Ann Hematol (2008) 87:755759

DOI 10.1007/s00277-008-0486-8

ORIGINAL ARTICLE

Malaria detection with the Sysmex XE-2100 hematology

analyzer using pseudoeosinophilia and abnormal WBC
scattergram
Hee Jin Huh & Gwi Young Oh & Jung Won Huh &
Seok Lae Chae

Received: 2 January 2008 / Accepted: 8 March 2008 / Published online: 22 April 2008
# Springer-Verlag 2008

Abstract Recent investigation using the Sysmex XE-2100

hematology analyzer (Sysmex Corporation, Japan) has
demonstrated erroneously high eosinophil counts and
abnormal white blood cell (WBC) scattergrams in malaria
cases. This study was conducted to assess the diagnostic
efficiency of the Sysmex XE-2100 analyzer for malaria.
One hundred forty-four patients initially diagnosed with
Plasmodium vivax infection, 319 patients with febrile
illness, and 24 patients who underwent malaria treatment
were analyzed. Atypical features on Sysmex XE-2100
analyzer were categorized as pseudoeosinophilia (a gap of
more than 5% in eosinophil counts between the Sysmex
XE-2100 analyzer and microscopic examination) and
abnormal WBC scattergram. Pseudoeosinophilia or abnormal WBC scattergram were detected in 100 of 144 malariapositive samples (sensitivity 69.4%, specificity 100%). The

H. J. Huh : S. L. Chae
Department of Laboratory Medicine, College of Medicine,
Dongguk University,
Goyang, Republic of Korea
G. Y. Oh
Department of Laboratory Medicine, Eone Reference Laboratory,
Seoul, Republic of Korea
J. W. Huh
Department of Laboratory Medicine, College of Medicine,
Ewha Womans University,
Seoul, Republic of Korea
S. L. Chae (*)
Department of Laboratory Medicine,
Dongguk University International Hospital,
Siksa-dong, Ilsandong-gu, Goyang-si,
Gyeonggi-do 411-773, Republic of Korea
e-mail: rocky@duih.org

samples with pseudoeosinophilia or abnormal WBC scattergrams showed significantly higher parasite counts than the
samples without pseudoeosinophilia or an abnormal WBC
scattergram (P<0.05). All 24 samples from patients for
whom the malaria smear was repeated after malaria
treatment was initiated showed a normalized eosinophil
count and a normal WBC histogram. In conclusion,
attention to differential count and WBC scattergrams
provided by the Sysmex XE-2100 would be a valuable
tool in malaria detection.
Keywords Malaria . Pseudoeosinophilia . Abnormal WBC
scattergram . Sysmex XE-2100 hematology analyzer

Introduction
Malaria is diagnosed using a combination of clinical
observations, case history, and diagnostic tests, principally
microscopic examination of stained slides. However,
classical microscopic examination is labor intensive and
time consuming. Limitations of malaria diagnosis using this
method have led to the development of several new
techniques that simplify and speed up diagnosis and
increase sensitivity [1]. However, these all rely on clinical
suspicion and, consequently, a clinical request. Although
some methods lend themselves to automation (e.g., polymerase chain reaction) [2], no technique can yet be used for
routine clinical automated screening.
There is growing interest in the use of routine hematological blood analysis as a component of measurement for
the presumptive diagnosis of malaria infection [1, 3]. It has
been reported that abnormal depolarizing patterns detected
by a Cell-Dyn hematology analyzer (Abbott Diagnostics,
Santa, CA, USA) showed high sensitivity for malaria

756

diagnosis [4]. One advantage of this method is its potential

to detect cases where clinical suspicion does not lead to a
request for a malaria test [5]. A few cases of malariainfected patients with pseudoeosinophilia on the Sysmex
XE-2100 hematology analyzer (Sysmex Corporation, Kobe,
Japan) were recently reported [6]. Moreover, abnormal
white blood cell (WBC) histograms were found in many
malaria-infected patients.
The aim of this study was to determine the usefulness of
pseudoeosinophilia and abnormal WBC scattergrams, as
detected by the Sysmex XE-2100 analyzer, for the
diagnosis of malaria in a routine laboratory setting.

Ann Hematol (2008) 87:755759

Parasitemia was indirectly calculated by assessing the

parasite numbers during the counting of 200 WBCs in the
thin blood film and, thereby, converting this to the number
of parasites per microliter of blood. Based on the parasite
number related with fever [7], samples from malaria
patients were subgrouped as a low parasitemia group
(<500 uL1) and a high parasitemia group (500 uL1).
Statistical analysis was performed using the Statistical
Package for the Social Sciences (SPSS) program (version
11.0 for Windows; SPSS Inc., Chicago, IL, USA). Pearson
2 test and Fishers exact test was used to compare
categorical variables. Students t-test was used to compare
continuous variables between groups. P values of <0.05
were considered significant.

Materials and methods

The study group comprised a total of 487 samples from
patients for whom complete blood cell analysis and malaria
smears had been requested. Malaria patient group contained
144 patients that were initially diagnosed with Plasmodium
vivax infection (114 males and 30 females) and 24 patients
who underwent malaria treatment (18 males and six
females) at Dongguk University International Hospital and
Eone Reference Laboratory between June 2006 and
October 2007. Two hundred forty-two patients who
presented to the emergency department with a febrile
illness without malaria at Dongguk University International
Hospital between July 2007 and January 2008 and 77
patients diagnosed as bacteremia at Dongguk University
International Hospital November 2007 and January 2008
were included in control group. The diagnosis of 242
febrile patients were as followed: acute gastroenteritis 30
patients; respiratory tract infection 92 patients; urinary tract
infection 29 patients; other bacterial infection 36 patients;
fever due to other causes 55 patients. The diagnosis of
malaria was made by examining Wright-stained thick and
thin blood smears.
For complete blood cell analysis, we used the Sysmex
XE-2100 hematology analyzer. This instrument differentiates WBCs using side fluorescence and side-scattered
light. An organic acid reagent binds specifically to the
granules of eosinophils and allows them to be discriminated
from neutrophils based on their higher side-scatter signal
intensities. WBC differential plots were examined for
abnormal WBC scattergrams. Manual WBC differential
counting was also performed by microscopic examination
of Wright-stained smears. Atypical features were categorized as pseudoeosinophilia (a gap of more than 5% in
eosinophil counts between the Sysmex XE-2100 analyzer
and microscopic examination) and abnormal WBC scattergram. The sensitivity and specificity of malaria detection
using pseudoeosinophilia and abnormal WBC scattergrams
were determined.

Results
Pseudoeosinophilia and abnormal WBC scattergrams
Of the 144 samples from malaria patients, 56 (38.9%)
showed pseudoeosinophilia. In samples with pseudoeosinophilia, the mean (standard deviation (SD)) neutrophil count
by microscopic examination and Sysmex XE-2100 analyzer
were 67.1% (17.9) and 56.4% (15.1), respectively. The
mean (SD) eosinophil counts determined by microscopic
examination and Sysmex XE-2100 analyzer were 0.6%
(1.0) and 15.9% (9.5), respectively.
When compared to the normal WBC differential scattergram (Fig. 1a), 75 samples from malaria patients showed
various abnormal features. The most strikingly noted
abnormal WBC scattergram was a nonclassified plot
extending from neutrophils toward the area of eosinophils
(n=33; Fig. 1b). In these cases, neutrophils and eosinophil
counts are displayed as bars on the Sysmex XE-2100
analyzer. Other features encountered in malaria-positive
samples were two atypical eosinophil populations (n=31;
Fig. 1c), overlapping neutrophil and eosinophil populations
(n=7; Fig. 1d), and two neutrophil populations (n=4;
Fig. 1e). Among the 31 samples with two atypical
eosinophil populations, four samples simultaneously
showed two neutrophil populations (Fig. 1c).
Sensitivity and specificity
Using the abnormal WBC scattergrams for the initial
diagnosis, the sensitivity and specificity were 52.1% and
100%, respectively (Table 1). Using pseudoeosinophilia for
the initial diagnosis, the sensitivity and specificity were
38.9% and 100%, respectively. Overall, pseudoeosinophilia
or an abnormal WBC scattergram was detected in 100 of
144 malaria-positive samples (sensitivity 69.4%, specificity
100%).

Ann Hematol (2008) 87:755759

Fig. 1 Abnormal WBC scattergrams generated by the Sysmex
XE-2100 hematology analyzer.
a Normal WBC differential
plots (blue neutrophil plots and
red eosinophil plots). b Nonclassified neutrophils and eosinophils with the plot extending
from neutrophils toward the area
of eosinophils. c Two atypical
eosinophil populations and two
neutrophil populations. d Overlapping neutrophil and eosinophil populations. e Two
neutrophil populations

757

DIFF

DIFF

According to Fishers exact test, there was a significant

difference in frequency of pseudoeosinophilia or abnormal
WBC scattergram between malaria-positive samples and
control samples (P<0.05).
Correlation of parasite count with pseudoeosinophilia
and abnormal WBC scattergram
The samples without pseudoeosinophilia or abnormal WBC
scattergrams showed significantly lower parasite counts
(517.9866.3 uL1 vs. 4,003.36,530.2 uL1) and higher
platelet count (80,977 42,914 uL 1 vs. 54,600
31,079 uL1) than the samples with pseudoeosinophilia or
an abnormal WBC scattergram (P<0.05; Table 2). Age,
sex, and WBC count were not different between the
samples with pseudoeosinophilia or an abnormal WBC

DIFF

DIFF

DIFF

scattergram and the samples without pseudoeosinophilia or

an abnormal WBC scattergram (P>0.05).
The sensitivity was 39.3% for the low parasitemia group
when pseudoeosinophilia or an abnormal WBC scattergram
was used for malaria detection, but this sensitivity increased
significantly, to 88.6%, for the high parasitemia group
(Table 3).
Follow-up results of malaria patients
During the treatment periods of 24 malaria patients, all cases
with an initial abnormal WBC scattergram and pseudoeosinophilia were changed to normal. Among 24 samples, all
nine samples from patients who underwent treatment for less
than 3 days and had atypical features at initial diagnosis had a
normalized eosinophil count and WBC histogram.

Table 1 Diagnostic efficiency

of the Sysmex-2100 analyzer
in malaria

Malaria
Negative
(n=319)
Pseudoeosinophilia
Positive
Negative
Abnormal WBC scattergram
Positive
Negative
Pseudoeosinophilia or abnormal WBC scattergram
Positive
Negative

Sensitivity
(%)

Specificity
(%)

38.9

100

52.1

100

69.4

100

Positive
(n=144)

0
319

56
88

0
319

75
69

0
319

100
44

758
Table 2 Comparison of
parasite counts between
samples with and without
atypical features on the
Sysmex XE-2100 analyzer

Ann Hematol (2008) 87:755759

Pseudoeosinophilia
Positive (n=56)
Negative (n=88)
Abnormal WBC scattergram
Positive (n=75)
Negative (n=69)
Pseudoeosinophilia or abnormal WBC scattergram
Positive (n=100)
Negative (n=44)

Discussion
Automated blood cell analyzers can detect cases of malaria
in the absence of clinical suspicion [1, 8]. Suspicious plots
should be checked by stained blood film. The Cell-Dyn
3500 was the first autoanalyzer that allowed the detection
of malaria during routine investigation by complete blood
cell analysis [5]. The Cell-Dyn 3500 appears to detect
hemozoin-containing leukocytes according to their depolarizing properties [3]. It is believed that hemozoin-laden
monocytes, which have ingested parasites, are the major
component of the defining abnormal population identified
using the Cell-Dyn method [8, 9]. Hemozoin-containing
neutrophils seemed to be misidentified as eosinophils [9].
Moreover, it was suggested that the Cell-Dyn 3500 could
detect intraerythrocytic pigmented malarial parasites in rare
circumstances, such as relatively high parasitemia or
osmotic resistance [3].
Pseudoeosinophilia was detected in samples from malaria patients using the Sysmex XE-2100 analyzer [6, 10].
The Sysmex XE-2100 analyzer cannot differentiate hemozoin-containing monocytes from normal monocytes. However, the instrument detects hemozoin-containing
neutrophils and classifies them incorrectly as eosinophils
due to considerable side light scattering, which causes a
diagnosis of pseudoeosinophilia [6]. In this study, the mean
difference in neutrophil counts between microscopic examination and the Sysmex XE-2100 analyzer was 11%, and
the mean difference in eosinophil counts was 15%. This
difference could be explained by the incorrect classification
of neutrophils as eosinophils. Eleven samples with a normal

Parasite count (Mean SD (uL1))

P value

2437.43452.2
376.71378.6

0.002

4659.57246.5
1067.52013.2

0.000

4003.36530.2
517.9866.3

0.000

eosinophil count and an abnormal WBC scattergram

showed two atypical eosinophil populations or two neutrophil populations. This suggests that the instrument also
shows hemozoin-containing neutrophils to be abnormally
scattered eosinophil or neutrophil populations without
pseudoeosinophilia. The number of hemozoin-containing
neutrophils has been identified as an adverse prognostic
indicator [11, 12]. This raises the possibility that the use of
the Sysmex XE-2100 analyzer for malaria diagnosis in our
study could also be useful prognostically.
The sensitivity of Cell-Dyn for the diagnosis of malaria
was variable, ranging from 4395% [4, 8, 13, 14], and the
specificity was reported to be as high as 96100%. This
study is the first evaluation of the sensitivity and specificity
of the Sysmex SE 2100 analyzer for malaria diagnosis. The
overall sensitivity and specificity were 69.4% (100/144)
and 100% (319/319), respectively, in our study. It has been
suggested that the kinetics of pigment-containing WBCs
may vary between different populations and could be
related to the severity of infection and host immunity
factors [4, 8, 13, 15]. In agreement with this, not all patients
in our study showed pseudoeosinophilia or abnormal WBC
scattergrams.
Although an abnormal WBC scattergram on the Sysmex
XE-2100 analyzer is specific for malaria diagnosis, it is
impossible to pay attention to all WBC histograms to detect
malaria in the laboratory. Among 100 samples with
pseudoeosinophilia or an abnormal WBC scattergram, 56
samples showed pseudoeosinophilia and 33 samples
showed blank eosinophil counts. Among the other 11
samples with a normal eosinophil count and an abnormal

Table 3 Comparison of pseudoeosinophilia and abnormal WBC scattergram between the low parasitemia group and the high parasitemia group

Pseudoeosinophilia (%)
Abnormal WBC scattergram (%)
Pseudoeosinophilia or abnormal WBC scattergram (%)

Low parasitemia group (n=56)

High parasitemia group (n=88)

17.8 (15/56)
17.8 (15/56)
39.3 (22/56)

34.1 (30/88)
68.2 (60/88)
88.6 (78/88)

P value
0.5
0.000
0.000

Ann Hematol (2008) 87:755759

WBC scattergram, ten samples showed thrombocytopenia.

High incidence of thrombocytopenia has been reported in
malaria patients [16]. Disadvantage of detecting malaria
parasite by microscopic examination of stained slides is that
expertise and continuous training are required. Therefore,
the advantage of the Sysmex XE-2100 rather than microscopic examination and Cell-Dyn is possible to suspect
malaria infection using high eosinophil count or uncounted
eosinophil before review of slide or WBC scattergram.
After suspecting malaria infection in febrile patients due to
eosinophilia or a blank eosinophil count, careful review of
WBC histograms could be helpful in the diagnosis of
malaria. Interestingly, using pseudoeosinophilia and abnormal WBC histograms, we detected several cases of malaria
during the recent 2 years without clinical suspicion.
This study demonstrated that samples with pseudoeosinophilia or abnormal WBC scattergrams showed significantly higher parasite counts than the samples without
pseudoeosinophilia or an abnormal WBC scattergram (P<
0.05). Furthermore, the high parasitemia group showed
higher sensitivity for malaria diagnosis than the low
parasitemia group. These results are different from previous
studies using Cell-Dyn, which showed no correlation
between the amount of parasites and the abnormal
depolarizing pattern [4, 8, 13]. This difference could be
explained by the short half-life (67 hours) of neutrophils
compared with that of monocytes (23 weeks) [17].
Therefore, it is possible that recent parasites counts could
be reflected by the Sysmex XE-2100 abnormal WBC
scattergram.
In previous reports related to follow-up evaluation with
Cell-Dyn, samples of treated malaria with no residual
parasitemia showed abnormal depolarization [14, 18]. In
this study, pseudoeosinophilia and abnormal WBC scattergrams became normal within 3 days after the initiation of
treatment. The normalized eosinophil count and WBC
scattergram within a few days could be also explained by
the kinetics of hemozoin clearance, as the removal of
hemoglobin-containing neutrophils is faster than that of
hemoglobin-containing monocytes. If Sysmex XE-2100
analyzer is used as a clinical follow-up parameter for
eradication of malaria after treatment, further evaluation to
be applied to a larger cohort of patients is needed.
In conclusion, the Sysmex XE-2100 analyzer has an
advantage in that it is able to detect unexpected malaria
cases. Considering its low sensitivity, hematological analysis performed with this instrument should not be considered an alternative to existing methods for the diagnosis of
malaria. However, attention to differential counts and WBC
scattergrams provided by Sysmex XE-2100 may be
valuable in the diagnosis of malaria.

759

References
1. Hanscheid T (1999) Diagnosis of malaria: a review of alternatives
to conventional microscopy. Clin Lab Haematol 21:235245
2. Zalis MG, Ferreira-da-Cruz MF, Balthazar-Guedes HC, Banic
DM, Alecrim W, Souza JM, Druilhe P, Daniel-Ribeiro CT (1996)
Malaria diagnosis: standardization of a polymerase chain reaction
for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia. Parasitol Res 82:612616
3. Hanscheid T, Pinto BG, Cristino JM, Grobusch MP (2000)
Malaria diagnosis with the haematology analyser Cell-Dyn
3500: what does the instrument detect. Clin Lab Haematol
22:259261
4. Hanscheid T, Melo-Cristino J, Pinto BG (2001) Automated
detection of malaria pigment in white blood cells for the diagnosis
of malaria in Portugal. Am J Trop Med Hyg 64:290292
5. Hanscheid T, Pinto BG, Pereira I, Cristino JM, Valadas E (1999)
Avoiding misdiagnosis of malaria: a novel automated method
allows specific diagnosis, even in the absence of clinical
suspicion. Emerg Infect Dis 5:836838
6. Huh J, Jung J, Yoon H, Chung W (2005) Pseudoeosinophilia
associated with malaria infection determined in the Sysmex XE2100 hematology analyzer. Ann Hematol 84:400402
7. Boisier P, Jambou R, Raharimalala L, Roux J (2002) Relationship
between parasite density and fever risk in a community exposed to
a low level of malaria transmission in Madagascar highlands. Am
J Trop Med Hyg 67:137140
8. Mendelow BV, Lyons C, Nhlangothi P, Tana M, Munster M,
Wypkema E, Liebowitz L, Marshall L, Scott S, Coetzer TL (1999)
Automated malaria detection by depolarization of laser light. Br J
Haematol 104:499503
9. Hanscheid T, Valadas E, Grobusch MP (2000) Automated malaria
diagnosis using pigment detection. Parasitol Today 16:549551
10. Park GB, Cha YJ (2006) Three cases of pseudoeosinophilia
associated with malaria determined in the Sysmex XE-2100
Automated Hematology Analyzer. Korean J Lab Med 26:7780
11. Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ (1995)
Intraleucocytic malaria pigment and prognosis in severe malaria.
Trans R Soc Trop Med Hyg 89:200204
12. Hanscheid T, Egan TJ, Grobusch MP (2007) Haemozoin: from
melatonin pigment to drug target, diagnostic tool, and immune
modulator. Lancet Infect Dis 7:675685
13. Grobusch MP, Hanscheid T, Kramer B, Neukammer J, May J,
Seybold J, Kun JF, Suttorp N (2003) Sensitivity of hemozoin
detection by automated flow cytometry in non- and semi-immune
malaria patients. Cytometry B Clin Cytom 55:4651
14. Suh IB, Kim HJ, Kim JY, Lee SW, An SS, Kim WJ, Lim CS
(2003) Evaluation of the Abbott Cell-Dyn 4000 hematology
analyzer for detection and therapeutic monitoring of Plasmodium
vivax in the Republic of Korea. Trop Med Int Health 8:1074
1081
15. Padial MM, Subirats M, Puente S, Lago M, Crespo S, Palacios G,
Baquero M (2005) Sensitivity of laser light depolarization analysis
for detection of malaria in blood samples. J Med Microbiol
54:449452
16. Chai JY (1999) Re-emerging Plasmodium vivax malaria in the
Republic of Korea. Korean J Parasitol 37:129143
17. Day NP, Pham TD, Phan TL, Dinh XS, Pham PL, Ly VC, Tran
TH, Nguyen TH, Bethell DB, Nguyan HP, White NJ (1996)
Clearance kinetics of parasites and pigment-containing leukocytes
in severe malaria. Blood 88:46944700
18. Metzger WG, Mordmuller BG, Kremsner PG (1995) Malaria
pigment in leucocytes. Trans R Soc Trop Med Hyg 89:637638