of blood films
l theblood sample
l squash method
l wedge method
The blood sample
l EDTA anticoagulated blood
» di or tri potassium salt of ethylenediaminetetraacetic acid
» minimises changes to cells
» labelled with sample identifiers, collection time and date
l Tube must be filled with the right amount of blood
» if there is an excess of anticoagulant then artefacts occur
» if insufficient anticoagulant then small clots can form
l smears made as soon as possible (<3 h)
l sample must be well mixed prior to sampling
Squash method
l squash method
» a drop of blood/marrow is placed on slide
Blood smear preparation
l squash method
» a drop of blood/marrow is placed on slide
» a second slide is placed on top
Blood smear preparation
l squash method
» a drop of blood/marrow is placed on slide
» a second slide is placed on top to form a cross
» the slides are pulled apart with a gentle sliding
action
Blood smear preparation
l squash method
» a drop of blood/marrow is placed on slide
» a second slide is placed on top to form a cross
» the slides are pulled apart with a gentle sliding
action
» air dry and label
Blood smear preparation
l squash method
» a drop of blood/marrow is placed on slide
» a second slide is placed on top to form a cross
» the slides are pulled apart with a gentle sliding
action
» air dry and label
» sounds simple, but it is tricky to get the
– right amount of blood,
– correct speed of separation, and
– a jerk-free sliding action.
Wedge method
A B C x 40
x 10
Thin, good and thick smears
Thick films
l caused by
» too large a drop of blood
» spreading is too fast
» angle of spreader is too great
l excess plasma causes nucleated cells to
shrink and stain intensely - difficult to identify
l erythrocytes form rouleaux (stacks of coins)
and can not be evaluated
Thin films
l caused by
» drop of blood too small
» spreading is too slow
» angle of spreader is too shallow
l smudge cells (broken cells) are increased
l erythrocytes are distorted and may appear
artificially spheroid
l greater numbers of nucleated cells are pushed
to the edges of the smear -inaccurate differentials
Straight vs bullet tail
Straight vs Bullet tails
Accumulation of WBC
at tail of smear
Staining blood smears
l Romanowsky group of stains
» eg. May-Grunwald-Giemsa, Jenner, Wright’s, Leishman’s
l pH dependent reactions (pH 6.4-6.8 is crucial)
l contain methanol to fix cells
» basic dye
– eg. methylene blue and its oxidation products
– to colour acidic cell components blue, eg nuclei
» acidic dye
– eg. eosin
– to colour haemoglobin and other basic cytoplasmic
components orange-pink
Staining blood smears
l films well air-dried before staining or will fall off
l stain solution applied to films
» must remain for at least 1 min to allow cells to be
fixed in methanol
» longer times if BM smear or high WCCs
l equal amount of buffer (pH 6.4-6.8) added, mixed
» metallic sheen develops if correct ratio achieved
» incubate 4-6 min
l wash in distilled water, air dry.
» do not over wash; neutral water is best
Staining of blood smears
l most numerous
l 7-8 um
l round
l pink coloured
l central pale area
less than one third
the diameter of the
cell
l no nucleus
Platelets
l 1-3 um
l generally ovoid in
shape but with wide
variation
l blue staining
l red or purple
granules throughout
cytoplasm
l no nucleus
Neutrophils
l 10-15 um
l cytoplasm: pink with fine
violet-pink granules
l nucleus: dark blue-purple,
clumped nuclear
chromatin, 2-5 lobes joined
by a thin chromatin strand
l women may show
drumstick
Small lymphocytes
l 10-12 um
l cytoplasm: very thin rim
around nucleus, light blue,
usually no granules,
sometimes a perinuclear
clear zone
l nucleus: eccentric, round,
no lobes, deep purple,
very clumped chromatin
Large lymphocytes
l 12-16 um
l cytoplasm: light blue,
abundant; occasionally can
see pink granules
l nucleus: eccentric, round
or slightly indented, no
lobes, less darkly staining,
clumped nuclear chromatin
Monocytes
l 12-20 um
l round, sometimes have
irregular edge
l cytoplasm: smoky blue,
may have fine pink
granules, may have
vacuoles
l nucleus: variable, large,
U-shaped, may be folded
or convoluted, fine
chromatin strands
Eosinophils
l 12-18 um
l cytoplasm: filled with large,
round, uniform, orange-red
granules, often no stained
cytoplasm between granules
l nucleus: usually bi-lobed,
less dark staining and less
intense nuclear chromatin
clumping than neutrophils
Basophils
l 10-15 um
l cytoplasm: filled with
densely-staining
variable, purplish-
black granules, often
cover nucleus
l nucleus: U-shaped,
fine nuclear
chromatin pattern,
often not visible.
EDTA Artefacts
l blood deteriorates in EDTA
l normal morphology for 5 h at 4oC
l at room temperature normal cells change
» monocytes accumulate vacuoles within 1 hr
» erythrocytes change shape after 6 hours
– crenated
» platelets swell and appear pale staining
» day old blood shows distributional abnormalities
– many nucleated cells carried to tail
Smear Artefacts
l slow air drying of smear
» moisture artefact in red cells
– appear hypochromic
– sharp edge between central pale area and Hb
» hairy appearance to lymphocyte cytoplasm
» shrinkage of normal leucocytes
l smudge cells
» leucocytes that were destroyed in the spreading
process
» eosinophilic, amorphous material or bare nuclei
References
l Practical Haematology
» Dacie and Lewis, Churchill-Livingstone
l Clinical haematology
» Stein-Martin et al. Lippincott Co, Chapter 3
l Clinical Haematology and Fundamentals of
Haemostasis
» Harmening, F. A. Davis
l http://www.grad.ttuhsc.edu/courses/histo/blood/
l http://www.med.nagoya-u.ac.jp/pathy/Pictures/atlas.html