Preparation and examination

of blood films

Diana Quinn 2000

 Lecture overview

l blood smear preparation techniques

l methods for staining blood smears
l evaluation of stained blood smear
» features of a good film
» common flaws
» normal cellular components of blood
» other arefacts in smears
 Blood smears

l prepared on all CBE blood samples that are shown

to have abnormalities in their complete blood count
or automated differential leucocyte count
l examined for
» manual differential leucocyte count
» morphology changes in
– erythrocytes
– leucocytes
– platelets
Blood smear preparation - Overview

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 mostly used for preparing bone marrow smears

l makes a pair of smears
l can use coverslips or glass slides
» slides are easier to handle
l superior leucocyte distribution
l platelets are unevenly distributed betweens pairs of
smears
l no defined area for detecting cell abnormalities
 Blood smear preparation

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

l most widely used

l also called push-smear and spreader slide smears
l inherently poor distribution of nucleated cells
» neutrophils and monocytes appear more at edges and at
tail of film which may lead to an overestimate of
lymphocytes in a leucocyte differential count
l trauma to cells
» strong forces may shear particularly weak cells causing
artefacts
l easiest and cheapest smear method
l SOP 1.3
 Blood smear preparation

l place clean slide on a flat surface

l transfer a sample of well-mixed fresh whole blood
onto the slide
» 2-3 mm diameter drop
» 1 cm away from end of slide that is closest to your writing
hand
l hold the other end of the slide steady with your non-
writing hand
l position a clean spreader at a 25 - 45o angle to the
stationary slide (for ‘thin’ blood use higher angle)
 Blood smear preparation

l holding the side edges of the spreader, pull it back

until it touches the drop of blood
l allow the blood drop to spread along the spreader
l immediately push the spreader slide forward with a
smooth rapid stroke, maintaining the same angle
and exerting very little pressure
l the blood should feather somewhere between 1/2 or
3/4 of the way along the stationary slide
l air dry and label with at least 1 unique identifier
Blood smear preparation
 The spreader slide

l clean between samples with water and tissues

l decontaminate after use

l quality of spreader directly effects the quality of

the smear and the distribution of leucocytes
l narrower than slide
l spreading edge should be clean, smooth, and
polished without scratches or chips
l if you’ve got a good spreader - look after it!
 Smears should......

l cover at least half of the length of the glass slide

l finish 1 cm from end of slide
l be narrower than slide so edges of smear can be
examined
l be smooth transition from thick to thin
» ie no waves, streaks, troughs, holes or bubbles
l have a straight feathered end (not bulletted)
l be thin enough to allow fixation to occur
l have a broad rainbow representing the area of ideal
thickness
 Good smear
A B C

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

l smears should have straight-ish tail ends

l bullet ends
» caused by spreading smear before drop has
spread completely along spreader edge
» high accumulation of leucocytes at edges
» high accumulation of leucocytes at tail
 Gritty tails

l a gritty tail indicates an accumulation of

nucleated cells
» large number of leucocytes (leukaemia)
» slow or delayed spreading of smear
» only using part of the blood drop
» rough edge on spreader
» dirty spreader
» heparin as an anticoagulant
 Gritty 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 stained, dried smears can be mounted

in PIX and coverslipped
l label slides to indicate the identity of the
sample (not just the patient)
» date of preparation
» unique reference eg. UR, internal code
» patient name
l SOP 1.4, 1.5
Examination of blood smears - overview

l low power (x 10) scan

l high power (x40) scan
l oil immersion (x100) examination
» SOP 1.13, 1.14
 Low power scan (x 10)
l determine the overall staining quality
» precipitation of stain, colour reactions
l determine if there is a good distribution of the cells
» scan edges and centre to ensure there are no clumps of
WBC, RBC or platelets or abnormal cells
l locate the area of ideal thickness
» 50% of the cells do not touch another cell, the others are in
groups of two or three
» RBC should have a graduated central pallor
 Low power scan (x 10)
» estimate white cell count (WCC)
– move to area adjacent to tail of film (holes of the tail
must be in adjacent field)
– count the number of nucleated cells using one button of
your differential cell counter
– repeat with 4 more fields using a separate button for
each field
– the five numbers should be close to each other in value
indicating good distribution of leucocytes
– the total should be divided by a factor (CH and CH2
microscopes = 18)
– compare smear WCC to automated WCC
 High power scan (x 40)
l Differential leucocyte count
» 2 x 100 cell counts; nucleated reds/100 WBC
l morphology of leucocytes
» staining patterns, abnormal granulation
l morphology of erythrocytes
» staining patterns, variation in size, shape,
haemoglobinisation, presence of inclusion bodies
l morphology of platelets
» staining patterns, variation in size, abnormal granulation
 Oil immersion scan (x 100)
l repeat a difficult differential leucocyte count
l reassess abnormal leucocyte, erythrocyte and
platelet morphology
l estimate platelet count
» count the number of platelets in 10 oil immersion
fields
» indirect platelet count = N x 109/L
» compare indirect count to automated count
Normal cellular components of a
blood film

l erythrocytes (red cells)

l platelets
l leucocytes (white cells)
» neutrophils
» lymphocytes (large and small)
» monocytes
» eosinophils
» basophils
Erythrocytes

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