Performance Testing of the ClearLLab LS (Lymphoid Screen)*

Bharathi Ravi1, Pooja Dalvi1, Badri Natrajan1, Neha Girish1, Diana B Careaga2 , Guoyan Cheng2

1Research & Development, Beckman Coulter Inc., Bangalore Development Center, India
2Clinical Research, Beckman Coulter, Inc. Miami, FL, USA

Abstract

Introduction

Use of single-color CE-IVD antibody-fluorochrome conjugates and laboratory developed tests (LDTs) for identification of hematolymphoid populations entails extensive sample preparation, verification and validation, thus increasing workflow time and complexity in inventory management. To simplify the process and to enable utility of a standardized panel, the ClearLLab LS Reagent*, a 10-color, 12-antibody dried lymphoid screening panel was designed for use on the Navios and Navios EX Flow Cytometers. Its performance was assessed on fresh and aged specimens collected in different anticoagulants. The repeatability, reproducibility and the limits of detection of the ClearLLab LS panel were also evaluated.

Method

Specimen age was evaluated for 20 blood specimens up to 26 hours for K2EDTA and K3EDTA and 48 hours for ACD and Heparin anticoagulants at room temperature (RT) (18-25°C). The performance of the processed specimens was evaluated for storage up to five hours at RT (18-25°C) and 26 hours at 2-8°C. The repeatability of the panel was determined on a total of nine specimens (three specimens each of blood, bone marrow and lymph node), with 10 replicates each. The reproducibility of the panel was evaluated by three operators over five days, at two time-points a day, two repeats per time-point on two Navios flow cytometers using control cells. The detection limits were calculated for the 72 blank and 120 low level samples.

Results

The percentage mean difference and 95% confidence interval (CI) of the percentage gated positive cells for each marker across all anticoagulants were within 5%. The drift over specimen age and prepared specimens was calculated for all anticoagulants. The drift and the 95% CI for B, T and NK lineage was within 5% of percentage positive gated populations. The repeatability and reproducibility of markers with greater than 20% of CD45+ positive population was under 5% and 10% respectively and markers with less than 20% CD45+ positive population was under 10% and 15% respectively. The limit of detection (LOD) for all markers in the panel was less than 1%.

Conclusion

Similar performance of biological specimens across anticoagulants and age as well as low coefficient of variation (CV) on clinical and normal specimens demonstrate the potential for adoption of panels such as the ClearLLab LS reagent panel across geographies for improved standardization.

Materials & Methods

The ClearLLab LS panel design (Figure 1) was dose optimized for a range of 2,000-20,000 cells per µL. Biological specimens were processed using a wash-lyse-wash-stain-wash protocol for optimized performance. Briefly, samples are washed with a buffer containing 2% Fetal Calf Serum (FCS), lysed with VersaLyse Lysing Solution (part number A09777) and washed again with buffer to remove any plasma/serum protein interferences. The washed specimens are stained with the ClearLLab LS dry reagent tube for 15 minutes, washed to remove excess antibody and re-suspended and acquired.

Figure 1. ClearLLab LS Panel Design

Fluorophore FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 Pacific-Blue Krome Orange
Specificity Kappa Lambda CD19 CD56 CD10 CD34 CD5 CD20 CD3 CD45
CD8 CD4                

The following gating strategy was used to assess the T, B and NK cell subpopulations.

Gating strategy to assess the T, B and NK cell subpopulations. FS vs SS dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. FS TOF vs TIME dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD45 vs SS dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD19 vs CD3 dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD8/kappa vs CD4/lambda dot plot gated on CD3+.
Gating strategy to assess the T, B and NK cell subpopulations. CD8/kappa vs CD4/lambda dot plot gated on CD19+.
Gating strategy to assess the T, B and NK cell subpopulations. CD5 vs CD3 dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD3 vs CD56 dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD20 vs CD10 dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD20 vs CD19 dot plot.
Gating strategy to assess the T, B and NK cell subpopulations. CD5 vs CD19 dot plot.

The reproducibility of ClearLLab LS was assessed with control cells (IMMUNO-TROL, part number 6607077, Stem-Trol, part number IM3632), over five days on two instruments by three operators with two runs per day. The overall reproducibility was evaluated along with the reproducibility based on instruments, operator, day of experiment and run. The results show that the CV is less than 10% for all CD markers.

Sample Type Kappa CD8 Lambda CD4 CD19 CD56 CD10 CD34 CD5 CD20 CD3 CD45
n 119 119 119 119 119 119 119 119 119 119 119 119
Repeatability CV 1.7% 3.3% 2.4% 2.2% 2.8% 5.8% 3.3% 6.8% 1.4% 2.4% 1.0% 0.9%
Reproducibility CV 2.1% 3.3% 3.0% 3.0% 4.6% 6.8% 3.3% 6.8% 1.8% 5.1% 1.1% 1.2%

A total of 72 blank and 120 data low level samples were used to calculate the LOD. Limit of blank (LOB) was calculated using a non-parametric approach and LOD was calculated using the classical approach as per the CLSI guideline, EP17-A2. LOD is <1% for 100,000 CD45+ WBCS.

  Kappa+ Lambda+ CD8+ CD4+ CD19+ CD56+ CD10+ CD34+ CD5+ CD20+ CD3+
LOB 0.00005 0.00001 0.00011 0.00027 0.00010 0.00003 0.00005 0.00050 0.00047 0.00014 0.00052
LOD 0.553 0.514 0.240 0.148 0.073 0.054 0.675 0.093 0.160 0.273 0.158

A total of 20 donors collected simultaneously in each anticoagulant, K2EDTA, K3EDTA, Heparin and ACD were used for evaluating anticoagulant equivalency. The drift of percentage positive gated cells in all specificities and 95% CI was calculated (y axis) for combinations of anticoagulants compared to each other. 95% CI and drift was within 5% for each CD marker's positive population compared to each anticoagulant.

Figure 2. Drift and 95% CI for anticoagulant comparison for 12 specificities

Drift and 95% CI for anticoagulant comparison for 12 specificities
Anticoagulant Day 1 Day 2 Day 3 Day 4
K2EDTA and K3EDTA S0T0

S26T0 (Morning-specimen age)

S26T5 (Evening-Prepared specimen at 18-25°C)

 S26T26 (Morning-Prepared specimen at 2-8°C)  
Heparin and ACD S0T0  

S50T0 (Morning-specimen age)

S50T5 (Evening-Prepared specimen at 18-25°C)

 S50T26 (Morning-Prepared specimen at 2-8°C)

A total of 80 whole blood specimens (N=20/anticoagulant) were collected for this study. The drift and 95% CI for each lineage is compared to the control (S0T0) for whole blood specimens. Lineages were grouped as: 1. B Cell Lineage: CD19, CD20, Kappa, Lambda and CD10 2. T Cell Lineage: CD3, CD5, CD4 and CD8 3. NK Cell Lineage: CD56 4. Immature Blasts: CD34.

Figure 3. Drift and 95% CI per lineage for specimen age and prepared specimen stability.

  Specimen age stability at 18-27°C Prepared Specimen stability at 18-25°C Prepared Specimen stability at 2-8°C
K2EDTA Drift 95% CI Drift 95% CI Drift 95% CI
B cell lineage 0.0 [-0.5 - 0.5] -0.4 [-1.0 - 0.1] -0.1 [-0.6 - 0.3]
CD34 -0.2 [-0.3 - -0.1] -0.3 [-0.6 - -0.1] -0.4 [-0.6 - -0.2]
CD45 -1.1 [-2.4 - 0.1] -0.1 [-1.4 - 1.2] -0.7 [-1.6 - 0.3]
NK cell lineage -0.6 [-1.8 - 0.5] -0.1 [-1.0 - 0.7] -0.7 [-1.6 - 0.3]
T cell lineage 1.4 [0.5 - 2.3] 0.7 [0.0 - 1.3] 0.6 [-0.2 - 1.3]
  Specimen age stability at 18-27°C Prepared Specimen stability at 18-25°C Prepared Specimen stability at 2-8°C
K3EDTA Drift 95% CI Drift 95% CI Drift 95% CI
B cell lineage -0.4 [-0.8 - 0.0] -0.2 [-0.7 - 0.2] 0.1 [-0.3 - 0.6]
CD34 -0.3 [-0.5 - -0.1] -0.3 [-0.5 - 0.0] -0.3 [-0.5 - 0.0]
CD45 1.0 [-0.5 - 2.5] -1.5 [-2.8 - -0.3] -0.8 [-2.6 - 1.1]
NK cell lineage -1.2 [-2.4 - 0.0] -0.1 [-1.1 - 0.9] -0.3 [-0.9 - 0.3]
T cell lineage 0.7 [-0.1 - 1.6] 0.6 [-0.1 - 1.3] 0.8 [0.0 - 1.6]
  Specimen age stability at 18-27°C Prepared Specimen stability at 18-25°C Prepared Specimen stability at 2-8°C
Heparin Drift 95% CI Drift 95% CI Drift 95% CI
B cell lineage -0.2 [-0.6 - 0.3] -0.2 [-0.2 - 0.6] -0.1 [-0.5 - 0.3]
CD34 -0.1 [-0.2 - 0.0] -0.1 [-0.1 - 0.0] -0.1 [-0.2 - -0.1]
CD45 1.4 [-0.6 - 3.4] 0.7 [-1.5 - 2.9] 0.6 [-0.7 - 1.8]
NK cell lineage -1.4 [-2.4 - -0.3] -0.3 [-1.2 - 0.5] -0.6 [-1.4 - 0.3]
T cell lineage 0.8 [0.0 - 1.5] 0.6 [-0.1 - 1.4] 1.3 [0.5 - 2.1]
  Specimen age stability at 18-27°C Prepared Specimen stability at 18-25°C Prepared Specimen stability at 2-8°C
ACD-A Drift 95% CI Drift 95% CI Drift 95% CI
B cell lineage -1.5 [-2.2 - -0.7] -1.6 [-2.3 - -0.9] -1.1 [-1.9 - -0.3]
CD34 -0.2 [-0.3 - -0.1] 0.0 [-0.1 - 0.1] -0.1 [-0.2 - 0.0]
CD45 3.2 [1.2 - 5.1] 2.6 [0.6 - 4.6] 2.6 [0.7 - 4.5]
NK cell lineage -0.7 [-1.6 - 0.2] -0.8 [-1.9 - 0.3] -0.6 [-1.9 - 0.6]
T cell lineage 1.1 [0.2 - 2.0] 0.6 [-0.2 - 1.4] 0.5 [-0.5 - 1.4]

9 specimens, 3 of each specimen type, namely peripheral blood, bone marrow and lymph node, were used to assess the repeatability of the ClearLLab LS. CV of markers with less than 1% positive cells was not calculated. The CV is less than 5% and 10% for CD markers with ≥ 20% and < 20% positive gated events.

Figure 4. Repeatability on nine specimens

  Peripheral Blood Bone Marrow Lymph Node
Marker Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3
Kappa+ 1.80% 1.50% 0.80% 2.80% 2.20% * 5.70% 0.80% 3.20%
CD8+ 7.50% 4.80% 1.20% 2.20% 2.30% 0.40% 4.40% 2.60% 3.50%
Lambda+ 3.20% 2.50% 1.70% 2.90% 7.50% * 1.20% 1.00% 5.20%
CD4+ 1.80% 0.90% 0.80% 0.70% 3.10% 6.30% 0.60% 0.50% 0.40%
CD19 2.40% 1.30% 2.80% 4.90% 4.30% * 4.90% 4.00% 5.90%
CD56+ 2.10% 2.60% 1.60% 4.80% 5.00% 8.70% * 6.70% *
CD10+ * * * 8.40% 1.50% 5.10% 3.70% * *
CD34+ * * * 7.70% 3.00% 4.80% * * *
CD5+ 2.20% 1.30% 0.50% 1.20% 1.90% 3.80% 5.00% 2.50% 0.50%
CD20+ 2.60% 1.30% 3.20% 6.60% 4.20% * 4.90% 4.30% 6.80%
CD3 2.20% 1.30% 0.30% 1.20% 2.30% 2.90% 4.80% 2.50% 0.60%
CD45+ Lymphs 2.70% 2.90% 2.40% 5.00% 2.00% 6.20% 2.60% 1.50% 0.70%

Conclusion

The consistent and versatile performance of the ClearLLab LS reagent demonstrates the advantage of using a standardized product compared to LDTs for screening of hematological neoplasia on the Navios Flow Cytometer. The specific choices and combinations in the ClearLLab LS reagent address the clinical indications, account for all major lymphoid cell populations present in the specimen and provide sufficiently comprehensive identification of all major categories of hematopoietic cell populations in both normal and neoplastic states.

*ClearLLab LS (Lymphoid Screen) is CE-IVD marked under Regulation (EU) 2017/746. For In Vitro Diagnostic Use. The Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.

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