The Cylex Immune Cell Function Assay measures the concentration of ATP from circulating CD4 cells following in vitro stimulation with phytohemagglutinin (PHA) as an indicator of immune cell function. This measurement is made on heparin anticoagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell mediated immune response in populations undergoing immunosuppressive therapy for organ transplant.

The Cylex Immune Cell Function Assay detects cell-mediated immunity (CMI) by measuring the concentration of ATP from CD4 cells following stimulation. This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell-mediated immunity in an immunosuppressed population.

Device Description

The Cylex Immune Cell Function Assay measures the concentration of ATP following in vitro stimulation with phytohemagglutinin (PHA) as an indicator of immune cell function. This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase. The assay is used for the detection of cell mediated immune response in populations undergoing immunosuppressive therapy for organ transplant.

The Cylex Immune Cell Function Assay detects cell-mediated immunity in whole blood. Following incubation, increased ATP synthesis occurs within the cells that respond to the stimulant phytohemagglutinin (PHA). Concurrently, whole blood to monoclonal antibody coated magnetic particles are added to immunoselect CD4 cells from both the stimulated and non-stimulated wells. After washing the selected CD4 cells on a magnet tray, Lysis Reagent is added to release intracellular ATP. Through the released ATP produces light according to the following equation: Luciferin + ATP + O2 Luciferase Mo Oxyluciferin + AMP + Pyrophosphate + CO2 + Light. The amount of light measured by a luminometer (emission maximum 562 nm) is proportional to the concentration of ATP. The concentration of ATP (ng/mL) is calculated from a calibration curve to characterize the cellular immune function of the sample.

AI/ML Overview

Here's a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Cylex Inc. Immune Cell Function Assay

1. Table of Acceptance Criteria and Reported Device Performance

The provided text does not explicitly state pre-defined "acceptance criteria" in terms of specific thresholds for sensitivity, specificity, or similar performance metrics that the device had to meet to be approved. Instead, it presents the results of a clinical study aimed at demonstrating the device's ability to differentiate between two distinct populations (healthy vs. immunosuppressed transplant recipients) and comparing its statistical performance to a predicate device.

The primary demonstration of performance is the statistical difference in ATP measurements between the two patient populations.

Performance Metric	Cylex ICF Assay	Comparator Assay (CD4 Count)
Statistical Difference between Healthy and Transplant Populations	p < 0.0001 (Highly significant) - Means are statistically significantly different.	p < 0.0001 (Highly significant) - Means are statistically significantly different.
Healthy (Non-immunusuppressed) Population
n	40	40
Mean ATP (ng/mL)	464	N/A
Mean CD4 Count (cells/µL)	N/A	746
SD ATP (ng/mL)	145	N/A
SD CD4 Count (cells/µL)	N/A	431
Median ATP (ng/mL)	443	N/A
Median CD4 Count (cells/µL)	N/A	654
Range of Values ATP (ng/mL)	243-967	N/A
Range of Values CD4 Count (cells/µL)	N/A	130-2659
Transplant (Immunosuppressed) Population
n	63	63
Mean ATP (ng/mL)	304	N/A
Mean CD4 Count (cells/µL)	N/A	542
SD ATP (ng/mL)	163	N/A
SD CD4 Count (cells/µL)	N/A	423
Median ATP (ng/mL)	293	N/A
Median CD4 Count (cells/µL)	N/A	503
Range of Values ATP (ng/mL)	58-759	N/A
Range of Values CD4 Count (cells/µL)	N/A	<68*-1904
Distribution of Results (Cylex ICF Assay)
Patients ≤225 ATP ng/mL (Transplant)	38% (24/63)	N/A
Patients ≤225 ATP ng/mL (Healthy)	0% (0/40)	N/A
Patients 226-524 ATP ng/mL (Transplant)	52% (33/63)	N/A
Patients 226-524 ATP ng/mL (Healthy)	67% (27/40)	N/A
Patients ≥525 ATP ng/mL (Transplant)	10% (6/63)	N/A
Patients ≥525 ATP ng/mL (Healthy)	33% (13/40)	N/A
Distribution of Results (Total CD4 Count)
Patients <410 cells/µL (Transplant)	N/A	46% (29/63)
Patients <410 cells/µL (Healthy)	N/A	20% (8/40)
Patients ≥410 cells/µL (Transplant)	N/A	54% (34/63)
Patients ≥410 cells/µL (Healthy)	N/A	80% (32/40)

Note: The primary "acceptance criteria" appear to be establishing statistical significance in differentiating between healthy individuals and immunosuppressed transplant recipients, which was met (p < 0.0001 for the Cylex assay and the comparator assay).

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

Test Set Sample Size:
- Apparently Healthy Adults: 44 (40 included in calculations after outlier exclusion)
- Transplant Recipients: 78 (63 included in calculations after outlier exclusion)
- Total: 122 (103 included in calculations after outlier exclusion)
Data Provenance: The study was a "multi-center study" but the specific country of origin is not explicitly stated. The context (FDA 510(k) submission) suggests it was conducted in the US or under US regulatory guidelines. The study used "freshly drawn blood," indicating a prospective data collection.

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

The document does not mention the use of experts to establish ground truth for the test set. The "ground truth" for classifying patients was based on their clinical status: "apparently healthy adults" vs. "transplant recipients (undergoing immunosuppressive therapy)". The comparator assay (Becton Dickinson TriTest™/MultiTest™ CD4 counts) also served as a comparison point, but not as an expert-adjudicated ground truth.

4. Adjudication Method for the Test Set

No explicit adjudication method (e.g., 2+1, 3+1) is mentioned. The classification was based on the patient's clinical status (healthy vs. transplant recipient). Samples with %CV > 20% were excluded as outliers, which could be considered a form of data quality control.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study is for an in vitro diagnostic (IVD) assay measuring a biomarker, not for an imaging device or AI algorithm requiring human interpretation. Therefore, there's no mention of human readers or improved performance with AI assistance.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, the study presents the standalone performance of the Cylex Immune Cell Function Assay. The reported ATP ng/mL values are solely generated by the device based on its intended mechanism (measuring ATP after PHA stimulation). There is no human interaction or interpretation involved in generating the primary raw data or the final ATP concentration.

7. The Type of Ground Truth Used

The ground truth used was clinical diagnosis/patient status:

"Apparently Healthy Adults" (non-immunosuppressed)
"Transplant Recipients" (immunosuppressed, undergoing therapy, with specific organ transplants listed).

8. The Sample Size for the Training Set

The document does not provide information about a separate "training set" or its sample size. This type of submission (510(k) for an IVD) focuses on the clinical performance study (test set) for device validation rather than an AI/machine learning model development process that typically involves distinct training, validation, and test sets.

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

Since no training set is mentioned for this IVD submission, information on how its ground truth was established is not provided.

Summary

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K 013169

APR 0 2 2002

March 21, 2002

510(K) SUMMARY

Cylex Inc. Immune Cell Function Assay

Submitted by:

Cylex Inc. 8980-1 Old Annapolis Road Columbia, MD 21045

Contact:

Dr. Judy Britz

Name of Device:

Trade Name:	Immune Cell Function Assay
Common Name:	CD4 Cell Stimulation Assay
Classification Name:	Automated Differential Cell Counter
Predicate Device:	Becton Dickinson TriTest™ CD4 FITC/CD8 PE/CD3PerCP Reagent;Becton Dickinson MultiTest™ CD3 FITC/CD8 PE/CD4PerCP/CD4 APC Reagent

Device Description:

Device Describitor.
Intended Use: The Cylex Immune Cell Function Assay measures the concentration of ATP intended Ose. The Gylox filmulation with phytohemagglutinin (PHA) as an indicator of immune cell function. This measurement is made on heparin anti-coagulated whole Indicator of inindile cell function: "This models on the assay is used for the detection of cell blood dailing a luminoneter and lablem. Include going immunosuppressive therapy for organ transplant.

Test Description: The Cylex Immune Cell Function Assay detects cell-mediated immunity in Test Description: The Oylex Mininens Collinitians Colling incubation, increased ATP writhesis occurs within the cells that respond to the stimulant phytohemagglutinin (PHA). synthesis occurs whall the oblished in the absence of stimulant for the purpose of assessing Concarrently, whole blood to mouslanal antibody coated magnetic particles are added to immunoselect CD4 cells from both the stimulated and non-stimulated wells. After washing the selected CD4 cells on a magnet tray, Lysis Reagent is added to release intracellular ATP. selected CD4 cells of a magnet tray, byels nought the released ATP produces light according to the following equation:

Mo Oxyluciferin +AMP +Pyrophosphate +CO2 + Light Luciferin +ATP +O2 Luciferase

The amount of light measured by a luminometer (emission maximum 562 nm) is propritional to The amount of light meadrou by a lantration of ATP (ng/mL) is calculated from a calibration the concentration of ATT : The concerners to characterize the cellular immune function of the sample.

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510(K) SUMMARY

Cylex Inc. Immune Cell Function Assay (cont.)

Substantial Equivalence:

The Cylex Inc. Immune Cell Function Assay has been found to be substantially equivalent to the Becton Dickinson TriTest™ CD4 FITC/CD8 PE/CD3 PerCP Reagent (K971205) and MultiTest™ CD3 FITC/CD8 PE/CD45 PerCP/CD4 ACP Reagent (K974360). All assays differentiate CD4 cells: the Cylex assay determines the responsiveness of those cells and the Becton Dickinson assays count the number of those cells.

A multi-center study was conducted on freshly drawn blood collected from 44 apparently healthy adults and 78 transplant recipients (17 at discharge from the hospital and 61 post-discharge follow-up). The samples were evaluated with the Cylex Immune Cell Function Assay. The apparently healthy adult population consisted of 11% (5) females, 86% (38) males and 3% (1) unknown, with an age range of 20 - 60 years. The ethnicity of the population was 80% (35) African American, 16% Caucasian (7), and 4% (2) other or unknown. The transplant population consisted of 33% (26) females and 67% (52) males, with an age range of 20 - 64 years. The ethnicity of the population was 15% (12) African American, 74% Caucasian (58), 10% (8) other or unknown. The organs transplanted were 55% (43) liver, 36% (28) kidney, 4% (3) pancreas, and 5% (4) multiple organs.

For purposes of these calculations, samples with %CV > 20% (after application of the outlier rule) were not included. The means of the two populations were found to be statically different; the results are summarized in the following table.

Population	Statistic	Cylex ICF Assay(ATP ng/mL)	Comparator AssayCD4 Count by FlowCytometry(cells/µL)
Apparently Healthy(Non-immunosuppressed)	n	40	40
	Mean	464	746
	SD	145	431
	Median	443	654
	Range of Values	243-967	130-2659
Transplant(immunosuppressed)	n	63	63
	Mean	304	542
	SD	163	423
	Median	293	503
	Range of Values	58-759	<68*-1904

Summary of Clinical Trial Results

*Lower linear limit of the flow cytometry assay

NOTE: The means of the Cylex ICF Assay results for the two populations are statistically significantly different (p <0.0001). The means of the comparator assay CD4 count results for the two populations are also statistically significantly different (p < 0.0001).

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510(K) SUMMARY

Cylex Inc. Immune Cell Function Assay (cont.)

	Patient Status
Cylex ICF AssayResult	TransplantSubjects	ApparentlyHealthyAdults	Total
≤225 ATP ng/mL	24 (38%)	0 (0%)	24
226 - 524 ATPng/mL	33 (52%)	27 (67%)	60
≥525 ATP ng/mL	6 (10%)	13 (33%)	19
Total	63	40	103

	Patient Status
Total CD4Count	TransplantSubjects	ApparentlyHealthyAdults	Total
<410 cells/µL	29 (46%)	8 (20%)	37
≥410 cells/µL	34 (54%)	32 (80%)	66
Total	63	40	103

The percentage of transplant patients with an assay result <525 ATP ng/mL was 90% (57/63, The percentage of transplant patients). The percentage of transplant subjects having an assay result ≤225 ATP ng/ml was 38% (24/63, 95% confidence interval 26.1 – 51.2%). The result =220 XTP Tighth was 00% (2525 ATP ng/mL was 10% (6/63, 95% confidence interval 3.6 - 19.6%).

The percentage of apparently healthy adults having an assay result <525 ATP ng/mL was 67% The percentage of apparently houlthy additioning and securities of apparently healthy adults (21140, 90% ounned finer of one of one of a sure of apparently healthy adults having an assay result ≥525 ATP ng/mL was 33% (13/40, 95% confidence interval 18.6 – 49.1%).

*U.S. Patent No. 5,773,232

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/3/Picture/1 description: The image shows the seal of the U.S. Department of Health and Human Services. The seal is circular and contains the department's name around the perimeter. In the center of the seal is an abstract design featuring a stylized caduceus, a symbol often associated with medicine and healthcare. The caduceus consists of a staff with two snakes coiled around it, topped with wings.

Food & Drug Administration 10903 New Hampshire Avenue Building 66 Silver Spring, MD 20993

Cylex Inc. c/o Ms. Judi Smith, Principal, Judi Smith, LLC 8980-I Old Annapolis Road Columbia, MD 21045

SEP 2 1 2010

Re: K013169

Trade/Device Name: Cylex Immune Cell Function Assay Regulation Number: 21 CFR 864.5220 Regulation Name: Automated Differential Cell Counter Regulatory Class: Class II Product Code: NID Dated: January 14, 2002 Received: January 15, 2002

Dear Ms. Smith:

This letter corrects our substantially equivalent letter of April 2, 2002.

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 (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 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

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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); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); 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.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please go to

http://www.fda.gov/AboutFDA/CentersOffices/CDRH/CDRHOffices/ucm115809.htm for the Center for Devices and Radiological Health's (CDRH's) Office of Compliance. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

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 (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm.

Sincerely yours,

for Maria M. Chan, Ph. D.

Maria M. Chan. 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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K013169 Cylex Corp.

Div/Branch	Last Name	Date
DIHD/IMDB	Reeves	09/21/2010
DIHD/IMPB	PHILIP	9-21-2010
DIHD/IMDB	CARRINGTON	9/21/2010

Date of Update	By	Description of Update
7/27/09	Brandi Stuart	Added Updates to Boiler Table
8/7/09	Brandi Stuart	Updated HFZ Table

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Page 1 of 1

510(k) Number (if known):

013169

Device Name: Immune Cell Function Assay

Indications For Use:

(PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Reena Philip

Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

510K k013/69

) Prescription Use (Per 21 CFR 801.109)

Over-The-Counter Use

(Optional Format 1-2-96)

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510(k) Number (if known):

K0i31i69

Device Name: Immune Cell Function Assay

Indications For Use:

(PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Dmu H

rision Sign Division of Clinical Laboratory Devices KG13169 510(k) Number

Prescription Use
(Per 21 CFR 801.109)

Over-The-Counter Use

(Optional Format 1-2-96)

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.”