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Automated detection of malaria with haematology ana indian.

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  • 1. 11/22/13 Automated detection of malaria with haematology analyzer sysmex xe-2100 :<b>Sarita Mohapatra<sup>1</sup>, Jyotish C Samantaray<sup>1</sup>, S Ar… Home ORIGINAL ARTICLE [Download PDF] Year : 2011 | Volume : 65 | Issue : 1 | Page : 26--31 Automated detection of malaria with haematology analyzer sysmex xe-2100 Sarita Mohapatra1, Jyotish C Sam antaray1, S Arulselvi2, Jitender Panda1, Khushboo Munot 1, Renu Saxena3, 1 Department of Microbiology, JPNA Trauma Centre, New Delhi, India 2 Department of Laboratory Medicine, JPNA Trauma Centre, New Delhi, India 3 Department of Haematology, All India Institute of Medical Sciences, New Delhi, India Correspondence Address: Sarita Mohapatra E-36, Ansari Nagar (West), AIIMS Residential Campus, New Delhi- 110 029 India 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. 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 How to cite this URL: 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 [serial online] 2011 [cited 2013 Nov 22 ];65:26-31 Available from: http://www.indianjmedsci.org/text.asp?2011/65/1/26/103163 Full Text Introduction www.indianjmedsci.org/printarticle.asp?issn=0019-5359;year=2011;volume=65;issue=1;spage=26;epage=31;aulast=Mohapatra 1/5
  • 2. 11/22/13 Automated detection of malaria with haematology analyzer sysmex xe-2100 :<b>Sarita Mohapatra<sup>1</sup>, Jyotish C Samantaray<sup>1</sup>, S Ar… Malaria is a common public health problem observed worldwide. The classical presentation includes fever with chill and rigor. Diagnosis is based using a combination of clinical history, travel history, and laboratory tests. Microscopy (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 XE-2100, 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 [Figure 1]a. [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 abovedescribed patterns and exhibits various abnormal scattergram during routine CBC analysis. [4] The abnormal patterns are observed as double neutrophil [Figure 1]b, extended neutrophil with a decrease space between neutrophil and eosinophil [Figure 1]c, grey zone [Figure 1]d double eosinophil [Figure 1]e, and a combination among the above. [1],[2],[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 [Figure 1]f. [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.{Figure 1} 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 XE-2100). A "positive 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 DIFF and WBC/BASO plot of SysmexXE-2100. Follow-up samples were taken after 24 h, 48 h, and 72 h 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. www.indianjmedsci.org/printarticle.asp?issn=0019-5359;year=2011;volume=65;issue=1;spage=26;epage=31;aulast=Mohapatra 2/5
  • 3. 11/22/13 Automated detection of malaria with haematology analyzer sysmex xe-2100 :<b>Sarita Mohapatra<sup>1</sup>, Jyotish C Samantaray<sup>1</sup>, S Ar… 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]. A maximum 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)] [Table 1]. Sensitivity of QBC was found to be highest (97.1%) followed by ICT (94.2%). Fifty-two out of 70 malaria 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) [Table 1]. An abnormal WBC/BASO plot [Figure 1]f was observed in 37 malaria 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 [Table 1]. Positive predictive value and negative predictive value were found to be 59.7% and 95.2% (95% CI), respectively [Table 1].{Table 1} Discussion 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 72 h 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 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, [Figure 1]), 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. CampuzanoZuluaga et al. found a significant correlation between the number of 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 www.indianjmedsci.org/printarticle.asp?issn=0019-5359;year=2011;volume=65;issue=1;spage=26;epage=31;aulast=Mohapatra 3/5
  • 4. 11/22/13 Automated detection of malaria with haematology analyzer sysmex xe-2100 :<b>Sarita Mohapatra<sup>1</sup>, Jyotish C Samantaray<sup>1</sup>, S Ar… person to person depending on the immune status of host and severity of infection. [7],[8],[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.{Table 2} Conclusion Sysmex XE-2100 is a rapid, automated, and high throughput device for detection of malaria. In comparison to other methods, it is easy to interpret and needs less technical expertise for malaria detection. Although, the sensitivity and specificity of Sysmex analyzer is found lesser than QBC and ICT, it is comparable with the most prevalent conventional diagnostic method i.e. Giemsa. In our study, normalization of scattergrams correlates with the microscopic parasite negativity. Hence, along with the malaria detection, it can be used as a clinical parameter to detect malaria eradication after treatment. Limitation of this study includes it 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. References 1 Hansceid T, Pito BG, Cristino JM, Grobusch MP. Malaria diagnosis with the haematology analyser CellDyn 3500: What does the instrument detect? Clin Lab Haematol 2000;22:259-61. 2 Scott CS, Van ZD, Ho E, Meyersfeld D, Ruivo L, Mendelow BV, et al. Automated detection of malaria associated intraleucocytic haemozoin by Cell-Dyn CD4000 depolarization analysis. Clin Lab Haematol 2003;25:77-86. 3 Huh HJ, Jung J, Yoon H, Chae SL. Malaria detection with the Sysmex XE-2100 haematology analyzer 4 using pseudoeusinophilia and abnormal WBC scatterogram. Ann Haematol 2008;87:755- 9. Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ. Intraleucocytic malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med Hyg 1995;89:200-4. 5 Hanscheid T, valadas E, Grobusch MP. Automated malaria diagnosis using pigment detection. 6 Parasitol Today 2000;16:549-51. Campuzano-Zuluaga G, Alvarez-Sanchez G, Escobar-Gallo GE, Valencia-Zuluaga LM, Rios-Orrego AM, Pabon-Vidal A, et al. Design of malaria diagnostic criteria for Sysmex XE-2100 haematology analyzer. Am J Trop Med 2010;82:402-11. 7 Amodu OK, Adeyemo AA, Olumese PE, Gbadegesin RA. Intraleucocytic malaria pigment and clinical severity of malaria in children. Trans R Soc Trop Med Hyg 1998;92:54-6. 8 Day NP, Pham TD, Phan TL, Dinh XS, Pham PL, Ly VC, et al. Clearance kinetics of parasites and pigment-containing leukocytes in severe malaria. Blood 1996;88:4694-700. 9 Metzger WG, Mord muller BG, Kremsner PG. Malaria pigment in leukocytes. Trans R Soc Trop Med Hyg 1995;89:637-8. 10 Huh J, Jung J, Yoon H, Chung W. Pseudoeosinophilia associated with malaria infection determined in the Sysmex XE-2100 haematology analyzer. Ann Haematol 2005;87:400-2. Friday, November 22, 2013 www.indianjmedsci.org/printarticle.asp?issn=0019-5359;year=2011;volume=65;issue=1;spage=26;epage=31;aulast=Mohapatra 4/5
  • 5. 11/22/13 Automated detection of malaria with haematology analyzer sysmex xe-2100 :<b>Sarita Mohapatra<sup>1</sup>, Jyotish C Samantaray<sup>1</sup>, S Ar… Site Map | Home | Contact Us | Feedback | Copyright and Disclaimer www.indianjmedsci.org/printarticle.asp?issn=0019-5359;year=2011;volume=65;issue=1;spage=26;epage=31;aulast=Mohapatra 5/5