[Elizabeth_Zeibig]_Clinical_Parasitology__A_Practi(z-lib.org)

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CHAPTER 2 Specimen Collection and Processing
smear preparation can lead to unsatisfactory
results. Capillary blood should be free-flowing
and not contaminated with the alcohol used to
cleanse the puncture site. Blood that is milked
from the finger will be diluted with tissue fluids,
making it difficult to detect the parasites. Anticoagulants
cause some distortion to the staining
process and subsequent parasite morphology but
most laboratories use venipuncture specimens
collected with an anticoagulant. Blood specimens
should be collected in tubes containing ethylenediaminetetraacetic
acid (EDTA). If malaria is suspected,
it is best to prepare smears within 1 hour
of collection, because storage of blood for a
longer period leads to distortion and possible
loss of malarial parasites. Similarly, malarial tests
should always be considered immediately because
this disease can rapidly progress to life-threatening
complications.
The timing of obtaining blood samples varies
with the parasite suspected. For example, the
malarial forms present in peripheral blood at a
given time correlate with the specific phase in the
organism’s life cycle. In general, the filarial parasites
have a certain periodicity, or time at which
the microfilariae are most likely to be present in
the peripheral blood. Specific details regarding
collection time are addressed on an individual
basis in the parasite chapters of this text.
Processing. Typical blood sample processing
for parasites consists of preparing thick and thin
blood smears, staining them using a permanent
stain, and examining them microscopically.
Blood samples may also be processed by performing
the Knott technique, examining buffy
coat slides, or setting up and reading cultures. A
description of each processing option follows.
Thick and Thin Smears. Once the blood
sample has been collected, two types of smears
may be made, thick and thin. Thick smears are
frequently satisfactory for screening purposes,
particularly when malaria is suspected. Thin
smears provide the best view of the malarial
parasites in red blood cells and are recommended
for species identification. It is important to note
that dehemoglobinized thick smears typically
have a much higher concentration of parasites
than thin smears. Thick smears are primarily
used when parasites are few in number or when
thin smears are negative. The advantage of the
thick smear is increased ability to detect the
malarial parasites; the disadvantage is that
the red blood cells have been lysed and it is not
possible to assess the morphology of parasites
that are detected. Suggested procedures for
making thick and thin smears are given in Procedures
2-10 and 2-11, respectively.
Permanent Stains. There are two permanent
stains commonly used for the detection of blood
parasites, Wright’s stain, which contains the fixative
and stain in one solution, and Giemsa stain,
in which the two are separate. Wright’s stain
typically yields only satisfactory results. Further
discussion of Wright’s stain may be found in
more comprehensive parasitology manuals and
in hematology texts. Giemsa stain is thus considered
the preferred stain because it allows for the
detection of parasite detail necessary for species
identification. A suggested procedure for staining
thick and thin smears with Giemsa stain is given
in Procedure 2-12. A synopsis of the expected
blood and tissue parasite colors and of background
material seen following Giemsa staining
is found in Table 2-7.
TABLE 2-7
Structure or Material
Leishmania, trypanosome,
malaria, and Babesia nuclear
structures
Cytoplasm
Schüffner’s dots
Filariae
Nuclei
Sheath
Background aterial
Red blood cells
White blood cells
Neutrophilic granules
Eosinophilic granules
Appearance of Select
Parasitic Structures and
Background Material on
Giemsa Stain
Appearance
Red
Blue
Red
Blue to purple
Clear; may not stain
Pale red
Purple
Pink-purple
Purple-red