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ORIGINAL ARTICLE
AUTOMATED DETECTION OF MALARIA WITH HAEMATOLOGY
ANALYZER SYSMEX XE-2100
SARITA MOHAPATRA, JYOTISH C.SAMANTARAY, S.ARULSELVI1, JITENDER PANDA,
KHUSHBOO MUNOT, RENU SAXENA2

ABSTRACT
BACKGROUND: Diagnosis of malaria is usually made by microscopy [Giemsa, Acridine
Orange (AO), and Quantitative Buffy Coat (QBC) assay], which requires expertise.
Currently, automated haematology analyzers are being used for complete blood count
(CBC), in all acute febrile and non-febrile illnesses which simultaneously detects malaria.
The normal scattergram by the analyzer (Sysmex 2100) comprises of five parameters
i.e.lymphocytes (pink), monocytes (green), neutrophils (blue), eosinophils (red) with
a space between the neutrophil and eosinophil populations. AIMS: We carried out a
prospective study to compare the efficacy of Sysmex XE-2100 (Sysmex Corporation, Kobe)
for detection of malaria in comparison to other conventional techniques. MATERIALS AND
METHODS: 430cases were analyzed for malaria by microscopy (QBC, AO, Giemsa), ICT
(Immunochromatography) and flowcytometric analyzer (Sysmex XE-2100). The abnormal
scattergrams were observed as double neutrophil, double eosinophil, grey zone, extended
neutrophil zone with a decrease space between eosinophil and neutrophil, and a
combination of above patterns. RESULTS: Out of 70 positive cases[49/70(70%) P.vivax,
18/70(25.7%) P.falciparum, and 3/70(4.2%) both P.vivax and P.falciparum], 52 showed
abnormal scattergrams by the analyzer. The sensitivity and specificity of hematology
analyzer found to be 74.2% and 88%, respectively. CONCLUSION: Flowcytometric
analyzer is a rapid, high throughput device which needs less expertization for the
diagnosis of malaria. Hence, it can be used in the diagnostic laboratories as an early
modality for diagnosis of malaria in suspected as well as clinically in apparent cases.
Key words: Automated hematology analyzer, malaria detection, Sysmex XE-2100

INTRODUCTION
Malaria is a common public health problem
observed worldwide. The classical presentation
Department of Microbiology, 1Department of Laboratory
Medicine, JPNA Trauma Centre, 2 Department of
Haematology, All India Institute of Medical Sciences,
NewDelhi, India
Address for correspondence:
Dr.Sarita Mohapatra,
E-36, Ansari Nagar (West), AIIMS Residential Campus,
NewDelhi- 110029, India.
E-mail:saritarath2005@yahoo.co.in

Indian Journal of Medical Sciences, Vol. 65, No. 1, January 2011

includes fever with chill and rigor. Diagnosis is

based using a combination of clinical history,
travel history, and laboratory tests. Microscopy
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DOI:
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AUTOMATED DETECTION OF MALARIA BY SYSMEX XE-2100

(using Giemsa stain, Acridine Orange (AO)

stain, Quantitative buffy coat (QBC) assay),
immunochromatographic test (ICT), and PCR
are performed only after the clinical diagnosis.
Among all, Giemsa stain is the most prevalent
method worldwide, and taken as the gold
standard for malaria diagnosis. Now-a-days,
hematology analyzers [Cell Dyn, (Abbott
Diagnostics, Santa Clara, CA), GEN.S, LH-750
(Beckman Coulter, Miami, FL), Sysmex XE2100, Sysmex XE-1800 (Sysmex corporation,
Kobe)] are also used for automated detection
of malaria in many febrile patients during
complete blood count (CBC) analysis.[1,2] Along
with hematological parameters these analyzers
can detect malaria parasites containing
hemozoin pigment. Sysmex analyzer works on
the principle of flowcytometry and measures
the different blood corpuscular elements.[3] It
uses a semiconductor laser to give three types
of optical information about the cells. Forward
scatter light (FSL) measures cell size, side
scatter (SSC) determines the granularity of the
internal structure, and side fluorescence light
(SFL) provides information about the nuclear
content.
In Sysmex XE-2100 analyzer (Sysmex
corporation, Kobe, Japan), the normal
scattergram in the DIFF plot is constituted
of five parameters; lymphocytes (pink),
monocytes (green), neutrophils+basophil
(blue), eosinophils (red) with a space between
the neutrophil and eosinophil populations
[Figure1a].[3] Hemozoin pigments produced
by malaria parasite have also the tendency to
depolarize the laser beam. So, the parasitized
RBC with various morphologic forms
(trophozoites, schizoints, gametocyte) and the
phagocytic cells (monocyte, macrophage, and

neutrophil containing the parasite) mimic the

above-described patterns and exhibits various
abnormal scattergram during routine CBC
analysis.[4] The abnormal patterns are observed
as double neutrophil [Figure1b], extended
neutrophil with a decrease space between
neutrophil and eosinophil [Figure1c], grey
zone [Figure1d] double eosinophil [Figure1e],
and a combination among the above.[1-3] In the
WBC/BASO plot, the basophils are selectively
separated from the rest WBCs without showing
any abnormal events above the x-axis. The
presence of events in this area is suggestive
of malarial infection [Figure1f].[3] The primary
aim of this study was to compare the efficacy
of hematology analyzer (Sysmex XE-2100)
for the detection of malaria with respect to the
conventional methods. The secondary aim
was to determine the different parasitic forms
that contribute to the different population in the
abnormal scattergrams.

MATERIALS AND METHODS

This prospective study was conducted in
the department of microbiology from August
to December, 2010. Patients with a clinical
suspicion of malaria were enrolled in the
study group. Two milliliters of blood was
collected in an EDTA vial and tested for malaria
parasite by the conventional methods [Giemsa,
AO, QBC and ICT (LDH based OptiMal)]
and the hematology analyzer (Sysmex XE2100). Apositive case for malaria in our
study was defined as positive by either of
the above described conventional methods
(i.e.microscopy and/or ICT). Blood samples
tested negative by the conventional methods
were taken as controls. The samples were
analyzed for abnormal scattergrams in the
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INDIAN JOURNAL OF MEDICAL SCIENCES
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Figure1: Scattergrams in Sysmex XE-2100; (a) normal DIFF plot, (b) double neutrophil, (c) extended neutrophil with
decreased space between neutrophil and eosinophil, (d) grey zone, (e) double eosinophil, (f) abnormal events above
the x-axis in the WBC/Baso plo

DIFF and WBC/BASO plot of SysmexXE-2100.

Follow-up samples were taken after 24h,
48h, and 72h of initiation of antimalarial
treatment and reviewed by all the methods.
The microscopic finding was matched with
the abnormal scattergrams produced by the
analyzer. Statistical analysis of all the methods
were calculated by 2/2 table analysis.

RESULTS
Out of 430 clinically suspected malaria cases,
70 were found to be positive by conventional
methods. P.vivax was the predominant species
followed by P.falciparum[49/70(70%) for
P.vivax, 18/70(25.7%) for P.falciparum,
and 3/70(4.2%) for both P.vivax and
P.falciparum]. Amaximum number of malaria
positive cases were detected by QBC followed
by other methods like ICT, AO, and Giemsa
[QBC (68/70), ICT (66/70), AO (51/70), Giemsa
(49/70)] [Table1]. Sensitivity of QBC was found
Indian Journal of Medical Sciences, Vol. 65, No. 1, January 2011

to be highest (97.1%) followed by ICT (94.2%).

Fifty-two out of 70malaria positive cases
showed abnormal scattergram in flowcytometric
analyzer. The positive samples showed various
abnormal patterns in DIFF scattergram such as
double neutrophil, double eosinophil, extended
neutrophil with decreased space between
eosinophil and neutrophil, grey zones, and
the combination among the above. Extended
neutrophil with decrease space between
neutrophil and eosinophil was observed to be
the commonest pattern (n=22) followed by
the grey zone (n=7) [Table1]. An abnormal
WBC/BASO plot [Figure1f] was observed in
37malaria positive cases. Thus, considering
atypical scattergram in the DIFF plot and
the WBC/BASO plot, overall sensitivity and
specificity of Sysmex XE-2100 was found to be
74.2% and 88%, respectively [Table1]. Positive
predictive value and negative predictive value
were found to be 59.7% and 95.2% (95% CI),
respectively [Table1].

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AUTOMATED DETECTION OF MALARIA BY SYSMEX XE-2100

DISCUSSION

cells, malaria parasites were picked up by the

flowcytometric analyzer and depending on their
size, nuclear and pigment content appeared in
the area of neutrophil and eosinophil. Neutrophils
possess more nucleic acid and lesser internal
granularity in comparison to eosinophils. Hence,
in the normal DIFF plot (side fluorescence
Vs side scatter, Figure1), they are placed
to the left to that of eosinophil population.
By careful observation and follow-up study
of various malaria samples, we observed
that gametocyte, mature schizonts, and late
trophozoites were contributed to the atypical
neutrophil patterns (i.e.extended neutrophil,
decreased space between neutrophil and
eosinophil and double neutrophil). Our finding
also suggested that blood samples with a
predominant form of gametocyte appear as the
extended neutrophil zone with decrease space
between neutrophil and eosinophil [Table 2].
It may appear as double neutrophil depending
on the stage of gametocyte (mature/immature),
schizoint, and late trophozoite present in the
blood sample. Campuzano-Zuluaga etal. found
a significant correlation between the number of

Sysmex hematology analyzer differentiates white

blood cell in blood by means of forward scatter,
side scatter, and side fluorescence scatter laser
beam. However, hemozoin pigments (because
of its birefringent nature) are also capable
of scattering the laser light. In heavy malaria
infection, these pigments were engulfed by
mononuclear cell (monocytes, macrophages)
and polymorphonuclear cells (neutrophils),
showing various atypical scattergrams.[3,4] There
are several evidences regarding the use of
these analyzers for detection of malaria.[5] To
the best of our knowledge, this is the first study
correlating abnormal scattergrams produced by
flowcytometric analyzer with various forms of
malaria parasite found during the microscopic
examination. Complete reversion of abnormal
to normal scattergram was observed after 48
to 72h of initiation of antimalarial treatment.
The disappearance of parasitic forms in the
follow-up blood samples were corresponded
with the resolution of atypical pattern from the
scattergram. We feel, apart from the phagocytic

Table1: Detection of malaria parasite by conventional methods and flowcytometric analyzer

Methods

Positive (n)

Negative (n)

Sensitivity (%)

Specificity (%)

PPV (%) (95%CI)

NPV (%) (95%CI)

Giemsa

49

21

70

100

100

94.8

AO

51

19

72.8

100

100

94.9

QBC

68

97.1

100

100

99.4

ICT

66

94.2

100

100

98.9

Sysmex XE-2100

52

18

74.2

91.1

59.7

95.2

n = Total number

Table2: Parasitic forms showing abnormal patterns in the DIFF plot in flow- cytometric analyzer
Parasitic forms (n)

Decreased space1

2EN

Grey

3DN

DE

Combination patterns

Normal scattergram

Gametocyte (47)

12

10

11

Schizonts (1)

E.trophozoites (9)

L.trophozoites (3)

Pigment containing cells (6)

EN = Extended neutrophil zone, DN=Double neutrophil, DE=Double eosinophil, 1Decreased space between neutrophil and
eosinophil

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INDIAN JOURNAL OF MEDICAL SCIENCES
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late trophozoites, schizonts, and gametocyte of

P.vivax and abnormalities formed in the DIFF,
WBC/BASO, and RET-EXT scattergrams.[6] In
malaria infection, monocytes and macrophages
remain the first line of defense followed by
neutrophils. Moreover, neutrophil-containing
pigment is observed under heavy parasitaemic
condition and considered as a poor prognostic
indicator.[5] Although, the kinetics of hemozoin
pigment containing WBC varies from person to
person depending on the immune status of host
and severity of infection.[7-9] Many authors have
suggested that pigment-containing neutrophils
are abnormally represented the as double
eosinophil population by the analyzer.[10] In
our study, double eosinophil population in the
DIFF plot was observed in blood samples with a
predominant stage of late trophozoite. Apart from
the DIFF plot, the WBC/Baso plot is an important
area where one should look for malaria. It is
more accurate and easy to identify.[6] It was the
first area to be normalized in follow-up samples
in our study.

has not been evaluated for detection of malaria

in endemic areas. Detection of malaria by
abnormal scattergram in the flowcytometric
analyzer is not only economical but also,
can be easily screened and documented
by nontechnical personnel. Therefore,
hematologist should be aware of this aspect
of abnormal scattergram so that an early and
prompt diagnosis of malaria can be made.

CONCLUSION

4. Nguyen PH, Day N, Pram TD, Ferguson DJ, White

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How to cite this article: Mohapatra S, Samantaray JC, Arulselvi
S, Panda J, Munot K, Saxena R. Automated detection of malaria
with haematology analyzer sysmex xe-2100. Indian J Med Sci
2011;65:26-31.
Source of Support: Nil. Conflict of Interest: None declared.

Indian Journal of Medical Sciences, Vol. 65, No. 1, January 2011