bloodfilms-ms felicia

8/9/2019 bloodfilms-ms felicia

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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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l squash method


a second slide is placed on top


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l squash method


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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l squash method



action

air dry and label


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l squash method



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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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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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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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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