Blood smear preparation

and staining
 Aim of blood smear

& Value oI‘ blood lilins:

›8 Examination of thin blood films is important

in the investigation and management of
anaemia, infections, and other conditions which
produce changes in the appearance of blood
cells and differential white cell count.

›# A blood film report can provide rapidly and at

low cost, useful information about a
patient's condition.
 Making blood films

G Three basic steps to make blood

i"ilm:

1. Preparation of blood smear.

2. Fixation of blood smear.

3. Staining of blood smear.

I. PREPARATION OF BLOOD SMEAR

M Specimen:

EDTA anticoagulated blood is preferred.

Blood smears can also be made from Pinger
stick blood directly onto slide.

• Reagents, equipment. and supplies:

(a) Spreaders
(b) Clean slides
 Making blood smear
'
8?, Three methods may be used to make
blood smears: $
1. The cover glass smear.
2. The wedge smear .
3. The spun smear.
• The spun smear requires an
automatic slide spinner. For the
purpose of this lab exercise, we will use
the wedge smear.
 WEDGE BLOOD SMEAR
é: Procedure:
1. Fill a capillary tube three-quarter full with the “.
anticoagulated specimen or a wooden stick. ›
[Link] a drop of blood, about 2 mm in diameter approximately
an inch from the frosted area of the slide.

[Link] the slide on a flat surface, and hold the narrow side of the
non frosted edge between your left thumb and forefinger.

[Link] your right hand, place the smooth clean edge of a second
(spreader) slide on the specimen slide, just in front of the blood
drop.

[Link] the spreader slide at a 30° angle, and draw it back against
the drop of blood.
6. Allow the blood to spread almost to the edges of the
slide.

[Link] the spread forward with one light, smooth, and fluid
motion. A thin film of blood in the shape of a bullet with a
feathered edge will remain on the slide.

[Link] the frosted edge with patient name, ID# and

date.

[Link] the blood film to air-dry completely before

staining. (Do not blow to dry. The moisture from your
breath will cause RBC artifact.)
STEPS FOR BLOOD FILM
tai bod hea
l y d
 Procedure notes
I. CHARACTERISTICS OF A GOOD
SMEAR:
1. A good blood film preparation
wilt be thick at the drop end and
thin at the opposite end.
Note: As soon as the drop of blood is
placed on the glass slide, the smear
should be made without delay. Any delay
results in anabnormal distribution of the ,
white blood cells, with many of the large
white cells accumulating at the thin edge
of the smear.
CHARACTERISTICS OF A
SMEAR:
GOOD
2. The blood smear should
occupy the central portion of the
slide.

[Link] blood smear should not touch

the edges. except for point of
application.

4. Should be margin free.

 n. The thickness of the spread
The thickness of the spread when pulling
the smear is determined by:

1. The angle of the spreader slide. (the |

greater the angle, thethicker and shorter .
the smear).

2. Size of the blood drop.

3. Speed of spreading.
 The thickness of the spread
,
Notes:
[Link] the hematocrit is increased, the
angle of the spreader slide should
be decreased.

[Link] the hematocrit is decreased, the

angle of the spreader slide should be
› increased.
high HCT

small angle

low HCT

large angle
Common causes of a poor blood smea
a. Drop of blood too large or too small.

b. Spreader slide pushed across the slide g

in a jerky manner.

c. Failure to keep the entire edge of the

spreader slide against the slide while
making the smear.

d. Failure to keep the spreader slide at a

300 angle with the slide.
 Common causes of a poor blood smear

e. Failure to push the spreader

slide completely across the slide.

f. Irregular spread with ridges

and long tail: Edge of
spreader dirty or chipped,
dusty slide.
 Common causes of a poor blood smear

g. Holes in film:
Slide contaminated with fat or grease and. air
•,
bubbles.

h. Cellular degenerative changes:

Delay in Cixing, inadequate fixing time or
methanol contaminated with water.
A: Blood film with jagged tail made
from a
spreader with achipped end.
B: Film which is too thick
C: Film which is too long, too wide,
uneven thickness and made on a
greasy slide.
Examples of
unacceptable
smears
Examples of unacceptable
smears
Notes:
1. Although this is the easiest and most popular methods
for producing a blood smear, it does not produce a quality
smear.

The WBCs are unevenly distributed and RBC distortion i

seen at the edges
Smaller WBCs such as lymphocytes tend to reside in the
middle of the feathered edge.

2. Large cells such as monocytes, immature cells and

abnormal cells can be found in the outer limits of this area

3. Spun smears produce the most uniform distribution

of blood cells.
 Biologic causes of a poor smear
, q
a. Cold agglutinin - RBCs will clump
together.
Warm the blood at 37o C for 5 minutes,
and then remake the smear.

b. Lipeiiiia - holes will appear in the smear.

There is nothing you can do to correct this. ,

c. Roulearix - RBC's will form into stacks

resembling coins.
There is nothing you can do to correct this.
SLIDE FIXATION” &
STAINING
 II. Fixing the films
&1 To preserve the morphology of the
cells, films must be fixed as soon as
possible after they have dried.

ér It is important to prevent contact

with water before fixation is complete.

£. Methyl alcohol (methanol) is the

choice,
alcohol") canethyl
although be [Link] (”absolute ,

G Methylated spirit (959< ethanol) must

not be used as it contains water.
 II. Fixing the films $
&To fix the films, place them in a '
covered staining jar or tray
containing the alcohol for 2-3
minutes. In humid climates it
might be necessary to replace
the methanol 2-3 times per
day, the old portions can be
used for storing clean slides.
 Staining the
ñ: Romanowsky staining:
Romanowsky stains are universally employed for
staining blood films and are generally very
satisfactory.

fi: There are a number of different combinations ›

of these dyes, which vary, in their
staining
characteristics.

1. May-Grunwald-Giemsa is a good method

for routine work.
2. Giemsa stain is thought to produce more
delicate
staining characteristics.
Romanowsky
staining: '
3. Wright's stain is a simpler method.
$
4. Leishman's is also a simple
method, which is especially suitable
when a stained blood film is required
urgently or the routine stain is not
available (e.g. at night).

5. Field's stain is a rapid stain

used primarily on thin films for
malarial parasites.
& The Princi
main gl
components of a
Romanowsky stain are:
&. A cationic e or basic dye (methylene blue
or its oxidation products such as azure
B), which binds to anionic sites and
gives a blue-grey color to nucleic acids
(DNA
RNA), or nucleoproteins, granules o
basophils and weakly to granules o
neutrophils
fl An anionic or acidic dye such as eosin
Y , or eosin B, which binds to
cationic sites on proteins and gives an
orange-red color to hemoglobin and p,
 Eosinophilic
granules

Blue Basophilic
nucleus granules
 Principle
éi Leishman's stain : a polychromatic stain
£1 Methanol : fixes cells to slide.
éi methylene blue stains RNA,DNA:
blue-grey color
h1. Eosin stains hemoglobin, eosin granules:
orange-red color
N. pH value of phosphate buffer is very
›,
important.
 STAINING
é[Link] smear arePROCEDURE
air dried.
9I. Flood the smear with stain.
B: Stain for 1-5 min.
Experience will indicate the
optimum time.
é. Add an equal amount of
buffer
solution and mix the stain by
blowing "
an eddy in the fluid. ”
é: Leave the mixture on the
slide for 10-
15 min.
é[Link] off by running water
When aminationdry, besamine
Excompletely lood smea r
the smear
’
with the microscope as follows:
Low power (10x) scan.
Determine the overall staining quality of
the blood smear.
a. Stain should not be too dark or too pale.

b. There should be no stain precipitate

present on smear.

[Link] should be appropriate color of

reddish pink.
 Examination blood
smear
[Link] have dark purple nuclei
with varying shades of blue cytoplasm.
e. Neutrophils have dark purple nuclei
with
reddish, granular cytoplasm.
[Link] have a lighter purple nucleus
with a gray-blue cytoplasm.
[Link] have bright
red/orange granules.
h. Basophils have dark purple nuclei
 Examination blood smear

BI. Determine if there is a good

distribution of the cells on the smear.
a. Scan the edges and center of the slide to
be sure there are no clumps of
RBCs, WBCs or platelets.
b. Scan the edges for abnormal cells.
c. High power (40 x) scan
& Find an optimal area for the detailed
examination and enumerations of cells.
a. The RBCs should not quite touch each
other.
b. There should be no area containing
large amounts of broken cells or
Precipitated stain.
c. The RBCs should have a
graduated central pallor.
d. Nuclei and cytoplasm of WBCs should be
‹
the proper color.
e. Platelets should be clearly visible.
Notes on the staining procedure:
›a Whichever method is used, it
is important to select dyes that are
not contaminated with other dyes
or metallic salts. !
›s Staining time must be specific
for each lot of stains and so we
must follow the kit procedure.
ts Bone marrow time staining must be );,
increased ”
Staining characteristics of a correctly stained
normal film:
n Nuclei Purple

» Erythrocytes Deep pink

• Neutrophils Orange-pink
• Blue; some small
Lymphocytes deep blue
lymphocytes Grey-blue
• Monocytes Blue
• Basophils
n C riiiiiiles Fine purple
» Neutrophils Red-orange
• Eosinophils Purple-black
• Basophils Fine reddish (azurophil)
• Monocytes Purple
Platelets
 Staining faults
BI. faint:
Too Staining time too short.
Excessive washing after
staining.

B Stain deposit:
Stain solution left inuncovered
jar. Stain solution not filtered.
Dirty slides.
 PH of the phosphate buffer
The phosphate buffer controls the PH
of the stain. If the PH is too acid, those
cells or cell parts taking up an acid dye
stain will stain pinker and the acid
components that stain with the basic
dye show very pale staining.
If the stain —buffer mixture is alkaline,
the red blood cells will appear grayish
blue and the white cell nuclei will stain
very deeply purple.
Therefore, to stain all cells and cell
parts well, the PH of the phosphate
buffer is critical.
Continue....
'
.. 1. The staining rack must be
exactly l o ‘ ainst
uneven se enin of $h sdea
2. Insuft"icient washing of the smears
when removing the stain and
buffer mixture may cause stain
precipitate on the dried smear.
3. Excessive rinsing of the
stained smear will cause the stain to
fade.
PERFORMING A MANUAL
DIFFERENTI&L
Noexw men•nRW
BLØØD
SMEAR

Eosl
Neutrop

Lymphosyțø
 MANUAL DIFFERENTIAL 1
Principle
A stained smearis examined
in order to determine the
leukocyte
percentage present
of and assesstype
each the oerythrocyte j
and platelet morphology.
Increases in any of the normal leukocyte types
or the presence of immature leukocytes or
erythrocytes in peripheral blood are important
diagnostically in a wide variety of inflammatory
disorders and leukemia.
Erythrocyte abnormalities are clinically
important in various anemia's.
R: Platelet size irregularities are
suggestive o particular thrombocyte disorders.
 MANUAL DIFFERENTIAL
& Specimen:
›a Peripheral blood smear made from g
EDTA- ,
anticoagulated blood.
›s blood
Smears collection
should from EDTA
be made specimens
within 1 hour
ofstored at 1room temperature to avoid
distortion of cell morphology.
›a Unstained smears can be stored
for indefinite periods in a dry environment,
but stained smears gradually fade unless
cover slipped.
 MANUAL DIFFERENTIAL

é: Reagents, supplies and

equipment:
[Link] cell counter
designed for differential
counts

[Link], immersion oil

and lens
paper
Procedure:
g: Focus the on the
microscope 10X
1. objective
Scan the(lowsmear to check for
power).
cell distribution, clumping, and
abnormal cells.
2. In scanning the mporta
smear i nt
irregular,
to such unusual
note anything as
rouleaux or RBC
clumping.
•Observe one field and record the
number of WBC according to the
different type then turn to another field
in the snake-liked direction.
•Avoid repeat or miss some cells
OBSERVATIONS UNDER X 4 OX : WBC
ESTIMATES

G Using the X 40 high dry with no

P Choose
oil. a portion of the peripheral
smear where there is only slight
overlapping of the RBCs.
£). Count 10 fields, take the total
number of white cells and divide by 10.
éi To do a WBC estimate by taking the
average number of white cells and
multiplying by 2000.
 OBSERVATIONS UNDER x 100:
PLATELET ESTIMATES
1. Use the oil immersion lens
estimate the number of platelets per
Pield.
2. Look at 5-6 fields and take
an !
average.

!
3. Multiply the average by 20,000.
81. Platelets per oil immersion field (OIF)
1) «8 platelets/OIF = decreased
MANUAL DIFFERENTIAL COUNTS
& These counts are done in the same
area as WBC and platelet estimates
with the red cells barely touching.
¥). This takes place under X 100 (oil) using |
› the zigzag method.
-hi Count 100 WBCs l
f
fl Reporting results
G Absolute number of cells/pl = 9o of cell
type in differential x white cell count.
 OBSERVING AND RECORDING
NUCLEATED RED BLOOD
CELLS (NRBCS)

8?, If 10 or more nucleated RBC's

(NRBC) are seen, correct the
White Count using this formula:

Corrected WBC Count =

WBC x 100/( NRBC +
100)
Reference values vary
depending on age:
 White blood
Leukocytes are classified into two main groups;
cells
granulocytes and nongranulocytes (also known
as agranulocytes).
1. The granulocytes, ( neutrophils, eosinophils,
and basophiles), have granules in their cell
cytoplasm.
Also have multilobed nucleus. As a result they
are also called polymorphonuclear leukocytes
or "polys"
The nuclei of neutrophils also appear
to be segmented, so
they may also be called " segmented
neutrophils or "segs".
2. The nongranuliicyte, (lymphocytes and
monocytes), do not have granules and have
nonlobular nuclei. They are sometimes referred
to as mononuclear leukocytes.
 Leukocytosis
% Leukocytosis, a WBC above 10,000 is q
usually due to an increase in one of the Live
types of white blood cells and is given the
name of the cell that shows the primary
increase.

1. Neutrophilic = neutrophilia
leuhocytosis
= lymphocytosis
2. Lymphocytic leukocytosis
= eosinophilia
[Link]
=monocytosis
IeuI‹ocytosis [Link]
= basophilia
leukocytosis [Link]

leukocytosis
 ta) Ensinnphil (c) N eutroph
il

Id) Small lymphocyte (e) Monocyte

[Link]
éi Neutrophils are so named because they
are not well stained by either eosin, a
red acidic stain, or by methylene blue, a j
basic or alkaline stain 3
'
’I
G Neutrophils are also known as "segs",
"PMNs" or "polys"
(polymorphonuclear).

éi They are the body's primary

defense against bacterial infection.
Neutrophils:
&Normally, most of the neutrophils
circulating in the bloodstream are in a
mature form, with the nucleus of the cell
being divided or segmented. Because of
the segmented appearance of the nucleus,
neutrophils are sometimes referred to as
"segs."

¥ The nucleus of less mature neutrophils is

not segmented, but has a band or rod-like
shape. Less mature neutrophils those
that have recently been released from the
bone marrow into the bloodstream - are
known as "bands" or "stabs".
 !\
n
Band Segmented
neutrophii neutrophile
Shift to left Increased bands mean acuti
infection, usually bacterial.
Shift to right Increased
hypersegmented neutrophile.
Increased neutrophils count (neutrophilia)
1. Acute bacterial infection.
2. Many inflammatory processes.
3. During physical stress.
4. With tissue necrosis.
5. Granulocytic leukemia.
Decreased neutrophil count (neutropenia)
6. Typhoid fever
7. Brucellosis
[Link] diseases, including hepatitis, influenza,
rubella, and mumps.
9.A great infection can also deplete the bone marrow
of neutrophils.
[Link] drugs used to treat cancer prodxce bone
marrow depression.
BAND NEUTROPHIL

Cytoplasm : pink

Granules: primary
secondary

Nucleus: dark purple blue

dense chromatin
SEGMENTED NEUTROPHIL

Cytoplasm : pink

Granules: primary
secondary

Nucleus: dark purple blue

dense chromatin
2-5 lobes
SEGMENTED NEUTROPHIL
2. Eosinophils

& The most common reasons for an

increase in the eosinophil count are
Allergic reactions such as hay fever,
asthma, or drug hypersensitivity.

[Link] infection
[Link]
leukemia
Eosinophils

Cytoplasm : full of granules •

Granules: large refractile, 1

orange-red

Nucleus: blue
dense chromatin
2 lobes like a pair of glass
3 Basophils
ñ The purpose of basophils is not completely
understood.

›a Basophils are phagocytes and contain heparin,

histamines, and serotonin.

›a Tissue basophils are also called” mast cells.

la Basophile counts are used to analyze allergic

reactions.

An alteration in bone marrow function such as

leukemia or Hodgkin's disease may cause an
increase in basophils.

›a Corticosteroid drugs may cause the body's

small
BASOPHIL

Cytoplasm : pink

Granules: dark blue

— black obscure
nucleus

Nucleus: blue
 [Link]
D: Lymphocytes are the primary components
of the body's immune system. They are the
source of serum immunoglobulins and of
cellular immune response.
X Two types of lymphocytes:

1. B lymphocyte : Humoral ¡
immunity
2. T lymphocyte : Cellular
immunity
Lymphocytes: $
Lymphocytes increase (lymphocytosis) in: '
[Link] viral infections
[Link].
[Link] fever
[Link] leukemia.
A decreased lymphocyte (lymphopenia)
count of less than 500 places a patient
at very high risk of infection,
particularly
viral infections.
Lymphocytes:

Diameter: small 7-9

large 12-16

Cytoplasm: medium blue

Granules: small agranular
Large a few primary granules

Nucleus: dark blue Yound

dense chromatin
 5. Monocytes
&). Monocytes are the largest cells in
normal blood. They act as phagocytes
in some inflammatory diseases and are
the body's second line of defense
against infection.
Diseases that cause monocytos
a include: is
•TubercuIosis
•BruceIlosis
•Malaria
•Rocky Mountain spotted
fever.
•Monocytic leukemia
•Chronic ulcerative colitis
Monocytes

Cytoplasm : grey blue

Granules: dust-like lilac

color granules
Nucleus: blue
large
irreg
ularl
y
shaped and
 Notes g
1.: A well-made and well-stained
smear is essential to the accuracy of the
differential count. The knowledge and ability
of the cell morphologist is critical to
high-quality results.

2. Before reporting significant

abnormalities such as blasts, malaria or
other significant finding on a patient's
differential, ask a more experienced tech
to review the smear for confirmation. In
clinical settings where a pathologist or
hematologist is present, the smear is set
aside for Pathologist Review.
3. If disrupted cells are present such as
smudge cells or basket cells, not them on the
It may be necessary to make an albumin
report.
smear to prevent the disruption of the cells.
RBC morphology and WBC morphology
must always be performed on the non-
albumin smear.

4. When the WBC is very

low (below 1,000/pL), it is difficult
to find enough WBCs
to perform a 100-
cell diGerential. In this situation,
a differential is
usually performed
by counting 50 cells. A notation on the
report must be made that only 50 white cells
5. When the WBC is very high (>50,000/pL), a
200-cell diG may be performed to increase
the accuracy of the diff.

6. Never hesitate to ask questions concerning

morphology or the identification of cells.
The diGerential is one of the most difficult
laboratory tests to learn. In fact, learning
about cells and their morphology is a process
that continues for as long as you perform
differentials.