ORTHO-MUNE OKB (CD19)- MONOCLONAL ANTIBODY (MURINE) PHYCOERYTHRINE CONJUGATE

K964754 · Ortho Diagnostic Systems, Inc. · GKZ · Jan 22, 1997 · Hematology

Device Facts

• Record ID: K964754
• Device Name: ORTHO-MUNE OKB (CD19)- MONOCLONAL ANTIBODY (MURINE) PHYCOERYTHRINE CONJUGATE
• Applicant: Ortho Diagnostic Systems, Inc.
• Product Code: GKZ · Hematology
• Decision Date: Jan 22, 1997
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Intended Use

Ortho-mune OKB19A PE Conjugate is intended for use in identification and enumeration of CD19+ human B lymphocytes in whole blood by flow cytometry. The intended use is the same as the intended use of the predicate device, Monoclonal Antibody Test for B Cells [Anti-Leu-12 (CD19) PE (Phycoerythrin)] commercially distributed by Becton Dickinson Immunocytometry Systems.

Device Story

Ortho-mune OKB19A (CD19) PE Conjugate is a murine monoclonal antibody conjugated to phycoerythrin (PE) fluorochrome. Used in clinical laboratories, the reagent is added to whole blood specimens for staining CD19+ B lymphocytes. Samples are analyzed via flow cytometry (specifically the ORTHO CYTORONABSOLUTE system). The device identifies and enumerates B cell populations; results are used by clinicians to assess lymphocyte subsets in patients, including those with AIDS/ARC. The output provides the percentage of CD19+ cells, aiding in the characterization of immune status.

Clinical Evidence

Performance compared against predicate at three sites using 205 normal donors and 87 AIDS/ARC patients. Linear regression analysis showed equivalence in identifying CD19+ cells. Reproducibility studies (within and between labs) across low, normal, and high concentrations demonstrated acceptable CVs. Linearity study across a range of 20-9000 cells/uL showed slopes near 1.0 and R=1.000.

Technological Characteristics

Murine monoclonal antibody (OKB19A) conjugated to phycoerythrin (PE) fluorochrome. Designed for use with flow cytometry systems. Reagent is a liquid conjugate for staining whole blood samples.

Indications for Use

Indicated for the identification and enumeration of CD19+ human B lymphocytes in whole blood samples from normal donors and patients 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

• Monoclonal Antibody Test for B Cells [Anti-Leu-12 (CD19) PE (Phycoerythrin)]

Submission Summary (Full Text)

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JAN 22 1997
K964754

# 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™ OKB™19A (CD19)
Monoclonal Antibody (Murine)
Phycoerythrin Conjugate | PREDICATE: | Monoclonal Antibody Test for B Cells
[Anti-Leu-12 (CD19) PE Phycoerythrin] |
| DATE: | May 14, 1996 | | |

## DEVICE DESCRIPTION

Ortho-mune OKB19A Monoclonal Antibody (Murine) Phycoerythrin (PE) Conjugate contains the purified monoclonal antibody OKB19A conjugated to the fluorochrome phycoerythrin.

## INTENDED USE

Ortho-mune OKB19A PE Conjugate is intended for use in identification and enumeration of CD19+ human B lymphocytes in whole blood by flow cytometry. The intended use is the same as the intended use of the predicate device, Monoclonal Antibody Test for B Cells [Anti-Leu-12 (CD19) PE (Phycoerythrin)] commercially distributed by Becton Dickinson Immunocytometry Systems.

## TECHNOLOGICAL CHARACTERISTICS

Both Ortho-mune OKB19A Monoclonal Antibody (Murine) PE Conjugate and Monoclonal Antibody Test for B Cells utilize monoclonal antibodies specific for human B cells (OKB19A/Leu-12) respectively, conjugated to the same fluorochrome, phycoerythrin.

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 96

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# PERFORMANCE CHARACTERISTICS

Performance of Ortho-mune OKB19A Monoclonal Antibody (Murine) PE Conjugate was compared with that of Monoclonal Antibody Test For B Cells at three external, geographically distinct sites. Whole blood specimens from 205 normal donors, and 87 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 CD19+ 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 OKB19A AND LEU-12 ASSAYED ON THE CYTORONABSOLUTE
N=205 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Ortho-mune Reagent | Mean % | Range % | BD Reagent | Mean % | Range % |
| OKB19A (CD19)+ | 13.3 | 1.3 - 31.1 | LEU-12 (CD19)+ | 14.1 | 0.1 - 31.3 |

TABLE 2

| PERCENT POSITIVE STAINED CELLS IN AIDS/ARC PATIENTS DETECTED BY OKB19A AND LEU-12 ASSAYED ON THE CYTORONABSOLUTE
N=87 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Ortho-mune Reagent | Mean % | Range % | BD Reagent | Mean % | Range % |
| OKB19A (CD19)+ | 10.2 | 0.4 - 41.9 | LEU-12 (CD19)+ | 11.1 | 0.8 - 38.7 |

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 97

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Linear regression analysis of total percent CD19+ cells from the combined normal and AIDS/ARC populations is found in Chart 1.

# CHART 1

Ortho-mune OKB19A (CD19) PE vs Monoclonal Antibody Test for B Cells (Leu-12)

Ortho-mune OKB19A(PE)

This study demonstrates that the performance of Ortho-mune OKB19A (CD19) Monoclonal Antibody (Murine) PE Conjugate is equivalent to Monoclonal Antibody Test for B Cells [Anti-Leu-12 (CD19) PE (Phycoerythrin)] reagent in identification and enumeration CD19+ human lymphocytes in whole blood by flow cytometry.

Ortho Diagnostic Systems Inc.

Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225

Additional Information Submitted May, 1996

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Reproducibility studies were performed at three independent laboratories using samples with low, normal, and high relative percent CD19+ cells.

Specimens from each of ten normal donors (whole blood, EDTA) were processed using monoclonal antibodies bound to microbeads to produce samples of low, normal, and high relative percent CD19+ cells. The samples were separated into aliquots for each laboratory. Samples were stained in replicates of 10 with Ortho-mune OKB19A (CD19) PE Conjugate 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 CD19+ cells are presented in Table 3.

TABLE 3

| WITHIN LABORATORY REPRODUCIBILITY
Ortho-mune OKB19A(PE) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| N = 10 donors | | | | | | | |
| Ortho-mune OKB19A(PE) | All SITES
Mean
Percent
Positive | Site A | | Site B | | Site C | |
| | | CV | #
Reps | CV | #
Reps | CV | #
Reps |
| TOTAL CD19+ Low | 1.134 | 24.182 | 100 | 20.793 | 100 | 22.106 | 100 |
| TOTAL CD19+ Normal | 15.330 | 5.082 | 100 | 4.635 | 98 | 4.545 | 100 |
| TOTAL CD19+ High | 26.760 | 3.438 | 99 | 3.401 | 100 | 3.447 | 100 |

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 99

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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 CD19+ cells are presented in Table 4.

TABLE 4

| BETWEEN LABORATORY REPRODUCIBILITY
Ortho-mune OKB19A(PE) | | | | | |
| --- | --- | --- | --- | --- | --- |
| N = 10 donors | | | | | |
| Ortho-mune OKB19A(PE) | 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 CD19+ Low | 1.132 | 1.152 | 1.119 | 1.466 | 300 |
| TOTAL CD19+ Normal | 15.395 | 15.410 | 15.144 | 0.976 | 298 |
| TOTAL CD19+ High | 26.518 | 26.787 | 26.930 | 0.782 | 299 |

Ortho-mune OKB19A (CD19) Monoclonal Antibody (Murine) PE Conjugate immunophenotyping reagent shows acceptable within and between laboratory reproducibility for determination of CD19+ lymphocyte percentages

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 100

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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 OKB19A (CD19) PE 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 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 Ortho-mune OKB19A (CD19) PE Conjugate reagent demonstrated linear performance for total CD19+ 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 CD19+ cells for each donor specimen are shown in. Regression analysis statistics are provided in Table 5.

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 101

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# CHART 2

Ortho-mune OKB19A(PE)


DONOR ←→ 81 ☐-☐-☐ 82 ●●●● 83 ●●●● 84

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 102

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TABLE 5

| LINEARITY
Ortho-mune OKB19A(PE) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| N = 4 | | | | | | |
| Ortho-mune
OKB19A(PE) | Donor | SLOPE | CI | INTERCEPT | CI | R |
| TOTAL CD19+ | 1 | 1.003 | 0.01 | -6.012 | 5.33 | 1.000 |
| TOTAL CD19+ | 2 | 1.004 | 0.00 | -4.017 | 2.59 | 1.000 |
| TOTAL CD19+ | 3 | 1.003 | 0.00 | -4.126 | 2.20 | 1.000 |
| TOTAL CD19+ | 4 | 1.001 | 0.00 | 0.311 | 1.36 | 1.000 |
| TOTAL CD19+ | All | 1.002 | 0.00 | -3.308 | 1.50 | 1.000 |

## CONCLUSION

Performance of Ortho-mune OKB19A (CD19) Monoclonal Antibody (Murine) Phycoerythrin Conjugate is substantially equivalent to Monoclonal Antibody Test for B Cells [Anti-Leu-12 (CD19) PE (Phycoerythrin)] in the identification and enumeration of CD19+ human T lymphocytes in whole blood by flow cytometry.

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 103

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Reproducibility studies were performed at three independent laboratories using samples with low, normal, and high relative percent CD19+ cells.

Specimens from each of ten normal donors (whole blood, EDTA) were processed using monoclonal antibodies bound to microbeads to produce samples of low, normal, and high relative percent CD19+ cells. The samples were separated into aliquots for each laboratory. Samples were stained in replicates of 10 with Ortho-mune OKB19A (CD19) PE Conjugate 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 CD19+ cells are presented in Table 3.

TABLE 3

| WITHIN LABORATORY REPRODUCIBILITY
Ortho-mune OKB19A(PE) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| N = 10 donors | | | | | | | |
| Ortho-mune OKB19A(PE) | All SITES
Mean
Percent
Positive | Site A | | Site B | | Site C | |
| | | CV | #
Reps | CV | #
Reps | CV | #
Reps |
| TOTAL CD19+ Low | 1.134 | 24.182 | 100 | 20.793 | 100 | 22.106 | 100 |
| TOTAL CD19+ Normal | 15.330 | 5.082 | 100 | 4.635 | 98 | 4.545 | 100 |
| TOTAL CD19+ High | 26.760 | 3.438 | 99 | 3.401 | 100 | 3.447 | 100 |

Ortho Diagnostic Systems Inc.
Ortho-mune OKB19A (CD19) PE Conjugate Ref. No. K95-1225
Additional Information Submitted May, 1996
Page 99