Drying is an important step in the production of good-quality blood films. Allow the blood film to thoroughly air-dry before
applying stain, or use a heat block (at the low setting) or a hair dryer to hasten the drying time, thus minimizing refractile
markings that can distort erythrocyte morphology. In addition, keep formalin and formalin-containing containers away from
all blood and cytology smears to prevent staining artifacts such as increased cytoplasmic granularity and basophilia.
STAINING THE BLOOD FILM
Most veterinary practices use a modified Wright's stain (Romanowsky stain) for both hematology and cytology. Modified Wright's
stains are available as either three- or two-solution kits. We prefer kits with three separate solutions: an alcohol fixative,
an eosinophilic staining solution, and a dark-blue staining solution. Veterinary practices should have two separate Coplin
stain jar sets—one for hematology and cytology samples and one for contaminated samples such as those collected for ear and
fecal cytology. For optimal results, replace staining solutions regularly.
Recommended blood staining protocol includes dipping the air-dried slide five to 10 times in each solution while blotting
one edge briefly in between each solution. Rinse the slide with distilled water after the final staining step, and allow the
blood film to air-dry before examination. Good staining technique is critical to identifying polychromatophils on a peripheral
blood film. If the staining is done improperly, many of the quick Romanowsky-type stains will not give the needed tinctorial
differences between mature RBCs (orange-red) and polychromatophils (bluish-pink). Typically, poor staining with the various
quick stains results in all RBCs having a bluish or muddy tint.
EVALUATING THE BLOOD FILM
A high-quality microscope is essential for hematology. We recommend a binocular microscope with a minimum of 10×, 20×, and
100× (oil-immersion) objectives and wide-field 10× oculars. When evaluating the slide, make sure maximum light reaches the
blood film. This means positioning the substage condenser as close as possible to the stage with the iris wide open. The light
source should be equipped with a variable rheostat to allow maximal control of light intensity. To ensure optimal focused
light for the microscopic evaluation, the microscope should be in Köhler illumination. Contact your microscope vendor, or
review the microscope manual to ensure proper illumination.
The three zones
A good-quality blood film has three zones: the body (near the point of blood application), the monolayer (the zone between
the body and feathered edge), and the feathered edge (the area most distant from the point of application) (Figure 2). The definition of the monolayer varies from institution to institution, but the definition used to ensure consistent semiquantification
of various morphologic abnormalities is the area where about half of the RBCs are touching one another without overlapping.
The monolayer is the area of the blood film where WBCs and platelet numbers are estimated and cell morphology is examined.
Although the monolayer is the only zone where morphologic evaluation of individual cells is performed at oil-immersion magnification,
systematic blood film evaluation includes assessing of all three zones.
Figure 2. The components of the peripheral blood film slide.