Glocyte Automated Cell Counter System

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K152776 B. Purpose for Submission: Clearance of new device C. Measurand: Total nucleated cells and erythrocytes in cerebrospinal fluid D. Type of Test: Quantitative determination of fluorescence labeled total nucleated cells and erythrocytes in cerebrospinal fluid E. Applicant: Advanced Instruments, Inc. F. Proprietary and Established Names: GloCyte® Automated Cell Counter System GloCyte® Low and High Level Controls G. Regulatory Information: 1. Regulation section: 21 CFR 864.5200, Automated cell counter 21 CFR 864.8625, Hematology quality control mixture 2. Classification: Class II (instrument, assay, and controls) 3. Product code: GKL, counter, cell, automated (particle counter) JPK, mixture, hematology quality control {1} 4. Panel: Hematology (81) H. Intended Use: 1. Intended use(s): The GloCyte® Automated Cell Counter System is intended for use by trained healthcare professionals in clinical laboratories to provide quantitative determination of fluorescence labeled total nucleated cells and erythrocytes in cerebrospinal fluid collected from adult and pediatric patients. The GloCyte® Low and High Level Controls are assayed hematology controls designed to monitor the performance of the GloCyte® Automated Cell Counter System. Assayed parameters include total nucleated cells and erythrocytes. 2. Indication(s) for use: Same as intended use 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: GloCyte Instrument I. Device Description: The GloCyte® Automated Cell Counter System is an automated cell counter that concentrates and enumerates total nucleated cells (TNCs) and red blood cells (RBCs) using fluorescent microscopy and digital image analysis principles. The test method uses one of two reagents to stain TNCs (propidium iodide with detergent) or RBCs (fluorochrome labeled anti-human RBC antibody in buffer with stabilizers), and a digital imaging system to count the cells. The image is captured by a digital CCD camera, and the fluorescent stained cells are counted via digital image processing. The GloCyte® Automated Cell Counter System includes the GloCyte instrument, computer (hardware and software), Vacuum Station, Sample Preparation Tray, barcode reader, pipettes (10 and 30 µL), test cartridge, TNC and RBC reagents, Low and High Level Controls. J. Substantial Equivalence Information: 1. Predicate device name(s): {2} Sysmex XN-10 2. Predicate 510(k) number(s): K112605 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device GloCyte® Automated Cell Counter System | Predicate Sysmex XN-10 | | Sample Type | Cerebrospinal fluid (CSF) | CSF (and other body fluids) | | Hardware | Semiconductor laser with optical components | Flow system, semiconductor laser with optical components | | Parameter(s) | TNC, RBC | WBC-BF#, RBC-BF# | | Differences | | | | --- | --- | --- | | Item | Device GloCyte® Automated Cell Counter System | Predicate Sysmex XN-10 | | Intended Use | The GloCyte® Automated Cell Counter System is intended for use by trained healthcare professionals in clinical laboratories to provide quantitative determination of fluorescence labeled total nucleated cells and erythrocytes in cerebrospinal fluid collected from adult and pediatric patients. | The XN-Series modules (XN-10, XN-20) are quantitative multi-parameter automated hematology analyzers intended for in vitro diagnostic use in screening patient populations found in clinical laboratories.The XN-Series modules classify and enumerate the following parameters in whole blood: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, NEUT%/#, LYMPH%/#, MONO%/#, EO%/#, BASO%/#, IG%/#, RDW-CV, RDW-SD, MPV, NRBC#/%, RET%/#, IPF, IRF, RET-He and has a Body Fluid mode for body fluids. The Body Fluid mode enumerates the WBC-BF, RBC-BF, MN%/#, PMN%/#, and TC-BF parameters in cerebrospinal fluid (CSF), serous fluids (peritoneal, pleural) and synovial fluids. Whole blood should be collected in K2 or K3EDTA anticoagulant and, Serous and Synovial fluids in K2EDTA anticoagulant to prevent clotting of fluid. The use of anticoagulants with CSF specimens is neither required nor recommended. | {3} | Differences | | | | --- | --- | --- | | Item | Device GloCyte® Automated Cell Counter System | Predicate Sysmex XN-10 | | Test Principles | Detection of fluorescence from stained TNCs and RBCs using a semiconductor laser and optical system to capture fluorescent cell images and calculate and display cell counts. | Performs hematology analyses according to the Hydro Dynamic Focusing (DC Detection), flow cytometry method (using a semiconductor laser), and SLS-hemoglobin method. | | Reagents | TNC Reagent (hemolyzes RBCs and stains nucleated cells). RBC Reagent (anti-human glycophorin A/B antibody fluorescent stain). | LYSERCELL WDF (Lyse) FLUOROCELL WDF (Stain) CELLPACK DCL (Diluent) CELLPACK™ DFL (Diluent) | | Calibrators | No external calibrator | XN-10 Calibrator (XN CAL) | | Sample/Fluidic Pathway | No fluidic pathway | Single fluidic pathway | | Throughput | 6–15 samples/hour | 40 samples/hour maximum | | Sample Volume | 30μL for TNC test 30μL for RBC test | 88μL | | Controls | GloCyte® Low and High Level Controls – 2 Levels | XN Check BF – 2 Levels | ## K. Standard/Guidance Document Referenced (if applicable): IEC61010-1:01 Safety requirements for electrical equipment for measurement, control and laboratory use - Part 1: General requirements. IEC61010-2-101:02 Safety requirements for electrical equipment for measurement, control and laboratory use - Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipment. IEC60825-1 Safety of laser products - Part 1: Equipment classification and requirements. IEC 62304: 2006 Medical device software - Software life cycle processes. ISO 14971: 2007 Medical devices - Application of risk management to medical devices. CLSI Document H26-A2 Validation, Verification, and Quality Assurance of Automated Hematology Analyzers; Approved Standard - Second Edition. 2010. CLSI Document EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline. 2014. CLSI Document EP09-A3 Method Comparison and Bias Estimation Using Patient Samples. 2013. CLSI Document EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. 2009. {4} CLSI Document EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline- Second Edition. 2012. CLSI Document EP06-A Evaluation of Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. 2003. CLSI Document H56-A Body Fluid Analysis for Cellular Composition; Approved Guideline. 2006. ## L. Test Principle: Detection of fluorescence from stained TNCs and RBCs using a semiconductor laser and optical system to capture fluorescent cell images and calculate and display cell counts. ## M. Performance Characteristics (if/when applicable): ### 1. Analytical performance: #### a. Precision/Reproducibility: **Repeatability** Repeatability was accessed in-house using two test cartridge lots, two reagent lots (one TNC and one RBC) and GloCyte® Low and High Level Controls. Fourteen manipulated human CSF samples (7 TNC samples and 7 RBC samples) with concentrations evenly distributed throughout the reportable range of the GloCyte® Automated Cell Counter System were tested. Ten replicates were performed on the GloCyte System per cell concentration level for a total of 70 tests each for the RBC counts and TNC counts. The SD and %CV were calculated for each study sample. The results of the repeatability met the performance specifications (%CV) for the GloCyte® Automated Cell Counter System. **Summary of Repeatability Results: In-house TNC** | Assay | Test Level | N | Dilution Performed | Mean | Median | Standard Deviation | %CV | | --- | --- | --- | --- | --- | --- | --- | --- | | TNC | TNC 1* | 10 | 1:1,000 | 10,313 | 10,500 | 874.4 | 8.5 | | | TNC 2* | 10 | 1:1,000 | 8,707 | 8,767 | 907.6 | 10.4 | | | TNC 3 | 10 | 1:1,000 | 6,807 | 7,000 | 492.3 | 7.2 | | | TNC 4 | 10 | 1:100 | 5,225 | 5,190 | 223.2 | 4.3 | | | TNC 5 | 10 | 1:100 | 2,846 | 2,873 | 176.6 | 6.2 | | | TNC 6 | 10 | 1:100 | 1,898 | 1,890 | 138.5 | 7.3 | | | TNC 7 | 10 | 1:10 | 426 | 427 | 13.6 | 3.2 | *indicates TNC concentration above the GloCyte System reportable range {5} Summary of Repeatability Results: In-house RBC | Assay | Test Level | N | Dilution Performed | Mean | Median | Standard Deviation | %CV | | --- | --- | --- | --- | --- | --- | --- | --- | | RBC | RBC 1* | 10 | 1:10,000 | 727,800 | 736,000 | 35,383 | 4.9 | | | RBC 2* | 10 | 1:10,000 | 647,800 | 650,000 | 45,200.30 | 7.0 | | | RBC 3 | 10 | 1:10,000 | 446,000 | 441,333 | 25,066.80 | 5.6 | | | RBC 4 | 10 | 1:10,000 | 263,800 | 261,667 | 14,429.30 | 5.5 | | | RBC 5 | 10 | 1:10,000 | 98,400 | 99,334 | 6,403.70 | 6.5 | | | RBC 6 | 10 | 1:1,000 | 49,467 | 49,567 | 1,751.10 | 3.5 | | | RBC 7 | 10 | 1:100 | 10,546 | 10,680 | 584.60 | 5.5 | *indicates RBC concentration above the GloCyte System reportable range An additional repeatability was conducted at three intended use sites using 13 TNC and 16 RBC clinical patient clinical CSF samples, nine test cartridges lots, six reagent lots (3 TNC and 3 RBC), and two lots of the GloCyte® Low and High Level Controls. Low Level Control samples (approximately 12 cells/μL of TNC and RBC) were tested using the 30 μL test volume; High Level Control samples (approximately 130 cells/μL of TNC and RBC) were tested using the 10 μL test volume. Each sample and control was run 10 times by a single operator for RBC and/or TNC counts. A total of 100 tests each for RBC counts and TNC counts were run at each site. The results of the repeatability met the performance specifications (%CV) for the GloCyte® Automated Cell Counter System. Repeatability Performance | Site 1 | | N | Mean | Median | Standard Deviation | %CV | | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Sample ID | | | | | | | TNC | 303 | 10 | 17.9 | 18.0 | 1.1 | 6.2 | | | 304 | 10 | 71.6 | 71.0 | 3.8 | 5.3 | | | 306 | 10 | 4.9 | 5.0 | 0.6 | 11.6 | | | H1103 | 10 | 123.2 | 123.5 | 3.1 | 2.5 | | | L1103 | 10 | 10.6 | 11.0 | 0.8 | 8.0 | | RBC | 301 | 10 | 17.1 | 17.0 | 1.0 | 5.8 | | | 302 | 10 | 75.7 | 74.5 | 4.5 | 5.9 | | | 305 | 10 | 148.3 | 149.0 | 7.9 | 5.3 | | | 307 | 10 | 101.3 | 101.0 | 6.5 | 6.4 | | | H1103 | 10 | 131.1 | 130.5 | 8.7 | 6.6 | | | L1103 | 10 | 11.7 | 11.5 | 1.1 | 9.1 | {6} | Site 2 | | N | Mean | Median | Standard Deviation | %CV | | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Sample ID | | | | | | | TNC | 300 | 10 | 4.1 | 4.0 | 0.7 | 18.0 | | | 304 | 10 | 147.8 | 152.0 | 10.5 | 7.1 | | | 305 | 10 | 78.5 | 79.0 | 2.2 | 2.8 | | | 308 | 10 | 1.2 | 1.0 | 0.4 | 35.1* | | | 310 | 10 | 11.7 | 12.0 | 0.9 | 8.1 | | | 313 | 10 | 177.6 | 171.0 | 29.4 | 16.5 | | | H1105 | 10 | 118.6 | 119.5 | 4.7 | 4.0 | | | L1105 | 10 | 10.3 | 10.5 | 0.9 | 9.2 | | RBC | 303 | 10 | 77.5 | 78.0 | 6.0 | 7.8 | | | 306 | 10 | 138.5 | 138.5 | 4.2 | 3.0 | | | 307 | 10 | 6.3 | 6.0 | 0.7 | 10.7 | | | 309 | 10 | 60.3 | 59.5 | 6.7 | 11.1 | | | 311 | 10 | 62.9 | 61.5 | 5.7 | 9.1 | | | 314 | 10 | 838.2 | 844.0 | 73.5 | 8.8 | | | H1105 | 10 | 134.2 | 134.0 | 3.8 | 2.9 | | | L1105 | 10 | 11.4 | 11.5 | 1.0 | 8.5 | *Sample diluted 1:10. The mean of this sample (1.2 cells/μL) is below the LoQ of the GloCyte System, and hence is not within the measuring range. | Site 4 | | N | Mean | Median | Standard Deviation | %CV | | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Sample ID | | | | | | | TNC | 125 | 10 | 2.1 | 2.0 | 0.3 | 15.1 | | | 128 | 10 | 7.8 | 8.0 | 0.9 | 11.8 | | | 129 | 10 | 5.5 | 5.5 | 0.9 | 15.5 | | | 198 | 10 | 65.9 | 65.0 | 4.3 | 6.5 | | | 199 | 10 | 7.9 | 8.0 | 1.0 | 12.6 | | | 200 | 10 | 183.3 | 182.0 | 7.4 | 4.0 | | | H1103 | 10 | 128.6 | 127.0 | 4.4 | 3.4 | | | L1103 | 10 | 9.8 | 10.0 | 0.9 | 9.4 | | RBC | 125 | 10 | 34.3 | 34.5 | 3.0 | 8.8 | | | 126 | 10 | 5.0 | 5.0 | 0.8 | 16.3 | | | 127 | 10 | 209.1 | 211.0 | 6.3 | 3.0 | | | 195 | 10 | 7.1 | 7.0 | 1.0 | 14.0 | | | 196 | 10 | 66.7 | 66.5 | 5.6 | 8.5 | | | 197 | 10 | 131.0 | 131.0 | 3.6 | 2.7 | | | H1103 | 10 | 125.3 | 127.5 | 6.8 | 5.4 | | | L1103 | 10 | 10.0 | 10.0 | 1.1 | 10.5 | {7} Reproducibility Reproducibility performance was conducted at three intended use sites over 20 operating days utilizing 15 test cartridges lots, six reagent lots (3 TNC and 3 RBC), and one lot of Low and High Level Controls for both the RBC and TNC assays. Testing was done twice daily using the same set of controls, for 20 days. Each control set was run, in duplicate, independently, by two operators at each site. Standard deviation and %CV were calculated for each measurand and the results obtained were within specifications. Reproducibility Performance | Combined Sites | | | | Within Run | | Between Run | | Between Day | | Between Site | | Between-Operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Control | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | TNC | Low | 480 | 10.6 | 1.1 | 10.1 | 0.0 | 0.0 | 0.2 | 1.6 | 0.4 | 3.9 | 0.3 | 2.4 | 1.2 | 11.2 | | | High | 480 | 122.9 | 7.3 | 5.9 | 0.0 | 0.0 | 0.0 | 0.0 | 3.8 | 3.1 | 1.8 | 1.5 | 8.4 | 6.9 | | RBC | Low | 480 | 11.3 | 1.0 | 9.2 | 0.0 | 0.0 | 0.2 | 1.9 | 0.4 | 3.8 | 0.3 | 2.7 | 1.2 | 10.5 | | | High | 480 | 130.0 | 6.8 | 5.3 | 0.0 | 0.0 | 0.9 | 0.7 | 2.2 | 1.7 | 1.3 | 1.0 | 7.4 | 5.7 | | Site 1 | | | | Within-Run | | Between-Run | | Between-Day | | Between-Operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Control | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | TNC | Low | 160 | 11.0 | 1.0 | 9.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.3 | 3.0 | 1.1 | 9.9 | | | High | 160 | 124.4 | 6.0 | 4.8 | 0.0 | 0.0 | 0.4 | 0.3 | 5.3 | 4.3 | 8.0 | 6.5 | | RBC | Low | 160 | 11.7 | 1.0 | 9.2 | 0.1 | 1.2 | 0.0 | 0.0 | 0.4 | 3.7 | 1.1 | 9.2 | | | High | 160 | 129.7 | 5.6 | 4.3 | 0.0 | 0.0 | 0.0 | 0.0 | 2.6 | 2.0 | 6.2 | 4.8 | | Site 2 | | | | Within-Run | | Between-Run | | Between-Day | | Between-Operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Control | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | TNC | Low | 160 | 10.6 | 1.0 | 9.8 | 0.1 | 1.0 | 0.1 | 1.0 | 0.0 | 0.0 | 1.1 | 9.1 | | | High | 160 | 125.7 | 7.4 | 5.9 | 0.0 | 0.0 | 0.2 | 0.1 | 0.0 | 0.0 | 7.4 | 5.9 | | RBC | Low | 160 | 10.8 | 1.0 | 9.2 | 0.2 | 1.9 | 0.2 | 1.4 | 0.3 | 2.7 | 1.1 | 9.9 | | | High | 160 | 132.4 | 6.9 | 5.2 | 1.3 | 1.0 | 0.6 | 0.4 | 3.6 | 2.7 | 7.9 | 6.0 | {8} 9 | Site 4 | | | | Within-Run | | Between-Run | | Between-Day | | Between-Operator | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Control | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | TNC | Low | 160 | 10.1 | 1.0 | 10.1 | 0.0 | 0.0 | 0.1 | 1.4 | 0.4 | 4.0 | 1.1 | 11.0 | | | High | 160 | 118.5 | 7.5 | 6.3 | 0.8 | 0.7 | 0.4 | 0.3 | 0.0 | 0.0 | 7.5 | 6.4 | | RBC | Low | 160 | 11.2 | 0.9 | 8.4 | 0.0 | 0.0 | 0.1 | 1.3 | 0.2 | 1.6 | 1.0 | 8.6 | | | High | 160 | 127.9 | 7.4 | 5.8 | 0.0 | 0.0 | 0.0 | 0.0 | 2.0 | 1.6 | 7.7 | 6.0 | A reproducibility study was also performed in-house over 25 days using two GloCyte instruments, 19 test cartridge lots, and 10 reagent lots (5 TNC and 5 RBC), one lot of GloCyte® Low and High Level Controls as samples as well as a Mid-Level Control sample that was made from a combination of the Low and High Level Control, and two operators. Three cell concentration levels were tested in duplicate for both TNCs and RBCs, twice daily (2 runs) by each operator on each GloCyte instrument. The Low Level Control targeted (approximately 12 cells/μL), the Mid-Level targeted (approximately 40–50 cells/μL), and the High Level targeted (approximately 120 cells/μL) each targeted cell concentration for each control level were for TNCs and RBCs. The Low Level Control was tested with the 30 μL test volume and the High Level Control was tested with the 10 μL test volume. The mid-level control was tested with both the 30 μL and 10 μL test volumes. To further demonstrate precision performance, SD and %CV of the within-run, between-run, within-day, between-instruments, between-operator, and between-day precision calculations were performed. The test results demonstrate that the %CV specifications were met for the Low, Mid, and High Level cell concentrations for both the TNC and RBC assays on the GloCyte® Automated Cell Counter System, at test volumes of 10 μL or 30 μL. Reproducibility Performance – In-house Study | In-house | | | | Within-Run | | Between-Run | | Between-Instrument | | Between-Operator | | Between-Day | | Total | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Cell Type | Level | Test Volume | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | TNC | Low | 30 | 10.8 | 1.1 | 10.0 | 0.0 | 0.0 | 0.1 | 0.2 | 0.0 | 0.0 | 0.3 | 2.4 | 1.1 | 10.3 | | | Mid | 10 | 42.9 | 3.3 | 7.7 | 0.2 | 0.4 | 0.6 | 1.4 | 1.1 | 2.5 | 1.5 | 3.4 | 3.8 | 8.9 | | | | 30 | 41.1 | 2.1 | 5.2 | 0.5 | 1.1 | 0.0 | 0.0 | 0.5 | 1.3 | 1.3 | 3.2 | 2.6 | 6.3 | | | High | 10 | 122.3 | 6.4 | 5.3 | 0.9 | 0.7 | 0.0 | 0.0 | 3.6 | 2.9 | 2.2 | 1.8 | 7.7 | 6.3 | | RBC | Low | 30 | 12.3 | 1.0 | 2.2 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.2 | 0.5 | 1.0 | 8.4 | | | Mid | 10 | 46.1 | 3.3 | 7.1 | 0.0 | 0.0 | 0.0 | 0.0 | 1.0 | 2.2 | 1.3 | 2.8 | 3.7 | 7.9 | | | | 30 | 45.0 | 2.1 | 4.7 | 0.4 | 0.9 | 0.2 | 0.5 | 1.1 | 2.4 | 1.1 | 2.4 | 2.7 | 5.9 | | | High | 10 | 133.6 | 7.2 | 5.4 | 0.0 | 0.0 | 0.4 | 0.3 | 2.9 | 2.2 | 1.3 | 1.0 | 7.8 | 5.9 | {9} # b. Linearity/assay reportable range: The linearity was conducted in-house using manipulated human CSF samples with concentrations spanning the entire reportable intervals (RI) of both TNC and RBC cell types on the GloCyte® Automated Cell Counter System. A total of 14 concentration levels ranging from roughly 0–8,000 TNCs/μL were tested for the TNC and a total of 15 concentration levels ranging from roughly 0–800,000 RBCs/μL for the RBC were tested on three GloCyte® Automated Cell Counter Systems by three operators. The RI consists of both the analytical measurement range (sample levels that do not require a dilution to be measured on the GloCyte® Automated Cell Counter System) and the extended measuring interval (sample levels that require dilution to be measured on the GloCyte® Automated Cell Counter System). Dilution guidelines based on the visual appearance of the samples as well as dilution/test volume recommendations provided by the GloCyte® Automated Cell Counter System software were used to run each of the test samples tested in the linearity study. The results of the GloCyte® Automated Cell Counter System linearity study support the following linearity claims: Analytical Measuring Range (AMR) | Cell Type | AMR GloCyte® Automated Cell Counter System [cells/μL] | | --- | --- | | TNC | 3–123 | | RBC | 2–123 | # c. Traceability, Stability, Expected values (controls, calibrators, or methods): # GloCyte® Low and High Level Controls Value Assignment Value assignment was conducted by two operators on two GloCyte® Automated Cell Counter Systems with three vials of each control level. Each vial is tested four times by each of the two operators. The vials are stored at $2 - 8^{\circ}\mathrm{C}$ and brought to room temperature for 15 minutes prior to testing. The mean and standard deviation for the low and high controls are calculated from the total number of tests for each control level. The final value assignment for each lot of controls is based on the mean count $\pm 3$ standard deviations. # Quality Control Stability To determine closed vial stability, three control lots were analyzed at several time points (0, 1, 3, 4, 5, 6, 7, 8, 9, 11, 13 months). The unopened GloCyte® Low and High Level Control vials from each lot were removed from $2 - 8^{\circ}\mathrm{C}$ storage conditions and tested with both TNC Reagent (10 replicates) and RBC Reagent (10 replicates) on a GloCyte® Automated Cell Counter System. GloCyte® Low and High Level Controls closed vial stability study data support a stability claim of 7 months when stored at {10} 2–8°C. To determine open vial stability two lots of GloCyte® Low and High Level Controls were tested over a 15 day period, with three replicates tested from each of three vials for a total of nine replicates for both TNC and RBC assays. The opened vial stability claim has been established at 8 days after opening when stored at 2–8°C after each use. ## CSF Sample Stability The sample stability study was conducted at three clinical laboratory sites by testing nine total nucleated cell (TNC) CSF samples and seven (7) red blood cell (RBC) CSF samples over the course of 5 hours, with three replicates tested each hour on the GloCyte® Automated Cell Counter System. The data collected in the CSF stability study was normalized due to the varying cell concentrations among samples tested by converting the cell counts into percent recoveries based on the cell count obtained for each sample at time zero. The result from each individual replicate tested beyond time zero was divided by the mean cell count produced at time zero and multiplied by 100%. The results of the CSF specimen stability study support a 4-hour timeframe for testing CSF specimens upon delivery to the clinical laboratory. ## GloCyte Reagent Stability Reagent stability was assessed using simulated spinal fluid (SSF). SSF samples were created on each day of testing using whole blood diluted into a simulated CSF matrix. The stability of the GloCyte TNC and RBC Reagents was established using both closed reagent vials (shelf life stability) and open reagent vials (in-use stability). ### i. TNC Reagent Stability #### Closed vial stability Three vials from each of three lots of TNC Reagent that were stored at two temperatures (4°C and 10°C) and tested at various time intervals (0, 1, 2, 3, 4, 5, 6, 7, 9 or 10, 13, 15 and 16 months). TNC Reagent vials were removed from their respective storage conditions at each time point and brought to room temperature before testing for a total of five replicates per TNC Reagent vial on the GloCyte® Automated Cell Counter System. The TNC closed vial study data supports stability claim of 13 months when stored at 2–8°C. #### Open vial stability Six TNC Reagent vials from two lots of reagent were brought to room temperature from 4°C storage conditions and tested on the GloCyte® Automated Cell Counter System at each test point (0, 4, 7, 13, and 15 months). Three replicates from each vial were tested with the normal saline and simulated spinal fluid samples. Three of the six TNC Reagent vials were then stored at 4°C and the other three TNC Reagent vials were stored at 22°C to simulate refrigerated and room temperature storage conditions, respectively. These opened TNC Reagent vials from both storage conditions were tested weekly for a total of 5 weeks. The 11 {11} TNC opened vial stability claim has been established at 5 days after opening when stored at room temperature. All acceptance criteria for the closed and open vial stability were met for the TNC Reagent. ## ii. RBC Reagent Stability ### Closed vial stability Three vials from each of three lots of RBC Reagent that were stored at two temperatures (4°C and 10°C) and tested at various time intervals (0, 1, 2, 3, 4, 5, 6, 7, 10, 13, 15 and 17 months). The RBC Reagent vials were removed from their respective storage conditions at each time point and brought to room temperature before testing on the GloCyte® Automated Cell Counter System. Normal saline and simulated spinal fluid samples were tested with the RBC Reagents for a total of five replicates per RBC Reagent vial on the GloCyte System. The RBC closed vial study data supports a preliminary stability claim of 13 months when stored at 2–8°C. ### Open vial stability Six RBC Reagent vials each from each of two lots of RBC Reagent were brought to room temperature from 4°C storage conditions and tested on the GloCyte® Automated Cell Counter System with normal saline and simulated spinal fluid at each test point (0, 4, 7, 13, and 15 months). Three replicates from each vial were tested with the normal saline and simulated spinal fluid samples. Three of the six RBC Reagent vials were then stored at 4°C and the other three RBC Reagent vials were stored at 22°C to simulate refrigerated and room temperature storage conditions, respectively. These opened RBC Reagent vials from both storage conditions were tested weekly for a total of five weeks. The RBC opened vial stability claim has been established at 5 days after opening when stored at room temperature. All acceptance criteria for the closed and open vial stability were met for the RBC Reagent. ### GloCyte Test Cartridge Stability To determine closed vial stability, three lots of GloCyte test cartridges were evaluated on the GloCyte System for both TNC and RBC assays at three different storage temperatures (8°C, 22°C, and 42°C). Testing was performed at various time points (0, 1, 2, 3, 4, 5, 6, 7, 8, 10 and 13 months). GloCyte test cartridges were brought to room temperature from the different storage conditions prior to testing on the GloCyte at each time point. The GloCyte test cartridges were tested with normal saline and simulated spinal fluid (both nucleated cells and red blood cells) for a total of 72 cartridges per cell type. The GloCyte test cartridge study data supports a stability claim of 10 months when stored at 10°C – 40°C. ## d. Detection limit: Limit of Blank (LoB) testing was performed using five blank human CSF samples with low concentrations (1–2 cells/μL) of TNCs and/or RBCs on two GloCyte® 12 {12} Automated Cell Counter Systems. Samples were tested four times with one reagent lot and one cartridge lot and four times with a second reagent and cartridge lot for a total of 40 tests per day. A total of 120 tests were performed on each instrument per assay (TNC or RBC) over a testing period of 3 days. LoD testing was performed using CSF specimens with low (RBC or TNC) count levels (1–2 cells/μL). Six RBC and six TNC CSF specimens were tested on each of two GloCyte instruments. The reference cell counts for each low-count CSF specimen were determined by manual hemocytometer cell counting. Each low-count CSF specimen was tested 10 times with one reagent lot and one cartridge lot and 10 times with a second reagent and cartridge lot. A total of 120 tests were performed on each instrument per assay. The LoQ was determined by graphically plotting the estimated percent total error (%TE) of the GloCyte® Automated Cell Counter System along the y-axis and the "true" manual count for each sample along the x-axis and then performing a linear regression. The LoQ is the manual count at which the linear regression intersects the 20% maximum allowable %TE acceptance criteria. | Analyte | LoB (Cells/μL) | LoD (Cells/μL) | LoQ (Cells/μL) | | --- | --- | --- | --- | | RBC | <1 | 1 | 2 | | TNC | <1 | 1 | 3 | These results indicate that the estimated LoB, LoD and LoQ for the GloCyte® Automated Cell Counter System TNC and RBC assays meet the expected performance criteria for background limits and low limits of the measuring ranges. e. Analytical specificity: The interference study was conducted to evaluate the potential of various endogenous substances present in CSF to affect RBC and TNC counts. Human whole blood was drawn on days of testing and mixed with pooled human CSF to create the Matrix/Base Pools containing RBCs or TNCs at two different cell concentrations: low (approximately 10 cells/μL) and high (approximately 120 cells/μL). A total of four Matrix/Base Pools were created, two per assay cell type (RBC or TNC cells/μL). Samples for interference testing were created using these Base Pools by spiking in a fixed volume of either the potential interferent or the solvent used to prepare the potential interferent stock solution. All the potential interferents, with the exception of nucleated red blood cells (NRBCs), were tested at high, "worst case" concentrations that a typical laboratory would observe among patient specimens submitted for analysis, and lower concentrations that are more prevalent in a normal population or in patients with only slightly elevated levels of that substance. Interference from NRBCs were evaluated using six clinical peripheral blood samples with varying NRBC concentrations used to create manipulated CSF samples to be tested using the TNC Reagent on the GloCyte® Automated Cell Counter System. 13 {13} Each NRBC test sample was run for a total of 10 replicates. The GloCyte® Automated Cell Counter System RBC and TNC Assays count immature cells including NRBCs. Interference Testing Summary | Potential Interferent | GloCyte Assay | Highest Concentration at which No Interference was observed | | --- | --- | --- | | Conjugated Bilirubin | TNC | 307.8 μmol/L (18.0 mg/dL) | | | RBC | 307.8 μmol/L (18.0 mg/dL) | | Unconjugated Bilirubin | TNC | 323.2 μmol/L (18.9 mg/dL) | | | RBC | 323.2 μmol/L (18.9 mg/dL) | | Hemolytic Hemoglobin | TNC | 1.1 g/dL | | | RBC | NONE | | Protein | TNC | 59.0 g/L | | | RBC | 59.0 g/L | | Lactate | TNC | 13.2 mmol/L | | | RBC | 13.2 mmol/L | | Haemophilus influenzae | TNC | 10^{8} CFU/mL | | | RBC | 10^{8} CFU/mL | | Streptococcus pneumoniae | TNC | 10^{8} CFU/mL | | | RBC | 10^{8} CFU/mL | | Neisseria lactamica | TNC | 10^{7} CFU/mL | | | RBC | 10^{8} CFU/mL | | Escherichia coli | TNC | 10^{8} CFU/mL | | | RBC | 10^{8} CFU/mL | | Candida albicans | TNC | 10^{8} CFU/mL | | | RBC | 10^{8} CFU/mL | | Platelets | TNC | 14.7 x 10^{3} Platelets/μL | | | RBC | 14.7 x 10^{3} Platelets/μL | | Nonspecific FcR Binding (Monocytes) | RBC | 1.7 x Monocytes/μL | | RBC Fragments | RBC | Could Not be Quantified | | Nucleated RBCs | TNC | None | f. Assay cut-off: Not applicable 2. Comparison studies: {14} # a. Method comparison with predicate device: A total of 321 CSF specimens (203 adult specimens and 118 pediatric specimens) for TNC and a total of 422 CSF specimens (243 adult specimens and 179 pediatric specimens) for RBC were analyzed at five clinical sites in the U.S. The CSF samples were tested for TNC and RBC counts using the Neubauer hemocytometer (reference method). Each sample was analyzed in duplicate on the Neubauer hemocytometer and on the GloCyte® Automated Cell Counter System. Results were compared by regressing the first of the duplicate measurements for the GloCyte® Automated Cell Counter System against the mean of the duplicate counts for the hemocytometer. An in-house method comparison was also performed by using manipulated CSF samples (31 samples for TNC and 41 samples for RBC) that were created by diluting human TNC and RBC into pooled blank human CSF. Pediatric samples (pediatric venous blood and pediatric CSF) and adult samples (adult venous blood and adult CSF) were contrived to allow for separate analyses of pediatric and adult data. Test sample levels were randomly tested. Two manual counts were performed using the Neubauer hemocytometer and two counts were performed using the GloCyte® Automated Cell Counter System. The external and in-house method comparison slope and intercept results of all regression analyses met the acceptance criteria. The TNC and RBC results demonstrate that the GloCyte Automated Cell Counter System yields results equivalent to manual counts using the Neubauer hemocytometer for the intended use, in both pediatric and adult populations. Summary of Method Comparison Clinical Data by Site | Cell Type | Population | N | Range [cells/μL] | Slope (95% CI) | Intercept (95% CI) | Differences within 95% CI | | --- | --- | --- | --- | --- | --- | --- | | Site 1 | | | | | | | | TNC | Pediatric | 59 | 0 – 1,329 | 1.066 (1.000 – 1.250) | 0.000 (-0.250 – 0.000) | 93% | | | Adult | 2 | NA | NA | NA | NA | | RBC | Pediatric | 88 | 0 – 758,125 | 0.874 (0.858 – 0.929) | 0.000 (-0.107 – 0.170) | 95% | | | Adult | 4 | NA | NA | NA | NA | | Site 2 | | | | | | | | TNC | Pediatric | 19 | 0 – 2,513 | 0.822 (0.740 – 0.890) | 1.000 (-0.781 – 1.781) | 95% | | | Adult | 90 | 0 - 601 | 0.962 (0.903 – 1.000) | 0.038 (0.000 – 0.193) | 96% | | RBC | Pediatric | 21 | 0 – 25,695 | 0.929 (0.876 – 1.039) | 0.000 (-1.039 – 0.000) | 95% | | | Adult | 111 | 0 – 49,000 | 0.943 (0.914 – 0.999) | 0.000 (0.000 – 0.086) | 98% | {15} | Cell Type | Population | N | Range [cells/μL] | Slope (95% CI) | Intercept (95% CI) | Differences within 95% CI | | --- | --- | --- | --- | --- | --- | --- | | Site 3 | | | | | | | | TNC | Pediatric | 9 | 0 - 261 | 1.000 (0.333 - 1.423) | 0.000 (-1.423 - 0.667) | 100% | | | Adult | 32 | 0 - 1,472 | 1.030 (0.969 - 1.167) | 0.864 (-0.167 - 1.031) | 97% | | RBC | Pediatric | 19 | 0 - 4,886 | 0.932 (0.823 - 1.074) | 0.000 (-1.074 - 4.354) | 95% | | | Adult | 39 | 0 - 59,722 | 1.029 (0.982 - 1.088) | 0.000 (-0.088 - 0.018) | 95% | | Site 4 | | | | | | | | TNC | Pediatric | 14 | 0 - 209 | 1.047 (0.933 - 1.458) | -0.047 (-1.687 - 0.067) | 93% | | | Adult | 77 | 0 - 6,306 | 1.042 (1.000 - 1.134) | -0.042 (-0.137 - 0.000) | 97% | | RBC | Pediatric | 16 | 0 - 251,112 | 0.895 (0.8202 - 1.074) | 1.053 (0.131 - 3.478) | 94% | | | Adult | 87 | 0 - 58,417 | 1.068 (1.021 - 1.114) | 0.000 (0.000 - 0.000) | 97% | | Site 5 | | | | | | | | TNC | Pediatric | 17 | 0 - 975 | 0.933 (0.831 - 1.147) | 0.067 (-7.448 - 4.471) | 94% | | | Adult | 2 | NA | NA | NA | NA | | RBC | Pediatric | 35 | 0 - 11,000 | 0.910 (0.835 - 0.935) | -0.281 (-1.442 - 1.000) | 97% | | | Adult | 2 | NA | NA | NA | NA | Summary of Method Comparison Studies | Parameter | | Range (cells/μL) | Slope (95% CI) | Intercept (95% CI) | | --- | --- | --- | --- | --- | | RBC | Pediatric | 0 – 817,500 | 0.910 (0.885 0.935) | 0.000 (-0.045, 0.058) | | | Adult | 0 – 901,250 | 1.000 (0.986, 1.007) | 0.000 (0.000, 0.014) | | TNC | Pediatric | 0 – 7,672 | 0.963 (0.909, 1.000) | 0.037 (0.000, 0.182) | | | Adult | 0 – 9,900 | 1.000 (1.000, 1.003) | 0.000 (-0.003, 0.000) | b. Matrix comparison: Not applicable 3. Clinical studies: {16} a. Clinical Sensitivity: Not applicable b. Clinical specificity: Not applicable c. Other clinical supportive data (when a. and b. are not applicable): 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: The reference ranges were based on the existing medically accepted published reference ranges. CSF Normal Reference Ranges | Demographic | Total WBC | RBC | | --- | --- | --- | | Neonates (<1 year) | 0–30 cells/μL | None | | Ages 1 to 4 years | 0–20 cells/μL | None | | Ages 5 to puberty (18 years) | 0–10 cells/μL | None | | Adults (>18 years) | 0–5 cells/μL | None | # N. Instrument Name: GloCyte® Instrument # O. System Descriptions: 1. Modes of Operation: Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device? Yes __x__ or No __ Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission? {17} Yes _______ or No _______ x _______ 2. Software: FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types: Yes _______ x _______ or No _______ 3. Specimen Identification: The personal computer and application software provide the primary user interface for interactions with the GloCyte Instrument. Test information and user commands are entered into the instrument program using a standard keyboard and mouse/pointing device. 4. Specimen Sampling and Handling: The Sample Preparation Tray includes locations for patient specimen tubes, dilution tubes, staining tubes, cartridges, controls, and either TNC or RBC reagents. CSF is stained with TNC or RBC reagents. The stained CSF sample is deposited manually onto the test cartridge, using either the 10 or 30 µL fixed volume pipette. The test cartridge is used in conjunction with the Vacuum Station, which draws the stained test sample liquid through the test cartridge membrane, and is analyzed in the GloCyte Instrument. 5. Calibration: Not applicable 6. Quality Control: GloCyte® Low and High Level Controls P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above: Not applicable Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 18