The Role of Automatic Hematology Analyzer in Diagnosing Malaria : Case Report

Malaria is a parasitic disease transmitted by female Anopheles mosquitoes and caused by blood protozoa of the genus Plasmodium. Indonesia, particularly its eastern regions, remains an endemic area for malaria.⁹ Plasmodium vivax, a malaria species infecting human worldwide, is found in 95 countries, putting around 2.85 billion people at risk of infection.¹⁰ Human infections with Plasmodium vivax occur across most of the Indonesian archipelago,⁵ accounting for up to 40% of malaria cases in the country.⁹

Malaria tertiana or vivax malaria may lead to relapse due to the presence of dormant hypnozoites in the liver.⁹ This aligns with the information gathered in this case. The patient exhibited fever symptoms for two weeks, accompanied by chills, and had a history of malaria one year prior. Clinicians routinely request a complete blood count (CBC) examination using an automated analyzer in cases where patients complain of fever.^11,12 Malaria infection can be anticipated in patients presenting with anemia and thrombocytopenia.³ These results align with observations in patients exhibiting anemia and thrombocytopenia. In 23% of cases, leukocytosis was identified,³ which is consistent with the mild leukocytosis and neutrophilia observed in this particular case.

Malaria diagnosis remains a challenging process in laboratory settings.⁶ The adoption of automated hematology analyzers for detecting Plasmodium parasites represents a novel approach to aid in malaria diagnosis.¹³ Numerous studies have explored the effectiveness of automated hematology analyzers in malaria detection.⁶ The Flow Cytometry method on these analyzers serves as a sensitive supplementary diagnostic tool by identifying the presence of Plasmodium in whole blood.^11,13 This aligns with the WHO guideline for malaria diagnosis, aiming for a 95% sensitivity in samples with parasite counts exceeding 100 parasites/μl.^11,14

Automated hematology analyzers have integrated diverse methods for characterizing blood cell populations and delivering precise CBC results.^1,11 These analyzers typically operate based on two main principles. Firstly, optical scattering assesses the deviation of light path caused by the size and granularity of cells. Secondly, the principle of electrical impedance measures the alteration in the electric current as blood cells pass through the analyzer.¹⁵

The Sysmex XN series, an automated hematology analyzer, analyzes leukocyte cell population data, scattergrams, White Cell Differential (WDF), and white cell nucleated (WNR) channels to aid in diagnosing malaria.⁶ A scattergram is a graphical representation of the distribution of two variables in a sample population, illustrating the relationship between these variables. The Sysmex analyzer utilizes the flow cytometry method, where each cell passes through laser light scattered from various directions. Forward scattered light (SFL) measures cell volume, while side scattered light (SSC) gauges cell granularity.³

Abnormalities in scattergrams related to malaria infection exhibit different levels of sensitivity and specificity.¹⁵ The detection of parasites relies on scattered light and fluorescence intensity (WDF).¹¹ Infected red blood cells with parasites resist lysis by the diluting solution and enter the white blood cell (WBC) counting chamber.³ Leukocytes ingest the hemozoin pigment after the rupture of the Plasmodium schizont. This system aids in characterizing the presence of malaria parasite infection and has the potential to significantly narrow down the screening space. However, the complete clinical utility of this system is yet to be fully determined.¹¹

Automated hematology analyzers utilize specific fluorescence markers to tag nucleic acids. The higher the intracellular nucleic acid content (DNA and RNA), the greater the fluorescence signal produced.⁷ The hemozoin pigment generated by Plasmodium parasites can depolarize laser light and is phagocytosed by neutrophils and monocytes. This results in abnormal scattergram images from infected red blood cells.^3,16 The presence of gametocytes and/or schizonts manifests as a distinctive blue "ghost" appearance, forming abnormal clusters in the WNR channel. Thus, the use of the WNR scattergram is considered more accurate ¹³

Abnormal scattergrams found in malaria cases from several previous studies are greying of neutrophils and eosinophil population, two eosinophil populations, overlapping of eosinophil and neutrophil populations, two neutrophils populations, greying of lymphocyte and monocyte groups, two lymphocyte populations and right shift of RBC ghost area.^3,8,16  WBC-DIFF scattergram abnormalities are useful in presumptive diagnosis of malaria along with other abnormal hematological parameters such as thrombocytopenia, pseudo eosinophilia, leukopenia.¹  

The abnormal scattergram image in the WDF channel for this case reveals the presence of two neutrophil populations, as indicated by the light blue color in Figure 6, suggesting a vivax malaria infection. Pillai et al⁸ reported that 65.4% of cases with abnormal scattergrams showing two neutrophil populations were associated with vivax malaria infections, with an odds ratio of 1.667 and a p-value <0.001. In a study by Merlyn et al¹³ conducted in Merauke, various abnormal images of WDF, WNR, and RET scattergrams were observed. In 92.3% of Plasmodium vivax infection cases, abnormal scattergram predominance was noted in WDF and WNR channels. WDF channel scattergrams display abnormal cell clusters when schizonts and/or gametocytes are present.¹³ The variation in WDF channel scattergrams depends on the type of parasite species, with adult parasites and hemozoin crystals more commonly found in Plasmodium vivax and Plasmodium ovale compared to Plasmodium falciparum parasites. Consequently, WDF channel scattergram abnormalities are often identified in cases involving these two species.¹³