Clin Chem Lab Med 2018; 56(8): 1277–1288
Simon Degandta, Bart Peetersa, Stijn Jughmans, Nancy Boeckxb and Xavier Bossuytb,*
Analytical performance of an automated
volumetric flow cytometer for quantitation
of T, B and natural killer lymphocytes
https://doi.org/10.1515/cclm-2017-0638
Received July 20, 2017; accepted January 19, 2018; previously
­published online February 21, 2018
Abstract
Background: Quantitation of lymphocyte subsets (B cells,
T cells, CD4 and CD8 T cells and NK cells) classically relies
on quantitation of lymphocytes and immunophenotyping
by flow cytometry. AQUIOS CL (Beckman Coulter) is a fully
automated system that performs an onboard volumetric
cell count, automatically processes the sample (staining,
lysing and fixation) and analyzes the results. We com-
pared AQUIOS CL to a dual-platform analysis and evalu-
ated analytical performance.
Methods: We evaluated precision, sample stability, inter-
sample carryover, linearity and interpanel consistency.
AQUIOS CL was compared to a dual-platform method
(Sysmex XE-5000 and BD FACSCanto-II). A total of 113
patient samples were included: 45 from posttransplant
patients, 44 from children and 24 random routine samples.
The degree of automation was scored through the need of
manual revisions triggered by AQUIOS CL run notifica-
tions and run flags.
Results: Intrarun and interrun variability was <9.1%
with dedicated control material and <32.1% with patient
samples. Relative values of lymphocyte subsets could
be determined up to 48 h after venipuncture when the
sample was kept at room temperature. There was no carry-
over and good linearity. Interpanel consistency was 3.3%
for relative values and 9.4% for absolute values. Method
comparison showed good analytical correlation between
AQUIOS CL and a dual-platform method. Thirty-five
percent of the samples triggered a run notification. In 74%
of these samples, the results could be accepted without
intervention, so in 26% of all samples, an unnecessary
notification was generated.
a
Simon Degandt and Bart Peeters share first authorship.
b
Nancy Boeckx and Xavier Bossuyt share senior authorship.
*Corresponding author: Prof. Dr. Xavier Bossuyt, PhD, MD,
Department of Laboratory Medicine, UZ Leuven, Herestraat 49,
3000 Leuven, Belgium, E-mail: xavier.bossuyt@uzleuven.be
Simon Degandt, Bart Peeters, Stijn Jughmans and Nancy Boeckx:
Department of Laboratory Medicine, UZ Leuven, Leuven, Belgium
Conclusions: AQUIOS CL allows for reliable fully auto-
mated immunophenotyping of lymphocyte subset quanti-
tation. Gating algorithms could be further improved.
Keywords: AQUIOS; flow cytometry; lymphocyte subsets.
Introduction
In clinical laboratories, quantitation of lymphocyte
subsets (helper and cytotoxic T cells, B cells and NK
cells) is performed (i) to monitor CD4 T-cell counts in
HIV-positive patients, (ii) to monitor immunosuppressive
or immunomodulatory therapy (e.g. in transplantation
and autoimmunity), (iii) to assess immune reconstitu-
tion post-hematopoietic cell transplantation, (iv) to assess
the immune status in immune-mediated diseases such as
primary immunodeficiencies and (v) to classify tumors of
hematopoietic and lymphoid tissues [1–4].
Quantitation of lymphocyte subsets classically relies
on (i) quantitation of the total number of lymphocytes
(e.g. by a hematology analyzer) and (ii) immunophenotyp-
ing of lymphocyte subsets by flow cytometry (FCM). For
immunophenotyping, labeled antibodies to a set of (cell
surface) markers is used, such as CD3 (T cells), CD4 (helper
T cells), CD8 (cytotoxic T cells), CD19 (B cells) and CD56/
CD16 (NK cells). Immunophenotyping is labor-intensive
involving several preanalytical preparation steps such as
sample lysis, cell staining and washing, which are prone
to human mistakes. Moreover, a dual-platform approach,
in which data obtained by a hematology analyzer are com-
bined with FCM data, suffers from high intra- and inter-
laboratory variation [5, 6].
During the past decade, manufacturers made efforts
to automate these processes and developed washing sta-
tions and loading racks for cytometers. Besides, tubes
precoated with monoclonal antibodies were introduced
as well as bead-based methods that use counting micro-
beads as counting reference allowing quantitation of cells
on the flow cytometer. Such bead-based methods elimi-
nate the use of a routine hematology analyzer for leuko-
cyte cell counting. In an attempt to maximally automate
the process, Beckman Coulter released a fully automated
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1278 Degandt et al.: Evaluation AQUIOS CL
volumetric flow cytometer, AQUIOS CL (Beckman Coulter
Life Sciences, Miami, FL, USA). This device is able to
perform an onboard volumetric cell count, to automati-
cally process the sample (staining, lysing and fixation), to
analyze the sample including acquisition of cells, gating
of leukocyte/lymphocyte populations, and to prepare a
report. AQUIOS CL is a closed system and runs tests pre-
configured by the manufacturer, or user-defined assays
through a more open configuration software. For now, two
panels are available, allowing quantitation of T cells, CD4
and CD8 T cells, B cells and NK cells.
So far, two studies that evaluated AQUIOS CL have
been reported. Gossez et al. [7] compared CD4+ T lym-
phocyte quantitation by volumetric AQUIOS CL to CD4 T
lymphocyte quantitation by bead-based single platform
analysis, whereas Grossi et al. [8] compared quantifica-
tion of B cells, T cells, T cell subsets and NK cells by volu-
metric AQUIOS CL to quantification by bead-based FCM.
Both studies showed good agreement between the two
methods, and Grossi et al. [8] reported a reduction in turn-
around time for the AQUIOS CL system.
In the present study, we compared immunophenotyp-
ing results obtained with AQUIOS CL to results obtained
with a dual-platform analysis and performed a more com-
prehensive analysis of the analytical performance, includ-
ing precision, sample stability, intersample carryover and
linearity of the analytical measurement range. For the
correlation study, specific attention was given to samples
with low levels of lymphocytes or lymphocyte subsets by
including samples from a posttransplant and pediatric
population. We also documented the type and number of
system errors.
Materials and methods
Samples
For precision studies, two commercial controls were used: the
AQUIOS IMMUNO-TROL Cells (Beckman Coulter) and the AQUIOS
IMMUNO-TROL Low Cells (Beckman Coulter). Both positive cell con-
trols were processed in the same manner as the whole blood samples.
A total of 113 peripheral blood samples were used for the evalu-
ation, including 45 samples from posttransplant patients, 44 pedi-
atric samples (age <16 years) and 24 other routine samples. In all
these patients, a routine FCM analysis of T-, B- and/or NK-subsets
was requested, and analysis on the AQUIOS CL was done on leftover
sample.
Peripheral blood was collected in K2-EDTA tubes and kept at
room temperature until analysis. The blood samples used for method
comparison were first analyzed with the current dual-platform
method and kept at room temperature until analysis on the AQUIOS
CL system. Time between the two analyses was kept to a minimum
and never surpassed 30 min. No more than 48 h delay was permit-
ted between sample collection and both measurements, except for
stability studies.
The study was approved by the Local Ethics Committee. A signed
informed consent was not needed as leftover samples were used.
Dual-platform approach: Sysmex XE-5000 and BD
FACSCanto-II
For the dual-platform approach, the leukocyte count was obtained
from a Sysmex XE-5000 device (Sysmex, Kobe, Japan) using light
scattering technology. Leukocyte differentiation was done automati-
cally by the instrument whenever possible. In case a sample was
flagged by the instrument and differentiation was not possible, the
sample was differentiated manually by an experienced technologist.
The system was calibrated and quality controlled according to the
manufacturer’s instructions.
A stain/lyse/wash procedure was used for immunophenotypic
analysis. Based on the physicians request, the following multicolor
reagent combinations were used: CD3-FITC/CD8-PE/CD45-PerCP/
CD4-APC and/or CD3-FITC/CD16.56-PE/CD45-PerCP/CD19-APC (BD
Biosciences). Samples were measured on a BD FACSCanto-II TM flow
cytometer (BD Biosciences, San Jose, CA, USA) by analyzing 10,000
events within the lymphocyte gate. Acquisition and analysis was
done by using the BD FACSDiva Software version 6 (BD Biosciences,
San Jose, CA, USA). Gating was done manually by experienced lab
technicians and verified by a medical supervisor. The following lym-
phocyte populations were enumerated: CD3+, CD3+/CD4+/CD8−,
CD3+/CD4−/CD8+, CD3−/CD19+ and CD3−/CD56CD16+.
Single platform volumetric FCM: AQUIOS CL
The AQUIOS CL system is based on a “Load & Go” principle, which
means that the system combines sample preparation and fluores-
cence-based cellular analysis into one system. Briefly, closed tubes
are loaded on the system. After a mixing step, the samples are cap-
pierced and 43 μL is pipetted into a 96-well microplate. The blood is
stained with 13 μL of a monoclonal antibody reagent, which is a com-
bination of four or five murine monoclonal antibodies. After 15 min
of incubation, the blood is lysed using 435 μL of lysing reagents A
and B. Lysing reagent A is a cyanide-free lytic reagent that lyses red
blood cells in preparation for white blood cell measurement. Lysing
reagent B slows the reaction caused by reagent A and preserves the
white blood cells for measurement in the flow cell. Finally, 100 μL of
prepared sample is aspirated for analysis. Absolute leukocyte count
is based on an electronic-volume measurement. For cell staining, two
ready-to-use mixes of antibodies were used: AQUIOS Tetra-1 Panel
(Beckman Coulter) (CD45 FITC/CD4 RD1/CD8 ECD/CD3 PC5) and
AQUIOS Tetra-2 Panel (Beckman Coulter) (CD45 FITC/[CD56+CD16]
RD1/CD19 ECD/CD3 PC5). A combination of both panels is referred
as AQUIOS Tetra Combo. These reagents provide identification and
enumeration of total CD3+, CD3+/CD4+/CD8−, CD3+/CD4−/CD8+,
CD4/CD8 ratio, CD3−/CD19+/CD56CD16−, CD3−/CD19−/CD56CD16+
lymphocytes in peripheral whole blood, together with CD45+ and
CD45+ Low SS (lymphocytes) count. Dual-positive CD4+/CD8+ cells,
however, are not taken into account by this gating strategy. Sample
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acquisition runs for 45 s. Relative (rel) values are measured as per-
centage of total lymphocytes and absolute (abs) counts are derived
from the absolute leukocyte count. Gating is done automatically by
the system’s software and should only be revised when run notifi-
cations and flags are shown on the result screen. Run notifications
are generated to convey an abnormal cell distribution or population,
which may be specimen specific. Run flags indicate an issue that
affects the results. Both notifications and flags need manual revision,
but run flags are considered more critical. During the evaluation, all
automatic gatings were checked visually, but no active interfering
regarding gating was allowed. In samples with a run notification or
flag, the reason was checked, and it was investigated whether these
samples should be excluded. Within the evaluation period, internal
quality control was assessed by running both AQUIOS IMMUNO-
TROL Cells and IMMUNO-TROL Low Cells daily.
AQUIOS CL uses a specific gating strategy for lymphocyte gating
(Figure 1, see Supplemental Material).
Analytical performance AQUIOS CL
Precision: To analyze precision, CLSI document EP15-A3 was
­followed [9].
Intrarun variability was measured by analyzing (Tetra Combo
analysis) the IMMUNO-TROL Cells (lot 6170007) 13 times in a row and
IMMUNO-TROL Low Cells (lot 6180010) 8 times in a row. Precision
was also measured using patient samples in order to exclude matrix
effects. The following samples were studied: (a) a sample with nor-
mal values was measured with Tetra Combo analysis, (b) a sample
with low CD3+/CD4+ cells was measured with Tetra-1 Panel and (c) a
sample with high CD3−/CD19+ cells and two samples with low CD3−/
CD19+ cells were measured with Tetra-2 Panel. The normal sample
was measured 20 times in a row, the other patient samples 10 times.
Interrun variability was measured by analyzing the IMMUNO-
TROL Cells and IMMUNO-TROL Low Cells with Tetra Combo analysis
on 20 different days.
Imprecision was calculated using the coefficient of variation (CV).
Sample stability: Sample stability was tested with a sample from a
healthy male volunteer. Between analyses, the sample was stored at
room temperature for five consecutive days (day 0 until day 4). Each
day, Tetra Combo analysis was performed in duplicate on the sample
with an interval of 24 h. For each parameter, the % difference with the
value measured on day 0 was calculated.
Intersample carryover: Carryover was evaluated with two patient
samples: a sample with low CD3−/CD19+ cells and a sample with
high CD3−/CD19+ cells. The Tetra-2 Panel was measured on each
sample separated by a system flush, to obtain reference values. Next,
the Tetra-2 Panel was measured 10 times alternately on both samples
without intermittent flushing of the tube system. Afterwards the 95%
confidence interval of each parameter in the 10-fold tandem run was
calculated and compared with the reference value.
Linearity analytical measurement range: Two pathological samples
were selected for linearity assessment: one sample with low CD3−/
CD19+ cells and one sample with high CD3−/CD19+ cells. Both sam-
ples were mixed in seven different ratios (10/0, 9/1, 8/2, 6/4, 4/6, 2/8,
0/10). The Tetra Combo analysis was performed in duplicate on all
Degandt et al.: Evaluation AQUIOS CL 1279
seven mixtures on the same day. For each parameter correlation coef-
ficients (R2) were calculated after linear regression.
Interpanel consistency: Both Tetra-1 and Tetra-2 Panels were ana-
lyzed whenever a Tetra Combo analysis was requested (n = 71). In
order to evaluate consistency between these panels in the same
sample, the difference of the common parameter, i.e. CD3+ cells, was
calculated, both rel and abs. Parallel to our calculation, AQUIOS CL
generated an error flag (“Inter panel count error”) whenever the dif-
ference of CD3+ cells (rel) of Tetra-1 and Tetra-2 in a Tetra Combo anal-
ysis was greater than 15%. However, AQUIOS Tetra Software System
Guide states that the difference of CD3+ cells (rel) in a Tetra Combo
analysis should not be greater than ±3.5%. Moreover, an interna-
tional guideline for performing single-platform absolute CD4+ T-cell
determinations states that the difference between replicate lineage
markers, such as CD3, should be ≤2% [10].
Method comparison between AQUIOS CL
and ­dual-­platform approach
In total, 113 patient samples were analyzed for comparison between
the current approach and AQUIOS CL. Only those tests that were
requested by the physician were performed with our current method
and repeated on the AQUIOS CL system. Therefore, only a part of the
samples (n = 71) were analyzed with the Tetra Combo panel, whereas
other samples were analyzed with either Tetra-1 (n = 36) or Tetra-2
Panel (n = 6) alone.
For each parameter (CD3+, CD3+/CD4+, CD3+/CD8+, CD3−/
CD19+ and CD3−/CD56CD16+, rel and abs values), the correlation
between both methods was evaluated by a Passing-Bablok regres-
sion. Confidence interval values of the slope and intercept were
calculated. All calculations were performed on the total cohort of
patient samples as well as on the pediatric and the posttransplant
subpopulations, both for Tetra-1 and Tetra-2 Panels.
To evaluate clinical agreement, all measured parameters of
each sample were categorized as low, normal or high for both meth-
ods based on published reference values [11]. A Cohen’s kappa coef-
ficient of agreement was calculated based on the classifications of
both methods.
Flagging and notifications of AQUIOS CL
All run flags and notifications were documented and listed in order
to give an idea in how many cases revision of the results was neces-
sary. All flags and notifications due to the evaluation setting, such as
“Missing QC” and “Sample ID re-used”, were omitted.
Statistics
Statistical analysis was performed using Microsoft Excel® 2013 (Micro-
soft Corp., Redmond, WA, USA) and Analyse-it® for Microsoft Excel
version 3.90 (Analyse-it Software Ltd., Leeds, UK). Imprecision was cal-
culated using the CV. Linearity was measured with linear regression and
R2. Correlation between methods was evaluated with Passing-Bablok
regression and Bland-Altman regression. Clinical agreement was evalu-
ated by Cohen’s kappa coefficient (observed unweighted kappa).
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1280 Degandt et al.: Evaluation AQUIOS CL

A
All data points
1000

800

600

FS-Lin
400

200

0
0 200 400 600 800 1000
SS-Lin
B C
Cells Cells
1000 1000

800 800

600 600
EV-Lin
SS-Lin

400 400

200
200

0 Lymphs (45)
0
100 101 102 103 104 Lymphs EV
CD45 0 200 400 600 800 1000
SS-Lin
OR AND

D Lymphs all E
50 Lymphs
50
CD3– All
CD3+ All CD3–
CD3+

25 25

0 0
–101100 101 102 103 104 –101 100101 102 103 104
CD3 CD3

F G
CD3+ All CD3–
104 104

103 103
CD56/16
CD4

102 102

101 101
100 100
–101
–101
–101 100 101 102 103 104
–101 100 101 102 103 104
CD8
CD19

Figure 1: Gating strategy on AQUIOS CL.

Leukocytes are selected on a SS/FS plot in order to exclude debris (A). Lymphocytes are gated on a CD45/SS plot (B) and on a SS/EV plot (C).
All events of both gates are combined in one gate, “Lymphs all” (D), and only joint events are combined in another gate, “Lymphs” (E). From
“Lymphs all”, CD3+ events are selected on a CD3 histogram (D), and from this gate, CD4+/CD8− and CD4−/CD8+ events are measured on
a CD4/CD8 plot (F). From “Lymphs”, CD3− events are selected on a CD3 histogram (E), and from this gate, CD19+/CD56CD16− and CD19−/
CD56CD16+ events are measured on a CD19/CD56CD16 plot (G).

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Results
Analytical performance AQUIOS CL
Precision
For IMMUNO-TROL Cells and IMMUNO-TROL Low Cells,
all CVs were ≤5% for high counts (≥300 cells/μL or >20%)
and ≤10% for low counts (<300 cells/μL or ≤20%) both for
intrarun and interrun precision (Table 1).
These criteria were not always met for the patient
samples (Table 1). For the normal sample, CD3+/CD8+ (rel
and abs) and CD3−/CD56CD16+ (abs) CVs were between
5% and 10%. The first CD3−/CD19+ low sample showed
a high CV for CD3−/CD19+ (abs) (10.76%), but the mean
value was situated around the manufacturer’s defined
lower limit of quantification (25 cells/μL). For the CD3−/
CD19+ high sample, all were below 10%. The second
CD3−/CD19+ low sample and the CD3+/CD4+ low sample
showed for most parameters high imprecision; however,
all values with a CV >15% were situated around the manu-
facturers defined limits of quantification.
Sample stability
Relative values for lymphocyte subsets remained stable
during the first 2 days but showed significant instability
on day 3 (−17.85%) for CD3−/CD19+ cells and on day 4 for
the other populations (Table 2). A difference was consid-
ered significant if it was higher than 10% (for values ≤20%
or <300 cells/μL) or higher than 5% (for values >20% or
≥300 cells/μL), according to the manufacturer’s impreci-
sion criteria.
All absolute values were unacceptable on day 3. Abso-
lute values for CD19+ cells were unacceptable after 1 day
and CD3+ cells of the Tetra-2 Panel after 2 days (Table 2).
The absolute lymphocyte count degraded rapidly,
whereas the relative values were stable for at least 3 days.
Intersample carryover
During the 10-fold tandem run, all obtained values
remained within the 95% confidence interval, so no carry­
over was observed (see Supplemental Material, Table 1).
Linearity analytical measurement range
All parameters showed excellent linearity with R2 ≥0.99.
For CD3−/CD56CD16+, however, no R2 was calculated as
the evaluated range was too narrow (Table 3, Figure 2).
Degandt et al.: Evaluation AQUIOS CL 1281
Interpanel consistency
Tetra Combo Analysis was performed on 71 samples. As
eight of these samples were excluded for method compari-
son, interpanel consistency was evaluated on the remain-
ing 63 samples. The mean difference between CD3+ cells
(rel) on Tetra-1 Panel and Tetra-2 Panel was 3.3% and
between CD3+ cells (abs) 9.4%. None of the differences
between CD3+ cells (rel) exceeded 15%, however, for CD3+
cells (abs), three samples exceeded the limit of 15% dif-
ference. These were all samples with low absolute CD3+
counts (114, 151 and 285 counts/μL). Applying the limit
of 2% difference as stated in international guidelines, as
many as 16 samples (or 25.4%) of the relative values and
44 samples (or 69.8%) of the absolute values should not
be accepted.
Method comparison between AQUIOS CL
and dual-platform approach
A total of 113 blood samples were initially included in the
method comparison study. However, 10 patient samples
were excluded from the statistical analysis for different
reasons. Two samples were highlighted by AQUIOS CL
with the run flag “Insufficient lymphocytes”. The first
sample had 132 lymphocytes/μL on AQUIOS CL and 442/μL
on XE-5000. The other sample had 85 lymphocytes/μL on
AQUIOS CL and 44/μL on XE-5000 combined with manual
differentiation. In a third sample, there was an incorrect
CD3+ gating in the Tetra-2 Panel, noticed through the error
flag “Inter Panel Count Error”. The fourth sample was a
sample mainly consisting of atypical blasts. The remain-
ing six samples showed suboptimal gating necessitat-
ing manual gating or a rerun. These were highlighted by
AQUIOS CL through the run notification “Potential sample
or gating issue”.
After exclusion, 103 samples were used for the com-
parison of which 63 were analyzed with Tetra Combo
panel, 34 with Tetra-1 Panel alone and six with Tetra-2
Panel alone. For the pediatric population, 42 samples
were compared, 40 with Tetra Combo panel, two with
Tetra-1 Panel alone and none with Tetra-2 Panel alone. For
the posttransplant population 42 samples were compared,
17 with Tetra Combo panel, 25 with Tetra-1 Panel alone
and none with Tetra-2 Panel alone.
Statistical comparison
For each parameter, correlation was measured for relative
values (percentage of lymphocytes) and for absolute values
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 1282

Table 1: Intra- and interrun precision of the AQUIOS CL Tetra-1 Panel (upper section) and the Tetra-2 Panel (lower section).

Precision Sample type Counts CD3+ T1 (rel) CD3+ T1 (abs) CD3+/CD4+ (rel) CD3+/CD4+ (abs) CD3+/CD8+ (rel) CD3+/CD8+ (abs)

Intrarun (CV%) Patient sample: normal 20 1.14 3.14 1.95 2.93 6.95 7.61
Patient sample: low CD3−/CD19+ (1) 10 n/a n/a n/a n/a n/a n/a
Patient sample: low CD3+/CD4+ 10 6.10 14.26 26.34 32.10 8.13 12.97
Degandt et al.: Evaluation AQUIOS CL

Patient sample: high CD3−/CD19+ 10 n/a n/a n/a n/a n/a n/a
Patient sample: low CD3−/CD19+ (2) 10 n/a n/a n/a n/a n/a n/a
IMMUNO-TROL 13 1.43 2.28 1.83 2.77 1.93 2.20
IMMUNO-TROL Low 8 2.33 2.63 4.10 4.85 2.58 2.65
Interrun (CV%) IMMUNO-TROL 20 2.54 3.16 1.52 3.68 3.88 3.93
IMMUNO-TROL Low 20 2.46 3.97 3.60 5.14 4.91 4.30
CD3+ T2 (rel) CD3+ T2 (abs) CD3−/CD19+ (rel) CD3−/CD19+ (abs) CD3−/CD56CD16+ (rel) CD3−/CD56CD16+ (abs)

Intrarun (CV%) Patient sample: normal 20 1.21 2.94 4.43 7.64 3.12 5.25
Patient sample: low CD3−/CD19+ (1) 10 2.35 1.98 9.59 10.76 6.44 8.79
Patient sample: low CD3+/CD4+ 10 n/a n/a n/a n/a n/a n/a
Patient sample: high CD3−/CD19+ 10 1.72 4.76 0.25 4.53 5.99 6.85
Patient sample: low CD3−/CD19+ (2) 10 1.18 3.51 13.82 13.87 16.38 18.86
IMMUNO-TROL 13 1.14 2.25 2.26 3.60 3.61 4.38
IMMUNO-TROL Low 8 2.17 3.75 4.97 6.02 4.82 9.09
Interrun (CV%) IMMUNO-TROL 20 1.76 4.88 4.08 4.86 4.69 7.60
IMMUNO-TROL Low 20 2.29 4.98 5.18 7.28 6.64 7.96

CV, coefficient of variation; n/a, not applicable.

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Table 2: AQUIOS CL Tetra-1 (upper section) and Tetra-2 (lower section) sample stability at room temperature for 4 days.

Day CD3+ T1 (rel) CD3+ T1 (abs) CD3+/CD4+ (rel) CD3+/CD4+ (abs) CD3+/CD8+ (rel) CD3+/CD8+ (abs)

1 1.31 −0.33 1.38 −0.26 2.71 1.12

2 −1.84 −2.51 −1.35 −0.78 −0.95 −1.36
3 2.13 −9.53 2.84 −8.94 2.87 −8.68
4 7.57 −10.89 7.29 −11.16 8.18 −10.3

CD3+ T2 (rel) CD3+ T2 (abs) CD3−/CD19+ (rel) CD3−/CD19+ (abs) CD3−/CD56CD16+ (rel) CD3−/CD56CD16+ (abs)

1 1.85 −2.48 −8.67 −12.50 −0.34 −4.67

2 −0.33 −7.75 −5.47 −12.50 2.19 −5.69
3 2.11 −12.63 −17.85 −29.50 −1.23 −15.45
4 7.81 −17.70 −36.28 −51.50 −20.34 −39.43

Relative differences (in %) compared to reference value on day 0. Bold: difference higher than 10% (for values ≤20% or <300 cells/μL) or
higher than 5% (for values >20% or ≥300 cells/μL).

Table 3: Linearity of AQUIOS CL analytical measuring range.

CD3+ T1 CD3+ T2 CD3+/CD4+ CD3+/CD8+ CD3−/CD19+ CD3−/CD56CD16+ Lymphocytosis

(abs) (abs) (abs) (abs) (abs) (abs)

Analytical measuring 55–4700 55–4700 35–3000 45–1600 25–1000 20–1000 20–4700

range (manufacturer)
Experimentally 27.5–2285 24–2632.5 12–1104 11–1206.5 3–692 308.5–444 495.5–5885.5
checked range
R2 0.99 1.00 0.99 0.99 1.00 n/a 1.00

n/a, not applicable.

Figure 2: Linearity of AQUIOS CL analytical measuring range.

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 1284 Degandt et al.: Evaluation AQUIOS CL

A (cell count/μL) (Figure 3). Tables with correlation values

4000 between AQUIOS CL and the dual-platform approach,
together with correlation plots are provided in the supple-
3500
mentary data (see Supplemental Material, Tables 2 and 3,
3000
Figures 1–24).
CD3+/CD4+ (count/µL): AQUIOS CL

The parameters in the Tetra-1 Panel of AQUIOS CL

2500 showed small but statistically significant proportional
negative biases for CD3+ (abs), CD3+/CD4+ (rel and abs)
2000
and CD3+/CD8+ (rel and abs) and a constant positive bias
for CD3+/CD4+ (rel) when compared to the dual-platform
1500
Passing-Bablok fit approach in 97 samples. The pediatric subpopulation
(y = 3.723 + 0.9271x)
1000 (n = 42) showed only a slight statistically significant pro-
portional negative bias for CD3+ (rel), CD3+/CD4+ (rel)
500 and CD3+/CD8+ (rel). The posttransplant subpopulation
(n = 42) showed proportional negative bias for CD3+ (abs)
0
0 500 1000 1500 2000 2500 3000 3500 4000 and CD3+/CD8+ (rel and abs), and a constant positive bias
CD3+/CD4+ (count/µL): dual-platform approach
for CD3+/CD4+ (rel).
B
The parameters in the Tetra-2 Panel of AQUIOS
400
CL showed significant proportional negative biases
200
for CD3+ (abs) and CD3−/CD56CD16+ (rel) and a con-
AQUIOS CL – dual-platform approach

0 stant positive bias for CD3−/CD56CD16+ (rel and abs)

CD3+/CD4+ (count/µL):

–200 in 69 samples. In the pediatric subpopulation (n = 40),

a proportional negative bias was observed for CD3+
–400
Mean
(rel and abs) and a constant positive bias for CD3−/
–600 (–62.5) CD56CD16+ (rel). In the posttransplant population
–800 95% LoA (n = 17), a proportional negative bias was observed for
(–358.7–233.7)
–1000 CD3−/CD56CD16+ (abs) and a constant positive bias for
CD3−/CD19+ (rel).
–1200
0 500 1000 1500 2000 2500 3000 3500 4000
CD3+/CD4+ (count/µL):
dual-platform approach + AQUIOS CL/2

Figure 3: Regression between dual-platform approach and AQUIOS CL. Clinical agreement
(A) Passing-Bablok regression (red line) between dual-platform
approach (x-axis) and AQUIOS CL (y-axis). Green lines indicate Kappa coefficients for clinical agreement between the two
reference values. This example shows Passing-Bablok regression
methods, together with the 95% confidence interval, are
for CD3+/CD4+ (abs); (B) Bland-Altman regression (full blue line)
between dual-platform approach and AQUIOS CL. Dotted blue lines
shown in Table 4.
indicate 95% limit of agreement. This example shows Bland-Altman All kappa coefficients were higher than 0.71 showing
regression for CD3+/CD4+ (abs). substantial agreement between both methods. For some

Table 4: Clinical comparison (kappa coefficient) between AQUIOS CL and the dual-platform approach.

CD3+ T1 (rel) CD3+ T1 (abs) CD3+/CD4+ (rel) CD3+/CD4+ CD3+/CD8+ (rel) CD3+/CD8+ (abs)
(abs)

Kappa coefficient 0.75 [0.62–0.88] 0.90 [0.82–0.99] 0.91 [0.83–0.99] 0.98 [0.94–1.00] 0.80 [0.67–0.92] 0.84 [0.73–0.95]
[95% CI]
CD3+ T2 (rel) CD3+ T2 (abs) CD3−/CD19+ CD3−/CD19+ CD3−/CD56CD16+ CD3−/CD56CD16+
(rel) (abs) (rel) (abs)

Kappa coefficient 0.79 [0.64–0.94] 0.97 [0.90–1.00] 0.94 [0.85–1.00] 1.00 [1.00–1.00] 0.81 [0.67–0.94] 0.71 [0.52–0.91]
[95% CI]

CI, confidence interval.

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parameters, kappa coefficient was higher than 0.90, indi-
cating an almost perfect clinical agreement.
Flagging and notifications of AQUIOS CL
Eight samples out of 113 (7.1%) had one or more run flag,
and 48 samples (42.5%) had one or more run notification.
Run flags were “Inter Panel Count Error” (n = 3),
“Insufficient Lymphs” (n = 2), “Over Lyse Incub” (n = 1),
“Clog or Bubble” (n = 1), “ReRun” (n = 1) and “High Count
Rate” (n = 1). The flag “Insufficient Lymphs” occurs when
less than 500 lymphocytes are analyzed. Samples with
this flag showed low lymphocytic counts with the current
method (442 lymphocytes/μL and 44 lymphocytes/μL).
These samples were excluded from method compari-
son as mentioned above. The sample with “High Count
Rate” had a leukocyte count of 213.36 × 109/L with the
current method and mainly consisted of lymphoblasts.
This sample was also excluded from method compari-
son. Three samples flagged an “Inter Panel Count Error”.
This flagging is specific for the Tetra Combo analysis
and occurs when there is a difference greater than 15%
between the corresponding populations of the Tetra-1 and
Tetra-2 Panels. In two samples, no abnormalities could be
seen, but in the third sample, there was an incorrect CD3+
gating in the Tetra-2 tube. Therefore, this sample was also
excluded from method comparison. The sample with run
flag “Clog or Bubble” also showed the flag “ReRun”, but
no abnormalities were noticed. The flag “Clog or Bubble”
is shown whenever there is an abrupt interruption of the
events over time. The sample was rerun and the same
values were obtained as before, but without run flags. The
flag “Over Lyse Incub” means that for some reason (e.g.
unforeseen shutdown of the system) the sample was too
long in the lysis solution before measurement. The sample
with this flag was rerun and the values corresponded to
those obtained before.
In all but one sample with run notifications (n = 47),
the notification was “Potential sample or gating issue”,
meaning that on one plot the gating could be possibly
incorrect. The other sample showed the notification “Low
CD8”. All notifications were reviewed and apparently a
minor problem with the device was noticed: a problem in
the flow cell rendered incorrect electronic volume (EV)/
side scatter (SS) plots, causing notifications in 13 consecu-
tive samples. AQUIOS CL uses this EV/SS plot for lympho-
cyte gating as an addition to lymphocyte gating on the
SS/CD45 plot. However, for these 13 samples, lymphocyte
counting was correct and all results correlated well with
Degandt et al.: Evaluation AQUIOS CL 1285
the current method. Therefore, results were not omitted
from method comparison. Nevertheless, when subtract-
ing these samples in counting of notifications, 35 (35%)
of 100 samples showed a notification which was not due
to mechanical problems of the device. After revision of
these 35 samples with notifications, 26 could be accepted
without any intervention, 2 should be rerun and in 7
samples the lymphocyte-gate on EV/SS plot and on SS/
CD45 plot should be optimized manually. As mentioned
above, these samples were excluded from analytical
method comparison.
Discussion
This study evaluated the analytical features of AQUIOS
CL and compared AQUIOS CL to a dual-platform method
(Sysmex XE-5000 and BD FACSCanto-II).
Precision study showed an acceptable intrarun and
interrun variability when dedicated control material was
used. Comparable results were obtained by Gossez et al.
[7] for CD3+/CD4+ (abs). However, our precision study
showed clearly higher CVs for patient samples (ranging
from 0.25% to 26.34% for relative values and from 1.98%
to 32.10% for absolute values). Few to no precision crite-
ria are available for FCM values. Ricos et al. defined a CV
of 12.5% as a desirable imprecision for only CD3+/CD4+
(abs), a criterion that was met for a patient sample with
normal values, but not for patient samples with low CD3+/
CD4+ cells. Moreover, high CVs were especially noticed for
low values, around the lower limit of the analytical meas-
uring range. Focusing on the concentrations of absolute
CD4+ T-cell count with the highest impact on clinical deci-
sions (100–300 counts/μL), a good precision was noticed:
dedicated control material, AQUIOS IMMUNO-TROL Low
Cells, showed a mean concentration of 147.50 counts/μL
with a CV of 4.85%.
Relative values of lymphocyte subset counts seemed
to be stable at least up to 48 h when the K2-EDTA whole
blood sample is stored at room temperature, which is
longer than demonstrated by other authors [12, 13] and
longer than the manufacturer’s recommendation of 24 h.
Next to CD19+ staining, especially leukocyte count was
most prone to deterioration over time, causing more insta-
ble absolute values [14]. These results imply that clinical
samples can be stored overnight before analysis without
causing significant mistakes. When only relative values
are considered, samples can even be stored for 2 days
before analysis.
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1286 Degandt et al.: Evaluation AQUIOS CL
Interpanel consistency was acceptable according to
the applied limit of 15%. However, when using the 2%
limit as described in MMWR guideline [10], the rejection
rate rose to 25.4% for CD3 (rel) and 69.8% for CD3 (abs).
Method comparison showed a good analytical corre-
lation between AQUIOS CL and our current dual-platform
method. Some small proportional and constant biases
were noticed, but these were too small to be of clinical
significance.
AQUIOS CL is presented as a “Load & Go” device.
During this study, there was indeed a clear reduction
in hands-on time. Lab technicians only had to load rea-
gents and racks with (closed) patient samples, while all
further preanalytical and analytical steps were done
by the AQUIOS CL system. Even gating of all popula-
tions of interest was done automatically through gating-­
algorithms by the software system. However, this last step
was the most cumbersome step, and manual review was
necessary for some samples. In order not to undermine
the “Load & Go” principle, these cases should be kept to
a minimum and the users should be properly warned if
manual revision is needed. AQUIOS CL warns for revision
using run flags and notifications. During this study 7.1%
of the samples showed a run flag. In most of the cases, a
rerun could resolve the problem (i.e. for flags “Inter Panel
Count Error”, “Over Lyse Incub” and “Clog or Bubble”). In
case of flag “Insufficient Lymphs”, there are insufficient
lymphocytes detected to obtain a trustworthy, representa-
tive lymphocyte subsets. Other flags, such as “High Count
Rate” are suspected for other hematological disorders and
need further, more elaborated immunophenotyping. In
this study, 35% of the samples showed a run notification
(35/100). In 26 out of these 35 samples (74.3%), the results
could be accepted without intervention, whereas in nine
samples out of these 35 samples (25.7%) with notifications
an action was needed: this included a rerun in two cases
(5.7%) and an optimization of the gating in seven cases
(20%). In 26% of all samples an unnecessary notifica-
tion was shown, so most notifications did not necessitate
manual revision. The relative high number of notifications
could lead to “revision fatigue” of the users, meaning loss
of concentration of the lab technician during revision
since most notification have minimal or no impact.
One sample showing an “Inter Panel Count Error”
flag, had an incorrect CD3+ gating in the Tetra-2 Panel
while there was a correct CD3+ gating in the Tetra-1
Panel. This sample was repeated, but instead of request-
ing a Tetra Combo panel, Tetra-1 and Tetra-2 Panels were
performed separately. Again one CD3+ gating was done
incorrectly, but this sample was not flagged as there was
no interpanel comparison. However, there was a run
notification “Potential sample or gating issue” which also
should trigger manual revision. This incorrect CD3-gating
is a consequence of the gating algorithm the AQUIOS CL
software uses. In order to distinguish between the nega-
tive and positive population, the software uses a histo-
gram CD3-plot and searches for a valley between the two
populations to place a cutoff between the positive and
the negative populations. In case only one population is
present (either positive or negative), the software cannot
find a valley and places its cutoff before or after the only
population present, together with a notification “Poten-
tial sample or gating issue”. A suggestion to optimize this
gating strategy is to use a cutoff based on absolute fluores-
cence intensity values when no valley is detected.
Another drawback of the gating strategy is neglecting
the possible presence of a dual-positive CD4+/CD8+ T-cell
population. The presence of less than 5% of this popula-
tion in peripheral blood is considered as premature release
of CD4+ CD8+ T-cells from the thymus into the periphery
[15]. However, more than 5% CD4+/CD8+ T cells should be
considered as abnormal and should be further explored.
Another important remark on the use of AQUIOS CL
is its incompatibility with the stabilized blood samples
universally used for certified external quality assessment
(EQA) schemes such as UNKEQAS Immune Monitoring.
On AQUIOS CL, manual gating was necessary in order to
prevent a slight negative bias as the AQUIOS algorithm
would exclude lymphocytes for not meeting gating crite-
ria (even though they are genuine cells). In order to prop-
erly evaluate the unique walkaway character of AQUIOS
CL, EQA providers are challenged to produce a material
that is both compatible with this system but also has
clinical relevance in terms of the cell counts used. To meet
these requirements the ISO 17043 accredited UK NEQAS
Immune Monitoring Alternative Technologies program (31
international participants as of June 2017) uses material
that is sourced from Beckman Coulter and manipulated to
ensure that the levels of CD4+ T lymphocytes (and other
populations) are unique to the EQA exercise but differ-
ent from the supplied material. The utilization of a com-
mercial source for the raw material from which the EQA
samples are produced complies with ISO 17043 and the
underlying principles of EQA.
This study has limitations. First, there is a selec-
tion bias as we enriched for samples from pediatric
patients and posttransplant patients. Second, we com-
pared AQUIOS CL with a dual-platform methodology
because in Belgium 82% of the clinical laboratories use
this method. This methodology, however, is known to be
less precise than single-platform methods [6] and more
and more abandoned. Nowadays, almost 90% of the UK
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NEQAS participants of the immune monitoring scheme
use a single platform method. Third, AQUIOS CL was only
evaluated for a period of 3 months. Therefore, robustness
of the device over a longer period of time was not evalu-
ated. We noticed a minor mechanical issue at the end of
the evaluation period, which caused run notifications.
The error was caused by a problem in the flow cell which
resulted in incorrect EV/SS plots. This issue had only a
small, insignificant impact on the results.
To the best of our knowledge, this is the first study
that demonstrates an acceptable analytical performance
of AQUIOS CL and good correlation with a dual-platform
method for the quantification of T (both CD4 and CD8),
B and NK lymphocyte subsets. Gossez et al. [7] focused
on absolute CD4 T cell enumeration and its population
mainly consisted of patients with HIV. A recent study
of Grossi et al. [8] compared AQUIOS CL to a bead-
based method using 224 routine samples. They focused
mainly on a comparison with a bead-based method and
the influence on turnaround times. Here we evaluated
both Tetra-1 and Tetra-2 Panels for enumeration of all
T, B and NK lymphocytes, partly by comparing it to a
dual-platform method, in a pediatric population, in
a ­posttransplant population and in samples from our
routine work load.
In conclusion, AQUIOS CL allows automated immu-
nophenotyping of lymphocyte subset quantitation. Ana-
lytical performance was acceptable and quantitative
results of the different lymphocyte subsets were compa-
rable to results obtained with a dual-platform method;
however, interpanel consistency is troublesome. Auto-
matic gating sometimes needed to be manually reviewed.
AQUIOS CL warns – through run flags and notifications –
whenever such a manual revision is needed. As the
number of notifications was relatively high (about one-
third of all samples), gating algorithms should be further
improved to reduce this number. Overall, the system looks
promising for quantification of lymphocyte subsets in
routine clinical practice.
Acknowledgments: This study was supported by Beck-
man Coulter through donations of laboratory equipment
and supplies. This private company had no role in study
design and the collection of data. They did help in inter-
preting gating results with run notifications. Beckman
Coulter had no role in the preparation of the manuscript
or the decision to submit it for publication. We thank
Professor David Barnett and Liam Whitby (UK National
External Quality Assessment Scheme [UK NEQAS], Shef-
field) for providing information and data regarding the UK
NEQAS immune monitoring scheme.
Degandt et al.: Evaluation AQUIOS CL 1287
Author contributions: BP, NB, XB: study design. SD:
drafted the manuscript. BP, SJ: patient recruitment. SD,
BP, SJ: data acquisition. SD, BP: statistical analysis. NB,XB
checked and improved content and English grammar
and style of the manuscript. NB, XB commented on and
edited the manuscript, which was read and approved by
all authors. All the authors have accepted responsibility
for the entire content of this submitted manuscript and
approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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