 5. Take 1 vial of the control material Thaw it at ambient temperature and mix well Immediately after thawing place the vial in the ice Transfer the entire amount (1.2mL) using the transfer pipette of the Xpert HIV-1 kit and add it to the Xpert HIV-1 sample chamber of the cartridge Close the lid and launch the test on GeneXpert - Many other vendors for quality control material are also available than the one outlined above. - External controls should be used in accordance with local, state accrediting organizations, as applicable 27 © Cepheid Result Interpretation 28 © Cepheid Result Interpretation Algorithm Sample Volume Adequacy (SVA) Pass Fail Probe Check Control (PCC) Fail ERROR Pass IQS-H & IQS-L Out of range Invalid HIV-1 Within the valid range Test Stopped anytime 29 © Cepheid * If copy/mL scale selected, otherwise scale is adapted to IU/mL No Result Copies/mL or IU • You can choose in what units your results are displayed ‒ Copies/mL or International Units/mL (1 copy/mL = 1.72 IU/mL) 1 Select Define Assays 2 Highlight Xpert HIV-1 Viral Load Assay 3 Choose units you prefer to see for the quantitative result 4 Save your settings 30 © Cepheid HIV-1 DETECTED > 1 × 107 copies/mL - The target HIV-1 is detected above the analytical measurement range - IQS-H: PASS - IQS-H has a Ct value within the valid range - IQS-L: PASS - IQS-L has a Ct value within the valid range - Probe Check: PASS Example calculation: 1x107 = 10 000 000 (million) copies/mL 31 © Cepheid HIV-1 DETECTED xx copies/mL - The target HIV-1 is detected at a quantitative value - IQS-H: PASS - IQS-H has a Ct value within the valid range - IQS-L: PASS - IQS-L has a Ct value within the valid range - Probe Check: PASS Example calculation: 6.72E03 = 6.72 x 103 = 6720 copies/mL 32 © Cepheid HIV-1 DETECTED < 40 copies/mL - The target HIV-1 is detected below the analytical measurement range - IQS-H: PASS - IQS-H has a Ct value within the valid range - IQS-L: PASS - IQS-L has a Ct value within the valid range - Probe Check: PASS 33 © Cepheid HIV-1 NOT DETECTED - The target HIV-1 is NOT detected - IQS-H: PASS - IQS-H has a Ct value within the valid range - IQS-L: PASS - IQS-L has a Ct value within the valid range - Probe Check: PASS 34 © Cepheid Patient Trend Report • You can monitor a specific patient over a period of time by creating a trend report 1 Select Reports and Patient Trend Report 2 Choose • Time range • Patient ID • Assay • Target preferences 4 Preview the Report 5 View your Report 35 © Cepheid Available only in Software version GxDx 4.6a and higher Troubleshooting 36 © Cepheid Factors That Negatively Affect Results • Improper specimen collection ‒ Performance with other collection devices and specimen types has not been assessed • Improper transport or storage of collected specimen ‒ Refer to the Package Insert for the appropriate handling instructions • Improper testing procedure ‒ Modification to the testing procedures, technical error and sample mix-up may impact the test results ‒ Careful compliance with the package insert is necessary to avoid erroneous results • Interfering substance ‒ False negative results or invalid results may be observed in the presence of interfering substance 37 © Cepheid INVALID Result Presence or absence of the HIV-1 target can not be determined - IQS-H and or IQS-L: FAIL Internal Quantitative Control Cycle thresholds are not within the valid range - Probe Check: PASS ‒ Cause ‒ Improper sample collection (using heparin tube for e.g) ‒ Incorrect sample preparation ‒ Improper storage of the cartridges ‒ Inefficient sample processing in cartridge ‒ Missing primer/probe or enzyme beads ‒ Presence of inhibitors in the sample ‒ Solution ‒ Repeat the test with a new cartridge and new sample 38 © Cepheid ERROR Result The Sample Volume Adequacy (SVA) passes if it meets the validated acceptance criteria. Error Code Cause Solution ‒ Ensure the Sample is added to cartridge 2096 2097 40 © Cepheid No sample added Not enough sample added ‒ Ensure cartridge is loaded within 30 min. after adding sample ‒ Ensure the minimum sample volume is added to the cartridge ‒ Ensure cartridge is loaded within 30 min. after adding sample NO RESULT Xpert HIV-1 Viral Load 1 – The presence or absence of HIV-1 cannot be determined. – A NO RESULT indicates that insufficient data were collected. – IQS-H or IQS-L: NO RESULT – Probe Check: NA (not applicable) ‒ Cause ‒ Test was stopped with stop test button ‒ Electrical failure ‒ Solution 41 © Cepheid ‒ Secure the power ‒ Repeat the test with a new cartridge Re-test Procedure 1 2 3 4 Discard used cartridge Follow your institution’s safety guidelines for disposal of cartridges Obtain the residual sample, mix according to Package Insert If the leftover sample volume is insufficient, or the retest continues to return an INVALID, ERROR, or NO RESULT, collect a new sample 42 © Cepheid Obtain a new cartridge Label appropriately as retest on the new cartridge Process the sample per the package insert Run the test on the GeneXpert System Technical Assistance • Before contacting Cepheid Technical Support, collect the following information: ‒ ‒ ‒ ‒ ‒ Product name Lot number Serial number of the System Error messages (if any) Software version and, if applicable, Computer Service Tag number • Log your complaint online using the following link http://www.cepheid.com/us/support : Create a Support Case 43 © Cepheid 44 © Cepheid