DAKO MOUSE ANTI-HUMAN T-CELL, CD3/RPE-CY5, CLONE UCHT1 & DAKO MOUSE ANTI-HUMAN T-CELL, CD3/RPE, CLONE UCHT1

K991402 · Dako Corp. · GKZ · Jun 21, 1999 · Hematology

Device Facts

• Record ID: K991402
• Device Name: DAKO MOUSE ANTI-HUMAN T-CELL, CD3/RPE-CY5, CLONE UCHT1 & DAKO MOUSE ANTI-HUMAN T-CELL, CD3/RPE, CLONE UCHT1
• Applicant: Dako Corp.
• Product Code: GKZ · Hematology
• Decision Date: Jun 21, 1999
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Intended Use

For In Vitro Diagnostic Use Monoclonal Mouse Anti-Human T-cell, CD3/RPE-Cy5 conjugated, Clone UCHT1 (Code No.C7067) (Anti-CD3/RPE-Cy5) and Monoclonal Mouse Anti-Human T-cell, CD3/RPE conjugated, Clone UCHT1 (Code No. R0810) (Anti-CD3/RPE) have been developed for use in flow cytometry for the analysis of T-cells in peripheral blood. These reagents allow simultaneous detection and quantification of CD3-positive cells (T-cells) in normal and pathological conditions such as immunodeficiency disorders. Each reagent is one component of the suggested monoclonal antibody (MAb) combinations for routine immunophenotyping of lymphocytes in peripheral blood.

Device Story

Monoclonal mouse anti-human CD3 antibodies (clone UCHT1) conjugated to R-phycoerythrin (RPE) or RPE-Cy5. Reagents bind specifically to T-lymphocyte cluster determinants. Used in clinical laboratories for flow cytometry analysis of peripheral blood samples. Operates by labeling CD3+ T-cells, allowing identification and quantification via flow cytometer. Healthcare providers use these results for immunophenotyping to assist in the diagnosis and monitoring of immunodeficiency disorders. Benefits include standardized, reliable identification of T-cell populations in patient blood samples.

Clinical Evidence

Bench testing only. Linearity established using cell lines (R2=0.999). Reproducibility tested across two flow cytometer platforms with CVs ranging from 0.01 to 0.05. Specificity verified in five healthy donors; antibody binding specific to lymphocytes. Clinical correlation performed on 177 samples (150 healthy, plus patients with illnesses) comparing subject reagents to predicate (Anti-CD3/FITC). Linear regression analysis showed high correlation (R2=0.9750 for RPE-Cy5; R2=0.9870 for RPE), confirming 1:1 comparability.

Technological Characteristics

Monoclonal mouse anti-human CD3 antibody (clone UCHT1). Conjugates: R-phycoerythrin (RPE) or RPE covalently coupled to cyanin 5 (Cy5). Formulation: 0.05M Tris-HCl buffer, pH 7.2, 15mM NaN3, 0.1M NaCl, 1% stabilizing protein. Analyte detection via flow cytometry. Standalone reagent for use with laboratory flow cytometers.

Indications for Use

Indicated for use in flow cytometry for the analysis of T-cells in peripheral blood. Used for the detection and quantification of CD3-positive cells in patients with normal or pathological conditions, including immunodeficiency disorders. Intended for professional use as part of routine lymphocyte immunophenotyping.

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

• DAKO Mouse anti-human T-cell, CD3/FITC, Clone UCHT1 Code No.F0818

Submission Summary (Full Text)

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JUN 21 1999

:

:

K991402

## 510(k) Summary

:

| Submitter: | DAKO Corporation<br>6392 Via Real<br>Carpinteria, CA 93013<br>805-566-6655 |
|---------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Contact: | Gretchen M. Murray, Ph.D. |
| Date Summary<br>Prepared: | April, 1999 |
| Device Names: | DAKO® Mouse Anti-Human T-cell, CD3/RPE-Cy5, Clone UCHT1<br>(Product Code No. C7067) and<br>DAKO® Mouse Anti-Human T-cell, CD3/RPE, clone UCHT1<br>(Product Code No. R0810) |
| Device<br>Classification: | Class II according to 21 CFR 864.5220, on the basis that monoclonal<br>antibodies are accessories for automated differential cell counters. |
| Panel: | The device classification is under the Hematology and Pathology Devices panel,<br>Division of Clinical Laboratory Devices. |
| Predicate Device: | DAKO Mouse anti-human T-cell, CD3/FITC, Clone UCHT1 Code No.F0818 |
| Devise<br>Description: | Monoclonal Mouse Anti-Human T-cell, CD3/RPE-Cy5 conjugated, Clone<br>UCHT1 (Code No. C7067) and Monoclonal Mouse Anti-Human T-cell, CD3/RPE<br>conjugated, Clone UCHT1 (Code No. R0810) are specific for T-lymphocyte<br>cluster determinants as evaluated by the International Workshop on Human<br>Leukocyte Differentiation Antigens. UCHT1 CD3-specific monoclonal antibody<br>was designated B28 antibody at the Second Workshop (Reinherz, EL, Haynes,<br>BF, Nadler, LM, Bernstein, ID, eds. Leukocyte Typing II, Vol. 2. New York-<br>Berlin-Heidelberg-Tokyo: Springer Verlag, 1986.) Purified monoclonal mouse<br>anti-human CD3 is produced in tissue culture, dialyzed and conjugated with<br>either R-phycoerythrin (RPE) covalently coupled to cyanin 5 (Cy5) or R-<br>phycoerythrin (RPE). One ml (1.0 ml) containing the conjugated antibody is<br>supplied in 0.05M Tris-HCl buffer, pH 7.2, 15mM NaN ₃ , 0.1M NaCl, stabilized<br>with 1% stabilzing protein. |
| Intended Use: | For <i>In Vitro</i> Diagnostic Use<br><br>Monoclonal Mouse Anti-Human T-cell, CD3/RPE-Cy5 conjugated, Clone UCHT1<br>(Code No.C7067) (Anti-CD3/RPE-Cy5) and Monoclonal Mouse Anti-Human T-<br>cell, CD3/RPE conjugated, Clone UCHT1 (Code No. R0810) (Anti-CD3/RPE)<br>have been developed for use in flow cytometry for the analysis of T-cells in<br>peripheral blood. These reagents allow simultaneous detection and<br>quantification of CD3-positive cells (T-cells) in normal and pathological<br>conditions such as immunodeficiency disorders. Each reagent is one<br>component of the suggested monoclonal antibody (MAb) combinations for<br>routine immunophenotyping of lymphocytes in peripheral blood. |

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Comparison of Technological Characteristics:

Linearity:

Binding linearity was determined over serial dilutions of a cell line known to express the antigen diluted with a cell line that has no antigenic sites. For Anti-CD3/RPE-Cy5, C7067 and Anti-CD3/RPE, R0810 the cell line without antigenic reactivity is Raji cells, while the cell line with antigenic sites is JM cells. Five dilutions were tested, with a linear equation calculated from the results. The equation for Anti-CD3/RPE-Cy5, C7067, y = -0.02 + 1.0x,

Image /page/1/Figure/3 description: The image contains two line graphs comparing '% Expected' to '% Recovered'. The graph on the left is titled 'CD3/RPE/Cy5, C7067 LINEARITY' and shows a linear relationship between the expected and recovered values. The graph on the right is titled 'CD3/RPE, R0810 LINEARITY' and shows two lines, one for '% Recovered' and one for '% Expected', which are very close to each other, indicating a strong correlation.

R2 = 0.999, and Anti-CD3/RPE y=2.84 + 1.0x, R2 = 0.999.

Reproducibility:

Ten replicates from peripheral blood of three donors were tested for reproducibility of Anti-CD3/RPE-Cy5 and run on two flow cytometers from different manufacturers at three concentrations of antigen. Different levels of CD3+ lymphocytes were selected from a population of normal and abnormal peripheral blood samples. Each level of CD3 was analyzed within one day on both machines.

| FACScan Anti-CD3/RPE-Cy5 | Mean % CD3+ | $\pm$ 1 SD | %CV | n |
|-----------------------------|--------------|------------|------|----|
| High Level | 71.51 | 0.53 | 0.01 | 10 |
| Medium Level | 37.54 | 0.58 | 0.02 | 10 |
| Low Level | 10.76 | 0.40 | 0.04 | 10 |
| Profile II Anti-CD3/RPE-Cy5 | Mean % CD3+ | $\pm$ 1 SD | %CV | n |
| High Level | 71.44 | 0.74 | 0.01 | 10 |
| Medium Level | 36.21 | 0.86 | 0.02 | 10 |
| Low Level | 12.35 | 0.32 | 0.03 | 10 |
| FACScan Anti-CD3/RPE | Mean % CD3 + | $\pm$ 1 SD | %CV | n |
| High Level | 72.07 | 1.11 | 0.02 | 10 |
| Medium Level | 38.50 | 0.87 | 0.02 | 10 |
| Low Level | 11.03 | 0.51 | 0.05 | 10 |
| Profile II Anti-CD3/RPE | Mean % CD3+ | $\pm$ 1 SD | %CV | n |
| High Level | 71.53 | 0.74 | 0.01 | 10 |
| Medium Level | 36.01 | 0.49 | 0.01 | 10 |
| Low Level | 10.79 | 0.53 | 0.05 | 10 |

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#### Specificity:

Specificity of Anti-CD3/RPE-Cy5 and Anti-CD3/RPE has been verified by tests performed on five apparently healthy adult donors of various races at DAKO Corporation. Cell populations tested were RBC's, granulocytes, monocytes, lymphocytes and platelets. The results indicate antibody binding of Anti-CD3/RPE-Cy5 and Anti-CD3/RPE are specific for lymphocytes. Lymphocytes bound to Anti-CD3/RPE-Cy5 antibodies on an average of 72%, representative of the T-cell population. Approximately 2% of monocytes bound with the Anti-CD3/RPE-Cy5. However, monocyte binding can be excluded from the lymphocyte analysis by proper gating on lymphocytes. Lymphocytes bound to Anti-CD3/RPE antibodies on an average of 70%, representative of the T-cell population. Approximately 9% of monocytes bound with the Anti-CD3/RPE.

| | %Positive Red<br>Blood Cells | % Positive<br>Granulocytes | % Positive<br>Monocytes | % Positive<br>Lymphocytes | % Positive<br>Platelets |
|---------------|------------------------------|----------------------------|-------------------------|---------------------------|-------------------------|
| Average (n=5) | 0.02 | 1.50 | 1.82 | 71.90 | 0.12 |
| (range) | (0.0-0.1) | (0.9-2.2) | (1.1-2.7) | (60.3-78.6) | (0.0-0.3) |

#### DAKO Anti-CD3/RPE-Cy5 Specificity

| | % Positive Red<br>Blood Cells | % Positive<br>Granulocytes | % Positive<br>Monocytes | % Positive<br>Lymphocytes | % Positive<br>Platelets |
|---------------|-------------------------------|----------------------------|-------------------------|---------------------------|-------------------------|
| Average (n=5) | 0.08 | 0.60 | 8.52 | 69.92 | 1.08 |
| (range) | 0.0-0.3 | 0.4-0.7<br>(n=4) | 1.6-16.3 | 58.4-75.9 | 0.1-3.8 |

#### DAKO Anti-CD3/RPE Specificity

Predicate Correlation: Correlation of Anti-CD3/RPE-Cy5, C7067 and Anti-CD3/RPE, R0810 to a predicate Anti-CD3/FITC, F0818 reagent, was determined by testing duplicate samples with each reagent across 153 normal, apparently healthy individuals at three geographically separate laboratories. Linear regression analysis of the data gave the following equations and Pearson correlation's.

Y(DAKO Anti-OD3/RPE-Cy5+ Lymphocytes) = 6.22 + 0.92 X(DAKO Anti-OD3/FITC + Lymphocytes); R2 = 0.9208 n = 150 y(DAKO Anti-CD3/RPE+ Lymphocytes) = 9.85 + 0.87 x(DAKO Anti-CD3/FITC + Lymphocytes). R2 = 0.8040.

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In addition, samples from patients with illnesses were compared, and their data added to the results of the testing of the 150 apparently healthy individuals. Linear correlation was performed on the total database. Linear regression analysis gave the following equation and R2:

y(DAKO Anti-CD3/RPE-Cy5+ Lymphocytes) = 2.43 + 0.98 x(DAKO Anti-CD3/FITC + Lymphocytes). R2 = 0.9750 n = 177 y(DAKO Anti-CD3/RPE+ Lymphocytes) = 1.20 + 0.99 x(DAKO Anti-CD3/FITC + Lymphocytes). $R^2$ = 0.9870 n = 176 These equations indicate that Anti-CD3/RPE-Cy5, C7067 and Anti-CD3/RPE, R0810 reagents are comparable on 1:1 basis to the Anti-CD3/FITC, F0818 reagent.

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#### Device Description

Monoclonal Mouse Anti-Human T-cell, CD3/RPE-Cy5-conjugated, C7067, and, Monoclonal Mouse Anti-Human T-cell, CD3/RPE, R0810 are specific for T-lymphocyte cluster determinants as evaluated by the International Workshop on Human Leukocyte Differentiation Antigens. UCHT1 CD3-specific monoclonal antibody was designated B28 antibody at the Second Workshop (Reinherz, EL, Haynes, BF, Nadler, LM, Bernstein, ID, eds. Leukocyte Typing II, Vol. 2. New York-Berlin-Heidelberg-Tokyo: Springer Verlag, 1986.) Purified monoclonal mouse anti-human CD3 is produced in tissue culture, dialyzed and conjugated with R-phycoerythrin (RPE) covalently coupled to cyanin 5 (Cy5), or conjugated with R-phycoerythrin (RPE). One ml (1.0 ml) containing the conjugated antibody is supplied in 0.05M Tris-HCl buffer, pH 7.2, 15mM NaNg, 0.1M NaCl, stabilized with 1% carrier protein.

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Image /page/4/Picture/1 description: The image is a black and white seal for the Department of Health and Human Services (HHS). The seal features a stylized eagle with three lines representing its body and wings. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" are arranged in a circular pattern around the eagle.

JUN 21 1999

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Gretchen M. Murray, Ph.D. Regulatory Affairs Manager DAKO Corporation 6392 Via Real Carpinteria, California 93013

Re: K991402

> Trade Name: Monoclonal Mouse Anti-Human T-cell, CD3 Clone UCHT1 RPE-CY5 or RPE Conjugated

Regulatory Class: II Product Code: GKZ Dated: April 20, 1999 Received: April 22, 1999

Dear Dr. Murray:

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 legally marketed predicate 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 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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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 [phone].

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 [phone]. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at [phone]. 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 [phone] or [phone], or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours,

Steven Putman

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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K 991402

510(k) Number: Device Name: Monoclonal Mouse Anti-Human T-cell, CD3 Clone UCHT I RPE-Cy5 Conjugated

Indications For Use:

Monoclonal Mouse Anti-Human T-cell, CD3, Clone UCHT1, RPE-Cy5 conjugated, has been developed for use in flow cytometry for the analysis of Tcells. This reagent allows simultaneous detection and quantification of CD3positive cells (T-cells) in normal and pathological conditions such as immunodeficiency disorders. It is one component of the suggested monoclonal antibody (MAb) combinations for routine immunophenotyping of lymphocytes in peripheral blood.

Immunophenotyping of lymphocytes is widely applied for diagnosis of immunodeficiencies. DAKO Anti-CD3/RPE-Cy5 is one of the reagents utilized when performing immunophenotyping of lymphocytes.

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

Concurrence of CDRH, Office of Device)Evalu

O(k) Num

Prescription Use V (Per 21 CFR 801.109) OR

Over-The-Counter Use (Per 21 CRF 801.110)

IVD Üse (Per 21 CFR 801.119)

(Optional Format 1-2-96)

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510(k) Number: Device Name: Monoclonal Mouse Anti-Human T-cell, CD3 Clone UCHTI, RPE Conjugated

# Indications For Use:

Monoclonal Mouse Anti-Human T-cell, CD3, Clone UCHT1, RPE conjugated, has been developed for use in flow cytometry for the analysis of T-cells. This reagent allows simultaneous detection and quantification of CD3-positive cells (T-cells) in normal and pathological conditions such as immunodeficiency disorders. It is one component of the suggested monoclonal antibody (MAb) combinations for routine immunophenotyping of lymphocytes in peripheral blood.

Immunophenotyping of lymphocytes is widely applied for diagnosis of immunodeficiencies. DAKO Anti-CD3/RPE is one of the reagents utilized when performing immunophenotyping of lymphocytes.

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

| | Concurrence of CDRH, Office of Device Evaluation (ODE) | |
|--|--------------------------------------------------------|--|
|--|--------------------------------------------------------|--|


| (Division Sign-Off) | |
|-----------------------------------------|---------|
| Division of Clinical Laboratory Devices | |
| 510(k) Number | K991402 |

OR

| Prescription Use | <div> <img alt="Check" src="check.jpg"/> </div> |
|----------------------|-------------------------------------------------|
| (Per 21 CFR 801.109) | |

| Over-The-Counter Use | |
|----------------------|--|
| (Per 21 CRF 801.110) | |

IVD Use

IVD USC (Per 21 CFR 801.119)

(Optional Format 1-2-96)