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Table of Contents
CASE REPORT
Year : 2022  |  Volume : 15  |  Issue : 2  |  Page : 101-103

Pseudoeosinophilia on automated analyser (Sysmex XN-1000) a guide to malaria detection - An incidental finding


Department of Laboratory Medicine, Yashoda Hospital, Hyderabad, Telangana, India

Date of Submission01-Oct-2021
Date of Decision13-Oct-2021
Date of Acceptance07-Nov-2021
Date of Web Publication04-Jul-2022

Correspondence Address:
Majed Abdul Basit Momin
Department of Laboratory Medicine, Yashoda Hospital, Malakpet, Nalgonda X-Roads, Hyderabad - 500 036, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/hmj.hmj_67_21

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  Abstract 


Rationale: Malaria is a protozoan disease which has a worldwide distribution in tropical and subtropical regions. The peripheral blood smear examination is a gold standard and an immunochromatographic rapid diagnostic test is an adjuvant diagnostic tool for the detection of malaria. For the past two decades, the utility of automated blood cell counters for the detection of malarial infection is growing in the presumptive diagnosis of malaria in developing countries. Patient Concerns: Here, we report a case in which a young male came for surgical evaluation for inguinal hernia surgery. On routine haemogram evaluation, malaria was suspected with the help of flow cytometry based haematology analyser (Sysmex XN 1000) generated histogram showing erroneously high eosinophil count (pseudoeosinophilia) and atypical white blood cell scattergram. Diagnosis: Peripheral smear and malarial antigen examinations confirmed Plasmodium vivax species. Outcomes: Patient was treated with antimalarial medications and elective surgery to repair inguinal hernia was planned after 10 days. Lessons: Here, we hope to increase the awareness of laboratory physicians to explore the possibilities of diagnosing malaria through hints provided by automated haematology analyser generated histograms.

Keywords: Flow cytometry, haematology analysers histogram, malaria, Plasmodium vivax, pseudoeosinophilia


How to cite this article:
Momin MA, Singh RD. Pseudoeosinophilia on automated analyser (Sysmex XN-1000) a guide to malaria detection - An incidental finding. Hamdan Med J 2022;15:101-3

How to cite this URL:
Momin MA, Singh RD. Pseudoeosinophilia on automated analyser (Sysmex XN-1000) a guide to malaria detection - An incidental finding. Hamdan Med J [serial online] 2022 [cited 2022 Aug 10];15:101-3. Available from: http://www.hamdanjournal.org/text.asp?2022/15/2/101/349783




  Introduction Top


Malaria is a vector-borne protozoan disease transmitted by the bite of infected Anopheles mosquitoes. The microscopy and rapid diagnostic test are two diagnostic tests which help in a prompt and accurate diagnosis and help in effective disease management.[1] However, it is impossible to screen all peripheral smears or to perform malarial antigen detection in all samples, especially in the absence of a clinical request. Diagnosis of malaria based on flow cytometry by haematology analysers can be an important adjuvant diagnostic tool in the detection of malaria even in the absence of clinical suspicion. A careful observation of white blood cells differential (WDF) and white blood cells and nucleated blood cells (WNR) scattergram provided by the analyser in the presence of eosinophilia gives a hint about the underlying pseudoeosinophilia and the presence of the parasite.[2] Here, we report a case of detection of malaria with pseudoeosinophilia in a young patient who came for a surgical opinion of inguinal region swelling without revealing a history of fever.


  Case Report Top


In the middle of COVID-19 pandemic cases, a 23-year-old male attended a surgical outpatient department with complaints of swelling in the right inguinal region. There was no other medical illness. His vital parameters showed the temperature of 98°F, pulse rate of 90 beats/min with normal respiratory rate and blood pressure (120/70 mm of Hg). On general examination, there was no pallor or icterus. Systemic examination of the cardiovascular, respiratory system and neurological examination was normal. The abdominal examination reveals soft, swelling in the right inguinal region with positive cough impulse suggestive of right inguinal hernia. Surgical intervention was planned, and he was advised to undergo routine laboratory and radiological examination.

At the initial laboratory examination, haemogram results were as follows: RBCs 4.78 million/cu mm (4–5.5 millions/cu mm), haemoglobin13.7 g/dl (12–15 g/dl), WBCs 4500/cumm (4000–11000/cumm) and platelets, 1.25 lakh/cumm (1.5–4.5 lakh/cumm). The differential count revealed neutrophils 60%, lymphocytes 24%, eosinophils 12% and monocytes 04%.

However, the WBC histogram (WDF) showed instead of one which is normally seen [Figure 1]a, double neutrophils and double eosinophil clusters and abnormal blue coded events below the neutrophil-eosinophil cluster in RBCs ghost region [Figure 1]b, while WNR scattergram which normally showed RBCs debris in side fluorescence light (SFL) (side fluorescent) plot [Figure 1]c, showed three abnormal distinctive clusters of RBCs debris at side scatter plot side scatter (SSC) [Figure 1]d. These histogram findings hint at the presence of parasites and pseudoeosinophilia. For further confirmation, we prepared thick and thin peripheral smears and stained them with Leishman stain and Giemsa stain, which revealed malarial parasite of Plasmodium vivax species with trophozoites and gametocytes forms of P. vivax [Figure 2]. The parasite index was 1%. A manual differential count showed eosinophils 3%. There was no evidence of haemolysis seen in the peripheral smear. Malarial parasite antigen testing was performed which showed a band positive for pan-lactate dehydrogenase.
Figure 1: Normal scattergram and Plasmodium vivax positive patient WDF and WNR scattergram; (a) Normal Wigner distribution function scattergram; (b) showing two neutrophil clusters (N), two oeosinophil clusters (E) and abnormal blue coded events below the neutrophil-eosinophil cluster in red blood cell-ghost region. (c) Normal WNR scattergram and (d) abnormal clusters of red blood cell debris at side scatter plot (SSC)

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Figure 2: Peripheral smear leishman stained image showed gametocytes form (a) trophozoites (b) and malarial antigen card test with positive PV and pan-lactate dehydrogenase band

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The blood coagulation studies including tests for prothrombin time, activated partial thromboplastin time and fibrinogen were normal. Erythrocyte sedimentation rate was 30 mm at the end of 1 h. Biochemical investigation showed normal liver function test and normal renal function tests. Viral serology was non-reactive for HIV, HBs Ag and HCV. The complete urine examination was normal. Radiological examination for chest X-ray and ultrasound abdomen showed the normal study. The details of the laboratory findings were shared with the consulting surgeon and on further referral consultation of the general physician were known history of fever and antipyretic medicine, which was hidden initially to avoid triage screening for COVID-19. Finally, the patient was treated with antimalarial medicine and elective surgery to repair inguinal hernia planned after 10 days.


  Discussion Top


The complete blood count (CBC) is one of the most commonly ordered blood tests. An automated haematology analyser (AHA) is now widely used for the complete blood count. Haematology analysers generate suspect flags or warning messages that enable the user (technician or laboratory physician) to detect positive samples and to react with the follow-up actions because of the warnings.[2] Sysmex analyser works on the principle of flow cytometry in which flow cells scatter a beam of laser light focussed on them displaying various scattergrams. Forward scatter light which indicates cell size, SSC indicates the complexity of internal structures such as granules and SFL indicate the nuclear content. These three signals are used to differentiate and count white cells, nucleated RBCs, reticulocytes and platelets and to detect abnormal or immature cells with the help of unique digital technology.[3]

The WDF channel differentiates and counts neutrophils, lymphocytes, monocytes, eosinophilia and debris. The WNR channel counts WBCs and nucleated RBCs. By the flow cytometry method, a two-dimensional scattergram is plotted with SSC representing X-axis and SFL representing the Y-axis. Eosinophilia are more granular and have less nuclear material compared to neutrophils. Hence, in the normal Differential (DIFF) scatter plot, they are placed to the right of neutrophils.[4] The malaria-related abnormalities were noted in comparison with normal scattergram as follows:[5]

  1. Greying of neutrophil and eosinophil populations (G of NE)
  2. Two eosinophil populations (Two E)
  3. Overlapping of eosinophil and neutrophil populations (O of EN)
  4. Two neutrophil populations (Two N)
  5. Greying of lymphocyte and monocyte groups (G of LM)
  6. Two lymphocyte populations (Two L)
  7. Abnormal blue-coded events below the neutrophil-eosinophil cluster.


In the present case, haemogram showed thrombocytopenia and increased eosinophils (12%) and the WDF scattergram revealed two eosinophil and two neutrophil populations and abnormal blue coded events below the neutrophil-eosinophil cluster (NE cluster) in the RBCs (ghost region). The WNR scattergram (SFL-SSC) showed three distinctive clusters more at SSC region. The reason behind these changes is secondary to extracellular pigment or hemozoin and resistant to RBCs lysis. Hemozoin is a crystalline, brown pigment formed from released toxic heme resulting from the digestion of host haemoglobin by the intraerythrocytic malarial parasite. Hemozoin within the phagocytes can be detected by depolarization of laser light due to its birefringent property as cells pass through a flow cytometer channel. As per the study conducted by Pillai et al. to assess the usefulness of Sysmex XN 1000 analyser scattergrams in the diagnosis of malaria, found that the WBC-DIFF channel abnormalities were seen in 28 of 31 cases (sensitivity 90%) among P. vivax patients compare to Plasmodium falciparum group (sensitivity 50%) and the most common abnormality was two neutrophil populations (69.1%) and right shift of RBCs ghost area was seen in 64.2% in vivax malaria.[5]

Pseudoeosinionophilia is defined when the difference between manual and automated differential counts is >5%. Pseudoeosinophilia is caused when the neutrophils containing malaria pigment are spuriously counted as oeosinophils because of increased granularity and plotted in the eosinophil area.[6] A study done by Huh and colleagues, pseudoeosinophilia and abnormal DIFF scatter-plot alone yielded sensitivities of 39% and 52%, respectively.[7] Sharma et al. found that the sensitivity in the detection of malaria is increased when both abnormal WBC scattergrams and haematological findings are considered together than only WBC scattergrams alone.[8] The most common haematological abnormality in malaria among formed elements is thrombocytopenia (92%). It is the strongest predictor of malaria. It is due to increased splenic clearance and a reduction in the lifespan of platelets.[9]


  Conclusion Top


To conclude, AHA with histogram, scattergram flags, pseudoeosinophilia and thrombocytopenia represents an advantage that aids in the presumptive diagnosis of malaria in particular when clinical suspicion is low or not requested by a clinician. It could become an important adjuvant diagnostic tool in addition to the gold standard peripheral smear and rapid malaria detection method.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initial will not be published and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tjitra E, Suprianto S, Mary D, Currie JB, Anstey N. Field evaluation of the ICT Malaria P.f/P.v immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptive clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol 1999;37:2412-7.  Back to cited text no. 1
    
2.
Green R, Wachsmann-Hogiu S. Development, history, and future of automated cell counters. Clin Lab Med 2015;35:1-10.  Back to cited text no. 2
    
3.
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.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Shariff MH, Muzamil Dar A, Vidya P. Malaria diagnosis by abnormal scattergrams in automated hematology analyzer. Int J Pharm Biol Sci 2016;6:55-9.  Back to cited text no. 4
    
5.
Pillai KR, Arvind P, Mukta Ramesh P. The utility of automated haematology analyser XN 1000 scattergrams in the diagnosis of malaria. Natl J Lab Med 2020;1:6-10.  Back to cited text no. 5
    
6.
Adlekha S, Jaiswal RM, Chadha T, Singla A. The correlation of spurious eosinophilia in automated hematological analyzer Sysmex XS-800i with Plasmodium infection diagnosis. Indian J Med Sci 2011;65:469-75.  Back to cited text no. 6
  [Full text]  
7.
Huh HJ, Oh GY, Huh JW, Chae SL. Malaria detection with the Sysmex XE-2100 hematology analyzer using pseudoeosinophilia and abnormal WBC scattergram. Ann Hematol 2008;87:755-9.  Back to cited text no. 7
    
8.
Sharma S, Sethi N, Pujani M, Kushwaha S, Sehgal S. Abnormal WBC scattergram: A clue to the diagnosis of malaria. Hematology 2013;18:101-5.  Back to cited text no. 8
    
9.
Singh A, Narang V, Sood N, Garg B, Gupta VK. Malaria diagnosis using automated analysers: A boon for hematopathologists in endemic areas. J Clin Diagn Res 2015;9:C05-8.  Back to cited text no. 9
    


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