Original research paper

Abnormal WBC scattergram: A clue to thediagnosis of malariaSunita Sharma1, Neha Sethi1, Mukta Pujani1, Shivani Kushwaha2,Shivali Sehgal2

1Department of Pathology, Lady Hardinge Medical College, New Delhi, India, 2Department of Pathology andBlood Bank, Lady Hardinge Medical College, New Delhi, India

Objective: Malaria is highly prevalent and endemic in tropical countries and carries a significant healthburden. 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, andleucopenia; however, none of these abnormalities are specific. The present study was undertaken toassess the utility of WBC scattergram in predicting the diagnosis of malaria.Methods: In this study all cases diagnosed as Plasmodium vivax/Plasmodium falciparum infection onperipheral smear examination were included. Their complete blood counts and WBC scattergramsobtained from XT2000i were critically evaluated. Accordingly, sensitivity, specificity, positive predictivevalue (PPV), and negative predictive value of detection of malaria by abnormal WBC scattergram with andwithout abnormal blood counts were also calculated.Results: A total of 2251 ethylendiaminetetraacetic acid samples were run on XT2000i hematologyautoanalyzer. Out of these 148 cases of malaria were diagnosed on peripheral smear (128 P. vivax and20 P. falciparum). While analyzing the WBC scattergrams, 233 cases including 124 (83.8%) malaria casesshowed different abnormalities. Sensitivity and PPV for the diagnosis of malaria by abnormal WBCscattergram 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 patientswith malaria. Therefore, the presence of an abnormal WBC scattergram with thrombocytopenia in a febrilepatient helps the pathologist to clinch the diagnosis of malaria.

Keywords: Autoanalyzer, Scattergram, Sysmex, Malaria

IntroductionMalaria is highly prevalent and endemic in tropicalcountries and carries a significant health burden.The detection of malaria by light microscopy ofGiemsa-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 tothe development of several new techniques for detec-tion of malaria like quantitative buffy coat examin-ation using fluorescent dyes,1 antigen-coated dipsticktests,2 and polymerase chain reaction.3 Althoughthese tests are fairly sensitive in detecting the malariaparasite, they are expensive and not routinelyavailable.There are many hematological abnormalities associ-

ated with malaria like anemia, thrombocytopenia, and

leucopenia; however, none of these abnormalities arespecific. As CBC 1is the baseline investigation to beordered in patients with fever, any malaria detectingmethod added to it, could help in early detection ofmalaria and reduce its complications.4

Many studies have been conducted all over theworld using the automated blood cell counters likecell dyne hematology analyzer,5 GEN S andLH750,6 XE2100 and XS100i7 in the evaluation ofmalarial detection. In the present study, the abnormal-ities of WBC scattergram by XT2000i were evaluated,which can give a clue to diagnosis of malaria andthereby alerting the pathologist to review the bloodsmears more carefully.

Materials and methodsIn this study, all cases diagnosed as Plasmodiumvivax/Plasmodium falciparum infection on peripheral

Correspondence to: Neha Sethi, Department of Pathology, Lady HardingeMedical College, New Delhi, India. Email: nehasethi3@gmail.com2

© W.S. Maney & Son Ltd 2012DOI 10.1179/1607845412Y.0000000029 Hematology 2012 VOL. 0 NO. 0 1

smear examination from June 2011 to September2011 were included. This is a descriptive study inwhich a retrospective analysis of all positive caseswas done. Their complete blood counts and WBCscattergrams obtained from XT2000i were criticallyevaluated for any abnormal findings. The parasitemiawas calculated on peripheral smear examination byindirectly counting the number of parasites in 25fields. The total parasite count per microliter ofblood was then calculated by:

Number of parasites observedTotal RBC in 25 fields

× total RBC count

= parasites/μl of blood

The degree of parasitemia was then correlated withany abnormal findings on differential WBC countsor abnormal WBC scattergram patterns. Also duringthe same period all samples run on XT2000i were ana-lyzed. The WBC scattergrams of non-malarial caseswere also evaluated.Accordingly, sensitivity, specificity, positive predic-

tive value (PPV), and negative predictive value(NPV) of detection of malaria by abnormalWBC scat-tergram with and without abnormal blood counts werealso calculated.

ResultsA total of 2251 ethylendiaminetetraacetic acid sampleswere run on XT2000i hematology autoanalyzer fromJune to September 2011. Out of these, 148 cases ofmalaria were diagnosed on peripheral smear (128P. vivax and 20 P. falciparum). Eighty-two were malesand 66 were females, the age range was between 5 and60 years with maximum cases falling between 14 and25 years of age.Bicytopenia was observed in 44.6% of cases fol-

lowed by pancytopenia (21.60%), isolated thrombocy-topenia (21.60%), and isolated anemia (9.40%;Table 1).While analyzing the WBC scattergrams, 233 cases

including 124 (83.80%) malaria cases showed differentabnormalities (Table 2). Some cases showed more thanone abnormality.

The most common abnormalities observed weregraying of both eosinophil and neutrophil groups(Fig. 1) (41.10%) followed by two eosinophil popu-lations (Fig. 2) (16.20%), overlapping of neutrophiland eosinophil groups (Fig. 3) (16.20%), two neutro-phil populations (Fig. 4) (14.70%), graying of bothlymphocyte and monocyte groups (Fig. 5) (8.50%),graying of all leucocyte groups (2.30%), and two lym-phocyte populations (Fig. 6) (0.7%). It was alsoobserved that as compared with only 4.90% abnormal-ities in P. vivax, 22.20% abnormalities of P. falciparumwere showing graying of lymphocyte and monocytegroups. In the present study rightward shift of RBCghost area in DIFF 3(Fig. 7) and WBC/BASO scatter-grams was also found in malaria cases (32.40 and30.40%, respectively). In six cases (4%), spuriouslyhigh eosinophil counts were found in WBC scatter-grams as compared with manual WBC differentialcount.

About 5.10% non-malaria cases like leukemias, tha-lassemias, septicemia, hemolytic diseases of newborn,and others also showed various abnormalities inWBC scattergrams as described above. Leukemiacases (31.20%) showed graying of all leucocytegroups in WBC scattergram. Diseases like thalasse-mias, septicemia and hemolytic diseases of newborn,having leucoerythroblastic blood picture (44%)showed graying of different leucocytes group, twoneutrophil populations, and overlapping of neutrophiland eosinophil groups. Some cases (24.80%) of

Table 1 Hemogram findings in malaria cases

Hemogram abnormality Number of malaria cases (%)

Anemia (exclusively) 14(9.40)Leucopenia (exclusively) –

Thrombocytopenia(exclusively)

32(21.60)

Bicytopenia 66(44.60)Pancytopenia 32(21.60)Leucocytosis 3(2.03)No abnormality 1(0.06)Total 148.00

Table 2 Different abnormalities in WBC scattergram inmalaria cases

Abnormalityin WBCscattergram

Plasmodiumvivax number

ofabnormalities

(%)

Plasmodiumfalciparumnumber of

abnormalities(%)

Total numberof

abnormalities(%)

Graying ofeosinophil,Neutrophilgroups

48(47.05) 5(18.50) 53(41.10)

2 eosinophilpopulation

13(12.70) 8(29.60) 21(16.20)

Overlapping ofeosinophilandneutrophilgroups

16(5.70) 5(18.50) 21(16.20)

2 neutrophilpopulation

16(15.70) 3(11.10) 19(14.70)

Graying oflymphocyte,monocytegroups

5(4.90) 6(22.20) 11(8.50)

Graying ofwhole area

3(2.90) – 3(2.30)

2 lymphocytepopulation

1(0.90) – 1(0.70)

Totalabnormalities

102 27 129

Sharma et al. Clue to the diagnosis of malaria

Hematology 2012 VOL. 0 NO. 02

eosinophilia and smear with platelet aggregates alsoshowed similar WBC scattergram abnormalities.The peripheral blood films of all malaria cases were

examined and total number of parasites was counted.However, no correlation was observed between degreeof parasitemia and different patterns of WBCscattergram.Sensitivity, specificity, PPV, and NPV were calcu-

lated on the basis of abnormal WBC scattergram find-ings with or without cytopenias in malarial positivecases (Table 3). The sensitivity and PPV for the diag-nosis of malaria by abnormal WBC scattergram were83.78 and 53.20%, respectively. This had increased to98.60 and 57.25%, respectively, when cytopeniaswere included.

DiscussionThe Sysmex XT2000i hematology autoanalyzermeasures different leucocytes by flow cytometryusing a semiconductor laser beam and separates thecell by using the three signals, forward scatter, sidescatter, and side fluorescence. The DIFF channel(differential count) uses surfactant to lyse red blood

cells and platelets and a polymethine dye to bind tonucleic acid of WBC to give fluorescence signal inten-sity proportional to nucleic acid content. Also it usesan organic acid which specifically bind to eosinophilgranules to be differentiated from neutrophils byhigh side scatter intensities. WBC/BASO channeluses forward and side scatter signals by using anacidic reagent which causes shrinkage of all cellsexcept for basophils and WBC (bare nucleii). Thusthe WBC and the basophil counts are derived fromthis channel.8

The first automated hematology analyzer to detectmalaria was Cell Dyn (CD) 3500 (Abott diagnostics,Santa, CA, USA) which detected hemozoin contain-ing leucocytes especially monocytes abnormaldepolarizing patterns during routine CBC analyses.This method was also beneficial in cases where therewas no clinical suspicion of malaria.5

Two case series from South Korea have shown theusefulness of pseudoeosinophilia and abnormalitiesin the DIFF scattergram in detecting malaria onSysmexXE-2100. Huh et al.9 (2008) in their study of

Figure 1 Graying of eosinophil and neutrophil groups.

Figure 2 Two eosinophil populations.

Figure 3 Overlapping of eosinophil, neutrophil groups.

Figure 4 Two neutrophil populations.

Sharma et al. Clue to the diagnosis of malaria

Hematology 2012 VOL. 0 NO. 0 3

144 malaria cases found 38.9% cases showing pseu-doeosinophilia and 52.10% cases showing abnormal-ities in WBC scattergram like non-classified potextending from neutrophils towards eosinophil area(22.90%), two eosinophil populations (21.50%), twoneutrophil populations (27.80%) and overlapping ofneutrophil and eosinophil populations (48.60%). Thisis caused when hemozoin containing particles interferewith the machine’s WBC detection system. Theseabnormalities are due to the abnormal counting ofhemozoin containing neutrophils as well as due totheir detection as eosinophils near the neutrophilcluster. Yoo et al. in 2010 also studied the importanceof pseudoeosinophilia and abnormal WBC scatter-gram in assessment of 413 malaria cases. They found39% cases with pseudoeosinophilia and 15.70% caseswith abnormal WBC scattergram like two neutrophiland two eosinophil populations.10

In a Colombian study by Campuzano Zuluagaet al.11 in 2010, the quantitative data, scatterplotsand histograms were used to construct predictivemodels for each malaria species, by using binary logis-tic regression.

The present study showed 83.8% malaria caseshaving abnormal WBC scattergram. Most commonabnormality was found to be graying of eosinophiland neutrophil populations (41.10%). Other commonabnormalities were overlapping of eosinophil and neu-trophil populations (16.20%) and two eosinophilpopulations (16.20%).

In the present study, rightward shift of RBC ghost inWBC/BASO and DIFF scattergrams were also verycommonly found in malaria cases. This can be attrib-uted to the presence of extracellular pigment and RBClysis which are reflected in that area.7

Although previous studies showed pseudoeosino-philia in approximately 39% of cases,8,9 the presentstudy had only 4% cases showing spuriously high eosi-nophil count on scattergram as compared with smears,but it has shown little extra significance as timedevoted for calculating it by microscopy will enablethe observer to look for the parasite.12

About six cases of P. falciparum showed graying oflymphocyte and monocyte groups, which might be dueto the interference in their detection by ring forms ofthe parasite.7

Huh et al.8 (2008) showed in their study that XE-2100 had sensitivity of 69.40% and specificity of100% while using pseudoeosinophilia and abnormalWBC scattergram in detection of malaria.

Figure 7 Rightward shift of RBC ghost area in DIFFscattergram.

Figure 5 Graying of lymphocyte and monocyte groups.

Figure 6 Two lymphocyte populations.

Table 3 Sensitivity, specificity, PPV, and NPV of abnormalscattergrams with or without cytopenias

Abnormalscattergram

(%)

(Abnormalscattergram + cytopenias)

(%)

Sensitivity 83.78 98.60Specificity 94.82 94.80PPV 53.20 57.25NPV 98.80 94.80

Sharma et al. Clue to the diagnosis of malaria

Hematology 2012 VOL. 0 NO. 04

Yoo et al. (2010) reported sensitivity of 46.20% andspecificity of 99.70% by using pseudoeosinophilia andabnormal WBC scattergram by Sysmex-2100. Theseparameters 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 abovestudies while using abnormal WBC scattergram indetection of malaria. This sensitivity increased to98.6% 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 beinganemias and thrombocytopenias. Several studies havebeen 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 thesehematological findings, the detection rate of malariawill definitely increase, as is shown in the present study.Several new techniques have come up for the

detection of malaria but requires special requisition bythe treating clinician in patients suspected of malariainfection; however, the abnormal WBC scattergramfindings may alert the pathologist to look for malariain subclinical cases also.15 As, CBC is the mostfrequently requested investigation in patients withfever, if the laboratory staff is aware of these WBCscattergram abnormalities, they will be more carefulto look for the presence of parasite in blood smears.

References1 Levine RA, Wardlaw SC, Patton CL. Detection of hematopara-sites using quantitative buffy coat analysis tubes. ParasitolToday. 1989;5:132–4.

2 A rapid dipstick antigen capture assay for the diagnosis of falci-parum malaria. Bull WHO. 1996;74:47–54. 4

3 Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN.Identification of the 4 human malaria parasite species in fieldsamples by the polymerase chain reaction and detection of ahigh prevalence of mixed infections. Mol Biochem Parasitol.1993;58:283–92.

4 Shapiro MF, Greenfield S. The complete blood count and leuko-cyte differential count. An approach to their rational application.Ann Intern Med. 1987;106:65–74.

5 Mendelow BV, Lyons C, Nhlangothi P, Tana M, Munster M,Wypkema E, et al. Automated malaria detection by depolariz-ation of laser light. Br J Haematol. 1999;104:499–503.

6 Fourcade C, Casbas MJ, Belaouni H, Gonzalez JJ, Garcia PJ,PepioMA. Automated detection of malaria by means of the hae-matology analyser Coulter GEN.S. Clin Lab Haematol. 2004;26:367–72.

7 Yan F, Dai Y, Zhang Z, Wan H. The correlation of abnormalinformation in Sysmex hematology analyzers XE-2100 andXS-1000i with diagnosis of Plasmodium infection. Sysmex JInt. 2008;18:50–3.

8 Fluorescence differentiation supports malaria diagnostics May2006. Sysmex.

9 Huh HJ, Oh GY, Huh JW, Chae SL. Malaria detection withthe Sysmex XE-2100 hematology analyzer using pseudoeosino-philia and abnormal WBC scattergram. Ann Hematol. 2008;87:755–9.

10 Yoo JH, Song J, Lee KA, Sun YK, Kim YA, Park TS, et al.Automated detection of malaria-associated pseudoeosinophiliaand abnormal WBC scattergram by the Sysmex XE-2100 hema-tology analyzer: a clinical study with 1801 patients and real-timequantitative PCR analysis in vivax malaria-endemic area. Am JTrop Med Hyg. 2010;82:412–4.

11 Zuluaga GC, Sanchez GA, Gallo GEE, Zuluaga LMV, OrregoAMR, Vidal AP, et al. Design of malaria diagnostic criteriafor the Sysmex XE-2100 hematology analyzer. Am J TropMed Hyg. 2010;82:402–11.

12 Zuluaga GC, Hänscheid T, GrobuschMP. Automated haematol-ogy analysis to diagnose Malaria. Malar J. 2010;9:346.

13 Chai JY. Re-emerging Plasmodium vivaxmalaria in the Republicof Korea. Korean J Parasitol. 1999;37:129–43.

14 Fialon P, Macaigne F, Becker M, Boisseau MR, Cazenave J,Ripert C, et al. Hematological features in imported malaria.Value for the diagnosis of forms with low parasitemia. PatholBiol (Paris). 1991;39:122–5.

15 Hanschied T, Pinto BG, Pereira I, Cristino JM, Valadas E.Avoiding misdiagnosis of malaria: a novel automated methodallows specific diagnosis, even in the absence of clinical suspi-cion. Emerg Infect Dis. 1999;5:836–8.

Sharma et al. Clue to the diagnosis of malaria

Hematology 2012 VOL. 0 NO. 0 5

Authors QueriesJournal: HematologyPaper: HEM49Article title: Abnormal WBC scattergram: A clue to the diagnosis of malaria

Dear AuthorDuring the preparation of your manuscript for publication, the questions listed below have arisen.Please attend to these matters and return this form with your proof. Many thanks for your assistance

QueryReference Query Remarks

1 Please provide the expansion of CBC.

2

Please provide complete academic orinstitutional address details including zip/postal code for correspondence.

3Please provide the expansion of DIFF andBASO.

4 Please provide authors name in reference 2.