Hematology

ISSN: (Print) 1607-8454 (Online) Journal homepage: https://www.tandfonline.com/loi/yhem20

Abnormal WBC scattergram: a clue to the

diagnosis of malaria

Sunita Sharma, Neha Sethi, Mukta Pujani, Shivani Kushwaha & Shivali
Sehgal

To cite this article: Sunita Sharma, Neha Sethi, Mukta Pujani, Shivani Kushwaha & Shivali Sehgal
(2013) Abnormal WBC scattergram: a clue to the diagnosis of malaria, Hematology, 18:2, 101-105,
DOI: 10.1179/1607845412Y.0000000029

To link to this article: https://doi.org/10.1179/1607845412Y.0000000029

Published online: 18 Jul 2013.

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Abnormal WBC scattergram: a clue to the
diagnosis of malaria
Sunita Sharma 1, Neha Sethi 1, Mukta Pujani 1, Shivani Kushwaha2,
Shivali Sehgal2
1
Department of Pathology, Lady Hardinge Medical College, New Delhi, India, 2Department of Pathology and
Blood Bank, Lady Hardinge Medical College, New Delhi, India

Objective: Malaria is highly prevalent and endemic in tropical countries and carries a significant health
burden. The detection of malaria by light microscopy of Giemsa-stained smears is the gold standard.
There are many hematological abnormalities associated with malaria like anemia, thrombocytopenia, and
leucopenia; however, none of these abnormalities are specific. The present study was undertaken to
assess the utility of WBC scattergram in predicting the diagnosis of malaria.
Methods: In this study all cases diagnosed as Plasmodium vivax/Plasmodium falciparum infection on
peripheral smear examination were included. Their complete blood counts and WBC scattergrams
obtained from XT2000i were critically evaluated. Accordingly, sensitivity, specificity, positive predictive
value (PPV), and negative predictive value of detection of malaria by abnormal WBC scattergram with and
without abnormal blood counts were also calculated.
Results: A total of 2251 ethylendiaminetetraacetic acid samples were run on XT2000i hematology
autoanalyzer. Out of these 148 cases of malaria were diagnosed on peripheral smear (128 P. vivax and
20 P. falciparum). While analyzing the WBC scattergrams, 233 cases including 124 (83.8%) malaria cases
showed different abnormalities. Sensitivity and PPV for the diagnosis of malaria by abnormal WBC
scattergram were 83.78 and 53.20%, respectively. This had increased to 98.60 and 57.25%, respectively,
when cytopenias were included.
Discussion: Sysmex XT-2000i is capable of detecting specific abnormalities in WBC scattergram in patients
with malaria. Therefore, the presence of an abnormal WBC scattergram with thrombocytopenia in a febrile
patient helps the pathologist to clinch the diagnosis of malaria.
Keywords: Autoanalyzer, Scattergram, Sysmex, Malaria

Introduction leucopenia; however, none of these abnormalities are

Malaria is highly prevalent and endemic in tropical
countries and carries a significant health burden.
The detection of malaria by light microscopy of
Giemsa-stained smears is the gold standard.
However, it has its own limitations of being time con-
suming and requires well-trained staff. This has led to
the development of several new techniques for detec-
tion of malaria like quantitative buffy coat examin-
ation using fluorescent dyes,1 antigen-coated dipstick
tests,2 and polymerase chain reaction.3 Although
these tests are fairly sensitive in detecting the malaria
parasite, they are expensive and not routinely
available.
There are many hematological abnormalities associ-
ated with malaria like anemia, thrombocytopenia, and
specific. As complete blood count (CBC) is the base-
line investigation to be ordered in patients with fever,
any malaria detecting method added to it, could
help in early detection of malaria and reduce its
complications.4
Many studies have been conducted all over the
world using the automated blood cell counters like
cell dyne hematology analyzer,5 GEN S and
LH750,6 XE2100 and XS100i7 in the evaluation of
malarial detection. In the present study, the abnormal-
ities of WBC scattergram by XT2000i were evaluated,
which can give a clue to diagnosis of malaria and
thereby alerting the pathologist to review the blood
smears more carefully.

Materials and methods

Correspondence to: Neha Sethi, Department of Pathology, Lady Hardinge In this study, all cases diagnosed as Plasmodium
Medical College, Bhagat Singh Marg, New Delhi 110001, India. Email:
nehasethi3@gmail.com vivax/Plasmodium falciparum infection on peripheral

© W. S. Maney & Son Ltd 2013

DOI 10.1179/1607845412Y.0000000029 Hematology 2013 VOL. 18 NO. 2 101
Sharma et al. Clue to the diagnosis of malaria

smear examination from June 2011 to September The most common abnormalities observed were
2011 were included. This is a descriptive study in graying of both eosinophil and neutrophil groups
which a retrospective analysis of all positive cases (Fig. 1) (41.10%) followed by two eosinophil popu-
was done. Their complete blood counts and WBC lations (Fig. 2) (16.20%), overlapping of neutrophil
scattergrams obtained from XT2000i were critically and eosinophil groups (Fig. 3) (16.20%), two neutro-
evaluated for any abnormal findings. The parasitemia phil populations (Fig. 4) (14.70%), graying of both
was indirectly calculated on peripheral smear examin- lymphocyte and monocyte groups (Fig. 5) (8.50%),
ation by counting the number of parasites in 25 fields. graying of all leucocyte groups (2.30%), and two lym-
The total parasite count per microliter of blood was phocyte populations (Fig. 6) (0.7%). It was also
then calculated by: observed that as compared with only 4.90% abnormal-
ities in P. vivax, 22.20% abnormalities of P. falciparum
Number of parasites observed were showing graying of lymphocyte and monocyte
× total RBC count
Total RBC in 25 fields groups. In the present study rightward shift of RBC
= parasites/μl of blood ghost area in differential count (DIFF) (Fig. 7) and
WBC/basophil (BASO) scattergrams was also found
The degree of parasitemia was then correlated with in malaria cases (32.40 and 30.40%, respectively). In
any abnormal findings on differential WBC counts six cases (4%), spuriously high eosinophil counts
or abnormal WBC scattergram patterns. Also during were found in WBC scattergrams as compared with
the same period all samples run on XT2000i were ana- manual WBC differential count.
lyzed. The WBC scattergrams of non-malarial cases About 5.10% non-malaria cases like leukemias, tha-
were also evaluated. lassemias, septicemia, hemolytic diseases of newborn,
Accordingly, sensitivity, specificity, positive predic- and others also showed various abnormalities in
tive value (PPV), and negative predictive value WBC scattergrams as described above. Leukemia
(NPV) of detection of malaria by abnormal WBC scat- cases (31.20%) showed graying of all leucocyte
tergram with and without abnormal blood counts were groups in WBC scattergram. Diseases like thalasse-
also calculated. mias, septicemia and hemolytic diseases of newborn,
having leucoerythroblastic blood picture (44%)
showed graying of different leucocytes group, two
Results
neutrophil populations, and overlapping of neutrophil
A total of 2251 ethylendiaminetetraacetic acid samples
and eosinophil groups. Some cases (24.80%) of
were run on XT2000i hematology autoanalyzer from
June to September 2011. Out of these, 148 cases of
malaria were diagnosed on peripheral smear (128
Table 2 Different abnormalities in WBC scattergram in
P. vivax and 20 P. falciparum). Eighty-two were males malaria cases
and 66 were females, the age range was between 5 and
Plasmodium Plasmodium
60 years with maximum cases falling between 14 and vivax number falciparum Total number
25 years of age. Abnormality of number of of
Bicytopenia was observed in 44.6% of cases fol- in WBC abnormalities abnormalities abnormalities
scattergram (%) (%) (%)
lowed by pancytopenia (21.60%), isolated thrombocy-
topenia (21.60%), and isolated anemia (9.40%; Graying of 48(47.06) 5(18.52) 53(41.08)
eosinophil,
Table 1). Neutrophil
While analyzing the WBC scattergrams, 233 cases groups
including 124 (83.80%) malaria cases showed different 2 eosinophil 13(12.7) 8(29.63) 21(16.28)
population
abnormalities (Table 2). Some cases showed more than Overlapping of 16(5.69) 5(18.52) 21(16.28)
one abnormality. eosinophil
and
neutrophil
Table 1 Hemogram findings in malaria cases groups
2 neutrophil 16(15.69) 3(11.11) 19(14.73)
Hemogram abnormality Number of malaria cases (%) population
Graying of 5(4.90) 6(22.22) 11(8.53)
Anemia (exclusively) 14(9.46) lymphocyte,
Leucopenia (exclusively) – monocyte
Thrombocytopenia 32(21.62) groups
(exclusively) Graying of 3(2.94) – 3(2.32)
Bicytopenia 66(44.59) whole area
Pancytopenia 32(21.62) 2 lymphocyte 1(0.98) – 1(0.78)
Leucocytosis 3(2.03) population
No abnormality 1(0.68) Total 102 27 129
Total 148.00 abnormalities

102 Hematology 2013 VOL. 18 NO. 2

 Sharma et al. Clue to the diagnosis of malaria

Figure 1 Graying of eosinophil and neutrophil groups.

Figure 3 Overlapping of eosinophil, neutrophil groups.

eosinophilia and smear with platelet aggregates also

cells and platelets and a polymethine dye to bind to
showed similar WBC scattergram abnormalities.
nucleic acid of WBC to give fluorescence signal inten-
The peripheral blood films of all malaria cases were
sity proportional to nucleic acid content. Also it uses
examined and total number of parasites was counted.
an organic acid which specifically bind to eosinophil
However, no correlation was observed between degree
granules to be differentiated from neutrophils by
of parasitemia and different patterns of WBC
high side scatter intensities. WBC/BASO channel
scattergram.
uses forward and side scatter signals by using an
Sensitivity, specificity, PPV, and NPV were calcu-
acidic reagent which causes shrinkage of all cells
lated on the basis of abnormal WBC scattergram find-
except for basophils and WBC (bare nucleii). Thus
ings with or without cytopenias in malarial positive
the WBC and the basophil counts are derived from
cases (Table 3). The sensitivity and PPV for the diag-
this channel.8
nosis of malaria by abnormal WBC scattergram were
The first automated hematology analyzer to detect
83.78 and 53.20%, respectively. This had increased to
malaria was Cell Dyn (CD) 3500 (Abott diagnostics,
98.60 and 57.25%, respectively, when cytopenias
Santa, CA, USA) which detected hemozoin contain-
were included.
ing leucocytes especially monocytes abnormal
depolarizing patterns during routine CBC analyses.
Discussion This method was also beneficial in cases where there
The Sysmex XT2000i hematology autoanalyzer was no clinical suspicion of malaria.5
measures different leucocytes by flow cytometry Two case series from South Korea have shown the
using a semiconductor laser beam and separates the usefulness of pseudoeosinophilia and abnormalities
cell by using the three signals, forward scatter, side in the DIFF scattergram in detecting malaria on
scatter, and side fluorescence. The DIFF channel SysmexXE-2100. Huh et al. 9 (2008) in their study of
(differential count) uses surfactant to lyse red blood

Figure 2 Two eosinophil populations. Figure 4 Two neutrophil populations.

Hematology 2013 VOL. 18 NO. 2 103

Sharma et al. Clue to the diagnosis of malaria
Figure 5 Graying of lymphocyte and monocyte groups.
144 malaria cases found 38.9% cases showing pseu-
doeosinophilia and 52.10% cases showing abnormal-
ities in WBC scattergram like non-classified pot
extending from neutrophils towards eosinophil area
(22.90%), two eosinophil populations (21.50%), two
neutrophil populations (27.80%) and overlapping of
neutrophil and eosinophil populations (48.60%). This
is caused when hemozoin containing particles interfere
with the machine’s WBC detection system. These
abnormalities are due to the abnormal counting of
hemozoin containing neutrophils as well as due to
their detection as eosinophils near the neutrophil
cluster. Yoo et al. in 2010 also studied the importance
of pseudoeosinophilia and abnormal WBC scatter-
gram in assessment of 413 malaria cases. They found
39% cases with pseudoeosinophilia and 15.70% cases
with abnormal WBC scattergram like two neutrophil
and two eosinophil populations.10
In a Colombian study by Campuzano Zuluaga
et al. 11 in 2010, the quantitative data, scatterplots
and histograms were used to construct predictive
models for each malaria species, by using binary logis-
tic regression.
Figure 6 Two lymphocyte populations.
104 Hematology 2013 VOL. 18 NO. 2
Figure 7 Rightward shift of RBC ghost area in DIFF
scattergram.
The present study showed 83.8% malaria cases
having abnormal WBC scattergram. Most common
abnormality was found to be graying of eosinophil
and neutrophil populations (41.10%). Other common
abnormalities were overlapping of eosinophil and neu-
trophil populations (16.20%) and two eosinophil
populations (16.20%).
In the present study, rightward shift of RBC ghost in
WBC/BASO and DIFF scattergrams were also very
commonly found in malaria cases. This can be attrib-
uted to the presence of extracellular pigment and RBC
lysis which are reflected in that area.7
Although previous studies showed pseudoeosino-
philia in approximately 39% of cases,8,9 the present
study had only 4% cases showing spuriously high eosi-
nophil count on scattergram as compared with smears,
but it has shown little extra significance as time
devoted for calculating it by microscopy will enable
the observer to look for the parasite.12
About six cases of P. falciparum showed graying of
lymphocyte and monocyte groups, which might be due
to the interference in their detection by ring forms of
the parasite.7
Huh et al. 8 (2008) showed in their study that XE-
2100 had sensitivity of 69.40% and specificity of
100% while using pseudoeosinophilia and abnormal
WBC scattergram in detection of malaria.
Table 3 Sensitivity, specificity, PPV, and NPV of abnormal
scattergrams with or without cytopenias
Abnormal (Abnormal
scattergram scattergram + cytopenias)
(%) (%)
Sensitivity 83.78 98.60
Specificity 94.82 94.80
PPV 53.20 57.25
NPV 98.80 94.80

Yoo et al. (2010) reported sensitivity of 46.20% and
specificity of 99.70% by using pseudoeosinophilia and
abnormal WBC scattergram by Sysmex-2100. These
parameters were low than above study because pro-
duction of hemozoin depends on the number of para-
sites, severity of infection and host immunity factors.9
The present study showed high sensitivity (83.78%)
and specificity (94.82%) as compared with above
studies while using abnormal WBC scattergram in
detection of malaria. This sensitivity increased to
98.6% when abnormal scattergram findings were com-
bined with cytopenias in the detection of malaria.
The present study showed that most cases had cyto-
penias in any of the blood cell lineage, common being
anemias and thrombocytopenias. Several studies have
been done which showed high incidence of thrombocy-
topenias and anemias in malaria cases.13,14 If the abnor-
mal WBC scattergram findings are included with these
hematological findings, the detection rate of malaria
will definitely increase, as is shown in the present study.
Several new techniques have come up for the
detection of malaria but requires special requisition by
the treating clinician in patients suspected of malaria
infection; however, the abnormal WBC scattergram
findings may alert the pathologist to look for malaria
in subclinical cases also.15 As, CBC is the most
frequently requested investigation in patients with
fever, if the laboratory staff is aware of these WBC
scattergram abnormalities, they will be more careful
to look for the presence of parasite in blood smears.
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