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Diana Careaga1, Robert Magari1, Karen Lo1, Michael Keeney2, Janice Popma2, Joanne Luider3, Aito Ueno3, Gerard Lozanski4, Bruce Briggs4, Elena Afonina5, Enrique Rabellino5, and Liliana Tejidor1

Beckman Coulter Diagnostics, Inc., 1Clinical Research, Miami, Florida, USA 2London Health Sciences Centre, London, Ontario, Canada 3Foothills Hospital, Calgary, Alberta, Canada 4Ohio State University Medical Center, Columbus, Ohio Beckman Coulter Life Science, Inc., 5Clinical Applications Development, Miami, Florida, USA

DEMONSTRATION OF THE ANALYTICAL PERFORMANCE OF THE NAVIOS FLOW CYTOMETER IN A MULTI-CENTER STUDY

Introduction

Objectives and Methods

The performance of the Navios™ cytometer* with CYTO-STATR tetraCHROME™ reagents compared to the Cytomics FC500 for lymphocyte subset analysis in patients suspected of immunodeficiency and apparently healthy individuals was evaluated in a multi-center study.

To assess accuracy, the Navios Tetra method was compared to both FC500 manual gating and the tetraCXP algorithm methods in ≥300 samples covering the CD4+ medical decision levels at 3 sites. Samples were prepared by using manual pipetting and TQ-Prep or Prep Plus 2 and TQ-Prep. Precision performance of the Navios CYTO-STATR tetraCHROME™ system was evaluated based on CLSI EP5-A2 using control materials. The adult reference interval was determined following CLSI EP28-A2.

The Navios™ cytometer showed excellent repeatability & reproducibility. Table 1 summarizes representative results for tetraCHROME tube 1 for all sites combined. The repeatability and reproducibility for very low CD4 levels (133 cells/µL) was <5% and <7%, respectively. Figure 1 illustrates a three-way comparison (Navios vs. FC500 manual and FC500 CXP vs. FC500 manual) confirming the improved comparability of Navios tetra to the manual reference method (FC500). The comparison of Navios Tetra to FC500 manual gating showed minimal positive bias at medical decision levels and slightly higher positive bias when compared to tetraCXP. Bias and confidence intervals for these comparisons are shown in Tables 2 & 3 while Bland Altman plots are illustrated in Figures 2 & 3. Observed differences are in part the result of modifications made to the Navios tetra algorithm compared to the FC500 algorithm (tetraCXP) to include lymphocyte populations with altered forward light scatter properties that maintain both side scatter and CD45 fluorescence intensity properties. The inclusion of these populations is consistent with published literature (Glencross1 et. al., ). The analysis method employed in Navios tetra presumes that the sample at time of collection contains minimal apoptosis-committed cells since apoptosis progresses quickly and apoptotic cells are cleared from the circulation very rapidly. Any apoptosis and eventual cell death in the specimen is more likely the result of pre-analytical conditions post-venipuncture such as temperature and the effects due to aging of the specimens. Some samples demonstrating decreased forward scatter properties that may be characteristic of dead or apoptotic cells may yield different results from previous tetra algorithms. Normal reference intervals for lymphocyte subsets (data not shown) were consistent with published values.

Conclusion

The Navios™ flow cytometer demonstrated excellent performance in lymphocyte subset analysis with CYTO-STATR tetraCHROME™. *Recently received clearance by the US FDA on Sept 24, 2013; available for in vitro diagnostic use within the U.S.A. 1Glencross, D.K. et. al. Large-scale Affordable Panleucogated CD4+

testing with Proactive Internal & External Quality Assessment: In Support of the South African National Comprehensive Care, treatment & Management Programme for HIV & AIDS

Table 2. Bias and Its Confidence Intervals at Medical Decision Points and Quartiles

Navios Tetra (Test) vs. FC 500 Manual (Reference) - tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 Reagent

Figure 2. CD3+CD4+ Absolute Count – Bland-Altman Plot

Table 3. Bias and Its Confidence Intervals at Medical Decision Points and Quartiles

Navios Tetra (Test) vs. FC 500 tetraCXP (Reference) - tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 Reagent

Figure 3. CD3+CD4+ Absolute Count – Bland-Altman Plot

Results

Figure 1. CD3+CD4+ Absolute Count – Estimated Bias & Confidence Intervals Plot

FC 500 Manual (Reference – FC Man) vs. Navios Tetra (Test – Navios) and FC500 tetraCXP (Test – FC)

Table 1. Precision results from the combined sites

Repeatability Within Site ReproducibilityAnalyte Unit N Mean %CV %CV %CV

cells/µL 244 746 3.696 5.106 5.360% 244 72.32 0.855 0.988 1.003

cells/µL 244 483 3.890 5.209 5.461% 244 46.80 1.520 1.606 1.655

cells/µL 244 247 4.569 5.831 6.092% 244 23.98 2.920 2.920 2.939

Repeatability Within Site ReproducibilityAnalyte Unit N Mean %CV %CV %CV

cells/µL 245 344 3.355 4.505 5.122% 245 57.82 1.632 1.661 1.669

cells/µL 245 133 4.422 5.712 6.858% 245 22.36 3.045 3.345 3.526

cells/µL 245 194 3.789 4.756 5.519% 245 32.66 2.545 2.556 2.565

Total CD3+

CD3+CD4+

CD3+CD8+

ImmunoTrol Low

Total CD3+

CD3+CD4+

CD3+CD8+

ImmunoTrolCombined Sites PrepPlus 2 and TQ-Prep

Level BiasLower

Confidence Limit

Upper Confidence

Limit25th Percentile 1050 -3.34 -8.83 2.14

50th Percentile 1345 -10.21 -17.07 -3.35

75th Percentile 1725 -19.03 -29.19 -8.8625th Percentile 71.84 0.33 0.18 0.4750th Percentile 77.44 0.40 0.28 0.5375th Percentile 82.12 0.47 0.32 0.62

25th Percentile 306 9.25 6.60 11.90

50th Percentile 531 8.07 3.35 12.7875th Percentile 827 6.51 -1.12 14.15

Medical Decision 50 10.59 3.01 18.18Medical Decision 100 10.33 3.80 16.86Medical Decision 350 9.02 6.12 11.91Medical Decision 500 8.23 3.77 12.6825th Percentile 21.96 0.83 0.68 0.9850th Percentile 30.84 0.79 0.66 0.9175th Percentile 42.31 0.73 0.58 0.89

25th Percentile 470 -4.74 -7.84 -1.6450th Percentile 695 -6.61 -11.09 -2.1375th Percentile 988 -9.04 -16.30 -1.7825th Percentile 29.64 -0.01 -0.20 0.1750th Percentile 42.37 0.11 -0.04 0.2575th Percentile 56.02 0.24 0.06 0.42

CD3+CD8+

cells/µL

%

Conducted at 3 sites in North America

CD3+CD4+

cells/µL

%

Total CD3+

cells/µL

%

Level BiasLower

Confidence Limit

Upper Confidence

Limit25th Percentile 1001 35.12 28.90 41.33

50th Percentile 1294 36.91 29.11 44.72

75th Percentile 1675 39.26 27.58 50.9425th Percentile 72.93 -0.85 -1.01 -0.6950th Percentile 78.61 -0.84 -0.98 -0.7075th Percentile 83.49 -0.82 -0.99 -0.66

25th Percentile 284 23.68 20.88 26.47

50th Percentile 507 33.72 28.60 38.84

75th Percentile 806 47.26 38.76 55.77

Medical Decision 50 13.10 4.50 21.69

Medical Decision 100 15.36 8.10 22.61Medical Decision 350 26.65 23.50 29.79

Medical Decision 500 33.42 27.52 39.33

25th Percentile 20.71 0.87 0.70 1.0350th Percentile 30.60 0.60 0.47 0.7475th Percentile 42.15 0.30 0.14 0.46

25th Percentile 460 12.57 9.40 15.7550th Percentile 682 16.87 12.25 21.48

75th Percentile 973 22.51 14.98 30.0525th Percentile 30.62 -0.48 -0.65 -0.3150th Percentile 43.38 -0.65 -0.78 -0.5175th Percentile 56.95 -0.83 -1.00 -0.66

CD3+CD8+

cells/µL

%

Conducted at 3 sites in North America

CD3+CD4+

cells/µL

%

Total CD3+

cells/µL

%

Navios Tetra (Test) vs. FC 500 Manual (Reference) Navios Tetra (Test) vs. FC 500 tetraCXP (Reference)

Navios Tetra Precision Performance using control materials - tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 Reagent