(211 days)
K93292
No
The document does not mention AI, ML, or related terms, and the description of the device and studies focuses on traditional flow cytometry methods and statistical analysis.
No
This device is for lymphocyte immunophenotyping, which is a diagnostic procedure, not a therapeutic one. It provides information about the patient's immune status but does not treat or cure any condition.
Yes
The device is intended for "lymphocyte immunophenotyping" and the results "may be reported as either percent positive cells, or as absolute counts of lymphocytes and lymphocyte subsets," which are used in medical diagnosis. The performance studies also involve "HIV-antibody positive donors," further indicating its use in diagnosing or monitoring specific health conditions.
No
The device description explicitly states that the system consists of hardware components (ORTHO CytoronAbsolute Laser Flow Cytometer, ORTHO TRIO Monoclonal Antibodies, and Ortho-Count Calibration Kit) in addition to the software (ImmunoCount II Software).
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states it is for "lymphocyte immunophenotyping," which is a diagnostic procedure performed on biological samples (blood).
- Device Description: The system includes components like a flow cytometer, software, monoclonal antibodies, and a calibration kit, all designed to analyze biological samples for diagnostic purposes.
- Input Imaging Modality: Flow cytometry is a technique used to analyze the physical and chemical characteristics of particles in a fluid as they pass through a laser beam. This is a common method in in vitro diagnostics.
- Description of the training set, sample size, data source, and annotation protocol: While not explicitly stated as "training set," the description of the blood specimens from normal and HIV-antibody positive donors used for performance studies indicates the use of biological samples for evaluating the device's diagnostic capabilities.
- Summary of Performance Studies: The performance studies compare the device to a "traditional method (flow cytometer + hematology analyzer)" for determining absolute counts and percentages of lymphocytes and subsets. This comparison to established diagnostic methods further supports its IVD nature.
- Key Metrics: The metrics reported (Mean%, Range%, Mean (cells/μl), Range (cells/μl), R, Slope, Intercept, CV, Mean Slope, CI) are all relevant to evaluating the performance of a diagnostic test.
- Predicate Device(s): The listed predicate devices are other flow cytometers and hematology analyzers, which are also IVD devices used for similar diagnostic purposes.
In summary, the device's intended use, components, methodology, and the context of its performance evaluation all clearly indicate that it is an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
ORTHO ImmunoCount Flow Cytometry System is intended to be used for lymphocyte immunophenotyping. Results may be reported as either percent positive cells, or as absolute counts of lymphocytes and lymphocyte subsets.
Product codes
Not Found
Device Description
ORTHO ImmunoCount Flow Cytometry System consists of ORTHO CytoronAbsolute Laser Flow Cytometer, ImmunoCount II Software, ORTHO TRIO Monoclonal Antibodies for lymphocyte immunophenotyping, and Ortho-Count Calibration Kit for calibration and verification of system calibration.
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
The donors were male (44.5%) and female (55.5%) with an age range of 21 years to 62 years.
The donors were male (88.1%) and female (11.9%) with an age range of 3 years.
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
A total of 200 normal whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for percent positive stained cell determination. A total of 151 normal whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for absolute lymphocyte count determination. These were collected from normal donors.
A total of 119 HIV-antibody positive whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for percent positive stained cell determination. A total of 89 HIV-antibody positive whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for absolute lymphocyte count determination. These were collected from HIV-antibody positive donors.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
The device was evaluated in a clinical study conducted at four laboratories, comparing the ORTHO ImmunoCount Flow Cytometry System to a traditional method combining flow cytometry and hematology analysis.
Study 1: Comparison with Predicate Method (Normal Donors)
- Study Type: Clinical comparison
- Sample Size: 200 normal whole blood specimens for percent positive cell determination, 151 normal whole blood specimens for absolute lymphocyte count determination.
- Key Results:
- For percent positive cells (N=200): Mean percentages for ImmunoCount were very close to Predicate for all lymphocyte subsets (e.g., CD3+CD4+: ImmunoCount 45.5%, Predicate 45.2%; Mean CD3+: ImmunoCount 74.8%, Predicate 73.4%).
- For absolute counts (N=151): Mean counts were comparable to Predicate (e.g., CD3+CD4+: ImmunoCount 855 cells/μl, Predicate 900 cells/μl; Mean CD3+: ImmunoCount 1423 cells/μl, Predicate 1473 cells/μl).
- Linear regression analysis (Table C) for absolute counts showed high R values (ranging from 0.83 to 1.00) and slopes generally close to 1, indicating strong correlation with the predicate method for normal donors.
Study 2: Comparison with Predicate Method (HIV-antibody Positive Donors)
- Study Type: Clinical comparison
- Sample Size: 119 HIV-antibody positive whole blood specimens for percent positive cell determination, 89 HIV-antibody positive whole blood specimens for absolute lymphocyte count determination.
- Key Results:
- For percent positive cells (N=119): Mean percentages were comparable to Predicate (e.g., CD3+CD4+: ImmunoCount 17.7%, Predicate 17.6%; Mean CD3+: ImmunoCount 80.5%, Predicate 78.1%).
- For absolute counts (N=89): Mean counts were comparable to Predicate, though some differences were noted (e.g., CD3+CD4+: ImmunoCount 260 cells/μl, Predicate 313 cells/μl; Mean CD3+: ImmunoCount 1078 cells/μl, Predicate 1261 cells/μl).
- Linear regression analysis (Table D) for absolute counts for combined normal and HIV-positive samples showed R values ranging from 0.88 to 0.96, indicating good correlation.
Study 3: Within Laboratory Reproducibility
- Study Type: Reproducibility study
- Sample Size: N(LOW) = 139; N(NORMAL) = 142; N(HIGH) = 146 (five normal whole blood specimens run in replicates of 10 at three labs in low, normal, and high concentrations).
- Key Results: Low coefficients of variation (CV) for absolute lymphocyte counts, demonstrating strong within-laboratory reproducibility. CVs for CD3+, CD3+CD4+, CD3+CD8+, CD3+(16/19/3) and ISUM Lymph Count were generally below 7%, while CD16+CD3- and CD19+ had higher CVs (up to 16.3%) especially at low counts due to their low population levels.
Study 4: Between Laboratory Reproducibility
- Study Type: Reproducibility study
- Sample Size: Data collected from three independent laboratories (N=139 for low, N=142 for normal, N=146 for high count preparations). Samples run in replicates of 10.
- Key Results: Low coefficients of variation (CV) for absolute lymphocyte counts, demonstrating acceptable between-laboratory reproducibility. Between-site CVs were generally low (e.g., CD3+(4/8/3) 0.9% for low, 1.6% for normal, 2.7% for high). Higher CVs for CD16+CD3- and CD19+ were noted, similar to within-lab results, but did not significantly affect the ImmunoSum (ISUM) CV.
Study 5: Specimen Age Criteria
- Study Type: Stability study
- Sample Size: 64 whole blood specimens (34 normal, 30 HIV-antibody positive)
- Key Results: Samples processed within 24 to 72 hours of collection produced comparable results for absolute counts. Mean slope values were very close to 0, and CI values were narrow, indicating stability over 72 hours.
Study 6: Linearity
- Study Type: Linearity study
- Sample Size: 4 normal whole blood specimens
- Key Results: Demonstrated excellent linearity (correlation coefficient R ranging from 0.99 to 1.00) across a wide range of absolute counts for each lymphocyte subset and ISUM.
Overall Conclusion: The performance of the ImmunoCount Flow Cytometry System was equivalent to the predicate method for determining absolute lymphocyte counts and lymphocyte subset percentages. It also demonstrated excellent within and between laboratory reproducibility, stability for samples up to 72 hours, and linearity over a wide range of counts.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Linear regression R values, Slopes, Intercepts, Mean Percentage, Range Percentage, Mean (cells/μl), Range (cells/μl), Coefficient of Variation (CV).
Predicate Device(s)
K93292
Reference Device(s)
Coulter T 540 K896873, Coulter JT2 K874383, AGROS K895127
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.”
0
(
K954570
510(k) SUMMARY
APR 3 0 1996
SUBMITTER: Ortho Diagnostic Systems Inc 1001 U.S. Highway 202 P.O. Box 350 Raritan, NJ 08869-0606
CONTACT: Joanne Harris Tel.: (908) 218-8404 (908) 218-8168 FAX .:
DEVICE NAME:
ORTHO™ ImmunoCount Flow Cytometry System
ORTHO ImmunoCount Flow Cytometry System includes:
ORTHO CytoronAbsolute™ Laser Flow Cytometer (optional AutoBioSampler)
ORTHO ImmunoCount II Software
ORTHO TRIO Monoclonal Antibodies (Murine)
ORTHO TRIO Control (FITC/PE/CyP) Monocional Antibodies (Murine)
ORTHO TRIO CD4/CD8/CD3 (OKT™4A/OKT8/OKT3) (FITC/PE/CyP) Monoclonal Antibodies (Murine)
ORTHO TRIO CD16/CD19/CD3 (NK/OKB™19A/OKT3) (FITC/PE/CyP) Monoclonal Antibodies (Murine)
Ortho-Count Calibration Kit (currently under review, Ref. No. K-935720)
PREDICATE:
FACScan™ Flow Cytometer Simulset Software Simultest™ reagents K93292
Simultest Control IgG1/IgG2a (IgG1 FITC/IgG2PE)
Simultest LeucoGATE™ (CD45/CD14[Anti-HLe 1/Leu™M3])
Simultest CD3/CD4 (Leu-4/3a)
Simultest CD3/CD8 (Leu-4/Leu-2a)
Simultest CD3/CD16+CD56 (Leu-4/11c+19)
Simultest T and B Cell Test CD3/CD19 (Leu4/Leu12)
Hematology Analyzers
| Coulter T 540 | K896873 |
|---|---|
| Coulter JT2 | K874383 |
| AGROS | K895127 |
DATE: September 29, 1995
1
DEVICE DESCRIPTION:
ORTHO ImmunoCount Flow Cytometry System consists of ORTHO CytoronAbsolute Laser Flow Cytometer, ImmunoCount II Software, ORTHO TRIO Monoclonal Antibodies for lymphocyte immunophenotyping, and Ortho-Count Calibration Kit for calibration and verification of system calibration.
INTENDED USE:
ORTHO ImmunoCount Flow Cytometry System is intended to be used for lymphocyte immunophenotyping. Results may be reported as either percent positive cells, or as absolute counts of lymphocytes and lymphocyte subsets.
TECHNOLOGICAL CHARACTERISTICS:
ORTHO ImmunoCount Flow Cytometry System is substantially equivalent to the combination of flow cytometer, lymphocyte immunophenotyping reagents, and hematology analyzer when used for determination of percentages and absolute counts of lymphocytes and lymphocyte subsets.
i
The traditional method for determination of absolute counts of lymphocyte subsets combines lymphocyte immunophenotyping reagents and a flow cytometer for determination of lymphocyte subset percentages, with a hematology analyzer for determination of total lymphocyte count, to obtain absolute counts of lymphocyte subsets.
Image /page/1/Figure/7 description: The image shows a diagram that outlines the process of calculating the absolute number of CDX+ cells. The process starts with a hematology analyzer to get the total white blood cell count. This value is then multiplied by the percentage of lymphocytes, which is then multiplied by the percentage of positive CDX lymphocytes, which is obtained using flow cytometry and immunophenotyping reagents. The final result is the absolute number of CDX+ cells.
2
PERFORMANCE DATA:
ORTHO ImmunoCount Flow Cytometry System was compared to the traditional method for determining absolute counts of lymphocytes and lymphocyte subsets in a clinical study conducted at four laboratories. The instruments and reagents used in the clinical evaluation of the ORTHO ImmunoCount Flow Cytometry System are:
FACScan™ Flow Cytometer with Simulset Software - Becton Dickinson Immunocytometry Systems
Simultest™ Monoclonal Antibody Immunophenotyping Reagents -Becton Dickinson Immunocytometery Systems
Simultest Control IgG1/1gG22 (IgG1 FITC/IgG22PE) Simultest LeucoGATE™ (CD45/CD14|Anti-HLe-I/LeuTM-M3 )) Simultest CD3/CD4 (Leu-4/3a) Simultest CD3/CD8 (Leu-4/Leu-2a) Simultest CD3/CD16+CD56 (Leu-4/11c+19) Simultest T and B Cell Test - CD3/CD19 (Leu4/Leu12)
Coulter® JT2 Automated Hematology Analyzer -Coulter Electronics
Coulter T Series Automated Hematology Analyzer -Coulter Electronics
Argos Hematology Analyzer -ABX France
Blood specimens were collected from 200 apparently healthy donors. Normal donors were defined as any person who was not clinically diagnosed with an immunodeficiency disease or hematological malignancy. The donors were male (44.5%) and female (55.5%) with an age range of 21 years to 62 years. Three geographically distinct clinical sites in the United States were used to determine absolute lymphocyte counts and four geographically distinct clinical sites in the United States were used to determine the percent positive stained cells. A total of 200 normal whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for percent positive stained cell determination. A total of 151 normal whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for absolute lymphocyte count determination.
3
| IMMUNOCOUNT SYSTEM
LYMPHOCYTE SUBSET PERCENT POSITIVE
| NORMAL DONORS N = 200 | |||||
|---|---|---|---|---|---|
| ImmunoCount | Mean% | Range% | Predicate | Mean% | Range% |
| CD3+CD4+ | 45.5 | 20 - 68 | CD3+CD4+ | 45.2 | 20 - 65 |
| CD3+ (4/8/3) | 74.5 | 36 - 101 | CD3+ (3/4) | 73.2 | 29 - 91 |
| CD3+CD8+ | 24.5 | 9 - 48 | CD3+CD8+ | 26.9 | 9 - 55 |
| CD3+ (4/8/3) | 74.5 | 36 - 101 | CD3+ (3/8) | 73.5 | 31 - 90 |
| CD16+CD3- | 11.6 | 1 - 58 | CD(16&56)+CD3- | 13.1 | 2 - 65 |
| CD3+ (16/19/3) | 75.1 | 39 - 89 | CD3+ (3/16&56) | 73.6 | 31 - 91 |
| CD19+ | 13.3 | 3 - 51 | CD19+ | 13.3 | 3 - 28 |
| CD3+ (16/19/3) | 75.1 | 39 - 89 | CD3+ (3/19) | 73.3 | 30 - 89 |
| Mean CD3+ | 74.8 | 38 - 95 | Mean CD3+ | 73.4 | 30 - 90 |
| IMMUNOCOUNT SYSTEM | |||||
| LYMPHOCYTE SUBSET ABSOLUTE COUNTS | |||||
| NORMAL DONORS N = 151 | |||||
| ImmunoCount | Mean (cells/μl) | Range (cells/μl) | Predicate | Mean (cells/μl) | Range (cells/μl) |
| CD3+CD4+ | 855 | 257 - 1785 | CD3+CD4+ | 900 | 250 - 1940 |
| CD3+ (4/8/3) | 1417 | 477 - 3372 | CD3+ (3/4) | 1472 | 480 - 3530 |
| CD3+CD8+ | 481 | 112 - 1588 | CD3+CD8+ | 551 | 100 - 1750 |
| CD3+ (4/8/3) | 1417 | 477 - 3372 | CD3+ (3/8) | 1477 | 470 - 3500 |
| CD16+CD3- | 221 | 19 - 1536 | CD(16&56)+CD3- | 266 | 60 - 2280 |
| CD3+ (16/19/3) | 1428 | 512 - 3423 | CD3+ (3/16&56) | 1476 | 470 - 3530 |
| CD19+ | 255 | 35 - 826 | CD19+ | 270 | 50 - 840 |
| CD3+ (16/19/3) | 1428 | 512 - 3423 | CD3+ (3/19) | 1469 | 480 - 3540 |
| Mean CD3+ | 1423 | 494 - 3398 | Mean CD3+ | 1473 | 475 - 3525 |
TABLE A
4
Blood specimens were collected from 119 HIV-antibody positive donors. HIV-antibody positive donors were defined as any individual positive for the HIV antibody inclusive of AIDS or AIDS Related Complex (ARC) according to the current criteria for these diseases, Morbidity Mortality Weekly Report, Vol. 41/No. 31, May 8, 1992). The donors were male (88.1%) and female (11.9%) with an age range of 3 years. Three geographically distinct clinical sites in the United States were used to determine absolute lymphocyte counts and four geographically distinct clinical sites in the United States were used to determine the percent positive stained cells. A total of 119 HIV-antibody positive whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for percent positive stained cell determination. A total of 89 HIV-antibody positive whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System for absolute lymphocyte count determination.
5
| IMMUNOCOUNT SYSTEM
| LYMPHOCYTE SUBSET PERCENT POSITIVE | |||||
|---|---|---|---|---|---|
| HIV ANTIBODY POSITIVE DONORS N = 119 | |||||
| ImmunoCount | Mean% | Range% | Predicate | Mean% | Range% |
| CD3+CD4+ | 17.7 | 0 - 55 | CD3+CD4+ | 17.6 | 0 - 56 |
| CD3+ (4/8/3) | 80.1 | 18 - 115 | CD3+ (3/4) | 78.1 | 24 - 96 |
| CD3+CD8+ | 57.3 | 7 - 89 | CD3+CD8+ | 58.6 | 16 - 91 |
| CD3+ (4/8/3) | 80.1 | 18 - 115 | CD3+ (3/8) | 78.3 | 21 - 96 |
| CD16+CD3- | 8.8 | 0 - 45 | CD(16&56)+CD3- | 10.6 | 1 - 50 |
| CD3+ (16/19/3) | 80.9 | 23 - 95 | CD3+ (3/16&56) | 78.4 | 14 - 97 |
| CD19+ | 10.4 | 2 - 42 | CD19+ | 10.8 | 2 - 40 |
| CD3+ (16/19/3) | 80.9 | 23 - 95 | CD3+ (3/19) | 77.4 | 22 - 95 |
| Mean CD3+ | 80.5 | 21 - 103 | Mean CD3+ | 78.1 | 20 - 96 |
| IMMUNOCOUNT SYSTEM | |||||
| LYMPHOCYTE SUBSET ABSOLUTE COUNTS | |||||
| HIV ANTIBODY POSITIVE DONORS N = 89 | |||||
| ImmunoCount | Mean | ||||
| (cells/µl) | Range | ||||
| (cells/µl) | Predicate | Mean | |||
| (cells/µl) | Range | ||||
| (cells/µl) | |||||
| CD3+CD4+ | 260 | 0 - 1029 | CD3+CD4+ | 313 | 0 - 1400 |
| CD3+ (4/8/3) | 1078 | 42 - 2986 | CD3+ (3/4) | 1262 | 50 - 3650 |
| CD3+CD8+ | 758 | 39 - 1961 | CD3+CD8+ | 931 | 40 - 2810 |
| CD3+ (4/8/3) | 1078 | 42 - 2986 | CD3+ (3/8) | 1267 | 40 - 3600 |
| CD16+CD3- | 93 | 5 - 572 | CD(16&56)+CD3- | 144 | 20 - 720 |
| CD3+ (16/19/3) | 1079 | 34 - 2927 | CD3+ (3/16&56) | 1267 | 30 - 3650 |
| CD19+ | 131 | 13 - 554 | CD19+ | 170 | 20 - 870 |
| CD3+ (16/19/3) | 1079 | 34 - 2927 | CD3+ (3/19) | 1248 | 40 - 3650 |
| Mean CD3+ | 1078 | 38 - 2957 | Mean CD3+ | 1261 | 40 - 3638 |
TABLE B
6
Linear regression analyses of absolute lymphocyte counts and percent positive cells obtained using the ORTHO ImmunoCount Flow Cytometry System and the traditional (predicate) method are presented below.
| ImmunoCount | Predicate | Graph | R | Slope | Intercept |
|---|---|---|---|---|---|
| CD4⁺ (4/8/3) | CD4⁺ (3/4) | 3a | 1.00 | 1.00 | 0 |
| CD3⁺ (4/8/3) | CD3⁺ (3/4) | 3b | 0.83 | 0.87 | 12 |
| CD8⁺ (4/8/3) | CD8⁺ (3/8) | 3c | 0.98 | 1.01 | -2 |
| CD3⁺ (4/8/3) | CD3⁺ (3/8) | 3d | 0.85 | 0.90 | 9 |
| CD16⁺ CD3⁺ (16/19/3) | CD16⁺ CD3⁺ (3/16+65) | 3e | 0.91 | 0.90 | 0 |
| CD3⁺ (16/19/3) | CD3⁺ (3/16+56) | 3f | 0.93 | 0.88 | 11 |
| CD19⁺ (16/19/3) | CD19⁺ (3/19) | 3g | 0.88 | 0.98 | 0 |
| CD3⁺ (16/19/3) | CD3⁺ (3/19) | 3h | 0.91 | 0.91 | 9 |
| Mean CD3⁺ | Mean CD3⁺ | 3i | 0.91 | 0.91 | 9 |
TABLE C
TABLE D
| NORMAL N = 151
AND
| HIV ANTIBODY POSITIVE N = 89 | |||||
|---|---|---|---|---|---|
| ImmunoCount | Predicate | Graph | R | Slope | Intercept |
| CD4+ (4/8/3) | CD4+ (3/4) | 3A | 0.94 | 0.89 | 25 |
| CD3- (4/8/3) | CD3- (3/4) | 3B | 0.89 | 0.84 | 119 |
| CD8+ (4/8/3) | CD8+ (3/8) | 3C | 0.92 | 0.78 | 43 |
| CD3+ (4/8/3) | CD3+ (3/8) | 3D | 0.89 | 0.84 | 112 |
| CD16+ CD3- (16/19/3) | CD16+ CD3- (3/16+65) | 3E | 0.94 | 0.76 | 7 |
| CD3- (16/19/3) | CD3+ (3/16+56) | 3F | 0.88 | 0.84 | 123 |
| CD19+ (16/19/3) | CD19+ (3/19) | 3G | 0.89 | 0.86 | 10 |
| CD3+ (16/19/3) | CD3+ (3/19) | 3H | 0.89 | 0.85 | 124 |
| Mean CD3+ | Mean CD3+ | 3I | 0.89 | 0.84 | 118 |
| ISUM | Lymphocyte | 3J | 0.88 | 0.86 | 87 |
| All lymphocyte subsets | All lymphocyte subsets | 3K | 0.96 | 0.89 | 3 |
7
WITHIN LABORATORY REPRODUCIBILITY
Five normal whole blood specimens were used in a within-laboratory reproducibility study at three independent laboratories. The normal whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and run in the ORTHO ImmunoCount Flow Cytometry System. The samples were run diluted (0.5X WBC), concentrated (4X WBC) and undiluted to simulate low, high and normal samples. The leukocyte-depleted blood was prepared using a Sepracell" unit (Baxter Fenwall). Buffy coat preparations were prepared from the donor samples, counted and adjusted to a 4X concentration. Serial dilutions were made from the 4X concentrate of each sample's buffy coat and diluting this concentrate with leukocyte depleted whole blood from the same sample. All samples were run in replicates of 10. The coefficients of variations (CV) for normal, concentrated and diluted samples were compared and are contained in Table E. The coefficient of variation was generated from variability between the ten replicates from each of the three laboratories. The low CV values demonstrate strong within-laboratory reproducibility for each lymphocyte subset. The CV values for normal and low counts for CD16+ and CD19+ values were higher due to the low counts for these populations. The higher CVs for CD16+ and CD19+ values do not significantly affect the ImmunoSum (ISUM) CV.
| TABLE E
ORTHO IMMUNOCOUNT SYSTEM
WITHIN-LABORATORY REPRODUCIBILITY
COEFFICIENT OF VARIATION OF ABSOLUTE LYMPHOCYTE COUNTS
| N(LOW) = 139; N(NORMAL) = 142; N(HIGH) = 146 | |||
|---|---|---|---|
| LOW | |||
| CV | NORMAL | ||
| CV | HIGH | ||
| CV | |||
| ORTHO ImmunoCount | |||
| System | |||
| CD3⁺ (4/8/3) | 5.2 | 4.9 | 4.3 |
| CD3⁺CD4⁺ | 6.2 | 5.2 | 4.5 |
| CD3⁺CD8⁺ | 6.4 | 6.4 | 4.5 |
| CD16⁺CD3⁻ | 16.3 | 10.1 | 6.0 |
| CD3⁺ (16/19/3) | 5.0 | 4.9 | 4.3 |
| CD19⁺ | 12.0 | 10.0 | 5.7 |
| ISUM Lymph Count | 5.0 | 4.9 | 4.2 |
8
BETWEEN LABORATORY REPRODUCIBILITY
Five normal whole blood specimens were compared between three independent laboratories. The whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and analyzed in the ORTHO ImmunoCount Flow Cytometry System. Low, normal and high count preparations were made according to the method described in the Within-Laboratory Reproducibility section. All samples were run in replicates of ten. The data collected from all laboratories are shown in Table F. The coefficient of variation was generated by comparing the three laboratories absolute lymphocyte subset means from the 10 replicates. The low CV values demonstrate acceptable between-laboratory reproducibility at all three laboratories. The CV values for normal and low counts for CD16+ and CD19+ values were higher due to the low counts for these populations. The higher CVs for CD16+ and CD19+ values do not significantly affect the ImmunoSum (ISUM) CV.
| TABLE F | ||||
|---|---|---|---|---|
| ORTHO IMMUNOCOUNT SYSTEM | ||||
| BETWEEN-LABORATORY REPRODUCIBILITY | ||||
| COEFFICIENT OF VARIATION OF ABSOLUTE LYMPHOCYTE COUNTS | ||||
| LOW COUNT PREPARATION : N = 139 | ||||
| ORTHO ImmunoCount | ||||
| System | Lab 1 Mean | |||
| Cells/μL | Lab 2 Mean | |||
| Cells/μL | Lab 3 Mean | |||
| Cells/μL | Between-site CV | |||
| CD3+(4/8/3) | 750 | 740 | 736 | 0.9 |
| CD3+CD4+ | 481 | 480 | 473 | 0.9 |
| CD3+CD8+ | 231 | 221 | 224 | 2.2 |
| CD16+CD3 | 146 | 122 | 160 | 13.1 |
| CD3+(16/19/3) | 754 | 748 | 740 | 0.9 |
| CD19+ | 116 | 98 | 121 | 10.3 |
| ISUM Lymph count | 1011 | 963 | 1009 | 2.6 |
| NORMAL COUNT PREPARATION: N = 142 | ||||
| ORTHO ImmunoCount | ||||
| System | Lab 1 Mean | |||
| Cells/μL | Lab 2 Mean | |||
| Cells/μL | Lab 3 Mean | |||
| Cells/μL | Between-site CV | |||
| CD3+(4/8/3) | 1494 | 1468 | 1445 | 1.6 |
| CD3+CD4+ | 958 | 953 | 933 | 1.3 |
| CD3+CD8+ | 456 | 434 | 437 | 2.6 |
| CD16+ CD3 | 363 | 290 | 309 | 11.5 |
| CD3+(16/19/3) | 1502 | 1515 | 1489 | 0.8 |
| CD19+ | 221 | 212 | 250 | 8.5 |
| ISUM Lymph count | 2067 | 2002 | 2028 | 1.6 |
| HIGH COUNT PREPARATION: N = 146 | ||||
| ORTHO ImmunoCount | ||||
| System | Lab 1 Mean | |||
| Cells/μL | Lab 2 Mean | |||
| Cells/μL | Lab 3 Mean | |||
| Cells/μL | Between-site CV | |||
| CD3+(4/8/3) | 5961 | 5718 | 5663 | 2.7 |
| CD3+CD4+ | 3786 | 3687 | 3667 | 1.7 |
| CD3+CD8+ | 1835 | 1693 | 1683 | 4.8 |
| CD16+ CD3 | 1548 | 1334 | 1347 | 8.4 |
| CD3+(16/19/3) | 5990 | 5929 | 5737 | 2.2 |
| CD19+ | 962 | 844 | 999 | 8.3 |
| ISUM Lymph count | 8417 | 8028 | 7959 | 3.0 |
Ortho Diagnostic Systems Inc. ORTHO™ ImmunoCount Flow Cytometry System Page 480
9
SPECIMEN AGE CRITERIA
A total of 64 whole blood specimens, normal (34 donors) and HIV-antibody positive (30 donors), were tested at three external clinical sites over an extended time period. The samples were collected in EDTA anticoagulant tubes. Samples were prepared and tested within 24 hours, 48 hours and 72 hours after collection. The absolute counts for each ORTHO TRIO Monclonal Antibody, run in the ORTHO ImmunoCount Flow Cytometry System, are compared in below. The data demonstrates that samples processed within 24 to 72 hours of collection produce comparable results. CI refers to confidence interval of the mean slope.
TABLE G
| ORTHO IMMUNOCOUNT SYSTEM
ABSOLUTE LYMPHOCYTE COUNTS OVER A 72-HOUR TIME PERIOD
| NORMAL DONORS N=34; HIV-ANTIBODY POSITIVE DONORS N=30 | |||||
|---|---|---|---|---|---|
| ORTHO | |||||
| ImmunoCount | |||||
| System | Mean Count | ||||
| 24 Hours | |||||
| (Cells/µL) | Mean Count | ||||
| 48 Hours | |||||
| (Cells/µL) | Mean Count | ||||
| 72 Hours | |||||
| (Cells/µL) | Mean Slope | CI | |||
| CD3+ (4/8/3) | 1367 | 1388 | 1362 | -1.5 | 2.3 |
| CD3+CD4+ | 615 | 631 | 619 | -0.4 | 1.0 |
| CD3+CD8+ | 664 | 665 | 653 | -1.2 | 1.3 |
| CD16+CD3- | 188 | 199 | 249 | 0.8 | 1.4 |
| CD3+ (16/19/3) | 1360 | 1356 | 1338 | -3.6 | 2.4 |
| CD19+ | 207 | 206 | 192 | -0.0 | 0.7 |
| ISUM | |||||
| Lymph Count | 1755 | 1761 | 1779 | -2.9 | 2.6 |
LINEARITY
Four normal whole blood specimens were used in a linearity study at Ortho Diagnostic Systems. The whole blood specimens were stained with ORTHO TRIO Monoclonal Antibodies and run in the ORTHO ImmunoCount Flow Cytometry System. The samples were run diluted (0.5X, 0.25X, 0.125X, 0.065X), concentrated (5X, 4X, 2X) and undiluted (1X) to yield low, high, and normal cell counts. The leukocyte depleted blood was prepared using a Sepracell™ unit (Baxter Fenwall.) Buffy coat preparations were prepared from the donor sample, counted and diluted to a 5X concentration. Serial dilutions were made fromt he 5X concentrate of each samples buffy coat and diluting this concentrate with leukocyte depleted whole blood from the same unit. Table H shows the correlation coefficient of the linear regression analysis of absolute counts for each marker and ISUM.
10
| TABLE
| p | B |
|---|---|
| ------------ | --- |
| ORTHO IMMUNOCOUNT SYSTEM
LINEAR RANGE OF ABSOLUTE COUNTS
| NORMAL DONORS N=4 | ||
|---|---|---|
| ORTHO | ||
| ImmunoCount | ||
| System | R | Range |
| Cells/µL | ||
| CD3+(4/8/3) | 1.00 | 75 - 9829 |
| CD3+CD4+ | 1.00 | 0 - 6300 |
| CD3+CD8+ | 1.00 | 0 - 2952 |
| CD16+CD3- | 0.99 | 16 - 1796 |
| CD3+ (16/19/3) | 1.00 | 59 - 10390 |
| CD19+ | 1.00 | 19 - 2507 |
| ISUM Lymph Count | 1.00 | 95 - 14694 |
CONCLUSION
- The performance of the ImmunoCount Flow Cytometry System was equivalent to ● the predicate method for the determination of absolute lymphocyte counts and lymphocyte subset percentages and absolute counts CD3+, CD3+CD4+, CD3+CD8+, CD16+CD3-, and CD19+.
- ORTHO ImmunoCount Flow Cytometry System demonstrated excellent within and between laboratory reproducibility for low, normal, and high absolute counts for total lymphocytes and lymphocyte subsets
- ORTHO ImmunoCount Flow Cytometry System, when used to assay samples at extended time points after collection (48 through 96 hours after collection), showed that samples assays up to 72 hours after collection produced equivalent absolute counts for total lymphocytes and for each of the lymphocyte subsets CD3+, CD3+CD4+, CD3+CD8+, CD16+CD3-, and CD19+
- ORTHO ImmunoCount Flow Cytometry System demonstrated excellent linearity . when compared against dilution over a wide range of absolute counts for each lymphocyte subset and ISUM.