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 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.

AI/ML Overview

Here's an analysis of the provided text, focusing on the acceptance criteria and study details for the ORTHO ImmunoCount Flow Cytometry System:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the comparison to a "predicate method" and the demonstration of linearity and reproducibility. The tables provided (TABLE A, B, C, D, E, F, G, H) present the performance data, which implicitly serves as the "reported device performance." Since explicit numerical acceptance criteria (e.g., "R-value > 0.95") are not stated, I will infer them from the data presented, particularly the R-values, slopes, and intercepts for comparability, and CVs for reproducibility. For comparability, "equivalent" means the values are close to the predicate. For reproducibility, "low CV" is the criterion. For linearity, an R-value close to 1.00 is the criterion.

Acceptance Criteria Category	Specific Metric	Implied Acceptance Criterion (from Predicate/Reproducibility)	Reported Device Performance (ORTHO ImmunoCount)	Met/Not Met (Based on typical expectations for medical devices)
Comparability (Predicate Method)
Percent Positive Cells (Normal Donors)	Mean % difference vs. Predicate (for various subsets like CD3+CD4+, CD16+CD3-, CD19+, Mean CD3+)	Mean % difference should be minimal, suggesting equivalence to the predicate. Range overlap.	See TABLE A. Differences in mean % are generally small (e.g., CD3+CD4+ ImmunoCount 45.5% vs. Predicate 45.2%; CD16+CD3- ImmunoCount 11.6% vs. Predicate 13.1%). Ranges largely overlap.	Met
Absolute Counts (Normal Donors)	Mean (cells/µL) difference vs. Predicate (for various subsets)	Mean (cells/µL) difference should be minimal, suggesting equivalence to the predicate. Range overlap.	See TABLE A. Differences in mean (cells/µL) are generally small (e.g., CD3+CD4+ ImmunoCount 855 vs. Predicate 900; CD16+CD3- ImmunoCount 221 vs. Predicate 266). Ranges largely overlap.	Met
Percent Positive Cells (HIV+ Donors)	Mean % difference vs. Predicate (for various subsets)	Mean % difference should be minimal, suggesting equivalence to the predicate. Range overlap.	See TABLE B. Differences in mean % are generally small (e.g., CD3+CD4+ ImmunoCount 17.7% vs. Predicate 17.6%; CD16+CD3- ImmunoCount 8.8% vs. Predicate 10.6%). Ranges largely overlap.	Met
Absolute Counts (HIV+ Donors)	Mean (cells/µL) difference vs. Predicate (for various subsets)	Mean (cells/µL) difference should be minimal, suggesting equivalence to the predicate. Range overlap.	See TABLE B. Differences in mean (cells/µL) are generally small (e.g., CD3+CD4+ ImmunoCount 260 vs. Predicate 313; CD16+CD3- ImmunoCount 93 vs. Predicate 144). Ranges largely overlap.	Met
Linear Regression (Normal Donors - Percent)	R-value between ImmunoCount and Predicate (for % positive cells)	R-value > 0.85 (typical for good correlation), Slope close to 1, Intercept close to 0.	See TABLE C. R-values range from 0.83 (CD3+ (4/8/3) vs CD3+ (3/4)) to 1.00 (CD4+ (4/8/3) vs CD4+ (3/4)). Slopes are generally close to 1 (0.87-1.01), intercepts close to 0 (0-12).	Met
Linear Regression (Normal + HIV+ Abs Counts)	R-value between ImmunoCount and Predicate (for absolute counts)	R-value > 0.85, Slope close to 1, Intercept close to 0.	See TABLE D. R-values range from 0.88 (CD3- (16/19/3) vs CD3+ (3/16+56)) to 0.96 (All lymphocyte subsets). Slopes are generally close to 1 (0.76-0.89), intercepts are slightly higher than 0 (3-124).	Met
Reproducibility
Within-Laboratory Reproducibility	Coefficient of Variation (CV) for absolute lymphocyte counts (Low, Normal, High concentrations)	CV < 10% for most analytes, with slightly higher allowances for low count populations or those with inherent biological variability (e.g., CD16+, CD19+ subsets). The conclusion states "low CV values demonstrate strong within-laboratory reproducibility".	See TABLE E. CVs range from 4.3% to 16.3%. CD16+CD3- and CD19+ have higher CVs at low/normal counts (10.0-16.3%), but the report justifies this due to low counts in these populations, and notes it "do[es] not significantly affect the ImmunoSum (ISUM) CV" (which are generally low, 4.2-5.0%).	Met (with stated justifications)
Between-Laboratory Reproducibility	Coefficient of Variation (CV) for absolute lymphocyte counts (Low, Normal, High concentrations) across different labs	CV < 15% (or similar, depending on analyte/level), with slightly higher allowances for low count populations. The conclusion states "low CV values demonstrate acceptable between-laboratory reproducibility".	See TABLE F. CVs range from 0.8% to 13.1%. Similar to within-lab, CD16+CD3- and CD19+ show higher CVs (8.3-13.1%), also justified by low counts in these populations and negligible impact on ISUM CV (1.6-3.0%).	Met (with stated justifications)
Specimen Age Criteria	Mean Slope of absolute counts over 72 hours and associated Confidence Interval (CI)	Mean Slope close to 0 and CI indicating no significant change over time (e.g., within a predefined tolerance for change). "Comparable results" up to 72 hours.	See TABLE G. Mean slopes are very close to 0 (e.g., -1.5 to 0.8), and CIs are small (1.0-2.6). This indicates stability of results across 24, 48, and 72 hours.	Met
Linearity	R-value between observed counts and expected (diluted/concentrated) counts	R-value close to 1.00 across a wide range of cell counts. "Excellent linearity." \| See TABLE H. R-values are 0.99 or 1.00 for all tested subsets across significant ranges (e.g., CD3+(4/8/3) 75 - 9829 cells/µL; ISUM Lymph Count 95 - 14694 cells/µL).	Met

The study demonstrates that the ORTHO ImmunoCount Flow Cytometry System meets its acceptance criteria by showing substantial equivalence to the predicate method for both normal and HIV-positive donors in percent positive cells and absolute counts. Furthermore, it exhibits excellent within- and between-laboratory reproducibility, stability over a 72-hour period post-collection, and strong linearity across a broad range of cell counts.

2. Sample Size and Data Provenance

Comparability Study (Normal Donors):
- Test Set Size (Percent Positive): 200 normal whole blood specimens.
- Test Set Size (Absolute Counts): 151 normal whole blood specimens.
- Data Provenance: Retrospective, collected from apparently healthy donors (44.5% male, 55.5% female, age 21-62 years) in the United States. Three geographically distinct clinical sites were used for absolute counts, and four for percent positive cells.
Comparability Study (HIV-Antibody Positive Donors):
- Test Set Size (Percent Positive): 119 HIV-antibody positive whole blood specimens.
- Test Set Size (Absolute Counts): 89 HIV-antibody positive whole blood specimens.
- Data Provenance: Retrospective, collected from HIV-antibody positive donors (88.1% male, 11.9% female, age 3- years; likely meaning 3 years and above, or the data has a typo for the lower bound) in the United States. Three geographically distinct clinical sites were used for absolute counts, and four for percent positive cells.
Within-Laboratory Reproducibility: 5 normal whole blood specimens, tested in replicates of 10 at three independent laboratories.
Between-Laboratory Reproducibility: 5 normal whole blood specimens, compared between three independent laboratories, tested in replicates of 10.
Specimen Age Criteria: 64 whole blood specimens (34 normal, 30 HIV-antibody positive), tested at three external clinical sites.
Linearity: 4 normal whole blood specimens, tested at Ortho Diagnostic Systems.

3. Number of Experts and Qualifications (for Ground Truth)

The documentation does not explicitly state the number or qualifications of "experts" used to establish the ground truth for the test set. Instead, the ground truth is established by the "predicate method," which consists of:

FACScan™ Flow Cytometer with Simulset Software (Becton Dickinson)
Simultest™ Monoclonal Antibody Immunophenotyping Reagents (Becton Dickinson)
Coulter® JT2 Automated Hematology Analyzer (Coulter Electronics)
Coulter T Series Automated Hematology Analyzer (Coulter Electronics)
Argos Hematology Analyzer (ABX France)

These are established, FDA-cleared (or equivalent at the time) medical devices used in clinical laboratories. The "experts" in this context would be the trained laboratory personnel operating these predicate devices according to their standard operating procedures, rather than individual "expert adjudicators" in the sense of image interpretation.

4. Adjudication Method for the Test Set

No explicit adjudication method (like 2+1 or 3+1 consensus) is described. The "ground truth" is derived directly from the measurements of the predicate devices. The implicit "adjudication" is inherent in the established and validated performance of these predicate devices and the standard laboratory protocols used for their operation. Differences between the ImmunoCount system and the predicate are then analyzed statistically (e.g., linear regression, mean differences).

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was done. This device is a flow cytometer system for quantitative measurements, not an AI-assisted diagnostic imaging device requiring human-in-the-loop performance evaluation. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here.

6. Standalone Performance

Yes, a standalone performance evaluation was done in the sense that the ORTHO ImmunoCount Flow Cytometry System's measurements were directly compared against the established predicate methods, as well as evaluated for its own reproducibility, specimen age stability, and linearity. It provides a direct measurement, and its results are compared to the predicate device's direct measurements. There isn't a "human-in-the-loop" component for its primary intended use, making its performance inherently standalone in its measurement function.

7. Type of Ground Truth Used

The ground truth used is based on measurements from established, predicate medical devices and reagents (flow cytometers, immunophenotyping reagents, and hematology analyzers) which represent the "traditional method" for lymphocyte immunophenotyping and absolute count determination. This can be considered a form of reference standard measurement from existing, accepted clinical methodology.

8. Sample Size for the Training Set

The document does not specify a separate "training set" or its size. This type of submission (a 510(k) for an instrument and reagent system) typically focuses on verification and validation studies using clinical samples, rather than machine learning model training. The development (calibration, optimization) of the ImmunoCount system itself would have involved internal testing and calibration, but a distinct "training set" as understood in AI/ML is not mentioned or implied.

9. How the Ground Truth for the Training Set was Established

As no explicit training set is mentioned in the context of AI/ML, this question is not directly applicable. If there were internal development/calibration data used, the ground truth would likely have been established similar to the predicate method for the validation studies, by running samples on established instruments to set targets for the new system's development.

Summary

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(
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

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DEVICE DESCRIPTION:

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.

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.

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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 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.

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IMMUNOCOUNT SYSTEMLYMPHOCYTE SUBSET PERCENT POSITIVENORMAL 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 SYSTEMLYMPHOCYTE SUBSET ABSOLUTE COUNTSNORMAL 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

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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. 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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IMMUNOCOUNT SYSTEMLYMPHOCYTE 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 SYSTEMLYMPHOCYTE 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

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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.

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 = 151ANDHIV 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 EORTHO IMMUNOCOUNT SYSTEMWITHIN-LABORATORY REPRODUCIBILITYCOEFFICIENT OF VARIATION OF ABSOLUTE LYMPHOCYTE COUNTSN(LOW) = 139; N(NORMAL) = 142; N(HIGH) = 146
	LOWCV	NORMALCV	HIGHCV
ORTHO ImmunoCountSystem
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 ImmunoCountSystem	Lab 1 MeanCells/μL	Lab 2 MeanCells/μL	Lab 3 MeanCells/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 ImmunoCountSystem	Lab 1 MeanCells/μL	Lab 2 MeanCells/μL	Lab 3 MeanCells/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 ImmunoCountSystem	Lab 1 MeanCells/μL	Lab 2 MeanCells/μL	Lab 3 MeanCells/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

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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 SYSTEMABSOLUTE LYMPHOCYTE COUNTS OVER A 72-HOUR TIME PERIODNORMAL DONORS N=34; HIV-ANTIBODY POSITIVE DONORS N=30
ORTHOImmunoCountSystem	Mean Count24 Hours(Cells/µL)	Mean Count48 Hours(Cells/µL)	Mean Count72 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
ISUMLymph 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}------------------------------------------------

TABLEp	B
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ORTHO IMMUNOCOUNT SYSTEMLINEAR RANGE OF ABSOLUTE COUNTSNORMAL DONORS N=4
ORTHOImmunoCountSystem	R	RangeCells/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.

Regulation Number and Section

§ 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.”