BD FACSCanto II flow cytometer (4-2-2 and 5-3 configurations), BD FACSCanto flow cytometer (4-3-3 configuration)

K141468 · Bd Biosciences · OYE · Feb 27, 2015 · Hematology

Device Facts

• Record ID: K141468
• Device Name: BD FACSCanto II flow cytometer (4-2-2 and 5-3 configurations), BD FACSCanto flow cytometer (4-3-3 configuration)
• Applicant: Bd Biosciences
• Product Code: OYE · Hematology
• Decision Date: Feb 27, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Indications for Use

The BD FACSCanto flow cytometer (4-3-3 configuration) functions as part of a system with dedicated clinical software intended for use with cleared or approved in vitro diagnostic (IVD) assays that are indicated for use with the instrument for the identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (640 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III. For in vitro diagnostic use. The BD FACSCanto II flow cytometers (4-2-2 and 5-3 configurations) function as part of a system with dedicated clinical software intended for use with cleared or approved in vitro diagnostic (IVD) assays that are indicated for use with the instrument for the identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (633 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III. For in vitro diagnostic use.

Device Story

Flow cytometer system for clinical immunophenotyping; utilizes fluidics, optics, and electronics subsystems to analyze human cell subsets. Input: biological samples (e.g., whole blood) processed via lyse/wash or lyse/no-wash methods. Operation: sample introduced manually or via BD FACSLoader; fluidics system transports sample to flow cell; lasers (blue 488nm, red 640nm/633nm) excite fluorochromes; photomultiplier tubes (PMTs) detect forward scatter, side scatter, and fluorescence signals. Software (BD FACSCanto clinical software) performs automated immunophenotyping; BD FACSDiva software used for setup/service. Output: quantitative cell subset data displayed on workstation. Used in clinical laboratories by trained personnel. Benefits: standardized, automated identification and enumeration of cell populations for diagnostic assessment.

Clinical Evidence

Bench testing only. Performance evaluated using CLSI guidelines (EP9-A2 for accuracy, EP5-A2 for precision, EP6-A for linearity). Studies compared subject configurations (4-2-2, 5-3, 4-3-3) against the predicate using BD Multitest IMK Kit (4-color) and BD Multitest 6-color TBNK assays. Results demonstrated equivalent performance across all configurations for IVD assays. Carryover testing met acceptance criteria.

Technological Characteristics

Flow cytometer with fluidics, optics, and electronics. Configurations include 488nm blue and 633/640nm red lasers. 4-2-2 and 4-3-3 models include an additional 405nm violet laser for research use. Detection via 7 to 11 PMTs; only 6 channels cleared for IVD. Connectivity: standalone workstation. Software: BD FACSCanto clinical software and BD FACSDiva. Automated sample prep via BD FACS Sample Prep Assistant III.

Indications for Use

Indicated for identification and enumeration of human cell subsets in clinical laboratories using cleared or approved IVD assays. For use with or without BD FACS Sample Prep Assistant III. Prescription use only.

Regulatory Classification

Identification

An automated differential cell counter is a device used to identify one or more of the formed elements of the blood. The device may also have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood, bone marrow, or other body fluids. These devices may combine an electronic particle counting method, optical method, or a flow cytometric method utilizing monoclonal CD (cluster designation) markers. The device includes accessory CD markers.

Special Controls

*Classification.* Class II (special controls). The special control for this device is the FDA document entitled “Class II Special Controls Guidance Document: Premarket Notifications for Automated Differential Cell Counters for Immature or Abnormal Blood Cells; Final Guidance for Industry and FDA.”

Predicate Devices

• BD FACSCanto system (4-2 configuration) with BD FACSCanto clinical software (k041074)
• BD FACSCanto system (4-2 configuration) with BD FACSDiva software (k040725)

Reference Devices

• BD FACSCanto II system (4-2 configuration) with BD FACSCanto clinical software and BD FACSDiva software (k062087)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM INSTRUMENT ONLY TEMPLATE

A. 510(k) Number:
k141468

B. Purpose for Submission:
New Submission

C. Manufacturer and Instrument Name:
BD Biosciences

BD FACSCanto™ II flow cytometers (4-2-2 and 5-3 configurations) with BD FACSCanto™ clinical software and BD FACSDiva™ software

BD FACSCanto™ flow cytometer (4-3-3 configuration) with BD FACSCanto™ clinical software and BD FACSDiva™ software

D. Type of Test or Tests Performed:
Flow Cytometric Immunoassays

E. System Descriptions:

1. Device Description:

The BD FACSCanto™ II system (4-2-2 and 5-3 configurations) and the BD FACSCanto™ (4-3-3 configuration) systems consist of a flow cytometer, a self-contained fluidics cart, and a computer workstation. System options include an automated sample loader, a barcode reader, and a sample preparation assistant.

All optical configurations of the FACSCanto™ II share the same dimensions: 25.2 inches in height by 35.7 inches in width by 24 inches in depth. The units are designed to fit a lab bench 22 inches in depth. Each system weighs less than 320 pounds, excluding the FACSLoader and computer workstation. They are able to accept 100/115/230 VAC (volts alternating current) at 50-60 Hertz. Power consumption is rated at 500 Watts.

The FACSCanto™ 4-3-3 configuration shares the same height and width as the FACSCanto™ II instrument but is 25 inches deep and designed to fit on a 27 inches deep lab bench. It weighs less than the FACSCanto™ II instrument (270 pounds without FACSLoader or workstation) and has similar power input and consumption specifications.

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2. Principles of Operation:

Flow cytometers combine fluidics, optics, and electronics subsystems to measure and analyze signals emitted when particles in a liquid stream flow through a glass cuvette, at which beams of laser light are directed. The scatter and fluorescence light signals from these particles provide information about cell size, internal complexity, and relative fluorescence intensity. The Instructions for Use for each BD FACSCanto™ II and BD FACSCanto™ instrument includes more details on the system components and theory of operations.

The instruments are intended for use with cleared or approved in vitro diagnostic (IVD) assays for the identification and enumeration of human cell subsets that are indicated for use with the instrument.

3. Modes of Operation:

Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes ☐ X ☐ or No ☐

Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes ☐ X ☐ or No ☐

4. Specimen Identification:

Barcode reader or manual entry.

5. Specimen Sampling and Handling:

The instruments may be used with or without the BD FACS Sample Prep Assistant III. Specimen handling should be performed according to the intended IVD assay intended for use with the instruments.

6. Calibration:

Calibration is performed with the IVD assay intended for use with the instruments.

7. Quality Control:

Quality control is performed with the intended IVD assay intended for use with the instruments.

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8. Software:

FDA has reviewed applicant’s Hazard Analysis and Software Development processes for this line of product types:

Yes ☐ X ☐ or No ☐

F. Regulatory Information:

1. Regulation section:

21 CFR § 864.5220, Automated Differential Cell Counter

2. Classification:

Class II

3. Product code:

OYE, flow cytometric reagents and accessories

4. Panel:

Hematology (81)

G. Intended Use:

1. Indication(s) for Use:

BD FACSCanto™ flow cytometer (4-3-3 configuration) with BD FACSCanto™ clinical software and BD FACSDiva™ software:

The BD FACSCanto flow cytometer (4-3-3 configuration) functions as part of a system with dedicated clinical software intended for use with cleared or approved in vitro diagnostic (IVD) assays that are indicated for use with the instrument for the identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (640 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III.

For in vitro diagnostic use.

BD FACSCanto™ II flow cytometers (4-2-2 and 5-3 configurations) with BD FACSCanto™ clinical software and BD FACSDiva™ software:

The BD FACSCanto II flow cytometers (4-2-2 and 5-3 configurations) function as part of a system with dedicated clinical software intended for use with cleared or approved in-

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vitro diagnostic (IVD) assays that are indicated for use with the instrument for the identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (633 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III.

For in vitro diagnostic use.

2. Special Conditions for Use Statement(s):

For Prescription Use Only.

H. Substantial Equivalence Information:

1. Predicate Device Name(s) and 510(k) numbers:

BD FACSCanto™ system (4-2 configuration) with BD FACSCanto™ clinical software, k041074

BD FACSCanto™ system (4-2 configuration) with BD FACSDiva™ software, k040725

BD FACSCanto™ II system (4-2 configuration) with BD FACSCanto™ clinical software and BD FACSDiva software, k062087 was also used a reference device.

2. Comparison with Predicate Device:

BD FACSCanto™ II 4-2-2 and 5-3 configurations with BD FACSCanto™ clinical software and BD FACSDiva™ software:

| Similarities and Differences | | | |
| --- | --- | --- | --- |
| Item | New Canto II 4-2-2 Configuration | New Canto II 5-3 Configuration | Predicate Canto 4-2 Configuration |
| Intended Use | The BD FACSCanto™ II flow cytometers (4-2-2 and 5-3 configurations) function as part of a system with dedicated clinical software intended for use with cleared or approved in vitro diagnostic (IVD) assays that are | Same | BD FACS Canto Flow Cytometer with BD FACS Canto Software [or BD FACSDiva Software] is used for immunophenotyping in clinical laboratories, using previously cleared IVD assays for flow cytometry that |

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| Similarities and Differences | | | |
| --- | --- | --- | --- |
| Item | New Canto II 4-2-2 Configuration | New Canto II 5-3 Configuration | Predicate Canto 4-2 Configuration |
| | indicated for use with the instrument for the identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (633 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III. | | utilize the lyse no-wash sample preparation method. The lymphocytes subsets include; CD3^{+}CD8^{+}, CD3^{+}CD4^{+}, CD3^{-} CD16^{+} and/or CD56^{+}, CD3^{-} CD19^{+}, and CD3^{+}. |
| Device Classification | 21 CFR § 864.5220, Automated Differential Cell Counter | Same | Same |
| Device Product Code | OYE, flow cytometric reagents and accessories | Same | GKZ, Counter Differential Cell |
| IVD Lasers/Excitation | Blue Laser: Blue/488 nm, 20mW
Red Laser: Red/633nm, 17mW HeNe | Blue Laser: Same
Red lasers: Same | Blue Laser: Same
Red lasers: Same |
| RUO Lasers | Violet laser: Violet/405 nm, 30mW | Not applicable | Not applicable |
| IVD Optical Detector Array Configurations* | 1st detector array*
FACSCanto™ 4-2 configuration modified to enable one additional detector in 5-3 configuration | 1st detector array*
Same, but one additional detector is enabled
2nd detector array*
Same, but one | 1st detector array: (blue “octagon”) receives 488nm excited and scattered light. The array is configured to detect SSC and 4 |

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| Similarities and Differences | | | |
| --- | --- | --- | --- |
| Item | New
Canto II 4-2-2
Configuration | New
Canto II 5-3
Configuration | Predicate
Canto 4-2
Configuration |
| | 2nd detector array*
FACSCanto™ 4-2
configuration
modified to enable
one additional
detector in 5-3
configuration | additional detector
is enabled | fluorochromes
(FITC, PE,
PerCP/PerCPCy5.5,
PE-Cy7)

2nd detector array:
(red “trigon”) receives 633nm
excited light. The
array is configured
to detect 2
fluorochromes
(APC, APCCy7) |
| IVD Collection
Optics:
Mirrors* | Blue*:
735LP
655LP
610LP
556LP
502LP
A pinhole was
added in front of
SSC to optimize the
system design for
improved
automated
adjustment of the
SSC parameter.

Red*:
735LP
685LP | Blue*:
Same

Red*:
Same | Blue:
735LP
655LP
556LP
502LP
Neutral Density
filter

Red:
735LP |
| IVD Collection
Optics:
Bandpass Filters | Blue:
780/60
670LP
585/42
530/30
488/10 (SSC)

Red:
780/60
660/20 | Blue:
Same

Red:
Same | Blue:
Same

Red:
Same |

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| Similarities and Differences | | | |
| --- | --- | --- | --- |
| Item | New Canto II 4-2-2 Configuration | New Canto II 5-3 Configuration | Predicate Canto 4-2 Configuration |
| Electronics* | One consolidated data acquisition electronics board with improved preamplifier circuitry. | Same | Multiple electronics boards containing acquisition electronics components |
| Fluidics* | Consists of a monolithic ported block that duplicates FACSCanto™ fluidic scheme. The manifold improves reliability and ease of serviceability. | Same | Consists of a pinch valve assembly which controls the flow of sample, saline, and waste fluids. Includes a separate wet cart assembly. |
| Fluidics Cart* | BD FACSCanto™ fluidics cart with the incorporation of a manifold assembly and improved chemical compatibility of valve material. | Same | Original FACSCanto™ design with FACSFlow™ sheath fluid cubitainer |
| Sample Introduction* | SIT with modified tube guide, sensor, and probe tip | Same | Original FACSCanto Sample injection tube (SIT) design |
| Automated Sample Presentation* | BD FACSLoader with updated motor, pneumatic actuation, and sliding access doors | Same | BD FACSLoader |
| Software | BD FACSCanto™ clinical software v2.1 or higher (currently v3.0)
BD FACSDiva™ software v5.0.1 or higher | Same | BD FACSCanto™ clinical software v1.0 or higher
BD FACSDiva™ software v4.0 or higher |
| Instrument Setup and Quality Control | Automated setup using BD | Same | Same |

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| Similarities and Differences | | | |
| --- | --- | --- | --- |
| Item | New Canto II 4-2-2 Configuration | New Canto II 5-3 Configuration | Predicate Canto 4-2 Configuration |
| | FACSCanto™ clinical software and BD FACS 7-color setup beads | | |
| Sample Preparation | Manual pipetting for the lyse/wash or lyse/no-wash methods, or automated with the BD FACS Sample Prep Assistant (SPA) for the lyse/no-wash method | Same | Same |
| Sample Type** | Assay-dependent | Same | Same |
| | | | |
| * Indicates change is same as cleared in FACS Canto II (4-2) configuration, k062087 (reference device) | | | |
| **Sample Types are assay-dependent; refer to FDA cleared or approved assays for designated sample types. For use with Multitest 6-color TBNK and IMK kit, whole blood is the indicated sample type in the assay’s IFU. | | | |
| Note: Seven to ten color immunophenotyping is for research use only (RUO). | | | |

BD FACSCanto™ 4-3-3 configuration with BD FACSCanto™ clinical software and BD FACSDiva™ software:

| Similarities and Differences | | |
| --- | --- | --- |
| Item | New Canto 4-3-3 Configuration | Predicate Canto 4-2 Configuration |
| Intended Use | The BD FACSCanto™ flow cytometers (4-3-3 configuration) function as part of a system with dedicated clinical software intended for use with cleared or approved in vitro diagnostic (IVD) assays that are indicated for use with the instrument for the | BD FACS Canto Flow Cytometer with BD FACS Canto Software [or BD FACSDiva Software] is used for immunophenotyping in clinical laboratories, using previously cleared IVD assays for flow cytometry that utilize the lyse no- |

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| Similarities and Differences | | |
| --- | --- | --- |
| Item | New
Canto 4-3-3
Configuration | Predicate
Canto 4-2
Configuration |
| | identification and enumeration of human cell subsets. Only six detection channels using a blue (488 nm) and a red (640 nm) laser have been cleared for in vitro diagnostic use. For use with or without the BD FACS Sample Prep Assistant III. | wash sample preparation method. The lymphocytes subsets include; CD3^{+}CD8^{+}, CD3^{+}CD4^{+}, CD3^{-}CD16^{+} and/or CD56^{+}, CD3^{-}CD19^{+}, and CD3^{+}. |
| Device Classification | 21 CFR § 864.5220, Automated Differential Cell Counter | Same |
| Device Product Code | OYE, flow cytometric reagents and accessories | GKZ, Counter Differential Cell |
| IVD Lasers/Excitation | Blue Laser: 488 nm, 20mW
Red Laser: 640nm, 40mW HeNe | Blue Laser: Same
Red lasers: 633nm, 17mW HeNe |
| RUO Lasers | Violet laser: Violet/405 nm, 30mW | Not applicable |
| IVD Optical Detector Array Configurations* | 1st detector array* Same as FACSCanto™ 4-2 (predicate) configuration with minor filter modification
2nd detector array* Same as FACSCanto™ 4-2 (predicate) configuration with minor filter modification, one additional detector enabled | 1st detector array: (blue “octagon”) receives 488nm excited and scattered light. The array is configured to detect SSC and 4 fluorochromes (FITC, PE, PerCP/PerCPCy5.5, PE-Cy7)
2nd detector array: (red “trigon”) receives 633nm excited light. The array is configured to detect 2 fluorochromes (APC, APCCy7) |
| IVD Collection Optics: Mirrors* | Blue*: 735LP
655LP
550LP
502LP | Blue: 735LP
655LP
556LP
502LP |

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| Similarities and Differences | | |
| --- | --- | --- |
| Item | New Canto 4-3-3 Configuration | Predicate Canto 4-2 Configuration |
| | A pinhole was added in front of SSC to optimize the system design for improved automated adjustment of the SSC parameter. Red*: 735LP 685LP | Neutral Density filter Red: 735LP |
| IVD Collection Optics: Bandpass Filters | Blue: 780/60 695/40 575/25 530/30 488/10 (SSC) Red: 780/60 670/30 | Blue: 780/60 670LP 585/42 530/30 488/10 (SSC) Red: 780/60 660/20 |
| Electronics | Similar to FACSCanto™ (predicate) 4-2 configuration with modification to the pre-amplifier circuitry for linearity improvements. | Multiple electronics boards containing acquisition electronics components |
| Fluidics* | Consists of a monolithic ported block that duplicates FACSCanto™ fluidic scheme. The manifold improves reliability and ease of serviceability. | Consists of a pinch valve assembly which controls the flow of sample, saline, and waste fluids. Includes a separate wet cart assembly. |
| Fluidics Cart* | BD FACSCanto™ fluidics cart with the incorporation of a manifold assembly and improved chemical compatibility of valve material. | Original FACSCanto™ design with FACSFlow™ sheath fluid cubitainer |
| Sample Introduction | Original FACSCanto™ Sample injection tube (SIT) design | Same |

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| Similarities and Differences | | |
| --- | --- | --- |
| Item | New
Canto 4-3-3
Configuration | Predicate
Canto 4-2
Configuration |
| Automated Sample Presentation | BD FACSLocader | Same |
| Software | BD FACSCanto™ clinical software v2.4 or higher (currently v3.0)
BD FACSDiva™ software v7.0 or higher | BD FACSCanto™ clinical software v1.0 or higher
BD FACSDiva™ software v4.0 or higher |
| Instrument Setup and Quality Control | Automated setup using BD FACSCanto™ clinical software and BD FACS 7-color setup beads | Same |
| Sample Preparation | Manual pipetting for the lyse/wash or lyse/no-wash methods, or automated with the BD FACS Sample Prep Assistant (SPA) for the lyse/no-wash method | Same |
| Sample Type** | Assay-dependent | Same |
| * Indicates change is same as cleared in FACS Canto II (4-2) configuration, k062087 (reference device) | | |
| **Sample Types are assay-dependent; refer to FDA cleared or approved assays for designated sample types. For use with Multitest 6-color TBNK and IMK kit, whole blood is the indicated sample type in the assay’s IFU. | | |
| Note: Seven to ten color immunophenotyping is for research use only (RUO). | | |

I. Special Control/Guidance Document Referenced (if applicable):

CLSI EP05-A2: Evaluation of Precision Performance of Quantitative Measurement Methods

CLSI EP06-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach

CLSI EP09-A2: Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline, Second Edition

J. Performance Characteristics:

For all population subsets referred to below, the following abbreviations are used:

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$\mathrm{CD4}^{+} =$ cells that positively express CD45 and CD3 and CD4
$\mathrm{CD8}^{+} =$ cells that positively express CD45 and CD3 and CD8
$\mathrm{CD19}^{+} =$ cells that positively express CD45 and CD19 and negatively express CD3
$\mathrm{CD16^{+}56^{+} =}$ cells that positively express CD45 and both CD16 and CD56 and negatively express CD3

Cleared IVD assays and reagents used to demonstrate equivalence across instrument configurations:

BD Multitest IMK kit (IMK; cleared k041074)
BD Multitest 6-color TBNK reagent (TBNK; cleared k090967)
BD FACS 7-color setup beads (k040026)

# 1. Analytical Performance:

# a. Accuracy:

The Accuracy study design was based on recommendations in the CLSI document EP9-A2. Method comparison with the predicate instrument configuration was used to determine accuracy through comparative evaluation of the same specimen stained with the BD Multitest IMK Kit (4-color; IMK) and the BD Multitest 6-color TBNK reagent (TBNK) and analyzed in parallel with the test method and predicate device. Surplus samples were obtained with IRB approval from Johns Hopkins Medical Institutions, Baltimore, MD, and from Biocollections Worldwide, Inc.

For each test system (BD FACSCanto™ II 4-2-2 configuration, BD FACSCanto™ II 5-3 configuration, and BD FACSCanto™ 4-3-3 configuration), the accuracy studies shared the same design and differed only by the system configuration under test, the source of samples, and total number of samples tested. For the purposes of the study, accuracy was defined as the relative or absolute difference between the two systems for each of the specified parameters.

Manufacture's pre-defined acceptance criteria were as follows:

| Lymphocyte Subset Percentage | Average Predicate Value <30% | Average Predicate Value >30% |
| --- | --- | --- |
| | Criteria = Absolute ±3% of average predicate Value | Criteria = Absolute ±10% of average predicate Value |
| | CD3+, CD4+, CD8+ | CD19+, CD16+ CD56+ |
| Lymphocyte Subset Absolute Counts | ≤ 10% relative to the predicate, i.e. ((test-pred)/pred)x100%)≤10% | ≤ 20% relative to the predicate |

All results for all three instrument configurations on both the IMK kit and TBNK kit were determined by assay, result type (absolute count or subset percentages), and subset in both Bland Altman (bias) plots and Deming Regression scatter plots. All summary results for the FACSCanto™ II 4-2-2 configuration on the TBNK kit for all

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outcomes and regression results for the FACSCanto™ II 5-3 configuration and FACSCanto™ 4-3-3 configuration on the TBNK kit are shown below:

BD FACSCanto™ II 4-2-2 – Lymphocyte Absolute Count Results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Absolute counts (cells/μL) | Mean Bias (%) | SD | 95% CI | Acceptance criterion (%) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- | --- |
| CD3+ | 104 | 217–3952 | -2.0 | 6.50 | -3.0, -0.9 | ±10% | Pass |
| CD4+ | | 6–2079 | 2.0 | 24.7 | -2.1, 6.0 | ±10% | Pass |
| CD8+ | | 62–3462 | -1.6 | 7.26 | -2.8, -0.4 | ±10% | Pass |
| CD19+ | | 0–820 | -0.9 | 24.0 | -4.8, 3.0 | ±20% | Pass |
| CD16+CD56+ | | 15–633 | -7.5 | 16.7 | -10.2, -4.8 | ±20% | Pass |

BD FACSCanto™ II 4-2-2 – Summary of the Deming regression absolute count results using BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3+ | 104 | 0.98 (0.96–1.00) | 0.5 (-17.11–18.10) | 0.99 |
| CD4+ | | 0.97 (0.94–1.01) | 3.42 (-1.80–8.64) | 0.99 |
| CD8+ | | 0.98 (0.95–1.01) | 0.81 (-15.6–16.99) | 0.99 |
| CD19+ | | 095 (0.90–1.01) | 1.48 (-4.64–7.60) | 0.95 |
| CD16+CD56+ | | 0.95 (0.90–0.99) | -4.86 (-10.51–0.80) | 0.95 |

BD FACSCanto™ II 4-2-2 – Configuration Lymphocyte Subset Percentage Results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Subset percentage (%) | Mean Bias (%) | SD | 95% CI | Acceptance criterion (%) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- | --- |
| CD3+ | 104 | 49.5–92.3 | 1.0 | 2.5 | 0.6, 1.4 | ±10% | Pass |
| CD4+ | | 0.9–57.0 | 0.6 | 1.2 | 0.4, 0.8 | ±3% | Pass |
| CD8+ | | 10.2–82.1 | 1.4 | 4.8 | 0.6, 2.0 | ±10% | Pass |

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BD FACSCanto™ II 4-2-2 – Summary of the Deming regression subset percentage results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3+ | 104 | 1.00 (0.96–1.04) | 0.68 (-2.05–3.42) | 0.97 |
| CD4+ | | 1.01 (0.99–1.02) | 0.38 (0.04–0.73) | 0.99 |
| CD8+ | | 1.01 (0.99–1.02) | 0.20 (-0.62–1.01) | 0.99 |
| CD19+ | | 1.01 (0.98–1.04) | -0.16 (-0.51–0.20) | 0.97 |
| CD16+CD56+ | 1.02 (0.98–1.06) | -0.73 (-1.23–0.23) | 0.96 | |

BD FACSCanto™ II 5-3 – Summary of the Deming regression absolute count results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3+ | 101 | 1.02 (0.99–1.05) | -11.86 (-47.24–23.52) | 0.99 |
| CD4+ | | 1.00 (0.98–1.02) | 3.24 (-3.09–9.57) | 0.99 |
| CD8+ | | 1.02 (1.00–1.04) | -5.81 (-15.64–4.02) | 0.99 |
| CD19+ | | 1.06 (1.03–1.08) | -4.25 (-6.33–-2.16) | 0.98 |
| CD16+CD56+ | | 1.01 (0.95–1.06) | 6.17 (-2.90–15.23) | 0.98 |

BD FACSCanto™ II 5-3 – Summary of the Deming regression subset percentage results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3+ | 101 | 0.98 (0.94–1.02) | 1.13 (1.94–4.21) | 0.95 |
| CD4+ | | 1.00 (0.99–1.02) | 0.27 (0.80–0.25) | 0.99 |
| CD8+ | 1.01 | 0.99 (0.98–1.01) | 0.13 (0.40–0.20) | 0.99 |
| CD19+ | 1.01 | 0.99 (0.98–1.01) | -0.13 (-0.40–0.10) | 0.99 |

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BD FACSCanto™ 4-3-3 – Summary of the Deming regression absolute count results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3⁺ | 108 | 1.0 (0.97–1.01) | 0.5 (-1.25–2.22) | 0.99 |
| CD4⁺ | | 1.0 (0.99–1.02) | -0.2 (-0.72–0.33) | 0.99 |
| CD8⁺ | | 1.0 (0.99–1.02) | -0.3 (-1.02–0.34) | 0.99 |
| CD19⁺ | | 1.0 (0.98–1.06) | -0.3 (-0.71–0.12) | 0.98 |
| CD16⁺CD56⁺ | | 1.0 (0.95–1.05) | 0.1 (-0.42–0.66) | 0.98 |

BD FACSCanto™ 4-3-3 – Summary of the Deming regression subset percentage results for BD Multitest 6-color TBNK Reagents:

| Subset | N | Slope (95% CI) | Intercept (95% C)I | R² |
| --- | --- | --- | --- | --- |
| CD3⁺ | 108 | 1.0 (0.94–0.99) | 2.1 (0.01–4.17) | 0.97 |
| CD4⁺ | | 1.0 (1.00–1.03) | -0.6 (-1.17–0.04) | 0.99 |
| CD8⁺ | | 1.0 (0.97–1.00) | 0.4 (-0.14–1.00) | 0.99 |
| CD19⁺ | | 1.0 (0.95–1.02) | 0.3 (-0.09–0.70) | 0.98 |
| CD16⁺CD56⁺ | | 1.0 (-0.95)1.03 | 0.3 (-0.13–0.71) | 0.97 |

The accuracy study for each new instrument configuration (BD FACSCanto™ II 4-2-2, BD FACSCanto™ II 5-3, and BD FACSCanto™ 4-3-3; test methods) demonstrates equivalence between the test system with BD FACSCanto™ clinical software and the BD FACSCanto™ 4-2 system (predicate) with BD FACSCanto™ clinical software.

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# b. Precision/Reproducibility:

Instrument precision is assay-dependent and should be performed with each IVD assay intended for use with the instruments.

To assess variability within the instrument configurations, two precision studies were conducted using control materials and whole blood.

Precision studies with control material were conducted in accordance with CLSI EP05-A2. Whole blood controls for lymphocyte subset enumeration were used to estimate precision for the following parameters:

- Percent and absolute count of CD4⁺ T lymphocytes
- Percent and absolute count of CD8⁺ T lymphocytes
- Percent and absolute count of CD3⁺ T lymphocytes
- Percent and absolute count of CD19⁺ B lymphocytes
- Percent and absolute count of CD16⁺56⁺ NK lymphocytes

Data were acquired on multiple test systems (BD FACSCanto™ II 4-2-2 configuration, BD FACSCanto™ II 5-3 configuration, BD FACSCanto™ 4-3-3 configuration) equipped with BD FACSLoaders. For each test system, instruments were rotated over the study duration for at least 21 days, using one instrument per day. Multiple different operators were rotated to collect data throughout the study.

Measurements were obtained from two separate runs per day. Each run was separated by a minimum of 4 hours. Only one operator collected samples per system per day for both reagents. The instruments were set up for acquisition of samples using BD FACS 7-color setup beads and the BD FACSCanto™ clinical software setup module. Tested samples consisted of two levels of whole blood control cells. Quality control samples were included in each run. Each tested sample was stained in duplicate, and each quality control sample was stained singly with the BD Multitest IMK Kit (4-color) and with the BD Multitest 6-color TBNK reagent in BD Trucount tubes.

The manufacture's pre-defined acceptance criteria for total precision and within-run precision values for subset percentages and for absolute counts were as follows:

- Subset percentages: the upper one-sided 95% confidence bound on the standard deviation should be ≤ 2.5% for CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes.
- Absolute counts: the upper one-sided 95% confidence bound of the percent CV should be ≤ 10% for T lymphocytes and ≤ 20% for CD19+ lymphocytes and CD16+56+ lymphocytes.

A second whole blood repeatability study was performed to assess instrument precision using patient samples. The whole blood specimens used in the accuracy

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study were used to determine repeatability (all samples done in duplicate) therefore results cannot be directly compared across instruments. Samples adequately represented the CD4 analytical measuring range and included concentrations around the medical decision points for each kit.

All parameters for the BD Multitest 6-color TBNK reagent and BD Multitest IMK Kit (4-color) met the precision/reproducibility (control material) and repeatability (whole-blood) criteria for the BD FACSCanto™ II 4-2-2 with BD FACSCanto™ clinical software, the BD FACSCanto™ II 5-3 configuration with BD FACSCanto™ clinical software, and the BD FACSCanto™ 4-3-3 configuration with BD FACSCanto™ clinical software.

c. Linearity:

Instrument linearity is assay-dependent and should be performed with each IVD assay intended for use with the instrument configurations.

Linearity studies were performed on each configuration to mathematically verify that the relationship between the observed values and the true concentrations of the analyte was linear for each configuration. The objective of each linearity study was to estimate the linearity of the test systems (BD FACSCanto™ II 4-2-2 configuration, BD FACSCanto™ II 5-3 configuration, and BD FACSCanto™ 4-3-3 configuration, each with BD FACSCanto™ clinical software) using the BD Multitest IMK Kit (4-color) and the BD Multitest 6-color TBNK reagent for the lyse/no-wash method of sample preparation.

Linearity was demonstrated in accordance with CLSI EP06-A section 5.3. Appropriate sample dilutions were chosen across the assays' ranges. Whole blood from one donor was collected; the mononuclear cells were harvested and stained to determine the subset cell count to ensure that the dilutions would cover the full linearity range. The autologous plasma was reserved and used to pool the specimen into 11 evenly spaced dilutions. Replicates of each dilution were stained with the BD Multitest IMK Kit (4-color) and BD Multitest 6-color TBNK Reagent in Trucount tubes.

For the absolute counts of each detailed subset (CD4⁺, CD8⁺, CD3⁺, CD19⁺, and CD16⁺CD56⁺), the system was considered linear if the higher order coefficient terms (2nd and 3rd order terms) of the polynomial model are not statistically significant.

Regression statistics, including 2-sided 95% confidence intervals and percent recovery were provided for all each cellular subset. The instrument system using the FACSCanto Clinical software for the IMK and TBNK assay was found to be linear for each parameter (absolute counts) for each configuration.

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d. Carryover:

Specimen carry over studies were performed on the BD FACSCanto™ II and BD FACSCanto™ 4-3-3 configuration to determine whether cellular results were materially affected by residue or other contamination from neighboring samples.

Carryover for both manual and BD FACSLoader acquisition was evaluated on each of three BD FACSCanto™ 4-3-3 configuration and three BD FACSCanto™ II instruments. Percent carryover was estimated by introducing three consecutive abnormally high leucocyte prepared samples followed by three consecutive abnormally low leucocyte prepared samples, and calculating the percent difference according to the formula % Carryover = [(L1-L3)/H3-L3]*100.

Manufactures predetermined acceptance criteria were as follows:

- For each donor on each BD FACSCanto™ II and each BD FACSCanto™ 4-3-3 configuration instrument used in the study, the leucocyte carryover percentage shall be ≤ ± 0.1% when the sample is loaded manually.
- For each donor on each BD FACSCanto™ II and each BD FACSCanto™ 4-3-3 configuration instrument used in the study, the leucocyte carryover percentage shall be ≤ ± 0.5%, when the sample is automatically loaded.

Carryover from one specimen to another was demonstrated to be less than the predetermined acceptance limits. The BD FACSCanto™ 4-2-2 and 5-3-configurations and the BD FACSCanto™ 4-3-3 configuration showed acceptable leucocyte carryover.

e. Interfering Substances:

Not applicable:

2. Other Supportive Instrument Performance Data Not Covered Above:

Not applicable.

K. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

L. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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