CellMek SPS System: Sample Preparation Performance

Percent gated populations from automated versus manual sample preparations using a wash/stain/lyse & fix/wash workflow with a 10-color antibody panel in liquid or dry format

Kelly Andrews, Gang Xu, Xizi Dai, Karen Lo, Jessica Ashbaugh, Jin Zhang, Ernesto Staroswiecki
Research and Development, Beckman Coulter Life Sciences, Miami, FL United States

Workflow

Sample Preparation

CellMek SPS Sample Preparation Station main unit

CellMek SPS

Sample Preparation System

DURACartridges with the packaging

DURACartridges

10-Color dry reagent cocktail

Sample Acquisition

Navios EX Flow Cytometer

Navios Flow Cytometer

Sample Analysis

Kaluza C Analysis Software 10 Color Data

Kaluza C

Flow Cytometry Analysis Software

Introduction

The CellMek SPS Instrument is an automated sample preparation system intended for in vitro diagnostic use in flow cytometry laboratories designed to process whole blood (WB), bone marrow (BM), and other relevant single-cell specimens for downstream flow cytometry analysis.

Refrigerated antibody carousel of CellMek SPS

Clinical workflows commonly consist of washing a specimen aliquot, staining with liquid or dry antibodies, lysing red blood cells and fixing stained white blood cells (WBC), washing lysed/fixed prepared sample, and reconstituting in buffer for downstream flow cytometry analysis. A workflow such as this, which utilizes all modules of the CellMek SPS instrument, was chosen to assess system performance compared to manually prepared samples. Modules used include the sample transport module, reaction plate module, the cell wash module, the dry reagent module, the liquid antibody module, the prep reagent module, and the output module.

Methods

WB, BM, and other single-cell suspensions (cerebral spinal fluid [CSF] or other body fluid) were obtained from normal and clinical donors (97 unique donors) and were processed using a representative wash/stain/lyse & fix/wash workflow with a custom 10-color antibody panel in either dry DURACartridge format or liquid cocktail format. The 10-color panel consisted of Kappa-FITC, Lambda-PE, CD10-ECD, CD5-PC5.5, CD200-PC7, CD34-APC, CD38-AA700, CD20-AA750, CD19-PB, and CD45-KrO.

Table 1. Custom 10-color antibody panel in liquid or DURACartridge dry reagent format.

Antibody Panel Marker and Fluorochrome Combination (Liquid or DURACartridge Format)
FITC PE ECD PC5.5 PC7 APC AA700 AA750 PB KrO
10-Color
Panel
Kappa Lambda CD10 CD5 CD200 CD34 CD38 CD20 CD19 CD45

Samples were prepared in duplicate on one of three CellMek SPS instruments, totaling 152 datapoints each for CD45+ WBC and gated sub-populations (liquid and dry pooled). Each sample processed with the CellMek SPS instrument was compared to a manually prepared matched-donor sample using equivalent workflow and reagents. All prepared sample data were acquired on a Navios flow cytometer and analyzed using Kaluza C software. Measurement Procedure Comparison and Bias Estimation was performed by biostatisticians.

Results

Figure 1. Representative overlay of dot plots from samples prepared automated with the CellMek SPS System (shown in green) or manually (shown in red). Dot sizes of Kappa/Lambda plots have been enlarged for better visualization. Plots are for illustrative purposes only.

Table 2. Clinical Donor

Specimens from patients having or suspected of having hematolymphoid malignancies; designated as abnormal by pathologist. These are obtained as spent samples from hospitals/clinics.

(Click on the population to see the Regression Plot)

Population N Manual Sample Preparation Automated Sample Preparation Difference 95% Confidence Limits
Mean Min Max Mean Min Max Lower Upper
Lymphocytes 67 29.08 5.35 80.16 27.49 5.83 75.83 -1.58 -1.99 -1.17
Monocytes 67 7.42 0.72 25.29 8.24 0.77 26.60 0.82 0.60 1.05
Granulocytes 67 61.71 7.28 88.61 62.06 6.53 87.80 0.35 -0.06 0.77
CD10+ 67 59.86 3.50 87.65 60.16 4.47 87.69 0.30 -0.11 0.71
CD34+ 67 1.60 0.01 95.10 1.61 0.01 94.93 0.00 -0.02 0.02
CD200+CD19+(Ly) 67 9.89 0.22 22.45 10.69 0.01 23.35 0.80 0.60 1.00
CD38+ 67 7.20 2.60 24.34 8.08 1.28 24.73 0.88 0.66 1.10
CD20+CD200+(Ly) 67 9.62 0.24 22.77 10.42 0.00 23.29 0.80 0.61 0.99
CD5+CD19-(Ly) 67 70.85 8.60 88.49 70.53 8.97 88.42 -0.32 -0.54 -0.10
CD19+CD20+ 67 12.99 0.22 27.30 13.24 0.00 27.33 0.26 0.06 0.46
Kappa+(CD19+CD20+) 66 59.26 42.32 71.25 59.23 42.17 72.60 -0.03 -0.40 0.35
Lambda+(CD19+CD20+) 66 40.04 27.35 57.26 40.03 26.60 57.40 -0.00 -0.39 0.38

Table 3. Clinical Donor

Specimens from patients having or suspected of having hematolymphoid malignancies; designated as abnormal by pathologist. These are obtained as spent samples from hospitals/clinics.

(Click on the population to see the Regression Plot)

Population N Manual Sample Preparation Automated Sample Preparation Difference 95% Confidence Limits
Mean Min Max Mean Min Max Lower Upper
Lymphocytes 31 17.55 3.03 54.91 17.69 2.83 50.76 -0.15 -0.37 0.66
Monocytes 31 10.47 1.45 89.78 10.97 1.61 88.52 0.51 0.18 0.83
Granulocytes 31 65.33 1.89 87.99 64.46 1.04 85.86 -0.87 -1.47 -0.28
CD10+ 31 35.84 1.30 81.81 37.53 1.52 83.00 1.69 1.06 2.33
CD34+ 31 4.19 0.02 54.05 4.29 0.02 53.83 0.10 -0.05 0.25
CD200+CD19+(Ly) 31 10.23 0.03 28.75 11.29 0.02 34.20 1.06 0.47 1.65
CD38+ 31 11.51 1.21 91.00 12.12 1.26 89.06 0.61 0.22 0.99
CD20+CD200+(Ly) 31 9.80 0.02 26.28 10.95 0.02 32.28 1.15 0.48 1.82
CD5+CD19-(Ly) 31 70.85 48.06 87.44 69.17 46.31 87.38 -1.67 -2.40 -0.95
CD19+CD20+ 31 11.42 0.07 30.65 12.12 0.06 35.77 0.70 0.11 1.28
Kappa+(CD19+CD20+) 29 59.80 42.21 88.97 60.30 45.73 86.74 0.50 -0.49 1.48
Lambda+(CD19+CD20+) 29 37.40 11.12 47.01 37.41 13.20 47.59 0.01 -1.02 1.05

The expected results for total bias from reference of percent positive:

  • ± 5% point difference for % positive subsets ≤ 20%
  • ± 8% point difference for % positive subsets > 20%

Table 4. Subcomponent bias from manually prepared reference of percent positive gated sub-populations at percentiles.

Sub-
component
Group Percentile Level Bias 95% Confidence Limits Acceptance
Limit (±)
Conclusion
Lower Upper
Sample
Preparation
Panel Design
Typical
Incubation
25 5.895 0.493 0.380 0.606 5 Pass
50 20.263 0.349 0.216 0.483 8 Pass
75 56.340 -0.012 -0.294 0.269 8 Pass
Throughput
Optimized
25 6.605 0.493 0.300 0.687 5 Pass
50 25.505 0.176 -0.030 0.381 8 Pass
75 58.765 -0.384 -0.713 -0.054 8 Pass
Instrument 1 25 5.740 0.593 0.471 0.714 5 Pass
50 20.740 0.323 0.201 0.446 8 Pass
75 57.820 -0.342 -0.550 -0.135 8 Pass
2 25 7.280 0.259 0.062 0.456 5 Pass
50 27.240 0.322 -0.062 0.707 8 Pass
75 54.820 0.410 -0.345 1.164 8 Pass
3 25 7.445 0.280 -0.011 0.571 5 Pass
50 18.920 0.233 -0.022 0.488 5 Pass
75 58.370 0.072 -0.346 0.491 8 Pass
Antibody
Format
Dry 25 6.238 0.387 0.272 0.502 5 Pass
50 21.193 0.228 0.083 0.373 8 Pass
75 57.135 -0.154 -0.459 0.151 8 Pass
Liquid 25 5.850 0.703 0.519 0.886 5 Pass
50 22.480 0.444 0.277 0.610 8 Pass
75 58.295 -0.113 -0.366 0.140 8 Pass
Anti-
coagulant
ACD 25 6.230 0.491 0.377 0.606 5 Pass
50 22.710 0.317 0.204 0.429 8 Pass
75 57.475 -0.052 -0.246 0.142 8 Pass
EDTA 25 7.565 0.523 0.416 0.631 5 Pass
50 25.138 0.250 0.154 0.345 8 Pass
75 58.345 -0.268 -0.427 -0.109 8 Pass
Heparin 25 6.855 0.130 -0.045 0.304 5 Pass
50 33.395 0.186 -0.002 0.374 8 Pass
75 56.605 0.236 -0.060 0.531 8 Pass
None 25 2.460 0.783 0.292 1.275 5 Pass
50 12.208 0.537 -0.056 1.130 5 Pass
75 50.065 -0.419 -1.758 0.919 8 Pass
Specimen Type BM 25 5.255 0.682 0.516 0.849 5 Pass
50 20.280 0.423 0.255 0.592 8 Pass
75 55.660 -0.187 -0.498 0.124 8 Pass
Body Fluid 25 2.213 0.933 0.390 1.476 5 Pass
50 10.648 0.699 0.040 1.357 5 Pass
75 48.283 -0.347 -1.932 1.237 8 Pass
CSF 25 4.140 0.010 -1.006 1.026 5 Pass
50 14.973 -0.130 -1.038 0.778 5 Pass
75 67.520 -0.812 -2.701 1.077 8 Pass
WB 25 7.390 0.347 0.266 0.428 5 Pass
50 28.270 0.195 0.118 0.272 8 Pass
75 58.055 -0.022 -0.145 0.102 8 Pass

Conclusion

There is equivalent sample preparation performance between the automated CellMek SPS and manual preparation process irrespective of common sources of variability. Total bias from manually prepared reference of percent positive gated sub-populations was within 5 percentage points for populations ≤ 20% and within 8 percentage points for populations > 20%. Sub-component analysis of sample preparation panel design (typical incubation or throughput optimized), instrument (1, 2, or 3), antibody format (liquid or dry), anticoagulant (EDTA, ACD, Heparin, or none), and specimen type (WB, BM, body fluid, or CSF) was assessed on a pooled marker dataset. All sub-component categories analyzed were within 5 percentage points for populations ≤ 20% and within 8 percentage points for populations > 20%.

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