For use with any flow cytometer with specified detection ranges.
For use with erythrocyte lysed whole blood.
For use with or without an isotype control.
For in vitro diagnostic use.
To identify and enumerate percentages and absolute counts of CD3+ and CD16+CD56+ lymphocytes in normal individuals and patients with certain tumors and viral infections

Device Description

The BDIS TriTEST CD3 fluorescein isothiocyanate (FITC)/CD16+CD56 phycoerythrin (PE)/CD45 peridinin chlorophyll protein (PeCP) reagent is a three-color, direct immunofluorescence reagent for identifying and enumerating percentages of T lymphocytes (CD3+) and Natural Killer lymphocytes (CD3- and CD16+ and/or CD56+) in erythrocytelysed whole blood (LWB). When used with TRUCOUNT Absolute Count Tubes, the product will yield absolute counts in cells/uL. The Becton Dickinson TriTEST/TRUCOUNT system for immunophenotyping consists of a flow cytometer (either from BDIS or from another manufacturer), conjugated monoclonal reagent (TriTEST CD3 FITC/CD16+CD56 PE/CD45 PerCP) and TRUCOUNT Absolute Count Tubes.

The process to obtain lymphocyte subset percentages includes: 1) obtaining a whole blood sample, 2) cell-surface antigen staining with three-color monoclonal antibody reagents, 3) erythrocyte lysis, and 4) flow cytometric acquisition and analysis of list mode data. Analysis involves computing the ratio of reagent-positive events to the CD45 positive events, and expressing the ratio as a percentage.

To obtain absolute counts, the TriTEST reagent and whole blood are added directly to an Absolute Count Tube prior to lysis. The remaining process, until analysis, is identical to that for percentages. Analysis for absolute counts requires that an additional region, the bead region, be identified and the events in this region counted. The proportion of reagent positive events to bead events (P) is computed. The absolute count is P x (beads/pellet)/(volume of blood sample).

When monoclonal antibody reagents are added to human whole blood, the fluorochromelabeled antibodies bind specifically to antigens on the surface of leukocytes, thus identifying lymphocyte populations. The patient blood sample is added to the counting bead pellet and is treated with fluorochrome-labeled antibodies and the erythrocytes are lysed with FACS® Lysing Solution. The flow cytometer is set up so that cell populations for most samples occupy approximately the same region of fluorescence space. The sample is then introduced into the flow cytometer and the stained cells and beads fluoresce when excited by a laser beam.

The three-color reagent permits identification of lymphocyte subsets using fluorescence gating instead of scatter gating. This three-color reagent allows direct gating on the CD45-positive population using a combination of fluorescence and side scatter parameters. By gating on the CD45-positive population, a maximum number of lymphocytes may be captured in the gate and non-lymphocyte contamination may be minimized.

AI/ML Overview

This document describes the Becton Dickinson TriTEST™ reagent CD3 FITC/CD16+CD56 PE/CD45 PaCP; TRUCOUNTTM Absolute Count Tubes.

Here's an analysis of the provided information:

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state quantitative acceptance criteria in a dedicated table. Instead, it describes performance characteristics and concludes that the device is "as safe and effective as the predicate device" based on these studies. The equivalence to predicate devices (Simultest IMK-Lymphocyte Tube F for percentages, and Simultest IMK-Lymphocyte Tube F plus ADCC for absolute counts) is the primary "acceptance criterion" indirectly.

Performance Characteristic	Reported Device Performance
Accuracy (Percentages)	Demonstrated equivalence to Simultest IMK-Lymphocyte Tube F.
Accuracy (Absolute Counts)	Demonstrated equivalence to Simultest IMK-Lymphocyte Tube F plus automated differential cell counter (ADCC).
Use of Isotype Control	Data indicated that the reagent may be used with or without an isotype control for setting lymphocyte gate and quadrant markers.
Reference Range Studies	Performed; acknowledged that variables like sex, age, and geographical location influence range, and each site must determine its own.
Stability (Time-from-draw & Time-from-sample preparation)	Samples should be stained and analyzed within 6 hours of draw.
Within-specimen Reproducibility	Acceptable reproducibility demonstrated (10 replicates from 3 specimens at BDIS; 3 aliquots from each donor at 3 clinical sites).
Linearity	Linear response observed over concentrations ranging from 16,700 to 200 lymphocytes/µL and from 31,000 to 2500 WBC/µL (tested with 3 normal donors diluted to 5 concentrations).
Cross Reactivity	Clones' cross-reactivity reported in literature; conjugation and product formulation have not changed their specificity.
Cross-platform Reproducibility	Indicated that the device can be used on flow cytometers not made by Becton Dickinson.

2. Sample Size Used for the Test Set and Data Provenance:

The document provides some information about sample sizes but is not comprehensive for all studies.

Accuracy Studies: Not explicitly stated. The studies were performed at "Cleveland Clinic, Johns Hopkins Hospital, Institute of Tropical Medicine, University of North Carolina, and at Becton Dickinson Immunocytometry Systems laboratories in San Jose, California." This implies a prospective design across multiple geographical locations (USA and potentially international due to "Institute of Tropical Medicine").
Within-specimen Reproducibility:
- BDIS: 10 replicates from 3 specimens.
- 3 Clinical Sites: 3 aliquots from each donor (number of donors not specified).
Linearity: Blood samples from 3 normal donors.

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

The document does not specify the number of experts or their qualifications used to establish ground truth. It mentions "testing at Cleveland Clinic, Johns Hopkins Hospital, Institute of Tropical Medicine, University of North Carolina, and at Becton Dickinson Immunocytometry Systems laboratories." This implies clinical laboratory professionals were involved, but their specific roles, number, and qualifications (e.g., medical technologists, pathologists, immunologists, experience levels) are not detailed.

4. Adjudication Method for the Test Set:

The document does not describe any specific adjudication method (e.g., 2+1, 3+1, none) for establishing ground truth or resolving discrepancies in the test results. The studies focused on demonstrating equivalence to predicate methods, implying direct comparison rather than a separate expert-driven ground truth adjudication process.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

This device is not an AI-assisted diagnostic tool. It is a reagent and system for flow cytometry. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not applicable and was not performed.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done:

The device is a system comprising reagents, tubes, and analysis performed on a flow cytometer. While the analysis part involves computing ratios and identifying regions, it's not described as a standalone "algorithm" in the modern AI sense. The performance assessment implicitly covers the entire system's ability to enumerate cell populations, which is an automated process once the sample is prepared and run on the flow cytometer.

7. The Type of Ground Truth Used:

The ground truth for establishing performance (particularly accuracy) was based on comparison to predicate devices/methods:

For percentages: Simultest IMK-Lymphocyte Tube F.
For absolute counts: Simultest IMK-Lymphocyte Tube F plus automated differential cell counter (ADCC).

This is a clinical comparison against established and predicate laboratory methods, rather than pathology, expert consensus (in the sense of independent review of an image), or outcomes data.

8. The Sample Size for the Training Set:

The concept of a "training set" in the context of machine learning or AI algorithms is not applicable here. This device is a biochemical reagent and flow cytometry system, not a learning algorithm. The studies described are performance validation studies, not training.

9. How the Ground Truth for the Training Set was Established:

As mentioned above, there is no "training set" in the AI sense for this device. The performance was validated against established predicate methods.

Summary

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NOV 25 1997

Attachment F

Summary of Safety and Effectiveness

Submitter Information (21 CFR 807.92(a)(1))

Submitter:	Becton Dickinson Immunocytometry Systems2350 Qume DriveSan Jose, CA 95131-1807
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Anna Longwell, Esq. Contact: Director, Regulatory Affairs - Corporate (408) 954-2254
Summary date: March 21, 1997

Name of Device and Classification (21 CFR 807.92(a)(2))

Becton Dickinson TriTEST™ reagent CD3 FITC/CD16+CD56 Name: PE/CD45 PaCP; TRUCOUNTTM Absolute Count Tubes

Classification: Class II

Predicate Device (21 CFR 807,92(a)(3))

The BDIS TriTEST™ CD3 FITC/CD16+CD56 PE/CD45 PerCP reagent with TRUCOUNT Absolute Count Tubes when used to enumerate percentages of T lymphocytes and Natural Killer lymphocytes (NK cells), is substantially equivalent to Simultest M IMK-Lymphocyte Tube F (cleared to market under 510(k) K913192). When used to enumerate absolute counts of T and NK cells, it is substantially equivalent to Simultest IMK-Lymphocyte Tube F plus automated differential cell counter (ADCC).

Description of the Device (21 CFR 807.92(a)(4))

323

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Summary of Safety and Effectiveness

events to bead events (P) is computed. The absolute count is P x (beads/pellet)/(volume of blood sample).

Intended Use (21 CFR 807.92(a)(5))

For in vitro diagnostic use to identify and enumerate percentages and absolute counts of T and NK lymphocytes in blood.

Indications for Use

For use with any flow cytometer with specified detection ranges .
For use with erythrocyte lysed whole blood .
For use with or without an isotype control .
For in vitro diagnostic use .
To identify and enumerate percentages and absolute counts of CD3+ and CD16+CD56+ .
lymphocytes in normal individuals and patients with certain tumors and viral infections

Comparison to Predicate Device (21 CFR 807.92(a)(6))

The three-color reagent (for percentages) is substantially equivalent to Simultest IMK-Lymphocyte Tube F in that they share the same intended uses and use essentially the same monoclonal antibody/flow cytometric methodology. The TriTEST and Simultest products differ in the steps used to determine analysis gates to identify the lymphocyte population.

The TriTEST / TRUCOUNT system (for absolute counts) is substantially equivalent to Simultest IMK-Lymphocyte Tube F plus ADCC in that they share the same intended uses. Compared to the predicate, the TriTEST/TRUCOUNT system requires less sample handling, a simpler sample preparation scheme and fewer sample tubes to achieve the same end result. Results demonstrate that the products yield essentially equivalent performance characteristics.

Performance Data (21 CFR 807.92(b)(2))

Performance of the product was established by testing at Cleveland Clinic, Johns Hopkins Hospital, Institute of Tropical Medicine, University of North Carolina, and at Becton Dickinson Immunocytometry Systems laboratories in San Jose, California.

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Summary of Safety and Effectiveness

Several studies were performed:

· Accuracy was determined for both percentages and absolute counts. Accuracy data demonstrated the TriTEST product's equivalence to Simultest and TriTEST/TRUCOUNT product's equivalence to Simultest/ADCC.
· Use of an Isotype Control was studied. Data was analyzed for percent T lymphocytes and percent NK lymphocytes first using a control to set lymphocyte gate and quadrant markers and then using only the stained sample to set a lymphocyte gate and quadrant markers. Data indicated that the reagent may be used with or without an isotype control.
· Reference range studies were performed. Many variables, such as sex, age and geographical location may influence the reference range. Each site must determine its own reference range.
· A stability study was conducted to assess the time effect relating to age of blood (timefrom-draw) and the time effect relating to the age of the stain (time-from-sample preparation), as well as the combined effect of both. Stability was determined for both percentages and absolute counts. Results indicate that samples should be stained and analyzed within 6 hours of draw.
· Within-specimen reproducibility was performed at BDIS; 10 replicates from 3 specimens were assessed. Within-specimen reproducibility was also performed at 3 clinical sites; 3 aliquots from each donor were assessed. Results demonstrated acceptable within-sample reproducibility.
· Linearity was determined using blood samples from 3 normal donors diluted to 5 concentrations, ranging from 16,700 to 200 lymphocytes/pL and from 31,000 to 2500 WBC/uL. Results indicate a linear response over this range.
· Cross reactivity of these clones is reported in the literature. Conjugation and product formulation have not changed their specificity.
· Results from a coss platform reproducibility study indicated that the TriTEST reagent CD3/CD16+CD56/CD45, with or without TRUCOUNT tubes, can be used on flow cytometers not made by Becton Dickinson.

Performance Data - Conclusions (21 CFR 807.92(b)(3))

The results of the clinical studies demonstrate that the device is as safe and effective as the predicate device.

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Image /page/3/Picture/2 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized depiction of an eagle with its wings spread, symbolizing protection and care. The eagle is positioned within a circular border that contains the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA". The text is arranged around the circumference of the circle, emphasizing the department's role and national scope.

Food and Drug Administration 2098 Gaither Road NOV 2 5 1997 Rockville MD 20850

Cindy Morrow Sr. Regulatory Specialist BECTON DICKINSON IMMUNOCYTOMETRY SYSTEMS 2350 Oume Drive San Jose, CA 95131-1807

K971110 Re: Trade Name: Becton Dickinson TriTEST Reagent CD3 FITC/CD16+CD56 PE/CD45 PerCP; TruCOUNT Absolute Count Tubes Regulatory Class: II Product Code: GKZ, 81 Dated: March 24, 1997 Received: March 26, 1997

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Dear Ms. Morrow:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General requlation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions.

Failure to comply with the GMP requlation may result in In addition, FDA may publish further requlatory action. announcements concerning your device in the Federal Register. this response to your premarket notification Please note: submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770)488-7655.

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Butman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page	of
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510(k) Number (if known): _ K 971110

Device Name:_

Indications For Use:

For use with any flow cytometer with specified detection ranges �
For use with erythrocyte lysed whole blood �
For use with or without an isotype control ◆
For in vitro diagnostic use ◆
To identify and enumerate percentages and absolute counts of CD3+ and CD16+CD56+ lymphocytes in normal individuals and patients with certain tumors and viral infections

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Peter E. Mulford

Prescription Use (Per 21 CFR 801.109)

Over-The-Counter Use

(Optional Format 1-2-96)

Regulation Number and Section

§ 864.5220 Automated differential cell counter.

(a)
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.(b)
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.”