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The AQUIOS CL Flow Cytometer is intended for use with in vitro diagnostic flow cytometric applications using up to four fluorescent detection channels using a blue (488 mm) laser, two light scatter detection volume (EV). It is used in conjunction with the following reagents and software package.

AQUIOS Tetra-1 Panel and AQUIOS Tetra-2+ Panel monoclonal antibody reagents are for use on the AQUIOS CL Flow Cytometer with peripheral whole blood for immunophenotyping. These reagents are indicated for use in the immunologic assessment of patients having, or suspected of having, immune deficiency. These reagents provide identification and enumeration of;

• AQUIOS Tetra-1 Panel Monoclonal Antibody Reagent

· Total CD3+, CD3+CD4+,CD3+CD3+, CD3+CD4+/CD3+CD8+ (ratio only) lymphocyte percentages and absolute counts.

• CD45+ absolute count

• · CD45+ Low SS (lymphocytes) percentage and absolute count
  AQUIOS Tetra-2+ Panel Monoclonal Antibody Reagent

• · Total CD3+, CD3-CD19+, CD3-CD56+ and/or CD16+ lymphocyte percentages and absolute counts.

• CD45+ absolute count

• · CD45+ Low SS (lymphocytes) percentage and absolute count

AQUIOS Flow Cytometry Software may be run on an independent computer workstation for off-line analysis of results generated by the AQUIOS CL Flow Cytometer with the monoclonal antibody reagents listed above. The off-line analysis must be performed in accordance with the product labeling.

AQUIOS Tetra-1 Panel and AQUIOS Tetra-2+ Panel monoclonal antibody reagents are for use on the AQUIOS CL Flow Cytometer with peripheral whole blood for immunophenotyping. These reagents are indicated for use in the immunologic assessment of patients having, or suspected of having, immune deficiency. These reagents provide identification and enumeration of;

• AQUIOS Tetra-1 Panel Monoclonal Antibody Reagent

· Total CD3+, CD3+CD4+,CD3+CD8+, CD3+CD4+/CD3+CD8+(ratio only) lymphocyte percentages and absolute

counts.

• CD45+ absolute count
• · CD45+ Low SS (lymphocytes) percentage and absolute count
• AQUIOS Tetra-2+ Panel Monoclonal Antibody Reagent
• · Total CD3+, CD3-CD19+, CD3-CD56+ and/or CD16+ lymphocyte percentages and absolute counts.
• CD45+ absolute count
• · CD45+ Low SS (lymphocytes) percentage and absolute count

AQUIOS IMMUNO-TROL Cells are assayed, lysable whole blood quality control product for immunophenotyping analysis using monoclonal antibody reagents and flow cytometry. It provides a positive cell control that is processed in the same manner as a whole blood sample. This allows verification of instrument and reagent performance. It also verifies the methods used for staining targeted cells, lysing erythrocytes, and analyzing samples by the AQUIOS CL Flow Cytometer.

AQUIOS IMMUNO-TROL Low Cells are assayed, lysable whole blood quality control product for immunophenotyping analysis using monoclonal antibody reagents and flow cytometry. It provides a positive cell control that is processed in the same manner as a whole blood sample. This allows verification of instrument and reagent performance. It also verifies the methods used for staining targeted cells, lysing erythrocytes, and analyzing samples by the AQUIOS CL Flow Cytometer.

AQUIOS Lysing Reagent Kit is used as part of the AQUIOS flow cytometer system. The kit consists used by AQUIOS flow cytometers to prepare whole blood samples for analysis of white blood cells.

The AQUIOS CL Flow Cytometry System is composed of the following components:

• . AQUIOS CL Flow Cytometer
• AQUIOS System Software ●
• AQUIOS Tetra-1 Panel CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 ●
• AQUIOS Tetra-2+ Panel CD45-FITC/(CD56+CD16)-RD1/CD19-ECD/CD3-PC5 ●
• AQUIOS Immuno-Trol Cells ●
• AQUIOS Immuno-Trol Low Cells ●
• . AQUIOS Lysing Reagent Kit

The AQUIOS CL Flow Cytometer uses flow cytometric principles to determine qualitative and quantitative measurements of biological and physical properties of cells and other particles. These properties are measured when the cells pass through the laser beam(s) in single file.

The AQUIOS System Software is designed for the AQUIOS CL flow cytometer. It includes the algorithms and test definitions that provide automated analysis and results for AQUIOS Tetra-1 and 2+ reagents; this application cannot be modified by the user.

The AQUIOS Flow Cytometry System also offers an optional standalone offline workstation. This workstation is identical to the workstation that is physically connected to the instrument and can be used for off-line analysis of results generated by the AQUIOS CL Flow Cytometer with AQUIOS Tetra-1 and Tetra-2+ reagents and AQUIOS System software according to the product labeling.

AQUIOS Tetra-1 Panel CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 reagent provides identification and enumeration of CD45+, CD45+ Low SS, and CD3+/CD4+, CD3+/CD8+, and CD3+ lymphocyte percentages and absolute counts in peripheral whole blood. AQUIOS Tetra-2+ Panel CD45-FITC/(CD56+CD16)-RD1/CD19-ECD/CD3-PC5 provides identification and enumeration of CD45+, CD45+ Low SS, and CD3+, CD3-/CD19+ and CD3-/CD56+CD16+ lymphocyte percentages and absolute counts in peripheral whole blood. Additionally, both panels provide for CD45+ absolute count and CD45+ Low SS absolute count and percentage.

AQUIOS Immuno-Trol and Immuno-Trol Low Cells are assayed, lysable whole blood quality control product for immunophenotyping analysis using monoclonal antibody reagents and flow cytometry. It provides a positive cell control that is processed in the same manner as a whole blood sample. This allows verification of instrument and reagent performance. It also verifies the methods used for staining targeted cells, lysing erythrocytes, and analyzing samples by the AQUIOS CL Flow Cytometer.

The AQUIOS CL Flow Cytometer uses on-board sample preparation as part of the overall system workflow. The AQUIOS Lysing Reagent Kit is comprised of two readyto-use reagents: Reagent A lyses the red blood cells, Reagent B quenches the solution, slowing the lyse reaction down in preparation for analysis. This reagent system provides a rapid, no-wash, standardized, whole blood lysing solution for sample to sample, and laboratory to laboratory reproducibility.

Here's an analysis of the acceptance criteria and study findings for the AQUIOS CL Flow Cytometry System, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document provides summary results rather than explicit, quantified acceptance criteria for many tests. However, it consistently states that the device "meets performance requirements" or "was demonstrated." Where specific performance measures or outcomes are mentioned, they are included below.

2. Sample Sizes Used for the Test Set and Data Provenance

The document does not explicitly state the sample sizes used for the test set portion of the studies (which would typically involve independent validation after development). Instead, it describes general testing approaches. It also does not specify the country of origin for the data or whether it was retrospective or prospective.

For example, for "Method Comparison," it merely states "To evaluate bias between the subject device versus the predicate" and uses CLSI EP09-A3 (Method Comparison and Bias Estimation Using Patient Samples), which implies patient samples were used, but no quantity or demographics are given. Similarly, "Adult Reference Intervals" implies a study on adults, but sample size and demographics are not specified.

3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications

The document does not mention the use of experts to establish a "ground truth" for a test set in the traditional sense of image interpretation or complex diagnostic decision-making. The AQUIOS CL Flow Cytometry System is an automated cell counter. Its "ground truth" for the test set would typically be established by established reference methods, predicate devices, or validated laboratory assays (e.g., manual differential counts, confirmed cell populations by expert flow cytometrists, or validated internal methods). The document refers to "predicate devices" for comparison, which themselves are legally marketed and validated, serving as a de facto "ground truth" in terms of established performance.

4. Adjudication Method for the Test Set

Not applicable. As "ground truth" is not explicitly established by human experts in a subjective interpretation process (like in radiology), an adjudication method in the sense of resolving inter-reader disagreements is not described or implied. The system relies on comparisons to predicate devices and validated methods.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No. An MRMC comparative effectiveness study, which typically compares human reader performance with and without AI assistance, was not conducted. This device is an automated in vitro diagnostic system for cell counting and immunophenotyping, not a system that assists human readers in interpreting complex cases. Its "effectiveness" is measured by its analytical performance (accuracy, precision, linearity, etc.) against established laboratory methods and predicate devices.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop) Performance Was Done

Yes, implicitly. The entire set of performance studies described (Fluorescence Linearity, Electronic Volume Linearity, Laser Performance, Analyzer Carryover, Instrument Settings Stability, Gravimetrics, Comparability, Assay Linearity, Assay Carryover, Detection Capability, Specimen and Prepared Sample Stability, Method Comparison, Precision, Adult Reference Intervals, Reagent Stability, Lot Variability) evaluates the device's autonomous performance. The AQUIOS CL Flow Cytometer, along with its reagents and software, is designed to perform these tasks automatically. While human operators are involved in sample preparation and loading, the core measurements and classifications are driven by the instrument's algorithms and hardware, making these standalone performance assessments. The "AQUIOS System Software" also includes algorithms for automated analysis.

7. The Type of Ground Truth Used

The ground truth for the performance studies appears to be based on:

• Comparison to predicate devices: For analytical accuracy (e.g., "Method Comparison" against FACSCalibur or UniCel DxH 800). This means the established, cleared performance of the predicate serves as the standard.
• Validated internal methods/specifications: For various instrument and assay characteristics like linearity, precision, detection limits, and stability.
• Published values: For establishing "Adult Reference Intervals," which were confirmed to be "consistent with published values for T, B, and NK lymphocyte subsets."
• CLSI (Clinical and Laboratory Standards Institute) guidelines and standards: These provide the methodological framework and often implicit performance thresholds for many tests (e.g., CLSI EP06-A for linearity, CLSI EP5-A2 for precision, CLSI H26-A2 for hematology analyzers).

8. The Sample Size for the Training Set

The document does not specify a separate "training set" or its size. For an IVD device like this, which performs quantitative measurements based on biophysical properties, the development process generally involves extensive internal testing and refinement (calibration, optimization, verification) using a wide range of samples, rather than a distinct "training set" in the machine learning sense. The performance studies described in the document are primarily for validation and verification.

9. How the Ground Truth for the Training Set Was Established

Since a "training set" in the AI/ML context is not explicitly mentioned, the concept of establishing ground truth for it is not directly addressed. Instead, the analytical methods (flow cytometry principles, reagent chemistries, software algorithms) are based on well-established scientific principles and calibrated against reference materials and methods during the device's development and manufacturing.

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The EZCD4 Assay is intended to be performed on a Guava PCA System with CytoSoft 2.3 version software which includes three modules; EZCD4, Guava Check and Clean and Shutdown. The system is intended to identify and quantify the absolute counts of CD4 T-Lymphocytes in EDTA whole blood. The GuavaEZCD4 system is intended for the ongoing monitoring of patients with documented diagnosis of an immunodeficiency disease. The Guava EZCD4 system is intended for use only by trained laboratory professionals.

The Guava EZCD4 System is an optimized cell analysis system consisting of the Guava PCA instrument, EZCD4 software for data acquisition and analysis and an EZCD4 Reagent Kit consisting of optimized reagents and protocols. The Guava EZCD4 Reagent Kit is a two-color direct immunofluorescence kit used for the enumeration of mature CD4 T lymphocytes in human blood. It consists of a monoclonal anti-human CD3 antibody conjugated to the tandem dye, phycoerythrin (PE)-Cy5 (PECy5) and a monoclonal anti-human CD4 antibody conjugated to PE. Guava 1X Lysing Solution and Guava Fixative are available in separate packaging and are added, after staining, to lyse erythrocytes and to preserve cells respectively. The Guava PCA instrument includes a laptop computer. The Guava PCA incorporates a new technology termed micro capillary cytometry. The device is a 3-parameter flow cytometer that utilizes a solid state 532 nm green laser, a fixed optical & fluidic system, a self-aligning flow cell and a micro capillary delivery system to perform cell analysis. The micro-syringe stepper pump allows the use of small sample volumes and volumetric measurements for the determination of absolute counts per microliter. The Guava PCA optic system detects forward scatter and two different wavelengths of fluorescence, 580/20 nm and 675/20 nm. It has a small foot print, 21.6 cm x 32 cm x 36.3 cm and is equipped with a network ready laptop computer with a 10GB hard drive and Windows 2000 operating system. The software is application specific with onscreen messaging enabling ease of use and facilitates short, effective training programs. Data is readily analyzed immediately after acquisition and is stored as electronic files on the hard drive.

{
  "acceptance_criteria_and_performance_table": {
    "title": "Summary of Guava EZCD4 System Performance Studies",
    "headers": [
      "Study Type",
      "Acceptance Criteria",
      "Reported Device Performance"
    ],
    "rows": [
      [
        "Correlation Study (vs. Predicate Devices)",
        "Not explicitly stated as numerical acceptance criteria, but implied demonstration of substantial equivalence via linear regression (R-squared, Slope, Intercept).",
        "**Site 2:** N=92, R-squared=0.95, Slope=+1.00, Intercept=18.64, Range=13-1465\n**Site 3:** N=91, R-squared=0.93, Slope=+0.96, Intercept=35.51, Range=17-1175\n**Site 4:** N=88, R-squared=0.98, Slope=+1.17, Intercept=18.46, Range=47-1439\n**Site 5:** N=94, R-squared=0.98, Slope=+0.95, Intercept=13.29, Range=8-1076\n(Conclusion: Results are substantially equivalent)"
      ],
      [
        "Linearity and Sensitivity Study",
        "Not explicitly stated as numerical acceptance criteria, but implied demonstration of linearity across a wide range of CD4+ T cell counts.",
        "Linear regression equation: y = 1.0118x - 45.237\nR-squared = 0.9866\n(Graph shows strong linearity from 0 to >2000 CD4+ T cells/µL)"
      ],
      [
        "Intra-Laboratory Reproducibility",
        "Not explicitly stated as numerical acceptance criteria (e.g., max CV%), but implied low variability across sites and ranges.",
        "**Site 2:** Low CV=13.50%, Mid CV=8.06%, High CV=4.75%\n**Site 3:** Low CV=10.45%, Mid CV=7.20%, High CV=4.92%\n**Site 4:** Low CV=11.05%, Mid CV=3.87%, High CV=3.56%\n**Site 5:** Low CV=4.69%, Mid CV=3.69%, High CV=3.23%"
      ],
      [
        "Carryover Study",
        "Absence of significant sample carryover, as determined by statistical comparison.",
        "Wilcoxon-Mann-Whitney test 1-sided p-value = 0.1474 (Conclusion: No significant sample carryover demonstrated)"
      ],
      [
        "Specificity Study (Guava anti-CD3)",
        "Expected specificity for peripheral blood lymphocytes.",
        "Mean % positive in Lymphocytes: 47.80%\nMean % positive in Monocytes: 3.52%\nMean % positive in Granulocytes: 1.52%\n(Conclusion: Expected specificity for lymphocytes)"
      ],
      [
        "Specificity Study (Guava anti-CD4)",
        "Expected specificity for peripheral blood lymphocytes, allowing for known weak positive staining of monocytes.",
        "Mean % positive in Lymphocytes: 23.58%\nMean % positive in Monocytes: 81.58%\nMean % positive in Granulocytes: 4.39%\n(Conclusion: Expected specificity for lymphocytes, with known weak positive for monocytes)"
      ],
      [
        "Blood Sample Aging (Pre-Staining)",
        "Storage of unstained EDTA whole blood for specific periods without significant impact on results.",
        "Unstained EDTA anti-coagulated whole blood specimens may be stored for periods up to and including 72 hours (at 18-25°C) prior to staining and analysis."
      ],
      [
        "Blood Sample Aging (Post-Staining)",
        "Storage of stained and fixed EDTA whole blood for specific periods without significant impact on results.",
        "Stained EDTA anti-coagulated whole blood specimens may be stored for periods up to and including 24 hours (at 18-25°C or 2-8°C) prior to analysis."
      ]
    ]
  },
  "study_details": {
    "test_set_sample_size": {
      "correlation_study": "365 abnormal whole blood samples (approximately 90 per site) from three CD4+ absolute count ranges (0-200, 201-500, 501-2000).",
      "linearity_sensitivity": "22 aliquots (11 pairs) prepared from bulk blood samples.",
      "intra_laboratory_reproducibility": "10 replicate whole blood samples from each of three abnormal donors (low, mid, high ranges) per site, across 4 sites. Total 120 samples (3 donors x 10 replicates x 4 sites).",
      "carryover_study": "21 low range sample replicates and 18 high range sample replicates in a specific alternating sequence.",
      "specificity_study": "5 healthy subjects, EDTA anti-coagulated whole blood samples, analyzed for 15,000 events per blood component for each donor.",
      "blood_sample_aging": "Pre-Staining: 5 healthy normal donors, 5 blood specimens each (total 25 specimens). Post-Staining: 5 healthy normal donors, 7 stained samples per blood sample, pooled (total 35 samples for 18-25°C and 35 for 2-8°C)."
    },
    "data_provenance": {
      "correlation_study": "Prospective, collected at four clinical trial sites in the US.",
      "specificity_study": "Prospective, collected from healthy subjects.",
      "blood_sample_aging": "Prospective, collected from healthy normal donors."
    },
    "number_of_experts_ground_truth_test_set": "Not applicable, as these studies primarily assessed device performance against predicate devices or expected biological/analytical characteristics rather than expert-derived ground truth.",
    "qualifications_of_experts": "Not applicable.",
    "adjudication_method": "Not applicable.",
    "mrmc_comparative_effectiveness_study": "No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly described, as this is a device for quantifying CD4+ T cells, not for image interpretation by human readers. The correlation study compared the device's output to predicate flow cytometry systems.",
    "standalone_performance_done": "Yes, various standalone performance metrics were evaluated, including linearity, reproducibility (intra-laboratory precision), carryover, and specificity. The correlation study also assessed its performance against predicate devices.",
    "type_of_ground_truth_test_set": {
      "correlation_study": "Reference measurements from predicate flow cytometry systems (Becton Dickinson FACSCalibur with MultiTest or TriTest reagents).",
      "linearity_sensitivity": "Known 'Expected values' derived from gravimetric preparation of blood cell aliquots with known high and low CD4+ T cell counts.",
      "intra_laboratory_reproducibility": "The device's own measurements across replicates and sites, assessing internal consistency; no external 'ground truth' in the context of diagnostic accuracy.",
      "carryover_study": "The device's own measurements of alternating low and high concentration samples, showing lack of influence.",
      "specificity_study": "The characteristic immunological staining patterns of known cell populations (lymphocytes, monocytes, granulocytes) as observed on a reference flow cytometer (FACSCalibur) with the study reagents.",
      "blood_sample_aging": "The device's own measurements at different time points, comparing to the initial (0 hour) measurement value as a reference for stability."
    },
    "training_set_sample_size": "Not specified. As this is a flow cytometer system with reagents and software for quantitative measurements, it's unlikely to have a 'training set' in the machine learning sense. The software's algorithms would likely be based on established flow cytometry principles and calibrated/validated during development.",
    "how_ground_truth_training_set_established": "Not applicable. The system's operation and algorithms for data acquisition and analysis are based on established principles of flow cytometry and are likely calibrated and validated through internal development and testing, rather than an external 'ground truth' for a machine learning training set."
  }
}

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The Cylex Immune Cell Function Assay measures the concentration of ATP from circulating CD4 cells following in vitro stimulation with phytohemagglutinin (PHA) as an indicator of immune cell function. This measurement is made on heparin anticoagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell mediated immune response in populations undergoing immunosuppressive therapy for organ transplant.

The Cylex Immune Cell Function Assay detects cell-mediated immunity (CMI) by measuring the concentration of ATP from CD4 cells following stimulation. This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell-mediated immunity in an immunosuppressed population.

The Cylex Immune Cell Function Assay measures the concentration of ATP following in vitro stimulation with phytohemagglutinin (PHA) as an indicator of immune cell function. This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell mediated immune response in populations undergoing immunosuppressive therapy for organ transplant.

The Cylex Immune Cell Function Assay detects cell-mediated immunity in whole blood. Following incubation, increased ATP synthesis occurs within the cells that respond to the stimulant phytohemagglutinin (PHA). Concurrently, whole blood to monoclonal antibody coated magnetic particles are added to immunoselect CD4 cells from both the stimulated and non-stimulated wells. After washing the selected CD4 cells on a magnet tray, Lysis Reagent is added to release intracellular ATP. Through the released ATP produces light according to the following equation: Luciferin + ATP + O2 Luciferase Mo Oxyluciferin + AMP + Pyrophosphate + CO2 + Light. The amount of light measured by a luminometer (emission maximum 562 nm) is proportional to the concentration of ATP. The concentration of ATP (ng/mL) is calculated from a calibration curve to characterize the cellular immune function of the sample.

Here's a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Cylex Inc. Immune Cell Function Assay

1. Table of Acceptance Criteria and Reported Device Performance

The provided text does not explicitly state pre-defined "acceptance criteria" in terms of specific thresholds for sensitivity, specificity, or similar performance metrics that the device had to meet to be approved. Instead, it presents the results of a clinical study aimed at demonstrating the device's ability to differentiate between two distinct populations (healthy vs. immunosuppressed transplant recipients) and comparing its statistical performance to a predicate device.

The primary demonstration of performance is the statistical difference in ATP measurements between the two patient populations.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study is for an in vitro diagnostic (IVD) assay measuring a biomarker, not for an imaging device or AI algorithm requiring human interpretation. Therefore, there's no mention of human readers or improved performance with AI assistance.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, the study presents the standalone performance of the Cylex Immune Cell Function Assay. The reported ATP ng/mL values are solely generated by the device based on its intended mechanism (measuring ATP after PHA stimulation). There is no human interaction or interpretation involved in generating the primary raw data or the final ATP concentration.

7. The Type of Ground Truth Used

The ground truth used was clinical diagnosis/patient status:

• "Apparently Healthy Adults" (non-immunosuppressed)
• "Transplant Recipients" (immunosuppressed, undergoing therapy, with specific organ transplants listed).

8. The Sample Size for the Training Set

The document does not provide information about a separate "training set" or its sample size. This type of submission (510(k) for an IVD) focuses on the clinical performance study (test set) for device validation rather than an AI/machine learning model development process that typically involves distinct training, validation, and test sets.

9. How the Ground Truth for the Training Set Was Established

Since no training set is mentioned for this IVD submission, information on how its ground truth was established is not provided.

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