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.
- First screen
the entire smear at a low magnification (10× or 20× objective lens),
to detect large parasites such as microfilaria.
- 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).
- 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.
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.
- Screen at low magnification
(10× or 20× objective lens) if this has not been done on the thick smears.
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.
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.
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
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
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.