(462 days)
Ortho-mune OKT3 Monoclonal Antibody (Murine) FITC Conjugate, Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin conjugate
Not Found
No
The summary describes a flow cytometry reagent and its performance characteristics, with no mention of AI or ML in the device description, intended use, or performance studies. The analysis focuses on antibody binding and flow cytometer performance comparisons.
No
The device is described as an in vitro diagnostic reagent used for the identification and enumeration of specific T lymphocytes in whole blood by flow cytometry, not for treating any condition.
Yes
Explanation: The "Intended Use / Indications for Use" section states that the device is "intended for use in identification and enumeration of CD3+ and CD8+ human T lymphocytes in whole blood by flow cytometry." Identifying and enumerating specific cell types in blood is a diagnostic function, as it provides information used to diagnose, treat, or prevent disease. The performance studies also involve "normal donors, and 77 AIDS/ARC patients," indicating its use in assessing disease states.
No
The device description clearly states it is a blend of monoclonal antibodies conjugated to fluorochromes, which are physical reagents, not software. The summary also describes performance studies involving staining and analyzing whole blood specimens using a flow cytometer, further indicating a hardware and reagent-based device.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states it's for "identification and enumeration of CD3+ and CD8+ human T lymphocytes in whole blood by flow cytometry." This is a diagnostic purpose performed in vitro (outside the body) on a biological sample (whole blood).
- Device Description: It's a reagent (monoclonal antibody blend) used to stain cells in a biological sample for analysis.
- Input Imaging Modality: Flow cytometry is a technique used to analyze characteristics of cells in a fluid, which is a common method in in vitro diagnostics.
- Anatomical Site: Whole blood is a biological specimen.
- Performance Studies: The studies described involve analyzing patient samples (normal donors and AIDS/ARC patients) to assess the device's performance in identifying and enumerating specific cell populations, which is typical for validating an IVD.
- Predicate Devices: The predicate devices are also described as monoclonal antibodies used for similar diagnostic purposes, further supporting the IVD classification.
The information provided clearly indicates that this device is a reagent intended for use in a laboratory setting to perform diagnostic tests on human biological samples.
N/A
Intended Use / Indications for 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 cvtometry. 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.
Product codes
- Not Found
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 isothiccyanate and phycoerythrin respectively.
Mentions image processing
- Not Found
Mentions AI, DNN, or ML
- Not Found
Input Imaging Modality
- Not Found
Anatomical Site
- Not Found
Indicated Patient Age Range
- Not Found
Intended User / Care Setting
- Not Found
Description of the training set, sample size, data source, and annotation protocol
- Not Found
Description of the test set, sample size, data source, and annotation protocol
- Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
- 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 Orthomune 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.
- These studies demonstrate that the performance of the two color reagent Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT3) Monoclonal Antibody (Murine) is equivalent to the single color reagents Ortho-mune OKT3 FITC Conjugate and OKT8 PE equiration to the single single of total percent CD3+ and total percent CD8+ cells in whole blood by flow cytometry.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
- 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 CYTORONABSOLUTE (Y axis), and for the COULTER EPICS (X axis) versus the CYTORONABSOLUTE (Y axis).
- 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.
- 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.
- 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.
- Chart 1: Y = -0.359 + 0.976(X), with an R-value of 0.928.
- Chart 2: Y = 5.547 + 0.882(X) and an R-value of 0.969.
- Chart 3: Y = -0.3519 + 1.03999(x), with an R-value of 0.9955.
- Chart 4: Y = 2.707 + 0.981(X) and a correlation coefficient R = 0.983.
- Tables 4 and 5 report CV values for within and between laboratory reproducibility.
- Tables 6 reports on linearity analysis, with slopes for TOTAL CD3+ ranging from 0.999 to 0.999, and R values of 1.000. For CD3+CD8+, slopes range from 0.999 to 1.001, with R values of 1.000.
Predicate Device(s)
- Ortho-mune OKT™3 Monoclonal Antibody (Murine) FITC Conjugate, Ortho-mune OKT8 Monoclonal Antibody (Murine) Phycoerythrin Conjugate
Reference Device(s)
- Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
- Not Found
§ 864.5220 Automated differential cell counter.
(a)
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.(b)
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.”
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REVISED 510(k) SUMMARY
MAY 10 1996
Joanne Harris CONTACT: SUBMITTER: Ortho Diagnostic Systems 1001 U.S. Hwy 202 Tel: (908) 218-8404 Fax: (908) 218-8168 Raritan, NJ 08869-0606
Ortho-mune™ OK-COMBO DEVICE NAME: CD3-FITC/CD8-PE (OKTTM3/OKT8) Monoclonal Antibody (Murine)
Ortho-mune™ OKT™3 PREDICATE: 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 isothiccyanate 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 cvtometry. 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
1
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 Orthomune 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.
| 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 |
TABLE 1
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K95-0482 Additional Information Submitted February, 1996 Page 187
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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 i
Image /page/3/Figure/1 description: The image shows the text "OK-COMBO CD3/CD8 vs Ortho-mune OKT3 FITC". The text is in bold and is centered in the image. The text appears to be a title or heading.
Image /page/3/Figure/2 description: This image is a scatter plot comparing "OK-COMBO Reagent" and "Predicate Reagent". The x-axis represents "Predicate Reagent", while the y-axis represents "OK-COMBO Reagent". The data points are scattered around a line, and the equation of the line is given as Y = -0.359 + 0.976(X), with an R-value of 0.928.
TOTAL CD3+ PERCENT
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K95-0482 Additional Information Submitted February, 1996 Page 189
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CHART 2
OK-COMBO CD3/CD8 vs Ortho-mune OKT8 PE
Image /page/4/Figure/2 description: This image is a scatter plot that compares the OK-COMBO Reagent to the Predicate Reagent. The x-axis represents the Predicate Reagent, and the y-axis represents the OK-COMBO Reagent. The plot shows a positive correlation between the two reagents, with the equation of the line of best fit being Y = 5.547 + 0.882(X) and an R-value of 0.969.
TOTAL CD8+ PERCENT
These studies demonstrate that the performance of the two color reagent Ortho-mune OK-COMBO CD3-FITC/CD8-PE (OKT3/OKT3) Monoclonal Antibody (Murine) is equivalent to the single color reagents Ortho-mune OKT3 FITC Conjugate and OKT8 PE equiration to the single single of total percent CD3+ and total percent CD8+ cells in whole blood by flow cytometry.
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. Nc. K95-0482 Additional Information Submitted February, 1996 Page 190
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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.
6
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 CYTORONABSOLUTE (Y axis), and for the COULTER EPICS (X axis) versus the CYTORONABSOLUTE (Y axis). Results for percent CD3+CD8+ cells using Ortho-mune OK-COMBO CD3-FITC/CD8-PE and the CYTORONABSOLUTE versus Simultest CD3/CD8 (Leu-4/2a) using the FACScan are presented in Chart 3.
CHART 3
Image /page/6/Figure/2 description: This image is a scatter plot comparing CYTORONABSOLUTE and FACScan measurements. The plot includes data points, a trendline, and an equation describing the relationship between the two variables. The equation is Y = -0.3519 + 1.03999(x), with an R-value of 0.9955, indicating a strong positive correlation. The plot is labeled as 'Site B' and measures 'Percent CD3+CD8+'.
7
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.
Image /page/7/Figure/1 description: The image is a scatter plot comparing CYTORONABSOLUTE and COULTER EPICS at Site A, measuring Percent CD3+CD8+. The x-axis represents COULTER EPICS, and the y-axis represents CYTORONABSOLUTE, with values ranging from 0 to 80. A regression line is plotted through the data points, with the equation Y = 2.707 + 0.981(X) and a correlation coefficient R = 0.983.
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.
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K95-0482 Additional Information Submitted February, 1996 Page 193
8
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.
| 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 |
TABLE 4
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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 | ||||
| Mean Percent | |||||
| Positive | |||||
| (All Donors) | SITE B | ||||
| Mean Percent | |||||
| Positive | |||||
| (All Donors) | SITE C | ||||
| Mean Percent | |||||
| Positive | |||||
| (All Donors) | ACROSS SITE | ||||
| 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.
10
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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Image /page/11/Figure/1 description: This image is a scatter plot titled "OK - COMBO CD3/CD3 CD3+". The x-axis is labeled "Expected CDS+ Counts" and ranges from 0 to 14000. The y-axis is labeled "Observed CDS+ Counts" and ranges from 0 to 14000. A line of best fit is plotted through the data points.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------DONOR *** 71 0-0-0 73 & & △ 74
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K4/5-0482 Additional Information Submitted February, 1996 Page 197
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CHART 6
OK - COMBO CD3/CD8 CD3+CD8+
Image /page/12/Figure/1 description: This image is a scatter plot with a line through the data. The x-axis is labeled "Expected CDS+CD8+ Counts" and ranges from 0 to 4000. The y-axis is labeled "Observed CDS+CD8+ Counts" and ranges from 0 to 4000. The data points are clustered around the line, indicating a strong correlation between the expected and observed counts.
DONOR *** 71 0-3 0 72 �-� 73 & & & 74
Ortho Diagnostic Systems Inc. Ortho-mune OK-COMBO CD3-FITC/CD8-PE Ref. No. K.95-0482 Additional Information Submitted February, 1996 Page 198
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TABLE 6
| OK-COMBO CD3/CD8 | ||||||
|---|---|---|---|---|---|---|
| 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.