Ortho-mune OKT4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate is used to identify and enumerate the percentage of CD4+ human T lymphocytes in whole blood by flow cytometry.

Identification and enumeration of abnormal levels of CD4lymphocytes may be clinically significant in the prognosis of secondary immunodeficiency diseases (acquired immunodeficiency syndrome [AIDS]).

AIDS is characterized by a severe reduction in T helper CD3+CD4+) lymphocytes and a decrease in the CD4:CD8 ratio. The CD4:CD8 ratio has been used as a predictor of time to the development of AIDS. CD4 counts should also be determined using a CD3/CD4 combination reagent to eliminate monocyte contamination (CD3-CD4+dim). CD4+lymphocytes (expressed as an absolute number, a percentage of lymphocytes or as a ratio of CD4+ to CD8+ T lymphocytes) represents the best single predictor of the progression of AIDS. CD4+ lymphocyte numbers usually start to decline relatively soon after human immunodeficiency virus (HIV) infection.

Device Description

Ortho-mune OKT4A Monoclonal Antibody (Murine) FITC Conjugate contains the purified monoclonal antibody OKT4A conjugated to the fluorochrome fluorescein isothiocyanate.

AI/ML Overview

Acceptance Criteria and Device Performance for Ortho-mune™OKT™4A (CD4) FITC Conjugate

This document outlines the acceptance criteria and performance data for the Ortho-mune™OKT™4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate, as submitted in K951459. The study aimed to demonstrate substantial equivalence to the predicate device, CD4 (Leu™-3a) FITC.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for substantial equivalence are inferred from the performance characteristics presented, specifically demonstrating comparable results to the predicate device and acceptable reproducibility and linearity. Given the nature of a 510(k) submission for substantial equivalence, the "acceptance criteria" are implicitly met by showing that the new device performs "as well as" or "equivalently to" the predicate device on key performance metrics.

Performance Characteristic	Acceptance Criteria (Implicit)	Reported Device Performance (Ortho-mune OKT4A)
Equivalence to Predicate	Mean and range of percent CD4+ cells for normal and AIDS/ARC populations should be comparable to the predicate device. Regression analysis (Ortho-mune vs. Predicate) should show a strong correlation (R-value close to 1) and a slope near 1 with a small intercept.	Normal Donors (N=206): Mean % CD4+ = 47.0 (Range: 12.4 - 68.7). Predicate (LEU-3a) Mean % CD4+ = 45.1 (Range: 14.6 - 64.6). AIDS/ARC Patients (N=88): Mean % CD4+ = 18.3 (Range: 1.0 - 55.1). Predicate (LEU-3A) Mean % CD4+ = 17.2 (Range: 0.6 - 51.6). Linear Regression: Y = 0.969 + 1.019(X), R = 0.985 (combining normal and AIDS/ARC populations). This demonstrates strong correlation and a slope very close to 1, indicating equivalence.
Within-Laboratory Reproducibility	Coefficients of Variation (CV) should be acceptably low across low, normal, and high CD4 levels, and 95% Confidence Intervals (CI) should demonstrate precision.	Mean Percent Positive & CV across 3 sites (N=10 per site, 10 replicates each):- Low CD4: Mean % (Site 1: 9.60, Site 2: 9.36, Site 3: 8.86); CV (Site 1: 7.60, Site 2: 6.12, Site 3: 5.47)- Normal CD4: Mean % (Site 1: 47.38, Site 2: 47.19, Site 3: 48.54); CV (Site 1: 2.33, Site 2: 2.00, Site 3: 2.21)- High CD4: Mean % (Site 1: 56.13, Site 2: 55.83, Site 3: 56.50); CV (Site 1: 1.93, Site 2: 1.79, Site 3: 2.09)Demonstrated excellent within-laboratory reproducibility.
Between-Laboratory Reproducibility	Coefficients of Variation (CV) should be acceptably low across low, normal, and high CD4 levels, and 95% Confidence Intervals (CI) should demonstrate precision across different laboratories.	Mean Percent Positive & CV across 3 sites (N=10 samples, 10 replicates each):- Low CD4: Mean % = 9.25; CV = 4.09; +/- 95% CI = 1.63- Normal CD4: Mean % = 47.70; CV = 1.53; +/- 95% CI = 3.14- High CD4: Mean % = 56.15; CV = 0.60; +/- 95% CI = 1.46Demonstrated excellent between-laboratory reproducibility.
Linearity	Linear regression analysis (observed vs. expected CD4+ counts) should show slopes indistinguishable from 1 and R-values close to 1,000 (R=1).	Slopes and R-values for 4 donors and overall:- Donor 1: Slope = 0.999 (CI 0.003), Intercept = 6.099 (CI 5.242), R = 1.000- Donor 2: Slope = 1.000 (CI 0.001), Intercept = 0.395 (CI 2.226), R = 1.000- Donor 3: Slope = 0.999 (CI 0.003), Intercept = 4.449 (CI 4.176), R = 1.000- Donor 4: Slope = 1.000 (CI 0.001), Intercept = 0.285 (CI 1.316), R = 1.000- All Donors: Slope = 0.999 (CI 0.001), Intercept = 2.777 (CI 1.655), R = 1.000Demonstrates linear performance across 20 to 9000 cells/uL.

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

Sample Size for Equivalence Study:
- 206 normal donors
- 88 AIDS/ARC patients
- Total: 294 whole blood specimens.
Sample Size for Reproducibility Studies:
- 10 normal donors for within-laboratory study (processed to create low, normal, high CD4 populations).
- 10 normal donors for between-laboratory study (processed to create low, normal, high CD4 populations).
Sample Size for Linearity Study:
- Specimens from 4 normal donors (processed to produce low, normal, high numbers of lymphocyte subsets via concentration/dilution).
Data Provenance: The studies were conducted at three external, geographically distinct sites. The data is prospective for the purpose of this submission, though the underlying specimens are from existing populations (normal donors and AIDS/ARC patients). The country of origin of the data is not explicitly stated but is implied to be the United States given the submission to the FDA.

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

No external experts were explicitly used to establish ground truth in the traditional sense of consensus reading. The "ground truth" for the comparative effectiveness study was the measurement obtained by the predicate device, CD4 (Leu-3a) FITC, using flow cytometry. For the reproducibility and linearity studies, the ground truth was derived from the measurements of the device itself (Ortho-mune OKT4A) against established laboratory methods (e.g., automated hematology analyzer for total lymphocyte count).

4. Adjudication Method for the Test Set

No adjudication method was used for the test set. The study directly compared the measurements obtained by the new device to those obtained by the predicate device for equivalence, and relied on statistical analysis of direct measurements for reproducibility and linearity.

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

No MRMC study was done, as this device is an immunophenotyping reagent for flow cytometry, not an imaging device requiring human reader interpretation in the same manner. The "human readers" (flow cytometry operators) would be operating the instruments rather than interpreting complex images. The focus was on the performance of the reagent itself. Therefore, comparing human reader improvement with and without AI assistance is not applicable here.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the studies evaluating the performance of the Ortho-mune OKT4A (CD4) FITC Conjugate can be considered "standalone" in the sense that they assess the direct analytical performance of the reagent itself, as measured by a flow cytometer. The flow cytometry process is automated in terms of cell counting and fluorescent signal detection, and the reagent's performance is objectively measured. While human operators are involved in sample preparation and instrument setup, the core performance metrics (staining, enumeration, reproducibility, linearity) characterize the reagent's analytical capability independently of direct "human interpretation" of results in the way an imaging AI might be evaluated.

7. The Type of Ground Truth Used

For Equivalence Study: The predicate device, CD4 (Leu-3a) FITC, was used as the reference standard. The goal was to establish substantial equivalence, meaning the new device performs comparably to a legally marketed device. This is a form of comparative ground truth.
For Reproducibility Studies: The measurements from the Ortho-mune OKT4A reagent itself, over multiple replicates and sites, were used to assess the consistency of its own performance. This relies on the inherent precision of the laboratory measurements.
For Linearity Study: A combination of an automated hematology analyzer (for total lymphocyte count) and the CYTORONABSOLUTE flow cytometer (with the Ortho-mune reagent for percent positive CDx cells) was used. The "expected" values were calculated based on serial dilutions and established counts, which serves as a form of derived or calculated ground truth.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning or AI development. This device is a monoclonal antibody reagent, and its development typically involves laboratory-based optimization (e.g., antibody titration, conjugation efficiency) rather than algorithm training with large datasets. The studies described here are for validation of the final product.

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

As noted above, the concept of a "training set" and associated ground truth establishment for AI/machine learning is not applicable to this type of medical device (an immunophenotyping reagent). The development process would have involved standard biological and chemical methods to characterize the antibody and its conjugate, ensuring specificity and reactivity, but not a "training set" with ground truth in the AI context.

Summary

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Ortho-munç OKT4A (CD4) FITC Conjugatc
Ref. No. K951459 - Submitted July, 1997

K951459

510(k) Summarv

JUL 1 4 1997

SUBMITTER: Ortho Diagnostic Systems Inc. 1001 U.S. Highway 202 Raritan, NJ 08869-0606

CONTACT: Gail Kromer Tcl: (908) 218-8179 Fax: (908) 218-8168

DEVICE NAME: Ortho-mune™OKT™4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate

PREDICATE: CD4(Leu™-3a) FITC

DATE: July 8, 1997

DEVICE DESCRIPTION

Ortho-mune OKT4A Monoclonal Antibody (Murine) FITC Conjugate contains the purified monoclonal antibody OKT4A conjugated to the fluorochrome fluorescein isothiocyanate.

INTENDED USE:

Ortho-mune OKT4A FITC Conjugate is intended for use in identification and enumeration of CD4+ human T lymphocytes in whole blood by flow cytometry. The intended use is the same as the intended use of the predicate device, CD4 (Leu-3a) FITC commercially distributed by Becton Dickinson Immunocytometry Systems.

TECHNOLOGICAL CHARACTERISTICS

Both Ortho-mune OKT4A Monoclonal Antibody (Murine) FITC Conjugate and CD4 (Leu-3a) FITC utilize monoclonal antibodies specific for human helper/inducer T cells OKT4A/Leu-3a) respectively, conjugated to the same fluorochrome, fluorescein isothiocyanate.

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PERFORMANCE CHARACTERISTICS

Performance of Ortho-mune OKT4A Monoclonal Antibody (Murine) FITC Conjugate was compared with that of CD4 (Leu-3a) FITC at three external, geographically distinct sites. Whole blood specimens from 206 normal donors, and 88 AIDS/ARC patients were stained and analyzed using the ORTHO CYTORONABSOLUTE™ flow cytometer, Ortho Diagnostic Systems Inc.

For each specimen, the percentage of gated cells which showed positive by each marker was calculated. The mean and range of the percent CD4+ cells for the normal donor and AIDS/ARC population are shown in Table I and Table 2 respectively.

PERCENT POSITIVE STAINED CELLS IN NORMAL DONORS DETECTED BYOKT4A AND LEU-3A ASSAYED ON THE CYTORONABSOLUTEN=206
Ortho-muneReagent	Mean %	Range %	BDReagent	Mean %	Range %
OKT4A(CD4)+	47.0	12.4 - 68.7	LEU-3a(CD4)+	45.1	14.6 - 64.6

TABLE 1

TABLE 2

Ortho-muneReagent	Mean %	Range %	BDReagent	Mean %	Range %
OKT4(CD4)+	18.3	1.0 - 55.1	LEU-3A(CD4)+	17.2	0.6 - 51.6

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Linear regression analysis of total percent CD4+ cells from the combined normal and AIDS/ARC populations is found in Chart 1.

CHART 1

Ortho-mune OKT4A (CD4) FITC vs CD4 (Leu-3a) FITC

Ortho-mune OKT4A(FITC)

Image /page/2/Figure/5 description: The image is a scatter plot comparing "Ortho-mune Reagent" and "Predicate Reagent". The x-axis represents "Predicate Reagent", and the y-axis represents "Ortho-mune Reagent", both ranging from 0 to 70. The scatter plot shows a strong positive correlation between the two reagents, with data points clustered closely around a regression line, which is defined by the equation Y = 0.969 + 1.019(X), and the R-value is 0.985.

This study demonstrates that the performance of Ortho-mune OKT4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate is equivalent to CD4 (Leu-38) FITC reagent in identification and enumeration CD4+ human lymphocytes in whole blood by flow cytometry.

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WITHIN-LABORATORY REPRODUCIBILITY

Ten normal donors were used in a within-laboratory reproducibility study at three independent laboratories. The samples were processed using antibody-coated magnetic microbeads to produce low, normal and high percent positive CD4 populations to simulate leukopenia and leukocytosis. The samples were stained with Ortho-mune OKT4A (CD4) FITC Conjugate and run on the ORTHO CYTORONABSOLUTE. All samples were analyzed in replicates of ten. The coefficients of variation, calculated from the standard deviation between replicates, were compared and demonstrated excellent within-laboratory reproducibility at all concentrations tested. The 95% confidence intervals were calculated from the standard error of the site mean which includes variance components from replicate and donor variability minimized by the number of donors (10). The comparison of normal, concentrated (high) and diluted (low) samples is contained in Table 3.

TABLE 3: N = 10 WITHIN-LABORATORY REPRODUCIBILITY Ortho-mune OKT4A (CD4) FITC CONJUGATE PERCENT POSITIVE RESULTS

CD4 Level	Low			Normal			High
Subset: OKT4A (CD4)	Site 1	Site 2	Site 3	Site 1	Site 2	Site 3	Site 1	Site 2	Site 3
Mean Percent Positive	9.60	9.36	8.86	47.38	47.19	48.54	56.13	55.83	56.50
CV	7.60	6.12	5.47	2.33	2.00	2.21	1.93	1.79	2.09
+/- 95% Confidence Interval	4.18	3.53	3.45	3.74	3.84	4.22	4.93	4.91	5.15

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BETWEEN-LABORATORY REPRODUCIBILITY

Ten normal donor samples were compared between three independent laboratories. The samples were processed using antibody-coated magnetic microbeads to produce low, normal and high percent positive CD4 populations. The samples were stained with Ortho-mune OKT4A (CD4) FITC Conjugate and run on the ORTHO CYTORONABSOLUTE. All samples were analyzed in replicates of ten. The betweenlaboratory coefficient of variation (CV) and the 95% confidence intervals were calculated from the standard deviation between site means. The data collected from all sites are shown in Table 4. The data show excellent between-laboratory reproducibility.

TABLE 4: N = 10BETWEEN-LABORATORY REPRODUCIBILITYOrtho-mune OKT4A (CD4) FITC CONJUGATEPERCENT POSITIVE RESULTS
HighCD4 LevelLowNormal
Mean Percent Positive	9.25	47.70	રહ. 15
CV	4.09	1.53	0-60
+/- 95% ConfidenceInterval	1.63	3.14	1.46

Ortho-mune OKT4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate immunophenotyping reagent shows acceptable within and between laboratory reproducibility for determination of CD4+ lymphocyte percentages.

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A linearity study was performed using an automated hematology analyzer to determine to tal lymphocyte count, and the CYTORONABSOLUTE flow cytometer to determine the percent positive CDx cells.

Specimens from four normal donors (whole blood, EDTA) were processed to produce samples with low, normal and high numbers of lymphocyte subsets. Each whole blood specimen was concentrated by harvesting the buffy coat to obtain a white blood cell count between 20,000 and 40,000 cells/ul and then diluting to produce samples of high, normal and low numbers of lymphocyte subsets. A portion of each sample was stained in triplicate using Ortho-mune OKT4A (CD4) FITC Conjugate immunophenotyping reagent and analyzed using the CYTORONABSOLUTE flow cytometer. The total lymphocyte count of the concentrated sample for each donor was obtained using an sutomated hematology analyzer.

Linear regression analyses were performed as follows. The expected (X axis ) values were calculated by multiplying the corresponding serial dilutions by the hematology analyzer derived buffy coat lymphocyte count and by the CYTORONABSOLUTE derived lymphocyte subser percent positive. The observed (Y axis) values were determined as the total lymphocyte count calculated from the hematology derived value of the concentrated sample times the CYTORONABSOLUTE derived lymphocyte subset percent positive at each dilution.

The Ortho-mune OKT4A (CD4) FITC Conjugate reagent demonstrated linear performance for total CD4+ lymphocyte subsets across a lymphocyte count range of 20 cells/uL to 9000 cells/uL as demonstrated with slopes indistinguishable from 1 and R values of 1,000.

Linear regression analyses of observed versus expected values for total percent CD4+ cells for each donor specimen are shown in. Regression analysis statistics are provided in Table 5.

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Ortho - mune OKT4A(FITC)

Image /page/6/Figure/4 description: The figure is a scatter plot showing the relationship between expected CD4+ counts and observed CD4+ counts. The x-axis represents the expected CD4+ counts, ranging from 0 to 5000. The y-axis represents the observed CD4+ counts, ranging from 0 to 5000. A strong positive correlation is observed between the expected and observed CD4+ counts, with data points clustered tightly around a straight line.

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. . . . . . . .

TABLE 5

Ortho-mune	Donor	SLOPE	CI	INTERCEPT	CI	R
TOTAL CD4	1	0.999	0.003	6.099	5.242	1.000
TOTAL CD4	2	1.000	0.001	0.395	2.226	1.000
TOTAL CD4	3	0.999	0.003	4.449	4.176	1.000
TOTAL CD4	4	1.000	0.001	0.285	1.316	1.000
TOTAL CD4	All	0.999	0.001	2.777	1.655	1.000

CONCLUSION

Performance of Ortho-mune OKT4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate is substantially equivalent to CD4 (Leu-3a) FITC reagent in the identification and enumeration of CD4+ human T lymphocytes in whole blood by flow cytometry.

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Image /page/8/Picture/1 description: The image is a black and white logo for the U.S. Department of Health and Human Services. The logo features the department's name in a circular arrangement around a stylized symbol. The symbol consists of three curved lines that resemble a stylized human figure.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Ms. Gail Kromer Manager, Regulatory Affairs 1 2000 1 4 1997 Ortho Diagnostic Systems, Inc. 1001 U.S. Hiqhway 202 Raritan, New Jersey 09969-0606

Re : K951459/S3 Trade Name: Ortho-mune™ OKT™4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate Requlatory Class: II Product Code: GKZ Dated: April 21, 1997 Received: April 23, 1997

Dear Ms. Kromer:

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 reqistration, listing of devices, good manufacturing practice, labelinq, 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 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 Register. 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 Autman

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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Indications for Use Statement

Page 1 of 1

510(k) Number (if known) K951459

Ortho-mune™OKT™4A (CD4) Device Name: Monoclonal Antibody (Murine) FITC Conjugate

Indications for Use:

Ortho-mune OKT4A (CD4) Monoclonal Antibody (Murine) FITC Conjugate is used to identify and enumerate the percentage of CD4+ human T lymphocytes in whole blood by flow cytometry.

Identification and enumeration of abnormal levels of CD4lymphocytes may be clinically significant in the prognosis of secondary immunodeficiency diseases (acquired immunodeficiency syndrome [AIDS]).

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

Retin E. Moti

Concurrence of CDRH, Office of Devicsider S Division of Clinics 510(k) Numb

Prescription Use
✓

(Per 21 CFR 801.109)

Over-The-Counter-Use

(Optional Format 1-2-96)

Ortho Diagnostic Systems Inc. Ortho-mune ThoKT Tha (CD4) FITC Conjugate Ref. No. K951459 Additional Information - Submitted April, 1997 Page 1

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.”