ORTHO-MUNE OK-COMBO CD40FITC/CD8-PE (OKT4A/OKT8) MONOCLONAL ANTIBODY (MURINE)

K950625 · Ortho Diagnostic Systems, Inc. · GKZ · May 13, 1996 · Hematology

Device Facts

• Record ID: K950625
• Device Name: ORTHO-MUNE OK-COMBO CD40FITC/CD8-PE (OKT4A/OKT8) MONOCLONAL ANTIBODY (MURINE)
• Applicant: Ortho Diagnostic Systems, Inc.
• Product Code: GKZ · Hematology
• Decision Date: May 13, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Intended Use

Ortho-mune OK-COMBO CD4-FITC/CD8-PE is intended for use in identification and enumeration of CD4+ 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, Simultest CD4/CD8 (Leu-3a/2a) commercially distributed by Becton Dickinson Immunocytometry Systems.

Device Story

Ortho-mune OK-COMBO CD4-FITC/CD8-PE is a blend of murine monoclonal antibodies (OKT4A and OKT8) conjugated to fluorochromes (FITC and PE). Used in clinical laboratories with flow cytometry systems (e.g., ORTHO CYTORONABSOLUTE) to identify and enumerate CD4+ and CD8+ T lymphocytes in whole blood. Input: whole blood specimen; Process: antibody-antigen binding followed by flow cytometric analysis; Output: percentage of CD4+ and CD8+ positive cells. Healthcare providers use these results for immunophenotyping, aiding in the assessment of immune status in patients, including those with AIDS/ARC.

Clinical Evidence

Clinical performance evaluated at three sites comparing Ortho-mune OK-COMBO to Simultest CD4/CD8. Study population included 190 normal donors and 83 AIDS/ARC patients. Results showed high correlation in CD4+ and CD8+ cell enumeration. Reproducibility studies (N=11 donors) demonstrated acceptable within-laboratory and between-laboratory CVs. Linearity studies across a range of 20 to 18,676 cells/ul showed slopes near 1.0 and R=1.000.

Technological Characteristics

Murine monoclonal antibodies (OKT4A and OKT8) conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE). Reagent is a two-color blend for flow cytometry. No specific materials of construction or software algorithm class provided; device is a chemical reagent for use with existing flow cytometer hardware.

Indications for Use

Indicated for identification and enumeration of CD4+ and CD8+ human T lymphocytes in whole blood samples from normal donors and patients with AIDS/ARC.

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

• Simultest™ CD4/CD8 (Leu™-3a/2a)

Submission Summary (Full Text)

{0}

K950625

# REVISED 510(k) SUMMARY

| 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
CD4-FITC/CD8-PE
(OKT™4A/OKT8)
Monoclonal Antibody (Murine) | PREDICATE: | Simultest™ CD4/CD8
(Leu™-3a/2a) |
| DATE: | February 16, 1996 | | |

## DEVICE DESCRIPTION

Ortho-mune OK-COMBO CD4-FITC/CD8-PE (OKT4A/OKT8) Monoclonal Antibody (Murine) is a blend of the individual purified monoclonal antibodies OKT4A and OKT8 conjugated to the fluorochromes fluorescein isothiocyanate and phycoerythrin respectively.

## INTENDED USE

Ortho-mune OK-COMBO CD4-FITC/CD8-PE is intended for use in identification and enumeration of CD4+ 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, Simultest CD4/CD8 (Leu-3a/2a) commercially distributed by Becton Dickinson Immunocytometry Systems.

## TECHNOLOGICAL CHARACTERISTICS

Both Ortho-mune OK-COMBO CD4-FITC/CD8-PE (OKT4A/OKT8) Monoclonal Antibody (Murine) and Simultest CD4/CD8(Leu-3a/2a) utilize monoclonal antibodies specific for human helper/inducer T cells (OKT4A/Leu 3a) and human suppressor/cytotoxic T cells (OKT8/Leu-2a) respectively, conjugated to the same fluorochromes, fluorescein isothiocyanate and phycoerythrin.

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 128
153

{1}

# PERFORMANCE CHARACTERISTICS

Performance of the two color reagent Ortho-mune OK-COMBO CD4-FITC/CD8-PE (OKT4A/OKT8) Monoclonal Antibody (Murine) was compared with that of Simultest CD4/CD8 (Leu-3a/2a) at three external, geographically distinct sites. Whole blood specimens from 190 normal donors, and 83 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+ and percent CD8+ cells for the normal donor and AIDS/ARC population are shown in Table 1 and Table 2 respectively.

TABLE 1

| PERCENT POSITIVE STAINED CELLS IN NORMAL DONORS DETECTED BY OKT4A/OKT8 AND LEU-3a/2a ASSAYED ON THE CYTORONABSOLUTE
N=190 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Ortho-mune Reagent | Mean % | Range % | BD Reagent | Mean % | Range % |
| CD4+ (OKT4A) | 47.5 | 11.6-70.8 | CD4+ (LEU3a) | 44.1 | 13.1-61.2 |
| CD8+ (OKT8) | 27.8 | 10.9-72.8 | CD8+ (LEU2a) | 29.5 | 12.5-69.1 |

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 129

{2}

TABLE 2

| PERCENT POSITIVE STAINED CELLS IN AIDS/ARC PATIENTS DETECTED BY OKT4A/OKT8 AND LEU-3a/2a ASSAYED ON THE CYTORONABSOLUTE N=83 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Ortho-mune Reagent | Mean % | Range % | BD Reagent | Mean % | Range % |
| CD4+ (OKT4A) | 18.2 | 0.1-60.4 | CD4+ (LEU3a) | 15.8 | 0.2-50.4 |
| CD8+ (OKT8) | 56.1 | 12.6-81.9 | CD8+ (LEU2a) | 58.9 | 22.5-88.4 |

Linear regression analysis of total percent CD4+ cells from the combined normal and AIDS/ARC populations is found in Chart 1. Likewise, linear regression analysis of percent CD8+ cells from the combined normal and AIDS/ARC populations is found in Chart 2.

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 130
155

{3}

# CHART 1

## OK-COMBO CD4/CD8 vs Simultest Leu-3a/2a


Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 131
156

{4}

CHART 2
OK-COMBO CD4/CD8 vs Simultest Leu-3a/2a

TOTAL CD8+ PERCENT

These studies demonstrate that the performance of the two color reagent Ortho-mune OK-COMBO CD4-FITC/CD8-PE (OKT4A/OKT8) Monoclonal Antibody (Murine) is equivalent to Simultest CD4/CD8 (Leu-3a/2a) reagent in identification and enumeration of CD4+ and CD8+ human lymphocytes in whole blood by flow cytometry.

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 132
157

{5}

Reproducibility studies were performed at three independent laboratories using samples with low, normal, and high relative percent CD4+ 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 CD4+ and CD8+ cells. The samples were separated into aliquots for each laboratory. Samples were stained in replicates of 10 with Ortho-mune OK-COMBO CD4/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 CD4+ and percent CD8+ cells are presented in Table 3.

TABLE 3

| WITHIN LABORATORY REPRODUCIBILITY
OK-COMBO CD4/CD8 | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| N = 11 donors | | | | | | | |
| OK-COMBO
CD4/CD8 | All SITES
Mean
Percent
Positive | Site A | | Site B | | Site C | |
| | | CV | #
Reps | CV | #
Reps | CV | #
Reps |
| TOTAL CD4+ Low | 11.232 | 8.268 | 98 | 7.358 | 109 | 6.701 | 109 |
| TOTAL CD4+ Normal | 49.455 | 3.118 | 110 | 2.443 | 110 | 2.736 | 109 |
| TOTAL CD4+ High | 67.528 | 3.049 | 110 | 2.093 | 110 | 3.360 | 104 |
| TOTAL CD8+ Low | 7.521 | 15.735 | 110 | 7.186 | 110 | 7.972 | 104 |
| TOTAL CD8+ Normal | 32.782 | 4.244 | 110 | 3.102 | 110 | 3.442 | 109 |
| TOTAL CD8+ High | 56.234 | 3.392 | 98 | 2.462 | 109 | 2.740 | 109 |

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 133

{6}

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 CD4+ and percent CD8+ cells are presented in Table 4.

TABLE 4

| BETWEEN LABORATORY REPRODUCIBILITY
OK-COMBO CD4/CD8 | | | | | |
| --- | --- | --- | --- | --- | --- |
| N = 11 donors | | | | | |
| OK-COMBO
CD4/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 CD4+ Low | 10.744 | 11.994 | 11.389 | 5.496 | 316 |
| TOTAL CD4+ Normal | 50.103 | 49.392 | 48.937 | 1.189 | 329 |
| TOTAL CD4+ High | 67.934 | 68.473 | 66.235 | 1.729 | 324 |
| TOTAL CD8+ Low | 7.908 | 7.623 | 7.097 | 5.453 | 324 |
| TOTAL CD8+ Normal | 32.636 | 32.913 | 32.741 | 0.427 | 329 |
| TOTAL CD8+ High | 55.979 | 56.787 | 55.271 | 1.354 | 316 |

Ortho-mune OK-COMBO CD4/CD8 immunophenotyping reagent shows acceptable within and between laboratory reproducibility for determination of total CD4+ and CD8+ lymphocyte percentages

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 134

{7}

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 CD4/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 CD4/CD8 reagent demonstrated linear performance for both total CD4+ and 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 CD4+ and percent CD8+ cells for each donor specimen are shown in Chart 3 and Chart 4 respectively. Regression analysis statistics are provided in Table 5.

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 135
160

{8}

161

# CHART 3

## OK-COMBO CD4/CD8

CD4+


DONOR 71 72 73

# CHART 4

## OK-COMBO CD4/CD8

CD8+


DONOR 71 72 73

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 136

{9}

Ortho Diagnostic Systems Inc.
Ortho-mune OK-COMBO CD4-FITC/CD8-PE Ref. No. K95-0625
Additional Information Submitted February, 1996
Page 137

TABLE 5

| INTERCEPT | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| OK-COMBO CD4/CD8 | | | | | | |
| N=4 | | | | | | |
| OK-COMBO CD4/CD8 | Donor | SLOPE | CI | INTERCEPT | CI | R |
| TOTAL CD4+ | 1 | 0.998 | 0.004 | 8.060 | 6.795 | 1.000 |
| TOTAL CD4+ | 2 | 1.000 | 0.002 | -0.558 | 1.592 | 1.000 |
| TOTAL CD4+ | 3 | 0.999 | 0.002 | 4.117 | 2.719 | 1.000 |
| TOTAL CD4+ | 4 | 0.999 | 0.003 | 11.075 | 9.120 | 1.000 |
| TOTAL CD4+ | All | 0.999 | 0.001 | 5.325 | 2.743 | 1.000 |
| TOTAL CD8+ | 1 | 0.999 | 0.003 | 2.380 | 2.334 | 1.000 |
| TOTAL CD8+ | 2 | 0.999 | 0.003 | 1.547 | 2.374 | 1.000 |
| TOTAL CD8+ | 3 | 1.000 | 0.004 | 2.859 | 4.226 | 1.000 |
| TOTAL CD8+ | 4 | 1.000 | 0.003 | 5.326 | 5.797 | 1.000 |
| TOTAL CD8+ | All | 0.999 | 0.001 | 2.943 | 1.553 | 1.000 |

## CONCLUSION

Performance of the two color reagent Ortho-mune OK-COMBO CD4-FITC/CD8-PE (OKT4A/OKT8) Monoclonal Antibody (Murine) is equivalent to Becton Dickinson Simultest CD4/CD8 (Leu-3a/2a) reagent for identification and determination of percent CD4+ and CD8+ lymphocytes in whole blood by flow cytometry.