WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT. Product: Muse Auto CD4/CD4% kit WHO reference number: PQDx

Transcription

1 PQDx WHO PQ Public Report May 2018, version 1.0 WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT Product: Muse Auto CD4/CD4% kit WHO reference number: PQDx Muse Auto CD4/CD4% kit with product codes MCA100101, MCA500101, MCA1XK101, manufactured by EMD Millipore Corporation, CE-marked regulatory version, was accepted for the WHO list of prequalified in vitro diagnostics and was listed on 23 May This public report was amended on 24 May 2018 to reflect the correction of a typographical error. Summary of WHO prequalification assessment for Muse Auto CD4/CD4% kit Date Outcome Amended PQ public report 24 May 2018 listed Correction of a typographical error PQ listing 23 May 2018 listed Dossier assessment 03 May 2018 MR Site inspection(s) of quality management system 3 August 2017 MR Product performance evaluation 5 December 2017 MR MR: Meets requirements Intended use: Muse Auto CD4/CD4% Assay is intended to be performed on a Muse Cell Analyzer with Muse Auto CD4/CD4% software, which includes Muse Auto CD4/CD4%, Muse System Check, and Muse system cleaning modules. The assay is intended to identify and quantify both absolute CD4 T-cell counts and CD4 percent values in venous and capillary whole blood samples. The CD4 percent values are the absolute counts of the CD4 T-helper cells, expressed as a percentage of total lymphocytes in EDTA whole blood specimens from adult and pediatric patients. For in vitro diagnostic use. Assay description: Muse Auto CD4/CD4% Kit consists of the Muse Auto CD4/CD4% antibody cocktail, a proprietary mixture of antihuman lymphocyte antibodies and a monoclonal anti-human Page 1 of 7

2 PQDx WHO PQ Public Report May 2018, version 1.0 CD4 antibody. The kit also contains Muse 1X Lysing Solution to lyse erythrocytes. The anti-human lymphocyte antibodies detect all human lymphocytes. The CD4 antibody identifies human CD4-positive helper/inducer T cells (HLA Class II reactive) and recognizes a 60-kilodalton (kda) surface antigen.15 Monocytes also express CD4 but at lower density and have no co-expression of the other lymphocyte markers detected by reagents in this kit. The Muse Auto CD4/CD4% Kit is intended to be used only with the Muse Cell Analyzer as described in this document, using the Muse Auto CD4/CD4% software module, which allows for automatic acquisition and gating, and provides test results. Test kit contents: Component Muse Auto CD4/CD4% Kit 100 tests (product code MCA100101) One 1-mL vial of Muse Auto CD4/CD4% Antibody Cocktail (part no ) One 40-mL bottle of Muse 1X Lysing Solution (part number ) 500 tests (product code MCA500101) Five 1-mL vials of Muse Auto CD4/CD4% Antibody Cocktail (part no ) Five 40-mL bottle of Muse 1X Lysing Solution (part number ) 1000 tests (product code MCA1XK101) Ten 1-mL vials of Muse Auto CD4/CD4% Antibody Cocktail (part no ) Ten 40-mL bottle of Muse 1X Lysing Solution (part number ) Items required but not provided: Item Consumables: Muse System Check [C] Kit Muse Instrument Cleaning Fluid (ICF) Sample acquisition tubes, 1.5-mL microcentrifuge tubes with screw caps Disposable gloves 100% bleach solution Deionized, distilled, or reverse osmosis water Durables: N/A Equipment: Muse Cell Analyzer with Muse Auto CD4/CD4% software Calibrated precision pipette with disposable pipette tips Vortex mixer Catalogue number MCA MCA or Equivalent N/A N/A N/A N/A N/A N/A Storage: The antibody cocktail should be stored at 2 C to 8 C, do not freeze. The lysing agent should be stored upon receipt at 18 C to 25 C, do not freeze. Page 2 of 7

3 PQDx WHO PQ Public Report May 2018, version 1.0 Shelf-life upon manufacture: 24 months. Warnings/limitations: Please refer most current version of instructions for use. Prioritization for prequalification Based on the established criteria, Muse Auto CD4/CD4% kit was given priority for WHO prequalification. Dossier assessment EMD Millipore Corporation submitted a product dossier for Muse Auto CD4/CD4% kit as per the Instructions for compilation of a product dossier (PQDx_018 v1). The information (data and documentation) submitted in the product dossier was reviewed by WHO staff and external technical experts (assessors) appointed by WHO. The manufacturer's responses to the nonconformities found during dossier screening and assessment findings were accepted on 03 May Commitments for prequalification: 1. The manufacturer will undertake in-use stability studies on test kits manufactured. 2. The manufacturer will conduct shipping stability studies on test kits manufactured as per the WHO Technical Guidance TGS-2. WHO will to follow-up on implementation of these commitments at agreed time intervals and next re-inspection. Based on the product dossier screening and assessment findings, the product dossier for Muse Auto CD4/CD4% kit meets WHO prequalification requirements. Manufacturing site inspection A comprehensive inspection was performed at the site of manufacture (28820 Single Oak Drive, Temecula, California 92590, USA) of Muse Auto CD4/CD4% kit in August 2017 as per the Information for manufacturers on prequalification inspection procedures for the sites of manufacture of diagnostics (PQDx_014 v1). The inspection found that the manufacturer had an acceptable quality management system and good manufacturing practices in place that ensured the consistent manufacture of a product of good quality. The manufacturer's responses to the nonconformities found at the time of the inspection were accepted on 26 January Page 3 of 7

4 PQDx WHO PQ Public Report May 2018, version 1.0 Commitments for prequalification: 1. Relevant suppliers of antibodies will be audited by 30 June Above-mentioned site audits will include a review of evidence of validation of the conjugated antibody manufacturing processes. Based on the site inspection and corrective action plan review, the quality management system for Muse Auto CD4/CD4% kit meets WHO prequalification requirements. Product performance evaluation Muse Auto CD4/CD4% System was evaluated at the Institute of Tropical Medicine (ITM) Antwerp, Belgium which is a WHO Evaluating Laboratory for CD4 Enumeration between August and October The evaluation was conducted using the WHO evaluation protocol (PQDx_114) which was also approved by in-country ethical review board in Belgium. A total of 250 fresh venous blood specimens were used to study failure rates, reproducibility (intra-assay variation, inter assay variation, inter-instrument variation, instrument precision) and agreement with the BD FACSCalibur TM as the reference method using an antibody panel including CD3/CD4/CD8/CD45 monoclonal antibodies (MultisetTM, BD Biosciences) with TrucountTM tubes. Lastly, ease to use was assessed. The acceptance criteria are as follows: Specimen failure should be less than 10%. For reproducibility studies, a percentage coefficient of variation (%CV) should be less than 15% for CD4+ T counts of less than or equal to 200/μL and %CV should be less than 10% for CD4 counts of more than 200 cells/μl. Compared to the reference method, the bias should be less than 10%. Specimen failure which was defined as failure of the instrument to provide valid results was 0 % for venous whole blood. Testing of fresh specimens was conducted to assess the ability of the Muse Auto CD4/CD4% System to provide reproducible results. The overall intra-assay variability was 4.4 % for CD4 absolute count and 3.6 % for CD4%. Individual CV s per CD4 category were all below 5 % for CD4 T cell counts >200 and less than 15% for CD4 T cell counts less than or equal to 200 cells/µl. The inter-instrument precision was excellent with an average inter-instrument variability of 3.8 % for CD4 counts and 3.1 % for CD4%. The inter-assay variation for specimens kept up to 24 hours after collection ranged from 5.2% to 8% for CD4 absolute counts and CD4 percentages. Lastly, Muse Auto CD4/CD4% System had precision of less than 6 % for both CD4 counts and CD4% on venous blood specimens. The average carry-over (κ) was 0.09 for absolute counts and 1.3 for CD4 percentages which is below the acceptable limit of 2%. Regarding agreement with the reference method, the correlation coefficients were high with minimal bias. Operational characteristics of the Muse Auto CD4/CD4% System were assessed using a structured questionnaire by testing personnel. The Muse Auto CD4/CD4% System was found to be simple to use. In conclusion, based on the results of evaluations conducted in Page 4 of 7

5 PQDx WHO PQ Public Report May 2018, version 1.0 ITM, the performance of the Muse Auto CD4/CD4 capillary whole blood specimens compared to BD FACSCalibur TM. Labelling 1. Labels 2. Instructions for use Page 5 of 7

6 PQDx WHO PQ Public Report May 2018, version Labels Page 6 of 7

7 PQDx WHO PQ Public Report May 2018, version Instructions for use Page 7 of 7

8 Operational Specification Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date AUTHORING SITE MM-Temecula AFFECTED SITES TITLE MCA MCA MCA1XK101_ A Muse Auto CD4/CD4% Kit Signed by ELECTRONIC SIGNATURES Meaning of Signature Server Date (dd-mmm-yyyy HH:mm z) CLOR Julie Author Signature 16-May :23 CEST Averbeck Jean Technical Approval 16-May :08 CEST SMITH-MCCOLLUM Robert Management Approval 16-May :28 CEST TYAGARAJAN Kamala Management Approval 16-May :47 CEST Olson Christopher Quality Approval 16-May :41 CEST 1 of 18

9 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Millipore (UK) Ltd. 2 Fleming Road, Kirkton Campus, Livingston, EH54 7BN United Kingdom TEL: FAX: Muse Auto CD4/CD4% Kit MCA MCA MCA1XK Tests 500 Tests 1,000 Tests For In Vitro Diagnostic Use in Enumerating CD4 + T Lymphocytes and Determining CD4 + T Cells as a Percent of Total Lymphocytes in Whole Blood IVD For In Vitro Diagnostic Use; Export Only 2 of 18

10 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date 3 of 18

11 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Intended Use The Muse Auto CD4/CD4% Assay is intended to be performed on a Muse Cell Analyzer with Muse Auto CD4/ CD4% software, which includes Muse Auto CD4/CD4%, Muse System Check, and Muse system cleaning modules. The assay is intended to identify and quantify both absolute CD4 T-cell counts and CD4 percent values in venous whole blood samples. The CD4 percent values are the absolute counts of the CD4 T-helper cells, expressed as a percentage of total lymphocytes in EDTA whole blood samples from adult and pediatric patients and donors. The Muse Auto CD4/CD4% Assay is intended for professional use. The intended user is a healthcare worker, laboratory technician, or medical technologist fully trained in processing blood specimens, typically in a centralized hospital laboratory. For in vitro diagnostic use. Summary and Explanation The characterization of CD4 T cells in whole blood is an essential part of staging immune status and has been found to be of importance in evaluating immunodeficiency diseases, assessment of immunodeficiency disorders, and of immune reconstitution following stem cell transplantation. 1-4 Absolute CD4 T-cell counts and CD4 percentages of lymphocytes in particular are considered useful parameters for characterizing, monitoring, and evaluating individuals with or suspected of developing autoimmune and immunodeficiency diseases Individuals with immunodeficiency diseases for example, typically exhibit a progressive and selective depletion of the CD4-positive T-lymphocyte populations. 11 The CD4 percentage of lymphocytes, has also been demonstrated to be a useful parameter for monitoring the immune status of pediatric populations with, or suspected of having, immune deficiency Test Principle The Muse Auto CD4/CD4% Kit consists of the Muse Auto CD4/ CD4% antibody cocktail, a proprietary mixture of antihuman lymphocyte antibodies and a monoclonal anti-human CD4 antibody. The kit also contains Muse 1X Lysing Solution to lyse erythrocytes. The anti-human lymphocyte antibodies detect all human lymphocytes. The CD4 antibody identifies human CD4-positive helper/ inducer T cells (HLA Class II reactive) and recognizes a 60-kilodalton (kda) surface antigen. 15 Monocytes also express CD4 but at lower density and have no co-expression of the other lymphocyte markers detected by reagents in this kit. The Muse Auto CD4/CD4% Kit is intended to be used only with the Muse Cell Analyzer as described in this document, using the Muse Auto CD4/CD4% software module, which allows for automatic acquisition and gating, and provides test results. 1 4 of 18

12 Kit Components Muse Auto CD4/CD4% Kit contains the following reagents: Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Reagent Kit MCA Muse Auto CD4/CD4% Kit 100 tests MCA Muse Auto CD4/CD4% Kit 500 tests MCA1XK101 Muse Auto CD4/CD4% Kit 1000 tests Contents Materials Required but Not Supplied One 1-mL vial of Muse Auto CD4/CD4% Antibody Cocktail (part no ) One 40-mL bottle of Muse 1X Lysing Solution (part number ) Five 1-mL vials of Muse Auto CD4/CD4% Antibody Cocktail (part no ) Five 40-mL bottle of Muse 1X Lysing Solution (part number ) Ten 1-mL vials of Muse Auto CD4/CD4% Antibody Cocktail (part no ) Ten 40-mL bottle of Muse 1X Lysing Solution (part number ) Muse Cell Analyzer with Muse Auto CD4/CD4% software (Catalog No ) Muse System Check [C] Kit (Catalog No. MCA100102) Calibrated pipettes and disposable pipette tips Sample acquisition tubes, 1.5-mL microcentrifuge tubes with screw caps, (VWR, Catalog No or equivalent) Vortex mixer Disposable gloves Muse Instrument Cleaning Fluid (ICF) [Catalog No. MCA100103] 100% bleach solution (typically % sodium hypochlorite) Deionized (DI), distilled, or reverse osmosis (RO) water Warnings Handle all specimens and materials coming in contact with them as biohazards and as potentially infectious, and dispose of with proper precautions. Never pipette by mouth. Wear suitable protective clothing and gloves. 16 Sodium azide (present in the Muse Auto CD4/CD4% antibody cocktail [0.08%] and Muse 1X Lysing Solution [0.1%]) is toxic if ingested. Reagents containing sodium azide should be considered a poison. If products containing sodium azide are swallowed, seek medical advice immediately and show product container or label. (Refer to NIOSH, National Institute for Occupational Safety and Health; CAS#: ; and also to GHS, The Globally Harmonized System of Classification and Labeling of Chemicals.) Aqueous solutions of sodium azide, when mixed with acids, may liberate toxic gas. Any reagents containing sodium azide should be evaluated for proper disposal. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, flush with large volumes of water to prevent build-up in plumbing. Check with regulatory agencies to determine at what concentration sodium azide may cause a product to be regulated as hazardous waste. Add approximately 10 ml of 100% bleach to the waste bottle prior to using the Muse Cell Analyzer. Refer to the Muse AutoCD4/CD4% System User's Guide for information. 2 5 of 18

13 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Precautions Use only venous whole blood samples collected with EDTA as an anticoagulant. Performance may be affected by the use of other anticoagulants. Blood samples that are hemolyzed, clotted, lipemic, or discolored should be discarded and not be used. 17 Blood samples should be stored in blood collection tubes at 18 to 25 C and tested within 48 hours after they are collected. 18 Do not use patient specimens that have been previously fixed. Do not refrigerate whole blood specimens before preparing. Leave the capped tubes of blood specimens standing upright or lying on their sides if they are stored overnight. Do not rock or agitate blood specimens in any way during storage. The pipette used for sample preparation should be properly calibrated to ensure it is dispensing exactly 10 μl of blood. Pipetting precision and accuracy should be verified. Do not dilute whole blood or use any volume other than 10 μl for the assay. The Muse Auto CD4/CD4% antibody cocktail and Muse 1X Lysing Solution contain sodium azide as a preservative; however, care should be taken to avoid microbial contamination, which could cause erroneous results. The reagents provided are light-sensitive. Minimize exposure of reagents to bright light during storage and incubation. Do not substitute reagents from other manufacturers. Reagents may be damaged if left outside the recommended storage temperature ranges for extended periods. Do not freeze. If properly stored, the reagent is stable until the expiration date shown on the label. Do not use after the expiration date. Pooling, dilution, or alteration of any reagent may cause erroneous results. This product is supplied ready to use. Do not mix components from multiple lots. Alteration in the appearance of the reagent, such as the precipitation or discoloration (normal color is colorless to a light pink), indicates instability or deterioration. In such cases, do not use the reagent. Avoid leaving blood to dry on the side of the tube as this may cause erroneous results. Alteration of the protocol provided may cause erroneous results. Incubation times or temperatures other than those specified may give erroneous results. Prolonged exposure of whole blood samples to lytic reagents may cause white cell destruction and loss of cells from the target population. Follow recommended incubation times. Acquire stained samples on the Muse Cell Analyzer within 4 hours of sample preparation. Use only the lysing solution provided. Do not add fixative to the assay. The Muse System Check [C] procedure (described in the Muse System Check [C] package insert and Muse Auto CD4/CD4% System User's Guide) must be run and pass on the Muse Cell Analyzer each day of sample analysis and before proceeding to sample acquisition steps. If the Muse Cell Analyzer is turned off or there is a power outage and the instrument turns back on, rerun the System Check procedure and confirm the result passes before proceeding to data acquisition. If the Muse Cell Analyzer has been subject to relocation or transportation, run the System Check procedure and confirm the result passes before proceeding to data acquisition. 3 6 of 18

14 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Stability and Storage Store the Muse Auto CD4/CD4% antibody cocktail refrigerated (2 to 8 C). Do not freeze. Refer to the expiration date on the package label. Do not use the reagent after the expiration date. Once opened, the Muse Auto CD4/ CD4% antibody cocktail must be used within 16 weeks. Store the Muse 1X Lysing Solution at room temperature (18 to 25 C) on receipt. Do not freeze. Refer to the expiration date on the package label. Do not use the reagent after the expiration date. Once opened, the Muse 1X Lysing Solution must be used within 16 weeks. Return the Muse Auto CD4/CD4% antibody cocktail to 2 to 8 C immediately after use at room temperature (18 to 25 C). Do not leave out at higher temperatures for extended periods of time. The Muse Auto CD4/CD4% antibody cocktail contains light-sensitive dyes. Avoid prolonged exposure to light during storage and incubation. During shipment, small volumes of the reagent can become entrapped in the vial lid. For complete recovery, briefly centrifuge the vial of Muse Auto CD4/CD4% antibody cocktail in a tabletop microcentrifuge/centrifuge at a minimum speed of 1,000 x g prior to opening. Alternatively, use a pipette tip to recover material entrapped in the cap. Before You Begin At the start of each day of testing: 1. Turn on the system by pressing the power button located on the front of the unit below the touch screen. 2. Select the button with your name or log on ID. 3. Enter your PIN and press OK. 4. Fill the cleaning solution bottle with Muse ICF (Instrument Cleaning Fluid). 5. Empty the waste bottle, then add 10 ml of bleach. 6. Reset the fluid levels. Refer to the Muse Auto CD4/CD4% System User s Guide for instructions. 7. Perform a Complete System Clean. Refer to the Muse Auto CD4/CD4% System User s Guide for instructions. 8. Perform a System Check [C] procedure. Refer to the Muse System Check [C] package insert and the Muse Auto CD4/CD4% System User s Guide for instructions. IMPORTANT: Always perform a System Check procedure. Ensure the result is PASS before running samples. 9. Perform a Quick Clean cleaning procedure after the System Check procedure and before running samples. Specimen Collection The blood used for the procedure should be collected by venipuncture into a sterile K3 EDTA (lavender top) blood collection tube A minimum of 10 μl of whole blood is required for this procedure. For optimal results, stain blood within 48 hours of collection. Unstained anti-coagulated blood should be stored at 18 to 25 C prior to sample processing. CAUTION: Blood samples that are hemolyzed, clotted, lipemic, or discolored should be discarded. 4 7 of 18

15 CAUTION: Leave the capped tubes of blood standing upright or lying on their sides if they are stored overnight. Do not rock or agitate the blood in any way during extended storage. Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Staining Samples 1. Label one 1.5-mL microcentrifuge sample tube for each sample with sample identification information, such as a donor number. 2. Pipette 10 μl of Muse Auto CD4/CD4% antibody cocktail into each sample tube. CAUTION: Put the stock bottle of Auto CD4/CD4% antibody cocktail back into the refrigerator or on ice immediately after use. Do not allow the bottle of Auto CD4/CD4% antibody cocktail to remain at elevated temperatures for extended times. 3. Thoroughly resuspend the blood by gentle agitation for a few minutes before removing an aliquot for sample preparation. 4. Reverse pipette 10 μl of whole blood (from a well-mixed EDTA lavender-top tube) into the bottom of each tube containing the Muse Auto CD4/CD4% antibody cocktail. NOTE: If necessary, batch your samples to avoid over incubation with the reagents. 5. Cap the tubes and then vortex each sample immediately at medium intensity for 3 to 5 seconds. CAUTION: Avoid leaving blood to dry on the side of the tube, as this may cause erroneous results. 6. Incubate samples for 15 minutes at room temperature (18 to 25 C) in the dark. 7. Pipette 380 μl of Muse 1X Lysing Solution directly into each sample tube to bring the total sample volume to 400 μl. Cap and vortex each tube immediately on medium intensity for 3 to 5 seconds. 8. Incubate the samples for 15 minutes at room temperature (18 to 25 C) in the dark. Samples are ready for acquisition on the Muse Cell Analyzer using Muse Auto CD4/CD4% software. NOTE: Acquire samples on the Muse Cell Analyzer within 4 hours after preparation. Acquiring Samples on the Muse Cell Analyzer Acquire samples using the Muse AutoCD4/CD4% software module. Refer to the Muse Auto CD4/CD4% System User s Guide for details on acquisition and analysis. 1. Vortex each stained sample thoroughly before loading it on the Muse Cell Analyzer. 2. Acquire each sample on the Muse Cell Analyzer using the operator or supervisor mode of the software. Make sure you enter the sample ID. NOTE: Do not let the instrument capillary dry out. Load a tube of deionized water on the instrument between runs. 3. When you finish acquiring samples for the day, run the Extreme Clean procedure. 4. Power off the instrument after the Extreme Clean Procedure has been completed. 5 8 of 18

16 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Results The Muse Auto CD4/CD4% software module adjusts settings, acquires samples, and provides test results automatically. The results are displayed on the screen after each sample is acquired. Results displayed are dependent upon whether the software is used in operator mode or supervisor mode. Results displayed include sample information, test results for CD4 T Cells in original sample (cells/μl), CD4% of Lymphocytes, and Total Lymphocytes in original sample (cells/μl), as shown below. Results displayed in supervisor mode also provide a dot plot showing the staining pattern for the sample. Example results displayed in operator mode Figure 1. Results screen from operator mode (left) and administrator/supervisor mode (right). Example results displayed in administrator/supervisor mode You can export the report as a PDF file to a USB drive. Refer to the Muse Auto CD4/CD4% System User s Guide for more information on printing. The software also displays error messages when it is unable to run or gate the samples as shown below. Refer to the Muse Auto CD4/CD4% System User s Guide for details on errors and warnings. 6 9 of 18

17 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Figure 2. Results screen showing warning. The software provides an option to change the gate by selecting Manual Gating from the Edit Gate screen and moving the markers. Refer to the Muse AutoCD4/CD4% System User's Guide for more information. Manual gating should be performed only if the software is unable to autogate the sample. Reports Individual Sample Report Individual sample results may be printed from the printer tab on the Results screen. The sample report can be viewed using the Print Preview button. The report contains sample information, patient information, reference ranges, CD4 T cell count, total lymphocyte count, and CD4% test results. The report also indicates if a sample was gated automatically or manually. Any errors or warnings that occurred are also displayed in the report of 18

18 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Test Site: Hayward Site Address: Industrial Blvd. Hayward, CA Site Phone Number: Muse Auto CD4/CD4% Sample Report Lab Director: K. Tyagarajan Physician: Dr. Smith Test Operator: Administrator Pa ent ID: J. Doe Acquisi on Date & Time: 23-OCT :59 Age: 36 years Reagent Kit Lot No: 12 Sex: Male Reagent Kit Exp. Date: 23-Jan-2014 Sample ID : Sample_001 I nstrument Serial Number : N/A Blood Draw Date & Time : 21-Oct : 50 File Name : ADM_23OCT2013_ Sample No : 1 Comments: Automated Ga ng Results: Original Sample Values CD4T Cells: cells/μl CD4%: 37.2% Total Lymphocytes: cells/μl Analysis Date & Time: 04-NOV :42:42 Print Date & Time: 04-NOV :43:12 Reference Range: Lower Limit - Upper Limit CD4T Cells: cells/μl CD4%: % Total Lymphocytes: cells/μl So ware: Muse Auto CD4/CD4% 1.0 Errors & Warnings: Reviewed by: Figure 3. Sample report from operator mode (left) and administrator/supervisor mode (right) Summary Report The Data Set Summary Report contains numeric results for all samples in the data set, as well as the sample and test information, patient ID, any errors or warning that occurred, and whether the sample was manually gated. The summary report can be generated by selecting Print Data Set Summary Report on the printer tab. Figure 4. Data Set Summary Report for all samples in a data set Page 1 Report from sample displayed in operator mode Report from sample displayed in administrator/supervisor mode 8 11 of 18

19 Troubleshooting Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Problem Suggested Solution Inconsistent or inaccurate results Check staining, count, and percentage results from a control sample(s) using normal adult blood or a commercially available whole blood control(s) following the protocol Check that the sample was completely mixed before sample acquisition. Day-to-day variation of results Sample too concentrated (Error message: E06: Run aborted. Sample too concentrated or E07: After acquisition, sample too concentrated or E10: After acquisition, sample too concentrated; gated manually) Software unable to autogate samples (Error message: W02: The sample may not be correctly autogated or A01: The sample could not be autogated ) Ensure that the Muse System Check procedure was run each day and passed prior to using the Muse Cell Analyzer to acquire blood samples. Prepared samples may need to be diluted. If you see such an error message, prepare samples again according to the procedure in Staining Samples on page 5; however, in step 7 add 580 μl of 1X Lysing Solution (instead of 380 μl), lyse for 15 minutes, and acquire the sample. At the Sample Info screen, be sure to change the dilution factor to 60 before acquisition. Proceed to the supervisor mode and view dot plots to determine if manual gating of the samples is possible to obtain results. Not staining or lysing for recommended times might give rise to poor staining patterns that cannot be autogated. Blood not stored at recommended times and temperatures can deteriorate and give rise to poor staining patterns. Results appear to be changing over time. Prepared samples may be too old. Make sure prepared samples have been acquired within 4 hours of sample preparation. Sample acquisition is taking longer than expected or progress bar stops during acquisition. Or you received an error message stating you might have a clog. Instrument clogs frequently During acquisition, the software pauses for extended periods. Instrument becomes non-responsive. Cell aggregates, cell debris, salt crystals, or other particulates can cause a clog or blockage of the flow system. Press Abort, then Clean, then Backflush. Install a tube containing 20% bleach and click Clean. Load a tube of deionized water and run a Quick Clean cleaning cycle to remove bleach residue. If this procedure does not alleviate the problem, refer to the Muse Auto CD4/CD4% System User's Guide for more troubleshooting information. Your samples may contain significant amounts of cellular aggregates and debris that might build up in the flow system. Periodically run a Quick Clean cleaning cycle using DI water (after every 10 sample acquisitions). Quick Clean cleaning may also be run with ICF. In that case, be sure to follow with a Quick Clean cycle using DI water to rinse. If needed run an Extreme Clean procedure. If this does not solve problem, you may need to change the capillary. Refer to the Muse Auto CD4/CD4% System User's Guide. The sample may contain excessive amounts of debris. Allow the software to recover for 30 to 60 seconds before terminating the run. If necessary, turn off system then turn it on again. Run System Check, ensuring that it passes, and proceed to sample acquisition again. If necessary, turn off the system then turn it on again. Run System Check, ensuring that it passes, and proceed to sample acquisition again of 18

20 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Performance Characteristics The performance of the Muse Auto CD4/CD4% Kit using the Muse Cell Analyzer and Muse Auto CD4/CD4% software module was established by testing at EMD Millipore in the US and at a clinical site in Cameroon. Accuracy (Comparison Data) A study was performed at a clinical site in Cameroon to evaluate the Muse Auto CD4/CD4% Kit on the Muse Cell Analyzer instrument using the Muse Auto CD4/CD4% software module to determine clinical accuracy for the enumeration of CD4 T-cell counts, total lymphocyte counts, and CD4 percentages. Comparative results were determined using a commercially available IVD test kit. In the clinical study, EDTA whole blood samples were collected from 111 donors of which there were 64 females (1 month to 82 years age) and 47 males (1 month to 65 years age). Of this sample set, there were 28 pediatrics (1 month to 14 years of age; average age 5.6 yr) and 83 adults (15 years to 82 years of age; average age 36.8 yr). Regression statistics for the comparative device versus the Muse Auto CD4/CD4% test results are summarized in Table 1. Table 1: Regression Statistics of Muse Auto CD4/CD4% Test vs Comparative Device Results Parameter n R 2 Slope Intercept Range CD4 T-cell count ,918.6 cells/μl CD4% (of lymphocytes) % Total lymphocyte count ,588 cells/μl Figure 5. Regression plot of CD4 T-cell count (cells/μl) data from the comparative device versus the Muse Auto CD4/CD4% test from human whole blood samples of 18

21 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Figure 6. Regression plot of CD4% data from the comparative device versus the Muse Auto CD4/CD4% test from human whole blood samples Precision Studies Intra-assay Intra-assay precision data was obtained for control blood samples. Ten replicates for each blood control were prepared using the Muse AutoCD4/CD4% assay and tested. The results for these studies are summarized in Table 2. Mean and coefficient of variation (%CV) are provided for CD4 T-cell counts, total lymphocyte counts, and CD4% for each measurement range. Table 2: Intra-assay Precision Data for Blood Control Samples CD4 T-cell Count CD4% Total Lymphocyte Count (cells/μl) Average SD %CV Average SD %CV Average SD %CV Control Control , Inter-assay Inter-assay precision data was obtained by preparing and analyzing three blood control samples spanning different count ranges on 5 separate days using the Muse AutoCD4/CD4% assay and system. The results for these studies are summarized in Table 3. Mean and coefficient of variation (%CV) are provided for CD4 T-cell counts, total lymphocyte counts, and CD4% for each measurement range. Table 3: Inter-assay Precision Data for Blood Control Samples CD4 T-cell Count CD4% Total Lymphocyte Count (cells/μl) Average SD %CV Average SD %CV Average SD %CV Control Control , Control , of 18

22 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Sample Aging The potential influence of blood sample aging was evaluated on five abnormal donors. Unstained EDTA anticoagulated whole blood was stored for defined periods of time, then stained and analyzed. Based on the data generated from this study EDTA anti-coagulated whole blood may be stored for up to 48 hours prior to staining and analysis for the Muse Auto CD4/CD4% assay. Unstained anti-coagulated blood should be maintained at 18 to 25 C prior to sample processing. Linearity Linearity was assessed by testing triplicates of 24 prepared blood cell aliquots with the Muse Auto CD4/CD4% Kit. Results were found to be linear within the entire measurement range of 45 to 3,811 CD4 T cells/μl. LOB, LOD, and LOQ Studies were performed on determination of the Limit of Blank (LOB), LOD (Limit of Detection), and LOQ (Limit of Quantitation) measurements for the Muse AutoCD4/CD4% Assay. Our results demonstrate that LOB is 6 CD4 T cells/μl, LOD is 13 CD4 T cells/μl, and LOQ is 16 CD4 T cells/μl with manual gating and 37 CD4 T cells/μl with automated gating. Specificity To assess cellular cross-reactivity, the individual antibodies comprising the Muse Auto CD4/CD4% antibody cocktail were screened on five normal human adult donor blood samples. Results consistently demonstrated that the monoclonal antibodies reacted specifically with the expected populations. The Muse Auto CD4/CD4% antibody cocktail also shows good specificity for the lymphocyte population with minimal to no staining of granulocyte and monocyte populations. Limitations The results are dependent upon proper use of reagents and instruments. Symbols The following symbols are found on the packaging: Symbol Description Symbol Description IVD Catalog number Manufacturer Caution, consult accompanying documents In vitro diagnostic medical device Consult instructions for use Use by date Authorized representative in the European Community Sufficient for <n> tests Temperature limitation Biological risks of 18

23 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Related Kits Muse System Check [C] Kit MCA Muse Instrument Cleaning Fluid (ICF) [C] MCA References 1. Nicholson JK. Use of flow cytometry in the evaluation of primary and secondary immunodeficiency diseases. Arch Pathol Lab Med. 1989;113: Fahey JL, Taylor JMG, Detels R, Hofmann B, Melmed R, Nishanian P, Giorgi JV. The prognostic value of cellular and serologic markers in infection with human immunodeficiency virus type 1. N Eng J Med.1990;322: Fauci AS, Macher AM, Longo DL, Lane HC, Rook AH, Masur H, Gelmann EP. Acquired immunodeficiency syndrome: Epidemiologic, clinical, immunologic, and therapeutic consideration. Ann Intern Med. 1984;100: Williams KM, Gress RE. Immune reconstitution and implications for immunotherapy following haematopoietic stem cell transplantation. Best Pract Res Clin Haematol. 2008;21(3): Pirzada Y, Khuder S, Donabedian H. Predicting AIDS-related events using CD4 percentage or CD4 absolute counts. AIDS Res Ther. 2006;3: Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of HIV/ AIDS, World Health Organization BCT_HLM-392.pdf. 7. Hammer SM, Eron JJ Jr, Reiss P, Schooley RT, Thompson MA, Walmsley S, Cahn P, Fischl MA, Gatell JM, Hirsch MS, Jacobsen DM, Montaner JS, Richman DD, Yeni PG, Volberding PA; International AIDS Society-USA. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA. 2008;300(5): Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. January 10, 2011; Accessed at 9. Centers for Disease Control and Prevention. Revised guidelines for performing CD4 T-cell determinations in persons infected with human immunodeficiency virus (HIV). Morbid Mortal Wkly Rep. 1997;46: Taylor JM, Fahey JL, Detels R, Giorgi JV. CD4 Percentage, CD4 number, and CD4:CD8 ratio in HIV infection, which to chose and how to use. J Acquir Immune Defic Syndr. 1989;2(2): Brinchmann JE, Vartdal F, Thorsby E.Dankner. T lymphocyte subset changes in human immunodeficiency virus infection. J Acquir Immune Defic Syndr. 1989;2(4): Dankner WM, Lindsey JC, Levin MJ, et al. Correlates of opportunistic infections in children infected with human immunodeficiency virus managed before highly active retroviral therapy. Pediatr Infect Dis J. 2001;20(1): World Health Organization.HIV/AIDS Programme, strengthening health services to fight HIV/AIDS. Antiretroviral therapy of HIV infection in infants and children: Towards universal access. Recommendations for public health approach Geneva, Switzerland: WHO; Available at paediatric pdf. 14. O'Gorman MR, Zijenah LS. CD4 T cell measurements in the management of antiretroviral therapy-a review with an emphasis on pediatric HIV-infected patients. CD4 T cell measurements in the management of antiretroviral of 18

24 therapy-a review with an emphasis on pediatric HIV-infected patients. Cytometry B Clin Cytom. 2008;74 Suppl 1:S Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date 15. Ledbetter JA, Evans RL, Lipinski M, Cunningham-Rundles C, Good RA, Herzenberg LA. Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man. J Exp Med. 1981;153: Centers for Disease Control. Update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37: Calvelli T, Denny TN, Paxton H, Gelman R, Kagan J, Nicholson JK, Green TA. Guideline for Flow Cytometric Immunophenotyping: A Report From The National Institute of Allergy and Infectious Diseases, Division of AIDS. Cytometry. 1993;14: Ekong T, Kupek E, Hill A, Clark C, Davies A, Pinching A. Technical influences on immunphenotyping by flow cytometry. The effect of time and temperature of storage on the viability of lymphocyte subsets. J Immunol Methods. 1993;164: Wayne PA. Clinical Applications in Flow Cytometry: Quality Assurance and Immunophenotyping of Lymphocytes: Approved Guideline. National Committee for Clinical Laboratory Standards NCCLS document H42-A. Warranty EMD Millipore Corporation warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for either: a) a period of one year from the date of shipment of the products; or b) a term less than one year, as such term is expressly stated on the product specification or product label. EMD MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications, and descriptions of EMD Millipore products appearing in EMD Millipore s published catalogs and product literature may not be altered except by express written agreement signed by an officer of EMD Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon. In the event of a breach of the foregoing warranty, EMD Millipore s sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies EMD Millipore promptly of any such breach. If after exercising reasonable efforts, EMD Millipore is unable to repair or replace the product or part, then EMD Millipore shall refund to the Company all monies paid for such applicable product. EMD MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL, OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS EMD Millipore Corporation. All rights reserved. No part of these works may be reproduced in any form without permission in writing of 18

25 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Contact Information For technical support and order information, contact: Millipore (UK) Ltd. 2 Fleming Road, Kirkton Campus, Livingston, EH54 7BN United Kingdom TEL: FAX: EMD Millipore Corporation Single Oak Drive Temecula, CA USA TEL: (US and Canada) MILLIPORE ( ) EMD Millipore, Muse, and the M logo are trademarks of Merck KGaA, Darmstadt Germany. 15 May 15, 2018 Part No MAN, Rev of 18

26 Operational Specification Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date AUTHORING SITE MM-Temecula AFFECTED SITES TITLE MCA100102_ Muse System Check [C] Kit Signed by ELECTRONIC SIGNATURES Meaning of Signature Server Date (dd-mmm-yyyy HH:mm z) CLOR Julie Author Signature 15-May :38 CEST Averbeck Jean Technical Approval 15-May :11 CEST TYAGARAJAN Kamala Technical Approval 15-May :37 CEST SMITH-MCCOLLUM Robert Management Approval 15-May :55 CEST Olson Christopher Quality Approval 16-May :11 CEST 1 of 11

27 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Millipore (UK) Ltd. 2 Fleming Road, Kirkton Campus, Livingston, EH54 7BN United Kingdom TEL: FAX: Muse System Check [C] Kit Catalog No. MCA (100 Tests) For In Vitro Diagnostic Use; Export Only IVD 2 of 11

28 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date 3 of 11

29 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Application The Muse System Check [C] Kit is used to verify the performance of your Muse System by assessing counting accuracy and fluorescence detection using a standardized fluorescent bead reagent. The kit contains Muse System Check [C] Bead Reagent and System Check [C] Diluent. To assure that the Muse System provides reliable, accurate results, run the System Check procedure every day that the instrument is used before performing any assay. Test Principle Muse System Check [C] Bead Reagent is provided as a concentrated fluorescent bead suspension of known concentration. To run the System Check procedure, mix the bead reagent with System Check [C] Diluent at the recommended dilution to yield a bead suspension at a specific target concentration. Run three replicates of the suspension on the Muse Cell Analyzer. The Muse System Check software module analyzes the results from the three data acquisitions of the bead suspension and displays the particle counts and a PASS/FAIL result. The average and %CV values for these results are also displayed. Particle counts that are outside the expected range are displayed in red. These results and the instrument settings are saved to a spreadsheet file. The results from all System Check procedures are appended to a file, providing a history of data that allows you to monitor the performance of your Muse System. Summary of Protocol Add 380 μl of System Check [C] Diluent to a tube. Kit Components Add 20 μl of System Check [C] Bead Reagent. Mix thoroughly and run on Muse Cell Analyzer. Muse System Check [C] Kit (Catalog No. MCA100102) contains the following reagents: Muse System Check [C] Beads (Part No ) Muse System Check [C] Diluent (Part No ) Muse System Check [C] Bead Information Card (Part No ) 1 4 of 11

30 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Materials Required but Not Supplied Muse Cell Analyzer Micropipettors Disposable micropipettor tips Microcentrifuge tubes with screw caps, 1.5 ml (VWR, Catalog No , or equivalent) Vortex mixer Disposable gloves 100% bleach solution (typically % sodium hypochlorite) Deionized water Muse Instrument Cleaning Fluid (ICF) [C], Catalog No. MCA Precautions The Muse System Check [C] Kit is intended for in vitro diagnostic use. Wear proper laboratory attire (lab coat, gloves, safety glasses) when handling or using this reagent. All materials should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Avoid specimen contact with skin and mucous membranes. The System Check [C] Diluent contains 0.05% sodium azide, which is toxic if ingested. Reagents containing sodium azide should be considered a poison. If products containing sodium azide are swallowed, seek medical advice immediately and show product container or label. (Refer to NIOSH, National Institute for Occupational Safety and Health; CAS#: ; and also to GHS, The Globally Harmonized System of Classification and Labeling of Chemicals.) Aqueous solutions of sodium azide, when mixed with acids, may liberate toxic gas. Any reagents containing sodium azide should be evaluated for proper disposal. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, flush with large volumes of water to prevent build-up in plumbing. Check with regulatory agencies to determine at what concentration sodium azide may cause a product to be regulated as hazardous. Avoid microbial contamination of the solution, which may cause erroneous results. Do not use the reagent beyond the expiration date. Material Safety Data Sheets (MSDS) for kit reagents are available from our website ( muse), by contacting EMD Millipore Technical Support, or from the Millipore technical library at Storage Store the Muse System Check [C] Kit refrigerated at 2 to 8 C. Do not freeze. Refer to the expiration date on the package label. Do not use the reagent after the expiration date. Shield the System Check [C] Bead Reagent from excessive exposure to light. 2 5 of 11

31 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date Before You Begin Allow the System Check [C] Beads and System Check [C] Diluent to warm to room temperature before use. Thoroughly mix the System Check [C] Beads vial for at least 5 seconds using a vortex mixer set at moderate speed. Take care to ensure that the beads are well mixed before removing an aliquot to prepare the diluted bead sample. Time considerations: Once diluted the beads are ready to acquire. No incubation is necessary. Acquiring three replicates of the diluted beads on the Muse Cell Analyzer takes approximately 5 minutes. Bead Preparation 1. Transfer 380 μl of the System Check [C] Diluent to a microcentrifuge tube or sample acquisition titer tube using a calibrated pipettor. 2. Thoroughly mix the System Check [C] Beads vial. Using a calibrated pipettor, pipette 20 μl of the System Check [C] Bead Reagent. Carefully wipe off excess solution adhering to the exterior of the pipette tip. Transfer the 20 μl of the bead reagent into the 380 μl of diluent. Mix the solution by repeatedly withdrawing and expelling a portion of the suspension with a 1,000-μL pipettor. This should sufficiently mix the beads. However, if vortexing is desired, be sure to cap the tube before vortexing. 3. The diluted beads are ready acquire. To ensure proper sampling and reliable results from the System Check procedure, thoroughly mix the bead suspension immediately before loading the tube on the Muse instrument, then mix again prior to the second and third acquisitions. Running the Beads on the Muse Cell Analyzer 1. Select System Check under Essential Tools at the main menu to display the System Check screen. 2. Check the fluid levels in the cleaning and waste bottles before proceeding. Select Close in the dialog that appears. 3 6 of 11

32 3. Select Run System Check. Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date 4. The first time you run the procedure, enter the bead lot number, expiration date, and check code. All values can be found on the information card that comes with the bead kit. Enter the Bead Lot # and press Done on the keypad. Touch the calendar icon in the Exp. Date field to open a calendar, allowing you to select the expiration date. Touch outside the calendar to close it. Touch the Check Code field and enter the code, then select Done on the keypad. Once you enter this information, it will remain in the software. Each time you run the procedure, check the information to ensure it is accurate. You will need to update the values for each new lot number. 5. Select Next. 6. Mix the tube of prepared beads and load it on the system. NOTE: To ensure accurate counts, mix the prepared beads immediately before running on the system. 7. Select Run. 4 7 of 11

33 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date The system performs a prime, then acquires the first replicate. The progress bar and fly wheel provide indicators as to the status of acquisition. The progress bar is divided into three sections one for each replicate. If the fly wheel is turning but the progress bar in not advancing, the fluid system may be clogged or the beads may have settled to the bottom of the tube. If this occurs, refer to Troubleshooting Tips on page 6 for more information. 8. Remove the tube and vortex it to resuspend the beads. 9. Load the tube and select Run. The system acquires the second replicate. 10. Repeat steps 8 and 9 to acquire the third replicate. The system displays a PASS/FAIL result and the Particles/mL results for each replicate. Log list button and arrows. The bead lot number, date, and time of the run appear just above the Bead Info and printer tabs. All runs in the log list are listed in this format. The Status field at the bottom of the screen indicates that the System Check was completed. 11. (Optional) Select the log list button ( ) to view past System Check results or use the arrow buttons to display past results in the Bead Info tab. 12. (Optional) Select Options in the title bar to clear or export the check log, export the service log, view a trend graph, or view the event log. Refer to the Muse Auto CD4/CD4% System User s Guide for more information. 5 8 of 11

34 Doc ID: Version: 5.0 Status: Effective Effective Date: 16-May :01 CEST (Server Date System Check Results The result appears as PASS or FAIL. The result is based on the mean fluorescence intensity (MFI) values as well as the bead count for each replicate. The bead count (Particles/mL) for each replicate and the average are displayed, as well as the %CV. The MFIs are not displayed but can be found in the service log. For information on accessing the service log, refer to the Muse Auto CD4/CD4% System User s Guide. If any result for Particles/mL falls outside ±10% of the expected value, the result is outside the acceptable range and appears in red. If the result fails, touch the Help button (?) to display troubleshooting information. Troubleshooting Tips Touch Help button to get troubleshooting information. 1. Ensure the correct Bead Lot # and Check Code are entered. Refer to the System Check [C] Beads vial label and information card for values. 2. Thoroughly mix all suspensions of the System Check [C] Beads before pipetting the reagent or loading the diluted sample onto the instrument for data acquisition. Inadequate mixing results in erroneous bead counts. 3. System Check reagents should be equilibrated to room temperature before preparing a dilution of the bead reagent. The test sample should be at room temperature before acquiring data on the Muse System. 4. Cover or cap all tubes when vortexing to avoid volume loss while mixing. Sample loss may affect the concentration of particles in the diluted bead suspension. 5. To ensure accurate results, use pipettors that have been properly maintained and calibrated. 6. If the 1st Replicate or 2nd Replicate result is out of range, you can discontinue the run immediately by selecting Abort. Perform a Complete System Clean and rerun the System Check procedure. If the results continue to fall outside the expected range, prepare a new dilution of System Check [C] Beads. Rerun the System Check procedure with the fresh samples. 7. If, during acquisition, the progress bar stops advancing, then the sample may be depleted. Check the volume left in the tube. If it is less than 50 μl, prepare a freshly diluted suspension. If the volume is still sufficient, then most likely a blockage of flow has occurred. Perform a Backflush as outlined in Tip 5. Then perform a Quick Clean cleaning cycle to remove bleach residue. You may then continue with acquisition. If this procedure does 6 9 of 11