ORTHO-MUNE OK-COMVO CD3-FITC/CD8-PE (OKT 3/OKT8) MONOCLONAL ANTIBODY (MURINE)

K950482 · Ortho Diagnostic Systems, Inc. · GKZ · May 10, 1996 · Hematology

Device Facts

• Record ID: K950482
• Device Name: ORTHO-MUNE OK-COMVO CD3-FITC/CD8-PE (OKT 3/OKT8) MONOCLONAL ANTIBODY (MURINE)
• Applicant: Ortho Diagnostic Systems, Inc.
• Product Code: GKZ · Hematology
• Decision Date: May 10, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Intended Use

Ortho-mune OK-COMBO CD3-FITC/CD8-PE is intended for use in identification and enumeration of CD3+ and CD8+ human T lymphocytes in whole blood by flow cytometry. The intended use is the same as the intended use of the predicate device(s), Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate, and Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin conjugate.

Device Story

Ortho-mune OK-COMBO CD3-FITC/CD8-PE is a dual-color reagent blend of murine monoclonal antibodies (OKT3 and OKT8) conjugated to fluorochromes (FITC and phycoerythrin). Used in clinical laboratories by technicians/operators with flow cytometry instrumentation (e.g., ORTHO CYTORONABSOLUTE). Input: whole blood samples. Process: antibody-antigen binding allows simultaneous identification of CD3+ and CD8+ T-lymphocyte populations. Output: flow cytometric data representing percentages of CD3+ and CD8+ cells. Clinicians use these results for immunophenotyping to assess T-cell subsets, aiding in the management and monitoring of patients with immune-related conditions like AIDS/ARC.

Clinical Evidence

Clinical study compared dual-color reagent to single-color predicates using 199 normal donors and 77 AIDS/ARC patients on the ORTHO CYTORONABSOLUTE flow cytometer. Performance metrics included mean percent positive cells and linear regression analysis (R values near 1.0). Reproducibility studies (within and between laboratories) demonstrated acceptable CVs for CD3+ and CD3+CD8+ cell percentages. Linearity studies across a range of 20 to 18,676 cells/ul showed slopes indistinguishable from 1 and R=1.000.

Technological Characteristics

Murine monoclonal antibodies (OKT3, OKT8) conjugated to fluorochromes (FITC, phycoerythrin). Dual-color immunophenotyping reagent for flow cytometry. Analyzed via laser flow cytometer. No specific ASTM material standards cited.

Indications for Use

Indicated for the identification and enumeration of CD3+ and CD8+ human T lymphocytes in whole blood specimens from patients, including normal donors and those with AIDS/ARC, using flow cytometry.

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

• Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate
• Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin Conjugate

Reference Devices

• Becton Dickinson FACScan
• COULTER EPICS

Submission Summary (Full Text)

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K950482

# REVISED 510(k) SUMMARY

MAY 10 1996

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

**CONTACT:** Joanne Harris
Tel: [phone]
Fax: [phone]

**DEVICE NAME:** Ortho-mune™ OK-COMBO
CD3-FITC/CD8-PE
(OKT™3/OKT8)
Monoclonal Antibody (Murine)

**PREDICATE:** Ortho-mune™ OKT™3
Monoclonal Antibody (Murine)
FITC Conjugate

Ortho-mune OKT8
Monoclonal Antibody (Murine)
Phycoerythrin Conjugate

**DATE:** February 29, 1996

## DEVICE DESCRIPTION

Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT8) Monoclonal Antibody (Murine) is a blend of the individual purified monoclonal antibodies OKT3 and OKT8 conjugated to the fluorochromes fluorescein isothiocyanate and phycoerythrin respectively.

## INTENDED USE

Ortho-mune OK-COMBO CD3-FITC/CD8-PE is intended for use in identification and enumeration of CD3+ and CD8+ human T lymphocytes in whole blood by flow cytometry. The intended use is the same as the intended use of the predicate device(s), Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate, and Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin conjugate.

## TECHNOLOGICAL CHARACTERISTICS

Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT8) Monoclonal Antibody (Murine), Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate, and Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin Conjugate all utilize

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monoclonal antibodies specific for human T cells (OKT3) and human suppresser/cytotoxic T cells (OKT8) respectively. The purified monoclonal antibodies are conjugated to the same fluorochromes, fluorescein isothiocyanate and phycoerythrin respectively.

# PERFORMANCE CHARACTERISTICS

Performance of the two color reagent Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT8) Monoclonal Antibody (Murine) was compared with that of single color reagents Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate and Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin Conjugate. Whole blood specimens from 199 normal donors, and 77 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 total percent CD3+ and total percent CD8+ for the normal population and the AIDS/ARC population are shown in Table 1 and Table 2, respectively.

TABLE 1

| PERCENT POSITIVE STAINED CELLS IN NORMAL DONORS DETECTED BY OKT3/OKT8 AND OKT3 FITC AND OKT8 PE ASSAYED ON THE CYTORONABSOLUTE
N=199 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dual Color Reagent | Mean % | Range % | Single Color Reagent | Mean % | Range % |
| CD3+ (OKT3) | 74.5 | 45.9-87.4 | CD3+ (OKT3) | 75.9 | 58.4-90.1 |
| CD8+ (OKT8) | 31.2 | 15.0-74.9 | CD8+ (OKT8) | 29.2 | 13.2-75.5 |

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TABLE 2

| PERCENT POSITIVE STAINED CELLS IN AIDS/ARC PATIENTS DETECTED BY OKT3/OKT8 AND OKT3 FITC AND OKT8 PE ASSAYED ON THE CYTORONABSOLUTE
N=77 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dual Color Reagent | Mean % | Range % | Single Color Reagent | Mean % | Range % |
| CD3+
(OKT3) | 73.5 | 27.5-90.7 | CD3+
(OKT3) | 77.7 | 35.6-96.4 |
| CD8+
(OKT8) | 59.1 | 25.6-89.8 | CD8+
(OKT8) | 60.3 | 18.1-92.1 |

Linear regression analysis of total percent CD3+ cells from the combined normal and AIDS/ARC populations is found in Chart 1. Likewise, linear regression analysis of total percent CD8+ cells from the combined normal and AIDS/ARC populations is found in Chart 2.

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# CHART 1

## OK-COMBO CD3/CD8 vs Ortho-mune OKT3 FITC

### TOTAL CD3+ PERCENT


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# CHART 2

## OK-COMBO CD3/CD8 vs Ortho-mune OKT8 PE

TOTAL CD8+ PERCENT

These studies demonstrate that the performance of the two color reagent Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT8) Monoclonal Antibody (Murine) is equivalent to the single color reagents Ortho-mune OKT3 FITC Conjugate and OKT8 PE Conjugate for determination of total percent CD3+ and total percent CD8+ cells in whole blood by flow cytometry.

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For the CD3+CD8+ cell population the mean percent and range of results for the normal and AIDS/ARC population obtained using Ortho-mune OK-COMBO CD3-FITC/CD8-PE are shown in Table 3. Cells positive for both markers could not be determined with the single color reagents.

TABLE 3

| PERCENT MEAN AND RANGE OF CD3+CD8+ CELLS IN NORMAL AND AIDS/ARC DONORS DETECTED BY OKT3/OKT8 ASSAYED ON THE CYTORONABSOLUTE | | | | | |
| --- | --- | --- | --- | --- | --- |
| Normal Donors | Mean % | Range % | AIDS/ARC Donors | Mean % | Range % |
| CD3+CD8+ (OKT3/OKT8) | 25.8 | 12.3-67.5 | CD3+CD8+ (OKT3/OKT8) | 53.3 | 5.1-86 |

ORTHO CYTORONABSOLUTE™ Laser Flow Cytometer was used in the clinical studies comparing the performance of Ortho-mune OK-COMBO CD3-FITC/CD8-PE immunophenotyping reagent to Ortho-mune OKT3 FITC conjugate and OKT8 Phycoerythrin conjugate

A study was done comparing the performance of the ORTHO CYTORONABSOLUTE flow cytometer to recognized flow cytometers, Becton Dickinson FACScan™ and COULTER EPICS™ flow cytometers.

Specimens from ten normal donors (whole blood, EDTA) were processed using monoclonal antibodies bound to magnetic microbeads to product samples with low, normal, and high relative percent positive CD8 cells. A portion of each sample was stained in triplicate using immunophenotyping reagents from each manufacturer. Specimens stained with Ortho-mune OK-COMBO reagents were analyzed using the ORTHO CYTORONABSOLUTE flow cytometer. Likewise, samples stained with Simultest reagents were analyzed using the FACScan flow cytometer, and samples stained with Cyto-Stat®/ COULTER CLONE® were analyzed using the COULTER EPICS flow cytometer.

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For each sample the mean of the triplicate results were calculated for the Becton Dickinson FACScan, COULTER EPICS, and CYTOTONABSOLUTE within concentration and donor. Least square regression analyses were performed on these mean results for the FACScan (X axis) versus the CYTOTONABSOLUTE (Y axis), and for the COULTER EPICS (X axis) versus the CYTOTONABSOLUTE (Y axis). Results for percent CD3+CD8+ cells using Ortho-mune OK-COMBO CD3-FITC/CD8-PE and the CYTOTONABSOLUTE versus Simultest CD3/CD8 (Leu-4/2a) using the FACScan are presented in Chart 3.

# CHART 3


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Results for percent CD3+CD8+ cells using Ortho-mune OK-COMBO CD3+CD8+ cells and the CYTORONABSOLUTE flow cytometer versus Cyto-Stat/ COULTER CLONE CD3(IgG1)-FITC/T8-RD1 and the COULTER EPICS flow cytometer are presented in Chart 4.

# CHART 4


These studies demonstrate the equivalent performance of the ORTHO CYTORONABSOLUTE flow cytometer to both the Becton Dickinson FACScan and the COULTER EPICS flow cytometer for determination of percent positive cells when used with appropriate immunophenotyping reagents.

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Reproducibility studies were performed at three independent laboratories using samples with low, normal, and high relative percent CD3+ and CD8+ cells.

Specimens from each of eleven normal donors (whole blood, EDTA) were processed using monoclonal antibodies bound to microbeads to produce samples of low, normal, and high relative percent CD3+ and CD8+ cells. The samples were separated into aliquots for each laboratory. Samples were stained in replicates of 10 with Ortho-mune OK-COMBO CD3/CD8 reagent and analyzed using the ORTHO CYTORONABSOLUTE flow cytometer.

For within laboratory reproducibility, the variance for the replicate results was calculated within site, concentration and donor. The variance was averaged across site, concentration and donor. The square root replicate variance (SD) was divided by the appropriate mean percent positive result (by site and concentration) and multiplied by 100 to obtain the CV. Within laboratory reproducibility results for determination of total percent CD3+ and percent CD3+CD8+ cells are presented in Table 4.

TABLE 4

| WITHIN LABORATORY REPRODUCIBILITY
OK-COMBO CD3/CD8 | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| N = 11 donors | | | | | | | |
| OK-COMBO
CD3/CD8 | All SITES
Mean
Percent
Positive | Site A | | Site B | | Site C | |
| | | CV | #
Reps | CV | #
Reps | CV | #
Reps |
| TOTAL CD3+ Low | 56.525 | 3.400 | 98 | 2.486 | 110 | 3.488 | 109 |
| TOTAL CD3+ Normal | 76.676 | 2.130 | 110 | 1.569 | 110 | 2.307 | 109 |
| TOTAL CD3+ High | 87.146 | 2.407 | 109 | 1.539 | 110 | 2.424 | 110 |
| CD3+ CD8+ Low | 4.010 | 11.214 | 108 | 9.430 | 110 | 11.151 | 104 |
| CD3+ CD8+ Normal | 28.053 | 4.793 | 110 | 3.610 | 110 | 5.250 | 109 |
| CD3+ CD8+ High | 44.311 | 4.359 | 98 | 2.904 | 110 | 4.147 | 109 |

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The between laboratory CV was computed as follows. The mean percent positive for each site within concentration was calculated. The SD was computed on the three site means within concentration and the CV was obtained by dividing the SD by the overall mean within concentration and multiplying by 100. Between laboratory reproducibility results for determination of total percent CD3+ and percent CD3+CD8+ cells are presented in Table 5.

TABLE 5

| BETWEEN LABORATORY REPRODUCIBILITY
OK-COMBO CD3/CD8 | | | | | |
| --- | --- | --- | --- | --- | --- |
| N = 11 donors | | | | | |
| OK-COMBO
CD3/CD8 | SITE A | SITE B | SITE C | ACROSS SITE | |
| | Mean Percent
Positive
(All Donors) | Mean Percent
Positive
(All Donors) | Mean Percent
Positive
(All Donors) | Coefficient
of
Variation | #
Reps |
| TOTAL CD3+ Low | 56.707 | 58.151 | 54.879 | 2.899 | 317 |
| TOTAL CD3+ Normal | 77.261 | 77.105 | 75.676 | 1.139 | 329 |
| TOTAL CD3+ High | 87.964 | 87.987 | 85.491 | 1.646 | 329 |
| CD3+ CD8+ Low | 4.088 | 3.467 | 4.439 | 12.309 | 322 |
| CD3+ CD8+ Normal | 28.451 | 27.058 | 28.614 | 3.051 | 329 |
| CD3+ CD8+ High | 45.009 | 44.386 | 43.396 | 1.837 | 317 |

Ortho-mune OK-COMBO CD3/CD8 immunophenotyping reagent shows acceptable within and between laboratory reproducibility for determination of total CD3+ and CD3+CD8+ lymphocyte percentages.

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A linearity study was performed using an automated hematology analyzer to determine total 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 OK-COMBO CD3/CD8 immunophenotyping reagent and analyzed using the CYTORONABSOLUTE flow cytometer. The total lymphocyte count of the concentrated sample for each donor was obtained using an automated 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 subset 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 OK-COMBO CD3/CD8 reagent demonstrated linear performance for both total CD3+ and CD3+CD8+ lymphocyte subsets across a lymphocyte count range of 20 cells/ul to 18,676 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 CD3+ and percent CD3+CD8+ cells for each donor specimen are shown in Chart 5 and Chart 6 respectively. Regression analysis statistics are provided in Table 6.

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CHART 5

OK–COMBO CD3/CD8
CD3+


DONOR ←→ 71 0-3 0 72 ←→ 73 △ △ △ 74

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# CHART 6

OK–COMBO CD3/CD8
CD3 + CD8 +


DONOR ←→ 71 □-□ □ 72 ←→ 73 △△ △ 74

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TABLE 6

| N = 4 | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| OK-COMBO CD3/CD8 | Donor | SLOPE | CI | INTERCEPT | CI | R |
| TOTAL CD3+ | 1 | 0.999 | 0.003 | 10.907 | 6.694 | 1.000 |
| TOTAL CD3+ | 2 | 0.999 | 0.003 | 4.833 | 4.327 | 1.000 |
| TOTAL CD3+ | 3 | 0.999 | 0.003 | 5.103 | 9.583 | 1.000 |
| TOTAL CD3+ | 4 | 0.999 | 0.003 | 12.567 | 12.589 | 1.000 |
| TOTAL CD3+ | All | 0.999 | 0.001 | 7.870 | 3.552 | 1.000 |
| CD3+CD8+ | 1 | 1.000 | 0.003 | -0.889 | 2.414 | 1.000 |
| CD3+CD8+ | 2 | 0.999 | 0.005 | -0.144 | 3.352 | 1.000 |
| CD3+CD8+ | 3 | 0.999 | 0.005 | -0.657 | 5.106 | 1.000 |
| CD3+CD8+ | 4 | 1.001 | 0.005 | -5.762 | 6.280 | 1.000 |
| CD3+CD8+ | All | 1.000 | 0.002 | -1.868 | 1.935 | 1.000 |

## CONCLUSION

Based upon the linear regression analysis of the clinical study data, and the supplemental studies performed demonstrating equivalence of the CYTORONABSOLUTE flow cytometer to the recognized flow cytometers, Becton Dickinson FACScan, and COULTER EPICS, Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT8) Monoclonal Antibody (Murine) is equivalent to the single color immunophenotyping reagents OKT3 Monoclonal Antibody (Murine) FITC Conjugate and Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin Conjugate for identification and determination of percent CD3+ and CD8+ lymphocytes in whole blood by flow cytometry.

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K95-0482
Additional Information Submitted February, 1996
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