Navios EX Flow Cytometer, 6 Color/2 Laser, Navios EX Flow Cytometer, 8 Color/2 Laser, Navios EX Flow Cytometer, 10 Color/3 Laser
Device Facts
| Record ID | K162897 |
|---|---|
| Device Name | Navios EX Flow Cytometer, 6 Color/2 Laser, Navios EX Flow Cytometer, 8 Color/2 Laser, Navios EX Flow Cytometer, 10 Color/3 Laser |
| Applicant | Beckman Coulter |
| Product Code | OYE · Hematology |
| Decision Date | Jun 23, 2017 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 864.5220 |
| Device Class | Class 2 |
Indications for Use
The Navios EX Flow Cytometer is intended for use as an in vitro diagnostic device for immunophenotyping using up to four fluorescent detection channels using a blue (488 nm) laser and two light scatter detection channels. It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument.
Device Story
Navios EX Flow Cytometer uses hydrodynamic focusing to align cells in single file through a 488nm laser beam. Scattered and fluorescent light is collected by photomultiplier tubes (PMTs), transduced to electronic pulses, and processed by the system computer. Input consists of whole blood samples stained with monoclonal antibody reagents (CYTO-STAT tetraCHROME) and lysed using the COULTER ImmunoPrep system. The device performs automated standardization of light scatter and fluorescence intensities, color compensation, and lymphocyte subpopulation analysis via Navios EX tetra Software. Output includes qualitative and quantitative cell counts (percentages and absolute counts). Used in clinical laboratories by trained personnel. Results assist clinicians in assessing immune status. Benefits include automated, standardized, and reproducible immunophenotyping.
Clinical Evidence
Prospective method comparison study (n=424) across four sites compared Navios EX to the predicate Navios. Primary endpoints included CD3+, CD3+CD4+, CD3+CD8+, CD19+, and CD3-CD56+ percentages and absolute counts. Results showed high correlation (r > 0.99) and acceptable bias at medical decision levels. Precision studies (repeatability and site-to-site) met acceptance criteria with total CVs generally <10%. Linearity and carryover studies confirmed performance within established ranges.
Technological Characteristics
Benchtop flow cytometer; 488nm blue laser; 6, 8, or 10-color configurations; hydrodynamic focusing; PMT-based detection; digital integrator circuitry (40 MHz sampling). Software-controlled automated compensation and gating. Sterilization: N/A (instrument). Connectivity: PC-based workstation (Windows 7).
Indications for Use
Indicated for immunophenotyping and enumeration of lymphocyte subsets (CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3-CD56+) in peripheral whole blood for patients suspected of immune deficiency, including HIV diagnosis/prognosis and post-transplant monitoring. For prescription use only.
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
- Navios Flow Cytometer (K130373)
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
- K130373 — NAVIOS FLOW CYTOMETER SYSTEM · Beckman Coulter, Inc. · Sep 18, 2013
Submission Summary (Full Text)
{0}
1
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
K162897
B. Purpose for Submission:
Instrument modification
C. Manufacturer and Instrument Name:
Beckman Coulter, Inc.
Navios EX Flow Cytometer
D. Type of Test or Tests Performed:
Flow Cytometric Immuno-assay: Cell identification with quantitative cell counts
E. System Descriptions:
1. Device Description:
The Navios EX uses flow cytometric principles to determine qualitative and quantitative measurements of biological and physical properties of cells and other particles when the cells pass through the laser beam(s) in single file.
The Navios EX Flow Cytometer includes the 488nm laser as part of three available manufactured instrument configurations (same as the predicate, K130373).
- Navios EX Flow Cytometer, 6 Color/2 Laser
- Navios EX Flow Cytometer, 8 Color/2 Laser
- Navios EX Flow Cytometer, 10 Color/3 Laser
The lower level configurations are upgradeable to higher level configurations by adding filters, photomultiplier tubes (PMTs) and activating a laser, if required. Only the 488nm laser is utilized for cleared IVD applications (tetraCHROME cleared in K130373) and for the 488nm laser, only fluorescence channel 1 (FL1) through FL4 are the subject of this 510(k) submission.
The Navios EX Flow Cytometer system consists of:
- Navios EX Flow Cytometer
- Navios EX tetra Software
- Navios EX Software (optional off-line)
- CYTO-STAT tetraCHROME reagents
- Flow-Set Pro Fluorospheres
- Flow-Check Pro Fluorospheres
{1}
- Flow-Count Fluorospheres
- Immuno-Trol Cells
- Immuno-Trol Low Cells
- COULTER IMMUNOPREP Reagent System
- QuickCOMP 2 and QuickCOMP 4 Kits
- CYTO-COMP Cell Kit
- ISOFLOW Sheath Fluid
- FlowClean Cleaning Reagent
- TQ-Prep Workstation (Accessory for Sample Preparation)
- PrepPlus 2 Workstation (Accessory for Sample Preparation)
The Navios EX Flow Cytometer is a modification to the Navios Flow Cytometer (K130373). There are no changes to the consumables or preparation devices. The changes include:
- Replacement of optical assemblies and flow cell
- Replacement of the existing 488nm laser with a comparable 488nm laser
- Replacement of the band-pass filter (FL3) used for ECD dye
- Replacement of the Forward Scatter mask with a new mask configuration
- Replacement of the existing fixed aspiration probe with an adjustable one to account for the sample tube employed at the customer's laboratory
- Change to the optics module temperature management hardware
- Minor change to the sheath pressure
- Minor changes to the Navios EX tetra algorithm
An update to the cytometer's indications for use, providing additional detail on the configuration of the device
Navios EX System Software (on-board): The Navios EX Flow Cytometer contains on-board software which enables the user to acquire data from the Navios EX Flow Cytometer and to analyze, display, print and export acquired listmode data. The software has the following functions:
- Daily Routine (Startup/Shutdown)
- Setup Mode & AutoSetup
- Quality Assurance (Controls/Calibration)
- Sample Analysis
- Data Review
- Listmode Replay
- Report Generation
Navios EX Software is also offered as an optional software package for use on an independent computer workstation for off-line analysis of listmode files generated by the Navios EX Flow Cytometer with the monoclonal antibody reagents. The off-line version utilizes the same executable software as the on-board software with the instrument control portion/features disabled. Analysis must be performed in accordance with the product labeling.
The Navios EX tetra Software is an optional software package that is separate from the
2
{2}
Navios EX System software. It is used for immunophenotyping with CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 monoclonal antibody reagents on the Navios EX Flow Cytometer. It provides automated analysis and results for the identification and enumeration of CD3+CD4+, CD3+CD8+, CD3+, CD19+ and CD3-CD56+ lymphocyte percentages and absolute counts in peripheral whole blood. Absolute counts may be determined by the Navios EX Flow Cytometers using Flow-Count Fluorospheres (Single Platform Technology (SPT) Method) or separate hematology results (Dual Platform Method).
## 2. Principles of Operation:
The Navios EX flow cytometer aligns cells in suspension by hydrodynamic focusing and passes them through a laser beam one cell at a time. The scattered and fluoresced light that emanates from these cells as they intersect the laser beam is collected by photomultiplier tubes and transduced to an electronic pulse and counted by the instrument's computer. The utility of this device depends on the ability of a monoclonal antibody to bind to the surface of cells expressing discrete antigenic determinants. Specific cell staining is accomplished by incubating whole blood with the monoclonal antibody reagent, the CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5. Monoclonal antibody reagents are each a combination of four murine monoclonal antibodies, each conjugated to a specific fluorochrome and specific for a different cell surface antigen. Red blood cells are lysed with the COULTER ImmunoPrep Reagent System. The remaining white blood cells are analyzed on a Navios EX flow cytometer with the CYTO-STAT tetraCHROME reagents and the Navios EX tetra Software package. Navios EX System Software and Navios EX tetra Software, in conjunction with quality control reagents, provide automated standardization of light scatter and fluorescence intensities and automated adjustment of color compensation settings. The Navios EX tetra Software, in conjunction with the Navios EX Software, and CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 reagents, provides automated analysis of lymphocyte subpopulations.
## 3. Modes of Operation:
Batch or single tube
## 4. Specimen Identification:
Barcode reader or manual entry
## 5. Specimen Sampling and Handling:
The automated sample loader for the instrument uses a carousel that holds thirty-two 12 x
3
{3}
75-mm test tubes and includes a barcode reader.
6. Calibration:
Fluorescence intensity is calibrated by adjusting photomultiplier voltage using Flow-Set Pro Fluorospheres (K130373). The absolute count of a population is based on the calibration factor (CAL Factor) and the number of Flow-Count fluorospheres particles within a user defined CAL region.
7. Quality Control:
IMMUNO-TROL Cells and IMMUNO-TROL Low Cells (K030828) are assayed, lyseable, whole-blood quality control products used to verify the activity of the CYTO-STAT tetraCHROME reagents and to verify the methods used for staining targeted cells, lysing erythrocytes, and analyzing samples with flow cytometry.
8. Software:
FDA has reviewed applicant’s Hazard Analysis and Software Development processes for this line of product types: Yes ☐ X or No ☐
F. Regulatory Information:
1. Regulation section:
21 CFR 864.5220 – Automated Differential Cell Counter
2. Classification:
Class II
3. Product code:
OYE- flow cytometric reagents and accessories
PDX- flow cytometry calibrator
4. Panel:
Hematology (81)
{4}
5
G. Intended Use:
1. Indication for Use:
Navios EX Flow Cytometer:
The Navios EX Flow Cytometer is intended for use as an in vitro diagnostic device for immunophenotyping using up to four fluorescent detection channels using a blue (488 nm) laser and two light scatter detection channels. It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument.
Flow-Set Pro Fluorospheres
Flow-Set Pro Fluorospheres is a suspension of fluorescent microspheres used as an aid in standardizing forward scatter, side scatter, and fluorescence detectors (FL1-4) on the Cytomics FC 500, Navios and Navios EX Flow Cytometers.
Flow-Count Fluorospheres
Flow-Count Fluorospheres is a fluorescent microsphere reagent for direct determination of lymphocytes and lymphocyte subsets cell population percentages and absolute counts in biological specimens using EPICS XL/XL-MCL, Cytomics FC500, Navios and Navios EX flow cytometry systems as well as CD34+ cell population percentages and absolute counts in biological specimens using EPICS XL/XL-MCL and Cytomics FC500 flow cytometry systems.
Special Conditions for Use Statement:
For prescription use only
Special instrument requirements:
Use with TQ-Prep Workstation (Accessory for Sample Preparation)
H. Substantial Equivalence Information:
1. Predicate Device Name and 510(k) number:
Navios Flow Cytometer, K130373
2. Comparison with Predicate Device:
Navios EX Flow Cytometer with Navios EX tetra Software and Navios EX Software
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Navios EX Flow Cytometer: The Navios EX Flow Cytometer is intended for use as an in vitro diagnostic device for immunophenotyping using up to four | Navios Flow Cytometer: The Navios Flow Cytometer is intended for use as an in vitro diagnostic device for immunophenotyping. It can be used |
{5}
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | fluorescent detection channels using a blue (488 nm) laser and two light scatter detection channels. It is intended for use with in vitro diagnostic (IVD) assays and software that are indicated for use with the instrument. | in conjunction with the following monoclonal antibody reagents and software package:
•CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 monoclonal antibody reagents. These reagents provide identification and enumeration of CD3+CD4+, CD3+CD8+, CD3+, CD19+ and CD3-CD56+ lymphocyte percentages and absolute counts in peripheral whole blood.
Absolute counts may be determined by the Navios flow cytometer using Flow-Count Fluorospheres (single platform technology method) or separate hematology results (dual platform method). These reagents are indicated for use in the immunologic assessment of patients having or suspected of having immune deficiency.
•Navios tetra Software for automated analysis and results with CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5monoclonal antibody reagents.
Navios Software may be installed on an independent computer workstation for off-line analysis of listmode files generated by the Navios Flow Cytometer with the monoclonal antibody reagents and software package listed above. The off-line analysis must be performed in accordance with the product labeling. |
| Regulation Number and Description, Product Code | 864.5220 –Automated Differential Cell Counter | Same |
| Product Code | OYE | Same |
{6}
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Safety Features | Interlocks and mitigation of hazards via software and hardware controls | Same |
| Controlling Software | The acquisition software enables the user to acquire data from the instrument and to analyze, display, print and export acquired listmode data. The embedded software resides in the instrument. It controls the instrument functionality including the multicarousel loader (MCL) for sample introduction. The embedded software controls the instruments’ lasers, acquisition system and fluidics. The instrument fluidics aspirates the sample, and performs instrument maintenance functions such as startup/shutdown. The embedded software also captures and provides the data to the workstation for processing. | Same |
| System Configuration | • Bench top
• Printer
• PC based workstation running Microsoft Windows WIN7 application specific software | Same except workstation runs on Microsoft Windows Vista or WIN7 application |
| Sample Preparation with Monoclonal Antibodies | Off-board sample preparation following instructions provided with cleared monoclonal antibody reagent | Same |
| Sample Presentation | Prepared sample added to a daughter tube | Same |
| Prepared Sample Mixing prior to Acquisition | Prepared sample is vortex mixed | Same |
| Sample Introduction | Tube sampler
• Automated presentation with Multi-tube Carousel Loader (MCL) from 32 test tube capacity carousel
• Manual presentation into a tube location on a MCL via tube access door | Same |
| Aspiration Pathway | Same aspiration pathway used for automated and manual presentation | Same |
| Sample Identification | Bar-code reading of carousel position and labeled sample tube. User may also identify samples based on carousel location with a work list. | Same |
{7}
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Maximum Parameter Detectors | IVD configuration – Six (FS, SS, FL1 – FL4) | Same |
| Electronics | 40 MHz sampling Digital integrator circuitry w/ early stage ADC DC Restoration Approach | Same |
| Photomulti-plier Tubes (PMTs)/Colors | IVD configuration – Standard 4 PMTs (FL1 - FL4) off of 488 nm laser | Same |
| Color Separation | Collimated beam is separated into desired components with dichroic filters. | Same |
| Software | The flow cytometer is run by the Navios EX System Software which comes in an on-board version and an optional off-line version that can be used for analysis of listmode files on a separate computer workstation. Also provided is the Navios EX tetra Software which is used for on-line acquisition of data with the tetraCHROME reagents and for off-line analysis of listmode files. | Same |
| Data Reporting | FlowPAGE, Panel Report, Plots, and Statistics printouts | Same |
| Cleaning Cycle Between Samples | Executed with IsoFlow Sheath Fluid ensuring carryover specification is met | Same |
| Cleanse Cycle | Cleaning cycle performed with FlowClean cleaning reagent as part of the daily shutdown process, before and after running samples with vital dyes that stain the tubing, and as part of troubleshooting. | Same |
| Quality Control | Daily Instrument Checks Commercial Controls Inter-laboratory Quality Assurance Program (IQAP) | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Sample Aspiration Probe | Adjustable by Beckman Coulter field service personnel | Fixed height |
| Lasers / Driver Boards | IVD configuration – 488 nm: Same with the exception of: • Laser Diode, 55mW | IVD configuration – 488 nm • Diode Pumped Solid State (DPSS), 22mW |
{8}
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | • Single custom laser driver board | • Vertical polarization
• 3 laser driver boards (488 nm, 638 nm, 405 nm) |
| Forward Angle Light Scatter | • The forward scatter mask was redesigned to obtain similar forward scatter patterns to the Navios.
• The same single lens is used to collimate the light.
• The band-pass filter was redesigned to accommodate the new laser.
• The same diode detector is used to convert the light into electrical signals. | • Forward scatter mask blocks un-scattered laser light
• Single lens collimates the light
• Band-pass filter passes only 488nm light
• Diode detector converts light into electrical signals |
| Optics | Free space delivery of excitation laser light. Collection optics uses fiber optics.
Spherical lens and cylindrical lens coupled to the flow-cell – 488 spot size = 8 x 60 μm | Achromatic Crossed Cylinder Lens |
| Flow Cell | New Flow Cell with modified channel size and sheath flow pressure. No gel coupling of the lens and optics module temperature management hardware modified. | • Channel Size: 140 x 460 microns
• Sheath Flow Pressure: 4 psi
• Thin wall to allow for gel coupling distance
• Optics module temperature management hardware: two
• thermoelectric devices at the top and bottom of the optics module box |
| Fluorescence collection | Collection lens and light path:
• Spherical mirror and aspheric Schmidt lens
• Operates at NA=1.2.
• Light passes through optical fiber to PMT area
• Light leaves the fiber and is collimated
• Collimated light is separated by filters into desired bands
• PMTs detect light and convert to electrical signals | Collection lens and light path:
• 1st lens set:
• Gel coupled to flow cell, operates at numerical aperture
• (NA) of 1.2
• Light passes through optical fiber to PMT area
• Light leaves the fiber and is collimated
• Collimated light is separated by filters into desired bands
• PMTs detect light and convert to electrical signals |
| Optical Filters | Optical Filters: (SP = Short Pass, BP = Band Pass)
• FL1 FITC 550 SP – 525 BP
• FL2 PE 595 SP – 575/30 BP | All filters same, except:
FL3 ECD 655 SP – 620/30 BP |
{9}
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | • FL3 ECD is 655 SP – 614/20 BP
• FL4 PC5 730 SP – 695/30 BP | |
| Side Angle Light Scatter | Side Scatter light is collected from the same side of the flow-cell as the fluorescence from the blue laser intersection point. It is focused onto the optical fiber by the spherical mirror attached to the surface of the flow cell. The side scatter is split off from the fluorescence signal by a dichroic mirror before entering the filter/PMT assembly. | • Separate lens system focuses the light into an optical fiber
• Light exiting the optical fiber is focused onto a diode through a filter to pass 488nm only |
| Sample Analysis | Principle of analysis is unchanged
• Detection hardware - Changes to the lasers, optical components, laser driver electronics, and flow cell as described in this table.
• Signal Conditioner Analyzer board firmware enhancement to avoid potential data swap errors when processing event rates >25,000 events/ sec.
• Sample analysis pathway and gating is unchanged with the exception of the minor algorithm changes described | Principle of analysis – Flow cytometric
• Detection hardware – Lasers, fluidics, optics, electronics
• Sample analysis pathway
• Manual gating of cellular populations per tetraCHROME
• reagent IFU or automated gating of cellular populations for
• tetraCHROME reagents with Navios tetra software |
| Gating Strategy | Minor changes to the algorithm to accommodate data pattern changes resulting from the new optics module. The changes allow for proper positioning of the dividers between the CD3+CD4- and CD3+CD4+ populations and the CD3-CD56- and CD3-CD56+ populations. | The main Lymphocyte population is determined through a gate including the LADJ and GADJ regions. All gated and reported results (CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3-CD56+) are based on LADJ (including events displaying lower forward scatter properties within the lymphocyte population) and GADJ regions. |
I. Special Control/Guidance Document Referenced:
CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods, Approved Guideline, Second Edition 2004.
CLSI C28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline.
CLSI EP09-A3, Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Second Edition (Interim Revision)
{10}
CLSI H26-A2, Validation, Verification, and Quality Assurance of Automated Hematology Analyzers; Approved Guideline-Second Edition; Section 5.7 – Carryover
CLSI EP17-A2 Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline, Second Edition
EN 61010-1:2001 - Safety requirements for electrical equipment for measurement, control and laboratory use. Part 1: General requirements
## J. Performance Characteristics:
For all studies, the performance data met the sponsor’s acceptance criteria and were found to be acceptable.
## 1. Method Comparisons:
a. Method comparison with predicate device:
A method comparison study was conducted in accordance with CLSI EP09-A3, Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline – Third Edition. This prospective study was conducted with specimens collected and tested at four sites. Subjects included the population of patients presenting for diagnosis and/or prognosis of HIV or being evaluated for immune competency, patients monitored following transplantation and immunosuppression, and apparently healthy, hematologically normal individuals between 18–85 years old. A total of 424 whole blood specimens combined from all clinical sites with emphasis on the medical decision ranges for CD3+CD4+ (50, 100, 200, 350 and 500 cells/μL) were tested on the instrument (Navios EX) and the predicate (Navios).
The test system consisted of the Navios EX Flow Cytometer with Navios EX tetra Software and tetraCHROME reagents for CD3+, CD3+CD4+, CD3+CD8+, CD3-CD56+, CD19+ percent and absolute count with a single platform approach for absolute count using Flow-Count Fluorospheres. The Predicate system consisted of the Navios Flow Cytometer with Navios tetra software and tetraCHROME reagents for CD3+, CD3+CD4+, CD3+CD8+, CD3-CD56+, CD19+ percent and absolute count with a single platform approach for absolute count using Flow-Count Fluorospheres.
Whole blood specimens were held at room temperature and tested at one site within 24 hours from venipuncture. Specimens were prepared in duplicate with tetraCHROME reagents (CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 [Tetra 1] and CD45-FTC/CD56-RD1/CD19-ECD/CD3-PC5 [Tetra 2]) using the TQPrep Workstation and PrepPlus 2 Workstation analyzed once on both the Navios (predicate) and Navios EX (test) flow cytometers. The same preparation was analyzed
11
{11}
on each cytometer. Testing between test and predicate systems was randomized daily to minimize system bias and was within 2.5 hours of each other. Three lots of reagent were used across the sites.
The data met the acceptance criteria for all parameters including CD3+CD4+ at medical decision levels when analyzed for each site individually as well as when all sites were combined (shown in table below).
| Marker Unit | Slope | 95% CI | Intercept | 95% CI | Correlation |
| --- | --- | --- | --- | --- | --- |
| CD3+ T % | 1.009 | 0.997 to 1.022 | -0.415 | -1.375 to 0.545 | 0.993 |
| CD3+ T cells/μL | 0.988 | 0.990 to 0.996 | 3.066 | -2.826 to 8.958 | 0.995 |
| CD3+CD4+ % | 1.002 | 0.996 to 1.008 | -0.072 | -0.244 to 0.101 | 0.998 |
| CD3+CD4+ cells/μL | 0.986 | 0.970 to 1.002 | 0.403 | -3.255 to 4.062 | 0.997 |
| CD3+CD8+ % | 1.004 | 0.998 to 1.009 | -0.133 | -0.379 to 0.112 | 0.998 |
| CD3+CD8+ cells/μL | 0.987 | 0.979 to 0.994 | 0.578 | -1.936 to 3.092 | 0.996 |
| CD3+T % | 1.010 | 1.000 to 1.021 | -0.512 | -1.331 to 0.306 | 0.995 |
| CD3+T cells/μL | 0.996 | 0.990 to 1.002 | 2.866 | -1.743 to 7.476 | 0.998 |
| CD19+ % | 1.014 | 1.004 to 1.024 | -0.147 | -0.264 to 0.030 | 0.996 |
| CD19+ cells/μL | 0.097 | 0.947 to 0.992 | 0.000 | 0.000 to 0.000 | 0.996 |
| CD3-CD56+ % | 1.021 | 0.970 to 1.008 | 0.918 | 0.037 to 1.799 | 0.993 |
| CD3-CD56+ cells/μL | 0.986 | 0.994 to 1.047 | -0.117 | -0.324 to 0.089 | 0.994 |
Bias between methods was calculated from the regression line at the 25-th, 50-th and 75-th percentile of the range of the comparator for all analytes and for different medical decision points of CD3+CD4+ counts. Confidence limits of bias estimates were calculated based on standard errors of bias and $95\%$ confidence. The upper/ lower confidence limits were compared to the acceptance criteria.
| Analyte | Measurand | Level | Bias | 95% CI |
| --- | --- | --- | --- | --- |
| CD3+CD4+ | Cells/uL | 50 | -0.32 | -3.25 to 2.61 |
| | | 100 | -1.01 | -3.25 to 1.24 |
| | | 200 | -2.38 | -3.71 to -1.05 |
| | | 350 | -4.44 | -6.95 to -1.94 |
| | | 500 | -6.51 | -11.21 to -1.80 |
Navios EX flow cytometry system demonstrated acceptable accuracy compared to the predicate, Navios flow cytometer and demonstrated performance across the analytical measuring interval (AMI).
# b. Instrument Configuration Comparisons
Comparability of the performance of the Navios EX flow cytometer with Navios EX tetra Software was verified for all three configurations (6 color/2 laser, 8 color/2 laser, and 10 color/3 laser) and relative to the predicate Navios flow cytometer with Navios tetra Software for absolute counts and percent positive recovery of tetraCHROME target populations. Forty-one specimens (targeting approximately $15\%$ normal and $85\%$ clinical) targeting medical decision points for $\mathrm{CD3 + CD4 + }$ at
{12}
50, 100, 200, 350 and 500 cells/μL were analyzed on three Navios EX flow cytometer configurations (6 color/2 laser, 8 color/2 laser and 10 color/3 laser) with Navios EX tetra Software and the predicate device, Navios flow cytometer with Navios tetra Software using tetraCHROME reagents and Flow-Count Fluorospheres. Specimens were analyzed within 24 of venipuncture. Clinical donors were presenting for diagnosis and/or prognosis of HIV infection. Specimen samples were prepared in quadruplicate using a TQ-Prep and PrepPlus 2 and analyzed singly on each flow cytometer.
6-Color Configuration vs Predicate Device
| Marker Unit | Slope | 95% CI | Intercept | 95% CI | Correlation |
| --- | --- | --- | --- | --- | --- |
| CD3+ T % | 1.013 | 0.979 to 1.048 | -0.505 | -3.194 to 2.184 | 0.995 |
| CD3+ T cells/μL | 0.987 | 0.949 to 1.026 | -3.736 | -47.412 to 39.940 | 0.994 |
| CD3+CD4+ % | 0.999 | 0.979 to 1.019 | 0.019 | -0.417 to 0.456 | 0.999 |
| CD3+CD4+ cells/μL | 0.981 | 0.956 to 1.005 | -1.019 | -4.733 to 2.694 | 0.997 |
| CD3+CD8+ % | 1.005 | 0.984 to 1.026 | 0.099 | -1.021 to 1.218 | 0.998 |
| CD3+CD8+ cells/μL | 0.996 | 0.960 to 1.031 | -6.987 | -31.153 to 17.178 | 0.996 |
| CD3+T % | 1.013 | 0.985 to 1.041 | -0.761 | -2.952 to 1.430 | 0.997 |
| CD3+T cells/μL | 1.011 | 0.971 to 1.052 | -21.934 | -64.865 to 20.997 | 0.995 |
| CD19+ % | 1.012 | 0.994 to 1.031 | -0.105 | -0.352 to 0.142 | 0.998 |
| CD19+ cells/μL | 1.005 | 0.979 to 1.031 | -2.609 | -5.930 to 0.713 | 0.998 |
| CD3-CD56+ % | 1.012 | 0.970 to 1.054 | -0.264 | -0.569 to 0.041 | 0.994 |
| CD3-CD56+ cells/μL | 0.962 | 0.937 to 0.987 | -0.479 | -1.380 to 0.421 | 0.995 |
8 to Color Configuration vs Predicate Device
| Marker Unit | Slope | 95% CI | Intercept | 95% CI | Correlation |
| --- | --- | --- | --- | --- | --- |
| CD3+ T % | 1.01 | 0.965 to 1.054 | -0.201 | -3.699 to 3.297 | 0.993 |
| CD3+ T cells/μL | 0.967 | 0.919 to 1.015 | 14.226 | -27.898 to 56.351 | 0.983 |
| CD3+CD4+ % | 0.993 | 0.962 to 1.025 | 0.258 | -0.348 to 0.865 | 0.998 |
| CD3+CD4+ cells/μL | 0.984 | 0.959 to 1.009 | -1.679 | -5.122 to 1.763 | 0.995 |
| CD3+CD8+ % | 1.007 | 0.981 to 1.033 | -0.121 | -1.542 to 1.3 | 0.997 |
| CD3+CD8+ cells/μL | 0.955 | 0.897 to 1.013 | 15.272 | -20.204 to 50.748 | 0.988 |
| CD3+T % | 1.014 | 0.976 to 1.052 | -0.684 | -3.597 to 2.228 | 0.996 |
| CD3+T cells/μL | 0.978 | 0.944 to 1.011 | 16.787 | -13.693 to 47.267 | 0.992 |
| CD19+ % | 1.011 | 0.983 to 1.039 | -0.149 | -0.574 to 0.276 | 0.996 |
| CD19+ cells/μL | 0.99 | 0.931 to 1.05 | -0.972 | -10.725 to 8.78 | 0.999 |
| CD3-CD56+ % | 0.988 | 0.888 to 1.088 | -0.318 | -0.932 to 0.296 | 0.991 |
| CD3-CD56+ cells/μL | 0.944 | 0.916 to 0.972 | -1.818 | -3.135 to -0.502 | 0.993 |
10-Color Configuration vs Predicate Device
| Marker Unit | Slope | 95% CI | Intercept | 95% CI | Correlation |
| --- | --- | --- | --- | --- | --- |
| CD3+ T % | 1.005 | 0.964 to 1.047 | 0.062 | -3.196 to 3.321 | 0.994 |
| CD3+ T cells/μL | 0.968 | 0.946 to 0.990 | 17.062 | -4.024 to 38.147 | 0.997 |
| CD3+CD4+ % | 1.019 | 1.001 to 1.037 | -0.294 | -0.660 to 0.073 | 0.999 |
| CD3+CD4+ cells/μL | 0.979 | 0.956 to 1.002 | -1.128 | -6.725 to 4.469 | 0.999 |
| CD3+CD8+ % | 1.007 | 0.986 to 1.028 | -0.329 | -1.331 to 0.672 | 0.998 |
{13}
# 2. Precision and Reproducibility
# a. Precision-Long Term Imprecision
Precision Study 1: Precision study with control material using Navios EX tetra software.
Precision studies with control materials were conducted in accordance with CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline. Two levels of control material (IMMUNO-TROL Cells and IMMUNO-TROL Low Cells) were evaluated in the study. The controls were prepared with tetraCHROME reagents (CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5) and Flow-Count Fluorospheres. At one external site, controls were run in duplicate twice each day (morning and afternoon) for 20 days. At two additional external sites, controls were run in triplicate twice each day (morning and afternoon) for five days. Percent positive and absolute counts were measured for $\mathrm{CD3 + }$ , $\mathrm{CD3 + CD4 + }$ , $\mathrm{CD3 + CD8 + }$ , $\mathrm{CD3 - CD56 + }$ , and $\mathrm{CD19 + }$ populations for each level of control product. Absolute counts were determined using Flow-Count Fluorospheres. A new vial of control material was opened each day of testing. The lot of control material for this testing was the same at each site so that data could be pooled during analysis. Three lots of tetraCHROME reagents, Flow-Count Fluorospheres, and ImmunoPrep reagents were tested across the external sites. All parameters met the precision and reproducibility criteria for each site when analyzed separately and with all sites combined.
Precision study using control materials (IMMUNO-TROL (IT) and IMMUNO-TROL Low (ITL); All sites combined)
| Control Level | Unit | Mean | Within Run %CV | Between Runs %CV | Between Days %CV | Between Instruments/ Sites %CV | Total %CV |
| --- | --- | --- | --- | --- | --- | --- | --- |
| IT | CD3 % | 75 | 0.8 | 0.0 | 0.0 | 0.1 | 0.8 |
| IT | CD3 Cells/uL | 868 | 3.7 | 4.8 | 0.0 | 0.0 | 6.1 |
| IT | CD3+CD4+ % | 50 | 1.6 | 0.0 | 0.0 | 0.2 | 1.6 |
| IT | CD3+CD4+ Cells/uL | 575 | 4.0 | 4.8 | 0.0 | 0.0 | 6.2 |
| IT | CD3+CD8+ % | 25 | 2.9 | 0.1 | 0.0 | 0.0 | 2.9 |
| IT | CD3+CD8+ Cells/uL | 286 | 4.8 | 4.4 | 0.0 | 0.0 | 6.5 |
| IT | CD3 % | 75 | 0.7 | 0.0 | 0.0 | 0.1 | 0.7 |
| IT | CD3 Cells/uL | 883 | 2.7 | 4.9 | 0.0 | 0.0 | 5.6 |
| IT | CD3+CD8+ % | 50 | 1.6 | 0.0 | 0.0 | 0.2 | 1.6 |
| IT | CD3+CD8+ Cells/uL | 575 | 4.0 | 4.8 | 0.0 | 0.0 | 6.5 |
{14}
| IT | CD19+% | 16 | 2.6 | 0.0 | 0.0 | 0.0 | 2.6 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| IT | CD19+Cells/uL | 185 | 3.9 | 5.0 | 0.0 | 0.0 | 6.3 |
| IT | CD3-CD56+% | 7 | 5.7 | 3.6 | 0.0 | 3.3 | 7.5 |
| IT | CD3-CD56+Cells/uL | 81 | 7.1 | 8.1 | 0.0 | 3.3 | 11.3 |
| ITL | CD3 % | 58 | 1.4 | 0.4 | 0.4 | 0.0 | 1.4 |
| ITL | CD3 Cells/uL | 421 | 3.9 | 4.4 | 0.0 | 0.7 | 6.0 |
| ITL | CD3+CD4+% | 17 | 3.1 | 0.3 | 0.2 | 0.6 | 3.2 |
| ITL | CD3+CD4+Cells/uL | 125 | 4.7 | 4.6 | 0.0 | 1.2 | 6.7 |
| ITL | CD3+CD8+% | 35 | 2.3 | 0.0 | 0.0 | 0.6 | 2.4 |
| ITL | CD3+CD8+Cells/uL | 251 | 4.5 | 4.0 | 0.0 | 0.7 | 6.1 |
| ITL | CD3 % | 59 | 1.1 | 0.0 | 0.4 | 0.3 | 1.2 |
| ITL | CD3 Cells/uL | 422 | 2.9 | 4.3 | 0.0 | 0.7 | 5.2 |
| ITL | CD19+% | 19 | 2.5 | 0.5 | 0.2 | 0.1 | 2.6 |
| ITL | CD19+Cells/uL | 140 | 3.9 | 4.7 | 0.0 | 0.1 | 6.1 |
| ITL | CD3-CD56+% | 17 | 4.0 | 3.0 | 0.0 | 4.8 | 6.9 |
| ITL | CD3-CD56+Cells/uL | 124 | 5.3 | 6.3 | 0.0 | 5.3 | 9.8 |
# b. Precision - Whole Blood Repeatability
The precision study was conducted with whole blood samples in accordance with CLSI guideline H26-A2, Validation, Verification, and Quality Assurance of Automated Hematology Analyzers; Approved Standard - Second Edition guideline, and CLSI guideline EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition guideline. Whole blood specimens $(n = 141)$ were selected to cover the $\mathrm{CD3 + CD4 + }$ analytical measuring interval with emphasis on the medical decision levels (50, 100, 200, 350, and 500 cells/ $\mu \mathrm{L}$ ). Four $\mathrm{CD3 + CD4 + }$ ranges were targeted: 20-100, 101-350, 351-500, and $>500$ . A minimum of ten clinical or hematologically normal specimens range were collected for each range across all sites. All other markers were assessed at the levels found in the clinical conditions representing the $\mathrm{CD3 + CD4 + }$ levels. Two preparations were made for each tetraCHROME reagent and then each preparation was analyzed three times on the Navios EX flow cytometer using Navios EX tetra Software and Flow-Count Fluorospheres. The study was conducted at four sites. A minimum of three lots of tetraCHROME reagents, Flow Count Fluorospheres, and ImmunoPrep reagents were tested across the clinical sites.
{15}
Precision profile using whole blood (by percentile)
| Reagent | Measurand | Analyte | Percentile | Mean | % CV |
| --- | --- | --- | --- | --- | --- |
| Tetra 1 | % | CD3+ | 25th | 70 | 0.9 |
| | | | 50th | 77 | 0.8 |
| | | | 75th | 83 | 0.7 |
| | | CD3+CD4+ | 25th | 15 | 2.9 |
| | | | 50th | 27 | 2.1 |
| | | | 75th | 40 | 1.7 |
| | | CD3+CD8+ | 25th | 31 | 1.9 |
| | | | 50th | 46 | 1.4 |
| | | | 75th | 57 | 1.2 |
| | # | CD3+ | 25th | 767 | 2.6 |
| | | | 50th | 1242 | 2.5 |
| | | | 75th | 1801 | 2.5 |
| | | CD3+CD4+ | 25th | 152 | 4.0 |
| | | | 50th | 400 | 3.3 |
| | | | 75th | 834 | 2.8 |
| | | CD3+CD8+ | 25th | 440 | 3.1 |
| | | | 50th | 674 | 2.9 |
| | | | 75th | 887 | 2.8 |
| Tetra 2 | % | CD3+ | 25th | 70.6 | 0.7 |
| | | | 50th | 76.9 | 0.6 |
| | | | 75th | 83.7 | 0.5 |
| | | CD19 | 25th | 8.4 | 4.3 |
| | | | 50th | 11.7 | 3.5 |
| | | | 75th | 16.3 | 2.9 |
| | | CD3-CD56+ | 25th | 4.2 | 5.8 |
| | | | 50th | 7.0 | 4.4 |
| | | | 75th | 11.3 | 3.4 |
| | # | CD3+ | 25th | 735 | 2.2 |
| | | | 50th | 1265 | 2.0 |
| | | | 75th | 1878 | 1.9 |
| | | CD19 | 25th | 98 | 3.9 |
| | | | 50th | 185 | 2.8 |
| | | | 75th | 292 | 2.1 |
| | | CD3-CD56+ | 25th | 44 | 6.7 |
| | | | 50th | 128 | 4.6 |
| | | | 75th | 201 | 3.9 |
{16}
Imprecision was also assessed at each medical decision point. Results are presented in the following table.
| Analyte | Measurand | Level | % CV |
| --- | --- | --- | --- |
| CD3+CD4+ | Cells/uL | 50 | 5.0 |
| | | 100 | 4.4 |
| | | 200 | 3.8 |
| | | 350 | 3.4 |
| | | 500 | 3.1 |
For all CD markers at all percentiles as well as for the CD4 marker at the medical decision points, the $\% \mathrm{CV}$ estimated from the precision profile model met the acceptance criteria.
# c. Precision - Whole Blood Site-to-Site Variability
The site-to-site variability study was conducted using the $\mathrm{CD3 + CD4 + }$ absolute count and percentage from a subset of the specimens collected for the whole blood repeatability study. Four different levels within the four $\mathrm{CD3 + CD4 + }$ ranges were targeted across three sites. Forty-three specimens were selected based on the recovery of the predicate Navios system. Specimens at each level were chosen so that similar measurand recoveries were achieved across the sites to ensure poolability of the data and to minimize the effect of biological variability on the variability measurement. A minimum of three specimens per $\mathrm{CD3 + CD4 + }$ level per site were targeted for a minimum of twelve specimens per site. Different specimens were used in the $\mathrm{CD3 + CD4 + }$ percentage and $\mathrm{CD3 + CD4 + }$ absolute count analyses. The study was conducted at three sites. Three lots of tetraCHROME reagents (Tetra 1), Flow Count Fluorospheres, and ImmunoPrep reagents were tested across the clinical sites.
CD4 Precision - Whole Blood Site-to-Site Variability
| Mean | Unit | n | Repeatability CV% | Site-to-Site Reproducibility CV% | Total Reproducibility CV% |
| --- | --- | --- | --- | --- | --- |
| 71 | Cells/uL | 54 | 4.2 | 6.2 | 7.5 |
| 242 | Cells/uL | 60 | 3.5 | 2.4 | 4.3 |
| 437 | Cells/uL | 72 | 3.9 | 2.0 | 4.4 |
| 808 | Cells/uL | 60 | 3.1 | 0.4 | 3.1 |
| 15.2 | % | 71 | 2.8 | 1.6 | 3.2 |
| 20.5 | % | 66 | 2.9 | 4.2 | 5.1 |
| 25.8 | % | 60 | 2.6 | 2.8 | 3.8 |
| 38.2 | % | 60 | 1.9 | 0.0 | 1.9 |
# 3. Linearity:
# 1. Instrument Linearity
{17}
Instrument linearity was assessed with two beads of different fluorescent intensities (SpheroTechTM Rainbow particles level 4 and 5) analyzed at five different voltages covering the dynamic range of each PMT from the first decade (values of 0.1 to 1) to the fourth decade (values of 100 to 1000). The study was conducted on four Navios EX flow cytometers. The beads were run and the voltage of each PMT was adjusted so that the lower of the two peaks falls in the first decade (mean channel $< 1.0$ ) of the histogram. The beads were then "marched" across the dynamic range of the instrument by increasing the voltage until the brightest bead was positioned in the upper half of the top decade. The mean channel fluorescent intensity ratios of pairs were calculated for each PMT and these met the manufacturer's acceptance criteria demonstrating that the intensity differences between the beads were constant across the range of voltages assessed. Linearity of the Navios flow cytometer, from the photomultiplier tubes (PMT) to the electronics, independent of the application and gating methodology was demonstrated.
# 2. Assay Linearity
Linearity of the tetraCHROME reagents on the Navios EX Flow Cytometer with Navios EX tetra software and the manual gating method was established in accordance with CLSI EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach. Linearity samples were prepared using White Cell Pool (WCP) that is used to manufacture IMMUNOTROL and mixed with a diluent prepared using Red Blood Cell pool (RBC pool). This study established that the Navios EX instrument was linear for the tested phenotypes over the following ranges:
| tetraCHROME Reagent | Marker | Linear Range Cells/μL |
| --- | --- | --- |
| tetraCHROME | Total CD3+ | 61–5050 |
| CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 | CD3+CD4+ | 32–3012 |
| | CD3+CD8+ | 8–2040 |
| tetraCHROME | Total CD3+ | 56–4703 |
| CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 | CD19+ | 23–1082 |
| | CD3-CD56+ | 23–949 |
# 3. Analytical Measuring Interval
The AMI of the Navios EX flow cytometer was established and verified to meet all criteria including values within the established Linearity range; AMI lower limits above the LOQ values; AMI lower limits no less than the Navios (predicate) lower limit for each interval; AMI representative of the intended use population. Based on these criteria, the following are reported as the analytical measuring range for this device.
{18}
| tetra | Parameters | Analytical Measuring Range |
| --- | --- | --- |
| CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 | CD3+/μL | 65–3200 |
| | CD3+/CD4+/μL | 35–2000 |
| | CD3+/CD8+/μL | 15–2000 |
| CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 | CD3+/μL | 25–3200 |
| | CD3-/CD56+/μL | 35–650 |
| | CD19+/μL | 35–900 |
4. Carryover:
a. Carryover Using Beads
A carryover study was performed using Flow Check Pro fluorospheres on each of four Navios EX flow cytometers according to the methodology presented in CLSI Document: H26-A2, Validation, Verification, and Quality Assurance of Automated Hematology Analyzers; Approved Standard – Second Edition, Section 5.7. Testing consisted of a high concentration sample (Flow- Check Pro Fluorospheres) analyzed consecutively three times (HTV1, HTV2, HTV3) followed immediately by testing a low concentration sample (IsoFlow Sheath Fluid) three times (LTV1, LTV2, LTV3). Acceptable carryover was demonstrated.
b. Carryover Using Reagent
Carryover studies were conducted on three Navios EX instruments according to the methodology presented in CLSI H26-A2: Validation, Verification, and Quality Assurance of Automated Hematology analyzers; Approved Standard-Second Edition, Section 5.7 with regard to high or low value sample order. Two studies were conducted to evaluate specimen and reagent carryover. Scatter and fluorescence carryover was demonstrated to be less than 1% from one specimen to another when the numbers of gated events was 10,000 or greater. Acceptable carryover was demonstrated.
e. Interfering Substances: Not applicable
5. Other Supportive Instrument Performance Data Not Covered Above:
a. Detection limit:
The LOB, LOD, and LOQ studies were conducted in accordance with CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline-2nd Edition.
i. Limit of Blank (LoB): The study design incorporated five repetitions of each of the three prepared blank samples which were collected each day over four days, yielding a total of 60 data collection points per measuring system for each tetraCHROME reagent. Samples were prepared using the PrepPlus 2 sample
{19}
preparation method (PrepPlus 2 with TQ-Prep). A total of 360 data points were collected for the study using three blank sample preparations, one lot of each reagent and three Navios EX flow cytometers. LOB was calculated for each marker as the upper $95\%$ confidence level of the data as recommended in the CLSI EP17-A2 Guideline. LoB was calculated separately for each of the three instruments. A nonparametric approach based on the ranks of observations was used in the study to calculate LoB for each tetraCHROME marker.
ii. Limit of Detection (LoD) and Limit of Quantitation (LoQ): LoD and LoQ experiments were designed and analyzed according to the variant (precision profile) approach of CLSI EP17-A2. Eight dilutions of ImmunoTrol cells were prepared in ImmunoTrol erythrocytes and were treated with each of the two tetraCHROME reagents, Immunoprep reagents and Flow-Count to measure the LoD and LoQ. Testing was conducted over five days on three Navios EX Flow Cytometers. Each day, one lot of cells was prepared for each cytometer and five replicates of each dilution were tested on each flow cytometer. This resulted in a total of 1200 data points. The results from these studies are presented in the table below:
| Navios EX tetra | CD3+ | CD3+/CD4+ | CD3+/CD8+ | CD3/CD56+ | CD19+ | CD3+ |
| --- | --- | --- | --- | --- | --- | --- |
| LoB (cells/uL) | 7 | 5 | 3 | 0 | 0 | 7 |
| LoD (cells/uL) | 8 | 6 | 4 | 1 | 1 | 8 |
| LoQ (cells/uL) | 8 | 6 | 4 | 4 | 4 | 8 |
# c. Specimen and Prepared Sample Stability
Forty specimens having CD4 counts across the AMI were prepared using CYTO-STAT tetraCHROME reagents in duplicate within eight hours and held at room temperature for 24 and 48 hours and prepared again in order to verify the expected stability as reported in the literature. Specimens held 24 and 48 hours were refrigerated and tested again after an additional 24 and 48 hours. Samples were prepared by a TQ-Prep and PrepPlus 2 in duplicate for each specimen for each time point tested and analyzed singly. Flow-Count Fluorospheres were used to determine absolute counts for all samples analyzed. Analysis was performed using two Navios EX flow cytometers running Navios EX tetra Software. All parameters were verified to demonstrate the claimed stability of 24 hours for whole blood stored at room temperature and 24 hours for prepared specimens stored at refrigerated temperature.
# d. Laser Performance:
Laser performance was assessed on two Navios EX Flow Cytometers using Flow-Check Pro Fluorospheres at one site and it was demonstrated that neither integral signal intensity nor half peak coefficient of variation (HPCV) for forward light scatter (FS) and each of the linear fluorescence parameters vary more than acceptable levels over a period of 24 hours for Short-term Laser Performance and 8 days for Long-term Laser Performance. Stability of the laser performance of the Navios EX flow cytometer over time was demonstrated.
{20}
e. Combined IVD/RUO Functionality
The Navios EX will be marketed in three different configurations, which contain the same IVD functionality, but differ each in their RUO functionality. These three configurations are:
- 2 lasers, 6 color (PMT) [blue (488 nm) and red (638 nm) solid-state diode lasers]
- 2 lasers, 8 color (PMT) [blue (488 nm) and red (638 nm) solid-state diode lasers]
- 3 lasers, 10 color (PMT) [blue (488 nm), red (638 nm), and violet (405nm) solid-state diode lasers]
### K. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
### L. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
