ORTHO IMMUNOCOUNT FLOW CYTOMETRY SYSTEM
Device Facts
| Record ID | K954570 |
|---|---|
| Device Name | ORTHO IMMUNOCOUNT FLOW CYTOMETRY SYSTEM |
| Applicant | Ortho Diagnostic Systems, Inc. |
| Product Code | GKZ · Hematology |
| Decision Date | Apr 30, 1996 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 864.5220 |
| Device Class | Class 2 |
| Attributes | Pediatric |
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.
Device Story
System performs lymphocyte immunophenotyping using laser flow cytometry. Inputs: whole blood specimens stained with ORTHO TRIO monoclonal antibodies (CD4/CD8/CD3, CD16/CD19/CD3). Operation: CytoronAbsolute flow cytometer measures cell populations; ImmunoCount II software processes signals to calculate percent positive cells and absolute counts of lymphocyte subsets. Output: quantitative lymphocyte subset data. Used in clinical laboratories by trained personnel. Replaces traditional method requiring separate flow cytometer and hematology analyzer. Benefits: provides direct absolute counts of lymphocyte subsets, streamlining diagnostic workflows for immunodeficiency and hematological monitoring.
Clinical Evidence
Clinical study at 4 laboratories compared ImmunoCount to traditional methods (flow cytometer + hematology analyzer). Evaluated 200 normal donors and 119 HIV-antibody positive donors. Primary endpoints: absolute lymphocyte counts and percent positive cells. Results showed high correlation (R=0.83-1.00) and equivalent performance. Reproducibility studies (within and between labs) showed low CVs for lymphocyte subsets. Linearity confirmed across wide cell count ranges. Samples stable for up to 72 hours post-collection.
Technological Characteristics
System includes CytoronAbsolute laser flow cytometer, ImmunoCount II software, and murine monoclonal antibodies. Measures lymphocyte subsets via fluorescence and light scatter. Connectivity: standalone system. Calibration via Ortho-Count Calibration Kit. Software performs automated calculation of absolute counts from flow cytometry data.
Indications for Use
Indicated for lymphocyte immunophenotyping in patients, including those with HIV-antibody positive status (AIDS/ARC) and healthy donors, aged 3 to 75 years.
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
- FACScan Flow Cytometer (K93292)
- Simulset Software (K93292)
- Simultest reagents (K93292)
- Coulter T 540 (K896873)
- Coulter JT2 (K874383)
- AGROS (K895127)
Reference Devices
- Ortho-Count Calibration Kit (K935720)
Related Devices
- K141932 — AQUIOS CL FLOW CYTOMETER, AQUIOS TETRA-1 PANEL,AQUIOS TETRA-2+PANEL, AQUIOS IMMUNO-TROL, AQUIOS IMMUNO-TROL LOW, AQUIOS · Beckman Coulter, Inc. · Apr 10, 2015
- K170974 — BD FACSLyric Flow Cytometer (3-1, 4-2, 4-2-2 and 4-3-3 optical configurations), BD FACSuite Clinical Software, BD Multitest 6-Color Assays, BD Multitest 4-Color Assays, BD Multitest 6-Color TBNK · Becton, Dickinson and Company · Jul 3, 2017
- K121445 — TETRACHROME REAGENTS AND TETRACXP SYSTEM · Beckman Coulter, Inc. · Jul 26, 2013
Submission Summary (Full Text)
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K954570
510(k) SUMMARY
APR 30 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
FAX.: (908) 218-8168
## 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)
Monoclonal 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/IgG₂ₐPE)
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
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# 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.
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.
| HEMATOLOGY ANALYZER | FLOW CYTOMETER
IMMUNOPHENOTYPING
REAGENTS |
| --- | --- |
| ↓
Total white blood cell count X % lymphocytes X % positive CDX lymphocytes = absolute number of CDX+cells
mm³
mm³ | |
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# 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 Immunocytometry Systems
Simultest Control IgG₁/IgG₂ₐ (IgG₁ FITC/IgG₂ₐPE)
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)
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.
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TABLE A
| 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 |
| ISUM | 1905 | 667 - 4241 | lymphocyte | 2010 | 700 - 4159 |
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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 to 75 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.
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TABLE B
| 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 |
| ISUM | 1303 | 118 - 3691 | lymphocyte | 1568 | 200 - 4600 |
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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.
TABLE C
| NORMAL N = 200
AND
HIV ANTIBODY POSITIVE N = 119 | | | | | |
| --- | --- | --- | --- | --- | --- |
| 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 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 |
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# 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 | NORMAL | HIGH |
| ORTHO ImmunoCount System | CV | CV | CV |
| 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 |
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# 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 |
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# 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 from the 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.
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# TABLE H
| 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.
