AJCP / Original Article

Performance and Abnormal Cell Flagging Comparisons

of Three Automated Blood Cell Counters
Cell-Dyn Sapphire, DxH-800, and XN-2000
Julie Hotton, PharmD, Jan Broothaers, Caroline Swaelens, and Brigitte Cantinieaux, MD
From the Haematology Laboratory, CHU Saint-Pierre & Bordet Institute, Universit Libre de Bruxelles, Belgium.
Key Words: Cell-Dyn Sapphire; DxH-800; XN-2000; HemaCAM; Flag; Blast; Abnormal lymphocytes; IG; NRBC
DOI: 10.1309/AJCPE5R4SOQBUULZ

Objectives: To compare two hematological analyzersthe

DxH-800 (DxH; Beckman-Coulter, Miami, FL) and XN-2000
(XN; Sysmex, Kobe, Japan)with the Cell-Dyn Sapphire
(SAPH; Abbott, Santa Clara, CA).
Methods: We analyzed 4,375 samples. Slide reviews were
made in the presence of blast, abnormal lymphocyte, and
immature granulocyte (IG) flags or nucleated RBC (NRBC)
count.
Results: The analyzers exhibited excellent correlations for
CBC and neutrophils but displayed a limit correlation for
lymphocytes. The XN did not miss circulating blasts (0.5%95% in microscopy). For NRBCs, the XN demonstrated a
sensitivity of 90%; DxH, 74%; and SAPH, 29%. Only the XN
demonstrated a correlation with microscopy, permitting a
WBC six-part differential until 15% of NRBCs. The XN and
DxH gave useful IG counts with a cutoff less than 5% and a
WBC level more than 2,500/mm3. For abnormal lymphocytes
detection, only XN demonstrated sensitivity of more than
95%, but its specificity of 54% requires adaptation.
Conclusion: The XN increases the sensitivity of abnormal
cell detection compared with the other counters, permitting a
seven-part differential between predefined levels, decreasing
the slide review from 20% to 9%.

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Recent hematology analyzers are becoming more efficient, and the number of blood cell parameters reported
increases constantly.1 Automated leukocyte differentials
show a good correlation with morphology (MO) and, if any
abnormal cells are present, demonstrate a more accurate
performance to detect these cells due to the smaller number
of cells counted in MO; the interobserver variability and
the lack of uniform WBC distribution on the blood film
are inherent to manual microscopy. Automated digital cell
morphology systems have been developed to solve these
deficiencies and improve the efficiency and reproducibility
of slide review.
Many hematology laboratories draw up rules based on
the leukocyte differential and the presence of flags to decrease
the number of microscopic reviews and to improve the quality
of these reviews, in agreement with the Clinical and Laboratory Standards Institute (CLSI) consensus rules.2,3
In this study, three analyzersthe Cell-Dyn Sapphire
(SAPH; Abbott, Santa Clara, CA), DxH-800 (DxH, software
1.1.3; Beckman-Coulter, Miami, FL), and XN-2000 (XN;
Sysmex, Kobe, Japan)are evaluated for repeatability,
linearity, and carryover. Then, multiple comparisons among
the analyzers themselves are performed for hemoglobin
(Hb), mean corpuscular volume (MCV), platelets (PLTs),
and WBCs, as well as among the five normal populations
of circulating WBCs. We evaluate the performance of the
three analyzers, focusing on the capability of flagging in the
presence of abnormal cells. Nucleated RBCs (NRBCs) are
quantified by these three counters. Immature granulocytes
(IGs) are quantified by the XN and by the updated software
(2.0) of the DxH. Countings and flagging are compared with
results obtained by MO.

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ABSTRACT

Hotton et al / Performance Efficiencies of Cell Counters

Materials and Methods

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Multiple comparisons including the three analyzers were

performed for Hb, MCV, PLTs, and WBCs and for the seven
subclasses of differential with MO. Morphologic flags were
compared for each analyzer using the microscopic criteria
defined above. Sensitivities, specificities, and efficiencies
for each flag were calculated between each analyzer and
MO. Statistical analysis was performed by GraphPad Prism
4 (GraphPad Software, La Jolla, CA) using the Wilcoxon
signed rank test for paired samples, the Spearman rank test
for linear regression analysis, the Bland-Altman plot for systematic differences, and the measure of agreement by the k
coefficient.

Results
Repeatability
The coefficient of variation (CV) was lower than 5%
except for results of low WBCs for the SAPH and XN, low
PLTs for the XN, IG counts in MO, and NRBC counts in the
DxH and MO Table 1.
Linearity
Linearity data for the three analyzers showed excellent
correlations (R2 > 0.99) among values for all sets of diluted
samples Table 2.
Carryover
Carryover was 0.9%, 0.3%, and 0% for Hb; 0%, 0.1%,
and 0% for PLTs; and 0.2%, 0%, and 0% for WBCs for the
SAPH, DxH, and XN, respectively.
Comparisons Among the Three Analyzers for Hb, MCV,
PLTs, and WBCs
Comparisons of Hb, MCV, PLT, and WBC counts on
500 samples, randomly selected, showed for the four parameters excellent correlations among the analyzers (all R > 0.97).
A significant biological bias was observed only for PLTs by
the Bland-Altman diagrams Figure 1. PLT counts used in
SAPH were obtained by an optical method and were slightly
higher than those obtained with the DxH and XN, both calculated by the impedance method.
Comparison Among the Three Analyzers and MO
for Differentials
Correlations between analyzers and MO counts were performed for the 444 samples requiring a slide review. Correlations among the analyzers and MO counts were very good for
neutrophils for the three analyzers (R > 0.8), displayed a limit
(R > 0.7) for lymphocytes among MO counts and the DxH
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Repeatability, linearity, and carryover were performed

for the three analyzers. During one month, 4,375 routine
laboratory samples were randomly run in parallel within 4
hours of collection in K2EDTA tubes on the three analyzers. Slide reviews were realized due to the presence of blast,
abnormal lymphocytes (AL), IG, or NRBC flags on at least
one analyzer. Moreover, the presence of blast and lymphomatous cells was assessed by flow cytometry (FCM) on the
basis of multiple clinical and/or biological criteria. In this
context, the probability of missing true-positive immature
cells was very low.
In total, 444 samples were flagged and analyzed by
a digital microscope (HemaCAM; Horn Imaging, Aalen,
Germany) on 230 cells, reviewed by three experienced
cytologists (J.H., J.B., and C.S.). The choice of microscopy
review by HemaCAM as a reference method is based on
the results from a study performed in our laboratory demonstrating the higher sensitivity of HemaCAM to detect
abnormal cells than manual microscopy.
Most analyzers have only one flag for atypical or
abnormal lymphocytes. In the XN, the WBC differential
(WDF) channel differentiates two types of flags: atypical
lymphocytes and blast or abnormal lymphocytes. If this last
flag is present, a subsequent analysis in the white progenitor cell (WPC) channel allows discrimination between blast
cells and abnormal lymphocytes because of their reactivity
to a fluorescent dye and their different patterns in biparametrical side scatter/side fluorescence and side scatter/
forward scatter.
In this study, these two lymphocyte flags, atypical and
abnormal, were assessed together to ensure homogeneity
with other instruments. The morphologic criteria to assess
abnormal lymphocytes are based on a size larger than a normal lymphocyte; some changes to the shape or chromatin
of the nucleus, with nucleoli occasionally present; and the
quantity or basophilic color of the cytoplasm, with the presence of vacuoles or azurophilic granules. The probability
of finding abnormal cells is expressed into the flag for the
SAPH from 0.5 to 0.9 and by the Q-flag for the XN, which
is positive from 100 to a maximum of 300. The DxH does
not give probabilities associated with flags.
The morphologic criterion used to determine a positive
smear finding, in relationship to flagging, for the study was
the presence of blast, plasma cells, and NRBCs. We used
the threshold of 1% for IGs and 4% or more for ALs, as
recommended by the International Society for Laboratory
Hematology. Updated software (2.0) for DxH was launched
in June 2012, giving an early granulocyted cell (EGC) count
for each sample. Using this new software, 38 samples positive in MO for IGs were analyzed.

Statistical Analysis

AJCP / Original Article

and XN, and were not present between MO counts and the
SAPH. There was no correlation for monocytes, eosinophils,
and basophils among the three analyzers and MO counts.
Correlations between the two new analyzers with SAPH
were very good (R > 0.9) for neutrophils and lymphocytes
but displayed a limit (R > 0.6) for monocyte and eosinophil
counts. Correlations between the DxH and XN counts were
very good (R > 0.9) for all classes except basophils.

NRBC Flags
Fifty-nine cases with more than 0.5% of NRBCs were
identified by MO (range, 0.5-35 NRBCs/100 WBCs), with 22
cases having more than 2% of NRBCs. The XN demonstrated
the highest sensitivity for NRBC detection (81%) (Table
4), while the DxH had a sensitivity of 63% with four cases
(NRBC>2%) missing in comparison to the XN. The SAPH
needed to use simultaneously the fluorescent population flag
to increase the number of samples detected for NRBCs, resulting in a sensitivity of 29%.
In contrast to the sensitivities, the specificities and
efficiency rates for the three devices were 90% or more
using both thresholds of NRBCs. Measures of association
demonstrated an almost perfect agreement for the DxH/XN
association, with a k coefficient superior to 0.8, and moderate
agreement for the two associations of SAPH/DxH and SAPH/
XN, with k coefficients of 0.45 and 0.5, respectively.
Figure 2 illustrates the regression curve of the NRBC
counts for the XN and the DxH with MO, demonstrating only
a biologically significant R between the XN and MO counts.
With the updated software (2.0), the DxH still demonstrated a
bad correlation with an R2 of 0.19.
Immature Granulocyte Flags
More than 1% of IGs were counted in 127 cases by MO,
including infectious and hematologic malignant diseases. The
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Hotton_2013040177.indd 847

Mean (SD)

Normal Hb, g/dL (n = 5)

Cell-Dyn Sapphire
14.5 (0.07)
DxH-800
14.36 (0.04)
XN-2000
14.38 (0.08)
Low Hb, g/dL (n = 5)
Cell-Dyn Sapphire
4.05 (0.05)
DxH-800
5.35 (0.02)
XN-2000
3.88 (0.04)
High Hb, g/dL (n = 5)
Cell-Dyn Sapphire
19.4 (1.03)
DxH-800
20.8 (0.11)
XN-2000
20.8 (0.08)
Normal WBCs, 109/L (n = 5)
Cell-Dyn Sapphire
5.2 (0.065)
DxH-800
5.8 (0.086)
XN-2000
5.2 (0.076)
Low WBCs, 109/L (n = 5)
Cell-Dyn Sapphire
0.16 (0.025)
DxH-800
0.28 (0.007)
0.14 (0.011)
XN-2000
High WBCs, 109/L (n = 5)
Cell-Dyn Sapphire
55.2 (0.260)
DxH-800
46.3 (0.104)
XN-2000
57.3 (0.123)
Normal PLTs, 109/L (n = 5)
Cell-Dyn Sapphire
193.2 (5.890)
DxH-800
205.6 (1.486)
XN-2000
203.2 (5.449)
Low PLTs, 109/L (n = 5)
Cell-Dyn Sapphire
3.17 (0.127)
DxH-800
8.10 (0.372)
XN-2000
6.00 (0.100)
High PLTs, 109/L (n = 5)
Cell-Dyn Sapphire
1,076.2 (12.397)
DxH-800
998.1 (2.283)
XN-2000
941.8 (3.834)
IGs, %
DxH-800 (n = 5)
11.3 (0.4)
XN-2000 (n = 5)
4 (0.1)
Morphology IG <2% (n = 10)
1.1 (0.8)
Morphology IG >2% (n = 10)
8 (1.7)
NRBCs, /100 WBCs
DxH-800 (n = 5)
3.7 (0.5)
XN-2000 (n = 5)
4.9 (0.2)
Morphology NRBCs <2% (n = 10)
0.9 (0.6)
Morphology NRBCs >2% (n = 10)
7.1 (2.7)

CV, %
0.49
0.29
0.58
1.34
0.34
1.15
5.35
0.56
0.4
1.25
1.47
1.46
15.49
2.65
8.38
0.47
0.22
0.21
3.05
0.72
2.68
4.01
4.6
16.6
1.15
0.23
0.4
3.8
2.2
75.5
28.2
12.9
3.6
66.2
37.4

CV, coefficient of variation; Hb, hemoglobin; IG, immature granulocytes; NRBC,

nucleated RBC; PLT, platelet.

sensitivities of flagging were 20%, 29%, and 88% for the

SAPH, DxH, and XN, respectively (Table 4). The SAPH and
DxH (software 1.1.3) had missed 96 and 86 cases, respectively, in comparison to XN. The specificities for the SAPH,
DxH, and XN were 90%, 87%, and 84% and the efficiency
rates 70%, 71%, and 85%, respectively. No satisfactory
agreement among the three devices was demonstrated for IGs
(Table 4).
Statistical tests of the IG count between XN and MO
were done and demonstrated an R of 0.5 for the 127 samples
analyzed. When some outlier samples were removed (false

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Blast Flags
Eighteen cases with circulating blast cells were identified
by MO and confirmed by FCM (range, 0.5%-94.7% of blasts),
seven cases of acute myeloid leukemia, three cases of B-cell
acute lymphoblastic leukemia, seven cases of postchemotherapy, and one case of severe infectious disease Table 3. The
sensitivities of the SAPH, DxH, and XN to detect blast cells
were 89%, 72%, and 100%, respectively, using the three flags
of blast, IG, and AL together Table 4. Despite this association of flags, the SAPH and DxH still missed two and five
cases, respectively. If considering only the flag blast, the
sensitivities were 61%, 55%, and 72% for SAPH, DxH, and
XN, respectively. The specificities for the SAPH, DxH, and
XN were 92%, 89%, and 96%, with efficiency rates of 92%,
88%, and 97%, respectively. The k coefficients demonstrated
an almost perfect agreement between each pair of analyzers.

Table 1
Repeatability Results for Five Repeated Analyses of Each
Sample, Except for Repeatability of IGs and NRBCs by
Morphology (n = 10)

Hotton et al / Performance Efficiencies of Cell Counters

Table 2
Linearity Results for Three Samples With High Values of Hb,
WBCs, and PLTs Diluted With Buffer of Each Analyzer
Sample

positives due to toxic granulations or hyposegmentation of

neutrophils), the R was increased to 0.65. When we calculated the positive predictive value for the presence of more
than 1% of IGs in MO counts, we found the highest value
(68%) when the IG rate given by the XN was superior or
equal to 2%.
We analyzed the impact of a high left-shift flag on
the IG rate given by the XN. For leucopenia inferior to 2.5
109/L, whatever the IG rate given by the analyzer, samples
were false positive in more than 75% of cases if there was a
presence of a left-shift flag with a probability level between
200 and 300. With the updated software (2.0) for the DxH, an
important increase in sensitivity was observed, 84% instead

R2

Regression Equation

Hb
Cell-Dyn Sapphire
y = 0.1794x 0.6929
y = 0.1684x + 0.0029
DxH-800
XN-2000
y = 0.2105x 0.125
WBCs
Cell-Dyn Sapphire
y = 594.76x 347.62
DxH-800
y = 0.3023x 0.1929
XN-2000
y = 574.08x + 783.25
PLTs
Cell-Dyn Sapphire
y = 10,251x + 714.29
DxH-800
y = 7,020x 1,400
XN-2000
y = 10,968x 12,643

0.994
0.999
0.999
0.999
0.998
0.999
0.999
0.997
0.997

Hb, hemoglobin; PLT, platelet.

115

10
15

Average MCV (fL)

Difference PLT

150

Difference WBC

100
50
0
50 150 350 550 750 950
50
Average PLT ( 103/L)

1.5
1.0
0.5
0
0.5 0
1.0
1.5
2.0

20

30

Average WBC ( 103/L)

Difference Hb

5
0
55
5

75

95

115

10
15

Average MCV (fL)

100
50
0
50
50

100

2.0
1.5
1.0
0.5
0
0.5 0
1.0
1.5

150

350

550

750

0.4
0.2
0
0.2

0.4

Average Hb (g/dL)

150

10

16

10

Difference PLT

Difference MCV

95

11

Difference MCV

75

Average Hb (g/dL)

5
0
55
5

0
1

0.6

Difference PLT

16

2
3

11

Difference Hb

Difference MCV

Difference Hb

0
1

950

8
6
4
2
0
255
4
6

40
60
80

10

20

30

Average WBC ( 103/L)

4
3
2
1
0
0
1
2

16

Average Hb (g/dL)

75

95

115

Average MCV (fL)

60
40
20
0
50 150
20

Average PLT ( 103/L)

11

350

550

750

950

Average PLT ( 103/L)

10

20

30

Average WBC ( 103/L)

Figure 1 Bland-Altman plots for hemoglobin (Hb), mean corpuscular volume (MCV), platelets (PLTs), and WBCs for the three
analyzers on 500 samples. A, Hb SAPH vs DxH. B, Hb SAPH vs XN. C, Hb DxH vs XN. D, MCV SAPH vs DxH. E, MCV SAPH vs XN.
F, MCV DxH vs XN. G, PLT SAPH vs DxH. H, PLT SAPH vs XN. I, PLT DxH vs XN. J, WBC SAPH vs DxH. K, WBC SAPH vs XN. L,
WBC DxH vs XN.
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Difference WBC

Difference WBC

AJCP / Original Article

Table 3
Flags (Blast, AL, or IG) Given by the Three Analyzers for the Cases With Blast Cells in Morphology, Probability for the Cell-Dyn
Sapphire, and Q-Flag Values for the XN-2000
Cell-Dyn

Sapphire Flags
Sample
No.
Blast AL

IG

DxH-800 Flags
Blast

AL

IG

Blast

300

110

>5

AL

IG, %

Morphology, %

Diagnosis

>2 1.1
>5 0.5


38.9 + 1 Promonocytes
59
AML-4

Postchemotherapy
Postchemotherapy
AML-5a

170 >5 1.6

Postchemotherapy
120
>5 0.5
Postchemotherapy
+
+
300

>5
25.9 + 6.9 Promonocytes AML-4
+ 300 >5 1.2
Postchemotherapy
+
+
300

>5
29.9 + 22.1 Promonocytes AML-4
+ + 250
31.9
AML-4
+ 170 220
86.7
B-ALL
+ 190
3.8
AML-4

+ 300 >2 49
AML-4
+ 220 300
94.7
B-ALL
+ 230 >5 0.7
Postchemotherapy

+
270 >5 2.6
Postchemotherapy

130
0.5
Infectious
+ + 300 300
87
B-ALL

AL, abnormal lymphocytes; AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; IG, immature granulocytes; LyBlast, lymphoblast (Beckman-Coulter,
Miami, FL); MoBlast, monoblast (Beckman-Coulter); NeBlast, myeloblast (Beckman-Coulter); , no flag present; +, presence of flag.

Table 4
Sensitivities, Specificities, and Efficiencies to Detect Blast, IG (Software 1.1.3 for DxH), NRBCs, and Abnormal Lymphocytes
for the Three Analyzers on 444 Samples Analyzed by Morphology (MO)


Characteristic

Coefficient
Cell-Dyn
Sapphire, %

DxH-800,
%

XN-2000,
%

S/D

S/X

Blast (0.5% MO; n = 18)

Sensitivity
89
72
100
Specificity
92
89
96
Efficiency
92
88
97
0.84
0.94
PPV
33
22
54
NPV
99
99
100
IG (1% MO; n =137)
Sensitivity
20
29
88
Specificity
90
87
84
Efficiency
70
71
85
0.41
0.19
PPV
45
48
68
NPV
74
75
95
NRBC (0.5% MO; n = 59)
Sensitivity
29
63
81
Specificity
100
98
98
Efficiency
90
93
96
PPV
100
84
91
NPV
90
94
97
NRBC (2.0% MO; n = 22)
0.45
0.50
Sensitivity
45
86
100
Specificity
98
94
93
Efficiency
96
94
93
PPV
59
43
43
NPV
97
99
100
AL (4% MO; n = 31)
Sensitivity
26
22
97
Specificity
94
92
54
Efficiency
89
87
60
0.15
0.05
PPV
23
17
14
NPV
94
94
99

D/X

0.83

0.23

0.85

<0

AL, abnormal lymphocytes; D, DxH-800; IG, immature granulocytes; NPV, negative predictive value; NRBC, nucleated RBC; PPV, positive predictive value; S, Cell-Dyn
Sapphire; X, XN-2000.

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1
2 0.9
3
0.7
0.7
0.5 MoBlast
4

0.8
0.7 Abnormal differential

pattern
5 0.5 0.8 0.6
6
7
0.9

0.9 MoBlast-LyBlast
8 0.7
NeBlast
9
0.9
0.6
0.7 MoBlast
10 0.9 0.7 MoBlast
11 0.9 0.9 LyBlast
12 0.7
13 0.9 0.7 MoBlast
14 0.7 0.9 LyBlast
15 0.8

16 0.6 0.9
17

0.6


18 0.7 0.9 LyBlast

XN-2000 Flags

Hotton et al / Performance Efficiencies of Cell Counters

40
35
30
25
20
15
10
5
0

B
DxH-800 (NRBC %)

XN-2000 (NRBC %)

10
20
30
40
Manual Count (NRBC %)

35
30
25
20
15
10
5
0

10
20
30
40
Manual Count (NRBC %)

Figure 2 Correlations of the percentage of nucleated RBCs (NRBCs) determined by the (A) XN-2000 and the (B) DxH-800 with
the manual count method on 200 cells. XN-2000: y = 0.9546x + 0.7278; R 2 = 0.77945. DxH-800: y = 0.4343x + 1.032; R 2 =
0.32455.

AL Flags
Thirty-one cases were positive in MO for ALs, either for
lymphomatous cells (two non-Hodgkin lymphoma [NHL] and
five B-cell chronic lymphoid leukemia [B-CLL]) or for more
than 4% of abnormal lymphocytes (n = 15) or for the presence
of plasma cells (n = 9). For all lymphomatous cell cases, a
monoclonal population was found by flow cytometry. For the
five cases of B-CLL, the AL flag was present in four cases
for the SAPH, in five cases for the DxH, and in four cases
for the XN, which gave a false blast flag for the fifth case.
Two cases of NHL (other than CLL) were analyzed, with the
AL flag present in one case for the SAPH and DxH and in the
two cases for the XN. Fifteen cases containing 4% or more
of ALs were detected, flagging in two cases for the SAPH, in
one case for the DxH, and in all cases for the XN. Nine cases
contained plasma cells (range, 0.5%-1.2%), with the AL flag
in one case for the SAPH, in none for the DxH, and in eight
cases for the XN.
The specificities for the SAPH, DxH, and XN were 94%,
92%, and 54%, with efficiency rates of 89%, 87%, and 60%,
respectively (Table 4). Measures of association demonstrated
a slight or poor agreement for the three associations, with k
coefficients of 0.15 or less.

Discussion
With the SAPH and XN, repeatability was not good for
low WBCs (<0.3 109/L) and low PLTs (<10.0 109/L) with
XN (Table 1). For these two analyzers (SAPH and XN), an
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extended count is possible for low WBC events samples, but it

was not used in this study. Without using the extended WBC
mode, the CVs were less than 15% for the XN and more than
20% for the SAPH for WBC counts inferior to 0.3 109/L,
measured by an optical method.
For PLTs, in the SAPH, both optical and impedance
methods can be used to give PLT counts; we therefore used
the optical counts, with counts slightly higher than impedance
counts and leading to the bias previously described. For the
XN, the impedance method is used in the first instance, and if
an abnormal scattergram is detected by information processing unit software, optical counts or PLT-F (fluorescent platelets, a new channel of the XN series) can be used. The reason
for this high CV for PLT counts is also due to the expression
of results. For the XN, results increase by a thousand increments, while for the DxH and SAPH, they increase by 100
and 10 increments, respectively.
In MO, neutrophil percentages significantly increased
and those of lymphocytes and monocytes decreased in comparison to values of analyzers. These underestimations are
explained by the well-known lysis of lymphocytes4 during the
smearing, with the presence of nuclei shadows not counted in
differentials. Monocytes5 are mainly present on the sides of
smearing, an area not scanned by digital microscopy.
The XN showed a higher sensitivity than the SAPH and
DxH for all flags of interest, allowing the detection of two
samples with blasts, 96 with IGs, 32 with NRBCs, and 16 with
ALs not detected by the SAPH, as well as five with blasts, 85
with IGs, eight with NRBCs, and 22 with ALs not detected
by the DxH.
Blast Cells
The XN, thanks to its new channel WPC, increases the
blast rate detection in comparison to the two other devices
and, moreover, avoids false-positive flags due to monocytosis,
as unfortunately observed in the SAPH and DxH. Our limited
population of 18 samples with circulating blast cells does
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of 29% with the version 1.1.3 software. The specificity was

decreased to 67%. Since an IG count was also given for all
blood samples, a statistical test was done with MO counts and
showed an R of 0.23 for the 38 samples studied, increasing
to 0.77 when outlier samples were removed (same criteria as
for the XN).

AJCP / Original Article

not allow reaching a significant difference between the pairs

of analyzers with the k coefficient, despite the difference in
sensitivity for the three analyzers. Further investigations with
a higher number of positive cases should be done.

IGs
Automated IG counts are now available on the XN and
DxH (software 2.0). For the XN, the combination of a lysing
product and a fluorescent stain in the WDF channel allows
differentiating the IG cluster right above the neutrophil cluster in the biparametric histogram side scatter/fluorescence.
The enumeration of EGCs is possible on the DxH by a
novel combination of volume, conductivity, and light scatter parameters, which provide increased separation between
EGCs and other WBCs.
Results of sensitivity for IGs with the XN are considerably better then those of SAPH and DxH (software 1.1.3),
but the absence of a perfect correlation with MO (R = 0.65)
requires some algorithmic rules. The new software (2.0) of
the DxH improves the sensitivity to detect IGs. Preliminary
results demonstrated a good correlation with MO, with an
R superior to the XN (R = 0.77). This weak correlation is
explained in different ways, including the smaller number of
cells counted by MO, the subjectivity involved in the morphologic classification (especially the difficulty in separating
metamyelocytes from band cells),7 the inhomogeneous repartition of cells on blood smears, and the well-known important
CV for these rare events on blood smearing.8
A previous study had reported a better correlation coefficient between the XE-IG Master (XE-2100 and XE-5000)
and FCM method (R2 = 0.92) than with MO (R2 = 0.60).9
In another article,10 the same authors have shown that for
all samples with an automated IG count of more than 3%,
IGs were found by morphology, unless the WBC count was
less than 0.5 109/L. Although the software is the same in
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Hotton_2013040177.indd 851

ALs
During our study, we used the AL flag without any
threshold of probability for the SAPH or the Q-flag for the
XN. Combining the new WDF channel with the WPC channel
of the XN, six cases of mature lymphocytic neoplasms out of
seven were detected by the XN. Despite this good sensitivity,
its poor specificity needs further investigation of the AL flags
in the WPC channel. The XN demonstrates good capability
to flag the presence of plasma cells, in contrast with the two
other devices, which are present in the AL flag and do not
require a reflex test in the WPC channel. The poor and slight
agreements between the pairs of analyzers reflect the different
technologies of each analyzer to detect ALs.

Conclusion
In conclusion, the XN, thanks to its new combination of
fluorescent dyes and specific lysing agents, provides significantly more efficient results than the DxH and the SAPH for
NRBCs and IGs, with the possibility of using a WBC sevenpart differential with a range from 1% to 5% for IGs when the
leukocytosis is 2.5 109/L or higher and a range of NRBCs
from 1% to 15%.
The new software (2.0) of the DxH gives good preliminary results for using EGCs in routine differential results with
the same criteria for leukocytosis, but the efficiency of NRBC
recognition seems to be not improved compared with the 1.1.3
version. Moreover, the AL flag in the XN seems to increase
significantly the detection of mature lymphoid neoplasms
with the restriction of bad specificity. Further tests are needed
with a better tuning of the Q-flag for ALs.
Finally, the XN allows reducing, in a general and
oncologic hospital, the level of slide reviews for blast, IGs,
and NRBCs, with an increased sensitivity of detecting
these abnormal circulating cells. For the first time, we have

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NRBCs
Today, it appears clearly that the new dedicated NRBC
channel (WNR channel) of the XN is the best actual method
to enumerate NRBCs, according to Rosenthal et al6 and our
study, in the range of 0.5% to 15% of NRBCs. This channel
performs the WBC count and specific differentiation and
enumeration of basophils and NRBCs using a patented cellspecific lysing agent, which preserves basophil integrity,
and a new fluorescent dye, which stains stronger WBCs
than NRBCs.
The DxH uses seven parametersimpedance, radiofrequency, and five laser light scatter measurements (VCS
technology)to enumerate NRBCs, but the results obtained
are disappointing in comparison to those given by the XN
(Figure 2). The moderate agreements between SAPH/DxH and
SAPH/XN reflect the bad sensitivity of the SAPH for NRBCs.

the XN series, we did not find the same results, because the
left shift and leucopenia demonstrated a great impact on the
predictive positive value.
Further investigations must be done to evaluate interferences in the IG count, such as the left-shift flag and granularity index.11-13 Nevertheless, the advantage given by the
XN and DxH (software 2.0) to get a seven-part leukocyte
differential provides great time savings for a laboratory when
a small IG count is detected, allowing the validation of a
small amount of IG, with a cutoff of 5% or less and with a
leukocytosis of more than 2.5 109/L, without slide review
if any other flag is present. Rosenthal et al14 determined the
same optimum IG cutoff of 5% or less to use without MO
verification, with a decrease of 30% of smear reviews in their
laboratory, confirming our results.

Hotton et al / Performance Efficiencies of Cell Counters

decreased the slide review for our laboratory from 20% with
the SAPH to 9.3% with the XN.
Address reprint requests to Dr Hotton: Hematology Laboratory,
Cliniques de lEurope, Avenue de Fr 206, 1180 Bruxelles,
Belgium; j.hotton@cliniquesdeleurope.be.

References

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