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BLOOD SMEAR

EXAMINATION
Making Blood smear

I- Preparation of blood smear


There are three types of blood
smears:
1. The cover glass smear.
2. The wedge smear .
3. The spun smear.
The are two additional types of blood
smear used for specific purposes

1. Buffy coat smear for WBCs < 1.0109/L


2. Thick blood smears for blood parasites .

WEDGE BLOOD SMEAR


Specimen : EDTA blood within 2 to 3
hours & collected to the mark on tube.
Not's : May change RBCs morphology
such as Spiculated (crenated) cells if :

1. Excessive amount of anticoagulant to


specimen
2. Old blood - long standing.

3. Warm environment (room temperature)


may hasten changes.

PROCEDURE
placing a drop of blood from mixed
sample on a clean glass slide.
Spreader slide using another clean glass
slide at 30-40 degree angle.

Control thickness of the smear by


changing the angle of spreader slide
Allow 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

The thickness of the spread


Notes:
1. If the hematocrit is increased, the angle of
the s preader slide should be decreased.
2. If the hematocrit is decreased, the angle of
the spreader slide should be increased.

high HCT

small angle
low HCT

large angle

CHARACTERISTICS OF A GOOD SMEAR


1. Thick at one end, thinning out to a smooth
rounded feather edge.
2. Should occupy 2/3 of the total slide area.

3. Should not touch any edge of the slide.


4. Should be margin free, except for point of
application.
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.

COMMON CAUSES OF A POOR BLOOD


SMEAR

1. Drop of blood too large or too small.

2. Spreader slide pushed across the slide in a jerky manner.


3. Failure to keep the entire edge of the spreader slide against
the slide while making the smear.

4. Failure to keep the spreader slide at a 30 angle with the slide

5. Failure to push the spreader slide completely across the slide.

6. Irregular spread with ridges and long tail: Edge of spreader


dirty or chipped; dusty slide
7. Holes in film: Slide contaminated with fat or grease

8. Cellular degenerative changes: delay in fixing, inadequate


fixing time or methanol contaminated with water.

Examples of unacceptable smears

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.
D: A well-made blood film.

Examples of unacceptable smears

BIOLOGIC CAUSES OF A POOR SMEAR


1. Cold agglutinin - RBCs will clump
together. Warm the blood at 37 C for 5
minutes, and then remake the smear.

2. Lipemia - holes will appear in the smear.


There is nothing you can do to correct this.

3. Rouleaux - RBCs will form into stacks


resembling coins. There is nothing you
can do to correct this

Notes:
1.

Although this is the easiest and most popular methods for


producing a blood smear, it does not produce a quality smear.

2.

The WBCs are unevenly distributed and RBC distortion is seen at


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

3.

Large cells such as monocytes, immature cells and abnormal cells


can be found in the outer limits of this area.

4.

Spun smears produce the most uniform distribution of blood


cells.

SLIDE FIXATION &


STAINING
LEISHMAN'S STAIN

II- Fixing the films


To preserve the morphology of the cells, films must be fixed as soon as
possible after they have dried.

It is important to prevent contact with water before fixation is complete.


Methyl alcohol (methanol) is the choice, although ethyl alcohol ("absolute
alcohol") can be used.
Methylated spirit (95% ethanol) must not
be used as it contains water.
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.

III. Staining the film


Romanowsky staining:
Romanowsky stains are universally employed for staining blood
films and are generally very satisfactory.
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.

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.

Principle
The main components of a Romanowsky stain are:
A cationic 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 or RNA),
nucleoproteins, granules of basophils and weakly to granules
of neutrophils
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 eosinophil granules.
pH value of phosphate buffer is very important.

Eosinophilic granules

Blue nucleus

Basophilic granules

Staining procedure (Leishmans stain)


Thin smear are air dried.
Flood the smear with stain.

Stain for 1-5 min. Experience will indicate the optimum time.
Add an equal amount of buffer solution and mix the stain by
blowing an eddy in the fluid.
Leave the mixture on the slide for 10-15 min.
Wash off by running water directly to the centre of the slide
to prevent a residue of precipitated stain.
Stand slide on end, and let dry in air.

Staining procedure
Thin smear are air dried.
Flood the smear with stain.
Stain for 1-5 min. Experience will indicate the
optimum time.
Add an equal amount of buffer solution and mix
the stain by blowing an eddy in the fluid.
Leave the mixture on the slide for 10-15 min.
Wash off by running water directly to the centre of
the slide to prevent a residue of precipitated stain.
Stand slide on end, and let dry in air.

TOO ACIDIC

SUITABLE

TOO BASIC

CAUSES & CORRECTION


Too Acid Stain:

1. insufficient staining time


2. prolonged buffering or washing
3. old stain

Correction:

1)

lengthen staining time

2)

check stain and buffer pH

3)

shorten buffering or wash time

Too Alkaline Stain:


1. thick blood smear
2. prolonged staining

3. insufficient washing
4. alkaline pH of stain components
Correction :

1) check pH
2) shorten stain time
3) prolong buffering time

PERFORMING A MANUAL
DIFFERENTIAL AND ASSESSING RBC
MORPHOLOGY

PRINCIPLE
White Blood Cells.
1. Check for even distribution and
estimate the number present (also,
look for any gross abnormalities
present on the smear).
2. Perform the differential count.

PRINCIPLE
Red Blood Cells, Examine for :
1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination

Platelets.
1. Estimate number present.
2. Examine for morphologic abnormalities.

PROCEDURES
Observations Under 10
1. Check to see if there are good counting
areas available free of ragged edges and cell
clumps.
2. Check the WBC distribution over the
smear.
3. Check that the slide is properly stained.
4. Check for the presence of large platelets,
platelet clumps, and fibrin strands.

OBSERVATIONS UNDER 40X : WBC


ESTIMATES
Using the 40 high dry with no oil.
Choose 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.
To do a WBC estimate by taking the average
number of white cells and multiplying by 2000.

OBSERVATIONS UNDER 100: PLATELET


ESTIMATES
1. Use the oil immersion lens estimate the
number of platelets per field.
2. Look at 5-6 fields and take an average.
3. Multiply the average by 20,000.

4. Note any macroplatelets.


Platelets per oil immersion field (OIF)
1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased

PLATELETS

OBSERVING AND RECORDING


NUCLEATED RED BLOOD CELLS (NRBCS)
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)


Example : If WBC = 5000 and 10 NRBCs have been
counted

Then

5,000 100/110 = 4545.50

The corrected white count is 4545.50.

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 100 (oil) using the
zigzag method.
Count 100 WBCs including all cell lines from
immature to mature.
Reporting results
Absolute number of cells/l = % of cell type in
differential x white cell count

Observing direction:

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

NORMAL PERIPHERAL BLOOD SMEAR

LEUKOCYTOSIS
Leukocytosis, a WBC above 10,000 is usually due to
an increase in one of the five types of white blood
cells and is given the name of the cell that shows the
primary increase.
1. Neutrophilic leukocytosis

= neutrophilia

2. Lymphocytic leukocytosis

= lymphocytosis

3. Eosinophilic leukocytosis

= eosinophilia

4.Monocytic leukocytosis

=monocytosis

5.Basophilic leukocytosis

= basophilia

STAB NEUTROPHIL

Diameter:12-16

Cytoplasm : pink

Granules: primary
secondary

Nucleus: dark purple blue

dense chromatin

BAND NEUTROPHIL

SEGMENTED NEUTROPHIL

Diameter: 12-16

Cytoplasm : pink

Granules: primary
secondary

Nucleus: dark purple blue


dense chromatin
2-5 lobes

SEGMENTED NEUTROPHIL

1.NEUTROPHILS
Neutrophils are so named because they are not well
stained by either eosin, a red acidic stain, or by
methylene blue, a basic or alkaline stain.
Neutrophils are also known as "segs", "PMNs" or
"polys" (polymorphonuclear).

They are the body's primary defense against bacterial


infection.

Increased neutrophils count (neutrophilia)


1.

Acute bacterial infection.

2.

Granulocytic leukemia.

Decreased neutrophil count (neutropenia)


1.

Typhoid fever

2.

Brucellosis

3.

Viral diseases, including hepatitis, influenza, rubella, and


mumps.

LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL


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".
Left-shift: non-segmented neutrophil > 5%
Right-shift: hypersegmented neutrophil >3%

Segmented neutrophile

Band neutrophil

Shift to left Increased bands mean acute infection, usually bacterial.


Shift to right Increased hypersegmented neutrophile.

EOSINOPHIL

Diameter: 14-16

Cytoplasm : full of granules

Granules: large refractile, orange-red

Nucleus: blue
dense chromatin
2 lobes like a pair of glass

EOSINOPHIL

The most common reasons for an increase in the


eosinophil count are
1. Allergic reactions such as hay fever, asthma, or drug
hypersensitivity.

2. Parasitic infection
3. Eosinophilic leukemia

BASOPHIL

Diameter: 14-16
Cytoplasm : pink

Granules: dark blue black


obscure nucleus
Nucleus: blue

BASOPHIL

Basophils
The purpose of basophils is not completely understood.

Basophile counts are used to analyze allergic reactions.


An alteration in bone marrow function such as leukemia
may cause an increase in basophils.

LYMPHOCYTE

Diameter: small 7-9


large 12-16

Cytoplasm: medium blue

Granules: small agranular


large a few
primary granules

Nucleus: dark blue \round


dense chromatin

LYMPHOCYTE

4.LYMPHOCYTES
Lymphocytes are the primary components of the
body's immune system. They are the source of
serum immunoglobulins and of cellular immune
response.
Two types of lymphocytes:
1. B lymphocyte : Humoral immunity

2. T lymphocyte : Cellular immunity

Lymphocytes increase (lymphocytosis) in:


1.Many viral infections
2.Tuberculosis.
3.Typhoid fever
4.Lymphocytic leukemia.
A decreased lymphocyte (lymphopenia) count of less than 500
places a patient at very high risk of infection, particularly
viral infections.

MONOCYTE
Diameter: 14-20
Cytoplasm : grey blue
Granules: dust-like lilac
color granules
Nucleus: blue
large irregularly shaped
and folded

MONOCYTE

Diseases that cause a monocytosis include:


Tuberculosis
Brucellosis
Malaria
Monocytic leukemia

NOTES
1. Do not count cells that are disintegrating

eosinophil with no cytoplasmic membrane and with


scattered granules

Pyknotic cell (nucleus extremely condensed and


degenerated, lobes condensed into small, round clumps with
no filaments interconnecting).

smudge cells
Basket cells
smudge cells

Basket cells

2- Abnormal differentials
1. 200 Cell diff:

a. WBC > 15.0 (>20.0 for babies under 1 month and

labor unit)

b. Three or more basophils seen.


2. If more than five immature WBC's are seen (or any blasts) let
someone else diff slide and average results.
3. Correct WBC for NRBC's if you seen ten or more NRBCs/100 WBC.
4. Always indicate number of cells counted on diff.

5. If any cell type is extremely elevated (such as bands, monos, or eos >
20) indicate that you are aware of the abnormality by circling or
checking on the card next to the results.

3-Morphologic Changes Due To Area Of Smear


Thin area- Spherocytes which are really
"spheroidocytes" or flattened red cells. True
spherocytes will be found in other (Good) areas of
smear.
Thick area - Rouleaux, which is normal in such
areas. Confirm by examining thin areas. If true
rouleaux, two-three RBC's will stick together in a
"stack of coins" fashion..

tail

body

head

4. 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.

5. Before reporting significant abnormalities such as blasts, malaria or


other significant finding on a patients 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.

6. Never hesitate to ask questions concerning morphology or the


identification of cells. The differential 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.