Blood Specimens – Microscopic Examination

Examining thick smears

Since the erythrocytes (RBCs) have been lysed and the parasites are more concentrated, the thick smear is useful for screening for parasites and for detecting mixed infections.

1. First screen the entire smear at a low magnification (10× or 20× objective lens), to detect large parasites such as microfilaria.
2. Then examine the smear using the 100× oil immersion objective lens. Select an area that is well-stained, free of stain precipitate, and well-populated with white blood cells (WBCs) (10-20 WBCs/field).
3. If you see parasites, make a tentative species determination on the thick smear and then examine the thin smear to determine the species present. Most often, the thin smear is the appropriate sample for species identification.
4. Determination of “No Parasites Found” (NPF): For malaria diagnosis, WHO recommends that at least 100 fields, each containing approximately 20 WBCs, be screened before calling a thick smear negative. Assuming an average WBC count of 8,000 per microliter of blood, this gives a threshold of sensitivity of 4 parasites per microliter of blood. In nonimmune patients, symptomatic malaria can occur at lower parasite densities, and screening more fields (e.g., 200, 300, or even the whole smear) might be warranted, depending on the clinical context and the availability of laboratory personnel and time. NCCLS standards recommend examination of at least 300 fields using the 100× oil immersion objective.

Examining thin smears

Thin smears are useful for species identification of parasites already detected on thick smears, screening for parasites if adequate thick smears are not available, and a rapid screen while the thick smear is still drying.

1. Screen at low magnification (10× or 20× objective lens) if this has not been done on the thick smears.
2. Carefully examine the smear using the 100× oil immersion objective lens. NCCLS standards recommend examination of at least 300 fields using the 100× oil immersion objective.

Quantifying parasites

In some cases (especially malaria) quantification of parasites yields clinically useful information. If this information is needed by the physician, malaria parasites can be quantified against blood elements such as RBCs or WBCs.

To quantify malaria parasites against RBCs, count the parasitized RBCs among 500-2,000 RBCs on the thin smear and express the results as % parasitemia.

% parasitemia = (parasitized RBCs/total RBCs) × 100

If the parasitemia is high (e.g., > 10%) examine 500 RBCs; if it is low (e.g., <1%) examine 2,000 RBCs (or more); count asexual blood stage parasites and gametocytes separately. Only the former are clinically important and gametocytes of P. falciparum can persist after elimination of asexual stages by drug treatment.

To quantify malaria parasites against WBCs: on the thick smear, tally the parasites against WBCs, until you have counted 500 parasites or 1,000 WBCs, whichever comes first; express the results as parasites per microliter of blood, using the WBC count if known, or otherwise assuming 8,000 WBCs per microliter blood.

Parasites/microliter blood=(parasites/WBCs) × WBC count per microliter<or 8,000>

Results in % parasitized RBCs and parasites per microliter blood can be interconverted if the WBC and RBC counts are known, or otherwise (less desirably) by assuming 8,000 WBCs and 4,000,000 RBCs per microliter blood.

Detection of blood parasites using fluorescent dyes

Fluorescent dyes that stain nucleic acids have been used in the detection of blood parasites. In the Kawamoto technique, blood smears on a slide are stained with acridine orange and examined with either a fluorescence microscope or a light microscope adapted with an interference filter system. This results in a differential staining of nuclear DNA in green and of cytoplasmic RNA in red, which allows recognition of the parasites. The method has been applied to malaria parasites (and to a lesser extent, African trypanosomes).

In the Quantitative Buffy Coat (QBC®; Becton Dickinson) method, blood samples are collected in a special tube containing acridine orange, an anticoagulant, and a float, and then are centrifuged in a microhematocrit centrifuge. After centrifugation, the tubes are examined using a fluorescence microscope with a stage adapter, or a light microscope with a customized fluorescence attachment. Malaria parasites concentrate below the granulocyte layer in the tube. The QBC method is reported to have a good sensitivity for detection of malaria parasites, and has also been applied (albeit to a lesser extent) to other parasites such as trypanosomes, microfilaria and Babesia spp.