CD 19 R-PE, CD19 TRI-COLOR MONOCLONAL ANTIBODY

K963954 · Caltag Laboratories, Inc. · GKZ · Nov 25, 1996 · Hematology

Device Facts

• Record ID: K963954
• Device Name: CD 19 R-PE, CD19 TRI-COLOR MONOCLONAL ANTIBODY
• Applicant: Caltag Laboratories, Inc.
• Product Code: GKZ · Hematology
• Decision Date: Nov 25, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.5220
• Device Class: Class 2

Intended Use

CALTAG CD19 R-PE and CD19 TRI-COLOR are fluorochrome conjugated monoclonal antibody reagents that may be used to enumerate CD19+ lymphocytes in human peripheral blood by flow cytometric methods.

Device Story

Device consists of fluorochrome-conjugated (R-PE or TRI-COLOR) mouse monoclonal antibodies targeting human CD19 cell surface antigens. Used in clinical laboratories by trained personnel. Workflow: peripheral blood leukocytes incubated with antibody; unbound antibody washed away; red blood cells lysed; fixative added. Stained, fixed cells analyzed via flow cytometry. Output is percentage of CD19+ lymphocytes. Clinicians use this data for immunophenotyping and assessment of B-cell populations in patient samples. Benefits include standardized identification of B-cell subsets for diagnostic evaluation.

Clinical Evidence

Bench testing and clinical correlation study performed. Study included 155 healthy donors (age 16-72) to establish expected values and 20 abnormal donors for correlation. Correlation study (n=175) compared Caltag antibodies against Coulter predicates, yielding r² values between 0.96 and 0.98. Intra-lab and inter-lab reproducibility studies (n=30 per antibody) demonstrated low CVs across high, medium, and low ranges. Specificity data confirmed binding to lymphocyte regions with minimal cross-reactivity to monocytes, granulocytes, platelets, or RBCs.

Technological Characteristics

Monoclonal antibodies conjugated to R-PE or TRI-COLOR fluorochromes. Supplied as liquid in PBS. Sensing principle: immunofluorescence via flow cytometry. Analysis involves gating on lymphocyte populations. No specific ASTM material standards cited. Software/algorithm: standard flow cytometric data analysis (gating).

Indications for Use

Indicated for the enumeration of CD19+ B lymphocytes in human peripheral blood using flow cytometry. No specific age or gender contraindications are listed.

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

• Coulter CD19 RD1 Monoclonal antibody
• Coulter CD19 FITC monoclonal antibody

Submission Summary (Full Text)

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CALTAG LABORATORIES
K963954
510(K) SUMMARY
SUMMARY OF SAFETY AND EFFECTIVENESS DATA
NOV 25 1996
CD19 R-PE, CD19 TRI-COLOR Mouse Monoclonal Antibodies
To Human Cell Surface Antigens by Flow Cytometry

## NAME AND LOCATION OF MANUFACTURER:

Caltag Laboratories, Inc.
1849 Old Bayshore Highway
Suite 200
Burlingame, CA 94010
[phone]

## NAME OF CONTACT PERSON:

Robert C. Johnson
Executive Vice President
Caltag Laboratories, Inc.

## DATE OF PREPARATION OF SUMMARY:

October 1, 1996

Caltag Laboratories Inc. • 1849 Bayshore Blvd., Suite #200 • Burlingame, CA 94010
Telephone [phone] • [phone] • FAX [phone]

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## TRADE NAME OF THE DEVICE:

Caltag CD19 R-PE, CD19 TRI-COLOR Mouse Monoclonal Antibodies To Human Cell Surface Antigens by Flow Cytometry

## COMMON NAME:

Caltag CD19 R-PE, CD19 TRI-COLOR Monoclonal Antibody

## CLASSIFICATION NAME:

Automated Differential Cell Coulter (21 CFR 864.5220)

## LEGALLY MARKETED DEVICE (PREDICATE DEVICE) TO WHICH THE MANUFACTURER IS CLAIMING SUBSTANTIAL EQUIVALENCE:

Caltag CD19 R-PE Monoclonal Antibody to Human Cell Surface Antigens is substantially equivalent to the Coulter CD19 RD1 Monoclonal antibody for in-vitro diagnostic use.

Caltag CD19 TRI-COLOR Monoclonal Antibody to Human Cell Surface Antigens is substantially equivalent to the Coulter CD19 FITC and CD19 RD1 monoclonal antibodies for in-vitro diagnostic use.

## DESCRIPTION OF THE DEVICE:

The CALTAG CD19 R-PE and CD19 TRI-COLOR monoclonal antibodies bind to the surfaces of viable blood cells that express the CD19 antigen. To identify cells bearing the CD19 determinant, peripheral blood leukocytes are incubated with the monoclonal antibody, and washed to remove unbound antibody. Prior to removal of unbound antibody, lysis solution is added to lyse red blood cells. An appropriate fixative solution is added to lysed and washed cells. Stained and fixed cells are subsequently analyzed by flow cytometric methods.

## INTENDED USE OF THE DEVICE:

CALTAG CD19 R-PE and CD19 TRI-COLOR are fluorochrome conjugated monoclonal antibody reagents that may be used to enumerate CD19+ lymphocytes in human peripheral blood by flow cytometric methods.

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SUMMARY OF THE TECHNICAL CHARACTERISTICS OF THE MANUFACTURER'S DEVICE COMPARED TO THE PREDICATE DEVICE:

## Comparisons of Caltag CD19 and Coulter CD19 Monoclonal Antibodies

| No. | Item | Caltag Antibodies | Coulter Antibodies | Comparison |
| --- | --- | --- | --- | --- |
| 1. | Intended Use | Flow Cytometry | Flow Cytometry
Immunofluorescence | Substantially equivalent |
| 2. | Specificity | CD19 | CD19 | Substantially equivalent |
| 3. | Target cell | B lymphocyte | B lymphocyte | Substantially equivalent |
| 4. | Chemical form | Monoclonal antibody | Monoclonal antibody | Substantially equivalent |
| 5. | Fluorochromes | R-PE,
TRI-COLOR | FITC,
RD1 | Substantially equivalent |
| 6. | Available forms
FITC
PE
TRI-COLOR | liquid, PBS
liquid, PBS
liquid, PBS | lyophilized
liquid, PBS
not available | Substantially equivalent |
| 7. | Sample prep. methods | whole blood | whole blood | Substantially equivalent |
| 8. | Expected values from this study (n=155)
R-PE
TRI-COLOR | 5-21%
4-24% | 4-21% (RD1)
3-23% (FITC) | Substantially equivalent |

**NON CLINICAL TESTS SUPPORTING A DETERMINATION OF SUBSTANTIAL EQUIVALENCE:**

### EXPECTED VALUE DATA

Blood samples were collected from a total of 155 apparently healthy normal donors in an age range of 16 to 72 with a mean age of 41. Samples were collected and analyzed in each of three independent laboratories. An approximately equal number of males and females were collected and analyzed in each laboratory.

The normal donor population included members of differing ethnic origins, including adult Caucasian, Black, Oriental and Hispanic.

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Donors in geographically diverse areas of the United States, including the Western, Eastern and SouthCentral regions, participated in this study. Blood samples collected from each donor were stained with the CALTAG CD19 R-PE and CD19 TRI-COLOR monoclonal antibodies.

Summary of expected values for CALTAG CD19 monoclonal antibodies for all normal donors:

| procedure | mean % positive | S.D. ±2 S.D. | Range | n |
| --- | --- | --- | --- | --- |
| CD19 R-PE | 13.0 | 4.2 | 5-21 | 155 |
| CD19 TRI-COLOR | 13.9 | 5.0 | 4-24 | 155 |

## SPECIFICITY DATA

Blood samples were obtained from healthy normal donors of Caucasian, Black, Hispanic and Oriental ethnic origins. Samples of each donor were stained with CALTAG CD19 R-PE and CD19 TRI-COLOR monoclonal antibodies. Cells contained in the lymphocyte, monocyte and granulocyte regions were selected for analysis. Separate samples from the same donors were prepared for analysis of red blood cells and platelets and stained with each of the CALTAG monoclonal antibodies.

### CD19 R-PE

| Ethnic
Origin | Lymph. | Percent of Stained Cells | | | |
| --- | --- | --- | --- | --- | --- |
| | | Mono. | Gran. | Plt. | RBC |
| Caucasian | 18.0 | 0.6 | 0.9 | 0.5 | 0.5 |
| Caucasian | 13.3 | 1.1 | 0.8 | 0.3 | 0.7 |
| Hispanic | 12.2 | 0.7 | 0.8 | 0.4 | 1.0 |
| Oriental | 11.2 | 1.6 | 1.3 | 0.4 | 0.5 |
| Black | 14.6 | 0.0 | 0.5 | 0.6 | 0.9 |
| Mean | 13.9 | 0.8 | 0.9 | 0.4 | 0.7 |
| ±1 S.D. | 2.6 | 0.6 | 0.3 | 0.1 | 0.2 |

### CD19 TRI-COLOR

| Ethnic
Origin | Lymph. | Percent of Stained Cells | | | |
| --- | --- | --- | --- | --- | --- |
| | | Mono. | Gran. | Plt. | RBC |
| Caucasian | 18.3 | 0.0 | 1.0 | 0.3 | 0.4 |
| Caucasian | 12.1 | 0.2 | 1.1 | 0.4 | 0.1 |
| Hispanic | 11.6 | 0.2 | 0.3 | 0.2 | 0.7 |
| Oriental | 11.0 | 1.0 | 0.9 | 0.3 | 0.2 |
| Black | 12.0 | 0.7 | 0.9 | 0.4 | 0.2 |
| Mean | 13.0 | 0.4 | 0.8 | 0.3 | 0.3 |
| ±1 S.D. | 3.0 | 0.4 | 0.3 | 0.1 | 0.2 |

Specific and/or nonspecific antibody Fc binding to monocytes in a patient sample can be excluded by proper gating on lymphocytes on the flow cytometer.

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# REPRODUCIBILITY DATA (INTRA-LAB)

Intra-lab reproducibility for the CALTAG CD19 R-PE and CD19 TRI-COLOR conjugated monoclonal antibodies was determined by performing 10 replicated determinations for each antibody in each of three ranges; high, medium and low. Thus, a total of 30 determinations were performed for each form of CD19. In this manner, reproducibility was demonstrated throughout the entire measuring range.

The 10 determinations for each range were performed by the staining, processing and analysis of 10 separate samples. Lymphocytes were selected for the analysis of percent cells stained in each of the three ranges.

To perform this study, anticoagulated blood was obtained from an abnormal donor expressing a high percentage of CD19+ cells. Mid range and low range samples were obtained by adding known CD19- cells in appropriate ratios, while maintaining approximately the same total cell concentrations for the three ranges.

The study was performed in each of three independent laboratories, in the manner that each laboratory obtained, stained and analyzed separate blood samples. The following data are representative:

| procedure | Level | mean % positive | S.D. | % CV | n |
| --- | --- | --- | --- | --- | --- |
| CD19 R-PE | high | 66.6 | 0.4 | 0.5 | 10 |
| | mid | 46.7 | 0.8 | 1.6 | 10 |
| | low | 14.9 | 0.6 | 4.3 | 10 |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 | high | 65.4 | 0.7 | 1.0 | 10 |
| TRI-COLOR | mid | 46.1 | 0.6 | 1.3 | 10 |
| | low | 15.5 | 0.4 | 2.7 | 10 |

# REPRODUCIBILITY, (INTER-LAB)

Inter-lab reproducibility for the CALTAG CD19 R-PE and CD19 TRI-COLOR conjugated monoclonal antibodies was determined by performing 10 replicated determinations for each antibody in each of three ranges; high, medium and low. Thus, a total of 30 determinations were performed for each form of CD19. In this manner, reproducibility was demonstrated throughout the entire measuring range.

The 10 determinations for each range were performed by the staining, processing and analysis of 10 separate samples. Lymphocytes were selected for the analysis of percent cells stained in each of the three ranges.

The study was performed in each of three laboratories. All laboratories stained and analyzed blood samples from the same blood donors. Lysed unstained samples containing cells in the appropriate ranges were prepared by one of the participating laboratories for staining and analysis in each of the laboratories. The following data were obtained:

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| SITE 1 | | | | | |
| --- | --- | --- | --- | --- | --- |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 R-PE | high | 84.7 | 4.4 | 5.2 | 10 |
| | mid | 70.4 | 1.2 | 1.8 | 10 |
| | low | 42.7 | 3.2 | 7.6 | 10 |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 | high | 86.7 | 1.0 | 1.2 | 10 |
| TRI-COLOR | mid | 70.9 | 1.3 | 1.9 | 10 |
| | low | 42.7 | 0.9 | 2.1 | 10 |
| SITE 2 | | | | | |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 R-PE | high | 83.9 | 1.5 | 1.7 | 10 |
| | mid | 71.1 | 2.8 | 3.9 | 10 |
| | low | 42.5 | 1.9 | 4.6 | 10 |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 | high | 85.3 | 2.2 | 2.6 | 11 |
| TRI-COLOR | mid | 65.7 | 3.1 | 4.8 | 9 |
| | low | 32.6 | 2.0 | 6.0 | 10 |
| SITE 3 | | | | | |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 R-PE | high | 87.5 | 0.6 | 0.7 | 10 |
| | mid | 69.9 | 0.8 | 1.1 | 10 |
| | low | 38.8 | 1.6 | 4.2 | 10 |
| procedure | Level | mean % positive | S.D. | % CV | n |
| CD19 | high | 85.3 | 0.9 | 1.0 | 10 |
| TRI-COLOR | mid | 66.1 | 1.0 | 1.5 | 10 |
| | low | 30.7 | 2.4 | 7.9 | 10 |

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# CLINICAL TESTS SUPPORTING A DETERMINATION OF SUBSTANTIAL EQUIVALENCE:

## CORRELATION DATA

The correlation study was performed on 175 donors, including 155 normal and 20 abnormal donors.

Comparison of the CALTAG CD19 R-PE conjugated monoclonal antibody with the Coulter CD19 RD1 conjugated monoclonal antibody:

| procedure | mean % positive | r² value | slope | Y intercept | n |
| --- | --- | --- | --- | --- | --- |
| CD19 R-PE | 16.4 | 97.7 | 0.92 | 1.30 | 175 |
| CD19 RD1 | 16.2 | | | | |

CD19 R-PE
Linear regression $y = 1.30 + 0.92x$

Comparison of the CALTAG CD19 R-PE conjugated monoclonal antibody with the Coulter CD19 FITC conjugated monoclonal antibody:

| procedure | mean % positive | r² value | slope | Y intercept | n |
| --- | --- | --- | --- | --- | --- |
| CD19 R-PE | 16.4 | 96.3 | 0.93 | 0.69 | 175 |
| CD19 FITC | 16.7 | | | | |

CD19 R-PE
Linear regression $y = 0.69 + 0.93x$

Comparison of the CALTAG CD19 TRI-COLOR conjugated monoclonal antibody with the Coulter CD19 RD1 conjugated monoclonal antibody:

| procedure | mean % positive | r² value | slope | Y intercept | n |
| --- | --- | --- | --- | --- | --- |
| CD19 TRI-COLOR | 17.1 | 96.7 | 0.92 | 2.14 | 175 |
| CD19 RD1 | 16.2 | | | | |

CD19 TRI-COLOR
Linear regression $y = 2.14 + 0.92x$

Comparison of the CALTAG CD19 TRI-COLOR conjugated monoclonal antibody with the Coulter CD19 FITC conjugated monoclonal antibody:

| procedure | mean % positive | r² value | slope | Y intercept | n |
| --- | --- | --- | --- | --- | --- |
| CD19 TRI-COLOR | 17.1 | 97.4 | 0.94 | 1.36 | 175 |
| CD19 FITC | 16.7 | | | | |

CD19 TRI-COLOR
Linear regression $y = 1.36 + 0.94x$

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Comparison of the CALTAG CD19 TRI-COLOR conjugated monoclonal antibody with the CALTAG CD19 R-PE conjugated monoclonal antibody:

| procedure | mean % positive | r² value | slope | Y intercept | n |
| --- | --- | --- | --- | --- | --- |
| CD19 TRI-COLOR | 17.1 | 97.6 | 0.99 | -0.56 | 175 |
| CD19 R-PE | 16.4 | | | | |

CD19 TRI-COLOR
Linear regression $y = -0.56 + 0.99x$

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