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Preparation and examination
of blood films
Diana Quinn 2000
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Lecture overview
l blood smear preparation techniques
l
methods for staining blood smearsl evaluation of stained blood smear
features of a good film
common flaws
normal cellular components of blood
other arefacts in smears
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Blood smears
l prepared on all CBE blood samples that are shownto have abnormalities in their complete blood countor automated differential leucocyte count
l examined for
manual differential leucocyte count
morphology changes in
erythrocytes leucocytes
platelets
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Blood smear preparation - Overview
l
the blood samplel squash method
lwedge method
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The blood samplel 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 (
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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
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Blood smear preparation
l squash method
a drop of blood/marrow is placed on slide
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Blood smear preparation
l squash method
a drop of blood/marrow is placed on slide
a second slide is placed on top
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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
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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
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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.
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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 oflymphocytes in a leucocyte differential count
l trauma to cells
strong forces may shear particularly weak cells causingartefacts
l easiest and cheapest smear method
l SOP1.3
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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 writinghand
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 thestationary slide (for thin blood use higher angle)
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Blood smear preparation
l holding the side edges of the spreader, pull it backuntil it touches the drop of blood
l allow the blood drop to spread along the spreader
l immediately push the spreader slide forward with asmooth rapid stroke, maintaining the same angle
and exerting very little pressurel 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
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Blood smear preparation
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The spreader slide
l clean between samples with water and tissues
l decontaminate after use
l quality of spreader directly effects the quality ofthe smear and the distribution of leucocytes
l narrower than slide
l spreading edge should be clean, smooth, andpolished without scratches or chips
l if youve got a good spreader - look after it!
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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 beexamined
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
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Good smear
A
A B C
B Cx40
x 10
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Thin, good and thick smears
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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 toshrink and stain intensely - difficult to identify
l erythrocytes form rouleaux (stacks of coins)
and can not be evaluated
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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 appearartificially spheroid
l greater numbers of nucleated cells are pushed
to the edges of the smear -inaccurate differentials
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Straight vs bullet tail
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Straight vs Bullet tails
l smears should have straight-ish tail ends
l bullet ends
caused by spreading smear before drop hasspread completely along spreader edge
high accumulation of leucocytes at edges
high accumulation of leucocytes at tail
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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
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Gritty tails
Accumulation of WBCat tail of smear
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Staining blood smears
l Romanowsky group of stains
eg. May-Grunwald-Giemsa, Jenner, Wrights, Leishmans
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
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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 befixed 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
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Staining of blood smears
l stained, dried smears can be mounted
in PIX and coverslipped
l label slides to indicate the identity of thesample (not just the patient)
date of preparation
unique reference eg. UR, internal code patient name
l SOP1.4, 1.5
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Examination of blood smears - overview
l low power (x 10) scan
l high power (x40) scan
l oil immersion (x100) examination
SOP1.13, 1.14
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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 ofWBC, 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 ingroups of two or three
RBC should have a graduated central pallor
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Low power scan (x 10)
estimate white cell count (WCC)
move to area adjacent to tail of film (holes of the tailmust be in adjacent field)
count the number of nucleated cells using one button ofyour differential cell counter
repeat with 4 more fields using a separate button foreach field
the five numbers should be close to each other in valueindicating good distribution of leucocytes
the total should be divided by a factor (CH and CH2microscopes = 18)
compare smear WCC to automated WCC
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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
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Oil immersion scan (x 100)
l repeat a difficult differential leucocyte count
l reassess abnormal leucocyte, erythrocyte and
platelet morphologyl estimate platelet count
count the number of platelets in 10 oil immersionfields
indirect platelet count = N x 109/L
compare indirect count to automated count
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Normal cellular components of ablood film
l erythrocytes (red cells)
l platelets
l leucocytes (white cells)
neutrophils
lymphocytes (large and small)
monocytes
eosinophils
basophils
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Erythrocytes
l most numerous
l 7-8 um
l round
l pink coloured
l central pale area
less than one thirdthe diameter of thecell
l no nucleus
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Platelets
l 1-3 um
l generally ovoid in
shape but with widevariation
l blue staining
l red or purplegranules throughoutcytoplasm
l no nucleus
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Neutrophils
l 10-15 um
l cytoplasm: pink with fine
violet-pink granulesl nucleus: dark blue-purple,
clumped nuclearchromatin, 2-5 lobes joined
by a thin chromatin strandl women may show
drumstick
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Small lymphocytes
l 10-12 um
l cytoplasm: very thin rim
around nucleus, light blue,usually no granules,sometimes a perinuclearclear zone
l nucleus: eccentric, round,no lobes, deep purple,very clumped chromatin
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Large lymphocytes
l 12-16 um
l cytoplasm: light blue,
abundant; occasionally cansee pink granules
l nucleus: eccentric, roundor slightly indented, no
lobes, less darkly staining,clumped nuclear chromatin
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Monocytes
l 12-20 um
l round, sometimes have
irregular edge
l cytoplasm: smoky blue,may have fine pinkgranules, may havevacuoles
l nucleus: variable, large,U-shaped, may be foldedor convoluted, finechromatin strands
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Eosinophils
l 12-18 um
l cytoplasm: filled with large,
round, uniform, orange-redgranules, often no stainedcytoplasm between granules
l nucleus: usually bi-lobed,less dark staining and lessintense nuclear chromatinclumping than neutrophils
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Basophils
l 10-15 um
l cytoplasm: filled with
densely-stainingvariable, purplish-black granules, oftencover nucleus
l nucleus: U-shaped,fine nuclearchromatin pattern,often not visible.
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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
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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 ofHaemostasis
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
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