BD OneFlow LST; BD OneFlow B-CLPD T1; BD FACSLyric Flow Cytometer, 8-color [4-2-2], 10-color [4-3-3], 12-color (4-3-5)

{0} [LOGO] FDA U.S. FOOD & DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION ## DECISION SUMMARY ASSAY AND INSTRUMENT ### I Background Information: A 510(k) Number K260215 B Applicant BD Biosciences C Proprietary and Established Names BD OneFlow LST BD OneFlow B-CLPD T1 BD FACSLyric Flow Cytometer, 8-color (4-2-2), 10-color (4-3-3), 12-color (4-3-5) D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | PWD | Class II | 21 CFR 864.7010 – Flow Cytometric Test System for Hematopoietic Neoplasms | HE - Hematology | | OYE | Class II | 21 CFR 864.5220 – Automated Differential Cell Counter | | ### II Submission/Device Overview: A Purpose for Submission: New device B Measurand: Cluster of Differentiation (CD) Surface Markers on white blood cells C Type of Test: Immunophenotyping, qualitative, flow cytometric assay Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 {1} ### III Intended Use/Indications for Use: #### A Intended Use(s): See Indications for Use below. #### B Indication(s) for Use: # **BD OneFlow LST** BD OneFlow LST (Lymphoid Survey Tube) is intended for in vitro diagnostic use as a panel of fluorochrome-conjugated antibodies for qualitative flow cytometric immunophenotyping of mature lymphocyte populations on the BD FACSLyric flow cytometer using BD FACSuite Clinical Application. BD OneFlow LST is used as an aid in identification of hematologically abnormal mature B-cell, mature T-cell, and mature NK-cell lineages in patients having, or suspected of having, lymphoid lineage malignancies or neoplasms when used in conjunction with lineage-specific BD OneFlow reagents. BD OneFlow LST can be used with peripheral blood specimens collected in EDTA (K2 or K3) or heparin (sodium or lithium) tubes, bone marrow specimens collected in K2 EDTA tubes, and lymph node specimens. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical or laboratory findings. BD OneFlow LST provides multi-parameter, qualitative results for the following antigens: CD3, CD4, CD5, CD8, CD19, CD20, CD38, CD45, CD56, Igκ, Igλ, and TCRγ/δ. # **BD OneFlow B-CLPD T1** BD OneFlow B-CLPD T1 (B-cell Chronic Lymphoproliferative Disease Tube 1) is intended for in vitro diagnostic use as a panel of fluorochrome-conjugated antibodies for qualitative flow cytometric immunophenotyping of mature B-lymphocyte populations on the BD FACSLyric flow cytometer using BD FACSuite Clinical Application. When used in conjunction with BD OneFlow LST, BD OneFlow B-CLPD T1 is used as an aid in the diagnosis of patients having, or suspected of having, a B-cell non-Hodgkin's lymphoma including B-cell chronic lymphocytic leukemia (B-CLL). BD OneFlow B-CLPD T1 can be used with peripheral blood specimens collected in EDTA (K2 or K3) or heparin (sodium or lithium) tubes, bone marrow specimens collected in K2 EDTA tubes, and lymph node specimens. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical or laboratory findings. BD OneFlow B-CLPD T1 provides multi-parameter, qualitative results for the following antigens: CD10, CD19, CD20, CD23, CD43, CD45, CD79b, and CD200. # **BD FACSLyric Flow Cytometer, 8-color (4-2-2), 10-color (4-3-3), 12-color (4-3-5)** The BD FACSLyric flow cytometer is intended for use as an in vitro diagnostic device for the following: - Immunophenotyping using up to eight fluorescence detection channels and two light scatter channels using a violet (405 nm), a blue (488 nm), and a red (640 nm) laser. - Enumeration of residual white blood cells (rWBCs) in leucoreduced blood products. K260215 - Page 2 of 27 {2} It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument. ### C Special Conditions for Use Statement(s): For Prescription Use Only ### D Special Instrument Requirements: BD FACSLyric Flow Cytometer ### IV Device/System Characteristics: ### A Device Description: 1. BD OneFlow LST and BD OneFlow B-CLPD T1 Reagents: The reagents of the OneFlow LST and B-CLPD T1 consist of single-use tubes containing the following fluorochrome-conjugated antibodies in an optimized dried formulation as shown in the following table: | Reagent | FITC | PE | PerCP-Cy5.5 | PE-Cy7 | APC | APCH7 | V450 | V500-C | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | LST | CD8 and Igλ | CD56 and Igκ | CD5 | CD19 and TCRγ/δ | CD3 | CD38 | CD20 and CD4 | CD45 | | B-CLPD T1 | CD23 | CD10 | CD79b | CD19 | CD200 | CD43 | CD20 | CD45 | Traceability of individual clones to Human Leukocyte Differentiation Antigen (HLDA) Workshops consensus nomenclatures, for individual monoclonal antibodies within these panels are summarized in the following table: | Marker | Clone ID | Subclass | Species | HLDA | | --- | --- | --- | --- | --- | | CD3 | SK7 | IgG1 | mouse | Second | | CD4 | SK3 | IgG1 | mouse | First\( ^{1} \) | | CD5 | L17F12 | IgG2a | mouse | Second\( ^{3} \) | | CD8 | SK1 | IgG1 | mouse | First | | CD10 | HI10a | IgG1 | mouse | Fifth\( ^{4} \) | | Marker | Clone ID | Subclass | Species | HLDA | | --- | --- | --- | --- | --- | | CD38 | HB7 | IgG1 | mouse | Third | | CD43 | IG10 | IgG1 | mouse | Sixth\( ^{2} \) | | CD45 | 2D1 | IgG1 | mouse | Third | | CD56 | MY31 | IgG1 | mouse | Fifth | | CD79b | SN8 | IgG1 | mouse | Fifth | \( ^{1} \) Bernard AR, Boumsell L, Dausset J, et al. eds. Leucocyte Typing: Human Leucocyte Differentiation Antigens Detected by Monoclonal antibodies. Berlin: Springer-Verlag, 1984. \( ^{2} \) Kishimoto T, Kikutani H, von dem Born AEGK, et al. eds. Leucocyte Typing VI. New York: Garland Publishing, Inc., 1997. \( ^{3} \) Reinherz EL, Haynes BF, Nadler L, Bernstein ID, eds. Leukocyte Typing II. New York: Springer-Verlag, 1985. \( ^{4} \) Schlossman SF, Boumsell L, Gilks W, et al. eds. Leucocyte Typing V: White cell differentiation antigens. Oxford: Oxford University Press, 1995. K260215 - Page 3 of 27 {3} | Marker | Clone ID | Subclass | Species | HLDA | | --- | --- | --- | --- | --- | | CD19 | SJ25C1 | IgG1 | mouse | Second | | CD20 | L27 | IgG1 | mouse | Third^{6} | | CD23 | EBVCS-5 | IgG1 | mouse | Fifth | | Marker | Clone ID | Subclass | Species | HLDA | | --- | --- | --- | --- | --- | | CD200 | MRC-OX-104 | IgG1 | mouse | Seventh^{5} | | anti-Lambda | 1-155-2 | IgG1 | mouse | N/A | | anti-Kappa | TB28-2 | IgG1 | mouse | N/A | | anti-TCRγδ | 11F2 | IgG1 | mouse | N/A | ## 2. Instrument: BD OneFlow LST and BD OneFlow B-CLPD T1 are used on the BD FACLyric system. Refer to K170974 for detailed description and functioning of the BD FACSLyric Flow Cytometer. The current clearance adds a 405-nm violet laser and two detection channels for two additional fluorochromes (V450 and V500-C) that are excited off the violet laser. Three new fluorescent beads (BD FC Beads APC-H7, BD FC Beads V450, BD FC Beads V500-C), which are required for setup of the eight FACSLyric Flow Cytometer fluorescence detection channels, are included. These beads are used in conjunction with previously cleared BD CS&T Beads BD FC Beads 7-Color Kit, for use in standardization of the instrument. The system for use with BD OneFlow LST and BD OneFlow B-CLPD T1 is summarized as follows: | BD FACSLyris System | | | | --- | --- | --- | | Setup beads | Setup Software | Analysis software | | BD CS&T Beads BD FC Beads 7-Color Kit BD FC Beads APC-H07 BD FC Beads V450 BD FC Beads V500-C | BD FACSuite Clinical Application, v1.5 | BD FACSuite Clinical Application, v1.5 | Prior to first use of the reagents, installation of the BD OneFlow LST and BD OneFlow B-CLPD T1 Assay Modules is required for running the BD OneFlow LST and BD OneFlow B-CLPD T1 Assays. ### 3. Reagents and Materials Required but not Provided - BD OneFlow Assays Installer - BD FACS Lysing Solution - BD CS&T Beads - BD FC Beads 7-Color Kit - BD FC Beads APC-H7 - BD FC Beads V450 - BD FC Beads V500-C 5 Mason D, Andre P, Bensussan A, et al. eds. Leucocyte Typing VII. Oxford: Oxford University Press, 2002. 6 McMichael AJ, Beverley PCL, Cobbold S, et al. eds. Leucocyte Typing III. White Cell Differentiation Antigens. Oxford: Oxford University Press, 1987. K260215 - Page 4 of 27 {4} - (Optional) BD FACS Universal Loader - Other materials: deionized water, pipette, cell strainer, centrifuge, wash buffer, etc. ### B Principle of Operation: The BD OneFlow reagents are composed of monoclonal antibodies, each conjugated to a specific fluorochrome. The specimen is added to the reagent tube and incubated, allowing each fluorochrome-conjugated monoclonal antibody in the reagent tube to bind to a specific antigen on the surface of the cells. After incubation, BD FACS Lysing Solution is added to lyse the red blood cells in the sample. Cells are acquired on a flow cytometer using BD FACSuite Clinical software. During acquisition, the cells travel past the laser beam and scatter the laser light. Excitation of any fluorochrome-labeled antibodies that are bound to the cells, causes the stained cells to fluoresce at specific wavelengths of light. These scatter and fluorescence signals are detected by the instrument and provide information about the cell's size, internal complexity and relative fluorescence intensity that permits assignment of the cells to a specific lineage or disease status. The instrument software is used to analyze the data and report the result. ### Specimen Collection and Sample Processing Peripheral blood (PB) specimens are collected in K2 EDTA, K3 EDTA, sodium heparin, or lithium heparin. Bone marrow (BM) specimens are collected in K2 EDTA. Lymph node (LN) specimens are collected in phosphate-buffered saline (PBS), PBS-soaked gauze, or cell culture media. PB and BM specimens should be processed within 26 hours of collection. Viability of specimens should be assessed, for example by using Trypan Blue staining and counting the cells using a hemocytometer or a cell counter. Samples are pre-washed three times with wash buffer, then incubate with antibody reagent 30–35 minutes at 20–25°C, followed by another wash. PB and BM samples should be acquired within 2 hours of staining when kept at room temperature, protected from light. LN samples should be acquired within 1 hour of staining when kept at room temperature, protected from light. ### Sample Acquisition and Data Analysis The sample acquisition and data analysis are controlled within the assay modules installed by the BD OneFlow Assays Installer CLPD Panel. The assay modules are predefined to target specific cell populations that can be identified by BD OneFlow LST and BD OneFlow B-CLPD T1. Acquisition is performed with the BD OneFlow Acquisition sheet, which contains dot plots and gates to identify the relevant cell populations for the assay and 100,000 events are acquired. After acquisition, results are displayed in reports within the software where they are analyzed directly, and data is automatically provided in Flow Cytometry Standard (FCS) files (FCS v3.1) that are saved at a designated location on the computer workstation hard drive. The reports contain several major components such as plots, gates, and statistics. Gates are defined by and contained within the assay module. Gates are used within the software to allow identification of cells of interest, classify events in populations, display cell populations in plots, and calculate population statistics. All gates are present in the plots and may be adjusted by the user as needed. ### Interpretation of Results The BD OneFlow Assay Modules automatically generate laboratory reports after sample acquisition. The user reviews and then approves (via signature) these reports and can add information to the supplemental report, if needed. Reports allow for identification of various lymphocyte populations using a defined set of plots and gating strategies. Normal cells, which K260215 - Page 5 of 27 {5} display highly reproducible patterns of cell surface marker expression, are used as both internal quality controls and as references to identify candidate abnormal cell populations. Identification of cell populations, as carried out by a trained specialist, identify abnormal cell populations with patterns of cell surface marker expression that may be associated with specific hematological malignancies. The results are interpreted by a pathologist or equivalent professional in conjunction with other clinical or laboratory findings to reach the final diagnosis. ### C Instrument Description Information: 1. Instrument Name: BD FACSLyric Flow Cytometer 2. Modes of Operation Refer to K170974 The current clearance adds a 405-nm violet laser and two detection channels for two additional fluorochromes (V450 and V500-C) that are excited off the violet laser. Three new fluorescent beads (BD FC Beads APC-H7, BD FC Beads V450, AND BD FC Beads V500-C), which are required for setup of the eight FACSLyric Flow Cytometer fluorescence detection channels, are included and used in conjunction with previously cleared BD CS&T Beads, BD FC Beads 7-Color Kit, for use in standardization of the instrument. The cleared instrument is indicated for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument. 3. Specimen Identification: Barcode reader or manual entry 4. Specimen Sampling and Handling: Manual loading onto the tube port of the flow cytometer and automated loading through a multi-tube FACS Universal Loader 5. Calibration: Calibration is performed with the IVD assay intended for use with the instruments. 6. Quality Control: Quality control is performed with IVD assays intended for use with the instruments. BD CS&T Beads are used to run the characterization quality control (CQC) and the daily performance quality control (PQC) for the cytometer. BD FC Beads enable the software to automatically determine the spillover values (SOVs) for fluorescence compensation in the reference settings. In addition, the BD OneFlow Application Guide for Chronic K260215 - Page 6 of 27 {6} Lymphoproliferative Diseases presents representative dot plots for the use of normal cell populations as internal controls to verify reagent performance. This medical device product has functions subject to FDA premarket review as well as functions that are not subject to FDA premarket review. For those functions that are not subject to FDA premarket review, FDA assessed those functions only to the extent that they either could adversely impact the safety and effectiveness of the functions subject to FDA premarket review or they are included as a labeled positive impact that was considered in the assessment of the functions subject to FDA premarket review. # V Substantial Equivalence Information: # A Predicate Device Name(s): ClearLLab 10C Panels (B, T, M1, M2) BD FACSLyric Flow Cytometer # B Predicate 510(k) Number(s): K183592 K201814 # C Comparison with Predicate(s): BD OneFlow LST | Device & Predicate: | K260215 (Candidate) | K183592 (Predicate) | | --- | --- | --- | | Device Trade Name | BD OneFlow LST | ClearLLab 10C Panels (B, T, M1, M2) | | **General Device Characteristic Similarities** | | | | Intended Use/Indications for Use | BD OneFlow LST (Lymphoid Survey Tube) is intended for in vitro diagnostic use as a panel of fluorochrome-conjugated antibodies for qualitative flow cytometric immunophenotyping of mature lymphocyte populations on the BD FACSLyric flow cytometer using BD FACSuite Clinical Application. BD OneFlow LST is used as an aid in identification of hematologically abnormal mature B-cell, mature T-cell, and mature NK-cell lineages in patients having, or suspected of having, lymphoid lineage malignancies or neoplasms when used in conjunction | The ClearLLab 10C Panels are intended for in vitro diagnostic use for qualitative identification of cell populations by multiparameter immunophenotyping on the Navios and Navios EX flow cytometers. These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients having, or suspected of having, the following hematopoietic neoplasms: chronic leukemia, acute leukemia, non-Hodgkin lymphoma, myeloma, myelodysplastic syndrome (MDS), and/or myeloproliferative neoplasms (MPN). The reagents can be used with peripheral whole blood (collected in K2EDTA, Acid Citrate Dextrose (ACD) or Heparin), bone marrow (collected in | K260215 - Page 7 of 27 {7} | Device & Predicate: | K260215 (Candidate) | K183592 (Predicate) | | --- | --- | --- | | | with lineage-specific BD OneFlow reagents. BD OneFlow LST can be used with peripheral blood specimens collected in EDTA (K2 or K3) or heparin (sodium or lithium) tubes, bone marrow specimens collected in K2 EDTA tubes, and lymph node specimens. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical or laboratory findings. BD OneFlow LST provides multi-parameter, qualitative results for the following antigens: CD3, CD4, CD5, CD8, CD19, CD20, CD38, CD45, Igκ, Igλ, and TCRγ/δ. | K2EDTA, ACD or Heparin) and lymph node specimens. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings. These reagents provide multiparameter, qualitative results for the surface antigens listed below: • ClearLLab 10C B Cell Tube: Kappa, Lambda, CD10, CD5, CD200, CD34, CD38, CD20, CD19, CD45 • ClearLLab 10C T Cell Tube: TCRγδ, CD4, CD2, CD56, CD5, CD34, CD3, CD8, CD7, CD45 • ClearLLab 10C M1 Cell Tube: CD16, CD7, CD10, CD13, CD64, CD34, CD14, HLADR, CD11b, CD45 • ClearLLab 10C M2 Cell Tube: CD15, CD123, CD117, CD13, CD33, CD34, CD38, HLA-DR, CD19, CD45 | | Specimen Type | Human whole blood, bone marrow, and lymph node | Same | | Technology | Flow cytometry | Same | | Reagent Form | Dried reagents in clear plastic single-use tubes | Same | | Sample Preparation | Wash and prepare samples manually | Same | | **General Device Characteristic Differences** | | | | Storage Conditions | 2–8°C 23–27°C | 18–30°C | | Instrument | BD FACSLyric Flow Cytometer | Navios Flow Cytometer | | Software Analysis | BD FACSuite Clinical Application (version 1.5) with the BD OneFlow Assay Modules | Kaluza C data analysis software | | Reagent Tubes Composition | CD3, CD4, CD5, CD8, CD19, CD20, CD38, CD45, CD56, Igκ, Igλ, TCRγδ | CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD38, CD45, CD56, CD64, CD123, CD117, CD11b, HLA-DR, Igκ, Igλ, TCRγδ | | Reagents: Color Compensation | BD FC Beads 7-Color Kit BD FC Beads APC-H7 BD FC Beads V450 BD FC Beads V500-C | ClearLLab Compensation Kit with ClearLLab Compensation Beads | K260215 - Page 8 of 27 {8} # **BD OneFlow B-CLPD T1** | Device & Predicate: | **K260215** **(Candidate)** | **K183592** **(Predicate)** | | --- | --- | --- | | Device Trade Name | **BD OneFlow B-CLPD T1** | **ClearLLab 10C Panels** **(B, T, M1, M2)** | | **General Device Characteristic Similarities** | | | | Intended Use/Indications for Use | BD OneFlow B-CLPD T1 (B-cell Chronic Lymphoproliferative Disease Tube 1) is intended for in vitro diagnostic use as a panel of fluorochrome-conjugated antibodies for qualitative flow cytometric immunophenotyping of mature B-lymphocyte populations on the BD FACSLyric flow cytometer using BD FACSuite Clinical Application. When used in conjunction with BD OneFlow LST, BD OneFlow B-CLPD T1 is used as an aid in the diagnosis of patients having, or suspected of having, a B-cell non-Hodgkin's lymphoma including B-cell chronic lymphocytic leukemia (B-CLL). BD OneFlow B-CLPD T1 can be used with peripheral blood specimens collected in EDTA (K2 or K3) or heparin (sodium or lithium) tubes, bone marrow specimens collected in K2 EDTA tubes, and lymph node specimens. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical or laboratory findings. BD OneFlow B-CLPD T1 provides multi-parameter, qualitative results for the following antigens: CD10, CD19, CD20, CD23, CD43, CD45, CD79b, and CD200. | The ClearLLab 10C Panels are intended for in vitro diagnostic use for qualitative identification of cell populations by multiparameter immunophenotyping on the Navios and Navios EX flow cytometers. These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients having, or suspected of having, the following hematopoietic neoplasms: chronic leukemia, acute leukemia, non-Hodgkin lymphoma, myeloma, myelodysplastic syndrome (MDS), and/or myeloproliferative neoplasms (MPN). The reagents can be used with peripheral whole blood (collected in K2EDTA, Acid Citrate Dextrose (ACD) or Heparin), bone marrow (collected in K2EDTA, ACD or Heparin) and lymph node specimens. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings. These reagents provide multiparameter, qualitative results for the surface antigens listed below: • ClearLLab 10C B Cell Tube: Kappa, Lambda, CD10, CD5, CD200, CD34, CD38, CD20, CD19, CD45 • ClearLLab 10C T Cell Tube: TCRγδ, CD4, CD2, CD56, CD5, CD34, CD3, CD8, CD7, CD45 • ClearLLab 10C M1 Cell Tube: CD16, CD7, CD10, CD13, CD64, CD34, CD14, HLADR, CD11b, CD45 • ClearLLab 10C M2 Cell Tube: CD15, CD123, CD117, CD13, CD33, CD34, CD38, HLA-DR, CD19, CD45 | | Specimen Type | Human whole blood, bone marrow, and lymph node | Same | | Technology | Flow cytometry | Same | | Reagent Form | Dried reagents in clear plastic single-use tubes | Same | K260215 - Page 9 of 27 {9} | Device & Predicate: | K260215 (Candidate) | K183592 (Predicate) | | --- | --- | --- | | Sample Preparation | Wash and prepare samples manually | Same | | **General Device Characteristic Differences** | | | | Storage Conditions | 2–8°C 23–27°C | 18–30°C | | Instrument | BD FACSLyric Flow Cytometer | Navios Flow Cytometer | | Software Analysis | BD FACSuite Clinical Application (version 1.5) with the BD OneFlow Assay Modules | Kaluza C data analysis software | | Reagent Tubes Composition | CD10, CD19, CD20, CD23, CD43, CD45, CD79b, CD200 | CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD38, CD45, CD56, CD64, CD123, CD117, CD11b, HLA-DR, Igκ, Igλ, TCRγδ | | Reagents: Color Compensation | BD FC Beads 7-Color Kit BD FC Beads APC-H7 BD FC Beads V450 BD FC Beads V500-C | ClearLLab Compensation Kit with ClearLLab Compensation Beads | # **BD FACSLyric Flow Cytometer(s)** | Device & Predicate: | K260215 (Candidate) | K201814 (Predicate) | | --- | --- | --- | | Device Trade Name | BD FACSLyric Flow Cytometer 8-Color (4-2-2), 10-Color (4-3-3), 12-Color (4-3-5) | BD FACSLyric Flow Cytometer (3-1, 4-2, 4-2-2, 4-3-3, 4-3-5 Configurations) | | **General Device Characteristic Similarities** | | | | Intended Use/Indications for Use | The BD FACSLyric flow cytometer is intended for use as an in vitro diagnostic device for the following: - Immunophenotyping using up to eight fluorescence detection channels and two light scatter channels using a violet (405 nm), a blue (488 nm), and a red (640 nm) laser. - Enumeration of residual white blood cells (rWBCs) in leucoreduced blood products.It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument. | The BD FACSLyric flow cytometer is intended for use as an in vitro diagnostic device for immunophenotyping using up to six fluorescence detection channels and two light scatter channels using a blue (488-nm) and a red (640-nm) laser. It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument. | | Detection Principle | Immunofluorescence | Same | | Electronics | Up to 35000 events/sec | Same | K260215 - Page 10 of 27 {10} | Device & Predicate: | K260215 (Candidate) | K201814 (Predicate) | | --- | --- | --- | | Device Trade Name | BD FACSLyric Flow Cytometer 8-Color (4-2-2), 10-Color (4-3-3), 12-Color (4-3-5) | BD FACSLyric Flow Cytometer (3-1, 4-2, 4-2-2, 4-3-3, 4-3-5 Configurations) | | Sample Introduction | • Manual • Automated loading through FACS Universal Loader | Same | | Fluidics | BD FACSLyric flow cytometer fluidics | Same | | **General Device Characteristic Differences** | | | | Maximum Parameter Detectors | IVD detection channels eight (8) Fluorescence channels plus forward scatter and side scatter | IVD detection channels six (6) Fluorescence channels plus forward scatter and side scatter | | IVD Lasers/ Excitation | Blue Laser: • Wavelength: 488 nm • Optical power: 20 mW Red Laser: • Wavelength: 640 nm • Optical power: 40 mW Violet Laser: • Wavelength: 405 nm • Optical power: 40 mW | Blue Laser: • Wavelength: 488 nm • Optical power: 20 mW Red Laser: • Wavelength: 640 nm • Optical power: 40 mW | | Optical Configurations | • 3-laser (blue, red, violet), 8-color (4-2-2) • 3-laser (blue, red, violet), 10-color (4-3-3) • 3 laser (blue, red, violet), 12-color (4-3-5) | • 2-laser (blue, red), 4-color (3-1) • 2-laser (blue, red), 6-color (4-2) • 3-laser (blue, red, violet), 8-color (4-2-2) • 3-laser (blue, red, violet), 10-color (4-3-3) • 3 laser (blue, red, violet), 12-color (4-3-5) configuration | | Software | BD FACSuite Clinical Application (version 1.5). | BD FACSuite Clinical Application (version 1.4) | ### VI Standards/Guidance Documents Referenced: The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used: - CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures - CLSI EP09c, 3rd Ed., Measurement Procedure Comparison and Bias Estimation Using Patient Samples - CLSI EP12, 3rd Ed., Evaluation of Qualitative, Binary Output Examination Performance - CLSI EP25, 2nd Ed., Evaluation of Stability of In Vitro Medical Laboratory Test Reagents - CLSI EP28-A3c, Defining Establishing and Verifying Reference Intervals in the Clinical Laboratory - CLSI H43-A2, Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells - CLSI H26-A2, Validation Verification and Quality Assurance of Automated Hematology Analyzers K260215 - Page 11 of 27 {11} # VII Performance Characteristics (if/when applicable): # A Analytical Performance: # 1. Precision/Reproducibility: Precision studies were conducted according to the methodology presented in CLSI Guideline EP05-A3. # a. Within-Laboratory Precision: Within-laboratory precision of hematologically abnormal samples having malignant and/or non-malignant cell populations was evaluated with the BD OneFlow LST and BD OneFlow B-CLPD T1 assays on the BD FACSLyric flow cytometer with BD FACSuite Clinical software. # i. Single-Analyst Within-Laboratory Precision: A panel of 36 clinical whole blood, bone marrow and lymph node samples were analyzed by a single analyst (× 2 replicates/run × 2 runs, each, randomized and blinded). Each sample that was assessed for the precision of the detection of the presence or absence of a composite malignant phenotype, demonstrated 100% qualitative agreement (144/144) across all specimens processed across sites, anticoagulants, specimen types, and phenotypes. # ii. Multi-Analyst Within-Laboratory Precision: A panel of 20 clinical samples were collected across five sites and assessed, with representation of all specimen types and anticoagulants, with each sample tested in two replicates per run and two runs on one cytometer at each site. Following acquisition, sample data files were analyzed by each of three independent analysts in a randomized and blinded fashion for a total of twelve independent evaluations per sample. Each sample was assessed for the precision of the detection of the presence or absence of a malignant phenotype, as defined in the following tables as a composite of markers across the LST and B-CLPD T1 tubes. | OneFlow Tube | Population | Immunophenotype of Normal Population | Markers associated with aberrancy | | --- | --- | --- | --- | | LST | B cells | CD45^{+} SSC^{low} CD19^{+} CD20^{Heterogenous} | Igκ, Igλ restriction; CD5^{+}; CD20^{Het} | | B-CLPD T1 | B cells | CD45^{+} SSc^{low} CD19^{+} CD20^{+} | CD10, CD23, CD200, CD79b, CD43, CD20 | In the following tables, qualitative precision was determined for the identification of the aberrant or non-aberrant phenotypes above, based upon the outputs from both BD OneFlow LST and B-CLPD T1, in conjunction. K260215 - Page 12 of 27 {12} Precision from Multiple-Analysts for BD OneFlow LST + B-CLPD T1 (Qualitative Analysis) | | Agreement | | | | --- | --- | --- | --- | | | | Subgroup | (n/N) | | Site | 1 | 32/36 | 88.9% | | | 2 | 24/24 | 100% | | | 3 | 32/36 | 88.9% | | | 4 | 128/132 | 97.0% | | | 5 | 12/12 | 100% | | Specimen Type | PB | 116/120 | 96.7% | | | BM | 68/72 | 94.4% | | | LN | 44/48 | 91.7% | | Anticoagulant | K2 EDTA | 80/84 | 95.2% | | | K3 EDTA | 48/48 | 100% | | | Lithium heparin | 44/48 | 91.7% | | | Sodium heparin | 12/12 | 100% | | Clinical Diagnosis | Malignant | 168/168 | 100% | | | Non-malignant | 60/72 | 83.3% | | | Total | 228/240 | 95.0% | Multi-Analyst Agreement by Anticoagulant and Specimen Type across Sites and Specimen (Qualitative Analysis) | Anticoagulant | Specimen Type | Expected Phenotype | Agreement | | | --- | --- | --- | --- | --- | | | | | (n/N) | (%) | | K2 EDTA | BM | Aberrant | 60/60 | 100% | | K2 EDTA | BM | Non-aberrant | 8/12 | 66.7% | | K2 EDTA | PB | Aberrant | 12/12 | 100% | | K3 EDTA | PB | Aberrant | 48/48 | 100% | | Lithium heparin | PB | Aberrant | 24/24 | 100% | | Lithium heparin | PB | Non-aberrant | 20/24 | 83.3% | | Sodium heparin | PB | Aberrant | 12/12 | 100% | | N/A | LN | Aberrant | 12/12 | 100% | | N/A | LN | Non-aberrant | 32/36 | 88.9% | From the precision study designs above representing twelve (12) total reads per clinical sample (three analysts × two replicates/run × two runs), semi-quantitative numerical values for target populations were also assessed for precision, using quantitative ANOVA methods. The following table presents the precision of reported semi-quantitative results from LST and B-CLPD T1: Precision of Multiple-Analyst for BD OneFlow LST + BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis) | Population | Type | Anticoagulant | (%)^{1}Mean | Within-Run | | Between-Run | | Between-Analyst | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | | | | | | | | | | | | Abnormal CD19+CD5+ B Cells | BM | K2 EDTA | 18.3 | 0.93 | 5.06 | 0.68 | 3.71 | 0.28 | 1.55 | 1.19 | 6.46 | | | | K2 EDTA | 53.6 | 4.69 | 8.76 | 3.99 | 7.45 | 0.00 | 0.00 | 6.16 | 11.5 | | | | K2 EDTA | 67.9 | 0.49 | 0.72 | 2.45 | 3.61 | 0.00 | 0.00 | 2.50 | 3.68 | | | | K2 EDTA | 70.6 | 0.77 | 1.09 | 1.08 | 1.53 | 2.40 | 3.40 | 2.75 | 3.89 | | | | K2 EDTA | 90.3 | 0.21 | 0.24 | 0.09 | 0.10 | 0.38 | 0.42 | 0.45 | 0.50 | K260215 - Page 13 of 27 {13} Precision of Multiple-Analyst for BD OneFlow LST + BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis) | Population | Type | Anticoagulant | (%)^{1}Mean | Within-Run | Between-Run | Between-Analyst | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | | PB | K3 EDTA | 3.90 | 8.00 | 205 | 0.00 | 0.00 | 1.74 | 44.7 | 8.19 | 210 | | K2 EDTA | 14.3 | 5.54 | 38.8 | 34.8 | 243 | 0.24 | 1.71 | 35.2 | 246 | | K3 EDTA | 44.7 | 3.40 | 7.62 | 5.24 | 11.7 | 5.38 | 12.0 | 8.24 | 18.5 | | Lithium heparin | 53.4 | 14.7 | 27.6 | 3.38 | 6.33 | 0.00 | 0.00 | 15.1 | 28.3 | | K3 EDTA | 75.6 | 0.69 | 0.91 | 0.93 | 1.23 | 0.00 | 0.00 | 1.16 | 1.53 | | Lithium heparin | 88.5 | 3.53 | 3.99 | 2.67 | 3.01 | 0.00 | 0.00 | 4.43 | 5.00 | | K3 EDTA | 94.0 | 0.28 | 0.29 | 0.00 | 0.00 | 0.04 | 0.05 | 0.28 | 0.30 | | CD19+CD20+Igκ/Igλ-restricted B Cells | BM | K2 EDTA | 6.15 | 0.84 | 13.6 | 0.00 | 0.00 | 10.7 | 173 | 10.7 | 173 | | K2 EDTA | 52.5 | 5.74 | 10.9 | 0.00 | 0.00 | 4.47 | 8.52 | 7.28 | 13.9 | | K2 EDTA | 68.0 | 0.43 | 0.63 | 2.49 | 3.66 | 0.00 | 0.00 | 2.53 | 3.72 | | K2 EDTA | 70.6 | 0.76 | 1.07 | 0.91 | 1.28 | 2.45 | 3.46 | 2.72 | 3.85 | | K2 EDTA | 90.2 | 0.23 | 0.26 | 0.00 | 0.00 | 0.32 | 0.36 | 0.40 | 0.44 | | LN | N/A | 13.7 | 8.57 | 62.4 | 3.72 | 27.1 | 17.0 | 123 | 19.4 | 141 | | PB | K3 EDTA | 20.7 | 0.66 | 3.18 | 0.00 | 0.00 | 0.00 | 0.00 | 0.66 | 3.18 | | K3 EDTA | 46.3 | 3.54 | 7.65 | 7.33 | 15.8 | 2.72 | 5.88 | 8.58 | 18.5 | | Lithium heparin | 53.4 | 14.7 | 27.6 | 3.41 | 6.38 | 0.00 | 0.00 | 15.1 | 28.4 | | Sodium heparin | 64.3 | 1.80 | 2.80 | 0.00 | 0.00 | 0.00 | 0.00 | 1.80 | 2.80 | | K3 EDTA | 75.7 | 0.69 | 0.92 | 0.92 | 1.21 | 0.00 | 0.00 | 1.15 | 1.52 | | Lithium heparin | 88.4 | 3.46 | 3.91 | 2.70 | 3.05 | 0.00 | 0.00 | 4.38 | 4.96 | | K2 EDTA | 91.7 | 8.97 | 9.79 | 0.00 | 0.00 | 6.24 | 6.81 | 10.9 | 11.9 | | K3 EDTA | 94.0 | 0.28 | 0.29 | 0.00 | 0.00 | 0.04 | 0.04 | 0.28 | 0.30 | | B cells | BM | K2 EDTA | 2.51 | 0.18 | 7.07 | 0.39 | 15.5 | 0.39 | 15.5 | 0.58 | 23.0 | | K2 EDTA | 19.4 | 1.04 | 5.38 | 0.69 | 3.55 | 0.24 | 1.23 | 1.27 | 6.56 | | K2 EDTA | 54.5 | 2.94 | 5.39 | 5.36 | 9.84 | 0.00 | 0.00 | 6.12 | 11.2 | | K2 EDTA | 68.1 | 1.51 | 2.21 | 1.73 | 2.53 | 0.00 | 0.00 | 2.29 | 3.37 | | K2 EDTA | 73.3 | 0.86 | 1.17 | 1.11 | 1.52 | 0.54 | 0.74 | 1.51 | 2.05 | | K2 EDTA | 90.4 | 0.24 | 0.26 | 0.00 | 0.00 | 0.39 | 0.43 | 0.46 | 0.50 | | LN | N/A | 22.0 | 2.38 | 10.8 | 3.55 | 16.1 | 18.9 | 85.8 | 19.4 | 87.9 | | 24.7 | 1.00 | 4.03 | 1.50 | 6.08 | 0.00 | 0.00 | 1.80 | 7.30 | | 58.3 | 0.69 | 1.18 | 0.00 | 0.00 | 0.45 | 0.78 | 0.82 | 1.41 | | 64.8 | 5.18 | 8.00 | 0.54 | 0.83 | 0.00 | 0.00 | 5.21 | 8.04 | | PB | Lithium heparin | 4.89 | 0.21 | 4.26 | 9.76 | 200 | 0.00 | 0.00 | 9.77 | 200 | | K3 EDTA | 22.3 | 0.68 | 3.05 | 0.00 | 0.00 | 0.00 | 0.00 | 0.68 | 3.05 | | Lithium heparin | 24.6 | 0.61 | 2.48 | 0.00 | 0.00 | 0.05 | 0.22 | 0.61 | 2.49 | | K3 EDTA | 46.0 | 3.31 | 7.19 | 5.26 | 11.4 | 5.58 | 12.1 | 8.35 | 18.1 | | Lithium heparin | 57.6 | 0.36 | 0.63 | 0.86 | 1.49 | 0.00 | 0.00 | 0.93 | 1.62 | | Sodium heparin | 64.3 | 1.81 | 2.81 | 0.00 | 0.00 | 0.00 | 0.00 | 1.81 | 2.81 | | K3 EDTA | 75.8 | 0.71 | 0.93 | 0.93 | 1.23 | 0.00 | 0.00 | 1.17 | 1.54 | | Lithium heparin | 88.6 | 3.50 | 3.95 | 2.65 | 2.99 | 0.00 | 0.00 | 4.39 | 4.96 | | K2 EDTA | 91.9 | 8.38 | 9.12 | 0.00 | 0.00 | 5.91 | 6.43 | 10.3 | 11.2 | | K3 EDTA | 94.1 | 0.28 | 0.30 | 0.00 | 0.00 | 0.04 | 0.05 | 0.28 | 0.30 | | NK cells | BM | K2 EDTA | 0.60 | 0.06 | 9.47 | 0.05 | 8.97 | 0.05 | 8.75 | 0.09 | 15.7 | | K2 EDTA | 0.77 | 0.06 | 7.22 | 0.00 | 0.00 | 0.04 | 5.22 | 0.07 | 8.91 | | K2 EDTA | 0.80 | 0.17 | 21.3 | 0.00 | 0.00 | 0.17 | 20.9 | 0.24 | 29.9 | | K2 EDTA | 1.27 | 0.07 | 5.49 | 0.21 | 16.8 | 0.00 | 0.00 | 0.22 | 17.7 | | K2 EDTA | 1.49 | 0.14 | 9.54 | 0.18 | 11.9 | 0.00 | 0.00 | 0.23 | 15.2 | | K2 EDTA | 1.87 | 0.15 | 8.04 | 0.00 | 0.00 | 0.04 | 2.33 | 0.16 | 8.37 | | LN | N/A | 0.80 | 0.17 | 20.7 | 0.00 | 0.00 | 0.05 | 6.76 | 0.17 | 21.8 | | 1.06 | 0.10 | 9.69 | 0.00 | 0.00 | 0.03 | 3.15 | 0.11 | 10.2 | | 1.15 | 0.08 | 7.34 | 0.17 | 14.5 | 0.00 | 0.00 | 0.19 | 16.3 | K260215 - Page 14 of 27 {14} Precision of Multiple-Analyst for BD OneFlow LST + BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis) | Population | Type | Anticoagulant | (%)^{1}Mean | Within-Run | | Between-Run | | Between-Analyst | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | | | | | | | | | | | | | | | 3.78 | 2.25 | 59.6 | 2.66 | 70.5 | 3.04 | 80.3 | 4.62 | 122 | | | PB | K2 EDTA | 0.23 | 0.06 | 27.1 | 0.00 | 0.00 | 0.01 | 3.88 | 0.06 | 27.4 | | | | K3 EDTA | 0.59 | 0.05 | 8.70 | 0.00 | 0.00 | 0.03 | 5.34 | 0.06 | 10.2 | | | | Lithium heparin | 0.70 | 0.09 | 13.3 | 0.05 | 6.89 | 0.00 | 0.00 | 0.11 | 15.0 | | | | K3 EDTA | 1.49 | 0.07 | 4.68 | 0.00 | 0.00 | 0.14 | 9.48 | 0.16 | 10.6 | | | | K3 EDTA | 1.83 | 0.09 | 4.82 | 0.00 | 0.00 | 0.08 | 4.56 | 0.12 | 6.63 | | | | Sodium heparin | 1.97 | 0.07 | 3.60 | 0.08 | 4.25 | 0.00 | 0.00 | 0.11 | 5.57 | | | | Lithium heparin | 4.01 | 0.24 | 6.04 | 1.11 | 27.7 | 0.45 | 11.3 | 1.22 | 30.5 | | | | Lithium heparin | 5.59 | 0.08 | 1.40 | 0.09 | 1.54 | 0.12 | 2.17 | 0.17 | 3.01 | | | | K3 EDTA | 6.04 | 0.66 | 10.9 | 0.00 | 0.00 | 0.46 | 7.64 | 0.80 | 13.3 | | | | Lithium heparin | 6.16 | 0.31 | 5.03 | 0.13 | 2.14 | 0.00 | 0.00 | 0.34 | 5.46 | | T cells | BM | K2 EDTA | 5.00 | 0.20 | 3.95 | 0.00 | 0.00 | 0.21 | 4.17 | 0.29 | 5.74 | | | | K2 EDTA | 7.42 | 0.17 | 2.33 | 0.23 | 3.08 | 0.27 | 3.60 | 0.39 | 5.28 | | | | K2 EDTA | 8.79 | 0.38 | 4.37 | 0.42 | 4.75 | 0.00 | 0.00 | 0.57 | 6.46 | | | | K2 EDTA | 12.9 | 0.35 | 2.74 | 0.28 | 2.21 | 0.00 | 0.00 | 0.45 | 3.52 | | | | K2 EDTA | 13.1 | 1.17 | 8.93 | 0.29 | 2.21 | 0.00 | 0.00 | 1.20 | 9.20 | | | | K2 EDTA | 25.7 | 0.68 | 2.63 | 0.63 | 2.45 | 36.3 | 141 | 36.3 | 141 | | | LN | N/A | 29.8 | 0.79 | 2.64 | 0.00 | 0.00 | 0.28 | 0.94 | 0.84 | 2.81 | | | | | 37.2 | 0.36 | 0.96 | 0.25 | 0.68 | 0.00 | 0.00 | 0.44 | 1.18 | | | | | 68.8 | 0.87 | 1.26 | 1.10 | 1.59 | 2.53 | 3.68 | 2.89 | 4.20 | | | | | 70.9 | 0.99 | 1.39 | 0.44 | 0.62 | 22.8 | 32.1 | 22.8 | 32.2 | | | PB | K2 EDTA | 1.04 | 0.09 | 9.10 | 0.13 | 12.9 | 0.00 | 0.00 | 0.16 | 15.8 | | | | K3 EDTA | 2.20 | 0.12 | 5.31 | 0.00 | 0.00 | 0.04 | 1.82 | 0.12 | 5.61 | | | | Lithium heparin | 2.89 | 0.29 | 10.0 | 0.26 | 8.95 | 0.00 | 0.00 | 0.39 | 13.4 | | | | K3 EDTA | 6.70 | 0.14 | 2.10 | 0.28 | 4.22 | 0.00 | 0.00 | 0.32 | 4.72 | | | | Lithium heparin | 8.82 | 0.12 | 1.37 | 0.00 | 0.00 | 0.00 | 0.00 | 0.12 | 1.37 | | | | Sodium heparin | 14.3 | 0.39 | 2.76 | 0.31 | 2.15 | 0.00 | 0.00 | 0.50 | 3.50 | | | | K3 EDTA | 19.5 | 0.48 | 2.45 | 0.00 | 0.00 | 0.00 | 0.00 | 0.48 | 2.45 | | | | K3 EDTA | 20.1 | 2.26 | 11.3 | 1.07 | 5.32 | 1.91 | 9.52 | 3.15 | 15.7 | | | | Lithium heparin | 42.1 | 0.42 | 0.99 | 3.44 | 8.18 | 0.00 | 0.00 | 3.47 | 8.24 | | | | Lithium heparin | 44.0 | 0.31 | 0.71 | 0.20 | 0.45 | 0.00 | 0.00 | 0.37 | 0.84 | | TCRγδ+ T cells | BM | K2 EDTA | 0.35 | 0.13 | 37.3 | 0.00 | 0.00 | 0.20 | 57.0 | 0.24 | 68.1 | | | | K2 EDTA | 0.36 | 0.03 | 7.02 | 0.03 | 7.12 | 0.00 | 0.00 | 0.04 | 10.0 | | | | K2 EDTA | 0.53 | 0.07 | 13.6 | 0.02 | 4.47 | 0.05 | 10.2 | 0.09 | 17.6 | | | | K2 EDTA | 1.39 | 0.08 | 5.57 | 0.03 | 1.92 | 0.00 | 0.00 | 0.08 | 5.89 | | | | K2 EDTA | 1.45 | 0.18 | 12.7 | 0.37 | 25.4 | 0.00 | 0.00 | 0.41 | 28.4 | | | | K2 EDTA | 1.71 | 0.11 | 6.33 | 0.05 | 2.88 | 2.28 | 133 | 2.28 | 133 | | | LN | N/A | 0.70 | 0.16 | 22.7 | 0.14 | 20.8 | 0.06 | 8.08 | 0.22 | 31.8 | | | | | 1.07 | 0.33 | 30.9 | 0.28 | 25.9 | 0.04 | 3.56 | 0.43 | 40.5 | | | | | 1.44 | 0.32 | 22.6 | 0.00 | 0.00 | 0.43 | 30.0 | 0.54 | 37.6 | | | | | 12.6 | 2.21 | 17.6 | 1.32 | 10.5 | 20.9 | 166 | 21.0 | 167 | | | PB | Lithium heparin | 0.11 | 0.06 | 53.0 | 0.00 | 0.00 | 0.04 | 40.1 | 0.07 | 66.4 | | | | K3 EDTA | 0.13 | 0.04 | 30.5 | 0.00 | 0.00 | 0.03 | 22.1 | 0.05 | 37.6 | | | | K2 EDTA | 0.33 | 0.09 | 25.7 | 0.00 | 0.00 | 0.02 | 6.39 | 0.09 | 26.5 | | | | Sodium heparin | 0.35 | 0.03 | 8.34 | 0.04 | 10.2 | 0.01 | 3.46 | 0.05 | 13.6 | | | | Lithium heparin | 0.37 | 0.01 | 2.78 | 0.42 | 113 | 0.00 | 0.00 | 0.42 | 113 | | | | Lithium heparin | 0.43 | 0.02 | 5.51 | 0.00 | 0.00 | 0.02 | 5.07 | 0.03 | 7.49 | | | | K3 EDTA | 0.45 | 0.03 | 7.60 | 0.00 | 0.00 | 0.00 | 0.77 | 0.03 | 7.64 | | | | K3 EDTA | 0.64 | 0.09 | 13.3 | 0.05 | 7.52 | 0.00 | 0.00 | 0.10 | 15.3 | | | | K3 EDTA | 0.86 | 0.04 | 5.11 | 0.00 | 0.00 | 0.02 | 2.03 | 0.05 | 5.50 | K260215 - Page 15 of 27 {15} Precision of Multiple-Analyst for BD OneFlow LST + BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis) | Population | Type | Anticoagulant | (%)^{1}Mean | Within-Run | | Between-Run | | Between-Analyst | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | | | | | | | | | | | | | | Lithium heparin | 1.26 | 0.04 | 3.48 | 0.00 | 0.00 | 0.03 | 2.53 | 0.05 | 4.31 | | BD OneFlow B-CLPD T1 | | | | | | | | | | | | | Abnormal CD19+CD20+ B Cells | BM | K2 EDTA | 1.05 | 0.20 | 19.1 | 0.00 | 0.00 | 1.81 | 173 | 1.82 | 174 | | | | K2 EDTA | 19.9 | 0.88 | 4.41 | 0.85 | 4.26 | 0.00 | 0.00 | 1.22 | 6.13 | | | | K2 EDTA | 54.9 | 6.82 | 12.4 | 0.00 | 0.00 | 3.87 | 7.06 | 7.84 | 14.3 | | | | K2 EDTA | 67.1 | 11.0 | 16.4 | 2.59 | 3.87 | 0.00 | 0.00 | 11.3 | 16.9 | | | | K2 EDTA | 72.1 | 1.27 | 1.77 | 1.00 | 1.39 | 0.00 | 0.00 | 1.62 | 2.25 | | | | K2 EDTA | 90.8 | 0.36 | 0.40 | 0.00 | 0.00 | 0.09 | 0.10 | 0.37 | 0.41 | | | LN | N/A | 8.26 | 0.28 | 3.43 | 0.00 | 0.00 | 14.3 | 173 | 14.3 | 173 | | | | | 22.0 | 2.27 | 10.3 | 0.00 | 0.00 | 19.1 | 86.8 | 19.2 | 87.4 | | | PB | K3 EDTA | 23.1 | 0.87 | 3.75 | 0.48 | 2.09 | 0.00 | 0.00 | 0.99 | 4.29 | | | | K3 EDTA | 44.2 | 5.36 | 12.1 | 5.47 | 12.4 | 3.84 | 8.68 | 8.56 | 19.4 | | | | Lithium heparin | 59.2 | 1.13 | 1.91 | 0.78 | 1.31 | 0.00 | 0.00 | 1.37 | 2.32 | | | | Sodium heparin | 64.8 | 0.16 | 0.24 | 0.62 | 0.96 | 0.00 | 0.00 | 0.64 | 0.99 | | | | K3 EDTA | 78.8 | 0.72 | 0.91 | 0.00 | 0.00 | 0.00 | 0.00 | 0.72 | 0.91 | | | | Lithium heparin | 90.2 | 1.08 | 1.20 | 2.42 | 2.68 | 0.00 | 0.00 | 2.65 | 2.94 | | | | K3 EDTA | 93.9 | 0.34 | 0.36 | 0.21 | 0.23 | 0.00 | 0.00 | 0.40 | 0.43 | | | | K2 EDTA | 95.7 | 0.09 | 0.09 | 0.15 | 0.15 | 0.00 | 0.00 | 0.17 | 0.18 | $^{1}$ All continuous parametric values are % of total CD45+ Leukocytes # b. Between-operator precision: A total of 17 hematologically abnormal clinical specimens representing the intended use matrices and anticoagulants were evaluated with the BD OneFlow LST and B-CLPD T1 reagent tubes, by a total of three laboratory operators (i.e., conducting specimen processing, staining, acquisition), utilizing the BD FACSLyric Flow Cytometer. Each laboratory operator performed separate complete assay protocol runs including sample preparations in triplicate, twice per day. Following acquisition, each FCS file was analyzed, using BD FACSuite Clinical Application and the applicable BD OneFlow Assay module, by a single analyst at each site. Of the 17 evaluable samples tested, all results showed 100% qualitative agreement across operators and replicates per sample. The number of samples tested is depicted in the tables below for all anticoagulants and all samples with 17 samples × 2 runs/sample × 3 replicates/run × 3 operators = 306 total analyses. Samples used and evaluated in the study | Sample Type | K2 EDTA | Sodium heparin | Lithium heparin | none | | --- | --- | --- | --- | --- | | BM (Bone Marrow) | 6 | 0 | 0 | 0 | | LN (Lymph Node) | 0 | 0 | 0 | 5 | | PB (Peripheral Blood) | 3 | 2 | 1 | 0 | Imprecision for parametric semi-quantitative values for critical measurands was calculated by ANOVA methods, following CLSI EP05-A3, and presented in the following table: K260215 - Page 16 of 27 {16} Between-Operator Precision for BD OneFlow LST and BD OneFlow B-CLPD T1 Reagents | Population | Type | Anticoagulant | (%)^{1} | Within-Run | Between-Run | Between-Operator | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | Abnormal κ-restricted CD19^{+}CD20^{+} B cells | BM | K2 EDTA | 1.68 | 0.17 | 10.4 | 0.13 | 7.62 | 0.10 | 5.73 | 0.22 | 12.9 | | K2 EDTA | 18.3 | 0.86 | 4.68 | 0.62 | 3.39 | 0.27 | 1.46 | 1.06 | 5.78 | | K2 EDTA | 73.2 | 1.02 | 1.39 | 0.00 | 0.00 | 0.00 | 0.00 | 1.02 | 1.39 | | PB | Lithium heparin | 28.6 | 1.37 | 4.79 | 0.49 | 1.72 | 0.00 | 0.00 | 1.46 | 5.09 | | K2 EDTA | 65.7 | 0.98 | 1.49 | 0.00 | 0.00 | 0.35 | 0.53 | 0.98 | 1.49 | | Abnormal λ-restricted CD19^{+}CD20^{+} B cells | BM | K2 EDTA | 63.0 | 3.72 | 5.91 | 2.82 | 4.48 | 3.00 | 4.77 | 4.67 | 7.41 | | K2 EDTA | 67.2 | 3.64 | 5.41 | 1.54 | 2.29 | 0.00 | 0.00 | 3.95 | 5.88 | | K2 EDTA | 81.9 | 0.57 | 0.70 | 0.00 | 0.00 | 0.00 | 0.00 | 0.57 | 0.70 | | PB | K2 EDTA | 74.8 | 1.09 | 1.46 | 1.10 | 1.47 | 1.09 | 1.45 | 1.55 | 2.07 | | Abnormal CD19^{+}CD5^{+} B cells | BM | K2 EDTA | 0.29 | 0.09 | 29.3 | 0.00 | 0.00 | 0.02 | 5.54 | 0.09 | 29.3 | | K2 EDTA | 2.41 | 1.44 | 59.8 | 0.83 | 34.3 | 0.91 | 37.6 | 1.66 | 68.9 | | K2 EDTA | 17.7 | 0.84 | 4.76 | 0.57 | 3.21 | 0.16 | 0.89 | 1.02 | 5.74 | | K2 EDTA | 71.4 | 0.62 | 0.87 | 0.38 | 0.53 | 0.00 | 0.00 | 0.73 | 1.02 | | K2 EDTA | 73.0 | 1.01 | 1.39 | 0.00 | 0.00 | 0.00 | 0.00 | 1.01 | 1.39 | | K2 EDTA | 81.9 | 1.90 | 2.32 | 0.00 | 0.00 | 0.00 | 0.00 | 1.90 | 2.32 | | PB | Lithium heparin | 28.6 | 1.38 | 4.82 | 0.49 | 1.73 | 0.00 | 0.00 | 1.46 | 5.12 | | K2 EDTA | 74.5 | 1.09 | 1.46 | 1.02 | 1.37 | 1.02 | 1.37 | 1.49 | 2.00 | | K2 EDTA | 81.0 | 0.71 | 0.87 | 0.00 | 0.00 | 0.00 | 0.00 | 0.71 | 0.87 | | B cells | BM | K2 EDTA | 2.46 | 0.19 | 7.69 | 0.10 | 4.14 | 0.08 | 3.17 | 0.22 | 8.74 | | K2 EDTA | 18.7 | 0.85 | 4.53 | 0.63 | 3.34 | 0.32 | 1.71 | 1.05 | 5.63 | | K2 EDTA | 72.0 | 0.62 | 0.86 | 0.43 | 0.60 | 0.12 | 0.16 | 0.76 | 1.05 | | K2 EDTA | 73.2 | 1.02 | 1.39 | 0.00 | 0.00 | 0.00 | 0.00 | 1.02 | 1.39 | | K2 EDTA | 73.7 | 1.15 | 1.56 | 2.46 | 3.34 | 2.36 | 3.20 | 2.72 | 3.69 | | K2 EDTA | 85.7 | 1.82 | 2.13 | 0.00 | 0.00 | 0.00 | 0.00 | 1.82 | 2.13 | | LN | N/A | 1.97 | 0.40 | 20.1 | 0.00 | 0.00 | 0.00 | 0.00 | 0.40 | 20.1 | | 28.3 | 1.32 | 4.65 | 0.28 | 1.01 | 0.45 | 1.58 | 1.35 | 4.76 | | 40.5 | 1.74 | 4.29 | 0.00 | 0.00 | 0.00 | 0.00 | 1.74 | 4.29 | | 56.4 | 1.05 | 1.86 | 0.52 | 0.93 | 0.58 | 1.03 | 1.17 | 2.08 | | 63.3 | 5.56 | 8.79 | 0.63 | 0.99 | 0.00 | 0.00 | 5.60 | 8.85 | | PB | Sodium heparin | 1.72 | 0.06 | 3.24 | 0.00 | 0.00 | 0.00 | 0.00 | 0.06 | 3.24 | | K2 EDTA | 2.23 | 0.08 | 3.53 | 0.00 | 0.00 | 0.00 | 0.00 | 0.08 | 3.53 | | Sodium heparin | 2.36 | 0.14 | 5.74 | 0.09 | 3.83 | 0.00 | 0.00 | 0.16 | 6.90 | | Lithium heparin | 28.7 | 1.38 | 4.81 | 0.50 | 1.73 | 0.00 | 0.00 | 1.46 | 5.11 | | K2 EDTA | 75.1 | 1.11 | 1.48 | 1.04 | 1.38 | 1.05 | 1.40 | 1.52 | 2.02 | | K2 EDTA | 81.2 | 0.69 | 0.84 | 0.00 | 0.00 | 0.00 | 0.00 | 0.69 | 0.84 | | NK cells | BM | K2 EDTA | 0.76 | 0.09 | 11.8 | 0.03 | 4.49 | 0.02 | 2.06 | 0.10 | 12.6 | | K2 EDTA | 0.86 | 0.50 | 57.7 | 0.32 | 37.5 | 0.00 | 0.00 | 0.59 | 68.8 | | K2 EDTA | 0.96 | 0.11 | 11.5 | 0.05 | 5.44 | 0.05 | 4.95 | 0.12 | 12.7 | | K2 EDTA | 1.46 | 0.15 | 10.5 | 0.21 | 14.6 | 0.20 | 14.0 | 0.26 | 18.0 | | K2 EDTA | 1.50 | 0.17 | 11.6 | 0.28 | 18.6 | 0.25 | 16.8 | 0.33 | 22.0 | | K2 EDTA | 2.04 | 0.24 | 11.8 | 0.21 | 10.2 | 0.20 | 9.77 | 0.32 | 15.6 | | LN | N/A | 0.86 | 0.16 | 18.8 | 0.00 | 0.00 | 0.00 | 0.00 | 0.16 | 18.8 | | 1.19 | 0.07 | 5.76 | 0.00 | 0.00 | 0.00 | 0.00 | 0.07 | 5.76 | | 1.49 | 0.07 | 4.95 | 0.01 | 0.94 | 0.02 | 1.07 | 0.08 | 5.04 | | 3.37 | 0.56 | 16.7 | 0.17 | 4.99 | 0.00 | 0.00 | 0.59 | 17.4 | | 12.4 | 1.60 | 12.9 | 1.11 | 8.91 | 0.90 | 7.26 | 1.95 | 15.6 | | PB | Sodium heparin | 1.56 | 0.09 | 5.83 | 0.00 | 0.00 | 0.00 | 0.00 | 0.09 | 5.83 | | Sodium heparin | 1.59 | 0.31 | 19.5 | 0.28 | 17.9 | 0.29 | 18.5 | 0.42 | 26.5 | | K2 EDTA | 1.97 | 0.07 | 3.42 | 0.07 | 3.55 | 0.07 | 3.65 | 0.10 | 4.93 | | K2 EDTA | 3.11 | 0.13 | 4.24 | 0.12 | 3.89 | 0.12 | 3.80 | 0.18 | 5.75 | | K2 EDTA | 3.28 | 0.19 | 5.76 | 0.03 | 0.85 | 0.00 | 0.00 | 0.19 | 5.82 | | Lithium heparin | 4.76 | 0.26 | 5.48 | 0.10 | 2.09 | 0.00 | 0.00 | 0.28 | 5.86 | K260215 - Page 17 of 27 {17} # Between-Operator Precision for BD OneFlow LST and BD OneFlow B-CLPD T1 Reagents | Population | Type | Anticoagulant | (%)^{1} | Within-Run | | Between-Run | | Between-Operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | **BD OneFlow LST** | | | | | | | | | | | | | T cells | BM | K2 EDTA | 2.11 | 0.09 | 4.28 | 0.11 | 5.38 | 0.11 | 5.26 | 0.14 | 6.87 | | | | K2 EDTA | 4.76 | 0.24 | 5.02 | 0.19 | 3.91 | 0.14 | 2.84 | 0.30 | 6.36 | | | | K2 EDTA | 7.13 | 0.22 | 3.08 | 0.00 | 0.00 | 0.00 | 0.00 | 0.22 | 3.08 | | | | K2 EDTA | 7.22 | 0.25 | 3.49 | 0.28 | 3.94 | 0.25 | 3.46 | 0.38 | 5.27 | | | | K2 EDTA | 7.78 | 0.18 | 2.37 | 0.12 | 1.60 | 0.10 | 1.24 | 0.22 | 2.86 | | | | K2 EDTA | 9.42 | 0.32 | 3.41 | 0.73 | 7.73 | 0.69 | 7.34 | 0.80 | 8.45 | | | LN | N/A | 24.0 | 3.60 | 15.0 | 1.05 | 4.39 | 0.00 | 0.00 | 3.75 | 15.6 | | | | | 30.8 | 0.95 | 3.08 | 0.00 | 0.00 | 0.00 | 0.00 | 0.95 | 3.08 | | | | | 37.9 | 0.80 | 2.11 | 0.47 | 1.25 | 0.44 | 1.16 | 0.93 | 2.45 | | | | | 53.3 | 2.11 | 3.96 | 0.00 | 0.00 | 0.00 | 0.00 | 2.11 | 3.96 | | | | | 54.5 | 1.51 | 2.77 | 1.56 | 2.87 | 1.13 | 2.07 | 2.17 | 3.99 | | | PB | K2 EDTA | 3.70 | 0.12 | 3.25 | 0.00 | 0.00 | 0.00 | 0.00 | 0.12 | 3.25 | | | | K2 EDTA | 7.72 | 0.31 | 3.97 | 0.00 | 0.00 | 0.00 | 0.00 | 0.31 | 3.97 | | | | Lithium heparin | 10.4 | 0.22 | 2.13 | 0.18 | 1.75 | 0.16 | 1.49 | 0.29 | 2.75 | | | | K2 EDTA | 14.1 | 0.46 | 3.27 | 0.00 | 0.00 | 0.00 | 0.00 | 0.46 | 3.27 | | | | Sodium heparin | 15.5 | 0.48 | 3.11 | 0.13 | 0.85 | 0.00 | 0.00 | 0.50 | 3.23 | | | | Sodium heparin | 17.9 | 0.32 | 1.76 | 0.14 | 0.76 | 0.00 | 0.00 | 0.34 | 1.92 | | TCRγδ+ T cells | BM | K2 EDTA | 0.27 | 0.06 | 20.9 | 0.02 | 8.32 | 0.00 | 0.00 | 0.06 | 22.5 | | | | K2 EDTA | 0.38 | 0.07 | 19.4 | 0.00 | 0.00 | 0.02 | 5.11 | 0.07 | 19.4 | | | | K2 EDTA | 0.56 | 0.03 | 6.19 | 0.06 | 11.0 | 0.06 | 10.8 | 0.07 | 12.6 | | | | K2 EDTA | 0.56 | 0.13 | 23.2 | 0.18 | 33.1 | 0.16 | 29.1 | 0.22 | 40.4 | | | | K2 EDTA | 0.94 | 0.06 | 6.54 | 0.03 | 2.88 | 0.04 | 3.79 | 0.07 | 7.15 | | | | K2 EDTA | 1.08 | 0.15 | 14.2 | 0.18 | 16.7 | 0.16 | 14.9 | 0.24 | 21.9 | | | LN | N/A | 0.79 | 0.24 | 29.7 | 0.00 | 0.00 | 0.00 | 0.00 | 0.24 | 29.7 | | | | | 1.04 | 0.28 | 27.3 | 0.00 | 0.00 | 0.00 | 0.00 | 0.28 | 27.3 | | | | | 1.08 | 0.33 | 30.9 | 0.26 | 23.8 | 0.27 | 24.9 | 0.42 | 39.0 | | | | | 2.38 | 1.10 | 46.3 | 0.39 | 16.6 | 0.00 | 0.00 | 1.17 | 49.2 | | | | | 3.16 | 0.68 | 21.4 | 0.42 | 13.4 | 0.44 | 13.8 | 0.80 | 25.2 | | | PB | K2 EDTA | 0.28 | 0.04 | 15.2 | 0.01 | 3.10 | 0.00 | 0.00 | 0.04 | 15.5 | | | | Sodium heparin | 0.35 | 0.02 | 5.06 | 0.01 | 1.43 | 0.00 | 0.00 | 0.02 | 5.25 | | | | K2 EDTA | 0.44 | 0.03 | 7.57 | 0.00 | 0.00 | 0.00 | 0.00 | 0.03 | 7.57 | | | | K2 EDTA | 0.49 | 0.03 | 5.42 | 0.01 | 1.98 | 0.00 | 0.00 | 0.03 | 5.77 | | | | Sodium heparin | 0.61 | 0.04 | 6.94 | 0.00 | 0.00 | 0.00 | 0.00 | 0.04 | 6.94 | | | | Lithium heparin | 1.63 | 0.05 | 2.98 | 0.06 | 3.95 | 0.06 | 3.91 | 0.08 | 4.95 | | **BD OneFlow B-CLPD T1** | | | | | | | | | | | | | Abnormal CD20^{+} B cells | BM | K2 EDTA | 1.33 | 0.07 | 5.38 | 0.05 | 3.65 | 0.05 | 4.00 | 0.09 | 6.51 | | | | K2 EDTA | 3.01 | 0.13 | 4.29 | 0.05 | 1.66 | 0.06 | 2.05 | 0.14 | 4.60 | | | | K2 EDTA | 18.6 | 2.01 | 10.8 | 0.00 | 0.00 | 0.00 | 0.00 | 2.01 | 10.8 | | | | K2 EDTA | 72.5 | 1.05 | 1.44 | 0.97 | 1.34 | 0.67 | 0.93 | 1.43 | 1.97 | | | | K2 EDTA | 74.8 | 1.06 | 1.42 | 0.91 | 1.22 | 0.93 | 1.24 | 1.40 | 1.87 | | | | K2 EDTA | 77.3 | 0.32 | 0.41 | 0.63 | 0.81 | 0.64 | 0.82 | 0.70 | 0.91 | | | | K2 EDTA | 87.7 | 0.59 | 0.68 | 0.36 | 0.41 | 0.00 | 0.00 | 0.69 | 0.79 | | | PB | Lithium heparin | 28.8 | 2.11 | 7.33 | 1.43 | 4.98 | 1.21 | 4.21 | 2.55 | 8.86 | | | | K2 EDTA | 74.9 | 0.76 | 1.02 | 1.34 | 1.79 | 1.26 | 1.68 | 1.54 | 2.06 | | | | K2 EDTA | 81.0 | 0.94 | 1.16 | 0.00 | 0.00 | 0.00 | 0.00 | 0.94 | 1.16 | # c. Site-to-Site Reproducibility: Multisite precision of the BD OneFlow LST and BD OneFlow B-CLPD T1 reagent tubes on the BD FACSLyric system was assessed by testing control material following EP05-A3. K260215 - Page 18 of 27 {18} Three sites each contributed one operator and one instrument, staining control materials in triplicate in two daily runs (AM/PM) on five non-consecutive days, yielding (N=90) data points per marker: 1 lot × 3 replicates × 3 sites (1 operator/site × 1 instrument/site) × 2 runs/day × 5 days. All 12 LST markers and all 8 B-CLPD T1 markers met repeatability and reproducibility acceptance criteria. For LST, the widest confidence intervals were observed for CD3 (upper 95% CI = 1.80% CV) and CD5 (95% CI = 1.67% CV). For OneFlow B-CLPD T1, the largest upper 95% confidence bounds were observed for CD200 (upper 95% CI = 0.75 SD), and CD23 (upper 95% CI = 0.32 SD), as evaluated under SD criteria for low measurand levels. ### d. Lot-to-Lot Precision: Lot-to-lot reproducibility of the BD OneFlow LST and BD OneFlow B-CLPD T1 reagent tubes was evaluated on the BD FACSLyric Flow Cytometer using BD FACSuite Clinical Application. Six hematologically abnormal peripheral specimens were tested using three reagent lots. Each individual sample was stained in duplicate across two runs (separated by a minimum of two hours) by a single operator on a single cytometer at one site, yielding six replicates per reagent tube per run, and twelve replicates per sample in total. Qualitative analysis assessed agreement of composite immunophenotypes within each tube across all replicates, reporting agreement counts, percentage agreement, lower 95% confidence bounds on percentage agreement, and any instances of disagreeing phenotypes. The qualitative analysis demonstrated 100% agreement across all samples, replicates and lots. Semi-quantitative parametric analysis was conducted for PB samples from above, and presented in the following table: Lot-to-Lot Precision of BD OneFlow LST and BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis | | Parent | (%)1 | Within-Run | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Mean | SD | %CV | SD | %CV | SD | %CV | | BD OneFlow LST | | | | | | | | | | Abnormal Igλ/Igκ-restricted B cells | % CD45+Leukocytes | 44.0 | 0.74 | 1.67 | 0.29 | 0.66 | 0.79 | 1.80 | | | | 53.3 | 0.70 | 1.31 | 0.63 | 1.17 | 0.94 | 1.76 | | | | 77.8 | 0.32 | 0.41 | 0.02 | 0.02 | 0.32 | 0.42 | | Abnormal CD19+CD5+B cells | % CD45+Leukocytes | 44.0 | 0.90 | 2.04 | 0.00 | 0.00 | 0.90 | 2.04 | | | | 53.1 | 0.65 | 1.22 | 0.67 | 1.25 | 0.93 | 1.75 | | B cells | % CD45+Leukocytes | 45.4 | 0.58 | 1.27 | 0.30 | 0.67 | 0.65 | 1.44 | | | | 53.5 | 0.69 | 1.28 | 0.63 | 1.17 | 0.93 | 1.74 | | | | 77.8 | 0.32 | 0.42 | 0.02 | 0.02 | 0.32 | 0.42 | | Igκ+ | % B cells | 0.30 | 0.03 | 10.7 | 0.04 | 12.9 | 0.05 | 16.8 | | | | 2.11 | 0.57 | 27.2 | 0.00 | 0.00 | 0.57 | 27.2 | | | | 64.2 | 4.54 | 7.08 | 0.97 | 1.51 | 4.65 | 7.24 | | | | 64.4 | 0.93 | 1.44 | 0.90 | 1.39 | 1.29 | 2.01 | | | | 99.6 | 0.06 | 0.06 | 0.00 | 0.01 | 0.06 | 0.06 | | Igλ+ | % B cells | 0.32 | 0.04 | 11.3 | 0.00 | 0.00 | 0.04 | 11.3 | | | | 35.8 | 4.54 | 12.7 | 0.97 | 2.71 | 4.65 | 13.0 | K260215 - Page 19 of 27 {19} Lot-to-Lot Precision of BD OneFlow LST and BD OneFlow B-CLPD T1 (Semi-Quantitative Analysis | | Parent | (%)1 | Within-Run | | Between-Lot | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Mean | SD | %CV | SD | %CV | SD | %CV | | | | 35.9 | 0.83 | 2.32 | 0.55 | 1.54 | 1.00 | 2.78 | | | | 97.8 | 0.49 | 0.50 | 0.00 | 0.00 | 0.49 | 0.50 | | | | 99.7 | 0.04 | 0.04 | 0.04 | 0.04 | 0.06 | 0.06 | | NK cells | % CD45+Leukocytes | 0.87 | 0.13 | 15.3 | 0.13 | 14.9 | 0.19 | 21.4 | | | | 1.34 | 0.09 | 6.74 | 0.06 | 4.75 | 0.11 | 8.24 | | | | 1.57 | 0.06 | 3.87 | 0.09 | 5.81 | 0.11 | 6.98 | | | | 2.49 | 0.11 | 4.41 | 0.03 | 1.16 | 0.11 | 4.56 | | | | 3.12 | 0.12 | 3.84 | 0.09 | 3.02 | 0.15 | 4.88 | | | | 4.94 | 0.35 | 7.12 | 0.55 | 11.2 | 0.65 | 13.2 | | T cells | % CD45+Leukocytes | 8.29 | 0.23 | 2.77 | 0.00 | 0.00 | 0.23 | 2.77 | | | | 8.63 | 0.16 | 1.85 | 0.17 | 1.97 | 0.23 | 2.70 | | | | 10.3 | 0.25 | 2.44 | 0.05 | 0.49 | 0.26 | 2.49 | | | | 15.3 | 0.15 | 0.98 | 0.20 | 1.32 | 0.25 | 1.64 | | | | 15.8 | 0.57 | 3.62 | 0.24 | 1.50 | 0.62 | 3.92 | | | | 28.0 | 0.61 | 2.19 | 0.00 | 0.00 | 0.61 | 2.19 | | TCRγδ+T cells | % CD45+Leukocytes | 0.15 | 0.02 | 14.5 | 0.01 | 10.0 | 0.03 | 17.6 | | | | 0.20 | 0.02 | 9.50 | 0.00 | 0.00 | 0.02 | 9.50 | | | | 0.34 | 0.03 | 8.49 | 0.03 | 7.32 | 0.04 | 11.2 | | | | 0.55 | 0.04 | 7.45 | 0.04 | 7.40 | 0.06 | 10.5 | | | | 0.61 | 0.05 | 8.74 | 0.03 | 5.06 | 0.06 | 10.1 | | | | 1.02 | 0.03 | 3.17 | 0.03 | 2.78 | 0.04 | 4.21 | | BD OneFlow B-CLPD T1 | | | | | | | | | | AbnormalCD19+CD20+ | % CD45+Leukocytes | 45.7 | 0.54 | 1.19 | 0.00 | 0.00 | 0.54 | 1.19 | | | | 53.9 | 0.73 | 1.35 | 0.50 | 0.92 | 0.88 | 1.64 | | | | 78.8 | 0.19 | 0.25 | 0.28 | 0.36 | 0.34 | 0.43 | | CD20+CD23+Lymphocytes | % B cells | 6.05 | 0.78 | 12.9 | 0.25 | 4.09 | 0.82 | 13.5 | | | | 58.6 | 4.56 | 7.78 | 2.53 | 4.32 | 5.22 | 8.90 | | | | 82.2 | 0.74 | 0.90 | 0.00 | 0.00 | 0.74 | 0.90 | | CD20+CD43+Lymphocytes | % B cells | 0.27 | 0.04 | 12.6 | 0.03 | 12.4 | 0.05 | 17.7 | | | | 80.7 | 0.79 | 0.97 | 0.45 | 0.56 | 0.90 | 1.12 | | | | 82.4 | 0.65 | 0.79 | 0.00 | 0.00 | 0.65 | 0.79 | | CD20+CD79b+Lymphocytes | % B cells | 0.22 | 0.03 | 11.8 | 0.01 | 4.86 | 0.03 | 12.7 | | | | 9.20 | 1.45 | 15.8 | 0.00 | 0.00 | 1.45 | 15.8 | | | | 81.8 | 0.82 | 1.00 | 0.33 | 0.40 | 0.88 | 1.08 | | | | 89.2 | 0.31 | 0.35 | 0.11 | 0.12 | 0.33 | 0.37 | | CD20+CD200+Lymphocytes | % B cells | 58.4 | 2.87 | 4.91 | 5.53 | 9.47 | 6.23 | 10.7 | | | | 78.1 | 0.46 | 0.58 | 0.15 | 0.19 | 0.48 | 0.61 | | | | 82.3 | 0.56 | 0.68 | 0.00 | 0.00 | 0.56 | 0.68 | ## 2. Linearity: Not applicable K260215 - Page 20 of 27 {20} # 3. Analytical Specificity/Interference: Adequate washing with phosphate buffered saline (PBS) before staining with reagents removes residual plasma and interfering substances. Labeling mitigations have been included for therapeutics derived from monoclonal antibodies (e.g., rituximab). # 4. Detection Limit: a. Limit of Blank (LoB): LoB was performed per CLSI EP17-A2 using two peripheral blood (PB) specimens from healthy donors, testing ten replicates/specimen, with 20 total acquisitions across two reagent lots by a single operator using four BD FACSLyric Flow Cytometers. A separate analyst conducted immunophenotype re-analysis using BD FACSuite Clinical Application. LoB was based on the 95th percentile of the percentage of apparent abnormal cells (% AbN) observed across all acquisitions. LoB is determined to be 0.01% from 10⁵ acquired events. b. Limit of Detection/Quantitation (LoD/Q): The LoD was determined following CLSI EP17-A2 using a precision profile method (refer to §VII.A.1.a.i above) for aberrant phenotypes from n=20 unique native individual samples evaluated in a total of 12 independent reads each. LoD/Q is determined to be 1.0% immunophenotypically aberrant cells as a proportion of 10⁵ acquired events. # 5. Assay Reportable Range: Not applicable # 6. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): a. Traceability: For monoclonal antibodies, Leukocyte Typing Workshop information for each antibody clone was provided. Each clone identity is controlled and is traced back to the original source; refer to Device Description. b. Reagent Stability: Reagent stability studies of the BD OneFlow LST and BD OneFlow B-CLPD T1 assay tubes were conducted in accordance with CLSI EP25-Ed2. i. Shelf-Life: Real-time stability studies were conducted for the shelf-life of the BD OneFlow LST and BD OneFlow B-CLPD T1 assay tubes stored at 5±3°C or 25±2°C. Three lots of reagents were tested for each device using normal peripheral blood samples. Each lot was evaluated at multiple time points, with a minimum of four time points. K260215 - Page 21 of 27 {21} # ii. In-Use Stability: In-use stability of the reagents of the BD OneFlow LST and BD OneFlow B-CLPD T1 was evaluated for two conditions: (1) Tube Out of Pouch (TOP), reflecting exposure to room temperature after removal from the sealed pouch; and (2) Open Pouch (OP), reflecting repeated open-and-close cycles during in-use storage at 2–8°C or 25–29°C. At each test point, for TOP, tubes were removed from the pouches and placed at room temperature, protected from light, for 1-3 hours prior to testing. For OP, pouches were opened and closed for 5 minutes at room temperature up to six times prior to testing. The stability of the BD OneFlow reagents are summarized in the following table: | Device | Shelf-Life | | In-Use (TOP) | | In-Use (OP) | | --- | --- | --- | --- | --- | --- | | BD OneFlow LST | 16 months | 2–8°C | 3 months | 2–8°C | 1 hour | | | | 23–27°C | | 25–29°C | | | BD OneFlow B-CLPD T1 | 24 months | 2–8°C | 3 months | 2–8°C | 1 hour | | | | 23–27°C | | 25–29°C | | # c. Specimen Stability: Age of Blood (AOB; time from collection to staining) and Age of Stain (AOS; time from staining to acquisition) were independently evaluated for peripheral blood (PB) and bone marrow (BM) specimens, and AOS alone for lymph node (LN) specimens, using BD OneFlow LST and BD OneFlow B-CLPD T1 reagents. Testing was performed on four BD FACSLyric flow cytometers by five operators across seven lots of BD OneFlow LST and five lots of BD OneFlow B-CLPD T1, using 27 PB, 4 BM, and 7 LN hematologically abnormal specimens representing all of the claimed anticoagulants (K2 EDTA, K3 EDTA, lithium heparin, and sodium heparin), with one to two replicates per time point per CLSI EP12-A2 and EP25-A2. Supported stability claims for each claimed specimen type are summarized in the following table: | Specimen Type | Temperature | Age of Blood (AOB) | Age of Stain (AOS) | | --- | --- | --- | --- | | BM | 23–27°C | 26 hours | 2 hours | | PB | 23–27°C | 26 hours | 2 hours | | LN | 23–27°C | n.d. | 1 hour | # 7. Assay Cut-Off: Not applicable. # 8. Accuracy (Instrument): a. Instrument Optical Equivalence: The BD FACSLyric flow cytometer is available in three optical configurations: 8-color (4-2-2), 10-color (4-3-3), and 12-color (4-3-5). An optical equivalence study was conducted to verify that immunophenotyping results obtained with BD OneFlow LST and BD OneFlow B-CLPD T1 are equivalent across all three configurations using 8-color configuration as K260215 - Page 22 of 27 {22} the reference. A panel of 34 clinical samples including peripheral blood and bone marrow collected in K2 EDTA, lithium heparin, and sodium heparin were tested for BD OneFlow LST and a panel of 33 of these samples were tested for B-CLPD T1. Each sample was stained in duplicate on all three instruments, producing six replicates per sample per assay. A minimum of three lots of each reagent tube were rotated throughout the study. Quantitative mean bias was assessed for ten cell populations in LST (Leukocytes, Lymphocytes, T Cells, CD4+CD8−, CD8+CD4−, B Cells, Igκ+, Igλ+, NK Cells, and TCRγδ+ Cells) and three populations in B-CLPD T1 (Leukocytes, Lymphocytes, and B Cells). Qualitative phenotypic concordance across three configurations was 100% across all 34 LST samples and all 33 B-CLPD T1 samples, including all subgroups evaluated (peripheral blood, bone marrow, hematologically abnormal, CLL, and follicular lymphoma). Deming regression analyses showed the slopes close to 1.0, intercepts close to 0 for most populations. # b. Carryover: Carryover studies were conducted on three BD FACSLyric flow cytometers, using three reagent lots, and two laboratory operators, for each of two acquisition modes: manual port and BD FACS Universal Loader, following CLSI H26-A2. # i. Specimen Carryover: Because both assays share identical sample preparation workflows, the BD OneFlow LST tube was used as the representative assay for the carryover assessment. High-concentration samples (HTV; >40 × 10³ WBC cells/μL) and low-concentration samples (LTV; <300 WBC cells/μL) were each prepared by diluting peripheral blood (PB) samples to prepare the desired concentration of WBCs. Each sample was stained in triplicate and acquired sequentially (H1, H2, H3, L1, L2, L3) across three runs on three BD FACSLyric flow cytometers, using one reagent lot per instrument and both the BD FACS Universal Loader and manual port acquisition modes. A daily clean procedure was performed after each set and between the H and L samples during manual acquisition runs, per the labeled recommendations. No sample carryover was observed for the assay. # ii. Reagent Carryover: The reagent carryover study was performed using Chronic Lymphocytic Leukemia (CLL) PB samples. Samples were prepared as high-signal (“H”) replicates stained with the relevant BD OneFlow tube (LST or B-CLPD T1), and low-signal (“L”) replicates prepared with 4× drying buffer and deionized water only, without antibodies. Both H and L tubes otherwise followed the standard staining protocol. Samples were acquired sequentially in sets of six (H1, H2, H3, L1, L2, L3) across three acquisition runs per assay, using three different BD FACSLyric instruments and one reagent lot per instrument. Sets 1 and 2 were performed via manual acquisition; Set 3 used the universal loader. A daily clean procedure was performed after each set and between the H and L samples during manual acquisition runs, per the labeled recommendations. No reagent carryover was observed across all acquisitions. K260215 - Page 23 of 27 {23} # **B Comparison Studies:** 1. Method Comparison with Predicate Device: Not applicable. See Clinical Studies below. 2. Matrix Comparison: The studies were conducted following CLSI EP35-Ed2, to assess the impact of anticoagulant upon peripheral blood. a. Study 1: Anticoagulant Equivalency in Normal Peripheral Blood A total of 22 normal (healthy) peripheral blood (PB) samples were collected in K2 EDTA and sodium heparin to assess the impact of anticoagulant upon BD OneFlow LST and BD OneFlow B-CLPD T1 reagent tubes on the BD FACSLyric Flow Cytometer. Each sample was stained in duplicate with both the BD OneFlow LST and B-CLPD T1 reagent tubes, yielding 88 data points per assay (22 samples × 2 anticoagulants × 2 replicates). Three BD FACSLyric Flow Cytometers, three operators, and six internal analysts participated. Three lots of B-CLPD T1 and five lots of LST reagent tubes were rotated throughout the study with site, instrument, reagent lot, and operator variance components controlled on a per-specimen basis. For all reported cell populations, including Leukocytes (% of SSC Singlets), Lymphocytes (% of Leukocytes), T Cells, CD4+CD8−, CD8+CD4−, B Cells, Igκ+, Igλ+, NK Cells, and TCRγδ+ Cells, qualitative and quantitative assessment demonstrated acceptable performance across the paired K2 EDTA and sodium heparin samples. b. Study 2: Anticoagulant Equivalency in Intended-Use Peripheral Blood A panel of 32 intended-use PB specimens were collected simultaneously into four anticoagulants: K2 EDTA, K3 EDTA, lithium heparin, and sodium heparin. Each sample was stained in duplicate per reagent tube, and run on the BD FACSLyric flow cytometer with BD OneFlow LST or BD OneFlow B-CLPD T1 reagent tubes, yielding eight acquisition runs per sample for each assay. A single operator processed and acquired each specimen's four anticoagulant aliquots on one instrument; seven analysts reviewed results. The final evaluable dataset comprised 31 samples for LST and 30 samples for B-CLPD T1. At least 10 specimens per anticoagulant (K2 EDTA, K3 EDTA, Na heparin, Li heparin) were represented. Among the 31 specimens for LST, 21 were classified as malignant and 10 as non-malignant. Among 21 specimens for B-CLPD T1, 20 were malignant and 10 non-malignant. For data analysis, six pairwise anticoagulant comparisons were evaluated: K2 EDTA vs. K3 EDTA, K2 EDTA vs. Li heparin, K2 EDTA vs. Na heparin, K3 EDTA vs. Li heparin, K3 EDTA vs. Na heparin, and Li heparin vs. Na heparin. All quantitative (mean bias and donor bias) and qualitative results demonstrated acceptable performance across all six pairwise anticoagulant comparisons. Individual donor bias values for all reported cell populations (e.g., abnormal CD19+CD5+, CD19+CD20+, CD20+CD10+ B cells; helper T cells; cytotoxic T cells; γδ T cells; NK cells; Igκ+/λ+ B cells) demonstrated ≤ ±20% measurand bias across all comparisons for populations within individual samples. K260215 - Page 24 of 27 {24} ## C Clinical Studies: ### 1. Clinical Sensitivity and Specificity: A multi-center study was conducted to evaluate the diagnostic accuracy of the BD OneFlow LST and BD OneFlow B-CLPD T1 reagents to detect the presence or absence of an abnormal phenotype, relative to the clinical outcome of “malignant” or “non-malignant”, based on the clinical sites’ final patient diagnosis. A total of 250 evaluable individual patient specimens were enrolled from five clinical sites including two United States (U.S.) sites and three outside the United States (OUS). All specimens were from hematologically abnormal patients having, or suspected of having, hematological malignancies, consistent with the intended use population. Specimens were classified by clinical diagnosis as either “malignant” or “non-malignant”, and further classified by diagnosis being a non-B cell lineage neoplasm (i.e., T- or NK-cell), B-CLL, or a chronic B-cell lymphoma or leukemia other than CLL. The “other than CLL” specimens included non-Hodgkin lymphomas that included diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), lymphoplastic lymphoma (LPL)/Waldenström’s macroglobulinemia, hairy cell leukemia (HCL), and Burkitt’s lymphoma (BL). The distribution of anticoagulants by specimen type and disease condition is shown in the table below: | | Malignant | | | Non-malignant | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | Anticoagulant | PB | BM | LN | PB | BM | LN | Total | | K2 EDTA | 51 | 18 | 0 | 58 | 12 | 0 | 139 | | K3 EDTA | 61 | 0 | 0 | 18 | 0 | 0 | 79 | | Heparin | 0 | 0 | 0 | 3 | 0 | 0 | 3 | | N/A (lymph node) | - | - | 19 | - | - | 10 | 29 | | Total | 112 | 18 | 19 | 79 | 12 | 10 | 250 | Distribution of Diagnostic Categories by Clinical Site | Site | Location | Non-Malignant | B-CLL | Other B-CLPD malig. | T or NK Cell | NK+B-CLL | Total | | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | US | 51 | 19 | 5 | 4 | 0 | 79 | | 2 | US | 28 | 7 | 12 | 3 | 0 | 50 | | 3 | OUS | 5 | 29 | 18 | 0 | 0 | 52 | | 4 | OUS | 14 | 11 | 7 | 6 | 2 | 40 | | 5 | OUS | 3 | 13 | 13 | 0 | 0 | 29 | | Total | | 101 | 79 | 55 | 13 | \( 2^1 \) | 250 | \( ^{1} \) n=2 patient subjects with biclonal CLL + NK malignancies were considered true positives in downstream performance calculations A qualified hematopathologist, blinded to the final clinical diagnosis, independently evaluated results at each site to determine the absence or presence of an abnormal phenotype from OneFlow results. The result was designated as “aberrant” if an abnormal population was identified, and “non-aberrant” if an abnormal population was not identified. Immunophenotypes were further classified by the aberrant populations identified by analysts: B-CLL associated phenotypes, or phenotypes consistent with other chronic B-cell dyscrasias. K260215 - Page 25 of 27 {25} To characterize the overall clinical performance of the BD OneFlow reagents, the composite immunophenotypes determined by site readers from LST and B-CLPD T1 were compared to final clinical diagnoses for each patient, represented in the following table: | | Clinical Diagnosis | | | | Totals | | --- | --- | --- | --- | --- | --- | | | B-CLL | Other B-CLPD malig. | T/NK malig. | no malignancy | | | B-CLL | 78 | 1 | 0 | 1 | 80 | | Other B-CLPD phenotypes | 2 | 52 | 0 | 4 | 69 | | No B-cell aberrancy | 1 | 2 | 13 | 96 | 101 | | Totals | 81 | 55 | 13 | 101 | 250 | The following clinical performance parameters have been calculated for the OneFlow B-CLPD T1 reagents used in conjunction with the OneFlow LST reagents: | | target | (n)/(N) | point est. | (95% CI)^{1} | | --- | --- | --- | --- | --- | | Sensitivity | B-CLL | 78/81 | 96.3% | (89.7–98.7%) | | Sensitivity | Other B-CLPD malignancy | 52/55 | 94.5% | (85.1–98.1%) | | Sensitivity | Overall | 130/136 | 95.6% | (90.7–98.0%) | | Specificity | Overall | 112/114 | 98.2% | (93.8–99.5%) | | PPV | Overall | 130/135 | 96.3% | (91.6–98.4%) | | NPV | Overall | 112/115 | 97.4% | (92.6–99.1%) | $^{1}$ two-sided confidence intervals calculated from Wilson methods ## 2. Clinical Cut-Off The clinical cutoff will depend on professional analysis of the target aberrancy; analytical validation of these aberrancies have been performed to 1.0% of total leukocytes in the bone marrow, whole blood, and lymph node specimen types. ### D Expected Values/Reference Range: The expected frequency of lymphocyte subpopulations in the peripheral blood (PB) samples of apparently healthy individuals was assessed by testing 69 donor samples using the BD OneFlow LST and B-CLPD T1 reagent tubes on the BD FACSLyric Flow Cytometer. The nonparametric central 95% interval (i.e., 2.5$^{th}$ through 97.5$^{th}$ percentiles) were calculated following CLSI EP28-A3c. Descriptive statistics and reference intervals for lymphocyte subpopulations measured with the BD OneFlow LST and B-CLPD T1 tubes are presented in the following table: Reference intervals for select populations from BD OneFlow LST and B-CLPD T1 reagents | Population | Parent | Median^{1} | Min–Max^{1} | 2.5^{th}–97.5^{th} Percentile Interval^{1} | | --- | --- | --- | --- | --- | | B cells | CD45+ | 3.88 | 0.25–8.26 | 0.67–7.89 | | Igκ+ B cells | CD19+ | 58.8 | 38.1–68.6 | 47.38–67.0 | | Igλ+ B cells | CD19+ | 40.8 | 31.4–61.5 | 33.1–52.5 | | T cells | CD45+ | 23.4 | 6.33–39.4 | 7.74–37.1 | | TCRγδ+ T cells | CD45+ | 0.91 | 0.06–4.35 | 0.08–3.66 | | CD4+CD8− T cells | CD45+ | 14.2 | 4.37–27.3 | 5.70–25.4 | | CD8+CD4− T cells | CD45+ | 7.70 | 1.61–15.1 | 1.68–15.0 | K260215 - Page 26 of 27 {26} Reference intervals for select populations from BD OneFlow LST and B-CLPD T1 reagents | Population | Parent | Median^{1} | Min-Max^{1} | 2.5^{th}-97.5^{th} Percentile Interval^{1} | | --- | --- | --- | --- | --- | | NK cells | CD45+ | 3.93 | 1.06-10.0 | 1.28-9.97 | | CD5+CD19+ B cells | CD45+ | 0.36 | 0.02-1.54 | 0.04-1.37 | | CD5-CD19+ B cells | CD45+ | 3.53 | 0.23-7.48 | 0.63-7.29 | | CD5+CD19+ B cells | CD19+ | 9.79 | 2.83-22.2 | 3.76-20.7 | | Igκ+ | CD5+CD19+ | 59.3 | 50.3-67.6 | 50.3-66.5 | | Igλ+ | CD5+CD19+ | 40.4 | 32.4-49.7 | 33.0-49.1 | | CD5-CD19+ B cells | CD19+ | 90.2 | 77.8-97.2 | 79.4-96.2 | | Igκ+ | CD5-CD19+ | 58.7 | 36.3-69.4 | 46.4-67.5 | | Igλ+ | CD5-CD19 | 40.7 | 30.6-63.4 | 32.5-53.5 | $^{1}$ Continuous parametric measurand values represent % of parent populations defined in "Parent" column ### E Other Supportive Instrument Performance Characteristics Data: Electrical safety and electromagnetic compatibility (EMC) testing were performed, and the system was found to be acceptable. Software and cybersecurity documentation was reviewed and found to be acceptable ### VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. ### IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K260215 - Page 27 of 27