For in vitro diagnostic use to identify and enumerate percentages and absolute counts of CD3+ and CD3+CD8+ lymphocytes in blood.
For use with any flow cytometer equipped with a 488 mm laser and capable of detection in the ranges: 515-545 nm, 562-607 nm, and > 650 nm.
For use with erythrocyte lysed whole blood.
For use with or without an isotype control.
To characterize and monitor forms of autoimmune diseases, such as lupus.
To characterize and monitor congenital or acquired immunodeficiencies, such as SCID or AIDS.

Device Description

The BDIS Trilles T CD3 flucescein isothiocyanate (FITC)/CD8 phycocrythrin (PE)/CD45 peridinin chlorophyll protein (PeCP) reagent is a three-color, direct immunoflucrescence reagent for identifying and enumerating percentages of T lymphocytes (CD3+) and Tsuppressor/cytotoxic (CD3+CD8+) cells in eythrocyte-lysed whole blood (LWB). When used with TRUCOUNT Absolute Count Tubes, the product will yield absolute counts in cells/JL. The Becon Dickinson Trill BT/TRUCOUNT system for immunophenoryping consists of a flow cycometer (either from BDIS or from another manufacture), conjugated monoclonal reagent (TriTEST CD3 FITC/CD8 PE/CD45 PerCP) and TRICOUNT 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 crometric 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 I x (beads/pellet)/(volume of blood sample).
When monoclonal antibody reagents are added to human whole blood, the flucrochrome-labeled 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 fluctochrome-labeled antibodies and the crythrocytes 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 excired by a laser beam.
The three-color reagent permits identification of lymphocyte subsers using fluorescence gating instead of forward scatter gating. This three-color reagent allows direct gating on the CD45positive population using a combination of fluorescence and side scatter parameters. By gaing on the CD45-positive population, a maximum number of lymphocyces may be caprured in the gate and non-lymphocyte contamination may be minimized.

AI/ML Overview

The provided text describes the Becton Dickinson TriTEST™ reagent and TRUCOUNT™ Absolute Count Tubes system. This device is intended for in vitro diagnostic use to identify and enumerate percentages and absolute counts of CD3+ and CD3+CD8+ lymphocytes in blood using flow cytometry. The primary claim in the document is about substantial equivalence to predicate devices rather than meeting specific performance criteria against predefined thresholds.

Therefore, the acceptance criteria are implicitly met by demonstrating substantial equivalence to the predicate devices, IMK-Lymphocyte Tube E and the FACSCount System. The "reported device performance" is framed as its equivalence to these predicate devices.

Here's the breakdown of the information requested:

1. A table of acceptance criteria and the reported device performance

Acceptance Criteria (Implicit from Substantial Equivalence)	Reported Device Performance
Accuracy (Percentages): Substantially equivalent to IMK-Lymphocyte Tube E for enumerating percentages of lymphocytes.	Data demonstrated TriTEST's equivalence to IMK-Lymphocyte Tube E.
Accuracy (Absolute Counts): Substantially equivalent to FACSCount system for enumerating absolute counts of CD3+ and CD3+CD8+.	Data demonstrated TriTEST/TRUCOUNT product's equivalence to FACSCount.
Reproducibility (Within-Specimen): Acceptable within-sample reproducibility for both percentage enumeration and absolute counts.	Results demonstrated acceptable within-sample reproducibility at BDIS and three clinical sites.
Linearity: Produces linear results over the intended range of lymphocyte and WBC concentrations.	Results indicate the product gives linear results over concentrations ranging from 16,700 to 200 lymphocytes/µL and from 31,000 to 2,500 WBC/µL.
Stability: Maintains performance over specified timeframes for blood draw and staining.	Staining samples within 24 hours of draw and analyzing within 24 hours of staining, OR staining within 48 hours of draw and analyzing within 6 hours, is recommended.
Cross-Platform Reproducibility: Acceptable performance across different flow cytometers.	For absolute count results, small (< 20%) non-zero bias but good correlation between Becton Dickinson and Coulter cytometers. Users advised to validate performance characteristics. Acceptable for percent positive results or absolute counts with non-BD flow cytometers.
Isotype Control Use: Performance with or without an isotype control.	Data indicated the reagent may be used with or without an isotype control.

2. Sample size used for the test set and the data provenance

Sample Size: The document does not explicitly state the numerical sample size for any of the performance studies. It mentions "blood samples from three normal donors" for linearity.
Data Provenance: The studies were conducted at:
- Cleveland Clinic (USA)
- Johns Hopkins Hospital (USA)
- Institute of Tropical Medicine (unspecified country, but often refers to institutions focused on tropical infectious diseases, frequently in developing countries or with international collaborations)
- University of North Carolina (USA)
- Becton Dickinson Immunocytometry Systems laboratories in San Jose, California (USA)
Retrospective/Prospective: The document does not specify whether the data was retrospective or prospective. Given the nature of a 510(k) submission and performance studies, it's highly likely to be prospective data collection for the purpose of demonstrating device performance, but this is not explicitly stated.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not mention the use of experts to establish ground truth or their qualifications. The "ground truth" for this device appears to be comparison against established predicate devices and standard laboratory methodologies rather than expert consensus on individual cases.

4. Adjudication method for the test set

The document does not mention any adjudication method. This type of device (flow cytometry reagent for enumerating cell populations) typically relies on quantitative measurements from instruments calibrated against established standards, rather than subjective interpretation requiring adjudication among experts.

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

No, an MRMC comparative effectiveness study was NOT done. This device is a diagnostic reagent and tube system for flow cytometry, which is an automated or semi-automated process for cell enumeration. It does not involve human "readers" in the way an imaging AI algorithm would.
Not applicable for AI assistance. The document predates the widespread regulatory consideration of AI-driven medical devices and does not mention any AI components. The "assistance" for human operators comes from the flow cytometer and associated software, which are standard tools.

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

Standalone performance was done. The performance studies described (accuracy, reproducibility, linearity, stability) assess the performance of the reagent and tube system in conjunction with a flow cytometer. While a human operates the flow cytometer and analyzes the data, the core "algorithm" and chemical reaction of the device itself are being evaluated. This is effectively a "standalone" evaluation of the device's technical specifications.

7. The type of ground truth used

The ground truth was established by comparison to predicate devices (IMK-Lymphocyte Tube E and the FACSCount System) and standard laboratory methodologies for cell enumeration and characterization. This implies that the predicate devices are considered the "gold standard" or accepted reference for the measurements, and their results serve as the ground truth against which the new device's performance is measured. Linearity and stability studies likely used controlled samples with known or precisely determined values.

8. The sample size for the training set

The document does not mention a "training set" or "training data." This is because the device is a chemical reagent and consumable, not a machine learning algorithm that requires training.

9. How the ground truth for the training set was established

Not applicable. As stated above, there is no mention of a training set as this is not an AI/ML device.

Summary

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4.005

K970326

Attachment B

Summary of Safery and Effectiveness

NOV 21 1997

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

Becton Dickinson Immunocytometry Systems Submitter: 2350 Qume Drive San Jose, CA 95131-1807

Anna Longwell, Esq. Contact: Director, Regulatory Affairs - Corporate (408) 954-2254
Summary date: November 19, 1997

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

Becton Dickinson TriTESTM reagent CD3 FITC/CD8 PE/CD45 PerCl'; Name: TRUCOUNTIM Absolute Count Tubes

Class II Classification:

Predicate Device (21 CFR 807.92(2)(3))

The BDIS TrillEST™ CD3 FITC/CD8 PE/CD45 PerCPreagent, when used to enumerate percentages of lymphootes is substantially equivalent to IMK-Lymphocyte Tube E that was cleared to market under 510(k) K913192. When the TriTEST reagent is used with TRUCOUNT Absolute Count Tubes, it is substantially equivalent to the BDIS FACSCount system cleared under 510(k) K933486.

Description of the Device (21 CFR 807.92(2)(4))

The BDIS Trilles T CD3 flucescein isothiocyanate (FITC)/CD8 phycocrythrin (PE)/CD45 peridinin chlorophyll protein (PeCP) reagent is a three-color, direct immunoflucescence reagent for identifying and enumerating percentages of T lymphocytes (CD3+) and Tsuppressor/cytotoxic (CD3+CD8+) cells in eythrocyte-lysed whole blood (LWB). When used with TRUCOUNT Absolute Count Tubes, the product will yield absolute counts in cells/JL. The Becon Dickinson Trill BT/TRUCOUNT system for immunophenoryping consists of a flow cycometer (either from BDIS or from another manufacture), conjugated monoclonal reagent (TriTEST CD3 FITC/CD8 PE/CD45 PerCP) and TRICOUNT 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 crometric acquisition and analysis of list mode data. Analysis involves computing the

CD3/CD8/CD45 510(k) Notification Attachment B: Summary of Safety and Effectiveness

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Summary of Safety and Effectiveness

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 I x (beads/pellet)/(volume of blood sample).

When monoclonal antibody reagents are added to human whole blood, the flucrochrome-labeled 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 fluctochrome-labeled antibodies and the crythrocytes 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 excired by a laser beam.

The three-color reagent permits identification of lymphocyte subsers using fluorescence gating instead of forward scatter gating. This three-color reagent allows direct gating on the CD45positive population using a combination of fluorescence and side scatter parameters. By gaing on the CD45-positive population, a maximum number of lymphocyces may be caprured in the gate and non-lymphocyte contamination may be minimized.

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

For in vitro diagnostic use to identify and enumerate percentages and absolute counts of CD3+ and CD3+CD8+ lymphocytes in blood.

Indications for Use

For use with any flow cytometer equipped with a 488 mm laser and capable of decection . in the ranges: 515-545 nm, 562-607 nm, and > 650 nm
. For use with erythrocyte lysed whole blood
For use with or without an isotype control .
To characterize and monitor forms of autoimmune diseases, such as lupus
. To characterize and monitor congenital or acquired immunodeficiencies, such as SCID or AIDS

CD3/CD8/CD45/TRUCOUNT 510(k) Nasification Assachment B: Summary of Safety and Effectiveness

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Summary of Safety and Effectiveness

Clinical Utility

The determination of CD3+ and CD3+CD8+ lymphocytes has been found useful in monitoring some forms of immunodeficiency and autoimmune disease.

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

The CD3 FITC/CD8 PE/CD45 PerCP TriTEST reagent, when used to enumerate percentages of lymphocytes, is substantially equivalent to the IMK-Lymphocyte Tube E that was cleared to market under 510(k) K913192. When used with TRUCOUNT Absolute Count Tubes to enumerate absolute counts, it is substantially equivalent to the FACSCount System (K933486) for CD3+and CD3+CD8+. Both the TriTEST product and the predicate devices yield equivalent results for the same analytes, and both are intended for use as an in vitro diagnostic test using, a flow cytometer-based instrument and recommended computer hardware and software. The products differ in the steps used to determine analysis gates to identify the lymphocyte popul:tion.

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

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

Several studies were performed:

Accuracy was determined by comparison to both IMK-Lymphocyte Tube E and the FACSCount . system. Accuracy data demonstrated the TriTEST and TriTEST/TRCOUNT product's equivalence to IMK-Lymphocyte Tube E and FACSCount, respectively.
Use of isony control was studied. Data were analyzed for percent CD3+ and pecent . CD3+CD8+ first using a control to set gates and markers and then using only the stained sample to set gates 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 (time-from-daw) . and the time effect relating to the age of the stain (time-from-sample preparation), as well :s the

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Summary of Safety and Effectiveness

combined effect of both. Stability was determined for both percentages and absolute counts. Stability was determined for whole blood samples at 6, 24, 48 and 72 hours post draw. Additionally, stability was measured for stained samples at 6 and 24 hours from time of staining. A combination of the two, time from draw plus time from sample preparation, was studied. Results indicated that either 1) staining the samples within 24 hours of craw and analyzing samples within 24 hours of staining or altenatively, 2) staining the samples with 48 hours of draw and analyzing them within 6 hours is recommended.

Within-soecimen reproducibility was performed at BDIS and at three clinical sites for both . percentage enumeration and absolute counts Results demonstrated acceptable within-sample reproducibility.
Linearity was determined using blood samples from three normal donors diluted to five t concentrations, ranging from 16,700 to 200 lymphocytes/JLL and from 31,000 to 2,500 WBC/UL. Results indicate the product gives linear results over this range.
Cross reactivity of these clones is reported in the literature. Conjugation and product famulation . have not changed their specificity.
Results from a cross platform reproducibility study indicated 1) for absolute count results there . was a small (< 20%) non-zero bias, but good corelation between results on a Becton Dickinson flow cytomere versus a Coulte cytometer. Therefore, users will be advised that they must validate performance characteristics for absolute counts. 2) For determining percent positive results or absolute counts, TriTEST reagent with RUCOUNT Absolute Count Tube may be used with 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 devices.

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Image /page/4/Picture/2 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo features a stylized eagle with three lines forming its body and wings. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" are arranged in a circular pattern around the eagle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

NOV 21 1997

Ms. Anna Longwell, Esq. Director, Requlatory Affairs - Corporate Becton Dickinson Immunocytometry Systems 2350 Qume Drive San Jose, California 95131-1807

Re : K970326 Becton Dickinson TriTEST™ Reagent CD3 FITC/CD8 Trade Name: PE/CD45 PerCP; TRUCOUNT™ Absolute Count Tubes Regulatory Class: II Product Code: GKZ Dated: August 28, 1997 Received: September 2, 1997 - .... - - ------

Dear Ms. Longwell:

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 aqainst 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 requirement, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (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 regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Reqister. Please note: this response to your premarket notification 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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Artachment A TriTEST CD3/CD45 with TRUCOUNT Control Beads & Absolute Count Tubes K970326

Indications for Use Statment

For use with any flow cytometer equipped with a 488 nm laser and capable of . detection in the ranges: 515-545 nm, 562-607 nm, and > 650 nm
For use with crythrocyte lysed whole blood .
For use with or without an isotype control .
To characterize and monitor forms of autoimmune diseases, such as lupus .
To characterize and monitor congenital or acquired immunodeficiencies, such as SCID or . AIDS

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

Concurrence of CDRH, Office of Device Evaluation (ODE)

Peter E. Maher

Division Sign-Off) Division of Clinical Laboratory Devices 510(k) Number

Prescription Use (Per 21 CFR 801.109) OR

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.”