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Overview :
Examination of a thick blood smear can be used to screen a
patient for low level malarial parasitemia. When properly made a thick smear can
be very helpful. Unfortunately, many people do not know how to make a good thick
smear.
Goals:
(1) To look at
a relatively large amount of blood as quickly as possible.
(2) To remove
hemoglobin from red cells which might otherwise obscure the parasites.
(3) To prevent
artifacts.
(4) To
properly stain the parasites.
Using proper slides:
(1) The slides should be clean and free of grease and dirt.
(2) The slides should be washed in alcohol and then dried prior
to use.
Determine the type of blood sample:
(1) If the
blood sample does not contain an anticoagulant (for example from a fingerstick)
then fibrin may form as the blood dries. It is important to stir these specimens
with a small stick while the blood is drying.
(2) Blood
collected into an anticoagulant can be applied directly to the slide.
Making the smear:
(1) It is
important that the smear not be too thick. You should barely be able to read
newsprint through the smear when complete.
(2) Blood can
be collected in a capillary tube, the tip of which can be moved in an increasing
spiral.
(3) If a
capillary tube is not available, then the edge of a slide or an applicator stick
can be used.
(4) If the
blood smear is too thick then the blood film will probably wash off as a sheet
or flake off during staining.
Handling the unstained smear:
(1) Let the
slides air dry. If the air is humid, then place the slide in a desiccator.
(2) Protect
the smear from heat, sunlight and insects (flies may try to feed on the
blood).
(3) Do not
heat or fix the smear since this will prevent laking of the hemoglobin out of
erythrocytes.
Removing the hemoglobin from the red blood cells (laking):
(1) It is
essential to remove the hemoglobin from within the erythrocytes, usually be
washing in a hypotonic solution.
(2) If the
smear is fixed, then hemoglobin will not elute out of the red cells and the
smear will be impossible to read.
(3) A dilute
saponin solution (0.5% to 1%) can be used to treat the smear very briefly (5
seconds) to help lyse red cells, after which the slide is rinsed.
(4) A dilute
stain can combine both laking and staining into a single step.
Staining the slide:
(1) The
staining solution must be buffered to the proper pH.
(2) The
appropriate stain should be prepared to the proper concentration.
(3) The slide
should be stained for the proper length of time, after which it is quickly
rinsed then allowed to air dry.
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