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SECTION 7
SOLVING PROBLEMS
7.2 Abnormal Staining Results
Smear
Separation
or Tearing
Losing blood cells from the slide surface during a staining cycle can happen for several
reasons:
Improper Alcohol Fixation
If the alcohol-fix phase of the cycle fails, there will be cell loss when stain is
applied. Verify that the alcohol nozzle is operating properly with normal spray
pattern and spray volume (Sections 6.4, 6.5). To identify the problem as a
fixation failure, fix some specimen slides in methanol prior to loading into the
carousel. Select a fixation setting that increases fixation (7, 8, or 9).
Wet Smears
Loading wet blood smears into the carousel may contribute to smear tearing.
Allow smears to dry for several minutes before beginning a stain cycle.
Thick Smears
Smears that are too thick can cause smear separation. If the smear has a thick
“wedge” cross section, cells can be torn away from the slide surface in the
thicker regions, while the thinner “monolayered” regions remain usable. Severe
tearing that starts in the thick region may also extend into the monolayer.
Dirty Slides
Dirty slides are a major source of cell loss during staining. We strongly
recommend that you use new, premium quality slides.
Even with premium quality slides, random loss may occur due to inadequate
slide surface quality. When the stainer is functioning properly, slide quality
accounts for most, if not all, cell loss problems.
Red Cell Artifacts
Refractile bodies or inclusions (seen as dark ringed objects on red cells) are
thought to be caused by the presence of water during fixation. Often called
“water spotting,” this effect is common in Romanowsky staining. Methanol
used for fixation must be anhydrous (0.5% or less water).
Methanol that is exposed to the atmosphere will absorb a considerable amount
of water, particularly when the relative humidity is high. The first line of
defense to avoid water spotting is to make certain the methanol used for fixing
smears is free of water. If in doubt, replace it with reagent known to be
anhydrous.
Wet Smears