TETRAONE SYSTEM FOR EPICS XL FLOW CYTOMETRY SYSTEMS AND CYTO-STAT TERTRACHROME CD45-FITC/CD4-RDI/CD8-ECD/CD3-PC5 by COULTER CORP.

The tetraONE™ SYSTEM for EPICS® XL Flow Cytometry Systems and CYTO-STAT® tetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 Monoclonal Antibody Reagent combines a four-color fluorescent monoclonal antibody reagent, four quality control reagents, an optional absolute count reagent, and software for automated analysis of lymphocyte populations in whole blood using EPICS® XL Flow Cytometry Systems with SYSTEM II™ Software. The tetraONE™ SYSTEM software is designed to further simplify flow cytometric analysis by increasing automated modes of operation and the accuracy, precision and reliability of results. The tetraONE™ SYSTEM does not require an isotypic control for monitoring and adjusting for non-specific and nontargeted monoclonal antibody binding to irrelevant cellular populations. The tetraONE™ SYSTEM software monitors and adjusts for such binding by automatically placing cursors based on the separation of positive and negative peaks. The CD45/CD4/CD3 reagent contains a monoclonal antibody, CD45, to identify a lymphocyte gate to allow CD3+, CD4+, CD8+, dual-positive CD3+/CD4+ and dualpositive CD3+/CD8+ measurements.

AI/ML Overview

The acceptance criteria and study proving the device meets them are summarized below:

Acceptance Criteria and Reported Device Performance

Acceptance Criteria	Reported Device Performance
Accuracy: CD3+, CD4+, CD8+, CD3+/CD4+ and CD3+/CD8+ percentages and absolute counts comparable to predicate devices (CD3/T4 and CD3/T8).	Results from comparison with CD3/T4 and CD3/T8 demonstrated that CD45/CD4/CD8/CD3, CD3/T4, and CD3/T8 identify and enumerate essentially identical numbers of targeted lymphocytes in whole blood specimens. Analysis included minimums, maximums, means ± 1 SD, 95% confidence intervals, regression and correlation analyses, and analyses of variance.
Linearity: The assay should demonstrate linearity over a range of lymphocyte concentrations.	Regression and correlation analyses for recovered versus expected absolute counts demonstrated linearity of the assay.
Within Run (Intralaboratory) Precision: Consistent measurements for lymphocyte concentrations within single laboratory runs.	Results for mean ± 1 SD and CV demonstrated Within Run (Intralaboratory) Precision of the assay.
Interlaboratory Precision: Consistent measurements for lymphocyte concentrations across different laboratories.	Results for mean ± 1 SD and CV demonstrated Interlaboratory Precision of the assay.

Study Information

Sample sizes used for the test set and the data provenance:
- Accuracy: Normal and abnormal (e.g., Human Immunodeficiency Virus, organ transplant, autoimmune disease, low white blood cell count) whole blood specimens. The exact number of specimens is not specified, but they were collected from "geographically diverse populations of males and females unselected as to race and ranging in age from 18 to 85 years."
- Linearity: One concentrated COULTER™ CYTO-TROL™ Control Cells sample, serially diluted to achieve a range of CD3+, CD4+ (CD3+/CD4+), and CD8+ (CD3+/CD8+) lymphocyte concentrations. Three replicate measurements were made.
- Within Run (Intralaboratory) Precision: A normal whole blood specimen selectively depleted to create three levels of CD3+, CD4+ (CD3+/CD4+), and CD8+ (CD3+/CD8+) lymphocyte concentrations. Ten replicate measurements were made for each level.
- Interlaboratory Precision: A single normal whole blood specimen. Ten replicate measurements were made on the same day using different laboratories and EPICS® XL-MCL flow cytometers.
- Data Provenance: Not explicitly stated, but "geographically diverse populations" and the mention of "COULTER CORPORATIONP.O. BOX 169015Miami, Florida 33116-9015 USA" and various international branches suggest data could be from multiple locations, but specific countries are not detailed. All studies appear to be prospective to assess the performance of the new device.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts: Not applicable. The ground truth for this device (a lymphocyte immunophenotyping system) is established through comparison with existing, well-validated methods (CD3/T4 and CD3/T8 reagents) and control materials (COULTER™ CYTO-TROL™ Control Cells). The assessment relies on the measurements obtained from these established methods and the inherent properties of the control cells, rather than expert consensus on individual specimens.
Adjudication method for the test set: Not applicable. The study involves direct comparison of measurements between the new device and established methods, or assessment against known concentrations (linearity) and variability (precision). There is no mention of human adjudication of results in the traditional sense.
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: Not applicable. This is a medical device for automated laboratory analysis (flow cytometry) and does not involve human readers interpreting images or data that an AI would assist with. The "software for automated analysis" refers to process automation, not AI-assisted human interpretation.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done: Yes. The device, the tetraONE™ SYSTEM, including its software, performs automated analysis. The studies described are evaluating the performance of this system directly, without human intervention during the measurement and analysis process beyond setting up the experiment and operating the flow cytometer. The software "is designed to further simplify flow cytometric analysis by increasing automated modes of operation and the accuracy, precision and reliability of results." The system "automatically placing cursors based on the separation of positive and negative peaks."
The type of ground truth used:
- Accuracy: The "ground truth" for accuracy was established by comparing the measurements from the new tetraONE™ SYSTEM with those obtained using predicate devices (CYTO-STAT®/COULTER CLONE® CD3(IgG1)-FITC/T4-RD1 and CD3(IgG1)-FITC/T8-RD1 Monoclonal Antibody Reagents), which are considered established and reliable methods for lymphocyte immunophenotyping. Lymphocyte purity corrections were applied to the comparator values.
- Linearity: The ground truth was based on expected concentrations from serially diluted control cell samples (COULTER™ CYTO-TROL™ Control Cells), implying known or reliably characterized target concentrations.
- Precision: The ground truth for precision was the measured concentrations themselves across multiple replicates, evaluated for consistency rather than absolute accuracy against an external standard.
The sample size for the training set: Not explicitly stated. The document describes product testing (validation) for the device, not the development or training of its internal models. Any development/training samples used for the software's automated analysis capabilities would likely have been part of an earlier phase of product development, not detailed in this premarket notification.
How the ground truth for the training set was established: Not explicitly stated as the document focuses on product testing/validation rather than the software development process. However, given the nature of flow cytometry and the comparison with predicate devices, it can be inferred that ground truth for any potential "training" (e.g., for automated cursor placement) would have been established through well-characterized samples, potentially validated by expert review of plots and comparison to established methods over time.

Summary

{0}------------------------------------------------

JAN 22 1997

COULTER CORPORATIONP.O. BOX 169015Miami, Florida 33116-9015 USA	Date:	August 16, 1996
Customer Service: (800) 526-7694Product Information: (800) 526-6932(305) 380-3800(800) 327-6531	Title:	Summary of Safety and Effectiveness Information For 510(k) Premarket Notification
Coulter CorporationMiami, Florida USA	Product:	tetraONE™ SYSTEM for EPICS® XL Flow Cytometry Systems andCYTO-STAT® tetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5Monoclonal Antibody Reagent
Coulter Leasing CorporationMiami, Florida USA	Company:	Coulter Corporation11800 SW 147 AvenueMiami, FL 33196-2500
Coulter Electronics, Pty. Ltd.Sydney, Australia	Contact:	Dr. Marion S. Gaide (M/C: 31-B06)Senior Regulatory Affairs SpecialistCorporate Regulatory Affairs
Coulter Electronics Ind. & Com.. Ltda.Rio de Janeiro, Brazil
Coulter Electronics of Canada. Ltd.Burlington, Ontario, Canada	Telephone:	305-380-2594
Coulter Electronics, Ltd.Luton Bedfordshire England	Common or Usual or Classification Name:	Lymphocyte Immunophenotyping Systemwith Reagents and Software for Flow Cytometry
Coultronics France, S.A.Margency. France
Coulter Electronics GmbHKrefeld, Germany
Coulter Electronics (HK), Ltd.Hong Kong	Product Classification:	Product Code: GKZ; C.F.R. Section: 864.5220; ClassificationPanel: Hematology and Pathology Devices; Device Class: II
Coulter K. K.Tokyo, Japan
Coulter de Mexico S.A., DE C.V.Mexico City, Mexico	Intended Use:	The tetraONE™ SYSTEM for EPICS® XL Flow Cytometry Systems and CYTO-STAT® tetraCHROME™ CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 MonoclonalAntibody Reagent combines a four-color fluorescent monoclonal antibody reagent,four quality control reagents, an optional absolute count reagent, and software forautomated analysis of lymphocyte populations in whole blood using EPICS® XLFlow Cytometry Systems with SYSTEM II™ Software. The system is intended "ForIn Vitro Diagnostic Use" and allows simultaneous identification and enumeration oftotal CD4+, total CD8+, total CD3+, dual-positive CD3+/CD4+ and dual-positive CD3+/CD8+ T lymphocytes percentages and absolute counts. The systemalso provides the T4/T8 ratio.
Coulter Electronics, Ltd.Mijdrecht. Netherlands	Substantial Equivalence:	510(k) Premarket Notification:K922745CYTO-STAT®/COULTER CLONE® CD3(IgG1)-FITC/T4-RD1Monoclonal Antibody Reagent
Coulter Electronics, Pty. LtdAuckland, New Zealand		510(k) Premarket Notification:K922744CYTO-STAT®/COULTER CLONE® CD3(IgG1)-FITC/T8-RD1Monoclonal Antibody Reagent
Coulter Electronics Sales of P.R. Inc.San Juan Puerto Rico	Product Differences:	CD45/CD4/CD8/CD3, CD3/T4 and CD3/T8 are essentially identical withrespect to features and principles of operation. The new and comparatorsystems use the same, well-established, state-of-the-art technologies ofimmunophenotyping with monoclonal antibodies and flow cytometry to
Coulter Electronics, Ltd.Venezuela
Coulter Elektronik A.S.Istanbul, Turkey
Coulter Electronics S.A.Caracas, Venezuela

. measure cellular components in whole blood via immunofluorescence analysis. Further, the intended use of the new and comparator systems is the same. Also, each liquid reagent allows simultaneous identification and enumeration of more than one lymphocyte population in a single specimen using a single reagent.

Science Serving Humanity

{1}------------------------------------------------

The new and comparator systems differ in only a few respects. One difference results from the more advanced software for the new tetraONE™ SYSTEM. The tetraONE™ SYSTEM software is designed to further simplify flow cytometric analysis by increasing automated modes of operation and the accuracy, precision and reliability of results. The new and comparator systems also differ in that the new tetraONE™ SYSTEM does not require an isotypic control for monitoring and adjusting for non-specific and nontargeted monoclonal antibody binding to irrelevant cellular populations. The tetraONE™ SYSTEM software monitors and adjusts for such binding by automatically placing cursors based on the separation of positive and negative peaks.

There are two differences between the reagents.

Lymphocyte Gating: The CD45/CD4/CD3 reagent contains a monoclonal antibody, CD45, to a. identify a lymphocyte gate to allow CD3+, CD4+, CD8+, dual-positive CD3+/CD4+ and dualpositive CD3+/CD8+ measurements. In contrast, CD3/T4 and CD3/T8 require a separate reagent, Mo2-RD1/KC56 (T-200)-FITC, for this purpose.

Fluorescent Labeling: b.

CD45/CD4-RD1/CD8-ECD/CD3-PC5:

CD45: FITC (Fluorescein Isothiocyanate); CD4: RD1 (Phycoerythrin); CD8: ECD (Phycoerythria-Texas Red); CD3: PC5 (Phycoerythrin-Cy5).

CD3/T4 and CD3/T8:

CD3: FITC (Fluorescein Isothiocyanate); T4: RD1; T8: RD1 (Phycoerythrin).

Product testing to assess the performance of CD45/CD4/CD3 is described below. Studies were Product Testing: designed in line with instructions for use given in the tetraONE™ SYSTEM Guide, Package Inserts, Product Manuals, and performance specifications. Specimens were assayed with CD3/T4 and CD3/CD8 for comparison purposes. The results of product testing demonstrated that CD45/CD4/CD8/CD3 met all performance specifications and provided mature T (CD3+), inducer T (CD4+; CD3+/CD4+) and suppressor/cytotoxic T (CD8+; CD3+/CD8+) lymphocyte values comparable to those of CD3/T4 and CD3/T8.
- 1. Accuracy:

Normal and abnormal (e.g., Human Immunodeficiency Virus, organ transplant, autoimmune disease, low white blood cell count) whole blood specimens were collected from geographically diverse populations of males and females unselected as to race and ranging in age from 18 to 85 years. Specimens were divided, processed as lysed preparations and assayed in parallel with CD45/CD4/CD8/CD3, CD3/T4 and CD3/T8. The CD3+, CD4+, CD8+, CD3+/CD4+ and CD3+/CD8+ percentages expressed in terms of the total lymphocyte count and absolute counts (cells/uL) were determined with EPICS® XL-MCL flow cytometers gated on lymphocytes. White blood cell counts and 5-part differentials were obtained for all specimens. CD3/T4 and CD3/T8 values were corrected for lymphocyte purity (Lymphocyte Gate Limits: lymphocyte recovery ≥ 90%; lymphocyte purity ≥ 85%).

Results analyzed in terms of minimums, maximums, means ± 1 SD, confidence intervals with 95% limits, regression and correlation analyses, and analyses of variance demonstrated that CD45/CD4/CD8/CD3, CD3/T4 and CD3/T8 identify and enumerate essentially identical numbers of the targeted lymphocytes in whole blood specimens.

1. Linearity:
  Three replicate measurements were made on a concentrated COULTER™ CYTO-TROL™ Control Cells sample serially diluted to achieve a range of CD3+, CD4+ (CD3+/CD4+) and CD8+ (CD3+/CD8+) lymphocyte concentrations. Samples were assayed with CD45/CD4/CD3 and analyzed on an EPICS® XL-MCL flow cytometer gated on lymphocytes. Values were expressed in terms of absolute counts (cells/uL).

Results analyzed in terms of regression and correlation analyses for recovered versus expected absolute counts demonstrated linearity of the assay.

.
.
.
.
.

{2}------------------------------------------------

Within Run (Intralaboratory) Precision: 3.
Ten replicate measurements were made for each of three levels of CD3+, CD4+ (CD3+/CD4+) and CD8+ (CD3+/CD8+) lymphocyte concentrations using a COULTER EPICS® XL-MCL flow cytometer gated on lymphocytes. Levels were obtained by selective depletions of a normal whole blood specimen and assayed with CD45/CD4/CD8/CD3. Values were expressed in terms of % of the total lymphocyte count.

Results analyzed in terms of mean ± 1 SD and CV demonstrated Within Run (Intralaboratory) Precision of the assay.

Interlaboratory Precision: 4.
Ten replicate measurements were made on the same day using different laboratories and EPICS® XL-MCL flow cytometers. All measurements were made on a single normal whole blood specimen divided and assayed with CD45/CD4/CD3. Values were expressed in terms of % of the total lymphocyte count.

Results analyzed in terms of mean ± 1 SD and CV demonstrated Interlaboratory Precision of the assay.

Marion S. Gaide, Ph.D.

Senior Regulatory Affairs Specialist Corporate Regulatory Affairs

August 16, 1996
Date

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.”