Screening for parasites: Since erythrocytes (RBCs) are lysed and the parasites become more concentrated, thick smears are especially useful for screening parasites and identifying mixed infections.

Begin by screening the entire smear at a low magnification (10× or 20× objective lens) to detect large parasites such as microfilaria.

Next, examine the smear using the 100× oil immersion objective lens.

Focus on areas that are well-stained, free of stain precipitate, and populated with white blood cells (WBCs) (10-20 WBCs/field).

Identifying parasites: If parasites are observed, make a tentative species determination using the thick smear. Then, examine the thin smear for more precise species identification, as the thin smear is typically more suitable for this purpose.

No Parasites Found (NPF): For malaria diagnosis, the World Health Organization (WHO) recommends screening at least 100 fields, each containing about 20 WBCs, before declaring a thick smear negative. This method ensures a sensitivity threshold of 4 parasites per microliter of blood, assuming an average WBC count of 8,000 per microliter of blood. In cases involving nonimmune patients with symptoms, lower parasite densities may still cause malaria, necessitating examination of additional fields (200, 300, or even the entire smear). NCCLS standards advise screening at least 300 fields using the 100× oil immersion objective lens.

Purpose: Thin smears are crucial for species identification of parasites detected on thick smears, screening when adequate thick smears are unavailable, and providing a rapid screen while thick smears dry.

Screening process:

Dear Lykkers, begin with low magnification (10× or 20× objective lens) if not already done for thick smears.

Carefully examine the smear at 100× oil immersion magnification. NCCLS standards recommend inspecting at least 300 fields using this lens.

Quantification for clinical use: In specific cases, such as malaria, quantifying parasites provides valuable clinical information. Malaria parasites can be quantified against RBCs or WBCs based on the physician’s needs.

Against RBCs:

Count parasitized RBCs among 500-2,000 RBCs on the thin smear, expressing results as a percentage of parasitemia: % parasitemia = (parasitized RBCs / total RBCs) × 100

If parasitemia is high (>10%), examine 500 RBCs.

If parasitemia is low (<1%), examine 2,000 RBCs or more.

Against WBCs: On the thick smear, tally parasites against WBCs until 500 parasites or 1,000 WBCs are counted, whichever occurs first.

Express results as parasites per microliter of blood:

Parasites/microliter blood = (parasites / WBCs) × WBC count per microliter (or 8,000, if unknown).

Interconversion between % parasitized RBCs and parasites per microliter blood is possible if WBC and RBC counts are known. Otherwise, assume 8,000 WBCs and 4,000,000 RBCs per microliter blood.

Kawamoto technique: This method uses acridine orange to stain nucleic acids in blood smears. Slides are examined with a fluorescence microscope or a light microscope equipped with an interference filter system. Nuclear DNA stains green, while cytoplasmic RNA stains red, facilitating parasite identification. This method is mainly used for malaria and, to a lesser extent, African trypanosomes.

Quantitative Buffy Coat (QBC®) method: Blood samples are collected in specialized tubes containing acridine orange, an anticoagulant, and a float. The tubes are centrifuged using a microhematocrit centrifuge and examined with a fluorescence microscope or a light microscope adapted for fluorescence. Malaria parasites concentrate below the granulocyte layer, and this method demonstrates high sensitivity for detecting malaria. It can also be applied to other parasites, including trypanosomes, microfilaria, and Babesia spp.