BD Multitest 6-Color TBNK reagent with BD Trucount tubes is intended for in vitro diagnostic use with the BD FACSCanto system to identify and determine the percentages and absolute counts of T, B, and natural killer (NK) cells as well as the CD4 and CD8 subpopulations of T cells in peripheral blood.

Device Description

The BD Multitest 6-Color TBNK reagent is a six-color direct immunofluorescence reagent for use with the BD FACSCanto system to identify and determine the percentages and absolute counts (using BD Trucount tubes) of T, B, and natural killer (NK) cells as well as the CD4 and CD8 subpopulations of T cells in peripheral blood.

When a known volume of whole blood is added to the reagent in a BD Trucount tube, the fluorochrome-labeled antibodies in the reagent bind specifically to leucocyte surface antigens. The stained samples are treated with BD FACS lysing solution to lyse erythrocytes, and the lyophilized pellet in the BD Trucount tube dissolves, releasing a known number of fluorescent beads.

During acquisition, the cells and beads travel past two laser beams and scatter the laser light. The stained cells and beads fluoresce at different intensities. These scatter and fluorescence signals, detected by the flow cytometer, provide information about each cell's size, internal complexity, and relative fluorescence intensity. During analysis, the absolute number (cells/uL) of positive cells in the sample can be determined by comparing cellular events to bead events.

AI/ML Overview

Here's an analysis of the provided text, focusing on the acceptance criteria and the study that proves the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device appear to be primarily focused on its accuracy, precision, linearity, and stability in comparison to a predicate device. The performance data is presented in terms of mean bias for accuracy and standard deviation/coefficient of variation for precision.

Metric (Acceptance Criteria)	Lymphocyte Subset	Reported Device Performance (Mean Bias % or Standard Deviation (SD) / %CV)
Accuracy (Mean Bias %) (Compared to Predicate Device)	CD4 (Percentage)	0.1 (-0.1, 0.2)
	CD8 (Percentage)	-0.8 (-1.0, -0.5)
	CD3 (Percentage)	-0.2 (-0.4, 0.0)
	CD19 (Percentage)	0.3 (0.2, 0.4)
	CD16+CD56 (Percentage)	-0.1 (-0.3, 0.0)
	CD4 (Absolute Count)	-2.7 (-4.2, -1.3)
	CD8 (Absolute Count)	-3.5 (-4.4, -2.6)
	CD3 (Absolute Count)	-1.9 (-2.6, -1.2)
	CD19 (Absolute Count)	1.8 (0.3, 3.3)
	CD16+CD56 (Absolute Count)	-2.3 (-4.1, 0.5)
Precision (Repeatability - SD) (Within-Device Precision for Percentages)	CD4 (Low Sample)	0.64
	CD4 (Normal Sample)	0.95
	CD8 (Low Sample)	1.07
	CD8 (Normal Sample)	0.65
	CD3 (Low Sample)	1.17
	CD3 (Normal Sample)	0.86
	CD19 (Low Sample)	0.89
	CD19 (Normal Sample)	0.62
	CD16+CD56 (Low Sample)	0.90
	CD16+CD56 (Normal Sample)	0.61
Precision (Repeatability - %CV) (Within-Device Precision for Absolute Counts)	CD4 (Low Sample)	7.6
	CD4 (Normal Sample)	4.7
	CD8 (Low Sample)	4.1
	CD8 (Normal Sample)	4.7
	CD3 (Low Sample)	4.0
	CD3 (Normal Sample)	4.2
	CD19 (Low Sample)	5.7
	CD19 (Normal Sample)	5.3
	CD16+CD56 (Low Sample)	7.0
	CD16+CD56 (Normal Sample)	7.9
Linearity (R2)	CD4	0.998
	CD8	0.991
	CD3	0.996
	CD19	0.989
	CD16+CD56	0.999
Sample Stability	Anticoagulated blood	Stained within 24 hours of draw and analyzed within 6 hours of staining
Stain Stability	Stained samples	Analyzed within 6 hours of staining (implied from above)

Note: The document states "Substantial equivalence and the performance of BD Multitest 6-color TBNK reagent with BD Trucount tubes have been demonstrated through accuracy, precision, linearity, and sample and stain stability studies." The acceptance criteria are implicitly met if the reported performance data falls within acceptable ranges relative to the predicate, as interpreted by the FDA's substantial equivalence determination.

2. Sample Size Used for the Test Set and Data Provenance

Sample Size for Accuracy Study (Test Set): n = 117 for all lymphocyte subsets (CD4, CD8, CD3, CD19, CD16+CD56) for both percentages and absolute counts.
Sample Size for Precision Study (Test Set): n = 42 for all lymphocyte subsets for both percentages and absolute counts across "Low Sample" and "Normal Sample" groups.
Data Provenance: The document does not explicitly state the country of origin or whether the data was retrospective or prospective. Given the nature of medical device submissions in the US, it is highly probable that the studies were conducted in the US, and typically prospective data is preferred for performance validation. However, this is not explicitly mentioned.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

This type of device (automated differential cell counter) does not rely on expert interpretation of images or other subjective data for ground truth. The "ground truth" for the accuracy study is established by comparison to a legally marketed predicate device (BD Multitest IMK kit with BD FACSCanto system and clinical software). The predicate device itself has established performance characteristics, and the ground truth for that device would have been established through methods like flow cytometry standards, reference methods, or clinical correlation. No human experts are used to establish ground truth in the way a radiologist might for an imaging AI.

4. Adjudication Method (for the test set)

Not applicable. As explained in point 3, the ground truth is established by the predicate device's measurements, not by human interpretation or adjudication.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is an automated diagnostic assay (a reagent/instrument system) for quantifying cell populations, not an AI-assisted diagnostic imaging tool or a system that requires human interpretation in the same way. Therefore, an MRMC study comparing human readers with AI assistance is not relevant to its evaluation.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the studies presented (accuracy, precision, linearity) represent the standalone performance of the device system (reagent + instrument + software). The results are generated directly by the system without human interpretation influencing the quantification of the lymphocyte subsets. The comparison is made against another device system (the predicate).

7. The Type of Ground Truth Used

The ground truth for the accuracy study was the measurements obtained from the predicate device, the BD Multitest IMK kit used with the BD FACSCanto system and BD FACSCanto clinical software (K041074). This is referred to as "method comparison."

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning. This device is a flow cytometry assay system, not an AI/ML algorithm that is typically "trained" on data. Its performance is based on the biochemical reactions of antibodies and the physical principles of flow cytometry. If there were any internal device calibration or optimization data, it is not described here as a "training set."

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

Not applicable, as there is no explicitly defined "training set" in the context of an AI/ML algorithm for this device. The system functions based on established biological and chemical principles, not on a machine learning model that requires a trained ground truth dataset in the conventional sense. The "ground truth" for the development of such assays would derive from established laboratory standards and reference methods for cell enumeration and phenotyping.

Summary

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510(k) Summary

This summary of safety and effectiveness is being submitted in accordance with the requirements of the Safe Medical Devices Act of 1990 and 21 CFR 807.92.

The assigned 510(k) number is ___

K060375.

JUN -6 2006

Submitter Information

Submitter: BD Biosciences 2350 Qume Drive San Jose, CA 95131
Contact: Carter Navarro Regulatory Affairs Specialist Tel: 408.954.2469 Fax: 408.954.2495 carter navarro@bd.com

Summary Date: February 9, 2006

Device Name and Classification

Trade Name:	BD Multitest 6-color TBNK reagent withBD Trucount tubes
Classification Name:	Automated Differential Cell Counter
Regulation Number:	21 CFR 862.5220
Product Code:	GKZ

Substantially Equivalent Predicate Device

BD Multitest 6-color TBNK reagent with BD Trucount tubes is substantially equivalent to BD Multitest IMK kit (K980858) when used with the BD FACSCanto system with BD FACSCanto clinical software (K041074).

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Device Description

Intended Use

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Comparison to Predicate Device

Characteristic	Predicate:BD Multitest IMK Kit withBD Trucount Tubes (K980858)and BD FACSCanto System withBD FACSCanto Clinical Software(K041074)	Candidate:BD Multitest 6-Color TBNKReagent with BD Trucount Tubes
Intended Use	Identification and determination ofpercentages and absolute counts of thefollowing mature human lymphocytesubsets in erythrocyte-lysed wholeblood: T lymphocytes (CD3+,CD3+CD4+, and CD3+CD8+), Blymphocytes (CD3-CD19+), and NKlymphocytes (CD3-CD16+CD56+).	Same.
Components(includingCluster Designation,Conjugation, andClone)	BD MultitestCD3/CD16+CD56/CD45/CD19(1 vial, 50 tests)CD3 FITC (SK7) CD16 PE (B73.1) +CD56 PE (NCAM16.2) CD45 PerCP (2D1) CD19 APC (SJ25C1) Buffer with 0.1% sodium azide BD MultitestCD3/CD8/CD45/CD4(1 vial, 50 tests) CD3 FITC (SK7) CD8 PE (SK1) CD45 PerCP (2D1) CD4 APC (SK3) Buffer with 0.1% sodium azide BD FACS Lysing SolutionBD Trucount Tubes (100 tubes)	BD Multitest 6-Color TBNK Reagent(1 vial, 50 tests) CD4 PE-Cy7 (SK3) CD8 APC-Cy7 (SK1) CD3 FITC (SK7) CD19 APC (SJ25C1) CD16 PE (B73.1) +CD56 PE (NCAM16.2) CD45 PerCP-Cy5.5 (2D1) Buffer with 0.1% sodiumazide BD Trucount Tubes (50 tubes)BD FACS Lysing Solution(not included with reagent)

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Characteristic	Predicate:BD Multitest IMK Kit withBD Trucount Tubes (K980858 )and BD FACSCanto System withBD FACSCanto Clinical Software(K041074)	Candidate:BD Multitest 6-Color TBNKReagent with BD Trucount Tubes
Specificity	Specificities of antibodies have beenverified by the International Workshopon Human Leukocyte DifferentiationAntigens. 1,2,3,4,5,6	Same.
Method to IdentifyPopulations ofInterest	Lyse/no-wash method using a two-tubepanel with four-color antibody reagents(one tube stained withCD3/CD16+CD56/CD45/CD19, theother with CD3/CD8/CD45/CD4) toidentify lymphocytes with specific cell-surface antigens, fluorescencetriggering, and CD45 vs. SSC forgating. Uses fluorescent beads toquantify absolute counts.	Same, except uses a one-tube panelwith six-color antibody reagent.

3 Ritz J, Trinchieri G, Lanier LL. NK-cell antigens: section report. In: Schlossman SF, Bournsell L, Gilks W, et al, eds. Leucocyte Typing V: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1995;2:1367-1372.

4 Cobbold SP, Hale G, Waldmann H. Non-lineage, LFA-1 family, and leucocyte common antigens: new and previously defined clusters. In: McMichael AJ, ed. Leucocyte Typing III: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1987:788-803.

5 Bernard A, Boumsell L, Hill C. Joint report of the first international workshop on human leucocyte differentiation antigens by the investigators of the participating laboratories: T2 protocol. In: Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman SF, eds. Leucocyte Typing. New York, NY: Springer-Verlag; 1984:25-60.

6 Nadler LM. B Cell/Leukemia Panel Workshop: summary and comments. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human B Lymphocytes. New York, NY: Springer-Verlag; 1986;2:3-43.

4 Haynes BF. Summary of T-cell studies performed during the Second International Workshop and Conference on Human Leukocyte Differentiation Antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer-Verlag; 1986;1:3-30.

2 Schmidt RE. Non-lineage/natural killer section report: new and previously defined clusters. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:517-542.

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Characteristic	Predicate:BD Multitest IMK Kit withBD Trucount Tubes (K980858)and BD FACSCanto System withBD FACSCanto Clinical Software(K041074)	Candidate:BD Multitest 6-Color TBNKReagent with BD Trucount Tubes
Control	Recommend use of commerciallyavailable whole blood control withestablished values for subsetpercentages and absolute counts.	Recommend use of two levels ofcommercially available whole bloodcontrols with established values forsubset percentages and absolutecounts.BD specifically recommends the useof BD Multi-Check and BD Multi-Check CD4 Low controls.
Instrument andSoftware	BD FACSCanto flow cytometer withBD FACSCanto clinical softwareversion 1.0.	Same, except uses version 2.0 of BDFACSCanto clinical software.
System Setup	BD FACSCanto flow cytometer withBD FACSCanto clinical softwareversion 1.0 andBD FACS 7-color setup beads.	Same, except uses version 2.0 of BDFACSCanto clinical software.
Sample and StainStability	Anticoagulated blood stored at roomtemperature (20-25°C) must be stainedwithin 48 hours of draw and thenanalyzed within 24 hours of staining.	Anticoagulated blood stored at roomtemperature (20-25°C) must bestained within 24 hours of draw andthen analyzed within 6 hours ofstaining.
Results	CD3+, CD3+CD4+, and CD3+CD8+ Tlymphocytes; CD3-CD19+ Blymphocytes, and CD3-CD16+CD56+NK lymphocytes expressed aspercentages of total lymphocytes or asabsolute counts (cells/\u03bcL) in wholeblood.	Same.
Sample Type	Erythrocyte-lysed whole blood,collected in K3 EDTA blood collectiontubes.	Same.

. ・・・・・・・・

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Summary of Performance Data

Substantial equivalence and the performance of BD Multitest 6-color TBNK reagent with BD Trucount tubes have been demonstrated through accuracy, precision, linearity, and sample and stain stability studies.

Accuracy

The accuracy study design was based on NCCLS document EP9-A2, Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline – Second Edition. The predicate method was BD Multitest IMK kit (K980858) on the BD FACSCanto system with BD FACSCanto clinical software (K041074).

LymphocyteSubset	n	Mean Bias (%)(95% CI)	Range for Test System(%)(Predicate System)
CD4	117	0.1(-0.1, 0.2)	0.51 - 66.86(0.81 - 68.67)
CD8	117	-0.8(-1.0, -0.5)	10.77 - 83.43(10.91 -83.09)
CD3	117	- 0.2(-0.4, 0.0)	33.81 - 88.45(33.12-89.51)
CD19	117	0.3(0.2, 0.4)	0.1 - 35.89(0.08-35.77)
CD16+CD56	117	-0.1(-0.3, 0.0)	2.44 - 51.47(3.04 - 50.84)

Lymphocyte Subset Percentages

. . . . . .

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LymphocyteSubset	n	Mean Bias (%)(95% CI)	Range for Test System (cells/µL)(Predicate System)
CD4	117	-2.7(-4.2, -1.3)	4.39 - 1592.79(6.23 - 1590.7)
CD8	117	-3.5(-4.4, -2.6)	50.79 - 2416.16(57.91 - 2194.02)
CD3	117	-1.9(-2.6, -1.2)	107.14 - 3403.08(108.74 - 3231.05)
CD19	117	1.8(0.3, 3.3)	0.5 - 1207.49(0.42 - 1199.38)
CD16+CD56	117	-2.3(-4.1, 0.5)	6.7 - 918.43(7.94 - 955)

Lymphocyte Subset Absolute Counts

Precision

The precision study design was based on NCCLS document EP5-A, Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline.

Lymphocyte Subset	n	Low Sample7SD	Normal Sample8SD
CD4	42	0.64	0.95
CD8	42	1.07	0.65
CD3	42	1.17	0.86
CD19	42	0.89	0.62
CD16+CD56	42	0.90	0.61

Repeatability of Lymphocyte Subset Percentages

7 Streck CD-Chex Plus CD4 Low controls.

8 Streck CD-Chex Plus controls.

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Lymphocyte Subset	n	Low SampleSD	Normal SampleSD
CD4	42	0.69	1.23
CD8	42	1.29	0.81
CD3	42	1.23	0.90
CD19	42	0.89	0.62
CD16+CD56	42	0.96	0.62

Within-Device Precision for Lymphocyte Subset Percentages

Repeatability of Lymphocyte Subset Absolute Counts

Lymphocyte Subset	n	Low Sample%CV	Normal Sample%CV
CD4	42	7.6	4.7
CD8	42	4.1	4.7
CD3	42	4.0	4.2
CD19	42	5.7	5.3
CD16+CD56	42	7.0	7.9

Within-Device Precision of Lymphocyte Subset Absolute Counts

Lymphocyte Subset	n	Low Sample%CV	Normal Sample%CV
CD4	42	8.0	4.8
CD8	42	5.0	5.4
CD3	42	4.4	4.2
CD19	42	6.0	5.7
CD16+CD56	42	8.0	7.9

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Linearity

The linearity study design was based on NCCLS document EP6-A, Evaluation of the Linearity of Quantitative Analytical Methods; Approved Guideline. Concentration levels were established based on an expected CD4+ range of 200 to 3,000 cells/uL.

LymphocyteSubset	Linear Range(cells/µL)	R2
CD4	4 - 2,234	0.998
CD8	158 - 1,125	0.991
CD3	498 - 3,356	0.996
CD19	71 - 447	0.989
CD16+CD56	0 - 1,559	0.999

Lymphocyte Subset Linear Ranges for Absolute Counts

Sample and Stain Stability

Whole blood should be collected aseptically by venipuncture using K3 EDTA blood collection tubes. Anticoagulated blood stored at room temperature (20 – 25° C) must be stained within 24 hours of draw and then analyzed within 6 hours of staining.

Conclusions from Performance Data

BD Multitest 6-color TBNK reagent with BD Trucount tubes is substantially equivalent to the predicate device.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/9/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. Inside the circle is an abstract symbol resembling an eagle or bird in flight, with stylized lines forming the body and wings.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

JUN -6 2006

BD Biosciences c/o Mr. Carter Navarro Regulatory Affairs Specialist 2350 Qume Dr. San Jose, CA 95131-1807

Re: K060375

Trade/Device Name: BD Multitest 6-color TBNK reagent with BD Trucount tubes Regulation Number: 21 CFR 864.5220 Regulation Name: Automated Differential Cell Counter Regulatory Class: Class II Product Code: GKZ Dated: February 10, 2006 Received: February 13, 2006

Dear Mr. Navarro:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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Page 2 -

This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Office of Compliance at (240) 276-0484. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Marie, than for
Dr. Robert Becker

Robert L. Becker, Jr., M.D., Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indications for Use Statement

510(k) Number: K060375

Device Name: BD Multitest 6-color TBNK reagent with BD Trucount tubes

Indications For Use:

For use with the BD FACSCanto flow cytometer. .
For use with whole blood collected in K3 EDTA tubes. .
For use in the identification and determination of percentages and absolute . counts of T, B, and natural killer (NK) cells as well as the CD4 and CD8 subpopulations of T cells in peripheral blood.
For in vitro diagnostic use. .

X Prescription Use _ (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use (21 CFR 801 Subpart C)

PLEASE DO NOT WRITE BELOW THIS LINE – CONTINUE ON ANOTHER PAGE OF NEEDED

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

Maria Chan for
Josephine Bautista
DIVISION SIGN-OFF

Office of In Vitro Diagnostic
Device Evaluation and Safety

51000 K260375

Page 1 of ____________________________________________________________________________________________________________________________________________________________________

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.”