[Downloaded free from http://www.indianjmedsci.org on Tuesday, March 12, 2013, IP: 125.16.60.178] || Click here to downl 26 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: 430 cases 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, New Delhi, India Address for correspondence: Dr. Sarita Mohapatra, E-36, Ansari Nagar (West), AIIMS Residential Campus, New Delhi- 110 029, India. E-mail:
[email protected] 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 Access this article online Quick Response Code: Website: www.indianjmedsci.org DOI: 10.4103/0019-5359.103163 PMID: ***************************** 178] || Click here to down 27 AUTOMATED DETECTION OF MALARIA BY SYSMEX XE-2100 (using Giemsa stain. 2013. Now-a-days. Santa Clara. Two milliliters of blood was collected in an EDTA vial and tested for malaria parasite by the conventional methods [Giemsa. microscopy and/or ICT). monocytes (green).[Downloaded free from http://www.e. immunochromatographic test (ICT). the parasitized RBC with various morphologic forms (trophozoites. MATERIALS AND METHODS This prospective study was conducted in the department of microbiology from August to December. Japan).indianjmedsci.[1-3] In the WBC/BASO plot. lymphocytes (pink). QBC and ICT (LDH based OptiMal)] and the hematology analyzer (Sysmex XE2100). So. January 2011 . Sysmex XE-1800 (Sysmex corporation. Quantitative buffy coat (QBC) assay). Blood samples tested negative by the conventional methods were taken as controls. schizoints.[3] Hemozoin pigments produced by malaria parasite have also the tendency to depolarize the laser beam. Vol. neutrophils + basophil (blue). (Abbott Diagnostics. CA).[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.[1. March 12. In Sysmex XE-2100 analyzer (Sysmex corporation. eosinophils (red) with a space between the neutrophil and eosinophil populations [Figure 1a].2] Along with hematological parameters these analyzers can detect malaria parasites containing hemozoin pigment. Patients with a clinical suspicion of malaria were enrolled in the study group.16. and taken as the gold standard for malaria diagnosis.org on Tuesday.[4] The abnormal patterns are observed as double neutrophil [Figure 1b]. The secondary aim was to determine the different parasitic forms that contribute to the different population in the abnormal scattergrams. gametocyte) and the phagocytic cells (monocyte. the normal scattergram in the DIFF plot is constituted of five parameters. grey zone [Figure 1d] double eosinophil [Figure 1e]. extended neutrophil with a decrease space between neutrophil and eosinophil [Figure 1c]. FL). side scatter (SSC) determines the granularity of the internal structure. Kobe)] are also used for automated detection of malaria in many febrile patients during complete blood count (CBC) analysis. and a combination among the above. 65. hematology analyzers [Cell Dyn. Among all. IP: 125. Forward scatter light (FSL) measures cell size. No.S. 1. and PCR are performed only after the clinical diagnosis. GEN. 2010.60. the basophils are selectively separated from the rest WBCs without showing any abnormal events above the x-axis. Sysmex analyzer works on the principle of flowcytometry and measures the different blood corpuscular elements. AO.[3] It uses a semiconductor laser to give three types of optical information about the cells. LH-750 (Beckman Coulter. The presence of events in this area is suggestive of malarial infection [Figure 1f]. Sysmex XE2100. Giemsa stain is the most prevalent method worldwide. Kobe. macrophage. and side fluorescence light (SFL) provides information about the nuclear content. Acridine Orange (AO) stain. and neutrophil containing the parasite) mimic the above-described patterns and exhibits various abnormal scattergram during routine CBC analysis. The samples were analyzed for abnormal scattergrams in the Indian Journal of Medical Sciences. A “positive case for malaria” in our study was defined as positive by either of the above described conventional methods (i. Miami. Thus. AO. respectively [Table 1]. and 72 h of initiation of antimalarial treatment and reviewed by all the methods. extended neutrophil with decreased space between eosinophil and neutrophil. P.indianjmedsci. 18/70 (25.1%) followed by ICT (94.2%) for both P. No. March 12. (c) extended neutrophil with decreased space between neutrophil and eosinophil. January 2011 to be highest (97. Fifty-two out of 70 malaria positive cases showed abnormal scattergram in flowcytometric analyzer. AO (51/70). and the combination among the above. A maximum number of malaria positive cases were detected by QBC followed by other methods like ICT. 70 were found to be positive by conventional methods. grey zones. . IP: 125. 65. double eosinophil. An abnormal WBC/BASO plot [Figure 1f] was observed in 37 malaria positive cases. (d) grey zone. RESULTS Out of 430 clinically suspected malaria cases.178] || Click here to downl INDIAN JOURNAL OF MEDICAL SCIENCES 28 a b c d e f Figure 1: Scattergrams in Sysmex XE-2100. falciparum.7% and 95. respectively [Table 1]. considering atypical scattergram in the DIFF plot and the WBC/BASO plot. (e) double eosinophil.org on Tuesday. Vol. and 3/70 (4. 2013. and Giemsa [QBC (68/70). (f) abnormal events above the x-axis in the WBC/Baso plo DIFF and WBC/BASO plot of SysmexXE-2100.[Downloaded free from http://www.2% and 88%. Giemsa (49/70)] [Table 1]. Statistical analysis of all the methods were calculated by 2/2 table analysis.16.2% (95% CI). Follow-up samples were taken after 24 h. The positive samples showed various abnormal patterns in DIFF scattergram such as double neutrophil. The microscopic finding was matched with the abnormal scattergrams produced by the analyzer. 48 h. overall sensitivity and specificity of Sysmex XE-2100 was found to be 74. vivax and P. (b) double neutrophil. ICT (66/70). 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) [Table 1]. falciparum [49/70 (70%) for P. (a) normal DIFF plot. falciparum]. 1. Sensitivity of QBC was found Indian Journal of Medical Sciences. vivax was the predominant species followed by P.60. Positive predictive value and negative predictive value were found to be 59. vivax.7%) for P.2%). e. Figure 1).1 100 100 99.cytometric analyzer Parasitic forms (n) Decreased space1 2EN Grey 3DN DE Combination patterns Normal scattergram Gametocyte (47) 12 10 7 0 4 3 11 Schizonts (1) 0 0 0 1 0 0 0 E. malaria parasites were picked up by the flowcytometric analyzer and depending on their size. side scatter.9 Sysmex XE-2100 52 18 74. By careful observation and follow-up study of various malaria samples. However. DE = Double eosinophil. Hence. showing various atypical scattergrams. Vol. these pigments were engulfed by mononuclear cell (monocytes.16. We feel. we observed that gametocyte.[5] To the best of our knowledge.60.4] There are several evidences regarding the use of these analyzers for detection of malaria. trophozoites (9) 4 0 0 0 0 4 1 L. It may appear as double neutrophil depending on the stage of gametocyte (mature/immature). 1. this is the first study correlating abnormal scattergrams produced by flowcytometric analyzer with various forms of malaria parasite found during the microscopic examination. IP: 125. DN = Double neutrophil. and late trophozoites were contributed to the atypical neutrophil patterns (i. 1Decreased space between neutrophil and eosinophil Indian Journal of Medical Sciences. in the normal DIFF plot (side fluorescence Vs side scatter. hemozoin pigments (because of its birefringent nature) are also capable of scattering the laser light.7 95. found a significant correlation between the number of Sysmex hematology analyzer differentiates white blood cell in blood by means of forward scatter.2 91.2 n = Total number Table 2: Parasitic forms showing abnormal patterns in the DIFF plot in flow.org on Tuesday. 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].1 59.9 QBC 68 2 97. and side fluorescence scatter laser beam. macrophages) and polymorphonuclear cells (neutrophils). 2013.indianjmedsci. extended neutrophil.8 100 100 94. Complete reversion of abnormal to normal scattergram was observed after 48 to 72 h of initiation of antimalarial treatment.178] || Click here to downl 29 AUTOMATED DETECTION OF MALARIA BY SYSMEX XE-2100 DISCUSSION cells. apart from the phagocytic Table 1: 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. they are placed to the left to that of eosinophil population.2 100 100 98.[Downloaded free from http://www.4 ICT 66 4 94. January 2011 . 65.8 AO 51 19 72. March 12. In heavy malaria infection. Campuzano-Zuluaga et al. Neutrophils possess more nucleic acid and lesser internal granularity in comparison to eosinophils. trophozoites (3) 0 1 0 0 1 1 0 Pigment containing cells (6) 1 1 0 0 0 0 4 EN = Extended neutrophil zone. schizoint. and late trophozoite present in the blood sample. nuclear and pigment content appeared in the area of neutrophil and eosinophil. No.[3. decreased space between neutrophil and eosinophil and double neutrophil). The disappearance of parasitic forms in the follow-up blood samples were corresponded with the resolution of atypical pattern from the scattergram. mature schizonts. Nguyen PH. It is more accurate and easy to identify. Ho E. Intraleucocytic malaria pigment and clinical severity of malaria in children. No.178] || Click here to downl INDIAN JOURNAL OF MEDICAL SCIENCES 30 late trophozoites. Although. Alvarez-Sanchez G. Valencia-Zuluaga LM. the WBC/Baso plot is an important area where one should look for malaria. Hanscheid T. can be easily screened and documented by nontechnical personnel. 65. it can be used as a clinical parameter to detect malaria eradication after treatment. Malaria detection with the Sysmex XE-2100 haematology analyzer using pseudoeusinophilia and abnormal WBC scatterogram.25:77-86. Scott CS.92:54-6. has not been evaluated for detection of malaria in endemic areas. Adeyemo AA. 5. Pito BG. and high throughput device for detection of malaria. Parasitol Today 2000.[7-9] Many authors have suggested that pigment-containing neutrophils are abnormally represented the as double eosinophil population by the analyzer. NJ. Jung J. WBC/BASO. along with the malaria detection. 2. Vol.[6] In malaria infection. Campuzano-Zuluaga G. Escobar-Gallo GE.22:259-61.[Downloaded free from http://www. Huh HJ. Detection of malaria by abnormal scattergram in the flowcytometric analyzer is not only economical but also. Olumese PE. Hansceid T. the kinetics of hemozoin pigment containing WBC varies from person to person depending on the immune status of host and severity of infection. neutrophil-containing pigment is observed under heavy parasitaemic condition and considered as a poor prognostic indicator. Ferguson DJ.60. Hence. Van ZD. monocytes and macrophages remain the first line of defense followed by neutrophils.87:755- 9. Automated detection of malaria associated intraleucocytic haemozoin by Cell-Dyn CD4000 depolarization analysis. Day N. . hematologist should be aware of this aspect of abnormal scattergram so that an early and prompt diagnosis of malaria can be made. Limitation of this study includes it Indian Journal of Medical Sciences. et al. Trans R Soc Trop Med Hyg 1998. it is comparable with the most prevalent conventional diagnostic method i. 7. January 2011 in severe malaria. Automated malaria diagnosis using pigment detection. In our study. the sensitivity and specificity of Sysmex analyzer is found lesser than QBC and ICT. Trans R Soc Trop Med Hyg 1995. Apart from the DIFF plot. Mendelow BV. Chae SL. Clin Lab Haematol 2003. In comparison to other methods. 1. Cristino JM. IP: 125. Yoon H. Grobusch MP.org on Tuesday. automated.indianjmedsci. Grobusch MP. Intraleucocytic malaria pigment and prognosis Sysmex XE-2100 is a rapid.16:549-51. 6. and gametocyte of P. normalization of scattergrams correlates with the microscopic parasite negativity. [10] In our study. et al. valadas E. vivax and abnormalities formed in the DIFF.82:402-11. Amodu OK. Gbadegesin RA. it is easy to interpret and needs less technical expertise for malaria detection. Ann Haematol 2008. Pabon-Vidal A. schizonts. March 12. CONCLUSION 4. White REFERENCES 1. Rios-Orrego AM.16. and RET-EXT scattergrams. 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